Neuropsychiatry: October 2008 Archives

Along with co-applicants Mark and David Geier, TAP Pharmaceuticals* filed an international patent application (PCT/US2007/082866), "Methods of Treating Autism and Autism Spectrum Disorders," in October of last year for the use of Lupron (leuprolide acetate, a GnRH analog), with or without chelation, in children with autism. (A big discovery hat tip for finding this patent application, along with related US patent applications, goes to Kathleen Seidel of the Neurodiversity blog.) The text of this application was published in August May of this year, and treatment descriptions in 7 children can be found if the reader is willing to wade through a lot of repetitive verbiage—including a seemingly endless string of "need in the art," "known in the art," and "skilled in the art."

Essentially, the treatment "invention" of TAP and the Geiers is intended to lower elevated mercury levels in autistic children by giving the chemical castrator Lupron. The applicants base this idea on a 1968 article, which showed that mercuric chloride complexes with testosterone in a hot benzene solution, a condition not possible in living organisms. The patent idea of TAP and the Geiers is to lower testosterone in autistic children by giving them Lupron, which then supposedly frees up toxic mercury. The idea is that the freed-up mercury can then be eliminated with the aid of chelation therapy, if necessary.

However, TAP and the Geiers don't limit their endocrinologic therapy to Lupron, nor their disease targets to autism. They also include the treatment possibilities of antiandrogen hormones (eg, cyproterone [Androcur; Schering-Plough AG]) and birth control pills and propose that mercury toxicity is implicated in Alzheimer's disease, diabetes, heart disease, obesity, amyotrophic lateral sclerosis, asthma, and immune disorders.

What follows is a summary of the pediatric "examples" of TAP's and the Geiers' so-called invention. In all cases of autism, remarkable improvements in gastrointestinal symptoms (if present) and social/cognitive skills are described, sometimes within days of what are described as well-tolerated injections of Lupron.

Subject ages: Four children were of prepubertal age (two 6-year-old boys; a 7-year-old girl; and an 8-year-old boy), and 3 children were within the age range of puberty or beyond (an 11-year-old girl, an 11-year-old boy, and an 18-year-old boy).

Previous treatments: Two of the children (a 6-year-old boy and the 8-year-old boy) had undergone previous chelation therapy with DMSA for approximately 11 and 15 months, respectively. Clinical improvement is described in the case of the 8-year-old boy; the 6-year-old boy did not demonstrate improvement with chelation, according to the patent application. The 11-year-old girl received prescription amphetamines (Adderall; Shire) for the diagnosis of attention deficit hyperactivity disorder, given at the age of 5 years.

Claims of clinical signs of precocious puberty: In no case of the prepubertal or pubertal children is the Tanner stage noted in the patent application. Signs of precocious puberty in 3 of the prepubertal children are vaguely described as increased body, leg, or facial hair; masturbation; "genital development"; or "early sexual behaviors." The 11-year-old girl showed "mild signs of precocious puberty" (whatever those may be) and "fully developed pubic hair" by 8 years of age. These descriptions were presumably obtained in retrospect by history. The girl also began menstruating at the approximate age of 10 years—which is earlier than average, but not precocious.

The performance of GnRH stimulation tests in the patient examples, as recommended by the Lupron Prescribing Information, is not described by the patent applicants. Other diagnostic criteria for central precocious puberty described in the PI, including the documentation of advanced bone age and a number of baseline tests (to exclude congenital adrenal hyperplasia, a chorionic gonadotropin-secreting tumor, a steroid-secreting testicular tumor, and an intracranial [eg, pituitary] tumor), are also not documented in the application. Addendum: Head MR imaging was performed in 2 individuals; however, the timing of the imaging is not provided by the patent applicants.

Mercury assessment: Mercury levels in the absence of chelation therapy are provided only in the case of the 11-year-old boy, whose blood showed "minimal" levels of mercury (1.5 μg/L; reference range, 0.0-14.9 μg/L) and whose urine did not reveal the presence of mercury. The patent applicants claim that urinary porphyrins (specifically urophorphyrin[sic] and hexacarboxylphorphyrins[sic]) were elevated in the 11-year-old girl. Measurements of urinary porphyrins have been proposed by the Geiers to be surrogate markers for mercury toxicity in autism on the basis of a rat study.

Thiol levels: Given that the Geiers have proposed a "decreased detoxification capacity" in children with autism, defined by certain thiol levels, these metabolites were measured in 2 children. Levels of plasma cysteine and reduced glutathione (GSH) were measured during and after the 6-year-old boy's initial chelation therapy, and selected thiol levels were measured in the 8-year-old boy after his Lupron/chelation therapy. The plasma cysteine levels, although below the reference range contained in the patent application, are within the control ranges of those in the literature. In the case of plasma GSH, the levels (when converted to units of μmol/L) are orders of magnitude greater than those found in relevant medical/science articles.

