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Serum levels of BDNF, folate and homocysteine: In relation to hippocampal volume and psychopathology in drug naïve, first episode schizophrenia
Schizophrenia Research 159 (2014) 51–55
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Serum levels of BDNF, folate and homocysteine: In relation to hippocampal volume and psychopathology in drug naïve, first episode schizophrenia Xueqin Song a,b,c,⁎,1, Xiaoduo Fan c,1, Xue Li a, David Kennedy c, Lijuan Pang a, Meina Quan c, Xumei Chen a, Jinsong Gao a, Wei Zhang a, Jianjiang Zhang a, Luxian Lv b,d,⁎⁎ a
The First Affiliated Hospital/Zhengzhou University, Zhengzhou, China Henan Province Biological Psychiatry Key Laboratory, Xinxiang Medical University, Xinxiang, China c Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA, USA d Henan Province Mental Hospital, The Second Affiliated Hospital/Xinxiang Medical University, Xinxiang, China b
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 15 July 2014 Accepted 25 July 2014 Available online 12 August 2014 Keywords: Schizophrenia Folate Homocysteine BDNF Hippocampus
a b s t r a c t Objective: The present study was to examine serum levels of brain-derived neurotrophic factor (BDNF), folate, homocysteine (Hcy), and their relationships with hippocampal volume and psychopathology in drug naïve, first episode schizophrenia. Method: Drug naïve, first episode schizophrenia patients and healthy controls were enrolled in the study. Serum levels of BDNF, folate and Hcy were measured using enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECLIA), and enzymatic cycling method respectively. Hippocampus was parcellated and bilateral hippocampal volumes were measured using FreeSurfer. Results: Forty-six patients with drug naïve, first episode schizophrenia (SZ group) and 30 healthy controls (control group) were enrolled. The SZ group had significantly lower serum levels of BDNF and folate, and significantly higher serum levels of Hcy compared with the control group (p = 0.013, p b 0.001, p = 0.003 respectively). There were no significant differences in hippocampal volumes between the two groups (ps N 0.2). Within the SZ group, there were significant positive relationships between serum levels of BDNF and both left and right hippocampal volumes (r = 0.327, p = 0.026; r = 0.338, p = 0.022 respectively). In contrast, such relationships did not exist in the control group. Within the SZ group, there were significant negative relationships between serum levels of folate and PANSS-total scores and PANSS-negative symptom scores (r = 0.319, p = 0.031; r = 0.321, p = 0.030 respectively); and there was a positive relationship between serum levels of Hcy and PANSS-total scores (r = 0.312, p = 0.035). Controlling for potential confounding variables resulted in similar findings. Conclusions: Drug naïve, first episode schizophrenia presents decreased serum levels of BDNF, folate and increased serum levels of Hcy, which may play an important role in the neurodevelopmental process and clinical manifestation of schizophrenia. © 2014 Published by Elsevier B.V.
1. Introduction Schizophrenia is a severe and common psychiatric disorder characterized by disturbed brain development; abnormalities in brain morphology have been found in various brain regions in patients with schizophrenia (Wright et al., 2000; Shenton et al., 2001; Honea et al., 2005; Ellison-Wright et al., 2008; Glahn et al., 2008). Temporal
⁎ Correspondence to: X. Song, The First Affiliated Hospital/Zhengzhou University, Zhengzhou, China, 450052. Tel.: +86 371 6796 6822. ⁎⁎ Correspondence to: L. Lv, Henan Mental Hospital, The Second Affiliated Hospital/ Xinxiang Medical University, Xinxiang, China, 453003. Tel.: +86 373 337 3969. E-mail addresses: [email protected] (X. Song), [email protected] (L. Lv). 1 Xueqin Song, M.D., PH.D. and Xiaoduo Fan M.D., M.P.H., M.S. share co-first authorship.
http://dx.doi.org/10.1016/j.schres.2014.07.033 0920-9964/© 2014 Published by Elsevier B.V.
