Chapter 3
Organic Acid Testing, Byproducts of Yeast and their Relationship to Autism
By Dr. William Shaw


 Metabolic Disease Testing: The History of Organic Acid Testing

My discovery about abnormal organic acids in autism 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. It seemed possible that this new technology 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, schizophrenia, and Alzheimer’s disease.
 
In 1991, I had accepted the job as Director of Clinical Chemistry, Endocrinology, and Toxicology at a children’s hospital because I wanted to improve upon what had been done previously in the field of metabolic diseases. I was also hoping to extend the existing technology to other diseases with unknown causes.

In the field of metabolic diseases, urine samples are analyzed for their chemical constituents after extracting the chemical compounds from the urine using organic solvents such as ether and ethyl acetate. It is preferable to test urine over blood because urine is a filtrate of blood in which much of the water has been removed, so that the concentration of a compound in urine might be 100 times more concentrated than it was in blood.  A very high concentration of characteristically abnormal chemical compounds would indicate the likely presence of a genetic disease. For example, when a child has PKU (or phenylketonuria), which is a genetic disease where a genetic mutation is present, very high concentrations of chemical compounds called phenylketones will appear in the urine. This mutant gene codes for an abnormal form of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine.  Since the enzyme is defective, phenylalanine is not converted to tyrosine and phenylalanine builds up in the blood just as a logjam begins in a narrow part of a stream. If a child with PKU is treated with a diet low in phenylalanine as an infant, the child will develop normally. However, if the diagnosis of PKU is not made until the child is much older; the child may be significantly impaired and suffer mental retardation (1). 
As a biochemist, I believed that any diseases which had caused devastating effects on an individual were bound to change their biochemistry. The presumption was that, if a person had a severe disease like autism, seizures, or cerebral palsy, there would have to be some change in one or more of the chemicals processed in the body. All of the body’s chemical processes proceed by particular metabolic routes or pathways. Allow me to use an analogy to the Los Angeles freeway system. If an accident happens in Anaheim (a suburb of Los Angeles), traffic may back up in downtown Los Angeles. After a while, alternate roads begin to be utilized and the traffic begins to move again but at a much slower rate. If you measured the number of cars taking different alternate routes, you could pinpoint exactly where the accident had occurred. Using this analogy, the chemicals we eat as food are the traffic, which proceeds along well-marked major highways called metabolic pathways until an accident occurs. The accident might be a mutation, an infectious disease, or a vitamin deficiency. As a result of the accident, the traffic flow of molecules is diverted onto the slow alternate routes instead of the twelve-lane expressway. The person with the slow traffic of molecules is alive but may not be functioning as well as individuals in which all the metabolic highways are open. The problem I was faced with, using the highway analogy, was “What if certain highways were not even listed on the highway map because the people who compiled them either didn’t know about them, or knew about them but didn’t include them on the map?”
In laboratories using the old organic acid technology, certain abnormal compounds in urine samples might be noted but the amount of the chemical compound would not usually be quantitated.  In essence, the record of the analysis called a chromatogram would be visually examined or eyeballed to determine if a markedly abnormal substance was present. Although this method of examination may have been adequate 20-30 years ago, it was not up to date with the best and current technology.

Let me give another analogy: You go into a bank, open an account and make deposits for several weeks.  After about a month you go back into the bank and you say, “I’d like to know my account balance.” The teller looks at you and says with a straight face, “A lot”.  You feel concerned about this lack of information and press her for more information, and she says, “You really have more than most people do”. That is still not satisfactory but there is no manager available so you walk away feeling confused and decide to go back later when a different teller is on duty. The next time you come in, you request the manager, asking again for your balance and this time the manager says, “Not much." Although this type of accounting may be adequate for comparing the assets of Bill Gates and a street person, it is not much help for those in the middle class. In essence, the majority of metabolic disease testing that was performed ten years ago was the “a lot” or “not much” variety, and this still exists in as much as 50% of the testing done today.


