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Analyses of Toxic Metals
and Essential Minerals in the Hair
of Arizona Children with Autism
and Associated Conditions,
and Their Mothers
J. B. ADAMS,*
,1
C. E. HOLLOWAY,
1
F. GEORGE,
2
AND
D. QUIG
3
1
Arizona State University, Tempe, AZ 85287-6006;
2
Holistic
Osteopathic Medical Care, Cave Creek, AZ; and
3
Doctor’s Data,
St. Charles, IL
Received April 4, 2005; Revised May 9, 2005; Accepted May 11, 2005
ABSTRACT
The objective of this study was to assess the levels of 39 toxic metals
and essential minerals in hair samples of children with autism spectrum
disorders and their mothers compared to controls. Inductively coupled
plasma–mass spectrometry was used to analyze the elemental content of
the hair of children with autism spectrum disorders (n=51), a subset of
their mothers (n=29), neurotypical children (n=40), and a subset of their
mothers (n=25). All participants were recruited from Arizona. Iodine levels
were 45% lower in the children with autism (p=0.005). Autistic children
with pica had a 38% lower level of chromium (p=0.002). Autistic children
with low muscle tone had very low levels of potassium (–66%, p=0.01) and
high zinc (31%, p=0.01). The mothers of young children with autism had
especially low levels of lithium (56% lower, p=0.005), and the young chil-
dren (ages 3–6 yr) with autism also had low lithium (–30%, p=0.04). Low
iodine levels are consistent with previous reports of abnormal thyroid
function, which likely affected development of speech and cognitive skills.
Low lithium in the mothers likely caused low levels of lithium in the
young children, which could have affected their neurological and
immunological development. Further investigations of iodine, lithium,
and other elements are warranted.
Index Entries: Autism; hair analysis; iodine; lithium; potassium.
Biological Trace Element Research 193 Vol. 110, 2006
© Copyright 2006 by Humana Press Inc.
All rights of any nature, whatsoever, reserved.
0163-4984/(Online)1559-0720/06/11003–0193 $30.00
*Author to whom all correspondence and reprint requests should be addressed.
INTRODUCTION
Autism is a developmental disorder that occurs in children under 3 yr
of age and involves three major areas: impaired language/communica-
tion, abnormal or stereotypic behaviors, and impaired social interactions.
Its diagnosis is based solely on the observation of these symptoms, as there
is no known cause or biological marker. It is a spectrum disorder, ranging
from classic autism (most severe), to Pervasive Developmental Disorder
Not Otherwise Specified (PDD/NOS), to Asperger’s syndrome (least
severe). Studies of identical twins have clearly demonstrated that both
genetic and environmental factors are important, but the genes and envi-
ronmental factors have not yet been identified.
Regarding environmental factors, either the exposure to toxins or lack
of essential nutrients could be important. There have been suggestions that
mercury or other toxic metals might play a role in the pathogenesis of
autism (1). Similarly, the lack of essential minerals is known by definition
to cause many health problems, and a lack of them (or in some cases, an
excess of them) could contribute to the etiology of autism. The amount of
toxic metals and essential minerals can be assessed by blood, urine, and
hair. Hair is useful for measuring metals, as they are much more concen-
trated in the hair. Because hair grows at a rate of 1–1.5 cm/mo, a 2- to 3-
cm sample from next to the scalp can provide an average over 2–4 mo. It
provides a measure of what is being transported in the body during that
time, but it will not be able to detect earlier exposures. There was a recent
well-publicized criticism of hair analysis studies (2), but the major criti-
cism centered on differences in results among different labs, some of which
were using inferior equipment and less rigorous preparation techniques.
Those concerns can be addressed by using a single high-quality lab, with
blinded testing of subjects vs controls. In a classic review of over 250
reports, the EPA (3) concluded that hair is “a meaningful and representa-
tive tissue” for measuring toxic metals and selected nutrients. Similarly,
the National Health and Nutrition Evaluation Study (4) continues to use
hair as one method to evaluate levels of metals such as mercury.
