ORGANIC ACIDS TEST (OAT) PROVIDER SUPPORT GUIDE

Mosaic Diagnostics | 800-288-0383 | customerservice@mosaicdx.com | MosaicDX.com | 8400 W 110th Street, Suite 500, Overland Park, KS 66210

Disclaimer: The content of this Provider Support Guide is for informational purposes only and is not intended to be a substitute for medical advice from a licensed healthcare practitioner. The statements in this report have not been evaluated by the Food and Drug Administration and are intended to be lifestyle choices for potential risk mitigation. Please consult a licensed healthcare practitioner for medication, treatment, diet, exercise or lifestyle management as appropriate. This product is not intended to diagnose, treat, or cure any disease or condition.

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Provider Support Guide

INTRODUCTION

What are Organic Acids

Organic acids are intermediates or end products of metabolic reactions that are excreted in urine and play essential roles in pathways related to energy production, detoxification, neurotransmitter synthesis, and amino acid breakdown. All organisms produce them, including specific ones from bacteria and fungi, allowing for additional insights into gut microbial activity.

When these metabolic pathways function efficiently, organic acids are processed and excreted at expected levels. However, when enzymatic function is impaired, nutrient cofactors are deficient, and/or inhibitors such as toxins or microbial byproducts are present, these pathways may become blocked, leading to abnormal accumulation of organic acids in the urine. Excess from organic acids that are specific to other organisms can also allude to overgrowth or infection from that organism.

How do they Apply to Integrative Health?

Clinically, the Organic Acids Test (OAT) offers valuable insight into a patient’s biochemical pathways and microbiome. It provides practitioners with a comprehensive snapshot of multiple body systems in a single test, making it especially useful in cases involving complex or multisystem complaints. The test can help identify imbalances in energy metabolism, mitochondrial dysfunction, oxidative stress, detoxification, neurotransmitter metabolism, nutrient deficiencies, microbial overgrowth, and even, in rare instances, inborn errors of metabolism. In addition to its diagnostic utility, repeat testing can be used to monitor therapeutic progress, such as the effectiveness of orthomolecular interventions, antibiotics, or anti-fungal therapies.

intake, or nutrient insufficiencies.

Organic acids are measured in urine and normalized to creatinine to account for differences in hydration status. Because urine reflects circulating metabolites over several hours, it provides a non-invasive and practical medium for evaluating systemic function. While dietary intake and supplement use near the time of collection can influence results, following test preparation instructions, including avoiding certain foods and supplements, helps improve validity and clinical interpretation.

The Organic Acids Test (OAT) from Mosaic Diagnostics analyzes metabolic byproducts to provide a comprehensive snapshot of nutritional and metabolic health, supporting a functional and integrative approach to patient care. This also helps practitioners better understand each person’s unique biochemistry to help optimize their health journey.

The underlying mechanism of OAT interpretation relies on the understanding that organic acid accumulation and patterns often signal a disruption in normal metabolic flow. This disruption may stem from genetic enzyme defects, toxic exposures, poor dietary

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SUMMARY PAGE

The Summary of Results page was developed to give clinicians a streamlined high-level overview of Organic acids Test (OAT) findings. The summary organizes abnormal organic acids into six key clinical categories, allowing practitioners to quickly identify patterns and prioritize areas of concern.

Each category is assessed and results color-coded to reflect the level of imbalance (green = optimal, yellow = marginal, red =significant), allowing clinicians to quickly prioritize areas of concern.

Category-specific icons are also introduced and carried through the interpretation section, to indicate the category(s) associated with each organic acid.

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KEY CLINICAL CATEGORIES

Allowing for easier visualization and interpretation of results quickly. Each flagged analyte is grouped within a category based on its clinical relevance, with accompanying comments that explain the significance of the findings.

These six clinical insight categories include:

Microbial Overgrowth

Toxic Exposure

Markers of fungal yeast and mold, bacteria, and Clostridia species, and their metabolic byproducts.

Organic acids associated with environmental toxins and toxicants, oxidative stress, and glutathione depletion.

Mitochondrial Health

Methylation/Detoxification

Intermediates of glucose, fatty acid, and amino acid metabolism involved in energy production.

Metabolites reflecting methyl group cycling, glutathione status, and nutrients involved in both methylation and detoxification.

Neurotransmitter Metabolites

Nutritional Needs

Dopamine, serotonin, and norepinephrine/epinephrine pathway metabolites, providing insights into neurotransmitter imbalances.

Direct and indirect biomarkers reflecting the functional demand for B-vitamins, antioxidants, and other key micronutrients.

This categorization system gives practitioners a clear, functional snapshot of metabolic imbalances, supporting efficient clinical decision-making and individualized patient care. It also coincides with the grouping of the interpretations in the next section, connecting how they influence the six categories of clinical insight, and summary page.

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EXPLANATION OF INTERPRETATIONS FORMAT

These interpretations are based on an extensive review of biochemical and scientific literature. These interpretations expand on the information provided on the OAT report. Each is structured in the following way:

6 Tartaric acid

Tartaric acid is a naturally occurring dicarboxylic acid associated with the activity of Aspergillus, Penicillium, and to a lesser extent, Candida and Saccharomyces.127 128 129 Elevated levels may indicate fungal dysbiosis.130 It can also inhibit the Krebs cycle by disrupting malic acid utilization, potentially impacting mitochondrial function.131 Additionally, dietary sources such as grapes, red wine, tamarind, and certain food additives may contribute to elevated levels.132 133 134

The icons indicate the clinically relevant categories associated with Tartaric Acid. The expanded interpretations below provide information related to each category.

Microbial Overgrowth (Mold and/or Yeast)

Elevated levels of tartaric acid are associated with mold and potentially yeast as it is known to be produced by Aspergillus and Penicillium and possibly Candida and Saccharomyces.10,39,40,41,42 Tartaric acid can also encourage growth of yeast.41

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Tartaric acid is an analog of malic acid and may inhibit this part of the Citric Acid Cycle.42 Evaluate Fumaric acid 25 and Malic acid 26 abnormalities for further insights if this is suspected.

Toxic Exposure

If tartaric acid is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic byproducts of mold (e.g., Aspergillus), pose significant health risks.19,20 Identifying specific mycotoxins can aid effective remediation and address mold- related health concerns.19,21 Regardless of source, tartaric acid can act as a muscle toxin in very high concentrations, inhibiting malic acid production and potentially causing adverse effects such as paralysis. These toxic effects are very rare and are usually associated with extremely elevated levels.46

Refer to the Mold/Mycotoxin Exposure Table for corresponding metabolites and patterns.

Additional Insights

Dietary: Elevated urinary tartaric acid levels can result from dietary intake of tartaric-rich foods, especially grapes and red wine, as well as tamarind and hawthorn.43,44 It is also used as a food additive for its antioxidant properties and tart flavor and can be found in candies (especially lime and grape flavors), mustards, jams, fruit juices, ice creams, and leavening agents (e.g., cream of tartar).43,45

Low Values There is no known clinical significance for low values.

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Helpful Reference Tables

The following tables are useful references and support the interpretations.

Microbial Overgrowth Tables

These subsequent tables highlight metabolites linked to specific microbial organisms and may support clinical interpretation by revealing patterns suggestive of overgrowth. Findings should be interpreted in context with the clinical presentation to support identification of potential microbial involvement.

TABLE 1: FUNGAL OVERGROWTH

Fungus Associated OAT Metabolite

Mold (Aspergillus, Penicillium, Cladosporium)

Citramalic acid 1 5-Hydroxymethyl-2-furoic acid 2 Furan-2,5-dicarboxylic acid 4 Furancarbonylglycine 5 Tartaric acid 6

Oxalic acid 21 Fumaric acid 25 Malic acid 26 Citric acid 29

Candida

Tartaric acid 6 Arabinose 7 Oxalic acid 21

Glutaric acid 53 Methylcitric (Biotin) 57 Orotic acid 60

Saccharomyces spp.

Tartaric acid 6 Glycolic acid 20 Oxalic acid 21

Lactic acid 22 Succinic acid 24

Various Yeast

3-Oxoglutaric acid 3 Carboxycitric acid 8 Glyceric acid 19

Glycolic acid 20 2-Oxoglutaric acid 27

TABLE 2: BACTERIAL OVERGROWTH

Bacteria Associated OAT Metabolite

Firmicutes phylum

Tricarballylic acid 9

Clostridiales and Faecalibacterium prausnitzii Various Bacteria

Hippuric acid 10

2-Hydroxyphenylacetic acid 11 5HIAA 38 Quinolinic acid 39

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Bifidobacterium, Clostridium, Escherichia coli, and Eubacterium

4-Hydroxybenzoic acid 12 4-Hydroxyhippuric acid 13

Bifidobacteria, E. coli, or Lactobacilli Various Clostridium spp.

DHPPA 14

4-Hydroxyphenylacetic acid 15 HPHPA 16 4 Cresol 17 3-Indoleacetic acid 18

HVA 33 VMA (low) 34 HVA/VMA ratio 35 DOPAC 36 Phenyllactic acid 69

Acetobacter and Gluconobacter Acetobacter, Escherichia, or Rhodococcus

Glyceric acid 19

Glycolic acid 20

Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus faecalis, Streptococcus group B, Bacillus

Lactic acid 22

Prevotellaceae, Veillonellaceae Gram-negative bacteria

Succinic acid 24

Malic acid 26

Corynebacterium glutamicum

2-Oxoglutaric acid 27

Butyrate-producing bacteria

Ethylmalonic acid 45

Propionic acid producing bacteria B. fragilis and P. copri, Bifidobacterium longum, Collinsella aerofaciens and H. Pylori, and arious Firmicutes

Methylmalonic acid 50

Pyridoxic acid 51

Lactobacillus plantarum

Pantothenic acid 52

Bifidobacterium longum, Lactobacillus spp.

Methylcitric acid (Biotin) 57

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Bacteroidetes spp. and thermophilic lactic acid producing bacteria

Pyroglutamic acid 58

Escherichia coli

Orotic acid 60

Pseudomonas, Bacillus, Azospirillum, Salmonella, Achromobacter, Vibrio, Yersinia, and Mycobacteria

2-Hydroxyhippuric acid 61

Lactic acid producing bacteria, and Clostridium species

2-Hydroxyisocaproic acid 65

Bifidobacteria, Lactobacillus, and Clostridium spp.

4-Hydroxyphenyllactic acid 72

Clostridium aminobutyricum, Pseudomonas spp

4-Hydroxybutyric acid 75

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Toxic Exposure Tables

The following tables highlight metabolites linked to specific toxic exposures (mycotoxins, toxicants, and heavy metals) and can support clinical interpretation by revealing patterns suggestive of exposure. Findings should be interpreted in context with the clinical presentation and the patient’s history.

TABLE 3: MOLD/MYCOTOXINS EXPOSURE Exposure Category Mold and Mycotoxin Association Associated OAT Metabolite

Mold and Mycotoxins (Aspergillus, Penicillium, Fusarium, Cladosporium)

Aspergillus spp

Citrimalic acid 1 Furancarbonylglycine 5 Fumaric acid 25 Citric acid 29

Aspergillus, Cladosporium spp

5-Hydroxymethyl-2-furoic acid 2

Aspergillus, Fusarium spp

Tricarballylic acid 9

Aspergillus, Penicillium spp

Furan-2,5-dicarboxylic acid 4 Tartaric acid 6 Oxalic acid 21 Malic acid 26

Aflatoxins and other Mycotoxins

Succinic acid 24

OTA, Fusaric acid (Fusarium)

HVA 33

Fusaric acid

VMA (Low) 34 HVA/VMA 35 DOPAC 36

Various Mycotoxins

Pyroglutamic acid 58 2-hydroxybutyric acid 59

*Of note this does not rule out mold or mycotoxin exposure, rather it serves as clues to potentially identify involvement.

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TABLE 4: TOXICANT EXPOSURE

Exposure Category Toxicant Associated OAT Metabolite

Industrial Solvents / VOCs / Parabens

Polyethylene furanoate (PEF)

Furan-2,5-dicarboxylic acid 4

Toluene

Hippuric acid 6

Parabens

4-Hydroxybenzoic acid 12 4-Hydroxyhippuric acid 13

Ethylene glycol, polyglycolate, trichloroacetic acid

Glycolic acid 20

Ethylene glycol, ethylene oxide

Oxalic acid 21

Polychlorinated Biphenyls (PCBs)

HVA/DOPAC ratio- Low 37

Styrene, benzene, acrylonitrile, 1-bromopropane, 1,3 butadiene, ethylene oxide, vinyl chloride

Pyroglutamic acid 58

Styrene and ethylbenzene

Mandelic acid 68

4-Hydroxyphenylacetic acid 15 HPHPA 16 4-Cresol 17 3-Indoleacetic acid 18

Pesticides

Glyphosate, Organophosphate

Numerous

HVA 33 DOPAC 36

Other

Ingredients in various industrial application

Sebacic acid 49

Acrylamide

5HIAA 38

Phthalates

Quinolinic acid 39

Organic solvents and pollutants

2-Hydroxyhippuric acid 61

Acrolein

Malonic acid 74

GHB

4-Hydroxybutyric acid 75

General Toxicant Exposure

Succinic acid 24 Pyroglutamic acid 58 2-Hydroxybutyric acid 59

*Note: this does not rule out exposure, rather it serves as clues to potentially identify toxicant involvement.

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TABLE 5: HEAVY METAL EXPOSURE

Exposure Category Associated OAT Metabolite Additional Details

Heavy Metals/ Elements Antimony, cadmium, and mercury

Lactic acid 22

Antimony, cadmium, and mercury

Pyruvic acid 23

Aluminum, Cadmium, and other heavy metals

Succinic acid 24

Aluminum, cadmium, gallium, mercury, silver, and zinc

Fumaric acid 25

Arsenic and cadmium

Malic acid 26

Aluminum, arsenic, and fluoride

Aconitic acid 28

Aluminum, arsenic, cadmium, mercury, and tin

Citric acid 29

Aluminum, arsenic, lead, manganese, and mercury

HVA 33

Aluminum, lead, manganese, and mercury

VMA (Low) 34

Aluminum and manganese

HVA/VMA 35

Aluminum, arsenic, lead, manganese, and mercury

DOPAC 36

Various Heavy Metals

Pyroglutamic acid 58

Various Heavy Metals

2-Hydroxybutyric acid 59

Lead and uranium

Phosphoric acid 76

*Of note this does not rule out exposure, rather it serves as clues to potentially identify heavy metal involvement.

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TABLE 6: NUTRIENT–MARKER REFERENCE TABLE

The following table highlights metabolites linked to specific nutrients and may support clinical interpretation by revealing patterns suggestive of insufficiencies.

Nutrient/Category Relevant OAT Marker(s) Interpretation

B1 (Thiamin)

Required for pyruvate dehydrogenase; elevation: impaired carbohydrate metabolism, potential thiamin need, or mitochondrial stress.

• Glycolytic markers 22 – 23 • 2-Oxoglutaric acid 27 • 2-Hydroxyhippuric acid 61 • BCKA Metabolites 62 – 66

B2 (Riboflavin) Needed for acyl-CoA dehydrogenase and glycolate oxidase; elevations in Krebs or fatty acid markers may reflect B2 need.

• Pyruvic acid 23 • Succinic acid 24 , 2-Oxoglutaric acid 27 • HVA 33 , VMA 34 , DOPAC 36 , HVA/DOPAC 36 • Tryptophan metabolites 38 – 40 • Folate metabolites 41 , 42 • Ketone and Fatty Acid markers 45 – 49 • Glutaric acid 53 • Indicators of Detoxification 59 , 60 • Amino Acid metabolites 62 – 66 68 – 77

B3 (Niacin)

Niacin is required for NAD+/ NADP+ production; abnormal tryptophan metabolites or Krebs cycle imbalances may indicate B3 insufficiency or inflammation- driven demand.

• Glyceric acid 19 • Glycolytic markers 22 , 23 • Malic acid 26 , 2-Oxoglutaric acid 27 • VMA 33 , HVA/VMA 35 • Quinolinic acid 39 , Kynurenic acid 40 • 3-Hydroxybutyric acid 43 , acetoacetic acid 44 • 2-Hydroxybutyric acid 59 • Amino acid metabolites 62 – 66 , 75

B5 (Pantothenic Acid) Pantothenic acid is a precursor to CoA; elevations in Krebs or fatty acid markers may suggest impaired energy metabolism and B5 need.

• Hippuric acid 10 • Pyruvic acid 23 • 2-Oxoglutaric acid 27 • Pantothenic acid 52 • BCKA metabolites 62 – 66

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B6 (Pyridoxine) Cofactor for transamination and neurotransmitter synthesis; elevations in oxalates and neurotransmitter markers may indicate a functional B6 deficiency. Influential in methylation and energy production.

• 2-Hydroxyphenylacetic acid 11 • Oxalate metabolites 20 – 21 • HVA 33 , VMA 34 , DOPAC 36 • Tryptophan metabolites 38 – 40 • Pyrimidine metabolites 41 , 42 • Pyridoxic acid 51 • Indicators of Detoxification 58 , 59 • 2-Hydroxyisocaproic 65 , 2-Oxo-4-methiolbutyric acids 67

Cofactor for carboxylase enzymes; elevations in methylmalonic or methylcitric acids may reflect biotin deficiency or dysbiosis- related interference.

B7 (Biotin)

• Methylmalonic acid 50 • Methylcitric acid 57

Essential for methylation and nucleotide synthesis; abnormalities in folate metabolites or 2-hydroxybutyric acid may suggest B9 deficiency or MTHFR-related need.

B9 (Folate)

• HVA/DOPAC 37 • Pyrimidine metabolites 41 – 42 • 2-Hydroxybutyric acid 59 • Amino Acid metabolites 68 – 70

Abnormal = functional or genetic B12 deficiency.

B12 (Cobalamin)

• Succinic acid (L) 24 • HVA/DOPAC 37 • Pyrimidine Metabolites 41 – 42 • Methylmalonic acid 50 • Methylcitric acid 57 • Indicators of Detoxification 59 , 60

Vitamin C Excess can potentially raise oxalates; deficiency may impact immunity and antioxidant defenses.

• Aconitic acid 28 • HVA 33 , VMA 34 , HVA/VMA 35 , DOPAC 36 • Ascorbic acid 54 • Amino Acid Metabolites 68 – 70

CoQ10 (Ubiquinone) Essential for the electron transport chain; low CoQ10 may impair mitochondrial function.

• 4-hydroxybenzoic acid 12 • Lactic acid 22 • Succinic acid 24 • 3-Hydroxy-3-Methylglutaric acid (CoQ10) 55

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Calcium Can be depleted by tricarballylic or oxalic acids, while an insufficiency can influence enzymatic function in mitochondrial function. Excess can impair neurotransmitter metabolism.

• Tricarballylic acid 9 • Oxalic acid 21 • 2-Oxoglutaric acid 27 • Citric acid 29 • HVA/DOPAC ratio 37

Magnesium

Needed for ATP-dependent enzymes; deficiency can impair oxalate, Krebs, and ketone metabolism. Also necessary for proper catecholamine metabolism.

• Tricarballylic acid 9 • Oxalic acid 21 • Pyruvic acid 23 • Citric Acid Cycle metabolites 26 , 27 , 29 • HVA 33 , VMA 34 , DOPAC 36 , HVA/DOPAC 37 • 5-HIAA 38 • Ketone and Fatty Acid markers 47 , 48 • Indicators of Detoxification 58 , 60 • Amino Acid metabolites 62 – 65

Zinc

Cofactor for kynureninase; deficiency may shift tryptophan pathway toward quinolinic, zinc can inhibit 27 as well as support reactions.

• Tricarballylic acid 9 • Lactic acid 22 • 2-Oxoglutaric acid 27 • 5-HIAA 38 • Ascorbic acid 54 • Orotic acid 60

Iron Cofactor in mitochondrial SOD and metabolism of oxalate precursors.

• Oxalic acid 21 • Lactic acid 22 • Mitochondrial – Citric Acid Cycle markers 24 , 25 , 26 , 28 , 29 • HVA 33 , VMA 34 , DOPAC 36 , HVA/DOPAC 37 • Quinolinic acid 39 • Ascorbic acid 54 • Amino Acid metabolites 68 – 70

Glutathione Markers of oxidative stress, methylation support; elevation: glutathione depletion or detox strain.

• Glycolic acid 20 • Aconitic acid 28 , Citric acid 29

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Glycine

Building block of glutathione; elevated pyroglutamic acid could indicate glycine deficiency.

• Hippuric acid 10 , 4-Hydroxyhippuric acid 13 • Pyroglutamic acid 58

• Ketone and Fatty Acid Oxidation markers 45 – 49

Carnitine

Needed for FA transport into mitochondria; elevations suggest carnitine need or mitochondrial block.

Phenylalanine/ Tyrosine

Involved in the synthesis of dopamine, norepinephrine, and epinephrine.

• 2-Hydroxyphenylacetic acid 11 • HVA 33 , VMA 34 , DOPAC 36 , Mandelic 68 , Phenyllactic acid 69 , Phenylpyruvic acid 70

• 5-Hydroxyindoleacetic acid (5-HIAA) 38 , Quinolinic acid 39 , Kynurenic acid 40

Tryptophan

Precursor to these neurotransmitters has the potential to go down kynuernic pathway vs serotonin.

Lipoic Acid

Coenzyme in pyruvate and a-KG dehydrogenase; need suspected if elevations + oxidative stress markers.

• Pyruvic acid 23 • 2-Oxoglutaric acid 27

Disclaimer: The content of this Provider Support Guide is for informational purposes only and is not intended to be a substitute for medical advice from a licensed healthcare practitioner. The statements in this report have not been evaluated by the Food and Drug Administration and are intended to be lifestyle choices for potential risk mitigation. Please consult a licensed healthcare practitioner for medication, treatment, diet, exercise or lifestyle management as appropriate. This product is not intended to diagnose, treat, or cure any disease or condition.

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INTERPRETATIONS

Intestinal Microbial Overgrowth

FIGURE 1

In additional to organic acids being biproducts of human metabolic pathways, various microorganisms, including bacteria and fungi, can metabolize certain substrates into organic acids through metabolic process such as fermentative and oxidative pathways. Once absorbed, these metabolic byproducts enter circulation and are transported to the liver, where partial metabolism may occur, and then eliminated via renal filtration and excreted in the urine. Measurement of these urinary organic acids can provide an indirect biomarker profile of microbial overgrowth or dysbiosis.

METABOLISM ORGANIC ACID PRODUCTION

BACTERIA FUNGUS HUMAN METABOLISM

URINE

e.g., Candida Arabinose Clostridia HPHPA

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YEAST AND FUNGAL MARKERS

1 Citramalic acid

Citramalic acid, produced by microorganisms such as Aspergillus niger and some bacteria, can be a marker of dysbiosis and fungal overgrowth, and may be linked to mold exposure.1-4 Elevated levels may impair energy production by disrupting the Citric Acid Cycle and are influenced by dietary intake of foods like apples, tomatoes, and wine.5-9

Microbial Overgrowth (Mold)

Citramalic acid may originate from fungi such as Aspergillus niger.1,5,10 While certain bacteria, including Rhodospirillum rubrum, Chloroflexus aurantiacus, and Alcaligenes xylosoxydans can also produce this metabolite, these organisms are not found in the human microbiome.11-15 Although Aspergillus is not a typical resident in the human GI Tract, it can be ingested, and there is potential to overgrow.16-18 Additionally, studies have identified citramalic acid as a marker of dysbiosis and an indicator of fungal overgrowth.3,4

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Citramalic acid is an analog of malic acid, with an additional CH3 group, and as an analog, it has the potential to inhibit malic acid utilization in the Citric Acid Cycle. This interference could potentially cause elevations in malic acid 26 while depleting oxalacetic acid, stressing the Citric Acid Cycle activity and impairing energy production.10

Toxic Exposure

If citramalic acid is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic byproducts of mold (e.g. Aspergillus), pose significant health risks.19, 20 Identifying specific mycotoxins can aid effective remediation and address mold- related health concerns.19, 21

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: Citramalic acid levels can also be influenced by dietary intake of ripe fruits such as apples, pitaya, tomatoes, and sugar beets, as well as wine.6-9

Low Values There is no known clinical significance for low values.

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2 5-Hydroxymethyl-2-furoic acid

5-Hydroxymethyl-2-furoic acid (HMFA) is a normal human urinary metabolite in minor amounts, but has also been identified as both Aspergillus and Cladosporium byproducts, linking it to mold exposure.22,23 It is found to have antimicrobial activity, particularly on bacteria (Staphylococcus aureus and Bacillus subtilis), which could impact the microbiome. Additionally, HMFA can form during cooking processes such as ”browning” food or the breakdown process of certain sugars, negatively impacting human health, even from food.24,25,26

Microbial Overgrowth (Mold)

Both Aspergillus and Cladosporium species, along with other molds such as Gibberella fujikuroi, Helminthosporium maydis, and Pyricularia grisea have all demonstrated production of this metabolite.22,23

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

If HMFA is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic byproducts of mold like Aspergillus and Cladosporium, pose significant health risks.19, 20 Identifying specific mycotoxins can aid effective remediation and address mold-related health concerns.21

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: Foods rich in HMFA, such as coffee, beer, fruit juices, dried fruit (especially plums and grapes), bread, honey, and breakfast cereals, can contribute to increased HMFA levels in urine.25-28 Of note is HMFA’s tendency to quickly degrade once exposed to additional heating methods, or aeration.29

Low Values There is no known clinical significance for low values.

3 3-Oxoglutaric acid

3-Oxoglutaric acid is a short-chain keto acid and a simple carboxylic acid. It is a microbial metabolite of yeast and is also an analog of 2-oxoglutarate (also known as alpha-ketoglutaric acid) from the Citric Acid Cycle, playing a potential role in mitochondrial health.4

Microbial Overgrowth (Mold)

Elevated levels of 3-oxoglutaric acid may indicate yeast overgrowth, or other dysbiotic gut flora, however, evidence linking this marker to dysbiosis is still limited.4,10

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Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

3-oxoglutaric acid is an analog of 2-oxoglutarate (also known as alpha-ketoglutaric acid), an intermediate in the Citric Acid Cycle. This analog can potentially interfere with the alpha- ketoglutaric dehydrogenase complex, which converts 2-oxoglutarate to succinyl CoA, thereby influencing mitochondrial activity and potentially causing elevations in 2-oxoglutaric acid 27 .10

Additional Insights

Low Values There is no known clinical significance for low values.

4 Furan-2,5-dicarboxylic acid

Furan-2,5-dicarboxylic acid (FDCA) is a dicarboxylic acid produced from hydroxymethylfurfural (HMF). Various microorganisms, including molds such as Aspergillus and Penicillium, can metabolize HMF into FDCA. FDCA can be generated by certain foods and beverages and is also a byproduct of polyethylene furanoate (PEF) polymer found in plastics.28,30-33

Microbial Overgrowth (Mold)

Elevated levels of FDCA in urine may suggest Aspergillus exposure or activity.31,34 Other organisms such as soil-based bacteria (e.g. Cupriavidus basilensis and Raoultella ornithinolytica) and other fungi such as Penicillium spp. and Pleurotus ostreatus (oyster mushroom), have also been known to produce FDCA.31,35

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

If FDCA is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic chemical byproducts produced by certain types of mold, such as Aspergillus and Pennicillium, pose significant health risks.19,20 Identifying specific mycotoxins can aid effective remediation and address mold-related health concerns.19,21 Additionally, FDCA is used as a monomer with ethylene glycol to produce polyethylene furanoate (PEF) in plastics, which can also potentially contaminate foods.33,36 There are other toxicants found in PEF containing plastics, and while FDCA itself is not yet assessed, frequent exposure to plastics may warrant a TOXDetect order.

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: FDCA has been shown to be from food sources containing furans, such as coffee, alcoholic beverages, preserved foods, and baked goods.28,32,37

Low Values There is no known clinical significance for low values.

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5 Furancarbonylglycine

Furancarbonylglycine, also known as 2-furoylglycine, is a furan-derived acyl glycine that may be linked to the fungus Aspergillus, serving as a marker for mold exposure or overgrowth. Additionally, dietary intake of high-temperature processed foods and beverages, such as coffee, can influence their levels. 34,38

Microbial Overgrowth (Mold)

Furancarbonylglycine, a urinary metabolite associated with the fungus Aspergillus may indicate fungal exposure or overgrowth when elevated. In one study, antifungal treatment lowered levels of furancarbonylglycine (along with 5-hydroxy-methyl-2-furoic acid 2 , furan-2,5- dicarboxylic acid 4 ), further suggesting its potential role as a marker for fungal dysbiosis.34

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

If Furancarbonylglycine is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic byproducts of mold (e.g. Aspergillus), pose significant health risks.19,20 Identifying specific mycotoxins can aid effective remediation and address mold-related health concerns.19,21

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: Intake of foods processed at high temperatures, such as coffee, can increase levels of this furan derivative.38

Low Values There is no known clinical significance for low values.

6 Tartaric Acid

Tartaric acid is a naturally occurring dicarboxylic acid associated with the activity of Aspergillus, Penicillium, and to a lesser extent, Candida and Saccharomyces.39-41 Elevated levels may indicate fungal dysbiosis. It can also inhibit the Citric Acid Cycle by disrupting malic acid utilization, potentially impacting mitochondrial function. Additionally, dietary sources such as grapes, red wine, tamarind, and certain food additives may contribute to elevated levels.43-45

Microbial Overgrowth (Mold and/or Yeast)

Elevated levels of tartaric acid are associated with mold and potentially yeast, as it is known to be produced by Aspergillus and Penicillium and possibly Candida and Saccharomyces.10,39-42 Tartaric acid can also encourage the growth of yeast.41

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Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Tartaric acid is an analog of malic acid and may inhibit this part of the Citric Acid Cycle. Evaluate Fumaric acid 25 and Malic acid 26 abnormalities for further insights if this is suspected.42

Toxic Exposure

If tartaric acid is thought to be coming from mold, it is important to evaluate the presence of mycotoxins. Mycotoxins, which are toxic byproducts of mold (e.g. Aspergillus), pose significant health risks.19,20 Identifying specific mycotoxins can aid effective remediation and address mold- related health concerns.19,21 Regardless of source, tartaric acid can act as a muscle toxin in very high concentrations, inhibiting malic acid production and potentially causing adverse effects such as paralysis.46 These toxic effects are very rare and are usually associated with extremely elevated levels.46

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: Elevated urinary tartaric acid levels can result from dietary intake of tartaric-rich foods, especially grapes and red wine, as well as tamarind and hawthorn.43,44 It is also used as a food additive for its antioxidant properties and tart flavor and can be found in candies (especially lime and grape flavors), mustards, jams, fruit juices, ice creams, and leavening agents (e.g., cream of tartar).43,45

Low Values There is no known clinical significance for low values.

7 Arabinose

Arabinose is an aldopentose sugar that can be metabolized by various organisms through pathways involving conversion into intermediates of the pentose phosphate pathway. It has been studied as a marker for intestinal yeast overgrowth and shown clinically to respond to antifungal therapies. 4,10 Arabinose is also a naturally occurring sugar compound found in numerous plants, such as beets and grains.41,48

Microbial Overgrowth(Yeast)

Elevated levels of L-arabinose suggest a potential yeast overgrowth such as Candida spp. or other dysbiotic gut flora.4,10 Case studies, including individuals with autism, have shown reductions in arabinose levels and symptom improvement following antifungal therapy, supporting its potential role in clinically significant fungal overgrowth.4,10

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

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Additional Insights

Dietary: Arabinose elevations can also originate from dietary sources. It is naturally found in the cell wall of plants (e.g. grains, beets, plant gums) or may be derived from the breakdown of hemicellulose and pectins found in numerous fruits and vegetables.48,49

Low Values There is no known clinical significance for low values.

8 Carboxycitric Acid

Carboxycitric acid is used to evaluate intestinal microbial overgrowth, particularly with yeast. While evidence supporting its role as a marker for Microbial Overgrowthis still developing, levels may provide insights into fungal activity.10,50

Microbial Overgrowth(Yeast)

Elevated carboxycitric acid levels in urine may indicate intestinal fungal overgrowth.50 Studies suggest that these levels can decrease following antifungal therapy, which highlights the potential association with yeast and fungal dysbiosis, however data is still limited.4,10

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Additional Insights

Low Values There is no known clinical significance for low values.

9 Tricarballylic Acid

Tricarballylic acid is often associated with the fumonisin class of mycotoxins produced mainly by various Fusarium species and Aspergillus section Nigri (black aspergilli).51-53 These mold exposures can come from indoor environments and contaminated dietary sources such as numerous grain products.53,54 This metabolite may also be produced from certain bacteria.50 Tricarballylic acid has been shown to bind to various minerals, influencing nutritional needs.51

Microbial Overgrowth(Mold or Bacteria)

Exposure to fumonisins, a mycotoxin class produced by various Fusarium species and Aspergillus section Nigri found in contaminated foods, releases tricarballylic acid during breakdown in the gastrointestinal tract.53,54 Certain gut bacteria, such as species from the Firmicutes phylum, can also produce tricarballylic acid during their metabolic processes.50,51

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

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Toxic Exposure

Fumonisins, mycotoxins linked to Fusarium and Aspergillus contamination, are major sources of tricarballylic acid.52,54 Fusarium is a mold that has been found in water-damaged buildings as well as food sources such as grains and grain products.53-55 Elevated levels of tricarballylic acid in urine may indicate mycotoxin exposure, suggesting the need for further assessment, such as urine mycotoxin testing.

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Tricarballylic acid has the ability to bind divalent ions, like magnesium, calcium, and zinc, potentially contributing to mineral imbalances.51

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Additional Insights

Low Values There is no known clinical significance for low values.

BACTERIAL MARKERS

10 Hippuric acid

Hippuric acid is a conjugate of glycine and benzoic acid, and may be influenced by gut bacteria, diet, and environmental exposures. It can be produced during the breakdown of benzene- type aromatic compounds from either microbial activity in the gut, exposure to toluene, or naturally produced in the liver from dietary intake of polyphenol-rich foods. Low levels can indicate poor microbial activity, glycine or B5 insufficiencies, and have also been associated with numerous chronic conditions.56-62 In rare cases, extreme elevations may be linked to genetic metabolic disorders.62,63

Microbial Overgrowth (non-specific Bacteria)

Hippuric acid can be the result of gut bacterial metabolism of phenylalanine, or from their metabolism of polyphenol-rich compounds that generate benzoic acid. 64 Microbial Overgrowths in the gut may influence hippuric acid levels, with elevated levels potentially indicating an overgrowth of certain bacteria, such as those from the order Clostridiales and the species Faecalibacterium prausnitzii, possibly due to their enhanced ability to metabolize polyphenols.64,65

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

Elevated hippuric acid can be indicative of exposure to toluene.66 Toluene is a solvent used

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in paints, dyes, fingernail polish, and gasoline, all of which can emit into the air, leading to exposures.67-70 Toluene metabolism produces benzoic acid via hydroxylation and P450 enzymes, leading to increased hippuric acid levels.71

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Theoretically, elevated hippuric acid levels may induce glycine depletion, as glycine is required for its production.

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

In rare instances, extremely elevated hippuric acid can be associated with certain genetic disorders including Phenylketonuria (PKU), hydroxymethylglutaryl-CoA lyase deficiency, tyrosinemia 1, Maple Syrup Urine Disease (MSUD), and propionic acidemia.72,73

Corresponding markers to the aforementioned genetic diseases include: 2-Hydroxyphenylacetic acid 11 , 3-Indoleacetic acid 18 , 2-Hydorxyisovaleric acid 62 , Mandelic acid 68 , Phenylactic acid 69 , or Phenylpyruvic acid 70 for PKU. 3-Hydroxyglutaric acid 31 , elevations for hydroxymethylglutaryl-CoA lyase deficiency, 2-Hydroxyphenylacetic acid 11 , 4-Hydroxyphenylacetic acid 15 , 2-Hydroxyisovaleric acid (62) and 4-Hydroxyphenyllactic acid 72 for tyrosinemia. 2-Hydroxyisovaleric acid 62 , 2-Oxoisovaleric acid 63 , 3-Methyl-2- oxovaleric acid 64 , 2-Hydroxyisocaproic acid 65 , or 2-Oxoisocaproic acid 66 for MSUD, or Methylmalonic acid 50 , Methylcitric acid 57 , Pyroglutamic acid 58 , 2-Hydroxybutryic acid 59 , and 2-Hydroxyisovaleric acid 62 for propionic acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Foods such as potatoes, apples, pears, blueberries, black currants, blackberries, and beverages like certain juices, coffee, and tea all have specific polyphenols (e.g. chlorogenic acids and epicatechins) that can be broken down into benzoic acid from gut bacteria and then conjugated with glycine in the liver to form hippuric acid.74,75 Benzoic acid is also naturally present in certain foods, such as dairy products and strawberries, and can also be used as a preservative (e.g. sodium benzoate) due to its antimicrobial properties.58,64,76

Other markers associated with intake of polyphenol-rich food include: 4-Hydroxybenzoic acid 12 , 4-Hydroxyhippuric acid 13 , DHPPA (3,4-dihydroxyphenylpropionic acid) 14 , and Hydroxyphenylacetic acid 15 .

