Inhibition of dopamine conversion to norepinephrine by Clostridia metabolites appears to be a (the) major cause of autism, schizophrenia, and other neuropsychiatric disorders.

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).

Figure 2. Effect of Clostridia metabolites on human catecholamine metabolism. DHPPA, 4-cresol, HPHPA, HVA, and VMA are all measured in The Great Plains Laboratory organic acid test.

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 intellectually disabled 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.

Reference:

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.

Integrative Treatments for Behavioral Problems in Children

By: James Greenblatt, MD

Attention deficit/hyperactivity disorder (ADHD) is a multifactorial condition that is influenced by genetic, biological, environmental, and nutritional factors. While there are numerous integrative therapies available including vitamins, minerals, herbs, neurofeedback, exercise, and meditation, individuals are unique and thus require personalized treatments based on their own biological needs identified through laboratory testing. In this article, we will discuss commonly overlooked mineral deficiencies and imbalances in the gastrointestinal flora that can exacerbate behavioral symptoms and impede the therapeutic effect of pharmacological treatment.

In the early 1960s, researchers discovered that zinc was an essential trace mineral necessary for normal growth and development. Zinc is also critical for immune function, and the activity of over 300 enzymes is dependent on zinc bioavailability. Zinc is a vital component of the central nervous system, maintaining neurotransmitter activity. This mineral enhances GABA, one of our main inhibitory/relaxation neurotransmitters. Moreover, zinc is needed as a co-factor to produce melatonin which helps regulate dopamine function.

Multiple studies have confirmed that not only are zinc levels lower in children with ADHD, but the extent of the deficiency is proportionately correlated with the severity of ADHD symptoms including inattention, hyperactivity, impulsivity, and conduct problems:

• Toren et al. (1996) found that almost one-third of 43 ADHD children aged 6-16 were severely deficient in serum zinc.

• Another study involving 48 ADHD children aged 5-10 demonstrated that most of the participants had serum zinc levels in the lowest 30% of the reference range.

• There is a highly significant inverse correlation between zinc level and parent and teacher ratings of inattention among children with ADHD (Arnold et al., 2005). A more recent study echoed the same findings, when researchers analyzed the zinc in the hair of 45 children with ADHD against 44 controls. They found that there was a relationship between hair zinc levels and worse overall ADHD symptoms (Shin et al., 2014).

• In a recent study, 70% of the 20 ADHD cases examined were zinc deficient. Those with lower hair zinc levels reported significantly increased symptoms of inattention, hyperactivity, and impulsivity (Elbaz et al., 2016).

• In a larger group of 118 children with ADHD, those with the lowest blood levels of zinc had the most severe conduct problems, anxiety, and hyperactivity as rated by their parents (Oner et al., 2010).

In children with ADHD, plasma zinc levels were shown to directly affect information processing via event related potentials which reflect brain activity. In ADHD children compared to controls, the amplitudes of P3 waves in frontal and parietal brain regions were significantly lower (worse working memory) and the latency of P3 in the parietal region was significantly longer (slower information processing). Unsurprisingly, plasma zinc levels were significantly lower in the ADHD children compared to the control children. When a low-zinc ADHD subgroup was compared to a nondeficient ADHD subgroup, the latencies of N2 in frontal and parietal brain regions were significantly shorter (worse information processing and inhibition) (Yorbik et al., 2008).

Supplementation with zinc is more effective at improving ADHD symptoms when compared to placebo, and can also be an effective adjuvant therapy to enhance the therapeutic effect of stimulant medication without increasing the dosage. When 400 ADHD children aged 6-14 were randomized to zinc sulfate 150 mg/day or placebo for 12 weeks, those taking zinc had significantly reduced symptoms of hyperactivity, impulsivity, and impaired socialization (Bilici et al., 2004). Similarly, when over 200 children were randomized to zinc 15 mg/day or to placebo for 10 weeks, those taking zinc saw significant improvement in attention, hyperactivity, oppositional behavior, and conduct disorder. And these children had normal zinc levels to begin with (Üçkardeş et al., 2009). In a small study of 18 boys with ADHD, higher baseline hair zinc levels predicted better behavioral response to amphetamine (Arnold et al., 1990). In a six-week double blind, placebo controlled trial, researchers assessed the effects of zinc in combination with methylphenidate (Ritalin). 44 children aged 5-11 were randomized to methylphenidate plus zinc sulfate 55 mg/day or methylphenidate plus placebo. At week 6, those taking zinc had significantly better scores on the Parent and Teacher ADHD Rating Scale (Akhondzadeh et al., 200452 children aged 6-14 with ADHD were randomized to zinc glycinate 15 mg/day or placebo for 13 weeks. For the first 8 weeks, they only took zinc then for the last 5 weeks they also took d-amphetamine. The optimal absolute mg/day amphetamine dose with zinc was 43% lower than with placebo (Arnold et al., 2011).

Copper is an essential trace mineral that plays an active role in the synthesis of dopamine and norepinephrine. However, excess copper can manifest as displays of aggression, hyperactivity, insomnia, and anxiety. Elevated copper levels can also cause low zinc levels and reduce the efficacy of medications commonly used to treat ADHD.

Copper may affect ADHD through its role in antioxidant status. Copper/Zinc superoxide dismutase (SOD-1) is a key enzyme in our antioxidant defense system. Both copper and zinc participate in SOD enzymatic activities that protect against free radical damage. In a study on 22 ADHD children and 20 controls, serum Copper/Zinc SOD levels of ADHD children were significantly lower in individuals with high serum copper when compared to controls. It is also hypothesized that excess copper can damage dopamine brain cells by destroying antioxidant defenses, such as lowering Copper/Zinc SOD levels (Russo, 2010).

In a randomized controlled trial on 80 adults with ADHD, lower baseline copper levels were associated with better response to treatment with a vitamin-mineral supplement (Rucklidge et al., 2014). Unfortunately, even copper levels that are considered normal can negatively affect cognition. In a group of 600 adolescents with normal copper levels, blood copper was associated with decreased sustained attention and short-term memory (Kicinski et al., 2015).

Magnesium is part of 300 enzymes that utilize ATP (cellular energy) and is important for nerve transmission. It is involved in the function of the serotonin, noradrenaline, and dopamine receptors. Magnesium has been progressively declining in our food supply due to increased consumption of processed foods. The use of medications, presence of stress, and caffeine and soft drink consumption also deplete magnesium, and it is estimated that 50% of Americans are deficient in magnesium (Mosfegh et al., 2009).

