oxidative stress in autism review article

22 ALTERNATIVE THERAPIES, nov/dec 2004, VOL. 10, NO. 6 CME: Oxidative Stress in Autism

DIRECT EVIDENCE OF OX I DATIVE INJURY IN AU T I S MBodily lipids, proteins, glycoproteins, and nucleic acids are

subject to oxidative injury, and a number of analytical methodsexist for measurement of oxidative by-products in urine, blood,breath, and organ tissue samples. Oxidized lipids and their pro-tein adducts are commonly used as oxidative biomarkers. Lipids,which comprise biological membranes, are easily oxidized, par-ticularly if highly unsaturated.

D i rect markers for lipox i d ation are higher in autism. In apublished study which carefully eliminated dietary and medici-nal confounders, re d-cell thiobarbituric reactive substance(TBARS, a measure of lipoxidation) was twice higher in autisticchildren than in age-matched controls.5 Other preliminary stud-ies found serum lipid perox i d e s6 and urinary isopro s t a n e s7 s i g-nificantly higher in autistic children.

Indirect markers are consistent with greater lipoxidation in

OXIDATIVE STRESS IN AUTISMWoody R. McGinnis, MD

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Wh en oxidants exceed th e ant iox i d a n tdefense, biological systems suffer ox i d at i v es t ress, with damage to biomolecules andfunctional impairment. Autism is a behav-i o ral disord e r, with hallmark communica-tion and social deficits. It has been suggested that ox i d at i v es t ress may play a role in the pathophysiology underlying thebehaviors that define autism.1 Another serious behavioral disor-der, schizophrenia, features high oxidative biomarkers 2 and doc-u m e n t ation o f cli nic al re spo nse to antiox i d a n t . 3 M a n yneuroleptic medications used in the treatment of schizophreniaare, in fact, potent antioxidants.4

CME continuing medical education

Woody R. McGinnis, M D, a former primary care physician,t re ats ADHD and autism with nutritional interventions. Heis currently organizing an international ox i d at i ve stress inautism symposium scheduled for 200 5.

I n n o Vision Communications is accredited by the Ac c re d i t at i o nCouncil for Continuing Medical Education to provide continu-ing medical education for physicians. The learner should studythe article and its figures or tables, if any, then complete the self-e va l u ation at the end of the activity. The activity and self-eva l u a-tion are expected to take a maximum of 2 hours.

STATEMENT OF PURPOSEI n d i rect markers are consistent with gre ater ox i d ative stre s s

in autism. They include gre ater fre e - radical pro d u c t i o n ,i m p a i red energetics and cholinergics, and higher exc i t o t ox i cm a rkers. Brain and gut, both abnormal in autism, are part i c u l a r-ly sensitive to ox i d ative injury.

Higher re d-cell lipid peroxides and urinary isoprostanes inautism signify gre ater ox i d ative damage to biomolecules. A pre-

l i m i n a ry study found accelerated lipofuscin depositionconsis-tent with ox i d ative injury to autistic brain in cortical areas serv-ing language and communication.

Double-blind, placebo-controlled trials of potent antioxi-dantsvitamin C or carnosinesignificantly improved autisticb e h av i o r. Benefits from these and other nutritional interv e n-tions may be due to reduction of ox i d ative stress. Un d e r s t a n d i n gthe role of ox i d ative stress may help illuminate the pat h o p h y s i o l-ogy of autism, its environmental and genetic influences, newt re atments, and pre v e n t i o n .

OBJECTIVESUpon completion of this article, participants should be able to:

1. Be aw a re of laborat o ry and clinical evidence of gre at e rox i d ative stress in autism.

2 . Understand how gut, brain, nutritional, and toxic status inautism are consistent with gre ater ox i d ative stress.

3. Describe how anti-oxidant nutrients are used in the con-t e m p o ra ry tre atment of autism.

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CME: Oxidative Stress in Autism ALTERNATIVE THERAPIES, nov/dec 2004, VOL. 10, NO. 6 23

autism. Low concentrations of highly unsaturated lipids in autis-tic red-cell membrane8 suggest oxidative depletion. Higher phos-pholipase A28 and loss of membrane lipoprotein asymmetry9 i nautism comport with oxidative effects.

Lipofuscin is a non-degradable matrix of oxidized lipid andc ro s s -linked protein which forms in tissue as a result of ox i d at i v ei n j u ry. Co-l o c a l i z ation of lipofuscin with specific subcellular com-ponents or injurious agents may provide clues to neuro p at h o g e n-esis. In Alzheimers disease, lipofuscin is associated with ox i d i z e dmitochondrial DNA.10 In a documented case of human merc u rypoisoning with psycho-organic symptoms, elevated merc u ry inb rain localized in lipofuscin 17 years after exposure .11

Lipofuscin is experimentally-induced by strong pro - ox i-dants such as iron12 or kainic acid. 13 In animals, lipofuscin formsinitially in hippocampus, and later in cortical brain.14 In animalexperiments, lipofuscin deposition is re t a rded by supplementa-tion with vitamins C and E15 or carnitine,16 and measurable brainactivity is inverse to lipofuscin content.17

Edith Lopez-Hurtado and Jorge Prieto found greater lipofus-cin in areas of autistic cortical brain concerned with languageand communication, deficits of which are integral to the diagno-sis of autism. After age-seven, in comparison to controls, greaterlipofuscin was measured in autis tics in Brodmann are a2 2

(We r n i c k es, speech recognition), are a3 9 ( re a d i n g) and are a4 4

(Brocas, language production).18 (See Figure 1.)

In both autistic and control subjects, lipofuscin was alwaysmore prominent in Brodmann area44 at all ages. Analysis by cor-tical layers showed that the number of cells (both pyramidal andnon-pyramidal neurons) containing lipofuscin was larger in lay-ers II and IV. A significant decrease in neuronal cell numbers wasfound in layers II and IV of autistic cortex, compared tocontrols.18 Greater lipofuscin also has been reported in Rett syn-drome,19 on the autistic spectrum.

Retina, a virtual extension of the brain, is very sensitive to

ox i d ative stress. Gre ater ox i d ative stress is associated with flat-tened electro re t i n o g rams and increased retinal lipid peroxides inanimal experiments.2 0 In autism, abnormal re t i n o g rams with flat-tened b-wav e s21-2 2 suggest ox i d ative retinal injury. Re t i n o g ra p h i cresponse to antioxidants has not been tested in autism.

D ata implying gre ater ox i d ation of biomolecules in autismare summarized in Table 1.

INDIRECT MARKERS ARE CONSISTENT WITH GREAT E ROX I DATIVE STRESS

I n d i rect markers for gre ater ox i d ative stress in autisminclude: 1) lower endogenous antioxidant enzymes and glu-t athione, 2) lower antioxidant nutrients, 3) higher organic tox i n sand heavy metals, 4) higher xanthine oxidase and cytokines, and5) higher production of nitric oxide (NO), a toxic fre e - radical.