Low Values

Microbial Overgrowth: Low levels have also been associated with dysbiosis and may be

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significant when evaluating for shifts in the microbiome.61

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Nutritional Needs: Since glycine conjugates with benzoic acid to form hippuric acid, low levels may be due to inadequate glycine, which would impact production.62 Additionally, Vitamin B5 itself plays a role by facilitating benzoic acid’s conversion into hippuric acid; therefore, if hippuric acid is low, B5 insufficiency may be an influence.62

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Associated Conditions: Very low hippuric acid may be used in evaluating renal clearance and hepatic function. Low levels have also been associated with high blood pressure, atherosclerosis, obesity, diabetes, autism, schizophrenia, and depression.56-60

11 2- Hydroxyphenylacetic acid

2-Hydroxyphenylacetic acid is a metabolite of phenylalanine and plays a role in microbial and metabolic processes.77 It is associated with microbial activity in the gut, phenylalanine utilization for neurotransmitter production, and certain genetic and dietary influences.77-79

Microbial Overgrowth(non-specific Bacteria)

2-Hydroxyphenylacetic acid is mainly discussed in the literature as a microbial metabolite that can indicate bacterial overgrowth or dysbiosis in the gastrointestinal tract.77 Various bacteria may produce this compound as part of the breakdown of phenolic containing compounds, although non-specific with current literature.56 Additionally, Caenorhabditis elegans, which is a nematode, and Penicillium chrysogenum have been shown to produce it, but evidence is limited.80-82

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

2-Hydroxyphenylacetic acid can be a byproduct of phenylalanine metabolism and may reflect altered phenylalanine hydroxylase function or biopterin pathway disruptions tied to neurotransmitter metabolism.78

Evaluate Homovanillic acid (HVA) 33 , Vanillylmandelic acid (VMA) 34 , Dihydroxyphenylacetic acid (DOPAC) 36 and 5-Hydroxyindoleacetic acid (5-HIAA) 38 abnormalities for further insights.

Nutritional Needs

Decreased vitamin B6 can impair tetrapyrrole biosynthesis, leading to potentially reduced biopterin synthesis, influencing phenylalanine’s conversion to dopamine.83

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

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Genetics

In rare instances, very elevated levels of 2-hydroxyphenylacetic acid are associated with Phenylketonuria (PKU), a genetic disorder caused by a deficiency of the enzyme phenylalanine hydroxylase. This condition results in the accumulation of phenylalanine and its metabolites, including Hippuric acid 10 , 2-Hydorxyisovaleric acid 62 , Mandelic acid 68 , Phenyllactic acid 69 , Phenylpyruvic acid 70 .79

Tyrosinemia, a group of rare disorders affecting tyrosine metabolism, can also result in extreme elevations due to impaired enzymatic breakdown of tyrosine and its derivatives. Additional markers associated with this disease include Hippuric acid 10 , 4-Hydroxyphenylacetic acid 15 , 2-Hydroxyisovaleric acid 62 , and 4–Hydroxphenyllactic acid 72 .84

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional insights

Dietary Sources: 2-Hydroxyphenylacetic acid is a metabolite produced when certain foods are consumed, such as blueberries, strawberries, apricots, tomatoes, chanterelle mushrooms, and grapes .77

Medications/Supplement Influences: Taking certain botanicals such as chaste tree (Vitex trifolia), parsley, oregano, and lemon verbena may increase 2-hydroxyphenylacetic acid levels due to their metabolism.77

Associated Conditions: 2-hydroxyphenylacetic acid has also been linked to specific conditions, including chronic constipation and atopic dermatitis.85,86

Low Values There is no known clinical significance for low values.

12 4- Hydroxybenzoic acid

4-Hydroxybenzoic acid (4-HBA) is an organic compound that can be formed through the bacterial metabolism of dietary polyphenols or certain amino acids.87 Natural elevations may also be linked to dietary polyphenol intake and can potentially support CoQ10 synthesis.88,89 However, exposure to endocrine-disrupting preservatives, known as parabens, has been shown to elevate 4-HBA.90

Microbial Overgrowth(non-specific Bacteria)

4-HBA can serve as a marker of gut microbiota activity. Elevated urinary levels may indicate intestinal dysbiosis or bacterial overgrowth. Certain bacterial genera, such as Clostridium, Eubacterium, Escherichia, and Bifidobacterium, are involved in metabolizing dietary polyphenols and amino acids into 4-HBA, contributing to its increased production.87

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

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Toxic Exposure

Parabens, commonly used as preservatives in personal care products, pharmaceuticals, and packaged foods, are absorbed through the skin or GI tract and metabolized into 4-HBA, increasing urinary levels.91 Recent studies link parabens to health concerns, including endocrine disruption, particularly with regard to estrogenic activity.90,92

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

4-HBA has been shown to support Coenzyme Q10 (CoQ10) biosynthesis.89

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Additional Insights

Dietary: Dietary intake of polyphenol-rich foods such as green tea, coconut, garlic, some berries, soybeans, and almonds can naturally elevate 4-HBA levels.88,93-95

Other markers associated with intake of polyphenol rich food include Hippuric acid 10 , 4-Hydroxyhippuric acid 13 , DHPPA (3,4 dihydroxyphenylpropionic acid) 14 , and Hydroxyphenylacetic acid 15 .

Medications/Supplement Influences: Supplements that contain botanicals with higher amounts of rosmarinic acid (e.g. oregano, rosemary, mint, lemon balm, holy basil) can metabolize into 4-Hydroxyhippuric acid.94,95

Associated Conditions: 4-HBA has been associated with various conditions, including celiac disease, cystic fibrosis, short bowel syndrome, and unclassified enteritis.96

Low Values There is no known clinical significance for low values.

13 4-Hydroxyhippuric acid

4-Hydroxyhippuric acid is a urinary metabolite formed by the conjugation of glycine with 4-hydroxybenzoic acid 12 , a compound produced from microbial metabolism of dietary polyphenols, amino acids, or parabens. Elevated levels may stem from gastrointestinal bacterial overgrowth, high intake of polyphenol-rich foods, or paraben exposure from personal care products, pharmaceuticals, and packaged foods.88,90 In the case of a low value, in theory, if 4-hydroxybenzoic acid 12 is elevated, and 4-hydroxyhippuric acid is low, it could be an indication of glycine insufficiency.97

Microbial Overgrowth(non-specific Bacteria)

Bacterial overgrowth in the GI tract can lead to increased metabolism of dietary compounds, resulting in elevated levels of 4-hydroxyhippuric acid.87 Microbial species such as

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Bifidobacterium, Clostridium, Escherichia coli, and Eubacterium have all been found to metabolize polyphenols and amino acids like tyrosine and phenylalanine into 4-hydroxybenzoic acid, which is subsequently conjugated to form 4-hydroxyhippuric acid.87

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

Parabens, commonly used as preservatives in personal care products, pharmaceuticals, and packaged foods, are absorbed through the skin or GI tract and metabolized into 4-hydroxybenzoic acid 12 , which is then conjugated to form 4-hydroxyhippuric acid. Recent studies link parabens to health concerns, including endocrine disruption, particularly with regard to estrogenic activity.90,92

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary: Dietary intake of polyphenol-rich foods such as green tea, coconut, garlic, some berries, soybeans, and almonds can naturally elevate 4-Hydroxyhippuric acid levels.88,93

Other markers associated with intake of polyphenol-rich food include Hippuric acid 12 , 4-Hydroxybenzoic acid 12 , DHPPA (3,4 dihydroxyphenylpropionic acid) 14 , and Hydroxyphenylacetic acid 15 .

Medications/Supplement Influences: Supplements that contain botanicals with higher amounts of rosmarinic acid (e.g. oregano, rosemary, mint, lemon balm, holy basil) can metabolize into 4-Hydroxyhippuric acid.94,95

Low Values Theoretically, if 4-hydroxybenzoic acid 12 is elevated, and 4-hydroxyhippuric acid is low, it could be an indication of glycine insufficiency, since 4-hydroxyhippuric acid conjugates with glycine to form it.97

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

14 3,4 dihydroxyphenylpropionic acid (DHPPA)

3,4-Dihydroxyphenylpropionic acid (DHPPA), also known as dihydrocaffeic acid, is a metabolite produced by gut microbiota through the breakdown of certain dietary polyphenols.98 DHPPA is primarily associated with Lactobacilli, Bifidobacteria, E. coli, and some Clostridium species, often identified as commensal.98-100 Elevated levels may indicate a polyphenol-rich diet or an abundance of these flora, while low levels suggest insufficient polyphenols or potentially reduced beneficial bacteria.101

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Microbial Overgrowth(Beneficial Bacteria)

Elevated DHPPA levels may indicate bacterial growth, particularly from the genera of Lactobacilli or Bifidobacteria, or species E. coli, which metabolize the polyphenol chlorogenic acid into DHPPA.98-100,102 Clostridium orbiscindens can also contribute to elevations by converting flavonoids like luteolin and quercetin into DHPPA, but this species is generally identified as commensal.102-104

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Additional Insights

Dietary Intake: A diet rich in chlorogenic acid can significantly elevate DHPPA levels. Beverages such as coffee and wine, fruits like apples, pears, peaches, blueberries, cherries, eggplants, and tomatoes, leafy greens like collard greens, and other foods such as chicory, beets, sweet and white potatoes, carrots, asparagus, olives, peas, sunflower seeds, mustard, and rosemary, are all abundant in chlorogenic acid.99,105 Consuming these foods increases the availability of substrates for bacterial metabolism, resulting in higher urinary DHPPA levels.99,105 Probiotic-rich foods may also influence levels.

Medications/Supplement Influences: Theoretically, probiotics containing Lactobacilli or Bifidobacteria may influence elevations.

Benefits: DHPPA elevations have been shown to directly inhibit pro-inflammatory cytokines.106 It has also been shown to have other potential health benefits including reducing oxidative stress and having cardio and neuroprotective properties.107

Low Values Reduced DHPPA may indicate insufficient dietary intake of polyphenol-rich foods or a depletion of beneficial gut bacteria.101 Factors such as dysbiosis, antibiotic use, or health conditions affecting the microbiome may limit the breakdown of chlorogenic acid into DHPPA.108

Other markers associated with intake of polyphenol rich foods include Hippuric acid 10 , 4-Hydroxybenzoic acid 12 , 4-Hydroxyhippuric acid 13 , and Hydroxyphenylacetic acid 15 .

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CLOSTRIDIA BACTERIAL MARKERS

15 4-Hydroxyphenylacetic acid

4-Hydroxyphenylacetic acid (4-HPAA) is a microbial metabolite derived from the bacterial transamination of tyrosine and polyphenols.109 Dysbiosis is often associated with Clostridia species, which may influence neurotransmitter balance.110,111 Elevations have also been observed in gastrointestinal disorders such as SIBO and may be affected by polyphenol consumption.112,113

Microbial Overgrowth(Clostridia Bacteria)

Elevated 4-HPAA levels are often associated with anaerobic bacterial overgrowth, particularly Clostridia spp. including C. difficile, C. stricklandii, C. lituseburense, C. subterminale, C. putrefaciens, and C. propionicum.114-116 Overgrowth can result from gut microbiota disruptions due to antibiotic use, glyphosate or organophosphate exposure, or similar factors.117,118 Other potential bacterial contributors include Acinetobacter, Klebsiella, Pseudomonas, and Proteus, however evidence is limited for these genera. Of note, it is common for only one Clostridia metabolite to be elevated and still be significant.119

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Theoretical and anecdotal evidence shows that phenylalanine and tyrosine related metabolites from Clostridia spp. may inhibit the conversion of dopamine to norepinephrine through blocking the dopamine beta hydroxylase (DBH) enzyme.120-123 C. difficile has the ability to decarboxylate 4-HPAA to produce 4-cresol 17 , and this clostridia metabolite has been shown to directly influence the DBH enzyme.110,111

When this occurs, a pattern of elevated Homovanillic acid (HVA) 33 , low Vanillylmandelic acid (VMA) 34 , and elevated HVA/VMA ratio 35 may be observed.

Toxic Exposure

Pesticides like glyphosate and organophosphates can disrupt the microbiome by reducing beneficial bacteria, creating conditions that favor pathogenic dysbiosis. These chemicals have been shown to promote Clostridia overgrowth.117, 118

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Genetics

Though rare, certain metabolic conditions, such as tyrosinemia, are associated with extreme elevations. Other markers associated with tyrosinemia include Hippuric acid 10 , 2-Hydroxyphenylacetic acid 11 , 2-Hydroxyisovaleric acid 62 , and 4-Hydroxyphenyllactic acid 72 .19

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Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: High levels of 4-HPAA may result from a diet rich in polyphenols, commonly found in fruits, vegetables, tea, coffee, wine, and whole grains.113 Gut microbes metabolize these compounds into 4-HPAA, which can then become elevated even in the absence of dysbiosis.113

Other markers associated with intake of polyphenol-rich foods include: Hippuric acid 10 , 4-Hydroxybenzoic acid 12 , 4-Hydroxyhippuric acid 13 , and DHPPA (3,4 dihydroxyphenylpropionic acid) 14 .

Associated Conditions: Elevated levels of 4-HPAA have been associated with conditions such as small intestinal bacterial overgrowth (SIBO), Giardia lamblia infections, celiac disease, cystic fibrosis, and post-surgical changes like ileal resection.112,124

Low Values There is no known clinical significance for low values.

16 HPHPA (3-(3-Hydroxyphenyl)-3-hydroxypropanoic acid)

HPHPA (3-(3-Hydroxyphenyl)-3-hydroxypropionic acid) is produced by various Clostridia bacteria through the metabolism of phenylalanine and may disrupt catecholamine metabolism and signaling.99,115 Elevated HPHPA levels are linked to numerous behavioral, gastrointestinal, and neuropsychiatric conditions.99

Microbial Overgrowth(Clostridia Bacteria)

Evidence suggest HPHPA may be produced by various Clostridia bacteria including C. sporogenes, C. botulinum, C. chlortoluron’s, C. mangenoti, C. ghoni, C. bifermentans, C. sordellii, and occasionally C. difficile via the metabolism of phenylalanine.99,115,125 Phenylpropionic or m-tyrosine, both of which can break down to HPHPA, have been shown to be neurotoxic and metabotoxic, and may disrupt catecholamine signaling or amino acid transport.99,126,127 Additionally, through beta-oxidation, HPHPA can be converted into hydroxybenzoic acid, and its glycine conjugate, hydroxyhippuric acid, meaning elevated levels of 4-Hydroxybenzoic acid 12 , and 4-hydroxyhippuric acid 13 may also provide insights .Of note, it is common for only one Clostridia metabolite to be elevated and still be significant.126

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

It has been theorized that HPHPA’s phenolic structure may disrupt catecholamine metabolism by inhibiting the conversion of dopamine to norepinephrine/epinephrine. Clinical observations have shown when HPHPA is elevated, urinary dopamine metabolites (Homovanillic acid (HVA)

33 ) become higher, and norepinephrine/epinephrine metabolites (Vanillylmandelic acid (VMA)

34 ) are reduced, creating an elevated HVA/VMA ratio 35 .120

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Toxic Exposure

Pesticides like glyphosate and organophosphates can disrupt the microbiome by reducing beneficial bacteria, creating conditions that favor pathogenic dysbiosis. These chemicals have been shown to promote Clostridia overgrowth.117,118

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Associated Conditions: Elevated levels of HPHPA are associated with a range of behavioral, gastrointestinal, and neuropsychiatric symptoms, including conditions such as autism, schizophrenia, ADHD, tic disorders, obsessive-compulsive disorder, chronic fatigue syndrome, and anorexia.99,126,128

Low Values There is no known clinical significance for low values.

17 4-Cresol

4-Cresol is primarily produced by Clostridium difficile and related bacteria through the metabolism of tyrosine.116 It is a significant marker of gut dysbiosis and can inhibit dopamine beta-hydroxylase (DBH), leading to elevated dopamine and toxic byproducts.116,123 It is linked with environmental toxicants such as toluene, creosote-treated wood, and pesticides, as well as attributed to more severe symptoms in autistic children.129-133 It has also been associated with cardiovascular, metabolic, and renal diseases.134

Microbial Overgrowth(Clostridia Bacteria)

4-Cresol is primarily produced by C. difficile and C. scatologenes, with C. difficile being the most common source.116 Other bacterial families, such as Coriobacteriaceae, Lactobacillus, and Enterobacteriaceae also could produce 4-cresol with certain substrates, but to a much lesser degree.135-137 Elevated levels often indicate gut dysbiosis, potentially caused by interventions or exposures that disrupt the microbiome.138 Moreover, 4-cresol has demonstrated antimicrobial properties that can potentially further dysbiosis.116

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

4-cresol has been shown to inhibit the dopamine beta hydroxylase enzyme (DBH), reducing the conversion of dopamine to norepinephrine.111,123 4-cresol can form covalent bonds with DBH, leading to elevated dopamine levels. An excessive increase in dopamine can result in unstable dopamine quinones, toxic dopamine adducts that disrupt the brain’s mitochondrial energy production, and oxygen superoxide, all of which can cause significant damage to the brain.120

Inhibition of DBH can cause the pattern of elevated Homovanillic acid (HVA) 33 and

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3,4-dihydroxyphenylacetic acid (DOPAC) 36 , along with low Vanillylmandelic acid (VMA) 34 and elevated HVA/VMA ratio 35 .

Toxic Exposure

Pesticides like glyphosate and organophosphates have been shown to promote Clostridia overgrowth.117,118 To a much lesser degree, 4-cresol can also be produced from the metabolism of toluene, menthofuran (from peppermint and pennyroyal essential oils), and creosote, a wood preservative containing polycyclic aromatic hydrocarbons (PAHs) and phenolic compounds.129-132 Another organic acid that can be a metabolite of Toluene is Hippuric acid 10 .

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Associated Conditions: Urinary levels of 4-cresol have been found to be significantly higher in autistic children compared to controls and are associated with greater clinical severity.133,139 This metabolite has also been associated with cardiovascular, metabolic, and renal diseases.134,140,141

Low Values There is no known clinical significance for low values.

18 3-Indoleacetic acid

3-Indoleacetic acid (IAA) is a metabolite of tryptophan that is produced primarily through the indolic pathway. It is often linked to Clostridia, though other bacteria and yeast may also contribute.142 While IAA has been associated with systemic inflammation, increased virulence of fungus, and various conditions, including autism, cardiovascular disease, kidney dysfunction, and psychological disorders, it has also been shown to exhibit certain beneficial properties.143-146

Microbial Overgrowth(Clostridia Bacteria)

IAA is mainly produced by intestinal bacteria through tryptophan breakdown with elevated levels being linked to C. stricklandii, C. lituseburense, C. subterminale, and C. putrefaciens.142 Other bacteria like Escherichia, Bacteroides, Lactobacillus, and Pseudomonas, as well as other fungi such as Saccharomyces cerevisiae and Candida tropicalis, may also produce IAA.144,147 Moreover, IAA has been shown to induce filamentation, and promote biofilm formation in fungus.144,148

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

Pesticides like glyphosate and organophosphates have been shown to promote Clostridia overgrowth.117,118

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

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Genetics

Extreme IAA levels are linked to Hartnup’s Disease, a rare genetic disorder that impairs the renal and intestinal transport of neutral amino acids (e.g. tryptophan), causing elevated urinary amino acids and IAA.149 Symptoms include skin rashes, ataxia, psychosis, and rashes resembling pellagra.150 IAA may also be elevated in phenylketonuria (PKU).151 Other associated metabolites of PKU include Hippuric acid 10 , 2-Hydroxyphenylacetic acid 11 , 2-Hydorxyisovaleric acid 62 , Mandelic acid 68 , Phenyllactic acid 69 , Phenylpyruvic acid 70 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Associated Conditions: Elevated IAA levels have been linked to systemic inflammation, autism, cardiovascular disease, kidney dysfunction, and psychological disorders like anxiety and depression.143,146,152

Benefits: The IAA metabolite has been shown to reduce oxidative stress, scavenge free radicals, lower pro-inflammatory cytokine production, and support intestinal homeostasis and mucosal immunity via activation of the aryl hydrocarbon receptor (AhR) pathway.145

Low Values There is no known clinical significance for low values.

OXALATE METABOLITES

Figure 2:

Citric acid

This figure explains how oxalic acid elevations can result from both direct and indirect microbial metabolism. Molds such as Aspergillus niger and Penicillium spp. are capable of direct oxalate production as part of their primary metabolic processes. In contrast, yeasts, including Candida, generate glyoxylate through the glyoxylate cycle, wherein isocitrate is cleaved by isocitrate lyase to yield glyoxylate. This intermediate can subsequently be converted in the liver to oxalic acid via lactate dehydrogenase (LDH)–mediated reactions. Glyoxylate can also be converted to glycine with the support of B6.

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19 Glyceric Acid

Elevated glyceric acid can result from Microbial Overgrowths, nutrient deficiencies, diet, or genetic mutations. Elevated glyceric acid from fungal or microbial overgrowth, may be through the glyoxylate pathway, where excess glyoxylate is converted to glyceric acid or from direct production.153-155 Insufficiencies in vitamin B3 or tryptophan, which are cofactors for glyoxylate reductase/hydroxypyruvate reductase (GRHPR), the enzyme regulating this pathway, may also contribute to increased levels.153,156 Additionally, high fructose intake, glycerol metabolism, and various genetic conditions can also lead to excess glyceric acid.157-160

Microbial Overgrowth(Fungal and Bacteria)

Elevated glyceric acid, may result from overgrowth from fungi or other organisms.153 It is theorized that elevated glyceric acid may stem from the intestinal microbes’ glyoxalate pathway, where excess glyoxylate can convert to glyceric acid.153,154 Other microbes shown to produce glyceric acid directly, include Acetobacter and Gluconobacter.155

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Nutritional Needs

The enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR) utilizes reduced forms of NAD as cofactors, requiring either Vitamin B3 (Niacin) or tryptophan for its production.156,161,162

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Primary Hyperoxaluria Type 2 (PH2) is linked to mutations in the GRHPR gene, which causes a deficiency of the enzyme GRHPR.163 The lack of GRHPR prevents glyoxylates conversion to glycolate, leading to an excess amount of glyceric acid and Oxalic acid 21 .157

D-glyceric aciduria is another genetic disorder associated with elevated glyceric acid and is caused by the deficiency of glycerate 2-kinase, though this disease is not well studied at this time.164

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: High fructose or fat intake can metabolize into glyceric acid.158 Metabolism of glycerol containing foods or beverages can form intermediate glyceric acid, before reacting with aldehyde dehydrogenase to generate glyceraldehyde.165

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Associated Conditions: Elevated glyceric acid has been shown to induce pancreatic islet cell damage, inducing glucose intolerance.158 It has also been associated with rheumatoid arthritis, schizophrenia, and bipolar disorder, though the mechanism has yet to be explained.159,160

Low Values There is no known clinical significance for low values.

20 Glycolic Acid

Glycolic acid is a small alpha-hydroxy acid involved in various metabolic pathways. Increased levels have been attributed to several different causes including overgrowth of bacteria and fungi, exposures to certain toxicants such as ethylene glycol (antifreeze), polyglycolate (PGA), and trichloroacetic acid, reduced B6 and glutathione levels, the use of supplemental collagen or skincare products, and certain rare genetic diseases.153,166-171 Regardless of the source, elevated levels in urine have been associated with oxidative stress.169

Microbial Overgrowth (Fungal and Bacteria)

Elevated glycolic acid, may result from overgrowth of fungi or other organisms.153 It is theorized that elevated glyceric acid may stem from the intestinal microbes’ glyoxalate pathway, where excess glyoxylate can convert to glycolic acid.153,172 Other microbes shown to produce glycolic acid directly, include Acetobacter, Escherichia, Cryptococcus, Rhodococcus, and Saccharomyces.155,167

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

Ethylene glycol, found in anti-freeze, can be metabolized into glycolic acid, as well as Oxalic acid

21 , Lactic acid 22 , and Pyruvic acid 23 .166

In addition, glycolic acid can be used as a component for creating polyglycolate (PGA), which is a synthetic polymer used in sutures and implantable medical devices.167,173 It can also be a metabolite of trichloroacetic acid exposures from certain metals, plastics, or dermatological agents to treat warts, dermal peeling or tattoo removal.174 Oxalic acid 21 is also a metabolite of exposure and may help with evaluation.174

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

The enzyme alanine:glyoxylate aminotransferase (AGT), which prevents excess glycolic acid buildup, is B6-dependent.168

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Moreover, oxidative stress depletes glutathione (GSH), which normally helps convert glycolic acid into less harmful compounds. When GSH is low, glycolic acid is diverted to oxalate

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production, increasing oxalic acid levels. Evaluate Pyroglutamic acid (58) for further clarification if GSH could be a factor.169

Genetics

Primary Hyperoxaluria Type 1 (PH1) is caused by mutations in the AGXT gene, leading to a defect in the liver specific enzyme AGT.171 This enzyme impairs the ability to break down oxalates, increasing levels of glycolic and Oxalic acids 21 .171,175

Furthermore, glycolic aciduria, another genetic condition linked to type 1 primary hyperoxaluria, can result from HAO1 gene mutations affecting glycolate oxidase or D-glycerate dehydrogenase, disrupting glycolic acid metabolism.176

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medications/Supplement Influences: Supplemental collagen can contribute significantly to urinary glycolic acid due to hydroxyproline metabolism.170 Dermal applications that use glycolic acid, such as certain facial regimens or chemical peels, may increase output of glycolic acid in the urine.177

Associated Conditions: Lipid peroxidation and protein glycation produce glyoxal, which is metabolized into glycolic acid.56 Elevated levels may indicate increased oxidative stress.178

Low Values There is no known clinical significance for low values.

21 Oxalic Acid

Oxalates, the anionic form of oxalic acid, are naturally occurring compounds that can form crystalline structures when combined with a cation (e.g. calcium, lead, etc.).179 Various organisms such as plants and fungi produce them for structure, defense, and function, but they are also a normal part of human metabolism.180,181 Molds and yeasts can produce oxalates directly and indirectly, respectively, while beneficial bacteria such as Oxalobacter formigenes help break them down.180,182 Additionally, exposure to certain toxicants such as ethylene oxide, ethylene glycol, and trichloroacetic acid can increase levels in the urine. When consumed from oxalate containing plants (e.g. spinach, soy, beets, tea. etc.), soluble and unbound oxalates are either absorbed in the colon or bind to minerals like calcium and form insoluble compounds in the intestinal lumen, followed by excretion in feces.183,184 Oxalates’ ability to bind essential minerals may contribute to deficiencies, while factors like vitamin B6 insufficiency, fat malabsorption, high-oxalate food consumption, collagen supplementation, and certain medications can further impact oxalate excretion.170,179,185,186 Regardless of source, in humans, excessive oxalates can potentially cause systemic problems and have been associated with numerous health conditions involving pain and inflammation.187-194

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Microbial Overgrowth (Fungal and Bacteria)

Oxalic acid can be elevated due to indirect or direct production from various organisms.180,182 Mold, particularly Aspergillus niger and Penicillium spp, have been shown to produce oxalates directly, while certain yeast such as Candida spp and Saccharomyces spp, produce glyoxylate via the glyoxylate pathway, which is then processed into either oxalates, glycine, glycerate 19 and glycolate 20 .40,172,180,181,195-197

Certain beneficial bacteria, including Oxalobacter formigenes, Lactobacillus plantarum, L. acidophilus, L. fermentum, L. gastricus, Bifidobacterium breve, and B. longum, are known to be oxalotrophic, meaning they metabolize oxalates as a source of carbon and electrons.180

An imbalance in beneficial gut bacteria can disrupt oxalate breakdown, leading to increased absorption from the gut and excess oxalates being filtered through urine instead of excreted in stool.180

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure

Ethylene oxide, or its hydrolyzed form ethylene glycol, can both metabolize to oxalic acid.198 These toxicants can be found in various plastics, cosmetics, the food industry, stain removers, and antifreeze.198 Additionally, trichloroacetic acid exposures through metals, plastics, or dermatological agent to treat warts, dermal peeling or tattoo removal, can contribute to oxalic acid in the urine. Glycolic acid 20 can also be a metabolite generated from both ethylene glycol and trichloroacetic acid exposures and may help with interpretation.166,174 If mold is a suspected source of oxalates, mycotoxin exposure may also need to be considered.174

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

High amounts of oxalates may contribute to mineral deficiencies due to their high affinity for certain minerals like calcium, magnesium, and iron.179

Insufficiencies in Vitamin B6 (pyridoxine) can cause glyoxylate to favor the oxalate pathway instead of glycine, so elevations in oxalic acid may be related to low B6.185

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Additionally, elevated urinary oxalate levels may be influenced by fat malabsorption, as unabsorbed fats bind calcium, increasing soluble oxalate absorption.186 Fat malabsorption can influence the status of fat-soluble vitamins, including Vitamins A, D, E, and K. Phosphoric acid

76 can be influenced by Vitamin D levels, and may help support running a serum Vitamin D, 25-Hydroxy.199

Genetics

Rare genetic hyperoxalurias, types I and II can both causes elevated urinary oxalic acid.175

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Type 1 is usually associated with elevated Glycolic acid 20 , while type II is associated with elevated Glyceric acid 19 .175

Aside from type I and II hyperoxalurias, other genetic SNPs have been shown to influence calcium oxalate formations. The UMOD gene codes for uromodulin, which inhibits calcium crystals in the urine.200 A SNP in this gene may increase oxalate formation risk and potentially be worsened by a SPP1 variant. 200 The calcium-sensing receptor (CaSR) gene codes for a receptor that senses extracellular calcium levels in the kidneys and has been shown to promote calcium oxalate crystal adhesion.201 The SLC26A4 gene provides instructions for making pendrin, which transports negative particles across cell membranes and influences mineral properties, and may consequently increase calcium oxalate formation, particularly in the vestibular system.202

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Elevations can be caused by consumption of high oxalate foods including spinach and other various greens, soy, beets, sweet potato, variety of berries, cocoa, black tea, nuts (especially almonds), tea, and various grains.183,203 This is not an exhaustive list, but it should be noted, oxalate abundance in food has biological variation including cultivation practices, time of harvest, and growing conditions, as well as cooking methods and amount consumed.204

Medication/ Supplement Influences: Supplemental collagen can contribute to elevated urinary oxalic acid and glycolic acid 20 due to hydroxyproline metabolism. Although studies on high doses of vitamin C (>500–1500 mg/day) contributing to oxalic acid are conflicting, ascorbate’s conversion to oxalate can be upregulated in the presence of high copper.179,205-208 Associated markers for Vitamin C 54 as well as HVA/VMA ratio 35 , which is influenced by copper, may provide additional insights.170

Associated Conditions: High oxalate levels have been linked to numerous conditions involving orthopedic, urogenital, cardiovascular, ocular, neuromuscular, skeletal, dental, pulmonary, neurological, endocrine, and gastrointestinal systems.187-194

Low Values There is no known clinical significance for low values.

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MITOCHONDRIAL METABOLITES

Figure 3:

This diagram presents key markers involved in glycolysis, fatty acid oxidation, and mitochondrial energy production, as depicted in the metabolic pathway diagram. Glucose is metabolized through glycolysis to pyruvic acid, which may convert to lactic acid or enter the mitochondria as acetyl-CoA to fuel the citric acid (Krebs) cycle. Fatty acids, shuttled into mitochondria via carnitine, are also oxidized to generate acetyl-CoA, supporting further energy production. As the cycle progresses through intermediates, such as aconitic, 2-oxoglutaric, succinic, fumaric, and malic acids, electrons are transferred to the electron transport chain (ETC), driving ATP synthesis.

The diagram also maps how organic acid levels can reflect underlying enzymatic function, highlighting areas of dysfunction and their potential causes. Inhibitors like heavy metals or mycotoxins, and deficiencies in cofactors such as vitamins and minerals, can impair key enzymes involved in these pathways. Metabolites shown in red and green, represent measured organic acids in the report, and are dynamic based on abnormal or normal values, respectively.

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GLYCOLYTIC CYCLE METABOLITES

22 Lactic acid

Lactic acid is primarily produced in the cytoplasm when mitochondrial oxidative phosphorylation is impaired, causing a shift toward anaerobic glycolysis. Elevations can indicate mitochondrial dysfunction, especially when seen with other mitochondrial metabolites.209 It may also be produced by various microbes—including E. coli, Streptococcus, and Saccharomyces—suggesting possible dysbiosis.209,210 Environmental toxins like cadmium, mercury, and mycotoxins can impair enzymes such as pyruvate dehydrogenase or lactate dehydrogenase, further contributing to lactic acid buildup.211-219 Nutrient insufficiencies in CoQ10, zinc, iron, or B vitamins may also drive elevations by impairing energy metabolism or increasing anaerobic glycolysis.223-229 In rare cases, genetic mitochondrial disorders or certain medications may be involved, while elevated levels are also associated with hypoxia, diabetes, and other chronic conditions.209,220-222

Microbial Overgrowth

Lactic acid can be produced by many different microbial organisms including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus faecalis, Streptococcus group B, Bacillus, Saccharomyces cerevisiae, Rhizopus spp., and more.209,210

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Cadmium, mercury, and antimony can impact enzymes such as pyruvate dehydrogenase (PDH), pyruvate carboxylase and lactate dehydrogenase (LDH).211-218 These heavy metals can interfere with enzyme function in several ways, typically by binding to thiol (sulfhydryl) groups in proteins or disrupting the enzymes’ normal cofactors, leading to altered activity. Mycotoxins can induce hypoxia through various mechanisms which can increase lactic acid levels.219

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Without sufficient CoQ10, the electron transport chain cannot function appropriately and may increase lactic acid levels.223 Zinc deficiency can also lead to increased lactate dehydrogenase activity promoting the conversion of pyruvate to lactate. Insufficient zinc also disrupts mitochondrial function leading to an increased reliance on anaerobic glycolysis and therefore

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increasing lactic acid levels.224,225 With low iron levels or in an anemic state, tissue hypoxia may be induced and can increase lactic acid levels.224,225 Lactic acidosis may be caused by B1 (thiamine) deficiency.227 When B3 levels are low, NAD+ levels decrease, leading to an impaired conversion of pyruvate to lactate by lactate dehydrogenase, thereby increasing lactic acid formation.228,229

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Some rare metabolic disorders may cause increased incidence of lactic acidosis due to defects in enzymes involved in carbohydrate metabolism and/or mitochondrial function. These disorders may include, among others, pyruvate dehydrogenase or pyruvate carboxylase deficiency, fumarase deficiency, or defects in the electron transport chain.230-232

For additional insight into rare mitochondrial disorders, reference pyruvic acid 23 , succinic acid

24 , and fumaric acid 25 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/Supplement Interactions: In some cases, a few medications may increase lactate levels, such as metformin (especially in those with renal impairment), aspirin, and nucleoside reverse transcriptase inhibitors (NRTIs) used in HIV treatment.220,221

Associated Conditions: Lactic acid buildup may result in cases of tissue hypoxia or hypoperfusion, such as with intense activity or exercise, with low iron levels or in an anemic state, in sleep-related breathing disorders such as sleep apnea, or with pulmonary or cardiac dysfunction.209,226,233,234 Increased lactic acid levels may occur with insulin or blood sugar dysregulations such as hyperglycemia or in the case of diabetes mellitus. This occurs due to high glucose levels increasing the rate of glycolysis and subsequent levels of pyruvate and lactate. Hyperglycemia may also impair the oxidation of lactate, leading to its accumulation.235 Lactic acid levels can be elevated in conditions like liver disease, alcohol dependence, seizure disorders, and severe infections due to impaired lactate clearance, increased lactate production, and metabolic disruptions.222

Low Values There is no known clinical significance for low values.

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23 Pyruvic acid

Pyruvic acid is produced during glycolysis and, depending on oxygen availability, is either converted into acetyl-CoA in the mitochondria or lactic acid under anaerobic conditions.236 Elevated pyruvate levels can indicate mitochondrial dysfunction or be associated with prolonged fasting, exercise, or certain toxic exposures (e.g., cadmium, mercury, antimony).211-217,237,238 Nutrient deficiencies in vitamins B1, B2, B3, B5, and magnesium can also lead to increased pyruvate levels.237-239 In rare genetic disorders, pyruvate accumulation may occur. Additional factors such as liver damage, alcoholism, and certain diets may also contribute to elevated pyruvate.240-245

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

Pyruvate levels also may increase through the Cahill cycle during prolonged fasting and exercise. In the liver, alanine derived from muscle is converted to pyruvate, which is then used to produce glucose via gluconeogenesis. This process helps maintain blood glucose levels when glycogen stores are low and supports energy production during physical activity, elevating pyruvate levels in the process.237,238

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Cadmium, mercury, and antimony can impact enzymes such as pyruvate dehydrogenase (PDH) and pyruvate carboxylase.211-217 These heavy metals can interfere with enzyme function in several ways, typically by binding to thiol (sulfhydryl) groups in proteins or disrupting the enzymes’ normal cofactors, leading to altered activity.

