The Organic Acid Test (OAT): What It Measures and Why It’s Used

The Organic Acid Test (OAT): What It Measures and Why It’s Used

Organic acid testing (OAT) has become very popular amongst functional medicine doctors and dietitians.

This urine-based test provides a wealth of information about the functioning of various bodily systems and can be used to identify possible nutrient deficiencies, gut dysbiosis, and more.

Keep reading to learn more about the science behind organic acid testing, who might benefit from it, and how to get tested.

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What Is an Organic Acid?

In humans, organic acids are byproducts or intermediates of chemical reactions that occur within the body (1).

The quantity (and sometimes the type) of organic acids produced are unique to each individual and can be influenced by a variety of factors.

Some factors that can influence organic acid production within the body include (2, 3):

    • Diet
    • Environmental toxins
    • Gut microbes
    • Genetics
    • Kidney and liver function
    • Medications
    • Nutrient status
    • Oxidative stress

In order for the body to run effectively, it needs proper amounts of vitamins and minerals to act as cofactors for enzymatic reactions. 

Without enough of these nutritional cofactors (or if someone has genetic mutations that affect how well their enzymes work), these chemical pathways are unable to proceed at their normal pace. 

As a result, organic acids begin to build up and are excreted in the urine:

Organic Acid Metabolism Visual Graphic

Our urinary organic acid profiles can give us clues as to what’s happening in our bodies, and this is the basis of organic acid testing.

What Does a Urine Organic Acids Test Evaluate?

Organic acid testing (OAT) is often compared to performing an emissions test on a car. 

For cars, the exhaust fumes that come out of the tailpipe are a byproduct of fuel combustion, and the composition can tell us how efficiently the engine burns fuel.

In the same way, organic acids in our urine are byproducts of our metabolism and can give us information about how efficiently our body systems are performing.

Reasons for Abnormal OAT Results

When the body isn’t functioning optimally, organic acids in the urine can become elevated or depleted, depending on what is occurring.

The main reasons for abnormal urinary organic acid results include:

    1. Inborn errors of metabolism
    2. B-vitamin deficiencies
    3. Gut dysbiosis
    4. Impaired fatty acid metabolism
    5. Ketosis or poor carbohydrate metabolism
    6. Neurotransmitter metabolism
    7. Mitochondrial dysfunction
    8. Oxidative stress
    9. Poor detoxification 
    10. Inflammation

We’ll review each of these scenarios in detail below.

Reason #1: Inborn Errors of Metabolism

Inborn errors of metabolism are genetic disorders that disrupt biochemical pathways in the body (1).

These disorders are rare, but their effects can be devastating (4).

Often, there is a defect in a gene that codes for a certain enzyme, causing the body to either produce malfunctioning enzymes or none of the enzymes at all.

These missing or malfunctioning enzymes prevent important biochemical reactions from occurring and cause metabolites (including organic acids) to accumulate (1).

For example, one of the better known inborn errors of metabolism is phenylketonuria (PKU).

In PKU, there is a defect in the gene for phenylalanine hydroxylase (PAH), which is responsible for converting the amino acid phenylalanine to tyrosine (5).

When PAH isn’t working properly, the body tries to metabolize phenylalanine through an alternative pathway, and organic acids like phenyllactate and phenylacetate are produced as a result (6, 7).

These organic acids can be measured in the urine via routine screening tests.

Other inborn errors of metabolism can also be detected by urinary organic acids, including:

    • Alkaptonuria: homogentisic acid (8, 9)
    • Canavan disease: N-acetylaspartic acid (10, 11)
    • Malonyl-CoA decarboxylase deficiency: malonic acid (12, 13)
    • Maple syrup urine disease (MSUD): 3-Methyl-2-oxovaleric acid (14)

In the United States, all newborns are screened for these diseases, so they’re usually diagnosed quickly (15).

However, about 50% of these disorders are not detected until later in childhood, and some aren’t diagnosed until adulthood (16).

Organic acid testing is still mostly used for the diagnosis of these disorders, but the science has progressed, and now we can use OAT to evaluate overall metabolic health as well.

Reason #2: B-Vitamin Deficiencies

Organic acid testing can also be used to detect deficiency (or insufficiency) of certain B vitamins.

For example, elevated levels of the organic acid methylmalonic acid (MMA) in the urine are a well-validated indicator of vitamin B12 deficiency (17).

If you’re deficient in vitamin B12, methylmalonyl CoA cannot be converted to succinyl CoA. MMA then builds up in the blood and is eventually excreted in the urine, where it can be detected by an organic acid test.

Organic acids that can indicate possible vitamin B deficiencies when out of range include:

    • General B-vitamins: Alpha-ketoisovalerate, alpha-ketoisocaproate, alpha-keto-beta-methylvalerate (18)
    • Biotin: Beta-hydroxyisovalerate (19)
    • Folate: Formiminoglutamate (20, 21)
    • Vitamin B12: Methylmalonate (17, 22)
    • Vitamin B6: Xanthurenate (23, 24)

However, many of these markers have other factors that can increase or decrease their levels, so proper test prep and interpretation is important.

Additionally, MMA and formiminoglutamate levels can provide insight into how well methylation pathways are functioning within the body since B12 and folate are important methyl donors.

Reason #3: Gut Dysbiosis

Organic acids are produced by bacteria in the gut as they metabolize various components of the foods we eat. 

A portion of these acids is absorbed and excreted into the urine, providing insight into diet and gut health.

Types of dysbiosis (and their associated urinary organic acids typically measured) include:

    • General bacterial overgrowth: benzoate, hippurate, indican, phenylacetate, phenylpropionate, p-cresol, p-hydroxybenzoate, p-hydroxyphenylacetate, and tricarballylate (25, 26, 27, 28, 29, 30, 31)
    • Lactic acid bacterial overgrowth: D-lactate (32, 33)
    • Clostridial overgrowth: 3,4-dihydroxyphenylpropionate (34)
    • Yeast overgrowth: D-arabinitol (35)

Elevated levels of the “general bacterial overgrowth” organic acids are not diagnostic of any particular overgrowth but can help guide further testing decisions. 

Additionally, since these markers are influenced by diet, it is important to follow test prep instructions carefully and work with an experienced practitioner for interpretation.

More human research is needed to better understand the links between gut microbiota, diet, urinary organic acids, and various medical conditions.

Reason #4: Impaired Fatty Acid Metabolism

If the body is struggling to burn fat for fuel via beta-oxidation, levels of some organic acids in the urine will rise as the mitochondria use alternative metabolic pathways.

The most commonly measured organic acids related to impaired fat metabolism are:

    • Adipate (36)
    • Ethylmalonate (37)
    • Suberate (38)

Possible nutrition-related reasons for poor fatty acid metabolism include carnitine and/or riboflavin deficiency or increased need for these nutrients due to genetic variants. Inborn errors of metabolism are another common cause (39, 40, 41).

Reason #5: Ketosis or Poor Carbohydrate Metabolism

When you fast or consume a very low-carbohydrate diet, the body naturally produces ketones to be used for energy instead.

These ketones can be detected in the urine with organic acid testing:

    • Acetoacetic acid (42)
    • 3-hydroxybutyric acid (43)

Even when not in ketosis, high levels of other urinary organic acids can identify possible issues with carbohydrate metabolism. These include:  

    • Pyruvic acid (44)
    • Lactic acid (45)

Elevated levels can be due to genetic mutations or deficiencies in required nutrients like thiamin, lipoic acid, CoQ10, pantothenic acid, riboflavin, or niacin (46, 47, 48, 49, 50, 51).

