Metabolic Profiling of Plasma from Discordant Schizophrenia Twins: Correlation between Lipid Signals and Global Functioning in Female Schizophrenia Patients Tsz M. Tsang,†,§ Jeffrey T.-J. Huang,‡,§ Elaine Holmes,†,§ and Sabine Bahn*,‡ Biological Chemistry, Biomedical Sciences Division, Faculty of Medicine, Imperial College, London, SW7 2AZ, United Kingdom and Institute of Biotechnology, University of Cambridge, Cambridge, CB2 1QT, United Kingdom Received November 4, 2005 1H
NMR spectroscopy-based metabonomic analysis was employed to investigate plasma samples from 21 pairs of monozygotic twins discordant for schizophrenia and 8 pairs of age-matched healthy twins in an effort to disentangle genetic and epigenetic components of schizophrenia. We identified alterations in the lipid profile of both affected and unaffected schizophrenia twins. Additionally, there is a close association of VLDL/LDL signals and Global Functioning Scores in female twins suffering from schizophrenia. Our results further support a link between metabolic disturbances and the etiopathology of schizophrenia.
Keywords: schizophrenia • lipid • LDL • VLDL • global functioning score • metabolic profiling
Introduction Twin studies are particularly powerful in trying to unravel genetic and environmental factors responsible for complex disorders such as schizophrenia. Previous studies have demonstrated that the likelihood of developing schizophrenia correlates highly with the level of consanguinity and reaches a concordance rate of about 30-50% for monozygotic twins.1,2 Thus, it is now widely accepted that nongenetic environmental factors contribute to the etiopathology of schizophrenia and/ or precipitate the onset of the syndrome. Numerous biological (viral exposure,3 illicit drug use,4 perinatal insults,5 etc.) and social stressors are being considered as environmental disease components, which are likely to interact with a predisposing genotype. Investigations of discordant twins may help to disentangle some of these components. Due to the difficulties of obtaining brain samples from discordant twins in sufficient numbers, studies of monozygotic twins discordant for schizophrenia have so far focused on brain imaging. Twin studies imply that one of the most consistently reported brain alterations in schizophrenia, i.e., lateral ventricular enlargement, can be attributed to environmental factors.6,7 Recent functional genomics studies suggest a metabolic component to the schizophrenia syndrome. Findings of abnormal glucoregulation, mitochondrial dysfunction, and lipid metabolism8-11 align well with previous evidence of metabolic alterations in schizophrenia and affective disorders. Antipsychotic drug action has been prominently linked to dyslipidemia, but reports of altered glucose metabolism predate the anti* To whom correspondence should be addressed. E-mail:
[email protected]. † Imperial College, London. ‡ University of Cambridge. § These authors contributed equally to the work.
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psychotic era (reviewed by Haupt and Newcomer12) and a recent report aimed at determining the rate of metabolic syndrome in long-term schizophrenia patients found the prevalence of metabolic syndrome to be inversely correlated to the daily dose of antipsychotic drugs.13 Here, we present results from an 1H NMR-based metabonomics study, where we identified alterations in the lipid profile of both affected and unaffected schizophrenia twins. Furthermore, we found that lipid levels correlated highly with global function scores for the affected female twins.