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

The conversion of pyruvic acid to acetyl-CoA by the pyruvate dehydrogenase complex (PDC) requires several essential cofactors vitamin B1 (Thiamine pyrophosphate), B2 (FAD), B3 (NAD+), B5 (coenzyme A), lipoic acid and magnesium. Deficiencies of these nutrients can cause an elevation of pyruvic acid.237-239

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Several rare metabolic disorders, including pyruvate dehydrogenase deficiency and disorders

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of sugar metabolism, can cause pyruvate accumulation due to defects in enzymes that normally convert pyruvate to other metabolites (e.g., acetyl-CoA, oxaloacetate). The common clinical feature is lactic acidosis, with symptoms ranging from developmental delays to muscle weakness and neurological issues. Proper diagnosis typically involves biochemical assays and genetic testing to identify the specific enzyme deficiencies or genetic mutations involved.240,246

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Pyruvic elevations can be caused by consuming a high-fat, low- carbohydrate diet or high-sucrose diet.244,245

Associated Conditions: Pyruvic acid levels may be elevated in conditions associated with liver damage or alcoholism.241-243

Low Values There is no known clinical significance for low values

CITRIC ACID CYCLE METABOLITES

24 Succinic acid

Succinic acid plays a key role in the Citric Acid Cycle and the electron transport chain, where it is oxidized to fumarate (fumaric acid) via succinate dehydrogenase (SDH), also known as mitochondrial Complex II. SDH is the only enzyme that participates in both the Citric Acid Cycle and the Electron Transport Chain (ETC), giving insights into both pathways.247 Elevated levels can indicate mitochondrial dysfunction, Microbial Overgrowth, or toxic exposures, such as heavy metals or certain fungicides.248-258 Deficiencies in nutrients such as iron, riboflavin (B2), and CoQ10 can also contribute to increased succinic acid levels.259-262 Low succinic acid levels are possible in rare metabolic disorders such as SSADH deficiency, and in theory could be reflective of impaired glycolysis, fatty acid oxidation, or vitamin B12 deficiency, all of which could reduce acetyl-CoA or succinyl-CoA availability for the citric acid cycle though evidence in the scientific literature is limited.263-266

Microbial Overgrowth

Imbalances in the gut microbiota are linked to higher levels of succinic acid, with conditions like inflammatory bowel disease and obesity showing increased levels due to shifts in succinate- producing and consuming bacteria. Dysbiosis, marked by an overgrowth of succinate- producing bacteria such as Prevotellaceae and Veillonellaceae, is associated with higher circulating succinate levels. Elevated succinate levels are also observed in animal models of intestinal inflammation and in patients with severe systemic inflammatory response

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syndrome (SIRS), suggesting that gastrointestinal dysbiosis plays a role in various pathological conditions.251-254 Fungi also produce succinic acid mainly through the Citric Acid Cycle, with anaerobic conditions or respiratory chain inhibition prompting the use of the reductive Citric Acid Cycle. Fungi like Penicillium simplicissimum and Aspergillus saccharolyticus produce succinate via fumarate reductase. Saccharomyces cerevisiae also produces succinic acid through both branches of the TCA cycle during anaerobic fermentation, with the reductive branch being the primary pathway.267-270

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevated succinic acid may indicate impaired activity of succinate dehydrogenase (SDH), a key enzyme that links the Citric Acid Cycle and electron transport chain (ETC) at Complex II.248-250 This disruption can create a bottleneck in electron flow, leading to reduced ATP production and increased oxidative stress.271,272 Succinate accumulation also reflects shifts in the mitochondrial redox state and can signal broader dysfunction in mitochondrial energy metabolism.273,274 Elevations, especially in the presence of other mitochondrial metabolite elevations can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposures

The succinate dehydrogenase enzyme is particularly vulnerable to toxic exposures that inhibit its function and can therefore increase succinic acid levels. Aluminum has been found to reduce the activity and expression of succinate dehydrogenase. Cadmium impairs enzymatic function by disrupting the electron transport chain, particularly at complexes II and III, while also promoting the production of reactive oxygen species (ROS).255,256 A class of fungicides known as succinate dehydrogenase inhibitors (SDHIs) includes boscalid and fluxapyroxad among others and are used widely in agriculture and inhibit succinate dehydrogenase activity which may increase succinic acid levels.257,258 Various mycotoxins, particularly Aflatoxin B1 (AFB1), has been demonstrated to inhibit succinate cytochrome c reductase, a part of the succinate dehydrogenase complex and potentially leading to an increased level of succinic acid as well as compromised mitochondrial function.275

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Succinate dehydrogenase contains three iron-sulfur clusters, making iron essential for proper electron transfer during the conversion of succinic acid to fumaric acid. In iron-deficient states, succinic acid may increase.259,260 FAD, which is synthesized by vitamin B2 (riboflavin), supports the activity of succinate dehydrogenase. In riboflavin deficiency, succinate dehydrogenase activity decreases, and succinic acid levels increase.261 CoQ10 accepts electrons from succinate dehydrogenase, enabling the electron transport chain to continue and prevents the buildup of

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succinate. Research shows that CoQ10 can also protect succinate dehydrogenase activity from being inhibited by L-malate.262

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Rare causes of elevated succinic acid include mitochondrial mutations, which can result from changes in nuclear or mitochondrial DNA affecting mitochondrial proteins, such as in Kearns-Sayre syndrome.276

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/ Supplement Interactions: Succinic acid is a byproduct of gamma-aminobutyric acid (GABA), and supplementation, as well as derivatives such as gabapentin, may increase levels.277,278

Associated Conditions: Lipopolysaccharides (LPS) increase succinic acid levels by triggering a metabolic shift in macrophages, boosting glycolysis and glutamine-dependent anaplerosis, which stabilizes HIF-1a and promotes inflammation.279,280 Under hypoxic conditions, succinate dehydrogenase activity is reversed, leading to the production of succinic acid, which is transported to oxygen-rich tissues.281 Elevated succinic acid levels are also found in autism spectrum disorder (ASD), linked to mitochondrial dysfunction and metabolic disturbances. Additionally, changes in the gut microbiome in ASD may contribute to increased succinic acid production and metabolism.282,283

Low Values

Mitochondrial Health: Theoretically, impaired glycolysis or fatty acid oxidation could lead to less acetyl-CoA entering the citric acid cycle and therefore, decreasing intermediate levels.264,265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

Nutrient Needs: Vitamin B12 is crucial for the enzyme methylmalonyl-CoA mutase, which converts methylmalonyl-CoA to succinyl-CoA. When vitamin B12 is deficient, methylmalonyl- CoA mutase activity is impaired, causing an accumulation of methylmalonic acid and a decrease in succinyl-CoA production. As succinyl-CoA is a precursor to succinic acid in the Citric Acid Cycle, this deficiency could possibly lead to lower succinic acid levels.266

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics: In the rare disorder known as Succinic semialdehyde dehydrogenase (SSADH) deficiency, the enzyme SSADH, which is involved in the breakdown of GABA to succinic acid

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via the intermediate succinic semialdehyde, is absent, and GABA is alternatively converted to GHB while succinic acid levels fall. For additional insights, refer to 4-hydroxybutyric acid 75 .263

Associated Conditions: Succinic acid levels are lower in individuals with attention-deficit/ hyperactivity disorder (ADHD), possibly in part due to the impact of antibiotics and stimulant medications on GI microbiota and short-chain fatty acid (SCFA) production. Age also influences SCFA profiles, with children showing different patterns from adults. Disruptions in the microbiota-gut-brain axis, influenced by diet, medication, and antibiotic use, may contribute to these lower succinic acid levels and the psychiatric symptoms in ADHD.284

25 Fumaric acid

Fumaric acid is a key intermediate in the Citric Acid Cycle, produced from succinic acid by succinate dehydrogenase (SDH) and then converted to malic acid by the enzyme fumarase.285 Elevated fumaric acid levels may indicate mitochondrial dysfunction, toxic exposure (e.g., to metals such as aluminum, mercury, and cadmium), or metabolic changes in immune cells.286-288 Iron is essential for the proper function of fumarase and succinate dehydrogenase, and deficiencies can lead to elevated fumaric acid.259,260,289 Fumaric acid and its derivatives have therapeutic potential, particularly in treating neurodegenerative diseases due to their antioxidant and anti-inflammatory properties.288,290,291 Low fumaric acid levels, in theory, may result from impaired glycolysis, fatty acid oxidation, or toxic exposures that inhibit key enzymes in the Citric Acid Cycle.255-258,264,265,275

Microbial Overgrowth

Fumaric acid is a metabolite of Aspergillus.285

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Some metals inhibit fumarase activity, especially by way of interfering with iron metabolism or targeting the iron-sulfur clusters of the enzyme, leading to inactivation or downregulation. Metals that impact fumarase include aluminum, gallium, silver, mercury, cadmium, and zinc, which can all lead to elevated levels of fumaric acid.286,287 Fumaric acid is also a metabolite of Aspergillus.285

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Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Iron is important in the proper functioning of iron-sulfur cluster-containing enzymes such as fumarase.289 Low iron levels can impair the enzymatic function of fumarase and lead to elevated levels of fumaric acid. However, succinate dehydrogenase also contains three iron-sulfur clusters, making iron essential for proper electron transfer during the conversion of succinic acid to fumaric acid.259,260

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Additional Insights

Benefits: When immune cells are activated, they undergo metabolic changes, including increased glycolysis and glutaminolysis, which lead to fumaric acid accumulation and epigenetic reprogramming, key to trained immunity.288 Fumarase can translocate to the nucleus in response to DNA damage, potentially acting as a tumor suppressor and contributing to elevated fumaric acid levels.290 Additionally, fumaric acid esters activate the Nrf2 pathway, reducing oxidative stress and neuroinflammation, with potential therapeutic benefits.291

Low Values

Mitochondrial Health: Theoretically, impaired glycolysis or fatty acid oxidation could lead to less acetyl-CoA entering the citric acid cycle and therefore, decreasing intermediate levels.264,265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

Toxic Exposure: The succinate dehydrogenase enzyme is particularly vulnerable to certain toxic exposures that inhibit its function and can therefore potentially lower fumaric acid levels, though this is theoretical. Aluminum has been found to reduce the activity and expression of succinate dehydrogenase. Cadmium impairs enzymatic function by disrupting the electron transport chain, particularly at complexes II and III, while also promoting the production of reactive oxygen species (ROS).255,256 A class of fungicides known as succinate dehydrogenase inhibitors (SDHIs) includes boscalid and fluxapyroxad among others and are used widely in agriculture and inhibit succinate dehydrogenase activity, which may decrease fumaric acid levels.257,258 Various mycotoxins, particularly, Aflatoxin B1 (AFB1) has been demonstrated to inhibit succinate cytochrome c reductase, a part of the succinate dehydrogenase complex and potentially leading to an increased level of succinic acid as well as compromised mitochondrial function.275

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

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26 Malic acid

Malic acid is an important metabolite in the Citric Acid Cycle, helping with energy production by transferring reducing equivalents in aerobic conditions and assisting glycolysis in anaerobic conditions.292 Elevated malic acid levels can result from Microbial Overgrowth, mitochondrial dysfunction, or toxic exposures such as arsenic and cadmium, which inhibit malate dehydrogenase.10,292-297 Nutrient deficiencies, particularly in vitamin B3, which is essential for malate dehydrogenase function, can also lead to increased malic acid levels.298,299 Malic acid is found in foods such as apples and wines and is sometimes used in supplements or medications.292,300,301 Low levels, in theory, could result from impaired metabolism, magnesium deficiency, or certain heavy metals that inhibit key enzymes in the Citric Acid Cycle.264,265,286,287,302,303

Microbial Overgrowth

LPS from Gram-negative bacteria promotes the growth of organic acid-producing bacteria, influencing metabolism and byproducts. Overgrowth increases malic acid production via MleR- regulated pathways, enhancing acid tolerance and further stimulating malic acid synthesis.293,294 Fungi such as Aspergillus and Penicillium can also produce malic acid.292,295 Additionally, citramalic acid 1 from fungus has the potential to inhibit malic acid utilization in the Citric Acid Cycle, causing interference leading to elevations in malic acid.10

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Arsenic and cadmium can both inhibit malate dehydrogenase activity, disrupting the citric acid cycle and electron transport chain, leading to increased malic acid levels.296,297 Mycotoxins from Aspergillus and Penicillium have also been shown to influence malic acid levels.292,295

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

NAD+, derived from Vitamin B3, is the main cofactor for the enzyme malate dehydrogenase, serving as an electron acceptor in the conversion of malate to oxaloacetate. The enzymatic function is highly dependent on the presence of NAD+, as it enables the transfer of electrons during the catalytic reaction.298,299 Without adequate B3, malic acid levels may increase.

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Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Additional Insights

Dietary Influences: Malic acid is a tart-tasting organic acid in some wines and foods such as apples, especially contributing to the sourness of green apples.292

Medication/ Supplement Interactions: Malic acid can be used in medications such as almotriptan malate for migraines or supplements such as Super Malic used in fibromyalgia.300,301

Associated Conditions: Oxidative stress can also increase malic acid levels as part of a broad metabolic response and the associated mitochondrial dysfunction.304

Low Values

Mitochondrial Health: Theoretically, impaired glycolysis or fatty acid oxidation could lead to less acetyl-CoA entering the citric acid cycle and therefore, decreasing intermediate levels.264,265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

Toxic Exposure: Some metals inhibit the fumarase activity, especially by way of interfering with iron metabolism or targeting the iron-sulfur clusters of the enzyme, leading to inactivation or downregulation. Metals that impact fumarase include aluminum, gallium, silver, mercury, and zinc, which can all lead to decreased levels of malic acid.286,287 Although cadmium can inhibit both malate dehydrogenase and fumarase, it has a stronger effect on inhibiting fumarase, which could conceptually reduce malic acid levels.303

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs: Magnesium can boost the activity of MDH by stabilizing the enzyme’s structure and playing a role in the catalytic process, hypothetically decreasing malic acid levels.302

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Associated conditions: Some studies have shown that only tissue malate is depleted after intense physical activity, while other crucial metabolites from the Citric Acid Cycle required for energy production remain unaffected. As a result, a malic acid deficiency has been proposed as a primary cause of physical exhaustion.292

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27 2-Oxoglutaric acid

2-Oxoglutaric acid, produced from isocitrate by isocitrate dehydrogenase in the Citric Acid Cycle, is crucial for energy production.305 Elevated levels of 2-oxoglutaric acid can indicate mitochondrial dysfunction, microbial overgrowth, or nutrient deficiencies, particularly in vitamins B1, B2, B3, B5, and magnesium, which are essential for its metabolism.305-308 Rare genetic disorders, such as fumarase deficiency, may also cause increased levels.305 2-Oxoglutaric acid plays a key role in nitrogen scavenging, protein synthesis, and collagen production, and is essential for bone tissue formation.305 Low levels, in theory, may result from manganese deficiency, impaired glycolysis, or fatty acid oxidation.264,265,309

Microbial Health

Some evidence has been found supporting 2-oxoglutaric acid production by some bacteria (Corynebacterium glutamicum) and yeasts.305,306

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Nutritional Needs

Vitamins B1 (thiamine, precursor of TPP), B2 (riboflavin, precursor of FAD), B3 (niacin, precursor of NAD+), B5 (pantothenic acid, a component of CoA), and calcium are essential for alpha-ketoglutarate dehydrogenase function.307 Lipoic acid supports the enzyme’s oxidative decarboxylation of 2-oxoglutaric acid, while magnesium stabilizes the enzyme complex and aids TPP binding. Deficiencies in these nutrients may elevate 2-oxoglutaric acid levels.307,308,310 Additionally, elevated zinc levels may inhibit alpha-ketoglutarate dehydrogenase, further increasing 2-oxoglutaric acid levels.311

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

2-oxoglutaric elevations may be linked to fumarase deficiency and 2-ketoglutarate dehydrogenase complex deficiency, both of which are rare inherited metabolic disorders.305

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

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Additional Insights

Medication/ Supplement Interactions: Supplemental alpha-ketoglutarate may also increase values.305

Associated Conditions: Both liver and kidney dysfunction or diseases may alter the activity of alpha-ketoglutarate dehydrogenase, leading to increased levels of 2-oxoglutaric acid.312 Alpha- ketoglutarate dehydrogenase is sensitive to downregulation in the presence of reactive oxygen species (ROS) and under oxidative stress.313 Conditions with elevated glutamate levels, such as cerebral ischemia, may lead to increased production of 2-oxoglutaric acid, serving as an alternative energy source for the Citric Acid Cycle during energy-deprived states.314

Benefits: 2-oxoglutaric acid acts as a nitrogen scavenger and a precursor to glutamate and glutamine, enhancing protein synthesis and inhibiting degradation in muscles. It supports bone tissue formation and is essential for collagen production by aiding proline hydroxylation. Additionally, it promotes the differentiation of regulatory T-cells.305

Low Values

Mitochondrial Health: Impaired glycolysis or fatty acid oxidation could hypothetically lead to less acetyl-CoA entering the citric acid cycle and therefore, decreasing intermediate levels.264,265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

Nutritional Needs: Manganese has been shown to inhibit the activity of glutamate dehydrogenase (GDH), the enzyme responsible for converting glutamate into 2-oxoglutaric acid, potentially leading to decreased levels.309

28 Aconitic acid

Aconitic acid is formed as an intermediate between citrate and isocitrate, where the enzyme aconitase catalyzes the conversion of citrate to aconitic acid and then aconitic acid to isocitrate.315 The enzyme requires variable cofactors, including an iron-sulfur cluster, for its activity and is sensitive to oxidative and nitrative stress, which can lead to elevated aconitic acid levels in the step from aconitic acid to isocitric acid.316,317 Elevated aconitic acid can indicate mitochondrial dysfunction, toxic exposures (such as arsenic, aluminum, or fluoride), or nutrient deficiencies, particularly in iron.316-322 Glutathione can protect aconitase from stress- induced inhibition, and iron deficiency can impair its function, leading to increased aconitic acid levels.316,317 Impaired glycolysis or fatty acid oxidation theoretically could result in decrease aconitic acid levels due to reduced acetyl-CoA availability in the citric acid cycle, though evidence is limited.264,265

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Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Toxic exposures, such as arsenic, aluminum, and tin, can inhibit aconitase activity and lead to an increase in aconitic acid levels.318-320 High intake of fluoride may also cause increased aconitic acid levels.321

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Methylation/Detoxification

Aconitase is particularly susceptible to stress and may become inhibited under conditions of oxidative and nitrative stress, leading to increased aconitic acid levels. Glutathione can modulate the inactivity and protect the enzymatic activity.317

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with glutathione.

Nutritional Needs

Aconitase enzymatic function is dependent on iron. In iron-deficient states, the enzyme’s activity is reduced and may lead to elevated levels of aconitic acid.145 Aconitase also requires glutathione to support its enzymatic activity by maintaining the redox state of the Fe-S cluster, along with vitamin C to preserve enzymatic activity and cysteine to contribute to the formation and maintenance of the Fe-S cluster.317,322

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients and glutathione.

Additional Insights

Low Values: Theoretically, impaired glycolysis or fatty acid oxidation could lead to less acetyl- CoA entering the citric acid cycle and therefore, decreasing intermediate levels.264,265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

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29 Citric acid

Citric acid is a key component of the Citric Acid Cycle, formed from acetyl-CoA and oxaloacetate by the enzyme citrate synthase. The enzyme aconitase, which requires iron for activity, converts citric acid to isocitrate, and its function is essential for proper citric acid metabolism.315,316 Elevated citric acid levels can indicate microbial overgrowth, mitochondrial dysfunction, or toxic exposure to substances such as arsenic or aluminum.318-321,323-325 Nutrient deficiencies, particularly in iron, can reduce aconitase activity, leading to increased citric acid levels, while oxidative stress can also inhibit the enzyme.316 Low citric acid levels may result from conditions such as hypokalemia, impaired metabolism, or iron overload, and can increase the risk of oxalate stone formation due to greater free calcium availability.265,326-329

Microbial Health

Many fungi, such as Aspergillus spp, and yeasts, produce citric acid, meaning overgrowths in the microbiome may lead to an increased value.323

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Toxic exposures, such as cadmium, mercury, arsenic, aluminum and tin, can inhibit aconitase activity and lead to an increase in citric acid levels.318-320,324,325 Though data is limited, studies suggest high intake of fluoride may also cause increased citric acid levels. Additionally, if suspicious of Aspergillus, may need to consider mycotoxins.321

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Aconitase requires iron to convert citric acid to aconitic acid. In iron deficient states, the enzyme’s activity is reduced and may lead to elevated levels of citric acid.316 Aconitase may become inhibited under conditions of oxidative and nitrative stress leading to increased citric acid levels. Glutathione can modulate inactivity and protect enzymatic activity.317

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient and glutathione.

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Additional Insights

Dietary Influences: Citrus fruits such as lemons, oranges, and limes contain citric acid, which may be reflected in the value.330 Citrate is commonly added to foods and products as a flavor additive and preservative. Diet sodas are a significant source of citrate, while other sources may include dietary supplements, processed foods, and skincare products or cosmetics.331-334 Given its antibacterial properties, citric acid may be used in cleaning products.335

Associated Conditions: Aconitase enzymatic activity is highly sensitive to oxidative stress, leading to its downregulation or inhibition and subsequent increase in citric acid levels.336

Low Values

Mitochondrial Health: Impaired glycolysis or fatty acid oxidation could lead to less acetyl-CoA entering the citric acid cycle and, therefore, decreasing citric acid levels.265

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 .

Nutritional Needs: Magnesium is required for citrate synthase as this enzyme requires magnesium to facilitate the binding of substrates and subsequent catalytic reactions, including the condensation of acetyl CoA and oxaloacetic to form citrate.337

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Associated Conditions: Hypokalemia can result in low levels of citric acid due to intracellular acidosis and decreased tubular pH, leading to increased citrate uptake and metabolism.326 Iron overload can reduce citric acid levels by increasing aconitase activity and promoting the formation of iron-citrate complexes, which disrupt citrate metabolism and availability.327 Low citric acid levels result in more free calcium availability and decreased inhibition of crystal formation, both of which may increase the likelihood of oxalate stone formation, especially in the presence of elevated oxalic acid 21 .328,329

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

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AMINO ACID METABOLITES

30 3- Methylglutaric acid

3-Methylglutaric acid is involved in leucine metabolism and often accumulates due to mitochondrial dysfunction, particularly when acetyl-CoA is rerouted through the “acetyl- CoA diversion pathway” due to impaired Citric Acid Cycle function.338 Elevated levels of this acid can indicate mitochondrial health issues, especially when the electron transport chain is compromised.338,339 Rare genetic disorders, such as deficiencies in HMGCL or AUH, can also cause its accumulation.338,339 Additionally, certain medications, the ketogenic diet, and components of parenteral nutrition can promote the formation of 3-methylglutaric acid.230

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production. 3-methylglutaric acid levels are particularly elevated when the electron transport chain is impaired, causing the “acetyl-CoA diversion pathway” to be activated. This process prevents acetyl-CoA from entering the Citric Acid Cycle, leading to an increase in 3-methylglutaric acid.338,339

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Genetics

In rare metabolic disorders such as deficiencies in 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) or 3-methylglutaconyl-CoA hydratase (AUH), the breakdown of leucine is disrupted, causing the accumulation of intermediates like 3-methylglutaconyl-CoA. This intermediate may undergo side reactions, leading to the production of 3-methylglutaric acid.338,339

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: The ketogenic diet can result in ketoaciduria and the accumulation of various organic acids, including 3-methylglutaric acid. This is especially significant for individuals with pre-existing mitochondrial dysfunction, as the diet may worsen the metabolic imbalance. Additionally, certain components of total parenteral nutrition (TPN), such as isolated amino acids, can cause abnormal organic acid profiles, including increased levels of 3-methylglutaric acid. This occurs because these substrates can be directly metabolized into 3-methylglutaric acid or related compounds.230

Medication/ Supplement Interactions: Some medications, such as valproate, may promote the formation of 3-methylglutaric acid. The American College of Medical Genetics and

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Genomics states that valproate inhibits 3-methylcrotonyl-CoA carboxylase, resulting in increased urinary excretion of 3-hydroxyisovaleric acid and other related metabolites, which may include 3-methylglutaric acid. Other medications, such as levetiracetam, aspirin, benzoic acid, ibuprofen, and acetaminophen, can also affect organic acid profiles, potentially leading to elevated excretion of various organic acids, including 3-methylglutaric acid.230

Low Values There is no known clinical significance for low values.

31 3-Hydroxyglutaric acid

3-hydroxyglutaric acid is produced during lysine degradation and relies on the enzyme glutaryl-CoA dehydrogenase for its breakdown. Elevated levels of this acid can indicate mitochondrial dysfunction and may act as both an acidogen and a metabotoxin, potentially causing acidosis and other health issues. Chronic elevations are linked to the rare genetic disorder glutaric aciduria type I, which results from a deficiency in glutaryl-CoA dehydrogenase.340 Dietary factors such as the ketogenic diet and medications such as valproate can also increase 3-hydroxyglutaric acid levels.230

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Genetics

Chronically elevated levels of 3-hydroxyglutaric acid may be linked to the rare inherited disorder, glutaric aciduria type I. This is a condition in which the body cannot fully metabolize the amino acids lysine, hydroxylysine, and tryptophan due to a deficiency of mitochondrial glutaryl-CoA dehydrogenase. As a result, excess intermediate breakdown products (such as glutaric acid, glutaryl-CoA, 3-hydroxyglutaric acid, and glutaconic acid) can accumulate, potentially causing damage to the brain, particularly the basal ganglia, as well as other organs.340

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

The American College of Medical Genetics and Genomics notes that dietary factors (i.e., ketogenic diet) and medications (i.e., valproate) can affect organic acid metabolism, potentially leading to elevated levels like 3-hydroxyglutaric acid.230

Dietary Influences: The American College of Medical Genetics and Genomics notes that dietary factors such as the ketogenic diet can impact organic acid metabolism and potentially increase levels of 3-hydroxyglutaric acid.230

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Medication/ Supplement Interactions: The American College of Medical Genetics and Genomics notes that some medications, such as valproate, may affect organic acid metabolism, potentially increasing levels of 3-hydroxyglutaric acid.230

Low Values There is no known clinical significance for low values.

32 3-Methylglutaconic acid

3-methylglutaconic acid is involved in leucine metabolism and the mevalonate shunt, connecting isoprenoid and mitochondrial acetyl-CoA metabolism. Elevated levels can indicate mitochondrial dysfunction and act as both an acidogen and a metabotoxin. Persistent elevations are linked to rare metabolic disorders such as 3-methylglutaconic aciduria and GAMT deficiency.341 Total parenteral nutrition, the ketogenic diet, and medications such as valproate can also increase its levels.230

Mitochondrial Health

Elevations, especially in the presence of other mitochondrial metabolite elevations, can indicate issues with overall mitochondrial health and energy production.

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Genetics

Persistently elevated levels of 3-methylglutaconic acid are linked to some rare inborn errors of metabolism, including 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, 3-methylglutaconic aciduria type I, 3-methylglutaconic aciduria type III, 3-methylglutaconic aciduria type IV, and guanidinoacetate methyltransferase deficiency (GAMT deficiency).341

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Some dietary factors can impact 3-methylglutaconic acid, such as total parenteral nutrition (TPN), certain infant formulas, and the ketogenic diet.230

Medication/ Supplement Interactions: Medications also may play a role in increased 3-methylglutaconic acid levels. Drugs such as valproate, levetiracetam, aspirin, ibuprofen, and acetaminophen can increase 30-methylglutaconic acid.230

Associated Conditions: Some clinical conditions, such as infections, fever, and other stressors, can cause temporary increases.230

Low Values There is no known clinical significance for low values.

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NEUROTRANSMITTER METABOLITES

Figure 4:

This diagram illustrates the interconnected biochemical pathways involved in neurotransmitter synthesis and metabolism, focusing on serotonin and catecholamines. It shows how tryptophan is converted through the biopterin cycle into serotonin to 5HIAA, while phenylalanine and tyrosine are transformed into dopamine, norepinephrine, and epinephrine, to ultimately form HVA and VMA, respectively. Key enzymes (e.g., TDO, IDO, TH, MAO, COMT) and cofactors (such as BH4, iron, copper, B vitamins, and SAMe) are required at multiple steps, with certain processes are inhibited by stress-related factors and microbial metabolites. This illustrates the complex regulation of mood-related neurotransmitters and their dependence on nutrient cofactors, enzymatic activity, and external influences.

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PHENYLALANINE AND TYROSINE METABOLITES

33 Homovanillic acid (HVA) (dopamine)

Homovanillic acid (HVA) is the primary urinary metabolite of dopamine and serves as an indirect marker of dopamine metabolism.342 Elevated HVA levels may suggest increased dopamine turnover, often due to Microbial Overgrowths (e.g., Clostridia or Fusarium species), toxic exposures (heavy metals, pesticides, mycotoxins), or nutritional deficiencies (e.g., vitamin C, copper, magnesium) that impair dopamine conversion via enzymes such as dopamine β-hydroxylase (DBH).120,343-357 Stress, stimulant medications, certain dietary patterns, and methylation status can also influence HVA by altering dopamine synthesis or degradation pathways.358-371 Low HVA may reflect impaired dopamine synthesis, enzyme dysfunction (COMT/ MAO), methylation disturbances, or deficiencies in cofactors such as vitamin B6, B2, C, iron, or magnesium.372-387 Genetic variations in DBH, COMT, or MAO may further modify dopamine metabolism and HVA levels.388,389 Evaluation of related metabolites and cofactors can help contextualize HVA findings within broader biochemical and neurological patterns.

Microbial Overgrowth

Clostridia overgrowth has been linked to inhibition of dopamine beta hydroxylase (DBH), an enzyme crucial for converting dopamine to norepinephrine. This inhibition may disrupt normal dopamine metabolism, potentially increasing HVA levels.120

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

vHVA levels reflect dopamine activity, with elevated levels suggesting increased dopamine metabolism and low levels proposing reduced dopamine turnover or metabolic dysfunction.342

For a more in-depth evaluation into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , Phenylpyruvic acid 70 , and 4-Hydroxyphenyllacetic acid 72 .

Toxic Exposures

Mycotoxins, such as ochratoxin A and fusaric acid, may cause elevated HVA levels by disrupting dopamine metabolism through mechanisms such as DBH inhibition, oxidative stress, and neuroinflammation.120,351-353 Some pesticides increase HVA levels, possibly by inducing oxidative stress or causing dopaminergic neurodegeneration.348-350 Exposure to heavy metals like lead, aluminum, manganese, arsenic, and mercury can result in elevated HVA levels, typically due to the inhibition of DBH activity.343-347

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

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Methylation/Detoxification

Methylation plays a crucial role in dopamine metabolism by influencing the expression of key enzymes such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO).374-376 Additionally, hypomethylation of dopamine-related regulatory elements, like the IGF2 enhancer, can upregulate dopamine synthesis by increasing tyrosine hydroxylase expression.390

VMA 34 and DOPAC 36 values may give additional insights into COMT as well as 5HIAA for MAO function. Additionally, refer to the Nutrient–Marker Cross-Reference Table for insights into corresponding organic acids that are associated with methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutritional Needs

Magnesium deficiency can raise HVA levels by increasing oxidative stress, which upregulates enzymes such as MAO and ALDH.356,357 Insufficient copper and vitamin C levels can also elevate HVA acid levels by reducing DBH activity.354,355

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Genetic single-nucleotide polymorphisms (SNPs) in the DBH enzyme can lead to elevated HVA levels by altering the enzyme’s activity, potentially affecting dopamine to norepinephrine conversion and influencing catecholamine balance.388 Variations in the COMT and/or MAO enzymes can increase HVA levels by altering the enzymatic activity involved in dopamine and norepinephrine metabolism, influencing their related metabolites.389

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Foods rich in phenylalanine and tyrosine, such as meat, fish, dairy, eggs, soy, legumes, and nuts, can elevate HVA levels by providing the substrates necessary for dopamine synthesis.362 Increased L-DOPA intake from foods can lead to elevated dopamine synthesis and subsequently higher HVA levels.363,364 Aspartame consumption may elevate HVA levels and influence dopamine metabolism through mechanisms such as phenylalanine competition, oxidative stress, and inflammation. Additionally, the byproduct phenolic acid of 2-hydroxyhippuric acid, along with other phenolic compounds, can potentially inhibit DBH function by inactivating it, likely through hydrogen atom abstraction, with the phenolic structure itself being critical for this inhibition mechanism.365-368 Salicylates raise HVA levels by inducing oxidative stress and enhancing dopamine metabolism through hydroxyl radical formation and cytochrome P450 enzyme activity, leading to increased dopamine turnover and higher metabolite levels.369,370 Flavanol-rich foods, such as dark chocolate, tea, and berries, can increase HVA levels by enhancing enzyme activity and expression, leading to higher HVA production.371

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Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients. For more insight into possible aspartame or salicylate intake, reference 2-Hydroxyhippuric acid 61 .

Medication/Supplement Interactions: Medications and supplements such as MAO inhibitors, tyrosine, L-dopa, dopamine medications (Levodopa, Sinemet, etc), SNRIs (Wellbutrin, etc), tricyclic antidepressants, amphetamines, appetite suppressants, caffeine, mucuna pruriens, and quercetin can increase HVA levels.360,361,363,364,391-394 Disulfiram and Etamicastat help treat alcohol, drug, and gambling addictions by inhibiting the DBH enzyme, which increases dopamine, decreases norepinephrine, and elevates HVA levels, ultimately reducing addictive behaviors by modulating dopaminergic and adrenergic systems.395,396

Associated Conditions: Dopamine dysregulation in ADHD is thought to result from genetic, neurobiological, and environmental factors affecting dopamine transmission and receptor functioning, leading to symptoms like inattention and hyperactivity; elevated HVA levels are often linked to this dysregulation.397 Elevated HVA levels may be found in autism, likely due to impaired dopamine metabolism, particularly the decreased conversion of dopamine to norepinephrine, which results in increased dopamine turnover and higher HVA production.398,399 Stress can cause elevated HVA levels by triggering increased catecholamine output from the adrenal glands, which is further amplified by vitamin C depletion, as this deficiency disrupts the normal conversion of dopamine to norepinephrine, resulting in heightened dopamine metabolism to HVA.358,359 Stress from post-traumatic stress disorder (PTSD) and general stress increases HVA levels, likely due to higher noradrenergic activity.400

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Low Values

Neurotransmitter Metabolites: Disruptions in COMT and MAO enzymes impair dopamine metabolism, leading to increased dopamine levels and the accumulation of toxic metabolites, which may explain the correlation with low HVA levels.372,373 Evaluating DOPAC 36 in particular can give additional insight into dopamine levels prior to metabolizing to HVA. Moreover, deficiencies in cofactors involved in the dopamine generation pathway, particularly with tetrahydrobiopterin (BH4), can impair synthesis, and cause low dopamine levels, and subsequently low HVA.401

For more insights into dopamine metabolites, assess the phenylalanine and tyrosine metabolites 33 – 37 , 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , and Phenylpyruvic acid 70 .

Methylation/Detoxification: Methylation of the COMT gene can alter its expression, affecting dopamine degradation, while CpG methylation in the MAO-A promoter can regulate dopamine breakdown.374-376 Increased methylation of dopamine-related genes, such as the dopamine transporter (DAT) gene, has been linked to reduced dopamine levels by enhancing DAT

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availability and reducing synaptic dopamine.377,378 Methylation also influences dopamine synthesis by regulating the availability of tetrahydrobiopterin (BH4), which is a critical cofactor for tyrosine hydroxylase (TH).379-381

VMA 34 and DOPAC 36 values make give additional insights into COMT, as well as 5HIAA 38 for MAO, and BH4 functions. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12 and magnesium).

