William Shaw, PhD
Concentrations of the dopamine metabolite homovanillic acid, or HVA, have been reported to be much higher in the urine of children with autism compared to controls. In the same study, severity of autism symptoms was directly related to the concentration of HVA. There was a relation between the urinary HVA concentration and increased agitation, stereotypical behaviors, and reduced spontaneous behavior. Furthermore, vitamin B6, which has been shown to decrease autistic symptoms, decreases urinary HVA concentrations. Excess dopamine has been implicated in the etiology of psychotic behavior and schizophrenia for over 40 years. Drugs that inhibit dopamine binding to dopaminergic receptors have been some of the most widely used pharmaceuticals used as antipsychotic drugs and have been widely used in the treatment of autism. Recent evidence reviewed below indicates that dopamine in high concentrations may be toxic to the brain.
Dopamine is a very reactive molecule compared with other neurotransmitters, and dopamine degradation naturally produces oxidative species (Figure 1). More than 90 percent of dopamine in dopaminergic neurons is stored in abundant terminal vesicles and is protected from degradation. However, a small fraction of dopamine is cytosolic, and it is the major source of dopamine metabolism and presumed toxicity. Cytosolic dopamine (Figure 1) undergoes degradation to form 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) via the monoamine oxidase pathway. Alternatively, dopamine undergoes oxidation in the presence of excess iron or copper (common in autism and schizophrenia) to form dopamine cyclized o-quinone, which is then converted to dopamine cyclized o-semiquinone, depleting NADPH in the process. Dopamine cyclized o-semiquinone then reacts with molecular oxygen to form oxygen superoxide free radical, an extremely toxic oxidizing agent. In the process, dopamine cyclized o-quinone is reformed, resulting in a vicious cycle extremely toxic to tissues producing dopamine, including the brain, peripheral nerves, and the adrenal gland.
It is estimated that each molecule of dopamine cyclized o-quinone produces thousands of molecules of oxygen superoxide free radical in addition to depleting NADPH. The o-quinone also reacts with cysteine residues on glutathione or proteins to form cysteinyl-dopamine conjugates (Figure 1). One of these dopamine conjugates is converted to N-acetylcysteinyl dopamine thioether, which causes apoptosis (programmed cell death) of dopaminergic cells. These biochemical abnormalities cause severe neurodegeneration in pathways that utilize dopamine as a neurotransmitter. Neurodegeneration is due to depletion of brain glutathione and NADPH as well as the overproduction of oxygen superoxide free radicals and neurotoxic N-acetylcysteinyl dopamine thioether. In addition, the depletion of NADPH also results in a diminished ability to convert oxidized glutathione back to its reduced form.
What is the likely cause of elevated dopamine in autism? A significant number of studies have documented increased incidence of stool cultures positive for certain species of Clostridia bacteria in the intestine in children with autism using culture and PCR techniques. All these studies have indicated a disproportionate increase in various Clostridia species in stool samples compared to normal controls. In addition, metabolic testing has identified the metabolites 3-(3-hydroxyphenl)-3-hydroxypropionic acid (HPHPA) and 4-cresol from Clostridia bacteria at significantly higher concentrations in the urine samples of children with autism and in schizophrenia.
Treatment with antibiotics against Clostridia species, such as metronidazole and vancomycin, eliminates these urinary metabolites with reported concomitant improvement in autistic symptoms. In addition, I had noticed a correlation between elevated HPHPA and elevated urine homovanillic acid (HVA). The probable mechanism for this correlation is that certain Clostridia metabolites have the ability to inactivate dopamine beta-hydroxylase, which is needed for the conversion of dopamine to norepinephrine (Figure 2).
Such metabolites are not found at only trace levels. The concentration of the Clostridia metabolite HPHPA in children with autism may sometimes exceed the urinary concentration of the norepinephrine metabolite vanillylmandelic acid (VMA) by a thousand fold on a molar basis and may be the major organic acid in urine in those with severe gastrointestinal Clostridia overgrowth, and even exceed the concentration of all the other organic acids combined. Dopamine beta hydroxylase that converts dopamine to norepinephrine in serum of severely retarded children with autism was much lower than in those who were higher functioning. Decreased urine output of the major norepinephrine metabolite meta-hydroxyphenolglycol (MHPG) was decreased in urine samples of children with autism, consistent with inhibition of dopamine beta hydroxylase.
Many physicians treating children with autism have noted that the severity of autistic symptoms is related to the concentration of the Clostridia marker 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) in urine. These are probably the children with autism with severe and even psychotic behavior treated with Risperdal® and other anti-psychotic drugs, which block the activation of dopamine receptors by excess dopamine. I have identified a number of species of Clostridia species that produce HPHPA including C. sporogenes, C.botulinum, C. caloritolerans, C. mangenoti, C. ghoni, C.bifermentans, C. difficile, and C. sordellii. All species of Clostridia are spore formers and thus may persist for long periods of time in the gastrointestinal tracts even after antibiotic treatment with oral vancomycin and metronidazole.
How do the changes in brain neurotransmitters caused by Clostridia metabolites alter behavior? The increase in phenolic Clostridia metabolites common in autism significantly decreases brain dopamine beta hydroxylase activity. This leads to overproduction of brain dopamine and reduced concentrations of brain norepinephrine, and can cause obsessive, compulsive, stereotypical behaviors associated with brain dopamine excess and reduced exploratory behavior and learning in novel environments that are associated with brain norepinephrine deficiency. Such increases in dopamine in autism have been verified by finding marked increases in the major dopamine metabolite homovanillic acid (HVA) in urine. The increased concentrations of HVA in urine samples of children with autism are directly related to the degree of abnormal behavior. The concentrations of HVA in the urine of some children with autism are markedly abnormal.
In addition to alteration of brain neurotransmitters, the inhibition of the production of norepinephrine and epinephrine by Clostridia metabolites may have a prominent effect on the production of neurotransmitters by the sympathetic nervous system and the adrenal gland. The major neurotransmitter of the sympathetic nervous system that regulates the eyes, sweat glands, blood vessels, heart, lungs, stomach, and intestine is norepinephrine. An inadequate supply of norepinephrine or a substitution of dopamine for norepinephrine might result in profound systemic effects on physiology. The adrenal gland which produces both norepinephrine and epinephrine might also begin to release dopamine instead, causing profound alteration in all physiological functions. In addition to abnormal physiology caused by dopamine substitution for norepinephrine and dopamine, dopamine excess causes free radical damage to the tissues producing it, perhaps leading to permanent damage of the brain, adrenal glands, and sympathetic nervous system if the Clostridia metabolites persist for prolonged periods of time, if glutathione is severely depleted, and if there is apoptotic damage caused by the dopamine metabolite N-acetylcysteinyl dopamine thioether.
Depletion of glutathione can be monitored in The Great Plains Laboratory organic acid test by tracking the metabolite pyroglutamic acid, which is increased in both blood and urine when glutathione is depleted. In addition, The Great Plains Laboratory also tests the other molecules involved in this toxic pathway, the dopamine metabolite homovanillic acid (HVA), the epinephrine and norepinephrine metabolite VMA and the Clostridia metabolites HPHPA and 4-cresol.
In summary, gastrointestinal Clostridia bacteria have the ability to markedly alter behavior in autism and other neuropsychiatric diseases by production of phenolic compounds that dramatically alter the balance of both dopamine and norepinephrine. Excess dopamine not only causes abnormal behavior but also depletes the brain of glutathione and NADPH and causes a vicious cycle producing large quantities of oxygen superoxide that causes severe brain damage. Such alterations appear to be a (the) major factor in the causation of autism and schizophrenia. The organic acid test (see sample organic acid test report below) now has the ability to unravel a major mystery in the causation of autism, schizophrenia, and other neuropsychiatric diseases, namely the reason for dopamine excess in these disorders.
In the past, some physicians would order the organic acid test once a year or less. With the new knowledge of the mechanism of Clostridia toxicity via inhibition of dopamine beta-hydroxylase, it seems that the control of such toxic organisms needs to monitored much more frequently to prevent serious brain, adrenal gland, and sympathetic nervous system damage caused by excess dopamine and oxygen superoxide. Below is a test report of a child with autism tested with The Great Plains Laboratory Organic acid test.
DISCUSSION OF PATIENT RESULTS
In the graph above, the vertical bar is the upper limit of normal and the patient’s value is plotted inside a diamond (red for abnormal, black for normal). The above results were from a boy with severe autism. The HPHPA Clostridia marker was very high (979 mmol/mol creatinine), about 4.5 times the upper limit of normal. However, the metabolite due to Clostridium difficile was in the normal range, indicating that Clostridium difficile was unlikely to be the Clostridium bacteria producing the high HPHPA. In other words, a different Clostridia species was implicated. The major dopamine metabolite homovanillic acid (HVA) was extremely high (87 mmol/mol creatinine), almost 7 times the upper limit of normal. The major metabolite of epinephrine and norepinephrine, VMA was in the normal range. The HVA/VMA ratio was 15, more than five times higher than the upper limit of normal, indicating a severe imbalance in the production of epinephrine/norepinephrine and that of dopamine. The very high dopamine metabolite, HVA, indicates that the brain, adrenal glands, and sympathetic nervous system may be subject to severe oxidative stress due to superoxide free radicals and that brain damage due to severe oxidative stress might result if the Clostridia bacteria are left untreated. Below the same patient’s results are displayed in a form that is related to the metabolic pathways. This graphical result now appears on all organic acid results from The Great Plains Laboratory, Inc.
Shaw W. Increased urinary excretion of a 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia spp. in the gastrointestinal tract, in urine samples from patients with autism and schizophrenia. Nutr Neurosci. 2010 Jun;13(3):135-43.
by William Shaw, PhD, and Matthew Pratt-Hyatt, PhD
Director and Associate Director of The Great Plains Laboratory, Inc.
Published in the January 2017 issue of Townsend Letter
Glyphosate is the world’s most widely produced herbicide and is the primary toxic chemical in Roundup™, as well as in many other herbicides. In addition, it is a broad-spectrum herbicide that is used in more than 700 different products from agriculture and forestry to home use. Glyphosate was introduced in the 1970s to kill weeds by targeting the enzymes that produce the amino acids tyrosine, tryptophan, and phenylalanine. This pathway (called the Shikimate Pathway) is also how bacteria, algae, and fungi produce the same amino acids. This pathway is not present in humans, so manufacturers of glyphosate claim this compound is “non-toxic” to humans. However, evidence shows there are indeed human consequences to the widespread use of this product when we consume plants that have been treated with it and animals who’ve also consumed food treated with it.
Usage of glyphosate amplified after the introduction of genetically modified (GMO) glyphosate-resistant crops that can grow well in the presence of this chemical in soil. In addition, toxicity of the surfactant commonly mixed with glyphosate, polyoxyethyleneamine (POEA), is greater than the toxicity of glyphosate alone.1 In 2014, Enlist Duo™, a herbicide product which contains a 2,4-dichlorophenoxyacetic acid (2,4- D) salt and glyphosate, was approved in Canada and the US for use on genetically modified soybeans and genetically modified maize, both of which were modified to be resistant to both 2,4-D and glyphosate. 2,4-D, which has many known toxic effects of its own is perhaps better known as a component of Agent Orange, an herbicide used by the United States during the Vietnam War to increase aerial visibility from war planes by destroying plant growth and crops.
Glyphosate and Chronic Health Conditions
Recent studies have discovered glyphosate exposure to be a cause of many chronic health problems. One specific scientific paper listed Roundup™ as one of the most toxic herbicides or insecticides tested.2 Exposure to glyphosate has been linked to autism, Alzheimer’s, anxiety, cancer, depression, fatigue, gluten sensitivity, inflammation, and Parkinson’s.3-4 A 54-year-old man who accidentally sprayed himself with glyphosate developed disseminated skin lesions six hours after the accident.6 One month later, he developed a symmetrical parkinsonian syndrome. Figure 1 shows the correlation between glyphosate usage and rates of autism, tracking services received by autistic children under the Individuals with Disabilities Education Act (IDEA). This data was originally collected by Dr. Nancy Swanson, along with similar data for many other chronic disorders.14 The causes for these disorders have been linked to glyphosate’s impact on gut bacteria, metal chelation, and P450 inactivation.5-6 It can enter the body by direct absorption through the skin, by eating foods treated with glyphosate, or by drinking water contaminated with glyphosate. A recent study stated that a coherent body of evidence indicates that glyphosate could be toxic below the regulatory lowest observed adverse effect level for chronic toxic effects, and that it has teratogenic, tumorigenic and hepatorenal effects that can be explained by endocrine disruption and oxidative stress, causing metabolic alterations, depending on dose and exposure time.7
Glyphosate, Cancer, and the Microbiome
The World Health Organization International Agency for Research on Cancer published a summary in March 2015 that classified glyphosate as a probable carcinogen in humans.8 Possible cancers linked to glyphosate exposure include non- Hodgkin lymphoma, renal tubule carcinoma, pancreatic islet-cell adenoma, and skin tumors.. Studies have also indicated that glyphosate disrupts the microbiome in the intestine, causing a decrease in the ratio of beneficial to harmful bacteria.9 Thus, highly pathogenic bacteria such as Salmonella entritidis, Salmonella gallinarum, Salmonella typhimurium, Clostridium perfringens, and Clostridium botulinum are highly resistant to glyphosate, but most beneficial bacteria such as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis, and Lactobacillus spp. were found to be moderately to highly susceptible. The relationship between the microbiome of the intestine and overall human health is still unclear, but current research indicates that disruption of the microbiome could cause diseases such as metabolic disorder, diabetes, depression, autism, cardiovascular disease, and autoimmune disease.
Glyphosate and Chelation
Another study found that glyphosate accumulated in bones. Considering the strong chelating ability of glyphosate for calcium, accumulation in bones is not surprising. Other results showed that glyphosate is detectable in intestine, liver, muscle, spleen and kidney tissue. 5 The chelating ability of glyphosate also extends to toxic metals.10 The high incidence of kidney disease of unknown etiology (renal tubular nephropathy) has reached epidemic proportions among young male farm workers in sub-regions of the Pacific coasts of the Central American countries of El Salvador, Nicaragua, Costa Rica, India, and Sri Lanka.11 The researchers propose that glyphosate forms stable chelates with a variety of toxic metals that are then ingested in the food and water or, in the case of rice paddy workers, may be absorbed through the skin. These glyphosate-heavy metal chelates reach the kidney where the toxic metals damage the kidney. These authors also propose that these chelates accumulate in hard water and clay soils and persist for years, compared to much shorter periods of persistence for non-chelated glyphosate. Furthermore, these chelates may not be detected by common analytical chemistry methods that only detect free glyphosate, thus dramatically reducing estimates of glyphosate persistence in the environment when metals are high (for example, in clay soil or hard water).
Testing for Glyphosate
Because glyphosate has been linked with many chronic health conditions, testing for glyphosate exposure and particularly the level of exposure is important. The lower limit of quantification (LLOQ) for The Great Plains Laboratory’s Glyphosate Test is 0.38 μg/g of creatinine. The Great Plains Laboratory is the only CLIA certified lab currently performing a test for glyphosate in urine. Our Glyphosate Test can be performed on the same urine sample as for some of our other comprehensive tests, including the Organic Acid Test (OAT) or GPL-TOX (Toxic Non-Metal Chemical Profile). See Figure 2 for an example of our Glyphosate Test report.
As previously mentioned, glyphosate works by inhibiting the synthesis of tryptophan, phenylalanine, and tyrosine in plants. Humans need to obtain these amino acids from food sources. When food sources have scarce amounts of these amino acids due to glyphosate use, humans are at risk for deficiency too. Humans also require bacteria to maintain a healthy immune system. Research indicates that glyphosate decreases the amount of good bacteria in the gut such as bifidobacteria and lactobacilli and allows for the overgrowth of harmful bacteria such as campylobacter and C. difficile.12 Our lab has observed this in patients. We had a female patient who was suffering from depression who did a Glyphosate Test and an Organic Acids Test. Her glyphosate results were 2.99, which was over the 95th percentile and can be seen in Figure 3.
Upon analyzing her OAT we noticed two things. The first was that her 4-cresol was extremely high. This increased 4-cresol can be seen in Figure 4. As stated earlier, glyphosate exposure decreases the good bacteria and allows C. difficile to invade. C. difficile produces a toxin called 4-cresol, which we measure in the OAT. Research has shown that 4-cresol inhibits dopamine beta-hydroxylase.13 Dopamine beta-hydroxylase converts dopamine to norepinephrine. In the OAT we measure both homovanillic acid (dopamine metabolite) and vanillylmandelic acid (norepinephrine metabolite). We have observed patients with a high 4-cresol value have elevated homovanillic acid, which indicates an inability to convert dopamine to norepinephrine. The results from our aforementioned patient were consistent with these other results and can be seen in Figure 5. The recommendations for this patient were to treat her glyphosate exposure and to treat her C. difficile infection.
The results from these tests are indicative of why using the Organic Acids Test and Glyphosate Test together is so valuable and can help you provide more focused treatment for your patients. Treatment of glyphosate toxicity should be centered on determining the route of introduction and avoiding future exposure. Eating organic, non-GMO (genetically modified organism) foods and drinking reverse osmosis water are two of the best ways to avoid glyphosate. A recent study showed that people eating organic food had considerably lower concentrations of glyphosate in the urine.7 Drinking extra water may also be beneficial since glyphosate is water soluble, but that water should be filtered to remove pesticides or, ideally, be treated by reverse osmosis. More than 90% of corn and soy used are now of the GMO type. In addition, non-GMO wheat is commonly treated with glyphosate as a drying procedure. Glyphosate is somewhat volatile and a high percentage of rain samples also contained glyphosate.7
High correlations exist between glyphosate usage and numerous chronic illnesses, including autism14. Other disease incidences with high correlations include hypertension, stroke, diabetes, obesity, lipoprotein metabolism disorder, Alzheimer’s, senile dementia, Parkinson’s, multiple sclerosis, inflammatory bowel disease, intestinal infections, end stage renal disease, acute kidney failure, cancers of the thyroid, liver, bladder, pancreas, kidney, and myeloid leukemia.14 Correlations are not causations, yet they raise concern over the use of a chemical to which all life on earth appears to be exposed. Testing for glyphosate along with specific markers in the Organic Acids Test can both help determine the level of exposure to glyphosate and guide you toward the most optimal treatment plans for your patients.
1. Bradberry SM, Proudfoot AT, Vale JA. Glyphosate poisoning. Toxicol Rev. 2004;23(3):159-67.
2. Mesnage R et al. Major pesticides are more toxic to human cells than their declared active principles. Biomed Res Int. 2014: 179691
3. Samsel A, Seneff S. Glyphosate, pathways to modern diseases II: Celiac sprue and gluten intolerance. Interdiscip Toxicol. 2013;6:159-184.
4. Samsel A, Seneff S. Glyphosate, pathways to modern diseases III: Manganese, neurological diseases, and associated pathologies. Surg Neurol Int. 2015; 6: 45.
5. Krüger M, Schledorn P, Schrödl W, Hoppe HW, Lutz W, Shehata AA. Detection of Glyphosate Residues in Animals and Humans. J Environ Anal Toxicol. 2014. 4:2 http://dx.doi.org/10.4172/2161- 0525.1000210
6. Barbosa ER, Leiros da Costa MD, Bacheschi LA, Scaff M, Leite CC. Parkinsonism after glycine-derivative exposure. Mov Disord. 2001. 16: 565-568.
7. Mesnage R, Defarge N, Spiroux de Vendômois J, Séralini GE. Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem Toxicol. 2015 Oct;84:133-53.
8. Guyton KZ, Loomis D, Grosse Y et al. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. Lancet Oncol. 2015 May;16(5):490-1
9. Shehata AA, Schrödl W, Aldin AA, Hafez HM, Krüger M. The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Curr Microbiol. 2013 Apr;66(4):350-8.
10. Jayasumana C, Gunatilake S, Siribaddana S. Simultaneous exposure to multiple heavy metals and glyphosate may contribute to Sri Lankan agricultural nephropathy. BMC Nephrology 2015;16:103. doi 10.1186/s12882-015-0109-2
11. Jayasumana C, Gunatilake S, Senanayake P. Glyphosate, hard water and nephrotoxic metals: Are they the culprits behind the epidemic of chronic kidney disease of unknown etiology in Sri Lanka? Int. J. Environ. Res. Public Health 2014;11:2125-2147.
12. Clair E et al. Effects of Roundup® and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus. Curr Microbiol. 2012;64: 486-491.
