Tests for Humans
Zonulin and Metabolic Syndrome
Zonulin is considered a key biomarker of increased intestinal permeability1, with elevated levels found in Celiac Disease, Type1 Diabetes2, Coronary Heart Disease3 and Obesity4. Recent studies have examined the association between serum Zonulin levels and gastrointestinal (GI) symptoms, anthropometric and metabolic factors5.
Zonulin is secreted from enterocytes, but also from the liver, adipose tissue, brain, heart, immune cells, lungs, kidney, and skin6. It is triggered mostly by gluten and bacteria7.
A very recent observational study in middle-aged subjects shows associations between increased Zonulin levels and larger waist circumference, diastolic blood pressure, and fasting plasma glucose levels. This is in accordance with previous smaller studies which have shown that Zonulin correlates with BMI, waist-to-hip ratio, plasma levels of glucose, cholesterol, triglycerides, systolic blood pressure, and insulin resistance8.
An increased release of Zonulin from abdominal fat tissue and the liver may explain the elevated serum levels in the overweight6, and its correlation with waist circumference. Obesity and metabolic syndrome are both considered capable of creating a low-grade systemic inflammation9.
Altogether, these recent findings suggest that elevated Zonulin levels may be a biomarker of obesity and metabolic syndrome. The degree of increased intestinal permeability during these conditions has to be determined, to try to identify the etiology and the source of the elevated Zonulin levels.
Nonetheless, the conclusion from this and other studies in the field, indicate elevated Zonulin levels to be a biomarker of poor health, and could possibly be used clinically as a marker of the metabolic syndrome.
1. Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiol Rev 2011; 91:151-75.
2. Sapone A., de Magistris L., Pietzak M., Clemente M.G., Tripathi A., Cucca F., Lampis R., Kryszak D., Carten M., Generoso M., et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 2006; 55:1443-9.
3. LiC., GaoM., ZhangW., Chen C., Faying F., Hu Z.& Zeng C. Zonulin Regulates Intestinal Permeability and Facilitates Enteric Bacteria Permeation in Coronary Artery Disease. Sci. Rep. 2016; 6:1-10.
4. Moreno-Navarrete J. M., Sabater M., Ortega F., Ricart W. & Fernández-Real J. M. Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance. PLoS One 2012; 7:e37160.
5. Ohlsson B., Orho-Melander M.and Nilsson P. M. Higher.Higher Levels of Serum Zonulin May Rather Be Associated with Increased Risk of Obesity and Hyperlipidemia, Than with Gastrointestinal Symptoms or Disease Manifestations.Int. J. Mol. Sci. 2017; 18: 10.3390.
6. Vanuytsel T.,Vermeire S.,Cleynen I.The role of haptoglobin and its related protein, zonulin, in inflammatory bowel disease. Tissue Barriers 2013;1: e27321.
7. Fasano, A. Intestinal permeability and its regulation by zonulin: Diagnostic and therapeutic implications. Ann. N. Y. Acad. Sci. 2012;1258: 25–33.
8. Zhang D., Zhang L., ZhengY., Yue F., Russell R.D., Zeng Y. Circulating zonulin levels in newly diagnosed Chinese type 2 diabetes patients. Diabetes Res. Clin. Pract. 2014;106: 312–318.
9. Qi L, Hu F.B. Dietary glycemic load, whole grains, and systemic inflammation in diabetes: The epidemiological evidence. Curr. Opin. Lipidol. 2007; 18: 3–8.
Zonulin and its relationship to Coronary Artery Disease (CAD)
Zonulin is an established biomarker of increased intestinal permeability (IP)1. Elevated circulating blood Zonulin levels are prominent in Celiac Disease1, and Non-Celiac Gluten Sensitivity2, as well as in Type1 Diabetes3 and obesity4, the latter two being major risk factors for atherosclerosis. In addition, these elevated levels often occur well before the appearance of the classic signs and symptoms of these diseases and conditions.
