The uses and limitations of organic acids testing for gut health: a complete guide

Top Posts

The uses and limitations of Organic Acids Tests for gut health: a complete guide

Several popular organic acid tests claim to detect imbalances in gut bacteria and fungi, but most of the “microbial” markers on these tests do not measure what they claim to. Read on to learn more about what these tests can, and cannot, tell you about your gut microbiome.

If you’ve been exploring the online functional medicine space for a while, you’ve probably heard of urine Organic Acids Tests (OAT). In addition to measures of detoxification, cellular metabolism, neurotransmitter balance, and nutrient deficiencies, these tests are suggested to assess the health of the gut microbiome. 

Popular companies such as Genova, Great Plains, and Diagnostic Solutions include sections on their reports for markers of bacterial and yeast overgrowth. But is this actually a reliable way to assess gut health? 

In this article, we break down the basics of organic acids testing, the science behind it, and take a comprehensive look at each of the gut-related markers. 

What is organic acids testing?

An Organic Acids Test (OAT) is a functional medicine test that purports to detect metabolic imbalances and abnormalities in the body. This involves analyzing the levels of small molecules called organic acids in a morning urine sample. 

Organic acids testing is currently offered by companies such as Genova, Great Plains, and Diagnostic Solutions. In addition to markers for bacterial and fungal overgrowth, it also includes markers for detoxification, cellular metabolism, neurotransmitter balance, nutrient deficiencies, and mitochondrial health. 

What is the science behind it?

The science behind OAT is metabolomics, the study of metabolic reactions in the body by way of measuring their byproducts and intermediates, or “metabolites”. Many of these metabolites are organic acids. Using a technique called mass spectrometry combined with liquid chromatography (LC/MS), lab technicians can separate metabolites based on their structure and measure their concentrations.  

Of course, our own human cells are not the sole contributors to the pool of total urine metabolites. The gut microbiome (often regarded as an organ itself with a metabolic activity possibly surpassing the liver) contributes significantly to this pool. 

Metabolomics is becoming an increasingly important part of scientific research on the microbiome. Whereas stool tests can tell us which microbes are present in the gut, metabolomics is an attempt to tell us what the microbes are actually doing

Are organic acids tests legitimate? 

Organic acids testing sounds great in theory – measure the activity of the microbes, and you can get a better idea of their activity in the gut. 

However, several factors complicate their ability to provide a clear clinical picture of the microbiome. Many common OAT “microbial” metabolites are found in the diet and others are generated endogenously (in the body). Still others appear to respond more to age, toxin exposure, or recent dietary intake than the presence of particular microbes in your gut. 

In fact, as you’ll soon see when you read on, very few of the microbial markers on OAT are reliable measures of what they purport to measure.

Who should get an organic acids test? 

Given all of these limitations, organic acids testing has limited use for assessing gut health in most cases. Stool testing currently offers a more reliable method to assess the overall state of the gut microbiome. 

However, a select few OAT markers are rooted in evidence and may be beneficial for specific use cases. In the sections that follow, we’ve outlined what current research says about the most common bacterial and fungal metabolites found on urine organic acids tests. 

If you just want a summary of the most useful markers, feel free to skip to the summary section at the end. 

Bacterial markers:

Markers in this section are purported markers of bacterial dysbiosis, bacterial overgrowth, or bacterial polyphenol metabolism. 

2-Hydroxyphenylacetic Acid (2HPAA)

Popular companies: Genova 

What the company says it indicates: Bacterial dysbiosis

The evidence: 2HPAA has been used historically as a biomarker for phenylketonuria, a rare inherited disorder that reduces the body’s ability to process the amino acid phenylalanine (). It is also a metabolite of the plant compound coumarin, found in cinnamon, strawberries, and apricots, as well as a metabolite of proanthocyanidins, polyphenols found in dark-colored berries, vegetables, and chocolate (). 2HPAA can be found in the urine after coumarin is either consumed or injected, suggesting that it may be a host metabolite in addition to a microbial one (). 

In a comprehensive analysis of blood metabolites, 2HPAA was among the most significantly decreased markers in individuals with non-alcoholic fatty liver disease (). In an open-label pilot study of athletes consuming a fermented soymilk extract for four weeks, during which they participated in one competitive race, urinary 2HPAA excretion decreased by 30 percent compared to baseline (). This suggests the possibility that microbiome changes may have some effect on urinary levels. However, this was not a randomized or placebo-controlled study and the researchers did not collect data on the participants’ dietary intake.

Bottom line: likely influenced by polyphenol consumption and amino acid metabolism and absorption; not a reliable marker of gut bacteria.

3-Hydroxyphenylacetic Acid (3HPAA)

Popular companies: Genova 

What the company says it indicates: Bacterial dysbiosis

The evidence: Like 2HPAA, 3HPAA is an established biomarker for disturbed phenylalanine metabolism (). It is also a well-established bacterial byproduct of polyphenol metabolism and has even been used as a biomarker of polyphenol intake in clinical research (), particularly for wine and grape consumption (). According to studies on laboratory models of human digestion, it is the most abundant bacterial metabolite of both tomato and “pressure-processed onion” (, ). Exercise increases 3HPAA in mice, and rodent research suggests it may contribute to the cardioprotective effects of exercise after a heart attack (). 

In comprehensive profiling of fecal metabolites, low fecal 3HPAA distinguished individuals with colorectal cancer or adenomas from healthy controls (). In a single study of children with autism, oral vancomycin decreased markedly elevated baseline levels of 3HPAA, suggesting that gut populations of vancomycin-sensitive microbes may contribute to pathologically elevated levels ().

Bottom line: likely reflects dietary intake, lifestyle, and phenylalanine metabolism more than bacterial populations.

4-Hydroxybenzoic acid

Popular companies: Great Plains, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: 4HBA is a microbial metabolite of dark-colored polyphenols called anthocyanins. It is found naturally in red wine and in the blood after consuming anthocyanin-rich foods. One study found blood 4HBA levels to correlate positively with vascular function in healthy adults consuming freeze-dried cranberries (). 4HBA is also a metabolite of catechins from green tea, found in human serum and urine after tea consumption, and is the major phenolic compound in coconut (, , ).

A small randomized crossover study of the microbiome effects of drinking wine found that those with the greatest increase in fecal Bifidobacterium also had the greatest increase in urinary 4HBA (). Another study found that lab mice given oral proanthocyanidins only had detectable 4HBA if they were also supplemented with a mixed probiotic containing Bifidobacterium, Lactobacillus, and Akkermansia muciniphila, suggesting that at least some of these taxa may be key metabolizers ().

In 37 patients with IBS, urinary 4HBA increased on a low FODMAP diet and decreased on a high FODMAP diet, and was one of just three metabolites that could distinguish participants between the two groups (). In this study, 4HBA correlated positively with Akkermansia muciniphila, Lachnospira, Eubacterium, and Ruminococcus species. 

Bottom line: this likely beneficial metabolite mostly likely reflects a combination of beneficial gut bacteria and polyphenol-rich plant intake. Optimal ranges are unknown. 

4-Hydroxyhippuric Acid (4HHA)

Popular companies: Great Plains

What the company says it indicates: Bacterial dysbiosis

The evidence: 4HHA is a product of liver phase II detoxification of 4HBA, discussed above, which is conjugated to glycine to form 4HHA (). 4HHA has received far less attention in research than 4HBA, but their microbial and dietary origin is the same. Theoretically, the sum of 4HHA and 4HBA may better reflect polyphenol intake and polyphenol-metabolizing microbes, since the rate of liver detoxification can vary between people based on environmental and genetic factors. 

Athletes consuming a lactic acid bacteria-fermented soymilk extract and participating in a competitive event had a 3-fold reduction in urinary 4HHA without any change in 4HBA, suggesting diet and/or exercise may directly shift liver 4HBA metabolism, or there may be additional underexplored microbial origins of 4HHA ().

Bottom line: should be considered similarly to 4HBA until more research is conducted. Optimal ranges are unknown.

Benzoic Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: Benzoic acid is a microbial product of phenylalanine (an amino acid) and polyphenol metabolism (, ). Recent genetic analysis of gut microbiome samples suggests benzoic acid production is spread across the Firmicutes, Actinobacteria, and Proteobacteria phyla (3). Benzoic acid can be found naturally in some foods, including berries and milk products (4). It is also a component of sodium benzoate, a common preservative.

