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Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
A Prospective Study of Oxidative Stress Biomarkers in Autistic Disorders
David A Geier (davidallengeier@comcast.net)
Institute of Chronic Illnesses, Inc., Silver Spring, Maryland, USA
CoMeD, Inc., Silver Spring, Maryland, USA
Janet K Kern (JKern@dfwair.net)
Genetic Consultants of Dallas, Allen, Texas, USA
University of Texas Southwestern Medical Center, Dallas, Texas, USA
Mark A Geier (mgeier@comcast.net)
ASD Centers, LLC, Silver Spring, Maryland, USA
Abstract
The aim of this study was to evaluate oxidative stress
(OS) biomarkers in a prospective, blinded cohort study
of participants diagnosed with autism spectrum disorders
(ASDs). OS biomarkers, including: blood glutathione
(GSH), urine lipid peroxide, blood superoxidase
dismutase (SOD), and blood GSH peroxidase (GPx)
among participants diagnosed with ASDs (n=28) were
evaluated in comparison to laboratory provided reference
ranges. Testing was conducted using Genova
Diagnostics (CLIA-approved). Participants diagnosed
with ASDs had significantly (p<.005) decreased blood
GSH and GPx relative to laboratory reference ranges. By
contrast, participants diagnosed with ASDs had
significantly (p<.000) increased urine lipid peroxide
levels relative to laboratory reference ranges. A bimodal
distribution of significant differences from the laboratory
reference for blood SOD levels were observed
(high=10.7%, low=14.3%). Finally, a significant (p=.05)
inverse correlation was observed between blood GSH
levels and ASD severity using Childhood Autism Rating
Scale scores. The present observations are compatible
with increased OS and a decreased detoxification
capacity, particularly of mercury, in patients diagnosed
with ASDs. Patients diagnosed with ASDs should be
routinely tested to evaluate OS biomarkers and potential
treatment protocols should be evaluated to potentially
correct the OS abnormalities observed
.
Keywords: Heavy metal; Metabolic; Endophenotype;
Sulfation; Sulfur
Introduction
Autism spectrum disorders (ASDs) are prevalent
neurodevelopmental disorders that affect an estimated 1
in 150 children in the US (Austin, 2008). ASDs are
characterized by severe impairments in socialization,
communication and behavior. Children diagnosed with
an ASD may display a range of problem behaviors such
as hyperactivity, poor attention, impulsivity, aggression,
self-injury and tantrums. In addition, these children
often display unusual responds to sensory stimuli such
as hypersensitivities to light or certain sounds, colours,
smells or touch and have a high threshold for pain
(Austin, 2008). Finally, common co-morbidity
conditions often associated with ASDs include
gastrointestinal disease and dysbiosis (White, 2003),
autoimmune disease (Sweeten, Bowyer, Posey,
Halberstadt, & McDougle, 2003), and mental
retardation (Bolte & Poustak, 2002).
In attempting to understand the underlying
pathogenesis of ASDs a considerable body of research
has been conducted to evaluate potential candidate
causal genes. Genetic studies, to date, have not
uncovered genes of strong effect. It was postulated that
increasing rates of ASDs and less than 100%
monozygotic concordance of ASDs support a more
inclusive reframing of ASDs as a multi-system disorder
with genetic influence and environmental contributors
(Herbert et al., 2006). Investigators suggested that
ASDs may result from an interaction between genetic,
environmental, and immunological factors, with
oxidative stress as a mechanism linking these risk
factors (James et al., 2006).
Given the well-established fact that mercury (Hg) is
known to significantly increase oxidative stress and that
fetuses and infants are routinely exposed to Hg from
environmental sources (i.e. fish, dental amalgams,
vaccines, etc.), investigators have described that many
ASDs may result from a combination of
genetic/biochemical susceptibility, specifically a
reduced ability to excrete Hg, and exposure to Hg at
critical developmental periods (Geier, King, Sykes, &
Geier, 2008). Further, it was reported that Hg can cause
immune, sensory, neurological, motor, and behavioural
dysfunctions similar to traits defining/associated with
ASDs, and that these similarities extend to
neuroanatomy, neurotransmitters, and biochemistry.