13. DeWolf WE Jr. Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides. Biochemistry. 1988;27: 9093-9101.
14. Swanson NL, Leu A, Abrahamson J, and Wallet B. Genetically engineered crops, glyphosate and the deterioration of health in the United States of America. Journal of Organic Systems. 2014; 9(2):6- 37.
15. Environmental Protection Agency. Pesticides Industry Sales & Usage. 2006 and 2007 Market Estimates. Available at https://www.epa.gov/sites/production/files/2015-10/documents/market_ estimates2007.pdf. Accessed July 15, 2015.
16. Shehata AA et al. The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Curr. Microbiol. 2013;66: 350-358.
17. Larsen K et al. Effects of sublethal exposure to a glyphosate-based herbicide formulation on metabolic activities of different xenobiotic-metabolizing enzymes in rats. Int J Toxicol. 2014;33: 307-318.
William Shaw, PhD, is board certified in the fields of clinical chemistry and toxicology by the American Board of Clinical Chemistry. Before he founded the Great Plains Laboratory Inc., Dr. Shaw worked for the Centers for Disease Control and Prevention (CDC), Children’s Mercy Hospital, University of Missouri at Kansas City School of Medicine, and Smith Kline Laboratories. He is the author of Biological Treatments for Autism and PDD, originally published in 1998, and Autism: Beyond the Basics, published in 2009. He is also a frequent speaker at conferences worldwide.
He is the stepfather of a child with autism and has helped thousands of patients and medical practitioners to successfully improve the lives of people with autism, AD(H)D, Alzheimer’s disease, arthritis, bipolar disorder, chronic fatigue, depression, fibromyalgia, immune deficiencies, multiple sclerosis, OCD, Parkinson’s disease, seizure disorders, tic disorders, Tourette syndrome, and other serious conditions.
Matthew Pratt-Hyatt, PhD, received his PhD in cellular and molecular biology from the University of Michigan. He has trained under Dr. Paul Hollenberg, a prominent researcher on drug metabolism, and Dr. Curtis Klaassen, one of the world’s leading toxicologists. He has over a dozen publications in well-known research journals such as the PNAS and Cell Metabolism. He is currently associate laboratory director at the Great Plains Laboratory Inc. in Lenexa, Kansas, focused on diagnosis and treatment of mitochondrial disorders, neurological diseases, chronic immune diseases, and more. He specializes in developing tools that examine factors at the interface between genetics and toxicology. His work is bringing new insight into how genes and toxicants interact and how that may lead to mental health disorders, chronic health issues, and metabolism disorders.
William Shaw, PhD
In response to the inaccurate, unscientific article by Thomas Lodi, M.D. on oxalates1 in the December 2015 issue of Townsend Letter, I will make the following point by point responses:
(1)Cartoons about Popeye.
I will not use any cartoons in my response. Anyone interested in cartoons should immediately stop reading this article and start reading their local paper’s comic section.
The tone for accuracy of the author is set in the very first paragraph of his article in which his first reference, #23, has nothing to do with my green smoothie article, which is reference #24. A better reference would actually be #2 from my article2. When the clock strikes 13, the accuracy of the other 12 hours of the clock is in serious question.
(3)Inaccuracy about the contribution of endogenous production to total oxalate load.
Lodi states that 80-90% of oxalates in the body are endogenously produced. Unfortunately, the best scientific study refutes his assertion. According to Holmes et al3, who did extremely well-controlled studies on every aspect of oxalate metabolism and has publishedforty-one scientific articles on oxalates in the peer reviewed literature, the mean dietary oxalate contribution to total oxalate in the diet is 52.6 % on a high oxalate diet which was defined as a diet of 250 mg oxalate per day. The person drinking a green smoothie with 2 cups of raw spinach ingests 1312 mg of oxalates or over five times the level of what Holmes considers a high-oxalate diet, just in the spinach consumption alone and over 26 times the amount of oxalates in a low oxalate diet (50 mg per day)4. The estimated human production of oxalates is 40 mg per day3. On a green smoothie diet with two cups of spinach, the diet in normal humans contains 33 times the endogenous human production of oxalates just based on the spinach alone.
All of Lodi’s assertions about the benefits of a vegetarian diet are meaningless since there is no single vegetarian diet; there are as many vegetarian diets as there are vegetarians.
(4)Inaccuracy about the availability of calcium and magnesium in spinach.
Lodi states that “every plant, green and otherwise (including spinach) has abundant magnesium and calcium and potassium”. Unfortunately, none of the calcium and magnesium in spinach or other high oxalate plants is bioavailable since it is strongly bound to oxalates. Furthermore, the average oxalate value of spinach is 7.5 times its calcium content, making spinach a very poor choice for someone to maintain adequate calcium stores5. According to Kohmani, who added a good deal of spinach, similar to the diet of a person ingesting a daily green smoothie or a large daily spinach salad, to the diet of rats to determine its effects5:
“If to a diet of meat, peas, carrots and sweet potatoes, relatively low in calcium but permitting good though not maximum growth and bone formation, spinach is added to the extent of about 8% to supply 60% of the calcium, a high percentage of deaths occurs among rats fed between the age of 21 and 90 days. Reproduction is impossible. The bones are extremely low in calcium, tooth structure is disorganized and dentine poorly calcified. Spinach not only supplies no available calcium but renders unavailable considerable of that of the other foods. Considerable of the oxalate appears in the urine, much more in the feces.”
(5)Lodi argues that his patients haven’t complained about kidney stones while drinking a lot of green smoothies so oxalates must not be problematic.
Lodi’s contention that his patients on a high oxalate diet don’t have kidney stones is anecdotal. He presents no data from active chart review of his patients to determine if questions about kidney stones were ever asked. Furthermore, it is doubtful that his patients would have even have connected their diet with their kidney stones. I have had numerous seminars on the connection between oxalates and kidney stones and it is common to get feedback from the audience members that they had kidney stones shortly after starting either a diet including a spinach green smoothie or a large spinach salad on a regular basis. Since these comments were not even solicited, it is likely that even a larger number of individuals may have experienced kidney stones but were shy to voice their experiences. A neurologist friend attributes his recent severely-disabling stroke to the dietary changes encouraged by his wife that placed him on a daily green spinach smoothie for a considerable time.
Furthermore, Lodi seems to think that a lack of kidney stones indicates a lack of oxalate problems. However, oxalates may form in virtually every organ of the body including the eyes, vulva, lymph nodes, liver, testes, skin, bones, gums, thyroid gland, heart, arteries, and muscles6-7. Oxalates may occur in these other organs without appearing in the urinary tract at all and in individuals without genetic hyperoxalurias7. Oxalates have been implicated in heart disease7, stroke, vulvodynia, and autism8-10. Women of child-bearing age need to be especially careful of the spinach green smoothie diet because of the autism oxalate connection and the negative effects of spinach containing oxalates on fertility5. Prisoners in the state prisons in Illinois were encouraged by the Weston-Price Nutrition Foundation to file a lawsuit against the state because of their deteriorating health due to a high amount of soy protein in the prison diet11. Soy protein is tied with spinach as the highest oxalate foods4. Oxalates are especially toxic to the endothelial cells of the arteries, leading to atherosclerosis12. Oxalate crystals are concentrated in the atherosclerotic lesions7. Such lesions have commonly been overlooked by the use of stains of atherosclerotic lesions that make the oxalate crystals difficult to visualize. The relatives of people consuming the green smoothie diet would only know of their loved ones’ oxalate deposits throughout their organs on the day of their autopsies which employed pathological examinations that can detect oxalates.
Primary genetic hyperoxaluria is not the major cause of kidney stones in adults since 80% of individuals died of this disorder before age 20 and it is so rare that it could not possibly be the cause of most cases of oxalate kidney stones13. However, a genetic polymorphism present in up to 20% of Caucasian groups called P11L codes for a protein with three times less activity of alanine: glyoxylate aminotransferase (AGT) than the predominant normal activity polymorphism, leading to excessive endogenous production of oxalates14. This substantial group of individuals would be even more susceptible to the harm of a high oxalate diet. Kidney stones were rampant in the United Kingdom during the World Wars when rhubarb, another high oxalate food, was recommended as a substitute for other low oxalate but unavailable vegetables13.
In summary, those who do not care for their health can eat or drink whatever they want. But they should realize that their diets are fad-based and/or based on quasi-religious ( “feasts” as part of the “awakening” according to Lodi) reasons, not based on hard scientific evidence. Furthermore, they should be aware that their diet may kill them15. The green smoothie fad will go down in medical history with the AMA journal allowing cigarette advertising with physician endorsements and the use of mercury-containing teething powder for babies as one of the greatest health follies in a considerable time.
1. Lodi, T. Green smoothie bliss: Was Popeye secretly on dialysis? Townsend Letter for Doctors. Dec 2015 pgs 28-39
2. Shaw, W. The Green Smoothie Health Fad: This Road to Health Hell is Paved with Toxic Oxalate Crystals. Townsend Letter for Doctors. Jan 2015 Available online at: http://www.townsendletter.com/Jan2015/green0115.html
3. Holmes RP, Goodman HO, and Assimos DG. Contribution of dietary oxalate to urinary oxalate excretion. Kidney International, Vol. 59 (2001), pp. 270–276
4. Harvard T.H. Chan School of Public Health Nutrition Department's File Download Site on oxalates in the diet. https://regepi.bwh.harvard.edu/health/Oxalate/files Accessed December 1,2015
5. Kohmani,EF. Oxalic acid in foods and its fate in the diet. Journal of Nutrition 18(3):233-246,1939
6. Jessica N. Lange, Kyle D.Wood, John Knight, Dean G. Assimos, and Ross P. Holmes. Glyoxal Formation and Its Role in Endogenous Oxalate Synthesis. Advances in Urology Volume 2012, Article ID 819202, 5 pages doi:10.1155/2012/819202
7. G.A. Fishbein, R. G. Micheletti, J. S. Currier, E. Singer, and M. C. Fishbein, Atherosclerotic oxalosis in coronary arteries, Cardiovascular Pathology, vol. 17, no. 2, pp. 117–123, 2008.
8. Giuseppe Di Pasquale, , Mariangela Ribani, Alvaro Andreoli, , Gian Angelo Zampa, and Giuseppe Pinelli, Cardioembolic Stroke in Primary Oxalosis With Cardiac Involvement. Stroke 1989, 20:1403-1406
9. Solomons CC, Melmed MH, Heitler SM.Calcium citrate for vulvar vestibulitis. A case report. J Reprod Med. 1991 Dec;36(12):879-82.
10. Konstantynowicz J, Porowski T, Zoch-Zwierz W, Wasilewska J, Kadziela-Olech H, Kulak W, Owens SC, Piotrowska-Jastrzebska J, Kaczmarski M. A potential pathogenic role of oxalate in autism. Eur J Paediatr Neurol. 2012 Sep;16(5):485-91.
11. Monica Eng, Chicago Tribune reporter. Soy in Illinois prison diets prompts lawsuit over health effects. December 21, 2009. http://articles.chicagotribune.com/2009-12-21/news/0912200121_1_soy-protein-soy-cheeses-soyfoods-association. Accessed December 2,2015
12. RI Levin, PW Kantoff and EA Jaffe Uremic levels of oxalic acid suppress replication and migration of human endothelial cells. Arterioscler Thromb Vasc Biol 1990, 10:198-207
13. A. J. Chaplin Histopathological occurrence and characterization of calcium oxalate: a review. J. Clin. Path., 1977, 30, 800-811
14. Michael J. Lumb and Christopher J. Danpure. Functional Synergism between the Most Common Polymorphism in Human Alanine:Glyoxylate Aminotransferase and Four of the Most Common Disease-causing Mutations. Journal of Biological Chemistry Vol. 275, No. 46, November 17, pp. 36415–36422, 2000
Sanz P, Reig R: Clinical and pathological findings in fatal plant oxalosis. Am J Forensic Med Pathol 13:342–345, 1992
William Shaw, PhD
Calcium is one of the most tightly regulated substances in the body. In addition to the role of calcium as a structural element in bones and teeth (99% of the body’s calcium is in the bones), calcium is critically needed for nerve function. When calcium in the plasma drops about 30%, the person may develop tetany, a condition that is often fatal due to overstimulation of the nerves in both the central nervous system and peripheral nervous system, leading to tetanic contraction of the skeletal muscles. The concentration of calcium in the plasma is one of the most constant laboratory values ever measured. In the great majority of normal people, calcium only varies from 9-11 mg per dL, regardless of the diet (1). The reason is a complex hormonal system that utilizes the bones as a source of calcium. This regulatory system employs the parathyroid gland that secretes parathyroid hormone or parathormone to digest the bones and release calcium when there is only a small decrease in the plasma calcium. Parathormone also increases the absorption of calcium from the gastrointestinal tract and the kidney tubules. When calcium rises in the plasma, parathormone secretion decreases, depositing more calcium in the bones while renal and gastrointestinal absorption are decreased. Calcitonin, a polypeptide hormone produced by the thyroid gland, opposes the effects of parathyroid hormone. In addition, vitamin D increases the absorption of calcium from the gastrointestinal tracts and the kidney tubules like parathyroid hormone but has little effect on digesting bones to release calcium. One of the most controversial and misunderstood topics is what is the optimum nutritional intake of calcium and vitamin D. In the center of the controversy is the role of calcium in the initiation of plaque in the arteries, leading to atherosclerosis and cardiovascular disease.
An average adult ingests about 750 mg per day of calcium and secretes about 625 mg of calcium in the intestinal juices. If all the ingested calcium is absorbed, there would be a net absorption of 125 mg per day of calcium. Since the average person excretes about 125 mg calcium per day in the urine, the average person has a zero net calcium balance except when bone is being deposited. If bone is being deposited due to the stress of exercise or following a fracture, the regulation of the amount of urinary calcium excretion is the major factor to allow for bone growth. One of the major factors that prevents calcium absorption is the presence of high amounts of oxalates in the diet. The human body has the ability to make some oxalate endogenously, perhaps about 40 mg per day in individuals with a favorable genetic makeup. A low oxalate diet contains less than 50 mg per day of oxalates while a high oxalate diet with two cups or more of spinach, nuts, and berries in a smoothie or salad per day could easily contain 1500 mg per day of oxalates. Such high amounts of oxalates readily use up the 125 mg of available calcium, forming insoluble calcium oxalate salts which can deposit in every organ of the body. These deposits can easily initiate endothelial damage that can lead to strokes and myocardial infarctions (heart attacks) and such oxalate deposits have been detected in atherosclerotic lesions. The person on a high oxalate diet will have a much greater need for calcium and/or magnesium than the person on a low oxalate diet.
Since urine is the major controlling element for maintaining calcium balance that is under tight hormonal control, it appears to me that urine calcium is the best indicator of adequate dietary calcium. The most common reasons for low urine calcium are inadequate dietary calcium and/or a high oxalate diet. Other reasons for calcium deficiency include hypoparathyroidism, pseudohypoparathyroidism, vitamin D deficiency, nephrosis, nephritis, bone cancer, hypothyroidism, celiac disease, and malabsorption disorders.
The most common reason for high urine calcium is a diet high in calcium. Other reasons for calcium excess are vitamin D intoxication, hyperparathyroidism, osteolytic bone metastases, myeloma, excessive immobilization, Cushing’s syndrome, acromegaly, distal renal tubular acidosis, thyrotoxicosis, Paget’s disease, Fanconi’s syndrome, schistosomiasis, breast and bladder cancers, and sarcoidosis.
Magnesium is an essential element like calcium and is also in the bones (66% of the body’s magnesium is in the bones). It is a cofactor with many enzymatic reactions especially those requiring vitamin B6. Like extremely low calcium, extremely low magnesium can also cause tetany of the muscles.
The most common reason for low urine magnesium is low magnesium in the diet. Low magnesium in the diet may increase the incidence of oxalate crystal formation in the tissues and kidney stones. Less common causes of low magnesium include celiac disease, other malabsorption disorders, dysbiosis, vitamin D deficiency, pancreatic insufficiency, and hypothyroidism. Early signs of magnesium deficiency include loss of appetite, nausea, vomiting, migraine headaches, fatigue, and weakness. As magnesium deficiency worsens, numbness, tingling, muscle contractions and cramps, seizures, personality changes, anxiety, depression, attention deficit, abnormal heart rhythms, and coronary spasms can occur. Low urinary magnesium for long time periods is associated with increased risk of ischemic heart disease.
The most common reason for high urine magnesium is high magnesium in the diet. Less common causes of high urine magnesium include alcoholism, diuretic use, primary aldosteronism, hyperthyroidism, vitamin D excess, gentamicin toxicity, and cis-platinum toxicity. Increased urinary magnesium excretion can occur in people with insulin resistance and/or type 2 diabetes. Symptoms of marked magnesium excess can include diarrhea, hypotension, nausea, vomiting, facial flushing, retention of urine, ileus, depression, lethargy before progressing to muscle weakness, difficulty breathing, extreme hypotension, irregular heartbeat, and cardiac arrest.
- Guyton, Arthur. Textbook of Medical Physiology,3rd edition. WB Saunders Co, Philadelphia, 1966,pgs1100-1118.
- Fleming, CR, et al. The importance of urinary magnesium values in patients with gut failure. Mayo Clinic Proceedings. 1996 Jan;71(1):21-4.
William Shaw, PhD
Failure to provide adequate calcium to persons on the autistic spectrum is very dangerous and could lead to the loss of the eyes due to severe eye-poking behavior. This is an especially important topic because some individuals like Amy Yasko warns that calcium may cause overstimulation of neurons. Every element in our food and drink including water may cause death with excess intake but you will not find skull and cross-bone warnings on bottled water at the supermarket. The most relevant question is: How much calcium in the diet and in supplements is excessive?
Calcium deficiency can be a severe problem in normal children on a milk-free and dairy-free diet since milk is a significant source of protein, vitamin D, and calcium needed for strong bones and teeth. Some physicians have reported that rickets (1), a severe bone deformity, occurred in children with autism on the gluten and casein-free diet who did not receive added calcium supplements. Calcium and vitamin D supplementation is essential to children on a casein-free diet since most children with autism do not eat substantial amounts of other calcium-rich foods. Failure to provide adequate calcium to children on casein-free diets leads physicians to view such parents as negligent and ignorant and leads to skepticism about other nonstandard treatments for autism.
Children with autism may have an even more severe problem with calcium deficiency. Mary Coleman, M.D. (2) reported that children with autism who are calcium deficient are much more likely to poke out their eyes and a substantial number of children with autism have done so. I have talked to numerous parents of children with autism that began to touch their eyes after starting the casein-free diet. This abnormal behavior is associated with low urine calcium; blood calcium levels were usually normal. Parathyroid hormone, calcitonin, and vitamin D were all normal in patients with autism but all of them had low urine calcium. Treatment with calcium supplementation prevents this behavior but dietary supplementation with high calcium foods does not. (I suspect that this behavior is due to increased eye pain due to high deposits of oxalate crystals in the eye. Oxalates are high in urine samples of children with autism and can deposit in many tissues including the eyes. Low calcium may act to intensify this pain and poking out the eye relieves the pain.) Dr. Coleman also found that speech developed very quickly after calcium supplementation in a portion of mute children with autism who had low urine calcium. In one case, according to a parent who contacted me, her child with autism persisted in poking at the eyes even after one eye had been partially poked out and surgically re-implanted. Calcium supplementation stopped this behavior immediately. I am aware of many other children with eye-poking behavior in which calcium supplements stopped this behavior in less than two days. Verbal autistic children say that their eye pain is severe and that calcium supplementation stopped their pain quickly. In Coleman’s study of 78 children with autism, 20% had urine calcium values two standard deviations below the normal child’s range for urine calcium. Clearly, this extremely low group requires supplementation with calcium. I would recommend calcium supplementation for any child below the mean value urine calcium for normal children of the same age.
Magnesium research in autism is often combined with research on vitamin B6 since the two nutritional factors work together in a host of biochemical reactions. In one study in France (4), children on the autistic spectrum were given 6 mg per kilogram of body weight per day of magnesium and 0.6 mg per kilogram body weight of vitamin B6. This supplementation improved autistic symptoms including the following: social interactions (23/33), communication (24/33), stereotyped restricted behavior (18/33), and abnormal/delayed functioning (17/33). When the Mg-B6 treatment was stopped, autistic symptoms reappeared in a few weeks. Low magnesium levels may be associated with restlessness, sensitivity to noise, poor attention span, poor concentration, irritability, aggressiveness, and anxiety.
From a parent- “Our daughter also used to look in the mirror all the time - really up close and wanting to look at herself and poke her eyes. I was so worried about it that I finally put pepper juice on her fingers so she would stop. I know that sounds awful - but she had really gotten bad. Dr. Shaw said that their eyes are hurting so much from lack of calcium. He recommended 1000mg. daily - our daughter was about 43 pounds at the time. I started giving it to her and her eye poking stopped and I noticed that so many of her other stimming behaviors also decreased.”