Nutritional Needs: Vitamin B6 deficiency can decrease HVA levels by impairing enzymes, including monoamine oxidase (MAO), which are essential for dopamine metabolism.382 Vitamin B2 (riboflavin) deficiency may decrease levels by impairing the activity of flavoprotein enzymes like MAO and aldehyde dehydrogenase (ALDH), which are essential for dopamine metabolism.383 Additionally, vitamin C, iron, copper, and magnesium, which are cofactors for MAO and COMT, can impair dopamine synthesis and metabolism, leading to reduced dopamine and norepinephrine levels, and consequently, lower HVA levels.384-387

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Dietary: Low levels of the precursors phenylalanine and tyrosine can lead to low HVA levels, as these amino acids are essential for dopamine synthesis, and their deficiency disrupts normal dopamine metabolism.402

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Medication/ Supplement Influences: Antidepressants like fluvoxamine and fluoxetine may decrease dopamine turnover and lower HVA levels through various mechanisms, such as reducing tetrahydrobiopterin levels and inhibiting dopamine release.403,404 Quercetin may also potentially influence HVA levels by competing with dopamine transporters or inhibiting catechol-O-methyltransferase (COMT) activity, reducing the methylation of dopamine and consequently lowering HVA levels.405-408

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34 Vanillylmandelic acid (VMA) (norepinephrine, epinephrine)

Vanillylmandelic acid (VMA) is a metabolite of catecholamines (dopamine, epinephrine, and norepinephrine), produced from the amino acids tyrosine and phenylalanine during their metabolism, and excreted in urine as a marker for catecholamine activity in the body. VMA levels serve as markers for catecholamine imbalances, with elevated levels indicating increased metabolism and low levels suggesting reduced turnover or metabolic dysfunction.409 Toxic exposures, including lead, mercury, manganese, and aluminum, can disrupt catecholamine metabolism, raising VMA levels.410-414 nutritional insufficiencies, such as low magnesium or iron, and deficiencies in vitamins B2, B3, B6, C, and copper, can also impact VMA production by affecting enzymes such as dopamine beta hydroxylase (DBH).386,415-422 Additionally, VMA levels can be affected by medications, stress, genetics, and metabolic disorders, with COMT/MAO impairment and mold toxins potentially causing low levels.120,230,345,351,423-437

Neurotransmitter Metabolites

VMA levels, along with other metabolites such as HVA, serve as markers for catecholamine imbalances and potential disruptions in the enzymes involved in their metabolic pathway. VMA levels reflect the activity of epinephrine and norepinephrine, with elevated levels indicating increased catecholamine metabolism and low levels suggesting reduced turnover or metabolic dysfunction (see below for more information on low values).

For a deeper look into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 .

Toxic Exposures

Exposure to lead, manganese, mercury, and aluminum influences catecholamine metabolism, including the metabolism of dopamine and epinephrine, resulting in increased HVA and VMA levels. Lead interferes with enzymes involved in catecholamine pathways, manganese affects neurochemical metabolism, mercury induces oxidative stress that alters enzyme activity, and aluminum may contribute through similar oxidative stress mechanisms, all of which may lead to elevated levels and highlight the impact of heavy metal exposure on catecholamine metabolism.410-414

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Low magnesium levels are linked to increased catecholamine excretion, which can elevate VMA levels, as magnesium plays a key role in regulating catecholamine release.415

Iron deficiency can increase DBH activity as a compensatory mechanism, which may lead to altered catecholamine metabolism and elevated VMA levels, as DBH is required for VMA synthesis.386,416

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Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Elevated VMA levels can result from some rare inborn errors of metabolism, such as organic acidurias and amino acid metabolism disorders. Additionally, conditions like Costello Syndrome and vitiligo, which involve increased catecholamine release, can also contribute to elevated VMA levels.230,432,433

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: VMA is used in artificial vanilla flavoring and intake can cause elevations.409

Medication/Supplement Interactions: Medications and supplements such as stimulants (amphetamines, ADHD medications, caffeine, ephedrine, appetite suppressants), and dopamine reuptake inhibitors (e.g., risperidone) may increase VMA levels. The use of L-DOPA, dopamine, phenylalanine, and tyrosine raises HVA levels by promoting dopamine synthesis and metabolism and therefore influences VMA.426,427

Associated Conditions: Increased stress activates the sympathetic nervous system and HPA axis, boosting catecholamine release and metabolism into VMA, which serves as a marker for stress-related sympathetic activity. Studies show a positive correlation between elevated VMA levels and both immediate and long-term stress responses, including psychological and physiological stress and PTSD.428-431

Low Values

Microbial Overgrowth: Clostridia overgrowth inhibits the DBH enzyme, which is essential for converting dopamine to norepinephrine, disrupting dopamine metabolism and potentially leading to low VMA levels due to impaired production of norepinephrine and its metabolites.120,351

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Toxic Exposure: The mycotoxin, fusaric acid from Fusarium spp, can also inhibit the DBH enzyme, as can heavy metals such as aluminum and manganese, causing reduced conversion of Dopamine to norepinephrine, leading to lower VMA.120,345,351,436,437

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Methylation/Detoxification: Downregulation of either the COMT or MAO enzyme leads to low VMA levels, as COMT activity is responsible for O-methylating catecholamines into metanephrines, and MAO further deaminates these intermediates into VMA.434,435

HVA 33 and DOPAC 36 values may give additional insights into COMT as well as 5HIAA 38 for MAO function. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding

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organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutritional Needs: Iron overload, along with deficiencies in vitamins B2, C, and copper, can decrease VMA levels by disrupting dopamine beta-hydroxylase (DBH) activity and impairing catecholamine metabolism through various mechanisms.417-419 Insufficient vitamin B3, B6, or tetrahydrobiopterin (BH4) can lead to decreased VMA levels by impairing catecholamine synthesis or enhancing their degradation.420-422

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics: Genetic SNPs that cause deficiencies in the DBH enzyme can also decrease VMA levels.438

Dietary: Low dietary intake of phenylalanine and tyrosine reduces catecholamine synthesis, leading to decreased production of dopamine, norepinephrine, and epinephrine, and consequently lower VMA levels.439

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Medication/ Supplement Influences: Some pharmaceuticals such as disulfiram and etamicastat inhibit the DBH enzyme, which reduces norepinephrine production and impairs dopamine metabolism, potentially leading to low VMA levels.363,364, 371

35 HVA/VMA Ratio

The HVA/VMA ratio measures the balance between dopamine and norepinephrine/epinephrine production by catecholamine-producing neurons in the central nervous system, sympathetic nervous system, and adrenal glands. It reflects the conversion of dopamine to HVA and the conversion of dopamine to norepinephrine, processes regulated by the enzyme dopamine beta-hydroxylase (DBH).342,409 An elevated HVA/VMA ratio typically suggests disruptions in dopamine metabolism, often due to impaired DBH activity, such as through Clostridia overgrowth, mycotoxin exposure, heavy metals, nutritional deficiencies in niacin, copper, or vitamin C, by medications, supplements, or rare genetic factors that affect dopamine to norepinephrine conversion.120,345,351,354,355,388,395,396,436,440,441 In contrast, a low HVA/VMA ratio is usually indicative of reduced dopamine synthesis and turnover, often due to high cortisol levels or other factors inhibiting dopamine production.442-444

Microbial Overgrowth

Clostridia overgrowth inhibits DBH activity through byproducts like HPHPA, 4-cresol, and 4-hydroxyphenylacetic acid, disrupting dopamine metabolism and increasing the HVA/VMA ratio.351

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Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

The HVA/VMA ratio serves as a marker for catecholamine metabolism imbalances, with an elevated ratio suggesting disruptions in the conversion of dopamine to HVA, norepinephrine/ epinephrine to VMA, and/or dopamine to norepinephrine, often due to impaired DBH activity.

For a more in-depth evaluation into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , Phenylpyruvic acid 70 , and 4-Hydroxyphenyllacetic acid 72 .

Toxic Exposure

Fusaric acid, a mycotoxin produced by Fusarium species, inhibits the DBH enzyme, disrupting dopamine conversion to norepinephrine and causing an elevated HVA/VMA ratio due to increased dopamine and its metabolites.120 Elements such as Aluminum and Manganese have been shown to inhibit DBH enzyme, which could cause an elevated HVA/VMA ratio.345,436

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Vitamin B3 (Niacin) supports dopamine to norepinephrine conversion by aiding in NAD/NADP production, and a deficiency could impair DBH activity, leading to an increased HVA/VMA ratio.440,441 A deficiency in copper and vitamin C can reduce DBH activity, leading to an elevated HVA/VMA ratio.354,355

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Genetic single nucleotide polymorphisms (SNPs) in the DBH enzyme can increase the HVA/ VMA ratio by altering enzyme activity, which disrupts dopamine to norepinephrine conversion and affects catecholamine balance.388 Other genetic diseases such as Menkes disease, a rare copper transport disorder that impairs DBH activity, lead to elevated HVA/VMA ratios.354

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Aspartame consumption may increase the HVA/VMA ratio by elevating HVA levels through mechanisms like phenylalanine competition, oxidative stress, and inflammation, which impact dopamine metabolism.363-365

Review 2-Hydroxyhippuric acid 61 for further insights on aspartame intake.

Medication/Supplement Interactions: Certain pharmaceuticals, like disulfiram and etamicastat, inhibit the DBH enzyme, disrupting dopamine metabolism and reducing norepinephrine production, which can increase the HVA/VMA.395,396

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Associated Conditions: Low cortisol levels may increase the HVA/VMA ratio by disrupting the normal regulation of dopamine metabolism, leading to elevated HVA levels due to the effects of HPA axis hormones.445

Low Values High cortisol output can lower HVA/VMA ratio by inhibiting dopamine synthesis and turnover.442 Similarly, by increasing cortisol output, medications and supplements such as some psychostimulants, grapefruit juice, and licorice may decrease HVA/VMA ratio.443,444

36 Dihydroxyphenylacetic acid (DOPAC) (dopamine)

Dihydroxyphenylacetic acid (DOPAC) is a key dopamine metabolite formed through the oxidative deamination of dopamine by monoamine oxidase (MAO), resulting in a neurotoxic byproduct, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized into DOPAC by aldehyde dehydrogenase enzyme (ALDH2). DOPAC, along with HVA and VMA, is used to assess dopamine and norepinephrine metabolism, with fluctuations in these metabolites reflecting alterations in catecholamine turnover and balance.342,409,446 DOPAC levels can be elevated due to disruptions in dopamine metabolism, which can be caused by DBH inhibition via clostridia or toxic exposures such as mycotoxins or heavy metals.120,343-353 Nutritional deficiencies in vitamin B6, B2, magnesium, copper, and vitamin C, along with genetic variations in COMT or DBH, can also impact DOPAC levels.354-357,382,383,388,438,447 Low precursors like phenylalanine and tyrosine, as well as deficiencies in cofactors like tetrahydrobiopterin and other enzymatic cofactors, can impair dopamine synthesis and metabolism, leading to lower DOPAC levels.354-357,382,383,402,435,438,448 Additionally, dietary factors, medications, and certain pharmaceuticals can further affect dopamine metabolism and lead to either elevated or reduced DOPAC levels.363,365-367,369,370,395,402-404,446,449-454

Microbial Overgrowth

DOPAC can become elevated due to disruptions in dopamine metabolism from Clostridia byproducts inhibiting the dopamine beta hydroxylase (DBH) enzyme. These disruptions lead to impaired dopamine conversion to norepinephrine, causing an increase in dopamine levels and its metabolites, including DOPAC.120,351

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Elevated DOPAC levels indicate increased dopamine breakdown, reflecting higher dopamine turnover. This can result from disruptions in dopamine metabolism and can contribute to alterations in catecholamine balance.

For a deeper look into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , as well as 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , and Phenylpyruvic acid 70 .

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Toxic Exposure

Mycotoxins, such as ochratoxin A and fusaric acid, may lead to elevated DOPAC levels by disrupting dopamine metabolism through mechanisms like DBH inhibition, oxidative stress, and neuroinflammation. Similarly, some pesticides may increase DOPAC levels by inducing oxidative stress or causing dopaminergic neurodegeneration. Exposure to heavy metals like lead, aluminum, manganese, arsenic, and mercury can also result in elevated DOPAC levels, often due to the inhibition of DBH activity, which interferes with dopamine conversion.120,343-350,352,353

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Deficiencies in vitamin B6, vitamin B2 (riboflavin), and magnesium can elevate DOPAC levels by impairing the activity of MAO, with magnesium deficiency also increasing oxidative stress and riboflavin influencing tetrahydrobiopterin (BH4). Additionally, insufficient levels of copper and vitamin C can reduce DBH activity, further contributing to higher DOPAC levels.354-357,382,383,438 Catechol-O-methyltransferase (COMT) facilitates the conversion of DOPAC to HVA, and downregulation of COMT can result in elevated DOPAC levels. SAMe (S-adenosyl- L-methionine) and magnesium are essential cofactors for optimal COMT activity.435,448

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Additional Insights

Dietary Influences: Certain foods like tea, citrus, and soy can increase DOPAC levels as they promote its formation through microbial fermentation.363,446,449-452 Aspartame and salicylates can increase DOPAC levels by promoting oxidative stress and enhancing dopamine metabolism, with aspartame affecting dopamine through phenylalanine competition and salicylates via cytochrome P450 enzyme activity and hydroxyl radical formation.365-367,369,370

Medications/Supplement Interactions: Certain pharmaceuticals, such as disulfiram and etamicastat, inhibit the DBH enzyme, increasing dopamine metabolism to DOPAC.395 Additionally, Lithium has been shown to modulate dopamine metabolism, and although it has been shown to inhibit COMT activity, it may vary depending on the current neurochemical state.453,454

Associated Conditions: Single-nucleotide polymorphisms (SNPs) in the DBH enzyme can impair dopamine-to-norepinephrine conversion, elevating DOPAC levels, and disrupting catecholamine balance.355,388,447

Low Values

Methylation/Detoxification: Inhibitions in dopamine-metabolizing enzymes such as MAO can result in elevated dopamine levels and the accumulation of toxic metabolites, preventing their conversion to DOPAC and ultimately lowering levels.372,373

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HVA (33), VMA (34), and 5HIAA (38) values may give additional insights into MAO function.

Nutritional Needs: Deficiencies in cofactors involved in the dopamine pathway, such as BH4, vitamin C, vitamin B6, iron, and the enzymatic cofactors for MAO and COMT, can impair dopamine synthesis and metabolism, leading to reduced dopamine and norepinephrine levels, and consequently, lower DOPAC levels.384-387

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Dietary: Low levels of the precursors phenylalanine and tyrosine can lead to low DOPAC levels, as these amino acids are essential for dopamine synthesis.402

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Medications/Supplement Interactions: Antidepressants like fluvoxamine and fluoxetine decrease dopamine turnover and lower DOPAC levels through various mechanisms such as reducing tetrahydrobiopterin levels and inhibiting dopamine release.403,404 Genetic SNPs in enzymes involved in dopamine metabolism can reduce DOPAC levels.404

37 HVA/DOPAC Ratio

The HVA/DOPAC ratio reflects the balance in dopamine metabolism, with dopamine being first broken down into DOPAC by the monoamine oxidase (MAO) enzyme, and then DOPAC being converted to HVA by catechol-O-methyltransferase (COMT). A higher ratio can indicate increased COMT activity and conversion of DOPAC to HVA or decreased activity of MAO and DOPAC levels, while a lower ratio may suggest reduced COMT activity and slower conversion.342,446 This ratio can be influenced by factors such as genetic variations, methylation donor availability, and nutrient levels such as magnesium and SAMe, which upregulate COMT activity.389,408,448,455-457

Medications, supplements, and environmental factors can also impact COMT and MAO enzyme activity, leading to changes in the HVA/DOPAC ratio and reflecting various metabolic and genetic influences.458-463

Neurotransmitter Metabolites

An elevated HVA/DOPAC ratio reflects increased dopamine metabolism and turnover, indicating more conversion of DOPAC to HVA. This can suggest heightened dopamine activity or a shift in the balance of metabolic processes involved in dopamine breakdown.

For a more in-depth evaluation into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , Phenylpyruvic acid 70 , and 4-Hydroxyphenyllacetic acid 72 .

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Methylation/Detoxification

An increased HVA/DOPAC ratio indicates enhanced methylation activity, as the enzyme COMT plays a key role in transferring a methyl group from S-adenosyl-L-methionine (SAMe) to DOPAC, converting it to HVA. This process reflects an upregulation of methylation, which is important for the inactivation and elimination of dopamine and its metabolites. Factors such as genetic variations or methyl donor availability can influence this ratio, affecting overall methylation efficiency.389,408,457

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutritional Needs

Increased levels or intake of methylation factors, such as magnesium and SAMe, can upregulate COMT activity and increase HVA/DOPAC ratio. COMT enzyme activity can be upregulated by methylation factors like magnesium, which stabilizes the enzyme, and S-adenosylmethionine (SAMe), which acts as a methyl donor. Additionally, methylcobalamin (B12) and methylfolate (B9) enhance COMT activity by promoting the conversion of homocysteine to methionine, supporting SAMe production.448,464

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

COMT enzymatic activity can be upregulated by factors such as genetic variants), while MAO activity may be downregulated, potentially resulting in elevated HVA/DOPAC ratio levels.455,456

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/Supplement Interactions: Some medications and supplements such as L-Dopa, insulin, dihydrotestosterone (DHT), all-trans retinoic acid (ATRA) from vitamin A, and epigallocatechin-3-gallate (EGCG) have been shown to upregulate COMT activity through various mechanisms leading to an increased HVA/DOPAC ratio.458-460 The use of MAO inhibiting medications such as MAOIs for depression and supplements such as curcumin, guaiacol, resveratrol, and zingerone can also lead to an increase in HVA/DOPAC ratio by decreasing conversion of dopamine to DOPAC.461,462

Low Values

Toxic Exposure: Certain environmental toxicants, particularly the Polychlorinated Biphenyls (PCBs) have been shown to influence COMT function, potentially causing low HVA/DOPAC ratios.463

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

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Methylation/Detoxification: Downregulation of the COMT enzyme from low methylation factors such as SAMe, or genetic variants, may lead to decreased HVA/DOPAC ratios.448,455,464 Upregulation of the MAO enzyme or genetic variants may also decrease the HVA/DOPAC ratio.465,466

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutrient Needs: High levels of calcium, iron, and boron may potentially impair COMT function leading to decreased HVA/DOPAC ratios.448,455,464 Additionally, decreased levels of magnesium, a major cofactor for COMT, as well as methylation supporting nutrients such as methylcobalamin, or methylfolate, may decrease COMT activity.448,455,464

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

TRYPTOPHAN METABOLITES

38 5-Hydroxindoleacetic acid (serotonin)

5-Hydroxy indoleacetic acid (5-HIAA) is the primary metabolite of serotonin, formed when serotonin is enzymatically broken down by monoamine oxidase A (MAO-A) in the liver or synaptic cleft. It is used as a marker for serotonin levels and plays a role in various metabolic processes.470,471 Elevated 5-HIAA levels reflect increased serotonin breakdown, which can be influenced by factors such as dysbiosis, toxic exposure, nutritional status, medications and supplements, and conditions such as celiac disease and in more rare instances, carcinoid syndrome.472-478 On the other hand, disruptions in the microbiome, nutrient deficiencies, and downregulation of MAO can result in lower serotonin and 5-HIAA levels.376,478-483

Microbial Overgrowth

Dysbiosis, or an imbalance in gut microbiota, can lead to elevated serotonin levels by influencing enterochromaffin (EC) cells, which produce serotonin in the gastrointestinal tract. Certain gut bacteria, through the production of short-chain fatty acids, promote the synthesis of serotonin, while other bacteria stimulate serotonin release directly from EC cells.484-486

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Elevated 5-HIAA levels serve as an indicator of serotonin status, reflecting the breakdown of serotonin by MAO-A.

For a deeper look into neurotransmitter metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 and Tryptophan metabolites 38 – 40 .

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Toxic Exposure

Toxic exposures, especially from acrylamide, cause elevated 5-HIAA levels through mechanisms involving oxidative stress, neuroinflammation, and impaired neurotransmitter metabolism.472-474

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Methylation/Detoxification

Hypomethylation may upregulate MAO-A activity and increase 5-HIAA levels.376

For additional insights into MAO, assess HVA 33 , VMA 34 , and DOPAC 36 . Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Additional Insights

Dietary Influences: Some foods, such as bananas, pineapple, walnuts, soybeans, chicken, and tuna, can elevate 5-HIAA levels because of their high tryptophan and serotonin content.475-478

Medications/Supplement Interactions: Some medications and supplements such as SSRIs and 5-HTP, among others can elevate 5-HIAA levels by increasing serotonin availability, which is then metabolized by MAO into 5-HIAA.487,488

Associated Conditions: Celiac disease and tropical sprue can raise 5-HIAA levels by causing hyperplasia of serotonin-producing enterochromaffin cells in the intestinal mucosa, leading to increased serotonin production and its subsequent conversion to 5-HIAA.489,490 Carcinoid syndrome, which is rare and requires a 24-hour urine collection, among other testing to further evaluate, causes elevated 5-HIAA levels due to increased serotonin production from neuroendocrine tumors.491

Low Values

Microbial Overgrowth: Disruption of the gut microbiome, such as through antibiotic treatment, can reduce serotonin production and therefore 5-HIAA levels, emphasizing the role of a balanced microbiome in regulating serotonin levels.479

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Methylation/Detoxification: Hypermethylation can lead to downregulated MAO-A and lower 5-HIAA levels.376 MAO downregulation due to medications such as MAOIs and genetic SNPs, can lead to lower 5-HIAA levels.456,461

For additional insights into MAO, assess HVA 33 , VMA 34 , and DOPAC 36 .

Nutritional Needs: Deficiencies in key nutrients like vitamin B6, tetrahydrobiopterin (BH4), vitamin B2, zinc, tryptophan, Vitamin D, and magnesium can impair serotonin synthesis by disrupting the enzymes, cofactors and precursors necessary for serotonin production, resulting in lower serotonin and 5-HIAA levels.478,480-483

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients. Phosphoric acid 76 may give insights into vitamin D status.

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39 Quinolinic acid

Quinolinic acid (QA) is a neuroactive metabolite that is primarily produced through the kynurenine pathway, where tryptophan is degraded by the enzyme indoleamine 2,3-dioxygenase (IDO). This pathway is activated during inflammatory responses, leading to the accumulation of quinolinic acid, which serves a crucial role in NAD+ synthesis but also acts as an NMDA receptor agonist and neurotoxin. Elevated quinolinic acid levels are associated with excitotoxicity, inflammation, neurodegeneration, and conditions such as neurological and psychiatric disorders.492,493 Factors such as Microbial Overgrowth, nutrient deficiencies, toxic exposures, immune modulation, high cortisol, and inflammatory states can modulate quinolinic acid production, increasing its neurotoxic effects.493-507

Microbial Overgrowth

The gut microbiome can influence elevated quinolinic acid levels by altering tryptophan metabolism through the kynurenine pathway, particularly by enzymatic modulation of IDO1, which is upregulated during inflammation and may lead to increased quinolinic acid levels. Additionally, systemic inflammation triggered by lipopolysaccharide (LPS) activates macrophages, which are a significant source of quinolinic acid production.494,495

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Elevated quinolinic acid levels indicate potential disruptions in neurotransmitter balance, as it acts as an NMDA receptor agonist, contributing to excitotoxicity, neuroinflammation, and neurodegeneration. Its presence suggests heightened activation of the kynurenine pathway, which may also affect NAD+ synthesis and cellular energy, as well as reduced serotonin production.

For a deeper look into neurotransmitter metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 and Tryptophan metabolites 38 – 40 .

Toxic Exposure

Phthalates, such as DEHP and MEHP, inhibit ACMS decarboxylase (ACMSD), disrupting tryptophan metabolism and leading to increased production of quinolinic. Elevated quinolinic levels impair NAD+ synthesis, as phthalate exposure also inhibits quinolinic acid phosphoribosyltransferase (QPRT), an enzyme crucial for NAD+ biosynthesis.497,498

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

B3 (niacin) deficiency can cause elevated quinolinic acid levels due to the disruption of the kynurenine pathway, where excessive inflammation-induced IDO enzymatic activity depletes tryptophan and leads to the accumulation of kynurenine and quinolinic acid. This can impair

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NAD+ production, as quinolinic acid is a precursor in the synthesis of nicotinamide.493 Low levels of vitamins B2 and B6 can elevate quinolinic acid levels by impairing the kynurenine pathway, as both vitamins are essential cofactors for its proper metabolism.496

Elevated quinolinic acid acts as a metal chelator, binding transition metals like iron and copper, reducing their availability. This can disrupt essential redox reactions and lead to oxidative stress, which not only contributes to reactive oxygen species (ROS) production but also potentially depletes key nutrients, further impairing metabolic and immune function.508

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Additional Insights

Dietary Influences: Foods rich in tryptophan, such as chicken, soy, tuna, certain cereals, nuts, and bananas, may potentially raise quinolinic acid levels. Quinolinic acid has also been found in foods like Ceylon cinnamon, pitanga, Oregon yampahs, red bell peppers, and durians.477,492

Refer to the Nutrient–Marker Cross-Reference Table for insights into corresponding organic acids that are associated with tryptophan.

Medications/Supplement Interactions: Some medications, such as interferon-gamma and pyrazinamide are known to increase quinolinic acid levels through their effects on the kynurenine pathway.509,510 Oral contraceptive use is associated with lower neuroprotective kynurenic acid, a decreased kynurenic acid to quinolinic acid ratio, and higher CRP levels, potentially contributing to increased quinolinic acid levels.511

Associated Conditions: Quinolinic acid is strongly associated with inflammation and immune modulation, playing a central role in several health conditions. Estrogen has been shown to increase quinolinic acid levels by boosting pro-inflammatory cytokines and neurotoxic kynurenine metabolites.503 Elevated quinolinic acid levels are linked to neurological and psychiatric disorders, such as Alzheimer’s disease, stroke, schizophrenia, depression, and possibly autism, where they contribute to neuroinflammation and excitotoxicity.504-507

Additionally, high quinolinic acid levels are implicated in cardiovascular conditions, particularly atherosclerosis and ischemic injury, due to their connection with oxidative stress, inflammation, and early signs of systemic atherosclerosis.512,513 Immune activation, such as from cytokines stimulates increased quinolinic acid production, which is linked to inflammation and neurological damage.499-501 High cortisol levels can exacerbate the damaging effects of quinolinic, suggesting that elevated cortisol may increase quinolinic acid’s neurotoxic effects.502 Furthermore, quinolinic acid inhibits gluconeogenesis by binding to divalent metal ions required for phosphoenolpyruvate carboxykinase (PEPCK), thereby reducing glucose production.514 Elevated levels may also be associated with Lyme disease and multiple sclerosis as well as general increased viral load due to interferons upregulating the IDO enzyme.515

Benefits: Quinolinic acid may play a beneficial role in certain contexts, such as being a precursor for NAD+ synthesis, which is essential for energy production, DNA repair, and enzyme activity,

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as well as helping modulate immune responses during neuroinflammation. However, despite these potential benefits, elevated quinolinic levels are also linked to neurotoxicity, contributing to excitotoxicity, neuroinflammation, and neuronal damage.506,516

Low Values Low quinolinic acid levels are less concerning than elevated levels and can result from factors affecting the kynurenine pathway, such as enzyme inhibition (e.g., kynureninase inhibitors), the use of Highly Active Antiretroviral Therapy (HAART) in HIV patients, genetic mutations like the knockout of the QPRT gene, or nutritional influences such as low protein intake influencing enzymes like ACMSD.517-520 These factors can reduce the synthesis or conversion of quinolinic acid, offering potential therapeutic avenues for modulating its levels in clinical settings.

40 Kynurenic acid

The kynurenine pathway is a major metabolic route for tryptophan, where it is converted into several metabolites, including kynurenic acid and quinolinic acid, and is a key source of NAD+, essential for cellular energy production. While the pathway is beneficial for NAD+ synthesis and regulating physiological functions, it can become problematic when upregulated due to inflammation or stress, potentially leading to a deficiency in serotonin production and the accumulation of neuroactive metabolites like quinolinic acid, which can contribute to neurotoxicity, excitotoxicity, and neuroinflammation.492,521-524 Kynurenic acid levels are influenced by factors such as inflammation, stress, and nutritional deficiencies in B vitamins (B3, B2, B6), which disrupt the kynurenine pathway, as well as dietary intake of tryptophan-rich foods and certain medications.477,493,496,522-528 Elevated kynurenic acid is associated with conditions such as neurodegenerative diseases, psychiatric disorders, and inflammatory diseases, while low levels can result from high estrogen states, certain treatments, or insufficient protein intake.503,518,520-523

Neurotransmitter Metabolites

Elevated kynurenic acid levels may indicate an upregulated kynurenine pathway due to inflammation or stress, leading to a disruption in serotonin production and an accumulation of neuroactive metabolites like quinolinic acid. While kynurenic acid has neuroprotective effects, increased levels could contribute to neuroinflammation, excitotoxicity, and neurodegenerative or psychiatric disorders.

For a deeper look into neurotransmitter metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 and Tryptophan metabolites 38 – 40 .

Nutritional Needs

Insufficient levels of B3 (Niacin), B2 (Riboflavin), and B6 can increase kynurenic acid levels by disrupting the kynurenine pathway.493,496

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

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Additional Insights

Dietary Influences: Foods rich in tryptophan, such as chicken, soy, tuna, certain cereals, nuts, and bananas, may potentially increase kynurenic acid levels.477 Some foods contain kynurenic acid and will increase levels such as cacao powder, fermented foods (beer, red wine, yeast- fermented foods), honeybee products (propolis honey, bee pollen), tea, and coffee.529-531

Refer to the Nutrient–Marker Reference Table for insights into corresponding organic acids that are associated with tryptophan.

Medications/Supplement Interactions: Medications that may increase kynurenic acid levels include anticonvulsants, antidepressants, beta-adrenergic agonists, and angiotensin-converting enzyme inhibitors.525-528

Associated Conditions: Elevated kynurenic acid levels can be influenced by neurodegenerative conditions such as Alzheimer’s, Parkinson’s, and Huntington’s disease, as well as by infections like HIV-1 and Lyme neuroborreliosis. Additionally, increased kynurenic acid metabolism is linked to cognitive decline in disorders like schizophrenia and Down syndrome, and it has been identified as a uremic toxin in renal dysfunction.521 Increased stress in conditions such as major depressive disorder and more activates the tryptophan-kynurenine pathway, suppressing enzymatic function and leading to elevated kynurenic acid levels, which are linked to stress-induced behavioral changes and cognitive impairments.293 High kynurenic levels are associated with various inflammatory diseases, including ulcerative colitis and systemic lupus erythematosus, where elevated levels correlate with disease severity, activity, and clinical manifestations.522,523

Benefits: Kynurenic acid may provide neuroprotection through multiple mechanisms, including NMDA receptor antagonism to reduce excitotoxicity, antioxidant properties to scavenge reactive oxygen species, and the induction of neprilysin to degrade amyloid-beta peptides. Additionally, it stimulates BDNF-TrkB signaling, protects mitochondria, and modulates redox homeostasis to maintain neuronal survival and prevent oxidative damage.532-536

Low Values

Nutrient Needs: Vitamin B6 is an important cofactor for kynurenine aminotransferase (KAT), an enzyme crucial for converting kynurenine to kynurenic acid in the tryptophan metabolism pathway. Deficiency may lead to low kynurenic acid levels.537

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Dietary: Low protein intake, influencing the availability of the precursor tryptophan, may influence Kynurenic acid to be produced.520

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Medications/Supplement Interactions: The use of treatments such as Highly Active

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Antiretroviral Therapy (HAART) in HIV patients has been associated with low Kynurenic acid.518 Oral contraceptive use is linked to decreased kynurenic acid levels and reduced kynurenic to quinolinic ratios, potentially contributing to altered immune function and increased systemic inflammation.511

Associated conditions: Low kynurenic levels can result from high estrogen states.503 A hormone panel may give further assessment, if this is a potential.

PYRIMIDINE METABOLITES – FOLATE METABOLISM Methylation/Detoxification

41 Uracil

Uracil is a pyrimidine primarily found in RNA, where it pairs with adenine during transcription and translation, and is replaced by thymine in DNA.538 It plays a vital role in enzyme synthesis, cellular function, and biochemical regulation by serving as a coenzyme, an allosteric regulator, and a precursor to phosphorylated nucleotides (UMP, UDP, and UTP), which influence carbamoyl phosphate synthetase II activity, polysaccharide biosynthesis, and sugar transport.539-541 The conversion of uracil to thymine 42 is dependent on folate and the methylation of uracil, with 5,10-methylene tetrahydrofolate donating a methyl group via thymidylate synthase.542,543 Elevated uracil levels can indicate impaired methylation due to folate deficiency and various genetic variants.542,544,545 Other contributing factors include liver dysfunction and certain cancer treatments such as 5-fluorouracil.546,547

Methylation/Detoxification

The conversion of uracil to thymine depends on 5,10-methylene tetrahydrofolate, which donates a methyl group via thymidylate synthase.543 When folate metabolism is impaired, methylation is disrupted, leading to uracil accumulation in the nucleus.543 Impacts on the conversion of 5-methyltetrahydrofolate into SAM, which regulates gene transcription, is driven by folate, and without it, SAM is depleted, leading to DNA hypomethylation and altered gene expression.548 Uracil has been used as a potential biomarker for folate and methylation status from either genetic or micronutrient deficiencies.548

Review of 2-hydroxybutyric acid 59 , a byproduct of the homocysteine to cystathionine pathway, may also provide insight, along with a homocysteine level. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutrient Needs

Folate (Vitamin B9) deficiency may lead to excessive uracil incorporation into DNA, causing chromosome breaks from impaired methylation of dUMP to dTMP.549 These damages have

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been linked to cognitive risks and defects in cellular replication, and folate supplementation has demonstrated reversal.549 Moreover, deficiencies in vitamins B12, B6 (pyridoxine), and B2 (riboflavin) can also impact methylation capacity, impairing uracil-to-thymine conversion, leading to uracil accumulation in DNA, which may contribute to genetic instability and disease risk.550,551 In the presence of folate deficiency, this pathway can be preserved at the expense of homocysteine re-methylation.552

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Urea cycle disorders, particularly Ornithine Transcarbamylase Deficiency (OTCD), can cause excess carbamoylphosphate diversion into pyrimidine synthesis, leading to elevated uracil and orotic acid 60 levels.545 Elevated uracil can also come from genetic variants in dihydropyrimidine dehydrogenase (DPD) enzyme through the DPYD gene, as it can impact the catabolism of uracil, thymine 42 , and the analog 5-fluorouracil.544,553 Genetic variations affecting thymidylate synthase (TYMS) activity, which is the enzyme responsible for converting deoxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), may also lead to elevated uracil levels.542 SNPs in the production of methyl tetrahydrofolate dehydrogenase I (MTHFD1) will influence the conversion of uracil to thymine as well as methionine.543

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/ Supplement Influences: Cancer treatments such as 5-fluorouracil (5-FU) can increase uracil excretion in the urine.547

Associated Conditions: Liver dysfunction can disrupt uracil metabolism, leading to elevated urinary uracil levels.546 Uracil elevations related to folate deficiency have been shown in neuronal damage.549, 554 MTHFD1 is folate dependent, and when deficient can play a role in megaloblastic anemia, and severe combined immunodeficiency (SCID).555

Low Values There is no known clinical significance for low values.damage.556

42 Thymine

Thymine, also known as 5-methyluracil, is one of the four essential nucleobases in DNA, where it pairs with adenine to stabilize the double-helix structure. Thymine is synthesized by uracil methylation, and when bound to deoxyribose, forms thymidine, which can be phosphorylated to dTDP for DNA incorporation. Thymine metabolism can be influenced by abnormalities in methylation pathways and mutations in dihydropyrimidine dehydrogenase (DPD) production.551,553 It has also been shown to act as a biomarker for UV-induced DNA damage.556

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Methylation/Detoxification

The conversion of uracil to thymine depends on 5,10-methylene tetrahydrofolate, which donates a methyl group via thymidylate synthase.552 Thymine can be influenced by the polymorphism MTHFR C677T at the de novo dTMP synthesis, which can influence uracil’s misincorporation into DNA.551

Nutrient Needs

Folate deficiency, along with deficiencies in vitamins B12, B6 (pyridoxine), and B2 (riboflavin) can all impact methylation capacity, impairing uracil-to-thymine conversion.550,551 In the presence of folate deficiency, this pathway can be preserved at the expense of homocysteine remethylation.552

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Dihydropyrimidine dehydrogenase (DPD) deficiency is a rare genetic condition that impairs the breakdown of pyrimidines, leading to elevated urinary levels of uracil 41 , thymine, and the analog 5-fluorouracil.544,553 The DPD enzyme relies on flavin and NADP (nicotinamide adenine dinucleotide phosphate) as cofactors for this process.557 While some individuals with DPD deficiency remain asymptomatic, severe cases have been associated with seizures, autism, microcephaly, hypotonia, and delayed myelination.558,559

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Associated Conditions: Thymine can act as a biomarker for UV-induced DNA damage in humans, as exposure to UV radiation can cause DNA lesions, such as thymine dimers, leading to elevated urinary thymine levels.556,560 Research studies have shown increased thymine levels in psoriasis patients receiving PUVA therapy (psoralen with UVA radiation), a treatment known to induce DNA damage, and in lifeguards and agricultural workers with UV exposure consecutively for 12-18 days.560,561

Low Values There is no known clinical significance for low values.