Now, a recent study highlights the association between serum Zonulin levels and Coronary Artery Disease(CAD), with these levels significantly higher in CAD patients5. Several studies have reported an association between enteric bacteria and atherosclerosis, with a high diversity of bacterial genes detected in as high as 95% of atherosclerotic plaque biopsies, most of them (99.4%) belonging to the Enterobacteriacea family. This indicates that, besides an indirect effect, bacteria themselves might be directly engaged in the pathogenesis of CAD6. Exposure to other gram negative bacilli, such as Pseudomonas fluorescens, significantly increased Zonulin expression and decreased IP in a time dependent manner. This, in turn, may facilitate enteric translocation by disassembling the tight junctions, which might explain the observed high diversity of bacterial genes in blood samples. The authors postulate that bacteria may act in a “hit and run” role, meaning that bacterial infection may initiate the atherosclerosis in the early stages, but not during the active part of disease progression. This opens the possibility that gut microbiota transplantation may be able to alter atherosclerosis susceptibility, and that this healthier gut bacteria flora might lead to an alleviation of CAD development7.
Given the fact that Zonulin is significantly elevated in atherosclerosis and increases intestinal IP, it is possible that targeting Zonulin, using monoclonal antibodies or inhibitors, maybe a provocatively new way to move forward in CAD prevention and treatment.
1. Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiol Rev 2011; 91:151-75.
2. Barbaro MR, Cremon C, Caio G, Bellacosa L, De Giorgio R, Volta U, et al. The role of zonulin in non-celiac gluten sensitivity and irritable bowel syndrome. United Euro Gastroenterol J 2015; 3: A87.
3. Sapone A, de Magistris L, Pietzak M, Clemente MG, Tripathi A, Cucca F, Lampis R, Kryszak D, CartenM, Generoso M, et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 2006; 55:1443-9.
4. Moreno-Navarrete, J. M., Sabater, M., Ortega, F., Ricart, W. &Fernández-Real, J. M. Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance. PLoS One2012;7:e37160.
5. LiC, GaoM, ZhangW, Chen C, Faying F, Hu Z& Zeng C. Zonulin Regulates Intestinal Permeability and Facilitates Enteric Bacteria Permeation in Coronary Artery Disease. Sci. Rep. 2016; 6:1-10.
6. Armingohar, Z., Jørgensen, J. J., Kristoersen, A. K., Abesha-Belay, E. & Olsen, I. Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis. J Oral Microbiol. 2014;6:10.3402.
7. Gregory, J. C. et al. Transmission of atherosclerosis susceptibility with gut microbial transplantation. J Biol Chem. 2015; 290:5647–60.
Mice, Zonulin and Chronic Inflammatory Diseases
Zonulin, originally discovered as a toxin produced by the Vibrio cholerae bacterium, is identical to the precursor of Haptoglobin-2 (pre-HP2) in humans. A small percentage of humans have a gene variant that keeps them from producing the pre-HP2 (Zonulin). More than 90% of Wild-type (WT) mice express that same variant and do not produce Zonulin. They therefore representan ideal animal model for those humans who do not produce Zonulin.
Why is this important?
As we know, the intestinal epithelium functions primarily to digest nutrients and absorb electrolytes, but as a barrier it also functions to control the passage of antigens from the intestinal lumen to the submucosa. Loss, or degradation of this barrier function, secondary to an increase in Zonulin, can lead to increased or uncontrolled entry of dietary and microbial antigens and/or their metabolites, due to an increase in intestinal permeability (IP). This Zonulin action has already been established as a key factor in the pathogenesis of Celiac Disease(1) and Type 1 Diabetes(2). Very recent evidence confirms that Zonulin, as a key regulator of IP, also plays a major role in the pathogenesis of a number of Chronic Inflammatory Diseases (CID)(3).
In a recent study(3), researchers were able to genetically modify mice so that they could generate Zonulin. These Zonulin transgenic HP2 mice (Ztm) were subjected to dextran sodium sulfate (DSS), which has been shown to produce colitis in mice. After a week of DDS treatment and 4–7 days of recovery they were compared to the native, or WT mice.