L ower levels of antioxidant enzymes and glutathione inautism (Table 2) may stem from lesser production or greater con-sumption, and imply gre ater vulnerability to oxidants. Low e rantioxidant nutrients (Table 3) may attribute to lower intake orabsorption and/or greater oxidative depletion. A substantial lit-erature documents increased oxidation of biomolecules and cellinjury in relevant nutrient-deficient states.29

FIGURE 1 Greater Lipofuscin in Autistic Brain

Greater lipofuscin, a biomarker for oxidative injury, is found in areas ofautistic cortex serving language and communication. 18

4422

39

22

TABLE 1 Oxidized biomolecules in groups ofautistic children versus controls

Elevation / abnormality Reference

Red-cell lipid peroxides by TBARS (5) Serum lipid peroxides (6)Urinary isoprostanes (7)Lipofuscin in cortical brain (18)Abnormal retinograms (21)(22)

TABLE 2 Lower antioxidant enzymes and glutathionein groups of autistic children versus controls

Lower in autism Reference

Red-cell GSHPx (23)(24)Plasma GSHPx (24)Red-cell SOD (24)Platelet SOD (23)Red-cell catalase (5)Total plasma glutathione (25)Plasma GSH/GSSG (25)

TABLE 3 Lower antioxidant nutrients in groups ofautistic children versus controls

Nutrient Reference

Plasma vitamins C, E, and A (26)Red-cell activated B6 (P5P) (27)Red-cell B6 acitivity by EGOT (26)Red-cell magnesium (26)Red-cell selenium (26)Plasma zinc (28)Red-cell zinc (26)


Nutrient levels affect the status of glutathione and antiox i-dant enzymes. The glutathione-boosting effect of vitamin C andvitamin E supplementation is well-known. Marginal deficiency ofvitamin B6 is associated with lower glutathione perox i d a s e(GSHPx) and glutathione re d u c t a s e .3 0 All forms of GSHPx con-tain selenium, and strong corre l ations exist between low andlow-normal blood selenium levels and GSHPx activity.31

Organic tox i n s3 3 -4 0 and heavy metals3 5 a re strongly pro - ox i-dant. These may accumulate (Table 4) due to impaired detoxifi-c ation, which is demonstrated in autism.41 Toxins incite thep roduction of ox i d ative species by various mechanisms. Thevo l atile organic compounds and insecticides stimulate nitricoxide synthase (NOS).4 2 Copper catalyzes the production ofpotent hydroxyl radical (OH), especially when catalase is insuffi -cient.32 Mercury is known to increase oxidative stress by blockingmitochondrial energy production and depleting glutathione.

C i rc u l ating cytokines4 0 and xanthine oxidase (XO )5 a reg re ater in autism, and both generate free radicals. XO actuallyresults from ox i d ative alteration of xanthine dehyd ro g e n a s e .Cytokines and XO can be both cause and effect of ox i d ative stre s s .

HIGHER FREE-RADICAL PRODUCTION IN AU T I S MNO, which is short-lived, is measured indirectly as total

n i t r i t e + n i t ra t e, which are stable derivatives of NO. In autism,red-cell39 and plasma37,38 total nitrite + nitrate is elevated, and plas-ma n i t r i t e + n i t ra t e c o r re l ates positively with TBARS.3 9 Exc e s sNO production is suspected to play a role in other neurobehav-i o ral disorders, including schizophre n i a ,4 3 A l z h e i m e r s disease,Downs syndrome,44 and multiple sclerosis.45

Its unknown whether production of excess NO in autism islocalized to specific organs or tissues. Cy t o k i n e - p roducing cellsa n y w h e re can stimulate NO. If excess NO localizes in autism,the brain and gut are plausible sources, as both are often abnor-mal in autism to gross and microscopic inspection, and behav-ioral and gastrointestinal symptoms predominate.

Excess NO in brain would be a serious matter, as it increas-es apoptosis,46 leaky blood-brain barrier (BBB), 47 neurodegenera-t i o n ,4 8 and demye l i n at i o n .4 9 Such mechanisms might effectneurodevelopment in autism.

Decreased activity of oxidation-sensitive receptors is foundin autistic brain, and it is possible that this may re l ate to localNO, or more generally to gre ater ox i d ative stress. Cholinergic

receptor activity is decreased in autistic cortex,50 and cholinergicreceptors are sensitive to NO tox i c i t y.51 Gamma aminob u t y r i cacid (GABA) receptors, generically sensitive to oxidative stress,52

are reduced in autistic hippocampus.53 It is conceivable that theGABA-polymorphism that is associated with autism may lead toan increase in the sensitivity of this receptor to oxidative stress.54

In the existing literature, lesser cerebellar Purkinje cell num-bers and smaller neurons in the entorhinal cortex and medialamygdala are consistent findings in autism5 5 with mark e dPu rkinje cell loss described as the most consistent finding.5 6

Stunted pyramidal neurons and decreased complexity andextent of dentritic spines are found in hippocampus.5 7 T h e s efindings are unexplained. Current technology would allow quan-t i f i c ation and localization of specific ox i d ative (and nitro s at i v e )b i o m a rkers in autistic brain, and possible elucidation of micro-scopic pathology.

The autistic gut is inflamed (Table 5), and there appears tobe a mutually amplifying positive feedback loop between guti n f l a m m ation and NO. Pain, constipation or diarrhea, gastro e-sophageal re f l u x ,61 and increased intestinal permeability5 8 a recommon. Variably, chronic inflammation ranges from esophagusto colon. Inflammation of the distal ileum with adenopathy isp ro m i n e n t .6 0,6 2 In other clinical conditions, inflammation of thegut associates with gre ater increased NO production. Plasmanitrite + nitrate is elevated in childhood colitis.63 In chronic diar-rhea, urinar y nitrite + nitrate levels correlate with leaky gut.64 Inprobability, the inflamed autistic gut produces more NO.

NO is potently antimicrobial.65 Certain viruses and bacteriap rovoke massive local production of NO in gut6 6 and bra i n .6 7

Unfortunately, massive NO also oxidizes host tissue.66,68 Thus, ingut, excess NO is known to increase inflammation and perme-ability.69 The young gut is uniquely sensitive to damage by NO,particularly the ileum.70,71 Too much NO can deplete the antioxi-dant defense, depressing levels of reduced glutathione (GSH).72,73

Low GSH, in turn, increases NO.74 Nitrite binds GSH.75

Excess NO leads to increased formation of perox y n i t r i t e(O N O O), which savages biomolecules. O N O O is formed by re a c-tion of NO with superoxide (O), and is much more reactive thanits parent radicals. Known targets of O N O O attack, with possiblere l e vance to autistic pat h o p h y s i o l o g y, include: tyrosine groups (asin glutamine synthetase and glutathione reductase inhibition),s u l f h yd ryl (-SH) groups, superoxide dismutase (SOD), neuro f i l a-ments, ceruloplasmin (releasing pro - oxidant copper), membra n e

TABLE 4 Higher pro-oxidants in groups of autistic children versus controls

Parameter Reference

Plasma perchlorethylene, hexane, pentane (26)Red-cell mercury, lead, and arsenic (26)Higher provoked urinary mercury (35)Plasma copper (36)Plasma nitrite + nitrate (37)(38)Red-cell nitrite + nitrate (39)Circulating cytokines (40)Red-cell xanthine oxidase (5)

TABLE 5 Gut abnormalities in subgroups of autistic children

Ab n o r m a l i t y Autistic Sub-gro u p Re f e re n c e

High intestinal permeability 4 2 % A s y m p t o m atic ( 5 8 )Reflux esophagitis 6 9 % Ab dominal symptoms ( 5 9 )C h ronic gastritis 4 2 % Ab dominal symptoms ( 5 9 )C h ronic duodenitis 6 7 % Ab dominal symptoms ( 5 9 )Ileal lymphonodular hyperplasia 8 9 % Re g ressed, gut symptoms ( 6 0 )C o l i t i s 8 8 % Re g ressed, gut symptoms ( 6 0 )


receptors, ion channels, G- p roteins, and methionine. O N O O

depletes antioxidants, peroxidizes lipids, and breaks DNA.31 , 7 6

NO is too ephemeral for distant transport. But hypotheti-cally, excess NO in one tissue may cause damage in a more dis-tant site via higher circ u l ating nitrite and nitrate. For example,experimental intravenous nitrite injures the BBB.77 Higher levelsof nitrite in autism may link chronic gut and brain injury.Inflamed gut may thus damage brain.

