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02/08/2002
Background: Autism is a neuropsychiatric disorder characterized by onset prior to age 3 of severe abnormalities of reciprocal social relatedness; communication/language deficits; and restricted, stereotyped patterns of interest and behaviors. It is a lifelong developmental disability, with no known cure. Although a minority of autistic patients may be capable of independent living, most require lifelong care. Autism is one of the most mysterious developmental disabilities. We do not know what causes it, and there is no medical treatment for it. Recently, its incidence has been rapidly increasing in Arizona and elsewhere. In 1996, the Arizona Department of Developmental Disabilities (DDD) served 633 people with autism, and in 1999 they served 1057 people with autism [1]. This was an increase of 67% in only three years, far above the growth rate of the population. DDD spent $20,000,000. in providing services to people with autism in 1999 [1], and we estimate that special education services accounted for another $10,000. per child, or another $10,000,000 (90% of the people with autism in Arizona are school-age (3-21 years) [2]. Similarly, 1 in 250 children are now being diagnosed with autism in California, using the strict DSM-IV criteria; if the definition were broadened to include the full autism spectrum (Asperger’s syndrome and Pervasive Developmental Disorder – Not Otherwise Specified (PDD-NOS), it is estimated that the numbers in California would become 1 in 150 [3]. Although some of the increase is due to better diagnosing, it appears that there may be a real increase in the incidence as well, suggesting an environmental cause. Regardless of the cause, there is no doubt that many more children are now being diagnosed with autism, both in Arizona and nationwide, and that increase has a large effect on state and federal budgets, not to mention the impact on hundreds of thousands of families. There are an estimated 500,000 individuals in the US who are diagnosed with autism. Mercury and Autism: The cause of autism is unknown, but there are suggestions that mercury or other heavy metals may play a role in its pathogenesis. A recent review by Bernard et. al. [4] discusses over 75 similarities between autism and prenatal/infant mercury exposure. Some of the major similarities include a much higher prevalence/sensitivity in males vs females (80% of people with autism are male), decreased language/communication, decreased social relatedness, repetitive behaviors, and gastrointestinal problems. It is useful to review one major case of mercury sensitivity which occurred in the US. Pink Disease, or Acrodynia, very seriously affected thousands of young children between 1890 and 1950 [5]. Aside from the pink integumentary changes, there is considerable overlap in the dominant behavioral findings in acrodynia and autism. Specifically, children with acrodynia often suffered severe language and social regression, as well as extreme intolerance of light and sound. In 1950 it became known that acrodynia was associated with the use of oral teething powders containing calomel, or mercurous chloride, and the illness, at least the one with the pink integumentary changes, rapidly ceased to appear with the discontinuation of the powders. Hallmarks of acrodynia included latency of onset after exposure and an estimated incidence of 1:500 exposures, suggesting a wide variation in individual sensitivity to the mercury compound [6]. Similarly, a study of monkeys who were prenatally exposed to mercury found that they exhibited decreased social play and increased passive behavior [7]. Laboratory studies of autism and mercury toxicity have interesting parallels. Mercury is known to bind to SH groups, and since mitochondria is particularly rich in SH groups, mercury impairs cellular respiration [8]. Impaired mitochondrial function in autism is suggested by a frequent association of lactic acidosis, carnitine deficiency, and abnormal PET and NMR scans [9]. Serotonin signaling has long been suspect in autism, and organic and inorganic mercury and lead are known to suppress serotonin-induced neurotransmission currents, irreversibly in the case of mercury. [10] Neonatal exposure of animals to methyl mercury for twenty-one days results in persistent decreased synaptosomal dopamine uptake after the exposure period [11]. Autoimmunity is a predominate and unexplained finding in autism, and could be related to mercury and other heavy metals. Positive auto-immune markers are found in up to 80% of autistic children [12-16]. Antibody to myelin basic protein (MBP) [12, 13, 15], neural filament protein and glial filament acidic protein [14] are common in autism. Thirteen of seventeen autistic children demonstrated abnormal cell-mediated immune response to MBP, as measured by the macrophage migration inhibition test [17] Mercury also blocks muscarinic receptors [18], and mercury or lead exposure of animals and industrial workers is associated with elevation of the same auto-antibodies seen in autism, and these elevations correlate with serum and urine heavy metal levels [19]. Most recently, in one of the largest evaluations of children with autism, the Pfeiffer Clinic has reported [20] that 99% of the 502 people with autism that they have examined have evidence of metal detoxification problems, especially in terms of finding a significantly elevated Cu:Zn ratio in red blood cells. In summary, there are many similarities between autism and fetal/infantile mercury toxicity, both in terms of symptoms and physiological markers. This data is only circumstantial, but it suggests that there may be a link between autism and mercury. Mercury Exposure: Is it possible that there is widespread mercury exposure in the United States and other countries today? In fact, there are at least six well-documented sources of mercury exposure that exist worldwide:
Thus, it is clear that there are many sources of mercury exposure. Everyone is exposed to small levels of mercury from coal sources (air and water contamination), most people are exposed to vaccines containing thimerosal, and many people are exposed to dental amalgams and fish. Since sensitivity to mercury exposure varies greatly (the 1 in 500 developing acrodynia is a good example), it is possible that a fraction of the population could suffer from mercury toxicity and/or allergies, while most of the population remains largely unaffected. Recently, as part of the ongoing National Health and Nutrition Examination Survey, CDC researchers measured levels of mercury in blood and hair samples from 700 women and 300 children [32/33]. They found that nearly 1 in 10 US women have levels of mercury in their blood that are close to hazardous. The levels were lower in the children, presumably because they eat less fish. According to Dr. Susan Schober of the CDC, “We did not find any women who were actually over the recommended levels, but the findings indicate that continued efforts to reduce mercury exposure to the US population are necessary.” Thus, it seems possible that some women who have high levels of mercury and other metals could pass those metals on to their fetuses and nursing infants. Since there is a wide variation in the susceptibility/sensitivity to mercury, it seems possible that some infants may suffer neurological damage. The reason for the wide variation in sensitivity to mercury is not known, but it may be genetic. A recent report by researchers at Johns Hopkins [33] found evidence that genetic factors significantly influence how susceptible a person is to lead exposure. Specifically, they found that a common defect in DNA sequence in ALAD and VDR can modify the uptake, distribution, and elimination of lead from the body. So, it is quite possible that there are similar genetic factors which affect sensitivity to other heavy metals. 1. Treatments: Since the release of the Bernard et al report in Spring 2000 on “Autism: A Unique Type of Mercury Poisoning”[4], there has been a tremendous increase in the number of people with autism who are attempting detoxification treatments, especially since the report included several case studies of children with autism who improved due to detoxification treatments. In an attempt to bring some order to the many different detoxification protocols that are being used, the Autism Research Institute (one of the oldest private autism research centers in the US) convened a “Consensus Conference on the Detoxification of Autistic Children” on February 9-11, 2001. The attendees were 25 selected physicians and scientists knowledgeable about mercury and mercury detoxification (two of our co-investigators, Dr. McGinnis and Dr. George, and our three consultants, Dr. Boyd Haley, Dr. Quig, and Dr. Jane El-Dahr, were among the attendees). “The fifteen physicians present included 7 who were parents of autistic children who had detoxified their own children with excellent results. The physician attendees present had treated well over 3,000 patients for heavy metal poisoning, with 1,500 of them being autistic children.” “The purpose of the meeting was to arrive at a consensus document which would delineate the safest and most effective methods of detoxifying autistic children. Nine candidate detoxification protocols, including five submitted by non-attendees, were considered in detail by the conferees.” “The participants resolved the controversial issues harmoniously and arrived at the consensus position that was hoped for. The Autism Research Institute will issue its consensus report on the detoxification of autistic children at the earliest possible date—most likely in mid-March--after the participants have had an opportunity to review and approve the document.” Thus, this meeting brought together many of the top people in the mercury-autism field, and developed a consensus treatment protocol. Of the 1,500 children with autism being treated, only a few have completed the 1-2 year treatment, but the preliminary reports are very encouraging. Several physicians reported “complete recovery” from the symptoms of autism for their youngest patients (under 7), and substantial improvement for even the adults with autism. However, these preliminary reports are from clinicians who are not carrying out a formal research study. Although their lab results do demonstrate that high amounts of many toxic metals are being excreted, there is no formal evaluation of changes in the symptoms of autism. In summary, the cause of autism is unknown and intolerance to mercury and other