Plasma Metabolite, μmol/L	8-Year-Old Boy	6-Year-Old Boy	Patent Reference	Literature Reference
Homocysteine	5	—	Not given	6.0 ± 1.3a
Cysteineb	226	212	255-320	207 ± 22a
Sulfateb	302	—	302	369-451c
Reduced GSHb	651	651	>1041	2.2 ± 0.9a

^a From James et al, 2006.
^b Presented by the patent applicants in units of mg/dL.
^c From Chattaraj and Das, 1992. 

Testosterone levels: According to the patent applicants, baseline serum testosterone levels were elevated in 2 of their subjects: a 6-year-old boy (23 ng/dL; reference range, 0-20 ng/dL) and the 7-year-old girl (18 ng/dL; reference range, 0-10 ng/dL). They also emphasize high-normal levels of serum testosterone in the 8-year-old boy (25 ng/dL; reference range, 0-25 ng/dL) and the other 6-year-old boy (20 ng/dL). Follow-up testosterone levels predictably rose and then dropped during the Lupron therapy. (According to the Lupron PI, "During the early phase of therapy, gonadotropins and sex steroids rise above baseline because of the natural stimulatory effect of the drug. Therefore, an increase in clinical signs and symptoms may be observed.")

Lupron therapy: The Lupron therapy for the 7 pediatric subjects is tabulated. Therapy was not uniform and, in some cases, involved supplementation with the non-depot (subcutaneous) formulation of Lupron. The recommended starting dosage for Lupron Depot-Ped, according to the PI, is 0.3 mg/kg every 4 weeks: 7.5 mg if ≤25 kg (≤55 lb); 11.25 mg if 25-37.5 kg (55-82.5 lb); and 15 mg if >37.5 kg (>82.5 lb). How the doses of 22.5 mg IM in the cases of the 8-year-old boy and a 6-year-old boy were derived is not stated by the patent applicants. The Lupron PI also indicates that, if downregulation is not achieved (via GnRH stimulation testing and Tanner staging), the dose should be titrated upward in increments of 3.75 mg every 4 weeks.

Subject	Lupron Therapy
8-year-old boy	Depot, 22.5 mg IM on 11/24/04, 1/20/05, 3/25/05, 5/25/05, and 7/14/05
6-year-old boy	Depot 22.5 mg IM on 4/2/05, 5/21/05, and 7/9/05
6-year-old boy	Depot 15 mg IM followed immediately by 0.2 mL (55 μg/kg) sq à gradually increased in 0.1-mL increments to final dose of 0.4 mL (83 μg/kg) sq qd
7-year-old girl	0.3 mL (55 μg/kg) sq qd à increased by using Depot 15 mg IM to a final dose of 2.0 mg/d (74 μg/kg)
18-year-old boy	Depot 15 mg IM; augmented with 0.2 mL sq qd à gradually increased in 0.1-mL increments to 0.5 mL (45 μg/kg) sq qd
11-year-old boy	Depot 15 mg IM; augmented with 0.4 mL sq qd à gradually increased in 0.1-mL increments to 0.7 mL (32 μg/kg) sq qd
11-year-old girl	Depot 15 mg IM q 28 d plus 3.5 mg sq qd

The Lupron PI states that therapy should be monitored with a GnRH stimulation test, measurements of sex steroids, and Tanner staging. Bone age for advancement should be assessed every 6-12 months. Confirming GnRH stimulation tests and Tanner staging are not described by the patent applicants, nor is there a discussion of the monitoring of bone age during therapy.

In 5 cases, the total duration of Lupron therapy is not specified in the patent application. In the case of the 18-year-old boy, his serum testosterone level dropped from a baseline of 559 ng/dL (reference range, 241-827 ng/dL) to a follow-up level of 28 ng/dL. The serum testosterone level of the 11-year-old boy dropped from a baseline of 153 ng/dL to 35 ng/dL after "several months of treatment." Essentially both boys were subjected by the applicants to chemical castration with Lupron at the end or beginning of puberty, respectively.

The 11-year-old girl, who began menstruating at the age of 10 years, most likely underwent chemically induced menopause with her Lupron therapy. This girl was also treated with "low dose birth control pills," presumably in conjunction with her Lupron therapy. The rationale for prescribing OCPs with Lupron in the 11-year-old girl is not stated by the applicants. (I'm out of my medical territory here, but I've only heard of prescribing Lupron with OCPs in the setting of infertility therapy.)

