Schizophrenia Research 63 (2003) 73 – 78
www.elsevier.com/locate/schres
Low maternal vitamin D as a risk factor for schizophrenia:
a pilot study using banked sera
John McGrath a,*, Darryl Eyles a, Bryan Mowry a, Robert Yolken b, Stephen Buka c,d,e
a
Department of Psychiatry, Queensland Centre for Schizophrenia Research, University of Queensland,
The Park Centre for Mental Health, Wacol Q4076, Australia
b
Stanley Division of Developmental Neurovirology, Johns Hopkins School of Medicine, Baltimore, MD, USA
c
Harvard School of Public Health, Boston, MA, USA
d
Harvard Medical School, Boston, MA, USA
e
Harvard Institute of Psychiatric Epidemiology and Genetics, Boston, MA, USA
Received 6 June 2002; accepted 2 October 2002
Abstract
Objective: Evidence from epidemiology suggests that low maternal vitamin D may be a risk factor for schizophrenia.
Method: Based on sera taken during the third trimester, we compared the level of 25 hydroxyvitamin D3 in mothers of
individuals with schizophrenia or schizoaffective disorders versus mothers of unaffected controls. For each case, we selected
two controls matched on race, gender and date of birth of the offspring. Results: There was no significant difference in third
trimester maternal vitamin D in the entire sample (cases = 26, controls = 51). Within the subgroup of black individuals (n = 21),
there was a trend level difference in the predicted direction. Conclusions: Maternal vitamin D does not operate as a continuous
graded risk factor for schizophrenia, however, the results in the black subgroup raise the possibility that below a certain critical
threshold, low levels of maternal vitamin D may be associated with an increased risk of schizophrenia.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Vitamin D; Schizophrenia; Banked sera
1. Introduction
Prenatal nutritional deficiencies are biologically
plausible risk factors for schizophrenia (Brown et
al., 1996). To date, evidence linking maternal nutrition
and schizophrenia is based on ecological studies
drawn from the Dutch Hunger Winter (Susser et al.,
1998). These studies have reported an association
* Corresponding author. Tel.: +61-7-3271-8694; fax: +61-73271-8698.
E-mail address: [email protected] (J. McGrath).
between exposure to prenatal famine and an increased
risk of schizophrenia and affective disorders in the
offspring (Susser and Lin, 1992; Susser et al., 1996;
Brown et al., 2000). The precise nature of the nutritional exposures (e.g. micronutrient deficiency, protein or calorie malnutrition, etc.) that may underlie
these findings has not been clarified.
One specific nutritional factor that has been linked
to the epidemiology of schizophrenia is vitamin D
(McGrath, 1999). As vitamin D levels fluctuate over
the seasons, commentators have suggested that this
factor may be associated with the excess of schizo-
0920-9964/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0920-9964(02)00435-8
74
J. McGrath et al. / Schizophrenia Research 63 (2003) 73–78
phrenia births noted in winter and spring (Pile, 1951;
Moskovitz, 1978). In addition to seasonality, low
prenatal vitamin D may also provide a parsimonious
explanation for two other curious epidemiological
features of schizophrenia. The risk of developing
schizophrenia is increased in (a) those born in urban
versus rural locations (Marcelis et al., 1998; Mortensen et al., 1998) and (b) dark-skinned second generation migrants to cold climates (Harrison, 1990;
Selten et al., 2001). Urban place of residence is
associated with lower vitamin D levels compared to
rural place of residence (McGrath et al., 2001b) and
dark-skinned individuals living in cold climates are
particularly prone to develop hypovitaminosis D
(Holick, 1995; Looker et al., 2002).
Vitamin D (cholecalciferol) is mainly derived from
the action of ultraviolet radiation on a cholesterol
metabolite in the skin. After hydroxylation in the liver
(to 25-hydroxyvitamin D3: calcidiol), a second hydroxylation produces the active 1,25-dihydroxyvitamin D3 (calcitriol). Calcitriol is a potent seco-steroid
that regulates cell differentiation and proliferation,
immune function and genomic stability, in addition
to its classical role in calcium metabolism and bone
growth (DeLuca and Zierold, 1998). Receptors for
calcitriol are widely distributed throughout the developing and adult brain (Sutherland et al., 1992; Veenstra et al., 1998; Prufer et al., 1999) and the
importance of vitamin D in brain functioning is receiving increasing recognition (McGrath et al., 2001a;
Garcion et al., 2002).