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I suspected that many subtle changes in the body’s metabolism were being overlooked in using this kind of technology as a result of the “not much” and “a lot” kind of interpretations.  What I set out to do was to quantitate the changes in the different molecules in the urine just as the bank accountants in a bank balance the money transactions. I was successful because of new computer software that allowed for the rapid quantitation of very complex data.  If it were not for this particular software, my work would not have been possible.

This computer software had originally been designed for the environmental field. Our drinking water, sewage and ground water can contain many kinds of pesticides, herbicides, and industrial chemicals. Testing for all of these chemicals requires very sophisticated computer software and this software was ideally suited for doing metabolic disease testing.  The goals I set out to achieve were (1) to identify every chemical that I could, and (2) to quantitate everything I possibly could and do it as accurately as possible.

If we could know everything possible about people, including what kinds of chemical compounds were normal for them, then it would be easier to identify what was going on in the metabolism of a patient with a particular disease. Prior to beginning testing, we sent samples out to another laboratory performing the “a lot, not so much” kind of testing and I was very surprised to see that about 98% of the samples came back with an interpretation of normal.  It does not seem possible, in my opinion, that someone could have a devastating disease, and not have it alter their metabolism in some way.
 
I continued to work on developing a more elaborate system of testing of my own. As a result, I found that, indeed, there was some increased detection (perhaps 5-10%) of certain known genetic diseases.  However, this was a smaller increase than I had anticipated. I also noticed that in many different diseases, there were abnormal elevations of certain compounds that nobody seemed to know or care much about. When I discussed these findings with colleagues in the field of metabolic disease all over the world, they responded that these particular chemical compounds are not important because they are most likely a result of microorganisms in the intestinal tract. 

After receiving this information and filing it away in my mind, I continued to remain skeptical of this common perception that microbial products were unimportant. The body did not have a metabolic segregation system in which human metabolites were allowed into certain areas of the body, while microbial products were separated into other compartments. All of these products were intermixed throughout the body.  Several months after initiating my new laboratory service, Enrique Chaves, MD., a colleague of mine from the University of Kansas Medical School, and a pediatric neurologist who was also interested in biochemistry (a rather rare occurrence in physicians as a group) referred a woman to me who had two children with severe muscle weakness. Dr. Chaves, who had also been using the old technology in his laboratory, could find nothing unusual in the two brothers. The muscle weakness was so severe that sometimes, for several hours, these children could not even stand up.  There had been an intensive search for the cause of this muscle weakness. When Dr. Chaves analyzed the lab results, he found no evidence of any genetic disease.  Since I had this new technology, I was very interested in trying to find out what was going on.  I told the mom that we would test samples of her children’s urine and see if we could figure out what was happening to them. 

 Evaluation of Two Brothers with Autism

In the field of metabolic diseases, it is well known that some disease abnormalities only show up at the time the child is severely ill, i.e., if the child has a severe cold, flu or chicken pox. The biochemical pattern may be close to normal while the child is well.  So when I spoke to the mom, I emphasized that we should get multiple samples rather than just a single one.  Several months later, the mom came back with a whole armful of samples saved in her freezer, which were actually more samples than we usually tested in an entire month. I talked to my technologist Ellen Kassen and told her we would have to bite the bullet and get these tests under way as best we could.  We began to test the samples. In each sample, I would see that there was no chemical compounds characteristic of any of the known genetic diseases, which are called inborn errors in metabolism. My overall impression however, was that these samples were still abnormal because there was a marked difference in the kinds of chemical compounds found in the urine samples of the two brothers compared to those found in the urine of normal children.

These compounds were the same ones that my colleagues said were not important because they were from microorganisms in the intestinal tract.  I was now very curious about what was going on. By this time, my colleague Dr. Chaves had moved from across town to the same institution where I was located. I was able to walk across the hall and further discuss with him any possible explanation as to why these brothers had these abnormal concentrations of chemicals resulting from microorganisms.  At that time, he also mentioned that, in addition to the profound muscle weakness, the brothers also had autism.  When I looked at their medical charts, I saw that they also had a history of frequent ear infections which is similar to many children with autism. A brief description of the technology used for testing the samples is appropriate at this point.