The Lupron PI states that discontinuation of the drug, when used for central precocious puberty "should be considered before age 11 for females and age 12 for males." This recommendation is presumably to allow timely puberty to begin. In adults, Lupron is FDA indicated for the treatment of prostate cancer, endometriosis, and uterine fibroids.

Chelation therapy: Only 2 subjects, the 8-year-old boy and a 6-year-old boy underwent chelation therapy. In the case of the 6-year-old boy, chelation preceded Lupron therapy. The 8-year-old boy underwent chelation after the initiation of Lupron therapy. The rationale for starting chelation therapy before Lupron injections is not stated by the applicants.

Other hormonal therapies: In addition to the OCPs prescribed for the 11-year-old girl, the 8-year-old boy began therapy with the antiandrogen cyproterone acetate (Androcur; Schering-Plough AG) 50 mg tid between his 2nd and 3rd doses of Lupron. Cyproterone acetate was prescribed for an unspecified period of time.

TAP's name* on this international patent application, along with the Geiers, is more than just a little troubling, given that pediatric subjects were treated with the company's proprietary drug in a maverick, off-label fashion on the basis of dubious theories about autism. Moreover, this off-label treatment, with TAP's evident involvement or knowledge, was performed without clinical-trial protocols (specifically those for the protection of human subjects) being noted in the patent application. In addition, adherence to on-label treatment guidelines, as recommended by the company's prescribing information, is not described. Clearly 3 autistic pediatric patients, who were at the beginning or end of puberty, received a drug that is known to disrupt reproductive function.

DMSA = dimercaptosuccinic acid; GnRH = gonadotropin-releasing hormone.

* Given the dissolution of TAP, it's unclear whether Abbott or Takeda is now the primary applicant for this international patent. Abbott has apparently taken over the Lupron franchise.

In their recently published study in the Journal of the Neurological Sciences, Geier et al not only presented values for transsulfuration metabolites in children with autism spectrum disorder (ASD); they also measured urinary porphyrins. Their rationale for doing so was to test a pet theory that autism is due to a reduced ability to excrete environmental mercury, as a result of an innate "decreased detoxification capacity"—which is proposed to be characterized by altered levels of transsulfuration metabolites. (The questionable values for some transsulfuration metabolites presented by Geier et al in the JNS article and elsewhere have been discussed here, here, here, and here.) In addition, because investigators have not been able to consistently find elevated mercury levels in children with ASD by direct measurement, Geier et al have turned to the use of "mercury intoxication-associated urinary porphyrins" as markers of mercury toxicity (most notably from thimerosal-containing vaccines) in children with ASD. In the JNS article, they write,

[I]t was previously demonstrated that the transsulfuration pathyway products of glutathione [15] and sulfate [16] were related to mercury excretion rates, and that the heme synthesis pathway products of urinary porphyrins can provide specific profiles that reflect mercury toxicity [17].

The references cited by Geier et al in this statement are worth examining.

First, reference "15" is a 1985 article from Ballatori and Clarkson, with the relatively straightforward (if not bone-dry) title, "Biliary secretion of glutathione and of glutathione-metal complexes." This study is an examination of the excretion of methylmercury in the bile in rats, which was found by the authors to closely parallel the biliary secretion of reduced glutathione (GSH). Important pharmacokinetic differences between methylmercury and ethylmercury (which is in the vaccine preservative thimerosal) have been discussed at this blog and by many others. Also the biochemical/biophysiologic leap from rodents to humans should have been acknowledged by Geier et al.

The same criticism can be applied to the use of reference "16," a 2004 review of organ systems in the American lobster (!) that regulate and detoxify environmental heavy metals.

Reference "17" is a 1996 study by James S. Woods from the University of Washington. In rats, Woods demonstrated changes in urinary porphyrins after prolonged exposure to—again—methylmercury. Specifically levels of 4- and 5-carboxyl porphyrins and the expression of precoproporphyrin were demonstrated in the exposed animals. Woods also conducted a possibly controversial pilot study in Mexican dental workers who were exposed to mercury-containing dental amalgam. Woods showed that the excretion of mercury increased and that levels of these urinary porphyrins decreased with chelation.*

In the JNS study, Geier et al measured urinary porphyrins in their 28 subjects with ASD (age range, 2-16 years), but not in their control children—as they did when assessing transsulfuration metabolites. (The reason for not measuring urinary porphyrins in the control group is not explained by the authors.) So without a true control group, Geier et al compared urinary porphyrin values** in their 14 children with "mild" ASD (CARS score ≤38.5) with those in 14 children with "severe" ASD (CARS score ≥38.5). (It's not clear what the authors did with the kids who hit the 38.5 mark.)