Symptoms of magnesium deficiency include irritability, difficulty with concentration, insomnia, depression, and anxiety. A prospective population-based cohort of over 600 adolescents at the 14- and 17-year follow-ups found that higher dietary intake of magnesium was significantly associated with reduced externalizing behaviors (attention problems, aggressiveness, delinquency) (Black et al., 2015). Because up to 95% of those with ADHD are deficient in magnesium, almost all ADHD children can benefit from magnesium supplementation (Kozielec & Starobrat-Hermelin, 1997).

In a recent study on 25 patients with ADHD aged 6-16, 72% of children were deficient in magnesium and there was a significant correlation between hair magnesium, total IQ, and hyperactivity. The magnesium deficient children were randomized to magnesium supplementation 200 mg/day plus standard medical treatment or to standard medical therapy alone for 8 weeks. Those taking magnesium saw a significant improvement in hyperactivity, impulsivity, inattention, opposition, and conceptual level while those taking medication alone did not see these improvements (El Baza et al., 2015).

Supplements of magnesium plus vitamin B6, which increases magnesium absorption, have shown promise for reducing ADHD symptoms. One study on 52 children with ADHD found that 58% had low red blood cell magnesium levels. All the children were given preparations of magnesium plus vitamin B6 100 mg/day for a period of 1 to 6 months. In all patients, physical aggression, instability, attention at school, muscle rigidity, spasms, and twitching were improved. One of the treated children was a six-year old identified as “J”. Initially, J suffered from aggressiveness, anxiety, inattention, and lack of self-control. After taking magnesium supplements, he reported better sleep and concentration and no methylphenidate was needed (Mousain-Bosc et al., 2004). A later study by the same researchers also found that 40 children with ADHD had significantly lower red blood cell magnesium values than control children. Likewise, a magnesium-vitamin B6 regimen for at least 2 months significantly improved hyperactivity, aggressiveness, and school attention. The researchers concluded, “As chronic magnesium deficiency was shown to be associated to hyperactivity, irritability, sleep disturbances, and poor attention at school, magnesium supplementation as well as other traditional therapeutic treatments, could be required in children with ADHD” (Mousain-Bosc et al., 2006). In a larger study of 122 children with ADHD aged 6-11, 30 days of magnesium-vitamin B6 supplementation led to improved anxiety, attention, and hyperactivity. On a battery of tests, magnesium treatment increased attention, work productivity, task performance, and decreased the proportion of errors. The EEG of treated children showed positive changes as well, with brain waves significantly normalizing (Nogovitsina & Levitina, 2007).

There has also been a considerable amount of research illustrating the symbiotic, bidirectional relationship between the brain and the gut, and animal studies have demonstrated how certain strains of bacteria, or lack thereof, can alter cognitive and emotional processes. In the presence of dysbiosis, where “bad” bacteria outnumber the “good,” harmful strains of bacteria can proliferate and cause behavioral disturbances.

HPHPA is a harmful byproduct of some strains of the bacterium Clostridium that can disrupt the normal gut environment. Elevated urinary levels are commonly seen in ADHD children, especially those with poor response to stimulants. HPHPA inhibits the conversion of dopamine to norepinephrine. This causes dopamine to accumulate, resulting in decreased attention and focus. A patient should especially be tested for HPHPA if he or she experiences stimulant side effects such as irritability, agitation, or anxiety. ADHD medications work by increasing dopamine. But high HPHPA levels prevent the breakdown of dopamine, exacerbating symptoms. HPHPA must be cleared before medications will be helpful. Probiotics, good bacteria found in fermented food such as yogurt, or antibiotics can be used to lower HPHPA.

Intestinal overgrowth of Candida yeast is seen in some children with ADHD, mostly in those with a diet high in sugar that feed Candida, or in those who have received many rounds of antibiotics for recurrent ear infections. Antibiotics are effective at resolving infections by eradicating all bacteria, including the good bacteria. An early study found that children with the greatest history of ear infections (and presumably the greatest frequency of antibiotic use) had an increased chance for developing hyperactivity later (Hagerman & Falkenstein, 1987). Toxins produced by Candida can enter the bloodstream and then enter the brain where they can cause changes leading to hyperactivity and poor attention span. Fortunately, the presence of HPHPA and other yeast overgrowth can be easily detected with an organic acids test or with a stool sample. Candida can be treated with probiotics, antifungal foods (e.g. garlic, oregano, ginger), and a lower sugar diet. In some cases, a regimen of antibiotics and probiotics can be useful in reestablishing a healthy gut flora.

Nutritional augmentation strategies are frequently used as part of the integrative clinician’s toolbox to treat behavioral disorders in children. It is important for healthcare providers to collaborate and communicate with caregivers of children with behavioral disorders to discern whether other complementary therapies could be incorporated into treatment. By carefully assessing a patient’s whole health history and conducting appropriate laboratory testing, providers can make informed treatment recommendations that is tailored specifically for the individual.

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References

Akhondzadeh, et al (2004). Zinc sulfate as an adjunct to methylphenidate for the treatment of attention deficit hyperactivity disorder in children: A double blind and randomized trial ISRCTN64132371. BMC Psychiatry, 4, 9.

Arnold et al. (1990). Does hair zinc predict amphetamine improvement of ADD/hyperactivity? The International Journal of Neuroscience, 50(1-2), 103-7.

Arnold et al. (2005). Serum zinc correlates with parent- and teacher- rated inattention in children with attention-deficit/hyperactivity disorder. Journal of Child and Adolescent Psychopharmacology, 15(4), 628-36.

Arnold et al. (2011). Zinc for attention-deficit/hyperactivity disorder: Placebo-controlled double-blind pilot trial alone and combined with amphetamine. Journal of Child and Adolescent Psychopharmacology, 21(1), 1-19.

Bilici et al. (2004). Double-blind, placebo-controlled study of zinc sulfate in the treatment of attention deficit hyperactivity disorder. Progress in Neuropsychopharmacology & Biological Psychiatry, 28(1), 181-190.

Black et al. (2015). Low dietary intake of magnesium is associated with increased externalising behaviours in adolescents. Public Health Nutrition, 18(10), 1824-30.

Elbaz et al. (2016). Magnesium, zinc and copper estimation in children with attention deficit hyperactivity disorder (ADHD). Egyptian Journal of Medical Human Genetics, Egyptian Journal of Medical Human Genetics, in press.