lobe structure abnormalities, including amygdala, hippocampus and parahippocampal gyrus, are among the most frequently reported findings in this patient population (Shenton et al., 2001; Honea et al., 2005; Buehlmann et al., 2010; Ebdrup et al., 2010). Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, mediates differentiation and survival of neurons as well as synaptic plasticity during the brain development (Lu and Chow, 1999; Poo, 2001; Lu, 2003). Several lines of evidence suggest an important role of BDNF in schizophrenia. Post-mortem studies have found reductions in BDNF mRNA expression and proteins in the prefrontal cortex and hippocampus of schizophrenia brain (Durany et al., 2001; Weickert et al., 2003; Hashimoto et al., 2005; Weickert et al., 2005). Studies have shown decreased serum levels of BDNF in chronic, first episode, and drug naïve schizophrenia patients
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(Buckley et al., 2007; Ikeda et al., 2008; Chen da et al., 2009; Xiu et al., 2009); however, the relationship between serum levels of BDNF and schizophrenia psychopathology is less clear (Martinotti et al., 2012). Folate provides the substrate for intracellular methylation reactions that are essential to normal brain development and function. Methylation is important for DNA synthesis and repair, gene expression, neurotransmission synthesis and degradation, and homocysteine (Hcy) metabolism (Frankenburg, 2007). Abnormal folate metabolism has been implicated in schizophrenia. For example, reduced maternal folate intake (St Clair et al., 2005) and increased maternal blood levels of Hcy (Brown et al., 2007) are both associated with an increased risk for schizophrenia. Low blood levels of folate have been reported in patients with schizophrenia, and are associated with clinical manifestation especially in the negative symptom domain (Goff et al., 2004; Roffman et al., 2008). Among various brain regions, hippocampus contains the highest levels of neurotrophic factors, which play an important role in the brain development (Rizos et al., 2011). Hippocampus is an area of high expression of BDNF and its TrkB receptor (Murer et al., 2001). Previous studies have found a positive relationship between hippocampal volume and serum levels of BDNF in both chronic and first episode schizophrenia (Erickson et al., 2010; Rizos et al., 2011). In addition, a negative relationship between hippocampal volume and symptom severity has been found in chronic schizophrenia (Flaum et al., 1995; Rajarethinam et al., 2001). The present study was to examine serum levels of BDNF, folate, homocysteine (Hcy), and their relationships with hippocampal volume and psychopathology in drug naïve, first episode schizophrenia. We hypothesized that higher serum levels of BDNF and folate, and lower serum levels of Hcy are associated with larger hippocampal volume and less severe psychopathology in this patient population. 2. Methods 2.1. Subjects All subjects provided written informed consent to participate in the study, which was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Inpatients 18 and 45 years old diagnosed with first-episode schizophrenia were recruited. The study sample was composed of a consecutive series of admissions between November 2011 and December 2012. Patients were diagnosed with first-episode schizophrenia according to the criteria of DSM-IV (Runeson and Rich, 1994), and were never previously treated with antipsychotic medications or other psychotropics. The diagnosis of schizophrenia was further determined by a research psychiatrist (X.S.) using the Structured Clinical Interview for DSM-IV Axis I Disorders. Exclusion criteria included ongoing infections or allergies, autoimmune disorders, pregnancy, use of folate supplement, being medically unstable, history of alcohol or other substance use, and history of significant neurological illness. Healthy controls were recruited from the local community through advertisement. A complete medical history, physical examination, and routine laboratory tests were obtained from all subjects to rule out possible medical conditions. None of them had a history of any psychiatric conditions, or history of alcohol or substance use. 2.2. Clinical symptoms Symptoms of schizophrenia were assessed for all patient subjects using the Positive and Negative Syndrome Scale (PANSS). The PANSS was administered by the same rater (J.G.) throughout the study. 2.3. Bioassay Venous blood (5 mL) was collected between 7:00 and 8:00 AM to avoid circadian fluctuation of the parameters to be measured. The