Not too surprising, Geier et al claimed significant differences in urinary levels of pentacarboxyporphyin (ie, 5-carboxyl porphyrins) and precoproporphyrin (Table 2) between the mild and severe ASD groups, which would be consistent with the methylmercury rat data of Woods. The authors also ostensibly monkeyed around with the various ratios of urinary porphyrins and found other significant differences between the 2 ASD groups. Additional fiddling demonstrated relationships between the CARS score and some porphyrin ratios. These data are intended to show that the authors' surrogate markers for mercury intoxication—urinary pentacarboxyporphyin and precoproporphyrin—are associated with the severity of autism.

Last, Geier et al assessed the plasma oxidized glutathione (GSSG) levels—as a "strong indicator of cellular oxidative stress"—among ASD children with low urinary porphyrins or high urinary porphyrins and claimed significantly increased levels of plasma GSSG in subjects with high urinary pentacarboxyporphyin or precoproporphyrin levels. Again, Geier et al intended to demonstrate that their surrogate markers for mercury intoxication are associated with a reduced capacity to excrete mercury, per the GSSG level, in ASD children. The main problem with this particular finding is that the plasma GSSG values presented by Geier et al are considerably different (eg, by 3 orders of magnitude) from those published elsewhere, including references cited by the authors.

Also published data suggest that Geier et al should have controlled for age-related differences in urinary porphyrin excretion—especially given that their subjects ranged in age from 2-16 years. A 1996 study by Minder and Schneider-Yin (Age-dependent reference values of urinary porphyrins in children) found distinctive age-related changes in the urinary excretion of 3 porphyrins, which may be explained—depending on the porphyrin—by age-related changes in the physiologic development of the excretion system and heme synthesis. For instance, their data showed that the urinary excretion of coproporphyrin III decreases steadily from the age of approximately 2 years to late adolescence. In an e-mail response, lead author Elisabeth Minder stated, in reference to the JNS study by Geier et al, that "one should control the data for age."

CARS = Childhood Autism Rating Scale. 

* Curiously enough, Woods is a coauthor of a 2006 JAMA article, which reported no significant differences in urinary mercury levels or neurologic function between Portuguese children who received dental amalgam and those who received a resin-based composite for routine dental work. The study's ethics were criticized in the Petition to Order Mercury Amalgam Withdrawn From Interstate Commerce. According to the JAMA article, "Urinary...porphyrins were monitored as indicators of renal responses to mercury..and will be reported separately."

** Urinary porphyrin levels from the subjects, who were recruited in Dallas, Texas, were shipped to and measured at, for some inexplicable reason, the Laboratoire Philipe Auguste in Paris. A coauthor of the JNS article is Robert Nataf from the same Paris lab. Nataf is the lead author of a retrospective 2006 study, which reported relatively elevated coproporphyrin and precoproporphyrin levels in children with autism.

Once again, more published laboratory data bolster the argument that some metabolite values presented by Mark and David Geier in their studies of control and ASD children are highly questionable—and therefore, undermine the validity of their study results and, consequently, their conclusions.

A 1999 article from Melnyk S et al (anchor author Jill James) provides mean plasma thiol levels in 11 healthy adult women (Table 3) as follows. These are contrasted with the very different levels obtained by Geier et al in 2 of their studies (all values presented here are in µmol/L [either as a range or mean value ± SD]).

Plasma Thiol	Melnyk et al, Control	Geier et al, Control	Geier et al, ASD
Methionine	41.1 ± 2.5	1.3-5.0*	1.2-1.9*
Cysteine	227.1 ± 4.8	23.2 ± 4.2**	17.8 ± 8.3**
GSH	6.9 ± 0.5	911-1431*	585-911*
GSSG	1.5 ± 0.1	0.00035 ± 0.00005**	0.00048 ± 0.00016**

• In the case of plasma methionine, the published value of Melnyk et al is at least 8 times the control value of Geier et al.
• In the case of plasma cysteine, the published value of Melnyk et al is approximately 10 times the control value of Geier et al.
• In the case of GSH, the published value of Geier et al is at least 84 times that of Melnyk et al.
• In the case of GSSG, the published value of Melnyk et al is at least 3100 times that of Geier et al.

* From Geier DA, Geier MR. A clinical and laboratory evaluation of methionine cycle-transsulfuration and androgen pathway markers in children with autistic disorder. Horm Res. 2006;66:183-188. 