Benzoic acid is largely metabolized in the liver to hippuric acid (discussed below), which is excreted in far larger quantities in urine (20-200 times higher). Urinary benzoic acid excretion declines dramatically with age regardless of polyphenol intake. However, microbial benzoic acid production likely increases as the simultaneous increase in urinary hippuric acid is 10-50 times greater than the decrease in urinary benzoic acid (). 

Bottom line: likely reflects a combination of diverse dietary inputs and metabolism by a wide range of gut bacteria. May reflect age more than anything else. 

Hippuric Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: Hippuric acid is primarily derived from the conjugation of benzoic acid (discussed above) with glycine in the liver (3). It is also the final product of the detoxification of toluene, a toxin found in paint thinners (which is also metabolized to benzoic acid in the process), and has been used to monitor toluene exposure in occupationally exposed workers (). Urinary hippuric acid has also been used to estimate fruit and vegetable intake in observational research (), and has also been proposed as a biomarker for aging, as it significantly increases with age regardless of fruit and vegetable intake (). 

High urinary hippuric acid has been positively associated with metabolic health and increased gut microbial diversity in middle-aged adults in Denmark (3). Hippuric acid has also been found to be markedly reduced in Crohn’s disease, which may be a result of reduced microbial benzoic acid production rather than reduced conjugation in the liver ().

Bottom line: like its precursor benzoic acid, hippuric acid is influenced by polyphenol and preservative intake, phenylalanine absorption, toxic exposures, and age. 

4-Hydroxyphenylacetic Acid (4HPAA)

Popular companies: Genova, Great Plains, Diagnostic Solutions

What the companies say it indicates: Bacterial dysbiosis; C. difficile, C. stricklandii, C. lituseburense & others 

The evidence: This close relative of 3HPAA (discussed above) is one of the primary microbial metabolites of resveratrol, an antioxidant known for occurring at high levels in red wine (). 4HPAA also increases when you eat strawberries () and can result from the bacterial metabolism of the amino acid tyrosine  (). 

Interestingly, in one study, 4HPAA was among the most significantly increased blood metabolites in individuals with ulcerative colitis (). On the other hand, a recent study found that 4HPAA alone was sufficient to reduce diet-induced cardiometabolic disease in mice (). No studies have directly correlated urinary levels with bacterial populations.

Bottom line: likely reflects dietary polyphenol and amino acid intake more than bacterial composition. More research is needed to clarify its health effects.

Dihydroxyphenylpropionic Acid (DHPPA)

Popular companies: Genova, Great Plains

What the companies say it indicates: Bacterial dysbiosis or beneficial bacteria (depending on the company!)

The evidence: In the past decade, DHPPA has emerged as a validated and objective marker of dietary whole-grain wheat and rye intake (, ). While DHPPA is a microbial metabolite and therefore theoretically impacted by gut microbiome composition, the effect appears to be negligible compared with the impact of diet. Therapeutically, DHPPA protects against liver injury in rats ().

Bottom line: a marker of whole grain intake, not microbial populations.

HPHPA (3-hydroxyphenyl-3-hydroxypropionic acid)

Popular companies: Great Plains

What the company says it indicates: Clostridium spp. (C. sporogenes, C. caloritolerans, C. botulinum & others)

The evidence: HPHPA has frequently been identified as an elevated urinary marker in autism spectrum disorders, as well as schizophrenia (, , ). Treatment of autistic children with the broad-spectrum antibiotic vancomycin dramatically reduced urinary HPHPA, suggesting a strong microbial link (). While HPHPA is often suggested to be a biomarker of Clostridium species, we were unable to find any evidence to support this.

Bottom line: influenced by the microbiome and elevated in psychiatric disorders. No evidence to support this as a marker of Clostridium overgrowth.

Phenylacetic Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Phenylalanine malabsorption

The evidence: Phenylacetic acid is a product of the microbial breakdown of the amino acid phenylalanine. Preliminary research suggests that predominant metabolizers include Bacteroides species, Eubacterium halii, and some Clostridium (). Like indoleacetic acid, it is also a natural signaling molecule in plants (). 

Increasing dietary protein intake increases fecal phenylacetic acid in human volunteers, regardless of the levels of carbohydrate and fat in the diet (). Acid-blocking drugs also significantly increase fecal phenylacetic acid, perhaps related to a reduction in protein absorption (). Another study found that increasing dietary protein increased urinary phenylacetic acid, but only in mice, not in humans (). Together, this suggests phenylacetic acid is likely metabolized further in the human liver. 

Blood levels of phenylacetic acid rise dramatically after consuming curry spices, suggesting it is also a polyphenol metabolite (). Additionally, fecal transplant from a healthy lean donor to obese adults significantly decreased fecal phenylacetic acid at three months after transplant. This corresponded with an increase in Bacteroides and Blautia, suggesting phenylacetic acid is also sensitive to microbiome changes (). 

Multiple early studies indicated that individuals with depressive symptoms excrete lower amounts of phenylacetic acid in urine than healthy controls (). These results have not been replicated in several decades, nor correlated with microbial populations.

Bottom line: likely reflects dietary protein intake, protein absorption, and spice consumption, as well as bacterial populations. Urinary output may be influenced by an unknown breakdown pathway in the liver.

Indoleacetic Acid (3-Indole acetic acid, Indole-3-acetic acid, IAA)

Popular companies: Genova, Great Plains, Diagnostic Solutions

What the companies say it indicates: Tryptophan malabsorption (Genova), Clostridium spp. (Great Plains)

The evidence: IAA is derived from tryptophan metabolism by gut bacteria. Preliminary research suggests IAA-producers include Clostridium (), Bacteroides, Alistipes, Prevotellaceae, () and yeasts (). Research on celiac disease patients has found that those experiencing malabsorption symptoms such as steatorrhea have higher baseline urinary IAA and a greater increase in levels after consuming 4 grams of tryptophan ().  IAA is also naturally found in many edible plants; however, whether this contributes appreciably to urinary levels is unclear (). Conventionally, indoleacetic acid is considered a uremic toxin – one of several nitrogen-containing compounds that build up in the blood when kidney function is compromised ().

Recently, lab research has identified numerous therapeutic benefits of indoleacetic acid, alleviating pathologies as diverse as depression, non-alcoholic fatty liver disease, and ankylosing spondylitis in mice, while significantly modulating the microbiome ( , ). Feeding sulforaphane to mice on a high-fat, high-sugar diet restored depleted blood levels of IAA, likely by upregulating IAA-producing species (). At least some of IAA’s therapeutic benefit may be because it is a potent stimulator of the aryl hydrocarbon receptor (AhR) (); at the same time, other research suggests AhR activation may be responsible for its uremic toxicity in chronic kidney disease (). Despite significant research on this organic acid, urinary levels have not been correlated with specific gut microbes or microbiota patterns.

Bottom line: likely reflects protein malabsorption as well as bacteria and fungal metabolism. Likely beneficial at moderate levels and pathogenic at very high levels, though little research exists to establish a healthy urinary reference range.

p-Cresol (4-Cresol)

Popular companies: Great Plains

What the company says it indicates: Clostridioides difficile (previously named Clostridium difficile) overgrowth

The evidence: p-cresol is a microbial metabolite of tyrosine, produced by many gut microbes but in particularly large quantities by C. difficile (). Proton pump inhibitor (PPI) treatment was shown to increase urinary p-cresol by reducing protein absorption, suggesting that this metabolite may also be influenced by the degree to which protein is digested (). 

A broad metabolomic study identified p-cresol as one of three serum metabolites inversely correlated with type II diabetes risk. The researchers further found that giving p-cresol to mice with cardiometabolic disease reduced body fat, glucose intolerance, and fatty liver, while increasing the function of insulin-secreting pancreatic beta cells ().

On the other hand, p-cresol has been found to be elevated in children with autism (), and administering a dose 100 times higher than in the cardiometabolic study mentioned above to healthy mice induced autistic-like behavior. This effect was reversible with microbiome transplantation (). None of these studies correlated p-cresol levels to levels of C. difficile or other gut bacteria. 

Notably, a sulfate group is attached to p-cresol in the body to form p-cresyl sulfate, a uremic toxin that is known to accumulate in chronic kidney disease due to impaired excretion and has potentially cancerous effects (). Research has scarcely measured both p-cresol and p-cresyl sulfate in the same human subjects, so it is possible that the rate of sulfation may influence p-cresol’s apparent bidirectional effects.