Results showed highly increased morbidity (greater weight loss and colitis), and mortality,in the Zonulin producing mice: 40–70% compared to 0% in WT.
To mechanistically link Zonulin-dependent impairment of the small intestinal barrier with clinical outcome, Ztm were given the Zonulin inhibitor AT1001 in their drinking water, in addition to DSS. Without the Zonulin inhibitor the Ztm showed an increased IP at baseline when compared to WT mice, and a 4-6 fold increase in cell response to Zonulin after DSS treatment.Treatment with AT1001 prevented the DSS-induced increase in IP and completely reversed the morbidity and mortality in the Ztm.
The data showed clearly that Zonulin-dependent impairment of the small intestinal barrier is an early step in the process leading to the break of mucosal tolerance, with subsequent development of CID.
This mouse study highlights the importance of Zonulin as a biomarker for patient-targeted intervention (precision medicine) and disease interception (primary prevention) in genetically predisposed individuals.
1. Fasano A, Shea-Donohue T. Mechanisms of disease: the role of intestinal barrier function in the pathogenesis of gastrointestinal autoimmune diseases. Nat ClinPract Gastroenterol Hepatol 2005; 2:416-22.
2. Sapone A, et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 2006; 55:1443-9.
3. Sturgeon C, Lan J, Fasano A.Zonulin transgenic mice show altered gut permeability and increased morbidity/mortality in the DSS colitis model. Ann NY Acad Sci 2017; 1397: 130–142.
DHEA and DHEA-S: Their Function and Importance in Brain Function
Dehydroepiandrosterone (DHEA), together with its sulfated form (DHEA-S), comprise the most abundant form of steroid hormone in the body, with the average blood concentration of DHEA-S being about 10,000 times greater thanestradiol, the most potent estrogen. DHEA is derived from cholesterol and is produced by the adrenal glands, brain, testes and ovaries. It has great importance as a precursor for estrogen, progesterone, and testosterone and has often been called the "anti-aging hormone".
As a function of age, DHEA-S reaches its highest levels in the mid-twenties and decreases to around 20-25% by the early 70s. With this decrease, the brain loses the protective effects of the sex hormones and becomes increasingly susceptible to neurotoxic effects of cortisol and other damaging factors. As DHEA-S levels decrease, withcortisol levels remaining constant, there is a resulting increase in the cortisol/DHEA-Sratio. This can contribute to neurodegenerative pathology, such as Alzheimer’s disease.1DHEA replacement therapy can certainly mitigate some of the effects of aging –by increasing the levels of downstream sex hormones, enhancing the ability to adapt to stress, boosting the immune system and improving the body fat ratio.
Why does FLUIDS iQmeasure DHEA-S?
FLUIDS iQ tests for DHEA-S, the sulfated form of DHEA. It represents about 98% of circulating DHEA in the bloodstream and binds more strongly to its carrier protein, albumin.As a result it has a longer biological half-life, due to its slower metabolic clearance. DHEA-S also does not have a strong diurnal rhythm, as is the case with DHEA and cortisol, and very little day to day variance. The DHEA-S levels also correlate closely with the various symptoms of androgen excess and deficiency. Therefore, whereas DHEA is an indicator of the body’s response to acute stress,DHEA-S is a more stable indicator of adrenal activity and long term stress.
DHEA-S & the Brain
Recent evidence highlights the importance of DHEA and DHEA-S, not only as hormonal precursors, but also as active hormones in many biological processes, in particular those in the brain. They play important roles in the development and survival of the brain, helping to modulate the synthesis and release of neurotransmitters, inflammation and immunity, cognitive function and neurogenesis. In fact, DHEA-S is one of the first neurosteroids to have been discovered.2
DHEA-S serum levels are positively correlated with healthy psychological effects, such as attention, concentration and an overall stress-buffering effect. Low DHEA-S levels have been shown in patients with anorexia nervosa, as well as in diagnosed cases of depression.3 High levels of DHEA-S,in the face of low cortisol, have also been shown in PTSD patients.4
Mechanisms of Action
Studies show that both DHEA and DHEA-S act by regulating synaptic transmission, modulating neuronal excitability by directly interacting with GABA, dopamine, serotonin, glutamate, and others. However, they perform these functions through separate and distinct mechanisms.5 For example, they both stimulate dopamine release, but DHEA-S uses slow mechanisms, upregulating tyrosine hydroxylase levels, the enzyme responsible for dopamine production. In contrast, DHEA helps increase dopamine levels in rapid, intense bursts.
