Autism Studies 2016;3:1-23  (O/A)


A ‘unified field theory’ of autism


[F]or every disease there is a single key mechanism that dominates all others. If one can find it, and then think one’s way around it, one can control the disorder.... In short, I believe that the major diseases of human beings have become approachable biological puzzles, ultimately solvable


Lewis Thomas  The Medusa and the Snail


ABSTRACT


Evidence implicating glutathione depletion as single key mechanism (Thomas) in autistic disorders has reached critical mass. Glutathione depletion (a) elevates prenatal/postnatal androgens and limits estrogens (b) delays or prevents maturation of brain myelin (c) limits release of primary brain vasodilator nitric oxide (d) depletes glutamine, thus arginine and nitric oxide. Low brain glutamine limits entry of serotonin precursor tryptophan. Most arginine ingested as protein by these children may be needed by the liver to detoxify ammonia from intestinal bacteria and yeast, limiting arginine as only substrate for nitric oxide. Primary agents depleting glutathione in these children (and their mothers) are proposed to be heavy metals – and the antipyretic/analgesic acetaminophen (Tylenol, paracetamol) implicated in our national autism epidemic. Glutathione depletion arguably explains dysconnected brain hemispheres, anomalous brain asymmetries, extreme male brain, low brain blood flow, glutamine paradox, and other enigmas of autistic disorders. Do free taurine and glutamine released by fever relieve autistic behavior dramatically by dehydrating immature brain myelin – or compensating it?


INTRODUCTION – GLUTATHIONE DEPLETION

AS “SINGLE KEY MECHANISM”


Einstein coined the term “unified field theory” to describe his attempts to explain electromagnetism and gravity as aspects of a single field. This paper interprets  autism neuropathology as aspects of Lewis Thomas’s “single key mechanism”[1] – proposed to be depletion of glutathione (GSH), primary antitoxin and antioxidant in all human cells, and especially the liver.


Glutathione depletion in autism was first reported by James and colleagues in 2004: “Relative to the control children, the children with autism had significantly lower baseline plasma concentrations of methionine, SAM, homocysteine, cystathionine, cysteine, and total glutathione and significantly higher concentrations of SAH, adenosine, and oxidized glutathione.”[2] Geier and Geier found pre-pubertal children who previously regressed into autistic disorders (ASD) had high serum or plasma concentrations of adrenal androgen dehydroepiandrosterone (DHEA) and testosterone, and low plasma concentrations of methionine, cysteine, and glutathione. They noted DHEA can convert to androstenedione and then testosterone, or be sulfated to the “normally favored storage molecule” DHEA sulfate (DHEAS). Because sulfa-tion of DHEA requires glutathione as cofactor, they proposed that glutathione depletion in these children caused less DHEA to become DHEAS and more to become androstenedione and testosterone.[3]


This is a critical observation, because DHEAS from the fetal adrenal cortex is the major precursor of placental estrogens essential for fetal growth and maturation [4] – notably maturation of myelin sheaths, the fatty insulators around nerve fibers that give white matter its name. Geier and Geier noted evidence DHEAS is also significantly low in adults with ASD. Are prenatal and postnatal DHEAS and estrogens limited by glutathione depletion in autism – and does that explain dysconnected brain hemispheres, anomalous brain asymmetries, and the extreme male brain?


Glutathione depletion also reduces brain blood flow – directly by limiting release of nitric oxide, and indirectly by depleting glutamine, thus nitric oxide precursors. Other studies corroborated glutathione depletion in ASD.[5,6] Frye and colleagues tested the benefit of methylcobalamin and folinic acid in children with autism: “A greater improvement in glutathione redox status was associated with a greater improvement in expressive commun-ication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills.”[7] Brain GSH levels in normally intelligent adult males with ASD, however, were normal.[8]


Various external and internal toxins, oxidants, and carcinogens that glutathione neutralizes may explain glutathione depletion in ASD.[9] Heavy metals are often implicated, notably mercury and aluminum in vaccines, and lead in air and water. Any proposed environmental cause, however, must account for the sudden dramatic increase in autism incidence in this country that began about 1980 and continues today – our national epidemic.


GLUTATHIONE DEPLETION LEAVES MYELIN SHEATHS IMMATURE AND BRAIN BLOOD FLOW LOW


IMPAIRED BRAIN CONNECTIVITY IN ASD


Much evidence of impaired brain connectivity in ASD has been presented, notably a smaller corpus callosum, primary pathway linking the brain’s two hemispheres. Herbert and colleagues found the rapid brain growth soon after birth in these children primarily involved larger white-matter tracts within each hemisphere, and smaller white-matter tracts between hemispheres: “Since the bulk of interhemispheric cortical communication relies on information transfer via the corpus callosum, these larger brains with their disproportionately smaller corpus callosum sizes may experience greater than normal constraints on interhemispheric transfer of information .... This possible disproportionate increase in intrahemispheric connections, along with a bottleneck in interhemispheric linkages, should further increase the likelihood of functional lateralization and anatomical asymmetry.”[10]


Abnormal asymmetry of brain hemispheres in children with autism is most obvious in delayed speech or loss of speech at an early age. Proponents of the “left-hemisphere hypothesis” of autism note the left hemisphere develops later in gestation, so vulnerable longer to harmful prenatal influences.[11,12] Children with classic autism are often highly emotional, with little impulse control; also musical and artistic, clearly right-hemisphere attributes. Children with the autistic disorder Asperger syndrome, by contrast, speak without delay, and often have excellent left-hemisphere verbal and cognitive skills.


Functional magnetic resonance imaging (fMRI) detected a pervasive rightward shift in hemispheric laterality in visual, auditory, motor, language, executive, and attentional networks in autistic children and adolescents.[13] Eyler and colleagues used fMRI to study lateralized responses to spoken language in sleeping 2–3-year-olds later diagnosed with autism. They found unusually weak responses in left temporal cortex, unusually strong responses in right temporal cortex: “This abnormal lateralization is consistent with studies of older autistic children and adults.”[14]


The perceptive study and explanation by Just and colleagues of language comprehension in high-functioning autism (HFA) speaks for itself: “One of the enigmas of autism in high-functioning individuals is the juxtaposition of some domains of preserved or even enhanced cognitive function, coupled with domains of deficit. In particular, previous behavioural studies of the processing of language in high-functioning autistic individuals have reported a preserved or even enhanced ability in the narrower-scope task of reading individual words, coupled with a deficit in the broader-scope task of processing grammatically complex verbal instructions ... thus epitomizing in microcosm the enigma of autism.... The autistic participants showed significantly more activation in LSTG [Broca’s area] and significantly less activation in LIFG [Wernicke’s area] .... A plausible interpretation of this finding is that ... autistic participants engage in more extensive processing of the meanings of the individual words that comprise a sentence, manifested as more LSTG .... At the same time, the autistic participants showed less activation in LIFG than the control group. LIFG ... is associated with semantic, syntactic and working memory processes, all of which serve to integrate the meanings of individual words into a coherent conceptual and syntactic structure. The reduced activation in this region is consistent with the finding that high functioning autistic participants are impaired in their ability to process the meaning of complex sentences.... We propose that autism is a cognitive and neurobiological disorder marked and caused by underfunctioning integrative circuitry that results in a deficit of integration of information at the neural and cognitive levels.”[15]


Fiebelkorn and colleagues: “Years of research have demonstrated hemispheric specialization in the processing of local and global stimulus properties, with the left hemisphere specialized toward scrutinizing constituent features and the right hemisphere toward grouping features into whole objects.... Here, we combine these previous observations with our own data to introduce a model where isolation of the cerebral hemispheres in ASD leads to functional separation of local (left hemisphere) and global (right hemisphere) processing.... The bias toward local stimulus properties (or constituent features) in ASD might therefore result from both under-connectivity among anatomically separated cortical regions and over-connectivity within local cortical regions.”[16] [my emphases] Sounds like too little interhemispheric white matter and too much intrahemispheric white matter, as Herbert et al. reported.[10]


Diffusion tensor imaging detected decreased fractional anisotropy in white matter connecting brain regions implicated in social behavior in older male children and adolescents with ASD.[17] Nordahl and colleagues noted: “In older children, adolescents and adults with ASD, the corpus callosum is consistently reported to be smaller, with decreased fractional anisotropy and reduced interhemispheric functional connectivity.”[18] [my emphasis] These are critical clues, because anisotropy – parallel layers of myelin re-straining perpendicular water diffusion – increases as myelin sheaths mature. Beaulieu: “[S]tudies of cerebral white matter development in human neonates and infants in vivo have shown, in general, a decrease in the mean diffusivity and an increase in the degree of anisotropy with maturation.”[19]