We examined maternal vitamin D as a risk for
schizophrenia using banked maternal sera kept as part
of the National Collaborative Perinatal Project (Buka
et al., 1999). In this pilot study, we hypothesised that
the mothers of those with schizophrenia and other
psychotic disorders would have significantly lower
third trimester vitamin D levels compared to the
mothers of unaffected controls.
2. Methods
2.1. Participants
The study sample was drawn from the Boston, MA
and Providence, RI, cohorts of the National Collaborative Perinatal Project. Full details of the method-
ology are published elsewhere (Buka et al., 1999;
Zornberg et al., 2000; Buka et al., 2001). Pregnant
women were enrolled during clinical visits between
1959 and 1966 and selected to be representative of
patients receiving prenatal care at each study site. The
New England cohorts include 15,721 surviving offspring of a sample of 11,971 pregnant women.
Maternal serum samples were collected at various
time points and stored at NIH repositories at 20 jC.
In this study, we examined calcidiol levels in maternal
sera taken during the third trimester.
Offspring with psychotic illness were identified
through (a) personal interviews and/or (b) record linkage with psychiatric treatment facilities. Subjects were
re-contacted and interviewed by a trained diagnostic
interviewer using the Structured Clinical Interview for
DSM-IV (First et al., 1996) followed by best-estimate
consensus diagnoses generated by four experienced
diagnosticians according to DSM-IV criteria, based on
interview data and medical chart review. From the
cohort of 15,721 individuals, 119 offspring were
identified with an adult psychotic disorder (0.76%).
Thirty-nine met definite criteria for schizophrenia or
schizoaffective disorder (depressed) (0.25%). For the
current analysis, we selected at random 27 of these
subjects with schizophrenia or schizoaffective disorder
and with available maternal serum. For each case
subject, two healthy matched controls were selected,
matched for gender, race/ethnicity, and date of birth.
Controls were selected from an unaffected subset of
subjects with no Axis I psychiatric diagnoses. Human
subjects approval was granted by human studies
review groups at Harvard University, the National
Institute of Child Health and Human Development,
and local psychiatric facilities. After complete description of the study to the subjects, written informed
consent was obtained.
2.2. Assay
Calcidiol levels were assessed blind to case status
by a radioimmunoassay (Diasorin Stillwater, Minnesota, USA). This precursor is regarded as the most
appropriate indicator of overall vitamin D status
(Hunter, 1998; Hollis, 2000). The stored samples
had been diluted 1:5 in phosphate buffered saline,
thus, in order to ensure that samples with low values
of calcidiol (< 15 ng/ml) could be reliably measured,
J. McGrath et al. / Schizophrenia Research 63 (2003) 73–78
an extraction step was used prior to the immunoassay.
Samples were evaporated to dryness and then restored
to the original extract volume prior to assay with the
standard technique. The precision of this technique
was assessed in 10 aged plasma samples by diluting
them 1:5 in phosphate-buffered saline with subsequent storage at
70j. These samples were reconstituted and then assayed and results compared with
the same nondiluted plasma extracted according to the
manufacturers instructions. When this experiment was
repeated three times, the results yielded excellent
precision between the methods (slope = 0.90 F 0.03,
r2 = 0.89). Finally, assay accuracy was assessed with
samples obtained from the Vitamin D External Quality Assurance Scheme (DEQAS) yielding excellent
correlation (slope = 1.11 F 0.05, r2 = 0.97).
2.3. Analysis
As a preliminary examination of the data, t-tests
were used to assess the impact of season (winter/
spring versus summer/autumn) and race (black versus white) on calcidiol levels. The main analysis
was assessed with conditional logistic regression for
case/control matching where the odds ratio is for
75
proportional change in caseness per unit of calcidiol
(ng/ml) (Breslow and Day, 1980) using SAS version
8. One planned subgroup analysis was undertaken
with black and white case/control trios assessed
separately.
