Potential Role of Glutathione in Autism Spectrum Disorders
Potential Role of Glutathione in Autism Spectrum Disorders
Sixty six abstracts were identified via the electronic and hand search strategy. Of these, 24 were ineligible for inclusion. Reasons for exclusion were: 1) the paper did not contain any relevant data; 2) the data was already published in another article identified in the search; 3) data did not include the proband with an autism spectrum disorder; 4) the paper was a review article, conference abstract or comment on a previously published article; 5) the authors did not separate data for autism spectrum disorders from other psychological conditions; or 6) they were not English language articles with the exception of a seminal French study widely referred to in English language papers (Figure 3).
(Enlarge Image)
Figure 3.
Flow diagram of research papers retrieved for potential inclusion in our study.
Forty two studies were included in the review (41 that met the inclusion criteria plus the French study). Of these, one provided data obtained from in vitro models of γ-glutamyl cycle metabolites, twenty nine provided data on metabolites and/or co-factors of the γ-glutamyl cycle or trans-sulphuration pathway, six provided the results of intervention studies, six included genetic data and eight studies provided data on enzyme activity.
An overview of the studies included in this review is presented in Table 3. The level of evidence, study size and ascertainment of cases and controls are indicated along with a quality assessment score and/or assessment of risk of bias. Most studies were of the case control design, however, additionally there were two double blinded and one open labelled randomised controlled trial, a case series and a case report.
An assessment of study quality is presented in Table 4, Table 5, Table 6 and Table 7. The case definition used to include participants in the studies varied over time. The case definition for autistic disorder was not standardised until 1980 when it was included in the DSM-III. Asperger's Syndrome and PDD-NOS were added to the DSM-IV in 1994 which broadened the definition to include many children who were previously undiagnosed. While early studies centred on cases obtained from institutionalised psychiatric settings, cases were later recruited through internal research registers, multiple centres or community advertisements. Although diagnosis was independently confirmed in several studies, most relied on medical records or parent reports. None of the studies had used a structured sampling frame for case ascertainment making them prone to selection bias. Information about case ascertainment was not provided for eight studies.
Ascertainment and definition of controls also varied widely. While two studies sourced their controls by community advertising, most were sourced from hospitals, clinics or schools and fourteen studies did not provide information on the source of their controls. With respect to definition of controls, most studies recruited healthy children with no information about family history of autism spectrum disorders, although, four studies did ensure that controls did not have either a family history or sibling with autism and one screened for autism traits. At the other end of the scale, controls for four studies were poorly defined potentially biasing the results. Control values from one of these studies were used for two later studies. Additionally, three studies relied on laboratory reference ranges.
Gender is a potential confounder in studies of autistic disorder because the condition is four times more common in males than females. Only five studies were gender matched, four did not provide the gender of cases or controls and nine provided the gender of cases but not controls. Age may also be a potential confounder as serum glutamate was elevated in adults with autistic disorder compared to adult controls but was not significantly different in children with autistic disorder compared to child controls. In contrast, serum glycine and serine were not significantly different in either adults or children when levels in autistic disorder were compared to controls. One study included a range of participants from childhood to early adulthood, however, the findings were not stratified according to age.
All studies included in the review treated cases and controls equally. Laboratory blinding as to case and control status occurred for only one research group, although others were blinded to case status but not controls, for example, where the laboratory provided the control data or reference ranges or where another study was used for controls. Most studies did not state whether the laboratory was blinded.
Genetic studies were assessed for quality using the Newcastle Ottawa Scale plus additional criteria that included consideration of Hardy Weinberg equilibrium, power of the study, population stratification and correction for multiple comparisons. All except one of the six genetic studies considered Hardy Weinberg equilibrium, two provided power calculations and two adjusted for multiple comparisons (although a footnote indicating that the associations were no longer statistically significant was not added in one case). While population stratification is not relevant for transmission linkage studies, neither of the remaining studies were adjusted for this.
Both of the double blinded randomised intervention trials provided information about concealment and the laboratory was blinded thereby reducing performance and detection bias. Neither provided information about the randomisation process, complete outcome data and full reporting of results. While Bertoglio et al. 2010 state that 30 children completed the 12-week trial, closer inspection of the paper suggests that at least 32 children started the trial (see Table 1 in Bertoglio et al. 2010), however, no information on dropout or loss to follow-up was provided. Furthermore outcome data was only provided for the 'responder' sub-group in a form that was difficult to interpret. Adams et al. 2009 randomised children to receive either topical glutathione or a placebo before being given one round of a chelating agent with erythrocyte glutathione tested at baseline and 1–2 months following the intervention. It is not clear whether it is a typographical error, however, Table 1 of the study states that 77 children participated in the first phase of the study, but baseline data for erythrocyte glutathione is only provided for 72 children. Although the paper states that 49 started the second phase of the study and therefore, according to the protocol, had a second glutathione measurement, pre- and post-intervention erythrocyte glutathione is only provided for 38 participants with no comparison between the two arms of the study with levels being compared to an adult reference range provided by the laboratory. The second phase of the study involved 'high excreters' of urinary metal ions being given a further 6 rounds of chelation if allocated to the topical glutathione arm or 6 rounds of placebo if previously allocated to the topical placebo arm of the study. Erythrocyte glutathione was not measured at the completion of the second phase of the study.