Also, it was reported when reviewing the molecular
mechanisms of Hg intoxication that it can induce death,
disorganization and/or damage to selected neurons in
the brain similar to that seen in recent ASD brain
pathology studies, and this alteration may likely
produce the symptoms by which ASDs are diagnosed
(Geier et al., 2008).
2
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
Under normal conditions, a dynamic equilibrium
exists between the production of reactive oxygen
species (ROS) and the antioxidant capacity of the cell
(Granot & Kohen, 2004; Stohs, 1995). ROS includes
superoxide, hydroxyl, peroxyl, alkoxy, and nitric oxide
(NO) free radicals (Stohs, 1995). Superoxide is the first
reduction product of molecular oxygen, and it is an
important source of hydroperoxides and deleterious free
radicals (Fridovich, 1986). Hydrogen peroxide (H
2
O
2
)
reacts with reduced transition metals such as iron, via
the Fenton reaction, to produce the highly reactive
hydroxyl radical (McCord & Day, 1978). Most toxic
effects are due to hydroxyl radical formation, which
also initiates lipid peroxidation (McCord & Day, 1978).
Some endogenous enzymes such as xanthine oxidase
(XO), NO synthase, and monoamine oxidase (MAO)
can directly produce ROS (Granot & Kohen, 2004;
Kellogg & Fridovich, 1975; Stohs, 1995). Normally, the
ROS within the cells are neutralized by antioxidant
defense mechanisms. Superoxide dismutase (SOD),
catalase, and glutathione peroxidase (GPx) are the
primary enzymes involved in direct elimination of
ROS, whereas glutathione reductase and glucose-6-
phosphate dehydrogenase are secondary antioxidant
enzymes, which help in maintaining a steady
concentration of glutathione (GSH) and NADPH
necessary for optimal functioning of the primary
antioxidant enzymes (Chance, 1954; Gutteridge, 1977;
Maddipati & Marnett, 1987; Vendemiale, Grattagliano,
& Altomare, 1999). These enzymes require
micronutrients as cofactors such as selenium, iron,
copper, zinc, and manganese for optimal catalytic
activity and effective antioxidative defense mechanism
(Halliwell & Gutteridge, 1992). Additionally, GSH,
iron-binding transferrin, copper-binding ceruloplasmin,
tocopherol (Vitamin E), carotenoids, and ascorbic acid
(Vitamin C) are also involved in the anti-ROS defense
system (Erden-Inal, Sunal, & Kanbak, 2002;
Gutteridge, Richmond, Halliwell, 1980; Loeffler et al.,
1995). GSH is the most important antioxidant for
detoxification and is important for the elimination of
environmental toxins. Oxidative stress occurs when
ROS levels exceed the antioxidant capacity of a cell.
These ROS are highly toxic and react with lipids,
proteins and nucleic acids, and lead to cell death via
apoptosis or necrosis (Kannan & Jain, 2000).
The brain is highly vulnerable to oxidative stress due
to its limited antioxidant capacity, higher energy
requirement, and higher amounts of lipids and iron
(Juurlink & Paterson, 1998). The brain makes up about
2% of body mass but consumes 20% of metabolic
oxygen. The vast majority of energy is used by the
neurons (Shulman, Rothman, Behar, & Hyder, 2004).
Due to the lack of GSH-producing capacity by neurons,
the brain has a limited capacity to detoxify ROS.
Therefore, neurons are the first cells to be affected by
the increase in ROS and shortage of antioxidants and,
as a result, are most susceptible to oxidative stress.
