Geiers' Metabolite Values Are Questioned

In July, the journal of Neurochemical Research published a study online by the father-and-son duo of Mark and David Geier (anchor and lead authors, respectively), which concludes that children with autism spectrum disorders (ASDs) "should be routinely tested to evaluate transsulfuration metabolites" and that "treatment protocols should be evaluated to potentially correct the transsulfuration abnormalities observed." These conclusions are based on the authors' determination that levels of these metabolites—as markers of oxidative stress and "decreased detoxification capacity"—in children with ASD are significantly different from those in children without ASD.

However, the references used by Geier et al to measure these metabolites don't necessarily agree with the control values they obtained (determined by the NJ-based Vitamin Diagnostics, Inc). In some cases, these discrepancies are very large and should have been noted by the authors. In other, less dramatic cases, such discrepancies between the published literature and the results of Geier et al may be the result of age-related differences in these metabolites. In other words, values in healthy adults may differ from those in healthy children. But age-related changes, if they exist,* should have been accommodated by the authors, if necessary—especially given that their subjects ranged in age from 2 to 16 years.

The following is a stepwise examination of the plasma metabolite values obtained by Geier et al in their study and those supplied by their cited references or other relevant sources.

Cysteine: Geier et al propose a statistically significant 33% reduction of plasma total cysteine (µmol/L) in their 38 children with ASD (17.8 ± 8.3 vs 23.3 ± 4.2 in 64 neurotypical controls). However, their reference for the measurement of plasma cysteine, Han et al, provides values that are much higher: from ~160 to ~360 µmol/L in 40 adults (figure 1A), about 7-15 times the mean control value obtained by Geier et al. Other references in the medical literature report mean fasting values of total cysteine in human plasma that are consistent with those of Han et al: ~250 µmol/L (Ueland); 268 µmol/L ± 25 in 10 healthy men (Andersson et al); 213.7 µmol/L ± 14.7 in 13 volunteers aged 24-29 years (Guttormsen et al); and 264 µmol/L ± 28 in 10 healthy subjects aged 31-52 years (Suliman et al).

And in a pediatric study (James et al), which is cited by Geier et al, the mean value of plasma total cysteine in 73 control children (mean age, 10.8 years ± 4.1) was 207 µmol/L ± 22 and that in 80 autistic children (mean age, 7.3 years ± 3.2) was 165 µmol/L ± 14. Very similar values of plasma total cysteine were calculated in a smaller pediatric study by James et al (also referenced by Geier et al). Mark and David Geier themselves reported a plasma cysteine range of 2.24-4.01 mg/dL in 10 boys with ASD (age range, 3-9 years), which is equivalent to 185-331 µmol/L (given the molecular weight of cysteine, 121.15 µg/µmol). Their reference range (from the Great Smokies Diagnostic Laboratory) was 2.70-4.30 mg/dL or 223-355 µmol/L. 

It should be noted that Geier et al published an article in the September online issue of the Journal of Neurological Sciences, in which they appear to use laboratory data from 28 of the 38 subjects examined in the Neurochem Res study, owing to very similar mean values and standard deviations of transsulfuration metabolites (compare Table 2 in the Neurochem Res article with Table 3 in the JNS article). In addition, sections of text are almost identical between the 2 articles.

An important difference, however, in the JNS article lies in the Materials and Methods section, in which Geier et al indicate that they attempted to measure free cysteine, not total cysteine. This distinction, which is not made in the Neurochem Res article, may account for the lower cysteine values obtained by Geier et al in their subjects. Nevertheless, other studies in the literature indicate that the free cysteine value in human plasma is approximately 50% of the total cysteine value and therefore remains substantially higher than those values obtained by Geier et al—for instance, 112 µmol/L ± 15.2 in 13 volunteers aged 24-29 years (Guttormsen et al) and 140 µmol/L ± 21 in 10 healthy fasting subjects aged 31-52 years (Suliman et al). 