1. Hediger ML, England LJ,Molloy CA, Yu KF, Manning-Courtney P, Mills JL. Reduced bone cortical thickness in boys with autism or autism spectrum disorder. J Autism Dev Disord. 2008;38(5):848–856
2. Coleman, M. Clinical presentations of patients with autism and hypocalcinuria. Develop. Brain Dys. 7: 63-70, 1994
3. Caudarella R, Vescini F, Buffa A, Stefoni S. Citrate and mineral metabolism: kidney stones and bone disease. Front Biosci. 2003 Sep 1;8:s1084-106.
4. M. Mousain-Bosc et al Improvement of neurobehavioral disorders in children supplemented with magnesium-vitamin B6 II. Pervasive developmental disorder-autism. Magnesium Research 2006;19(1): 53-62
5. Fleming, CR, et al. The importance of urinary magnesium values in patients with gut failure. Mayo Clinic Proceedings. 1996 Jan;71(1):21-4
William Shaw Ph.D
Immunoglobulin G (IgG) food allergy testing has made vast advancements since the year 2003 when the American Academy of Allergy, Asthma, and Immunology published a statement that "Measurement of specific IgG antibodies to foods is also unproven as a diagnostic tool"(1) Most of the IgG food allergy throughout the world is done using the same immunochemical technique. First, soluble food proteins in solution are reacted to a solid phase that chemically binds to a variety of proteins. The use of plastic microtiter trays with one to several hundred wells has become the most common material used as the solid phase. Then these trays are washed, dried, and stored for later use. A sample of diluted serum is then added to each of the wells. Antibodies of all types in the diluted serum bind to the specific food molecules that are attached to the plastic wells of the tray. Next, the plates are washed to remove any nonspecific antibodies in the diluted serum. At this time, food antibodies from all of the five major immunoglobulin classes called G, A, M, E, and D may be attached to the food antigens on the plate. The next step confers specificity on the assay. Antisera from sheep, goats, rabbits, or other animals that specifically binds to IgG is added to microtiter wells and only binds to IgG, not to IgA, IgM, IgE, or IgD. This antibody to IgG has previously been modified by the attachment of an enzyme that can be measured conveniently. The amount of enzyme bound to food antigen-IgG complexes on the plate is directly related to how much IgG antibody is attached to a given food. The overall technique is termed Enzyme Linked Immuno Assay or ELISA. If IgG4 is measured, an antiserum specific for IgG4 only must be used for the final step.
The clinical usefulness of IgG testing in an array of illnesses is illustrated in an early article published by an otolaryngologist who reported that the majority of his patients had substantial health improvements after an elimination of foods positive by IgG food allergy tests (2). The overall results demonstrated a 71% success rate for all symptoms achieving at least a 75% improvement level. Of particular interest was the group of patients with chronic, disabling symptoms, unresponsive to other intensive treatments. Whereas 70% obtained 75% or more improvement, 20% of these patients obtained 100% relief. Symptoms most commonly improved 75%-100% on the elimination diets included asthma, coughing, ringing in the ears, chronic fatigue, all types of headaches, gas, bloating, diarrhea, skin rash and itching, and nasal congestion. The most common IgG food allergies were cow's milk, garlic, mustard, egg yolk, tea, and chocolate.
The usefulness of IgG food allergy to design customized elimination diets has now been documented in scientific studies. Irritable bowel syndrome (IBS) is a common, costly, and potentially disabling gastrointestinal (GI) disorder characterized by abdominal pain/discomfort with altered bowel habits (e.g., diarrhea, constipation). The major symptoms of IBS are (1) abnormality of bowel movement, (2) reduction in bowel sensitivity thresholds, and (3) psychological abnormality. Many IBS patients have psychological symptoms including depression, anxiety, tension, insomnia, frustration, hypochondria. psychosocial factors (3). Atkinson et al (4)evaluated a total of 150 outpatients with irritable bowel syndrome (IBS) who were randomized to receive, for three months, either a diet excluding all foods to which they had raised IgG antibodies (ELISA test) or a sham diet excluding the same number of foods but not those to which they had antibodies . Patients on the diet dictated by IgG testing had significantly less symptoms than those on the sham diet after 120 days on the diets. Patients who adhered closely to the diet had a marked improvement in symptoms while those with moderate or low adherence to the IgG test dictated diets had poorer response. Similar results were also obtained by Drisko et al (5). They used both elimination diet and probiotic treatment in an open label study of 20 patients with irritable bowel syndrome diagnosed at a medical school gastroenterology department. The most frequent positive serologic IgG antigen-antibody complexes found on the food IgG tests were: baker's yeast, 17 out of 20 (85%); onion mix, 13 out of 20 (65%); pork, 12 out of 20 (60%); peanut 12 out of 20 (60%); corn, 11 out of 20 (55%);wheat, 10 out of 20 (50%); soybean, 10 (50%); carrot, 9 out of 20 (45%); cheddar cheese, 8 out of 20 (40%); egg white, 8 out of 20 (40%). Only 5 out of 20 reacted by IgG antibody production to dairy; however the majority of patients reported eliminating dairy prior to trial enrollment presumably clearing antigen-antibody complexes prior to testing. Significant improvements were seen in stool frequency, pain, and IBS quality of life scores. Imbalances of beneficial flora and dysbiotic flora were identified in 100% of subjects by comprehensive stool analysis. There was a trend to improvement of beneficial flora after treatment but no change in dysbiotic flora. The one-year follow up demonstrated significant continued adherence to the food rotation diet, minimal symptomatic problems with IBS, and perception of control over IBS. The continued use of probiotics was considered less helpful.
IgG food allergy testing was also proved effective in the gastrointestinal disorder Crohn's disease. Bentz et al (6) found that an elimination diet dictated by IgG food allergy testing resulted in a marked reduction of stool frequency in a double blind cross-over study in which the IgG-dictated diet was compared to a sham diet in 40 patients with Crohn's disease. IgG food allergies were significantly elevated compared to normal controls. Cheese and baker's yeast (Saccharomyces cerevisiae) allergies were extremely common with rates of 83% and 84% respectively. Main et al (7), focusing on the baker's yeast allergy, also found extremely high prevalence of IgG allergy in patients with Crohn's disease. Titers of both IgG and IgA to S. cerevisiae in the patients with Crohn's disease were significantly higher than those in the controls. In contrast, antibody titers in the patients with ulcerative colitis were not significantly different from those in the controls. Among the patients with Crohn's disease there was no significant difference in antibody titers between patients with disease of the small or large bowel. Since IgG antibodies to S. cerevisiae cross react with Candida albicans (8), Candida species colonization might be a trigger for the development of Crohn's disease.
IgG food allergy to wheat, gluten, gliadin, rye, and barley are prevalent in the gastrointestinal disorder celiac disease. Virtually all patients with celiac disease have elevated IgG antibodies to gliadin if they currently have wheat or related grains in their diet. The confirmation of celiac disease is confirmed by the presence of flattened mucosa with a lack of villi when a biopsy sample of the small intestine is examined microscopically. Another confirmation test with equal sensitivity is a blood test for IgA transglutaminase antibodies. The antibody confirmation test is equal in accuracy to the biopsy test with the exception that individuals with IgA deficiency may have false negative results. However, I would estimate that only 1% of people with elevated IgG antibodies to gliadin and other grains related to wheat have celiac disease. If the individual is negative for the confirmation tests for celiac disease, many patients are frequently erroneously advised that that have no problem with wheat. Hadjivassiliou et al argued that it is a significant clinical error to classify wheat allergy through the filter of celiac disease (9) and argue that celiac disease is a subtype of wheat sensitivity. Many of their patients with wheat allergy but celiac disease negative had remission of severe neurological illnesses when they adopted a gluten free diet and expressed that in these patients the gluten molecule causes an autoimmune reaction in the brain rather than in the intestinal tract, likely against the Purkinje cells that are predominant in the cerebellum.
A wide range of additional studies has proven the clinical value of IgG antibodies in autism (10), bipolar depression (11), schizophrenia (12), migraine headaches (13), asthma (14), and obesity (15).
Total IgG Versus IgG4 Food Allergy
Immunoglobulin G (IgG) is classified into several subclasses termed 1, 2, 3, and 4. IgGs are composed of two heavy chain–light chain pairs (half-molecules), which are connected via inter–heavy chain disulfide bonds situated in the hinge region (Figure 1). IgG4 antibodies usually represent less than 6% of the total IgG antibodies. IgG4 antibodies differ functionally from other IgG subclasses in their lack of inflammatory activity, which includes a poor ability to induce complement and immune cell activation because of low affinity for C1q (the q fragment of the first component of complement). Consequently, IgG4 has become the preferred subclass for immunotherapy, in which IgG4 antibodies to antigens are increased to reduce severe antigen reactions mediated by IgE. If antigens preferentially react with IgG4 antibodies, the antigens cannot react with IgE antibodies that might cause anaphylaxis or other severe reactions. Thus, IgG4 antibodies are often termed blocking antibodies. Another property of blood-derived IgG4 is its inability to cross-link identical antigens, which is referred to as "functional monovalency". IgG4 antibodies are dynamic molecules that exchange half of the antibody molecule specific for one antigen with a heavy-light chain pair from another molecule specific for a different antigen, resulting in bi-specific antibodies that are unable to form large cross-linked antibodies that bind complement and thus cause subsequent inflammation(16). In specific immunotherapy with allergen in allergic rhinitis, for example, increases in allergen-specific IgG4 levels indeed correlate with improved clinical responses. IgG4 antibodies not only block IgE mediated food allergies but also block the reactions of food antigens with other IgG subclasses, reducing inflammatory reactions caused by the other IgG subclasses of antibodies to food antigens.
In IgG mediated food allergy testing, the goal is to identify foods that are capable of causing inflammation that can trigger a large number of adverse reactions. IgG1, IgG2, and IgG3 all are capable of causing inflammation because these antibodies do not exchange heavy and light chains with other antibodies to form bispecific antibodies. Thus, IgG1, IgG2, and IgG3 antibodies to food antigens can and do form large immune complexes or lattices that fix complement and increase inflammation. The presence of IgG4 antibodies to food antigens indicates the presence of antibodies to foods that will not usually cause inflammation even though high amounts of these antibodies do indicate the presence of immune reactions against food antigens. Testing only for IgG4 antibodies in foods limits the ability of the clinician to determine those foods that are causing significant clinical reactions that are affecting their patients. The importance of measuring other subtypes of IgG antibodies is highlighted in an article by Kemeny et al. (17). They found that IgG1 antibodies to gluten were elevated in all 20 patients with celiac disease but none of the patients had elevated IgG4 antibodies to gluten.
- 1. Statement of the AAAAI Work Group Report: Current Approach to the Diagnosis and Management of Adverse Reactions to Foods, October 2003. http://www.aaaai.org/ask-the-expert/usefulness-of-measurements-of-IgG-antibody.aspx (Accessed October 27,2013).
- 2. Dixon H, Treatment of delayed food allergy based on specific immunoglobulin G RAST testing relief. Otoloryngol Head Neck Surg 2000;123:48-54.
- 3. Nagisa Sugaya N and Nomura S, Relationship between cognitive appraisals of symptoms and negative mood for subtypes of irritable bowel syndrome. BioPsychoSocial Medicine 2008;2:9-14
- 4.Atkinson, W et al. Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial Gut 2004;53:1459-1464
- 5. Drisko J, Bischoff B, Hall M, McCallum R, Treating Irritable Bowel Syndrome with a Food Elimination Diet Followed by Food Challenge and Probiotics. Journal of the American College of Nutrition 2006; 25: 514–522
- 6. Bentz S, et al. Clinical relevance of IgG antibodies against food antigens in Crohn's disease: a double-blind cross-over diet intervention study. Digestion. 2010;81:252-64.
- 7.Janice Main, Hamish McKenzie, Grant R Yeaman, Michael A Kerr, Deborah Robson, Christopher R Pennington, David Parratt Antibody to Saccharomyces cerevisiae (bakers' yeast) in Crohn's disease BMJ 1988;297:1105-1106
- 8. Thomas Schaffer, Stefan Mueller, , Beatrice Flogerzi, , Beatrice Seibold-Schmid,Alain M. Schoepfer, and Frank Seibold Anti-Saccharomyces cerevisiae Mannan Antibodies (ASCA) of Crohn's Patients Crossreact with Mannan from Other Yeast Strains, and Murine ASCA IgM Can Be Experimentally Induced with Candida albicans Inflamm Bowel Dis 2007;13:1339 –1346
- 9. M Hadjivassiliou, R A Grünewald, G A B Davies-Jones Gluten sensitivity as a neurological illness. Neurol Neurosurg Psychiatry 2002;72:560–563
- 10. Vladimir T et al Higher Plasma Concentration of Food-Specific Antibodies in Persons With Autistic Disorder in Comparison to Their Siblings. Focus Autism Other Dev Disabl 2008; 23: 176-185
- 11. Severance EG et al Immune activation by casein dietary antigens in bipolar disorder. Bipolar Disord 2010;12: 834–842
- 12. Severance EG, et al Subunit and whole molecule specificity of the anti-bovine casein immune response in recent onset psychosis and schizophrenia. Schizophr Res. 2010;118:240-7.
- 13.Huber A, et al Diet restriction in migraine, based on IgG against foods: a clinical double-blind, randomised, cross-over trial. Int Arch Allergy Immunol. 1998; 115:67-72.
- 14.Vance G. et al. Ovalbumin specific immunoglobulin G and subclass responses through the first five years of life in relation to duration of sensitization and the development of asthma. Clia Exp Allergy 2004;34:1452-1459
- 15.Wilders-Truschnig M et al. IgG Antibodies Against Food Antigens are Correlated with Inflammation and Intima Media Thickness in Obese Juveniles. Exp Clin Endocrinol Diabetes 2008;116:241-5.
- 16. Marijn van der Neut Kolfschoten, et al Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange. Science 2007;317:1554-1555
- 17. Kemeny DM, et al Sub-class of IgG in allergic disease. I. IgG sub-class antibodies in immediate and non-immediate food allergy. Clin Allergy. 1986; 16:571-81.
William Shaw, PhD.
Recent internet news indicated the conviction of an oncologist who attempted to kill her boyfriend who was involved with another woman. The weapon of choice was ethylene glycol, popularly known as antifreeze, which had been placed in his coffee just after coitus. Although emergency measures saved the boyfriend's life, extensive deposits of oxalate crystals, the main toxic metabolite of ethylene glycol, had caused extensive kidney and liver damage, reducing the man's lifespan by about half.
Similar results in sabotaging your own health can occur through the regular consumption of a popular concoction called a "green smoothie". A recent Google search for "green smoothie" yielded 609,000 hits. In addition, a recent "improving your diet" seminar I attended promoted this same idea. Interestingly, on the same day, I reviewed test results of a urine organic acid test of a woman with oxalate values three times the upper limit of normal. A conversation with the patient indicated that she had recently turned to consuming daily "green smoothies" to "clean up her diet". The most common "green" components of the most popular green smoothies are spinach, kale, Swiss chard, and arugula. Each of these greens is loaded with oxalates. A typical internet recipe advises that two cups of packed raw spinach leaves is a good starting point for a good smoothie. In addition to the high oxalate greens added to the blender, green smoothie proponents frequently recommend adding a variety of berries or almonds, also containing high oxalate amounts. Similar high urine oxalate results were found in organic acid tests of a number of patients with kidney stones who had decided to eat large spinach salads daily as a "move to clean up my unhealthy diet". Unfortunately kidney stones are not the only health problems that people who regularly consume green smoothies and large spinach salads will experience with their new "clean" diet.
Seventy-five years ago, a food scientist of the Campbell Soup Company (1) reported: "Only a few foods, notably spinach, Swiss Chard, New Zealand spinach, beet tops, lamb's quarter, poke, purslane, and rhubarb have high oxalate content. In them, expressed as anhydrous oxalic acid, it is often considerably over 10% on a dry basis. In fifty-three samples, including practically all commercial and many experimental varieties grown in California and in Maryland as well as those shipped from Texas, Florida and Carolina, the average anhydrous oxalic acid content was 9.02% on the dry basis (maximum 12.6, minimum 4.5). Whereas spinach greatly increases the calcium content of the low calcium but well performing basal diet, it decidedly interferes with both growth and bone formation. If to a diet of meat, peas, carrots and sweet potatoes, relatively low in calcium but permitting good though not maximum growth and bone formation, spinach is added to the extent of about 8% to supply 60% of the calcium, a high percentage of deaths occurs among rats fed between the age of 21 and 90 days. Reproduction is impossible. The bones are extremely low in calcium, tooth structure is disorganized and dentine poorly calcified. Spinach not only supplies no available calcium but renders unavailable a considerable amount of the calcium in the other foods. Considerable amounts of the oxalate appear in the urine, much more in the feces."
The author also discovered that in addition to leading to excessive death and defective reproduction in the rats, high oxalate foods also cause soft and pliable bones and defective teeth.
Oxalate and its acid form oxalic acid are organic acids that come from three sources: the diet, fungus infections such as Aspergillus and Penicillium and possibly Candida (2-10), and also human metabolism (11).
Oxalic acid is the most acidic organic acid in body fluids and is used commercially to remove rust from car radiators. Antifreeze (ethylene glycol) is toxic primarily because it is converted to oxalate. Two different types of genetic diseases are known in which oxalates are high in the urine. The genetic types of hyperoxalurias (type I and type II) can be determined from the organic acid test done at The Great Plains Laboratory. Foods especially high in oxalates include spinach and similar leafy vegetables, beets, chocolate, soy, peanuts, wheat bran, tea, cashews, pecans, almonds, berries, and many others. Oxalates are not found in meat or fish at significant concentrations. Daily adult oxalate intake is usually 80-120 mg/d but it can range from 44-1000 mg/d in individuals who eat a typical Western diet. I estimate that the person who consumes a green smoothie with two cups (about 150 grams) of spinach leaves is consuming about 15 grams or 15,000 mg of oxalates or about 150 times the average daily oxalate intake. A complete list of high oxalate foods is available on the Internet at http://www.upmc.com/patients-visitors/education/nutrition/pages/low-oxalate-diet.aspx
High oxalate in urine and plasma was first found in people who were susceptible to kidney stones. Most kidney stones are composed of calcium oxalate. Stones can range in size from the diameter of a grain of rice to the width of a golf ball. It is estimated that 10% of males may have kidney stones some time in their lives. Because many kidney stones contain calcium, some people with kidney stones think they should avoid calcium supplements. However, the opposite is true. When calcium and magnesium are taken with foods that are high in oxalates, oxalic acid in the intestine combines with these minerals to form insoluble calcium and magnesium oxalate crystals that are eliminated in the stool. These forms of oxalate cannot be absorbed into the body. When calcium and/or magnesium are low in the diet, oxalic acid is soluble in the liquid portion of the contents of the intestine (called chyme) and is readily absorbed from the intestine into the bloodstream. If oxalic acid is very high in the blood being filtered by the kidney, it may combine with calcium and other metals, including heavy metals like lead and mercury to form crystals that may block urine flow, damage the kidney, and cause severe pain. These oxalate crystals can also form in the bones, skin, joints, eyes, thyroid gland, blood vessels, lungs, and even the brain (11-14). Oxalate crystals in the bone may crowd out the bone marrow cells, leading to anemia and immunosuppression (14). In addition to individuals with autism and kidney disease, individuals with fibromyalgia and women with vulvar pain (vulvodynia) may also suffer from the effects of excess oxalates (15-18).
Recent evidence also points to the involvement of oxalates in stroke, atherosclerosis, and in endothelial cell dysfunction (19-21). High amounts of oxalates were found concentrated in atherosclerotic lesions of the aortas and coronary arteries of a number of individuals at autopsy. These individuals did not have oxalate deposits in the kidney but did have oxalate deposits in other organs such as the thyroid gland and testis. Since the stains used by most pathologists examining atherosclerotic lesions cannot readily determine the presence of oxalates in diseased arteries, it seems possible that this cause of atherosclerosis may be much more common than previously realized. I suspect that oxalates are a much more common cause of atherosclerosis than high cholesterol. Furthermore, since ethylenediaminetetraacetic acid (EDTA) is effective in the removal of oxalate crystals deposited in the tissues (22,23), the benefits of intravenous EDTA in the treatment of cardiovascular disease may be mediated largely by the removal of oxalate crystals and their associated heavy metals from the tissues in which they are deposited.