Various microbial products and diets have been shown to influence p-cresol concentrations. Lactic acid bacteria-fermented soy extract, wheat bran extract, and a raw vegan diet have all been shown to significantly reduce urinary p-cresol in various studies (). Similarly, yeast mannan extract () and a low-fat diet have also been shown to reduce fecal p-cresol (). On the other hand, in a subgroup from the well-known PREDIMED trial, increased p-cresol was one of a group of three urinary metabolites that could distinguish people with high adherence to a Mediterranean diet with 97 percent accuracy ().

Bottom line: an impactful bacterial metabolite derived from diverse sources. Further research is needed to clarify its effects and optimal urinary ranges.

Yeast/fungal markers

Markers included in this section are purported markers of fungal dysbiosis or overgrowth.

3-Oxoglutaric Acid

Popular companies: Great Plains

What the company says it indicates:  Fungal overgrowth

The evidence: This organic acid is not known to be produced by the body, found in the diet, or associated with the consumption of specific foods, so it has promise as a microbial indicator. However, there is no evidence for the significance of high or low levels. 

Pharmaceutically, 3-oxoglutaric acid has anti-cancer properties (). One open-label pilot study found that endurance athletes had reduced urinary excretion of 3-oxoglutaric acid after consuming a lactic acid bacteria-fermented soybean extract(). Another study found that 3-oxoglutaric acid was decreased in a cohort of children in China with autism, a condition known to be associated with perturbed gut fungi (). 

Bottom line: likely of microbial origin, but has not been well-established as a bacterial or fungal marker.

5-Hydroxymethyl-2-furoic Acid (HMFA)

Popular companies: Great Plains, Diagnostic Solutions

What the company says it indicates: Aspergillus overgrowth

The evidence: Aspergillus does produce this organic acid. However, Aspergillus species are not considered true residents of the human gut and are instead thought to be transient from dietary and environmental sources (). HMFA is also a natural constituent of coffee (). Furthermore, organic acid tests may not properly distinguish HMFA from its close relative, 5-hydroxymethyl-2-furfural, which is increased in urine after dried plum consumption (). 

One study found that a three-month diet and exercise program increased fasting blood levels of HMFA by 20 percent in obese women (). While the study lacked a placebo group, the urine tests both before and after the intervention were taken after participants consumed a strict control diet for a week, suggesting that the change can likely be attributed to shifts in the microbiome. However, gut microbes were not directly assessed in this study.

Bottom line: may indicate dietary intake as much as gut microbes; which microbes it may indicate is currently unclear.

Furan-2,5-dicarboxylic Acid (FDCA)

Popular companies: Great Plains

What the company says it indicates: Aspergillus overgrowth

The evidence: In addition to microbial sources, FDCA is formed from the oxidation of 5-hydroxymethyl-2-furfural, the dietary constituent discussed above. A three-month diet and exercise program increased fasting blood levels of FDCA by 10 percent in obese women (). This change was likely entirely attributable to bacterial shifts rather than diet because the post-intervention test was taken after both groups consumed an identical standardized diet for one week. No research has correlated FDCA with specific gut bacteria or fungi.

Bottom line: limited research suggests modestly lower levels may indicate a metabolically healthy microbiome overall; however, correlations with specific taxa cannot be made.

Carboxycitric Acid

Popular companies: Great Plains

What the company says it indicates: Fungal overgrowth

The evidence: In one recent study, urinary carboxycitric acid was significantly reduced in children with autism compared with healthy controls; however, the researchers suggest this may reflect increased uptake into the brain rather than decreased gut microbial production (). 

In young athletes, urinary carboxycitric acid positively correlated with the proportion of fecal Firmicutes (). However, this was not a randomized or placebo-controlled study and the researchers did not collect data on the participants’ dietary intake.

Bottom line: may reflect both fungi and bacteria; however, its significance is unclear.

Citramalic Acid

Popular companies: Genova, Great Plains

What the company says it indicates: Fungal dysbiosis/overgrowth

The evidence: Citramalic acid is synthesized by many different bacteria in addition to Aspergillus species. It is also a natural constituent in apples, sugar beets, tomatoes, and other fruits (, , ). 

In elderly patients with chronic heart failure, increased fecal Klebsiella correlated negatively with blood levels of citramalic acid (). Conversely, citramalic acid has been found to be significantly elevated in serum in individuals with diabetic kidney disease, a pathology commonly associated with microbiota disturbances ().

Bottom line: may reflect generalized microbiome imbalances when elevated or depressed, but with unclear significance. Also influenced by fruit consumption.

Furancarbonylglycine

Popular companies: Great Plains

What the company says it indicates: Aspergillus overgrowth

The evidence: Extensive database searches did not turn up a single study mentioning this metabolite (If you find one, let us know!)

Bottom line: ignore this marker.

Tartaric Acid

Popular companies: Genova, Great Plains

What the company says it indicates: Fungal dysbiosis; Aspergillus overgrowth

The evidence: Urinary tartaric acid has been suggested as a biomarker of red wine intake, and when used in this way, has been correlated with lower LDL cholesterol (). Genova notes that chocolate intake may increase levels as well. 

No studies to date have provided direct evidence of a fungal origin for tartaric acid. One study in young athletes found that a reduction in tartaric acid levels paralleled reductions in other purported gut fungal markers and positively correlated with the proportion of Firmicutes (). However, the athletes in this open-label study consumed a fermented soymilk extract and competed in a race event, which confound the interpretation of these results. Urinary tartaric acid has also been found significantly reduced in children with autism ().

Bottom line: likely indicates wine and other polyphenol consumption; no direct evidence of fungal origin. 

Tricarballylic Acid

Popular companies: Great Plains

What the company says it indicates: Fusarium overgrowth

The evidence: Tricarballylic acid is a microbial derivative of dietary aconitate and citrate, found in diverse plant foods (). Fusarium species residing on corn have indeed been found to produce tricarballylic esters (9); however, so do many gut-resident microbes in rats (Fusarium species are considered transient and do not typically colonize the human gut. 

Accordingly, no evidence exists that urinary excretion in humans represents gastrointestinal overgrowth. In fact, research suggests it could represent beneficial shifts in the microbiome. A three-month diet and exercise program markedly increased the blood levels of tricarballylic acid in obese women during an oral glucose tolerance test, taken under diet-controlled conditions both before and after the intervention (). 

Recently, a large study found reduced tricarballylic acid among children with autism (). Another study found that endurance athletes consuming a lactic acid bacteria-fermented soybean extract had reduced urinary excretion of tricarballylic acid (). However, as mentioned previously, this was not a randomized or placebo-controlled study.

Bottom line: produced by many bacteria in addition to Fusarium species. More research is needed on this microbial metabolite, which may be beneficial rather than dysbiotic.

Arabinose

Popular companies: Great Plains

What the company says it indicates: Candida overgrowth

The evidence: Compared with its relative D-arabinitol, discussed below, arabinose is a much broader metabolite with more diverse endogenous and microbial sources. One study found that reductions in urinary arabinose paralleled other purported markers of fungal overgrowth (3-oxoglutaric acid, tartaric acid, tricarballylic acid), but gut fungi were not directly assessed (). A case report found similar parallel elevations in a pair of brothers with autism, also without fungal stool analysis ().

Bottom line: D-arabinitol is the better evidence-based alternative.

D-Arabinitol (DA)

Popular companies: Genova (D-arabinitol), Diagnostic Solutions (Arabinitol)

What the company says it indicates: Candida overgrowth

The evidence: Over a decade ago, a comprehensive review determined DA to be the only urinary marker of Candida overgrowth with substantial scientific evidence (). Today, this still appears to be the case. However, most research on DA has been in systemic and invasive candidiasis, more serious infections common in immunocompromised individuals. While this has helped establish DA as a marker of Candida, it provides little insight into how urinary levels might reflect gastrointestinal colonization or overgrowth. No studies to date have linked DA levels to fecal levels of Candida

Many Candida species have been found to produce DA, including C. albicans, C. parapsilosis, C. tropicalis, and C. guillermondii (). C. glabrata and C. krusei do not produce DA in vitro, though infections with C. glabrata have been associated with high urinary levels. While no human synthetic pathway has been identified, 10-fold higher levels of DA have been found in human cerebrospinal fluid versus blood, suggesting the nervous system may contribute to urinary excretion, albeit nominally (). On the other hand, human tissues readily metabolize the isomer L-arabinitol (LA) from dietary sugar alcohols (). L-arabinitol is also produced by some Aspergillus and Saccharomyces species (, ).