Reason #6: Neurotransmitter Metabolism

In order to communicate between neurons, the nervous system produces chemical messengers called neurotransmitters.

When these neurotransmitters are metabolized, organic acids are produced as a byproduct.

Functional markers of neurotransmitter metabolism that can be measured in the urine include: 

    • Norepinephrine/epinephrine: vanilmandelate (VMA) (52, 53)
    • Dopamine: homovanillate (HVA) (54, 55)
    • Serotonin: 5-hydroxyindoleacetate (5-HIAA) (56, 57)

Again, these results are just a starting point for further investigation. They are not diagnostic of neurotransmitter levels in the brain or spinal cord.

Urinary levels of these organic acids can be impacted by things like genetic snps, nutrient deficiencies, diet, exposure to environmental toxins, stress, sleep, neuroendocrine tumors, supplements, and adrenal function (58, 59, 60, 61, 62, 63, 64)

A skilled practitioner can look at the bigger picture to find patterns that point to possible root causes.

Reason #7: Mitochondrial Dysfunction

Mitochondrial dysfunction (when mitochondria do not produce energy at their optimal rate) can be caused by genetic disorders, inadequate nutrition, inflammation, environmental toxins, medications, viral infections, and DNA damage (65, 66, 67, 68).

In severe cases, mitochondrial deficiency disorders cause significant symptoms, such as poor muscle tone, weakness, impaired growth, enlarged heart or brain, and liver failure.

More recently, scientists have learned that less severe mitochondrial dysfunction plays a role in many chronic conditions, including cancer, diabetes, epilepsy, and neurodegenerative diseases like Parkinson’s, Alzheimer’s, and Huntington’s disease (69, 70, 71, 72, 73).

Organic acid testing is one of the best ways to test for mitochondrial dysfunction, since it measures most of the intermediates in the citric acid cycle (65, 74, 75, 76).

Urinary organic acids typically tested that involve the mitochondria include:

    • Alpha-ketoglutarate (77)
    • Cis-aconitate (78)
    • Citrate (79)
    • Fumarate (80)
    • Hydroxymethylglutarate (HMG) (81)
    • Isocitrate (82)
    • Malate (83)
    • Succinate (84)

When these organic acids are out of balance, this clues the clinician to search for factors that may be impairing mitochondrial function.

Nutritional factors include deficiency in coenzyme Q10, carnitine, riboflavin, thiamin, niacin, vitamin B12, iron, magnesium, or manganese, all of which are required for cellular respiration (energy production) in the mitochondria (85, 86, 87, 88).

Reason #8: Oxidative Stress

Oxidative stress occurs when there is an imbalance between free radicals and antioxidants within the body (89).

Free radicals are unstable atoms that steal electrons from other molecules, leading to cellular damage (90).

High levels of free radicals and oxidative stress contribute to chronic diseases like cancer and heart disease (91).

Thankfully, our bodies can use antioxidants to destroy free radicals and counteract oxidative stress (92, 93).

Antioxidant levels that are indirectly measured via urinary organic acid testing (along with their corresponding organic acids) include: 

    • Glutathione: pyroglutamic acid and 2-hydroxybutyric acid (94, 95, 96)
    • CoQ10: 3-hydroxy-3-methylglutaric acid (97, 98, 99)

It is also possible to indirectly measure DNA damage caused by high levels of oxidative stress via the following urinary marker:

    • DNA damage: 8-OH-dg (100)

Reason #9: Poor Detoxification

Some substances that our bodies absorb within the gut are shuttled to our liver, where they are transformed into water-soluble molecules that can be excreted in the sweat, urine, or bile. 

By measuring the levels of organic acids created during these processes, we can get insights into how well the liver and kidney’s detoxification processes are working.

Urinary organic acids related to detoxification and/or liver and kidney function that are commonly measured on OATs include (101):

    • Amino acid conjugation: benzoate, hippuric acid (102)
    • Liver enzyme function: Glucarate (103, 104, 105, 106)
    • Liver antioxidant synthesis: alpha-hydroxybutyrate (107)
    • Kidney recovery of amino acids: pyroglutamate (108, 109, 110)
    • Overall detox and antioxidant function: sulfate (111)
    • Ammonia clearance: Orotate (112)
    • Xylene exposure: 2-methylhippurate (113, 114)

Depending on which organic acid levels are out of range, nutrition and lifestyle interventions may be recommended, including:

    • Reducing exposure to environmental toxins
    • Correcting nutrient deficiencies that may impair these pathways (glycine, glutamine, taurine, cysteine, methionine, pantothenic acid, riboflavin, niacin, vitamin B6, folate, vitamin B12)  (115, 116, 117, 118, 119, 120).
    • Adding foods or supplements to the diet that enhance the body’s natural detoxification abilities (magnesium, NAC, zinc, selenium, copper, manganese, antioxidants, thiols, etc.) (121, 122, 123, 124, 125, 126)

A practitioner well-versed in functional nutrition can best assist in these recommendations.

Reason #10: Inflammation

Chronic inflammation can push the metabolism of tryptophan down the kynurenine pathway,  increasing levels of certain organic acids in the nervous system and urine, including (127):

    • Kynurenate (128)
    • Quinolinate (129)
    • Picolinate (130)

These organic acids can bind to NMDA receptors within the nervous system and increase (quinolinate and picolinate) or decrease (kynurenate) their excitatory activity.

More research is needed to understand the health implications of elevated levels of these organic acids, but it can indicate a possible issue with inflammation.

10 Best Insights From Organic Acid Testing Inforgraphic

Who Might Benefit from Organic Acid Testing?

Organic acid testing may be useful for people whose symptoms haven’t been explained through other blood work, stool tests, or urinary hormone tests.

It helps provide a bigger picture of mitochondrial function, nutrient deficiencies, neurotransmitter metabolism, antioxidant status, detoxification abilities, and gut health, which trained and experienced clinicians can use to dig deeper.

It can also be used to identify subclinical (non-symptomatic) metabolic imbalances in order to PREVENT chronic illness from developing.

However, there are some limitations to consider when deciding whether OAT is right for you:

1. Individual organic acids can be falsely elevated by diet, medications, or other factors, so proper test prep and working with an experienced practitioner is vital.

2. While OAT can evaluate the status of some vitamins, it does not provide a complete assessment of micronutrient status. Minerals and fat-soluble vitamin status cannot be measured via organic acids.

3. OAT is not able to detect intestinal parasites that might be contributing to your symptoms. 

4. OAT is relatively expensive and often isn’t covered by insurance, so it’s not a reasonable option for everyone.

Overall, organic acid testing can be a good starting point for complicated cases. It is rarely used in isolation, and additional testing is usually ordered after looking at the results.

How Is an Organic Acid Test Performed?

Performing an organic acid test is relatively easy. Your healthcare practitioner can order it for you, but it is not always covered by insurance.

To collect your sample, you are required to urinate into a collection cup first thing in the morning, before you’ve had anything to eat or drink.

Depending on which OAs are being measured, the instructions might require you to avoid certain foods and supplements for a couple of days leading up to the test as well.