Urine samples are extracted to obtain a purified extract for analysis by a gas chromatograph-mass spectrometer (GC/MS).  Samples are loaded onto a sampler tray of the GC/MS. The sample is then injected into a hollow tube in the oven of this instrument called a column. The different molecules in the sample go around and around in coils of this column just like a group of horses going around a racetrack and then come out at the finish line.  At the finish line the sample is bombarded by a beam of electrons that break the molecules into pieces of different sizes and shapes. The molecules can be identified because each molecule has a characteristic way of breaking up or fingerprint.  The data from this fingerprint is then transferred into a computer. Then the computer analyzes all that data, makes sense out of it, identifies it and quantifies how much of each kind of molecule is in the urine sample. The increase in the capability of this technology has been phenomenal. When I first started in this field, the analysis of a single chemical compound would have taken most of the day. Now we can identify a thousand different compounds in a single afternoon.

Figure 1 shows a typical chromatogram for the analysis of the urine sample of a normal child.   This profile is called a total ion chromato-gram. People who work in the field call each one of these blips that you see a peak. A peak is detected when identical molecules in the sample are swept by the pressure of an inert gas around the circular column and finish at a particular time. The size of this peak is proportional to how much of a particular kind of molecule there is. Small fast molecules cross the finish line faster than big slow molecules just as fast horses have the fastest race times. Fast molecules have the smallest transit time, which is called a retention time. The bigger the peak, the more of a compound is there. Conversely, the smaller the peak, the smaller the amount of compound.
A urine chromatogram of a normal child has many peaks, some of which are small and some of which are large. Contrast this chromatogram of a normal child with the child that has autism (Figure 2). An examination of this figure reveals that there are many more chemical products present in the urine sample of the child with autism. In retrospect, it was fortunate that the children I initially tested were more abnormal than the average child with autism since it helped me to notice the marked differences. There is both more of certain molecules (higher concentrations) indicated by larger peaks as well as more peaks (more compounds). In addition, some of the peaks found in the urine sample of the child with autism are nearly absent in the normal child.

What I found is that there was a consistent pattern of abnormally elevated chemicals in the urine samples of the two brothers with autism that were known to be derived from the intestinal microorganisms. So virtually all of the big changes that you see in the chromatogram of the child with autism (Figure 2) were due to the fact that they had much higher concentrations of the chemicals that were produced by microorganisms residing in their intestinal tracts.

 

 Evaluation of a Third Child with Autism

Based on all the information that I had gathered, I reasoned that if the abnormal compounds from the intestinal tract had something to do with causing autism, then treatment of the microorganisms that produced these byproducts should improve the behavior of the child. I only had to wait a short time before I got the opportunity to test out my hypothesis. A child had been referred to the Neurology Department of the hospital to confirm a case of autism and organic acid testing had been requested. This child had the kind of history that is very frequent in autism.
The child was developing completely normally when the child began to have ear infections. The ear infections continued, and they came one after another. The child developed a thrush or yeast infection of the mouth that occurs because antibiotics have killed off the normal bacteria that keep the yeast population in check.  Prior to the recurrent ear infections, the child had a vocabulary of about 150 to 200 words. Following the antibiotics and the yeast infection, the child’s development began to slow and then regressed. The child no longer spoke any words. The child became extremely hyperactive, was no longer social, no longer made eye contact, and had a very disruptive sleep pattern.  I have seen this particular pattern in many children with autism, but not in all. In some cases, the child may have been treated with antibiotics for recurrent streptococcal throat infections, urinary tract infections, sinusitis, or recurrent bronchitis.
I explained my theory to the mother of the child whom I’ll call Bruce. The mother was a nurse at another nearby hospital and understood about thrush and antibiotics and was willing to give the antifungal drugs a try. The patient “belonged to” the chief of neurology and his approval would be necessary to get a prescription for the drug, which he declined.  Being a nurse and knowing that the antifungal drug nystatin had no serious side-effects, she decided to obtain a prescription for nystatin from her family doctor, who was in private practice and not associated with the hospital. Within a couple of days of starting nystatin, Bruce, who had lost most of his normal development, began to improve. His eye contact came back, his extreme hyperactivity began to dissipate and he began to have a greater amount of focus.  The sleep pattern improved as well and Bruce slept through the night for the first time in months.
At day zero, the day that Bruce first came in and had the organic acid test done, the tartaric acid value in urine was 300 mmol/mol creatinine, a very abnormal value that was about twenty times the median 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, which was one of the compounds that I suspected was derived from the microorganisms, decreased considerably and continued to decrease as the nystatin was continued (Figure 3). Since nystatin is an antifungal drug, this indicated to me that a yeast or fungus (these terms are somewhat interchangeable in that they are biologically very closely related) was causing the secretion of this compound in the intestinal tract.