The open-label study design used in the remaining three intervention studies left them at high risk of selection, performance and detection bias, however, all studies provided complete outcome data and full reporting of results.
A kappa score of 0.87 was obtained which indicates a high level of agreement between raters for the assessment of quality of articles.
Table 8 summarises the findings of an in vitro study of γ-glutamyl cycle metabolites. Decreased free glutathione (fGSH) and increased GSSG were observed in both cytosol and mitochondrial extracts obtained from lymphoblastoid cell lines derived from children with autistic disorder compared to unaffected controls resulting in a decreased GSH:GSSG. Exposure to physiological levels of nitrosative stress showed no difference in the magnitude of GSH:GSSG from cells derived from children with autistic disorder compared to healthy controls, however, the baseline GSH:GSSG was significantly lower (by 30%) in cells from children with autistic disorder.
Data from key studies of metabolites of the γ-glutamyl cycle and trans-sulphuration pathway is shown in Figures 4, 5 and 6 and a summary of additional studies presented in Table 9.
(Enlarge Image)
Figure 4.
Meta-analysis of studies that compared plasma homocysteine in children with autism spectrum disorders to healthy controls.
(Enlarge Image)
Figure 5.
Heterogeneity of studies that compared plasma cystathione in children with autistic spectrum disorder and healthy controls.
(Enlarge Image)
Figure 6.
Heterogeneity of studies that compared plasma cysteine in children with autistic spectrum disorder and healthy controls.
The largest and most comprehensive study to date provided data for multiple metabolites of the γ-glutamyl cycle and trans-sulphuration pathway. This study reported significantly lower levels of GSH (by 32%) and higher levels of GSSG (by 66%) in plasma of children with autistic disorder compared to controls, together with significantly lower homocysteine and cysteine levels, while cystathione levels were significantly higher and cysteinyl-glycine levels were not significantly different. These findings confirm those of an earlier pilot study by the same researchers with the exception that cystathione was found to be lower in children with autistic disorder in the pilot study, as well as a later study by the same research group which focussed on a subgroup of children with autistic disorder who had abnormal methylation and/or GSH:GSSG.
Plasma homocysteine levels for the above studies showed that there was no statistically significant difference between children with autistic disorder and controls which has been replicated by a number of other research groups for children with autistic disorder, PDD-NOS and Asperger's syndrome as well as a mixed sample of children with autism spectrum disorders. The only study to report a significant increase in plasma homocysteine in children with autistic disorder was not replicated by the same research group using a fasted sample. Examination of statistical heterogeneity showed low heterogeneity overall (I = 34%) and no heterogeneity between diagnostic subgroups (I = 0%) (Figure 4). Meta-analysis resulted in a standardised mean difference (SMD) of −0.18 (95%CI −0.46−0.10) across 199 cases and 185 controls using a random effects model. Data from James et al. 2009 was not included in the analysis because the cases were selected for low methylation ratio or GSH:GSSG, however, the data is presented in Figure 4.
Similarly, no significant difference was observed in plasma cystathione from children with autistic disorder, PDD-NOS, Asperger's Syndrome or mixed autism spectrum disorders, although another study report it to be significantly higher in children with autistic disorder than controls (Figure 5). Examination of statistical heterogeneity showed that there was substantial overall heterogeneity (I = 70%) with moderate heterogeneity between diagnostic subgroups (I = 41.4%). It is hard to explain the heterogeneity given that two of the larger studies were conducted by the same research group using the same methodology.
Serine is required for synthesis of cystathione from homocysteine. Four studies found no significant difference in serum or plasma serine levels between children and adults with or without autistic disorder, PDD-NOS, Asperger's Syndrome or autism spectrum disorders (mixed sample), one study showed a trend towards a decrease in children with autistic disorder and another reported significantly increased plasma serine in children with autism spectrum disorders and significantly lower levels of serine were reported for platelet poor plasma in autism spectrum disorders. Factors that may have contributed to the heterogeneity between studies include fasting status, differing laboratory methods and varied selection of controls as well as correction for multiple comparisons.