Oxalate crystals may cause damage to various tissues due to their sharp physical structure and they may increase inflammation. Iron oxalate crystals may also cause significant oxidative damage and diminish iron stores needed for red blood cell formation (11). Oxalates may also function as chelating agents and may chelate many toxic metals such as mercury and lead. However, unlike common chelating agents like EDTA and DMSA that cause metals to be excreted, a reaction of oxalate with heavy metals like mercury and lead leads to the precipitation of the heavy metal oxalate complex in the tissues, increasing the toxicity of heavy metals by delaying their excretion (24).
What steps can be taken to control excessive oxalates?
- Use antifungal drugs to reduce yeast and fungi that may be causing high oxalates. Children with autism frequently require years of antifungal treatment. I have noticed that arabinose, a marker indicating yeast/fungal overgrowth on the organic acid test at The Great Plains Laboratory, is correlated with high amounts of oxalates (Figure 1). Candida albicans produces high amounts of the enzyme collagenase (25), which breaks down collagen in the gastrointestinal tract to form the amino acid hydroxyproline, which in a series of reactions is converted to oxalates, especially in people with low vitamin B6. Candida organisms have also been found surrounding oxalate stones in the kidney (10).
- Give supplements of calcium citrate and magnesium citrate to reduce oxalate absorption from the intestine. Citrate is the preferred calcium form to reduce oxalate because citrate also inhibits oxalate absorption from the intestinal tract. The best way to administer calcium citrate would be to give it with each meal. Children over the age of 2 need about 1000 mg of calcium per day. Of course, calcium supplementation may need to be increased if the child is on a milk-free diet. The most serious error in adopting the gluten-free, casein-free diet is the failure to adequately supplement with calcium.
- Give chondroitin sulfate to prevent the formation of calcium oxalate crystals (26).
- Vitamin B6 is a cofactor for one of the enzymes that degrades oxalate in the body and has been shown to reduce oxalate production (27).
- Consume a low-oxalate diet, avoiding high-oxalate foods such as leafy greens, beans, berries, nuts, tea, chocolate, wheat germ, and soy. Dr. Clare Morrison, a general practitioner from the U.K. who has fibromyalgia found relief from symptoms after changing to a low-oxalate diet. In a 2012 article in the Daily Mail, she said, "I cut these out of my diet and overnight my symptoms disappeared — the disabling muscle pains, tingling legs, fatigue and inability to concentrate all went" (28).
- Increase water intake to help eliminate oxalates.
Measuring oxalate toxicity
The organic acid test (Table 1) is one of the best measures for determination of both genetic and nutritional factors that lead to toxic oxalates. The organic acid test includes two additional markers, glycolic and glyceric acids, that are markedly elevated in genetic causes of excessive oxalate, the hyperoxalurias I and II. In addition, the organic acid test includes factors such as high fungal and Candida markers that make oxalate (fungus) or their precursors (Candida). Finally, although vitamin C poses little risk of excess oxalates at doses up to 2000 mg per day, I have measured marked increases in oxalates (more than ten times the upper limit of normal) in a child with impaired kidney function after a 50,000 mg intravenous vitamin C megadose. The organic acid test also includes the main vitamin B6 metabolite pyridoxic acid that diminishes the body's own production of oxalates.
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- Fomina M, Hillier S, Charnock JM, Melville K, Alexander IJ, Gadd GM. Role of oxalic acid overexcretion in transformations of toxic metal minerals by Beauveria caledonica. Appl Environ Microbiol. (2005) Jan;71(1):371-81.
- Ruijter GJG, van de Vondervoort PJI, Visser J. Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese. Microbiology (1999) 145, 2569–2576.
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- Kaminishi H, Hagihara Y, Hayashi S, Cho T. Isolation and characteristics of collagenolytic enzyme produced by Candida albicans. Infect Immun. (1986) August; 53(2): 312–316.
- Shirane Y, Kurokawa Y, Miyashita S, Komatsu H, Kagawa S. Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals by atomic force microscopy. Urol Res. (1999) Dec; 27(6):426-31.
- Chetyrkin SV, Kim D, Belmont JM, Scheinman JI, Hudson BG, Voziyan PA. Pyridoxamine lowers kidney crystals in experimental hyperoxaluria: a potential therapy for primary hyperoxaluria. Kidney Int. (2005) Jan;67(1):53-60.
- Morrison C. Ditch healthy berries to beat muscle pain: The eating plan that helped me cure my aches and pains. The Daily Mail Online. August 13, 2012. http://www.dailymail.co.uk/health/article-2187890/Ditch-healthy-berries-beat-muscle-pain-The-eating-plan-helped-cure-aches-pains.html. (Accessed November 21, 2014)
William Shaw, Ph.D., Lab Director and Matt Pratt-Hyatt, Ph.D., Associate Lab Director
The Great Plains Laboratory is excited to announce a new test for PLA2 activity, and we are the only commercial lab currently offering this particular test in urine. PLA2 is elevated in a wide range of inflammatory disorders from multiple sclerosis to cancer. This test can be easily added to the organic acids test to provide powerful new clinical insights and treatments for a variety of serious illnesses.
Chronic diseases are caused by many different biological imbalances, but they almost all create and have inflammation as a cornerstone. Inflammation plays a major part in most of the disorders that we spend billions of dollars to combat, searching for relief from the pain, swelling, and other symptoms that inflammation causes. Inflammation is the immune system's natural response to infection and injury. Phospholipase A2 (PLA2) is one of the key biochemical factors produced in the inflammation response. It is commonly found in human tissues, as well as insect and snake venom. In normal amounts, PLA2 is involved in remodeling cell membranes and changing cell architecture. In infections, PLA2 can break down the phospholipids in the membranes of bacteria, fungi, and parasites leading to their death. However, inflammation, like many other biological processes often has negative effects. The same phospholipase that attacks infectious agents may also attack the cell membranes of the human host, damaging or killing those cells. In addition, the products of the PLA2 reaction, lysolecithins and free fatty acids (Figure 1) are powerful detergents that have the ability to denature proteins and destroy their biological functions. The lysolecithins produced by PLA2 initiate the pain response.
The most common free fatty acid produced by PLA2 is arachidonic acid which can increase the production of powerful mediators of inflammation called prostaglandins, leukotrienes, and thromboxanes, collectively known as eicosanoids. These mediators play an important role in the generation and maintenance of inflammation in neural cells. In addition, arachidonic acid can be converted to 4-hydroxynonenal (4-NE), which can be very toxic due to covalent modification of important biomolecules including proteins, DNA, and phospholipids containing amino groups. In addition to PLA2 causing local damage, it may be transported by the blood vessels to other parts of the body, causing widespread tissue damage.
Diseases Associated with PLA2
Increased levels of PLA2 have been observed in most systemic inflammatory diseases. Studies have linked elevated PLA2 activity with multiple sclerosis, rheumatoid arthritis, Crohn's disease, pancreatitis, ulcerative colitis, allergies, atherosclerosis and cardiovascular disease, lung, prostate, small intestine, and large intestine cancers, with increased susceptibility to metastases, Candida infection, asthma, autism, chronic pulmonary obstructive disorder (COPD), and sepsis.
What Causes Elevated PLA2?
Phospholipase A2 is produced by the pancreas and released into the small intestine following a fatty meal. Infection or trauma of the pancreas may result in the release of phospholipase into the circulation, causing widespread damage or even death. Activation by viruses of proenzymes of PLA2 within the pancreas instead of, as normally, in the intestine, may cause pancreatitis. Phosholipase may be produced by cells of the immune system in response to bacterial antigens, especially those containing certain lipopolysaccharides (LPS). Allergies, especially those to house dust and cats, have been implicated as a trigger for PLA2 synthesis and release. Venoms from snakes, spiders, and bees contain high amounts of PLA2, which is responsible for much of the toxicity of these venoms. In addition, microorganisms such as Candida albicans and certain Clostridia species produce PLA2 which increases the ability of the microorganism to infect the host. Trauma may also cause significant increases in PLA2 and result in brain injury.
PLA2 and Inflammatory Disease
Research has implicated PLA2 in the pathophysiology of neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer's disease (AD). Multiple sclerosis involves both antigen-specific mechanisms and components of the innate immune system that result in inflammatory response. Elevated PLA2 activity was found to be ongoing among MS patients, with the highest levels measured in patients with progressive disease. In the development of Alzheimer's disease, the abnormal PLA2 levels appear to be related to oxidative signaling pathways involving NADPH oxidase and production of ROS species that lead to impairment and destruction of neurons and inflammation of glial cells.
Inflammation is the hallmark of rheumatoid arthritis (RA), a joint-destructive autoimmune disease. PLA2 is found in synovial fluid of RA-affected individuals and in the cartilage of RA patients as compared to cartilage from osteoarthritic and normal individuals.
Measurement of PLA2 is emerging as an important tool for evaluating the chance of cardiovascular disease (CVD), including future stroke, myocardial infarction, heart failure, and other vascular events. PLA2 appears to be more specific than hsCRP for CVD risk and may also have a pivotal role as a mediator of cardiovascular pathology. In atherosclerosis, PLA2 not only activates macrophages and formation of foam cells, but it also hydrolyzes LDL and HDL, spawning increased numbers of pro-atherogenic small LDL particles, and impairing anti-atherogenic HDL. PLA2 activity may even precipitate bleeding from atherosclerotic plaques.
PLA2 is expressed normally in pancreatic, gall bladder, and GI epithelial cells, but is significantly increased in inflammatory gastrointestinal disorders. In ulcerative colitis and Crohn's disease, all intestinal cell types increase expression of PLA2, which increases gut permeability and may actually contribute to infectivity.
PLA2 and Cancer
Elevations of PLA2 have been found in gastrointestinal cancers including colonic adenomas and carcinomas and pancreatic ductogenic carcinomas, among others. Patients with lung tumors positive for PLA2 had a greatly increased tumor growth rate and a markedly reduced survival rate. Patients with lung cancer also had higher plasma levels of PLA2 than patients with benign nodules. A similar pattern has been observed in prostate cancer, although metastatic tumors expressed lower PLA2 than primary tumors. As PLA2 releases arachidonic acid and other fatty acids from cell membranes, they initiate downstream production of tumor-promoting eicosanoids. In cancer, the spread of tumor cells from a primary tumor to the secondary sites within the body is a complicated process involving cell proliferation and migration, degradation of basement membranes, invasion, adhesion, and angiogenesis. Continued research on PLA2 expression in cancer will certainly reveal valuable new insights.
What lowers PLA2?
There has been a great deal of research done by both academia and pharmaceutical companies to find chemical inhibitors to PLA2. However, there has also been research on more natural methods for inhibiting PLA2. Glucocorticoids such as the natural hormone cortisol and pharmaceutical agents such as dexamethasone inhibit the production of phospholipase, decreasing harm caused by the enzyme but also decreasing the benefits of the enzyme in killing harmful microorganisms. Thus, excess glucocorticoids can reduce inflammation in a patient with tuberculosis while reducing the effects of PLA2 against the bacteria resulting in spread of the illness. Lithium at pharmacological doses, carbamazepine, and the antimalarial drug chloroquine are all PLA2inhibitors. Vitamin E is also an inhibitor of PLA2. In addition, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) (belonging to the omega-3 class of fatty acids) inhibit PLA2. Analysis has shown that treatment with supplements of Cytidine 5'-Diphosphocholine (CDP-choline) can limit the ability of PLA2 to promote inflammation. CDP-choline is a precursor in the formation of phospholipids and has been used as a nutritional supplement at doses ranging from 500-4000 mg per day in the treatment of patients with a variety of disorders including Parkinson's disease, memory disorders, vascular cognitive impairment, vascular dementia, senile dementia, schizophrenia, Alzheimer's disease (especially effective in those with the epsilon-4 apolipoprotein E genotype), head trauma, and ischemic stroke. A trial in patients with Alzheimer's disease indicated that CDP-choline (1,000 mg/day) is well tolerated and improves cognitive performance, cerebral blood perfusion, and the brain bioelectrical activity pattern. No side effects were noticed at the lower doses of CDP-choline and only some mild gastrointestinal symptoms were found using higher doses. No abnormal blood chemistry or hematology values were found after the use of CDP-choline.
Testing for PLA2
Because PLA2 is a relatively small enzyme (about 14 KD), it is able to be excreted in urine. 10 mL of the first morning urine before food or drink is suggested for testing. There are no dietary restrictions. This test is convenient to include with other urine tests such as organic acids, amino acids, and peptides. Since chelating agents might interfere with the test, they should not be used for at least 48 hours prior to testing. PLA2 testing is recommended for the following disorders:
- Multiple sclerosis
- Rheumatoid arthritis
- Crohn's disease
- Ulcerative colitis
- Cardiovascular disease including atherosclerosis
- Neurodegenerative diseases
- Bipolar depression, subtype with psychosis
- Candida infection
- Long term depression
- Chronic obstructive pulmonary disease (COPD)
Inflammation plays such a key role in so many diseases, and we believe this new PLA2 test will be a valuable tool in the treatment of patients suffering from numerous disorders. The test is now available and we hope you will integrate it into your practice. For more information about PLA2 and possible treatments, please see the references below.
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William Shaw, Ph.D.
Continued research at The Great Plains Laboratory has resulted in new information on Clostridia bacteria markers that will soon be available for the urine organic acid test. New information will soon be available for the organic acid interpretations of 3 (3 hydroxyphenyl)-3 hydroxypropionic acid (HPHPA), 4-hydroxyphenylacetic acid, phenyllactic acid, and 3-indoleacetic acid at the beginning of 2015.
In addition, this article will help to clarify information about the increased value of organic acid testing compared to stool testing for assessing Clostridia species.
First, the species that are the major producers of the precursors of HPHPA have been identified and include C. botulinum, C. sporogenes, and C.caloritolerans. (It is common to use the abbreviation for the Clostridia genus "C" when giving the genus and species designation.)
C. botulinum is a gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce the neurotoxin botulinum. The botulinum toxin can cause a severe flaccid paralytic disease in humans and animals and is the most potent toxin known to humankind (natural or synthetic) with a lethal dose of less than 1 μg (microgram) in humans. Symptoms of botulism include weakness, trouble seeing, feeling tired, and trouble speaking. This may then be followed by weakness of the arms, chest muscles, and legs. In food borne botulism, symptoms generally begin 18 to 36 hours after eating a contaminated food, but they can occur as early as 6 hours or as late as 10 days after eating the food.
It is interesting that the symptoms of botulism vary widely from a mild illness for which the patient may seek no medical treatment to a fulminant disease, killing within 24 hours (1). Since laboratory testing for this organism is only available at state health departments, it seems likely that many cases of botulism, especially the mild cases, may be undiagnosed. I suspect that some children with autistic behavior,with extremely high urine HPHPA, little or no speech, and extremely severe low muscle tone might actually have undiagnosed botulism, and further research on this possibility is warranted.
C. sporogenes is virtually identical to C. botulinum except it is lacking the gene for the botulinum neurotoxin. Like C. botulinum, it is an anaerobic gram-positive, rod-shaped bacterium that produces oval, subterminal endospores, and is commonly found in soil.
C. caloritolerans is named after its extreme heat (calor) resistance (tolerans). It can survive at the boiling point for 8 hours (2); its ability to resist heat may allow transmission even in well-cooked food. No scientific papers on any disease associations (other than my own articles dealing with its production of HPHPA) were found, which means there is still a great deal of research opportunity for microbiologists in the future.
High 4-hydroxyphenylacetic acid is associated with small intestinal bacteria overgrowth due to its production by the following Clostridia bacteria: C. diificile, C. stricklandii, C. lituseburense, C. subterminale, C. putrefaciens, and C. propionicum. C. difficile can be distinguished from the other species by its production of 4-cresol; none of the other species produce 4-cresol. No information on the pathogenicity of the other species producing 4-hydroxyphenylacetic acid is available. However, it is likely that 4-hydroxyphenylacetic is also an inhibitor of dopamine-beta-hydroxylase and appropriate treatment with probiotics or antibiotics may be clinically useful. 4-hydroxyphenylacetic acid is associated with bacterial overgrowth of the small intestine (3). Elevated values are common in celiac disease and cystic fibrosis, and have also been reported in jejuna web, transient lactose intolerance, Giardia infection, ileal resection, ileo-colic intersusseception, septicemia, and projectile vomiting. The elevations of 4-hydroxyphenylacetic acid in celiac disease and cystic fibrosis are so prevalent that involvement of these Clostridia bacteria may play a role in these illnesses. In C. difficileinfections 4-hydroxyphenylacetic acid is utilized by this bacteria to produce 4-cresol.
Very high amounts of phenyllactic acid are found in the rare genetic disease phenylketonuria (PKU). Moderate amounts of phenyllactic acid may be due to gastrointestinal overgrowth of the intestine of the following Clostridia bacteria: C. sordellii, C. stricklandii, C. mangenoti, C. ghoni, and C. bifermentans. C sordellii is usually considered a nonpathogen except in immunocompromised people, but has been implicated in catastrophic infectious gynecologic illnesses among women of childbearing age. The other species have rarely or never been reported to be pathogenic.
High 3-indoleacetic acid in urine is a byproduct of C. stricklandii, C. lituseburense, C. subterminale, and C. putrefaciens. No information on the pathogenicity of these species producing indoleacetic acid is available. However, very high amounts of this metabolite derived from tryptophan might indicate a depletion of tryptophan needed for other physiological functions.
4-cresol is predominantly produced by C. difficile, a pathogenic bacteria, that is one of the most common pathogens spread in hospitals. Toxin-producing strains of C. difficile can cause illness ranging from mild or moderate diarrhea to pseudomembranous colitis, which can lead to toxic dilatation of the colon (megacolon), sepsis, and death (4). 4-cresol (para-cresol) has been used as a specific marker for Clostridium difficile (5). 4-Cresol, a phenolic compound, is classified as a type-B toxic agent and can cause rapid circulatory collapse and death in humans (6). Yokoyama et al. (7) have recently proposed that intestinal production of 4-cresol may be responsible for a growth-depressing effect on animals. Signs of acute toxicity in animals typically include hypoactivity, salivation, tremors and convulsions. High amounts of 4-cresol have been found in autism (8); the amount of 4- cresol in the urine has been found elevated in baseline samples and in replica samples of autistic children. Higher values of 4-cresol are found in girls with autism compared to boys with autism and higher values are associated with greater clinical severity of autistic symptoms and history of behavioral regression. 4-cresol is apparently produced by Clostridia difficile as an antimicrobial compound that kills other species of bacteria in the gastrointestinal tract, allowing the Clostridia difficile to proliferate and predominate.
Organic acid test superior to stool testing for Clostridia testing
C. difficile is the only species of 100 species of Clostridia from the gastrointestinal tract to be commonly tested in hospital laboratories throughout the world. However, this species is not commonly cultured, but rather is detected by its toxin formation. The gastrointestinal damage caused by C. difficile is thought to be due to exposure to two toxins produced by C. difficile, toxin A and toxin B, with toxin B considered to be more toxic (4). The toxins can be tested by immunoassay of stool samples which is a fairly rapid test. Toxigenic stool culture, which requires growing the bacteria in a culture and detecting the presence of the toxins, is the most sensitive test for C. difficile, and it is still considered to be the gold standard (4). However, it can take 2 to 3 days for results. Polymerase chain reaction (PCR) evaluation of the C. difficile toxins is also becoming more available. Virtually all of the research on C. difficile is related to the effects of this species of bacteria on the intestinal tract. Toxin-negative C. difficile strains are considered nonpathogenic for the infection of the intestine (4) but cresol producing strains that don't produce toxins and B may be pathogenic due to their effects on brain metabolism and for the inherent toxicity of 4-cresol itself.
In addition, urinary 4-cresol elevations associated with C. difficileovergrowth are much less common than urinary HPHPA elevations associated with other Clostridia species. In a survey of 1000 consecutive samples submitted for urine organic acids tests, The Great Plains Laboratory found that 15.2% were abnormally elevated for HPHPA, 6.8% were abnormally elevated for 4-cresol, and 1.6% were abnormally elevated for both HPHPA and 4-cresol for a total positive percentage of 23.6%. Thus, if only stool testing for Clostridium difficile is performed on a patient, at least 15.2/23.6 or 64.4% (nearly two-thirds) of patients with clinically significant infections with other types of Clostridia might be missed.
Sometimes total Clostridia are tested using culture methods or PCR (polymerase chain reaction) technology. In one case, a parent showed me the stool test results of their child with autism. They had done a stool test with a laboratory using PCR technology to determine both C. difficile and total Clostridia. The total Clostridia was reported as extremely low and the C. difficile negative, but The Great Plains Laboratory organic acid test found high levels of the HPHPA marker. If the parent had relied on the stool test alone, their child might have missed an important therapeutic intervention that can restore normal neurotransmitter balance. The advantage of The Great Plains Laboratory organic acid test is that it is not necessary to determine particular species of Clostridia because it is the HPHPA and/or 4-cresol that are neurotoxic.