Figure 1
Urine of Normal Child GC/MS chromatogram

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Figure 2
Urine of Child with Autism GC/MS chromatogram

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Figure 3
Effect of Nystatin on Urine Tartaric Acid
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After 68 days Bruce’s mother started running out of nystatin and began giving only 1/2 doses so that she didn’t run out of it completely. During that time the tartaric acid starting going back up and when she got the nystatin prescription refilled, the tartaric acid went back down.  What this indicated to me was the fact that the nystatin was causing a marked reduction in this urinary 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. In some cases, reports have been received of this same phenomenon in hundreds of other cases. Even after six months and after two or three years of antifungal treatment, there is often a biochemical “rebound” and loss of improvements after discontinuing the antifungal therapy. Several explanations are possible for this phenomenon:

  • As a result of one or more defects in the immune system (see chapter on the immune system), the yeast that are everywhere in our environment including the food we eat repopulate the intestinal tract very rapidly. Early antibiotic use may alter the normal microorganisms in the intestinal tract into an abnormal pattern that the immune system recognizes as normal and will not attack these organisms. (See chapter on gastrointestinal microorganisms.)
  • The yeast are very resistant and have not been completely eliminated even after six months of antifungal therapy. Some of this resistant yeast might be the cell-wall deficient yeast described in the chapter on yeast.
  • 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 is 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.

 Properties of Tartaric Acid

What is tartaric acid and what is known about this product? A toxicology manual (3) indicates that tartaric acid is a highly toxic substance. As little as 12g has caused human fatality with death occurring within 12 hours to nine days after ingestion. Since this compound especially damages the muscles and the kidney (4,5) and may even cause fatal human nephropathy (kidney damage)(6), it was of particular interest to me since the two brothers with autism’s initial symptoms were extreme muscle weakness as well as evidence of impaired renal function.  Gastrointestinal symptoms were marked (violent vomiting and diarrhea, abdominal pain, thirst) and followed by cardiovascular collapse and/or acute renal failure (3). (A gram is approximately the weight of a cigarette.)
Interestingly, I have found that tartaric acid is 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 (7, 8) may help relieve the symptoms of this disorder.)  Values for tartaric acid in urine may be extremely elevated in autism. 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. (The child’s value returned to normal after a few weeks of antifungal treatment.) 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! Proponents of the theory that wheat gluten sensitivity is the main biochemical abnormality in autism would have difficulty in explaining this case, since rice was the only grain in this child’s diet. (Gluten and casein restriction is a very important therapy in most cases of autism and is dealt with in the chapters by Lisa Lewis, Pamela Scott, and Karyn Seroussi, as well as in the chapter on the digestive system).