Or conceiva b l y, distant NO production may raise levels ofc i rc u l ating stable products of NO (nitrite, nitrate, and S-n i t ro s o h e m o g l obin), which may in turn lead to gre ater gutNO, and resultant inflammation of the gut.7 8 -7 9 Nitrite andn i t rate are selectively re m oved from the circ u l ation by theg u t .8 0, 81 Various bowel flora convert nitrite and nitrate to NO bye n z y m atic re d u c t i o n ,8 2 , 8 3 which is catalytically favo red by lowoxygen tension,84 as found in gut. NO from distant pro d u c t i o nalso circ u l ates as S-nitro s o h e m o g l obin, and the release of NOin the bowel from this carrier protein is facilitated by low ox y-gen tension and the presence of sulphides produced by cert a i nb a c t e r i a .78 As modulated by flora, excess production of NOf rom anywhere in the bodyincluding brainmight serve toinflame the gut.

BRAIN AND BBB SENSITIVITY TO OX I DATIVE STRESS The brain is inherently sensitive to ox i d ative stress due to

higher energy re q u i rement, higher amounts of lipids and iro nand autooxidizable catecholamines, and lower levels of cert a i ne n dogenous antioxidant molecules.8 5, 8 6 The protective BBB alsois relatively sensitive to oxidative damage.87 Clinical and labora-tory findings suggest a leaky BBB in autism. (See Table 6.)

Rapid behavioral response to treatment with GSH hints of aleaky BBB in autism. In healthy animals with intact BBB, bra i np e n e t ration by GSH is practically nonexistent. Ye t , c l i n i c i a n sreport immediate behavioral improvement in some autistic chil-dren simultaneous with GSH infusion,93 suggesting a direct effecton the central nervous system.

In animals, experimental oxidative injury to the BBB prefer-entially injures the reticular format i o n .9 4 , 9 5 In autism, widelyre p o rted difficulty falling asleep or staying asleep9 2 suggests thepossibility of reticular formation dysfunction. The specificn at u re of rapid eye movement (REM) abnormalities found inautistic sleep disturbance is more typically associated with neu-

rodegenerative diseases in which oxidative stress has been docu-m e n t e d .9 6 Other than melatonin, which has proven effective inautistic sleep disord e r s ,9 7 the effect of antioxidants on autisticsleep disturbance has not been investigated.

L a b o rat o ry ob s e rvations suggest leaky BBB in autism.Pe r i vascular lymphocytic cuffs re p o rted in three of seven autis-tic bra i n s ,3 8 a re sentinel, though nonspecific. High autoim-mune titers to central nervous system proteins in autism8 8 - 91

suggest abnormal exposure of the immune system to bra i nantigens via leaky BBB.

The autoimmune response to brain antigen also may bepromoted by oxidative generation of neoepitopes, which occursvia oxidative alteration of host proteins.98 If they co-exist, autoim-mune and ox i d ative mechanisms in the autistic brain may bemutually re i n f o rcing, as NO production is significantlyincreased in central nervous autoimmune disease.67

Conditions which have been documented in autism areassociated with porous BBB in animals. Higher levels of circulat-ing cytokines,9 9 h e avy metals,10 0 N O ,101 and nitrite10 2 p ro d u c eleaky BBB in animals. Lower zinc status in autism27,29 may be rele-vant. Zinc at physiological concentrations protects the BBB frominjury,101 and zinc deficiency increases BBB permeability, particu -larly in conjunction with oxidative stress.103

I n t r i g u i n g l y, pre l i m i n a ry data find ov e r g rowth of gra m -n e g ative aerobes in autistic thro at and rectal culture s .10 4 T h e s eorganisms produce endo t oxin, re n owned for permeabilizat i o nof the BBB.

I n v e s t i g ation of the autistic BBB is warranted. Enhancedmagnetic resonance imaging demonstrates BBB leaks,10 5 -10 6 a n dscanning electron microscopy visualizes BBB injury, includingluminal pro t rusion, endothelial craters, va c u o l ation, inclusionbodies and necrosis, though such lesions may be sparse.100

G R E ATER OX I DATIVE STRESS AND THE GUTIs c h e m i a / re p e rfusion studies demonstrate that the gut is

very sensitive to oxidative injury.31,107 Ingested toxins (peroxidizedfats, electrophilic food contaminants) and microbial metabolitesp resent a large ox i d ative burden to the intestinal epithelium.10 8

Sufficient quantities of GSHPx (to reduce peroxides), GST (toreduce electrophiles), and GSH (to facilitate both GSHPx andGST), are required to protect the gut from oxidation.

As indicated earlier, ileal inflammation and adenopathy areconspicuous in autistic children with gastrointestinal symptoms.Ileum appears more vulnerable to ox i d ative injury. In animals,GST is 36-fold lower in the distal ileum than proximal intes-tine.109 Double knock-out genes for gastrointestinal GSHPx resultin mucosal inflammation of the ileum, but not other parts of theintestine.110 In human inflammatory bowel disease, NOS expres-sion is most prominent in the ileum, and ileum is most sensitiveto NO-dependent oxidative injury.111

Excess NO is a plausible mediator for autistic gastro i n-testinal symptoms. (See Table 7.) NOdegrades mucin, whichp rotects the gut from a wide variety of irritants.10 8 Excess NOi n c reases intestinal permeability,11 2 p re valent in autism.5 8 I n

TABLE 6 Leaky Blood-Brain Barrier in Autism?

Clues and Predisposing Factors Reference

High antibodies to brain proteins (88)(89)(90)(91)Sleep disturbance in autism (59)(92)Perivascular lymphocytic cuffing (38)Higher NO / nitrite (37)(38)(39)Lower zinc (26)(28)Higher circulating cytokines (40)Higher heavy metals (26)(35)


e xcess, NO re l a xes the esophageal sphincter,113 and two-third sof autistic children with gastrointestinal symptoms have re f l u xe s o p h a g i t i s . 5 9 Excessive NO inhibits gallbladder contra c-t i o n ,114 perhaps accounting for lighter-c o l o red stools ob s e rv e dby parents and clinicians in many autistic children. Poor bilef l ow impairs nutrition and limits delivery of protective GSH tothe gut mucosa.

Excess NO also mediates slow - t ransit constipat i o n .7 0

Many autistic children are constipated, some with very largecaliber stools. It is possible that malabsorption and floral ov e r-g rowths belie a tendency to constipation in even larger num-bers of autistic childre n .