heavy metals may be a possible cause. The symptoms of low-level mercury toxicity in infants and young children are very similar to the symptoms of autism. The wide variation in autism spectrum disorder could be explained by different levels of exposure to mercury at different stages of development, and by different sensitivities to it, possibly along with the effect of other heavy metals. There is ample evidence that most children in the US are exposed to significant levels of mercury, and the amount of exposure has been increasing (at least in the case of vaccines, and possibly from other sources). Finally, physicians are currently using detoxification treatments for over 1,500 people with autism, and preliminary results appear promising. Therefore, we feel that mercury toxicity should be formally investigated as a possible cause of autism. We propose to first determine if there is evidence that children with autism do suffer from mercury/metal intolerance. If we find that to in fact be the case, then we propose a formal evaluation of the Consensus Protocol which was developed at the Feb 2001 detoxification conference. 2. Approach: Participant Selection: For participant selection, we will send a mass mailing to 1000 families of people with autism in Arizona, using a mailing list compiled from the six major autism support groups in the state. As with our pilot study, this will be an ethnically diverse group, with Hispanics being the largest minority group represented. The initial criteria for inclusion in the study will be:
From the number of people who respond to our mailing and meet the above criteria, we will select a total of 50 children with autism, 50 age-and sex-matched controls, and their mothers (a total of 100 children and 100 mothers), giving preference to the youngest children. Each child with autism will undergo psychological testing first (see below), to evaluate their score on several standardized tests. Only those participants with a rating characteristic of autism will be allowed to participate in the study. Each participant will then undergo a physical exam, to confirm that they are in good physical health before conducting any other tests. Again, if anyone is rejected, replacement participants will be randomly selected. Phase One: Testing Once we have a group of participants, we will begin the following set of testing and evaluations, to determine if there is substantial evidence of mercury/metal intolerance in children with autism. Based on those findings, we will consider extending our work to include evaluation of the consensus detoxification protocol. Physical Examination/ Medical History: Each participant will be first be examined by a physician (Dr. George and/or Dr. McGinnis), to determine that they are in good health. We will compile a full medical history, with a focus on developmental milestones (age of talking, walking, possible regression, etc.), vaccination history (to evaluate mercury and aluminum exposure), medical problems (especially ear infections and gastrointestinal problems which are often elevated in autism), and a brief family medical history focusing on autoimmune function. This exam will screen for somatic manifestations of heavy metal poisoning in all subjects. Cholinergic signs such as pupillary size may be of particular interest in the context of mercury. Tremulousness and specific gait disturbance will be systematically recorded. Blood pressure measurement, never documented for autism, will be included, and may be of particular interest in relation to the PHF testing. Heavy Metal Exposure Questionnaire: We have developed a 6-page questionnaire to assess exposure to over 100 possible sources of heavy metals, including dietary intake of seafood, vaccination history, and many other factors. This questionnaire will be critical in helping us determine the major possible source(s) of heavy metals, and to determine if people with autism were exposed to unusually high levels: Heavy Metal Testing: We will conduct a variety of tests to evaluate heavy metal levels in the mothers and their children. We wish to test the mothers because we believe that one possibility is that they have high levels of heavy metals in their bodies, and animal studies have shown that mothers can pass heavy metals on to their children during pregnancy and breastfeeding, which is the most dangerous time period for exposure. Also, hypersensitivity to metals may be an inherited trait. These tests will be carried out by Doctor’s Data, and will include a morning urine sample, a packed red blood cell sample, and a hair sample. For the hair sample, we will require that the participants use no hair care products for two months previous except for Johnson and Johnson Baby Shampoo, to avoid contamination problems. Because mercury binds strongly to tissue, we may not find any unless the people have dental amalgams (which continuously release small amounts of mercury). However, that is why we are doing additional tests (MELISA, autoimmune and PHF) which will allow us to look for the possible effects of mercury and other heavy metals. MELISA Test: The Memory Lymphocyte Immunostimulation Assay (MELISA) is a new technique to measure the reactivity of hapten-specific lymphocytes to various allergens, including heavy metals [34-38]. MELISA measures the proliferative response of lymphocytes to heavy metals, and would appear to be the best technology available to assess “allergic” response to heavy metals. MELISA also grades the histological degree of direct cell injury with exposure to heavy metals, and may reflect individual susceptibility to toxic effect of metals [34]. In one study [35], adults with clinical symptoms suggestive of intoxication from dental amalgams had pathological findings on MRI scans in 81% and pathological findings by MELISA in 88%, 60% of who demonstrated immune sensitivity to mercury. Individuals with high reactivity on initial MELISA testing may enjoy decreasing reactivity and reversion to non-reactive status after removal of mercury sources and detoxification [39]. MELISA testing may therefore reflect certain threshold metal levels for on-going activation of the auto-immune response. The developer of the test, Dr. Stejskal, will be taking an active role in this project and will be serving as a Scientific Advisor and Immuno-toxicologist. Most relevant to this proposal, Dr. Stejskal recently began testing children with autism. Her preliminary results on 18 autistic children and 11 controls, found that 5 of 18 autistic children had a positive proliferative ("allergic") response on MELISA to Thimerosal, vs. 1/11 controls. Similar results were recently found for methyl mercury (6/10 autistics vs 0/11 controls) and inorganic mercury (6/18 autistics, vs 0/11 controls). Most importantly, 13/16 autistics tested positive for reactivity to the mercury-MBP vs. only 3/10 controls. The mercury-MBP reactivity is presumed to be caused by the mercury reconfiguring the three-dimensional MBP, to which the body generates the allergic (autoimmune) response. PHF testing: Parathyroid Hypertensive Factor (PHF) is produced by the parathyroid gland and is measurable by the University of Alberta. Preliminary PHF determinations on over 100 patients through the Pfieffer Treatment Center have revealed very high levels for autistic patients [20]. Heavy metals are known to block calcium L-channels at the cell membrane, whereas PHF is known to open calcium L-channels [40] and stimulate phosphodiesterase [41]. Calcium L-channels perform numerous functions, including initiation of transcriptional events which support learning, memory and endocrine secretion. Mercury inhibits L-channels at micromolar concentration [42] in an irreversible manner in hippocampal neurons. Hypothetically, elevated PHF may serve to at least partially compensate Hg-inhibition of L-channels. Mercury is also a potent inhibitor of cAMP [43], cellular levels of which presumably further decrease with PHF-stimulation of phosphodiesterase. Thus, in the context of mercury toxicity, PHF may play both adaptive and maladaptive roles. The very mechanism of mercury-induced auto-immune disease in mercury-sensitive rats is related to L-channel signaling. This process involves induction of interleukin-4 gene expression, which is mediated by protein kinase C-dependent calcium influx through L-channels [44]. PHF hypothetically may affect the auto-immune response. In conclusion, we think that it is important to measure PHF levels because they may be indicative of mercury toxicity and auto-immune response. Prof. Christina Benishin at the University of Alberta has volunteered to provide the testing at no charge. Vitamin/Mineral Testing: The blood samples will also be used for analyzing the level of several essential vitamins and minerals which Prof. Johnston’s lab is experienced with measuring, including: vitamin C, folic acid, vitamin B12, zinc, and iron. We have found vitamin C to be low in children with autism [45], and we have shown C to have a strong affect on glutathione levels [46], which is an important defense against heavy metals. We suspect that other vitamins and minerals can affect or be affected by heavy metals. We will also determine what supplements, if any, they are taking. Glutathione Levels: Glutathione is a key natural defense against the toxic effects of heavy metals, and one recent published study found lower levels of glutathione in autistics. Correlations between glutathione levels and tissue levels of one or more heavy metals, or between glutathione levels and cytotoxicity on MELISA, may be found. Prof. Carol Johnston will measure glutathione levels, as her group is experienced with that test. In addition, we will measure the level of glutathione transferase enzymes – GSTT1 and GSTTM1. A recent study found that the combined deletion of glutathione transferase enzymes-GSTT1 and GSTTM1 were found in 91 patients with thimerosal allergy measured by patch test and compared to the level of enzymes in 169 healthy controls and 114 patients with allergy to para-substituted aryl compounds [47]. This indicates that thimerosal sensitized subjects may have biochemical impairment in the functional capacity to detoxify thimerosal. The levels of GSTT1 and GSTM1 will be measured by polymerase chain reaction by Labor Schiwara in Bremen, and the test can be done with the same sample of lymphocytes used for the MELISA test. Alpha Lipoic Acid: Another of the body’s defenses against heavy metals is alpha lipoic acid, and it is commonly used in detoxification treatments. Alpha lipoic acid is an essential