The sample is then frozen and shipped to the laboratory, where organic acids are measured from the urine sample using liquid or gas chromatography-mass spectrometry (131).

Chromatography separates all of the different components found in the urine sample, while mass spectrometry is a tool used to identify and quantify each component by looking at its chemical makeup (132). 

After the lab receives your sample, it typically takes about 2 weeks to get your results.

Your practitioner will receive a report that lists the levels of individual organic acids as either being high, low, or within the normal range.

While it might be tempting to focus on individual OAs that show abnormal results, the overall pattern of abnormalities is more important (133).

In some ways, interpreting OAT results is a lot like working on a puzzle – you can’t fully understand what you’re seeing until you’ve put all the pieces together.

Where to Get an Organic Acid Test

While many labs offer a variety of organic acid tests, two companies are most popular:

1. Great Plains Laboratory: The Organic Acids Test

The Organic Acids Test from Great Plains Laboratory evaluates over 70 different organic acids.

They also offer a Microbial Organic Acids Test (MOAT) that can be used as a follow-up to the OAT for patients who have been treated for dysbiosis.

2. Genova Diagnostics: Organix 

Genova Diagnostics has a test called the Organix Basic Profile that includes more than 30 different OAs.

The Organix Comprehensive Profile offers a more detailed evaluation, with over 40 OAs.

The main difference between the two is that the Basic Profile does not include any of the OAs that serve as markers for bacteria and fungi.

However, they also offer Organix Dysbiosis, which is a separate test for just for microbial OAs (similar to MOAT from Great Plains).

If someone is only interested in looking at organic acids related to metabolism, the Metabolic Analysis Profile may be a good option.

Further Education on Organic Acid Testing

For healthcare practitioners interested in learning more about organic acid testing, Great Plains Laboratory regularly holds in-person trainings around the world. 

You can find a schedule of their upcoming training here

They also have a variety of free webinars available on their website.

Our own membership site, The Functional Nutrition Library, currently includes in-depth notes on interpreting the neurotransmitter-related organic acids, and additional notes will be added in the future.

Final Thoughts

Organic acids are byproducts of metabolism that provide insights on how different bodily systems are performing.

Through organic acid testing, we can detect possible nutrient deficiencies, gut dysbiosis, and other imbalances in metabolism.

Anyone who suffers from a chronic health condition or who simply wants to gain insight into their overall metabolic health is likely to benefit from OAT.

Because it only requires the collection of a urine sample, OAT is quick and painless for patients.

Interpreting OAT results is very complicated, so it’s best to review your results with a health professional who has experience with OAT.

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Organic Acid Test - What it measures and why it's used
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What Is the Best SIBO Test?

What Is the Best SIBO Test?

SIBO stands for small intestinal bacterial overgrowth – a condition in which bacteria overgrow within the small intestine, where they normally don’t exist in large numbers.

When bacteria overpopulate the small intestine, they ferment carbohydrates from the food we eat, causing uncomfortable symptoms.

Because the symptoms of SIBO are so general (abdominal pain, diarrhea and/or constipation, bloating), it can be difficult to correctly diagnose without using tests.

The most common way to test for SIBO is through breath testing, but there are several different types.

This article will explain the science behind testing for SIBO, the pros and cons of each method, and which test is best.

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What is SIBO?

Typically, we think of having a lot of gut bacteria as a good thing, and it usually is!

The right balance of gut bacteria provides a long list of benefits – it aids in digestion, nutrient production, and immunity, to name a few (1).

But sometimes there are TOO MANY bacteria hanging out in places they don’t belong, like the small intestine. This is called small intestinal bacterial overgrowth, or SIBO.

In a healthy gut, bacteria are prevented from entering and thriving in the small intestine through several defense mechanisms (2, 3):

  • Hydrochloric acid in the stomach kills bacteria that we’ve ingested.
  • Pancreatic enzymes and bile destroy bacteria that have survived the stomach.
  • The cells that line the small intestine (and probiotics) release antimicrobial peptides that fight infection.
  • Intestinal mucus traps bacteria while intestinal contractions (peristalsis) transport them out of the small intestine.
  • Immune cells attack pathogenic bacteria.
  • The ileocecal valve (between the small intestine and colon) prevents colonic bacteria from moving backward into the small intestine.

SIBO occurs when one or more of these defense mechanisms fail, and bacteria begin to colonize the small intestine in large numbers.

When bacteria are in the small intestine, they digest carbohydrates from our food, producing large amounts of gas that can cause VERY unpleasant symptoms.

Symptoms can vary, but these are some of the most common (2, 3):

  • Abdominal pain or cramping
  • Belching
  • Bloating (often extreme)
  • Brain fog
  • Depression and anxiety
  • Diarrhea, constipation, or both
  • Fat malabsorption
  • Fatigue
  • Flatulence
  • Food sensitivities or intolerances
  • Gastroparesis
  • Heartburn
  • Nausea
  • Nutrient deficiencies
  • Weight loss

Unfortunately, SIBO can’t be diagnosed by symptoms alone, because the symptoms are too general.

Instead, diagnostic tests should be used to rule in or rule out SIBO.

Who Should Be Tested for SIBO?

Anyone who has persistent unexplained gastrointestinal symptoms like bloating, gas, and diarrhea and has not had success with medical or dietary interventions should consider being tested for SIBO.

The best candidates for testing are those who, in addition to displaying the symptoms listed previously, have one or more of the following conditions linked with SIBO (2, 4).

  • Autonomic neuropathy in diabetes (5)
  • Celiac disease (6, 7)
  • Chronic kidney disease (8)
  • Chronic pancreatitis (9)
  • Crohn’s disease (10, 11)
  • Cystic fibrosis (12)
  • Deep vein thrombosis (13)
  • Diverticulitis (14)
  • Fibromyalgia (15)
  • Gallbladder removal (16)
  • Gallstones (17)
  • H. pylori infection (18)
  • Hypothyroidism (19)
  • Immunodeficiency syndromes (like IgA deficiency, AIDS, etc.) (20, 21)
  • Interstitial cystitis (22)
  • Irritable bowel syndrome (IBS) (23)
  • Liver cirrhosis (24, 25)
  • Multiple sclerosis (26)
  • Non-alcoholic steatohepatitis (NASH) (27)
  • Obesity (28, 29)
  • Parkinson’s disease (30, 31)
  • Radiation enteropathy (32, 33)
  • Restless leg syndrome (34)
  • Rheumatoid arthritis (35)
  • Rosacea (36)
  • Scleroderma (37, 38)
  • Short bowel syndrome (39, 40)

Of course, SIBO is not present in everyone who has one of these conditions, and we still don’t know whether it is a cause, consequence, or unrelated co-occurring phenomenon.

Since testing can be expensive out of pocket, some practitioners choose to treat suspected SIBO with antibiotics (prescription or herbal), based on symptoms alone.

Ultimately, it’s up to the practitioner to screen patients and determine who might benefit most from SIBO testing.

What Types of SIBO Tests Are Available?

To diagnose SIBO, we have to be able to measure whether there are large amounts of bacteria present in the small intestine.

Currently, this can be done either by performing a small intestine aspirate and culture or breath testing.

1. Small Intestine Aspirate and Culture

A “small intestine aspirate and culture” is considered the gold standard for diagnosing SIBO (2, 4).

A sample of fluid from the small intestine (usually the duodenum or jejunum) is collected using a flexible tube called an endoscope (41).

