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Organic acid testing, byproducts of yeast and their relationship to autism.
by William Shaw Ph.D.
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 do a better job than what had been done previously in the field of metabolic diseases. I wanted 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. Urine is preferentially tested instead of 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 characteristic abnormal chemical compounds would indicate the likely presence of a genetic disease. For example, in the genetic disease PKU or phenylketonuria, very high concentrations of chemical compounds called phenylketones appear in the urine because the child with PKU has a genetic mutation. This mutation in DNA 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 with significant mental retardation (1).
As a biochemist I thought that diseases which had very devastating effects on the individual were bound to change that particular individual's 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 were from out of town and 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. I didn't think this method of examination was the very best system. It was adequate when this field was starting off about 20-30 years ago, but I didn't think it was up to date with the best 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 no manager is on duty
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 ask for the manager and
again ask for your balance and this time the manager says, "Not much." This
type of accounting may be adequate for comparing the assets of Bill Gates
and a person who is collecting cans out on the street. But this type of accounting
is not good for much the in-between: the middle class. Now I think that after
a while you would stop going to that bank. In essence, the majority of metabolic
disease testing that was performed five years ago and perhaps 50% of the testing
done today was the "a lot or not so much" kind.
I suspected that there were many subtle changes in the metabolism of the body that were being overlooked by using the kind of technology that resulted in 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 able to do that because of new computer software that allowed for the rapid quantitation of very complex data. If it was not for this particular software, my work would not have been possible.
This computer software had originally been designed for the environmental field. In our drinking water, our sewage and in our ground water there may be many kinds of pesticides, herbicides, and industrial chemicals. Testing for all of these chemicals requires very sophisticated computer software. This software was ideally suited for doing metabolic disease testing. I set about achieving several goals including being able to quantitate everything that I possibly could, as accurately as I could, and to be able to identify every chemical that I could.
If we knew every single possible thing about an individual and if we knew what kind of chemical compounds were normal, then we would be able to easily say what was going on in the metabolism of a patient that had a particular disease. Prior to beginning testing, we sent samples out to a 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 was very surprising and confusing to me how devastating diseases would not alter metabolism in some way.
I continued the development of my own, more elaborate testing. I found that, indeed, there was some increased detection (perhaps 5-10%) of certain of the known genetic diseases. However, this was a smaller increase than I had anticipated. I also noticed that in many different diseases that there were abnormal elevations of certain compounds that nobody seemed to know too much about or care too much about. When I talked to colleagues in the field of metabolic disease all over the United States and even in other countries, they would say that these particular chemical compounds that you are finding are probably due to microorganisms in the intestinal tract and, therefore, they are not important.
And so I filed that kind of information away in my mind but continued to be skeptical of the non-importance of microbial products 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 shunted into other compartments. All of these products were intermixed throughout the body. Several months after initiating my new laboratory service, a colleague of mine from the University of Kansas Medical School, Enrique Chaves MD, a pediatric neurologist who was also interested in biochemistry (a rather rare occurrence in physicians as a group) referred to me a woman who had two children with severe muscle weakness. Dr. Chaves had also been using the old technology in his laboratory for genetic diseases and could find nothing unusual in the two brothers. The muscle weakness was so severe that sometimes, for several hours, they could not stand up. There had been an intensive search for the cause of this muscle weakness. When Dr. Chaves analyzed the samples, 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 help her out in finding out what was happening to her children.
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 or the 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, indeed, there were no chemical compounds characteristic of any of the known inborn errors in metabolism. But my overall impression was that these samples were still abnormal. When the samples were all tested, there was not any consistent abnormality in any of the known genetic diseases which are called inborn errors in metabolism. However, there was a marked difference in the kinds of chemical compounds that were present in the urine samples of the two brothers with the muscle weakness and 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 from across town Dr. Chaves had moved into the same institution. I just had to walk across the hall and ask him some more about what might be going on with these brothers that might explain why they had these abnormal concentrations of chemicals that are characteristic of 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 had a history that is similar to many children with autism which is that they had a history of frequent ear infections. A brief description of the technology used for testing the samples is appropriate at this point.
We use an instrument called a gas chromatograph-mass spectrometer. Samples are loaded on this module. The sample is injected into this instrument. The different molecules in the sample go around and around in a large circle called a column just like a group of horses going around a race track 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 are able to 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 is 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.
We also use a chromatogram for the analysis of the urine sample of a normal child. This profile is called a total ion chromatogram. Each one of these blips that you see is called a peak by people who work in the field. 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 by people in the field. The bigger the peak, the more of a compound is there. Conversely, the smaller the peak, the smaller the amount of compound is there.
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 3). 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 lucky 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. 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 or later on proved to be due to intestinal microorganisms. So virtually all of the big changes that you see in the chromatogram of the child with autism (2) were due to the fact that they had much higher concentrations of the chemicals that were produced by microorganisms that were 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 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 Dept of the hospital to confirm a case of autism and the 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. They came one after another. The child developed a thrush or yeast infection of the mouth which 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 - 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, or recurrent bronchitis.
I explained my theory to the mother of the child whom I'll call Bruce. She was a nurse at another nearby hospital and understood about thrush and antibiotics and wanted 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. He declined. I explained the situation to the child's mother . Since she was a nurse and knew that the antifungal drug nystatin had no serious side-effects, she decided to obtain a prescription for nystatin from her family doctor in private practice who was not associated with the hospital.
Within a couple of days of starting the antifungal drug nystatin, Bruce who had lost most of normal development began to improve and eye contact came back. Bruce's extreme hyperactivity began to go away 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 4). Nystatin is an antifungal drug which indicated to me that a yeast or fungus (these terms are somewhat interchangeable in that these they are very closely related biologically) was causing the secretion of this compound in the intestinal tract.
Figure
4
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. 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 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 received reports of this same phenomenon in hundreds of other cases. Even after six months and sometimes even after two or three years of antifungal treatment, there is often a biochemical "rebound" and loss of improvements after discontinuing antifungal therapy. Several explanations are possible for this phenomenon:
Because of one or more defects in the immune system(See chapter on the immune system.), the yeast which 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 these 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 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. (Continue to Part II.)
Buy The newest version (2002) of this book online!
Biological Treatments for Autism and PDD Online > Chapter 3: Part I | Part II | Part III