Methods and Materials Plasma samples from 21 pairs of monozygotic twins discordant for schizophrenia and 8 pairs of matched control twins were collected under standardized conditions by Dr. Fuller Torrey, Stanley Medical Research Institute, Bethesda, USA. All study participants gave their written informed consent and the original study was approved by an Institutional Review Board. The GAF (Global Assessment of Functioning) of each individual was derived by consensus of two interviewers using the Structured Clinical Interview for DSM-IV. The plasma was obtained from both twins simultaneously as part of a lymphocyte collection aphoresis procedure carried out at mid-morning, with both twins having been on similar diets and residing in a hotel together. Twin samples were divided into aliquots and stored at -80 °C. None of the samples underwent more than three freezethaw cycles prior to acquisition of NMR spectra. All experiments were performed under blind and randomized conditions. Plasma samples were diluted to a final volume of 550 µL by the addition of isotonic saline solution containing 10% D2O for the NMR field-frequency lock. 10.1021/pr0503782 CCC: $33.50
2006 American Chemical Society
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Metabolic Profiling of Discordant Schizophrenia Twins
Figure 1. Metabonomic analysis of plasma samples from monozygotic twins discordant for schizophrenia and control twins. (A) Partial 1H NMR spectrum of plasma samples from a representative pair of twins discordant for schizophrenia (affected co-twin in red and the unaffected in black) illustrate changes in lipid regions-(CH2)n and CH3 lipids. (B) A PLS-DA scores plot showing differentiation of control twins from twins with schizophrenia and their unaffected co-twins as determined by 1H NMR spectroscopy of plasma. Note that the unaffected co-twin shows an intermediate position between controls and the schizophrenic twin. (C) PLS-DA loadings plot revealing the spectroscopic regions attributable to distinction of disease classes. Note that the variables representing the aromatic region (7.029.10 ppm) are positively correlated with the control twins, most likely reflecting higher protein levels in this group. 1H NMR Spectroscopy of Plasma Samples. Standard 1-D 600 MHz 1H NMR spectra were acquired for all samples using a presaturation pulse sequence to effect suppression of the water resonance (pulse sequence: relaxation delay-90°-t1-90°-tm-90°acquire FID; Bruker Analytische GmbH, Rheinstetten, Germany). In this pulse sequence, a secondary radio frequency irradiation is applied specifically at the water resonance frequency during the relaxation delay of 2 s and the mixing period (tm ) 100 ms), with t1 fixed at 3 µs. Typically 256 transients were acquired at 300 K into 32 K data points, with a spectral width of 6000 Hz and an acquisition time of 1.36 s per scan. Prior to Fourier transformation, the free induction decays were multiplied by an exponential weighting function corresponding to a line-broadening factor of 0.3 Hz. Data Reduction and Pattern Recognition Procedures. To evaluate efficiently the metabolic variability within and between biofluids derived from patients and controls, spectra were data reduced using the software program AMIX (Analysis of MIXtures version 2.5, Bruker Rheinstetten, Germany) and exported into SIMCA-P (version 10.5, Umetrics AB, Umeå, Sweden) where a range of multivariate statistical analyses were conducted. Initially, principal components analysis (PCA) was applied to the data in order to discern the presence of inherent similarities in spectral profiles. Where the classification of 1H NMR spectra was influenced by exogenous contaminants, the
spectral regions containing those signals were removed from statistical analysis. To confirm the biomarkers differentiating between the schizophrenia patients and matched controls, projection to latent structure discriminant analysis (PLS-DA) was employed. Where appropriate, data were subjected to oneway analysis of variance (ANOVA) using the Statistical Package for Social Scientists (SPSS/PC 13; SPSS, Chicago). Where the F ratio gave p < 0.05, comparisons between individual group means were made by Dunnett T3 test at significance levels of p ) 0.05.
Results Plots of PLS-DA scores, based on 1H NMR spectra of plasma from 21 pairs of monozygotic twins discordant for schizophrenia and 8 pairs of matched control twins, differentiated the affected and unaffected twins from age-matched control twins (Figure 1, A and B). The loading coefficients indicated that resonances from very low-density lipoproteins (VLDL) (0.920.88 ppm and 1.28-1.32 ppm), low-density lipoproteins (LDL) (0.84-0.88 ppm and 1.24-1.28 ppm) and aromatic groups (∼7.5 ppm; most likely deriving from plasma proteins) were predominantly responsible for the separation (Table 2; Figure 1C). Co-twins with schizophrenia showed a 23% (p ) 0.015; ANOVA) increase in plasma VLDL signals (1.28-1.32 ppm) compared to control twins. Corresponding unaffected co-twins Journal of Proteome Research • Vol. 5, No. 4, 2006 757
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Table 1. Demographic Details of Monozygotic Twins
twins discordant for schizophrenia affected unaffected control twins MALE twins discordant for schizophrenia affected unaffected control twins
total
age
drug treatmentb
duration of illness (yrs)
DSM IV (Axis V)
gender (m/f)
21 21 16
33.0 ( 6.1 33.0 ( 6.1 32.1 ( 7.5
26757 ( 27320 0 0
12.4 ( 7.0 0 0
40.1 ( 13.7c 82.5 ( 5.0d 86.8 ( 4.5
13/8 13/8 6/10
13 13 6
32.5 ( 6.2 32.5 ( 6.2 38.7 ( 6.7a
27430 ( 32607 0 0
13.4 ( 6.9 0 0
43.4 ( 11.9c 82.1 ( 4.8d 88.7 ( 1.5
8 8 10
33.9 ( 6.4 33.9 ( 6.4 29.3 ( 6.4
25662 ( 17537 0 0
11.8 ( 7.3 0 0
34.8 ( 15.5c 83.3 ( 5.4 85.6 ( 5.4
FEMALE twins discordant for schizophrenia affected unaffected control twins
a p ) 0.04, control twins vs discordant twins with schizophrenia; Oneway ANOVA. b Fluphenezine equivalent; these patients only received typical antipsychotics at time of testing. c p < 0.01, vs the unaffected and control twins; Oneway ANOVA. d p < 0.05, vs control twins; Oneway ANOVA.