Figure 4
Figure-4

Surprisingly, the Food and Drug Administration lists tartaric acid in the Generally Recognized As Safe or GRAS category (9), which means this product, can be freely used as an additive in processed foods. Unless a food additive is put on the GRAS list, the food company using the product may have to spend thousands or even millions of dollars to prove its safety. Therefore, the political pressure to get a product on this GRAS list is intense. Tartaric acid is a byproduct of the wine industry and a tremendous amount of tartaric acid sludge has to be removed from the wine after the grape juice yeast fermentation. This sludge is the primary source of tartaric acid used as a food additive.
I have not yet found tartaric acid in Candida culture media but individuals with high amounts of tartaric acid in the urine also have high Candida counts in the stool. Tartaric acid is most likely a product of the breakdown of arabinose that may form in the body and/or during sample transport.  In the example below, the normalization of tartaric acid in the urine after antifungal treatment was associated with a reduced Candida yeast count in the stool. The antifungal treatment used was nystatin and Lactobacillus acidophilus for two months.

Status

Candida
Krusei stool

Yeast
tartaric urine*

Lactobacillus
stool

Before
antifungal
treatment

4+

993

0

After
antifungal treatment

0

1

4+

Normal
range

 

0-1+

0-15

3+ - 4+

*mmol/mol creatinine

Tartaric acid is available as a food additive in baking powder, grape and lime flavored beverages, and poultry. It may also be found in grapes and grape products. Cream of tartar, which may be used for baking, is nearly pure tartaric acid. It is used in the food industry as a firming agent, flavor enhancer, flavoring agent, humectant, acidity control agent, and sequestant (9). There is no evidence that any mammals can produce it, so it is most likely, purely a yeast by-product. Tartaric acid may only be formed in the absence of oxygen and it is an analog of the Krebs cycle compound malic acid (Figure 4). An analog is a chemical compound that closely resembles but is not identical to another chemical compound. The atoms that differ in the two molecules are shaded in gray. The reason an analog is important is that the analog may prevent the normal biochemical from completing its normal biochemical function.

I would use this analogy to explain the analogs. You live in a neighborhood in which the same builder used the same locksmith who put a lock in each house that is just a little different. There have been a few burglaries in your neighborhood recently, so when you go to visit your neighbor next door, you decide to lock your door before going to your neighbor’s house.  When you arrive at your neighbor’s house, your neighbor hands you a cup of coffee and you put your key on the kitchen counter right next to your neighbor’s key.  You drink the cup of coffee, chat for a while and when you decide to go home, you unknowingly reach down and pick up your neighbor’s key.  Then you take your neighbor’s key, which looks almost exactly like yours, go back to your house and put it into the lock.  It goes in but when you start to turn it, nothing happens.

On a molecular level, the same kind of thing happens.  Probably in some of the cases, the analog or false copy of the molecule breaks off and is stuck in the biological keyhole, which may be the critical part of an enzyme or cell receptor. These analogs then prevent the biochemical functioning from occurring.  In some cases, the key eventually comes out and the right one is able to perform its biochemical function, however, your metabolism has experienced some degree of delay and lacks efficiency.  This lack of efficiency can have a big impact if a high percentage of your metabolic processes are being affected simultaneously. Organs like the brain with a high rate of metabolism may be affected more than other organs. Think of how your TV set runs during a brownout when the supply of electricity is too low. If your metabolic processes are not efficient and are not producing sufficient energy, the brain may not process information efficiently.

Let’s return to tartaric acid and its specific role as an analog. Tartaric acid inhibits the enzyme fumarase (10), which is important in the function of the Krebs cycle, the biochemical process that produces most of the body’s energy. In addition, the inhibition of fumarase also decreases the supply of malic acid for other functions of the cell. The proper function of the Krebs cycle depends on a continuing supply of malic acid. If malic acid is not provided in sufficient quantities, the Krebs cycle is short-circuited.