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KETONE AND FATTY ACID OXIDATION

Glucose

THE CELL

Lipids

C

Y

T

O

P

L

A

S

M

Lactic Acid

Proteins

As, Cd

LDH

Fatty Acids

Pyruvic Acid

Amino Acids

Adipic Acid

PDC

Carnitine

Suberic Acid

Shuttle

Cd, Hg, Sb

B2, B3, CoA L-Carnitine

Ni, Cd

– OXIDATION

Acetyl-CoA

A

RI

D

N

O

CH

TO

MI

This diagram shows how lipids are broken down into fatty acids, which require the carnitine shuttle to cross into the mitochondria for β-oxidation. In the cytoplasm, fatty acids are first transported with the help of L-carnitine and converted into acylcarnitine, which is shuttled across the mitochondrial membranes and reconverted to acyl-CoA. Once inside the mitochondrial matrix, β-oxidation removes two-carbon units to form acetyl-CoA, a critical substrate for the TCA cycle and ketone production. If the carnitine shuttle is impaired or insufficient, fatty acid transport into mitochondria is blocked, leading to an accumulation of intermediates such as adipic and suberic acids and reduced energy generation from fats. Additionally, when fatty acid oxidation is limited, the body may rely more heavily on glycolysis and amino acid metabolism, with ketone body production (like 3-hydroxybutyric acid) reduced as an alternative energy source.

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43 3-Hydroxybutyric acid

3-Hydroxybutyric acid (β-hydroxybutyrate) and acetoacetic acid 42 are two main ketones involved in fatty acid metabolism and energy production.562 Their levels increase during fasting, carbohydrate-restricted diets, diabetes, or metabolic disorders. 563 Ketones serve as an essential alternative energy source, particularly for the brain and muscles, and play a role in lipid biosynthesis (e.g. cholesterol, phospholipids, etc.) and myelination.564 Elevated ketone levels can indicate metabolic shifts, such as prolonged fasting, vigorous exercise, or more serious conditions such as uncontrolled diabetes, or in rare cases, genetic disorders.564

Mitochondrial Health

Ketone production occurs in the mitochondria, after coenzyme A bound fatty acids (Fatty acyl CoA) are transported into the mitochondrial membrane via the carnitine shuttle.565 Once inside the mitochondria, the bound fatty acids undergo beta- oxidation generating acetyl CoA, for the citric acid cycle for ATP production, lipid biosynthesis, or for the synthesis of acetoacetate 44 .562 Additionally, certain amino acids, like leucine, can metabolize into acetoacetate and acetyl CoA.566 Acetoacetate can be further reduced to 3-hydroxybutyrate (3HB) via 3HB dehydrogenase, which is reversible based on NADH levels, and tends to be higher than acetoacetate in prolonged fasted states.564 Mitochondrial function regulates ketogenesis, and disruptions in this process, such as mitochondrial disorders or fatty acid oxidation defects, can lead to excessive ketone accumulation.564

Additional insights into the cascading effects of ketone generation can be found in acetoacetic acid 44 , the fatty acid oxidation markers 45 – 49 , and the other mitochondrial markers 22 – 32 .

Genetics

Two common inherited genetic conditions associated with abnormal ketone metabolism are systemic primary carnitine deficiency (CDSP), preventing fatty acyl CoA molecules from being transported into the mitochondria, and medium-chain acyl CoA dehydrogenase (MCAD) deficiency, a disorder that impairs the conversion of 4–12 carbon fatty acyl CoA molecules into acetyl CoA.567,568

Markers also associated with MCAD, include Acetoacetic acid 44 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Information

Dietary: Restricted diets such as strict ketogenic, GAPS, and other diets with elimination of carbohydrates can increase ketone production.563

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Medication/ Supplement Influence: Valproate has been shown to elevate ketones.183 Exogenous ketone supplementation, typically a variation of beta-hydroxybutyrate, has been shown to significantly increase blood and urinary levels.570-573

Associated Conditions: Ketones, though not diagnostic, can be associated with dysglycemia, diabetes, alcohol intoxication, strenuous exercise, corticoid steroid deficiencies, and infections.574,575

Developing Discussions: When evaluating 3HB and acetoacetate, blood tests are beginning to use their ratio to evaluate abnormal redox states of the mitochondria and give insights into the respiratory chain.190 Since the conversion of acetoacetate to 3HB is driven by NADH, if 3HB is higher than acetoacetate, Vitamin B3 (niacin), may be helpful in supporting the reaction.577,578 Furthermore, lactate and pyruvate ratio analysis are being evaluated to additionally evaluate the cystolic redox state since pyruvate is converted to lactate to re-oxidize NADH.578

For additional insights into NAD, one can also assess Quinolinic acid 39 elevations, as well as Lactic acid 22 and Pyruvic acid 23 for redox associations. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Low Values There is no known clinical significance for low values.

44 Acetoacetic acid

Acetoacetic acid and 3-Hydroxybutyric acid (3HB) 43 are two main ketones involved in fatty acid metabolism and energy production.562 Their levels increase during fasting, carbohydrate- restricted diets, diabetes, or metabolic disorders.563 Ketones serve as an essential alternative energy source, particularly for the brain and muscles, and play a role in lipid biosynthesis (e.g., cholesterol, phospholipids, etc.) and myelination.564 Elevated ketone levels can indicate metabolic shifts, such as prolonged fasting, vigorous exercise, or more serious conditions, such as uncontrolled diabetes, or in rare cases, genetic disorders.564

Mitochondrial Health

Ketone production occurs in the mitochondria after fatty acyl-CoA is transported across the mitochondrial membrane via the carnitine shuttle.565 Once inside the mitochondria, fatty acids undergo beta-oxidation, generating acetyl-CoA, which can enter the citric acid cycle, contribute to lipid biosynthesis, or serve as a precursor for acetoacetate synthesis.562 Additionally, certain amino acids, like leucine, can metabolize into acetoacetate and acetyl CoA.566 Acetyl CoA is then converted to acetoacetyl CoA by 3-ketothiolase, to HMG CoA by HMG CoA synthase, and finally broken down into acetoacetate by HMG CoA lyase.65 Acetoacetate can be further reduced to 3-hydroxybutyrate (3HB) via 3HB dehydrogenase, which is reversible based on NADH levels, and tends to be higher than acetoacetate in

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prolonged fasted states.202 Mitochondrial function regulates ketogenesis, and disruptions in this process, such as mitochondrial disorders or fatty acid oxidation defects, can lead to excessive ketone accumulation.564

Additional insights into the cascading effects of ketone generation can be found in 3HB 43 , the fatty acid oxidation markers 45 – 49 , the other mitochondrial markers 22 – 32 , and 3-hydroxy-3- methylglutaric acid 55 .

Genetics

Two common inherited genetic conditions associated with abnormal ketone metabolism are systemic primary carnitine deficiency (CDSP), preventing fatty acyl CoA molecules from being transported into the mitochondria, and medium-chain acyl CoA dehydrogenase (MCAD) deficiency, a disorder that impairs the conversion of 4–12 carbon fatty acyl CoA molecules into acetyl CoA.567,568

Markers also associated with MCAD, include 3-Hydroxybutyric acid 43 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Information

Dietary: Restricted diets such as strict ketogenic, GAPS, and other diets with elimination of carbohydrates can increase ketone production.563

Medication/Supplementation Influences: Valproate has been shown to elevate ketones.569

Associated Conditions: Ketones, though not diagnostic, can be associated with dysglycemia, diabetes, alcohol intoxication, strenuous exercise, corticoid steroid deficiencies, and infections.574,575

Developing Discussions: When evaluating 3HB and acetoacetate, blood tests are beginning to use their ratio to evaluate abnormal redox states of the mitochondria, and give insights into the respiratory chain.576 Since the conversion of acetoacetate to 3HB is driven by NADH, if 3HB is higher than acetoacetate, Vitamin B3 (niacin), may be helpful in supporting the reaction.577,578 Furthermore, lactate and pyruvate ratio analysis are being evaluated to additionally evaluate the cystolic redox state since pyruvate is converted to lactate to re-oxidize NADH.578

For additional insights into NAD, one can also assess Quinolinic acid 39 elevations, as well as Lactic acid 22 and Pyruvic acid 23 for redox associations. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Low Values There is no known clinical significance for low values.

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45 Ethylmalonic acid

Ethylmalonic acid (EMA) is a key metabolic marker associated with fatty acid oxidation disorders and mitochondrial dysfunction.579 It is derived from butyrate metabolism and can accumulate due to high-fat or vegan diets, riboflavin or carnitine insufficiencies, or in more rare instances, genetic mutations.580-582

Microbial Overgrowth

Although there is no direct association between butyrate-producing organisms, butyrate production, and ethylmalonic acid, theoretically, if there is a significant amount of butyrate production, it may have an influence on ethylmalonic levels.583,584

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Since EMA is linked to fatty acid oxidation and energy metabolism, reduced function of the pathway can have significant impacts on mitochondrial processes 62 EMA can also disrupt mitochondrial function by inhibiting key complexes within the electron transport chain (ETC), leading to impaired ATP production and increased oxidative stress, leading to further mitochondrial inefficiencies and imbalances.581,585 It can also interfere with succinate and glutamate, partly by inhibiting α-ketoglutarate dehydrogenase (KGDH) and compromising membrane potential.586,587

The mitochondrial metabolite 2-Oxoglutaric acid 27 is also influenced by KGDH, and additional changes in Succinic acid 24 may give further insights into EMA’s direct influence on the Citric Acid Cycle. Insights into the cascading effects on the other mitochondrial markers 22 – 32 might also be supportive.

Nutritional Needs

Deficiencies in carnitine or Vitamin B2 (Riboflavin) can lead to abnormal EMA levels, as carnitine is required for transporting fatty acids for oxidation and riboflavin is crucial for flavin adenine dinucleotide (FAD)-dependent enzymes involved in fatty acid metabolism.588,589

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Conditions like MCAD deficiency, glutaric acidemia II, ethylmalonic acidemia, and ethylmalonic encephalopathy caused by mutations in ETHE1 or SLC25A32 genes that disrupt butyrate metabolism can all disrupt fatty acid metabolism and lead to EMA accumulation.589-592 Genetic mutations in the short-chain acyl-CoA dehydrogenase (SCAD) enzyme that controls butyrate oxidative pathways could also increase ethylmalonic acid.579

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Markers associated with MCAD include 3-Hydroxybutyric acid 43 , Acetoacetic acid 44 , Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 . Glutaric acidemia is associated with 3-hydroxyglutaric acid 31 , Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , Sebacic acid 49 , and Glutaric acid 53 . Methylsuccinic acid 46 , together with EMA, can be associated with ethylmalonic acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Very high-fat diets (Ketogenic, GAPS, etc.) or extreme carbohydrate restriction, without proper intake of carnitine and Vitamin B2 (Riboflavin) can cause altered fatty acid metabolism and may influence elevations in EMA.582 Additionally, vegan diets without proper protein complementing may also increase the risk of carnitine deficiency, and stress fatty acid metabolism. Additional fatty acid oxidation markers that could also be influenced include Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 .582

Medication/ Supplement Influences: Since EMA is derived from butyrate, supplemental butyrate intake may increase EMA excretion levels.580 Medium-chain triglyceride (MCT) supplements and coconut oil may also influence elevations due to their ability to promote rapid oxidation of medium-chain fatty acids.582,593

Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 can also be influenced by MCT-containing foods and supplements.

Low Values There is no known clinical significance for low values.

46 Methylsuccinic acid

Methylsuccinic acid (MSA), is a fatty acid metabolite involved in isoleucine metabolism.594 It is associated with mitochondrial dysfunction from abnormal fatty acid utilization and has been associated with type 2 diabetes and various inborn errors of metabolism.595,596

Mitochondrial Health

MSA is linked to fatty acid oxidation and energy metabolism, and reduced function of the pathway can have significant impacts on mitochondrial dysfunction and cellular health. In reducing oxidative phosphorylation (OXPHOS) efficiency of fatty acids, ATP production is decreased, and energy deficits occur.597,598 This dysfunction also causes structural mitochondrial abnormalities, calcium homeostasis disruption, and increased reactive oxygen species (ROS), further damaging mitochondrial integrity.595,599

Additional insights into the cascading effects of mitochondrial issues include the other fatty acid oxidation markers 45 – 49 , and mitochondrial markers 22 – 32 .

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Nutritional Needs

Deficiencies in carnitine or Vitamin B2 (Riboflavin) can lead to abnormal MSA levels, as carnitine is required for transporting fatty acids for oxidation and riboflavin is crucial for flavin adenine dinucleotide (FAD)-dependent enzymes involved in fatty acid metabolism.600,601

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Conditions such as medium-chain acyl-CoA dehydrogenase deficiency (MCAD), ethylmalonic encephalopathy, glutaric acidemia, and isovaleric acidemia, have all been associated with MSA elevations.591,592,602 Short-chain acyl-CoA dehydrogenase (SCAD) enzyme defects can also cause elevations in MSA, along with ethylmalonic acid 45 .603

Markers also associated with MCAD include 3-Hydroxybutyric acid 43 , Acetoacetic acid 44 , Ethylmalonic acid 45 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 . Ethylmalonic acid 45 combined with MSA is associated with ethylmalonic encephalopathy. 3-Hydroxyglutaric acid 31 , Ethylmalonic acid 45 , Adipic acid 47 , Suberic acid 48 , Sebacic acid 49 , and Glutaric acid 53 are all associated with glutaric acidemia. 3-Hydroxy-3-methylglutaric acid 55 , Methylcitric acid 57 , 2-Hydroxyisovaleric acid 62 , and 3-Methyl- 2-oxovaleric acid 64 are also associated with isovaleric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Very high fat diets (Ketogenic, GAPS, etc.) or extreme carbohydrate restriction, without proper intake of carnitine and Vitamin B2 (Riboflavin) can cause altered fatty acid metabolism and may influence elevations in EMA.582 Additionally vegan diets with improper protein complementing may also increase the risk of carnitine deficiency, and stress fatty acid metabolism.582 Additional fatty acid oxidation markers that could also be influenced include; Ethylmalonic acid 45 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 .

Medication/ Supplement Influences: Medium chain triglyceride (MCT) supplements and coconut oil may influence elevations due to their ability to promote rapid oxidation of medium- chain fatty acids.582,593

Ethylmalonic acid 45 , Adipic acid 47 , Suberic acid 48 , and Sebacic acid 49 can also be influenced by MCT containing foods and supplements.

Associated Conditions: MSA has been shown to be associated with type 2 diabetes.596

Low Values There is no known clinical significance for low values.

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47 Adipic acid

Adipic acid is a dicarboxylic acid that serves as a key marker of fatty acid metabolism that undergoes omega-oxidation, when beta oxidation is impaired.579 High adipic acid may indicate mitochondrial dysfunction, carnitine or Vitamin B2 (Riboflavin) insufficiencies, metabolic stressors, dietary intake, or, in rare cases, genetic abnormalities.582,592,600,601,603

Mitochondrial Health

When fatty acid oxidation is impaired, alternative metabolic pathways such as beta and omega oxidation become active, leading to the potential accumulation of Adipic and Suberic acids 48 .579 Deficiencies in key mitochondrial enzymes or disruptions in oxidative phosphorylation can also contribute to elevations.604 Furthermore, the buildup of adipic and suberic acid as byproducts of lipid peroxidation and fatty acid oxidation can generate additional oxidative stress, leading to more stress on the mitochondria.605

Additional insights into the cascading effects of mitochondrial issues include the other fatty acid oxidation markers 45 – 49 and mitochondrial markers 22 – 32 .

Nutritional Needs

Deficiencies in carnitine or Vitamin B2 (Riboflavin) can lead to abnormal adipic levels, as carnitine is required for transporting fatty acids for oxidation and riboflavin is crucial for flavin adenine dinucleotide (FAD)-dependent enzymes involved in fatty acid metabolism.600,601 Moreover, magnesium can also influence the optimization of omega oxidation directly or through reducing the effects of oxidative stress.606,607

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

There are numerous genetic and mitochondrial disorders that can influence both adipic and suberic acids 48 , and are classified into mitochondrial, peroxisomal, and fatty acid oxidation disorders 62 The more well known conditions that can influence adipic acid include medium- chain acyl-CoA dehydrogenase deficiency (MCAD), multiple acyl-CoA dehydrogenase deficiency (MADD), glutaric acidemia, and short-chain acyl-CoA dehydrogenase (SCAD).592,603 Markers also associated with MCAD, include 3-Hydroxybutyric acid 43 , Acetoacetic acid 44 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Suberic acid 48 , and Sebacic acid 49 . 3-hydroxyglutaric acid 31 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Suberic acid 48 , Sebacic acid 49 , and Glutaric acid 53 are all associated with glutaric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

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Additional Insights

Dietary: Intake of gelatin, processed foods that contain it, and coconut oil can all increase adipic, as can very high-fat diets (Ketogenic, GAPS, etc.), fasting, or very limited carbohydrate intake without proper intake of carnitine and Vitamin B2 (Riboflavin).08

Additionally, vegan diets with improper protein complementing may also increase the risk of carnitine deficiency, and stress fatty acid metabolism.582 Additional fatty acid oxidation markers that could also be influenced include Ethylmalonic acid 45 , Methylsuccinic acid 46 , Suberic acid

48 , and Sebacic acid 49 .

Medication/Supplementation Influences: Valproate has been shown to elevate ketones.569

Associated Conditions: Adipic acid has been shown to be commonly elevated in children with autism spectrum disorders (ASD).609,610

Low Values There is no known clinical significance for low values.

48 Suberic acid

Suberic acid is a dicarboxylic acid that serves as a key marker of fatty acid metabolism that undergoes omega-oxidation when beta oxidation is impaired.579 High suberic acid may indicate mitochondrial dysfunction, carnitine or Vitamin B2 (Riboflavin) insufficiencies, metabolic stressors, dietary intake, or in rare cases, genetic abnormalities.582,592,600,601,603

Mitochondrial Health

When fatty acid oxidation is impaired, alternative metabolic pathways such as beta and omega oxidation become active, leading to the accumulation of Suberic and Adipic acids 47 .579 Deficiencies in key mitochondrial enzymes or disruptions in oxidative phosphorylation can also contribute to elevations.604 Furthermore, the buildup of adipic and suberic acid as byproducts of lipid peroxidation and fatty acid oxidation can generate additional oxidative stress, leading to more stress on the mitochondria.605

Additional insights into the cascading effects of mitochondrial issues include the other fatty acid oxidation markers 45 – 49 and mitochondrial markers 22 – 32 .

Nutritional Needs

Deficiencies in carnitine or Vitamin B2 (Riboflavin) can lead to abnormal adipic levels, as carnitine is required for transporting fatty acids for oxidation and riboflavin is crucial for flavin adenine dinucleotide (FAD)-dependent enzymes involved in fatty acid metabolism.600,601 Moreover, magnesium can also influence the optimization of omega oxidation directly, or through reducing the effects of oxidative stress.606,607

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

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Genetics

There are numerous genetic and mitochondrial disorders that can influence both adipic 47 and suberic acids, and are classified into mitochondrial, peroxisomal, and fatty acid oxidation disorders.62 The more well-known conditions that can influence adipic acid include medium- chain acyl-CoA dehydrogenase deficiency (MCAD), multiple acyl-CoA dehydrogenase deficiency (MADD), glutaric acidemia, and short-chain acyl-CoA dehydrogenase (SCAD).592,603 Markers also associated with MCAD, include 3-Hydroxybutyric acid 43 , Acetoacetic acid 44 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , and Sebacic acid 49 . 3-hydroxyglutaric acid 31 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , Sebacic acid 49 and Glutaric acid 53 are all associated with glutaric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Intake of gelatin, processed foods, and coconut oil can increase suberic acid, as can very high-fat diets (Ketogenic, GAPS, etc.), fasting, or very limited carbohydrate intake without proper intake of carnitine and Vitamin B2 (Riboflavin).608 Additionally, vegan diets with improper protein complementing may also increase the risk of carnitine deficiency, and stress fatty acid metabolism.582 Additional fatty acid oxidation markers that could also be influenced include Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , and Sebacic acid 49 .

Associated Conditions: Suberic acid has been shown to be commonly elevated in children with autism spectrum disorders (ASD).610

Low Values There is no known clinical significance for low values.

49 Sebacic acid

Sebacic acid is a medium-chain dicarboxylic acid obtained by oxidizing a fatty acid called ricinoleic acid (castor oil).611 It is produced through the oxidation of decanoyl-CoA to sebacoyl- CoA, a process facilitated by acyl-CoA dehydrogenase.611 Elevated sebacic acid is impaired fatty acid oxidation due to mitochondrial dysfunction, carnitine or riboflavin insufficiencies, or certain rare genetic disorders. Additionally, certain toxicants, high-fat diets, and MCT supplementation may impact its levels.595,598, 600,601,612,613

Mitochondrial Health

Linked to fatty acid oxidation and energy metabolism, reduced function of these pathways can have significant impacts on mitochondrial dysfunction and cellular health. In reducing oxidative phosphorylation (OXPHOS) efficiency of fatty acids, ATP production is decreased, and energy deficits occur.597,598 This dysfunction also causes structural mitochondrial abnormalities, calcium

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homeostasis disruption, and increased reactive oxygen species (ROS), further damaging mitochondrial integrity.595,599

Additional insights into the cascading effects of mitochondrial issues include the other fatty acid oxidation metabolites 45 – 49 , and mitochondrial metabolites 22 – 32 .

Toxic Exposure

Sebacic acid and its derivatives, such as pimelic acid, have diverse industrial applications, including use in plasticizers, lubricants, hydraulic fluids, cosmetics, and candles.612 It also plays a role in producing polyester and epoxy resins and serves as an intermediate in the synthesis of fragrances, disinfectants, and coating materials.614 It has not been well studied if sebacic acid is found in urine from these exposures, and itself is not toxic; however, it may help support investigations into exposures to toxicants found in the aforementioned products.

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Deficiencies in carnitine or Vitamin B2 (Riboflavin) can lead to abnormal sebacic acid levels, as carnitine is required for transporting fatty acids for oxidation and riboflavin is crucial for flavin adenine dinucleotide (FAD)-dependent enzymes involved in fatty acid metabolism.600,601

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Genetic conditions including multiple acyl-CoA dehydrogenase deficiency (MADD), medium- chain acyl-CoA dehydrogenase deficiency, carnitine-acylcarnitine translocase deficiency, Adrenoleukodystrophy (ALD), and peroxisome disorders such as Zellweger syndrome and neonatal adrenoleukodystrophy, can cause elevated sebacic acid levels.613,615,616

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Very high-fat diets (Ketogenic, GAPS, etc.) or extreme carbohydrate restriction, without proper intake of carnitine and Vitamin B2 (Riboflavin), can cause altered fatty acid metabolism and may influence elevations in Sebacic acid.82 Additionally, vegan diets with improper protein complementing may also increase the risk of carnitine deficiency, and stress fatty acid metabolism.82 Additional fatty acid oxidation markers that could also be influenced include Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , and Suberic acid 48 .

Medication/ Supplement Influences: Medium-chain triglyceride (MCT) supplements and coconut oil may influence elevations due to their ability to promote rapid oxidation of medium- chain fatty acids.582,593 Consumption of castor oil may also influence elevations.611 Ethylmalonic acid (45), Methylsuccinic acid 46 , Adipic acid 47 , and Suberic acid 48 may also be influenced by MCT-containing foods and supplements.

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Low Values There is no known clinical significance for low values.

NUTRITIONAL MARKERS

50 Methylmalonic acid

Vitamin B12 is essential for propionic acid metabolism, as it enables the conversion of methylmalonyl-CoA to succinyl-CoA through the B12-dependent enzyme methylmalonyl-CoA mutase (MMUT).617 When B12 is deficient, this process is disrupted, causing the accumulation of methylmalonic acid (MMA). Propionic acid metabolism connects branched-chain amino acid and odd-chain fatty acid oxidation to the Citric Acid Cycle, and disturbances in this pathway can have widespread metabolic consequences.618,619 Vitamin B12 not only plays a role in mitochondrial function, but it can also disrupt the methylation processes, which can have cascading effects, particularly in neurotransmitter metabolism and detoxification pathways.617

Microbial Overgrowth

Elevated MMA levels may signal gut microbiota imbalances due to an increase in the production of the short-chain fatty acid, propionic acid, which can interfere with metabolism.230,620

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Vitamin B12, in the form of adenosylcobalamin, is essential for mitochondrial health as it serves as a cofactor for methylmalonyl-CoA mutase (MUT), an enzyme that catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA.617 This step in propionate catabolism enables the breakdown of branched-chain amino acids, odd-chain fatty acids, and cholesterol, allowing them to be funneled into the citric acid cycle.618

Review Markers 24 – 29 for insights on the citric acid cycle, 3-methylglutaric acid 30 , 3 Methylglutaconic acid 32 , and Markers 62 – 67 for further evaluation of branched-chain amino acid utilization, and Markers 43 – 49 for fatty acid utilization for energy.

Neurotransmitter Metabolites

Vitamin B12 acts as a cofactor for methionine synthase in the conversion of homocysteine to methionine, which is then used to produce S-adenosylmethionine (SAM), a key methyl donor for neurotransmitter methylation via COMT.621,622 It also modulates cholinergic signaling by influencing choline availability for acetylcholine synthesis and helps regulate glutamate release to prevent excitotoxicity.623-625 Deficiency can impair myelin integrity, elevate neurotoxic homocysteine levels, and disrupt neurotransmitter metabolism.622,626

Review neurotransmitter metabolites 33 – 39 for further insights.

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Methylation/Detoxification

Vitamin B12 is essential for coordinating the folate and methionine cycles, which regulate methylation.617 As a cofactor for methionine synthase (MS), B12 enables the conversion of homocysteine to methionine using 5-methyltetrahydrofolate (5-mTHF), and sustains S-adenosylmethionine (SAM) production, which is a primary methyl donor for DNA, protein, and neurotransmitter methylation.617

For additional markers influencing methylation and SAM, look to HVA/DOPAC ratio 37 , Uracil 41 , and 2-Hydroxybutryic acid 58 . A homocysteine serum test may be helpful. Additionally, refer to the Nutrient–Marker Cross-Reference Table for insights into corresponding organic acids that are associated with methylation support (B2, B3, B6, B9, and magnesium).

Nutritional Needs

MMA serves as a functional biomarker for B12 deficiency since vitamin B12 is the cofactor for the enzyme methylmalonyl-CoA mutase, and its absence leads to the accumulation of MMA instead of the formation of succinyl-CoA.19 Biotin also plays a role in the metabolism of propionic acid, potentially aiding in reducing MMA accumulation.620

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Conditions such as methylmalonic acidemia and propionic acidemia are inherited metabolic disorders that severely disrupt detoxification and cellular pathways, leading to extreme developmental and neurological issues.620

Methylmalonic acidemia is also associated with Methyl citric acid 57 and 3-Methyl-2- oxovaleric acid 64 , but a normal homocysteine. Pyroglutamic acid 58 , 2-Hydroxybutryic acid 59 , 2-Hydroxyisovaleric acid 62 , and 3-Methyl-2-oxovaleric acid 64 are associated with propionic acidemia.

Elevated MMA may also indicate mitochondrial disorders such as Pearson syndrome, which affects the respiratory chain, along with mitochondrial DNA mutations further impairing energy metabolism.620 Other associated genetic disorders include Malonyl CoA decarboxylase deficiency, Malonic Aciduria, Methylmalonate Semialdehyde Dehydrogenase Deficiency, and Methylmalonic Aciduria.627-629 Malonyl CoA decarboxylase deficiency is also associated with malonic acid 74 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Individuals who do not consume animal products and do not supplement with B12 are at risk for insufficiencies.630,631

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Medication/Supplement Influences: Medications such as metformin, proton pump inhibitors, or H2 receptor blockers may impair B12 absorption.632,633 There is also some evidence that oral contraceptives may influence B12 insufficiencies.634

Associated Conditions: MMA elevations have been associated with Alzheimer’s disease, advanced age, pregnancy, and kidney disease.635-637 Individuals with short bowel syndrome, SIBO, gastritis, or other gastrointestinal conditions that impair B12 absorption, as well as those who have undergone bariatric surgery, are at a significantly higher risk for B12 deficiencies.638-640

Low Values There is no known clinical significance for low values.

51 Pyridoxic Acid

Pyridoxic acid is the primary metabolite of vitamin B6, serving as a biomarker for assessing status.641 Abnormal vitamin B6 levels may indicate excessive intake, medication influences affecting metabolism and excretion, or, in rare instances, genetic disorders such as pyridoxine- dependent epilepsy.642,643 Low levels may stem from inadequate intake, Microbial Overgrowths, or mitochondrial dysfunction, and may impact neurotransmitter synthesis, methylation, and detoxification pathways.230,421,644-646 Moreover, certain medications, hormonal factors, and conditions such as celiac disease, diabetes, or chronic kidney disease may further influence B6 status.647,648

Nutritional Needs

Elevated levels may indicate excessive vitamin B6 intake from diet or supplements.641

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics

Pyridoxine-dependent epilepsy, a rare genetic disorder caused by mutations in the PNPO gene encoding pyridox(am)ine 5’-phosphate oxidase, disrupts the rate-limiting step in B6 metabolism, leading to excess pyridoxic acid elimination.642

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Supplement/Medications Influences: Isoniazid and penicillamine may increase excretion of pyridoxic acid.643

Associated conditions: Pyridoxic acid levels can accumulate due to impaired renal clearance and an increase in catabolic states such as fever, fasting, or metabolic acidosis, which accelerate protein turnover and amino acid oxidation.230

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Low Values

Microbial Overgrowth: There is evidence that B6 can be produced by various bacteria in the microbiome (e.g. B. fragilis and P. copri (Bacteroidetes), Bifidobacterium longum and Collinsella aerofaciens (Actinobacteria), and H. Pylori), and if reduced in abundance, may influence insufficiencies.644,649 There is also some data that suggests a lack of B6 can influence the microbiome, as various Firmicutes (Veillonella, Ruminococcus, Faecalibacterium, and Lactobacillus spp.) are unable to produce their own and require their hosts to provide it.649

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health: Vitamin B6 supports Coenzyme A production, which is essential for energy metabolism.645 It also aids in the absorption and storage of vitamin B12, a key cofactor in mitochondrial pathways.645 Additionally, B6 influences hormone synthesis, including cortisol and glucagon, which regulate mitochondrial energy balance and metabolic processes.645

Assessing Markers 22 – 32 for mitochondrial insights, as well as refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Neurotransmitter Metabolites: Vitamin B6 is essential for neurotransmitter synthesis, serving as a cofactor for aromatic L-amino acid decarboxylase (AADC), which converts L-DOPA to dopamine and 5-hydroxytryptophan to serotonin.21 Deficiency in B6 can impair AADC activity, leading to reduced dopamine and serotonin levels.385

Evaluate HVA 33 , VMA 34 , DOPAC 37 , and 5-HIAA 38 for further insights.

B6 also supports the biopterin cycle by aiding in the regeneration of tetrahydrobiopterin (BH4), which is the enzyme involved in dopamine, norepinephrine, and serotonin synthesis.650,651

For additional insights into Biopterin pathways, review phenylalanine and tyrosine metabolites 33 – 37 , 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylacetic acid 69 , and Phenylpyruvic acid 70 .

B6 is a cofactor for various enzymes involved in the tryptophan-to-kynurenine pathway, and insufficient levels may lead to abnormal levels of Quinolinic acid 39 and Kynurenic acid 40 .652

Methylation/Detoxification: In the methylation pathway, B6 supports serine hydroxymethyltransferase (SHMT), facilitating the conversion of serine to glycine and providing one-carbon units for methionine and S-adenosylmethionine (SAM) synthesis, crucial for methylation reactions.646,653 Vitamin B6 is also essential for the transsulfuration pathway as a coenzyme for cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which convert homocysteine to cysteine for glutathione production.653-655

Review 2-hydroxybutyric acid 58 and Pyroglutamic acid 59 to gain further insights into methylation and detoxification components, as well as refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium) and glutathione.

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Nutritional Needs: Measuring pyridoxic acid may reflect B6 insufficiency.641 Moreover, vitamin B2 (Riboflavin) insufficiency can influence low B6 levels as it affects B6 activation.

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Dietary: Restrictions in intake of fish or beef, starchy vegetables, and grains may increase risk of inadequate intake.648,656,657

Supplement/Medications Influences: Cycloserine, various antiepileptic medications, and theophylline may influence B6 insufficiencies.647,658,659 Various birth controls or supplemental estrogen or progesterone may require an increased need for B6.634,660

Associated Conditions: Maladies such as celiac disease, diabetes, inflammatory bowel disease, or chronic renal failure can influence B6 absorption.648 Low B6 levels may contribute to calcium oxalate stone formation, as B6 helps convert glyoxylate into glycine instead of oxalate.185

Evaluate Oxalic acid 21 for further insights into oxalates.

52 Pantothenic Acid

Pantothenic acid (B5) plays a crucial role in metabolism, primarily through its involvement in Coenzyme A (CoA) synthesis, which is essential for energy production.661 Levels of B5 can be influenced by dietary intake, microbial activity, supplementation, including products containing B5 derivatives or royal jelly.662-664 Rare genetic mutations, such as those in the PANK2 gene linked to PKAN, can lead to significantly elevated levels due to impaired conversion to CoA.665

Additionally, low B5 levels may have a significant influence on mitochondrial dysfunction, potentially affecting the Citric Acid Cycle and energy metabolism.666

Microbial Overgrowth

There is evidence that suggests B5, in the form of pantothenic acid phosphate, can be produced by Lactobacillus plantarum, and if in high abundance, may influence high levels.662

Evaluate DHPPA 14 , 2-Hydroxyisocaproic acid 65 , and 4-Hydroxyphenyllactic acid 72 for more insights into Lactobacillus activity.

Nutritional Needs

High levels may reflect recent intake and may give insights into nutritional status.663

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics

Genetic mutations in the PANK2 gene are linked to PKAN, a disorder that impairs the

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conversion of pantothenic acid to its active form, CoA, causing significant elevations of pantothenic acid.665

Evaluate Lactic acid 22 , Pyruvic acid 23 , and the Citric Acid Cycle metabolites 24 – 29 , particularly 2-oxoglutaric acid 27 , for further insights.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: Pantothenic acid is abundant in whole grains, legumes, eggs, meat, shitake mushrooms, and high intakes of these foods the day before may influence levels.656

Medication/Supplement Influences: Supplements that contain royal jelly may influence elevations, as can any supplement containing Vitamin B5 (various forms).664

Low Values

Microbial Overgrowth: Pantothenic acid phosphate is produced by Lactobacillus, and if in insufficient amounts, may influence low levels.662

Evaluate DHPPA 14 , 2-Hydroxyisocaproic acid 65 , and 4-Hydroxyphenyllactic acid 72 for more insights into Lactobacillus activity.

Mitochondrial Health: Pantothenic acid aids in the synthesis of coenzyme A (CoA), which is required for the Citric Acid Cycle and energy production. If deficiencies are present, significant mitochondrial influences may occur.666

Evaluate Lactic acid 22 , Pyruvic acid 23 , and the Citric Acid Cycle markers 24 – 29 , particularly 2-oxoglutaric acid 27 , for further insights.

Nutritional Needs: Low levels may suggest insufficient intake.

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

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53 Glutaric Acid

Glutaric acid metabolism relies on riboflavin (B2) as a cofactor for glutaryl-CoA dehydrogenase, and a deficiency can lead to its accumulation, impacting amino acid and fatty acid metabolism.667 Riboflavin is essential for mitochondrial energy production, neurotransmitter synthesis, and detoxification through its role in FAD-dependent enzymes.401,668,669 Insufficient B2 levels can affect ATP synthesis, methylation, and oxidative stress balance, potentially contributing to metabolic dysfunction.668,670,671 Dietary restrictions, medications such as oral contraceptives, and inadequate riboflavin intake may further influence these processes, increasing the risk of deficiency-related complications.672,673

Mitochondrial Health

Riboflavin is absorbed from the diet, transported into mitochondria, and converted into FAD through the Rf-FAD cycle.668 The various FAD-dependent enzymes involved in fatty acid metabolism support the acetyl-CoA generation for the citric acid cycle.668 B2 also plays a role in the electron transport chain (ECT), which can have significant impacts on ATP production.668

Evaluate other fatty acid oxidation markers 45 – 49 for B2 influence, and Succinic acid 24 for further insights into the ECT.