3. Results
Serum from one control was not available. Visual
inspection and subsequent testing demonstrated that
one sample had undergone deterioration. The level of
calcidiol was >250 ng/ml, a level substantially above
levels reported in the literature (Vieth, 1999) and
nearly five times higher than the next highest sample
in the study. This sample was excluded, and as it was
maternal sera from one of the cases, the maternal sera
from the two matched unaffected controls were also
excluded. Thus, maternal sera from 26 cases and 51
unaffected controls were available. There were 68 male
and 9 female subjects. Seven of the cases and 14 of the
unaffected controls were black, while the remaining
subjects (19 cases, 37 controls) were white (Fig. 1).
Overall, there were seasonal differences in the level
of calcidiol, with calcidiol levels measured in sera
Fig. 1. Calcidol levels (ng/ml) in all subjects, white subjects only and black subjects only. The box plots show the median (central line), 25th and
75th quartiles (box), and the highest and lowest values (bars) excluding outliers (circles).
76
J. McGrath et al. / Schizophrenia Research 63 (2003) 73–78
obtained during winter/spring being significantly lower
than levels measured in sera obtained during summer/
autumn (mean ng/ml (S.D.) = 15.25 (8.18); 25.59
(10.02) respectively; t = 4.50, df 75, p < 0.001).
Overall, black mothers had significantly lower levels
of calcidiol compared to white mothers (mean ng/ml
(S.D.) = 11.76 (6.48); 22.06 (9.80) respectively,
t = 4.46, df 75, p < 0.001).
There was no significant difference in maternal
calcidiol levels between cases and matched controls
(Odds ratio = 0.98, 95% CI 0.92– 1.05). Within the
white subjects, there was no significant difference
(OR = 1.01, 95% CI 0.94 – 1.10), however, a trend
level difference in the predicted direction emerged
when black individuals were examined separately
(Case Mean (S.D.)= 8.44 (3.25); Control Mean
(S.D.) = 13.42 (7.13); OR = 0.78, 95% CI 0.55 –1.08).
In order to further explore this finding, we undertook a post hoc assessment of maternal calcidiol levels
in cases versus controls in those with calcidiol level
less than 15 ng/ml (a standard definition of vitamin D
deficiency (Vieth, 1999)). A total of 12 of the 26
mothers of cases and 15 of the 51 mothers of controls
had levels below 15 ng/ml. (Odds ratio = 2.06, 95% CI
0.77– 5.47), however, there was no significant group
difference in the mean calcidiol level between cases
and controls in this ‘deficient’ subgroup (data not
shown).
increased rates of hypovitaminosis D in dark-skinned
migrants to cold climates with (b) the increased risk of
schizophrenia in these ethnic groups. Black infants
growing up in cold climates have a higher prevalence
of rickets compared to white infants, as a result of the
influence of skin pigmentation on actinic production
of cholecalciferol (Holick, 1995; Kreiter et al., 2000;
Abrams, 2002). Perhaps hypovitaminosis D during
early postnatal life, which is more common in black
versus white infants, warrants inspection in future
studies. Alternatively, perhaps only marked prenatal
vitamin D deficiency operates as a risk-modifying
factor, in which case this study lacked sufficient power
to confidently detect this effect. These revised hypotheses are consistent with recent animal experiments
demonstrating (a) an association between the very low
prenatal vitamin D and altered brain development in
the rat (Eyles et al., 2002; Feron et al., 2002) and (b)
an association between combined prenatal and postnatal vitamin D depletion and altered prepulse inhibition in the rat (Mackay-Sim et al., 2002).
Future studies of the role of maternal vitamin D
and schizophrenia may wish to focus specifically on
individuals born during winter and spring, and darkskinned individuals living in cold climates. Based on
the group difference and variance found in the maternal calcidiol levels from the black individuals, over 18
case/control matched pairs of black individuals would
be required to confidently detect a group difference
(1:1 matching, alpha = 0.05, Power = 0.8).
4. Discussion
4.1. Limitations of the study
Maternal calcidiol levels during the third trimester
were not associated with risk of schizophrenia in the
offspring in this pilot study based on banked sera.
Thus, the hypothesis linking low prenatal vitamin D
and schizophrenia is weakened. In particular, the
results of this study allow us to reject the hypothesis
that maternal calcidiol operates as a continuous
graded risk factor for schizophrenia.