In the early 1980s, there were several studies of the levels of toxic and
essential minerals in the hair of children with autism (5–8). Several abnor-
malities were reported, but the results of the studies are generally incon-
sistent. There was also one study (9) that measured only vanadium
concentrations in 10 autistic adults vs 10 controls; no significant difference
in vanadium levels was found. Most of these studies suffer from a rela-
tively small sample size. Also, they did not differentiate between the chil-
dren who had pica (eating nonfood items) and those who did not, which
is common in autism and could account for an increased toxic metal bur-
den. A limitation with one study was that most of the controls were sib-
lings of children with autism. Also, the studies were done before 1985,
when measurement techniques were not as advanced as they are today.
Only one of the studies (8) mentioned the use of a special common sham-
poo, but the subjects only used it for 3 d prior to collecting the hair sam-
194 Adams et al.
Biological Trace Element Research Vol. 110, 2006
ple; this is important to avoid contaminants through hair care products,
which can invalidate results. Therefore, it is clear that a larger study, with
more rigorous avoidance of contaminants and an assessment of pica, is
necessary to resolve the discrepancies in the previous studies.
A recent study (10) measured mercury levels in the blood and hair of
children with autism (n=82; meanage =7.2 yr) compared to controls (n=55)
living in Hong Kong. The authors found that children with autism had
slightly elevated levels of mercury in their blood, but the difference was
not statistically significant (p=0.15). They did not find a significant differ-
ence in hair mercury levels, but the levels were unusually high in both
populations (2.26 and 2.07 ppm for the autism and control groups, respec-
tively); the authors suggested that the reason for the high levels is that the
“Hong Kong Chinese are famous for eating seafood.”
In addition, there was a recent study (11) of the level of mercury in the
baby hair of infants (aged 12–24 mo) who later were diagnosed with
autism compared to controls (n=94 and 45, respectively). This study found
that the autism group had one-eighth of the normal amount of mercury in
their baby hair compared to controls. In the control group, the amount of
mercury correlated with the mother’s seafood consumption and number
of mercury amalgam dental fillings, but that was not true of the children
with autism, who had a low level regardless of their mother’s seafood con-
sumption or number of dental fillings, which suggests a general inability
to excrete mercury. They also found that the severity of autism had a
strong inverse relationship with the level of mercury, with the most severe
group having the lowest levels of mercury in their hair. This is consistent
with the hypothesis that the group with the most inhibition of mercury
excretion would be the most severely affected.
There has never before been an attempt to analyze the hair of the
mothers of children with autism. Because the mothers are the major source
of exposure to toxic metals and also the source of essential minerals dur-
ing gestation and breastfeeding, we felt that it would be important to also
analyze the levels in their hair.
Therefore, this study was designed to investigate the hypothesis that
children with autism and/or their mothers had abnormal levels of toxic
metals or essential minerals in their hair. This will not necessarily indicate
cause and effect, but it will allow us to look for possible associations that
can be further investigated.
PARTICIPANT SELECTION
This study was conducted with the approval of the Human Subjects
Institutional Review Board of Arizona State University. All parents and
(where possible) children signed informed consent forms. The autism partic-
ipants were families of people with autism in the State of Arizona, contacted
using the mailing lists of the Greater Phoenix Chapter and the Pima
County (Tucson) Chapter of the Autism Society of America. The inclusion
Elements in Hair of Autistic Children 195
Biological Trace Element Research Vol. 110, 2006
criteria for the children was age 3–15 yr, with a diagnosis by a psychiatrist
or developmental pediatrician of Autism Spectrum Disorder (ASD),
including autism, PDD/NOS, and Asperger’s syndrome.
Parents of the participants with ASD asked friends and neighbors to
act as controls for the study. The criteria for the controls were that they (1)
be mentally and physically healthy individuals aged 3–15 yr without any
developmental delays, illness, or other medical conditions and (2) be unre-
lated to a person with ASD. Also, there was an attempt to match the ages
and genders as closely as possible. Table 1 lists the characteristics of the
cases and controls.
The children with ASD were also divided into several subgroups
based on other symptoms, so that it could be determined if any of the
196 Adams et al.
Biological Trace Element Research Vol. 110, 2006
Table 1
Characteristics of Participants
symptoms were associated with abnormal levels of elements in their hair.