Table 2. Statistical Analysis of Major Chemical Shifts that Are Changed in Plasma from Female Monozygotic Twinsa chemical shift (ppm)
0.84-0.88 0.88-0.92 1.24-1.28 1.28-1.32 7.5
assignmentb
affected twins
unaffected twins
control twins
lipid (LDL mainly) lipid (VLDL mainly) lipid (LDL mainly) lipid (VLDL mainly) aromatic groups
2.62 ( 1.91 ( 0.16c 3.72 ( 0.62c 3.15 ( 0.98c 0.142 ( 0.009c
2.37 ( 0.12 1.72 ( 0.10 2.96 ( 0.23d 2.31 ( 0.36 0.153 ( 0.007d
2.25 ( 0.18 1.61 ( 0.10 2.64 ( 0.28 1.97 ( 0.19 0.166 ( 0.008
0.12c
a Data are shown as mean ( S. D. b The assignments of signals are based on a study by Nicholson et al.21 Oneway ANOVA. d p < 0.05 vs control twins; Oneway ANOVA.
were also found to have increased 1.28-1.32 ppm signals, however, differences were not significant for the unaffected group (p ) 0.18; ANOVA). LDL levels in the three groups showed a trend similar to that of the VLDL signals but, again, did not reach statistical significance (data not shown). In addition, discordant schizophrenia twins had lower plasma protein levels represented by broad signals in the aromatic region around 7.5 ppm (14% and 8% reduction for the affected and unaffected co-twins respectively; p < 0.01). No difference was observed in high-density lipoprotein (HDL) signals (0.60.7 ppm) between the groups. Further analyses showed a much more pronounced differentiation of female twins (Figure 2). The key chemical shifts that differentiated the groups are listed in Table 2. Interestingly, PLS-DA analyses of the female affected and healthy discordant twins alone showed that the same scores and loading plots that significantly separated the discordant twins from control twins is responsible for the separation between the discordant twins themselves. This implies that the identified metabolic alterations are a genuine disease-related signature (see Supporting Information Figure 1). The sensitivity and specificity of signatures in distinguishing between affected and unaffected female co-twins for schizophrenia were 87.5% (7/8) and 75% (6/8), respectively using a PLS model built on the affected and unaffected groups based on the first component (data not shown). Furthermore, signals between 1.24 and 1.28 ppm (mainly LDL) correlated strongly with scores obtained from the DSM-IV Axis V Global Assessment of Functioning (GAF) Scale (R2 ) 0.62, Figure 3), which represents one of the most widely used methods for assessing impairment among patients with psychiatric disorders. The rating is made on a scale from 1 to 100 with ratings of 1-10 representing severe impairment and ratings of 90 or more 758
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c
p < 0.05 vs unaffected twins and control twins;
indicating superior functioning (DSM-IV). Plasma VLDL signals (1.28-1.32 ppm) of female twins show a strong correlation with GAF scores (R2 ) 0.54; Figure 3). No correlation was found when all twins or male twins alone were considered (data not shown). Age did not appear to have an effect on disease-related chemical shifts. However, antipsychotic drug exposure (measured as fluphenezine equivalent) also correlated with GAF scores and the metabolic signature of the female twins, respectively. On the other hand, corresponding plots of PLS-DA scores of plasma 1H NMR spectra derived from male twins discordant for schizophrenia showed a less prominent differentiation between affected and unaffected twins (Supporting Information, Figure 2A). Unlike the female twins, the loading coefficients indicated that resonances from the aromatic region, corresponding to plasma proteins, are mainly responsible for the separation among male twins (Supporting Information, Figure 2B). No correlation was found between the glucose signals and antipsychotic treatment, age, duration of illness, substance abuse and GAF scores (data not shown) for male twins. No significant difference was found between male control twins and unaffected co-twins (Supporting Information, Figure 2A).