Neurotransmitter Metabolites

Tetrahydrobiopterin (BH4), a key cofactor for enzymes involved in dopamine, norepinephrine, and serotonin production, requires FAD for dihydropteridine reductase (DHPR) to recycle BH4 and maintain neurotransmitter synthesis.379,401,600,650,651 Moreover, FAD plays a role in monamine oxidase (MAO), the enzyme involved in the metabolism of dopamine, norepinephrine, and serotonin.674 B2 has also been shown to help restore dopamine levels and reduce oxidative stress in the brain.671

For further insights into influences on biopterin cycles and MAO pathways, look to 2-Hydroxyphenylacetic acid 11 , HVA 33 , VMA 34 , 5HIAA 38 , Mandelic acid 68 , Phenylacetic acid

69 , and Phenylpyruvic acid 70 .

Methylation/Detoxification

Vitamin B2 activates methylenetetrahydrofolate reductase (MTHFR), which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, facilitating homocysteine remethylation and sustaining S-adenosylmethionine (SAM) levels for DNA and protein methylation.669,670 Moreover, FAD-dependent enzymes, such as dimethylglycine dehydrogenase, drive oxidative demethylation processes that generate tetrahydrofolate, linking riboflavin to one-carbon metabolism and xenobiotic clearance.675

Review of HVA/DOPAC ratio 37 , Uracil 41 , and 2-hydroxybutyric acid 59 for additional insights into methylation dysfunction. Additionally, refer to the Nutrient–Marker Reference Table for the

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corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutritional Needs

Glutaric acid metabolism relies on riboflavin (B2) as a cofactor for the enzyme glutaryl-CoA dehydrogenase.672 A deficiency in vitamin B2 can impair this enzyme’s function, leading to the accumulation of glutaric acid.667,676

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics

Glutaric acidemia type I (GA-1) and glutaryl-CoA dehydrogenase deficiency are autosomal recessive disorders caused by a deficiency in glutaryl-CoA dehydrogenase, which influences the breakdown of amino acids lysine, hydroxylysine, and tryptophan. 3-hydroxyglutaric acid (31) may also be influenced by GA-1.677

Glutaric acidemia type II (GA-2), or multiple acyl-CoA dehydrogenase deficiency (MADD), arises from a defect in either electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH), leading to impaired fatty acid, amino acid, and choline metabolism, which disrupts ATP synthesis, reduces gluconeogenesis, and causes excessive lipid accumulation.677

3-hydroxyglutaric acid 31 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , and Sebacic acid 49 are all associated with glutaric acidemias.

An additional rare genetic disorder, Riboflavin transporter deficiency (formerly known as Brown-Vialetto-Van Laere or Fazio-Londe syndrome), is caused by mutations in the SLC52A3 or SLC52A2 genes, which encode riboflavin transporters.678

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary: A diet that excludes meat, dairy, and fortified grains may lack a significant amount of Vitamin B2 (Riboflavin), increasing the risk of deficiencies.679

Medication/Supplement Influences: Valproic acid and oral contraceptives may influence B2 insufficiencies.673,680

Low Values There is no known clinical significance for low values.

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54 Ascorbic Acid (Vitamin C)

Ascorbic acid, also known as vitamin C, is a water-soluble nutrient found in citrus fruits and vegetables, essential for collagen synthesis, various biological functions, and antioxidant protection.681 Ascorbic acid levels are influenced by factors such as Microbial Overgrowth, mitochondrial health, neurotransmitter metabolism, and nutritional needs such as iron or zinc deficiency.682-689 Foods rich in vitamin C and supplements, as well as medications, can increase its levels, while conditions such as critical illness, infections, and smoking raise the body’s demand for it.689-698 Vitamin C levels are commonly low, due to its ability to be rapidly metabolized and excreted as well as its short half-life.699

Microbial Overgrowth

Fungal species, including yeast such as Saccharomyces cerevisiae, produce the stereoisomer D-erythroascorbic acid through a biosynthesis pathway similar to L-ascorbic acid, starting from D-arabinose.682

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Nutritional Needs

Iron deficiency increases the need for vitamin C, as it enhances the absorption of non-heme iron, with studies showing that iron regulates the uptake of ascorbic acid in the intestines. Similarly, zinc deficiency can impact vitamin C requirements due to zinc’s role in immune function and wound healing, processes also supported by vitamin C.688,689

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Additional Insights

Dietary Influences: Foods high in vitamin C, such as citrus fruits, strawberries, kiwi, bell peppers, and acerola juice, can increase ascorbic acid levels.690

Medication/Supplement Interactions: Medications and supplements such as microsomal enzyme inducers (e.g., phenobarbital and diallyl sulfide), aspirin, certain types of dietary fiber, such as hemicellulose, and vitamin C supplementation have been shown to boost urinary excretion of ascorbic acid, likely by affecting its metabolism or enhancing its absorption.691-694

Associated Conditions: High doses of vitamin C, particularly in the presence of copper, can increase oxalate levels in the body, as it is metabolized to oxalate, which is then excreted in the urine.700

To further assess oxalate status, reference glyceric 19 , glycolic 20 , and oxalic 21 .

Low Values

Mitochondrial Health: Insufficiencies in ascorbic acid levels could impair mitochondrial function

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by increasing oxidative stress, increase the potential for damage to the mitochondrial DNA, reduce ATP production, and disrupt energy metabolism, ultimately compromising overall mitochondrial efficiency.683-686

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Neurotransmitter Metabolites: Ascorbic acid is essential for the proper function of dopamine beta-hydroxylase (DBH), as it acts as a cofactor for converting dopamine to norepinephrine. Deficiency in vitamin C impairs DBH activity, leading to decreased norepinephrine synthesis.687 For more insight into neurotransmitter activity, reference the phenylalanine and tyrosine metabolites 33 – 37 .

Nutritional Needs: Ascorbic acid is rapidly metabolized and excreted primarily due to its renal handling, which involves a renal threshold mechanism, where excess vitamin C is cleared in the urine once plasma levels exceed a certain threshold. Its conversion to oxalate, short half-life, and saturable reabsorption in the kidneys contribute to this quick clearance from the body.699

If deficiency is suspected, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Associated Conditions: Conditions such as cirrhosis, malnutrition, critical illness, trauma, infections, diabetes, obesity, smoking, and strenuous exercise increase the need for vitamin C due to heightened oxidative stress, inflammation, and metabolic demands. Patients with these conditions often require higher vitamin C intake or supplementation to support immune function, wound healing, and to counteract oxidative damage. Smokers and athletes, in particular, have higher daily requirements, while individuals with chronic illnesses may need vitamin C to compensate for impaired absorption or increased turnover.689,695-698

55 3-Hydroxy-3-methylglutaric Acid * (Vitamin Q10 (CoQ10))

3-Hydroxy-3-methylglutaric acid (HMG) is an intermediate in leucine degradation, formed when the enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase) is deficient or impaired. This enzymatic influence disrupts ketone body synthesis and leads to the accumulation of HMG, contributing to metabolic imbalances. Low coenzyme Q10 (CoQ10) levels can exacerbate this by impairing mitochondrial function, leading to increased acetyl-CoA accumulation and further diversion into pathways such as those that produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is a substrate for HMG-CoA lyase. The disruption in metabolism also impacts cholesterol and CoQ10 synthesis, as the accumulation of HMG-CoA, a precursor in both pathways, can impair their downstream processes. Similarly, disruptions in the mevalonate pathway, where HMG-CoA is a key intermediate, hinder the synthesis of CoQ10.388,701-705

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Mitochondrial Health

Mitochondrial dysfunction caused by low CoQ10 levels may impair the electron transport chain, reduce ATP production, and increase oxidative stress. This inefficiency leads to acetyl-CoA accumulation, which is diverted into pathways that produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), affecting the activity of HMG-CoA lyase and increasing levels of 3-hydroxy-3- methylglutaric acid.338,702,705

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Nutritional Needs

CoQ10 deficiency or insufficiency can impair mitochondrial function and ATP production, leading to increased oxidative stress. This dysfunction results in acetyl-CoA accumulation, which is diverted into pathways that may increase levels of 3-hydroxy-3-methylglutaric acid.338,702,705

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics

Elevated levels of 3-hydroxy-3-methylglutaric acid are often seen in individuals with the rare condition, 3-hydroxy-3-methylglutaric aciduria, a disorder caused by a deficiency in the enzyme HMG-CoA lyase. This enzyme defect disrupts leucine degradation, leading to the accumulation of HMG in the mitochondria and urine. Other disorders, such as maple syrup urine disease, isovaleric acidemia, and methylcrotonylglycinemia, which also affect leucine degradation, as well as mitochondrial disorders, can lead to increased levels of 3-hydroxy-3-methylglutaric acid due to disrupted enzyme activity in the metabolic pathways.701

Other markers associated with MSUD include; 2-Hydroxyisovaleric acid 62 , 2-Oxoisovaleric acid 63 , 3-Methyl-2-oxovaleric acid 64 , 2-Hydroxyisocaproic acid 65 , and 2-Oxoisocaproic acid 66 , while Methylsuccinic acid 46 , Methylcitric acid 57 , 2-Hydroxyisovaleric acid 62 , and 3-Methyl-2-oxovaleric acid 64 are associated with isovaleric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/Supplement Interactions: Statins main mechanism of action is blocking HMG-CoA reductase, which blocks the conversion of HMG-CoA to mevalonate.706

Associated Conditions: 3-hydroxy-3-methylglutaric acid has been found to be significantly more elevated in patients with diabetes and those using statins.707,708

Low Values There is no known clinical significance for low values.

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56 N-Acetylcysteine (NAC) (Glutathione Precursor and Chelating Agent)

N-Acetylcysteine (NAC) is a derivative of L-cysteine and a precursor to the antioxidant glutathione. It helps replenish glutathione levels, neutralizes free radicals, and protects cells from oxidative damage.709 Elevated NAC levels may result from high supplemental intake, deficiencies, or rare conditions involved in acylation. Low levels can be physiologically normal and expected.709-714

Methylation/Detoxification

Being an important cofactor to the antioxidant glutathione, assessing NAC levels and conversion can provide more insight into detoxification, especially under circumstances of oxidative stress.709

Refer to the Nutrient–Marker Reference Table for insights into corresponding organic acids that are associated with glutathione.

Genetics

It is theorized that when NAC is elevated alongside pyroglutamic acid 58 , there may be a dysfunction in an acylase enzyme, which would compromise NAC to cysteine conversion, potentially influencing glutathione status.714

For more insight into this conversion, assess with pyroglutamic acid 58 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medication/Supplement Interactions: NAC is mostly converted to glutathione in the body, therefore, elevated levels are often related to intake, such as through intravenous formulations, oral supplementation.710-713

Additional Benefits: NAC offers several benefits, including replenishing glutathione levels to protect against liver damage from acetaminophen overdose, reducing the viscosity of mucus in respiratory conditions such as COPD, and providing antioxidant effects by reducing free radicals. It also shows promise in managing psychiatric disorders, such as bipolar disorder and schizophrenia, and may have antiviral properties.709

Low Values: Low values of NAC are often less clinically significant as most is converted efficiently, but may indicate an increase in utilization of antioxidants such as glutathione in the context of supplementation.709

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57 Methylcitric Acid* Biotin (Vitamin B7)

Methylcitric acid serves as a key marker for assessing biotin sufficiency, as biotin is essential for the propionyl-CoA carboxylase (PCC)-catalyzed conversion of propionyl-CoA to methylmalonyl- CoA.167 If biotin is insufficient, methylcitrate from propionyl CoA is elevated, and prevents succinyl CoA formation to support the citric acid cycle.715 Increased levels of methylcitric acid can also be influenced by Microbial Overgrowths that affect biotin synthesis or by biotinidase deficiency, which impairs biotin recycling and biotin-dependent enzymes. Additionally, conditions such as vitamin B12 deficiency, Crohn’s disease, and certain dietary factors can also contribute to elevated methylcitric acid levels.716-719

Microbial Overgrowth

The microbiome influences methylcitric acid levels through biotin synthesis, with specific bacteria such as Bifidobacterium longum shown to enhance biotin production. Other gut bacteria, such as Lactobacillus species, also contribute to biotin availability, affecting biotin- dependent enzymes and, consequently, methylcitric acid accumulation.720,721

Candida species, such as Candida albicans, are unable to synthesize biotin and rely on external sources for its acquisition, using specialized transporters to ensure survival and virulence.722

Moreover, biotin availability can promote growth and proliferation.723

Additionally, excessive production of the short-chain fatty acid propionic acid by gut microbiota can contribute to increased propionyl-CoA levels, diverting its metabolism towards methylcitric acid accumulation.177 This imbalance can result from dysbiosis or an overgrowth of specific bacteria that produce propionic acid during the breakdown of dietary components.724

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Propionyl-CoA levels, produced by certain amino acids and fatty acids, are converted to succinyl–CoA to be utilized in the Citric Acid Cycle, and this conversion is influenced by biotin. A backup in this pathway can disrupt the function of pyruvate dehydrogenase and reduce the synthesis of key metabolic compounds like citrate and ATP.725

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Nutritional Needs

Biotin (Vitamin B7) is a critical cofactor for propionyl-CoA carboxylase, and its deficiency leads to impaired metabolism of propionyl-CoA, causing an increase in methylcitric acid.715

Vitamin B12 deficiency impairs the enzyme methylmalonyl-CoA mutase, leading to the accumulation of methylmalonyl-CoA, which is converted into both methylmalonic acid 50 and

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methylcitric acid. Elevated levels of both metabolites may serve as markers for vitamin B12 deficiency, as well.726-728

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Biotinidase deficiency impairs biotin recycling, leading to secondary deficiencies in biotin- dependent enzymes such as propionyl-CoA carboxylase, which results in elevated levels of methylcitric acid.729 Another condition, methylcitric acidemia, results from a deficiency in methylcitrate synthase, an enzyme involved in propionate metabolism.725,730 Methylcitric acid is also associated with methylmalonic acidemia as well as isovaleric acidemia.230

Other metabolites associated with methylmalonic acidemia include methylmalonic acid 50 , 3-Methyl-2-oxovaleric acid 64 . For isovaleric acidemia, associated metabolites include Methylsuccinic acid 46 , 3-Hydroxy-3-methylglutaric acid 55 , 2-Hydroxyisovaleric acid 62 , and 3-Methyl-2-oxovaleric acid 64 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Consuming raw egg whites can affect biotin levels because they contain avidin, a protein that binds to biotin and influences its bioavailability.731

Methylcitric acid has also been detected in foods such as duck, chicken, swine, and cow’s milk, suggesting it could serve as a potential biomarker for the consumption of these foods.716

Associated Conditions: In Crohn’s disease, dysbiosis and the presence of adherent-invasive Escherichia coli (AIEC) bacteria lead to the degradation of propionate, resulting in increased methylcitric acid production. This accumulation is linked to the metabolic shifts and inflammation characteristic of the disease.732,733

Low Values There is no known clinical significance for low values.

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INDICATORS OF DETOXIFICATION

Figure 6:

METHYLATION

This figure explains the interconnections between the detoxification, methylation, biopterin, and urea cycles, displaying the collective role these pathways play in maintaining cellular health and supporting detoxification. Methylation is essential for DNA repair, neurotransmitter synthesis, and detoxification, relying on enzymes like MTHFR and BHMT to convert homocysteine into methionine, which produces S-Adenosyl Methionine (SAMe), a universal methyl donor. Biopterin, specifically tetrahydrobiopterin (BH4), is a critical cofactor in several enzymatic processes, including the synthesis of neurotransmitters and nitric oxide, and is influenced by MTHFR. The urea cycle also complements these processes by removing excess ammonia, a toxic byproduct of protein metabolism, through its conversion into urea for excretion. Detoxification pathways, particularly glutathione metabolism, are also critical, as described by elevated pyroglutamic acid levels, which may indicate impaired glutathione synthesis needed to neutralize free radicals and process toxins. Nutritional cofactors such as vitamins B6, B12, magnesium, and folate are essential for supporting methylation, detoxification, and the urea cycle. Disruptions in any of these pathways, whether due to genetic mutations, Microbial Overgrowths, or toxic exposures, can lead to oxidative stress, impaired cellular function, and toxin accumulation, emphasizing the interconnected nature of these biochemical processes.

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58 Pyroglutamic acid (Glutathione)

* A high value for this marker may indicate a Glutathione deficiency.

Pyroglutamic acid (5-oxoproline) is formed from glutamate, glutamine, and gamma-glutamylated peptides through gamma-glutamylcyclotransferase and is involved in glutathione metabolism. When intracellular glutathione synthesis is impaired, pyroglutamic acid levels rise, prompting increased cystine uptake to support restoration and reduce its accumulation.734-736 Factors such as Microbial Overgrowth, mitochondrial dysfunction, toxic exposures, methylation, and nutritional deficiencies (e.g., magnesium, vitamin B6, precursor amino acids) can elevate pyroglutamic acid levels by disrupting glutathione metabolism and related cycles.735,737-745 Conversely, genetic mutations, severe nutritional deficiencies, and metabolic disorders may influence low levels, though this is mainly theoretical.744,746,747

Microbial Overgrowth

Some bacteria of the microbiome, including Bacteroidetes species and thermophilic lactic acid bacteria, can increase pyroglutamic acid levels by converting glutamine to pyroglutamate, potentially affecting its accumulation in the gastrointestinal system.737,738

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Mitochondrial dysfunction related to ATP depletion disrupts the gamma-glutamyl cycle, impairing glutathione synthesis, leading to the accumulation of pyroglutamic acid. This dysfunction also affects glutamine metabolism, further contributing to increased pyroglutamic acid levels.735,739

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposure

Various toxins and toxicants can influence pyroglutamic acid levels by disrupting glutathione metabolism, leading to oxidative stress, redox imbalances, and impaired glutathione recycling. These disturbances also affect amino acid and glutathione intermediates, further elevating pyroglutamic acid levels.740,748 Metals, mycotoxins, and various toxicants like styrene, benzene, acrylonitrile, 1-bromopropane, 1,3 butadiene, ethylene oxide, vinyl chloride, and acrylamide can increase pyroglutamic acid levels by inducing oxidative stress and depleting glutathione. Specific mycotoxins, such as gliotoxin and roridin, either use or deplete glutathione, which impacts the formation of pyroglutamic acid.741,749,750

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Methylation/Detoxification

Methylation processes, particularly those involving S-adenosylmethionine (SAMe) and

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S-adenosylhomocysteine (SAH), influence amino acid metabolism and may elevate pyroglutamic acid levels by disrupting the regulation of key intermediates involved in the glutathione cycle, such as glutamate, glycine, and cysteine.742

Refer to the Nutrient–Marker Reference Table for insights into corresponding organic acids that are associated with glycine and glutathione. Additionally, reviewing HVA/DOPAC ratio 37 may give additional information with regard to SAMe, and 2-Hydroxybutyric acid 59 for insights into homocysteine’s conversion to cysteine.

Nutritional Needs

Deficiencies of magnesium, vitamin B6, glycine, cysteine, and glutamine can lead to increased pyroglutamic acid levels by disrupting the gamma-glutamyl cycle and glutathione synthesis. These nutrients are essential for ATP production, enzymatic reactions in amino acid metabolism, and the synthesis of glutathione, and their deficiencies can impair glutathione production, resulting in the accumulation of pyroglutamic acid.735,743-745 Low antioxidant levels can increase pyroglutamic acid levels by activating a futile ATP-depleting cycle in the gamma- glutamyl pathway.751

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Conditions such as nephropathic cystinosis, 5-oxoprolinase deficiency, glutathione synthetase deficiency (GSSD), urea cycle disorders, hawkinsinuria, and homocystinuria can elevate pyroglutamic acid levels by disrupting glutathione metabolic processes.734,751

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Low protein intake, especially of cysteine, glycine, and glutamine, can elevate pyroglutamic acid levels by disrupting the gamma-glutamyl cycle and impairing glutathione synthesis. Additionally, a vegetarian diet may increase pyroglutamic acid excretion due to differences in protein intake and nutrient synthesis compared to omnivorous diets.735,752 Long-ripened cheeses like Grana Padano and Parmigiano Reggiano Thermophilic can cause elevations due to the lactic acid bacteria that are important in processing, cyclizing glutamine through the action of glutamine to pGlu cyclase.738

Medication/Supplement Interactions: Use of certain medications such as acetaminophen, vigabatrin, flucloxacillin, and more can lead to increased pyroglutamic acid levels by depleting key metabolites like glutathione and cysteine and disrupting metabolic cycles.744,753,754 Theoretically, any medication or supplement that undergoes glutathione conjugation may stress glutathione levels and therefore influence pyroglutamic acid levels depending on dose, frequence, and other comorbidities.

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Associated Conditions: Type 2 diabetes and Alzheimer’s may contribute to elevated pyroglutamic acid through glutathione depletion or impaired synthesis.746,755-761

Additional benefits: Pyroglutamate supplementation may offer benefits such as improving glucose tolerance, cholesterol levels, and cognitive function, as well as providing anti- inflammatory and antidepressant-like effects. However, it may also disrupt glutathione homeostasis, potentially increasing risks.762-766

Low Values Low pyroglutamic acid levels and their clinical significance are largely theoretical, but may result from genetic mutations affecting enzymes in the gamma-glutamyl cycle, severe nutritional deficiencies (particularly in amino acid precursors), metabolic disorders that impact amino acid synthesis, or pharmacological interventions that disrupt glutathione metabolism, such as acetaminophen.744,746,747 Some studies have observed low levels of pyroglutamic acid, along with decreased levels of essential amino acids in autism spectrum disorder (ASD).767 Additional amino acid testing may give further insights, if this were the case.

59 2-Hydroxybutyric acid** (Methylation, Toxic Exposure)

** High values may indicate methylation defects and/or toxic exposures.

2-Hydroxybutyric acid, a byproduct of metabolism during oxidative stress or detoxification, is produced from alpha-ketobutyrate, which forms during the catabolism of threonine and methionine or glutathione synthesis, reflecting shifts in glutathione production.768 Changes in 2-hydroxybutyric acid levels can result from toxic exposures that induce oxidative stress and disrupt amino acid metabolism, impairing detoxification and promoting its accumulation.769-771 Deficiencies in methylation nutrients (e.g., folate, B12, methionine, B6, B2, and B3) and factors such as low protein intake, ketosis, diabetes, alcohol abuse, and genetic disorders can all elevate 2-hydroxybutyric acid by disrupting amino acid catabolism and metabolism.496,743,767,768,770-780

Toxic Exposure

Oxidative stress, potentially from toxic exposures, increases 2-hydroxybutyric acid levels by disrupting amino acid metabolism through the conversion of methionine and threonine to 2-oxobutyric acid, which is reduced to 2-hydroxybutyric acid. Additionally, oxidative stress depletes glutathione, impairing detoxification pathways and contributing to the accumulation of 2-hydroxybutyric acid as a byproduct.769-771

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Methylation/Detoxification

Impaired methylation, caused by deficiencies in methyl donors like folate (B9), B12, and methionine or genetic mutations in methylation enzymes, disrupts the methionine cycle and increases levels of S-adenosylhomocysteine (SAH). Elevated SAH inhibits methylation

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processes, leading to a buildup of homocysteine. Through the transsulfuration pathway, homocysteine is converted into cystathionine and then into alpha-ketobutyrate, which is reduced to 2-hydroxybutyric acid, thereby increasing its levels.781-784

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium). A serum homocysteine test may also provide additional insight.

Nutritional Needs

Deficiencies in folate, B12, or methionine can elevate 2-hydroxybutyric acid levels by increasing homocysteine. B6, B2, and B3 deficiencies, along with low cysteine and taurine, can further disrupt amino acid metabolism and redox balance, leading to the accumulation of 2-hydroxybutyric acid through impaired transsulfuration and enzyme regulation.496,743,767,770-773

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Several genetic disorders such as 3-hydroxyisobutyryl-CoA hydrolase deficiency (HIBCHD), primary hyperoxaluria type 2, and a genetic variant in the cystathionine beta-synthase (CBS) enzyme among others involving metabolic disruptions leading to the accumulation of 2-hydroxybutyric acid.778-780

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Low protein intake can increase 2-hydroxybutyric acid levels by promoting the catabolism of amino acids like methionine and threonine, inducing a metabolic shift towards ketogenesis, and impairing the breakdown of branched-chain amino acids, which leads to their conversion into 2-hydroxybutyric.774,775

Associated Conditions: In ketosis, diabetes, and alcohol abuse, elevated fatty acid oxidation increases ketone bodies and raises the NADH/NAD ratio, promoting the conversion of 2-oxobutyric acid to 2-hydroxybutyric acid. This shift occurs in response to insulin deficiency, chronic alcohol consumption, and metabolic states like diabetic ketoacidosis and alcoholic ketoacidosis.768,776,777

Review 3-hydroxybutyric acid 43 and Acetoacetic acid 44 for insights into ketones.

Low Values

Toxic Exposure: Phthalate exposure can decrease 2-hydroxybutyric acid levels by disrupting metabolic pathways related to oxidative stress and fatty acid oxidation.785 Quinolinic acid 39 can also be influenced by Phthalates. Look to Markers 44 – 49 for more insights into fatty acid oxidation disruptions. Refer to the Toxin Exposure Tables for additional insights into

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corresponding metabolites and patterns.

Dietary: High cysteine intake can decrease 2-hydroxybutyric acid levels by inhibiting some enzymatic activity and the ability to produce metabolites like 2-oxobutanoate, a precursor of 2-hydroxybutyric acid. Disruptions in the transsulfuration pathway can lead to increased 2-hydroxybutyric acid levels due to the accumulation of metabolic intermediates like 2-oxobutyric acid.786

60 Orotic acid (Ammonia Excess)

Orotic acid is a key intermediate in pyrimidine nucleotide synthesis, formed in the body from carbamoyl phosphate and aspartic.787 Elevated levels can indicate urea cycle dysfunction, where excess carbamoyl phosphate is diverted into orotic acid synthesis.784,788,789 Imbalances in the gut microbiome, particularly overgrowth of E. coli or Candida, may raise orotic acid levels, while low arginine, B12, B2, magnesium, or zinc can impair enzymes needed to process orotic acid.790-795 Disruptions in methylation pathways and BH4 deficiency can also lead to elevated levels.792,796-798 Liver dysfunction, high-protein diets, and certain medications or supplements (e.g., diuretics, lithium/magnesium orotate) can further contribute to orotic acid accumulation.784,788,789,791,799-801 Orotic acid elevations have been linked to hyperammonemia, oxidative stress, and impaired nitric oxide signaling.784,788,789,792,796,797,802

Microbial Overgrowth

Disruptions in the microbiome may increase orotic acid levels through bacteria like Escherichia coli, which produce orotic acid via the pyrimidine biosynthesis pathway, converting carbamoyl phosphate and aspartate into orotate and then orotic acid. Additionally, some Candida albicans strains can generate orotic acid.790,791

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Elevated orotic acid can result from tetrahydrobiopterin (BH4) deficiency, particularly in dihydropteridine reductase (DHPR) deficiency, which impairs BH4 recycling.798 This disruption affects the urea cycle, leading to secondary hyperammonemia and increased orotic acid levels.03 Since BH4 is crucial for neurotransmitter synthesis, its deficiency can also impact dopamine, serotonin, and nitric oxide production.804

Evaluate HVA 33 , VMA 34 , 5HIAA 38 to gain further insights into insufficient biopterin pathways. 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylactic acid 69 , and Phenylpyruvic acid 70 may also be influenced.

Methylation/Detoxification

Methylation impairments can elevate orotic acid levels by disrupting the one-carbon

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metabolism pathway, which affects the availability of S-adenosylmethionine (SAMe), a key methyl donor. Deficiencies in methylation cofactors, or genetic variations in the MTHFR enzyme, can reduce SAMe levels, impair methylation, and may lead to the accumulation of intermediates like orotic acid.792,796,797

For more insight into methylation, refer to the Uracil 41 , 2-hydroxybutyric 59 , as well as HVA/ DOPAC ratio 37 may give additional information with regards to SAMe. Additionally, refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with nutrients related to methylation support (B2, B3, B6, B9, B12, and magnesium).

Nutritional Needs

Ornithine deficiency and low arginine intake can both lead to the accumulation of carbamoyl phosphate, which is shunted into the pyrimidine biosynthesis pathway, resulting in increased orotic acid production and excretion. Low vitamin B12 causes high orotic acid levels by impairing the conversion of orotic acid to uridine monophosphate (UMP) due to reduced activity of key enzymes in pyrimidine metabolism. Deficiencies in vitamins and minerals such as magnesium, B2 (riboflavin), and zinc can impair key enzymes in the pyrimidine biosynthesis pathway, leading to the accumulation of intermediates like orotic acid.791-795

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Rare conditions, such as urea cycle defects (e.g., OTC deficiency, citrullinemia, and argininemia), Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome, hereditary orotic aciduria, and megaloblastic anemia from pyrimidine synthesis defects, can result in elevated orotic acid levels due to impaired urea cycle function or defective enzymes in the pyrimidine biosynthesis pathway.805-807

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influences: Orotic acid levels may be influenced by some dietary factors, such as cow’s milk and dairy products or a high-protein diet.799,800

Medication/Supplement Interactions: Some medications, such as diuretics like thiazides, some chemotherapy agents, and allopurinol, may increase orotic acid levels by either inhibiting enzymes in the pyrimidine biosynthetic pathway or disrupting the urea cycle, leading to the accumulation of intermediates.791,801 Because lithium or magnesium orotate is made up of a mineral salt and orotic acid, their intake may lead to increased levels of orotic acid being eliminated.

Associated Conditions: Conditions like liver cirrhosis, liver failure, chronic liver disease, gastrointestinal bleeding, and systemic portal shunting increase orotic acid levels due to

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hyperammonemia, which overwhelms the urea cycle and causes excess carbamoyl phosphate to enter the pyrimidine biosynthesis pathway.784,788,789 This leads to elevated orotic acid production and may contribute to hypertension and insulin resistance by impairing endothelial nitric oxide synthesis and disrupting insulin-stimulated NO production.802

Low Values: Low protein intake can result in reduced urinary orotic acid levels due to the decreased availability of nitrogen required for carbamoyl phosphate synthesis, a precursor in the pyrimidine biosynthesis pathway.800,808

61 2-Hydroxyhippuric acid (61) (Aspartame, Salicylates, or GI Bacteria)

2-hydroxyhippuric acid is a compound formed in the body to eliminate excess salicylates by conjugating with glycine after salicylic acid undergoes hydrolysis and further metabolism, and has no anti-inflammatory effects. Salicylates, including salicylic acid and its derivatives like aspirin, are commonly used in medicine as antipyretics, analgesics, and anti-inflammatory agents, but they also occur naturally in foods and can cause hypersensitivity reactions in some.809-811 Elevated 2-hydroxyhippuric acid levels can result from dysbiosis or certain medications and dietary factors, including foods high in salicylates and aspartame intake, topical treatments that contain salicylic acid, environmental pollutants, and medications such as aspirin may potentially alter metabolic pathways.61,805,812-824

Microbial Overgrowth

The microbiome increases 2-hydroxyhippuric acid levels by metabolizing dietary phenolic compounds and aromatic amino acids, such as phenylalanine, into intermediates like phenylpropionic acid and benzoic acid. Specific gut bacteria, including Pseudomonas, Bacillus, Azospirillum, Salmonella, Achromobacter, Vibrio, Yersinia, and Mycobacteria, play a crucial role in this process by reducing phenylalanine to phenylpropionic acid, which is further metabolized in the liver to form 2-hydroxyhippuric.812-814 Clostridia overgrowth can lead to elevated 2-hydroxyhippuric acid levels by metabolizing dietary aromatic compounds into benzoate, which is then conjugated with glycine to form hippurate and its derivatives.61

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

While there is no direct evidence that 2-hydroxyhippuric acid inhibits dopamine beta- hydroxylase (DBH) activity, its structural similarity to other known DBH inhibitors, such as 4-hydroxypyrazole, suggests it could potentially act through mechanisms like competitive inhibition or interference with the enzyme’s electron donors.123,825

For more insight into neurotransmitter activity, reference the phenylalanine and tyrosine metabolites 33 – 37 .

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Toxic Exposures

Environmental toxicants, such as organic solvents and pollutants, can elevate 2-hydroxyhippuric acid levels by altering metabolic pathways. Exposure to toxicants may lead to increased levels of 2-hydroxyhippuric acid due to changes in renal metabolism and the uptake of these compounds by the kidneys. This process can contribute to oxidative stress and kidney injury.823,824

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Additional Insights

Dietary Influences: Foods high in salicylates include various fruits such as blueberries, oranges, tangerines, tomatoes, and lemons, and beverages like tea and wine.816-819 Aspartame intake can increase 2-hydroxyhippuric acid levels by influencing metabolic pathways through its byproducts, methanol and formaldehyde, which may alter aromatic compound metabolism and contribute to oxidative stress.820-822 Glycine enhances the production and excretion of 2-hydroxyhippuric acid by conjugating with aromatic acids like benzoic acid, with higher glycine intake increasing this process.826,827

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Medication/ Supplement Interactions: Aspirin increases 2-hydroxyhippuric acid levels through its metabolism, where salicylic acid is hydroxylated by cytochrome P450 enzymes to form dihydroxybenzoic acids, which are then conjugated with glycine to produce 2-hydroxyhippuric acid. The use of salicylic acid, such as in skincare products, may also increase 2-hydroxyhippuric acid levels.805,815 High salicylate herb concentrations, including basil, cumin, oregano, and clove, may cause elevations.810, 817

Low Values There is no known clinical significance for low values.

AMINO ACID METABOLITES

62 2-Hydroxyisovaleric acid

2-Hydroxyisovaleric acid (2-HIVA) is a catabolite of the branched-chain amino acid (BCAA) valine. It is formed from α-ketoisovalerate (also known as 2-oxoisovalerate 63 ) via 2-hydroxyisovalerate dehydrogenase, a component of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex on the mitochondrial inner membrane.828-830 Elevated levels can provide insights into mitochondrial health, nutritional needs such as Vitamin B1 (Thiamin), and potential genetic predispositions.828,831,832 2-HIVA has been associated with conditions such as lactic acidosis and alcoholic or diabetic ketoacidosis, and with extreme elevations, which can give insights into metabolic disorders such as maple syrup urine disease (MSUD), and other inborn errors of metabolism.230,832-834

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Mitochondrial Health

2-HIVA is a product of ketogenesis and BCAA metabolism, which are crucial for mitochondrial energy production. 2-hydroxyisovaleric acid is metabolized into acetyl-CoA and acetoacetate via the BCKDH complex, which can enter both the ketogenic pathway and the citric acid cycle for energy production.828-830,835

For additional insights, evaluate the mitochondrial markers 22 – 32 , fatty acid oxidation section

45 – 49 , and the other amino acid metabolites 63 – 66 influenced by BCKDH complex.

Nutritional Needs

The diphosphate form of thiamine (known as TPP) serves as a major cofactor for BCKDH, which catalyzes the oxidation of BCAA valine, as well as the other BCAA isoleucine and leucine, to form the respective thioesters with acetyl CoA.831 Additional nutrients also influential for BCKDH include Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin B5 (pantothenic acid), alpha-lipoic acid, and magnesium.836

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Elevated 2-HIVA is associated with several genetic metabolic disorders, primarily involving defects in branched-chain amino acid metabolism from enzyme deficiencies or impairment. These include maple syrup urine disease (MSUD), phenylketonuria (PKU), methylmalonic acidemia, propionic acidemia, isovaleric acidemia, and multiple carboxylase deficiency, among others.832

Corresponding markers to the aforementioned genetic diseases include; Hippuric acid (10), 2-Oxoisovaleric acid 63 , 3-Methyl-2-oxovaleric acid 64 , 2-Hydroxyisocaproic acid 65 , or 2-Oxoisocaproic acid 66 for MSUD, Hippuric acid 10 , 2-Hydroxyphenylacetic acid 11 , 3-Indoleacetic acid 18 , 2-hydorxyisovaleric acid 62 , Mandelic acid 68 , Phenylactic acid 69 , or Phenylpyruvic acid 70 for PKU, Methylmalonic acid 50 , Pyroglutamic acid 58 , 2-Hydroxybutryic acid 59 , and 3-Methyl-2-oxovaleric acid 64 for propionic acidemia.

Additionally, 2-HIVA elevations have been noted in genetic disorders affecting mitochondrial function, such as glutaric aciduria and ornithine transcarbamylase deficiency.832

Glutaric acidemia is associated with 3-hydroxyglutaric acid 31 , Ethylmalonic acid 45 , Methylsuccinic acid 46 , Adipic acid 47 , Suberic acid 48 , Sebacic acid 49 , and Glutaric acid 53

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: Consumption of BCAA supplementation containing valine may increase this metabolite, particularly if there is a dysfunction in the enzyme complex.837-840

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Medication/Supplement Influences: Certain drugs, such as antivirals and valproic acid, can potentially elevate 2-oxoisovaleric acids, and ultimately 2-hydroxyisovaleric acid, due to their impact on mitochondrial function or the blocking of certain enzymes associated with BCAA pathways.230,841 Medications that can cause thiamin depletion, such as loop diuretics, chemotherapy agents, and certain antibiotics, may impair BCAA metabolism.227,666,842 BCAA supplementation may also cause elevations.838

Associated Conditions: Extreme exercise increases flux through the BCKDH pathway; however, if cofactors are insufficient, it can lead to a buildup of 2-oxoisovaleric (63) and/or 2-hydroxyisovaleric acids.843 Elevated levels have also been observed in individuals with severe neonatal asphyxia, lactic acidosis, and diabetic or alcoholic ketoacidosis.230,833,834,844

Low Values There is no known clinical significance for low values.