In a study comparing serum DA in 61 urology patients with and without candiduria (meaning live Candida species were isolated from their urine), only patients with candiduria and a high fever had elevated DA (7). Similar results were replicated in a study three years later (). However, the average DA levels among the participants without fevers were between 68-95mmol/mol creatinine, more than twice Genova’s optimal cutoff of 36. (This is an imperfect comparison – these studies use serum while Genova uses urine, but the ratio between arabinitol and creatinine has been found to be consistently similar between the two ().) Indeed, both the control and feverless candiduria patients had recently undergone major surgery and taken antibiotics, suggesting even those without candiduria may have had gastrointestinal Candida overgrowth. DA elevations as high as 192mmol/mol creatinine have been attributed to non-invasive GI Candida (0).

While little data exists on healthy levels, non-hospitalized healthy controls had an average serum DA of 18±6.7 mmol/mol creatinine in one study (), while another study found averages of 45±26 in men and 59±33 in women (). However, these control populations did not have strict inclusion criteria, which may explain the wide difference between these “normal” levels. For invasive candidiasis, DA cutoffs between 160-440 mmol/mol creatinine have been proposed; however, serum levels in diagnosed patients vary between 60-2200 in the published literature (, ). As the studies discussed in the previous paragraph suggest, the clearest elevations may only occur in the context of a fever. The evidence to date suggests, however, that DA levels are unlikely to distinguish invasive candidiasis specifically. Nonetheless, elevated DA likely reflects total Candida burden, whether invasive or not.

In a single-arm clinical study of 22 children with autism and GI disorders, the well-researched probiotic strain Lactobacillus rhamnosus Rosell-11 reduced urinary DA from 160 to 89.5 mmol/mol creatinine, which corresponded with an increased ability of the children to concentrate (). Autism has been associated with higher fecal levels of Candida (), and many Lactobacillus species demonstrate anti-Candida activity in vitro (). Once again, the DA levels in this study are far higher than Genova’s cutoff, suggesting that monitoring changes in your values over time may be more relevant than absolute levels.

The most recent research, largely in pediatric populations, has focused on the DA:LA ratio as a more sensitive and specific marker of candidiasis than DA alone (, ). Unfortunately, DA:LA is not currently offered on commercially available urinary organic acid tests.

Bottom line: D-arabinitol is a well-established metabolite of Candida species strongly elevated during fever-inducing invasive candidiasis or extreme gastrointestinal Candida colonization. At lower levels, however, it is unclear what value distinguishes normal DA from Candida overgrowth. Test results should be interpreted in context clinically. Undifferentiated “Arabinitol” (as on the Diagnostic Solutions OMX test), on the other hand, is useless.

Other microbial metabolites:

Markers included in this section are not specific to bacteria or fungi but are purported markers of microbial metabolism.

Equol

Popular companies: Diagnostic Solutions

What the company says it indicates: Bacterial isoflavone metabolism

The evidence: Equol is a specific bacterial metabolite of the isoflavone daidzein, found in soy and soy products. Daidzein metabolism is relatively rare in the human gut microbiome. More than half of people ostensibly lack equol-producing microbes and do not excrete equol regardless of soy intake. Equol production is best characterized in two species from the low abundance gut resident genus Slackia: S. isoflavoniconvertens and S. equolifaciens (100, 101Adlercreutzia equolifaciens, Adlercreutzia hattori, Asaccharobacter celatus, Bacteroides ovatus, Finegoldia magna, Lactobacillus mucosae, and Streptococcus intermedius have been identified as equol producers (102, 103). However, in a large observational study of over 1,000 participants in Japan, many of these species were present in fecal samples in similar amounts regardless of equol excretion (102). This suggests equol production may be a strain-specific trait, which is supported by recent laboratory research (104). Equol production is a two-step process, which has also been demonstrated to take place collaboratively between multiple species incapable of producing equol on their own (105). The Japanese study also found that equol excretors had 17 percent higher soy intake than non-excretors, suggesting daidzein intake may support the persistence of equol producers in the gut (102). Other dietary components may influence equol production from daidzein as well – a greater proportion of vegetarians than omnivores are equol excretors (105). A yeast mannan supplement significantly increased urinary equol after an isoflavone challenge in a randomized controlled trial in healthy adult women ().

However, whether or not metabolizing daidzein to equol is necessary to maximize the health-promoting benefits of soy isoflavones is controversial. A systematic review of 42 studies found that two-thirds of clinical trials studying the effect of soy isoflavones on coronary heart disease risk factors found no increased benefit in equol excretors versus non-excretors (106). On the other hand, oral equol supplements have been found in numerous clinical studies to reduce hot flashes in menopausal women (107), and an observational study found that high daidzein intake correlated with lower vasomotor symptoms only in equol excretors (108). However, supplementing soy isoflavones does not have a greater benefit for menopausal symptoms in equol excretors (107). Two recent observational studies found a lower risk for non-alcoholic fatty liver or less severe disease in equol excretors, however, one found a significant effect only in men and the other only in women (109, 110).

You are unlikely to have significant urinary equol if you haven’t eaten soy in the last few days whether or not you have the gut bacteria that produce it, so if you want to know your equol status be sure to eat some soy before your urine test (111).

Bottom line: urinary equol is the best way to tell if you have the gut bacteria that produce it, as long as you’ve eaten soy within a few days of your test. It is possible that equol excretors have an enhanced therapeutic benefit from eating soy products, though results from clinical research is mixed.

3,4-Dihydroxyhydrocinnamic Acid

Popular companies: Diagnostic Solutions

What the company says it indicates: Microbial polyphenol metabolism

The evidence: Despite being listed in some published studies as a dysbiosis marker (112), extensive database searches did not turn up a single study connecting this metabolite to microbial populations or even to diet.

Bottom line: not a reliable marker

3,5-Dihydroxybenzoic Acid (3,5-DHBA)

Popular companies: Diagnostic Solutions

What the company says it indicates: Microbial polyphenol metabolism

The evidence: 3,5-DHBA is both a naturally-occurring phytonutrient in apples, honey, peanuts, chickpeas, nettle, and dandelion, as well as a microbial metabolite of alkylresorcinols found primarily in wheat, barley, and rye (113). This organic acid is highly bioactive in humans, stimulating the lactate receptor HCAR1. It is unclear whether the analytical methods used by popular tests can unambiguously distinguish 3,5-DHBA from its common isomers pyrocatechuic acid (2,3-DHBA) and gentisic acid (2,5-DHBA) among others, which have vastly different origins and physiological significance. Regardless, there is no evidence to suggest that 3,5-DHBA is an indicator of gut microbes.

Bottom line: likely a highly non-specific marker of dietary intake. Ignore this marker.

Summary and conclusions

In conclusion, while metabolomics is a promising and growing area of research, the majority of microbial markers on popular urine Organic Acids Tests are not reliable markers of gut dysbiosis or overgrowth. Most are influenced or confounded by diet, effects of aging, toxin exposure, and/or are produced by a diverse range of microbes. 

Only a select few OAT markers are true microbial metabolites and show some promise to guide clinical practice:

  • D-arabinitol (offered on the Genova Organix test) is a well-established marker of Candida. Levels upwards of 160 mmol/creatine may indicate invasive Candidiasis or a high total Candida burden. More modest elevations may indicate GI overgrowth, but current research is unable to distinguish normal commensal levels of DA from overgrowth. Results should be interpreted in the context of signs and symptoms. For more on Candida testing and treatment, stay tuned for our upcoming course on gut fungi, yeast overgrowth, and the mycobiome!
  • P-cresol and indoleacetic acid, microbial metabolites of the amino acids tyrosine and tryptophan, respectively, also show some potential for clinical use, but more research is needed to fully understand their nuanced health effects and optimal concentrations. While significant elevations of p-cresol may indicate C. difficile overgrowth, this would still be better looked at with a stool test, as a variety of microbes can produce this metabolite. 

Given the significant limitations discussed here, most people would not benefit from a urine Organic Acids Test to assess gut health. Comprehensive stool testing, along with signs and symptoms, offers a much more reliable way to assess the current state of your gut health. 

Have you had an organic acids test? Did you find this article helpful? Share your thoughts or experience in the comments below!

Want to support this blog? 

If you appreciated this article, please consider joining my Patreon community! Patrons get perks like bonus research content, discounts on my online courses, and access to member-only Q&A sessions.

The uses and limitations of Organic Acids Tests for gut health: a complete guide

Several popular organic acid tests claim to detect imbalances in gut bacteria and fungi, but most of the “microbial” markers on these tests do not measure what they claim to. Read on to learn more about what these tests can, and cannot, tell you about your gut microbiome.