People sometimes assume that a test using DNA is more accurate than other types of testing. However, DNA testing is fraught with complexities. The nucleic acids of Clostridia are extremely diverse. The content of the nucleic acid bases guanosine and cytosine (G+C) is used to classify bacteria species. The G+C content of DNA of Clostridia species ranges from 21-54 % (9). The majority of intestinal species have G+C contents in the lower half of this range. Ribosomal RNA cataloging confirms that Clostridia occupy six independent sublines with multiple branches including non-Clostridia species. The failure to offer documentation on which species are being detected and how validation was performed should lead to caution by the user of such testing, especially when such tests may be labeled "experimental". Similar complexities exist with traditional culture methods for Clostridia since results are commonly reported from 0 to 4+. Since many Clostridia are not pathogenic, what does a high Clostridia level of 4+ indicate since beneficial, neutral, and harmful species are lumped together in one category? In reality, the results of stool tests for total Clostridia are virtually meaningless and may lead to inappropriate patient treatment.
It is estimated that there are about 10 billion cells of Clostridia per gram of stool. Clostridium ramosum is the most common (53% of all subjects tested) with a mean count of about one billion per gram of stool (9). The prevalence of some Clostridia species is highly dependent on diet. Stool samples of vegetarians did not contain Clostridium perfringens whereas meat and fish eaters had high amounts (10).
Since HPHPA is associated with multiple species of Clostridia but not Clostridium difficile, there is really no available confirmation test for determining the specific species of Clostridium producing HPHPA. As mentioned above, stool testing for total Clostridia is useless since it cannot currently differentiate between harmful or beneficial species. Since HPHPA, in my experience, disappears after treatment with vancomycin or metronidazole, I always recommend treatment based on the HPHPA value with a follow-up test 30 days after completion of treatment.
Confirmation testing of Clostridium difficile could be performed when 4-cresol is elevated. However, the prevalent testing for Clostridium difficile toxins A and B are focused on strains that cause gastrointestinal damage. Strains that produce 4-cresol but not toxins A or B may still cause significant psychiatric disease, so performing these toxin tests may muddy the interpretation of the clinical situation if these tests are negative. I think that it is easier to treat based on the 4-cresol results and then do follow-up testing of the 4-cresol on the organic acid test 30 days after completion of treatment.
- Beatty, H. Botulism. In: Harrison's Principles of Internal Medicine, 10th edition, ed. R. Petersdorf, et al. McGraw Hill. New York. 1983. Pages 1009-1013.
- Meyer, K.F. and Lang, O.W. A highly heat-resistant sporulating anaerobic bacterium: Clostridium caloritolerans, N. SP. The Journal of Infectious Diseases Vol. 39, No. 4 (Oct., 1926), pp. 321-327
- Chalmers, R.A., Valman. H.B., and Liberman, M.M., Measurement of 4-hydroxyphenylacetic aciduria as a screening test for small-bowel disease. Clin Chem 25:1791, 1979
- Carrico, R.M. Association for Professionals in Infection Control and Epidemiology (APIC) Implementation Guide to Preventing Clostridium difficile Infections http://apic.org/Resource_/EliminationGuideForm/59397fc6-3f90-43d1-9325-e8be75d86888/File/2013CDiffFinal.pdf (accessed Oct 30,2014)
- Sivsammye, G. and Sims, H.V. Presumptive identification of Clostridium difficile by detection of p-cresol (4-cresol) in prepared peptone yeast glucose broth supplemented with p-hydroxyphenylacetic acid. J Clin Microbiol. Aug 1990; 28(8): 1851–1853.
- Phua, T.J., Rogers, T.R., and Pallett, A.P. Prospective study of Clostridium difficile colonization and paracresol detection in the stools of babies on a special care unit. J. Hyg., Camb. (1984). 93. 17-25 17
- Yokoyama, M. T., Tabori, C., Miller, E. R. and Hogberg, M. G. (1982). The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites. The American Journal of Clinical Nutrition 35, 1417-1424.
- Persico, A.M. and Napolioni, V. Urinary p-cresol (4-cresol) in autism spectrum disorder. Neurotoxicology and Teratology 36 (2012) 82–90
- Wells, J.M. and Allison, C. Molecular genetics of intestinal anaerobes. In: Human Colonic Bacteria. Role in Nutrition, Physiology, and Pathology. Gibson and MacFarlane, ed. CRC Press. Ann Arbor. 1995. Page28
- 10. Conway, P. Microbial ecology of the human large intestine. In: Human Colonic Bacteria. Role in Nutrition, Physiology, and Pathology. Gibson and MacFarlane, ed. CRC Press. Ann Arbor. 1995. Pages 1-24
William Shaw Ph.D
I strongly disagree with the assertion by some individuals in the autism field that calcium is a problem to individuals on the autistic spectrum. Failure to provide adequate calcium is very dangerous and could lead to the loss of the eyes due to severe eye-poking behavior. Calcium deficiency can be a severe problem in normal children on a milk free diet since milk is a significant source of protein, vitamin D, and calcium needed for strong bones and teeth. Some physicians have reported rickets (1), a severe bone deformity, occurs in children with autism on the gluten and casein free diet who did not receive added calcium supplements. Calcium and vitamin D supplementation is essential to children on a casein free diet since most children with autism do not eat substantial amounts of other calcium-rich foods. Use of milk substitutes like B-Unique® provides adequate calcium, protein, and fat comparable to whole milk without the presence of casein and lactose that are problematic in most children with autism.
Children with autism may have an even more severe problem with calcium deficiency. Mary Coleman, M.D. (2) reported that children with autism who are calcium deficient are much more likely to poke out their eyes and a substantial number of children with autism have done so. I have talked to numerous parents of children with autism that began to touch their eyes after starting the casein-free diet. This abnormal behavior is associated with low urine calcium; blood calcium levels were usually normal. Treatment with calcium supplementation prevents this behavior. (I suspect that this behavior is due to increased eye pain due to high substance P or to deposits of oxalate crystals in the eye. Low calcium may act to intensify this pain and poking out the eye relieves the pain.) Dr. Coleman also found that speech developed very quickly after calcium supplementation in a portion of mute children with autism who had low urine calcium. Parathyroid hormone, calcitonin, and vitamin D were all normal in patients with autism but all of them had low urine calcium. In one case, according to a parent who contacted me, her child with autism persisted in poking at the eyes even after one eye had been poked out and surgically replaced. Calcium supplementation stopped this behavior immediately. I am aware of many other children with eye-poking behavior in which calcium supplements stopped this behavior in less than two days. Verbal autistic children say that their eye pain is severe and that calcium supplementation stopped their pain quickly. The urine calcium and magnesium tests will soon be a part of the Organic Acids Test offered by The Great Plains Laboratory, Inc.
It is important that calcium, magnesium, and zinc be in balance for optimal nutrition. Vitamin D supplementation may also be needed when milk is eliminated unless other sources of vitamin D are included in the diet or the child is exposed to adequate sunlight. Children with autism also need additional calcium to prevent oxalate deposition in the tissues. Although sardines and dark leafy greens like spinach, kale, turnips, and collard greens are high in calcium, all of these foods except sardines are high in oxalates. High oxalates can be fatal if formed in the renal tract. Oxalates in the urine are much higher in individuals with autism than in normal children. As a matter of fact, 36% of the children on the autistic spectrum had values higher than 90 mmol/mol creatinine, the value consistent with a diagnosis of genetic hyperoxalurias while none of the normal children had values this high. 84% of the children on the autistic spectrum had oxalate values outside the normal range (mean ± 2 std dev). None of the children on the autistic spectrum had elevations of the other organic acids (glyceric and glycolic acids) associated with genetic diseases of oxalate metabolism, indicating that oxalates are high due to external sources. When calcium is taken with foods that are high in oxalates, oxalic acid in the intestine combines with calcium to form insoluble calcium oxalate crystals that are eliminated in the stool. This form of oxalate cannot be absorbed into the body. When calcium is low in the diet, oxalic acid is soluble in the liquid portion of the contents of the intestine (called chyme) and is readily absorbed from the intestine into the bloodstream. If oxalic acid is very high in the blood being filtered by the kidney, it may combine with calcium to form crystals that may block urine flow and cause severe pain. However, such crystals may also form in the bones, joints, blood vessels, lungs, eyes, skin, heart, thymus, skeletal muscle, joints, fat, teeth, mouth, nerves, and even the brain. In addition, oxalate crystals in the bone may crowd out the bone marrow cells, leading to anemia and immunosuppression. Calcium citrate is the best source of calcium to prevent oxalate absorption because citrate ion blocks the absorption of oxalates in the intestinal tract (3). A combination of calcium citrate and magnesium citrate is the best form of supplement to provide calcium and magnesium needs while preventing excess oxalate buildups in the body.
- 1. Hediger ML, England LJ,Molloy CA, Yu KF, Manning-Courtney P, Mills JL. Reduced bone cortical thickness in boys with autism or autism spectrum disorder. J Autism Dev Disord. 2008;38(5):848–856
- 2. Coleman, M. Clinical presentations of patients with autism and hypocalcinuria. Develop. Brain Dys. 7: 63-70, 1994
- 3. Caudarella R, Vescini F, Buffa A, Stefoni S. Citrate and mineral metabolism: kidney stones and bone disease. Front Biosci. 2003 Sep 1;8:s1084-106.
William Shaw, Ph.D.
One of the puzzling aspects of autism is the marked increase in the incidence of autism that began in the United States in the early 1980s and has appeared to increase continuously since then. The highest incidence of autism has been reported to be South Korea, where the incidence is now reported to be one in 38 boys. Increased incidence of autism due to more effective diagnosis was disproved in the study of Irva Hertz-Picciotto who showed that perhaps 12% of the increased autism diagnoses could be attributed to improved diagnosis . A wide range of environmental factors has been associated with increased autism incidence, including pesticides, chemicals, phthalates, polychlorinated biphenyls, solvents, heavy metals or other pollutants. Although toxic chemicals are undoubtedly not beneficial for the health of any person, is there any information that indicates that a toxic avalanche of chemicals inundated the United States in the early 1980s? Indeed a wealth of knowledge about environmental chemicals has led to marked reductions in exposure to chemicals such as lead and dichlorodiphenyltrichloroethane (DDT) in the United States over the past 50 years. For example, acceptable safe limits for levels of lead in the blood have decreased from 60 μg/dL in 1960 to <5 μg/dL in 2010 .
Making a connection between disease appearance and causative agent is important. Clinical studies, epidemiological studies and post-market pharmacovigilence are of utmost importance in recognizing signals and drug-induced side effects. One of the most notable cases of serious adverse effects caused by a pharmaceutical agent was the terrible developmental epidemic of the birth of children with seal-like arms and legs (phocomelia) that was linked to the maternal use of the sedative thalidomide 20–35 days after conception . What would have happened if the thalidomide connection had never been made? One of the difficulties with chemical studies of autism associations is that most chemicals are used worldwide, making it difficult to find a "clean" environment where autism might be less prevalent. One of the clues that led to the discovery of thalidomide as the causative agent of deformed limbs was that it was much more commonly used in Europe than in the United States. Countries with the greatest use of thalidomide by pregnant women during pregnancy were those with the highest incidence of deformed babies. If there was a geographic region in the world in which the incidence of autism was much lower than that in the United States, a comparison of medical or dietary differences might provide a significant clue to the major cause of autism.
Such a country is Cuba. The highest estimate of the total incidence of autism in Cuba is 185 cases out of a total population of 11,000,000 (0.00168% of the population) compared with an estimate of as high as 1.5 million in a total United States population of 300 million (0.50%) [6, 7]. The percentage of the population with autism in the United States is thus 298 times higher than in Cuba. Cuba is much more economically challenged than the United States, with the per capita income of Cuba approximately eight times lower than that in the United States. Despite the economic challenges presented to the communist government of Cuba, basic healthcare is readily available and there are a large number of physicians trained in 14 different medical schools. Unlike the United States, where vaccines are optional in many states, vaccines are compulsory in Cuba and Cuba has one of the most highly vaccinated populations in the world against a wide variety of infectious agents. For example, the vaccination rate for measles was reported to be 99.7%. The association of autism with various vaccines has had a very controversial history with inflamed passions on both sides of the debate and will not be examined here.
However, a topic much less frequently addressed in association with autism is the therapies that are given in conjunction with vaccines. The practice of prescribing acetaminophen as a prophylactic fever preventative is widespread in the United States but is very uncommon in Cuba (personal communications, Dr Olympio Rodriquez Santos MD, MSc, Allergist, Camaguey, Cuba). In the United States, some physicians have started to advise parents to begin to take acetaminophen prophylactically daily 5 days prior to childhood vaccines; some children on such prophylactic treatment had an autistic regression that began prior to vaccination (personal communication, Kerry Scott Lane MD, Anesthesiologist, West Palm Beach, Florida, USA). In Cuba, acetaminophen is not approved as an over-the-counter (OTC) product, however, it has been available as an OTC product since 1959 in the United States. Furthermore, in Cuba, prophylactic use of antipyretic drugs is not the standard medical treatment for vaccine-related fever (personal communications with Dr Olympio Rodriquez Santos). If high fever continues after vaccination in Cuba for more than 2 days, the parents are advised to visit the physician's office where the drug metamizole is most commonly prescribed. Prescription of acetaminophen in such cases is rare. Metamizole is used in many countries throughout the world but is banned in the United States and some other countries because of a rare association with agranulocytosis.
Could the Use of Certain Antipyretic Drugs, Especially in Conjunction with Vaccines, be a Cause of Autism?
Torres was the first to ask if the suppression of fever by antipyretic drugs commonly used at the time of vaccination might cause the severe immune abnormalities that are prevalent in autism. Schultz et al. were even more specific when indicating in a series of articles that biochemical and immune disorders caused by increased use of the common drug acetaminophen may have caused the autism epidemic (Figure 1).[11–14]
Acetaminophen is also termed paracetamol and N-acetyl-p-aminophenol (AAP or APAP). More than 70% of the population in western countries has taken acetaminophen at least once, and a relevant percentage takes the drug chronically as a mild pain reliever and antipyretic. Acetaminophen is used to treat pain and fever and it has become one of the most popular OTC non-narcotic analgesic agents. For example, this compound has been taken at least once by >85% of children under the age of 91 months in the UK.15 In the US, approximately 79% of the general population regularly takes acetaminophen, including more than 35% of pregnant women.Acetaminophen has grown in popularity in large part due to its reputation for safety. For generations, Tylenol® (a popular brand of acetaminophen) advertisements have painted it as "the pain reliever hospitals use most." Acetaminophen is in >600 OTC and prescription products, from headache and cold remedies to cough syrups and sleep aids.
The study by Schultz et al. was the first to specifically link increased acetaminophen use to increased autism.This study included a graph similar to Figure 1 temporally relating increased autism incidence in California with increased acetaminophen use in the United States and decreased acetaminophen use with decreased rate of autism in California. In addition, similar increases in the rates of asthma correlated with the usage of acetaminophen were also noted by Becker and Schultz. They noted that the rates of autism incidence and asthma stopped increasing in the months following two attempted extortion events in which acetaminophen was deliberately laced with cyanide. The changes in the incidence of these very different diseases at exactly the time acetaminophen use dropped for a significant period of time is remarkable and may indicate the same factor as causing the two diseases. In addition, the autism paper by Schultz et al. included the results of an online survey of parents who had given their child the combined measles, mumps, rubella (MMR) vaccine, which revealed that children with autism had more adverse reactions to the MMR vaccine and were more likely to have been given acetaminophen than ibuprofen for those reactions. Compared with controls, children aged 1–5 years with autism were eight times more likely to have become unwell after the MMR vaccine, and were six times more likely to have taken acetaminophen. Children with autism who regressed in development were four times more likely to have taken acetaminophen after the vaccine. Illnesses concurrent with the MMR vaccine were nine times more likely in autistic children when all cases were considered, and 17 times more likely after limiting cases to children who regressed. There was no increased incidence of autism associated with ibuprofen use.
The incidence of attention deficit with hyperactivity over the last 50 years follows patterns similar to those of autism and asthma, although the data for attention deficit-hyperactivity disorder (ADHD) are not available to the same depth as the data for autism and asthma. Before 1970, the diagnosis of ADHD was relatively rare for schoolchildren and almost nonexistent for adolescents and adults. Between 1980 and 2007, there was an almost 8-fold increase of ADHD prevalence in the United States compared with rates of 40 years ago.Prevalence of ADHD in American schoolchildren was 1% in the 1970s, 3–5% in the 1980s, and 4–5% in the mid-to-late 1990s.[18–24] A study of hospital discharge rates for ADHD between 1989 and 2000 found a 381% increase over the study period.
Use of acetaminophen dramatically increased in the United States in the 1980s due to a concern over an association of aspirin with Reye's syndrome, although a number of critics reject this hypothesis.[26–28] For example, the current recommendations for the management of children with Kawasaki disease include treatment with high-dose aspirin in the acute phase, and low-dose aspirin during the period of thrombocytosis. For those with residual coronary problems, low-dose aspirin is often given over an even longer term. In Japan alone, up to 200,000 children have received aspirin for Kawasaki disease. Interestingly, only one case of Reye's syndrome associated with Kawasaki disease has ever been reported, and only in the Japanese literature, giving an incidence of 0.005%. In addition, retrospective reevaluation of patients with a diagnosis of Reye's syndrome who survived has revealed that many, if not most, had an underlying inborn error of metabolism (IEM). Many of these IEMs had not even been described when the diagnosis of Reye's syndrome was made. Inborn errors that may mimic Reye's syndrome include fatty-acid oxidation defects, amino and organic acidopathies, urea-cycle defects, and disorders of carbohydrate metabolism. Future discovery of other IEMs may ultimately explain even more of these cases. Additional etiologies that may mimic Reye's syndrome include viral infections, neuromuscular diseases, adverse drug reactions, and exposure to toxic chemicals and plants that cause hepatocellular damage and encephalopathy. Diagnostic methods such as GC/MS became more widely available in the 1980s and later so that the patients with IEMs were diagnosed with an IEM instead of Reye's syndrome. The main cause of Reye's syndrome appears to be the accumulation of nonesterified fatty acids and lysolecithins that have a high detergent activity and thus denature all proteins.
It is interesting that Cuba, which has a lower autism rate than the United States, allows the use of acetaminophen only by prescription, therefore, the use of acetaminophen in Cuba is only a minuscule fraction of acetaminophen use in the United States and many other countries throughout the world. When acetaminophen use was limited by a prescription requirement in the United States, the rate of autism was a small fraction of current rates of autism.
Unlike thalidomide, which was once promoted for its extreme safety prior to the discovery of its teratogenicity, acetaminophen has a long history of serious side effects associated with its use (Table 1).
Acetaminophen produces neurotoxic effects on rat brain neurons both in vitro and in vivo, its use during pregnancy is associated with teratogenic defects in testicular function and the gastrointestinal tract, and there is increased incidence of asthma in maternally exposed and postnatally exposed children.[13–15, 30, 31, 44-46]Acetominophen is converted to the very toxic metabolite N-acetyl-pbenzoquinone imine (NAPQI; Figure 2), which can cause oxidative damage to proteins, nucleic acids, amino acids, and lipids, in addition to increased mitochondrial and cellular damage and death.[32–35]
Acetaminophen also causes severe immune abnormalities at doses that do not damage the liver, depresses the immune response to vaccination, can cause severe metabolic acidosis when glutathione (GSH) is depleted, is the leading cause of liver failure and death in the United States, is associated with increased rates of certain blood cancers, and results in tens of thousands of visits to the emergency room and hospitalizations in the United States.[14, 36–43] A PubMed search of the scientific literature indicated the presence of 2685 articles regarding acetaminophen toxicity.
An article with the title, "Did acetaminophen cause the autism epidemic?" was more pointed. The rest of this article will deal with the known toxicity of acetaminophen and how other known anatomical, immunologic, biochemical, and infectious aspects of autism can be related to the effects of acetaminophen.
The metabolism of acetaminophen is shown in Figure 2. There are four major pathways for its detoxification. Acetaminophen can be converted to acetaminophen sulfate by phenol sulfotransferase (PST). It can also be converted to a glucuronide or deacetylated to a phenol. In addition, it can be converted to its extremely toxic metabolite NAPQI by the cytochrome P450 (CYP)2E1 enzyme. Several factors that increase the induction of CYP2E1, including smoking, obesity, vinyl chloride, and exposure to trichloroethylene are associated with increased incidence of autism. In addition, acetaminophen exposure itself induces increased CYP2E1, thus increasing the amount of NAPQI formed with each exposure to the drug.[49, 53] Posadas et al. found that acetaminophen exposure to rats resulted in a concentration-related increase in CYP2E1 in rat brains.