Monin and colleagues recently reported glutathione is necessary to mature myelin in children. “We found that GSH levels measured in the medial prefrontal cortex are positively associated with white matter integrity in the cingulum bundle of young healthy subjects and early psychosis patients.... Taken all together, these data suggest the presence of a critical developmental period during which a proper redox regulation and GSH levels are required for myelination .... This also suggests that there are several critical periods during which environmental risk factors could impact the normal development of myelin. Indeed, transient changes in GSH levels induced by environmental insults during pre-, peri- and postnatal periods may have an impact on oligodendrocyte maturation, consequently affecting later structural connectivity.”[20] [my emphasis]


LOW BRAIN BLOOD FLOW DESPITE HYPEREXCITABILITY


Autistic children are often hyperactive and excitable, plausibly from high brain glutamate.[21] Yet curiously, their brain blood flow is usually low. Zilbovicius and colleagues detected reduced cerebral blood flow (CBF) in the frontal cortex of autistic children 3–4 years old resembling blood flow in normal children half their age. Three years later frontal perfusion was normal: “Since CBF patterns in children are related to maturational changes in brain function, these results indicate a delayed frontal maturation in childhood autism.”[22] [my emphasis]


Ohnishi and colleagues determined regional cerebral blood flow (rCBF) in relation to symptoms: “Decreases in rCBF in autistic patients ... were identified in the bilateral insula, superior temporal gyri and left prefrontal cortex.... [P]atients’ behaviours could be classified into two syndromes: (i) impairments in communication and social interaction; and (ii) an obsessive desire for sameness. Factor I ... was associated with altered cCBF in the left medial prefrontal regions, including the anterior cingulate gyrus.”[23] Meresse and colleagues found blood flow low in the superior gyrus of the left temporal lobe: “The more severe the autistic syndrome, the more rCBF is low in this region, suggesting that left superior temporal hypoperfusion is related to autistic behavior severity.”[24]


Burroni and colleagues detected global and asymmetric low brain blood flow in autistic children: “A significant difference was also observed for the right-to-left asymmetry of hemispheric perfusion between the control group and autistic patients with a right prevalence greater in autistic ... children. Our data show a significant decrease of global cerebral perfusion in autistic children ....”[25] Degirmenci and colleagues detected asymmetric hypoperfusion in autistic children (AC) and their family members: “Hypoperfusion was seen in the right posterior parietal cortex in three AC, in bilateral parietal cortex in one AC, bilateral frontal cortex in two AC, left parietal and temporal cortex in one AC, and right parietal and temporal cortex in one AC.... In parents of AC, significant hypoperfusion was noted in the right parietal and bilateral inferior frontal cortex. In siblings of AC, perfusion in the right frontal cortex, right nucleus caudate and left parietal cortex was ... decreased.[26]


Floris and colleagues: “These alterations of typically occurring asymmetries are corroborated by findings based on single photon emission computed tomography [SPECT], and positron emission tomography [PET] showing atypical or reversed cerebral blood flow in frontal language regions.... Computerized tomography scanning reveals increased cerebral blood flow in the right temporal and right parietal lobes.”[27] Herbert suggested swollen astrocytes and microglia constricting capillaries might explain brain hypoperfusion.[28]


FAILURE OF NEUROVASCULAR COUPLING


A more pragmatic explanation for low brain blood flow in these children may be failure of neurovascular coupling. When neurons fire they release molecules that dilate nearby capillaries, notably neuronal nitric oxide (NO). Attwell and colleagues: “Synaptic release of glutamate activates neuronal NMDA (N-methyl-D-aspartate) receptors, resulting in Ca2+ entry into neurons and activation of neuronal nitric oxide synthase (nNOS). This releases NO, which dilates vessels ....”[29]


Reynell and Harris discussed explanations for the apparent failure of neurovascular coupling in autism, including depletion of neuronal nitric oxide.[30] The dilemma is that nitric oxide appears high in these children, to judge from high levels of metabolites nitrite and nitrate in blood.[31] The source of this nitric oxide is thought to be inducible nitric oxide synthase (iNOS) induced in brain microglia, astrocytes, and other cells as part of the inflammatory/immune response.[32] Two constitutive forms of nitric oxide synthase continuously present in blood vessel endothelial cells (eNOS) and neurons (nNOS) normally synthesize and release endothelial and neuronal nitric oxide, respectively.


Frye and colleagues recently tested nitric oxide metabolism in ASD children using sapropterin, a synthetic form of tetrahydrobiopterin (BH4), a cofactor for NOS.[32] Confirming the successes of Naruse and colleagues, Frye et al. found sapropterin improved communicative language in these children, which they attributed to stabilization of nitric oxide metabolism. But sapropterin also stimulates release of neuronal and endothelial nitric oxide.[33] Furthermore, induced nitric oxide commonly compensates depletion of constitutive nitric oxide [34] and nitrite serves as a reservoir “pool” to regenerate nitric oxide by reduction.[35]


Most intriguing is fresh evidence that glutathione sustains release of nitric oxide. McKinley-Barnard and colleagues studied effects of combined citrulline and glutathione supplements on nitric oxide synthesis in healthy athletes: “Nitric oxide (NO) is endogenously synthesized from L-arginine and L-citrulline. Due to its effects on nitric oxide synthase (NOS), reduced glutathione (GSH) may protect against the oxidative reduction of NO.... Intracellular glutathione exists in both the oxidized disulfide form (GSSG) or in reduced (GSH) state; the ratio between GSH and GSSG is held in dynamic balance depending on many factors including the tissue of interest, intracellular demand for conjugation reactions, intracellular demand for reducing power, and extracellular demand for reducing potential. In some cell types, GSH appears to be necessary for NO synthesis and NO has been shown to be correlated with intracellular GSH. GSH stimulates total L-arginine turnover and, in the presence of GSH, NOS activity is increased. This suggests that GSH may play an important role in protection against oxidative reaction of NO, thus contributing to the sustained release of NO. Therefore, combining L-citrulline with GSH may augment the production of NO.”[36] [my emphasis]


GLUTAMINE – A CRITICAL AMINO ACID DEPLETED IN AUTISM


The low level of plasma glutamine ... is suggested as a screening test for detecting autism in children especially those with normal IQ. The decreased level has been reported before in all children with autism. Ghanizadeh 2013 [37]


Glutamine is normally the most abundant amino acid (AA) in blood, but usually low in plasma of ASD children and often low in their brain [38] – although Pangborn reported some with high plasma glutamine from ammonia.[39] He noted high ammonia in autistic children was first detected in the early 1980s [personal communication 2010] – also more recently [40,41]. Wakefield and colleagues [42] proposed the diseased intestines of ASD children generate more ammonia than their impaired liver can clear at first pass, which enters the brain readily as ammonia gas, less readily as ammonium ion (NH4+).


Ammonia (NH3) is a consistent byproduct of amino acid metabolism (amino group: NH2) and protein degradation. Ammonia generated in the large and small intestine is carried to the liver via the portal vein for arginine-dependent conversion to urea, excreted in urine. Ammonia in the brain is trapped by astrocytes that combine it with excitatory amino acid transmitter glutamate (via enzyme glutamine synthetase) to form nontoxic glutamine – released to neurons to reform glutamate, and released into the bloodstream.[43]


Although highly toxic to the brain, Souba pointed out ammonia is “an essential nutrient and a key component of all proteins, nucleic acids, and amino acids … [and] a vital and major source of interorgan nitrogen transfer.”[43] This transfer usually begins with glutamine synthetase in tissues that generate (and trap) much ammonia (notably skeletal muscles, brain, and lungs). Glutamine safely carries two molecules of ammonia to the intestines for conversion to urea by the liver – and nourishes many cells and tissues along the way. Glutamine is alternative fuel in brain neurons and astrocytes, especially during hypoglycemia [44], primary fuel in rapidly replicating cells, e.g. blood vessel endothelial cells and intestinal entero-cytes.[45,46], and a primary brain osmolyte. Children with high brain glutamine from urea cycle disorders or propionic acidemia rarely show autistic behavior.[47,48] High blood/brain ammonia in ASD children without high blood/brain glutamine is paradoxical (glutamine paradox).