If you’ve been exploring the online functional medicine space for a while, you’ve probably heard of urine Organic Acids Tests (OAT). In addition to measures of detoxification, cellular metabolism, neurotransmitter balance, and nutrient deficiencies, these tests are suggested to assess the health of the gut microbiome. 

Popular companies such as Genova, Great Plains, and Diagnostic Solutions include sections on their reports for markers of bacterial and yeast overgrowth. But is this actually a reliable way to assess gut health? 

In this article, we break down the basics of organic acids testing, the science behind it, and take a comprehensive look at each of the gut-related markers. 

What is organic acids testing?

An Organic Acids Test (OAT) is a functional medicine test that purports to detect metabolic imbalances and abnormalities in the body. This involves analyzing the levels of small molecules called organic acids in a morning urine sample. 

Organic acids testing is currently offered by companies such as Genova, Great Plains, and Diagnostic Solutions. In addition to markers for bacterial and fungal overgrowth, it also includes markers for detoxification, cellular metabolism, neurotransmitter balance, nutrient deficiencies, and mitochondrial health. 

What is the science behind it?

The science behind OAT is metabolomics, the study of metabolic reactions in the body by way of measuring their byproducts and intermediates, or “metabolites”. Many of these metabolites are organic acids. Using a technique called mass spectrometry combined with liquid chromatography (LC/MS), lab technicians can separate metabolites based on their structure and measure their concentrations.  

Of course, our own human cells are not the sole contributors to the pool of total urine metabolites. The gut microbiome (often regarded as an organ itself with a metabolic activity possibly surpassing the liver) contributes significantly to this pool. 

Metabolomics is becoming an increasingly important part of scientific research on the microbiome. Whereas stool tests can tell us which microbes are present in the gut, metabolomics is an attempt to tell us what the microbes are actually doing

Are organic acids tests legitimate? 

Organic acids testing sounds great in theory – measure the activity of the microbes, and you can get a better idea of their activity in the gut. 

However, several factors complicate their ability to provide a clear clinical picture of the microbiome. Many common OAT “microbial” metabolites are found in the diet and others are generated endogenously (in the body). Still others appear to respond more to age, toxin exposure, or recent dietary intake than the presence of particular microbes in your gut. 

In fact, as you’ll soon see when you read on, very few of the microbial markers on OAT are reliable measures of what they purport to measure.

Who should get an organic acids test? 

Given all of these limitations, organic acids testing has limited use for assessing gut health in most cases. Stool testing currently offers a more reliable method to assess the overall state of the gut microbiome. 

However, a select few OAT markers are rooted in evidence and may be beneficial for specific use cases. In the sections that follow, we’ve outlined what current research says about the most common bacterial and fungal metabolites found on urine organic acids tests. 

If you just want a summary of the most useful markers, feel free to skip to the summary section at the end. 

Bacterial markers:

Markers in this section are purported markers of bacterial dysbiosis, bacterial overgrowth, or bacterial polyphenol metabolism. 

2-Hydroxyphenylacetic Acid (2HPAA)

Popular companies: Genova 

What the company says it indicates: Bacterial dysbiosis

The evidence: 2HPAA has been used historically as a biomarker for phenylketonuria, a rare inherited disorder that reduces the body’s ability to process the amino acid phenylalanine (). It is also a metabolite of the plant compound coumarin, found in cinnamon, strawberries, and apricots, as well as a metabolite of proanthocyanidins, polyphenols found in dark-colored berries, vegetables, and chocolate (). 2HPAA can be found in the urine after coumarin is either consumed or injected, suggesting that it may be a host metabolite in addition to a microbial one (). 

In a comprehensive analysis of blood metabolites, 2HPAA was among the most significantly decreased markers in individuals with non-alcoholic fatty liver disease (). In an open-label pilot study of athletes consuming a fermented soymilk extract for four weeks, during which they participated in one competitive race, urinary 2HPAA excretion decreased by 30 percent compared to baseline (). This suggests the possibility that microbiome changes may have some effect on urinary levels. However, this was not a randomized or placebo-controlled study and the researchers did not collect data on the participants’ dietary intake.

Bottom line: likely influenced by polyphenol consumption and amino acid metabolism and absorption; not a reliable marker of gut bacteria.

3-Hydroxyphenylacetic Acid (3HPAA)

Popular companies: Genova 

What the company says it indicates: Bacterial dysbiosis

The evidence: Like 2HPAA, 3HPAA is an established biomarker for disturbed phenylalanine metabolism (). It is also a well-established bacterial byproduct of polyphenol metabolism and has even been used as a biomarker of polyphenol intake in clinical research (), particularly for wine and grape consumption (). According to studies on laboratory models of human digestion, it is the most abundant bacterial metabolite of both tomato and “pressure-processed onion” (, ). Exercise increases 3HPAA in mice, and rodent research suggests it may contribute to the cardioprotective effects of exercise after a heart attack (). 

In comprehensive profiling of fecal metabolites, low fecal 3HPAA distinguished individuals with colorectal cancer or adenomas from healthy controls (). In a single study of children with autism, oral vancomycin decreased markedly elevated baseline levels of 3HPAA, suggesting that gut populations of vancomycin-sensitive microbes may contribute to pathologically elevated levels ().

Bottom line: likely reflects dietary intake, lifestyle, and phenylalanine metabolism more than bacterial populations.

4-Hydroxybenzoic acid

Popular companies: Great Plains, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: 4HBA is a microbial metabolite of dark-colored polyphenols called anthocyanins. It is found naturally in red wine and in the blood after consuming anthocyanin-rich foods. One study found blood 4HBA levels to correlate positively with vascular function in healthy adults consuming freeze-dried cranberries (). 4HBA is also a metabolite of catechins from green tea, found in human serum and urine after tea consumption, and is the major phenolic compound in coconut (, , ).

A small randomized crossover study of the microbiome effects of drinking wine found that those with the greatest increase in fecal Bifidobacterium also had the greatest increase in urinary 4HBA (). Another study found that lab mice given oral proanthocyanidins only had detectable 4HBA if they were also supplemented with a mixed probiotic containing Bifidobacterium, Lactobacillus, and Akkermansia muciniphila, suggesting that at least some of these taxa may be key metabolizers ().

In 37 patients with IBS, urinary 4HBA increased on a low FODMAP diet and decreased on a high FODMAP diet, and was one of just three metabolites that could distinguish participants between the two groups (). In this study, 4HBA correlated positively with Akkermansia muciniphila, Lachnospira, Eubacterium, and Ruminococcus species. 

Bottom line: this likely beneficial metabolite mostly likely reflects a combination of beneficial gut bacteria and polyphenol-rich plant intake. Optimal ranges are unknown. 

4-Hydroxyhippuric Acid (4HHA)

Popular companies: Great Plains

What the company says it indicates: Bacterial dysbiosis

The evidence: 4HHA is a product of liver phase II detoxification of 4HBA, discussed above, which is conjugated to glycine to form 4HHA (). 4HHA has received far less attention in research than 4HBA, but their microbial and dietary origin is the same. Theoretically, the sum of 4HHA and 4HBA may better reflect polyphenol intake and polyphenol-metabolizing microbes, since the rate of liver detoxification can vary between people based on environmental and genetic factors. 

Athletes consuming a lactic acid bacteria-fermented soymilk extract and participating in a competitive event had a 3-fold reduction in urinary 4HHA without any change in 4HBA, suggesting diet and/or exercise may directly shift liver 4HBA metabolism, or there may be additional underexplored microbial origins of 4HHA ().

Bottom line: should be considered similarly to 4HBA until more research is conducted. Optimal ranges are unknown.

Benzoic Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: Benzoic acid is a microbial product of phenylalanine (an amino acid) and polyphenol metabolism (, ). Recent genetic analysis of gut microbiome samples suggests benzoic acid production is spread across the Firmicutes, Actinobacteria, and Proteobacteria phyla (3). Benzoic acid can be found naturally in some foods, including berries and milk products (4). It is also a component of sodium benzoate, a common preservative.

Benzoic acid is largely metabolized in the liver to hippuric acid (discussed below), which is excreted in far larger quantities in urine (20-200 times higher). Urinary benzoic acid excretion declines dramatically with age regardless of polyphenol intake. However, microbial benzoic acid production likely increases as the simultaneous increase in urinary hippuric acid is 10-50 times greater than the decrease in urinary benzoic acid (). 