El Baza et al. (2016). Magnesium supplementation in children with attention deficit hyperactivity disorder. Egyptian Journal of Medical Human Genetics, 17(1), 63-70.

Hagerman & Falkenstein. (1987). An Association Between Recurrent Otitis Media in Infancy and Later Hyperactivity. Clinical Pediatrics, 26(5), 253.

Kicinski et al. (2015). Neurobehavioral function and low-level metal exposure in adolescents. International Journal of Hygiene and Environmental Health, 218(1), 139-146.

Kozielec & Starobrat-Hermelin. (1997). Assessment of magnesium levels in children with attention deficit hyperactivity disorder (ADHD). Magnesium Research: Official Organ Of The International Society For The Development Of Research On Magnesium, 10(2), 143-148.

Moshfegh et al. (2009). What We Eat in America, NHANES 2005–2006: Usual Nutrient Intakes from Food and Water Compared to 1997 Dietary Reference Intakes for Vitamin D, Calcium, Phosphorus, and Magnesium. U.S. Department of Agriculture, Agricultural Research Service: Washington, DC, USA.

Mousain-Bosc et al. (2004). Magnesium VitB6 intake reduces central nervous system hyperexcitability in children. Journal Of The American College Of Nutrition, 23(5), 545S-548S.

Mousain-Bosc et al. (2006). Improvement of neurobehavioral disorders in children supplemented with magnesium-vitamin B6. I. Attention deficit hyperactivity disorders. Magnesium Research: Official Organ Of The International Society For The Development Of Research On Magnesium, 19(1), 46-52.

Nogovitsina & Levitina. (2007). Neurological aspects of the clinical features, pathophysiology, and corrections of impairments in attention deficit hyperactivity disorder. Neuroscience and Behavioral Physiology, 37(3), 199-202.

Oner et al. (2010). Effects of Zinc and Ferritin Levels on Parent and Teacher Reported Symptom Scores in Attention Deficit Hyperactivity Disorder. Child Psychiatry and Human Development, 41(4), 441-447.

Rucklidge et al. (2014). Moderators of treatment response in adults with ADHD treated with a vitamin–mineral supplement. Progress in Neuropsychopharmacology & Biological Psychiatry, 50, 163-171.

Russo, A. (2010). Decreased Serum Cu/Zn SOD Associated with High Copper in Children with Attention Deficit Hyperactivity Disorder (ADHD). Journal of Central Nervous System Disease, 2, 9-14.

Shin et al. (2014). The Relationship between Hair Zinc and Lead Levels and Clinical Features of Attention-Deficit Hyperactivity Disorder. Journal of the Korean Academy of Child and Adolescent Psychiatry, 25(1), 28-36.

Toren et al. (1996). Zinc deficiency in attention-deficit hyperactivity disorder. Biological Psychiatry, 40(12), 1308-1310.

Üçkardeş et al. (2009). Effects of zinc supplementation on parent and teacher behaviour rating scores in low socioeconomic level Turkish primary school children. Acta Paediatrica, 98(4), 731-736.

Yorbik et al. (2008). Potential effects of zinc on information processing in boys with attention deficit hyperactivity disorder. Progress in Neuropsychopharmacology & Biological Psychiatry, 32(3), 662-667.

Clostridia difficile - The Role of Toxin A and B in its Pathogenicity

Kurt Woeller, DO

There are approximately 100 species of clostridia bacteria that can inhabit the gastrointestinal tract of humans. Not all of these clostridia are disease causing, but a certain few can lead to serious illness in susceptible individuals. There are 5 main species of clostridia known to cause disease: Clostridia botulinum, Clostridia perfringens, Clostridia tetani, Clostridia sordellii, and Clostridia difficile. This article will focus on the role of Clostridia difficile (C. difficile), and its production of various gastrointestinal toxins in human illness.

What Are Clostridia Bacteria?
Clostridia difficile, like all clostridia bacteria, is an obligate anaerobe. This means it is an organism that thrives in an oxygen devoid environment and is susceptible to being killed by normal atmospheric oxygen. It is unique in its ability to survive in hostile environments in part because of its spore development. Clostridia spores, the reproductive cell of clostridia, have thick cell walls which resist heat and antimicrobial compounds. These spores of clostridia are highly contagious and can be spread person to person even from individuals without symptoms of clostridia overgrowth.

C. difficile is a complex species of clostridia because of the various toxins it can produce. There are certain strains of C. difficile that produce compounds known to alter mitochondrial function by interfering with various steps in Kreb Cycle metabolism decreasing the amount of nicotinamide adenine-dinucleotide (NADH) used by the electron transport chain for adenosine-triphosphate (ATP) production (1). Other C. difficile strains create neurochemical compounds that disrupt dopamine production leading to various issues with regards to mental health. However, the most commonly known toxins produced by C. difficile are those that disrupt gastrointestinal function and in some individuals can lead to serious health problems.

The Prevalence of C. difficile Associated Disease
The rates of C. difficile infections leading to serious illness and death have been on the rise. According to the Centers for Disease Control even as recent as 2011 there were approximately 450,000+ documented case of Clostridium difficile infections (CDI) and upwards of 29,000 deaths (2). A large number of individuals who have an initial episode of CDI will develop at least one recurrence of the disease. The recurrence rates for CDI are high, in part because of the complex nature of C. difficile, and its spore forms that resist antibiotic intervention.

One of the problems with C. difficile is that not only can it lead to serious illness in susceptible individuals, but that it can be found in people, even children, who may not necessarily be suffering with primary issues related to C. difficile. For example, a paper in 2010 out of Poland (3) discussed the prevalence of C. difficile in fecal samples taken from 178 children ages 2 months to 2 years who were hospitalized for a variety of reasons. Their stools where examined for the presence of C. difficile Toxin A and B, the two main intestinal toxins known to trigger chronic diarrhea and bowel inflammation. The percentage of children infected with C. difficile was 68.6% and many of these children were not acutely sick from C. difficile. However, as mentioned previously, toxin A and toxin B from certain strains of C. difficile can lead to serious problems.

The Role of Toxin A and Toxin B
These two toxins are the main virulence factors related to mucosal damage from C. difficile. Toxin A and B are capable of causing mucosal damage resulting in digestive tract inflammation leading to either clostridia difficile associated diarrhea (CDAD) or Pseudomembranous colitis (4).