63 2-Oxoisovaleric acid

2-Oxoisovaleric acid (also known as alpha-ketoisovaleric acid) is a metabolite of the branched- chain amino acid valine.838 The enzyme branched-chain aminotransferase (BCAT) catalyzes the transfer of the amino group from valine to alpha-ketoglutarate, producing the branched- chain keto acid 2-oxoisovaleric acid, ultimately to be used for energy production.828,845 Impaired metabolism causing elevations can often be due to deficiencies in key cofactors such as Vitamin B1 (Thiamine) or enzymatic dysfunction, leading to metabolic imbalances, energy deficits, and increased risk for disorders such as insulin resistance.842,846 This metabolite provides insight into mitochondrial function, nutritional status, and potential genetic predispositions.847-849

Mitochondrial Health

Once 2-oxoisovaleric acid is formed, it undergoes oxidative decarboxylation by the branched chain alpha ketoacid dehydrogenase (BCKDH) complex in the mitochondrial matrix, yielding isobutyryl-CoA, which then enters further catabolic pathways leading to the production of succinyl-CoA, a citric acid cycle intermediate.830,845,848,850 If unable to support this pathway, it may lead to decreased activity of respiratory chain complexes, reduced ATP synthesis, and increased oxidative stress.847,851

For additional insights, evaluate the mitochondrial markers 22 – 32 , fatty acid oxidation section

45 – 49 , and the other amino acid metabolites 62 – 66 influenced by BCKDH complex.

Nutritional Needs

Several key nutrients are required for the optimal function of the BCKDH complex, particularly in the subunits E1, E2, and E3. Vitamin B1 (Thiamine) is essential for the E1 subunit, Vitamin B2 (Riboflavin) is required for the FAD-dependent E3 component, and Vitamin B3 (Niacin) is necessary for NAD+ synthesis, which is also a cofactor for E3. Pantothenic acid (vitamin B5) is

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the precursor for coenzyme A, required for the E2 component. Alpha-lipoic acid is a coenzyme for the E2 component, and magnesium is necessary for the proper function of thiamine pyrophosphate-dependent enzymes.666,842,846,848,852

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Genetic mutations affecting the BCKDH complex can lead to metabolic disorders such as MSUD, where BCAA and keto acid accumulation resulting in neurotoxicity, metabolic acidosis, and severe developmental impairments.230,849

Additional markers associated with MSUD include Hippuric acid 10 , 3-Methyl-2-oxovaleric acid

64 , 2-Hydroxyisocaproic acid 65 , and 2-Oxoisocaproic acid 66 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: Excessive dietary intake of BCAAs can potentially overwhelm BCKA metabolism, especially if the necessary micronutrients that support the BCKDH enzyme complex are lacking.837-840

Medication/Supplement Influences: Certain drugs, such as antivirals and valproic acid, can potentially elevate 2-oxoisocaporic acid 66 and 2-oxoisovaleric acids due to their impact on mitochondrial function.841 Medications that can cause thiamin depletion, such as loop diuretics, chemotherapy agents, and certain antibiotics, may impair BCAA metabolism.227,666,842 BCAA supplementation may also cause elevations.838

Associated Conditions: Elevated levels have been linked to insulin resistance and type 2 diabetes, potentially due to impaired BCKDH function.852 While exercise enhances BCAA breakdown, if there are insufficiencies in enzyme cofactors or increased inhibitory products such as NADH and acyl-CoA esters, it can lead to a buildup of 2-oxoisovaleric acid, especially if the exercise is excessive.843,852 Liver cirrhosis, chronic kidney disease, and hyperthyroidism can disrupt BCAA metabolism, contributing to accumulation.52 Malabsorption disorders, inflammatory bowel disease, or gut dysbiosis may impact the metabolism of BCAA, contributing to elevated levels.853

Low Values There is no known clinical significance for low values.

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64 3-Methyl-2-oxovaleric acid

3-Methyl-2-oxovaleric acid (also known as α-keto-β-Methylvaleric acid-KMV) is a branched- chain keto acid (BCKA) derived from isoleucine metabolism.828 Elevations can reflect impaired mitochondrial function, with disruptions in the citric acid cycle and therefore reduced ATP production.854 Inadequate activity of the branched chain alpha ketoacid dehydrogenase (BCKDH) enzyme complex, often due to nutrient deficiencies such as Vitamin B1 (thiamine), can contribute to these elevations.227,846 Additional factors to consider for elevations include excessive BCAA intake, certain medications that impair mitochondrial or BCAA metabolism, or, in atypical instances, genetic disorders such as Maple Syrup Urine Disease (MSUD).227,230,839

Mitochondrial Health

3-methyl-2-oxovaleric acid (KMV), from isoleucine, can further metabolize to form acetyl CoA and Succinyl CoA, and can be both glycogenic and ketogenic.855,856 It modulates mitochondrial respiration by competitively inhibiting the alpha-ketoglutarate dehydrogenase complex (KGDHC), impairing the citric acid cycle, and reducing ATP production.854 Accumulation of KMV and related BCKAs further disrupts mitochondrial energy homeostasis, promotes oxidative stress, and can alter calcium signaling, contributing to neurometabolic dysfunction.855,857

For additional insights, evaluate the mitochondrial markers 22 – 32 , fatty acid oxidation section

45 – 49 , and the other amino acid metabolites 62 – 66 influenced by BCKDH complex

Nutritional Needs

Several key nutrients are required for the optimal function of the branched-chain alpha- ketoacid dehydrogenase (BCKDH) complex, particularly in the subunits E1, E2, and E3. Vitamin B1 (Thiamine) is essential for the E1 subunit, Vitamin B2 (Riboflavin) is required for the FAD-dependent E3 component, and Vitamin B3 (Niacin) is necessary for NAD+ synthesis, which is also a cofactor for E3. Pantothenic acid (vitamin B5) is the precursor for coenzyme A, required for the E2 component. Alpha-lipoic acid is a coenzyme for the E2 component, and magnesium is necessary for the proper function of thiamine pyrophosphate-dependent enzymes.666,842,846,848,852

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Severe elevations of 3-methyl-2-oxovaleric acid are typically associated with Maple Syrup Urine Disease (MSUD), which can present in infancy or later in life during periods of metabolic stress.87 Other genetic diseases, including methylmalonic acidemia (MMA), propionic acidemia (PA), and isovaleric acidemia (IVA), have been associated with elevations.230

Other associated metabolites with MSUD include, Hippuric acid 10 , 2-Hydroxyisovaleric acid 62 , 2-Oxoisovaleric acid 63 , 2-Hydroxyisocaproic acid 65 , or 2-Oxoisocaproic acid 66 .

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MMA can be also associated with Methylmalonic acid 50 and Methylcitric acid 57 metabolites. Methylmalonic acid 50 , Pyroglutamic acid 58 , 2-Hydroxybutryic acid 59 , and 2-Hydroxyisovaleric acid 62 are associated with propionic acidemia, and Methylsuccinic acid 46 , 3-Hydroxy-3-methylglutaric acid 55 , Methylcitric acid 57 , and 2-Hydroxyisovaleric acid 62 , are associated with isovaleric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: Excessive dietary intake of BCAAs can potentially overwhelm BCKA metabolism, especially if the necessary micronutrients that support the BCKDH enzyme complex are lacking.837-840

Medication/Supplement Influences: Certain drugs, such as antivirals and valproic acid, can potentially elevate BCKAs due to their impact on mitochondrial function.841 Medications that can cause thiamin depletion, such as loop diuretics, chemotherapy agents, and certain antibiotics, may impair BCAA metabolism.227,666,842 BCAA supplementation may also cause elevations.838

Associated conditions: Elevated levels have been linked to insulin resistance and type 2 diabetes, potentially due to impaired BCKDH function.852 While exercise enhances BCAA breakdown, if there are insufficiencies in enzyme cofactors or increased inhibitory products such as NADH and acyl-CoA esters, it may lead to a buildup of BCKA, especially if the exercise is excessive.843,852 Liver cirrhosis, chronic kidney disease, and hyperthyroidism can disrupt BCAA metabolism, contributing to accumulation.852 Malabsorption disorders, inflammatory bowel disease, or gut dysbiosis may impact the metabolism of BCAA, contributing to elevated levels.853 Additionally, elevations in BCAA metabolites have been associated with cardiometabolic risks, though varying in age and disease state.858

Low Values There is no known clinical significance for low values.

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65 2- Hydroxyisocaproic acid

2-Hydroxyisocaproic acid (HICA) is a downstream metabolite of the branched-chain amino acid leucine, formed when leucine is first transaminated by branched-chain aminotransferase (BCAT) into alpha-ketoisocaproic acid (KIC), which is then reduced by cytosolic alpha-ketoisocaproate reductase (KICD).828-830 Elevations in HICA can arise from both endogenous metabolic shifts and microbial activity. Mitochondrial dysfunction or nutrient deficiencies affecting the BCKDH complex (e.g. thiamine, riboflavin, niacin, B5, B6, magnesium) can impair the normal conversion of leucine-derived KIC to isovaleryl-CoA, instead favoring its reduction to HICA. Additionally, certain microbial species (fungi, lactic acid bacteria, and Clostridium) can produce HICA, and high intake of fermented foods or probiotics may further contribute. Genetic conditions such as MSUD or dihydrolipoyl dehydrogenase (E3) deficiency can also elevate HICA, though they are less likely.

Microbial Overgrowth

2-Hydroxyisocaproic acid has been identified as a microbial metabolite produced by certain fungi, lactic acid bacteria, and Clostridium species, and also possesses antimicrobial properties.859,860

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Mitochondrial Health

Leucine is transaminated by BCAT to form KIC, which can either convert to isovaleryl-CoA via BCKDH complex or to HICA via KICD; dysfunction of branched-chain alpha ketoacid dehydrogenase (BCKDH) complex could theoretically shift excess KIC toward HICA production, with excess KIC potentially inhibiting alpha-ketoglutarate dehydrogenase, reducing mitochondrial membrane potential, and increasing reactive oxygen species.835,854,861,862 Moreover, if Isovaleryl-CoA was less available, it could influence yields of acetyl-CoA and acetoacetate 44 for energy synthesis.830,852,863

For additional insights, evaluate the mitochondrial markers 22 – 32 , fatty acid oxidation section

45 – 49 , and the other amino acid metabolites 62 – 66 influenced by BCKDH complex.

Nutritional Needs

The main enzymes involved in leucine to get to HICA are branched-chain aminotransferase (BCAT) and cystosolic alpha-ketoisocaproate reductase (KICD). BCAT requires pyridoxal 5’-phosphate (PLP), which is associated with Vitamin B6, and KICD requires NADH or NADPH, which is associated with Vitamin B3.829,864-867

Moreover, the diphosphate form of thiamine (known as TPP), serves as a major cofactor for BCKDH, which catalyzes the oxidation of KIC to isovaleryl-CoA.831 Additional nutrients also influential for BCKDH include Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin B5

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(pantothenic acid), alpha-lipoic acid, and magnesium.122

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

HICA has been associated with MSUD.230,849

Additional markers associated with MSUD include Hippuric acid 10 , 3-Methyl-2-oxovaleric acid 64 , 2-Hydroxyisocaproic acid 65 , and 2-Oxoisocaproic acid 66 .

HICA has also been found to be elevated in individuals with dihydrolipoyl dehydrogenase (E3) deficiency.868

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: High intake of fermented foods, which serve as dietary sources of certain bacteria that may produce this metabolite.859,860

Medication/Supplement Influences: Probiotics that contain lactic acid bacteria, may influence elevations.859,860

Associated Conditions: HICA has been observed in individuals with short bowel syndrome.869

Low Values There is no known clinical significance for low values.

66 2-Oxoisoaproic acid

2-Oxoisocaproic acid (also known as alpha ketoisocaporic acid (KIC)) is the transamination product of leucine and is a key intermediate in branched-chain amino acid (BCAA) catabolism. Elevated levels suggest dysfunction in this pathway—whether due to nutrient deficiencies (e.g., thiamin, riboflavin, niacin, B6, magnesium), metabolic stress, or in rare instances, genetic conditions such as Maple Syrup Urine Disease (MSUD). Excess KIC can impair mitochondrial oxidative phosphorylation and lead to reduced energy production and neurological or metabolic disturbances. Contributing factors may also include high-protein or leucine-rich diets, BCAA supplementation, certain medications (e.g., valproic acid, chemotherapy), or underlying conditions such as insulin resistance, liver or kidney dysfunction, or gut dysbiosis.870

Mitochondrial Health

2-oxoisocaproic acid is primarily metabolized in the mitochondria by the branched-chain α-keto acid dehydrogenase complex (BCKDC) to produce isovaleryl-CoA, ultimately contributing to acetyl-CoA, acetoacetate, and energy production via the TCA cycle and

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ketogenesis.132,133,134 At physiological concentrations, it can modulate mitochondrial respiration, but at pathophysiological levels (such as in maple syrup urine disease (MSUD)), KIC inhibits mitochondrial oxidative phosphorylation, particularly by inhibiting α-ketoglutarate dehydrogenase, leading to impaired TCA cycle flux, reduced NAD(P)H, and decreased mitochondrial membrane potential.854,861,862

For additional insights, evaluate the mitochondrial markers 22 – 32 , fatty acid oxidation section

45 – 49 , and the other amino acid metabolites 62 – 66 influenced by BCKDH complex.

Nutritional Needs

Several nutrients and cofactors impact these pathways. Vitamin B6 is required for the initial transamination step, while BCKDC activity depends on thiamine (vitamin B1), Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin B5 (pantothenic acid), alpha-lipoic acid, and magnesium.829

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Marked elevations in 2-oxoisocaproic and related BCAA metabolites can signal inborn errors of metabolism, especially Maple Syrup Urine Disease (MSUD). MSUD is caused by a deficiency in the BCKDH complex and can range in severity from classic infantile onset to milder intermittent forms230. This condition results in a toxic accumulation of BCAAs and their corresponding ketoacids in the brain and blood, leading to symptoms like seizures, ataxia, encephalopathy, behavioral changes, and metabolic crises.230

Additional markers associated with MSUD include Hippuric acid 10 , 3-Methyl-2-oxovaleric acid

64 , 2-Hydroxyisocaproic acid 65 , and 2-Oxoisocaproic acid 66 .

In other rare cases, isovaleric acidemia (IVA), a branched-chain organic aciduria caused by deficiency of isovaleryl-CoA dehydrogenase.230

Methylsuccinic acid 46 , 3-Hydroxy-3-methylglutaric acid 55 , Methylcitric acid 57 , and 2-Hydroxyisovaleric acid 62 are also associated with isovaleric acidemia.

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: High-protein diets, especially if predominating with leucine-rich foods, can overload the system and lead to elevations, especially in individuals with impaired BCAA processing capacity.837-840

Medication/ Supplement Influences: Certain drugs, such as antivirals and valproic acid, can potentially elevate 2-oxoisocaporic acid and 2-oxoisovaleric acid 63 due to their impact on mitochondrial function.841 Medications that can cause thiamin depletion, such as loop diuretics,

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chemotherapy agents, and certain antibiotics, may impair BCAA metabolism.227,666,842 BCAA supplementation may also cause elevations.838

Associated Conditions: Elevated levels have been linked to insulin resistance and type 2 diabetes, potentially due to impaired BCKDH function.852 While exercise enhances BCAA breakdown, if there are insufficiencies in enzyme cofactors or increased inhibitory products such as NADH and acyl-CoA esters, it may lead to a buildup of BCKA, especially if the exercise is excessive.843,852 Liver cirrhosis, chronic kidney disease, and hyperthyroidism can disrupt BCAA metabolism, contributing to accumulation.852 Malabsorption disorders, inflammatory bowel disease, or gut dysbiosis may impact the metabolism of BCAA, contributing to elevated levels.853

Low Values There is no known clinical significance for low values.

67 2-Oxo-4-methiolbutyric acid

2-oxo-4-methylthiobutyric acid (also known as 4-methylthio-2-oxobutyric acid or MTOB), an intermediate in methionine metabolism, suggests a disruption in the transamination pathway and may lead to the accumulation of toxic byproducts such as 3-methylthiobutyrate, methanethiol, and hydrogen sulfide.156 This metabolite may accumulate due to excessive methionine intake, vitamin B6 deficiency, or genetic defects in sulfur amino acid metabolism. If not properly metabolized, KMBA can give rise to toxic byproducts that impact cellular health, liver function, and energy regulation.872

Its elevation is often seen in the context of hypermethioninemia or inherited genetic disorders.

Methylation/Detoxification

MTBO is a key intermediate in the methionine salvage pathway, which maintains methionine and S-adenosylmethionine pools necessary for cellular methylation reactions.873,874 If elevated, it could give insights into methylation and SAM homeostasis.873

For additional markers influencing methylation and SAM, look to HVA/DOPAC ratio 37 and 2-Hydroxybutryic acid 58 .

Nutritional Needs

Vitamin B6 (pyridoxal phosphate) is a key cofactor required for directing methionine metabolism through the transsulfuration pathway.875 A deficiency can shift methionine metabolism toward the transamination route, increasing KMBA production.875

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

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Genetics

Various different genetic SNPs involved in methionine recycling or clearance could potentially influence elevations, and include S-adenosylhomocysteine (SAH) hydrolase, Methionine adenosyltransferase (MAT), Methylenetetrahydrofolate reductase (MTHFR), Glycine N-methyltransferase (GNMT), Cystathionine beta-synthase (CBS).876

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary influence: While a high-protein or methionine-rich diet may contribute to elevated MTBO, dietary intake alone is generally not the primary driver unless paired with other metabolic impairments.875

Medication/Supplement Influences: Methionine supplements in theory could potentially elevate KMBA, though it is not usually the primary influence.875

Associated Conditions: Elevations have been shown in hypermethioninemia, which is linked to changes in liver function, hemolytic anemia, and glucose regulation.872,877

Low Values There is no known clinical significance for low values.

68 Mandelic acid

Mandelic acid is a primary metabolite of phenylethylamine, which is formed from phenylalanine in an alterative pathway to phenylacetic acid 69 . Elevated mandelic acid is most commonly associated with toxic exposure to styrene or ethylbenzene, which are metabolized in the body and excreted as mandelic acid. These compounds are found in tobacco smoke, vehicle exhaust, synthetic rubber, plastics, resins, and food containers, especially when heated, making environmental exposure a primary concern. As a secondary mechanism, mandelic acid may also result from altered phenylalanine metabolism, where phenylalanine is diverted to phenylethylamine and subsequently to mandelic acid, particularly when dopamine metabolism is impaired. This diversion may be slightly influenced by deficiencies in iron or vitamin B2, which are cofactors for phenylalanine hydroxylase; however, genetic factors such as PKU, although rare, can also elevate mandelic acid when phenylalanine processing is disrupted.

Neurotransmitter Metabolites

When phenylalanine is unable to convert to tyrosine via phenylalanine hydroxylase (PAH) to generate dopamine, norepinephrine, and epinephrine, it goes an alternate path to ultimately generate mandelic acid.875,879

Additionally, neurotoxic aromatic hydrocarbons from styrene exposure (see below) can impair dopamine metabolism.880

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For a more in-depth evaluation into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Phenylacetic acid 69 , Phenylpyruvic acid 70 , and 4-Hydroxyphenyllacetic acid 72 .

Toxic Exposure

Mandelic acid is a major urinary metabolite formed from styrene and ethylbenzene metabolism via cytochrome P450 oxidation and subsequent enzymatic steps, making it a widely used biomarker for assessing contact.880-883 Exposures to these toxicants can occur through inhalation via tobacco smoke, off-gassing of synthetic rubber, resins, polyesters, vehicle exhaust, and food contact materials like Styrofoam or plastic containers, especially when heated.884

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Nutritional Needs

Iron and B2 influence PAH, and if deficient, may reduce the processing of phenylalanine to tyrosine, shunting phenylalanine back to phenylethylamine.885-887

BH4 synthesis and regeneration is essential to optimize phenylalanine metabolism, with folate (especially folinic acid in DHPR deficiency) aiding its regeneration and vitamin C and other antioxidants stabilizing BH4 by preventing oxidation and preserving its role in neurotransmitter and endothelial function.888-890

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

In rare cases, inborn errors like PKU (phenylketonuria) may elevate mandelic acid due to impaired metabolism of phenylalanine. However, this is usually accompanied by elevations in Hippuric acid 10 , 2-hydroxyphenylacetic acid 11 , 2-Hydorxyisovaleric acid 62 , phenylacetic acid

69 , and phenylpyruvic acid 70 .230

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influence: Dietary intake of phenylethylamine or excessive grain consumption can influence elevations.884,887

Medication/Supplement Influences: Skincare products or dyes may contain mandelic acid derivatives, but these are less common contributors.891

Low Values There is no known clinical significance for low values.

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69 Phenyllactic acid

Phenyllactic acid is formed when phenylalanine is transaminated to phenylpyruvic acid and then reduced. Elevated phenyllactic acid may result from impaired phenylalanine metabolism, particularly when phenylalanine hydroxylase (PAH) activity is reduced, diverting phenylalanine away from tyrosine and neurotransmitter synthesis toward alternative pathways. This can occur due to disruptions in BH4, a critical cofactor for PAH, which may be influenced by deficiencies in iron, vitamin B2, folate, or antioxidants such as vitamin C. Gastrointestinal overgrowth of certain Clostridium species may also contribute to mild elevations, although this connection is less established than with other phenylalanine metabolites. Genetic conditions such as phenylketonuria (PKU) and BH4 synthesis defects can lead to significant increases in phenyllactic acid and related metabolites.

Microbial Overgrowth

Slight elevations of phenyllactic acid can potentially be due to gastrointestinal overgrowth of Clostridium spp, including C. botulinium and C. sporogenes as well as other species.115,892

For additional insights into clostridia metabolites, review 4-Hydroxyphenylacetic acid 15 , HPHPA (3-(3-hydroxyphenyl)-3-hydroxypropionic acid) 16 , 4-cresol 17 , and 3-Indolacetic acid 18 .

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

The pathway that generates phenyllactic acid from phenylalanine becomes more prominent when phenylalanine hydroxylase (PAH) activity is impaired, diverting phenylalanine away from its primary role in producing tyrosine, a critical precursor for dopamine, norepinephrine, and epinephrine synthesis.893-896 Disruptions in Tetrahydrobiopterin (BH4), an essential cofactor for PAH, may also be a factor.

For a more in-depth evaluation into dopamine metabolites, assess the Phenylalanine and Tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenylpyruvic acid 70 , and 4-Hydroxyphenyllacetic acid 72 .

Nutritional Needs

Iron and B2 influence PAH, and if deficient, may reduce the processing of phenylalanine to tyrosine, shunting phenylalanine back to phenyllactic acid.885-887 Support for BH4 synthesis and regeneration is essential to optimize phenylalanine metabolism and reduce phenyllactic acid accumulation, with folate (especially folinic acid in DHPR deficiency) aiding its regeneration and vitamin C and other antioxidants stabilizing BH4 by preventing oxidation and preserving its role in neurotransmitter and endothelial function.803,889,890

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

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Genetics

Inborn errors such as phenylketonuria (PKU), a genetic deficiency of PAH, can lead to the accumulation of phenylalanine and its byproducts, including phenyllactic acid. Genetic factors affecting BH4 production may also contribute.230

Metabolites also associated with PKU include Hippuric acid 10 , 2-hydroxyphenylacetic acid 11 , 2-Hydorxyisovaleric acid 62 , and phenylpyruvic acid 70 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influence: Dietary intake of excessive phenylalanine, especially in the presence of decreased PAH function, could elevate phenyllactic acid.897

Low Values: There is no known clinical significance for low values.

70 Phenylpyruvic acid

Phenylpyruvic acid is formed from phenylalanine by transamination, in which phenylalanine is converted to phenylpyruvate by aminotransferase enzymes. This reaction occurs when the primary pathway for phenylalanine metabolism, hydroxylation to tyrosine by phenylalanine hydroxylase (PAH), is impaired. Elevations typically indicate impaired metabolism of phenylalanine due to reduced activity of phenylalanine hydroxylase (PAH), often caused by nutrient deficiencies (iron, B2) or dysfunction in its cofactor tetrahydrobiopterin (BH4). This diversion away from tyrosine production affects neurotransmitter synthesis, particularly dopamine, norepinephrine, and epinephrine. Nutritional support for BH4 regeneration, such as folate, vitamin C, and antioxidants, may help reduce phenylpyruvic acid accumulation. Genetic conditions such as phenylketonuria (PKU), which involves a deficiency of PAH, are a potential cause, as are variants affecting BH4 metabolism. Excessive dietary phenylalanine may also contribute, especially when PAH activity is already compromised.

Neurotransmitter Metabolites

The pathway that generates phenylpyruvic acid from phenylalanine becomes more prominent when phenylalanine hydroxylase (PAH) activity is impaired, diverting phenylalanine away from its primary role in producing tyrosine, a critical precursor for dopamine, norepinephrine, and epinephrine synthesis.893-896 Disruptions in Tetrahydrobiopterin (BH4), an essential cofactor for PAH, is also a factor.

For a more in-depth evaluation into dopamine metabolites, assess the phenylalanine and tyrosine metabolites 33 – 37 , along with 2-Hydroxyphenylacetic acid 11 , Mandelic acid 68 , Phenyllactic acid 69 , and 4-Hydroxyphenyllacetic acid 72 .

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Nutritional Needs

Iron and B2 influence PAH, and if deficient, may reduce the processing of phenylalanine to tyrosine, shunting phenylalanine back to phenylpyruvic acid.885-887 Support for BH4 synthesis and regeneration is essential to optimize phenylalanine metabolism and reduce phenypyruvic acid accumulation, with folate (especially folinic acid in DHPR deficiency) aiding its regeneration and vitamin C and other antioxidants stabilizing BH4 by preventing oxidation and preserving its role in neurotransmitter and endothelial function.803,889,890

Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with those nutrients.

Genetics

Inborn errors such as phenylketonuria (PKU), a genetic deficiency of PAH, can lead to the accumulation of phenylalanine and its byproducts, including phenyllactic acid. Genetic factors affecting BH4 production may also contribute.230

Metabolites also associated with PKU include: Hippuric acid 10 , 2-Hydroxyphenylacetic acid 11 , 2-Hydorxyisovaleric acid 62 , and Phenyllacetic acid 69 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influence: Dietary intake of excessive phenylalanine, especially in the presence of decreased PAH function, could elevate phenylpyruvic acid.897

Low Values There is no known clinical significance for low values.

71 Homogentisic acid

Homogentisic acid (HGA) is a byproduct of tyrosine breakdown via the enzyme homogentisate 1,2-dioxygenase (HGD), which processes tyrosine when it diverges from neurotransmitter synthesis. A deficiency in HGD, as seen in the genetic disorder alkaptonuria, impairs this catabolic pathway, disrupting energy metabolism and leading to elevated HGA levels.

Mitochondrial Health

From tyrosine, HGA converts to maleylacetoacetate via homogentisate 1,2-dioxygenase, which is ultimately further degraded to fumarate and acetoacetate, entering the Citric Acid Cycle for energy production.898

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

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Neurotransmitter Metabolites

Homogentisic acid is a key intermediate in the breakdown of tyrosine, when it diverges from the pathway of synthesizing dopamine, norepinephrine, and epinephrine.898

For a more in-depth evaluation of dopamine metabolites, assess the phenylalanine and tyrosine metabolites 33 – 37 .

Genetics

Alkaptonuria is inherited in an autosomal recessive pattern that involves a mutation in the HGD gene, impairing the conversion of HGA to maleylacetoacetate, leading to systemic buildup of toxic byproducts like benzoquinone acetic acid.899

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Associated Conditions: Elevated HGA has also been observed in individuals with uncontrolled hyperglycemia.900

Low Values There is no known clinical significance for low values.

72 4-Hydroxyphenllactic acid

4-Hydroxyphenyllactic acid (4-HPLA) is formed when L-tyrosine is first transaminated into 4-hydroxyphenylpyruvic acid, which is then reduced by the enzyme hydroxyphenylpyruvate reductase. Certain gut bacteria, such as Bifidobacteria, Lactobacillus, and Clostridium, can produce D-4-HPLA, linking elevations to microbial overgrowth or dysbiosis, while excessive dietary tyrosine intake may further contribute. In rare cases, elevations may be associated with tyrosinemia due to mutations in the HPD gene.

Microbial Overgrowth

4-HPLA (D form) is produced by various gut bacteria, including Bifidobacteria, Lactobacillus, and Clostridium, and elevations may reflect bacterial overgrowth or dysbiosis.115,901

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

Markedly elevated plasma tyrosine, as seen in tyrosinemia, has been shown in computational and clinical studies to competitively inhibit other large neutral amino acids (LNAAs) at the LAT1 transporter, reducing their entry into the brain. This may disrupt neurotransmitter synthesis and amino acid balance, particularly by limiting tryptophan availability, ultimately influencing the serotonin synthesis pathway.902,903 Additionally, excessive tyrosine levels have been shown to cause neurotoxicity.904

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For a more in-depth evaluation of dopamine metabolites, assess the phenylalanine and tyrosine metabolites 33 – 37 .

Genetics

Elevated 4-hydroxyphenyllactic acid (4-HPLA) is most directly linked to mutations in the HPD gene, which impair the enzyme 4-hydroxyphenylpyruvate dioxygenase, leading to tyrosinemia types I- III. This enzymatic block causes upstream accumulation of 4-HPLA.230

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Dietary Influence: Dietary intake of excessive tyrosine, especially in the presence of decreased enzyme function, could elevate 4-HPLA.905

Low Values There is no known clinical significance for low values.

73 N-Acetylaspartic acid

N-Acetylaspartic acid (NAA) is synthesized in neurons from aspartate and acetyl-CoA and plays roles in fluid balance, myelin synthesis, and neurotransmitter function. Elevated NAA is most commonly associated with Canavan disease, a rare autosomal recessive neurodegenerative disorder caused by mutations in the ASPA gene, leading to a deficiency of the enzyme aspartoacylase.

Genetics

N-acetylaspartic acid (NAA) is most commonly associated with Canavan disease, a rare autosomal recessive neurodegenerative disorder caused by mutations in the ASPA gene, leading to a deficiency of the enzyme aspartoacylase.906

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Low Values There is no known clinical significance for low values.

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74 Malonic acid

Malonic acid is a dicarboxylic acid involved in fatty acid metabolism and energy production. Elevations may result from impaired mitochondrial function or acrolein exposure from polluted air or burning cooking oils past their smoke point. Markedly high levels are associated with malonyl-CoA decarboxylase deficiency, often alongside elevated methylmalonic acid.

Mitochondrial Health

Elevated malonic acid can impair energy production by inhibiting both succinic acid dehydrogenase in the citric acid cycle and carnitine palmitoyltransferase I (CPT-I), limiting mitochondrial fatty acid oxidation.907-910

For more insight into mitochondrial health, reference the other mitochondrial markers 22 – 32 and fatty acid oxidation markers 45 – 49 .

Toxic Exposures

Acrolein can be metabolized into malonic acid through oxidative degradation pathways following initial epoxidation or hydration.911 While acrolein is primarily detoxified via glutathione conjugation and excreted as mercapturic acids, its conversion to small dicarboxylic acids like malonic acid and oxalic acid is a recognized secondary route.912 Acrolein exposure can occur through inhalation of polluted air from sources like tobacco smoke, vehicle exhaust, industrial emissions, and burning fuels, with smaller amounts also found in fried foods and cooking oils, though it is rarely present in drinking or surface water.913

Refer to the Toxin Exposure Tables for corresponding metabolites and patterns.

Genetics

In very high elevations, malonic acid is associated with the rare genetic metabolic disorder, malonyl-CoA decarboxylase deficiency.230

Malonyl CoA decarboxylase deficiency is also associated with methylmalonic acid 50 .

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Associated Conditions: Malonic acid has been associated with several conditions, including eosinophilic esophagitis and early preeclampsia.

Low Values There is no known clinical significance for low values.

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75 4-Hydroxybutyric acid

4-Hydroxybutyric acid (4-HBA), also known as gamma-hydroxybutyric acid (GHB), is primarily produced in the brain and liver through the degradation of gamma-aminobutyric acid (GABA), where GABA is converted to succinic semialdehyde (SSA) and then is either reduced to GHB or oxidized to succinate.914-917 Elevations in 4-HBA may result from microbial production by species like Clostridium aminobutyricum, Pseudomonas, and Saccharomyces cerevisiae, or GABA degradation in the brain and liver. As a neurotransmitter metabolite, it influences GABA and glutamate signaling by acting on GABA receptors, affecting inhibitory and excitatory balance. 4-HBA can also be elevated due to GHB ingestion, whether illicit or pharmaceutical, and its metabolism depends on NAD+, making B3 status a potential modulating factor. Genetically, elevations may be associated with inherited metabolic disorders such as SSADH deficiency, though this is rare.

Microbial Overgrowth

4-Hydroxybutyric acid (4-HBA) has been shown to be produced by various microbes, including Clostridium aminobutyricum, Saccharomyces cerevisiae, and Pseudomonas spp, through the conversion of GABA or related precursors.918

Refer to Microbial Overgrowth Tables for corresponding metabolites and patterns.

Neurotransmitter Metabolites

4-HBA is produced during gamma-aminobutyric acid (GABA) degradation and can influence both GABA and glutamate signaling, potentially disrupting excitatory and inhibitory neurotransmission. Gamma-hydroxybutyric acid (GHB) can cycle back to GABA and modulate neurotransmission by acting on both GABA B and high-affinity GHB receptors, while also indirectly affecting GABAA signaling by reducing presynaptic GABA release.919-921 This influences both inhibitory (GABAergic) and excitatory (glutamatergic) pathways in a region- and dose- dependent manner.922

Refer to phenylalanine and tyrosine 33 – 37 and tryptophan 38 – 40 metabolites for further review of neurotransmitters.

Toxic Exposures

4-HBA is chemically identical to GHB, which is a prototypical date rape drug used in drug- facilitated sexual assault due to its sedative, amnestic, and incapacitating effects.923,924

Nutritional Needs

The oxidation of 4-hydroxybutyric acid to succinic semialdehyde is catalyzed by 4-hydroxybutyrate dehydrogenase, an NAD+-dependent enzyme.925,926 Considering Vitamin B3 (Niacin) is a precursor to NAD, slight elevations may be influenced by B3 insufficiencies.927

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Refer to the Nutrient–Marker Reference Table for the corresponding organic acids associated with that nutrient.

Genetics

Elevated 4-HBA is most closely linked to inherited disorders such as SSADH deficiency, GABA transaminase deficiency, and 4-hydroxybutyric acid-CoA transferase deficiency.230

Note: This test is not diagnostic for genetic conditions, and specialized genetic testing may be warranted if a disorder is suspected.

Additional Insights

Medications/Supplements: Illicit or pharmaceutical use of GHB can significantly increase urinary 4-HBA.

Low Values There is no known clinical significance for low values.

MINERAL METABOLISM

76 Phosphoric

Phosphoric acid forms phosphate ions, which are essential for energy metabolism, DNA/ RNA structure, and bone homeostasis.928 High phosphoric acid levels can be caused by toxic exposures such as uranium or lead, high vitamin D and phosphorus intake, certain medications, and conditions that enhance bone resorption or impair phosphate reabsorption.821,928-941 Low phosphate levels stimulate the production of 1,25-dihydroxyvitamin D3, to regulate phosphate and calcium levels, supporting the relationship between low vitamin D and low phosphate levels. Otherwise, low values may suggest low dietary intake or absorption, kidney disease, liver disease, and conditions such as hypoparathyroidism.815,821,827,934,942-945

Toxic Exposure

Toxic exposures, such as heavy metals like uranium and lead, can increase phosphoric acid levels by causing metabolic acidosis and renal tubular dysfunction, which reduces phosphate reabsorption and leads to increased urinary phosphate excretion.929,930

Refer to the Toxic Marker Comparison Table for corresponding metabolites and patterns.