Transsulfuration metabolites in this study were measured at the Great Smokies Diagnostic Laboratory, a laboratory of dubious reputation. Methionine levels were originally presented in units of µmol/L. GSH values were originally presented in units of mg/dL (18-24 in children with ASD), which were then converted here to units of µmol/L for purposes of comparison (molecular weight of GSH, 307.43 µg/µmol).

** From Geier DA, Kern JK, Garver CR, Adams JB, Audhya T, Geier MR. A prospective study of transsulfuration biomarkers in autistic disorders. Neurochem Res. 2008;Jul 9. [Epub ahead of print]

Transsulfuration metabolites in this study were measured at Vitamin Diagnostics, Inc. Levels of GSSG were originally presented in units of nmol/L, which were converted here to units of µmol/L for purposes of comparison.

ASD = autism spectrum disorder; GSH = reduced glutathione; GSSG = oxidized glutathione.

Following an examination of questionable transsulfuration metabolite values published by Geier et al (summary table below), e-mails were sent to the editors of Neurochemical Research and the Journal of the Neurological Sciences, alerting them to the discrepancies between these data and very different values in the published literature.

Plasma Test	Mean ± SD (Geier et al)		Reference Control Values in Literature
	ASD Subjects (N = 38)	Controls
Cysteine, µmol/L	17.8 ± 8.3	23.2 ± 4.2	160-360* 268 ± 25 213 ± 14.7 264 ± 28 207 ± 22*
Oxidized glutathione (GSSG), nmol/L	0.48 ± 0.16	0.35 ± 0.05	200 240 ± 100* 646 ± 55 1400
Total sulfate, µmol/g protein	934 ± 252	1930 ± 184	4.61-7.52*

The associate editor of Neurochemical Research, Henry Sershon, forwarded my e-mail to Mark Geier (anchor author), who reviewed the information and responded to Dr. Sershon that the published values for GSSG were consistent with those of James et al in a 2004 study (which was also cited by Geier et al). In fact, a review of this study reveals that the printed mean value for plasma GSSG in control children, 0.32 nmol/L ± 0.1 (range, 0.11-0.43 nmol/L), is very similar to the control value of Geier et al (0.35 nmol/L ± 0.05). But it is also very different from the control value published by James et al in their 2006 study, 0.24 µmol/L ± 0.1, or 240 nmol/L ± 100.

To that end, I e-mailed Dr. James asking for clarification of the plasma GSSG values in her articles, and she responded that the units in the 2004 article were incorrect and should have been µmol/L, not nmol/L. Consequently this correction negates Dr. Geier's rebuttal that his values for plasma GSSG are consistent with those of James et al.

Dr. James added that she rarely sees GSSG levels greater than 0.4 µmol/L [or 400 nmol/L] in control children. Others indicate that measurements of plasma glutathione, reduced and oxidized, are highly dependent on testing procedures and vary substantially with even minor hemolysis. Still others indicate that "[i]t is not possible to distinguish between oxidized and reduced glutathione in serum or plasma," owing to very low concentrations.

* Reference cited by Geier et al.

In their 2005 Medical Hypothesis article, which inspired their questionable 2006 Hormone Research study (background here), Mark and David Geier cited a 1997 French case series (really a letter to the editor by Tordjman et al in the American Journal of Psychiatry) to support their measurement of serum testosterone in children with autism. Geier and Geier wrote...

In addition, Tordjman et al have reported on a case-series of 12 prepubertal autistic children (6-10 years old) in their inpatient child psychiatry department, four of whom the researchers observed to have precocious secondary sexual characteristics (growth of pubic hair, increase of testis volume) that suggest high androgenic activity in autistic disorders.

What Tordjman et al actually did, on the basis of their observation of 4 autistic children with precocious secondary sexual characteristics in their practice, was to measure plasma testosterone and adrenal androgen (presumably DHEA or DHEA-S) in 9 pre- or postpubertal inpatients with autism and 62 matched control children. Because of the possible positive correlation between testosterone and aggression, the investigators divided the 9 autistic children into 3 groups according to their aggressive behaviors. Notably, they observed that autistic children who displayed aggression against others were less likely to demonstrate the typical core symptoms of autism (withdrawal, stereotypy, language dysfunction)—which suggests, perhaps, that these children may actually have an alternative behavioral disorder.

Three of their 9 autistic subjects had abnormally high plasma testosterone levels (Table), given the study's matched reference values. These 3 children all showed aggression against others—meaning, according to the authors, they were less likely to demonstrate typical, core autistic symptoms.