Studies showing that plasma cysteine is significantly lower in children with autistic disorder are dominated by one research group that published three studies (one in children with abnormal methylation or GSH:GSSG) and their findings have been replicated by another research group. The same study found no significant difference in plasma cysteine levels for children with PDD-NOS or Asperger's Syndrome, as did a study comparing autism spectrum disorders (sample composition unknown) compared to controls. Plasma cysteine was significantly lower in a study comprising 28 children with autistic disorder and 10 children with PDD-NOS. Serum cysteine levels of children with autistic disorder compared to controls were not significantly different from controls. Overall statistical heterogeneity for plasma cysteine was considerable (I = 92%) and low to moderate between diagnostic subgroups (I = 39.8%). Again, factors that may have led to the high level of heterogeneity between studies include fasting status, differing laboratory methods and varied selection of controls.
A significant decrease in plasma total glutathione (tGSH) reported in four studies from the one research group in children with autistic disorder compared to controls have been confirmed by another two research groups with respect to autistic disorder as well as study of low functioning children with autism spectrum disorders (Figure 7). Reduced glutathione has also been reported to be lower in the plasma of children with autism spectrum disorders. In contrast, no significant difference for plasma tGSH or erythrocyte tGSH was reported for autism spectrum disorders (mixed diagnoses). The later study compared cases to an adult reference range while noting that the paediatric range is lower. Whole blood tGSH was reported to be lower in autistic disorder but not significantly different for PDD-NOS or Asperger's Disorder. Overall statistical heterogeneity was substantial for plasma tGSH (I = 93%) however there was no statistical heterogeneity between diagnostic sub-groups (I = 0%). Varying definition of cases and controls, laboratory and analytical methods may account for the range of heterogeneity.
(Enlarge Image)
Figure 7.
Heterogeneity of studies that compared tGSH in children with autistic spectrum disorder and healthy controls.
The same major research group published four studies showing a significant increase in plasma oxidised glutathione in autistic disorder which has been replicated by a further two research groups for autism spectrum disorders (Figure 8). Overall statistical heterogeneity was substantial (I = 67%), however, there was no statistical heterogeneity between diagnostic subgroups (I = 0%). Meta-analysis resulted in a SMD of 1.25 (95% CI 0.87–1.62) across 203 cases and 184 controls using a random effects model. As stated above, data from James et al. 2009 was not included in the analysis but is included in the tables accompanying the Figure.
(Enlarge Image)
Figure 8.
Meta-analysis of studies that compared GSSG in children with autistic spectrum disorder and healthy controls.
The same research group published 5 studies reporting significantly lower plasma tGSH:GSSG ratios in children with autistic disorder. The same controls were used for three of the studies. One of these studies tGSH and tGSH:GSSG were significantly lower (by 10.7% and 14.9% respectively) in children with autism who were heterozygous or homozygous for the delta aminolevulinic acid dehydratase (ALAD) 177 GC mutation, whereas there was no difference in GSSG. This polymorphism is found in the heme biosynthesis pathway where it has been associated with altered toxicokinetics of lead levels and elevated blood levels of lead. Three studies have shown that GSH:GSSG is lower in children with autism spectrum disorders lending credence to the original findings.
The findings for cysteinyl-glycine, a breakdown product of glutathione, were inconsistent. Initially it was reported that there was no significant difference in cysteinyl-glycine in children with autistic disorder compared to controls. A later study by the same research group was limited to children with abnormal methylation or GSH:GSSG showed that it had a significantly lower level in autistic disorder than in controls, however, a subsequent study of children with autism spectrum disorders showed that it was significantly higher than in controls. Differences in inclusion criteria, laboratory methods and control of confounding variables may account for the difference in the findings.
Finally, the relationship between GSH and the immune system was clearly demonstrated in a large study which showed that children with autistic disorder universally have lower natural killer cell activity in peripheral mononuclear cells than those without the disorder which correlated with low intracellular levels of GSH as shown in Table 10. Furthermore, when GSH was added to the culture medium, natural killer cell activity increased more in lymphocytes obtained from children with low natural killer cell activity than those with normal activity. Corresponding data were not provided for control children.
Glycine and glutamine are key compounds for the biosynthesis of glutathione obtained through dietary sources. Eight studies showed no significant difference in serum or plasma glycine in children with autistic disorder, autism spectrum disorders (mixed diagnoses) PDD-NOS or Asperger's Syndrome compared to controls. There was no statistical heterogeneity overall (I = 0) or between diagnoses (I = 0) for plasma glycine. The data was not pooled because there were only two studies for plasma and two for serum with data in a suitable form to combine.
In contrast the findings for glutamine were inconsistent with two studies reporting no significant difference in serum glutamine in children with autistic disorder compared with controls, a later study reporting a significant decrease in platelet glutamine in children with autistic disorder and further studies that reported serum glutamate to be significantly higher in adults with autistic disorder and children with autism spectrum disorders (mixed diagnoses) than controls.