Discussion The present study examined the metabolic plasma profiles of a total of 42 monozygotic twins discordant for schizophrenia and 16 matched control twins using 1H NMR spectroscopy in order to explore the role of genetic and environmental factors contributing to schizophrenia. Our result show that signals from VLDL, LDL and aromatic regions are the most important factors
Metabolic Profiling of Discordant Schizophrenia Twins
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Figure 2. Metabonomic analysis of plasma samples from female twins discordant for schizophrenia and female control twins. A PLSDA scores plot (Figure 2A) of female monozygotic twins showing a clear differentiation of control twins, unaffected twins and the schizophrenic twins as determined by 1H NMR spectroscopy of plasma. The loading plot (Figure 2B) demonstrates that LDL (0.86 and 1.26), VLDL (0.9 and 1.3) and aromatic regions (∼7.5) are the key chemical shifts that contribute to the separation. Note the high similarity between Figure 1C and Figure 2B.
differentiating ill and healthy co-twins discordant for schizophrenia from control twins. Interestingly, we found that this differentiation was much more pronounced for female twins. A similar finding was reported in a recent study on patients with acute-phase schizophrenia (drug free for at least 1 week) where the female schizophrenia group had much higher increases in serum LDL and VLDL levels compared to the male group.14 The major function of VLDL/LDL is cholesterol transport. In this study, we observed differences in both affected and unaffected twins in serum VLDL/LDL levels, suggesting alterations in lipid metabolism/transport in the pathophysiology of schizophrenia. Indeed, increased levels of apolipoprotein E have been reported in the prefrontal cortex of subjects with schizophrenia15,16 and a recent epidemiological study found that 37% of schizophrenia patients suffer from metabolic syndrome, which is characterized by disturbances in glucose and lipid regulation as well as hypertension. Importantly, the authors of this study found an inverse correlation of the daily dose of antipsychotics with metabolic syndrome in schizophrenia patients.13 In addition, recent studies from our laboratory using several high-throughput proteomic techniques on brain tissue,
cerebrospinal fluid as well as peripheral tissue of patients and matched controls, i.e., liver, red blood cells, and serum showed a consistent and significant reduction in the levels of apolipoproteins that are a major components of the HDL fraction (unpublished). Our current study adds further weight to this hypothesis. Although overall similar metabolic changes were observed in male and female schizophrenia twins, only in the female group could we discern a potential predisposing disease signature in unaffected co-twins. This could imply a greater genetic loading for female twins. A marked sex difference in schizophrenia is a well-established fact; female schizophrenia patients have on average a later age of onset and better prognosis. This has been attributed to protective effects of oestrogens. Women suffering from acute psychotic episodes have been shown to exhibit lower levels of oestrogen,17 which are known to have neuroprotective properties and may reduce cell death associated with excitotoxicity as well as oxidative stress.18 In female twins suffering from schizophrenia, alterations were highly associated with disease severity as well as exposure to typical antipsychotics, making it difficult to disentangle Journal of Proteome Research • Vol. 5, No. 4, 2006 759
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correlation between VLDL/LDL signals and GAF scores in female schizophrenia twins remains however unclear and warrants further investigation.
Acknowledgment. The authors would like to thank the Stanley Medical Research Institute and The Henry Smith Charity for financial support, special thanks to Dr Fuller Torrey for providing the twin samples and supporting this study. Thanks also to Dr Robert H. Yolken and Professor Chris Lowe for intellectual input. Thanks to all other members of the Bahn laboratory for discussions, help and encouragement. Most of all, thanks to all patients and healthy volunteers for their selfless donation of blood for this study. Supporting Information Available: Supporting Information Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References
Figure 3. Negative correlations between global functioning score (DSM IV, Axis V) and two key chemical shifts (1.24-1.28 ppm; A and 1.28-1.32 ppm; B) primarily corresponding to LDL and LDL levels in female twin plasma. The R2 are shown in each plots.