All of the subgroupings were based on their mothers’ reports of symptoms
and included pica (eating of nonfood items, such as paper, sand, etc.),
major developmental regression (occurring on average at 18 mo), exces-
sive ear infections (>8 during first 3 yr of life), gastrointestinal problems
(chronic diarrhea/constipation), sleep problems (moderate/severe), and
low muscle tone (moderate/severe). Table 1 gives the numbers in each
subgroup. Also, a subgroup of children aged 3–6 yr was analyzed, which
included 23 children with autism and 16 controls.
Mothers were asked to participate as well, but their participation was
optional. Only mothers who had not dyed or permed their hair within 2
mo of collecting samples were included in the data. (None of the children
had had their hair permed or dyed.) A subgroup of mothers of younger
children (ages 3–8 yr) was also analyzed, and it included 22 mothers of
children with autism and 15 controls.
HAIR SAMPLING AND ANALYSIS
All participants (children and mothers) were asked to wash their hair
for 2 wk with Johnson’s and Johnson’s “No More Tears” Formula Baby
Shampoo, without the use of any other hair care products (no conditioner,
gel, hairspray, etc.). After 2 wk, a sample of hair was cut using stainless-
steel scissors. The hair sample was taken from the nape of the neck, taking
the 1 in. closest to the neck.
The samples were sent to Doctors Data Lab for analysis in a blinded
fashion. In the laboratory, the hair specimens were further cut and washed
using a modified method developed by the International Atomic Energy
Agency (12). The hair specimens were cut into approx 0.3-cm pieces and
mixed to allow a representative subsampling of the hair specimen. After
cutting, each sample was washed four times with a 1 : 200 (v/v) dilution
of Triton X-100 and then rinsed with acetone and allowed to drain. Sam-
ples were then rinsed three times with ultrapure deionized water and two
times with acetone. The dried samples were weighed prior to nitric
acid/microwave digestion as described in detail by Puchyr et al. (13). After
digestion, the samples were cooled and a 500-µL aliquot of an internal
standard was added and mixed with 50 mL of ultrapure deionized water.
The samples were then analyzed for element content using inductively
coupled plasma–mass spectrometry. To ensure validity, calibration verifi-
cations, a certified hair reference control, in-house controls, spiked hair
samples, and other appropriate control samples were analyzed. Results are
expressed as micrograms per gram, equivalent to parts per million.
The results are reported in Tables 2–5. Statistical analysis of the data
was carried out with an unmatched t-test, assuming two-sided Normal
distributions of unequal variance. Because a total of 39 elements were
examined, only p-values of 0.01 or lower will be discussed, although
Tables 2–5 highlight values up to p=0.05.
Elements in Hair of Autistic Children 197
Biological Trace Element Research Vol. 110, 2006
198 Adams et al.
Biological Trace Element Research Vol. 110, 2006
Table 2
Toxic Metals: Children with Autism vs Controls
Note: Units are micrograms per gram. t-Test values are given if they statistically significant. The pica subgroup is a subgroup of
the total.
Table 3
Essential Minerals: Children with Autism vs Controls
Note: Units are micrograms per gram. t-Test values are given if they were statistically significant. The pica subgroup is a subgroup of the total.
200 Adams et al.
Biological Trace Element Research Vol. 110, 2006
Table 4
Toxic Metals: Mothers of Children with Autism vs Controls
Note: Units in micrograms per gram. t-Test values are blank because none were statistically significant.
Table 5
Essential Minerals; Mothers of Children with Autism vs Controls
Note: Units in micrograms per gram. Only the Li values were statistically significant.
LIMITATIONS OF THE PRESENT STUDY
This study has several limitations, including the following:
1. Sample size: A larger sample size, from multiple sites, is
needed to improve the statistical power of this study and val-
idate or refute its findings.