Hong and colleagues noted glutamine often provides glutamate to synthesize glutathione because glutamine enters cells more freely. They found glutamine ameliorated acetaminophen toxicity in rats: “Acetamino-phen toxicity causes hepatic GSH depletion and hepatic necrosis.... The authors conclude that glutamine-supplemented nutrition preserves hepatic glutathione, protects the liver, and improves survival during acetaminophen toxicity.”[49]


GLUTAMINE = CITRULLINE = ARGININE = NITRIC OXIDE


Glutamine is also critical in ASD as immediate precursor of the free (nonprotein) AA citrulline – precursor of the protein AA arginine, only substrate for primary vasodilator nitric oxide. Most ingested arginine is taken up by the liver; citrulline bypasses the liver and forms arginine in the kidneys, increasing systemic arginine.[50] Deutz observed that normally about 30% of intestinal glutamine becomes citrulline which the kidneys convert to arginine in one pass of blood through the circulation.[personal communication 2014] Deutz also noted: “About 80–90% of the citrulline is derived from the gut glutamine to citrulline conversion. Therefore, whole body citrulline production is related to the quantity of gut glutamine conversion to citrulline, and is most likely influenced by the amount of active gut tissue.”[51] Does intestinal inflammation in ASD children limit conversion of glutamine to citrulline?


Autism Research Institute (ARI) practitioners commonly give ASD children and adults oral glutamine to heal their intestines – from 250mg to 8g/day – but only two of ten reported improved behavior.[38] Verzella (MD) gives 5–7g/day of glutamine after cleansing their intestines of pathogens like bacteria and yeast: “Multifactorial and multisystemic is the condition, so that the improvement has different aspects in different children. Most common: sedation, less stereotypes, better sleep, more concentration.”[personal communication 2013] Pangborn recommended natural substances thyme, oregano, and Goldenseal to cleanse intestines.[52] Practitioners reported increased excitability from glutamine in a few children (one reported seiz-ures) – plausible because some glutamine breaks down to glutamate and ammonia (and perhaps other toxins) in the intestines.


BRAIN SEROTONIN ELEVATED BY GLUTAMINE AND PROLONGED BY nNOS


Serotonin (5-HT) is an unusual neurotransmitter because inhibits distally as well as locally. Inactivation of serotonin at synapses requires reuptake by the serotonin transporter (SERT) – why uptake inhibitors are calming. High brain serotonin triggers release of GABA, the brain’s primary inhibitory trans-mitter.[12] Reynell and Harris: “[G]lobal serotonin synthesis is abnormally low in children with autism but, in adolescence, it increases gradually to 1.5 times the level in adult controls.... Because serotonin is thought to produce a basal constriction of blood vessels, either a decrease or an increase in serotonergic activity could change vessel tone, and thus alter the vessel response to the vasodilators released by a given amount of neuronal activity.[30] [my emphasis] Is brain serotonin low in ASD because neuronal nitric oxide is low?


Zürcher and colleagues recently reviewed PET and SPECT studies of ASD, concluding: “The majority of ... studies reported decreases in 5-HT2a receptor and SERT compared to controls in various brain regions including the cingulate cortex, the medial prefrontal cortex, thalamus, temporal and parietal lobes ....”[53] Girgis and colleagues used PET to measure the brain serotonin receptor and SERT in Asperger adults: “5-HT2A receptor availability was numerically, but not statistically, greater in the Asperger’s Disorder group in every ROI [region of interest].[54]


Albrecht and Jones noted glutamine accumulation in the brain stimulates entry of serotonin’s precursor tryptophan and other neutral amino acids (NAA): “In particular, there is strong evidence for enhanced Gln/tryptophan exchange across the BBB in acute and chronic liver failure.[55] They cited evidence high intracellular concentrations of glutamine in brain microvessels stimulated uptake of NAA. Is brain serotonin low in ASD because brain glutamine is low?


Chanrion and colleagues detected reciprocal interaction between the serotonin transporter and neuronal nitric oxide synthase: “The present study ... demonstrates that SERT-mediated 5-HT uptake enhances the enzymatic activity of nNOS in cells coexpressing SERT and nNOS, revealing a reciprocal functional interaction between these protein partners.... Interestingly, NO production induced by 5-HT uptake was not mediated by an influx of Ca2+.... According to the present findings, increased nNOS levels would lead to intracellular sequestration of SERT, thereby preventing excessive 5-HT uptake and enhancing 5-HT neurotransmission.... A loss of the inhibitory influence of nNOS on the activity of SERT ... may conceivably be involved in the pathogenesis of psychiatric disorders, including depressive states and enhanced aggressiveness and impulsivity ....”[56]


Garthwaite commented on this study: “[T]he results of Chanrion et al. raise a number of important questions about the role of nNOS in the regulation of 5-HT transmission. 5-HT neurons appear to fire mainly in a regular slow manner in vivo. This firing pattern should lead to a tonic level of extracellular 5-HT within the brain regions innervated by the axons, the concentration being set by the transporter activity. By diminishing the amount of SERT in the cell membrane, binding of nNOS should adjust extracellular 5-HT upward. In effect, nNOS would be acting like an endo-genous antidepressant.[57] [my emphasis]


DISCUSSION – ENIGMAS OF AUTISM SEEN AS GLUTATHIONE DEPLETION


Thomas wrote of his single key mechanism: “This generalization is harder to prove, and arguable – it is more like a strong hunch than a scientific assertion – but I believe that the record thus far tends to support it. The most complicated, multicell, multitissue, and multiorgan diseases I know of are tertiary syphilis, chronic tuberculosis, and pernicious anemia. In each, there are at least five major organs and tissues involved, and each appears to be affected by a variety of environmental influences. Before they came under scientific appraisal each was thought to be what we now call a ‘multifactorial’ disease, far too complex to allow for any single causative mechanism. Yet when all the necessary facts were in, it was clear that by simply switching off one thing–the spirochete, the tubercle bacillus, or a single vitamin deficiency–the whole array of disordered and seemingly unrelated pathologic mechanisms could be switched off at once.”[1]


One could readily argue “whole array of disordered and seemingly unrelated pathologic mechanisms” aptly describes autism pathology. Various major enigmas have been recognized for years yet little understood – including their relation to each other:

  1. (1)abnormal/impaired white-matter connections

    within/between brain hemispheres

  1. (2)extreme male brainpronounced androgenic characteristics

    (e.g. enlarged intrahemispheric WM, aggression)

(3) hyperexcitability yet low brain blood flow

  1. (4)high blood/brain ammonia yet low blood/brain glutamine

    (glutamine paradox)

(5) altered intestinal microbiome from antibiotics

  1. (6)low sulfur amino acids and metabolites,

    e.g. methionine, cysteine, taurine, glutathione

(7) high blood serotonin yet low brain serotonin

(8) dramatic relief of autistic behavior by infectious fever

(9) Asperger children so different from autistic children – yet so alike

  1. (10) our autism epidemic that began about 1980 and now afflicts

    1 in 45 children [58] mostly boys


Certain associations are obvious at first glance. When glutamine is low, citrulline is probably low, thus arginine and nitric oxide, primary brain vasodilator. But why is blood/brain glutamine usually low when blood/brain ammonia is often high (glutamine paradox)? For one, magnesium and vitamin B6 – cofactors for glutamine synthetase [44,59] – are consistently low in these children.[60] Much glutamine may be needed to feed (and heal) intestinal enterocytes. A similar glutamine paradox occurs in the inborn metabolic disorder propionic acidemia [61]; one explanation is that glutamine synthe-tase requires ATP.[62] Low concentrations of plasma ATP/precursors were detected in ASD children.[59] More brain glutamate than glutamine may explain hyperexcitability.[21] Glutamine depletion depletes glutathione be-cause glutamine often provides glutamate to synthesize glutathione [49]; glutathione depletion depletes glutamine for the same reason.


High blood ammonia from a microbiome altered by antibiotics readily enters the brain, which has no urea cycle but converts ammonia to glutamate and then glutamine. Chronic ammonia accumulation shifts brain metabolism and blood flow from cortical to subcortical structures.[63]


IS MYELIN IMMATURE?