To get a more reliable result, samples are taken from multiple sites throughout the small intestine, because the bacteria can vary depending on the location (4).

The samples are then taken to a microbiology lab where they can be cultured and the bacteria can be quantified.

For a diagnosis of SIBO, there must be greater than 100,000 CFU (colony-forming units) per milliliter of sample fluid (although some researchers prefer a much lower cut-off of 1,000 CFU per mL) (4, 42).

Unfortunately, experts disagree about the validity of using these cultures to diagnose SIBO because they have several issues that may interfere with the results (43).

For example, samples can be contaminated by bacteria from the mouth or stomach as the endoscope passes through the GI tract (42).

In addition, some species of bacteria found in the small intestine can’t actually be grown outside the gut on a culture medium, so the results may be falsely low or skewed (44).

Despite these issues, small intestine aspiration and culture is still considered the gold standard for SIBO diagnosis.

However, it isn’t commonly used in clinical settings because it is a very expensive and invasive procedure. Instead, most practitioners prefer to use non-invasive breath tests.

2. SIBO Breath Testing

Breath testing is based on the fact that gut bacteria produce gases when they ferment carbohydrates.

The main gas produced is hydrogen (H2), but some gut microbes can convert hydrogen into methane (CH4), an odorless gas, or hydrogen sulfide (H2S), which smells like rotten eggs (45, 46).

About 80% of gases formed in the gut are expelled when we pass gas. The other 20% diffuse into the bloodstream and travel to the lungs, where they are exhaled and can be measured in our breath (2).

Any hydrogen, methane, or hydrogen sulfide gas found in the breath can be assumed to be a product of bacterial or archaeal fermentation because there are no other sources of these gases in the human body (45).

Breath levels of these gases serve as INDIRECT measurements of gut microbes because they only estimate the number of bacteria based on the amount of gas produced. They do not measure bacteria levels directly (47).

Some tests only measure hydrogen, but it’s better to measure BOTH hydrogen and methane since hydrogen can be converted into methane by archaea in the gut (42, 48, 49).

It would be ideal to measure hydrogen sulfide levels as well, but as of early 2019, no tests are commercially available (50).

In general, methane-dominant SIBO is linked to constipation, while hydrogen and hydrogen sulfide-dominant SIBO are linked to diarrhea.

Understanding which type of bacteria (methane, hydrogen, and/or hydrogen-sulfide producing) are colonizing the small intestine can guide treatment recommendations since different antimicrobials are better at eradicating each type.

For diagnosing SIBO, breath tests are recommended because they are non-invasive, inexpensive, and simple to perform (42, 45).

How does SIBO breath testing work?

During a breath test, you will be asked to first provide a breath sample to establish your baseline breath hydrogen levels.

Then, you will consume a liquid carbohydrate substrate (either glucose, lactulose, or both) and continue to provide breath samples every 15-20 minutes for 2-3 hours (42).

The breath samples are later sent to a lab where the gases can be measured and interpreted.

Is breath testing reliable?

Of course, there are some issues that may limit the usefulness of breath testing.

The biggest problem with breath testing is that it’s not standardized, so results may differ based on how the test was performed and interpreted by your provider (47).

New data also suggests that breath testing does not correlate well with the supposed “gold standard” jejunal aspirate testing (51).

Despite these issues, breath testing is still the best clinical tool available for diagnosing SIBO at the moment (42).

Which SIBO Breath Test is Best?

All breath tests measure hydrogen (and sometimes methane), but different carbohydrate substrates can be used in the testing beverage.

The two main carbohydrates used are glucose and lactulose (47).

Other substrates, like fructose, lactose, and sorbitol are available but used to diagnose carbohydrate malabsorption, not SIBO (47).

1. Glucose Breath Test

How does it work?

Glucose is a monosaccharide (the simplest form of carbohydrate) that is primarily absorbed in the proximal (first section) of the small intestine (4).

This means that glucose typically does not reach the colon where bacteria are, and thus, does not lead to the production of hydrogen or methane gas.

However, in someone with SIBO, there are bacteria within the small intestine (where they really shouldn’t be in) that WILL ferment the glucose and produce gas (52).

Thus, if breath hydrogen or methane levels increase significantly after consuming glucose, then SIBO is likely present (47).

Pros of glucose breath testing:

The main benefit of using glucose is that it is less likely to produce false positive results (47, 53, 54).

Since glucose is usually completely absorbed in the small intestine, it is less likely that fermentation from bacteria in the colon will interfere with results (47).

If a large increase in gas production occurs during the testing period, you can be fairly confident that there is SIBO.

Cons of glucose breath testing:

However, there is a potential downside to using glucose.

Since glucose is rapidly absorbed in the small intestine, it may not be able to identify SIBO that occurs in the distal (last section) of the small intestine (47, 54).

People with this type of overgrowth may show false-negative results on a glucose breath test because the sugar never reaches the bacteria for fermentation.

While this is the general consensus amongst most healthcare practitioners, new data suggests that glucose actually can reach the colon in some people.

One study traced the path of glucose through the intestines while simultaneously doing a breath test, and found that it WAS able to reach the colon and be fermented by colonic bacteria in 13% of the study population (55).

These participants had glucose breath tests that looked positive for SIBO, but were actually indicative of normal colonic fermentation.

Because of these findings, some researchers argue that glucose breath tests may be better indicators of glucose malabsorption than actual SIBO (51).

Additionally, there is some evidence from animal studies that diet may impact how well the gut absorbs glucose. Those who consume a lot of glucose are better able to absorb it than those who eat low-carb, which could also impact results (56, 57, 58).

Still, glucose is preferred by many experts (4, 59, 60).

Overall, the diagnostic accuracy of the glucose breath test is estimated to be around 72% (4).

2. Lactulose Breath Test

How does it work?

Lactulose is a disaccharide (consisting of fructose and galactose) that is poorly absorbed by the intestines (47).

When consumed by healthy individuals, lactulose makes its way to the large intestine, where it is metabolized by colonic bacteria and hydrogen and methane gases are produced (4).

In those with SIBO, however, the large number of bacteria in the small intestine begin to metabolize lactulose before it reaches the large intestine, causing an early increase in gas production (47).

Any remaining lactulose then travels to the large intestine, where it is metabolized by colonic bacteria, causing a second increase in gas production (47).

Pros of lactulose breath testing:

The benefit of using lactulose is that it travels through the entire small intestine, so you can get a better idea of what’s happening in the ileum (the final section of the small intestine), where SIBO sometimes occurs (61).

Cons of lactulose breath testing:

The downside to using lactulose is that it’s especially prone to false positive results (47, 54).

If intestinal transit time (the time it takes for food to move through the intestines) is faster than average, lactulose might reach the colon too soon.

When this happens, the gas production that occurs looks very similar to a SIBO positive test result.

Those who experience frequent diarrhea typically have a faster transit time, so lactulose breath tests aren’t as reliable for these patients (47).

Also, lactulose itself has been shown to reduce transit time and has been used as a laxative (62, 63).

Additionally, concerns have been raised about the reproducibility of lactulose test results.

One study found no significant correlation between test results conducted on the same people just two weeks apart (64).

Overall, the diagnostic accuracy of the lactulose breath test is estimated to be around 55% (4).