part of the ATP cycle (energy production) in every cell. We believe that individuals with autism may have reduced levels of alpha lipoic acid, since supplements of it increase the amount of toxic metal excretion [48]. Therefore, we will measure its level in the blood, probably using the test at Vitamin Diagnostics. Urinary Sulfate: Urinary sulfate levels will be measured because mercury collects in relatively high concentration in the kidney. Dr. Waring found that ninety-two percent of autistic children have reduced plasma levels of sulfate, but urinary sulfate excretion is elevated by a factor of two. [49]. The renal sulfate / bicarbonate anion exchanger is inhibited by mercury and associated with sulfaturia. [50] Sulphate status affects activation of neurotransmitters, thyroid hormone and detoxification. Dr. Waring of the University of Birmingham, U.K., has offered to test the subjects in our study at cost ($10 per subject for chemicals), including a measure of their urinary sulfate, sulfite, thiosulfate and thiocyanate. She has found all of them to be altered in autism, possibly due to inhibition of rhodanese and sulfite oxidase. Proteinuria: Dr. Waring has also consistently found elevated protein in urine of people with autism, and suspects that this is due to nephrotoxicity of Hg. Mercury and mercury compounds are well known to be nephrotoxic in humans and cause loss of protein in urine. This is probably due to binding of Hg with thiol (-SH) groups on the proteins of the kidney. Rats have an autoimmune reaction to mercury chloride which affects the kidneys, and this may also be the case in humans. [51,52] Therefore, we will also measure protein level in urine. Psychological Testing: Prof. Castro will supervise a series of standardized tests to evaluate the social, cognitive, and developmental skills of the children with autism. Since the tests are very well-standardized, he will assess only the children with autism, and not the typical controls. His evaluations will be “double-blinded” from the others, so that neither he nor the parents will know any of the other results. These tests will include the following: The Gilliam Autism Rating Scale is used to identify and diagnose autism and estimate the severity of the disorder. Items are based on the definition on autism adopted by the Autism Society of America and the Diagnostic and Statistical manual of Mental Disorders – Fourth Edition (DSM-IV). The items are grouped into three core subtests (stereotyped behaviors, communication, and social interaction) and one optional subtest (developmental disturbances). The Vineland Adaptive Behavior Scale is a developmental scale that assesses personal and social sufficiencies of individuals from birth to adulthood. It measures adaptive behavior in the communication, daily living skills, socialization, and motor skill domains. In addition, it can give an Adaptive Behavior Compose, which comprises the four adaptive behavior domains, as well as the maladaptive behavior domain. The TONI-3 assesses the intellectual functioning of the individual, and since it is a non-verbal test it is especially useful in children with autism who may have little or no speech. The Child Behavior Rating Scale is designed to assess the adaptive behavior of the child and its variation from the norm. The Conner’s Rating Scale Rev (CRS-R) is designed to assess the behavior and level of activity and hyperactivity of the child. The Autism Diagnostic Observation Schedule (ADOS), which involves a standardized evaluation of a child’s response to a standardized set of structured activities. The graduate student will attend a 1-week workshop for training in this technique. Dental Amalgams: Dr. Margolis, DDS, will carry out a dental exam and a careful review of dental records to retroactively evaluate the mother’s oral conditions during pregnancy and (if applicable) breastfeeding. This will include an evaluation of the total surface area of silver-mercury dental amalgams, the stability of the amalgams, and the likelihood of galvanic corrosion due to other types of metals in the mouth (gold fillings, stainless steel caps, etc.). A similar dental exam and review of dental records will be carried out on the children. This will help us to determine if there is any correlation between the amount of maternal amalgams and the level of heavy metal toxicity in the mothers and children. This evaluation will be double-blinded from the others to ensure reliability. The reliability of retroactive evaluations is limited, but we will do our best to obtain full dental records so that we can be as accurate as possible. Treatment: If the results of the above tests demonstrate significant evidence of mercury/metal toxicity/intolerance in the children with autism, then we propose to carry out a study of the effectiveness of the “best practices” treatment available at that time. It will likely be very similar to the Consensus Detoxification Protocol developed at the February 2001 workshop, but we will incorporate future changes to that protocol. The treatment stage will only include the children with autism, and will not include the “typical” control children. That Consensus Protocol involves approximately the following (the formal report will be released in mid-March 2001)