Dobbing and Sands studied changes in brain weight, cholesterol and water during the normal growth spurt in human brains from 10 weeks gestation to seven years old. The human brain growth spurt begins at midgestation, although five-sixths of the growth is postnatal, they concluded, which may extend into the second year and beyond. The growth spurt takes place in stages: first, multiplication of neurons, then multiplication of glia (predom-inantly oligodendroglia to generate myelin sheaths), then deposition of lipids in the sheaths. The decline in water content of the brain reciprocally parallels the increase in lipids. They described two components of normal myelination – longitudinal growth of sheaths as axons lengthen, and maturational growth as lipids are deposited, water is displaced, and sheaths thicken around axons: “The process of myelination may have two overlapping components: a ‘maturational’ one consisting of gradual thickening of the laminated sheaths around existing lengths of axon and a ‘growth’ process consisting of myelination concurrent with growth in axonal length.... Thus, in this general sense, myelination as a developmental, maturational process will be more reflected by increased lipid concentration; and the concurrent and later myelination of growth will be reflected by increased total amount.”[64] [my emphasis]


To judge from high levels of 5α-reductase in myelin membranes [65] longitudinal growth of myelin sheaths depends on testosterone becoming dihydrotestosterone. Maturational growth of myelin sheaths appears to depend on estrogens depositing lipids that displace water. Because myelin sheaths compact as water is displaced, the proportion of lipids to water may be a better measure of maturity than the thickness of sheaths, Dobbing and Sands concluded. Hendry and colleagues, using transverse relaxation time imaging, found brain white matter in autistic boys contained more water than normal globally and regionally.[66] Decreased anisotropy [17,18] also argues water distribution in myelin sheaths is immature.


Deoni and colleagues [67] investigated white matter in adult males with autism using multi-component relaxation analysis: “Individuals with autism show evidence of altered structural and functional ‘connectivity’ across large-scale brain systems.... A recurrent finding ... is that of increased overall brain volume, which has been suggested to result from differences in early brain development and may be caused by differential effects driving white matter (WM) to be larger in the autistic brain. Specifically, those brain regions exhibiting the greatest volume increases correspond to later and prolonged myelinating pathways. Further, these WM volume differences persist into young adulthood .... Myelin plays a critical role in establishing and maintaining congruent brain communication, and contributes substan-tively to WM volume. Histological evidence for abnormal myelination, myelin content, or myelin structure in the pathogenesis of ASD is derived from ex vivo post-mortem studies showing altered myelin composition with delayed compaction in the sheaths .... [my emphasis]


“Currently, the most robust approach to quantitatively estimating myelin content is through multi-component relaxation analysis (MCR). Within brain tissue, MCR aims to decompose the measured MR signal into contributions from two anatomically distinct water compartments: the slow relaxing intra- and extra-axonal water; and the faster relaxing water trapped between the myelin bilayers.... Our results show that individuals with autism have widespread MWF [myelin water fraction] reductions in brain regions previously implicated in ASD.... MWF is believed to be more specific to changes in lipid myelin content.... Our study has revealed widespread myelin alteration through MWF reduction in the brain of adults with autism.... Altered myelin, therefore, is likely associated with reduced connectivity. Our results are consistent with the current hypothesis that neural disconnectivity underpins ASD, as supported by structural imaging studies; functional imaging studies; and electroencephalography investigations. In each of these prior studies, abnormal connectivity was observed in frontal and temporal regions, consistent with our findings of lower myelin content in these areas.”[my emphases] Croteau-Chonka and colleagues: “Quantification of the myelin-associated signal, the MWF, is a useful metric for tracking white matter maturation and its relationship to cognitive development in the developing brain.”[68] [my emphasis] Deoni confirmed that “decreased MWF is associated with lower myelin content and immaturity.”[personal commu-nication 2016]


ESTROGENS MATURE MYELIN


Guyton and Hall: “[T]oward the end of pregnancy the daily production of placental estrogens increases to about 30 times the mother’s normal level of production.”[69] This is when fetal fat accumulation and brain development are most rapid.[70] Elitt and Rosenberg: “During pregnancy, the placenta synthesizes estrogens, including estradiol, estrone and estriol, from precursors of fetal and maternal origin ultimately producing levels that are 100 fold higher than in non-pregnant women.... Estrogen receptors are expressed on the oligodendrocyte plasma membrane and within the myelin sheath and estradiol induces morphological changes and increases myelin basic protein expression”[71] The brain is 60% lipid [72], much in myelin sheaths.[73] Geschwind and Galaburda: “At the time in the early postnatal period in which one finds the maximum ... estradiol receptors in the rodent brain and peak estradiol levels there is also intense sprouting of neurites and myelination of axons. [74] Prayer and colleagues used MRI sensitive to anisotropy to measure white-matter maturation in newborn rats: “[E]strogen-treated animals showed accelerated, and testosterone-treated animals delayed, maturation ....”[75]


Jamnadass and colleagues compared autistic traits in young adults to concentrations of testosterone, androstenedione, DHEA, and estrogens in umbilical cord blood sampled at their birth. Expecting to find a high ratio of androgens to estrogens, they were surprised to find no association with androgens or the androgen/estrogen ratio: “The current study provides no evidence to suggest that perinatal androgens, estrogens, or the A to E ratio sampled via cord blood predicts autistic-like traits in young adulthood.... It may be that different organizational effects occur early in gestation to those of late gestation, and that these are not evident in the circulating steroid profile at delivery.”[76]


Indeed, Swaab noted prenatal organizational effects of testosterone are greatest during the second trimester: “The early development of boys shows two periods during which the testosterone levels are high. The first peak occurs during mid-pregnancy. Testosterone levels peak in the fetal serum between weeks 12 and 18 of pregnancy. In weeks 34–41 of pregnancy the testosterone levels of boys are 10 times higher than those of girls.The second peak takes place in the first 3 months after birth.”[77] Auyeung and colleagues, however, did measure second-trimester fetal testosterone and estradiol concentrations in amniotic fluid. They concluded fetal testosterone was associated with autistic traits at 18–24 months – but fetal estrogen was not.[78] The most critical estrogen concentrations, however, are probably just before birth – yet Jamnadass et al. concluded autistic traits were not associated with the androgen/estrogen ratio at birth.


Lutchmaya and colleagues, however, found fetal testosterone (FT) high and fetal estradiol (FE) low in second-trimester amniotic fluid in children with low 2D:4D ratios at two years old.[79] Furthermore, autistic brains are smaller at birth [80] and have less myelin even in adulthood.[67] Because estrogens induce rapid brain growth and myelination just before birth in both genders, is it likely they are adequate in these children? But then why does the rapid brain overgrowth after birth favor testosterone-dependent white matter?


Why is brain myelin immature? The most obvious explanation is DHEAS/estrogen deficiencies.[3] Prenatal DHEAS deficiency limits placental estro-gens; postnatal DHEAS/estrogen deficiencies further delay/prevent matura-tion of myelin. Other factors may be inhibition by mercury and other metals of the enzyme aromatase [81], which converts androgens to estrogen, and the zinc enzyme carbonic anhydrase, which dehydrates myelin sheaths throughout life.[82]



EXTREME MALE BRAIN


Asperger observed that the autistic personality is an extreme variant of normal male intelligence.[83] Pursuing the implications, Baron-Cohen and colleagues presented six clues that fetal testosterone (FT) is high in children with autism [84]: (a) FT induces ring fingers longer than index fingers (low 2D:4D ratio); (b) girls with high levels of adrenal testosterone before birth have more autistic traits than their sisters; (c) as FT increases, autistic behaviors increase; (d) hypermasculinization; (e) precocious puberty in boys; (f) elevated serotonin. Excessive testosterone before birth, they explained, exaggerates the normal tendency of male brains to be larger than female brains – a difference caused by white-matter tracts within cerebral hemispheres enlarging at the expense of white-matter tracts connecting the hemispheres, as Herbert et al. detected in these children soon after birth.[10]


Despite extensive observations corroborating this extreme male brain theory of autism, however, one anomaly remains unexplained. Konner [85] noted the Tinbergens (early pioneers of autism research) detected signs of fear in these children in social situations, and “reasoned that exceptionally timid children might be at risk for developing the disorder if they grew up in a sufficiently threatening – or perhaps for them, merely a very intrusive – social environment.” Konner noted infants do not fear strangers nor separation from mother until about four to six months old, when the nerve tracts of the limbic system myelinate rapidly.