OX I DATIVE STRESS, LOW ENERGY PRODUCTION, ANDE XC I T OX I C I TY

Oxidative stress, impaired energy production and excitotox-icity are dynamically re l ated. For instance, energy-pro d u c i n gmitochondria are sensitive to oxidative injury,86, 115-120 and injuredmitochondria leak more oxidants.121-123 Also, impaired energy pro-duction predisposes to activation of exc i t at o ry re c e p t o r s ,d e c reased intracellular calcium buffering, increased ox i d i z i n gspecies, and apoptosis.86,124

Overstimulation of excitatory receptors results in oxidativeneuronal injury,125,126 and greater oxidative stress increases releaseof gl utamate and subse que nt stim ulat ion o f exc i t at o r yreceptors.127,128 Subcellular anatomy correlates with this function-al relationship: excitatory glutamate receptors and NOS in brainand gut129 are co-localized.

As a general rule, ox i d ative biochemistry adheres to the follow-ing construct, which is both consistent and useful:

Accordingly, greater oxidative stress in autism implies pos-sible problems in energy production and excitotoxicity.

I M PAIRED ENERGETICS IN AU T I S MMagnetic resonance imaging has demonstrated decre a s e d

ATP levels in autistic bra i n .13 0 Higher lactat e ,131-13 2 higher pyru-vat e ,13 3 higher ammonia, and lower carnitine13 4 a re do c u m e n t e din autistic children as a group, although not all autistic childrenh ave lower measurements in some or all of these para m e t e r s .

Collectively, these differences do suggest impaired mitochondrialfunction in autism, and in fact, mitochondrial abnormalities arereported in autistic case studies.135-136

Excess NO in autism may impair energy production, direct-ly or via ONOO . Excess NO reduces oxidative phosphorylation,l owering ATP and increasing lactat e .13 7 NO directly inhibitscomplex IV, causing leakage of superoxide and inhibition ofGSHPx.31 ONOO selectively damages complexes I and III.138 NOi n a c t i vates coenzyme A (CoA), depriving mitochondria of thisprecious energy currency.78 (See Figure 2.)

E XC I T O T OXIC MARKERS IN AU T I S MHigher extracellular glutamate in the brain is associated with

e xc i t o t ox i c i t y, especially if energy metabolism is compro m i s e d .13 9

Glutamic acid decarboxylase (GAD) converts glutamate to GA B A ,and GABA lessens exc i t o t ox i c i t y. A decrement in brain GAD favo r se xc i t o t ox i c i t y, by increasing glutamate and decreasing GABA.

T h e re is ample suggestion that GAD is deficient in autism.The quantity of GAD in post- m o rtem autistic brain is decreased byh a l f .14 0 Pe r i p h e ral measurements are consistent with GAD impair-ment. Re d-cell GAD binding affinity is low e r,2 6 plasma glutamat eh i g h e r,13 3, 141 and plasma glutamine low e r141 in autism. GA D,14 2 g l u t a-mine synthetase,143 the glutamate tra n s p o rt e r,144 and inhibitoryGABA re c e p t o r s9 a re sensitive to ox i d ative stre s s .5 2 (See Fi g u re 3. )

Whether cause or effect of greater oxidative stress in autism,g re ater exc i t o t oxicity is a reasonable hypothetical and clinicalconcern. Exc i t o t oxicity can also be aggravated by oral ingestion

TABLE 7 Autistic Gut Abnormalities Possibly Mediated by Excess NO

Abnormality in autism Reference

Inflammation (66)(68)(70)(71)Increased intestinal permeability (69)Low esophageal sphincter tone (113)Poor gall-bladder contraction (105)Slow-transit constipation (70)

Oxidative Stress

Excitotoxicity Poor Energetics

FIGURE 2 Impaired Energetics in AutismExcess NO inhibits CoA by conversion to metabollically inactive S-nitro s o -C o A ,78 with resultant reduction of acetylcholine and ATP production. Low e rAT P,13 0higher lactat e ,131 , 13 2 and pyru vat e13 3a re found in autism.

Glucose

Pyruvate

Acetyl CoA

CoANO

Lactic Acid

S-nitroso-CoA(metabolically inactive)

Acetylcholine

Krebs Cycle

FIGURE 3 Excitotoxicity in Autism?

Higher extra -cellular glutamate and lower GABA increase exc i t o t ox i c i t y. GAD quantity14 0 and activity2 6 a re lower in autism. GA D,14 2g l u t a m i n es y n t h e t a s e ,14 3and the glutamate tra n s p o rt e r14 4a re sensitive to ox i d ative stre s s .

Glutamate+NH3

Glutamine

Glutamine

synthetase

Glutamate

GABA

GAD


of exc i t o t ox i n s .145 The author joins other clinicians in advisingautistic patients to avoid exc i t o t oxic flavor enhancers such asMSG and aspartame in food and drink.

I M PAIRED CHOLINERGICS IN AU T I S ML a b o rat o ry and clinical ob s e rvations suggest a significant

cholinergic deficit in autism. Cholinergic receptor activity isl ower in autistic cere b ral cort e x .5 0 Tre atment with cholinergicagonists146-147 or precursor (deanol) for acetylcholine,148 is associat-ed with improved behavior in autism.

Response to bethanecol, a specific agonist for the muscarinicsubtype of cholinergic receptors, invokes muscarinic impairmentin autism. Oral bethanecol (2.5-12.5 mg b.i.d.) normalizes dilat e dpupils, increases bowel motility, and improves sleep pattern andb e h avior in many autistic children. Occasionally, sudden behav-i o ral improvement is re p o rted with the first dose of bethanecol,14 6

and the author confirms this ob s e rvat i o n .Ne u roimaging reveals cere b ral hypoperfusion in autism,14 9 -15 0

worsening with age,151 and vasodilatation of cerebral microvesselsis the province of muscarinic receptors.152-153 A possible explana-tion for the rapid bethanecol response is sudden improvement inc e re b ral perfusion. Bethanecol may stimulate readily accessiblemuscarinic receptors in the small blood vessels which perfuse thebrain. This hypothesis may be simple to test.

Muscarinic impairment in autism may potentiate gre at e rox i d ative stress. Ex p e r i m e n t a l l y, muscarinic signals are neuro-protective, shielding cells from oxidative stress and apoptosis.154

Muscarinic receptor numbers are decreased by oxidative stress.155

Muscarinic receptors are sensitive to NO tox i c i t y,15 6 a n dre l ative to other receptor subtypes, muscarinics are pre f e re n t i a l-ly sensitive to inhibition by ONOO 15 7 and other ox i d a n t s .15 8 A sdiscussed earlier, excess NO in autism may depress CoA.Besides its importance in energy production, CoA is a necessaryp recursor for the cholinergic neuro t ra n s m i t t e r, acetylcholine.(See Fi g u re 2.)

Insufficient CoA renders cholinergic neurons more vulnera-ble to a variety of toxic insults, including excess NO.51 L ow CoAdoes appear to play an important role in other encephalopat h i e s .5 4

A N T I OX I DANT NUTRIENTS IN THE TREATMENT OF AU T I S MHigh-dose vitamin C

A do u b l e -blind, placebo-c o n t rolled university trial utilized8 grams per 70 kg body weight per day of oral vitamin C in twoor three divided doses in institutionalized autistic childre n .15 9

Some of the cohort had been on doses of up to 4 grams of vita-min C prior to the trial. The cro s s - over design, comprised oft h ree 10-week periods, included tre atment of all subjects withvitamin C for the first 10 weeks. In the second and third phasesof the trial, half the cohorts received placebo and then vitamin C.The other half received vitamin C and then placebo.

Ps ychometric testing was performed after each 10 - w e e kphase of the study, but not prior. Total scores on the Ritvo -Freeman (RF) scale, which rates 47 social, affective, sensory, and

language behaviors, demonstrated improvement in the gro u pgoing from placebo to vitamin C, and worsening in the gro u pgoing from vitamin C to placebo (P=0.02).