Patient	Serum Testosterone, ng/mL
	Level	Ref Mean ± SD (range)	U Iowa Range
10-year-old boy	0.64	0.06 ± 0.03 (0.01-0.15)	0.18-1.50
17-year-old boy	8.8	5.51 ± 1.27 (0.27-7.50)	3.50-9.70
13-year-old girl	0.5	0.12 ± 0.09 (0.01-0.25)	0.15-0.35

The authors noted that the 10-year-old boy exhibited pubic-hair growth, which is probably not a sign of precocious puberty in boys aged 9 years or older. The 13-year-old girl, whose serum testosterone level exceeded the reference range in the study and that provided by the University of Iowa, also demonstrated a very high level of adrenal androgen, 4.40 ng/mL, at least according to the mean level in the study's control population (mean study reference, 0.88 ng/mL ± 0.39 [range, 0.36-1.70]; U Iowa reference range for DHEA, 1.5-5.7 ng/mL).

What Geier and Geier failed to note, however, in both their Medical Hypothesis and Hormone Research articles, is that a previous study by Tordjman et al (1995) did not find elevated testosterone levels in 31 prepubertal children with autism, when compared with 12 prepubertal subjects who had mental retardation/cognitive impairment* or 10 prepubertal control subjects. The mean levels of plasma testosterone and DHEA-S in 8 postpubertal autistic children were also similar to those in 11 postpubertal normal controls. Tordjman et al concluded from this study that "altered secretion of the androgens is not a common feature of autism."

A PubMed search by this blogger failed to disclose any other studies that assessed testosterone or other androgen levels in pre- or postpubertal children with autism.

DHEA = dehydroepiandrosterone; DHEA-S = dehydroepiandrosterone sulfate.

* The study did find increased plasma DHEA-S levels in prepubertal children with cerebral palsy (among those with mental retardation/cognitive impairment).

The discovery of questionable values for transsulfuration metabolites in the recently published studies by Geier et al (in Neurochemical Research and the Journal of the Neurological Sciences) prompts further investigation into the published work of the respective lead and anchor authors, David and Mark Geier. Specifically both recent articles reference a study of the father-son duo that was published in Hormone Research in 2006.

Information in this article has been dissected in detail at the neurodiversity blog, particularly with respect to the validity of son David's academic affiliation, the curious makeup of the IRB that approved the study, and father Mark Geier's limited experience in pediatric endocrinology, to name just three.

Evidently one of the Geiers' favored hypotheses in the pathogenesis of autism concerns the nexus of transsulfuration metabolism, androgen synthesis, and mercury toxicity, and they refer to their speculation on the subject in a 2005 Medical Hypothesis article, which was used to justify the measurement of several hormone levels and transsulfuration metabolites in the Hormone Research study.

In this study, the Geiers examined 16 children (14 boys, 2 girls) with autism spectrum disorder (ASD) who presented to the Genetic Centers of America (which is evidently located at the residential address of Mark Geier, 14 Redgate Ct, Silver Spring, MD).* The children underwent clinical examination, presumably by Mark Geier, to assess traits consistent with "hyperandrogenicity." These traits included, according to the Geiers, "early growth spurt, increased body and facial hair, aggressive behavior, and early secondary sexual changes." The Geiers also measured selected hormone levels (at LabCorp, Inc) and transsulfuration metabolites (at the Great Smokies Diagnostic Laboratory) in these children. A control population was not included in the study. The serum/plasma values of the study subjects were compared with reference values from the particular laboratory used.

So, in essence, the Geiers assessed selected serum/plasma values in 16 children with ASD, from 3 to 10 years of age, whom they believed to display signs of androgen excess (in 15 of the 16 children). However, several of the clinical traits noted by the authors do not, in and of themselves, necessarily indicate endocrine abnormalities in children—including masturbation, growth spurt, and an interest in female sexual organs among boys. A 10-year-old girl is noted to exhibit body hair and sexual development, which is not unusual given the age range of normal sexual development in girls. A 4-year-old boy is noted to exhibit male-pattern baldness, a dubious proposition without further clinical information or documentation. In only one case is the Tanner stage noted by the authors (although it is not indicated whether it is in respect to pubic hair or genitalia).

The subjects of Geier and Geier were tested for "androgen metabolites," including serum testosterone (n = 16), serum/plasma DHEA (n = 11), and serum FSH (n = 14). (Although FSH has androgenic properties in males, the glycoprotein is not, despite the Geiers' description, an androgen metabolite.)