Clinical Usage of Neurosteroids
Neurosteroids play an important role in brain disorders. Understanding the mechanisms that underlie neurosteroid function in mood pathologies may help in its use in the treatment of conditions entities such as anxiety and mood disorders, sleep disorders, chronic pain, traumatic brain injury and various neurodegenerative disorders.6
In summary, DHEA, and its sulphated form, are being shown to have clear therapeutic potential in helping regenerate theaging brain.
1. Murialdo G, Nobili F, Rollero A, Gianelli MV, Copello F, Rodriguez G, Polleri A: Hippocampal perfusion and pituitary-adrenal axis in Alzheimer's disease. Neuropsychobiology 2000;42:51-57.
2. Baulieu EE, Robel P: Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive neurosteroids. Proc Natl AcadSci U S A 1998;95:4089-4091.
3. Michael A, Jenaway A, Paykel ES, Herbert J: Altered salivary dehydroepiandrosterone levels in major depression in adults. Biol Psychiatry 2000;48:989-995.
4. Maninger N, Wolkowitz OM, Reus VI, Epel ES, Mellon SH: Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Front Neuroendocrinol 2009;30:65-91.
5. Perez-Neri I, Montes S, Ojeda-Lopez C, Ramirez-Bermudez J, Rios C: Modulation of neurotransmitter systems by dehydroepiandrosterone and dehydroepiandrosterone sulfate: mechanism of action and relevance to psychiatric disorders. ProgNeuropsychopharmacolBiol Psychiatry 2008;32:1118-1130.
6. Zorumski CF, Paul SM, Izumi Y, Covey DF, Mennerick S: Neurosteroids, stress and depression: potential therapeutic opportunities. NeurosciBiobehav Rev 2013;37:109-122.
Hormones and the Gastro-Intestinal Tract
The digestive system is responsible for the breakdown, absorption and assimilation of nutrients. A very significant control over these digestive processes is provided by hormone-secreting cells, the endocrinocytes, that secrete acid and mucous into the lumen or take up nutrients from the lumen. These intestinally-produced hormones are secreted into blood and saliva. They circulate systemically and affect other parts of the digestive tract, the liver, pancreas and brain. For example, Gastrin stimulates gastric acid secretion and proliferation of gastric epithelium, Secretin stimulates secretion of water and bicarbonate from the pancreas and bile ducts and Ghrelin is a strong stimulant for appetite and feeding. However, the hormones produced elsewhere in the body can also have a profound effect on GI function.
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As we know, there are many tests that have been used over the years to try to determine intestinal permeability. Unfortunately most of these tests are not validated for that purpose. These include the well-known lactulose/mannitol test; stool tests, such as the alpha 1-antitrypsin; the Indican (Opermayer) urine test; and of course tests for antibodies against the components that make up the tight junctions of the intestinal barrier.
However, the primary screening test that determines tight junction damage is the level of serum Zonulin. It is easy to perform and actually tests for changes in the tight junctions. Changes in Zonulin levels are directly correlated to changes in intestinal permeability and the penetration of macromolecules through the intercellular intestinal barrier.
Click here to learn more about how the Zonulin test compares to what has previously been available and the remarkable opportunities it offers to change patient treatment protocols.
Zonulin and Polycystic Ovary Syndrome
The protein Zonulin is a biomarker in the assessment of intestinal permeability. It is now regularly used when screening individuals with chronic inflammatory bowel disease and autoimmune conditions such as Celiac Disease, non-Celiac Gluten Sensitivity and Diabetes type 1.
A very recent article by Polak et al (1) highlights the use of Zonulin as a screening biomarker for Polycystic Ovary Syndrome (PCOS).