Of the six studies that measured co-factors of the γ-glutamyl cycle or trans-sulphuration pathway, five studies showed elevated levels of vitamin B6 in children with autistic disorder or autism spectrum disorders compared to controls, one showed a decrease in erythrocyte selenium in children with autistic disorder and another showed no change in whole blood selenium in children with an autism spectrum disorder compared to controls.
The findings of the six studies that report the outcome of interventions in autism focussed on normalising abnormalities in γ-glutamyl cycle or trans-sulphuration pathway metabolites are presented in Table 11. An initial pilot study showed that supplementation of children with autistic disorder with 800 μg folinic acid and 1,000 μg betaine per day for two months normalised homocysteine levels and improved GSH:GSSG. The addition of 75 μg/kg methyl-cobalamin injected twice weekly for one month further normalised GSH:GSSG. The same researchers conducted a larger intervention in 42 children with autistic disorder who had evidence of reduced methylation capacity or GSH:GSSG in which they were supplemented with folinic acid and methyl-cobalamin for 3 months. The new regimen, which used half the dose of folinic acid of that used in the pilot study, resulted in significant increases in metabolites of the trans-sulphuration pathway as well as GSH:GSSG, although they remained below those of the control children. Objective behavioural measures showed an improvement, although all participants were still well below normal (data not published).
Recently, a double blinded randomised controlled trial was published in which participants were administered either methyl-cobalamin or placebo for 6 weeks and then their treatment switched without washout for a further 6 weeks. Overall, there was no significant change in GSH, GSH:GSSG or behaviour. Thirty percent of participants, however, showed a significant improvement in objective behavioural measures which correlated with improved plasma GSH and GSH:GSSG levels. Interpretation of the findings is difficult because data was only provided for the 'responder' subgroup and this did not include standard deviations or units for plasma GSH or GSH:GSSG, nor did it state whether the GSH values reported in Figure 4 of their paper represented tGSH or reduced glutathione. Furthermore, data showing whether 'responders' had lower baseline concentrations of GSH or GSH:GSSG were not provided.
Additionally, a case report of a child with autistic disorder and cerebral folate deficiency showed the normalisation of low cerebral spinal fluid homocysteine following 2 weeks supplementation with 0.5 mg folinic acid/kg/day increasing to 1.0/kg/day for 3 months. Finally, a 40 session trial of hyperbaric oxygen therapy showed that it has no effect on plasma GSH:GSSG in children with autistic disorder and, as discussed above, incomplete data and selective reporting make it hard to interpret the findings of a randomised trial of topical glutathione before chelation.
The six studies that presented data on genetic polymorphisms of the γ-glutamyl cycle or trans-sulphuration pathway are summarised in Table 12. The best powered of these studies examined genetic variation in 42 genes (308 single nucleotide polymorphisms (SNPs)) related to glutathione, including those coding for enzymes that use glutathione as a co-factor (not included in this review), in 318 families from the Autism Genetic Resource Exchange repository. Several SNPs located in the genes for cystathionine γ-ligase (CTH), alcohol dehydrogenase 5, GCL and glutaredoxin showed significant or suggestive associations with autism spectrum disorders. Interaction models confirmed a significant association between CTH, glutaredoxin and glutaredoxin 3 and autism (OR = 3.78 (95% CI 2.36–6.04).
The study found no association between any of the GST genes and autism. This is in contrast to a previous study of case parent trios that found that homozygote cases for the GST-M*1 gene deletion (GST-M1*0) showed increased risk of autistic disorder and a later case control study that reported a borderline association between the GST-M*1 gene deletion (GST-M1*0) and autistic disorder and a significant interaction between the GST-M1*0 deletion and the reduced folate carrier 80A > G. Previous studies failed to find an association between GST-T1 or GST-P1 and autism.
Ming et al. 2010 Found that a polyalanine repeat polymorphism in the GPx gene (GPx-1) was associated with autistic disorder. Under-transmission of the variation encoding six alanine residues (ALA6) was observed in the families with autistic disorder, suggesting that this allele may be protective. The authors acknowledge that their interpretation is limited by inadequate knowledge of the function of the ALA alleles of GPx-1 gene.
As shown in Table 13 GPx-1 activity has been the subject of seven studies. The findings were inconsistent in plasma where two studies reported higher activity in cases than controls and two reported lower activity. A further four studies examined GPx-1 activity in erythrocytes. Of these, two reported lower activity and two reported no significant difference between cases and controls. No significant difference between cases and controls was reported for GPx-1 activity in platelets. In addition, a recently published study showed that glutathione-S-transferase activity was significantly reduced in children with low functioning autism spectrum disorders and there was a trend towards lower activity of glutathione reductase.