Pacing, flapping, rocking, and whirling behaviors, in partic-u l a r, corresponded to vitamin C manipulation, and a group ofstrong responders were described as obvious to the investiga-tors. No serious side effects were reported in this study, but clini-cians report excessive stool softening can limit vitamin C dosingin some autistic children.

Vitamin C is strongly antioxidant. This suggestsbut doesnot provean antioxidant mechanism for its therapeutic effect.A n t i oxidant effects of vitamin C do seem neatly tailored toautism. Vitamin C provides good protection against NO andONOO.31 Vitamin C is known to protect neurons from glutamateneurotoxicity,160 as glutamate re-uptake involves exchange for vit-amin C.161 Vitamin C blocks the inhibition of glutamate transportby NO,162 an effect seen particularly in the presence of copper,163

which is higher in autistic blood.28

CarnosineCarnosine, a nat u rally occurring amino acid found in high

c o n c e n t rations in the brain, is a strong antioxidant and neuro-p ro t e c t a n t .16 4 -16 6 A do u b l e -blind, placebo-c o n t rolled 8-week trialof carnosine 400 mg by mouth twice daily produced significanti m p rovement in autistic children compared with placebo.Psychometric testing demonstrated improvements in vocabulary(P= 0.01), socialization (P= 0.01), communication (P= 0.03), andbehavior (P=0.04).167 Side effects were inconsequential: sporadich y p e ractivity responded to lowering the dose of carnosine, andno child had to discontinue the study due to side effects.

Possible physiological mechanisms for the carnosine effectin autism include its prevention of NOtoxicity,168 the binding offree radicals and reactive hydroperoxides, and the ability to com-plex with metals such as copper.16 9 The copper:carnosine com-plex demonstrates antioxidant, SOD-like activity in vitro.170

Vitamin B6Any mechanistic hypothesis for autism should accommo-

date the successful application of high-dose vitamin B6 pioneeredby Bernard Rimland. Multiple controlled trials demonstrate thatin combination with magnesium, B6 improves behavior in manyautistic children.148,171-172 While serum B6 levels usually are normal,B6 a c t i v i t y, as reflected by ery t h rocyte glutamic ox a l o a c e t i ctransaminase (EGOT) assay, was significantly lower in a group ofautistic children than in controls.26

Py r i doxal kinase, which converts B6 to its active form,p y r i dox a l- 5 - p h o s p h ate (P5P), can be impaired in autism. A pre-l i m i n a ry study suggests very poor binding affinity of pyridox a lkinase in autistic red cells, as reflected by high Km ( M i c h a e l isc o n s t a n t ) .2 6 P5P activity in blood is below normal in over 40%of autistic subjects.27

Py r i doxal kinase impairment in autism is unexplained.Lower zinc26,28 and energy status in autism are attractive explana-tions, as pyridoxal kinase requires ATP-facilitated release of zinc


f rom metallothionein for activat i o n .17 3 Inhibiting agents alsoshould be considered. The strongest pyridoxal kinase inhibitorsare the carbonyl agents, which are exogenous chemicals such ash yd razine, from jet fuel.17 4 E n dogenous carbonyls are potentialinhibitors. They result from oxidative alteration of bodily lipids,proteins, and sugars, and are broadly elevated in clinical condi-tions associated with excess NO.22

While the cause of poor B6 function in autism is uncertain, wecan be sure that B6 impairment is an ox i d ative influence. As dis-cussed earlier, even marginal B6 deficiency is associated with low e rGSHPx and gluathione reductase activity, lower re d u c e d / ox i d i z e dg l u t athione ratios, and higher lipid peroxide levels.3 0

Mitochondrial decay results from B6 deficiency, and is asso-ciated with increased oxidative stress. 175-176 P5P is required for thesynthesis of key mitochondrial components: iron-sulfur crystals(for complex I, II, and III), heme (for complex IV) ,17 7 and coen-zyme Q10.17 8 Ex p e r i m e n t a l l y, P5P protects neurons from ox i d a-tive stress, apparently by increasing ATP production andstemming extracellular glutamate.179

Lagging B6 function lowers the excitotoxic threshold. P5P isa necessary cofactor for GAD, impairment of which can increaseg l u t a m ate receptor activation, NO, and ox i d ative stre s s .18 0 P 5 Pprotects GAD, which is sensitive to oxidative impairment,142 fromi n a c t i vat i o n .181 (P5P also protects gastrointestinal GSHPx bycomplex format i o n . )18 2 Pre d i c t a b l y, P5P administration to ani-mals increases brain GAD activity.183

Thus, high doses of B6 m ay benefit autistic patients byincreasing energy production, lessening excitotoxicity, increasingGABA, and reducing ox i d ative stress. Tre atment with B6 a l s om ay relieve a state of functional B6 deficiency caused by exc e s soxidants. The B6 vitamers are highly vulnerable to damage byox i d ative species such as hyd roxyl (OH) and singlet ox y g e n(1O2) .

18 4 -18 6 O x i d ative impairment of B6 could impair myriadenzymes and neurotransmitters in autism.

MagnesiumIn animal experiments, magnesium deficiency incre a s e s

N O ,18 7 i n c reases lipid perox i d e s ,18 8 and lowers plasma antiox i-d a n t s .18 9 L ower magnesium is clearly pro - oxidant. Magnesiums u p p l e m e n t ation lowers ox i d ative stress experimentally in ani-mals with higher oxidative stress.190

As a gro u p, autistic children have lower magnesium, as mea-s u red sensitively in red cells.2 6 Do u b l e -blind trials demonstrat eb e h av i o ral improvement and normalization of evoked potentialre c o rdings in autistic children receiving combined high-dose B6 a n dmagnesium, but no significant improvement with high-dose B6 o rmagnesium alone.191 The synergism may be a cofactor function. Fo rinstance, B6-dependent kinase, which affects diverse muscarinic andGA B A-nergic functions, re q u i res both B6 and magnesium.

13 9

Magnesium also protects against ox i d ative stress via func-tions unrelated to B6.

Production of NADPH, for reduction of glutat h i o n e ,requires magnesium. ATP synthase, which catalyzes energy pro-duction by oxidative phosphorylation, is magnesium-sensitive.192

In brain, magnesium normally blocks ov e ractivity of exc i t at o ryreceptors by modulating calcium channels.193

ZincL ower zinc status in autism is clearly established. Re d-c e l l

zinc, a sensitive indicator of zinc sufficiency, is significantlyl ower in the autistic gro u p,2 6 and in individual cases may be asl ow as half the lower limit for age-matched contro l s .19 4 P l a s m azinc is sub-normal in 40% of autistic children.28

L ow zinc potentiates ox i d ative stress. In animals, zinc-d e f i c i e n t diet decreases total glutathione, vitamin E, GST,GSHPx, and SOD levels, while increasing lipid peroxides andfree radicals in tissue, mitochondria, and cell membranes.195-198 Inelderly adults, zinc supplementation decreases lipid peroxides.197

In diabetics with re t i n o p at h y, zinc supplementation incre a s e sGSHPx and decreases lipid peroxides.200

Zinc status affects the intestine. Zinc deficiency in animalsi n c reases gastrointestinal NOS and susceptibility to gastro i n-testinal infection.2 01 C o n v e r s e l y, supplemental zinc decre a s e sintestinal lipoxidation202 and lessens intestinal permeability.203