Antioxidants are required for neuronal survival during
the early critical period (Perry, Norman, Litzburg, &
Gelbard, 2004). Children are more vulnerable than
adults to oxidative stress because of their naturally low
GSH levels from conception through infancy (Erden-
Inal, Sunal & Kanbak, 2002; Ono, Sakamoto, & Sakura,
2001). The risk created by this natural deficit in
detoxification capacity in infants is increased by the
fact that some environmental factors that induce
oxidative stress are found at higher concentrations in
developing infants than in their mothers, and
accumulate in the placenta. Taken together, these
studies suggest that the brain is highly vulnerable to
oxidative stress, particularly during the early part of
development that may result in neurodevelopmental
disorders such as ASDs.
The purpose of the present study was to provide
greater mechanistic insight into ASD associated disease
pathology by evaluating biomarkers of oxidative stress
in a cohort of participants diagnosed with ASDs. The
overall hypothesis of the present study was that there
would be increased clinically identifiable biomarkers of
oxidative stress and susceptibility to oxidative stress in
a cohort of participants diagnosed with ASDs. Further,
it was hypothesized that susceptibility factors
associated with increased oxidative stress would
significantly correlate with the clinical severity of the
participants diagnosed with ASDs examined.
Method
The study was conducted at the Autism Treatment
Center (ATC; Dallas, Texas). Phlebotomy took place at
Medical Center Plano, Outpatient Phlebotomy (Plano,
Texas).
The study protocol received Institutional Review
Board (IRB) approval from Liberty IRB, Inc. (Deland,
Florida). All parents signed a consent and Health
Insurance Portability and Accountability Act (HIPAA)
form and all received a copy. Children were in the
presence of one or both parents at all times during the
study.
Participants
The present study looked at qualifying participants (n
= 28) who were prospectively recruited from the
community of Dallas/Fort Worth, Texas area. All of the
children had a diagnosis of autism or pervasive
developmental disorder (PDD). Children included in the
present study were between 2 – 16 years of age and had
an initial Childhood Autism Rating Scale (CARS) score
≥ 30. A child with a CARS score ≥ 30 is considered to
have autism (Schopler, Reichler, DeVellis & Daly,
1980). This study excluded children who had a history
of Fragile X disorder, tuberous sclerosis,
phenylketonuria (PKU), Lesch-Nyhan syndrome, fetal
alcohol syndrome, or history of maternal illicit drug
3
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
use. In addition, this study excluded any children who
had a history of chelation therapy.
Clinical Evaluation
As a baseline, the researchers obtained information
regarding demographics, formal diagnosis, age at
diagnosis, age of apparent onset, information regarding
delay or regression, any current medical issues,
medications, and allergies on each child. A baseline
CARS evaluation was performed by Dr. Kern, who was
trained in the use of CARS, and has 12 years experience
in using the CARS to evaluate more than 300 persons
with an ASD diagnosis. Dr. Kern interviewed the
parents and observed each child. Table 1 summarizes
the pertinent demographics of the participants included
in the present study.
Table 1: A summary of the participants with ASD
included in the present study
Descriptive Information
Sex / Age
Male / Female (ratio) 23 / 5 (4.6 : 1)
Mean Age in Years ± Std (range) 5.8 ± 2.6 (2 – 13)
Race (n)
Caucasian 60.7% (17)
Hispanic 10.7% (3)
Black 10.7% (3)
Asian 10.7% (3)
Mixed 7.1% (2)
Autistic Disorder Characteristics
Mean CARS Score ± Std (range) 38.9 ± 6.4 (30 – 51)
Regressive (n)
1
60.7% (17)
Non-Regressive (n) 39.3% (11)
Autism (n) 67.9% (19)
Autism Spectrum Disorders (n)
2
32.1% (9)
Previous Treatments
Supplements (n) 32.1% (9)
Chelation (n) 0% (0)
Supplements + Chelation (n) 0% (0)
Note: Std = standard deviation; All participants examined in the
present study were living in the state of Texas:
1
Includes participants that had a regressive event in development at
any time following birth.
2
Autism spectrum disorders include participants diagnosed with
pervasive developmental disorder – not otherwise specified (PDD-
NOS) and Asperger’s disorder.