The pilot study has also provided an interesting
clue for future research. The trend level group difference found in the small black sample (7 cases and 14
controls) leaves open the possibility that factors associated with race may influence the putative association
between vitamin D and schizophrenia. This would be
congruent with one of the core elements of the original
hypothesis (McGrath, 1999), which linked (a) the
Apart from the small sample size, the study is also
limited by our ability to predict overall maternal
vitamin D status based on only one 3rd trimester
blood sample. It is likely that maternal calcidiol levels
fluctuate from week to week in response to outdoor
behaviour, dress, climate and diet.
Also, the interpretation of the absolute calcidiol
levels in this study requires caution. The calcidiol
levels detected in the stored sera were comparable to
the winter –summer fluctuations reported for nonpregnant women aged between 20 and 40 years living in
Boston, MA (12 –16 ng/ml for black women, 24– 34
ng/ml for white women; (Harris and Dawson-Hughes,
1998)). Groups interested in the association between
vitamin D and cancer have also reported that calcidiol
J. McGrath et al. / Schizophrenia Research 63 (2003) 73–78
levels as measured in sera stored over two to three
decades appear valid when compared to levels
detected in comparable fresh sera (Corder et al.,
1993; Braun et al., 1995; Nomura et al., 1998).
However, the long storage of the sera might have
resulted in a slight reduction in calcidiol levels due to
precipitation of vitamin D binding protein. We expect
that this artefact would impact on case and control
sera equally, but may reduce the range of calcidiol
levels and thus further reduce the power to detect true
but small group differences.
If the offspring of dark-skinned mothers living in
cold climates are more vulnerable to low prenatal
vitamin D, then the hypothesis would suggest that the
incidence of schizophrenia should also be higher in
these groups. While there are no US-based incidence
studies available to clarify this issue, two major
community-based studies of the prevalence of schizophrenia in the United States (Regier et al., 1993;
Kendler et al., 1996) have reported nonsignificant but
numerically higher prevalence rates for schizophrenia
in black individuals. Gallagher et al. (1984) also
reported a more prominent winter-birth excess for
schizophrenia in black patients compared to white
patients, and linked this effect to poorer maternal
nutritional during winter in black women.
The opportunity to measure candidate exposures
such as calcidiol in banked sera provides a powerful
tool to evaluate novel risk factors that ordinarily rely
on ecological studies. In this case, the results of this
pilot study have (a) allowed us to reject the proposition that vitamin D operates as continuous graded risk
factor for schizophrenia and (b) provided clues for
future refinements of the hypothesis.
Acknowledgements
The Stanley Medical Research Institute supported
this project. We are grateful to Graham Carter from
DEQAS for advice on the assay.
References
Abrams, S.A., 2002. Nutritional rickets: an old disease returns.
Nutr. Rev. 60, 111 – 115.
Braun, M.M., Helzlsouer, K.J., Hollis, B.W., Comstock, G.W.,
77
1995. Prostate cancer and prediagnostic levels of serum vitamin
D metabolites (Maryland, United States). CCC, Cancer Causes
Control 6, 235 – 239.
Breslow, N.E., Day, N.E., 1980. Statistical Methods in Cancer Research, Vol. 1: The Analysis of Case-Control Studies, IARC
Scientific Publications No. 32, International Agency of Research on Cancer, Lyon, France.
Brown, A.S., Susser, E.S., Butler, P.D., Richardson, A.R., Kaufmann, C.A., Gorman, J.M., 1996. Neurobiological plausibility
of prenatal nutritional deprivation as a risk factor for schizophrenia. J. of Nerv. Ment. Dis. 184, 71 – 85.
Brown, A.S., van Os, J., Driessens, C., Hoek, H.W., Susser, E.S.,
2000. Further evidence of relation between prenatal famine and
major affective disorder. Am. J. Psychiatry 157, 190 – 195.
Buka, S.L., Goldstein, J.M., Seidman, L.J., Zornberg, G.L., Donatelli, J.A., Denny, L.R., Tsuang, M.T., 1999. Prenatal complications, genetic vulnerability, and schizophrenia: the New
England longitudinal studies of schizophrenia. Psychiatr. Ann.
29, 151 – 156.