Results
Sixty six abstracts were identified via the electronic and hand search strategy. Of these, 24 were ineligible for inclusion. Reasons for exclusion were: 1) the paper did not contain any relevant data; 2) the data was already published in another article identified in the search; 3) data did not include the proband with an autism spectrum disorder; 4) the paper was a review article, conference abstract or comment on a previously published article; 5) the authors did not separate data for autism spectrum disorders from other psychological conditions; or 6) they were not English language articles with the exception of a seminal French study widely referred to in English language papers (Figure 3).
(Enlarge Image)
Figure 3.
Flow diagram of research papers retrieved for potential inclusion in our study.
Forty two studies were included in the review (41 that met the inclusion criteria plus the French study). Of these, one provided data obtained from in vitro models of γ-glutamyl cycle metabolites, twenty nine provided data on metabolites and/or co-factors of the γ-glutamyl cycle or trans-sulphuration pathway, six provided the results of intervention studies, six included genetic data and eight studies provided data on enzyme activity.
An overview of the studies included in this review is presented in Table 3. The level of evidence, study size and ascertainment of cases and controls are indicated along with a quality assessment score and/or assessment of risk of bias. Most studies were of the case control design, however, additionally there were two double blinded and one open labelled randomised controlled trial, a case series and a case report.
An assessment of study quality is presented in Table 4, Table 5, Table 6 and Table 7. The case definition used to include participants in the studies varied over time. The case definition for autistic disorder was not standardised until 1980 when it was included in the DSM-III. Asperger's Syndrome and PDD-NOS were added to the DSM-IV in 1994 which broadened the definition to include many children who were previously undiagnosed. While early studies centred on cases obtained from institutionalised psychiatric settings, cases were later recruited through internal research registers, multiple centres or community advertisements. Although diagnosis was independently confirmed in several studies, most relied on medical records or parent reports. None of the studies had used a structured sampling frame for case ascertainment making them prone to selection bias. Information about case ascertainment was not provided for eight studies.
Ascertainment and definition of controls also varied widely. While two studies sourced their controls by community advertising, most were sourced from hospitals, clinics or schools and fourteen studies did not provide information on the source of their controls. With respect to definition of controls, most studies recruited healthy children with no information about family history of autism spectrum disorders, although, four studies did ensure that controls did not have either a family history or sibling with autism and one screened for autism traits. At the other end of the scale, controls for four studies were poorly defined potentially biasing the results. Control values from one of these studies were used for two later studies. Additionally, three studies relied on laboratory reference ranges.
Gender is a potential confounder in studies of autistic disorder because the condition is four times more common in males than females. Only five studies were gender matched, four did not provide the gender of cases or controls and nine provided the gender of cases but not controls. Age may also be a potential confounder as serum glutamate was elevated in adults with autistic disorder compared to adult controls but was not significantly different in children with autistic disorder compared to child controls. In contrast, serum glycine and serine were not significantly different in either adults or children when levels in autistic disorder were compared to controls. One study included a range of participants from childhood to early adulthood, however, the findings were not stratified according to age.
All studies included in the review treated cases and controls equally. Laboratory blinding as to case and control status occurred for only one research group, although others were blinded to case status but not controls, for example, where the laboratory provided the control data or reference ranges or where another study was used for controls. Most studies did not state whether the laboratory was blinded.
Genetic studies were assessed for quality using the Newcastle Ottawa Scale plus additional criteria that included consideration of Hardy Weinberg equilibrium, power of the study, population stratification and correction for multiple comparisons. All except one of the six genetic studies considered Hardy Weinberg equilibrium, two provided power calculations and two adjusted for multiple comparisons (although a footnote indicating that the associations were no longer statistically significant was not added in one case). While population stratification is not relevant for transmission linkage studies, neither of the remaining studies were adjusted for this.
Both of the double blinded randomised intervention trials provided information about concealment and the laboratory was blinded thereby reducing performance and detection bias. Neither provided information about the randomisation process, complete outcome data and full reporting of results. While Bertoglio et al. 2010 state that 30 children completed the 12-week trial, closer inspection of the paper suggests that at least 32 children started the trial (see Table 1 in Bertoglio et al. 2010), however, no information on dropout or loss to follow-up was provided. Furthermore outcome data was only provided for the 'responder' sub-group in a form that was difficult to interpret. Adams et al. 2009 randomised children to receive either topical glutathione or a placebo before being given one round of a chelating agent with erythrocyte glutathione tested at baseline and 1–2 months following the intervention. It is not clear whether it is a typographical error, however, Table 1 of the study states that 77 children participated in the first phase of the study, but baseline data for erythrocyte glutathione is only provided for 72 children. Although the paper states that 49 started the second phase of the study and therefore, according to the protocol, had a second glutathione measurement, pre- and post-intervention erythrocyte glutathione is only provided for 38 participants with no comparison between the two arms of the study with levels being compared to an adult reference range provided by the laboratory. The second phase of the study involved 'high excreters' of urinary metal ions being given a further 6 rounds of chelation if allocated to the topical glutathione arm or 6 rounds of placebo if previously allocated to the topical placebo arm of the study. Erythrocyte glutathione was not measured at the completion of the second phase of the study.