Background on DMSA: DMSA is a prescription medicine with FDA approval for treating lead poisoning. It is approved for use in children as young as 2 years old. The recommended dose in the Physician’s Desk Reference (under Chemet in the PDR) is 30 mg/kg bodyweight per day (in 3 divided doses), with treatment times as long as 5 weeks for high levels of lead. Repeat dosing is often required, as metals slowly move from body tissue into the blood where they can bind with DMSA. It is frequently used “off-label” for removal of other heavy metals, since it also strongly binds them. Phase Two: Single Dose of DMSA If we find evidence of metal poisoning in a significant number of the children with autism, then we propose to determine the effectiveness of DMSA in removing those heavy metals. We will first measure kidney and liver function, and check for the possibility glucose-6-phosphate deficiency, to determine that administration of DMSA is safe. Proposed test of DMSA: Give each child with autism an oral dose of DMSA, at the recommended (PDR) dose of 30 mg/kg bodyweight per day (in 3 divided doses, eight hours apart) for only one day. We will then collect all urine samples for the day of the dosing and the following day, and measure the heavy metal levels. The urine results will determine whether or not there is a significant increase in excretion of heavy metals, and it will also provide valuable information on how rapidly the DMSA is absorbed and excreted in this population. Finally, only those children who show evidence of heavy metal excretion will continue on to phase three. Phase Three: Pre-treatment of nutritional and intestinal issues If the results of phase two indicate that DMSA causes a significant increase in the excretion of mercury and other heavy metals excreted in the urine of children with autism, then we propose to conduct a long-term treatment study for only those children, beginning with a pre-treatment of vitamin/mineral deficiencies and intestinal issues. First, we will carry out a complete analysis of vitamin/mineral status in the children. The reason is that a recent study by Dr. Vogelaar at Vitamin Diagnostics has found severe nutritional deficiencies in most children with autism. Specifically, he found that over 50% of his patients had low levels of the following vitamins/minerals: A, B1, B3, B5, biotin, selenium, zinc, and magnesium. He also found that over 50% of the patients had low levels of essential amino acids and two essential fatty acids (EPA and DGLA). Therefore, we think it will be important to pre-treat the children with a multi-vitamin/mineral supplement for three months. The vitamin/mineral supplement will be chosen based on the lab results, and two manufacturers (Kirkman Labs and Brainchild) have offered to design a vitamin/mineral supplement specifically for this study based on our vitamin/mineral results. At the end of the pretreatment, we will then retest, to determine if there has been a significant improvement in nutritional status. Only those children who have reasonably normal nutritional and intestinal status will continue on to phase four. Simultaneously, we will also use a urinary organic acid analysis to look for evidence of intestinal yeast infections, which is common in autism. Vogelaar’s study of urinary organic acids found that 14 of 20 children with autism had significant yeast infections, and 10 of 20 had significant bacterial infections. Therefore, all children will be provided with a probiotic (Culturelle), which in our clinical experience has proven effective at combating bacterial infections. If there is evidence of a yeast infection, the children will also be treated with a course of Nystatin, a standard anti-yeast medication which is not absorbed by the body, but rather passes through the gastrointestinal tract without being absorbed. Phase Four: Treatment with DMSA We plan to carry out a double-blind, placebo-controlled test of the latest Consensus Detoxification Protocol, to formally determine its safety and efficacy. This will include:
At this point we will break our “code” and determine who received the placebo and who received the DMSA. Phase 5: Single Use of Alpha Lipoic Acid If Phase 4 demonstrates both safety and efficacy in removing heavy metals, then we will test the use of alpha lipoic acid. For those participants who received the DMSA and whose heavy metals were reduced by 80% from their maximum, we will initiate a three-day treatment of alpha lipoic acid. We will collect pre and post urine and feces samples (alpha lipoic acid is primarily excreted in the feces). We expect to see a large increase in the amount of metals being excreted. Phase 6: Extended DMSA/Alpha Lipoic Acid treatment – future work If Phase 5 demonstrates safety and efficacy of the Alpha Lipoic Acid, then we will continue treatment for those participants who showed an increase in metal excretion. This treatment will consist of the following:
Note: Our current budget will not allow us to go beyond phase 5. However, we are sending similar versions of this proposal to other agencies, and so that funding combined with this would allow us to complete Phase 6. Statistical Analysis: The statistical analyses for this study will be supervised by Professor Richard Fabes at Arizona State University. Because we will be collecting a wide variety of data for each phase of this study, several statistical techniques and strategies will be used. First, the interrelations among many of the measures will be examined to ascertain the degree to which they may measure similar processes. Because of the need to understand how our measures vary by age of the children, we also will examine the degree to which the measures relate to age and the degree to which age of children affects the reliability of the measures. If we find significant relations with age, we will use age as a control variable in many of the following analyses. For the main analyses comparing two groups (e.g., autism and typically-developing, and then DMSA vs placebo), we will rely on a series of univariate and multivariate analyses of variance with group and sex of children as between-subject factors. Because of the large amount of data we will collect, the potential for obtaining significant results due to chance increases as the number of analyses increases. Therefore, we will adjust the significance level for the number of analyses we will compute. We also will examine the relation between severity of symptoms of autism with metal levels and sensitivity. For each child, we will estimate the total amount of heavy metals excreted based on their urine and feces levels. Using all of our data, we will explore the degree to which autism correlates with our various indexes of heavy metal levels and sensitivity (perhaps controlling for age and sex). We will examine the relations between the measures obtained from mothers and the severity of children’s autism. Correlations between maternal and child mercury levels/sensitivity will be explored, as will the relation between maternal data and children’s autistic symptoms and severity. By determining if these patterns also exist in typically-developing children, we will obtain a better understanding of the potential role that maternal mercury exposure/excretion may play in the onset and severity of autism. Finally, a careful analysis of our treatment study will allow us to determine if detoxification treatments do help some children with autism, and allow us to determine the factors which are most predictive of the likelihood of successful treatment. Thus, we believe that these analyses will allow us to determine whether or not there are statistically significant and reliable differences between the autistic and control groups in terms of their metal levels and/or sensitivity. We also will be able to determine which specific aspects of autism are related to mercury and other heavy metal levels, and whether maternal indexes relate to those of their children in specific and predictable ways. Finally, we will be able to evaluate the effectiveness of detoxification treatments. 3. Expected Benefits or Results This study will provide the most extensive investigation to date of the possible link between autism and mercury/metal toxicity. If such a link is found, it will help determine what are the primary source(s) of mercury and other metals, based on our Heavy Metal Exposure questionnaire, medical history, dental evaluations, and the MELISA test that can distinguish between sensitivities to different forms of mercury. This will be critical in learning how to reduce or prevent future exposures to metal toxicity. Also, if a link between autism and metal toxicity is found, then the proposed treatment study will investigate the Consensus Detoxification Protocol for Children with Autism, and determine its safety and effectiveness in terms of excretion of metals and the effect on autistic symptoms. The results of our study will be very valuable to the rapidly growing number of parents and physicians who are considering detoxification treatments. 4. General Project Information Research Team and Preliminary Research: Our research team consists of a broad and diverse group of researchers and clinicians with a complementary set of skills. Altogether, we believe we have all of the skills and experience necessary for the proposed work. Prof. Adams will coordinate the project, but will rely heavily on the individual expertise of every member of the team. Below we provide a brief overview of their experience and explain how it is relevant to our proposed research project. Dr. Woody McGinnis, MD, was a co-author and leading medical consultant on the report “Autism: A Unique Case of Mercury Poisoning.” He is currently involved with four autism research studies, several of which closely relate to this proposal, including a cod liver oil/bethanecol study, a PHF study, a mercury/autoimmunity study, and a metallothionein study. Three members of our research group (Profs. Adams, Johnston, and Fabes) are working with Dr. Raun Melmed and Dr. Cindy Schneider (the co-medical directors of the Southwest Autism Research Center) on a study of the effect of vitamin/mineral supplements on children with autism. This was a double-blind, placebo controlled study which lasted for 3 months and involved 24 children. The results of the study are still being analyzed, but our preliminary analysis shows that the vitamin/mineral supplement was somewhat effective in reducing some of the symptoms of autism, with improvements in sleep and gastrointestinal disorders being the most