Buka, S.L., Tsuang, M.T., Torrey, E.F., Klebanoff, M.A., Bernstein, D., Yolken, R.H., 2001. Maternal infections and subsequent psychosis among offspring. Arch. Gen. Psychiatry 58,
1032 – 1037.
Corder, E.H., Guess, H.A., Hulka, B.S., Friedman, G.D., Sadler,
M., Vollmer, R.T., Lobaugh, B., Drezner, M.K., Vogelman, J.H.,
Orentreich, N., 1993. Vitamin D and prostate cancer: a prediagnostic study with stored sera. Cancer Epidemiol. Biomark. Prev.
2, 467 – 472.
DeLuca, H.F., Zierold, C., 1998. Mechanisms and functions of
vitamin D. Nutr. Rev. 56, S4 – S10 (discussion S 54-75).
Eyles, D., Feron, F., Brown, J., Mackay-Sim, A., McGrath, J., 2002.
Low maternal vitamin D3 disrupts foetal brain maturation.
Schizophr. Res. 53, 228.
Feron, F., Mackay-Sim, A., Eyles, D., Brown, J., McGrath, J., 2002.
Vitamin D depletion during development markedly alters gene
expression in adult rat brain: gene array assessment of a novel
risk factor for schizophrenia. Schizophr. Res. 53, 83.
First, M.B., Spitzer, R.L., Gibbon, M., Williams, J.B.W., 1996.
Structured Clinical Interview for DSM-IV Axis I Disorders,
Clinician Version (SCID-CV) American Psychiatric Press,
Washington, DC.
Gallagher, B.J., Jones, B.J., McFalls, J.A., 1984. The ‘‘winter born
phenomenon’’ among schizophrenics: differences between
Blacks and Whites. J. Clin. Psychol. 40, 1151 – 1159.
Garcion, E., Wion-Barbot, N., Montero-Menei, C.N., Berger, F.,
Wion, D., 2002. New clues about vitamin D functions in the
nervous system. Trends Endocrinol. Metab. 13, 100 – 105.
Harris, S.S., Dawson-Hughes, B., 1998. Seasonal changes in plasma 25-hydroxyvitamin D concentrations of young American
black and white women. Am. J. Clin. Nutr. 67, 1232 – 1236.
Harrison, G., 1990. Searching for the causes of schizophrenia: the
role of migrant studies. Schizophr. Bull. 16, 663 – 671.
Holick, M.F., 1995. Environmental factors that influence the cutaneous production of vitamin D. Am. J. Clin. Nutr. 61, 638S – 645S.
Hollis, B.W., 2000. Comparison of commercially available (125)Ibased RIA methods for the determination of circulating 25-hydroxyvitamin D. Clin. Chem. 46, 1657 – 1661.
78
J. McGrath et al. / Schizophrenia Research 63 (2003) 73–78
Hunter, D., 1998. Biochemical indicators of dietary information. In:
Willett, W. (Ed.), Nutritional Epidemiology. Oxford Univ. Press,
New York, pp. 174 – 243.
Kendler, K.S., Gallagher, T.J., Abelson, J.M., Kessler, R.C., 1996.
Lifetime prevalence, demographic risk factors, and diagnostic
validity of nonaffective psychosis as assessed in a US community sample. The National Comorbidity Survey. Arch. Gen. Psychiatry 53, 1022 – 1031.
Kreiter, S.R., Schwartz, R.P., Kirkman Jr., H.N., Charlton, P.A.,
Calikoglu, A.S., Davenport, M.L., 2000. Nutritional rickets in
African American breast-fed infants. J. Pediatr. 137, 153 – 157.
Looker, A.C., Dawson-Hughes, B., Calvo, M.S., Gunter, E.W.,
Sahyoun, N.R., 2002. Serum 25-hydroxyvitamin D status of
adolescents and adults in two seasonal subpopulations from
NHANES III. Bone 30, 771 – 777.
Mackay-Sim, A., Feron, F., Eyles, D., Brown, J., McGrath, J., 2002.
Low vitamin D disrupts prepulse inhibition in adult but not
young rats: a new animal model of schizophrenia. Schizophr.
Res. 53, 216.