Reduced glutathione: Geier et al propose a statistically significant 25% reduction of reduced glutathione (µmol/L) in their children with ASD (3.14 ± 0.56 vs 4.2 ± 0.72 in 120 neurotypical controls). Their reference for the measurement of reduced glutathione, Bouligand et al, reported the substance in mouse liver, not human plasma. Therefore confirmatory reference values for reduced glutathione in human plasma must be obtained from other sources. Andersson et al measured the mean value of reduced glutathione in the plasma of 10 healthy men at 3.4 µmol/L ± 0.9—a value within the standard-deviation range of the mean values obtained by Geier et al in both children with ASD and neurotypical controls. In 27 healthy men (age range, 22-34 years), Curello et al calculated a mean reduced glutathione level of 4.2 µmol/L in plasma. In 13 children aged 10-17 years, Michelet et al calculated a mean reduced glutathione level in plasma of 3.57 µmol/L ± 0.74, which did not differ significantly from values in older subjects.

James et al (again, cited by Geier et al) calculated mean reduced glutathione levels in plasma of 2.2 µmol/L ± 0.9 (73 control children; mean age, 10.8 years ± 4.1) and 1.4 µmol/L ± 0.5 (80 autistic children; mean age, 7.3 years ± 3.2). Mark and David Geier previously reported a plasma reduced glutathione range of 18-28 mg/dL in 10 boys with ASD (age range, 3-9 years), which is equivalent to the stratospheric range of 585-911 µmol/L (given the molecular weight of reduced glutathione, 307.43 µg/µmol). Their reference range (again, from the Great Smokies Diagnostic Laboratory) was 28-44 mg/dL or 911-1431 µmol/L. 

Oxidized glutathione: Geier et al propose a statistically significant 37% increase in oxidized glutathione (nmol/L [not µmol/L]) in their children with ASD (0.48 ± 0.16 vs 0.35 ± 0.05 in 120 neurotypical controls). As in the case of reduced glutathione, Geier et al cite Bouligand et al as their reference, which poses the same problem previously mentioned. Williams et al found a mean, fasting plasma level of oxidized glutathione in 20 healthy adults of 1.4 µmol/L ± 0.1 or 1400 nmol/L ± 100—a value that is substantially greater than the mean values obtained by Geier et al. Curello et al reported a plasma level of 0.2 µmol/L (200 nmol/L) in volunteers, and a Chinese study determined the mean plasma level of oxidized glutathione in persons aged 20-29 years at 0.646 µmol/L ± 0.055 (646 nmol/L ± 55), with no significant differences among older age groups. James et al (cited by Geier et al) determined mean oxidized glutathione levels in plasma of 0.24 µmol/L ± 0.1 or 240 nmol/L ± 100 (73 control children; mean age, 10.8 years ± 4.1) and 0.40 µmol/L ± 0.2 or 400 nmol/L ± 200 (80 autistic children; mean age, 7.3 years ± 3.2). These reference data suggest a wide variation of levels of oxidized glutathione in plasma, from 200 to 1400 nmol/L, which are several orders of magnitude higher than those obtained by Geier et al. (Data also indicate that measurements of plasma glutathione, reduced and oxidized, are highly dependent on testing procedures and vary substantially with even minor hemolysis.)

Taurine: Geier et al propose an approximately 50% relative drop in the mean plasma taurine level (µmol/L) in their children with ASD (48.6 ± 14.0 vs 97.5 ± 8.8 in 27 neurotypical controls). Their reference for the measurement of plasma taurine, Hopkins et al (no PMID available), provides a mean taurine value of 129 µmol/L in platelet-rich plasma and a value of 84 µmol/L in platelet-poor plasma. Trautwein and Hayes calculated a normal plasma taurine level of 44 µmol/L ± 9 in fasting subjects, which is close to the mean level in the ASD subjects of Geier et al. Suliman et al calculated a mean value of 50 µmol/L ± 10 in 10 healthy fasting subjects aged 31-52 years. The AMA Manual of Style (10th ed) provides a wide reference range for plasma taurine: 24-168 µmol/L.

Total sulfate: Geier et al propose a significant 50% reduction of the mean level of plasma total sulfate (µmol per g of protein) in their children with ASD (934 ± 252 vs 1930 ± 184 in 82 neurotypical controls). Their reference for this measurement, Chattaraj and Das, provides a total sulfate range of 35.4-43.3 µg/mL or 35,400-43,300 µg/L in human serum from 6 subjects. This range is equivalent to 369-451 µmol/L, given the molecular weight of the sulfate ion, 96 daltons (µg/µmol). (The AMA reference range for total sulfate in serum is 310-990 µmol/L). To further convert this range to units used by Geier et al,** these values are divided by the normal plasma concentration of protein, 60-80 g/L, to calculate a liberal reference range for total sulfate in plasma of 4.61-7.52 µmol/g protein. This range, determined by using the values from Chattaraj and Das, is inexplicably much lower than both the neurotypical and ASD values reported by Geier et al.