PCOS is the most common hormonal disorder of women of reproductive age, with one in ten developing the syndrome and with estimates that more than half are unaware, during their entire lives, that they have it. Although the cause of PCOS is unknown, the diagnostic criteria include two of these three features: Increased levels of male hormone (hyperandrogenism), polycystic ovaries and menstrual irregularities, often due to an imbalance in estrogens and progesterone.
In addition, PCOS is often associated with Insulin Resistance (IR), which is thought to be due to abnormalities in cell insulin receptor signaling. This abnormality is the probable cause both of IR and elevated male hormone levels. The clinical importance of these findings is that women with PCOS, even if young and of normal weight, are five times more likely to develop type 2 diabetes and heart disease, the leading killer of women.
In PCOS, an overabundance of male hormones can result in hirsutism, when excessive amounts of hair grow on areas where men typically have hair, including the face, chest, stomach, back, thumbs, or toes. These elevated male hormone levels, which increase oil production, can lead to a host of dermal dilemmas, including dandruff and acne.
Women with PCOS tend to gain weight easily and losing weight can be very difficult. This is due to insulin and leptin resistance, two of the central factors involved in PCOS, since IR causes your body to store fat, and leptin resistance makes your brain think you’re in starvation mode. With IR, weight is easily gained around the abdomen, and this type of fat is the riskiest when it comes to cardiac health.
Finally, there is consistently higher levels of emotional distress in women with PCOS, when compared to controls. Psychological issues like depression, panic disorder, obsessive-compulsive disorder, and bipolar depression are related to a variety of underlying causes and exacerbated by PCOS.
Recent studies (2),(3) show evidence of significantly elevated Zonulin levels in women with PCOS compared to controls and demonstrate a strong correlation with insulin resistance, obesity, high blood lipid levels and severity of menstrual disorders. The speculation is that increased gut permeability may alter the intestinal barrier determining an inflammatory state that leads to insulin resistance. Further studies are ongoing.
1. Polak K, Czyzyk A, Simoncini T, Meczekalski B. "New markers of insulin resistance in polycystic ovary syndrome." J Endocrinol Invest, July, 2016.
2. Moreno-Navarrete JM, Sabater M, Ortega F, Ricart W, Fernández- Real JM. (2012) "Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance." PLoS One, 7(5), 2012.
3. Zhang D, Zhang L, Yue F, Zheng Y, Russell R. "Serum zonulin is elevated in women with polycystic ovary syndrome and correlates with insulin resistance and severity of anovulation." Eur J Endocrinol, 172(1):29–36, 2015.
Zonulin (prehaptoglobin-2) is a key protein found in intestinal cells, or enterocytes. Zonulin is the only physiological modulator of intercellular tight junctions described so far, which is involved in the control of the movement of macromolecules into the intestine and increased permeability, often called "Leaky Gut" (1).
Celiac Disease: Gliadin, the glycoprotein in wheat, is present in the intestinal lumen and is able to activate both the signaling and the release of zonulin. Activation of zonulin causes the opening of tight junctions, permitting the passage of gliadin through the tight junction barriers in subjects with dysregulation of the zonulin system, leading to a possible immune response.
This gliadin-zonulin leakage effect is longer and more pronounced (greater than 5 times) in the enterocytes of people with celiac disease (2). Other studies have shown that celiacs produce 30 times more zonulin than non celiacs, and have three times greater permeability. This strongly suggests that something more than gluten is contributing to "Leaky Gut" in people with celiac disease. It may be that certain types of intestinal dysbiosis (poor balance of bacteria and yeast in the intestines) makes some people, who are genetically predisposed, more likely to develop celiac disease in response to gluten.
Diabetes Mellitus Type 1: High zonulin levels and an increased intestinal permeability precede type 1 diabetes. In contrast, type 1 diabetes could be prevented by the inhibition of zonulin (3). In addition, many people who suffer from celiac disease also suffer from other autoimmune diseases. It has been suggested that increased levels of zonulin are also involved in the pathogenesis of multiple sclerosis and rheumatoid arthritis (3).