Lab Evaluation
Following the intake evaluation, each participant in
the present study had blood samples collected. The
laboratory specimens were all collected in the morning
following an overnight fast. Specimens were
immediately taken to and processed at LabCorp in
Medical City Hospital (Dallas, Texas) and then shipped
to Genova Diagnostics (Asheville, North Carolina). The
lab used in the present study was blinded and received
no information regarding the clinical status of the
participants examined or their CARS scores prior to
their testing of each sample.
Participants were tested for the following at Genova
Diagnostics (all CLIA-approved): blood GSH, blood
SOD, blood GPx, and urine lipid peroxides.
Controls
In order to evaluate each of the oxidative stress
biomarkers measured among the study participants
diagnosed with ASDs examined in the present study,
reference ranges for each test from Genova Diagnostics
were utilized.
Statistical Analyses
In the present study, the statistical package contained
in Microsoft Excel 2002 and StatsDirect (Version 2.7.2)
were utilized. For each study participant, their
biomarkers of oxidative stress were evaluated in
relation to the mean level from the reference range for
each test, so as to convert each patient’s measured test
values into a percent of the mean value ([patient’s
laboratory value / mean level from the reference range]
× 100 = percent of the pertinent mean). For each type of
biomarker of oxidative stress examined, the individual
results were then averaged to compute an overall
average percent of the pertinent means, and the standard
deviations for each attribute were calculated. Using the
two-sample heteroscedastic t-test statistic, these
“normalized” results from the study participants
diagnosed with an ASD were then statistically
compared to the corresponding data from the normal
control populations that comprised the laboratory
reference ranges. The null hypothesis was that there
should be no difference in the normalized means
between the study participants diagnosed with an ASD
for each biomarker of oxidative stress examined and the
corresponding means from the control populations
(derived from laboratory reference ranges). Further, the
blood GSH values obtained for each study participants
diagnosed with an ASD were evaluated for their
correlation with the severity of the disorder as derived
from the CARS scoring conducted on each study
participants using the unweighted least squares test
statistic. The null hypothesis was that the slope of the
line would be equal to zero. For all the statistical tests
in the present study, a two-tailed p-value ≤ .05 was
considered statistically significant.
Results
Table 2 summarizes an assessment of biomarkers of
oxidative stress among the study subjects with ASD in
comparison to the laboratory reference ranges. Overall,
the ASD group means for the biomarkers of oxidative
stress did not fall outside the laboratory reference
4
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
Table 2. An assessment of oxidative stress biomarkers among the participants diagnosed with an ASD in comparison
to laboratory reference ranges
Lab Test Mean ± Std
(% of Pertinent Mean ±
SEM)
Laboratory
Reference
Range Limits
P-value
1
% >
Reference
Range
Upper Limit
% <
Reference Range
Lower Limit
Urine
Lipid Peroxides
(µmol / g creatinine)
8.7 ± 2.4
(174 ± 9.1)
0 – 10 <.00 32.1 (9) 0 (0)
Blood Glutathione
(µmol / L)
1,004 ± 320
(82 ± 4.9)
669 – 1,793 <.00 3.6 (1) 7.1 (2)
Blood Glutathione
Peroxidase
(U / g Hb)
22.5 ± 5.2
(78 ± 3.4)
20 – 38 <.00 0 (0) 35.7 (10)
Blood Superoxidase
Dismutase
(U / g Hb)
9,767 ± 4,375
(89 ± 7.6)
5,275 –
16,662
.20 10.7 (3) 14.3 (4)
Note: Std = standard deviation; SEM = standard error of the mean
1
The unpaired t-test statistic was utilized
.
ranges (even though a sizeable percentage of individual
scores did so). It was observed that the study subjects
with an ASD had significantly decreased levels of
blood GSH and GPx. By contrast, the study subjects
with an ASD had significantly increased levels of urine
lipid peroxides. No overall significant differences were
observed for the blood level of SOD among study
subjects with an ASD and the laboratory reference
ranges, but a bimodal distribution of significant
differences from the laboratory reference for blood
SOD levels were observed (high=10.7%, low=14.3%).