Thus, the prophylactic use of acetaminophen for days before vaccination and for multiple vaccinations would likely greatly increase the conversion of acetaminophen to its extremely toxic NAPQI metabolite. Glucuronidation is commonly present at low capacity in the fetus, newborns, and young infants, such that exposure to acetaminophen at these times leads to greater metabolism by other pathways. Acetaminophen can also be deacetylated to form p-aminophenol, which can also be sulfated or converted to an active cannabinoid substance by being conjugated with arachidonic acid to form the conjugate termed AM404. p-Aminophenol may also be detoxified by PST.
Acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 by AM404.[12,13] If the sulfation pathway is defective, as has been shown in autism, and/or there is impaired glucuronidation, acetaminophen will be increasingly converted to the alternative metabolic routes, increasing production of its more toxic compounds NAPQI and AM404.[56, 57]
In addition to liver toxicity caused by acetaminophen, individuals without liver toxicity may have severe metabolic acidosis if they are poorly nourished due to illness or dietary insufficiency.[39, 40] In each of these cases, extremely high levels of pyroglutamic acid (also termed 5-oxoproline) are found in both the urine and blood serum. The connection between the elevation of this organic acid and acetaminophen use was first reported by Pitt et al. who suggested that acetaminophen ingestion depletes intracellular GSH stores, which then causes loss of feedback inhibition of γ-glutamylcysteine synthetase activity (Figures 3a and 3b). This increases production of γ-glutamylcysteine, which is partially converted to pyroglutamic acid.
CYP enzymes are a superfamily of hemoproteins that carry out oxidative metabolism of many endogenous and foreign chemicals. CYP2E1 is the principal CYP responsible for the metabolism of ethanol and is considered to be a major component of the microsomal ethanol-oxidizing system. Among xenobiotics metabolized by CYP2E1 are acetaldehyde, acetaminophen, acrylamide, aniline, benzene, butanol, carbon tetrachloride, diethylether, dimethyl sulfoxide, ethyl carbamate, ethylene chloride, halothane, glycerol, ethylene glycol, N-nitrosodimethylamine, 4-nitrophenol, pyrazole, pyridine, and vinyl chloride. Many of these chemicals are known toxins, established chemical carcinogens, or suspected carcinogens.
When cyp2e1 knockout mice that lack the ability to produce the CYP2E1 enzyme were challenged with acetaminophen, they were found to be considerably less sensitive to its hepatotoxic effects than wild-type animals, indicating that this CYP is the principal enzyme responsible for the metabolic conversion of the drug acetaminophen to its active hepatotoxic metabolite NAPQI (Figure 2). During fasting and diabetic ketosis, serum acetone, acetol, and 1,2-propanediol are elevated. CYP2E1 is concomitantly induced due to protein stabilization by acetone.35 Acetone is primarily oxidized to acetol by CYP2E1. As fasting increases ketone production with a concomitant increase in CYP2E1 activity, this in turn increases the production of NAPQI and decreases amino acid substrates for GSH production. Administering acetaminophen is likely to be much more toxic under fasting conditions, such as when a child has an illness that decreases appetite. Children who are sick and fasting and are administered vaccines with prophylactic acetaminophen are much more likely to suffer acetaminophen toxicity.
An examination of the importance of GSH and its biosynthesis is important to an understanding of acetaminophen toxicity. GSH is a tripeptide composed of the amino acids glutamate, cysteine and glycine (Figure 3a). It is present in virtually all aerobic cells at millimolar concentrations where it takes part in numerous fundamental processes. It is a very important antioxidant, participates in detoxification of certain drugs, toxic environmental chemicals, protects against lipid peroxidation and electrophiles, has antiviral effects, is involved in the biosynthesis of DNA, proteins, and leukotrienes, cell proliferation, apoptosis, neurotransmission, and neuromodulation. Decreased levels of GSH are found in several diseases such as liver cirrhosis, pulmonary disease, gastrointestinal or pancreatic inflammation, diabetes, HIV infection, and neurodegenerative diseases. In the normal physiological state (Figure 3a), GSH is produced by the condensation of the amino acids glutamate and cysteine to form the dipeptide γ-glutamylcysteine, which is catalyzed by the enzyme, γ-glutamyl cysteine synthetase. The dipeptide then condenses with the amino acid glycine to form the tripeptide GSH which is catalyzed by the enzyme GSH synthetase. The end product, GSH, exhibits negative feedback inhibition of γ-glutamylsynthetase to prevent the overproduction of GSH. If the amino acid glycine is present at high concentrations, γ-glutamylcysteine is converted in small amounts to pyroglutamic acid that can be recycled to form glutamate.
If GSH is severely depleted by the toxic metabolite of acetaminophen, NAPQI (Figure 3b), and/or there is inadequate glycine to produce GSH due to illness or nutritional deficiency, there is a lack of negative feedback of GSH on γ-glutamylsynthetase.
This severe depletion of GSH results in the synthesis of large amounts of γ-glutamylcysteine, which is increasingly converted to pyroglutamate when glycine is depleted, leading to metabolic acidosis.
As organic acid testing to determine pyroglutamate is usually performed only at specialized pediatric hospitals with specialized biochemical genetics services, the diagnosis of this metabolic disorder in patients may frequently be missed. In addition, NAPQI also deactivates some of the enzymes that recycle GSH, such as GSH peroxidase. The depletion of GSH diminishes the ability of the body to detoxify toxic chemicals. As of 2012, there were 170 articles that indicated an association between toxic chemical exposure and autism. The depletion of GSH will lead to enhanced toxicity of a large number of toxic chemicals. For example, Adams et al. found that variations in the severity of autism measurements could be explained, in part, by regression analyses of urinary excretion of toxic metals before and after DMSA, the chelating agent dimercaptosuccinic acid, and the level of red blood cell glutathione. Thus, partial depletion of GSH by moderate increases in NAPQI could lead to enhanced toxicity of heavy metals and perhaps many other toxic chemicals.
Depletion of GSH as a consequence of acetaminophen toxicity to the liver has attracted the most attention in the medical scientific community, as it can frequently be fatal or require a liver transplant or emergency treatment to prevent liver failure (the liver is the organ with the greatest concentration of GSH). However, acetaminophen toxicity has been implicated in a wide range of other disorders in humans and/or experimental animals including cancer, birth defects, asthma, allergies, and brain toxicity (Table 1).
Concentrations of p-aminophenol (which is converted to the cannabinoid substance AM404; Figure 2) from 1 to 100 μg/mL produced significant loss of mouse cortical neuron viability at 24 h compared with the controls.11 The naturally occurring endocannabinoid anandamide also caused similar 24 h loss of cell viability in developing mouse cortical neurons at concentrations ranging from 1 to 100 μg/mL, indicating the mechanism of cell death could be mediated through the cannabinoid receptors. Defective glucuronidation would also increase the conversion of acetaminophen to its more toxic metabolites, NAPQI and AM 404. Such defects in glucuronidation are common in the developing fetus and in newborns.
Posadas et al. found that acetaminophen causes concentration-dependent neuronal death in vitro at concentrations that are reached in human plasma during acetaminophen overdose, and are reached in the cerebrospinal fluid of rats for 3 h following doses that are below those required to induce acute hepatic failure in rats. Acetaminophen also increases both neuronal CYP2E1 enzymatic activity and protein levels, as determined by western blot, leading to neuronal death through mitochondrialmediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that acetaminophen injection induces neuronal death in the rat cortex. Posadas et al. established a direct neurotoxic action by acetaminophen both in vitro and in vivo in rats at doses below those required to produce hepatotoxicity and suggested that this neurotoxicity might be involved in the general toxic syndrome observed during patient acetaminophen overdose and, possibly, when acetaminophen doses in the upper dosing schedule are used, especially if other risk factors (moderate alcohol drinking, fasting, nutritional impairment) are present.
Purkinje Cell Abnormalities, Autism, and GSH Depletion
Ritvo et al. were the first to report abnormalities of Purkinje cells in the cerebella of people with autism. These cells are some of the largest neurons in the human brain, with an intricately elaborate dendritic arbor, characterized by a large number of dendritic spines; these neurons utilize γ-aminobutyric acid as their neurotransmitter and send inhibitory projections to the deep cerebellar nuclei, and constitute the sole output of all motor coordination in the cerebellar cortex. The initial study of Ritvo et al. indicated that the number of Purkinje cells in the vermis of the cerebellum was 15 standard deviations below the mean and approximately 8 standard deviations below the mean bilaterally in the cerebral hemispheres of the individuals with autism compared with controls. Vargas et al. found that brain tissues of autistic patients showed extensive neuroglial responses characterized by microglial and astroglial activation. In the brains of autistic patients, the most prominent histological changes were observed in the cerebellum, characterized by a patchy loss of neurons in the Purkinje cell layer and granular cell layer in nine out of ten cerebella; one of these cerebella also showed an almost complete loss of Purkinje cells from the Purkinje cell layer, as well as a marked loss of granular cells. Kern and Jones have summarized the important role of Purkinje cell abnormalities in autism, especially the susceptibility of these cells to oxidative stress during GSH depletion. Such depletion can be due to reduction of GSH due to excessive NAPQI exposure and/or to GSH depletion associated with elevated dopamine secondary to dopamine β-hydroxylase inhibition by phenolic Clostridia metabolites. The metabolite of acetaminophen, 4-amino phenol, also caused depletion of GSH.
Immune Abnormalities Associated with Acetaminophen Use
An important but under-appreciated aspect of acetaminophen toxicity is that direct, drug-induced harm accounts for only part of the overall syndrome of acetaminophen-induced liver injury. The reason for this is that the initial wave of drug-induced hepatocellular destruction is followed by a robust innate immune response, in which invading inflammatory cells release toxic oxidants and cause a second wave of destruction. The collateral damage inflicted by inflammatory cells can be so severe as to double the degree of tissue injury caused by acetaminophen alone.
Prymula and colleagues compared post-vaccine fever and post-vaccine changes in vaccine-specific antibody titers in children who either did or did not receive scheduled acetaminophen with immunizations. Four hundred and fifty nine healthy infants undergoing initial and booster vaccinations for a variety of vaccines were randomized to receive or not to receive prophylactic acetaminophen at the time of and for 24 h after vaccinations. Fever was significantly less common in acetaminophen-treated children after initial and booster vaccination. Children who received acetaminophen had significantly lower antibody titers to each vaccine-related antigen, although it was judged that the titers would probably be protective against the diseases for which they had been immunized. In children with severe sulfation deficiency with a predisposition to autism, vaccination without an adequate immune response might lead to viral infection even with the attenuated strains of the viral vaccines due to a lack of GSH needed for effective immune response.
Acetaminophen sales were high in Englishspeaking countries, and were positively associated with asthma symptoms, eczema and allergic rhinoconjunctivitis in children aged 13–14, and with wheeze, diagnosed asthma, rhinitis and bronchial responsiveness in adults. The prevalence of wheeze increased by 0.52% in 13–14 year olds and by 0.26% in adults (p < 0.0005) for each gram increase in per capita acetaminophen sales. Between 1980 and 2003, the prevalence of pediatric asthma in the United States increased from 3.6% to 5.8%, and similar increases were observed throughout the world. It has been speculated that frequent use of acetaminophen might influence asthma and rhinitis by depleting levels of reduced GSH in the nose and airways, thus shifting the oxidant/antioxidant balance in favor of oxidative stress and increasing inflammation.
Acetaminophen decreases GSH levels, principally in the liver and kidneys, but also in the lungs. These decreases are dose dependent; overdose levels of acetaminophen are cytotoxic to pneumocytes and cause acute lung injury, whereas nontoxic, therapeutic doses produce smaller, but significant reductions in GSH levels in type II pneumocytes and alveolar macrophages. Among healthy young volunteers, significantly lower serum antioxidant capacity has been seen within 2 weeks of ingestion of 1 g of acetaminophen. By depleting GSH levels, acetaminophen weakens the ability of the host to mitigate oxidative stress produced by reactive oxygen species (ROS) such as superoxide anions (O2 −), hydroxyl (•OH), and peroxyl (ROO−) radicals.Finally, when GSH levels are low, defective processing of disulfide bonds that are key in antigen presentation has been hypothesized. It is conceivable that decreased levels of GSH guide the expression of T-helper cell pathways by altering antigen presentation and recognition, thereby favoring the T2 allergic-dominant pathway. In a study of children with autism spectrum disorders (ASDs) in Sweden, airway symptoms of wheezing and physician-diagnosed asthma in the baseline investigation in infants and toddlers were associated with ASD 5 years later.
Skewed T1 or T2 responses were also indicated in ASD children.[65, 66] Analysis by Jyonouchi et al. of adaptive immune responses revealed markedly variable T1rT2 cytokine levels in ASD children compared with control siblings. It has been reported that immune responses in autistic children are relatively skewed to T2 on the basis of intracellular staining of T1rT2 cytokines. Higher levels of IgG, IgA, and IgE allergies to various foods, but especially milk and wheat, have been reported in children with autism, together with abnormal cellular immune responses to milk, wheat, and soy. It has been shown that depleting GSH in brain microglia and astroglia induces a neuroinflammatory response that results in both significant cytokine release and the release of material that is toxic to neurons.
In addition, the ability of GSH to downregulate nuclear factor-kB, and the inverse association between alveolar GSH levels and bronchial responsiveness, suggests that GSH may modify asthma inflammation. Secondly, studies in animals have found that acetaminophen can deplete the lung of GSH. These effects in macrophages raise the possibility that acetaminophen might also influence atopic diseases more generally through another mechanism, namely the promotion of atopy, since depletion of GSH in antigen-presenting cells promotes T-helper cell 2-type cytokine responses. This might explain why, in children, acetaminophen sales were associated with atopic eczema as well as with asthma and rhinitis. As of 2012, 416 studies had demonstrated an association between autism and immune abnormalities and/or inflammation.
The beginning of the rapid increase in autism in around 1980 coincides with the rapid increase in asthma, both of which coincide with the rapid increase in the use of acetaminophen following the Reye's syndrome scare over a possible association with aspirin.
It would seem likely that perhaps some children on the autistic spectrum might have both brain and lung abnormalities caused by acetaminophen. Is there any evidence for such combined abnormalities? Indeed, at the annual meeting of the American College of Chest Physicians in Honolulu, Hawaii in 2011, Barbara Stewart, a pediatric pulmonologist, found that a significant lung abnormality was present in 100% of children (n = 47) on the autistic spectrum who were examined, but in none of <300 children without autism.68 Most of the children with autism had been referred to her clinic due to persistent cough unresponsive to treatment. She noticed during bronchoscopy examinations, in which a lighted tube is inserted into the lungs, that, although the airways of the children initially appeared normal, the lower airway had doubled branches, or "doublets". Dr. Stewart said "when airways divide beyond the first generation, they typically branch like a tree, with one branch on one side and one on the other. A doublet occurs when there are twin branches that come off together instead of one, which are exactly symmetrical, in each of the lower locations that can be seen." In a study of ASD children in Sweden, airway symptoms of wheezing and physician-diagnosed asthma in the baseline investigation in infants and toddlers were associated with ASD 5 years later. It would seem very useful to examine the use of acetaminophen both prenatally and postnatally in the children with the abnormal lung anatomy.
As of 2012, 145 studies had shown an association between mitochondria function and autism. The data of Cover et al. showed that nitration of mitochondrial proteins and depletion of mitochondrial DNA after acetaminophen overdose in mice was due to peroxynitrite formation. In contrast, nDNA damage was not directly caused by peroxynitrite. Nuclear DNA damage after acetaminophen overdose is likely to be caused by DNase(s) unrelated to the caspase-activated DNase, which is typically responsible for DNA fragmentation during apoptosis. The data from Cover et al. suggest that the activation of these DNase(s) is dependent on the mitochondrial oxidant stress and peroxynitrite formation.
Recent data have demonstrated that nitrated tyrosine residues, as well as acetaminophen adducts, occur in the necrotic cells following toxic doses of acetaminophen. Nitrotyrosine was postulated to be mediated by peroxynitrite, a reactive nitrogen species formed by the very rapid reaction of superoxide and nitric oxide (NO). Peroxynitrite is normally detoxified by GSH, which is depleted in acetaminophen toxicity. NO synthesis (serum nitrate plus nitrite) was dramatically increased following acetaminophen.
The metabolism of acetaminophen (Figure 2) by CYP2E1 forms NAPQI, a reactive metabolite that binds to cysteine residues in cellular proteins and forms acetaminophen protein adducts, referred to as acetaminophen-cysteine complexes (APAP-CYS).
Heard et al. demonstrated that low concentrations of APAP-CYS are detectable in serum following therapeutic dosing with acetaminophen in the vast majority of individuals. The APAP-CYS concentrations following acetaminophen overdose varied widely. Serum APAP-CYS concentrations in patients with hepatic injury following acetaminophen overdose were generally much higher than those observed during therapeutic dosing. However, three overdose patients had APAP-CYS concentrations that were of the same order of magnitude as those observed with therapeutic dosing. Adduct concentrations varied according to the degree of exposure. Currently, an absolute adduct level exceeding 1.1 µmol/L appears consistent with acetaminophen toxicity. While therapeutic dosing generally produces concentrations below 0.5 µmol/L, values in people taking recommended dosages have been reported to be very close to the lower limit of APAP-CYS concentration associated with acetaminophen toxic overdoses (1.0 µmol/L).
NAPQI and Anomalous Hair Metal Values in Autism
Concentrations of the NAPQI metabolite increased to values as high as 1.0 µmol/L or 1000 nmol/L serum following therapeutic doses of acetaminophen. Hair proteins contain a considerable amount of cysteine, and hair proteins in hair follicles and would be expected to react with NAPQI to form NAPQI adducts with cysteine sulfhydryl groups in hair. Such cysteine groups also react strongly with heavy metals such as mercury. In comparison, the mean blood mercury concentration of children with autism was 19.53 nmol/L. Thus the concentration of NAPQI might be as much as 51 times the concentration of total mercury. Since NAPQI amounts are so much higher than mercury values, it would be expected that NAPQI might significantly reduce the capacity of hair proteins to bind mercury if the binding capacity of the hair sulfhydryl groups was exceeded by the previous binding of NAPQI. Such inhibition by NAPQI may help to explain anomalous data in the measurement of mercury in hair samples of children with autism. In two baby hair studies in which samples were obtained at first haircuts, mercury values in hair were much lower in children with autism compared with those of normal controls.[69, 70] In addition, children with the most severe symptoms of autism had the lowest amount of hair mercury. Studies in Kuwait and Saudi Arabia found opposite results in children with autism; children with autism had much higher amounts of hair mercury as well as other heavy metals, but in both of these studies the children were much older (mean ages 8.8 years in Saudi Arabia and 4.2 years in Kuwait) than those in the baby hair studies (12–24 months of age).[71, 72] All of this data could be explained by patterns of acetaminophen drug use in children. Acetaminophen is commonly used prophylactically to prevent fever in infants and toddlers who receive the bulk of their vaccines in the first 2 years of life. In contrast, few vaccines and attendant prophylactic acetaminophen are administered to older children with autism in the age range in which hair mercury values were elevated compared with controls. In these older children, hair metals such as mercury would be considerably higher due to a lack of competition from NAPQI for reactive sulfhydryl groups in hair proteins in the hair follicles.
Special Concerns About the Long-Term Defective Quality Control of Acetaminophen Products
All of the information to this point has been directed to the toxicity of pure acetaminophen. Toxicity is usually due to exceeding the recommended daily dosage, often occurring due to accidental combination use of multiple products containing acetaminophen or through changes in dosage amounts by manufacturers. Adding to this are reports that acetaminophen products have been contaminated at various times, potentially increasing health risks. These problems have been documented in Food and Drug Administration inspection reports and subsequent recalls.
From the time it was introduced in 1955, acetaminophen has become one of the most successful OTC drugs, and has earned the primary manufacturer, Johnson & Johnson, an estimated US$1.3 billion every year.Within the last 10 years, the FDA has chronicled manufacturing problems including mislabeling of children's tablets, where packages of the product listed an incorrect amount of the ingredients per tablet, inadequate cleaning of equipment used for manufacturing, insufficient follow-up in investigating consumer complaints, product mix-ups, and contamination with metal fragments and bacteria.[74, 75] The latter resulted in a recall of 135 million bottles of children's medications containing acetaminophen.