3. Doing Both Breath Tests

Since both glucose and lactulose breath tests have limitations, some practitioners choose to do both tests in order to aid in making a diagnosis.

Labs that offer both glucose and lactulose testing kits typically offer a discount when purchased together.

Pros of Glucose + Lactulose Breath Testing:

By using both substrates, it may be easier to detect SIBO located at either end of the small intestine, potentially reducing the likelihood of false-negative test results.

Cons of Glucose + Lactulose Breath Testing:

Ordering both tests is more expensive than using a single-substrate test and is not guaranteed to provide more clarity.

Most practitioners prefer one test over the other (usually glucose) and only order a single substrate test kit.

Comparison Chart of Glucose vs Lactulose Breath Testing

Where to Get Tested

Physicians’ orders are required to obtain a lactulose breath test, but glucose breath tests can be ordered directly, without a physician.

Laboratories like QuinTron and Aerodiagnostics offer glucose or lactulose breath testing, while BioHealth offers dual glucose and lactulose kits.

Make sure that whatever testing company you choose reports BOTH hydrogen and methane levels for the most accurate results.

How to Interpret Test Results

Unfortunately, there are no universally accepted standards for interpreting SIBO breath tests (4, 42, 47).

Interpretation can be difficult, so we’ve provided a few examples of what positive and negative test results may look like for both glucose and lactulose breath tests.

It’s important to note that these are just examples and results can vary quite a bit, so it’s best to follow interpretation guidelines and consult with experienced practitioners.

General Guidelines for Interpretation

The first step in interpreting a breath test is to check baseline hydrogen and methane levels.

A baseline hydrogen level greater than 16 ppm is considered high, which may be a sign of hydrogen-dominant SIBO, but could also be a sign of improper test prep (59, 65).

A baseline methane level greater than 10 ppm is suggestive of methane-dominant SIBO (42).

The next step is to look at the pattern of hydrogen and methane levels over the two or three hour breath collection period.

When hydrogen levels rise from baseline by more than 20 ppm within 90 minutes, this is typically considered a positive test result (42).

If methane levels are greater than or equal to 10 ppm at any time, including at baseline, it is considered a positive test result (42).

However, it should be noted that interpretation of methane levels is not very well studied and consensus is lacking (42).

Sometimes, hydrogen and methane levels remain low or nonexistent throughout the duration of the test, but it’s unclear why this happens in some people.

If this occurs in someone who displays SIBO-like symptoms, they may have an overgrowth of bacteria that convert hydrogen to hydrogen sulfide, which is not yet detectable on commercially-available breath tests (66).

Sample SIBO Test Results

1) Glucose Breath Test: Possible Negative for SIBO

If someone without SIBO takes a glucose breath test, it should look like this (59, 67):

Glucose Breath Test - Negative for SIBO

As you can see, hydrogen does not increase more than <20 ppm after the glucose is consumed, which usually means there is no bacterial overgrowth in the proximal small intestine (47).

However, if the person has symptoms (especially diarrhea and rotten egg smelling gas), they may have an overgrowth of hydrogen sulfide-producing bacteria that is not detectable on current breath tests.

2) Glucose Breath Test: Positive for SIBO

If someone with proximal SIBO takes a glucose breath test, it might look similar to this (59):

Glucose Breath Test - Positive for SIBO

Here, you can see that hydrogen increases to form a single peak after the glucose is ingested, showing that bacteria in the small intestine are likely metabolizing it (4, 47, 67).

Because the rise in hydrogen is greater than 20 ppm within the first 90 minutes, this is considered a positive test result (42).

However, if someone is a methane-producer, a positive result might look like this instead (67):

Glucose Breath Test - Positive for SIBO - Methane Producer

You see methane levels greater than 10 ppm throughout the test, so this is considered a positive result as well (42).

Note that methane production reduces hydrogen levels in the gut, so low hydrogen levels on a methane-positive test does not exclude the possibility of hydrogen-producing bacteria in the small intestine.

No firm guidelines exist for the interpretation of methane levels, so most practitioners follow the generic recommendation that anything above 10 ppm is positive.

3) Lactulose Breath Test: Negative for SIBO

If someone without SIBO takes a lactulose breath test, it should look like this (68):

Lactulose Breath Test Results - Negative for SIBO

Because this person’s gut bacteria are residing where they should (in the colon), you only see an increase in hydrogen once, when the lactulose reaches the colon and is metabolized.

4) Lactulose Breath Test: Positive for SIBO

If someone with SIBO takes a lactulose breath test, it might look a little like this (45, 68):


In this case, you see two peaks: one when the lactulose reaches the small intestine and begins to be metabolized by bacteria there, and another when the remaining lactulose reaches the large intestine and is metabolized by colonic bacteria (4).

The rise in hydrogen is greater than 20 ppm within the first 90 minutes, so this is considered a positive test result (42).

However, often a positive SIBO lactulose breath test won’t have a double-peak and will look like this instead (68):

Lactulose Breath Test Results - Positive for SIBO - Single Peak

Here, you can see that lactulose quickly begins to be metabolized by bacteria in the small intestine and hydrogen levels continue to rise throughout the test.

In the past, many experts agreed that two peaks were required for a lactulose breath test to be positive for SIBO (42).

However, this single peak presentation is actually more common, and newer recommendations suggest that two peaks are no longer required for the diagnosis of SIBO (42, 68).

Issues with SIBO Breath Testing

Although breath testing is the current preferred diagnostic tool for SIBO, there are several factors that may alter test results.

Fortunately, many of these can be minimized by following a few simple guidelines to prepare for the test.

1. Antibiotic Interference

Antibiotics can interfere with breath testing by killing bacteria and therefore depressing hydrogen and methane production (69).

Some antibiotics are more effective at killing gut bacteria than others, so the effects on hydrogen or methane production will vary (70).

Because it’s still unclear exactly to what degree antibiotics affect breath test results, it’s best to avoid them entirely before taking the test (42, 71).

Antibiotics are often used to treat SIBO, so sometimes a breath test is performed again immediately after finishing treatment to confirm that it was successful (42, 70, 71).

To improve test reliability: Wait at least 4 weeks after finishing any antimicrobials (including herbal treatments) before testing for SIBO (4).

2. Laxative Interference

When laxatives or bowel cleanses are used, large numbers of gut bacteria are eliminated through diarrhea (72).

Because there are fewer bacteria in the gut available to produce gas, excretion in the breath is reduced (4).

In fact, breath hydrogen tests have been used in research settings to evaluate the effectiveness of different laxatives used for colonoscopy preparation. Low breath hydrogen test results show that the preparation was adequate (73).

Unfortunately, taking any type of laxative before breath testing may cause a false negative result (4).

Before testing, patients should be asked if they’ve taken any laxatives, including supplements that have a laxative effect, like magnesium oxide.

Sometimes patients don’t realize that they have been given laxatives for a procedure, so it is a good idea to ask if they’ve had a colonoscopy or barium enema recently as well (74, 75).

To improve test reliability: Wait at least 1 week (but preferably 4 weeks) after using any laxatives or other colon-cleansing solutions before breath testing (4, 42). However, this may not be practical for patients with severe constipation or gastroparesis.

3. Prebiotic and Probiotic Effects

Prebiotics and probiotics both have the potential to interfere with breath test results.

Prebiotics are carbohydrates that humans cannot digest, but gut bacteria can. They remain in the gut until they reach the colon, where the bacteria that live there can consume them as food.