The abnormalities observed were greatest for urine lipid
peroxides (ASD mean was 174% of the control mean),
followed by blood GPx and blood GSH (ASD means
were 78% and 82% of the control means, respectively).
Additionally, as show in Figure 1, a significant inverse
correlation was observed between GSH levels and ASD
severity.
Discussion
The overall results of the present study showed
significant abnormalities in the biomarkers of oxidative
stress in a cohort of study participants diagnosed with
ASDs relative to laboratory provided reference ranges.
Further, the results suggest that there was a significant
inverse correlation between blood GSH levels and ASD
severity measured using CARS scoring. The oxidative
stress in autism may be caused by an imbalance
between the generation of ROS by
endogenous/exogenous pro-oxidants and the defense
mechanism against ROS by antioxidants.
Figure 1. A summary of the correlation between blood
glutathione and ASD severity
Note: The unweighted least squares statistic was used to evaluate the
relationship between blood glutathione levels and ASD severity.
ASD severity was measured blinded to the level of blood glutathione
using Childhood Autism Rating Scale (CARS) scoring.
Investigators have previously reported that lipid
peroxidation is increased in the plasma of children with
autism as compared to their developmentally normal,
non-autistic siblings (Chauhan, Chauhan, Brown, &
Cohen, 2004). Lipid peroxidation is a chain reaction
between polyunsaturated fatty acids and ROS, and it
produces lipid peroxides and hydrocarbon polymers
that are both highly toxic to the cell (Horton &
Total CARS Score
Blood Glutathione (umol / L)
R
2
= .14, p = .05
5
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
Fairhurst, 1987). Malonyldialdehyde (MDA) is an end
product of peroxidation of polyunsaturated fatty acids
and related esters, and is, therefore, used as a marker of
lipid peroxidation (Jain, 1984). The plasma MDA
contents measured by reaction with thiobarbituric acid
(TBA) were higher in 13 of 15 (87%) of autistic
subjects (Chauhan et al., 2004).
Recent reports also indicate increased levels of other
lipid peroxidation markers in autism, thus confirming
an increased oxidative stress in autism. For instance,
Zoroglu et al. (2004) have reported increased TBA-
reactive substances in erythrocytes of patients with
autism as compared to normal controls. Ming et al.
(2005) reported increased excretion of 8-isoprostane-
F2alpha in the urine of children with autism.
Isoprostanes are produced from the free radical
oxidation of arachidonic acid through non-enzymatic
oxidation of cell membrane lipids. Evans et al. (2008)
evaluated the oxidative stress metabolites of
carboxyethyl pyrrole (CEP) and iso[4]levuglandin
(iso[4]LG) E2-protein adducts in cortical brain tissues
in subjects diagnosed with autism. Significant
immunoreactivity toward all these markers of oxidative
damage in the white matter and often extending well
into the grey matter of axons was found in every case of
autism examined. These investigators reported that the
striking thread-like pattern appears to be a hallmark of
the autistic brain as it was not seen in any control brain,
young or aged, used as controls for the oxidative assays.
In another study, the density of lipofuscin, a matrix of
oxidized lipid and cross-linked protein that forms as a
result of oxidative injury in the tissues, was observed to
be greater in cortical brain areas concerned with
communication in subjects diagnosed with autism
(Lopez-Hurtado & Prieto, 2008). Lipofuscin was
previously demonstrated to be a depot for mercury in
human brain autopsy specimens from mercury
intoxicated patients (Opitz, Schweinsberg, Grossman,
Wendt-Gallitelli, & Meyermann, 1996). Finally, and
perhaps most importantly, Sajdel-Sulkowska, Lipinski,
Windom, Audhya, and McGinnis (2008), evaluated
cerebellar levels of the oxidative stress marker 3-
nitrotyrosine (3-NT), mercury, and the antioxidant
selenium levels between subjects diagnosed with autism
in comparison to controls. It was observed that there
were significant increases in the mean cerebellar levels
of 3-NT and the ratio of mercury/selenium in the brains
of subjects diagnosed with autism in comparison to
controls. It was also observed that there was a
significant positive correlation between cerebellar 3-NT
and mercury levels.