Markram and colleagues too observed that children with autism are often extremely anxious. They proposed an alternative theory of autism – intense world syndrome: “The current version of the amygdala theory of autism assumes a hypofunctional amygdala, which leads to lack or inappropriate-ness of social behavior in autism. In this view, autists fail to assign emotional significance to their environment and for this reason are not interested in others, do not attend to faces, and fail to engage in normal social interaction. However, based on the result in the VPA [valproic acid] model of autism and observations obtained in autistic humans, we propose that this view may be not correct and that quite to the contrary, the amygdala in the autistic individual may be hyper-reactive which leads to rapid excessive responses to socio-emotional stimuli. In this view, the autistic person would be overwhelmed with emotional significance and salience. As a consequence, the subject would want to avoid this emotional overload and would have to withdraw from situations, such as social encounters, which are rich in complex stimuli.’[86] [my emphases]


Because testosterone allays fear [85], why are autistic children anxious, even timid, around others? One explanation may be prenatal stress, which elevates maternal and fetal adrenal androstenedione, a weak androgen precursor of testosterone (and estrogen) that suppresses testosterone release from the fetal testes. Ward concluded: “[I]t appears that stress causes an increase in the weak adrenal androgen, androstenedione, from the maternal or fetal adrenal cortices, or from both, and a concurrent decrease in the potent gonadal androgen, testosterone.”[86] This happens because release of androstenedione and testosterone from the testes is triggered by luteinizing hormone from the pituitary – which high adrenal androstenedione suppresses by negative feedback. Androstenedione becomes testosterone in peripheral tissues including the brain, or becomes estrone then estradiol, the primary estrogen.[90] Jacklin and colleagues assessed timidity in infants by their reaction to fear-provoking toys. Low timidity in boys was associated with higher levels of testosterone at birth – but not androstenedione.[91] Taylor and colleagues studied effects of androstenedione (4-A) and DHEA on sexual behavior in male rats: “These results suggest that DHEA and 4-A are not merely precursors of sex hormones, and provide support for these steroids influencing the brain and behavior in a unique fashion that is dissimilar from the effects of testosterone on male sexual behavior.”[90]


Do prenatal stress, sulfate/glutathione depletion, and inhibition of aromatase  masculinize the brain of a male fetus via weak androgens – and myelinate via inadequate estrogens? Is a brain differentiated by androstenedione and DHEA more timid – and dysconnected – than a brain differentiated by testosterone and matured by estradiol? Does brain overgrowth after birth favor testosterone-dependent structures because prenatal testosterone was low?


ASPERGER SYNDROME vs, HIGH-FUNCTIONING AUTISM


Although the current Diagnostic and statistical manual of mental disorders (DSM-V) no longer distinguishes Asperger syndrome from other forms of autism, their differences have been much studied.[91-93] Most obvious is that Asperger children speak without delay; thus often diagnosed much later, when their social discomfort becomes troubling. Asperger children and adults appear unemotional and detached [94] without empathy [91] – yet that may mask an intense world.[95] Autistic children and adults, by contrast, are often highly emotional, with little impulse control – yet also lack empathy. These and other observations argue that children and adults with autism – including high-functioning autism (HFA) – are largely right-hemisphere lateralized, while children and adults with Asperger syndrome (AS) are largely left-hemisphere lateralized. Ozonoff and Griffith, however, conclud-ed: “The proposed visual–spatial deficits of AS individuals, as well as their difficulties producing and interpreting facial expressions, gestures, and prosody, have led to the hypothesis that the right hemisphere is dysfunctional in AS. Conversely, lateralization work has suggested that the left hemisphere is damaged in autism. This suggests a very appealing hypothesis, namely that AS and HFA result from different patterns of unilateral brain dysfunction. Unfortunately, however, this right-hemisphere–left-hemisphere dichotomy does not account for all data. First, it has long been evident that classically autistic children exhibit deficits typically considered right hemisphere in origin …. Second, recent studies have documented left-hemisphere damage in AS.”[96]


McAlonan and colleagues found Asperger children and high-functioning autistic children both had less gray matter than normal, but in different regions of the brain.[99] Next they measured white matter: “White-matter volumes around the basal ganglia were higher in the HFA group than ASP and higher in both autism groups than controls. Compared with controls, children with HFA had less frontal and corpus callosal white matter in the left hemisphere; those with ASP had less frontal and corpus callosal white matter in the right hemisphere with more white matter in the left parietal lobe.... HFA involved mainly left hemisphere white-matter systems; ASP affected predominantly right hemisphere white-matter systems.”[98] [my emphasis] Yang and colleagues used SPECT to measure rCBF in children with Asperger syndrome or high- functioning autism: “The decrease in rCBF in ASD is a global event, which involves the bilateral frontal, temporal, limbic system and basal ganglias. Asymmetry of hemispheric hypoperfusion was more obvious in the Asperger group than the autism group, which indicates a different neurobiological mechanism from that of autism.”[99] [my emphasis]


WHAT PROVOKED OUR AUTISM EPIDEMIC?


Navy scientist Stephen Schultz asked whether parents’ reports of their child’s regression into autism soon after receiving the measles-mumps-rubella vaccine (MMR) might implicate acetaminophen (Tylenol) given for its pain and fever. An online parent survey found that children given acetaminophen for the MMR became autistic much more often than children given ibuprofen.[100] Schultz and colleagues concluded our national autism epidemic began in 1980, when the Centers for Disease Control and Prevention (CDC) warned the American public that giving children aspirin might induce Reye’s syndrome (a rare but often fatal disorder of high ammonia) and everyone – parents, pediatricians, and hospitals – switched to acetaminophen. Schultz et al. explained that detoxifying acetaminophen in children under ten requires sulfation by the liver – consistently limited in ASD children.[101] When sulfation is impaired acetaminophen becomes a toxic metabolite that requires glutathione to detoxify. In a second study Schultz and colleagues found a close association between rapidly rising national sales of Tylenol after the CDC warning, and the rapidly rising incidence of autism in California.[102]


Biochemist Jon Pangborn of the Autism Research Institute (ARI) also implicated 1980 as the year our epidemic began, based on thousands of parents’ reports since the 1960s. Before 1980, approximately 50–60% of autistic children were abnormal from birth, and 40–50% regressed into autism at about 18 months. “Around 1980,” Pangborn reported, “all this began to change. The total frequency of occurrence doubled, doubled again, and by 1995 was approximately 10 times that of 1980. Furthermore, while the onset-at-birth type had increased 3 to 4 times, the onset-at-18-months type had skyrocketed to considerably more than 10 times its 1980 level.” Pangborn concluded that most of the autistic population now appeared to have “an acquired disease caused by something that we were not doing 20 years ago.”[39] Previc: “The incidence of autism has risen 10-fold since the early 1980s, with most of this rise not explainable by changing diagnostic criteria.”[105] Orlowski and colleagues compellingly debunked the associa-tion of aspirin with Reye’s syndrome.[103,104]


Biochemist William Shaw of Great Plains Laboratory recently confirmed that acetaminophen depletes glutathione in ASD: “The characteristic loss of Purkinje cells in the brains of people with autism is consistent with depletion of brain glutathione due to excess acetaminophen usage, which leads to premature brain Purkinje cell death.” Shaw noted that Cuba vaccinates all their children, especially against measles [with MMR since 1986], yet their autism incidence is only 1/300th of ours in the U.S. What’s the difference, according to Shaw? Cuba prohibits over-the-counter Tylenol, and only rarely allows acetaminophen prescribed for vaccinations, because acetaminophen is limited by the U.S. embargo.[106] Biochemist Richard Deth noted that acetaminophen (like mercury) readily binds selenium-containing proteins that underlie the glutathione system.[personal communication 2010]


Epidemiologists Bauer and Kriebel reported recommendations that aceta-minophen (paracetamol in the UK) be given before and after circumcision: “These guidelines include the suggestion of a first dose ... two hours prior to the procedure, and doses every 4–6 hours for 24 hours following the procedure. Thus newborn males often receive 5–7 doses ... during the developmentally vulnerable initial days of life.” They also cited evidence that may explain more children born autistic – by the early 1980s about 42% of American women used acetaminophen during the first trimester of pregnancy: “The rate climbed to over 65% in the early 1990’s, where it has essentially remained through 2004.”[107]


Kerry Scott Lane (MD) recently reported online: “Having studied the toxicology of acetaminophen and how it depletes glutathione ... it is clear as day to me, the trigger for regressive autism is tylenol/acetaminophen/paracetamol .... With limited or near zero glutathione in the brain at time of vaccination, the body is unable to detoxify the metals in the vaccines via the metallothionein system.... I spoke before an FDA panel of 30 plus experts in 2009 explaining my theory of autism causality and acetaminophen. My talk pointed directly at the synergism between acetaminophen and gliotoxin, a fungal/yeast toxin produced by Candida and Aspergillus, which has the unfortunate side effect of binding to and depleting glutathione, like aceta-minophen.”[108-110]