Consuming prebiotics (via supplements or food) may promote the growth of any bacteria currently living in the small intestine, increasing gas production and making SIBO symptoms worse.

It is generally advised to avoid prebiotic-rich foods or supplements prior to testing for SIBO (4).

Probiotics, on the other hand, are living bacteria that can be consumed via fermented foods or supplements.

They can alter the composition of gut bacteria, which in turn, may change the amount of hydrogen or methane produced (4).

However, actual scientific studies are inconclusive as to whether probiotics actually alter breath test results (76, 77).

Align probiotic has been shown to increase methane gas levels in some (but not all) healthy people undergoing a lactulose breath test, without causing symptoms (78).

This suggests that the probiotics caused a transient increase in bacteria in the small intestine as they moved through the digestive tract, but did cause a true overgrowth.

To improve test reliability: To be on the safe side, it might be worth discontinuing any prebiotic or probiotic supplements for up to 4 weeks before taking the test, but it’s unclear if this is absolutely necessary (4, 42).

4. Effects of fermentable carbohydrates (FODMAPs)

As discussed earlier, breath testing depends on the ability of bacteria in the small intestine to ferment a set dose of carbohydrates.

If any other carbohydrates are available for the bacteria to ferment, excess hydrogen will be produced, and the test results won’t be reliable (42).

Fermentable carbohydrates (also known as FODMAPs) include fermentable oligosaccharides (fructans and GOS), disaccharides (lactose), monosaccharides (fructose), and polyols (sugar alcohols) (79).

They can be found in a variety of whole grains, legumes, nuts, seeds, fruit, vegetables, dairy, and artificial sweeteners (80).

Some practitioners disagree about which foods are acceptable to consume the day before the test, but most recommend following a low-fiber diet with foods such as: (81, 82, 83, 84)

  • Baked or broiled chicken or fish
  • Eggs
  • Plain steamed white rice
  • Limited fat and oils (no butter or margarine)
  • Salt and pepper (no other herbs or spices)
  • Non-flavored black coffee or black tea (no sweeteners of any kind)
  • Plain water

Certain testing centers also allow white potatoes and white bread, but these may cause greater hydrogen excretion than plain white rice, so it’s probably best to avoid them (85).

For best results, it’s also recommended to complete an overnight fast from ALL food and drink (except for water) before the test (42).

To improve test reliability: Consume a diet low in fermentable carbohydrates the day before the test. Fast from all food and drink (except for water) 8-12 hours before the test (42).

5. Cigarette smoking interference

Several gases, including hydrogen and methane, are produced during the combustion of tobacco (4).

Increased hydrogen or methane from smoking could interfere with the test and potentially cause a false-positive test result.

It’s not clear how long it takes for hydrogen levels to return to normal after smoking, but one study found that it took only 15 minutes for hydrogen levels to normalize in young, healthy volunteers (86).

However, hydrogen levels may remain high for longer periods of time in individuals who have chronic lung disease, because normal lung function is impaired (87).

To improve test reliability: At the very least, smoking should be avoided for 15 minutes prior to taking a breath test, but avoiding it entirely on the day of the test is better (4, 42).

6. Impact of oral hygiene products

Over 700 species of bacteria have been detected in the oral cavity, many of which produce gases, such as hydrogen, when they ferment sugar from the food we eat (88, 89).

During a breath hydrogen test, these bacteria will produce some hydrogen gas from the glucose or lactulose substrate in the mouth (89)

Mouthwash kills these bacteria and may prevent interfering hydrogen excretion, reducing the risk of a false positive result.

However, some labs ask that you don’t use mouthwash before the test, so it is best to follow their guidelines.

To improve test reliability: Follow the lab instructions to know whether or not to use mouthwash before the test (4).

7. Impact of physical activity

Studies have shown that hydrogen excretion can vary depending on your breathing rate (90).

Hyperventilation (rapid breathing), such as occurs during exercise, leads to decreased hydrogen levels in your breath, which could cause a false negative test result during a breath hydrogen test (91).

To improve test reliability: Do not perform any activity that increases your breathing rate, such as exercise, right before or during the test (4, 42).

7 things that interfere with sibo breath tests

Future Directions for SIBO Testing

There have been recent advances in SIBO testing methods and exciting developments are on the horizon.

1. Testing for Hydrogen Sulfide SIBO

As mentioned above, a new breath testing device that is capable of measuring hydrogen, methane, and hydrogen sulfide is slated to hit the market within the next year or so.

High tech devices used in research studies suggest that hydrogen sulfide levels above 1.2 ppm likely indicate the presence of SIBO (92).

2. Antibody Testing

In some cases, food poisoning may trigger an autoimmune attack on the migrating motor complex (MMC) that messes up gut motility, increasing the risk of SIBO.

Researchers believe this occurs when pathogenic bacteria secrete a toxin known as CDT-B.

CDT-B is very similar in structure to a protein called vinculin, which is involved in coordinating peristalsis waves in the gut.

This similarity can trigger the immune system to accidentally attack vinculin, disrupting the migrating motor complex (the electrical signals that control gut peristalsis between meals) (9394).

There are tests available to check if the body is creating antibodies to CDT-B and vinculin. If it is, antimicrobial treatment and a prokinetic are often used to eliminate the infection and restore proper gut motility.

Antibody testing can be done through IBS-Smart, Cyrex Array-22, Commonwealth Diagnostics IBSchek, or Vibrant America IBSSure.

However, food poisoning (and a disordered MMC) is just one possible cause of SIBO, so a negative antibody test does not rule out SIBO caused by other factors.

Additionally, a positive test does not guarantee the presence of SIBO, since it is possible for a disordered MMC to trigger IBS without an overgrowth of bacteria in the small intestine.

This test is most helpful in determining whether disordered MMC may be a root cause of a person’s SIBO so it can be addressed accordingly, not for diagnosing SIBO itself.

3. Capsule Based Testing

One of the most exciting innovations in the SIBO testing space is the development of a smart capsule that can be swallowed and detect hydrogen levels every 5 minutes as it travels throughout the entire GI tract.

What’s even cooler, is that the data is automatically sent to an app on your phone so you can see what’s happening in real time!

This technology is currently being piloted with promising results and is slated to hit the market in the next 2 to 3 years (95).

4. Microbial Sequencing

Ideally, we would be able to assess both the amount and type of bacteria present in the small intestine.

This can be done by taking a sample of the fluid in the small intestines and performing PCR (polymerase chain reaction) analysis to detect microbes in the gut based on their DNA (96).

This methodology allows us to identify microbes that don’t grow well on a typical growth medium and wouldn’t show up in the “gold standard” aspirate and culture method.

PCR analysis of small intestinal aspirates is not routinely done at the present time, but holds a lot of promise for furthering our understanding of the gut microbiome.

Future Directions for SIBO Testing

Final Thoughts

Breath tests are currently the simplest, most convenient method for diagnosing SIBO.

Tests that use glucose as a substrate have the highest diagnostic accuracy but may be unable to detect SIBO that occurs in the distal small intestine.

Lactulose, on the other hand, can detect SIBO in any part of the small intestine but has lower diagnostic accuracy and is more prone to false-positive results.

Choosing a test that combines both glucose and lactulose is an excellent way to gain a better understanding of the patient’s clinical picture and can make interpretation easier.