The open-label study design used in the remaining three intervention studies left them at high risk of selection, performance and detection bias, however, all studies provided complete outcome data and full reporting of results.
A kappa score of 0.87 was obtained which indicates a high level of agreement between raters for the assessment of quality of articles.
In Vitro Studies of the γ-glutamyl Cycle
Table 8 summarises the findings of an in vitro study of γ-glutamyl cycle metabolites. Decreased free glutathione (fGSH) and increased GSSG were observed in both cytosol and mitochondrial extracts obtained from lymphoblastoid cell lines derived from children with autistic disorder compared to unaffected controls resulting in a decreased GSH:GSSG. Exposure to physiological levels of nitrosative stress showed no difference in the magnitude of GSH:GSSG from cells derived from children with autistic disorder compared to healthy controls, however, the baseline GSH:GSSG was significantly lower (by 30%) in cells from children with autistic disorder.
Metabolites and Cofactors of the γ-glutamyl Cycle and Trans-sulphuration Pathway
Data from key studies of metabolites of the γ-glutamyl cycle and trans-sulphuration pathway is shown in Figures 4, 5 and 6 and a summary of additional studies presented in Table 9.
(Enlarge Image)
Figure 4.
Meta-analysis of studies that compared plasma homocysteine in children with autism spectrum disorders to healthy controls.
(Enlarge Image)
Figure 5.
Heterogeneity of studies that compared plasma cystathione in children with autistic spectrum disorder and healthy controls.
(Enlarge Image)
Figure 6.
Heterogeneity of studies that compared plasma cysteine in children with autistic spectrum disorder and healthy controls.
The largest and most comprehensive study to date provided data for multiple metabolites of the γ-glutamyl cycle and trans-sulphuration pathway. This study reported significantly lower levels of GSH (by 32%) and higher levels of GSSG (by 66%) in plasma of children with autistic disorder compared to controls, together with significantly lower homocysteine and cysteine levels, while cystathione levels were significantly higher and cysteinyl-glycine levels were not significantly different. These findings confirm those of an earlier pilot study by the same researchers with the exception that cystathione was found to be lower in children with autistic disorder in the pilot study, as well as a later study by the same research group which focussed on a subgroup of children with autistic disorder who had abnormal methylation and/or GSH:GSSG.
Plasma homocysteine levels for the above studies showed that there was no statistically significant difference between children with autistic disorder and controls which has been replicated by a number of other research groups for children with autistic disorder, PDD-NOS and Asperger's syndrome as well as a mixed sample of children with autism spectrum disorders. The only study to report a significant increase in plasma homocysteine in children with autistic disorder was not replicated by the same research group using a fasted sample. Examination of statistical heterogeneity showed low heterogeneity overall (I = 34%) and no heterogeneity between diagnostic subgroups (I = 0%) (Figure 4). Meta-analysis resulted in a standardised mean difference (SMD) of −0.18 (95%CI −0.46−0.10) across 199 cases and 185 controls using a random effects model. Data from James et al. 2009 was not included in the analysis because the cases were selected for low methylation ratio or GSH:GSSG, however, the data is presented in Figure 4.
Similarly, no significant difference was observed in plasma cystathione from children with autistic disorder, PDD-NOS, Asperger's Syndrome or mixed autism spectrum disorders, although another study report it to be significantly higher in children with autistic disorder than controls (Figure 5). Examination of statistical heterogeneity showed that there was substantial overall heterogeneity (I = 70%) with moderate heterogeneity between diagnostic subgroups (I = 41.4%). It is hard to explain the heterogeneity given that two of the larger studies were conducted by the same research group using the same methodology.
Serine is required for synthesis of cystathione from homocysteine. Four studies found no significant difference in serum or plasma serine levels between children and adults with or without autistic disorder, PDD-NOS, Asperger's Syndrome or autism spectrum disorders (mixed sample), one study showed a trend towards a decrease in children with autistic disorder and another reported significantly increased plasma serine in children with autism spectrum disorders and significantly lower levels of serine were reported for platelet poor plasma in autism spectrum disorders. Factors that may have contributed to the heterogeneity between studies include fasting status, differing laboratory methods and varied selection of controls as well as correction for multiple comparisons.