Nutritional Needs

High phosphorus and vitamin D intake can increase phosphoric acid levels by enhancing the absorption and reabsorption of phosphate in the intestine and kidneys. Vitamin D, through its active metabolite 1,25-dihydroxyvitamin D3, stimulates phosphate absorption and helps regulate phosphate homeostasis. This leads to increased levels of phosphate in the blood, which, in turn, can elevate phosphoric acid levels, particularly in the context of dietary phosphate loading.821,928

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Additional Insights

Dietary Influences: Meat and poultry, dairy products, and grains and cereals contribute to increased phosphoric acid levels by providing natural phosphorus.946 Processed foods increase phosphoric acid levels through the use of phosphate additives, which are commonly found in bakery items, processed meats, cheeses, and beverages like colas. These additives, containing highly bioavailable inorganic phosphorus, and are rapidly absorbed by the body, leading to elevated phosphoric acid levels.947,948

Medication/Supplement Interactions: Medications that increase phosphoric acid levels include phosphate-containing laxatives, which can significantly raise serum phosphate levels, especially in individuals with compromised renal function. Additionally, drugs like K-Phos No. 2, certain proton pump inhibitors (e.g., omeprazole), calcium carbonate therapy with famotidine or lansoprazole, and medications with phosphate as an excipient, such as some antidiabetic, antidepressant, and antihypertensive medications, can contribute to elevated phosphate levels and potentially cause hyperphosphatemia.931-935

Associated Conditions: Conditions like hyperparathyroidism, vitamin D-resistant rickets, increased bone turnover, renal tubular damage, and kidney disease increase phosphoric acid levels by either enhancing bone resorption or impairing phosphate reabsorption in the kidneys. Other factors like familial hypophosphatemia, immobilization, metabolic acidosis, and active bone growth during pediatric development also contribute by promoting phosphate release from bones or impairing phosphate balance, resulting in higher phosphoric acid levels.936-941

Low Values

Nutritional Needs: Low phosphoric acid levels can be linked to low vitamin D and low phosphorus due to inadequate dietary intake or malabsorption.

Associated Conditions: Kidney disease may exacerbate low phosphoric acid levels due to impairments in phosphate reabsorption. Liver disease, acute liver failure, and conditions like hypoparathyroidism or pseudohypoparathyroidism reduce phosphate excretion, contributing to low phosphate levels.815,821,827,934,942-945

Disclaimer: The content of this Provider Support Guide is for informational purposes only and is not intended to be a substitute for medical advice from a licensed healthcare practitioner. The statements in this report have not been evaluated by the Food and Drug Administration and are intended to be lifestyle choices for potential risk mitigation. Please consult a licensed healthcare practitioner for medication, treatment, diet, exercise or lifestyle management as appropriate. This product is not intended to diagnose, treat, or cure any disease or condition.

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THERAPEUTIC CONSIDERATIONS

This section provides a structured framework to support clinicians when any of the six categories of clinical insight on the summary page are found to be imbalanced. These include Microbial Overgrowth, Mitochondrial Health, Neurotransmitter Metabolites, Methylation/ Detoxification, Toxic Exposure, and Nutritional Needs. For each category, targeted interventions are presented, outlining key metabolites associated with certain clinical insights and suggested supports for those particular markers and patterns. This systematic approach is intended to facilitate the development by practitioners of individualized care plans aimed at addressing underlying dysfunction and improving clinical outcomes.

MICROBIAL OVERGROWTH

THE 5RS FOR GENERAL SUPPORT

• Antimicrobials: Oregano oil (150-200 mg BID), Garlic extract (Allicin 300-600 mg/day), Berberine (500 mg TID), Caprylic acid (500 mg BID)949-956

Remove

Reduce microbial load

Rotate antimicrobials every 2-4 weeks to minimize resistance

• Binders (if mycotoxins suspected or possible Herxheimer): Activated charcoal (500-1000 mg/ day), Zeolite clay (1 tsp/day)957-960

Replace

• Digestive Enzymes such as Amylase, Lipase, Protease, or HCl (1-2 caps with meals)961, 962

Support digestion

Helps reduce fermentable substrates for microbial overgrowth

• Bile salts (250-500 mg with fatty meals if needed)963, 964

Reinoculate

• Probiotics: Saccharomyces boulardii (5-10 billion CFU BID for yeast overgrowth), Lactobacillus & Bifido blends (20-50 billion CFU/day)965-969

Restore healthy flora

Start after 2 weeks of antimicrobials

Repair

Gut lining support

• L-Glutamine (5 g/day)970-972

Begin mid- protocol to aid mucosal healing

• Zinc carnosine (75 mg BID)973,974

• Omega-3 fatty acids (1000-3000mg/day)975-978

Rebalance

Support systemic health

• Lifestyle: Low sugar, varied, anti-inflammatory, high-fiber diet • Stress management

Avoid rapid die-off with aggressive interventions

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SPECIFIC ORGANISM/MARKER CONSIDERATIONS

Marker Type

Elevated Findings Suggest

Therapeutic Focus

Example Interventions

Yeast/Mold (e.g., Arabinose, Furan-2,5- Dicarboxylic acid)

Candida or other fungal overgrowth

Antifungal botanicals, Pharmaceuticals, Probiotics, Mycotoxin clearance

• Caprylic acid, Cinnamon/ cinnamon leaf extract, Thyme essential oil, Oil of Oregano955,956,979-986

• Nystatin, Fluconazole, Itraconazole, or Amphotericin B.987-990

• Probiotics: Saccharomyces boulardii, Lactobacillus kefiri, L. Pentosis, L. Brevis, L. plantarum and Bacillus coagulans and B. subtilis.966,991-998

Bacterial (e.g., Hippuric, Benzoic, DHPPA)

Bacterial imbalance

Probiotics Fiber for SCFA production

• Lactobacillus spp, Bifidobacterium spp, and Bacillus spp.1001-1004

Clostridial (e.g., HPHPA, 4-Cresol)

Clostridia overgrowth (esp. neurotoxic species)

Targeted antimicrobials, Toxin neutralization

• High-dose S. boulardii, Lactobacillus species966,1005-1007

• Ginger, garlic, Oil of Oregano1008-1016

• Focus on detox pathways (e.g., NAC 600-1200 mg/day)1017, 1018

Oxalates (e.g., Glyceric, Glycolic, Oxalic)

Endogenous production of oxalate (often yeast or mold-related) or exogenous sources, such a certain plant-based foods

Microbial + oxalate- lowering support

• Address fungal overgrowth • Calcium + Magnesium citrate; 3:1 ratio with medium-high oxalates foods1019-1023

• Vitamin B61024-1026

• Low-oxalate diet if sensitive to medium-high oxalate foods • Low sodium intake

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ASSOCIATED PATTERNS FROM OTHER CATEGORIES

Microbial Impacts

Category

Markers

Neurotransmitter Metabolites

Clostridia inhibits dopamine → norepinephrine conversion.

HVA 33 , VMA 34 , HVA/VMA ratio 35

Toxic Exposure

Various herbicides can impact the microbiome. Mycotoxins can be antimicrobial and impact imbalances in the microbiome.

Succinic 24 , Pyroglutamic 58 , 2-Hydroxybutyric 59 acids

CLINICAL TIPS *See Disclaimer on Page 154

• Rotational Strategy: Rotate antimicrobials every 2-4 weeks to reduce development of resistance.

• Die-off Awareness: Always start low and go slow—support detox pathways with adequate hydration, increased fiber intake, sweating, magnesium, and vitamin C.

• Retesting Window: Consider retesting OAT after 8–12 weeks of intervention.

• Combination Therapy: Use synergistic approaches—e.g., berberine + probiotic + glutamine— rather than mono-strategy.

• Oxalate Alert: Elevations in this section may reflect yeast or mold-associated endogenous oxalate production. Correlate with other yeast and fungal markers.

• Environmental Cross-Talk: If markers 1-9 and 19-21 persist, screen for mold/mycotoxins (MycoTOX) and environmental toxicants (TOXDetect) to address upstream root causes.

TESTING CONSIDERATIONS

Consider the following testing for further insights:

•GI360 (with Zonulin and H. Pylori add-ons): A complement to the OAT, it can further assess additional organisms contributing to dysbiosis, as well as insights into inflammation and digestion.

• IgG Food Map: Food sensitivities are common with dysbiosis and may also give insights into immune response to Candida, if present.

• MycoTOX: Elevated mold-related metabolites or clinical signs of mycotoxin-induced microbiome impacts may warrant additional assessment.

• IgE Mold: If mold is suspected, this panel may provide insights into how the immune system is responding to the exposure.

• TOXDetect: Consider when microbial patterns, particularly with Clostridia, allude to possible toxicant- related influences, based on clinical history.

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MITOCHONDRIAL HEALTH

KEY ORGANIC ACID MARKERS

Category Marker(s) Clinical Insight

Glycolytic Markers

Could indicate mitochondrial dysfunction, impaired glucose metabolism, or tissue hypoxia.

Lactic 22 , Pyruvic 23 acids

Citric Acid Cycle Intermediates

Abnormal values may indicate impaired enzyme function or cofactor deficiencies (e.g., Mg, B vitamins).

Succinic 24 , Fumaric 25 , Malic 26 , 2-Oxoglutaric 27 , Citric 29 , Aconitic 28 , Citric 29 acids

Amino Acid Metabolism

Impaired amino acid utilization for energy production, potentially causing oxidative stress.

3-Methylglutaric 30 , 3-Hydroxyglutaric 31 , 3-Methylglutaconic 32 acids

Fatty Acid Metabolism

Elevated in carnitine or B2 deficiencies, or β-oxidation impairments, leading to increased oxidative stress and reduced energy production.

Ethylmalonic 45 , Methylsuccinic 46 , Adipic 47 , Suberic 48 , Sebacic 49 acids

Ketone Metabolism

May reflect impaired carbohydrate utilization, ketogenic adaptation, or mitochondrial overload.

3-Hydroxybutyric 43 , Acetoacetic 44 acids

NUTRIENT COFACTOR ASSOCIATIONS *See disclaimer on Page 154

Organic Acid Marker Suggested Support Likely Cofactor Deficiency General Dosage Range Guideline*

Benfotiamine or thiamine HCl, R-lipoic acid

Benfotiamine: 150-600 mg/day; Thiamine HCl: 50-100 mg/day; R-lipoic acid: 100-300 mg/day

B1 (Thiamine), Lipoic Acid846,1027-

Pyruvic Acid 23

10310

Riboflavin 5’-phosphate, ubiquinol

Riboflavin (as R5P or Riboflavin HCl): 10-30 mg/day; Ubiquinol: 100-300 mg/day

B2 (Riboflavin), CoQ101032-1035

Succinic 24 , Fumaric 25 , Malic 26 acids

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B3 (Niacin), Mg, B6772,829,830,1036,1037

Niacinamide, magnesium glycinate, P5P/ Pyridoxine HCl

Niacinamide: 50-150 mg/day; Magnesium glycinate: 200-400 mg/day; P5P: 25-75 mg/day (or Pyridoxine HCl: 50-150 mg/day)

3-Methylglutaric 30 , 3-Hydroxyglutaric 31 , 3-Methylglutaconic 32 acids

Carnitine, B21033,1038-1041

Acetyl-L-carnitine or L-carnitine tartrate; Riboflavin

Acetyl-L-carnitine: 500-2,000 mg/day; L-carnitine tartrate: 1-3 g/day; Riboflavin 10-30 mg/day

Ethylmalonic 45 , Methylsuccinic 46 , Adipic 47 , Suberic 48 , Sebacic 49 acids

* Dosages represent typical adult clinical ranges used in mitochondrial and metabolic support protocols. Dosing should be individualized based on patient characteristics, such as age, weight, comorbidities, efficacy, and contraindications, as well as practitioner judgment.

THERAPEUTIC INTERVENTION GRID *See disclaimer on Page 154

Clinical Scenario OAT Pattern Intervention Strategy

Mitochondrial nutrient blend (CoQ10, carnitine+ ALA, Mg, B-complex)

Fatigue, muscle weakness, Mild Cognitive Impairment

Elevated Citric Acid Cycle 24 – 29 + fatty acid 44 – 49 metabolites

Add carnitine + B2, optimize blood sugar using diet strategies, and if able, a Continuous Glucose Monitoring (CGM) device.1042-1044

Hypoglycemia vs hyperglycemia-related symptoms

Elevated glycolic 22 , 23 and ketone 43 ,44 and/or fatty acid

44 – 49 metabolites

Elevated multiple metabolites + history of exposure

Toxin-induced mitochondrial disruption

Mitochondrial repair + toxin binders + detox support (sweating, GI clearance, antioxidants)

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ASSOCIATED PATTERNS FROM OTHER SECTIONS

Influencing Section Marker(s) Mitochondrial Impact

Can produce similar metabolites, impacting elevations.

Microbial Overgrowth

Markers: 1 – 18 ; molds, yeast, and various bacteria

Direct mitochondrial impairment or contributes to associated oxidative stress.

Toxic Exposure

Succinic 24 , Pyroglutamic 58 , 2-Hydroxybutyric 59 acids

Markers associated with low iron, magnesium, amino acids, B vitamins (see nutrient chart)

May generate a higher demand for these nutrients, or insufficiencies can impact the enzymatic reactions for these pathways.

Nutrient Needs

CLINICAL TIPS *See disclaimer on Page 154

• Assess nutrient status before assuming mitochondrial dysfunction — many abnormalities reflect cofactor depletion.

• Consider toxic exposure interference (e.g., mold, heavy metals, environmental toxicants, certain medications) when multiple energy metabolism markers are elevated.

• Evaluate upstream fuel delivery issues, such as blood sugar dysregulation or gut malabsorption, which can impair mitochondrial input.

• Fatty acid and ketone metabolism markers can be early indicators of mitochondrial stress, especially in neurologic or fatigue cases.

• Use a layered support approach: mitochondrial nutrient repletion → reducing toxic burden → metabolic rehabilitation (e.g., gentle exercise, PQQ, NAD+).

TESTING CONSIDERATIONS

Consider the following testing for further insights:

• Metal-Toxic + Nutrient Elements, TOXDetect, and/or MycoTOX: Various toxic exposures can influence several mitochondrial enzymes in addition to contributing to a significant amount of oxidative stress that can impact the health of the mitochondria.

• Amino Acid Profile: Some data suggest, in extreme cases, insufficient amino acids can influence the mitochondrial pathways.

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NEUROTRANSMITTER PATHWAYS

KEY NEUROTRANSMITTER MARKERS

Neurotransmitter Key Marker(s) Interpretive Notes

Dopamine

HVA: dopamine turnover. Low: depletion or impaired synthesis; high: increased breakdown/stimulation.

HVA 33 , DOPAC 36 , HVA/ DOPAC ratio 37

Norepinephrine/ Epinephrine

High VMA: stress response, sympathetic activation. Low: catecholamine depletion or decreased breakdown.

VMA 34 , HVA/VMA ratio 35

Serotonin

Low: precursor depletion, poor synthesis. High: rapid production/metabolism.

5-HIAA 38

Kynurenine Pathway

High quinolinic: neurotoxicity, inflammation, need for NAD+; High kynurenic: neuroprotection.

Quinolinic 39 , Kynurenic 40 acids

COFACTORS & INFLUENCING FACTORS *See Disclaimer on Page 154

Pathway General Dosage Range Guideline* Nutrient Cofactors Clinical Influences

Dopamine/ Norepinephrine Iron (as ferritin repletion, typically 50- 200 mg elemental/day if deficient); P5P: 25–75 mg/day (or pyridoxine HCl: 10-25 mg/day); Vitamin C: 100-200 mg/day; Copper (only if deficient): 5-10 mg/day; Riboflavin (R-5-P or HCl): 10-50 mg/day; Magnesium (glycinate or malate): 300– 400 mg/day; SAMe: 400–1,200 mg/day

Iron, B6, Vit C, Copper, B2, Mg, SAMe671,1045-1053

Stimulants, stress, infection, MAO inhibitors, COMT polymorphisms

Serotonin 5-HTP: 50–300 mg/day (often divided); Pyridoxine HCl: 10-25 mg/day; Folate (L-5-MTHF): 400–1,000 mcg/day; Magnesium (glycinate or malate): 200– 400 mg/day; Riboflavin: 5-15 mg/day; Iron (if deficient): 50-200 mg elemental/day

5HTP, B6, Folate, Mg, B2, Iron1032,1045,1054-1060

SSRIs, inflammation, gut dysbiosis, IDO activation

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Riboflavin: 5-10 mg/day; v: 25–75 mg/ day; Niacinamide: 250–500 mg/day

Kynurenine Pathway (to assist shifting tryptophan towards serotonin instead of Kynurenic)

B2, B6, Niacin (B3)496,1061-1064

Inflammation, stress, infection (e.g. viral, Lyme), chronic illness, upregulation of IDO/TDO

Other supportive nutrients: Zinc, Omega 3s, Rosmarinic acid, Curcumin1065-1072

Other supportive nutrients: Zinc (5-15 mg/day); Omega-3s (EPA+DHA): 1–3 g/day Rosmarinic acid: 200–500 mg/day; Curcumin (500-1500 mg/day)

Dosages represent typical adult clinical ranges used in mitochondrial and metabolic support protocols. Dosing should be individualized based on patient characteristics, such as age, weight, comorbidities, efficacy, and contraindications, as well as practitioner judgment.

ASSOCIATED PATTERNS FROM OTHER SECTIONS

Influencing Section Neurotransmitter Impact Marker(s)

Microbial Overgrowth

Inhibition of dopamine → norepinephrine conversion

Elevated Clostridia markers 15 – 18

Markers associated with heavy metals, solvents, mycotoxins (see Mold, Toxicant, and Heavy Metal charts) and Markers 24 , 58 , 59

Toxic Exposure

Direct neurotoxicity, mitochondrial impairment

Methylation/ Detoxification

Could cause levels to look low, when they could actually be elevated.

Markers 41 , 50 , 51 , 53 , and 59 are associated with methylation Methylation can impact Biopterin and COMT, major cofactors in the generation and metabolism of NTs

Markers associated with low iron, magnesium, amino acids, and B vitamins (see nutrient chart)

Nutrient Needs

Limits neurotransmitter precursor availability and enzymatic activity

CLINICAL TIPS *See Disclaimer on Page 154

• Inflammation drives tryptophan down the kynurenine pathway, potentially raising quinolinic acid.

• 5-HIAA may be low due to precursor issues, high cortisol dampening serotonin synthesis, or excessive dopamine.

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• Review mitochondrial and microbial sections for upstream drivers of neurotransmitter disruption (e.g., Clostridia can inhibit Dopamine Beta Hydroxylase)

TESTING CONSIDERATIONS

Consider the following testing for further insights:

• Amino Acid Profile: Assessing amino acid precursors for neurotransmitter synthesis provides insight into potential limitations or excesses in the pathways governing production.

• DNA Methylation Pathway Profile: If metabolite patterns suggest disruption of methylation, COMT, or MAO pathways, this testing may help identify genetic influences.

• Saliva Hormone Profile: Provides insights into the neuroendocrine influences on certain neurotransmitter metabolites.

• Lyme and Co-infection testing (Lyme Direct Detect, Tickborne BB Direct Detect, and Bartonella IgG Detect): If Quinolinic acid 39 is elevated, and clinical history aligns with potential for Lyme- and co-infections, testing may be warranted.

METHYLATION & DETOXIFICATION

KEY ORGANIC ACID MARKERS

Marker Primary Pathway Clinical Significance

Glutathione recycling

Elevated with glutathione depletion or oxidative stress; common in toxic burden or chronic illness.

Pyroglutamic acid 58

Transsulfuration (CBS path)

Reflects increased demand for glutathione or methylation defects; rises with oxidative stress, methylation impairment, or increased detox demands. Elevated with ammonia overload, urea cycle dysfunction, or methylation impairments.

2-Hydroxybutyric acid 59

Urea cycle & nucleotide synthesis

Orotic acid 60

Glycine conjugation (Phase II)

May indicate xenobiotic exposure or impaired Phase II liver detox; also gut microbiome-influenced.

2-Hydroxyhippuric acid 61

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FUNCTIONAL NUTRIENT & DETOX SUPPORT*See Disclaimer on Page 154

Marker General Dosage Range Guideline* Suggested Support Mechanistic Focus

Pyroglutamic acid 58

NAC, glycine, glutamine, liposomal glutathione740,751,1073-1079

NAC: 600–1,800 mg/day (divided doses); Glycine: 1–3 g/ day; L-Glutamine: 2–5 g/day; Liposomal Glutathione: 250– 500 mg/day

Supports glutathione regeneration; key in redox homeostasis.

2-Hydroxybutyric acid 59

Taurine: 1–3 g/day; L-Methionine: 500–2,000 mg/ day; P5P: 25–75 mg/day (or pyridoxine HCl: 50–150 mg/day); Selenium (selenomethionine or yeast-bound): 100–200 mcg/day

Supports transsulfuration; indicates stress on glutathione demand.

Sulfur amino acids (taurine, methionine), B6, selenium655,1080-1090

L-Citrulline: 2–6 g/day; L-Arginine: 3–6 g/day; L-Ornithine: 1–3 g/day; Riboflavin: 20–100 mg/ day; P5P: 25–75 mg/day; Methylcobalamin: 1,000–5,000 mcg/day

Supports urea cycle and methylation

Citrulline, arginine, methylation support (B2,B6, B12)669,670,782,1086,1091-1096

Orotic acid 60

2-Hydroxyhippuric acid 61

Glycine: 1–3 g/day; Milk thistle extract (80% silymarin): 200– 600 mg/day; Dandelion root extract: 500–1,500 mg/day; Probiotics (multi-strain): 10–50 billion CFU/day

Reflects need for Phase II conjugation or microbial rebalancing.

Glycine, liver detox herbs (milk thistle, dandelion), probiotics75,827,1097-1108

*Dosages reflect typical adult integrative medicine ranges; pediatric dosing should be weight-adjusted. All supplementation should be guided by clinical context, laboratory monitoring, and patient tolerability.

PATTERN INTERVENTION GRID

Pattern on OAT Targeted Support Strategy

↑ Pyroglutamic (58), ↑ 2-Hydroxybutyric (59) acids

NAC or glutathione, Methylation support, mitochondrial antioxidants, sauna/sweating therapy, binders

↑ Orotic acid (60)

Citrulline, arginine, liver support, methylation nutrients

↑ 2- Hydroxybutyric acid (59)

Support Methylation, B6 for CBS enzyme, and may consider Homocysteine panel.

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ASSOCIATED PATTERNS FROM OTHER SECTIONS

Influencing Section Neurotransmitter Impact Marker(s)

Markers associated with heavy metals, toxicants, mycotoxins (see Mold, Toxicant, and Heavy Metal charts) and Markers 24 , 58 , 59

Toxic Exposure

Direct neurotoxicity, mitochondrial impairment

Nutrient Needs

Without proper support for methylation, dysfunction is likely to occur.

Markers 41 , 50 , 51 , and 53 are associated with nutrients that are needed for methylation.

CLINICAL TIPS *See Disclaimer on Page 154

• High pyroglutamate + fatigue → Consider glutathione depletion or oxidative load from toxic exposure or infections.

• 2-hydroxybutyric elevation → Suggests acute glutathione demand, transsulfuration pathway overload, or methylation stress.

• Orotic acid spike → Screen for ammonia overload (especially with fatigue, cognitive impairment); support urea cycle and methylation.

• Elevated 2-hydroxyhippuric → Consider gut dysbiosis or toxic exposure; enhance glycine and Phase II liver detox.

• Combine markers with clinical presentation: e.g., Pyroglutamate + 2-HB in a mold-exposed patient often suggests oxidative + detoxification burden.

TESTING CONSIDERATIONS

Consider the following testing for further insights:

• Amino Acid Profile: Assesses the status of amino acids involved in detoxification, methylation, and the urea cycle.

• DNA Methylation Pathway Panel: Can give insights into genetic SNPs associated with various points in the methylation cycle and transsulfuration pathway.

• Homocysteine: Provides more insight into the function of the methylation and transsulfuration pathways. *This is also on the amino acid profiles (both urine and plasma), but can also be a separate test if needed, particularly for reassessment or monitoring.

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TOXIC EXPOSURE

KEY ORGANIC ACID MARKERS

Organic Acid Marker Potential Toxic Exposure Clinical Insight

Mold-related metabolites (Markers 1 , 2 , 4 , 5 , 6 , 9 )

Mold activity or exposure

May indicate mold overgrowth or mycotoxin exposure.

Toxicants, heavy metals, and/or mycotoxins

Suggests mitochondrial impacts from toxic exposures

Succinic acid 24

Glutathione depletion from toxic burden

Reflects oxidative stress and detox demand

Pyroglutamic acid 58

Oxidative stress from toxic or inflammatory sources

Homocysteine favoring the transsulfuration pathway either to support GSH production or as a result of methylation stress.

2-Hydroxybutyric acid 59

FUNDAMENTAL DETOX STRATEGIES *See Disclaimer on Page 154

1. Reduce ongoing exposure by identifying and eliminating sources (e.g., moldy environments, contaminated water, occupational chemicals) and prioritizing organic foods to reduce pesticide intake, using HEPA air filtration and remove dust to limit airborne pollutants and mold spores, and avoiding plastics, synthetic fragrances, and other common environmental toxic exposures in the home and workplace.1109-1116

2. Support biotransformation pathways, especially glutathione conjugation, glucuronidation, sulfation, and methylation, using nutrients such as NAC, glycine, selenium, B6, B12, folate, and magnesium. More detailed information can be found in the following blog: The Liver: Supportive Nutrients in Detoxification – MosaicDX.

3. Enhance glutathione status via liposomal glutathione, N-acetyl cysteine (NAC), or other precursors like glycine

4. Target mitochondrial repair with CoQ10, acetyl-L-carnitine, alpha-lipoic acid, and B complex.

5. Bind and eliminate toxins and toxicants using agents like activated charcoal, zeolite clay, chlorella, humic and fulvic acids, fibers, or prescription binders (e.g., cholestyramine), depending on toxic exposure type.

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6. Support Phase II detoxification with sulforaphane, calcium-D-glucarate, taurine, and cruciferous vegetables. Additional information can be found in the following blog: The Liver: Its Important Role in Detoxification – MosaicDX

7. Address secondary dysbiosis or fungal overgrowth, which may be exacerbated by toxin- induced immune suppression or antimicrobial activity, using targeted botanicals and gut repair strategies.

8. Replenish key vitamins (e.g. B vitamins, C, etc.) and minerals (e.g., zinc, magnesium, molybdenum) displaced by toxic exposure to restore enzyme function.

9. Consider adjunct therapies such as sauna, lymphatic support, and detox protocols based on individual tolerance and clinical presentation.

CLINICAL TIPS *See Disclaimer on Page 154

• Elevated pyroglutamic acid often signals glutathione depletion—consider supporting glutathione synthesis via NAC, glutathione, or precursors.

• Elevations in succinic acid and related Citric Acid Cycle markers warrant assessment for toxic exposures that influence mitochondria (heavy metals, toxicants, mycotoxins).

• Fungal and Mycotoxin-Linked Markers to Screen for Mold Exposure: Elevated levels of 5-Hydroxymethyl-2-furoic acid, Furan-2,5-dicarboxylic acid, Furancarbonylglycine, Citramalic, Tartaric, and Tricarballylic acid can indicate mold exposure (especially Aspergillus or Fusarium species). Evaluate these in the context of clinical history and potential environmental exposure.

• Identify Environmental and Heavy Metal Toxicity Through Patterned Marker Changes: Look for elevated HVA, VMA, pyruvic, citric, quinolinic, mandelic, hippuric, oxalic, and malonic acids as possible indicators of toxic exposures from heavy metals (arsenic, mercury, cadmium), pesticides, phthalates, solvents, parabens, and combustion byproducts. Assess in conjunction with symptomatology and known or suspected exposure sources.

• Integrate these markers with clinical context—lab abnormalities alone don’t confirm exposure, but guide further investigation.

TESTING CONSIDERATIONS

Consider the following testing for further insights:

• Metal-Toxic + Nutrient Elements, TOXDetect, and/or MycoTOX: Based on clinical presentation, history, and patterns with OAT metabolites, one or all of the toxic exposure testing modalities may be warranted.

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NUTRIENT NEEDS

The following table highlights metabolites linked to specific nutrients and may support clinical interpretation by revealing patterns suggestive of insufficiencies.

NUTRIENT–MARKER REFERENCE TABLE

Nutrient / Category Relevant OAT Marker(s) Interpretation B1 (Thiamin)

Required for pyruvate dehydrogenase; elevation: impaired carbohydrate metabolism, potential thiamin need, or mitochondrial stress.

• Glycolytic markers 22 , 23 • 2-Oxoglutaric acid 27 • 2-Hydroxyhippuric acid 61 • BCKA Metabolites 62 – 66

B2 (Riboflavin)

Needed for acyl-CoA dehydrogenase and glycolate oxidase; elevations in Krebs or fatty acid markers may reflect B2 need.

• Pyruvic acid 23 • Succinic acid 24 , 2-Oxoglutaric acid 27 • HVA 33 , VMA 34 , DOPAC 36 , HVA/DOPAC 37 • Tryptophan metabolites 38 – 40 • Folate metabolites 41 , 42 • Ketone and Fatty Acid markers 45 – 49 • Glutaric acid 53 • Indicators of Detoxification 59 – 60 • Amino Acid metabolites 62 – 66 , 68 – 70

B3 (Niacin)

Niacin is required for NAD+/ NADP+ production; abnormal tryptophan metabolites or Citric Acid Cycle imbalances may indicate B3 insufficiency or inflammation-driven demand.

• Glyceric acid 19 • Glycolytic markers 22 – 23 • Malic acid 26 , 2-Oxoglutaric acid 27 • VMA 33 , HVA/VMA 35 • Quinolinic acid 39 , Kynurenic acid 40 • 3-Hydroxybutyric acid 43 , acetoacetic acid 44 • 2-Hydroxybutyric acid 59 • Amino acid metabolites 62 – 66 , 75

B5 (Pantothenic Acid)

Pantothenic acid is a precursor to CoA; elevations in Krebs or fatty acid markers may suggest impaired energy metabolism and B5 need.

• Hippuric acid 10 • Pyruvic acid 23 • 2-Oxoglutaric acid 27 • Pantothenic acid 52 • BCKA metabolites 62 – 66

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B6 (Pyridoxine)

Cofactor for transamination and neurotransmitter synthesis; elevations in oxalates and neurotransmitter markers may indicate a functional B6 deficiency. Influential in methylation and energy production.

• 2-Hydroxyphenylacetic acid 11 • Oxalate metabolites 20 , 21 • HVA 33 , VMA 34 , DOPAC 36 • Tryptophan metabolites 38 – 40 • Pyrimidine metabolites 41 – 42 • Pyridoxic acid 51 • Indicators of Detoxification 58 , 59 • 2-Hydroxyisocaproic 65 and 2-Oxo-4-methiolbutyric acids 67

B7 (Biotin)

Cofactor for carboxylase enzymes; elevations in methylmalonic or methylcitric acids may reflect biotin deficiency or dysbiosis- related interference.

• Methylmalonic acid 50 , • Methylcitric acid 57

B9 (Folate)

Essential for methylation and nucleotide synthesis; abnormalities in folate metabolites or 2-hydroxybutyric acid may suggest B9 deficiency or MTHFR-related need.

• HVA/DOPAC 37 • Pyrimidine metabolites 41 – 42 • 2-Hydroxybutyric acid 59 • Amino Acid metabolites 68 – 70

B12 (Cobalamin)

Abnormal: functional or genetic B12 deficiency.

• Succinic acid (L) 24 • HVA/DOPAC 37 • Pyrimidine Metabolites 41 – 42 • Methylmalonic acid 50 • Methylcitric acid 57 • Indicators of Detoxification 59 , 60

Vitamin C

Excess can potentially raise oxalates; deficiency may impact immunity and antioxidant defenses.

• Aconitic acid 28 • HVA 33 , VMA 34 , HVA/VMA 35 , DOPAC 36 • Ascorbic acid 54 • Amino Acid Metabolites 68 – 70

CoQ10 (Ubiquinone)

Essential for the electron transport chain; low CoQ10 may impair mitochondrial function.

• 4-hydroxybenzoic acid 12 • Lactic acid 22 • Succinic acid 24 • 3-Hydroxy-3-Methylglutaric acid (CoQ10) 55

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Calcium

Can be depleted by tricarballylic or oxalic acids, while an insufficiency can influence enzymatic function in mitochondrial function. Excess can impair neurotransmitter metabolism.

• Tricarballylic acid 9 • Oxalic acid 21 • 2-Oxoglutaric acid 27 • Citric acid 29 • HVA/DOPAC ratio 37

Magnesium

Needed for ATP-dependent enzymes; deficiency can impair oxalate, Krebs, and ketone metabolism. Also necessary for proper catecholamine metabolism.

• Tricarballylic acid 9 • Oxalic acid 21 • Pyruvic acid 23 • Citric Acid Cycle metabolites 26 , 27 , 29 • HVA 33 , VMA 34 , DOPAC 36 , HVA/DOPAC 37 • 5-HIAA 38 • Ketone and Fatty Acid markers 47 , 48 • Indicators of Detoxification 58 , 60 • Amino Acid metabolites 62 – 65

Zinc

Cofactor for kynureninase; deficiency may shift tryptophan pathway toward quinolinic, zinc can inhibit 27 as well as support reactions.

• Tricarballylic acid 9 • Lactic acid 22 • 2-Oxoglutaric acid 27 • 5-HIAA 38 • Ascorbic acid 54 • Orotic acid 60

Iron

Cofactor in mitochondrial SOD and metabolism of oxalate precursors.

• Oxalic acid 21 • Lactic acid 22 • Mitochondrial – Citric Acid Cycle markers 24 , 25 , 26 , 28 , 29 • HVA 33 , VMA 34 , DOPAC 36 , HVA/ DOPAC 37 • Quinolinic acid 39 • Ascorbic acid 54 • Amino Acid metabolites 68 – 70

Glutathione

Markers of oxidative stress, methylation support; elevation: tglutathione depletion or detox strain.

• Glycolic acid 20 • Aconitic acid 28 , Citric acid 29

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Glycine

Building block of glutathione; elevated pyroglutamic acid could indicate glycine deficiency.

• Hippuric acid 10 , 4-Hydroxyhippuric acid 13 • Pyroglutamic acid 58

Carnitine

• Ketone and Fatty Acid Oxidation markers 45 – 49

Needed for FA transport into mitochondria; elevations suggest carnitine need or mitochondrial block.

Phenylalanine/Tyrosine

Involved in the synthesis of dopamine, norepinephrine, and epinephrine.

• 2-Hydroxyphenylacetic acid 11 • HVA 33 , VMA 34 , DOPAC 36 , Mandelic 68 , Phenyllactic acid 69 , Phenylpyruvic acid 70

Tryptophan

Precursor to these neurotransmitters has the potential to go down the kynurenine pathway vs serotonin. Coenzyme in pyruvate and α-KG dehydrogenase; need suspected if elevations + oxidative stress markers.

• 5-Hydroxyindoleacetic acid (5-HIAA) 38

Quinolinic acid 39 , Kynurenic acid 40

Lipoic Acid

• Pyruvic acid 23 • 2-Oxoglutaric acid 27

CLINICAL TIPS *See Disclaimer on Page 154

• Elevations in 2-hydroxybutyric acid, pyrimidine metabolites, and imbalance in HVA/DOPAC may be related to Vitamin B9 insufficiencies.

• Imbalances in tryptophan metabolites (↑ quinolinic and ↓ kynurenic acids), and mitochondrial dysfunction (Citric Acid Cycle, ketones) → often linked to Vitamin B3 demand.

• Imbalanced fatty acid metabolism markers –> information on carnitine or Vitamin B2 needs

• Abnormal oxalic 21 , mitochondrial and fatty acid metabolites 23 , 26 , 27 , 29 , 47 , 48 , and catecholamine metabolites 33 , 34 , 36 , 37 , 38 may suggest magnesium deficiency.

• Elevations in oxalic acid 21 , lactic acid 22 , Citric Acid Cycle intermediates 24 , 25 , and amino acid markers 68 , 69 , 70 may suggest Iron deficiency.

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TESTING CONSIDERATIONS

Consider the following testing for further insights;

• Omega-3 Index Complete: Offers a more comprehensive review of essential fatty acids that are vital in cellular health, neurological function, regulating inflammation, hormonal imbalance, and many other biological functions.

• Amino Acid Profile: Provides a more complete assessment of amino acids.

• Metals –Toxic + Nutrient Elements: If minerals are suspected to be inadequate based on corresponding markers, may use this testing for further insights.

• Vitamin D: When Phosphoric acid 76 is abnormal, may need further review of Vitamin D status.

• GI360 (with H. Pylori add-ons): Can provide insights into deficiencies that may be related to insufficient digestion or inadequate microbial abundance and/or diversity.

Disclaimer: The content of this Provider Support Guide is for informational purposes only and is not intended to be a substitute for medical advice from a licensed healthcare practitioner. The statements in this report have not been evaluated by the Food and Drug Administration and are intended to be lifestyle choices for potential risk mitigation. Please consult a licensed healthcare practitioner for medication, treatment, diet, exercise or lifestyle management as appropriate. This product is not intended to diagnose, treat, or cure any disease or condition.

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