To improve the reliability of breath tests, it’s necessary to follow test preparation guidelines, which includes fasting 8-12 hours prior to the test.

Because of the limitations of breath testing, some practitioners may choose to do a “therapeutic trial,” which involves treating for SIBO even without a positive test result, if the patient has a predisposing condition and is presenting with many of the symptoms.

Navigating this can be tricky, and ultimately, only a physician is qualified to make a definitive diagnosis of SIBO.

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How to Test for Leaky Gut

How to Test for Leaky Gut

Have you heard the phrase “leaky gut” thrown around on the internet, but you’re not quite sure what it means?

Does it conjure up images of your intestines leaking out their contents, but you feel like that can’t be what everyone is talking about??

Don’t worry, it’s a little less dramatic (and a little more complicated) than that!

“Leaky gut” is actually a simpler term for “increased intestinal permeability” – a fairly common phenomenon associated with many chronic conditions (1).

In this article, we’ll provide a brief overview of what leaky gut is, why it occurs, and how medical professionals can actually test for it.

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What Is Leaky Gut?

The proper scientific term for leaky gut is “increased intestinal permeability” or “intestinal hyperpermeability” (2).

When the gut is “leaky”, it allows molecules that are normally kept out (like larger food particles or endotoxins) to pass through the lining of the intestines and into the bloodstream.

To understand why this might happen, first, we need to explore a little bit about the anatomy and physiology of the small intestine. We’ve broken it down into simple terms below.

The Anatomy of the Small Intestinal Barrier

Cells that make up the lining of the small intestine (where we absorb the bulk of our nutrients) are called enterocytes.

They form a barrier between the lumen (inside cavity) of the intestines and the rest of the body (3).

Their purpose is to regulate the absorption of vital nutrients and prevent anything dangerous (like bacteria, undigested food particles, or toxic substances) from being absorbed into the bloodstream (3).

In a healthy gut, most molecules (about 90%) are absorbed THROUGH the enterocytes, processed as needed, and then passed on into the bloodstream (4).

This is called the “transcellular” pathway and is tightly regulated to keep out anything harmful (5).

However, some molecules, especially small polar molecules like water, can also pass between the intestinal cells directly into the bloodstream (5).

This is known as the paracellular pathway.

Understanding the Paracellular Pathway

Enterocytes are held together by connections known as tight-junctions, adherens junctions, and desmosomes (6).

Normally, the permeability between enterocytes is very low and not many molecules are allowed to pass.

However, the permeability of these junctions can be influenced by a variety of factors, including regulatory proteins, nutrients, inflammatory cytokines, and bacteria (7).

When these factors cause tight junctions to stay open for too long, substances that otherwise wouldn’t be allowed to cross the intestinal barrier are able to cross, which can trigger an inflammatory immune response (4, 8).

And ultimately, when our bodies are exposed to inflammation over a long period of time, we are more likely to develop a variety of chronic diseases (9, 10, 11).

Demonstration of Trans and Paracellular Transportation Pathways

Causes of Leaky Gut

Potential causes of leaky gut that are currently being investigated include:

  • Alcohol consumption (12, 13)
  • Food components and additives (14)
  • High-fat diets (15)
  • Infections in the gut (16, 17)
  • Inflammation (18)
  • NSAID use (19, 20)
  • SIBO (21)
  • Stress (22, 23)
  • Vitamin A, Vitamin D, or zinc deficiency (24, 25)

9 Potential Causes of Leaky Gut - Functional Nutrition Answers

Conditions Linked to Leaky Gut

There are many conditions associated with leaky gut, but it is still debated whether increased permeability is a cause or consequence of these conditions.

These are some of the conditions that have been associated with leaky gut:

  • Alcohol-related liver disease (25)
  • Alzheimer’s disease (26)
  • Ankylosing spondylitis (27)
  • Asthma (28)
  • Autism (29)
  • Celiac disease (30, 31)
  • Chronic fatigue syndrome (32)
  • Critical illness or trauma (33, 34)
  • Depression (35, 36)
  • Fibromyalgia (37)
  • Food allergies or sensitivities (38, 39)
  • Inflammatory bowel disease (40, 41)
  • Irritable bowel syndrome (42)
  • Multiple sclerosis (43, 44)
  • NASH (45, 46)
  • Obesity (47, 48)
  • Parkinson’s disease (49)
  • Polycystic ovary syndrome (50, 51)
  • Rheumatoid arthritis (52)
  • Type 1 diabetes (53, 54)

What Types of Tests Are Available?

There are a few different ways to measure intestinal permeability, so let’s review the most common methods below.

1. Differential Sugar Test (Lactulose & Mannitol Test)

The differential sugar test has been used since the 1970s and is still the most popular way to test intestinal permeability (55).

There are technically a variety of sugars that can be used in this test, but lactulose and mannitol are most common and will be the focus of this review.

How does it work?

To perform the test, a sweet solution containing lactulose and mannitol is consumed after an overnight fast.

Then, all excreted urine is collected for 6 hours in order to measure the amount of lactulose and mannitol it contains (56).

Lactulose is a larger sugar, so it is not normally absorbed whole. If lactulose is found in the urine, it is assumed to have passed between the cells, indicating a “leaky” gut (56).

In contrast, mannitol is a smaller sugar alcohol that can be absorbed through enterocytes, particularly via small pores at the top of the villi of the small intestine.

The purpose of including mannitol in the drink is two-fold:

  1. It controls for any extraneous factors that might affect absorption or excretion (like gut motility or kidney function) since those factors would affect both types of sugars equally (57, 58).
  2. It can detect villous atrophy in the small intestine, since mannitol is primarily absorbed there. Poor mannitol absorption and excretion (but normal or high lactulose) suggests probable damage to the small intestinal villi.

This test is typically performed at hospitals or clinics, but health practitioners can also order at-home test kits for patients. Genova Diagnostics is a popular choice for these home testing kits, as well as Doctor’s Data.

Interpreting the Results.

The test results are usually expressed as a lactulose-to-mannitol ratio (LMR), in which the percentages (compared to the original oral dose) of lactulose and mannitol present in the urine are compared to each other (LMR = %lactulose / %mannitol) (57).

A higher LMR is associated with increased gut permeability (aka leaky gut).

It is important to note that this test may not be accurate when someone has SIBO (small intestinal bacterial overgrowth) because bacteria in the small intestine can metabolize these sugars before they are absorbed (59).

This could lead to low levels of lactulose and mannitol in the urine and potentially false-negative results.

Is there evidence to support this test?

Numerous studies have shown that LMR correctly identifies increased intestinal permeability, especially in patients with gastrointestinal diseases like Celiac disease and Crohn’s disease (60, 61, 62, 63).

However, lactulose is still a relatively small molecule (molecular weight of 342 Da), so it may not actually be a good indicator for how larger food antigens or bacterial toxins (which usually have a molecular weight of 5,000+ Da) would pass through the gut (64).

It is also important to note that other areas of the digestive tract can also become “leaky”, but different types of molecules are used to test the permeability of those regions (sucrose for the stomach, sucralose for the colon, or PEG or EDTA for the whole tract) (2459).

The Verdict: The lactulose & mannitol urine test is a popular way to evaluate permeability of the small intestine and is backed by decades of research. However, it’s important to note that this test may not be accurate for people with SIBO.

2. Serum Zonulin Testing

Zonulin is a regulator protein that basically acts like a gatekeeper of the intestines.