Studies showing that plasma cysteine is significantly lower in children with autistic disorder are dominated by one research group that published three studies (one in children with abnormal methylation or GSH:GSSG) and their findings have been replicated by another research group. The same study found no significant difference in plasma cysteine levels for children with PDD-NOS or Asperger's Syndrome, as did a study comparing autism spectrum disorders (sample composition unknown) compared to controls. Plasma cysteine was significantly lower in a study comprising 28 children with autistic disorder and 10 children with PDD-NOS. Serum cysteine levels of children with autistic disorder compared to controls were not significantly different from controls. Overall statistical heterogeneity for plasma cysteine was considerable (I = 92%) and low to moderate between diagnostic subgroups (I = 39.8%). Again, factors that may have led to the high level of heterogeneity between studies include fasting status, differing laboratory methods and varied selection of controls.
A significant decrease in plasma total glutathione (tGSH) reported in four studies from the one research group in children with autistic disorder compared to controls have been confirmed by another two research groups with respect to autistic disorder as well as study of low functioning children with autism spectrum disorders (Figure 7). Reduced glutathione has also been reported to be lower in the plasma of children with autism spectrum disorders. In contrast, no significant difference for plasma tGSH or erythrocyte tGSH was reported for autism spectrum disorders (mixed diagnoses). The later study compared cases to an adult reference range while noting that the paediatric range is lower. Whole blood tGSH was reported to be lower in autistic disorder but not significantly different for PDD-NOS or Asperger's Disorder. Overall statistical heterogeneity was substantial for plasma tGSH (I = 93%) however there was no statistical heterogeneity between diagnostic sub-groups (I = 0%). Varying definition of cases and controls, laboratory and analytical methods may account for the range of heterogeneity.
(Enlarge Image)
Figure 7.
Heterogeneity of studies that compared tGSH in children with autistic spectrum disorder and healthy controls.
The same major research group published four studies showing a significant increase in plasma oxidised glutathione in autistic disorder which has been replicated by a further two research groups for autism spectrum disorders (Figure 8). Overall statistical heterogeneity was substantial (I = 67%), however, there was no statistical heterogeneity between diagnostic subgroups (I = 0%). Meta-analysis resulted in a SMD of 1.25 (95% CI 0.87–1.62) across 203 cases and 184 controls using a random effects model. As stated above, data from James et al. 2009 was not included in the analysis but is included in the tables accompanying the Figure.
(Enlarge Image)
Figure 8.
Meta-analysis of studies that compared GSSG in children with autistic spectrum disorder and healthy controls.
The same research group published 5 studies reporting significantly lower plasma tGSH:GSSG ratios in children with autistic disorder. The same controls were used for three of the studies. One of these studies tGSH and tGSH:GSSG were significantly lower (by 10.7% and 14.9% respectively) in children with autism who were heterozygous or homozygous for the delta aminolevulinic acid dehydratase (ALAD) 177 GC mutation, whereas there was no difference in GSSG. This polymorphism is found in the heme biosynthesis pathway where it has been associated with altered toxicokinetics of lead levels and elevated blood levels of lead. Three studies have shown that GSH:GSSG is lower in children with autism spectrum disorders lending credence to the original findings.
The findings for cysteinyl-glycine, a breakdown product of glutathione, were inconsistent. Initially it was reported that there was no significant difference in cysteinyl-glycine in children with autistic disorder compared to controls. A later study by the same research group was limited to children with abnormal methylation or GSH:GSSG showed that it had a significantly lower level in autistic disorder than in controls, however, a subsequent study of children with autism spectrum disorders showed that it was significantly higher than in controls. Differences in inclusion criteria, laboratory methods and control of confounding variables may account for the difference in the findings.
Finally, the relationship between GSH and the immune system was clearly demonstrated in a large study which showed that children with autistic disorder universally have lower natural killer cell activity in peripheral mononuclear cells than those without the disorder which correlated with low intracellular levels of GSH as shown in Table 10. Furthermore, when GSH was added to the culture medium, natural killer cell activity increased more in lymphocytes obtained from children with low natural killer cell activity than those with normal activity. Corresponding data were not provided for control children.
Glycine and glutamine are key compounds for the biosynthesis of glutathione obtained through dietary sources. Eight studies showed no significant difference in serum or plasma glycine in children with autistic disorder, autism spectrum disorders (mixed diagnoses) PDD-NOS or Asperger's Syndrome compared to controls. There was no statistical heterogeneity overall (I = 0) or between diagnoses (I = 0) for plasma glycine. The data was not pooled because there were only two studies for plasma and two for serum with data in a suitable form to combine.
In contrast the findings for glutamine were inconsistent with two studies reporting no significant difference in serum glutamine in children with autistic disorder compared with controls, a later study reporting a significant decrease in platelet glutamine in children with autistic disorder and further studies that reported serum glutamate to be significantly higher in adults with autistic disorder and children with autism spectrum disorders (mixed diagnoses) than controls.