Clinicians increasingly appre c i ate zinc as a mainstay in thet re atment of autism. William Walsh, who has organized zincand copper data on more than 3,500 autistic children at thePfeiffer Tre atment Center, finds that high doses of zinc (2-3mg/kg body weight/day, as highly absorbable zinc picolinat e )a re often needed to normalize zinc levels and achieve optimalclinical re s p o n s e .2 0 4

Periodic measurement of plasma zinc is used to assure thatzinc is not pushed above the normal laborat o ry range. Zinc iswithheld on the day of testing to avoid artifact. Zinc supplemen-tation lowers copper. Serum copper monitoring is used to avoidsub-normal levels.205

Copper excess is evident in autism. Higher total serum cop-per,36 lower ceruloplasmin,6 and higher unbound serum copper 205

a re found in groups of autistic children. Copper, especiallyunbound, is highly pro - oxidant. Supplemental copper is ra re l yneeded in autism, and even small doses of copper have beenobserved to produce negative behavioral effects.205

Higher serum copper/plasma zinc ratios (in autism,mean 1.63 v 1.15 in controls, P< 0.0 0 01 ) ,3 6 a re significantly cor-re l ated with systemic ox i d ative stress in neuro d e g e n e rat i v ed i s e a s e .2 0 6 Sufficient zinc supplementation normalizes cop-per/zinc rat i o s .2 0 5

High zinc dosing can suppress manganese. A balancing do s eof manganese, administered separately from zinc at approx i m at e-ly 5 mg manganese/30 mg of zinc, is often beneficial, and seru mmanganese levels also may be monitored to avoid exc e s s .2 0 5

The antioxidant function of zinc is prodigious. There areseveral important mechanisms:

Zinc protects SH groups against oxidation, as for example,in the protection of the key antioxidant enzyme, GSHPx.19 5 T h einitial event in experimental zinc deficiency is loss of membraneSH groups, with consequent membrane fragility.207


Zinc competes with pro - oxidant metals such as copper andi ron for bindings sites, preventing metal-c atalyzed fre e - ra d i c a lformation.200 Copper-containing enzymes are inherently prone toa u t o ox i d ation, which is prevented by zinc.19 8 C o p p e r- i n d u c e dmembrane oxidation is prevented by zinc.208

Zinc is an essential constituent of copper-zinc S O D, a keya n t i oxidant enzyme. Even marginal zinc deficiency in humansd e c reases SOD activity.2 0 9 Z i n c-deficient SOD becomes pro - ox i-dant, catalyzing biomolecular attack by O N O O.14 8 Z i n c-l e s sSOD is neuro t ox i c .210

Zinc induces the synthesis of metallothionein (MT),211 a neffective scavenger of free radicals (including O N O O)21 2 a n ds e q u e s t rant for copper and other heavy metals.213,214 In animals,h i g h - dose zinc induces measurably higher gastrointestinal MTlevels.215 Moderate zinc deficiency in animals, in which overt neg-ative health effects are not manifest, is associated with significantreduction of retinal MT.213

MT normally increases as a protective response to ox i d a-tive stress, but actually decreases in response to oxidants whenzinc is deficient.214

MT blocks copper tox i c i t y, but this protective effect is lostin the presence of excess NO, which releases copper from MT,causing lipid perox i d ation and apoptosis.4 6 In brain, MTIII, an e u ronal growth inhibiting factor, is particularly sensitive tocopper displacement by oxidants.216 Such a mechanism could berelated to greater gross brain size in younger autistic children.204

Zinc exerts physiological glutamate receptor blockade,139 less-ening excitotoxicity.

Diverse biomolecules are protected from oxidation by zinc.By complexing with phospholipids,217 zinc blocks ox i d ation offatty membranes.209 Zinc blocks peroxidation of polyunsaturatedf at unbound to membra n e .218 Zinc generally inhibits the ox i d a-tion of enzymes and other proteins,198 including those with func-tional SH groups vulnerable to mild ox i d ative conditions:Na , K-AT Pase, Ca-AT Pase, aquaporin, the vo l t a g e - g ated calciumchannel, and the NMDA-calcium channel.207

Hy p o t h e t i c a l l y, ox i d ative stress may decrease clinical zincretention. At a molecular level, oxidants (including NO), dis-place zinc from proteins, including MT.19 7,216, 219,2 2 0 Re s e a rch isneeded to determine if this phenomenon extra p o l ates to lesserw h o l e -body zinc retention under gre ater ox i d ative stre s s .S c h i z o p h renics do h ave diminis hed ur inary zinc loss inresponse to high doses of B6,

2 21 possibly associated with anti-ox i-dant effects of B6.

Selenium in autismMean red-cell selenium levels are lower in autistic children26

and this may contribute to reported lower levels of GSHPx.23-24 Ass t ated pre v i o u s l y, GSHPx activity corre l ates with low - n o r m a land borderline selenium levels.31 Clinicians frequently treat autis-

tic children with oral selenium, 50-300 mcg daily.GSHPx is irreplaceable in the antioxidant defense, especial-

ly for protection of mitochondria, which do not contain catalasefor protection from peroxide.222 In addition, GSHPx confers soleprotection from organic hydroperoxides, which sustain the dev-astating lipoxidation chain reaction.85,222

Lower GSHPx activity in frank selenium deficiency is associ-ated with peroxidative damage and mitochondrial dysfunction.29

The physiological effects of selenium deficiency can be compen-sated partially by administration of vitamin E.31

GSHPx is sensitive to ina ct ivati on by copper1 8 2 a n dm e rc u ry. 2 2 3 Human merc u ry exposure is associate d withdecreased GSHPx activity and increased lipid peroxides.224 In ani-mals, GSHPx is protected by P5P223 and zinc supplementation.225

Lesser GSHPx in autism favors greater membrane lipid per-oxidation, which is known to impair receptor and enzyme func-tion, presumably due to conformational changes and altere db i n d i n g .2 2 6 Lipid perox i d ation has been shown to inhibit mus-carinic, adrenergic, serotonergic, and insulin receptors, as well asNa,K-ATPase and glutamine synthase.227

Reduced glutathione in the treatment of autismIn an open-label trial, daily intravenous GSH improv e d

p atients with early Pa rk i n s o n s disease.10 5 L i k ewise, intrav e n o u sGSH improves behavior in many autistic children, including veryrapid extinction of perseverative behaviors such as hand- f l a p-ping. Rare l y, apparent histamine-mediated reactions (sneez i n g ,coughing, pruritic eyes) are noted.93

Oral GSH, up to 30 mg/kg body weight/day in divided do s e s ,has been beneficial in some children with cystic fibrosis, a high-oxidant stat e .1 2 6 The author finds similar doses of oral GSH helpfulin some autistic children. Reversible adverse behav i o ral re a c t i o n sto oral GSH have been re p o rted in children with low plasma zinclevels. The adverse reactions may result from rapid induction ofmetallothionein by GSH, with tempora ry zinc depletion.2 2 8

Oral GSH is well-absorbed. In animals, plasma GSH do u b l e swithin 2 hours of a large oral dose, mostly from absorption ofintact GSH.2 2 9 I n c reased animal organ levels of GSH are at t r i b u t-able to absorption of intact GSH.2 3 0 In healthy humans, a 15 mg/kgo ral dose of GSH increases plasma GSH levels by 2- to 5-fold.2 2 9

Intestinal mucosal demand for GSH can exceed syntheticcapacity when demand is high,229 as would be expected in autism.Intestinal mucosa imports intact GSH from both the intestinallumen231-232 and the plasma231 to combat oxidation. In the normalphysiology, biliary excretion of GSH represents a significant por-tion of total hepatic GSH production, and bile regularly bat h e sthe intestinal mucosa with GSH.