Several studies have suggested alterations in the
enzymes that play a vital role in the defense mechanism
against damage by ROS in autism. For instance,
compared to controls, patients with autism showed
decreased activity of GPx in plasma and in erythrocytes
(Yorbik, Sayal, Akay, Akbiyik, & Sohmen, 2002),
reduced levels of total GSH and lower redox ratio of
reduced GSH to oxidized glutathione (GSSG) in plasma
(Geier & Geier, 2006; Geier & Geier 2007; Geier et al.,
2009a; James et al., 2006), and decreased catalase
(Zoroglu et al., 2004) and SOD (Yorbik et al., 2002)
activity in erythrocytes. In contrast, Sogut et al. (2003)
reported unchanged plasma SOD activity and increased
GPx activity in autism.
The significantly decreased blood GSH levels among
the participants diagnosed with ASDs in the present
study is of concern. GSH is a tripeptide of cysteine,
glycine, and glutamate that is synthesized in every cell
of the body. The essential intracellular reducing
environment is maintained by the high ratio of reduced
GSH to GSSG (Schafer & Buettner, 2001). The GSH
redox equilibrium regulates a wide range of functions
that include nitrogen and oxygen free radical scavenger
(Dickinson et al., 2003), protein redox status and
enzyme activity (Klatt & Lamas, 2000), cell membrane
integrity and signal transduction (Dickson & Forman,
2002; Sagrista, Garcia, Africa De Madariaga, & Mora,
2002), transcription factor binding and gene expression
(Deplancke & Gaskins, 2002), phase II detoxification
(Pastore, Federici, Bertini, & Piemonte, 2003), and
apoptosis (Hall, 1999).
Consistent with low total GSH levels and increased
oxidative stress, autistic children would be expected to
have difficulty resisting infection, resolving
inflammation, and detoxifying environmental
contaminants. Indeed, patients diagnosed with ASDs
were reported to suffer from recurrent infections
(Konstantareas & Homatidis, 1987), neuroinflammation
(Zimmerman et al., 2005), gastrointestinal
inflammation (Horvath & Perman, 2002; Jyonouchi,
Geng, Ruby, & Zimmerman-Bier, 2005), and impaired
antioxidant and detoxification capacity (Chauhan et al.,
2004; Geier & Geier, 2006; Geier & Geier 2007; Geier
et al., 2009a; James et al., 2006; Yorbik et al., 2002;
Zoroglu et al., 2004).
Further, an important relationship between GSH
availability and mercury excretion has been found
(Ballatori & Clarkson, 1985). Bile is the main route of
elimination for many metals, and the rate of secretion of
methyl and inorganic mercury into bile was low in
suckling rats but rapidly increased to adult rates soon
after weaning. These changes closely paralleled similar
developmental changes in the biliary secretion of
reduced GSH. It was observed that when reduced GSH
secretion into bile was completely inhibited, without
changing hepatic levels of reduced GSH or mercury,
mercury secretion was also completely blocked. These
researchers concluded that their results indicated a close
correspondence between the secretion of mercury and
reduced GSH.
Because GSH is essential for effective detoxification,
the effects of a lack of availablity total GSH on
detoxification are far-reaching. Exposure to toxins in
children with compromised detoxification capability
has an even greater potential to disrupt critical
6
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
developmental processes and result in developmental
neurotoxicity (Rice & Barone, 2000).