Toxin A is categorized as an enterotoxin, which means it is a toxin released by microorganisms that target the digestive system. It functions by changing host cell metabolism and tight junction formation. This can lead to mucosal cell damage, fluid accumulation and even cell death. Toxin A is considered to be the main cause of CDAD as it causes intestinal villi and brush border destruction. In severe cases of Toxin A production, it can lead to ulceration formation seen in Pseudomembranous colitis.

Pseudomembranous colitis is a type of inflammatory bowel disease of the colon that manifests with various ulcerations from mucosal damage and the development of a “pseudo” membrane (aka. ‘inflammatory membrane’), that overlays the site of mucosal injury. This inflammatory membrane is an accumulation of fibrin and inflammatory and necrotic cells that appear as a yellowish globule spread throughout the colon. In the late 1970s it was determined that C. difficile via the production of various toxins, was the causative organism for Pseudomembranous colitis.

Like Toxin A, Toxin B also plays a significant role in damage to the mucosal lining of the digestive system. Toxin B is categorized as a cytotoxin which means it is toxic to cells. Examples of cytotoxins would be chemicals produced by the immune system that damage other cells in the body. Bee venom, as well as poisons from spiders or snakes, are all classified as cytotoxins.

Toxin B causes major cellular disruption by interfering with signaling pathways, tight junction formation, formation of the cytoskeleton of the cell, and derangement of overall cell structure. It is a major virulence factor of C. difficile leading to vascular swelling and hemorrhaging. Also, Toxin B can not only have local inflammatory effects in the digestive system, but also systemic effects through its production of proinflammatory cytokines such as Tumor Necrosis Factor-αlpha.

It was felt for many years that serious bowel inflammation from C. difficile was generated by a single toxin, but both toxins are now known to be capable of causing mucosal damage.

Treatment and Testing
Treatment of C. difficile infections are mostly done by antibiotics. The two most common antibiotics are oral Flagyl (metronidazole) and oral Vancomycin (vancocin). Traditional intervention calls for 7 to 10 days of either antibiotic. As mentioned previously recurrence rates for C. difficile can be high, primarily because of C. difficile spore formation. There is a trend in C. difficile treatment to use cyclical courses of either antibiotic to aide in reduction of recurrence rates and increase clinical outcomes.

Stool testing for C. difficile is effective and is used as a primary diagnostic tool analyzing for the presence of Toxin A and Toxin B. Prior to stool testing direct visualization of inflammatory membranes was used through colonoscopy or sigmoidoscopy.

If either Toxin A or B is detected on stool pathogen screening the practitioner needs to correlate the information to the clinical presentation of the individual and treat accordingly or refer to a specialist for further evaluation. Not everyone with C. difficile will be symptomatic of intestinal disease so each situation needs to be evaluated individually. However, the presence of Toxin A or Toxin B found on stool pathogen testing certainly documents the presence of a strain of C. difficile. The individual should be treated appropriately.

Dr. Kurt N. Woeller is an author, international speaker, practicing clinician and founder of Integrative Medicine Academy (www.IntegrativeMedicineAcademy.com), which is an online training academy that provides various courses for health practitioners interested in integrative medicine.

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REFERENECS

1.      Richard E. Frye, et. al. Gastrointestinal dysfunction in autism spectrum disorders: the role of the mitochondria and the enteric microbiome. Microbial Ecology in Health and Disease. Volume 26, 2015.

2.      Lessa, FC, et.al. Burden of Clostridium difficile infection in the United States. N. Engl J Med, 2015;372:825-834.

3.      Prevalence of Clostridium difficile in the gastrointestinal tract of hospitalized children under two years of age. Med Dosw Mikrobiol; 2010;62(1):77-84 (Poland).

4.      (Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP; October 2010. "The role of toxin A and toxin B in Clostridium difficile infection". Nature 467 (7316): 711–3.

Clinical Usefulness of IgG Food Allergy Testing

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.

Clinical References

• 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.

 

Clostridia Detection and Comparison of Organic Acid Detection Versus Stool Testing

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.

HPHPA

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.

4-Hydroxyphenylacetic Acid

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.

Phenyllactic Acid

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.

3-Indoleacetic Acid

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

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.

Clinical References

• 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

The Role of Diet and the Gut in Mental Health

Terri Hirning

While the traditional mental health model focuses on brain function, neurotransmitters and potentially pharmaceutical medications, the ever burgeoning integrative mental health field understands there is more to it than that. Even mainstream media is starting to get the hint. Our gut influences our mind, emotions, cognition and mental health more than we've given it credit for in recent history. Whether we want to focus on the role food allergies play on mental health (1), (2) or how the gut-brain axis impacts our mental health (3), or even how the microbiome shapes our mental functioning (4) we can see the trend in research confirming what many integrative physicians and clinicians know: the gut matters

When we talk about the gut, we must cover diet. Some literature even suggests that a debilitating mental health disorder like Alzheimer's now be called "Type 3 Diabetes" (5) because of its links to certain kinds of foods and a generally poor diet. What is causing the alarming trend of food allergies, food sensitivities and the increase in auto-immune conditions? Is it GMO's? Is it Glyphosate (the herbicide used in products like Monsanto's Roundup)? Is it the prevalence of processed grains in our diets now? It may be all of thesethings, or none of these things, but as physicians and clinicians, the data suggests we take a closer look at our patients' diets and here are some things to consider:

Is there an underlying food allergy or multiple allergies? This can be an easy and yet very powerful place to start. Research shows that food allergies can indeed cause manifestations of mental health disorders. Running a simple IgG food allergy test from the Great Plains Laboratory, which also includes markers for Candida (harmful fungus in the gut) can be a great first step. More mainstream information on the treatment of Celiac disease can be also helpful in finding its connections to many mental health disorders like dementia, seizures, schizophrenia, etc.(6), and one does not have to be diagnosed with Celiac disease to be sensitive and reactive to gluten.

What about healthy gut function and microbiome population? Our microbiome is sensitive to our diets, and quickly reactive to changes. Looking at potential gut dysbiosis and the levels of beneficial flora in the gut is very important. An organic acids test will show you a wide range of metabolic markers, including several for bacteria (like Clostridia) and fungus (like Candida albicans) in the gut. If a patient has high levels of these, a course of treatment can be started to rid them of these invaders, possibly including dietary restrictions (like a low sugar, low carb diet) and adding helpful antibacterial or antifungal supplements. Then, to assess the beneficial bacteria in the gut, you may want to run a comprehensive stool analysis. This will help determine whether a patient needs to add a high-quality probiotic supplement to their diet and possibly increase his/her intake of probiotic-rich and fermented foods like kefir and sauerkraut.