A large percentage of patients with the disorder fibromyalgia, who have high amounts of tartaric acid in the urine, respond favorably to treatment with malic acid (11-13). I presume that supplements of malic acid are able to overcome the toxic effects of tartaric acid by increasing deficient malic acid. Fifty percent of the patients with fibromyalgia, who also have elevated yeast metabolites, also suffer from hypoglycemia (low blood sugar) even though their diet may have adequate or even excessive sugar (14). The reason for this 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 5) when the body uses up its supply. (The technical name for this process is gluconeogenesis or “new formation of glucose”.) If sufficient malic acid cannot be produced, the body cannot produce the sugar glucose which is the main fuel for the brain. Therefore, the person with hypoglycemia feels weak and their thinking is foggy because there is insufficient fuel for their brain.  If adults with elevated values of tartaric acid in the urine have foggy thinking, little energy, and are so depressed that they may seek out Dr. Kevorkian, imagine what a similarly affected young child, who has yet to form a clear concept of the world, must feel like.

Citramalic acid, like tartaric acid, is another analog of the normal compound malic acid. Citramalic acid is exactly the same (Figure 4) as malic acid except it has an extra CH3 group (called a methyl group) on it. Presumably, citramalic acid acts like tartaric acid in inhibiting the production of malic acid. There are two different types of citramalic acid called isomers. Both types of citramalic acid are probably in the urine of children with autism (2).

Figure 5
Krebs Cycle for Energy Production in Cell
Figure-5


 Arabinose and Candida

Figure 6 shows the chemical structure of a compound called arabinose, which is a sugar. (Arabinose is not an organic acid but is a chemical that we detect with our test.) This is not the same kind of sugar as kitchen table sugar but it is chemically very closely related.  Like all sugars, it is sweet, which is what makes it a sugar. I found that, in the two brothers with autism, some of the values were much higher than in normal children.

Figure 6
Sugars Related to Yeast Overgrowth

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In a study that was reported in the journal Science (a magazine in which experts report their findings to one another in highly technical language), Kiehn (15) reported information about a very closely related sugar called arabitol.  Normal individuals have very low values of arabitol in the blood serum, but as people got sicker (or colonized) with the yeast, the values of arabitol increased.  As the colonization worsened to a state called invasive candidiasis, the arabitol values could get extremely high: over a 1000 times the values found in the normal or control individuals. Many other papers have confirmed that high levels of this compound in both humans and animals were associated with Candida overload (16-18).
Figure 7 shows the distribution of arabinose values among two different groups. Each dot represents a different individual value for the urine concentration of this product. In children with autism, the values can be extremely high. Although there is some degree of overlap between the children with autism and the control group (normal children of the same age range), the mean and median values of urine arabinose for children with autism are much higher than those of normal children. The mean arabinose concentration in the urine samples of males with autism was over five times that of the normal male controls and the median value was six times that of the normal male controls. In infants (data not shown), arabinose values are extremely low, presumably because the intestinal tracts of newborn babies are nearly free of yeast.
Arabinose is a type of yeast sugar called an aldose that it is not known to be produced by humans.  Arabitol (the alternative name for it is arabinitol) is a closely related yeast carbohydrate that is produced by Candida. I suspect that humans may possess the ability to convert arabitol to arabinose. Bacteria in the intestine may also convert arabitol to arabinose. We find a compound that is identified as arabinose in very high levels in urine of children with autism. A child with autism with the highest level of urine arabinose (over 40 times the upper limit of normal) had chronic hypoglycemia following antibiotic treatment for a throat infection as an infant (see chapter on gastrointestinal tract). Below is a comparison of the amount of arabinose in the urine of a child with autism with the amount of yeast in the stool taken at the same time. After four months of treatment with the antifungal drugs fluconazole and nystatin, both the arabinose in the urine and the Candida in the stool significantly decreased. This child had a severe Candida problem due to a complete deficiency of the antibody IgA that normally protects the intestine from Candida infections. This child had only two uses of antibiotics in his life, which was apparently sufficient to set up the yeast overgrowth. After treatment with antifungal drugs, the child had a marked reduction in autistic symptoms with a CARS (Childhood Autism Rating Scale) score in the normal range. Previous CARS scores indicated moderate to severe autism.