Of the six studies that measured co-factors of the γ-glutamyl cycle or trans-sulphuration pathway, five studies showed elevated levels of vitamin B6 in children with autistic disorder or autism spectrum disorders compared to controls, one showed a decrease in erythrocyte selenium in children with autistic disorder and another showed no change in whole blood selenium in children with an autism spectrum disorder compared to controls.
Intervention Studies
The findings of the six studies that report the outcome of interventions in autism focussed on normalising abnormalities in γ-glutamyl cycle or trans-sulphuration pathway metabolites are presented in Table 11. An initial pilot study showed that supplementation of children with autistic disorder with 800 μg folinic acid and 1,000 μg betaine per day for two months normalised homocysteine levels and improved GSH:GSSG. The addition of 75 μg/kg methyl-cobalamin injected twice weekly for one month further normalised GSH:GSSG. The same researchers conducted a larger intervention in 42 children with autistic disorder who had evidence of reduced methylation capacity or GSH:GSSG in which they were supplemented with folinic acid and methyl-cobalamin for 3 months. The new regimen, which used half the dose of folinic acid of that used in the pilot study, resulted in significant increases in metabolites of the trans-sulphuration pathway as well as GSH:GSSG, although they remained below those of the control children. Objective behavioural measures showed an improvement, although all participants were still well below normal (data not published).
Recently, a double blinded randomised controlled trial was published in which participants were administered either methyl-cobalamin or placebo for 6 weeks and then their treatment switched without washout for a further 6 weeks. Overall, there was no significant change in GSH, GSH:GSSG or behaviour. Thirty percent of participants, however, showed a significant improvement in objective behavioural measures which correlated with improved plasma GSH and GSH:GSSG levels. Interpretation of the findings is difficult because data was only provided for the 'responder' subgroup and this did not include standard deviations or units for plasma GSH or GSH:GSSG, nor did it state whether the GSH values reported in Figure 4 of their paper represented tGSH or reduced glutathione. Furthermore, data showing whether 'responders' had lower baseline concentrations of GSH or GSH:GSSG were not provided.
Additionally, a case report of a child with autistic disorder and cerebral folate deficiency showed the normalisation of low cerebral spinal fluid homocysteine following 2 weeks supplementation with 0.5 mg folinic acid/kg/day increasing to 1.0/kg/day for 3 months. Finally, a 40 session trial of hyperbaric oxygen therapy showed that it has no effect on plasma GSH:GSSG in children with autistic disorder and, as discussed above, incomplete data and selective reporting make it hard to interpret the findings of a randomised trial of topical glutathione before chelation.
Genetic Studies of the γ-glutamyl Cycle and Trans-sulphuration Pathway
The six studies that presented data on genetic polymorphisms of the γ-glutamyl cycle or trans-sulphuration pathway are summarised in Table 12. The best powered of these studies examined genetic variation in 42 genes (308 single nucleotide polymorphisms (SNPs)) related to glutathione, including those coding for enzymes that use glutathione as a co-factor (not included in this review), in 318 families from the Autism Genetic Resource Exchange repository. Several SNPs located in the genes for cystathionine γ-ligase (CTH), alcohol dehydrogenase 5, GCL and glutaredoxin showed significant or suggestive associations with autism spectrum disorders. Interaction models confirmed a significant association between CTH, glutaredoxin and glutaredoxin 3 and autism (OR = 3.78 (95% CI 2.36–6.04).
The study found no association between any of the GST genes and autism. This is in contrast to a previous study of case parent trios that found that homozygote cases for the GST-M*1 gene deletion (GST-M1*0) showed increased risk of autistic disorder and a later case control study that reported a borderline association between the GST-M*1 gene deletion (GST-M1*0) and autistic disorder and a significant interaction between the GST-M1*0 deletion and the reduced folate carrier 80A > G. Previous studies failed to find an association between GST-T1 or GST-P1 and autism.
Ming et al. 2010 Found that a polyalanine repeat polymorphism in the GPx gene (GPx-1) was associated with autistic disorder. Under-transmission of the variation encoding six alanine residues (ALA6) was observed in the families with autistic disorder, suggesting that this allele may be protective. The authors acknowledge that their interpretation is limited by inadequate knowledge of the function of the ALA alleles of GPx-1 gene.
Studies of Glutathione Related Enzyme Activity
As shown in Table 13 GPx-1 activity has been the subject of seven studies. The findings were inconsistent in plasma where two studies reported higher activity in cases than controls and two reported lower activity. A further four studies examined GPx-1 activity in erythrocytes. Of these, two reported lower activity and two reported no significant difference between cases and controls. No significant difference between cases and controls was reported for GPx-1 activity in platelets. In addition, a recently published study showed that glutathione-S-transferase activity was significantly reduced in children with low functioning autism spectrum disorders and there was a trend towards lower activity of glutathione reductase.
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