Lack of availability of GSH may be only one part of
the issue. Examination of the effects of heavy metals
reveals that the presence of heavy metals, e.g., mercury,
can disrupt the very processes needed to excrete the
metals. Evidence shows that metal ions disrupt
methionine synthestase which then, results in the
inhibition of GSH production (Mutter, Naumann,
Schneider, Walach, & Haley, 2005). In addition, the
presence of metals causes oxidative stress, and since
GSH has the dual function of both reducing of oxidative
stress and detoxifying heavy metals, GSH may be
become rapidly depleted as a result of demand. This
situation may be further compounded in ASDs due to
the significant reduction in GPx (which further helps to
reduce oxidative stress).
Also, consistent with the results observed in the
present study, Pasca et al. (in press) observed a
significant inverse relationship between blood GSH
levels and diagnosed autism severity. These
investigators concluded that variations in GSH
abnormalities across the autism spectrum suggests the
possibility that it might be functionally significant in
subjects with mercury intoxication. Further, another
recent study extended the observation to observe that
the severity of ASDs were significantly positively
correlated with biomarkers of mercury intoxication, and
subjects diagnosed with an ASD were observed to have
a significant positive correlation between increasing
biomarkers of mercury intoxication and increased
plasma GSSG levels (Geier et al., 2009b). Taken
collectively, the present results, and those observed in
previous studies (Geier et al., 2009b; Pasca et al. in
press), indicate that GSH levels play a particularly
important functional role in determining the severity of
an ASD diagnosis, when mercury plays a causal role.
Strengths and Limitations
The present study has number of potential strengths
that help to support the observations made. First, the
design of the present study, as a prospective, blinded
study, helps to minimize the chance for selection bias of
study participants. In addition, the blinded nature of the
study ensured that biasing factors regarding clinical or
lab assessments of individual participants were
minimized because neither group was aware of the
other’s results.
Second, since the present study was conducted at the
ATC, a non-biomedical intervention center, the patients
examined in the present study were a priori not skewed
toward those seeking biomedical interventions at a
physician’s office. The participants examined in the
present study were selected from community contacts.
Third, and most importantly, the consistency and
specificity of the results observed were strengths of the
present study. It was observed that for each biomarker
of oxidative stress, with the exception of blood SOD,
there were significant overall differences relative to the
laboratory provided reference ranges.
Finally, since two-tailed p-values were used, and the
directions of the significant effects observed were in the
biologically plausible directions, with the mere chance
occurrence of observing the results found in the present
study being minimal. Furthermore, since < 20 total
statistical tests were generated in the present study, a
two-tailed p-value ≤ .05 was considered significant, and
most of the p-values calculated were < .01, it is
reasonable to conclude that the results observed were
not due to statistical chance.
In considering the potential limitations of the present
study, the number of study participants was of moderate
size. Despite this potential limitation of the present
study, it was observed that there were consistent
statistical effects observed. It would be worthwhile to
evaluate the consistency of the results observed here
with those in different and expanded cohorts of
individuals diagnosed with ASDs. In addition, the
present study did not examine a recruited cohort of
neurotypical children, but instead utilized the laboratory
reference ranges. It would be worthwhile to evaluate the
consistency of the results observed here with a cohort of
neurotypical children, but the results observed in the
present study were consistent with observations made
by other researchers in previous study (Geier et al.,
2008). Another potential limitation of the present study
was that the biomarkers analyzed were from peripheral
sources, and not directly measured in the brain of the
patients examined. In future studies, it would be of
interest to evaluate the correlation between peripheral
biomarkers with those measured in the brain of patients
diagnosed with an ASD. Finally, in the present study,
data was not evaluated concerning other biomarkers of
oxidative stress present in the study participants
examined. It would be of value in future studies to
examine if there was a potential correlation between
other biomarkers of oxidative stress among individuals
diagnosed with ASDs, and to evaluate their potential
correlations with ASD severity.
Conclusion
The present study is a novel prospective study
conducted to evaluate biomarkers of oxidative stress in
a cohort of patients diagnosed with ASDs using
routinely available clinical lab testing. For the study
participants examined, this study found that they had
significant evidence of decreased blood GSH and GPx.