ORAL ANTIBIOTICS IMPLICATED IN OUR EPIDEMIC


Parents’ reports of their child’s regression into autism soon after a course of broad-spectrum oral antibiotics – usually to treat otitis media (middle ear infection) – led Sandler and colleagues to investigate gut bacteria in these children. Oral vancomycin (antibiotic minimally absorbed) improved autistic behavior transiently but impressively.[111] Different species of intestinal bacteria have been detected in ASD children.[112] Adams and colleagues explained: “Commonly used oral antibiotics eliminate almost all of the normal gut microbiota.... Loss of normal gut flora can result in the overgrowth of pathogenic flora, which can in turn cause constipation [ammonia] and other problems.”[113] Autistic regression between 12 and 18 months was commonly associated with gastrointestinal symptoms.[114] Fallon found many autistic children under three with otitis media treated with the antibiotic amoxicillin/clavulanate (Augmentin), made with ammonia.[115] ASD children had more ear infections than typical children, and were treated with more antibiotics.[116] Greenberg and colleagues noted the association of acute otitis media, day care centers (DCC), antibiotic-resistant bacteria, and pediatric-group recommendations that high-dose amoxicillin/clavulanate was “the first therapeutic choice” for children in DCC: “The development and spread of resistant organisms are facilitated in DCCs as a result of the following: (i) large numbers of children; (ii) frequent close person-to-person contact; and (iii) a wide use of antimicrobial medications. Intensive antimicrobial usage provides the selection pressure that favors the emergence of resistant organisms, while DCCs provide an ideal environment for transmission ....”[117] Ball noted Augmentin was launched in 1981 to treat “upper and lower respiratory tract infections, urinary tract infections, skin and soft tissue infections and obstetric, gynaecological and intra-abdominal infections.”[118] Another reason our epidemic began in the early 1980s?


EVALUATION – GLUTATHIONE DEPLETION


Consistent findings were evidence of a curtailment of maturation in the forebrain limbic system, abnormalities in the cerebellar circuits, and an unusual pattern of change of postnatal brain size.... It is difficult to say at what period of brain development the curtailed maturation arose. However, the presence of malformations in the cerebral cortex indicates a pathology dating to the period of fetal development.   Kemper & Bauman 2002 [119]


Adams et al. found plasma arginine normal in ASD children.[59] Kuwabara and colleagues found high-functioning adult males with autism had high plasma arginine and taurine.[120] Cynober and colleagues, however, noted plasma concentrations of amino acids are hard to interpret, they are so small compared to intracellular and muscle concentrations.[121] Pangborn found urinary arginine low in 25% of 61 ASD children [52], urinary taurine low or wasted in 62%.[39,52] Geier and Geier found significantly lower plasma taurine, cysteine, and sulfate relative to controls.[122]


How can neuronal nitric oxide be low in ASD when blood nitrite is high? About 70% of nitrite normally derives from endothelial nitric oxide [123] – and induced nitric oxide appears high in these children [31,32] – so neuronal nitric oxide may well be low. Furthermore, nitrite reduces to nitric oxide, which glutathione depletion may limit. Neurovascular coupling also implies coordination between neurons firing and capillaries dilating – coordination nitrite may not provide. Some fear NOS produces reactive oxygen species (ROS) superoxide and peroxynitrite, but that happens when arginine [or BH4] is deficient, not adequate.[124]


Reynell and Harris considered other explanations for failure of neurovascular coupling in ASD: “Decreased inhibition is observed in the brains of autistic patients: the expression of the enzymes that synthesise the inhibitory neurotransmitter, GABA, and the expression of the receptors on which GABA acts are both reduced …. A further complication comes from the recent suggestion that GABA released from interneurons may act on glial cells or directly on microvessels to increase blood flow.”[30]


Does glutathione depletion after birth continue to limit estrogens required to mature myelin? DHEAS and DHEA both aromatize to estrogens outside the placenta, but DHEAS is normally many times more abundant (and longer-lasting) in blood than DHEA [125] – yet was low in ASD children [3] and adults.[126] 


Why hasn’t oral glutamine improved behavior in more ASD children? Gut tissue able to convert glutamine to citrulline may be lacking. Not all intestines may have been cleansed. Most gut glutamine in ASD children may be needed to feed – and heal – enterocytes.


Hindfelt noted an early manifestation of chronic ammonia accumulation in the brain is a “frontal lobe syndrome” – loss of cortical executive functions.[127] How common is chronic high blood ammonia in ASD? Shaw (Great Plains Laboratory) reported that about 250,000 tests of ASD children and adults found the organic acid orotate (orotic acid) in urine – a marker of chronic high blood ammonia – often slightly elevated: “The orotate is commonly abnormal in autism. The degree of abnormality is slight, usually 5–10% above reference range. I would estimate that perhaps 10–15% of cases of autism have this slight abnormality.”[personal communication 2014] Woeller (DO) concluded: “I think the vast majority of kids on the spectrum have ammonia issues of some sort because first, their guts are full of yeast and bacteria .... Second, the yeast and bacterial toxins can interfere with Kreb Cycle function, and third the methylation and urea cycles are impacted by infectious toxins .... I rarely see orotic acid high on the Organic Acids Test. Perhaps it takes a lot of imbalances to push this particular biochemical pathway to get it to appear elevated. When I do see it high it is usually a mild elevation.”[personal communication 2014] [128]


Ammonia may also be high in children weaned before six months old [129] because solid food has much more protein than breast milk. Axelsson: “When other foods are introduced during the weaning period the protein intake increases remarkably to 3–4 g/kg/day in spite of the fact that the protein requirement is decreasing.... The metabolic effects with high levels of urea in serum and urine, and the high levels of many amino acids may exceed the capacity of the hepatic and renal systems to metabolize and excrete the excess of nitrogen.”[130] Many infant formulas contain more protein than breast milk.


White-matter differences between Asperger children and high-functioning autistic children argue their similar behavior depends on reduced frontal and corpus callosal WM, while their unique behavior depends on which hemisphere has less frontal and corpus callosal WM. Yet the same argument can be made for their gray matter and blood flow differences.


One insightful explanation of hemispheric differences by Geschwind and Galaburda noted the left hemisphere is ‘wired’ to process information serially, while the right hemisphere is wired in parallel.[77] Current in series circuits flows through one component at a time, in sequence – useful for close analysis of information and sequential operations. Any faulty component impairs flow to downstream components. Sequential flow emphasizes process. Current in parallel circuits takes multiple pathways simultaneously at identical voltage – synthesizing information into gestalts.


If glutathione synthesis depends significantly on plasma glutamine, glutamine depletion in ASD children may deplete glutathione as effectively as glutathione depletion depletes glutamine. Is high ammonia as decisive as glutathione depletion? When ammonia is high and glutamine low, ammonia and glutathione depletion may synergize. Yet glutathione depletion explains more neuropathology. [Fig.1. SYNOPSIS].




























FIG. 1.  SYNOPSIS. HOW GLUTATHIONE DEPLETION PROVOKES AUTISM


Glutathione (GSH) – tripeptide of cysteine, glycine, glutamate – is primary antitoxin/antioxidant in all human cells and especially the liver. GSH neutralizes heavy metals (e.g. mercury/aluminum in vaccines, lead in air/water) and other toxins (notably acetaminophen/paracetamol/Tylenol), oxidants (e.g. hydrogen peroxide), and carcinogens. GSH is also cofactor for sulfation of fetal adrenal androgen dehydroepiandrosterone (DHEA) to DHEASmajor precursor of placental estrogens (EST). Because estrogens mature myelin sheaths, prenatal/postnatal DHEAS/estrogen deficiencies explain dysconnected brain hemispheres, anomalous brain asymmetries, and the extreme male brain. Because GSH sustains release of primary brain vasodilator nitric oxide (NO), GSH depletion limits brain blood flow. Because glutamine (GLN) enters cells more freely, glutamine often provides glutamate (GLU) to synthesize glutathione – depleting glutamine as precursor of citrulline (CIT) then arginine (ARG), only substrate for NO, and required to detoxify ammonia (NH3) in the liver. Glutamine is also primary fuel in rapidly replicating cells, alternate fuel in brain neurons and astrocytes. Blood and brain ammonia are often high in these children from early weaning (too much protein), constipation, and a gut microbiome altered by oral antibiotics – yet blood and brain glutamine are usually low (glutamine paradox). Explanations are (a) lack of magnesium (Mg) and B6 as cofactors for glutamine synthetase (GS), which turns ammonia + glutamate to glutamine, (b) lack of taurine (TAU) to assist this conversion, (c) low ATP, and (d) much glutamine needed to feed (and heal) intestinal enterocytes. Low brain glutamine limits entry of tryptophan (TRP), precursor of serotonin (5HT). Glutamine depletion depletes glutathione because glutamine often provides glutamate to synthesize glutathione; glutathione depletion depletes glutamine for the same reason. Do free taurine and glutamine – primary organic brain osmolytes – released by fever relieve autistic behavior dramatically by dehy-drating immature brain myelin – or compensating it?