significant. This study is relevant because our proposed work involves some of the same tests, as well as many others. In addition, Drs. Adams, George, and Johnston are just beginning a small pilot study to measure the heavy metal levels in mothers of children with autism. This study is a preliminary, highly-simplified version of the proposed study. This study involves 30 mothers of people with autism (any age), and 30 mothers of age-matched typical sons/daughters. We will measure the levels of heavy metals in the morning urine of the participants. Our hypothesis is that some of the mothers of children with autism excrete high levels of mercury, which could have affected their children during pregnancy and breastfeeding. We will use the Gilliam Autism Rating Scale (GARS) to assess the level of severity of autism in the children, and search for a correlation between the severity of autism and the levels of each heavy metal excreted by the mothers. This study of the mothers will complement the proposed study, which is much more comprehensive. This pilot study will be completed by June 2001, so that we will be fully ready for the proposed study. Prof. Carol Johnston has also carried out many research studies on nutrition and vitamins/minerals. One of the most relevant studies showed that consumption of 500 mg of vitamin C by college students resulted in a 50% increase of glutathione levels [6]. Glutathione is important in the removal of heavy metals, and our vitamin/mineral study found that children with autism have reduced levels of vitamin C. Therefore, in this proposal we plan to measure both vitamin C and glutathione levels, and Prof. Johnston has all the expertise and equipment necessary for this testing. Prof. Richard Fabes is the Chair of Family and Human Development at Arizona State University. He has extensive experience with statistical analysis of a wide range of research studies, including several related to children with autism. Dr. Frank George, D.O., M.D. (H) has a large clinical practice and is very experienced with metal detoxification testing and treatment, having treated over 200 people including 15 children with autism. Prof. Vera Stejskal is an Associate Professor of Immunology at the University of Stockholm. She developed the MELISA test, will conduct those tests for this study. Prof. Christina Benishin is an Associate Professor in the Department of Physiology in the medical school at the University of Alberta. She will perform the blood tests for PHF, as she has extensive experience with those tests. Prof. Felipe Castro is a Professor of Psychology at Arizona State University, and he will supervise the evaluations of the severity of autism. Dr. Mike Margolis, D.D.S., has a large dental practice with many years of experience dealing with children and adults. He will reviewing the dental history of the mothers and children and evaluating the total surface area of their dental amalgams. Finally, three researchers will serve as scientific consultants for this project. Prof. Boyd Haley, Chair of Chemistry at the University of Kentucky, will act as a scientific adviser on mercury testing, treatment, and dental amalgams. David Quig, Ph.D., will serve as a scientific adviser on heavy metal testing; he is the chief scientist at Doctor’s Data, which is one of the oldest and largest commercial labs for measuring heavy metal levels. Their lab will carry out the testing for heavy metals in blood, urine, hair, and feces. Jane El-Dahr, M.D., Tulane University, has been treating 40 children with autism using a protocol very similar to the Consensus Detoxification Prototcol. They have all reviewed this proposal and provided technical guidance. Thus, we feel that our diverse team has all the necessary experience for this proposal, and that our complementary skills will allow a thorough investigation of our hypothesis that heavy metals may contribute to the cause or symptoms of autism. References
BUDGET JUSTIFICATION PAGE MODULAR RESEARCH GRANT APPLICATION Total Direct Costs for Enitre Proposed Period of Support: $300,000. Initial Budget Period Second Year of Support Third Year of Support $100,000. $100,000. $100,000. Personnel Profs. Adams, Fabes, Johnston, and Castro will be volunteering their time to this project. Prof. Adams will lead the project, coordinating the efforts of the entire team and focusing on data analysis and interpretation. (15% of his time). Prof. Fabes will supervise the statistical analysis (5% effort). Prof. Johnston will assist with the vitamin/mineral testing, treatment, and analysis. Prof. Castro will supervise the psychological testing (5% effort). Two graduate students (50% and 25% Research Assistants) will coordinate all the logistics for the study, assist with the patient meetings, conduct the heavy metal analysis, and collect the data and perform a full statistical analysis. Subcontract to Arizona Biological Research Foundation: Year 1: $70,000. Year 2: $68,000. Year 3: $66,000. Dr. George and Dr. McGinnis will conduct the physical examinations, work with Dr. George’s nursing staff on the numerous medical tests, and help analyze the results (10% effort each). One graduate student will conduct all the psychological testing (25% time). All the medical tests will be paid for through this subcontract. |