S e v e re degeneration of the epithelium of the small intes-tine and colon, with mitochondrial swelling and degenerat i o n ,results from experimental GSH deficiency; these changes arep revented by the administration of oral GSH, which is associat-ed with increased mucosal GSH levels.2 3 3 In animals, mucosalGSH levels increase rapidly and significantly after oral GSH,but less in the ileum than elsew h e re .234 Oral GSH may low e r

CME: Oxidative Stress in Autism

ox i d ative stress in the autistic gut.S t rong in vitro a n t i - v i ral pro p e rties of GSH2 3 5 h ave been noted.

An oxidative perspective on emerging new treatmentsSubcutaneous vitamin B12 i n j e c t i o n s , as pre s e rvat i v e - f re e

m e t h y l c obalamin 1,2 5 0 -7,500 mcg weekly to daily, re p o rt e d l yi m p rove behavior in autistic childre n .2 3 6 One B1 2 i n t e r m e d i at e ,c ob(I)alamin, is exquisitely sensitive to ox i d ative inaction,2 3 7 s ofunctional B12 deficiency may result from greater oxidative stressin autism.

NO and nitrite elevations in autism send up a B12 red flag.An intermediate form of B1 2 reacts specifically with NO,

2 3 8 -2 4 0

and nitrite inactivates methylcob a l a m i n .2 41 NO binds B1 2 t oimpair enzyme function, as with in vivo inhibition of methioninesynthase by NO at physiological concentrat i o n s .2 4 2 Large par-enteral doses of B12 may scavenge and reverse the physiologicaleffects of excess NO.243

O ral folinic acid supplementation i m p roves subnormal glu-t athione and GSH / GSSG ratios in autistic childre n ,2 5 t h u squestioning functional folate status in autism. Te t ra h yd ro f o l at eis very sensitive to ox i d at i o n ,2 41 ,2 4 4 and degra d ation may be sign-ficant under conditions of gre ater ox i d ative stre s s .2 4 5 Fo l at edeficiency (which may be aggravated by B1 2 impairment) isk n own to decrease ATP levels and increase reactive ox y g e nspecies and exc i t o t ox i c i t y.246

Amino acid supplementation may be useful in autism. Plasmacysteine levels were much lower in a series of 286 supplement-nave autistic childre n .2 3 6 Cysteine is produced endo g e n o u s l yf rom methionine, and provides a full third of the substituentmolecules in glutathione and metallothionein.

Oral n-acetyl-cysteine (NAC), as a source of cysteine, is gener-ally well-tolerated in autistic children, while direct supplementa-tion with cysteine is not. Intravenous NAC (150-600 mg NAC +1000-2000 mg vitamin C + 1 ml sodium bicarbonate) treatmentsreportedly improve behavior in autistic children.236

The Pfeiffer Treatment Center follows generous zinc loadingwith a proprietary oral supplement247 which includes the aminoacid constitutents of MT. Initial data suggest that this so-c a l l e dMetallothionein Promotion formula increases levels of MT.3 7

Some parents re p o rt improvement in autistic behavior coinci-dent with greater exposure to natural sunlight. Ultraviolet radia-tion induces rapid induction of metallothionein,248 so may be ofbenefit if zinc is sufficient.

Thiamine tetra h y d ro f u rf u ryl disulfide (TT F D ) via rectal sup-p o s i t o ry improves behavior and increases heavy metals clear-ance in autistic childre n .2 4 9 TTFD provides high cellular levelsof thiamine, which boosts three mitochondrial enzymes know nto be especially sensitive to ox i d ative stre s s .1 2 0,2 5 0 One of these,a - k e t o g l u t a rate dehyd rogenase complex (KGDHC), is a rat e -limiter in energy metabolism and is inactivated by NO, bothd i rectly and via ONOO.1 2 0

C a s e i n- and gluten- f ree diet i m p roved behavior in autisticc h i l d ren, possibly by reducing excess central opioid effects.2 51

Higher peripheral opioid peptides from casein and gluten are

demonstrable in autistic urine,252 possibly due to oxidative inhi-bition of the enzyme needed to complete digestion of dietarycasein and gluten. 253 In addition, a shift towards oxidation in theredox environment is known to strengthen opioid binding, andGSH to weaken it.254

Fa t ty acid supplementation is beneficial in autism.2 5 5 L ow e rconcentrations of highly unsatur ated fatty acids in plasma256 andre d-cell membra n e s8,2 5 7 suggest ox i d ative depletion of these keym e m b rane building blocks and prostaglandin pre c u r s o r s .Depletion of omega-3 and omega-6 polyunsaturated fatty acids isalso seen in schizophrenia, and these changes are associated withincreased lipid peroxide levels.258

Eicosapentaenoic acid (EPA, an omega-3) is lower in red-cellmembranes of autistic children generally, arachidonic acid (AA,omega-6 )lower in the regressed subgroup.8 Fish oil, high in EPA,s u p p resses production of NO and other free ra d i c a l s3 0,2 5 9 a n dincreases expression of GST and mitochondrial SOD.259 Brain lev-els of NO and lipid peroxides are less in animals on diets supple-mented with fish oil.260

Ad m i n i s t ration of fish oil to even marginally B6- d e f i c i e n tanimals can result in increased tissue lipid peroxide levels.2 61

Prior administration of vitamin B 6 and other antioxidants is sug-gested in autism, to avoid generation of toxic lipid peroxides.

Ongoing administration of fish oil to autistic children isassociated with significant lowering of red-cell membrane diho-mo gamma linolenic acid (DGLA).8 DGLA is an essential o-6 pre-cursor for prostaglandin-1, which tightens leaky gut and boostsi m m u n i t y. Ac c o rd i n g l y, autistic children receiving fish oil maybenefit from a balancing dose of evening primrose oil, whichprovides gamma linolenic acid (GLA), DGLA precursor.

Clinical and laborat o ry assessment helps titrate fatty aciddosing. After antioxidant loading, many autistic children do wellon an initial dose of 3 grams fish oil and 1 gram evening prim-rose oil.262 Optimal doses vary individually, and over time.

LA B O R AT O RY ASSESSMENT OF OX I DATIVE STRESSThe use of oxidative biomarkers in the clinical management

of autism is just beginning. Various blood, urine, stool, andb re ath assay s2 6 3 m ay prove useful in determining optimal do s e sand combinations of nutrients and other interventions.

Some available assays include lipid peroxides, 4-h yd rox y -nonenal (4-HNE), malondialdehyde (MDA), isoprostanes, levug -landin adducts, nitro t y rosine, oxidized nucleic acids, pro t e i ncarbonyls, advanced glyc ation end- p roducts, cellular apoptosis,nutrient and antioxidant enzyme concentrations, total n i t r i t e +n i t ra t e, enzyme-binding affinities, and luminal NO by re c t a lc at h e t e r. Ten-fold higher levels of neopterin,2 6 4 a marker forupregulation of NO synthesis,76 suggest a possible clinical utilityof this measurement.