Today's mental health disorders are very complex. Their treatment requires a well-rounded look at the many factors impacting the body and brain, including diet, lifestyle, the microbiome, and more. When an integrative approach is used and these many factors considered when creating a treatment plan, time and time again we see improvements in functioning and a reduction in clinical symptoms.

Clinical References:

• Jackson J1, Eaton W2, Cascella N3, Fasano A4, Santora D5, Sullivan K6, Feldman S6, Raley H7, McMahon RP6, Carpenter WT Jr6, Demyanovich H6, Kelly DL8.Gluten sensitivity and relationship to psychiatric symptoms in people with schizophrenia Schizophr Res. (2014) Oct 10. pii: S0920-9964(14)00511-8. doi: 10.1016/j.schres.2014.09.023.

• Genuis SJ1, Lobo RA2. Gluten sensitivity presenting as a neuropsychiatric disorder . Gastroenterol Res Pract. (2014);2014:293206. doi: 10.1155/2014/293206.

• Nemani K1, Hosseini Ghomi R2, McCormick B3, Fan X3. Schizophrenia and the gut-brain axis. Prog Neuropsychopharmacol Biol Psychiatry. (2014) Sep 19;56C:155-160. doi: 10.1016/j.pnpbp.2014.08.018.

• Severance EG1, Yolken RH2, Eaton WW3. Autoimmune diseases, gastrointestinal disorders and the microbiome in schizophrenia: more than a gut feeling. Schizophr Res. (2014) Jul 14. pii: S0920-9964(14)00319-3. doi: 10.1016/j.schres.2014.06.027.

• De la Monte S, Wands J. Alzheimer's Disease Is Type 3 Diabetes–Evidence Reviewed. J Diabetes Sci Technol. (2008) 2(6): 1101–1113.

• Velasquez-Manoff Moises (2014 October 12). Can Celiac Disease Affect the Brain? The New York Times. Retrieved from:http://www.nytimes.com/2014/10/12/opinion/sunday/can-celiac-disease-affect-the-brain.html?smid=tw-share&_r=0

Tiny Friends in Hidden Places (But Sometimes They "Unfriend" You): Man's Evolving Relationship with the Microbiome

Pamela Gilford, MA, CCN

Acceptance of the germ theory of disease unleashed a two-century war between humanity and the unseen microbial world. First, better sanitation, and after WWII, the advent of antibiotic treatment, saved millions of lives. Bacteria were the enemy; babies were even bathed in a toxic, chlorinated wash now withdrawn from the market. The presence of bacteria in the GI tract was treated as nature's "mistake." Who cared that oral antibiotics killed off some gut flora? Oral delivery avoided that unpleasant shot in the buttocks.

The mysterious rise in autoimmune disease since the development of modern antibiotic therapy has been one clue that humans might not be winning the war against microbes (see references below). A slow reassessment of the microbes, grudgingly referred to as "GI commensals" (harmless organisms) began. The genesis of pathogenicity has been discovered to be a two-way street between the human immune system and microbes, now referred to as a "relationship". Multi-celled, "higher" organisms clearly coevolved with one-celled organisms to mutual benefit. Microbes help us digest our food and provide certain vitamins. The microbes that line most of our GI tract defend our GI mucosal lining against invading pathogens, and as they begin to colonize the sterile gut of a newborn, they "educate" the child's immune system.

Multi-celled host organisms (like us) provide food, water, physical safety, and transportation. How else could microorganisms have extended their range without transportation? Humans have been particularly helpful. If this notion seems odd, think about syphilis coming to the New World with the first ocean explorers, and more recently, HIV and ebola making the crossing.

Although the variety of our GI flora has been known for a long time (See Theodor Rosebury's Life on Man, 1969), the advent of DNA tools to catalog species has spurred new interest in how human metabolism interacts with GI flora. The National Institute of Health's huge Human Microbiome Project is now nearing completion (seehttp://www.hmpdacc.org/). Evidence-based probiotic supplements for medical use are eagerly anticipated, although the first truly "evidence-based"probiotic was probably Lactobacillus GG, or Culturelle, discovered in cheek swabs by two researchers from Tufts University (see references below). Goldin and Gorbach patented Culturelle in 1985 for treatment of diarrhea caused by Clostridia difficile in children and has since been the subject of several hundred studies. Subsequent DNA analysis has granted this strain of bacteria a species designation of Lactobacillus rhamnosus and it is now incorporated into many commercial probiotic blends.

Studies have shown that variations in the species composition of gut microflora are related to the risk of obesity and that probiotic treatment often improves mood (see references below). Inflammation, which can be modulated by healthy gut flora, has become definitively linked to the epidemic of depression worldwide, as well as the genesis of many chronic diseases (see www.Medscape.com – many articles).

Clinical Endocrinology News reports that 64% of family physicians are stressed and uncomfortable when faced with treating autoimmune disease (see reference below) The fact that available allopathic therapies come with substantial side effects does not improve the practitioners' or patients' comfort levels. The frustration of both in finding safe therapies for autoimmune and chronic disease has certainly contributed to the growing popularity of integrative or CAM approaches. An article in Science magazine from November 2013 (see reference below) cited research by Mathis and Littman who found that Prevotella copri was present in 75% of RA patients' intestines. Later, they were able to trigger inflammation in mice by inoculating with the bacterium. Such studies add weight to clinicians' observations that probiotic treatments and even fecal implants can often halt the progress of chronic diseases.

We have been discussing our microbial passengers and an important part of the organic acids test is the assessment of the level of metabolites of pathogenic microbes, including yeast and Clostridia bacteria. The COMP stool test cultures both beneficial and potentially pathogenic microbes and microscopic examination can catch both yeast and one-celled parasites. When we added the Candida marker to the IgG food allergy test, we discovered a very useful tool for determining if a patient has become sensitive to his/her own native fungal flora. At The Great Plains Laboratory, we believe that the metabolic and and toxic element assessments we offer assist in directing treatment protocols that can mediate many chronic conditions, eliminate harmful microbes, and reinforce beneficial gut flora for improvement in overall health.