Additional recalls occurred in 2009 and 2010 due to trace amounts of the chemical 2,4,6-tribromoanisole found in infant, children, and adult OTC products. This recall was prompted by 775 consumer reports of nausea, vomiting, stomach pains and diarrhea received by FDA due to the contamination. It was found that the chemical 2,4,6-tribromoanisole (a metabolite of a chemical fungicide tribromophenol) was being used in a manufacturing facility to treat the wood used in pallets used for transporting packaged materials.Tribromoanisole is produced when naturally-occurring airborne fungi and/or bacteria (usually Aspergillus sp.,Penicillium sp., Actinomycetes, Botrytis cinerea, Rhizobium sp., or Streptomyces) are presented with brominated phenolic compounds, which they then convert into bromoanisole derivatives.
Other FDA-publicized inspection reports of acetaminophen-manufacturing facilities have listed inadequate quality controls, lack of safeguards to isolate "rejected" raw materials and other drugs, and several human errors resulting in product mix-ups. Several recalls have occurred due to failed FDA manufacturing inspections and contaminated products affecting >300 million bottles of adult and children's medicines.[79, 82] Manufacturing controls are an integral aspect of pharmaceutical production to ensure public safety, and disregard or deficiency can lead to serious outcomes. In response to the high number of inadequacies found through inspections and numerous recalls, US health authorities have taken over supervision of three acetaminophen manufacturing plants to mitigate risks.
A Call to Action
A large-scale, long-term study of both prenatal and postnatal effects of acetaminophen exposure on the incidence of autism, attention deficit with hyperactivity, and asthma should be conducted. However, such a study would probably take at least 5 years. If acetaminophen is indeed the cause of all of these illnesses, should acetaminophen use continue for such a long period of time while millions of additional children are further affected? Respected physicians consider that the connection of acetaminophen with asthma has been proven beyond a reasonable doubt. Dr McBride, Professor of Pediatrics at Department of Pediatrics, Northeast Ohio Medical University, Rootstown, Ohio summarizes the evidence for the acetaminophen asthma findings: "There remains a possibility that confounding variables might explain some or all of the association between acetaminophen and asthma. For this reason we need further studies. At present, however, I need further studies not to prove that acetaminophen is dangerous but, rather, to prove that it is safe. Until such evidence is forthcoming, I will recommend avoidance of acetaminophen by all children with asthma or those at risk for asthma and will work to make patients, parents, and primary care providers aware of the possibility that acetaminophen is detrimental to children with asthma."
Since all children may be at risk from asthma, Dr. McBride is in effect saying that acetaminophen is contraindicated for the treatment of any children. Although the case for acetaminophen being a cause of autism and attention deficit with hyperactivity may not be as strong as the case for asthma, the severe asthma risk combined with the risks of autism and attention deficit with hyperactivity are so severe that we as a society should maintain a degree of caution with acetaminophen given the proven overall toxicity due to accidental overdose of the drug, and the availability of ibuprofen or abstaining from treatment as alternatives. A large-scale trial of acetaminophen restriction in pregnancy and the first 3 years of life is warranted to test the hypothesis that acetaminophen is a causative agent in autism, asthma, and attention deficit with hyperactivity. Due to the increased risks associated with accidental overuse of acetaminophen, increased public awareness of such risks is paramount.
Given associations of acetaminophen with increased rates of cancer, increased testicular damage, increased rates of asthma, and allergy, plausible causation of autism, and in vitro evidence of brain damage associated with metabolites of acetaminophen, new assessments of the relative risk of aspirin causing Reye's syndrome versus the risks of acetaminophen in children should be undertaken. If a clear link between acetaminophen use pre- or post-natally and autism is established, medical practice guidelines may need to be adjusted and alternative analgesics or antipyretics, such as ibuprofen, recommended. However, such a relationship may be difficult to establish, as studies may have to account for variations in dosage amounts, formats, combination product use, and for the possibility of product recalls given the recent reported manufacturing issues surrounding acetaminophen.
Note Added in Proof
After this article was submitted for publication on January 2, 2013 the following article was published prior to publication which strongly supports the hypothesis in the current article.
In 2013, Bauer and Kriebel reported that prenatal use of acetaminophen was strongly correlated with autism/Autism Spectrum Disorder prevalence (r = 0.80) using all available country-level data (n = 8) for the period 1984 to 2005. In addition, the authors found that after acetaminophen became commonly used to treat circumcision pain after 1995, there was a strong correlation between countrylevel (n = 9) autism/ASD prevalence in males and a country's circumcision rate (r = 0.98). A very similar pattern was seen among US states and when comparing the three main racial/ethnic groups in the US.
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Bauer AZ, Kriebel D. Prenatal and perinatal analgesic exposure and autism: an ecological link. Environ Health. 2013;12:41. doi: 10.1186/1476-069X-12-41.
Shaw, William Ph.D
More than 50 phenotypically different organic acidemias have been discovered since the first known disease of this type, isovaleric academia, was described in 1966. An organic acid is any compound that generates protons at the prevailing pH of human blood. Although some organic acidemias result in lowered blood pH, other organic acidemias are associated with relatively weak organic acids that do not typically cause acidosis.
Organic acidemias are disorders of intermediary metabolism that lead to the accumulation of toxic compounds that derange multiple intracellular biochemical pathways, including glucose catabolism (glycolysis), glucose synthesis (gluconeogenesis), amino acid and ammonia metabolism, purine and pyrimidine metabolism, and fat metabolism. The accumulation of an organic acid in cells and fluids (plasma, cerebrospinal fluid, or urine) leads to a disease called organic academia, or organic aciduria.
Although this technology has commonly been used for diagnosing genetic disease in children, genetic diseases in adults have also been detected with it. New applications of organic acid testing include detection of factors in psychiatric disorders, mitochondrial disease and dysfunction, and exposure to a wide variety of toxic chemicals from the environment, and assessment of inflammation due to overproduction of quinolinic acid from tryptophan.
Testing now includes markers for the following metabolites:
- Krebs Cycle
- Amino acid Metabolism
- DNA, RNA metabolism
- Neurotransmitter metabolism
- Organophosphate metabolism
- Yeast, fungal markers
- Markers for beneficial bacteria
- Oxalate markers for kidney stones, genetic disease
- Specific marker for ammonia toxicity
- Markers of fatty acid catabolism
- Metabolic diseases causing autism spectrum disorders
- Dry cleaning solvents
- Vinyl chloride
- Specific Clostridia marker
- Specific mitochondrial disease markers
- Vitamin deficiency markers
- Phosphate marker of bone function
- Marker for glutathione precursor
- Genetic screening with extremely sensitive markers
Organic acids are most commonly analyzed in urine because they are not extensively reabsorbed in the kidney tubules after glomerular filtration. Thus, organic acids in urine are often present at 100 times their concentration in the blood serum and thus are detected in urine with greater accuracy and precision than with blood samples. The number of organic acids found in urine is enormous: over 1000 have been detected since this kind of testing started 25 years ago. The challenge of dealing with so many compounds led to the use of gas chromatography-mass spectrometry (GC/MS) to analyze these complex body fluids.
With GC/MS, organic acids are chromatographically separated on the basis of their polarity and volatility and then bombarded by an electron beam that fragments the eluting molecules in a characteristic pattern. The patterns, or spectra, are stored by a computer system and then compared with known spectra that are compiled in a spectral "library." The software then compares an unknown spectrum to all the spectra on the hard drive and prints out those with the best fit. Since a single organic acid analysis generates over 1000 spectra, and each spectrum may consist of 600 ions, the software must be optimized to analyze the data in the most efficient and clinically relevant manner. Recently, the Great Plains Laboratory Inc. increased the sensitivity of this technology by approximately 1000-fold with the use of new triple-quadrupole MS technology so that a large number of toxic compounds can be measured at levels of micromoles/mole creatinine compared with urine compounds, such as vanillylmandelic (VMA), which is measured at levels of millimoles/mole creatinine.
The scope of organic acid testing has been markedly widened by commercial laboratories such that it can monitor physiological changes in nongenetic diseases and offer tremendous help in diagnosis and treatment of most chronic illnesses. Some examples are given below:
An adult with a movement disorder and bilateral temporal arachnoid cysts by brain imaging was found to have very elevated glutaric acid, indicating the presence of the genetic disease glutaric aciduria type 1.1Symptoms of this potentially fatal disorder include headaches, ataxia, memory loss, and many other neurological effects. Treatment with high doses of carnitine may be helpful in relieving symptoms in such cases, and of course such information is important for genetic counseling.
High levels of urine oxalates in an adult with frequent kidney stones led to a closer examination of the patient's dietary history. The patient ate a large spinach salad with pecans almost every day. Spinach is one of the foods highest in oxalates, and all nuts are high in oxalates as well. Treatment is directed at reducing dietary oxalates as well as calcium citrate and vitamin B6 supplementation.
After organic acid testing, a child with autism was found to have very high values (more than four times the upper limit of age-appropiate normals) of the catecholamine metabolites VMA and HVA, indicating a possible neuroblastoma. Follow-up imaging near the spine confirmed the presence of a previously undiagnosed neuroblastoma, likely saving the child's life.
Another child thought to have autism had very low amino acids, and the neurologist recommended high doses of amino acid supplements, which made the child severely ill. Organic acid testing revealed a massive excretion of methylmalonic acid, indicating that the child had methlmalonic aciduria, a severe genetic disorder. Treatment of this disorder requires extensive supplementation with vitamin B12 and a low-protein diet. Continued amino acid supplementation or a high-protein diet might have been fatal.
A person with severe depression was found to have low amounts of the serotonin metabolite 5-hydroxy-indoleacetic acid, which is derived from tryptophan. Depression is associated with decreased brain serotonin. However, the tryptophan metabolite by an alternate pathway, quinolinic acid, was much higher. Quinolinic acid is associated with inflammation such as arthritis and is considered to be neurotoxic, with a probable role in Parkinson's syndrome, Alzheimer's disease, Huntington's disease, and schizophrenia.2,3 The condition eosinophilia myalgia syndrome (EMS), associated with excessive tryptphan intake, is probably not due to tryptophan itself but to the inflammatory effects of its major metabolite quinolinic acid. Quinolinc acid administered by itself generated all of the symptoms of EMS.4,5 This research indicates that various conspiracy theories about contaminated tryptophan batches as the cause of EMS are unnecessary and probably wrong. 100% pure tryptophan at high enough doses will produce significant quantities of toxic quinolinic acid and EMS in susceptible individuals. Administration of 5-hydroxytryptophan (5-HT or 5-HTP) is a much safer option than tryptophan since 5-HT cannot be converted to the neurotoxic quinolinic acid, whereas only about 1% of tryptophan is converted to serotonin.6 Both the serotonin metabolite and quinolinic acid are measured by organic acid testing (Figure 1).
I recently proved that the dibiosis marker 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), the predominant dihydroxy-phenylpropionic acid isomer in urine measured in the organic acid test, is due to a combination of human metabolism and the metabolism by a group of Clostridia species, including but not limited to C. difficile.7 The same article indicates that 3,4-dihydroxyphenylpropionic acid (DHPPA) is a marker for beneficial bacteria in the gastrointestinal tract such as Lactobacilli, Bifidobacteria, and E. coli. The exception is one species of Clostridia orbiscindens that can convert the flavonoids luteolin and eriodictyol (which occur only in a relatively small food group that includes parsley, thyme, celery, and sweet red pepper) to DHPPA. The quantity of C. orbiscindens in the gastrointestinal tract is negligible (approximately 0.1% of the total bacteria) compared with the predominant flora of Lactobacilli, Bifidobacteria, and E. coli.7 DHPPA is an antioxidant that lowers cholesterol, reduces proinflammatory cytokines, and protects against pathogenic bacteria.
Outdated literature has referred to HPHPA as due to dietary origin based mainly on conjecture, but this conjecture was clearly disproved by my 2010 article which indicates that the metabolite is abolished by the antibiotic metronidazole.8 DHPPA, a different isomer, has been claimed to be a metabolite of Pseudomonas species, but the literature indicates that this compound is formed by the in vitro action of these species on quinolone, a component of coal tar – a substance missing from the diet of virtually all humans.9
HPHPA has been one of the most useful clinical markers in recent medical history. Treatment of individuals with high urinary values with metronidazole, vancomycin, or high doses of probiotics has led to significant clinical improvements or remissions of psychosis, tic disorders, seizures, autistic behaviors, hyperactivity, chronic fatigue syndrome, and obsessive compulsive behavior.
One of the newest aspects of organic acid testing is the screening for 74 different toxic chemicals in the environment by testing their organic acid metabolites. Solvents such as benzene, toluene, styrene, and others may be present for only short periods in body fluids and may also be lost in transit due to their volatility, but their metabolites are very stable. Using this screening technique, most metabolites of different organophosphates and pyrethrins can be measured as well as trichloroethylene, tetrachloroethylene, and vinyl chloride. Phthalates, an extremely toxic group of compounds implicated in infertility and abnormal sexual development in both males and females, can be measured by their metabolite phthalate, a specific chemical entity.
The chemical structure of phthalic acid (or phthalates) is nearly identical to quinolinic acid. A toxic effect occurs when phthalic acid competitively inhibits the reaction by which quinolinic acid is converted to the beneficial coenzyme NAD. High concentrations of phthalic acid or quinolinic acid may be associated with increased toxicity due to phthalate blockage of NAD formation and potential mitochondrial dysfunction due to deficient NAD for mitochondrial energy production.
One of the most important advances in the organic acid test is the addition of a biochemical marker, tiglylglycine, as a specific indicator for mitochondrial dysfunction.11 Mitochondrial dysfunction has been implicated in Parkinson's and Alzheimer's syndromes, diabetes, autism, chronic fatigue syndrome, aging, and many others. Tiglylglycine has been shown to be elevated in the urine in mitochondrial disorders involving defects of complexes I, II, III, and IV, protein complexes attached to the mitochondrial membrane that are involved in energy production. In addition to mutations in mitochondrial or nuclear DNA, mitochondrial dysfunction may also be due to exposures to toxic chemicals such as organophosphates and the solvent trichloroethylene. The advantage of the organic acid test is that if a mitochondrial dysfunction is detected, a number of different toxic chemicals implicated in mitochondrial damage can be reviewed to find the potential cause. Trichloroethylene has been found as a contaminant in the municipal water supply of many cities in both the US and Canada, and is used as a degreaser military bases and as a common solvent throughout the chemical industry. Mitochondrial disorders can be treated with a cocktail of nutritional substances including coenzyme Q10, carnitine, riboflavin, and others, when chemical exposure is not detected. If toxic chemicals are found, treatment with the Hubbard protocol can be highly successful for the removal of a wide array of toxic substances.12
- 1. Martinez-Lage J et al. Macrocephaly, dystonia, and bilateral temporal arachnoid cysts: glutaric aciduria type 1. Childs Nerv Sys. 1994;10(3): 198-203.
- 2. Guillemin GJ et al. Quinolinic acid in the pathogenesis of Alzheimer's disease. Adv Exp Med Biol. 2003;527:167-176.
- 3. Stoy N et al. Tryptophan metabolism and oxidative stress in patients with Huntington's disease. J Neurochem. 20015;93:611-623.
- 4. Silver RM et al. Scleroderma, fasciitis, and eosinophilia associated with the ingestion of tryptophan. N Engl J Med. 1990;322(13):874-881.
- 5. Noakes R, Spelman L, Williamson R. Is the L-tryptophan metabolite quinolinic acid responsible for eosinophilic fasciitis? Clin Exp Med. 2006;6(2):60-64.
- 6. Shah GM et al. Biochemical assessment of niacin efficiency among carcinoid cancer patients. Am J Gastroenterol. 2005;100:2307-2314.
- 7. Shaw W. Increased urinary excretion of a 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia species in the gastrointestinal tract, in urine samples from patients with autism and schizophrenia. Nutr Neurosci. 2010;13(3):1-10.
- 8. Kumps A, Duez P, Mardens Y. Metabolic, nutritional, latrogenic, and artifactual sources of urinary organic acids: a comprehensive table. Clin Chem. 2002,48:708-717.
- 9. Shukla OP. Microbial transformation of quinolone by a pseudomonas sp. Appl Environ Microbiol. 1986;51(6):1332-1342.
- 10. Fukuwatari T et al. Phthalate esters enhance quinolinate production by inhibiting alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarbocylase (ACMSD), a key enzyme of the tryptophan pathway. Toxicol Sci. 2004;81:302-308.
- 11. Bennett M et al. Tiglylglycine excreted in urine in disorders of isoleucine metabolism and the respiratory chain measured by stable isotope dilution GC-MS. Clin Chem. 1994;40(10):1879-1833.
- 12. Shaw W. The unique vulnerability of the human brain to toxic chemical exposure and the importance of toxic chemical evaluation and treatment in orthomolecular psychiatry. J Orthomol Med. 2010;25(3).
William Shaw, Ph.D.
The dysbiosis marker 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), the predominant dihydroxyphenylpropionic acid isomer in urine, is also measured in the Organic Acids Test offered by The Great Plains Laboratory. This marker was proven by Dr. William Shaw to be due to a combination of human metabolism and the metabolism by a group of Clostridia species, including but not limited to C. difficile.
HPHPA has been one of the most useful clinical markers in recent medical history. Treatment with metronidazole, vancomycin, or high doses of probiotics of individuals with high urinary values has led to significant clinical improvements or remissions of psychosis.
The biochemical role of Clostridia in altering brain neurotransmitters is due to the fact that Clostridia metabolites inactivate dopamine beta-hydroxylase, leading to an excess production of brain dopamine and reduced levels of the neurotransmitter norepinephrine. Excess dopamine is associated with abnormal or psychotic behavior. This imbalance can be demonstrated in the Organic Acids Urine Test by observing the ratio of the major dopamine metabolite, homovanillic acid (HVA), to that of the major norepinephrine metabolite, vanillylmandelic acid (VMA) when the Clostridia marker HPHPA is elevated. After treatment with metronidazole or vancomycin, HPHPA values return to normal along with normal ratios of HVA/VMA and normal behavior.
The highest value of HPHPA was measured in the urine of a young woman with first onset of schizophrenia. Treatment of Clostridia bacteria resulted in loss of auditory hallucinations. In autism, children with gastrointestinal Clostridia commonly exhibit aggressive behavior, agitation, obsessive compulsive behavior, and irritability. They may have very foul stools with diarrhea with mucus in the stools although some individuals may be constipated. Stool testing for Clostridia is usually of limited usefulness since most Clostridia species are considered probiotics or beneficial. There are about 100 species of Clostridia that are commonly found in the gastrointestinal tract. Only seven of these species are producers of HPHPA including C. sporogenes, C.botulinum, C. caloritolerans, C. angenoti, C. ghoni, C.bifermentans, C. difficile, and C. sordellii while C. tetani,C. sticklandii, C. lituseburense, C. subterminale, C.putifaciens, C. propionicum, C. malenomenatum, C.limosum, C. lentoputrescens, C. tetanomorphum, C.coclearium, C. histolyticum, C. aminovalericum, and C.sporospheroides do not produce compounds that are converted to HPHPA.
The same article by Dr. Shaw indicates that 3,4-dihydroxyphenylpropionic acid (DHPPA) is a marker for beneficial bacteria in the gastrointestinal tract such as Lactobacilli, Bifidobacteria, and E. coli. The exception is one species of Clostridia orbiscindens that can convert the flavanoids luteolin and eriodictyol, that occur only in a relatively small food group that includes parsley, thyme, celery, and sweet red pepper to 3,4-dihydroxyphenylpropionic acid. The quantity of C. orbiscindens in the gastrointestinal tract is negligible (approximately 0.1% of the total bacteria) compared to the predominant flora of Lactobacilli, Bifidobacteria, and E. coli (7). DHPPA is an antioxidant that lowers cholesterol, reduces proinflammatory cytokines, and protects against pathogenic bacteria. 2,3-Dihydroxyphenypropionic acid, a different isomer has been claimed to be a metabolite of Pseudomonas species but the literature indicates that this compound is formed by the in vitro action of these species on quinoline, a component of coal tar, a substance missing from the diet of virtually all humans.
1. Shaw W. Increased urinary excretion of a 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia spp. in the gastrointestinal tract, in urine samples from patients with autism and schizophrenia. Nutr Neurosci. 2010 Jun;13(3):135-43.