2. Sample bias for cases: The participants represented 51 families
out of approx 1000 in the Arizona area who were contacted by
mail. The reason for the modest participation rate was that the
study included several other parts [reported in a separate arti-
cle, (14)] and a modest participation rate is typical for medical
studies that require substantial time commitment. Because
participants generally did not know their elemental status
prior to the study, this was probably a minor effect.
3. Sample bias for controls: The controls were chosen from the
friends and neighbors of the cases, which allows for an easy
way to obtain a reasonable match of geographic location and
socioeconomic status, but is not the most rigorous method.
4. Prevalent vs incident cases: This study involves prevalent cases,
not incident ones, so the results are not necessarily indicative of
mineral status during the development of autism.
5. Autism severity: This study did not independently evaluate
the severity of autism. It would be interesting to determine if
the severity of certain symptoms of autism correlated with
hair levels.
6. Contamination: Contamination of hair samples is always a
concern. This study involved washing with a common sham-
poo and washing by the lab, which reduces those concerns.
With a large enough sample size, contamination of samples
should be fairly similar for the autism and control groups.
7. Correlation of hair levels with body levels: Although hair is
known to be a good indicator for body levels of some toxic
metals and essential minerals, that is not true for all cases, and
in some cases we do not know.
RESULTS
Toxic Metals in Children with Autism
As shown in Tables 2 and 3, none of the children with autism had
abnormal levels of toxic metals with a p-value of 0.01 or less. Some possi-
bly abnormal levels (p=0.05) are listed in Tables 2 and 3 and a larger study
is needed to investigate those.
202 Adams et al.
Biological Trace Element Research Vol. 110, 2006
Toxic Metals in the Mothers
In the mothers of children with ASD, there was no statistically signif-
icant difference in the level of heavy metals in their hair.
Essential Minerals in the Children with Autism
Iodine: For the children with ASD, the mean level of iodine was
much lower (45%) than for the control children, and the differ-
ence was highly statistically significant (p=0.005). When the
subgroup of age 3–6 yr was considered, the magnitude of the
difference was almost identical (47%), although the difference
was not statistically significant because of the smaller number
of children in the subgroup.
Phosphorus: There was a small, but very statistically significant,
difference in the level of phosphorus, with the children with
ASD having a 12% lower value than the controls, with a
p=0.001. When the subgroup of children age 3–6 yr was con-
sidered, the magnitude of the difference was almost identical
(11% lower), although the difference was not statistically sig-
nificant because of the smaller number of children in the sub-
group.
Lithium: In the subgroup of children aged 3–6 yr, the children
with ASD had a 30% lower level of lithium with a marginal sta-
tistical significance (p=0.04). For the full group of children
(aged 3–15 yr), the difference was less (15%) and it was not sta-
tistically significant.
Chromium: The pica subgroup had much less chromium than the
nonpica subgroup (0.28 vs 0.42, p=0.004) or the typical children
(0.28 vs 0.45, p=0.002).
In terms of other subgroups, there were some differences in levels of
essential minerals:
1. For the regression and gastrointestinal subgroups, there were
no differences that reached a statistical significance of p=0.01
or lower.
2. Sleep: The autistic children with sleep disorders had lower lev-
els of selenium than the autistic children without sleep disor-
ders (0.99 vs 1.19 PPM, p=0.007, with controls, p=1.06 PPM).
3. Ear infections: The autistic children with fewer infections had
slightly lower levels of phosphorus than the autistic children
with more infections (175 vs 200 PPM, p=0.004, controls
p=213 PPM).
4. Muscle tone: The autistic children with low muscle tone had
very low potassium compared to the autistic children with nor-
mal muscle tone (16 vs 61 PPM, p=0.01, vs controls =47 PPM)
and high zinc (193 vs 150 PPM, p=0.01, controls =147 PPM).
Elements in Hair of Autistic Children 203
Biological Trace Element Research Vol. 110, 2006
Essential Minerals in the Mothers
The only statistically significant differences in the levels of essential
minerals between the mothers of children with ASD and mothers of typical
children was in their lithium levels. In the mothers of children with ASD,
the level of lithium was 40% lower than the mothers of typical children,
and the result was marginally statistically significant (p=0.05). When the
subgroup of mothers of children aged 3–8 yr was considered, the differ-
ence was more pronounced (56% lower) and more statistically significant
(p=0.005).