Bottom line: likely reflects a combination of diverse dietary inputs and metabolism by a wide range of gut bacteria. May reflect age more than anything else. 

Hippuric Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Bacterial dysbiosis

The evidence: Hippuric acid is primarily derived from the conjugation of benzoic acid (discussed above) with glycine in the liver (3). It is also the final product of the detoxification of toluene, a toxin found in paint thinners (which is also metabolized to benzoic acid in the process), and has been used to monitor toluene exposure in occupationally exposed workers (). Urinary hippuric acid has also been used to estimate fruit and vegetable intake in observational research (), and has also been proposed as a biomarker for aging, as it significantly increases with age regardless of fruit and vegetable intake (). 

High urinary hippuric acid has been positively associated with metabolic health and increased gut microbial diversity in middle-aged adults in Denmark (3). Hippuric acid has also been found to be markedly reduced in Crohn’s disease, which may be a result of reduced microbial benzoic acid production rather than reduced conjugation in the liver ().

Bottom line: like its precursor benzoic acid, hippuric acid is influenced by polyphenol and preservative intake, phenylalanine absorption, toxic exposures, and age. 

4-Hydroxyphenylacetic Acid (4HPAA)

Popular companies: Genova, Great Plains, Diagnostic Solutions

What the companies say it indicates: Bacterial dysbiosis; C. difficile, C. stricklandii, C. lituseburense & others 

The evidence: This close relative of 3HPAA (discussed above) is one of the primary microbial metabolites of resveratrol, an antioxidant known for occurring at high levels in red wine (). 4HPAA also increases when you eat strawberries () and can result from the bacterial metabolism of the amino acid tyrosine  (). 

Interestingly, in one study, 4HPAA was among the most significantly increased blood metabolites in individuals with ulcerative colitis (). On the other hand, a recent study found that 4HPAA alone was sufficient to reduce diet-induced cardiometabolic disease in mice (). No studies have directly correlated urinary levels with bacterial populations.

Bottom line: likely reflects dietary polyphenol and amino acid intake more than bacterial composition. More research is needed to clarify its health effects.

Dihydroxyphenylpropionic Acid (DHPPA)

Popular companies: Genova, Great Plains

What the companies say it indicates: Bacterial dysbiosis or beneficial bacteria (depending on the company!)

The evidence: In the past decade, DHPPA has emerged as a validated and objective marker of dietary whole-grain wheat and rye intake (, ). While DHPPA is a microbial metabolite and therefore theoretically impacted by gut microbiome composition, the effect appears to be negligible compared with the impact of diet. Therapeutically, DHPPA protects against liver injury in rats ().

Bottom line: a marker of whole grain intake, not microbial populations.

HPHPA (3-hydroxyphenyl-3-hydroxypropionic acid)

Popular companies: Great Plains

What the company says it indicates: Clostridium spp. (C. sporogenes, C. caloritolerans, C. botulinum & others)

The evidence: HPHPA has frequently been identified as an elevated urinary marker in autism spectrum disorders, as well as schizophrenia (, , ). Treatment of autistic children with the broad-spectrum antibiotic vancomycin dramatically reduced urinary HPHPA, suggesting a strong microbial link (). While HPHPA is often suggested to be a biomarker of Clostridium species, we were unable to find any evidence to support this.

Bottom line: influenced by the microbiome and elevated in psychiatric disorders. No evidence to support this as a marker of Clostridium overgrowth.

Phenylacetic Acid

Popular companies: Genova, Diagnostic Solutions

What the company says it indicates: Phenylalanine malabsorption

The evidence: Phenylacetic acid is a product of the microbial breakdown of the amino acid phenylalanine. Preliminary research suggests that predominant metabolizers include Bacteroides species, Eubacterium halii, and some Clostridium (). Like indoleacetic acid, it is also a natural signaling molecule in plants (). 

Increasing dietary protein intake increases fecal phenylacetic acid in human volunteers, regardless of the levels of carbohydrate and fat in the diet (). Acid-blocking drugs also significantly increase fecal phenylacetic acid, perhaps related to a reduction in protein absorption (). Another study found that increasing dietary protein increased urinary phenylacetic acid, but only in mice, not in humans (). Together, this suggests phenylacetic acid is likely metabolized further in the human liver. 

Blood levels of phenylacetic acid rise dramatically after consuming curry spices, suggesting it is also a polyphenol metabolite (). Additionally, fecal transplant from a healthy lean donor to obese adults significantly decreased fecal phenylacetic acid at three months after transplant. This corresponded with an increase in Bacteroides and Blautia, suggesting phenylacetic acid is also sensitive to microbiome changes (). 

Multiple early studies indicated that individuals with depressive symptoms excrete lower amounts of phenylacetic acid in urine than healthy controls (). These results have not been replicated in several decades, nor correlated with microbial populations.

Bottom line: likely reflects dietary protein intake, protein absorption, and spice consumption, as well as bacterial populations. Urinary output may be influenced by an unknown breakdown pathway in the liver.

Indoleacetic Acid (3-Indole acetic acid, Indole-3-acetic acid, IAA)

Popular companies: Genova, Great Plains, Diagnostic Solutions

What the companies say it indicates: Tryptophan malabsorption (Genova), Clostridium spp. (Great Plains)

The evidence: IAA is derived from tryptophan metabolism by gut bacteria. Preliminary research suggests IAA-producers include Clostridium (), Bacteroides, Alistipes, Prevotellaceae, () and yeasts (). Research on celiac disease patients has found that those experiencing malabsorption symptoms such as steatorrhea have higher baseline urinary IAA and a greater increase in levels after consuming 4 grams of tryptophan ().  IAA is also naturally found in many edible plants; however, whether this contributes appreciably to urinary levels is unclear (). Conventionally, indoleacetic acid is considered a uremic toxin – one of several nitrogen-containing compounds that build up in the blood when kidney function is compromised ().

Recently, lab research has identified numerous therapeutic benefits of indoleacetic acid, alleviating pathologies as diverse as depression, non-alcoholic fatty liver disease, and ankylosing spondylitis in mice, while significantly modulating the microbiome ( , ). Feeding sulforaphane to mice on a high-fat, high-sugar diet restored depleted blood levels of IAA, likely by upregulating IAA-producing species (). At least some of IAA’s therapeutic benefit may be because it is a potent stimulator of the aryl hydrocarbon receptor (AhR) (); at the same time, other research suggests AhR activation may be responsible for its uremic toxicity in chronic kidney disease (). Despite significant research on this organic acid, urinary levels have not been correlated with specific gut microbes or microbiota patterns.

Bottom line: likely reflects protein malabsorption as well as bacteria and fungal metabolism. Likely beneficial at moderate levels and pathogenic at very high levels, though little research exists to establish a healthy urinary reference range.

p-Cresol (4-Cresol)

Popular companies: Great Plains

What the company says it indicates: Clostridioides difficile (previously named Clostridium difficile) overgrowth

The evidence: p-cresol is a microbial metabolite of tyrosine, produced by many gut microbes but in particularly large quantities by C. difficile (). Proton pump inhibitor (PPI) treatment was shown to increase urinary p-cresol by reducing protein absorption, suggesting that this metabolite may also be influenced by the degree to which protein is digested (). 

A broad metabolomic study identified p-cresol as one of three serum metabolites inversely correlated with type II diabetes risk. The researchers further found that giving p-cresol to mice with cardiometabolic disease reduced body fat, glucose intolerance, and fatty liver, while increasing the function of insulin-secreting pancreatic beta cells ().

On the other hand, p-cresol has been found to be elevated in children with autism (), and administering a dose 100 times higher than in the cardiometabolic study mentioned above to healthy mice induced autistic-like behavior. This effect was reversible with microbiome transplantation (). None of these studies correlated p-cresol levels to levels of C. difficile or other gut bacteria. 

Notably, a sulfate group is attached to p-cresol in the body to form p-cresyl sulfate, a uremic toxin that is known to accumulate in chronic kidney disease due to impaired excretion and has potentially cancerous effects (). Research has scarcely measured both p-cresol and p-cresyl sulfate in the same human subjects, so it is possible that the rate of sulfation may influence p-cresol’s apparent bidirectional effects.

Various microbial products and diets have been shown to influence p-cresol concentrations. Lactic acid bacteria-fermented soy extract, wheat bran extract, and a raw vegan diet have all been shown to significantly reduce urinary p-cresol in various studies (). Similarly, yeast mannan extract () and a low-fat diet have also been shown to reduce fecal p-cresol (). On the other hand, in a subgroup from the well-known PREDIMED trial, increased p-cresol was one of a group of three urinary metabolites that could distinguish people with high adherence to a Mediterranean diet with 97 percent accuracy ().