Clinical References:

• Hunter, P. 2012. The changing hypothesis of the gut. The intestinal microbiome is increasingly seen as vital to human health. Science and Society DOI 10.1038/embor.2012.68 |Published online 15.05.2012, EMBO reports(2012)13,498-500

• Thomas Jefferson University. 2014. Can antibiotics cause autoimmunity? ScienceDaily. March 31, 2014:http://www.sciencedaily.com/releases/2014/03/140331153520.htm

• 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

• Whiteman, H. 2014. Antibiotic use in children linked to juvenile idiopathic arthritis. Medical News Today. November 16, 2014:http://www.medicalnewstoday.com/articles/285453.php

• Parry, W. 2011. Overuse of antibiotics is seen behind many human ills. LiveScience. August 24, 2011:http://www.livescience.com/15740-helpful-bacteria-antibiotics.html

• Golden, B.R. and Gorbach, S.L. 2008. Clinical indications for probiotics: An overview. Clinical Infectious Dieases 46(12): S96-S100.

• Mason, J. 2013. Can probiotics keep my gastrointestinal system happy? Tufts Now. September 16, 2013:http://now.tufts.edu/articles/can-probiotics-keep-gastrointestinal-health

• Baumler, M.D. 2013. Gut bacteria. Today's Dietitian. June 2013: http://todaysdietitian.com/newarchives/060113p46.shtml

• 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

Usefulness of HPHPA marker in a wide range of neurological, gastrointestinal, and psychiatric disorders

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. 

References:

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.

Immunodeficiency, gastrointestinal Candidiasis, wheat and dairy sensitivity, abnormal urine arabinose, and autism: a case study

William Shaw, PhD

Abstract

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.

Introduction

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.

Therapy

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.

Discussion

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; negative impacts on development occur 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.

Clinical References

• 1. Warren R, Odell J, Warren W, Burger R, Maciulis A, Daniels W, Torres A. Immunoglobulin A deficiency in a subset of autistic subjects. J. Autism Develop Dis. 27:187-192,1997.

• 2. Warren R, Margaretten N, Pace N, Foster A. Immune abnormalities in patients with autism. J. Autism Develop Dis. 16, 189-197, 1986.

• 3. Warren R, Singh V, Cole P, Odell J, Pingree C, Warren L, DeWitt C, and McCullough M. Possible association of the extended MHC haplotvpe B44-SC30-DR4 with autism. Immunogenetics 36: 203-207, 1992.

• 4. Warren R, Yonk J, Burger R, Odell J, Warren W. DR positive T cells in autism: association with decreased plasma levels of the complement C4B protein. Neuropsychobiology 31: 53-57, 1995.

• 5. Singh, V, Warren, RP, Odell, JD, and Cole, P. Changes in soluble interleukin-2, interleukin-2 receptor, T8 antigen, and interleukin-I in the serum of autistic children. Clin. Immunol. Immunopath. 61: 448-455, 1991.

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Candida and Overgrowth - The Problem & Bacteria By-products

My discovery about the role of abnormal organic acids from gastrointestinal microorganisms in human disease began as many discoveries do, as an accident.

In the 1960’s, a great deal of progress had been made in discovering the biochemical abnormalities that caused a number of diseases called "inborn errors of metabolism" using a technology called gas chromatography-mass spectrometry. These diseases included phenylketonuria (PKU), tyrosinemia, maple syrup urine disease and many others.

It seemed possible that this new testing called organic acid testing or metabolic profiling might be applied to any disease. However, thirty years later, very little progress had been made in solving the mystery of a number of diseases like autism, fibromyalgia, schizophrenia, and Alzheimer’s disease.

There is a long list of diseases in which a substantial number of patients excrete elevated microbial metabolites in the urine which have been detected by urine organic acid testing:

• Autism

• Attention deficit disorder (ADD)

• Rett's syndrome

• Ulcerative colitis

• Seizures

• Depression

• Child Psychosis

• Fibromyalgia

• Chronic fatigue syndrome

• Pervasive developmental disorder (PDD)

• Colitis

• Schizophrenia

• Migraine headache

• Alzheimer's disease

• Systemic Lupus Erythematosus (SLE)

• Obsessive compulsive disorder (OCD)

• Tourette's syndrome

• Inflammatory bowel disease

• Down syndrome

• Crohn's disease

Microbial Ecology of the GI Tract

The number of microorganisms in the GI tract approaches the total number of cells in the body. Approximately 500 species of bacteria are present, of which 30-40 species of bacteria predominate, including several species of yeast and fungi.

The greatest number of species are anaerobic or facultative anaerobes. Yeast/fungi and Clostridia species are widely known to accompany the use of broad spectrum antibiotics. Furthermore, recent research indicates that the growth of certain Candida types is markedly stimulated by antibiotic addition to the culture media.

Books like The Yeast Connection and Yeast Syndrome have spread the knowledge about the health effects of Candida to the general public, but are widely ignored by a large segment of the medical community.

Detecting GI Microbial Overgrowth (Dysbiosis)

I became interested in using gas chromatography-mass spectrometry (GC/MS) to detect abnormal microbial metabolites when I worked at the Center for Disease Control (CDC). At CDC, GC/MS was used to identify the species of pure cultures of isolated bacteria. I wondered why you couldn’t directly test human body fluids directly for products of microorganisms.

Later, while working at Children’s Mercy Hospital, the pediatric hospital for the University of Missouri at Kansas City Medical School, I became interested in the role of abnormal urinary metabolites detected by GC/MS while evaluating two brothers who had autism as well as occasional severe muscle weakness.

Since some inborn errors of metabolism are associated with muscle weakness, I was really looking for metabolites characteristic of genetic diseases which were all negative. Instead, I noticed that several unusual compounds were consistently elevated in urine samples. None were adequately described in the medical literature. Colleagues in the field of metabolic diseases said they were probably from gut flora (microorganisms) and were therefore unimportant. Since several of these compounds were analogs (or altered forms) of normal Krebs cycle compounds, I thought these compounds might be significant, perhaps as anti-metabolites.

During the same time period, I was testing the culture media of a large number of different yeast and bacteria strains from the human gastrointestinal tract in order to find out which compounds in the human might be derived from the yeast and bacteria in the gastrointestinal tract.

Yeast Metabolites in the Urine of Children with Autism

The compound that led to the discovery of yeast as the source of many of these compounds in the urine of autistic children was tartaric acid.

The brothers with autism and severe muscle weakness had extremely high values of tartaric acid in their urine. Another child with autism had a urine value of tartaric acid 600 times that of normal children.

The only source of tartaric acid is yeast. This compound forms a sludge in the wine brewing process and has to be removed. Wine is sugar-water fermented by yeast (Saccharomyces cerevisiae) to alcohol and other yeast products. Humans do not produce this chemical.