DISCUSSION
Toxic Metals in the Children with Autism
Overall, it appears that the children with autism do not have major
differences in their levels of toxic metals compared to controls. Because
mercury toxicity has been suggested as a cause of autism, it is worthwhile
to note that the autistic children in this study had levels that were very
similar to those of the typical children. In terms of the validity of our test-
ing, it should be pointed out that the mean values we found for the typi-
cal children aged 3–6 yr (0.21 µg/g) are similar but somewhat higher than
those of the 1999–2000 NHANES study (4) of 838 children aged 1–5 yr (0.12
µg/g). In terms of our results, our finding of similar values of mercury for
autistic children and controls is consistent with the study by Ip et al. (10),
which found similar (albeit much higher) levels in the autism and control
children in Hong Kong. However, it should be pointed out that this is long
past their primary exposure to mercury (from thimerosal-containing vac-
cines, maternal seafood consumption, and maternal mercury dental fill-
ings), so this hair measurement would not reflect such a long previous
exposure.
Thus, our results are not necessarily inconsistent with the results of
Holmes et al. (11), which found unusually low levels in baby hair, as the
ages of their group (12–24 mo) are quite different than ours (age 3–15 yr).
Actually, if both sets of data are valid, then they suggest a temporary loss
of the ability to excrete mercury in young infants. This temporary loss
could be explained by higher use of oral antibiotics (for ear infections) in
children with autism as we found here, as Rowland et al. (15) showed that
oral antibiotics dramatically inhibit mercury excretion to one-tenth of nor-
mal in rats.
Toxic Metals in the Mothers
Overall, the mothers of children with autism did not have any statis-
tically significant differences in the level of toxic minerals in their hair.
Because mercury is of great interest as a possible cause of ASD, it is worth-
while to note that the mothers of children with ASD had 57% more mer-
204 Adams et al.
Biological Trace Element Research Vol. 110, 2006
cury in their hair on average than the typical mothers, but this difference
was not statistically significant (p=0.22). When the subgroup of mothers of
young children was considered, there was less difference. In terms of the
validity of our testing, it should be pointed out that the mean values we
found for the typical mothers (0.35 µg/g) were similar but somewhat
higher than those of the 1999–2000 NHANES study (4) of 1726 women of
age 16–49 yr (0.20 µg/g). Because the average value of the autism mothers
is somewhat higher than that of the controls in our study and the sample
size is small, a larger study might be warranted.
Essential Minerals in Children with Autism
Iodine: The low levels of iodine in the hair of children with autism
suggests that iodine could be important in the etiology of
autism, presumably through its effect on thyroid function. There
have been several studies of thyroid function and autism,
including a report of a high incidence of thyroid abnormalities in
parents of children with autism (16), abnormal thyroid function
in young adults with severe autism correlating with impaired
verbal communication (17), and reduced thyroid-stimulating
hormone (TSH) levels in children with autism (n=41) vs controls
(18). There was also one small study (n=14) that found normal
levels of TSH in children with autism, but that study did not
have a control group (19). Overall, the reports of abnormal thy-
roid function in most of the studies are consistent with our find-
ings of low iodine, and it is possible that impaired thyroid
function is a cause of some of the symptoms of autism, especially
language impairment and mental retardation.
Iodine deficiency was extremely common in parts of the
United States in the early 1900s and caused many cases of goi-
ters (enlarged thyroid) and cretinism (a form of mental retar-
dation resulting from iodine deficiency). This prompted the
federal government to encourage the addition of iodine to salt
(iodinized salt). According to Hollowell et al.’s analysis (20) of
the NHANES surveys I and III, average iodine levels in the
United States (measured in the urine) have declined more than
50% during the 20-yr period from 1971 to 1974 to 1988–1994,
presumably resulting from decreased use of table salt (which is
one of the major sources of iodinized salt). It is possible that the
decreasing level of iodine in the United States is causally
related to the large increase in autism during the last 20 yr.