Bottom line: an impactful bacterial metabolite derived from diverse sources. Further research is needed to clarify its effects and optimal urinary ranges.

Yeast/fungal markers

Markers included in this section are purported markers of fungal dysbiosis or overgrowth.

3-Oxoglutaric Acid

Popular companies: Great Plains

What the company says it indicates:  Fungal overgrowth

The evidence: This organic acid is not known to be produced by the body, found in the diet, or associated with the consumption of specific foods, so it has promise as a microbial indicator. However, there is no evidence for the significance of high or low levels. 

Pharmaceutically, 3-oxoglutaric acid has anti-cancer properties (). One open-label pilot study found that endurance athletes had reduced urinary excretion of 3-oxoglutaric acid after consuming a lactic acid bacteria-fermented soybean extract(). Another study found that 3-oxoglutaric acid was decreased in a cohort of children in China with autism, a condition known to be associated with perturbed gut fungi (). 

Bottom line: likely of microbial origin, but has not been well-established as a bacterial or fungal marker.

5-Hydroxymethyl-2-furoic Acid (HMFA)

Popular companies: Great Plains, Diagnostic Solutions

What the company says it indicates: Aspergillus overgrowth

The evidence: Aspergillus does produce this organic acid. However, Aspergillus species are not considered true residents of the human gut and are instead thought to be transient from dietary and environmental sources (). HMFA is also a natural constituent of coffee (). Furthermore, organic acid tests may not properly distinguish HMFA from its close relative, 5-hydroxymethyl-2-furfural, which is increased in urine after dried plum consumption (). 

One study found that a three-month diet and exercise program increased fasting blood levels of HMFA by 20 percent in obese women (). While the study lacked a placebo group, the urine tests both before and after the intervention were taken after participants consumed a strict control diet for a week, suggesting that the change can likely be attributed to shifts in the microbiome. However, gut microbes were not directly assessed in this study.

Bottom line: may indicate dietary intake as much as gut microbes; which microbes it may indicate is currently unclear.

Furan-2,5-dicarboxylic Acid (FDCA)

Popular companies: Great Plains

What the company says it indicates: Aspergillus overgrowth

The evidence: In addition to microbial sources, FDCA is formed from the oxidation of 5-hydroxymethyl-2-furfural, the dietary constituent discussed above. A three-month diet and exercise program increased fasting blood levels of FDCA by 10 percent in obese women (). This change was likely entirely attributable to bacterial shifts rather than diet because the post-intervention test was taken after both groups consumed an identical standardized diet for one week. No research has correlated FDCA with specific gut bacteria or fungi.

Bottom line: limited research suggests modestly lower levels may indicate a metabolically healthy microbiome overall; however, correlations with specific taxa cannot be made.

Carboxycitric Acid

Popular companies: Great Plains

What the company says it indicates: Fungal overgrowth

The evidence: In one recent study, urinary carboxycitric acid was significantly reduced in children with autism compared with healthy controls; however, the researchers suggest this may reflect increased uptake into the brain rather than decreased gut microbial production (). 

In young athletes, urinary carboxycitric acid positively correlated with the proportion of fecal Firmicutes (). However, this was not a randomized or placebo-controlled study and the researchers did not collect data on the participants’ dietary intake.

Bottom line: may reflect both fungi and bacteria; however, its significance is unclear.

Citramalic Acid

Popular companies: Genova, Great Plains

What the company says it indicates: Fungal dysbiosis/overgrowth

The evidence: Citramalic acid is synthesized by many different bacteria in addition to Aspergillus species. It is also a natural constituent in apples, sugar beets, tomatoes, and other fruits (, , ). 

In elderly patients with chronic heart failure, increased fecal Klebsiella correlated negatively with blood levels of citramalic acid (). Conversely, citramalic acid has been found to be significantly elevated in serum in individuals with diabetic kidney disease, a pathology commonly associated with microbiota disturbances ().

Bottom line: may reflect generalized microbiome imbalances when elevated or depressed, but with unclear significance. Also influenced by fruit consumption.

Furancarbonylglycine

Popular companies: Great Plains

What the company says it indicates: Aspergillus overgrowth

The evidence: Extensive database searches did not turn up a single study mentioning this metabolite (If you find one, let us know!)

Bottom line: ignore this marker.

Tartaric Acid

Popular companies: Genova, Great Plains

What the company says it indicates: Fungal dysbiosis; Aspergillus overgrowth

The evidence: Urinary tartaric acid has been suggested as a biomarker of red wine intake, and when used in this way, has been correlated with lower LDL cholesterol (). Genova notes that chocolate intake may increase levels as well. 

No studies to date have provided direct evidence of a fungal origin for tartaric acid. One study in young athletes found that a reduction in tartaric acid levels paralleled reductions in other purported gut fungal markers and positively correlated with the proportion of Firmicutes (). However, the athletes in this open-label study consumed a fermented soymilk extract and competed in a race event, which confound the interpretation of these results. Urinary tartaric acid has also been found significantly reduced in children with autism ().

Bottom line: likely indicates wine and other polyphenol consumption; no direct evidence of fungal origin. 

Tricarballylic Acid

Popular companies: Great Plains

What the company says it indicates: Fusarium overgrowth

The evidence: Tricarballylic acid is a microbial derivative of dietary aconitate and citrate, found in diverse plant foods (). Fusarium species residing on corn have indeed been found to produce tricarballylic esters (9); however, so do many gut-resident microbes in rats (Fusarium species are considered transient and do not typically colonize the human gut. 

Accordingly, no evidence exists that urinary excretion in humans represents gastrointestinal overgrowth. In fact, research suggests it could represent beneficial shifts in the microbiome. A three-month diet and exercise program markedly increased the blood levels of tricarballylic acid in obese women during an oral glucose tolerance test, taken under diet-controlled conditions both before and after the intervention (). 

Recently, a large study found reduced tricarballylic acid among children with autism (). Another study found that endurance athletes consuming a lactic acid bacteria-fermented soybean extract had reduced urinary excretion of tricarballylic acid (). However, as mentioned previously, this was not a randomized or placebo-controlled study.

Bottom line: produced by many bacteria in addition to Fusarium species. More research is needed on this microbial metabolite, which may be beneficial rather than dysbiotic.

Arabinose

Popular companies: Great Plains

What the company says it indicates: Candida overgrowth

The evidence: Compared with its relative D-arabinitol, discussed below, arabinose is a much broader metabolite with more diverse endogenous and microbial sources. One study found that reductions in urinary arabinose paralleled other purported markers of fungal overgrowth (3-oxoglutaric acid, tartaric acid, tricarballylic acid), but gut fungi were not directly assessed (). A case report found similar parallel elevations in a pair of brothers with autism, also without fungal stool analysis ().

Bottom line: D-arabinitol is the better evidence-based alternative.

D-Arabinitol (DA)

Popular companies: Genova (D-arabinitol), Diagnostic Solutions (Arabinitol)

What the company says it indicates: Candida overgrowth

The evidence: Over a decade ago, a comprehensive review determined DA to be the only urinary marker of Candida overgrowth with substantial scientific evidence (). Today, this still appears to be the case. However, most research on DA has been in systemic and invasive candidiasis, more serious infections common in immunocompromised individuals. While this has helped establish DA as a marker of Candida, it provides little insight into how urinary levels might reflect gastrointestinal colonization or overgrowth. No studies to date have linked DA levels to fecal levels of Candida

Many Candida species have been found to produce DA, including C. albicans, C. parapsilosis, C. tropicalis, and C. guillermondii (). C. glabrata and C. krusei do not produce DA in vitro, though infections with C. glabrata have been associated with high urinary levels. While no human synthetic pathway has been identified, 10-fold higher levels of DA have been found in human cerebrospinal fluid versus blood, suggesting the nervous system may contribute to urinary excretion, albeit nominally (). On the other hand, human tissues readily metabolize the isomer L-arabinitol (LA) from dietary sugar alcohols (). L-arabinitol is also produced by some Aspergillus and Saccharomyces species (, ).