When I pulled the medical charts of several other children with autism, they had similar abnormalities and immediately I wondered about a possible causal connection. The next step seemed obvious. If these compounds were from yeast and were causing some of the symptoms of autism, antifungal drugs which kill yeast should reduce some of the symptoms of autism. However, the two brothers did not receive antifungal therapy until nearly a year later.

At nearly the same time, a two-year-old boy was currently being evaluated for autism in the neurology department at the children’s hospital where I worked and I had just done the urine organic acid test. The child had been developing normally up to about 18 months of age and had a vocabulary of about 200 words.

He was treated several times for ear infections with antibiotics and developed thrush (a Candida or yeast infection of the mouth and tongue). His behavior deteriorated quickly after that. He lost all speech, became extremely hyperactive, woke up all night long, lost eye contact with his parents and was diagnosed with autism.

This pattern of normal development and regression associated with antibiotics is extremely common in autism and is especially common in males with autism (about a 20/1 male/female ratio). His organic acids were very elevated, which I thought was due to the yeast, including tartaric acid. The neurologist at the hospital would not prescribe the antifungal drug Nystatin for the child so the parents and I convinced their family physician to prescribe it.

The child’s eye contact returned by the following day and the elevated organic acids decreased markedly, although it took over 60 days for the urine tartaric acid to return to the normal range (Figure 1).

At day zero (Figure 1), the day that the child had the organic acid test done, the tartaric acid value in urine was over 300 mmol/mol creatinine, a very high value--about twenty times the normal value. (Most chemicals measured in urine are divided by the urine creatinine concentration to compensate for different amounts of fluid intake in different individuals.) Following the treatment with the Nystatin, the level of the tartaric acid decreased considerably and continued to decrease as the Nystatin was continued.

Within a couple of days of starting the antifungal drug Nystatin, the child--who had lost most of normal development--began to improve and eye contact came back. His extreme hyperactivity began to go away and he began to have a greater amount of focus. The sleep pattern improved as well. Nystatin is an antifungal drug which indicated to me that a yeast or fungus (these terms are somewhat interchangeable in that they are very closely related biologically) was causing the secretion of this compound in the intestinal tract.

After 68 days the child’s mother started running out of Nystatin and began giving only half doses so that she didn’t run out of it. During that time the tartaric acid starting increasing. When she got the Nystatin prescription refilled and restored the full dose of Nystatin, the tartaric acid decreased.

What this indicated to me was that the Nystatin was causing a marked reduction in this urine tartaric acid. The other significant finding was that even after two months of Nystatin, the biochemical abnormality would reappear within a short time of stopping the antifungal drug.

I have now detected this same phenomenon in hundreds of other cases. Even after six months of antifungal treatment, there is often a biochemical "rebound" and loss of improvements after discontinuing antifungal therapy. This rebound also occurs after other antifungal drugs as well.

Several explanations are possible for this phenomenon:

• Because of one or more defects in the immune system such as IgA deficiency, IgG deficiency, or severe combined immunodeficiency disease (SCID) which are found in most children with autism, the yeast, which are everywhere in our environment including the food we eat, repopulate the intestinal tract very rapidly.

• The yeast are very resistant and have not been completely eliminated even after six months of antifungal therapy

• The yeast have genetically transformed some of the human cells that line the intestinal tract so that some of the human cells now contain yeast DNA. These genetically transformed human cells produce both yeast and human products and are somewhat sensitive to antifungal drugs but are not killed by them and produce yeast products whenever antifungal drugs are absent.

• Some of the yeast are hidden in recesses of the intestinal tract or in the deeper layers of the mucosa that lines the intestine where they are relatively safe from the drug. Although their numbers are small, they readily repopulate the intestine after antifungals are stopped.

• In addition to the immune system taking inventory of its own cells, it seems increasingly likely that the immune system also takes an inventory of bacteria and yeast cells present in the intestinal tract soon after birth. This inventory is performed by a group of cells called the CD5+ B-cells, which are among the very first immunological cells to appear in the developing embryo and appear to play a role in tolerance to intestinal microorganisms in postnatal life. These cells may play a role in regulating the secretion of IgA, the antibody class that is secreted into the intestinal tract and which may select which microorganisms are tolerated in the intestinal tract. Furthermore, the eradication of normal flora especially when antibiotics are administered repetitively during infancy may cause the CD5+ cells to reject these normal organisms at a later age. Any cells that are on this early inventory may be awarded immune tolerance and will not be attacked later on by the immune system. Either antibiotic use in infancy or yeast infection of the mother during pregnancy may result in later immune tolerance to yeast.

Response of Children with Autism to Antifungal Therapy

Improvements commonly cited by parents of autistic children treated with antifungal therapy include: decreased hyperactivity, more eye contact, increased vocalization (more words and more usage), better sleep patterns, better concentration, increased imaginative play, reduced stereotypical behaviors (such as spinning objects), and better academic performance. Two children with autism treated with a combination of antifungal therapy, EPD treatment for food allergies, and gluten and casein free diet have completely recovered from autism.

More than 1000 children with autism have been treated with a wide variety of antifungal agents such as Nystatin, Lamisil, Sporanox, Nizoral, Diflucan, caprylic acid, grapefruit seed extract, and garlic extract with good clinical response in perhaps 80-90%. A survey of parents of autistic children by Rimland reports that antifungal therapy is ranked the most effective (by a wide margin) of all drug therapies used for the reduction of autistic symptoms.

Molecular Basis of Tartaric Acid Toxicity

The abnormal metabolites derived from yeast were first discovered in two brothers with autism. In addition to their autism, these brothers had a muscle weakness that was so severe that sometimes they could not even stand up. A neurologist colleague, Enrique Chaves, MD, told me that virtually all the autistic children he examined had hypotonia.

Both of these brothers excreted high amounts of tartaric acid in the urine. Biopsy of the muscle and examination by electron microscopy revealed normal structural features except for an unexplained "granularity." Electromyography, EEG’s, brain scans, and nerve conduction velocities were all normal.

A toxicology manual indicates that tartaric acid is a highly toxic substance. As little as 12 gm has caused human fatality with death occurring from 12 hours to 9 days after ingestion. Gastrointestinal symptoms were marked (violent vomiting and diarrhea, abdominal pain, thirst) and followed by cardiovascular collapse and/or acute renal failure. A gram is approximately the weight of a cigarette.