Free sulfate: Geier et al propose a 66% drop in the mean level of plasma free sulfate (µmol per g protein) in their children with ASD (1.37 ± 0.48 vs 4.1 ± 0.46 in 67 neurotypical controls). The reference for this measurement, Boismenu et al, provides a range of 200-650 µmol/L from 40 human serum samples. When this range is divided by the normal plasma concentration of protein, 60-80 g/L, the calculated reference for free sulfate is 2.5-10.8 µmol/g protein. In this case, the mean value of plasma free sulfate obtained by Geier et al in their subjects with ASD is below the lower limit calculated from the cited reference.

Geier et al present metabolite values in children with or without ASD that are questionable. In particular, some values measured by Geier et al—for example, cysteine (whether total or free), oxidized glutathione, and total sulfate—are considerably different from those published elsewhere, including those values obtained or calculated from references cited by the authors. Other values from ASD or neurotypical subjects—for example, reduced glutathione and taurine—are within the reference ranges published in the literature.*** Only in the case of plasma free sulfate was the mean level in ASD subjects outside of the normal range provided by the substantiating literature.

Plasma Test	Mean ± SD (Geier et al)		Reference Values in Literature
	ASD Subjects	Controls
Cysteine, µmol/L	17.8 ± 8.3	23.2 ± 4.2	160-360 268 ± 25 213 ± 14.7 264 ± 28 207 ± 22
Reduced glutathione, µmol/L	3.14 ± 0.56	4.2 ± 0.72	3.4 ± 0.9 3.57 ± 0.74 2.2 ± 0.9
Oxidized glutathione, nmol/L	0.48 ± 0.16	0.35 ± 0.05	200-1400
Taurine, µmol/L	48.6 ± 14.0	97.5 ± 8.8	84 or 129 44 ± 9 50 ± 10 24-168
Total sulfate, µmol/g protein	934 ± 252	1930 ± 184	4.61-7.52
Free sulfate, µmol/g protein	1.37 ± 0.48	4.1 ± 0.46	2.5-10.8

Before any diagnostic or treatment recommendations can be made on the basis of this study (or any study, for that matter), results must be shown to be reliably reproducible by a different set of authors using more than one experienced, reputable laboratory, and any discrepancies between control values and those in the literature must be noted and explained. It should also be determined whether tighter controls, particularly in the form of age matching between autistic and neurotypical subjects, should be performed when comparing these metabolite levels. Last, the significance of any mean values in autistic children that lie within the published reference ranges, although they may be statistically different from a given study's control values, must be considered cautiously.

* For instance, total glutathione levels, but not reduced glutathione levels, in whole blood have been noted to be lower in children than adults (see Michelet et al).

** Geier et al do not explain why they converted the values for both total and free sulfate to units expressing micromoles per g of protein.

*** Excepting the report by Mark and David Geier regarding reduced glutathione in children with ASD.

Photo: iStockPhoto

10/17/08 update: Emails have been sent to the editors of Neurochemical Research and the Journal of the Neurological Sciences, alerting them to the noted discrepancies in their peer-reviewed journals of the metabolite values of Geier et al. No responses have been received as yet. The editors may be contacted by email as follows.

Editorial Board for Neurochemical Research

Editor-in-Chief: Abel Lajtha, Nathan S. Kline Institute, Orangeburg, NY, Lajtha@NKI.RFMH.ORG 

Associate Editor: Nicolas Bazan, Louisiana State University, New Orleans, nbazan@lsuhsc.edu  

Associate Editor: Henry Sershen, Nathan S. Kline Institute, Orangeburg, NY, Sershen@NKI.RFMH.ORG

Editorial Board for JNS

Editor-in-Chief: Robert P. Lisak, Wayne State University, Detroit, MI, rlisak@wayne.edu

Deputy Editor: Paula A. Dore-Duffy, Wayne State University, Detroit, MI, pdduffy@wayne.edu

Deputy Editor: Richard A. Lewis, Wayne State University, Detroit, MI, ralewis@wayne.edu