One study found that iodine levels in hair did increase after
exposure to iodine, but the hair was also susceptible to external
contamination (such as from sweat) and loss of iodine because
of washing (21). In the present study, all samples were washed
in the same manner, so external contamination effects were
Elements in Hair of Autistic Children 205
Biological Trace Element Research Vol. 110, 2006
minimized; thus, the results should be reasonably reflective of
excretion rates of iodine. Future studies should use blood,
urine, or saliva for increased reliability.
Lithium: In the subgroup of children aged 3–6 yr, the children
with ASD had a 30% lower level of lithium with a marginal sta-
tistical significance (p=0.04). For the full group of children
(aged 3–15 yr), the difference was less (15%) and it was not sta-
tistically significant. Low lithium was the only statistically sig-
nificant finding in the mothers of children with autism, so we
hypothesize that low levels in the mothers was the cause of low
lithium in the young children, which tended to normalize as
they grew older. Anke et al. (22) found that hair is a reliable
method to assess lithium deficiency in goats, in agreement with
measurements of blood, milk, and several other organs.
Lithium concentrations are highest in the brain (23) and are
highest during the first trimester (24), so a deficiency of it dur-
ing pregnancy could adversely affect fetal development and
especially brain development. Also, low levels of lithium in
humans have been found to correlate with a wide range of
behavioral problems, including aggression and decreased
sociability (25–27). One placebo-controlled treatment study by
Schrauzer and de Vroey (28) found that low-dose supplemen-
tation (400 µg/day) was beneficial to drug addicts, resulting in
increases in the subcategories of happiness, friendliness, and
energy. It should be noted that lithium is also used at dramati-
cally higher doses (of the order of 500,000 µg/day) as a psychi-
atric medication for bipolar disorder.
In addition, goats on a lithium-deficient diet were found to
suffer from lowered immunological status and chronic inflam-
mations, they had less lithium in their milk, and their infants
were found to have reduced growth rates. We hypothesize that
the low levels of lithium in the ASD mothers result in lower
levels in their children, which might explain why the children
suffer from a higher level of ear infections in their first 3 yr of
life. In turn, higher level of ear infections results in higher oral
antibiotic use, which results in a temporary decrease in the
ability to excrete mercury and can also contribute to gastroin-
testinal problems by eliminating normal gastrointestinal flora.
So, a low lithium level is plausible as an important factor in the
etiology of autism.
Phosphorus: The significance of the slightly lower phosphorus
level in children with autism is unclear. It might be an indica-
tor that children with autism tend to consume foods with lower
nutritional content (less fruits and vegetables).
Chromium: The finding of low chromium in the pica subgroup of
autistic children was highly statistically significant. However,
206 Adams et al.
Biological Trace Element Research Vol. 110, 2006
it is unclear if it is a cause of pica or the result of it. Low levels
of iron and zinc have previously been reported as being associ-
ated with pica, possibly as a cause of it (29).
Overall, the pica subgroup had low levels of sodium, chromium, and
sulfur, with the low chromium level being the most statistically significant
and, hence, most likely to be a possible factor in the etiology of pica. The
pica group also had elevated levels of strontium and copper, presumably
because of increased consumption.
For the subgroup with low muscle tone, the finding of low
potassium was large and statistically significant. Potassium is
needed for muscle contractions and is released during periods
of activity. Low potassium in the hair is an indication of low
muscle activity. The low muscle activity could be the result of
low potassium in the body overall, but hair measurements are
inconclusive regarding this point.
For the other subgroups, the differences in essential minerals
were generally minor.
Essential Minerals in the Mothers
The very low level of lithium in the mothers of children with autism
is interesting because that was the only abnormal finding in the mothers.
The significance of this is discussed in the preceding subsection.
ACKNOWLEDGMENTS
First and foremost, we thank the many autism families and their
friends who volunteered as participants in this research study. We thank
the Greater Phoenix Chapter and the Tucson Chapter of the Autism Soci-
ety of America for their financial support and for help with recruiting par-
ticipants. We thank Arizona State University for financial support. We
thank Mike Margolis for his assistance. We thank Jon Pangborn and Bob
Smith for their useful comments.
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