FSH: In all 16 cases, serum FSH levels were within age- and sex-dependent reference ranges of LabCorp, which are consistent with pediatric FSH levels found elsewhere (eg, Pediatric Reference Ranges). However, the authors inexplicably concluded that the mean value of FSH in the 16 children with ASD was significantly decreased (35% of the normal reference). It should be noted that in 5 of these cases, LabCorp was unable to detect FSH in serum samples, and the authors therefore assumed that the value was equal to the lowest level that could be measured by LabCorp.

Testosterone: Geier and Geier reported elevated serum testosterone levels (ng/dL) in 12 of the 16 subjects (all boys). The reference ranges provided by LabCorp are consistent with pediatric ranges provided online by the University of Iowa. Addendum: According to Kaplowitz, prepubertal testosterone levels are generally less than 30 ng/dL; although levels ranging from 11 to 30 ng/dL may represent early puberty. The testosterone levels in all subjects of Geier and Geier were 25 ng/dL or lower.

DHEA: Geier and Geier reported elevated DHEA levels (ng/dL) in 10 (8 boys, 2 girls) of 11 subjects (Table). However, when these values are compared with age-dependent reference values from the University of Iowa, instead of LabCorp values, all 11 subjects fall within normal ranges. The Iowa reference advises that DHEA values "begin to increase progressively at about six years of age prior to any physical evidence of puberty."

Patient No.	Age (y)	Sex	Geier and Geier DHEA (ng/dL)	LabCorp Ref (ng/dL)	U Iowa Ref (ng/dL)
7	3	M	107	26-72	20-130
9	3	M	85	26-72	20-130
4	4	M	120	26-72	20-130
16	4	F	94	19-42	20-130
10	5	M	118	29-66	20-130
12	5	M	100	26-72	20-130
13	7	M	148	29-66	20-275
15	7	M	67	29-66	20-275
11	8	M	181	53-135	20-275
6	9	M	284	53-135	31-345
8	10	F	251	234-529	150-570

A summary of the Geiers' transsulfuration metabolite values is shown below (from Table 3). These values are compared with pediatric values from James et al (2006), a reference that was cited by Geier et al in their 2 recently published articles. In the cases of methionine and reduced glutathione, the values reported by the Geiers in Hormone Research (including reference values) are 1 and 2-3 orders of magnitude off, respectively, from those published in the literature.

Test	Geier and Geier, Hormone Research (Reference Values)	James et al, 2006 Mean Control Values in Children (n = 73)
Plasma reduced glutathione, µmol/L (n = 10, 3-9 y)	585-911a (911-1431)	2.2 ± 0.9
Plasma cysteine, µmol/L (n = 10, 3-9 y)	185-331b (223-355)	207 ± 22
Serum cystathione, µmol/L (n = 11, 3-10 y, includes 2 girls)	0.072-0.309 (0.044-0.342)	0.19 ± 0.1
Serum homocysteine, µmol/L (n = 12, 2-10 y, includes 2 girls)	2.9-9.9 (5.1-13.9)	6.0 ± 1.3 (5-15)c
Plasma methionine, µmol/L (n = 9, 3-10 y, includes 2 girls)	1.2-1.9 (1.3-5.0)	28.0 ± 6.5 (6-40)c

a Equivalent to the reported 18-28 mg/dL, given the molecular weight of reduced glutathione, 307.43 µg/µmol.
b Equivalent to the reported 2.24-4.01 mg/dL, given the molecular weight of cysteine, 121.15 µg/µmol.

c AMA reference values.

The plasma cysteine levels of 4 of 10 children were below the lower limit of the Geiers' laboratory reference range; but in all cases, they are well within the normal reference ranges provided by others in the literature (eg, see Han et al, figure 1A, along with others). In the case of serum cystathione, the values measured in the children with ASD were within the Geiers' reference ranges. Nevertheless, the Geiers went on to conclude that serum cystathione was significantly decreased in these children. In the case of serum homocysteine, 4 children demonstrated values below the lower limit of the laboratory reference range (2.9, 4.0, 4.5, 5.0 µmol/L). The University of Iowa indicates a normal value for serum homocysteine of <10 µmol/L, and the AMA provides a range of 5-15 µmol/L. An assessment of serum homocysteine levels in more than 3500 adolescent children, published in JAMA, determined a range of 0.1-25.7 µmol/L, with a median value of 4.9 µmol/L.