EVALUATION – OTHER THEORIES


ARE GENES DECISIVE?


[Y]ou cannot have a genetic epidemic . . . . Martha Herbert [131]


Previc [103] interpreted the genetic evidence in autistic disorders in light of epigenetic evidence: “While behavioral genetics has flourished during the past several decades and the number of genes linked to various normal and abnormal behavioral traits has multiplied, standard theories of genetic transmission have increasingly been challenged. A myriad of nongenetic factors ... termed ‘epigenetic’ have been shown to substantially modify or override genetic inheritance. Many if not most epigenetic effects occur prenatally and include maternal nutritional status, maternal exposure to drugs, maternal fever, and maternal psychosocial stress. The importance of the prenatal environment to brain development has even challenged the basic assumptions of behavioral genetics. The concordance rate between monozygotic [single egg] versus same-sexed dizygotic [two egg] twins and other same-sexed siblings has long been considered the single most important measure of genetic influence. It is assumed that monozygotic and dizygotic twins share the same prenatal environment and that the difference between their concordance rates is genetic; whereas, a difference in concordance rates for dizygotic versus regular siblings is caused by the greater shared prenatal and/or postnatal environments of the former. Using these methods, the genetic influence has been estimated at >50% in the case of intelligence and schizophrenia and 90% in the case of autism, although no gene or set of genes has ever been discovered that is specific to autism.


“In actuality, the prenatal environments of monozygotic and dizygotic twins are not identical, and the importance of shared zygosity appears exag-gerated.... [T]wo-thirds of monozygotic twins share the same placenta (chorion), whereas dizygotic twins never do so. Monochorionic twins are to varying degrees more similar than dichorionic twins on a host of behavioral and physiological measures including intelligence, birthweight, and risk for psychopathology.... Hence, it may be concluded that the chorion effect is greatest when prenatal influences are strongest. The increasing acceptance of the role of prenatal influences on genetically regulated brain development and the corresponding challenge to genetic estimates by the placental findings together suggest that prenatal factors should be considered more important than previously suspected in the etiology of many disorders”[103]


IS PRENATAL DOPAMINE DECISIVE?


Previc’s theory of dopamine hyperactivity emphasized our national epidemic is a critical clue: “The incidence of autism has risen 10-fold since the early 1980s, with most of this rise not explainable by changing diagnostic criteria. The rise in autism is paradoxical in that autism is considered to be one of the most genetically determined of the major neurodevelopmental disorders and should accordingly either be stable or even declining. Because a variety of epigenetic influences, particularly those occurring during the prenatal period, can override or masquerade as genetic influences, these should be considered as prime contributors to the recent increase of autism. Prenatal influences on dopamine activity are especially well-documented, including the effects of maternal psychosocial stress, maternal fever, maternal genetic and hormonal status, use of certain medications, urban birth, and fetal hypoxia. All of these factors have been implicated in the genesis of autism, which is characterized by a ‘hyperdopaminergic’ state based on evidence from monkey and human behavioral studies, pharmacological studies in humans, and a left-hemispheric predominance of both dopamine and autistic-like symptoms. Chronically high maternal levels of dopamine caused by the pressures of increasingly urbanized societies and by changing mater-nal demographics such as increased workforce participation, educational achievement level, and age at first birth, may be especially significant epigenetic contributors to the recent autism rise.... Partly by default, then, prenatal factors must be considered paramount in explaining the rise in autism during the past 20–30 years, although which specific prenatal influences may be involved is less clear.”[103]


ARE REACTIVE OXYGEN SPECIES (ROS) DECISIVE?


Despite evidence that reactive oxygen species damage mitochondria in ASD [132], Naviaux argued ROS are protective: “ROS and oxidative changes in chronic disease are the symptoms of disease and not the cause. Indeed, transient and regular stimulation of ROS production is required for [mitochondria] …. Catalogs of the many forms of oxidative changes that are found in 100 chronic diseases provide no insight into the underlying pathogenesis of disease.”[133]


IS AUTISM REVERSIBLE?


Herbert argued autism cannot be a permanent structural disorder because infectious fever often relieves autistic behavior dramatically.[28,134] Fluid diets before surgery had similar effect [135] as does severe stress – e.g. panic[136] or a broken bone.[137] Herbert concluded autism is a “chronic dynamic encephalopathy” – an ongoing active reversible brain pathology.[28,134] Fever’s phenomenal benefit, first reported by Sullivan in 1980 [138] still tantalizes parents and practitioners [139,140] – yet its physiology and biochemistry have never been investigated.[38] Zimmerman noted 80% of parents in Curran et al. [140] reported their child improved during fever on one or more Autism Behavior Checklist categories: “In clinical care, approximately 30% of parents report that their children with ASD improved dramatically during fever ... their symptoms are so obvious the family recognize them immediately.”[personal communication 2014] Informal parent surveys concluded fever helps 30–40% of autistic children.[141]


Evidence of improvements hours before fever’s onset [137] implicate release of free glutamine and taurine from skeletal muscles.[128] Fasting (e.g. fluid diets) also releases glutamine as provisional fuel. More challenging to explain is brief dramatic relief of autistic behavior by severe stress [136,138] since ordinary stress aggravates [142]. Ordinary stress too releases free glutamine, but plasma glutamine falls because so many cells require it. Does severe stress release sufficient glutamine to tip the balance? Or is the decisive factor epinephrine? Sympathetic beta-stimulation by epinephrine moves calcium and taurine into excitable cells; intense beta-stimulation by epinephrine [e.g. fever, panic] reverses this shift.143 Yet epinephrine hardly explains improvements hours before fever – unless low-grade fever.


The simplest explanation for fever’s benefit may be that free glutamine and taurineprimary organic brain osmolytesdraw water out of immature overhydrated myelin. Bergström and colleagues biopsied muscle tissue in healthy adults, precipitated out the proteins, then measured remaining free amino acids: “The concentration gradient was especially high for taurine, glutamic acid, and glutamine.... For a normal man with a body weight of 70 kg and a muscle mass of 40% of the body weight, the total volume of intracellular muscle water is 18.2 liters and the total intracellular amino acid content in muscle is 86.5g [plus 35g taurine]. Of this total pool of free amino acids ... free glutamine constitutes 61% ....”[143]


Sixty-one percent of 86.5g of intramuscular free amino acids equals almost 53g of free glutamine in adult muscles. Deutz estimated the muscles of a child weighing 25kg (55lb) contain about 20g free glutamine [personal communication 2016]. If the ratio of taurine to glutamine in adult muscles (35g/53g) holds for children’s muscles, a 25kg child will have about 13g free taurine.


But if glutamine and taurine relieve autistic behavior by drawing water out of immature brain myelin, why do improvements in most children end when fever ends? Does water return to the brain? How does that happen?


Another possibility is that water is carried out of the brain by taurine. Frosini found fever in rabbits released taurine from brain to CSF.[145] Is taurine (and its water) released from the brain more critical to fever’s benefit than glutamine and taurine released from muscles? Do taurine and its water return to the brain when fever ends?