 

Arabinose* in urine

Candida
parapsilosis in stool

Before therapy

341

4+

After antifungal therapy

51

0

Normal range

0-47

0-1+

*mmol/mol creatinine

Women with vulvovaginitis due to Candida were found to have elevated arabinose in the urine (20) and restriction of dietary sugar brought about a dramatic reduction in the incidence and severity of the vulvovaginitis. Thus, antifungal drug therapy for children with autism could be useful to reduce the concentration of a yeast produced abnormal carbohydrate that cannot 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.

Elevated protein-bound arabinose has been found in the serum proteins of schizophrenics (21) and in children with conduct disorders (22) and arabinose’s ability to alter protein function might be another mechanism by which arabinose might affect biochemical processes in autism and other diseases.


Figure 7
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 Other Sources of Arabinose

Arabinose may be found in some other foods in small quantities but the most significant source of dietary arabinose appears to be apples and pears. Arabinose values may be very elevated after drinking apple or pear juice or products such as applesauce or pear sauce (Figure 8). Therefore, apple and pear products should be restricted for a couple of days prior to testing. Several parents have reported severe worsening of autistic symptoms within a short time after their children ate apples. It is likely that the arabinose from apple products is responsible for this reaction.


Figure 8
Sources of Arabinose
Figure-8

Arabinose may also be formed from the breakdown of the sugar glucose (23) and antioxidants such as glutathione may inhibit this conversion (24). The breakdown of glucose also results in the formation of an aldehyde called glyoxal, which can also react with and modify protein structure and function. Glyoxal may be converted in the body to glycolic acid, glyoxylic acid, or oxalic acid. (Figure 8)


 Arabinose, Pentosidine, and Protein Crosslinks

The aldehyde group of arabinose can react with the extra amino chemical group (called an epsilon amino group) of an amino acid called lysine that is present in a wide variety of proteins. This combined arabinose-lysine molecule may then form cross-links with an amino acid called arginine in an adjoining protein (25), forming a compound called a pentosidine (Figures 9 A, B). The formation of a pentosidine may cross-link different proteins (Figure 10) and may alter both the biological structure and function of a wide variety of proteins (25). The effect on all of the body’s functions may be devastating.


Figure 9A
Figure-9A


Figure 9B
Figure-9B


Figure 9C
Figure-9C


Figure 10
Figure-10

Let’s use the LA freeway analogy again to understand. Suppose that on a very foggy day during rush hour, gremlins hiding under your moving car and those of your neighbors took strong steel bars and welded them to the frames of the moving cars. The steel bars stick out at a perpendicular angle for about three feet from the side of your car without you or any other driver noticing, because of the fog. Arabinose would be like the steel bar and the proteins would be like the cars. Next, the gremlins welded the other end of the steel bars to the frames of neighboring cars and they welded a bar between that neighboring car and a third car and so forth. Furthermore, some cars might be welded to each other by their bumpers in addition to their sides. Now imagine what will happen when one or more of the drivers wanted to exit or change lanes. Chaos and carnage would ensue. The combined molecule of arabinose, lysine, and arginine is called a pentosidine and is like the two cars welded together. The undoing of these cross-links will become a major challenge in the future for treating older individuals with autism in which many of these cross-links have already been established.

I suspect that autism was reversed in the children of Pam Scott and Karyn Seroussi because they started therapy at a very young age. However, there have been many reports of improvements in people with autism in their twenties after antifungal therapy. Antifungal therapy cannot undo any of the existing cross-links, but can only prevent the formation of new cross-links by reducing the production of yeast arabinose. The tissue concentration of this combined molecule is almost linearly related to age (25); the increase in crosslinks (steel bars) in this molecule is one of the main reasons we lose flexibility as we age.