William Shaw, PhD
A child with autism was found to have complete IgA deficiency (serum IgA < 6 mg/dL; normal 33-235 mg/dL), Candidiasis of the gastrointestinal tract based on evaluation of stool testing and elevated urine arabinose, and elevated serum antibodies to wheat and dairy products. The pretreatment urinary arabinose concentration (341 mmol/mol creatinine in this child was nearly six times the mean value (60.4 mmol/mol creatinine, n=20) of normal children and over ten times the median value (31.0 mmol/mol creatinine) of normal controls. After antifungal therapy for four months, the urine was retested. At that time the urine arabinose was measured at 51 mmol/mol creatinine, a value only 15% of the baseline sample. Restriction of wheat and dairy products from the diet and antifungal therapy led to a significant decrease in autistic behaviors and increased rate of learning. The Childhood Autism Rating Scale (CARS), an observational measure of various aspects of autism, for the child has decreased from a rating of 43 (severely autistic) prior to introduction of these therapies to a value of 29 (non-autistic) after therapy.
Studies done by the late Reed Warren Ph.D. at Utah State University and others indicate that most children with autism have a substantial immune abnormality of some type (1-20). Kontstantareas and Homatidis (21) at the University of Guelph in Ontario, Canada found a high correlation between the prevalence of ear infections and the incidence of autism. They found that the earlier the child had an ear infection, the more likely that child had a more severe form of autism. They also found that increased incidence of ear infections was associated with a more severe rather than a mild form of autism. Candida infection has been reported as a consequence of frequent antibiotic usage in both humans and animals (22-30) and an abnormal increase in the sugar arabinose probably from Candida has been reported in urine samples of two siblings with autism (31). However, Candida infection may also be common in children with immunodeficiencies who do not have an unusually high number of infections treated with antibiotics. The pattern of gastrointestinal Candida overgrowth, immunodeficiency, metabolic disorder and autism is well illustrated in the medical history of the child evaluated by us.
Previous medical evaluation
The child evaluated is a five-year-old Caucasian male with a normal birth at term and normal apgar scores. Newborn metabolic screens for phenylketonuria, hypothyroidism, galactosemia, and sickle-cell disease were within normal limits. Both parents are college graduates; both parents are considered socially well adjusted. The maternal grandmother suffered from multiple sclerosis and is now deceased. The maternal grandfather died secondary to viral cardiomyopathy; as a child he did not speak until three years of age but then talked and developed normally. The paternal grandparents are in good health.
A pediatric ophthalmologist evaluated the child at six months of age for intermittent crusting and tearing of the left eye, which was non-responsive to antibiotic drops. The patient had surgery for the blocked tear-duct and a possible undescended testicle at 16 months. Exploratory surgery did not locate the missing testicle; the patient was put on prophylactic antibiotics after surgery. Up to the age of three years, the patient had had only one or two ear infections treated by antibiotics, a couple of colds, and an upper respiratory infection. Immunizations were all on schedule. A routine physical examination at 15 months of age assessed development as normal although parents expressed concerns about lack of speech. The MMR vaccine was administered at this checkup. Assessment by the pediatrician at 18 months was "healthy 1.5 year old" who "does not need to return until 2 years of age." Deficiency of expressive language was noted in the medical record but the parents were not advised to seek additional consultation.
At a pediatric evaluation at 2 years of age, lack of expressive language (only 5 words) was again noted but no follow-up was recommended. At a pediatric evaluation at 2.5 years of age, no expressive language was noted and the child was referred to a hearing and speech clinic for evaluation. Diet was noted to consist of bread, pancakes, milk, peanut butter, and chicken. He was noted to always have loose stools. The subsequent hearing evaluation revealed normal hearing but recommended a developmental assessment of the child. Three months later at the age of 27 months, the child was diagnosed with autism by a developmental pediatrician at a university autism clinic using DSM-IV diagnostic criteria; developmental age was assessed as at the 19-20 month level. At this exam, otitis media was diagnosed and treated with Amoxicillin. A MRI scan of the head revealed some atrophy of the frontotemporal lobe. EEG and fragile X chromosome studies were normal. The child was seen by a second university autism clinic in another state, which confirmed the original diagnosis. The parents of the child were referred to support groups, to speech therapists, and to special schools for education and behavioral modification but were not referred for any evaluation of the child's immune or gastrointestinal function.
When the child was 4.75 years of age, the parents decided to embark on additional biochemical assessments of their child including allergy assessment, routine chemistry and hematology, evaluation of stool microorganisms, evaluation of immune function, and urine organic acid testing.
Comprehensive food allergy testing for 96 foods was performed using IgG specific enzyme linked immunoassay. The following allergens were positive by IgG-specific enzyme linked immunoassay: barley, gluten, wheat, bran, cow's milk, cheeses (cheddar, cottage, and Swiss), beef, grapefruit, orange, peanut, soybean, and sugar. The IgA endomysial antibody test, which is considered to be specific for celiac disease, was negative in this child.
Normal serum values were found for all of the following: glucose, urea nitrogen, creatinine, total protein, albumin, total bilirubin, alkaline phosphatase, AST, ALT, LDH, calcium, phosphorus, blood lead, sodium, potassium, chloride, bicarbonate, uric acid, triglycerides, cholesterol, anion gap, thyroxine, antinuclear antibodies, thyrotropin (highly sensitive), iron, copper, magnesium, cortisol, zinc, and ferritin. White cell count was slightly low (4900/mm3); normal: 5500-15,500/mm.3 Hemoglobin and hematocrit were normal. The white cell differential was normal except for a slight elevation of atypical lymphocytes. The absolute number and percentage of CD3, CD4, and CD8 cells, evaluated by flow cytometry were all within normal limits.
Analysis of serum immunoglobulins revealed normal values for serum IgG, IgM, IgE, and IgG subclasses but undetectable values for serum IgA (Table 1). Stool analysis revealed a 4+ overgrowth of Candida parapsilosis; normal is 0 and the highest possible overgrowth is 4+. Antifungal sensitivity of the organism indicated sensitivity to fluconazole, itraconazole, nystatin, and ketoconazole. Bacteria in the stool sample usually considered beneficial were Lactobacillus (2+) and Bifidobacteria (4+). Stool analysis also revealed 3+ levels of gamma streptococci and 4+ hemolytic E. coli. An evaluation of a urine sample by gas chromatography-mass spectrometry as described previously (31) taken at the same time indicated significant increases of the sugar arabinose as the major abnormality; there were no abnormalities associated with any recognized inborn error of metabolism.
Because of elevated Candida in the stool sample, indicating a gastrointestinal yeast overgrowth, the child was placed on 100,000 Units nystatin four times a day plus alternating weeks of Nizoral or Diflucan (2 mg/kg) and was also placed on a gluten and casein free diet approximately two months after beginning antifungal therapy. Both dietary and antifungal therapies are continuing five months later. The pretreatment urinary arabinose concentration (341 mmol/mol creatinine in this child was nearly six times the mean value (60.4 mmol/mol creatinine, n = 20) of normal children and over ten times the median value (31.0 mmol/mol creatinine) of normal controls. After antifungal therapy for four months, the urine was retested. At that time the urine arabinose was measured at 51 mmol/mol creatinine, a value only 15% of the baseline sample. With two additional months of antifungal treatment, the urine arabinose value decreased to 26 mmol/mol creatinine. A follow-up stool test indicated the absence of Candida in the sample.
Results of therapeutic interventions
The mother of the child reports a significant increase in eye contact, a significant decrease in self-stimulatory behavior, and increased use of spontaneous language shortly after beginning antifungal therapy. After beginning the casein and gluten free diet, the mother reports the child was able to follow three step verbal directions versus only one step directions previously. The mother also reported increased learning speed in the schooling program, increased verbal labeling, and increased spontaneous verbal initiations. The score for the child on the Childhood Autism Rating Scale (CARS), an observational measure of various aspects of autism has decreased from a rating of 43 (severely autistic) prior to introduction of these therapies to a value of 29 (non-autistic) after therapy. Cutoff for autism is 30 or above. The child is now considered by the assessment team at the state university autism clinic to be a high-functioning individual with autism. The child can now parallel play with other children in class, demonstrates an interest in peers, shares toys, and is engaging in some imaginative play.
Selective IgA deficiency
The most striking laboratory abnormality of this child is the absence of detectable IgA. IgA is the antibody that is involved with protection of the lining of the nasal passages and intestinal lining from microorganisms. Secretory IgA or sIgA is a special form of the IgA antibody that is secreted to protect the mucosa, which is the lining of the intestinal tract. Secretory IgA on a stool sample from this child was also noted as deficient. Secretory IgA is apparently secreted by the gall bladder and then trickles down the bile ducts into the small intestine. Some children with autism such as this one have very low or even completely absent levels of IgA (1,20); in such cases there is probably also a deficiency of a secretory IgA since secretory IgA is derived from IgA.
This extremely common immunodeficiency occurs in 1 in 600-1000 persons of European ancestry (32). The causes of IgA deficiency are not completely known. There are some cases in which the deficiency runs in families while in other cases it does not. It has been reported in association with abnormalities of chromosome 18, but most individuals with IgA deficiency have no detectable chromosomal abnormalities (32). IgA deficiency may also be caused by drugs or viral infection (rubella, cytomegalovirus, toxoplasmosis) and may be also be associated with intrauterine infections. Patients with IgA deficiency are usually deficient in both subtypes of IgA, IgA1 and IgA2.
In Gupta's study (20), 20% of the children with autism had a deficiency of IgA and 8% lacked it completely. Reed Warren and his colleagues (1) also found that 20% of individuals with autism had low serum IgA compared with none of the normal controls. Thus, complete IgA deficiency in autism is somewhere between 48 and 80 times higher in the autism population compared to a normal Caucasian population.
IgA replacement therapy cannot be used currently because the short half-life of IgA would make it an extremely expensive therapy. However, bovine colostrum, which is commercially available, is high in IgA and might be considered as a possible therapy for IgA-deficient patients. IgG therapy can be used with patients with low IgA values. If the IgA values are so low that they cannot even be detected, however, giving IgG therapy is too risky. It is possible that the immunodeficient person's body would produce antibodies against IgA present in gamma globulin, causing potentially fatal anaphylactic shock.
The clinical consequences of IgA deficiency range from severe systemic infection to a perfectly healthy state. Many IgA-deficient persons are never aware of their antibody deficiency while others may have recurrent infections, allergic diseases, and autoimmune diseases (32). This child with autism had significant Candidiasis of the gastrointestinal tract despite the fact that the child had only two courses of antibiotics during his lifetime. Thus, intestinal Candidiasis following antibiotic therapy appears to be a much greater risk in a child with immunodeficiency. The decrease in symptoms of autism after antifungal therapy and gluten and casein restriction has been noted in many children with autism (33). (The authors are aware of three children with autism diagnosed at university autism centers who are now considered symptom-free after antifungal treatment and gluten and casein restriction.)
The child being presented was never considered a "sickly" child by the parents. It is possible that the difficult to treat eye crusting may have been related to the IgA deficiency since IgA is secreted in tears, saliva, and gastric juice; the deficient IgA in the tears may have led to a greater number of eye infections. The occurrence of multiple sclerosis in the maternal grandmother might be of significance but she was never evaluated for IgA deficiency. The remarkable spectrum of clinical manifestations of this immunodeficiency may be related to variations in the ability to replace IgA antibodies in the mucous secretions with IgM antibodies. IgG2 and IgG4 subclass deficiencies are common in IgA deficiency but were not present in this individual.
IgA Deficiency and Celiac Disease
The incidence of selective IgA deficiency is 10 times higher in patients with celiac disease compared to the general population (34). The diagnosis of celiac disease cannot be excluded in an IgA deficient child because the endomysial antibody test uses IgA antibody specificity and may yield false negative results in such cases (35) so the possibility that the child may have celiac disease cannot be excluded. The parents elected to place the child on a wheat and dairy-free diet based on the ELISA-allergy test results so a diagnosis of celiac disease by intestinal biopsy would not be valid for this child. Positive IgG antibodies to gluten were found in 100% of IgA-deficient persons with biopsy proven celiac disease but who were negative by the endomysial antibody test (35). Most children with autism are sensitive to both gluten, the major protein in wheat and barley, and to casein, the major protein in cow's milk (36-40). Elevation of IgG antibodies to wheat, barley, and several dairy products is common in autism even though most children with autism do not have celiac disease (36-40). Behavioral improvements after restriction of gluten and casein are attributed to a decrease in peptides (casomorphin and gliadorphin) derived from gluten and casein that have central nervous system opioid effects (36-40).
Candidiasis and abnormal arabinose: possible implications in brain structure and function
The exact biochemical role of elevated arabinose is unknown but a closely related sugar alcohol, arabitol, has been used as a biochemical indicator of invasive candidiasis (41-43). We have never found elevated arabitol in thousands of urine samples tested, including many samples with elevated arabinose and high yeast counts in the stool. Elevated arabinose in the urine of two brothers with autism was first reported by Shaw et al. in 1995 (31) and has since then has been reported to be prevalent in urine samples from people with autism (33); values as high as 4000 mmol/mol creatinine have been found in children with autism (unpublished data). We have found arabitol but not arabinose in the culture media of multiple isolates of Candida albicans isolated from stool samples of autistic children (unpublished data). Presumably elevated arabitol in the urine may only occur in systemic rather than gastrointestinal Candidiasis since arabitol in portal blood is converted to arabinose in the liver. Arabinose in the urine decreased markedly after antifungal therapy, concomitantly with an elimination of stool Candida. Arabinose, a sugar aldehyde or aldose reacts with the epsilon amino group of lysine in a wide variety of proteins and may then form cross-links with arginine residues in an adjoining protein (44), thereby cross-linking the proteins and altering both biological structures and functions of a wide variety of proteins (Figure 1) including proteins involved in the interconnection of neurons. Decreased clinical symptoms of autism after antifungal treatment would be due to decreased arabinose and pentosidine formation, resulting in fewer random neural connections (neural noise) and increased numbers of neural connections that are oriented to the child's environment.
This adduct of arabinose, lysine, and arginine is called a pentosidine (Figure 1). The epsilon amino group of lysine is a critical functional group of many enzymes to which pyridoxal (vitamin B-6), biotin, and lipoic acid are covalently bonded during coenzymatic reactions (45); the blockage of these active lysine sites by pentosidine formation may cause functional vitamin deficiencies even when nutritional intake is adequate. In addition, this epsilon amino group of lysine may also be important in the active catalytic site of many enzymes. Protein modification caused by pentosidine formation is associated with crosslink formation, decreased protein solubility, and increased protease resistance. The characteristic pathological structures called neurofibrillary tangles associated with Alzheimer disease contain modifications typical of pentosidine formation. Specifically, antibodies against pentosidine react strongly to neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease (46). In contrast, little or no reaction is observed in apparently healthy neurons of the same brain.
Thus, it appears that the neurofibrillary tangles of Alzheimer's disease may be caused by the pentosidines. The modification of protein structure and function caused by arabinose could account for the biochemical and insolubility properties of the lesions of Alzheimer disease through the formation of protein crosslinks. Similar damage to the brains of autistic children might also be due to the pentosidines; neurofibrillary tangles have also been reported in the brain tissue of an individual with autism (47). Improvement of symptoms of autism after antifungal therapy might be mainly due to a decrease in the concentration of arabinose and a concomitant decrease in the production of pentosidine cross-links. Since pyridoxal (vitamin B-6) reacts with the same critical epsilon amino group of lysine, it is possible that the beneficial effects of vitamin B-6 in autism reported in multiple studies (48) may be mediated by prevention of further pentosidine formation. Analysis of brain tissue of people with autism for increased brain pentosidines could be invaluable in the confirmation of this hypothesis.
Women with vulvovaginitis due to Candida were found to have elevated arabinose in the urine (49); restriction of dietary sugar brought about a dramatic reduction in the incidence and severity of the vulvovaginitis. Thus, one of the mechanisms of action of antifungal drug therapy for autism might be to reduce the concentration of an abnormal carbohydrate produced by the yeast that can not be tolerated by the child with defective pentose metabolism or an inability to remove harmful pentosidines. Arabinose tolerance tests should be able to rapidly determine if such biochemical defects are present in children with autism.
A Model for Autism
The success of Gupta (20) in treating the autistic symptoms of children with autism with gamma globulin therapy indicates an immune abnormality in autism. Based on these findings and our findings of abnormal arabinose and other organic acids in other children with autism (31,33), we propose the following model for autism (Figure 2). According to this model, immune deficiencies, which may be genetic or acquired, lead to an increased frequency of infections, which in the United States are almost always treated with broad-spectrum oral antibiotics that result in intestinal yeast overgrowth. Furthermore, many isolates of Candida albicans produce gliotoxins (50,51) and other immunotoxins (52,53) which impair the immune system and increase the likelihood of additional infections which lead to additional antibiotic usage and greater proliferation of yeasts and antibiotic-resistant bacteria, setting up a vicious cycle. These organisms produce high amounts of abnormal carbohydrates such as arabinose and Krebs cycle analogs such as citramalic and tartaric acids (31).
There is no inherent reason that dramatic biochemical changes in multiple biochemical systems caused by microorganisms would not be expected to alter brain structure and function. In PKU, correction of the metabolic defect by restriction of phenylalanine during infancy allows for normal development; retardation occurs if dietary intervention occurs too late. If abnormally elevated metabolites cause autism, then it is reasonable to think that elevations of these compounds would have maximum negative impact during periods of critical brain growth and development. As in PKU, metabolic intervention in autism might only be possible in the early stages of the disorder before the brain has matured. The differences in severity of disease and individual differences in symptoms might be due to different combinations of metabolites, how elevated they are, the duration of the elevation, the age at which the metabolites become abnormally elevated, and the susceptibility of the individual developing nervous system to the different microbial metabolites.
Some children with autism have a history of frequent infections: two different parents of children with autism indicated to the authors that their children had over 50 consecutive infections (predominantly otitis media) treated with antibiotics. However, some children with autism such as the child presented here did not have excessive use of oral antibiotics and was not considered to be a "sickly child" by the parents or attending physicians. In this child the underlying immune deficiency and two uses of antibiotics apparently led to a persistent yeast overgrowth of the intestinal tract.
Genetic immunodeficiencies proposed as the major genetic factors in autism
Ritvo et al. (54) found a concordance rate for autism of 23.5% in dizygotic twins and 95.7% in monozygotic twins, indicating a strong genetic basis for autism. However, the results of the Stanford autism genetics study of 90 families affected by autism (55) indicate " that there are no genes with a major effect for autism. That is, our analyses show that autism is almost surely not a simple single major gene disorder, such as Huntington disease. Rather, the analyses from these 90 families indicate that there are likely to be a relatively large number of different genes related to the susceptibility for autism, each with a minor effect." We suspect that many of these "relatively large number of genes" are those that regulate the immune system. We have been impressed with the large number of studies that have indicated a wide number of abnormalities of the immune system in autism (1-20) including IgG deficiency, IgA deficiency, IgG subclass deficiency, myeloperoxidase deficiency (a genetic defect in an enzyme of the leukocytes that produces hypochlorite ion to kill yeast), reduced natural killer cell activity, markedly elevated serum levels of the cytokines interleukin-12 and interferon-gamma, increased anti-myelin and serotonin receptor antibodies, increased DR+ T cells, and a deficiency in complement C4b. In addition, some immune abnormalities in autism have been linked to adverse reactions to vaccinations (56). The two brothers with autism in which abnormal arabinose and abnormal organic acids were first reported (31) both had abnormally low concentrations of serum IgG. Autism has also been diagnosed in other children with defined inborn errors of metabolism such as biotinidase deficiency and isovaleric acidemia (Lombard, Personal Communication) in which yeast infections are common.
Efforts to locate a single autism gene would fail since any genetic factor that severely impairs the immune system may eventually lead to the proliferation of antibiotic-resistant yeasts and bacteria which then alter behavior of children at critical periods of development through the excretion of abnormal microbial metabolic products. Thus, autism appears to be a complex metabolic disorder involving immune deficiencies, autoimmune abnormalities, abnormal food sensitivities, and gastrointestinal microbial overgrowths that may result in altered human metabolism and protein function.
Figure 1. Reaction of arabinose from yeast with amino groups of lysine to form a Schiff base adduct. The rearranged Schiff base then reacts with a guanido group on an arginine residue of a second protein, resulting in two different proteins crosslinked through a pentosidine moiety.
Figure 2. Immunodeficiency model for autism. In this model, immunodeficiencies lead to antibiotic use that stimulates yeast overgrowth (primarily Candida) of the gastrointestinal tract. Certain strains of Candida produce immunosuppressant compounds called gliotoxins that further weaken the immune system and may lead to additional infections. Arabitol produced by Candida in the gastrointestinal tract is converted to arabinose by the liver. Elevated arabinose then leads to pentosidine formation, leading to increased neurofibrillary tangles in the brain.
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