By contrast, it was found that they had significant
evidence of increased levels of urine lipid peroxides.
Finally, it was also observed that blood SOD levels
were found to be significantly outside of the laboratory
reference range in a bimodal fashion. We recommend
that future studies should focus on further evaluating
biomarkers of oxidative stress in an expanded cohort of
individuals diagnosed with ASDs, and potential
treatment protocols be evaluated to potentially correct
7
Geier, Kern, & Geier: A prospective study of oxidative stress biomarkers in autistic disorders.
Electronic Journal of Applied Psychology: Innovations in Autism. 5(1): 2-10 (2009)
the oxidative stress abnormalities observed in the
present study. Additionally, we suggest that future
studies of individuals diagnosed with ASDs should
examine further biomarkers of oxidative stress. Finally,
we recommend, since the lab testing employed in the
present study for examining biomarkers of oxidative
stress are clinically available, relatively inexpensive,
and relatively noninvasive, that patients diagnosed with
ASDs be routinely tested to evaluate them.
Acknowledgements
This research was funded by a grant from the Autism
Research Institute, non-profit CoMeD, Inc., and by the
non-profit Institute of Chronic Illnesses, Inc. through a
grant from the Brenen Hornstein Autism Research &
Education (BHARE) Foundation.
The authors wish to acknowledge the generous help
of Brandon Work at LabCorp, Dallas and the
phlebotomists at Medical Center Plano, Outpatient
Phlebotomy. The authors wish to acknowledge the help
of the parents and children who participated in the
study; without their participation this type of
investigation would not be possible.
Conflict of Interest
David Geier, Janet Kern, and Mark Geier have been
involved in vaccine/biologic litigation. David Geier and
Mark Geier have a patent pending for the treatment of
autistic disorders. The authors have no financial
relationship with the laboratory utilized in the present
study.
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Correspondence to:
Mark R. Geier, M.D., Ph.D., FABMG, FACE
14 Redgate Ct.
Silver Spring, MD 20905
mgeier@comcast.net
Research Profile
Mark Geier has a M.D. and PhD. in genetics. He is
both board certified in genetics by the American Board
of Medical Genetics and a Fellow of the American
College of Epidemiology. In clinical practice for more
than 29 years, Dr. Geier is a founder, and presently the
medical director, of ASD Centers, LLC. Dr. Geier has
authored more than 20 peer-reviewed medical studies
on patients diagnosed with an autism spectrum disorder.
Dr. Geier’s research has specifically focused on
environmental exposures linked with autistic disorders,
as well as the underlying biochemistry of autistic
disorders. Over 600 patients diagnosed with an autism
spectrum disorder have been evaluated and treated with
the clinical protocols Dr. Geier has developed. Dr.
Geier is the co-holder of a patent pending for the
treatment of patients diagnosed with an autism
spectrum disorder.
David Geier is a founder of the non-profit 501(c)3
institute of Chronic Illnesses, Inc. as well CoMeD, Inc.,
also a 501(c) 3. Studying and publishing on the
relationship of genetic, biochemical and hormonal
changes in autism David's research has resulted in new
insights to the causes and treatment of autism and other
chronic illnesses. Overall, David has authored more
than 20 peer-reviewed medical studies on patients
diagnosed with autistic disorders. David is a co-holder
of a patent-pending for the treatment of patients
diagnosed with an autism spectrum disorder.
Janet Kern has an MS in Neuroscience and a PhD in
Human Development. She is an RN with 28 years of
experience in medicine. For the last 14 years Dr. Kern
has been doing research in autism. She specializes in
autism clinical research with a current focus of
biomedical vulnerability, toxicity, biomarkers, and
treatment efficacy. Dr. Kern has 21 peer-reviewed
publications in autism, 17 of which she is first author.
She began her post-doctorate in 1999 at the University
of Texas Southwestern Medical Center (UTSW) and
was promoted to assistant professor. Dr. Kern became
an independent research investigator in 2006 and
remains an adjunct assistant professor at UTSW.
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