This compound especially damages the muscles and the kidney and may even cause fatal human nephropathy (kidney damage) which was of interest to me, since the two brothers with autism had the extreme muscle weakness as well as evidence of impaired renal function.

A young Korean child with autism had a value of 6000 mmol/mol creatinine, a value that is about 600 times the median normal value. Assuming that the yeast in the intestine of the child were producing tartaric acid at a constant rate, this child would be exposed to 4.5 grams per day of tartaric acid, over one-third of the reported lethal dose of tartaric acid! (The child’s value returned to normal after a few weeks of antifungal treatment.)

Tartaric acid is an analog (a close chemical relative) of malic acid (Figure 2). Malic acid is a key intermediate in the Krebs cycle, a biochemical process used for the extraction of most of the energy from our food. Presumably tartaric acid is toxic because it inhibits the biochemical production of the normal compound, malic acid (Figure 3). Tartaric acid is a known inhibitor of the Krebs cycle enzyme fumarase which produces malic acid from fumaric acid.

Yeast Byproducts, Malic Acid Supplementation, & Fibromyalgia

Interestingly, I have found that tartaric acid and/or other yeast byproducts are also elevated in urine samples of adults with the disorder fibromyalgia, a debilitating disease associated with muscle and joint pain, depression, foggy thinking, and chronic fatigue. (Dr. Kevorkian has assisted in the suicide of two people with this disorder, which is tragic since a simple antiyeast treatment may help relieve the symptoms of this disorder.)

A large percentage of patients with fibromyalgia respond favorably to treatment with malic acid which is present in health food supplements such as Fibrocare and Supermalic. I presume that supplements of malic acid are able to overcome the toxic effects of tartaric acid by supplying deficient malic acid.

Treatment with the antifungal drug Nystatin kills the yeast and values for tartaric acid steadily diminish with antifungal treatment. Fifty percent of patients with fibromyalgia often suffer from hypoglycemia even though their diet may have adequate or even excessive sugar.

One of the reasons for the hypoglycemia may be due to the inhibition of the Krebs cycle by tartaric acid. The Krebs cycle is the main provider of raw materials such as malic acid that can be converted to blood sugar (Figure 3) by the process called gluconeogenesis when the body uses up its glucose supply.

If sufficient malic acid cannot be produced, the body cannot produce the sugar glucose, which is the main fuel for the brain. The person with hypoglycemia feels weak and their thinking is foggy because there is insufficient fuel for their brain. Of course, consumption of sugar may provide short-term relief but which also stimulates yeast overgrowth and within a short time symptoms are even worse.

Arabinose, Yeast, Autism, Alzheimer's Disease & Schizophrenia

Arabinose is a five carbon sugar with an aldehyde function called an aldose, which is frequently elevated in autism.

In some children with autism, arabinose concentrations may exceed 50 times the upper limit of normal. A scattergram of urine arabinose values comparing normal and autistic children in the same age range is given below (Figure 4).

The exact biochemical role of arabinose is unknown, but a closely related yeast alcohol arabitol has been used as a biochemical indicator of invasive candidiasis. We have never found elevated arabitol in any of the urine samples tested and we have not found arabinose in the culture media of multiple isolates of Candida albicans isolated from stool samples of autistic children (unpublished data).

We suspect that arabitol produced by yeast in the intestinal tract is absorbed into the portal circulation and then converted to arabinose by the liver. Hypoglycemia occurs in inborn errors of fructose metabolism in which fructose inhibits gluconeogenesis and it is possible that children with autism might be deficient in one or more enzymes involved in the metabolism of pentoses. Elevated protein-bound arabinose has been found in the serum glycoproteins of schizophrenics and in children with conduct disorders and alteration of protein function by arabinose is another mechanism by which arabinose might effect biochemical processes.

Women with vulvovaginitis due to Candida were found to have elevated arabinose in the urine; 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. Arabinose tolerance tests should be able to rapidly determine if such biochemical defects are present in children with autism.

Arabinose, Pentosidine, Protein Modification & Vitamin Deficiencies

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, thereby cross-linking the proteins and altering both biological structures and functions of a wide variety of proteins.

This adduct of arabinose, lysine, and arginine is called a pentosidine (Figure 5). The tissue concentration of this adduct is almost linearly related to age (Figure 6). 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; 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.

Pentosidines, Autism, Alzheimer’s & Neurofibrillary Tangles in the Brain

Protein modification caused by pentosidine formation is associated with crosslink formation (Figure 7), decreased protein solubility, and increased protease resistance. The characteristic pathological structures associated with Alzheimer disease contain modifications typical of pentosidine formation. Specifically, antibodies against pentosidine immunocytochemically label neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease.

In contrast, little or no staining with anti-pentosidine antibodies is observed in apparently healthy neurons of the same brain. 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.

Since the process of pentosidine formation is an oxidative one, the use of antioxidants as well as antifungal therapy appears to be a promising therapy for Alzheimer’s disease. Glutathione has been reported to inhibit pentosidine formation. Supplementation with the vitamins biotin, pyridoxal (B-6), and lipoic acid (whose function at protein epsilon amino groups may be blocked by pentosidines derived from arabinose) might also be beneficial because of functional deficiencies due to pentosidine formation (Figure 8).

Not surprisingly, neurofibrillary tangles similar to those found in the brains of Alzheimer’s victims have also been reported in the brain of an autistic person at autopsy. It has been reported that frequent urinary tract infections and high amounts of circulating immune complexes are associated with more severe Alzheimer disease. The use of antibiotics to treat urinary tract infections would of course lead to yeast overgrowth of the gastrointestinal tract.

Summary

Elevation of yeast metabolites such as tartaric acid and arabinose are found in many of the same disorders and are even more common in autism, SLE, Alzheimer’s disease, fibromyalgia, attention deficit hyperactivity, and chronic fatigue syndrome. The arabinose may interfere with gluconeogenesis and also may through pentosidine formation significantly alter protein structure, transport, solubility, and enzymatic activity as well as triggering autoimmune reactions to the modified proteins.

The finding of pentosidine in the neurofibrillary tangles of Alzheimer’s brains and its absence from normal areas of the brain may indicate a direct role of a yeast byproduct in accelerating the normal aging process. Tartaric acid from yeast overgrowth has a direct toxic effect on muscles and is an inhibitor of a key Krebs cycle enzyme that supplies raw materials for gluconeogenesis and offers an explanation for many of the symptoms of fibromyalgia.