In the re s e a rch arena, autistic brain and gut should beexamined for very specific ox i d ative and nitro s ative mark e r s .Conventional pathologic assessment of autistic brain tissue maynot detect neuronal loss due to apoptosis, a marker for oxidativestress,265 since removal of apoptotic cells can be rapid.266


FUTURE DIRECTIONSThis article has outlined data and concepts which suggest

t h at gre ater ox i d ative stress in autism may be important in thee x p ression of autistic symptoms, and perhaps the pat h o g e n e s i sof autism. If ox i d ative stress proves important in autism, thenthe nutritional management of autism,2 6 7 because it modulat e soxidative stress, presumably gains importance, too.

U l t i m at e l y, in order to tre at or prevent autism, we mayneed to reconsider some ingrained living habits. Consumptionof free radicals via foods fried in polyunsat u rated oils2 6 8 m ayneed to be curbed. Ingestion of exc i t o t oxic flavor enhancers,chlorine, nitrite, nitrate, and copper in water may need re a s s e s s-ment. Pro - ox i d a n t2 6 9,2 71 and antiox i d a n t2 7 2 ,2 7 5 d rug profiles maybecome more pert i n e n t .

While ox i d ative stress may be a persistent and tre at a b l eproblem, its impact may begin in utero. We may need to considerhow oxidative influences during pregnancy alter development top roduce re l e vant post- p a rtum effects. Gestational zinc deficien-c y, for instance, produces ox i d ative DNA damage in new b o r np r i m at e s .2 7 6 The ubiquitous flavor enhancer and exc i t o t ox i n ,monosodium glutamate, traverses the placenta and causes fetalneurotoxicity in rodents.277

Higher NO is a fact of autism which may provide clues tospecific etiologies, aggravants, and tre atments. Vi ral infectionscan increase greatly NO production in brain and other tissues,so higher NO production in autism makes systematic examina-tion of autistic brain and other tissues for viral antigen moreurgent than ever.

Higher NO in autism might productively focus at t e n t i o non treatment with antioxidants with specificity for NO. VitaminC is a good NO-quencher,278 and so are melatonin and uric acid.M e l atonin effectively scavenges both NO and ONOO.2 7 9

Melatonin has excellent potential for relief of oxidative stress inboth brain and gut,2 8 0 -2 81 i n c reases expression of GSHPx,2 8 2 a n dhas proven effective in autistic sleep disorders.97

Uric acid normally re p resents up to 60% of total plasmaantioxidant capacity.283 It effectively binds transition metals andreactive species, and is especially effective quenching NO31 andO N O O.2 8 4 C a reful upw a rd titration of uric acid levels with ora linosine, a uric acid pre c u r s o r, may be of benefit in high-NOstates such as multiple sclerosis,45 and autism.

Testing and tre ating mitochondrial function to improv eenergy production probably merits a higher priority in autism.Ac e t y l- L -carnitine (ALC) and a -l ipoic acid (ALA ) e n h a n c emitochondrial function and reduce ox i d ative stress in senes-cent animals.11 9 Oral administration of the mitochondrialmetabolite, L-carnitine, has improved behavior in childre nwith Rett syndro m e ,2 8 5 and initial testing of high carnitinedoses in autism is underw ay.

One university center often treats patients referred for sus-pected mitochondrial disease with a combination of CoQ10, vita-min E, and balanced B vitamins.2 8 6 C o Q 10, in combination oralone, is an at t ractive potential intervention in autism. It facili-t ates ATP production by carrying electrons and protons in the

electron transport chain, and also acts in its reduced form to pro-tect mitochondria by quenching oxidants.287 Vitamin B3 is crucialto mitochondrial energy production and effective in the high-ox i d ative state of schizophre n i a ,2 8 8 but has received little at t e n-tion in autism.

The clinical re l e vance of enzyme, re c e p t o r, G- p rotein, and vit-amin cofactor sensitivity to ox i d ative stress (Table 8) is uninvesti-g ated. Gl u c o s e - 6 - p h o s p h ate dehyd rogenase (G-6-PD) activity,given its central role in the reduction of GSH, is but one of manyox i d a n t-sensitive functions which deserve scrutiny in autism.

OV E RV I EWThe data demonstrate gre ater ox i d ative stress in autism.

The clinical response to antioxidant nutrients suggests thatox i d ative stress is important in the expression of autistic symp-toms. The question is whether ox i d ative stress is very impor-tant mechanistically.

Antioxidant therapeutic trials, which measure oxidative bio-m a rkers, could help elucidate the importance of an ox i d at i v emechanism. While we wait for this research, clinicians and par-ents are advised to implement safe nutritional interv e n t i o n s s o o n e r, rather than lat e r. It would not seem pre m at u re to startapplying laborat o ry biomarkers for ox i d ative stress to optimizedoses and combinations of nutrients.

The preliminary lipofuscin data are potentially very impor-tant, and replicative and expansive studies should be performedas soon as possible. It is conceivable that lipofuscin analysis willidentify specific toxic or infectious etiology. At the very least,lipofuscin is a strong hint that neurodevelopment in autism maybe altered by oxidative influences.

In this respect, chronic neonatal vitamin E deficiency mayhelp us understand the potential effect of excess ox i d ative stre s s

TABLE 8 Sensitive to ox i d ative / nitro s ative degra d at i o n

Enzyme or Co-Factor Reference

Glutamic acid decarboxylase (142)Glutamate transporter (144)Glutamine synthetase (143)(227)GABA channels (289)

B6 vitamers (184)(185)(186) Pyridoxal kinase by carbonyl inhibition (174)B6-dependent enzymes by carbonyl (290)

inhibitionTetrahydrofolate (241)(244)Methionine synthase (291)B12 vitamers (237)(238)(239)(240)

(241)Glucose-6-phosphate dehydrogenase (292)Coenzyme A (78)a -KGDHC (31)(250)(251) Na,K-ATPase, Ca++ channels, Aquaporin (207)(227)Catalase (293)

Glutathione peroxidase (182)


on neurodevelopment. Vitamin E deficiency is a neurological dis-ease which clearly results from low antioxidant protection fro mb i rt h .2 9 4 ,2 9 5 Lipofuscin deposition is pro m i n e n t .2 9 4 ,2 9 6 Ne u ro l o g i c a lsymptomsgait disturbance, abnormal ocular movements pre-sent at 18 -24 months,2 9 4 the same chronology as autistic re g re s s i o n.

Beyond analogy, vitamin E may have concrete implicationsin autism or in healthy children prior to regression. Neurologicalc o m p l i c ations of vitamin E deficiency are seen in patients withcommon variable immune deficiency (CVID) and entero p at h y,and vitamin E screening has been recommended for pat i e n t swith these conditions.297 The immune profile in autism approxi-m ates CVID,2 9 6 and there is no question about entero p at h y.Preliminary data suggest lower plasma vitamin E levels in autis-tic childre n .2 6 We need more vitamin E assessment, includingfunctional testing by red-cell hemolysis.

Optimistically, it is noted that oxidative damage to biomole-cules is often at least partially reversible. Oxidative inactivat i o nof enzymes, for instance, is reversed when sufficient antioxidantis provided.174 Even structural elements such as cytoskeleton canundergo restoration by GSH.299

If we learn that oxidative stress is an important mechanismin autism, then our search for the genetic and enviro n m e n t a lcauses becomes much more focused. From the oxidative wounds,our science may more rapidly deduce the cause, treatment, andprevention of autism.

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