TAURINE IN THE BRAIN


The intracellular amino acid pool is large in both astrocytes and neurons and enriched in taurine and glutamate, whose concentrations can reach 30–40 mM. Massieu et al. [145]


[A]cute ammonia challenge appears to release taurine more readily than any other amino acid studied. Albrecht & Schousboe [146]


The nonprotein sulfur amino acid taurine serves a variety of critical functions in the body, notably inhibitory transmitter and cryogen, regulator of active intracellular calcium, magnesium complement, and major organic brain osmolyte.[147–149] Release of taurine from brain to interstitial fluid/CSF is a common response to various provocations. Foos and Wu found most brain taurine was released by ischemia, free radicals, metabolic poisons, excessive glutamate, and ammonia.[147]


As noted, Pangborn found taurine was the AA most wasted or depleted in urine of ASD children.[39] He cited evidence taurine assists conversion of glutamate to glutamine, perhaps why taurine is so effective against seizures.[52] Taurine is most vulnerable to abbreviated breastfeeding [150], dietary deficiencies of its precursors methionine and cysteine [151], impaired synthesis from lack of bioactive B6 (pyridoxal phosphate) [39,52] and preemptory requirements for sulfate and glutathione.[52] Mother’s milk is rich in taurine; cow’s milk is low after calves are weaned [150]. Schultz and colleagues found longer breastfeeding was associated with less likelihood of developing autism [152] – yet many (but not all) infant formulas have been fortified with taurine since the mid-1980s.[52,153]


Although Pangborn concluded taurine was the first (and safest) amino acid to supplement in ASD children, and essential in light of the quantity of taurine the fetus receives in placental blood, and the infant in breast milk – but noted clinical researchers recommended no more than 2g/day of taurine for these children.[52]


Does taurine released from brain to CSF by fever carry more water than glutamine and taurine released from muscles to blood draw water? Osmotic pressure of a molecule depends on its resistance to crossing a membrane. If it doesn’t cross readily, it forces water to move. Oja and colleagues: “It appears that the plasma to tissue exchange of taurine is lowest in the brain, while taurine penetrates very fast for instance from plasma to liver (and also to kidney).”[154] Schaffer and colleagues observed: “[T]he size of the intracellular taurine pool is very large while the rate of taurine uptake via the beta amino acid transporter is slow. Thus changes in the size of the intracellular taurine pool only occur after chronic taurine exposure. By inference, the effects seen seconds or minutes after taurine addition to the extracellular medium cannot be attributed to altered intracellular taurine levels. A more likely candidate for these acute effects is the rise in extracellular osmolality.”[155] [my emphasis]


Taurine also carries calcium into excitable cells, as part of its role to regulate active concentrations. Ji and colleagues found high concentrations of sodium/calcium–ATPase and calcium/magnesium–ATPase in autistic brains postmortem: “Increased activity of these enzymes in the frontal cortex and cerebellum may be due to compensatory responses to increased intracellular calcium concentration in autism.”[156] Furthermore, fever elevates the temperature set point by shifting sodium ions from CSF to brain (notably the hypothalamus), displacing calcium ions.[157]


WHAT ELSE CAN BE DONE?


Kern and colleagues reported oral and transdermal glutathione elevated reduced glutathione, sulfate, cysteine, and taurine in ASD children. They noted oral glutathione is poorly absorbed and can’t enter cells; glutathione must be synthesized within cells.[156] A pharmacologist suggested whey protein or N-acetylcysteine (NAC) as precursors.[personal communication 2015] My 2006 PDR warned against denatured whey protein: “When subject to heat or shearing forces (inherent in most extraction processes), the fragile disulfide bonds within the peptides are broken and the bio-availability of cysteine [rate-limiting amino acid in GSH] is greatly diminished.”[157] Glycine and glutamine are key dietary substrates for glutathione.[9] Methionine can replace cysteine to synthesize glutathione.[158]


Oregon ARI pediatrician John Green (MD) has used NAC extensively for ASD for 15 years – orally, iv, and topically: “It is a potent GSH support … and is consumed molecule for molecule in detoxification. Hardan at Stanford did a study of pharmaNAC in ASD children, dosing from 900 to 2700 mg/day in stepwise fashion, and demonstrated significant neurologic improvements. It is also a competitive amino acid in glutamate transport, and could thus be calming .... Clinically, responses are very split, with an almost equal number of patients showing agitation vs. enhanced cognitive function in others. I start with 900 mg and move up as able. We tested five different preparations with a biochemist, and found the commercial preparations have very little actual NAC, being composed instead of congeners, cysteine, and degra-dation products. It is very fragile, oxidizes upon opening the bottle, so is of limited antioxidant value, though may still contribute to GSH synthesis. The pharmaNAC preparation is blister packed, so that the stability is much more secure.”[personal communication 2015]


Low-protein diets are usually recommended when blood ammonia is high, but Córdoba and colleagues concluded a normal-protein diet was safe for persons with episodic hepatic encephalopathy [152], as others have. Because the brain first detoxifies ammonia with α-ketoglutarate (forming glutamate) Pangborn prompted development of buffered α-ketoglutarate supplements.[52] Creatine (arginine + glycine) also detoxifies ammonia [160] – and is low in ASD.[38] Woeller has given ASD children 5–10g/day of creatine without adverse effects.[161]


Most ingested arginine is taken up by the liver, which requires arginine to detoxify ammonia. Citrulline bypasses the liver and forms arginine in the kidneys, increasing systemic arginine.[50,51] Citrulline has other advantages as a source of arginine/nitric oxide for ASD. Citrulline stimulates protein synthesis when dietary protein is low [e.g. casein/gluten-free diets] and provides sufficient arginine for constitutive nitric oxide but not induced nitric oxide.[162] Citrulline is also safer in large doses than arginine.[50] Watermelon and its juice are rich in citrulline and arginine [163].


Foods high in glutamine include cabbage, beets, beef, chicken, fish, beans, and dairy products.[164] Woeller recommended 500mg–1000mg oral gluta-mine daily (between meals) for ASD children, to improve gut health, muscle tone, and overall metabolism.[165] Oral glutamine may be more stable as the dipeptide glutamine/alanine (e.g. Sustamine). Branched-chain AAs are good glutamine precursors.


Taurine too dilates blood vessels, by stimulating endothelial nitric oxide.[166] Taurine is highest in clams, oysters, tuna (be wary of mercury), pork, lamb and other meats [52] – also sports drinks to balance caffeine The ARI recommended 250–500mg/day of taurine for ASD children, up to 2g/day for adults and adult-sized children.[167]


CONCLUSIONS – RES IPSA LOQUITUR


[T]he challenge posed by improvement of ASD with fever [is] thinking in terms of measures that can be dynamical. From there it is a short step to thinking about mechanisms amenable to intervention. Measuring taurine in children with autism while following their response to fever is one route of investi-gation.  Martha Herbert 2011 [168]


Autism is called heterogeneous because it takes many forms – suggesting multiple causes or mechanisms. Yet Lewis Thomas pointed out several diseases with multiple environmental causes and multiple pathologies linked to a “single key mechanism.” That key mechanism in autistic disorders is proposed to be glutathione depletion. As primary antitoxin/antioxidant, glutathione neutralizes heavy metals (e.g. mercury/aluminum in vaccines, lead in air/water) and other toxins implicated in autism. Less well-known are glutathione’s key roles in prenatal/postnatal androgen/estrogen balance, maturation of brain myelin, and brain blood flow. Prenatal glutathione depletion limits placental estrogens by limiting sulfation of fetal adrenal DHEA. Prenatal/postnatal estrogen deficiencies leave brain myelin immature and overhydrated. Glutathione depletion limits release of primary vasodilator nitric oxide, and depletes glutamine – precursor of citrulline/arginine/nitric oxide.These pathologies arguably explain white-matter dysconnection, anomalous brain asymmetry, low brain blood flow, and extreme male brain.


Glutathione depletion also explains the glutamine paradox – high blood/brain ammonia yet low blood/brain glutamine – because glutamine often provides glutamate to synthesize glutathione. Yet glutamine is also low because glutamine synthetase requires magnesium and B6 as cofactors – consistently low in these children – and because diseased intestines require glutamine for healing and fuel. Is high brain ammonia as decisive as low brain glutathione? Glutathione depletion explains more neuropathology. Compelling evidence argues our national autism epidemic began in 1980, when the CDC warned against aspirin for children, and everyone switched to acetaminophen (Tylenol) – which depletes glutathione.


If fever relieves autistic behavior dramatically despite persistently immature myelin sheaths, as seems likely, does that imply immature myelin is not the problem? Much evidence argues it is – implying a dynamic factor (Herbert) compensates during fever. This hopeful scenario makes the search for the dynamic factor even more pressing. Do free taurine and  glutamine released by fever draw (or carry) water out of immature overhydrated brain myelin? Can this benefit be replicated with oral taurine and glutamine? Fever’s benefit was first reported formally 35 years ago; what intrepid researchers will be first to conduct the obvious critical experiments?


Feynman’s reinvention of quantum mechanics did not so much explain how the world was, or why it was that way, as tell how to confront the world. It was not knowledge of or knowledge about. It was knowledge how to.... There were other kinds of scientific knowledge, but pragmatic knowledge was Feynman’s specialty. For him knowledge did not describe; it acted and accomplished.  James Gleick  Genius


acknowledgments


I’m most grateful to


Jimmy Harduvel of the Deschutes County Library in Bend, Oregon

(in memory)


William Ellis of St. John’s Cathedral, Spokane


Helen Emily Couch (in memory)


and the many researchers and practitioners who offered evidence and insight to this enterprise. you know who you are. many thanks . . .


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