1. Fasano A and Shea-Donohue T. Nat Clin Pract Gastroenterol & Hepatol. 2005; 2: 416-422.
2. Vanuytsel T, Vermeire S and Cleynenet I. Tissue Barriers. 2013; 1: 1-9.
3. Sapone A et al. Diabetes. 2006; 55: 1443-1449.
In their recent paper, “Saliva as an Emerging Biofluid for Clinical Diagnosis and Applications of MEMS/NEMS in Salivary Diagnostics” Dr. Chamandie Punyadeera and Paul Slowey, CEO of Oasis Diagnostics® Corporation, refer to saliva as “a miracle biofluid”. Why is that?
It’s quite simple: “Saliva mirrors the body’s health and well-being. And most of the bio-molecules found in blood in urine”, can also be found in saliva. And the fact that saliva collection is simple, non-invasive and with minimal risk of disease transfer, helps explain the reasons why, at FLUIDS iQ® , we consider saliva to be the blood of the 21st century™.
Given that we know that, in saliva, we can find almost all the biomolecules that are found in blood and urine, the clinical applications are myriad, especially in the detection of hormone levels. Anxiety, stress, behavioural disorders, reproductive issues, menopause...all of these and more are affected by levels of bio-molecules such as Cortisol, Testosterone, Progesterone, DHEA and Melatonin, which are easily found in saliva. FLUIDS iQ® ’s suite of hormone tests, to be introduced this fall, will be simple, easy to use saliva based tests.
No needles, just spit. A miracle biofluid indeed!
The fascinating story of the recently discovered protein, Zonulin, has recently advanced another chapter. Italian researchers announced the results of their latest research linking Zonulin with two common inflammatory conditions of the intestine. The researchers found that people with a Non Celiac Gluten Sensitivity (NCGS) and Irritable Bowel Syndrome (IBS) have higher than normal blood levels of Zonulin, suggesting an important role for the protein in the development of these conditions.
Speaking at the 23rd United European Gastroenterology Week in October of 2015 in Barcelona, Spain, Professor Giovanni Barbara of Bologna University said, "We were intrigued to find that blood levels of Zonulin were almost as high in patients with NCGS as in those with Celiac Disease. " Dr Barbara said that the findings may lead to new treatment strategies for these conditions.
Microbiome Effects of Vaginal vs Cesarean Delivery
Babies that are born vaginally have a microbiome edge over those born by cesarean (C) section. The bacteria they pick up from their mother’s birth canal seems to help protect them, since babies born by C-section have slightly higher rates of obesity, asthma and other health problems as they get older.1
Now a 2016 study offers preliminary evidence that when researchers have altered the microbiome of babies born through C-section, by swabbing them with microbes from their mothers’ vaginas, they have given these babies the same benefits as vaginal birth babies.2
Just prior to surgery, the researchers collected the mother’s vaginal microbes with sterile gauze. Within 2 minutes of birth, this gauze was swabbed all over the newborns’ bodies. The babies who received the swabs harbored skin, gut, anal and oral bacterial communities that were more like those of infants delivered naturally. This was in contrast to the C-section delivered babies who did not go through the swabbing procedure.
Repeated sampling of bacteria on the skin, in the anus, and in the mouth over the next 30 days revealed a shift toward a typical microbiome seen with vaginal birth, especially on the skin and mouth. In particular, there was “early enrichment” of Lactobacillus bacteria, followed in week two by Bacteroides - two types of healthy bacteria that are noticeably diminished in babies born by C-section.
The researchers note that larger studies will be needed to determine whether this bacterial seeding and the resulting microbiome alterations has any lasting effect on health later in life.
1. Maria G. Dominguez-Bello, Elizabeth K. Costello, Monica Contreras, Magda Magris, Glida Hidalgo, Noah Fierere, and Rob Knight. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. PNAS. 2010, 107, 11971-11975.
2. Maria G. Dominguez-Bello, Kassandra M. De Jesus-Laboy, Jose C. Clemente. Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer. Nature Medicine. 2016, 22, 250–253.