In a study comparing serum DA in 61 urology patients with and without candiduria (meaning live Candida species were isolated from their urine), only patients with candiduria and a high fever had elevated DA (7). Similar results were replicated in a study three years later (). However, the average DA levels among the participants without fevers were between 68-95mmol/mol creatinine, more than twice Genova’s optimal cutoff of 36. (This is an imperfect comparison – these studies use serum while Genova uses urine, but the ratio between arabinitol and creatinine has been found to be consistently similar between the two ().) Indeed, both the control and feverless candiduria patients had recently undergone major surgery and taken antibiotics, suggesting even those without candiduria may have had gastrointestinal Candida overgrowth. DA elevations as high as 192mmol/mol creatinine have been attributed to non-invasive GI Candida (0).

While little data exists on healthy levels, non-hospitalized healthy controls had an average serum DA of 18±6.7 mmol/mol creatinine in one study (), while another study found averages of 45±26 in men and 59±33 in women (). However, these control populations did not have strict inclusion criteria, which may explain the wide difference between these “normal” levels. For invasive candidiasis, DA cutoffs between 160-440 mmol/mol creatinine have been proposed; however, serum levels in diagnosed patients vary between 60-2200 in the published literature (, ). As the studies discussed in the previous paragraph suggest, the clearest elevations may only occur in the context of a fever. The evidence to date suggests, however, that DA levels are unlikely to distinguish invasive candidiasis specifically. Nonetheless, elevated DA likely reflects total Candida burden, whether invasive or not.

In a single-arm clinical study of 22 children with autism and GI disorders, the well-researched probiotic strain Lactobacillus rhamnosus Rosell-11 reduced urinary DA from 160 to 89.5 mmol/mol creatinine, which corresponded with an increased ability of the children to concentrate (). Autism has been associated with higher fecal levels of Candida (), and many Lactobacillus species demonstrate anti-Candida activity in vitro (). Once again, the DA levels in this study are far higher than Genova’s cutoff, suggesting that monitoring changes in your values over time may be more relevant than absolute levels.

The most recent research, largely in pediatric populations, has focused on the DA:LA ratio as a more sensitive and specific marker of candidiasis than DA alone (, ). Unfortunately, DA:LA is not currently offered on commercially available urinary organic acid tests.

Bottom line: D-arabinitol is a well-established metabolite of Candida species strongly elevated during fever-inducing invasive candidiasis or extreme gastrointestinal Candida colonization. At lower levels, however, it is unclear what value distinguishes normal DA from Candida overgrowth. Test results should be interpreted in context clinically. Undifferentiated “Arabinitol” (as on the Diagnostic Solutions OMX test), on the other hand, is useless.

Other microbial metabolites:

Markers included in this section are not specific to bacteria or fungi but are purported markers of microbial metabolism.

Equol

Popular companies: Diagnostic Solutions

What the company says it indicates: Bacterial isoflavone metabolism

The evidence: Equol is a specific bacterial metabolite of the isoflavone daidzein, found in soy and soy products. Daidzein metabolism is relatively rare in the human gut microbiome. More than half of people ostensibly lack equol-producing microbes and do not excrete equol regardless of soy intake. Equol production is best characterized in two species from the low abundance gut resident genus Slackia: S. isoflavoniconvertens and S. equolifaciens (100, 101Adlercreutzia equolifaciens, Adlercreutzia hattori, Asaccharobacter celatus, Bacteroides ovatus, Finegoldia magna, Lactobacillus mucosae, and Streptococcus intermedius have been identified as equol producers (102, 103). However, in a large observational study of over 1,000 participants in Japan, many of these species were present in fecal samples in similar amounts regardless of equol excretion (102). This suggests equol production may be a strain-specific trait, which is supported by recent laboratory research (104). Equol production is a two-step process, which has also been demonstrated to take place collaboratively between multiple species incapable of producing equol on their own (105). The Japanese study also found that equol excretors had 17 percent higher soy intake than non-excretors, suggesting daidzein intake may support the persistence of equol producers in the gut (102). Other dietary components may influence equol production from daidzein as well – a greater proportion of vegetarians than omnivores are equol excretors (105). A yeast mannan supplement significantly increased urinary equol after an isoflavone challenge in a randomized controlled trial in healthy adult women ().

However, whether or not metabolizing daidzein to equol is necessary to maximize the health-promoting benefits of soy isoflavones is controversial. A systematic review of 42 studies found that two-thirds of clinical trials studying the effect of soy isoflavones on coronary heart disease risk factors found no increased benefit in equol excretors versus non-excretors (106). On the other hand, oral equol supplements have been found in numerous clinical studies to reduce hot flashes in menopausal women (107), and an observational study found that high daidzein intake correlated with lower vasomotor symptoms only in equol excretors (108). However, supplementing soy isoflavones does not have a greater benefit for menopausal symptoms in equol excretors (107). Two recent observational studies found a lower risk for non-alcoholic fatty liver or less severe disease in equol excretors, however, one found a significant effect only in men and the other only in women (109, 110).

You are unlikely to have significant urinary equol if you haven’t eaten soy in the last few days whether or not you have the gut bacteria that produce it, so if you want to know your equol status be sure to eat some soy before your urine test (111).

Bottom line: urinary equol is the best way to tell if you have the gut bacteria that produce it, as long as you’ve eaten soy within a few days of your test. It is possible that equol excretors have an enhanced therapeutic benefit from eating soy products, though results from clinical research is mixed.

3,4-Dihydroxyhydrocinnamic Acid

Popular companies: Diagnostic Solutions

What the company says it indicates: Microbial polyphenol metabolism

The evidence: Despite being listed in some published studies as a dysbiosis marker (112), extensive database searches did not turn up a single study connecting this metabolite to microbial populations or even to diet.

Bottom line: not a reliable marker

3,5-Dihydroxybenzoic Acid (3,5-DHBA)

Popular companies: Diagnostic Solutions

What the company says it indicates: Microbial polyphenol metabolism

The evidence: 3,5-DHBA is both a naturally-occurring phytonutrient in apples, honey, peanuts, chickpeas, nettle, and dandelion, as well as a microbial metabolite of alkylresorcinols found primarily in wheat, barley, and rye (113). This organic acid is highly bioactive in humans, stimulating the lactate receptor HCAR1. It is unclear whether the analytical methods used by popular tests can unambiguously distinguish 3,5-DHBA from its common isomers pyrocatechuic acid (2,3-DHBA) and gentisic acid (2,5-DHBA) among others, which have vastly different origins and physiological significance. Regardless, there is no evidence to suggest that 3,5-DHBA is an indicator of gut microbes.

Bottom line: likely a highly non-specific marker of dietary intake. Ignore this marker.

Summary and conclusions

In conclusion, while metabolomics is a promising and growing area of research, the majority of microbial markers on popular urine Organic Acids Tests are not reliable markers of gut dysbiosis or overgrowth. Most are influenced or confounded by diet, effects of aging, toxin exposure, and/or are produced by a diverse range of microbes. 

Only a select few OAT markers are true microbial metabolites and show some promise to guide clinical practice:

  • D-arabinitol (offered on the Genova Organix test) is a well-established marker of Candida. Levels upwards of 160 mmol/creatine may indicate invasive Candidiasis or a high total Candida burden. More modest elevations may indicate GI overgrowth, but current research is unable to distinguish normal commensal levels of DA from overgrowth. Results should be interpreted in the context of signs and symptoms. For more on Candida testing and treatment, stay tuned for our upcoming course on gut fungi, yeast overgrowth, and the mycobiome!
  • P-cresol and indoleacetic acid, microbial metabolites of the amino acids tyrosine and tryptophan, respectively, also show some potential for clinical use, but more research is needed to fully understand their nuanced health effects and optimal concentrations. While significant elevations of p-cresol may indicate C. difficile overgrowth, this would still be better looked at with a stool test, as a variety of microbes can produce this metabolite. 

Given the significant limitations discussed here, most people would not benefit from a urine Organic Acids Test to assess gut health. Comprehensive stool testing, along with signs and symptoms, offers a much more reliable way to assess the current state of your gut health. 

Have you had an organic acids test? Did you find this article helpful? Share your thoughts or experience in the comments below!

Want to support this blog? 

If you appreciated this article, please consider joining my Patreon community! Patrons get perks like bonus research content, discounts on my online courses, and access to member-only Q&A sessions.

The post The uses and limitations of organic acids testing for gut health: a complete guide appeared first on Lucy Mailing, PhD.

This content was originally published here.

Can't Get enough Freebie, Subscribe

We will send you the latest digital Marketing technology and methods that should help you grow your business.

Subscribe to Our list

Custom Keto Diet

 

Exipure

 

All day slimming tea

 

ikaria Juice

 

Apple Cider Vinegar Ebook Membership