confidently the contribution of environmental factors and drug effects. Particularly, some of the typical antipsychotics such as phenothiazines and butyrophenones have been reported to increase serum LDL and/or VLDL levels.19,20 However, several lines of evidence suggest that our observations are not a drug effect: (1) Similar changes were identified in the unaffected co-twins. (2) most affected male twins also received phenothiazines or butyrophenones at the time of testing but did not show a similar correlation between LDL/VLDL levels and antipsychotics as found in females. One of the most interesting findings in this study is the close association of VLDL/LDL signals and Global Functioning Scores (DSM-IV, Axis V). This is, to our knowledge, the first report showing a strong correlation between a subjectively derived clinical rating score and an objective biomarker; the hope is that in future biomarker tests may aid psychiatrists in diagnosis and establishing clinical response. The reason for the high
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(1) Gottesman, I. I. Schizophrenia Genesis: The Origins of the Madness; Henry Holt & Co. Inc.: New York, 1991; p 296. (2) Kringlen, E. Am. J. Med. Genet. 2000, 97 (1), 4-11. (3) Yolken, R. H.; Torrey, E. F. Clin. Microbiol. Rev. 1995, 8 (1), 131145. (4) Kosten, T. R.; Ziedonis, D. M. Schizophr. Bull. 1997, 23 (2), 181186. (5) Kendler, K. S. Br. J. Psychiatry 1982, 141, 186-190. (6) Reveley, A. M.; Reveley, M. A.; Clifford, C. A.; Murray, R. M. Lancet 1982, 1 (8271), 540-541. (7) Suddath, R. L.; Christison, G. W.; Torrey, E. F.; Casanova, M. F.; Weinberger, D. R. N. Engl. J. Med. 1990, 322 (12), 789-794. (8) Iwamoto, K.; Bundo, M.; Kato, T. Hum. Mol. Genet. 2005, 14 (2), 241-253. (9) Middleton, F. A.; Mirnics, K.; Pierri, J. N.; Lewis, D. A.; Levitt, P. J. Neurosci. 2002, 22 (7), 2718-2729. (10) Mimmack, M. L.; Ryan, M.; Baba, H.; Navarro-Ruiz, J.; Iritani, S.; Faull, R. L.; McKenna, P. J.; Jones, P. B.; Arai, H.; Starkey, M.; Emson, P. C.; Bahn, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (7), 4680-4685. (11) Prabakaran, S.; Swatton, J. E.; Ryan, M. M.; Huffaker, S. J.; Huang, J. T.; Griffin, J. L.; Wayland, M.; Freeman, T.; Dudbridge, F.; Lilley, K. S.; Karp, N. A.; Hester, S.; Tkachev, D.; Mimmack, M. L.; Yolken, R. H.; Webster, M. J.; Torrey, E. F.; Bahn, S. Mol. Psychiatry. 2004, 9 (7), 684-697, 643. (12) Haupt, D. W.; Newcomer, J. W. J. Psychosom. Res. 2002, 53 (4), 925-933. (13) Heiskanen, T.; Niskanen, L.; Lyytikainen, R.; Saarinen, P. I.; Hintikka, J. J. Clin. Psychiatry 2003, 64 (5), 575-579. (14) Huang, T. L.; Chen, J. F. Schizophr. Res. 2005, 80 (1), 55-59. (15) Dean, B.; Laws, S. M.; Hone, E.; Taddei, K.; Scarr, E.; Thomas, E. A.; Harper, C.; McClean, C.; Masters, C.; Lautenschlager, N.; Gandy, S. E.; Martins, R. N. Biol. Psychiatry. 2003, 54 (6), 616622. (16) Digney, A.; Keriakous, D.; Scarr, E.; Thomas, E.; Dean, B. Biol. Psychiatry 2005, 57 (7), 711-715. (17) Huber, T. J.; Borsutzky, M.; Schneider, U.; Emrich, H. M. Acta Psychiatr. Scand. 2004, 109 (4), 269-274. (18) Behl, C.; Moosmann, B.; Manthey, D.; Heck, S. Novartis Found. Symp. 2000, 230, 221-234; discussion 234-238. (19) Sasaki, J.; Funakoshi, M.; Arakawa, K. Clin. Pharmacol. Ther. 1985, 37 (6), 684-687. (20) Shafique, M.; Khan, I. A.; Akhtar, M. H.; Hussain, I. J. Pak. Med. Assoc. 1988, 38 (10), 259-261. (21) Nicholson, J. K.; Foxall, P. J.; Spraul, M.; Farrant, R. D.; Lindon, J. C. Anal. Chem. 1995, 67 (5), 793-811.
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