The conclusions offered by Geier and Geier in Hormone Research, specifically that serum FSH, cysteine, cystathione, and homocysteine levels are significantly decreased and that DHEA levels are significantly increased in their subjects with ASD, are dubious—given that these levels (in every one of their ASD subjects) were within the normal laboratory ranges used by the Geiers or, in the case of DHEA, cysteine, and homocysteine, were within the normal ranges published by other sources. (Moreover, if the Geiers were proposing a kind of precocious puberty in children with ASD, then FSH would be expected to be elevated, not decreased.) In addition, the Geiers' values for plasma reduced glutathione and methionine are suspect, given that they are at least 1 order of magnitude off those found in other sources.

In the case of serum testosterone, the individual values in the majority of Geiers' ASD subjects were outside of the normal reference ranges (However, see Addendum above). Nevertheless, because of the noted concerns regarding some laboratory values in the Hormone Research article and the very liberal conclusions that Geier and Geier make from their data, confirmation of elevated testosterone levels in children with ASD is especially needed.

DHEA = dehydroepiandrosterone; FSH = follicle-stimulating hormone.

* And is also the address of the Institute of Chronic Illness.

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

Roger Brumback, editor in chief of the Journal of Child Neurology, is not happy. Neurologist Jon Poling, the lead author of the 2006 case report in JCN, "Developmental regression and mitochondrial dysfunction in a child with autism," did not inform Brumback's editorial board that he is the father of the girl described in the report, and moreover, that he petitioned the National Vaccine Injury Compensation Program (VICP) in 2003, claiming his daughter's alleged injury (ie, autism) was due to vaccination.

In a letter published in last month's issue of the JCN (BIG HT to Kathleen Seidel at Neurodiversity), Brumback describes the authors' lack of full disclosure "an appallingly troubling issue." The JCN editors were evidently troubled enough to determine if the case report was used to support the favorable and heavily publicized VICP ruling for the Polings earlier this year (it was not), but Brumback proposes that "media linkage of the published article to the legal outcome implies scientific support from JCN for this legal opinion." He now advises:

Beginning in January 2009, statements from all authors concerning potential conflicts of interest will be published as a part of each article. However, no written statement can substitute for honesty, good faith, and integrity on the part of the authors.

In their defense, the report's coauthors Richard Frye, Andrew Zimmerman, and John Shoffner claim in a separate letter that they did not know of Poling's pending VICP claim at the time of the report's submission to JCN. (However, it is unclear, as Seidel points out, whether the coauthors became aware of Poling's claim sometime later.) The coauthors did know that the report's subject is Poling's daughter.

Poling himself acknowledges, in yet another separate letter to the JCN, that he should have declared his daughter's identity to the JCN editors, but that he withheld her name "to protect a 6-year-old child." Poling confirms that the JCN report was not used to support the VICP claim and characterizes his involvement in the case as minimal—consisting of signing a "short original petition and submitting a required sworn parental affidavit." Poling also reveals that Zimmerman submitted an expert opinion to the VICP court in December 2007 at Poling's request.

Poling further claims, "There are certainly other physicians who have chosen not to publish promising leads or discoveries involving family members, out of respect for privacy or fear of the kind of criticism our article has generated," and suggests that "the JCN explore ways to encourage these helpful contributions, even when the patient is a family member."

An alternative suggestion is to require that any physician-author recuse himself from submitting a case report of a relative to medical journals. Let more objective physicians assess and submit this information for peer review—in an effort to eliminate conflicts of interest and, most important, to ensure the privacy and appropriate care of the patient.

* Poling also presented preliminary findings in his daughter's case in June 2001 at the Johns Hopkins Neurology Grand Rounds.

If anyone doubted the intellectual limits of Jenny McCarthy, the Weiners actress, here's unmitigated proof.

In response to Amanda Peet's characterization of antivax parents as "parasites" (which is not a far-off description), McCarthy—a supporting actress in Larry the Cable Guy's Witless Protection—tells Spectrum magazine, "I am so proud to be a parasite."

In all mustered graciousness, maybe McCarthy, featured in the straight-to-video Python, got "parasite" confused with "paragon"? Paralegal? Pair of shoes?

In any case, the same smarts that led McCarthy to accept a starring role in Dirty Love has evidently resulted in an embarrassingly limited vocabulary. Perhaps Jim Carrey will pull aside his girlfriend, who bumped boobs with Pam Anderson in Scary Movie 3, and whisper something corrective in her ear. Or maybe not. After all, he delivered that tin-ear you're-getting-on-my-nerves-like-Robin-Williams performance in Horton Hears a Who!

Peet evidently apologized for using the word "parasites" to describe antivax parents. But why is an apology necessary to someone like McCarthy, a cast member of the Untitled Patricia Heaton Project, when she views the characterization as favorable? Or maybe McCarthy (aka Yvette Denslow in BASEketball) is confused about the meaning of "proud."