One child with autism with a very high urine arabinose (1144 mmol/mol creatinine) was examined by MRI (a type of brain scan) and found to have diffuse demyelination (loss of myelin) of the white matter of the brain. (Values as high as 4000 mmol/mol creatinine have been found in children with autism who have not been eating apple products.) It is possible that pentosidine formation could account for this demyelination. Myelin is the material that covers the axons of the brain in much the same way that plastic insulating material is wrapped around copper electrical wire. Without an intact myelin cover, the nerve impulses in the brain are short-circuited just like an electrical wire with torn insulation. Most children with autism are not examined by their physicians with MRI but, on a research basis such an examination of children with high urine arabinose values might be helpful to prove a link between high arabinose and demyelination. A summary of the possible adverse effects of pentosidine is given in Table 1.


Table 1


Effects of Pentosidine Crosslinks

Decreased solubility-neurofibrillary tangles

Increased resistance to proteases

Decreased enzyme activity

Decreased access to coenzymes requiring
free amino: B-6, lipoic acid, biotin

Crosslinks decrease flexibility of structural
proteins in collagen and muscles

Stimulation of autoimmune disease by
crosslink and glycosylated proteins

 

 Arabinose and Impaired Vitamin Function

The epsilon amino group of lysine is a critical functional group of many enzymes to which the vitamins pyridoxal (vitamin B-6), biotin, and lipoic acid are covalently bonded during coenzymatic reactions (26); the blockage of these active lysine sites by pentosidine formation may cause functional vitamin deficiencies (Figure 11) even when nutritional intake is adequate. In addition, the epsilon amino groups of lysine may also be important in the active catalytic site of many enzymes.


 Pentosidines, Tangled Nerves, Alzheimer’s Disease, and Autism

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 (27). 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 children with autism might also be due to the pentosidines and neurofibrillary tangles have also been reported in the brain tissue of an individual with autism (28). It has been reported that frequent urinary tract infections are associated with more severe Alzheimer’s disease (29). The use of antibiotics to treat urinary tract infections would of course lead to yeast overgrowth. I have found that urine arabinose is elevated in some cases of Alzheimer’s disease and have received a report of a favorable response from antifungal therapy to treat Alzheimer’s disease from a woman with a child with autism and a father with Alzheimer’s disease.

 

 Prevention of Pentosidine Formation with High Doses of Vitamin B-6 and Other Vitamins?

Glutathione has been reported to inhibit pentosidine formation (24). 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. Addition of vitamin B-6 derivatives or vitamin C to proteins helps to prevent pentosidine formation (30).  In fact, I suspect that the beneficial effects of vitamin B-6 in autism reported in multiple studies (31) may be mediated by prevention of pentosidine formation. Pamela Scott used high amounts of vitamin B-6, for her child who recovered from autism prior to starting antifungal therapy. I suspect that this reduced somewhat the effects of the yeast die-off reaction. One way to test this idea would be to do a formal study to see if vitamin B-6 supplementation was less effective in treating autistic symptoms after antifungal therapy compared to supplementation before antifungal therapy.


Figure 11
Figure-11

Other compounds called furans that are occasionally elevated in the urine of children with autism are probably derived from fungus such as Aspergillus (32-34) rather than yeast although it is possible they may be produced by yeast as well. The names of these compounds are called 5-hydroxymethyl-2-furoic acid, furan-2, 5-carboxylic acid, and furancarbonylglycine. The concentration of furan compounds in the urine also dropped markedly in children with elevated values after nystatin therapy, indicating to me a probable yeast and/or fungal origin of these compounds. Other investigators (35, 36) noted that these compounds increased after sugar consumption and assumed that these compounds were sugar products of human metabolism but neglected to take into account the Japanese work and the role of gastrointestinal microorganisms in modification of sugars in the food. My interpretation is that these compounds may be derived from sugar but that they are converted to these furan products by the metabolism of yeast and/or fungi in the intestinal tract.


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