When zonulin levels rise, the tight junctions between enterocytes open slightly in order to allow nutrients and other molecules to move between them (1).

Since the discovery of the regulatory protein zonulin in the year 2000, scientists have been measuring serum zonulin levels to determine the likelihood of increased intestinal permeability (30).

How does it work?

Testing for zonulin is a much simpler process than the differential sugar test.

If you choose to have this test done, your blood will be drawn and sent to a lab where it will be evaluated for the amount of zonulin it contains.

High levels of zonulin in the blood means too much zonulin has been released in the gut, which probably signifies increased permeability (1).

There are several labs that offer zonulin testing:

  • The “Advanced Intestinal Barrier” test from Dunwoody Labs evaluates serum zonulin, DAO, and histamine levels.
  • The popular “GI-Map” from Diagnostic Solutions Laboratory is a stool test designed to evaluate your gut microbiome. It also offers fecal zonulin as an add-on.

Is there evidence to support this type of test?

While many studies have found high zonulin levels in patients with conditions associated with leaky gut, there are a few issues with using this lab test as a way to diagnose leaky gut (3065, 66).

Problem #1: Unstable zonulin levels.

Zonulin levels are prone to large fluctuations because zonulin is targeted by the immune system.

Once released into the bloodstream, macrophages target and destroy zonulin, so blood levels will vary for each person from day to day, based on their immune response (67, 68).

Problem #2: Measurement error.

Another issue is that most labs use a technique called ELISA (enzyme-linked immunosorbent assay) to measure zonulin levels in blood samples.

However, a study released in 2018 found that ELISA often improperly identified other types of proteins as zonulin, leading to falsely elevated results (69).

Problem #3: Poor correlation with differential sugar tests.

In one study, researchers actually tested patients using both lactulose/mannitol and zonulin testing.

The results of the two tests didn’t correlate very well, so some scientists have questioned the usefulness of serum zonulin testing (65, 67).

Zonulin Antibodies – A better choice?

Testing for zonulin antibodies might be a better alternative.

A recent study found that zonulin antibody testing had better reproducibility (meaning that it consistently showed the same results for the same patient) than serum zonulin levels (67).

Basically, when zonulin is released into the bloodstream, it triggers certain immune cells to produce specific antibodies (also called immunoglobulins) that recognize zonulin.

Because these zonulin antibodies are more stable and remain in the bloodstream longer, they are thought to be a better measurement of zonulin release than serum zonulin  (70).

The Verdict: Testing for zonulin antibodies (through labs like Cyrex or Vibrant Wellness) is more accurate than testing serum zonulin alone. However, it’s unclear whether this is better than lactulose/mannitol testing.

3. Other Types of Antibody Testing

In addition to testing for zonulin antibodies, other types of antibodies are being used as indirect measures of gut permeability.

These include:

  • LPS antibodies: Lipopolysaccharide is a type of sugar found on the cell walls of some gram-negative bacteria. LPS molecules are released when the bacteria die, but since they are relatively large in size they do not normally pass through the lining of the small intestine. However, when the gut is “leaky” some LPS can pass between the cells and stimulate the immune system. Therefore, the presence of LPS antibodies in the blood is an indirect marker of gut permeability (71).
  • Occludin antibodies: Occludin is one of the proteins that make up the tight-junctions between enterocytes. If occludin breaks down, the gut becomes more permeable, and if occludin fragments cross the intestinal barrier, they can stimulate the immune system to form antibodies against them. The presence of occludin antibodies in the blood suggests leaky gut (72).
  • Actomyosin network antibodies: Similarly, actomyosin proteins are found between the cells of the small intestinal lining and play a role in opening up the tight junctions. If the actomyosin network begins to break down and antibodies are formed, it suggests increased gut permeability as well (72, 73).

Testing options:

  • Cyrex Labs has a test called “Array 2” that analyzes zonulin antibodies as well as antibodies for occludin and LPS.
  • Vibrant Wellness has a test called “Wheat Zoomer” that checks for zonulin, actin, and LPS antibodies and also includes serum zonulin levels.

It is important to note that any testing that involves antibodies can be blunted by steroids or immunosuppressants.

While these tests don’t directly measure intestinal permeability, if larger molecules like LPS are able to pass through the intestinal barrier and be flagged by the immune system, chances are that other large molecules like food antigens are able to pass through as well.

4. Intestinal Biopsy

Technically, the most direct way to test for leaky gut is to have an intestinal biopsy done (24).

After removing a sample of intestinal tissue, a device called an “Ussing chamber” is used to measure the transport of molecules across the lining of the intestines (74).

Using this information, it can be determined whether there is actually increased permeability.

Biopsy was the first method used when scientists began studying the concept of intestinal permeability. However, since this an expensive and invasive method, it is not typically used in clinical practice.

Instead, scientists used the information they learned from studying intestinal permeability to create less invasive tests, like the differential sugar test (75).

The Verdict: Intestinal biopsies for the evaluation of leaky gut may be accurate, but this method is far too invasive, expensive, and labor-intensive to be used regularly. It is typically only used in research settings.

Should You Get Tested for Leaky Gut?

Well…maybe not.

Many practitioners believe that testing for leaky gut is a waste of time and money for several reasons.

Downside #1: Leaky gut is probably pretty common.

We know that leaky gut is linked with MANY health conditions (see above), so if you have any of them, there’s a very good chance you have leaky gut as well.

Downside #2: It doesn’t get to the root cause.

Sure, testing for leaky gut can tell you whether or not you have increased intestinal permeability, but many practitioners believe that leaky gut is more of a symptom than a root cause.

Ultimately, we need to know what actually triggered the leaky gut so that we can address that directly.

That’s why many practitioners prefer to evaluate or test for things that are likely to trigger leaky gut, like infections, stress, nutrient deficiencies, or alcohol and medication use.

Downside #3: Knowing if you have leaky gut will probably not affect your treatment.

Knowing whether or not you have leaky gut will probably not affect your treatment protocol.

Testing for leaky gut doesn’t get you any closer to figuring out the root cause (which can then be addressed directly).

Someone on a limited budget is probably better off requesting more targeted tests to determine why their gut might be extra permeable.

However, some practitioners do like to test for leaky gut to evaluate the effectiveness of their interventions.

So, for example, if intestinal permeability is high at the beginning of treatment and then reduced after therapy, this suggests that the treatment protocol was probably effective.

If you do choose to get tested for leaky gut, the best options are the differential sugar test (lactulose/mannitol test) and zonulin antibody testing.

Final Thoughts

Leaky gut (or, more technically, intestinal hyperpermeability) is a real phenomenon associated with many health conditions.

It occurs when the tight junctions between the cells that line our small intestines open up slightly, allowing larger molecules to “leak” through into the bloodstream.

When the immune system encounters these unexpected molecules, it can trigger an inflammatory response, which over time, is believed to increase the risk of many chronic illnesses.

However, leaky gut is probably more of a symptom than a root cause. Digging deeper to find the things that trigger leaky gut may be a better use of time and resources.

Because of this, most healthcare practitioners don’t specifically test for leaky gut unless they plan to use it as a marker of treatment effectiveness.

If you do plan on testing for leaky gut, the differential sugar test or serum zonulin antibodies are the preferred methods.

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How to Test for Leaky Gut - Functional Nutrition Answers