Marcelis, M., Navarro-Mateu, F., Murray, R., Selten, J.-P., van Os,
J., 1998. Urbanization and psychosis: a stud of 1942 – 1978 birth
cohorts in The Netherlands. Psychol. Med. 28, 871 – 879.
McGrath, J., 1999. Hypothesis: is low prenatal vitamin D a
risk-modifying factor for schizophrenia? Schizophr. Res. 40,
173 – 177.
McGrath, J., Feron, F., Eyles, D., 2001a. Vitamin D: the neglected
neurosteroid? Trends Neurosci. 24, 570 – 572.
McGrath, J.J., Kimlin, M.G., Saha, S., Eyles, D.W., Parisi, A.V.,
2001b. Vitamin D insufficiency in south-east Queensland. Med.
J. Aust. 174, 150 – 151.
Mortensen, P.B., Pedersen, C.B., Westergaard, T., Wohlfahrt, J.,
Ewald, H., Mors, O., Andersen, P.K., Melbye, M., 1998. Familial and non-familial risk factors for schizophrenia: a population-based study. Schizophr. Res. 29, 13.
Moskovitz, R.A., 1978. Seasonality in schizophrenia (letter). Lancet
1, 664.
Nomura, A.M., Stemmermann, G.N., Lee, J., Kolonel, L.N., Chen,
T.C., Turner, A., Holick, M.F., 1998. Serum vitamin D metabolite
levels and the subsequent development of prostate cancer (Hawaii, United States). CCC, Cancer Causes Control 9, 425 – 432.
Pile, W.J., 1951. A study of the correlation between dementia praecox and the month of birth. Va. Med. Mon. 78, 438 – 440.
Prufer, K., Veenstra, T.D., Jirikowski, G.F., Kumar, R., 1999.
Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord. J. Chem. Neuroanat. 16, 135 – 145.
Regier, D.A., Farmer, M.E., Rae, D.S., Myers, J.K., Kramer, M.,
Robins, L.N., George, L.K., Karno, M., Locke, B.Z., 1993.
One-month prevalence of mental disorders in the United States
and sociodemographic characteristics: the Epidemiologic Catchment Area study. Acta Psychiatr. Scand. 88, 35 – 47.
Selten, J.P., Veen, N., Feller, W., Blom, J.D., Schols, D., Camoenie,
W., Oolders, J., van der Velden, M., Hoek, H.W., Rivero, V.M.,
van der Graaf, Y., Kahn, R., 2001. Incidence of psychotic disorders in immigrant groups to The Netherlands. Br. J. Psychiatry
178, 367 – 372.
Susser, E.S., Lin, S.P., 1992. Schizophrenia after prenatal exposure
to the Dutch hunger winter of 1944 – 1945. Arch. Gen. Psychiatry 49, 938 – 988.
Susser, E., Neugebauer, R., Hoek, H., Brown, A.S., Lin, S., Labovitz, D., Gorman, J.M., 1996. Schizophrenia after prenatal
famine: further evidence. Arch. Gen. Psychiatry 53, 25 – 31.
Susser, E., Hoek, H.W., Brown, A., 1998. Neurodevelopmental
disorders after prenatal famine: the story of the Dutch Famine
Study. Am. J. Epidemiol. 147, 213 – 216.
Sutherland, M.K., Somerville, M.J., Yoong, L.K., Bergeron, C.,
Haussler, M.R., McLachlan, D.R., 1992. Reduction of vitamin
D hormone receptor mRNA levels in Alzheimer as compared to
Huntington hippocampus: correlation with calbindin-28 k
mRNA levels. Brain Res. Mol. Brain Res. 13, 239 – 250.
Veenstra, T.D., Prüfer, K., Koenigsberger, C., Brimijoin, S.W.,
Grande, J.P., Kumar, R., 1998. 1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo. Brain
Res. 804, 193 – 205.
Vieth, R., 1999. Vitamin D supplementation, 25-hydroxyvitamin D
concentrations, and safety. Am. J. Clin. Nutr. 69, 842 – 856.
Zornberg, G.L., Buka, S.L., Tsuang, M.T., 2000. Hypoxic-ischemia-related fetal/neonatal complications and risk of schizophrenia and other nonaffective psychoses: a 19-year longitudinal
study. Am. J. Psychiatry 157, 196 – 202.