blood was put into a glass tube and allowed to clot at room temperature. Serum was obtained through centrifugation at 3000 rpm for 10 min, and was then divided into 3 Eppendorf tubes, and stored at −70 °C for assay. Serum levels of BDNF were measured by enzyme linked immunosorbent assay (ELISA) (R&D Systems, USA). Serum levels of folate were measured by electrochemiluminescence immunoassay (ECLIA) (Abbott Laboratories, USA). Serum levels of Hcy were measured by enzymatic cycling method (Kangte Bio-Tech, China). All assays were performed according to manufacturers' instructions. The sensitivity for BDNF was 20 pg/mL with inter-assay variation coefficient of 9.0% and intra-assay variation coefficient of 5.0%. Standard curve concentrations were calculated in triplicate for each plate. Absorbencies were measured by a microtiter plate reader (absorbency at 492 nm). All assays were carried out at the same time, and were conducted blinded to the subjects' group status. 2.4. Image acquisition and processing T1-weighted Spoiled Gradient Echo (SPGR) images were collected for all subjects on a 3.0 Tesla Scanner (Signa HDxt 3 T GEHCGEHC) at the Magnetic Resonance Center of the First Affiliated Hospital of Zhengzhou University. The acquisition protocol included the following pulse sequence and parameters: repetition time (TR) = 12 ms, echo time (TE) = 4.5 ms, inversion time (TI) = 1100 ms, flip angle: 7°, field of view (FOV) = 256 × 256 × 220 mm3, matrix size: 256 × 256, slice thickness: 1 mm without spacing, scan piles: 192, and scan time: 17 min. All data acquisitions were performed in the coronal plane, which was perpendicular to the anterior commissure–posterior commissure (AC–PC) line. Scan results were optimized by high contrast between gray matter and white matter, and between gray matter and cerebral spinal fluid (CSF) to obtain the best structure and surface images. MRI data were coded and cataloged, and transferred to the Center for Morphometric Analysis (CMA), Department of Psychiatry at University of Massachusetts Medical School (UMMS) for analysis with subject's identity and clinical information blinded. Images were realigned using the line between the anterior and posterior commissures and the sagittal sulcus to correct for head tilt. The FreeSurfer software (http://surfer. nmr.mgh.harvard.edu/) was used to parcellate T1-weighted SPGR images into cortical and subcortical gray and white matter regions for each subject, based on probabilistic information automatically
Table 1 Demographic and clinical characteristics of the study sample. Characteristics
Schizophrenia group (N = 46) (Mean ± SD)
Control group (N = 30) (Mean ± SD)
t
p
Age (years) Education (years) Age of illness onset (years) Disease duration (months) PANSS-positive PANSS-negative PANSS-general PANSS-total
22.54 12.63 21.57 7.79 21.85 18.13 38.78 78.72
24.33 ± 7.42 13.73 ± 3.51
−1.118 −1.550
0.269 0.129
N (%)
χ2
p
± ± ± ± ± ± ± ±
N (%) Gender Male Female Smoking status Yes No
5.79 2.09 5.14 7.54 3.17 2.28 2.30 4.74
28 (61%) 18 (39%)
17 (57%) 13 (43%)
9 (20%) 37 (80%)
7 (23%) 23 (77%)
0.133
0.716
0.155
0.694
PANSS, the Positive and Negative Syndrome Scale, including positive symptoms, negative symptoms, general psychopathology subscales.
X. Song et al. / Schizophrenia Research 159 (2014) 51–55 Table 2 Comparison of serum levels of BDNF, folate and homocysteine between the schizophrenia group and the control group. Characteristics
Hcy (μmol/L) Folate (ng/mL) BDNF (pg/mL)
Schizophrenia group (N = 46)
Control group (N = 30)
t
p
Effect size (Cohen d)
(Mean ± SD)
(Mean ± SD)
25.31 ± 9.30
19.12 ± 7.02
3.109
0.003
0.75
5.62 ± 2.61
7.66 ± 1.70
−3.784
b0.001
−0.93
746.31 ± 171.89
828.67 ± 109.30
−2.553
0.013
−0.57
Hcy, homocysteine; BDNF, brain derived neurotrophic factor.
estimated from a manually labeled training set (Fischl et al., 2002). Hippocampus is one of the regions that have shown comparable accuracy as with manual labeling.
2.5. Statistical analysis The data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL). Demographics, clinical measures and laboratory values were reported using descriptive statistics. Group comparisons were performed using the Student's t-test for continuous variables and χ2 test for categorical variables. Pearson correlation was used to examine the relationships between variables of interest. Further, partial correlation was used to examine the relationships between variables of interest after controlling for potential confounding variables. A p value of less than 0.05 (2-tailed) was used for statistical significance.
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3. Results Fifty-four patients were approached for the study, 5 refused to participate in the study, and 3 didn't fit the inclusion/exclusion criteria. Forty-six first episode, drug naïve schizophrenia patients (SZ group) and 30 healthy controls (control group) were enrolled in the study. The age range for patients enrolled in the study was 18–36 years old. There were no significant differences in age, gender, education, and smoking status between the two groups (ps N 0.1, Table 1). The SZ group had significantly lower serum levels of BDNF and folate and higher serum levels of Hcy and was compared with the control group (p = 0.013, p b 0.001, p = 0.003 respectively, Table 2). However, there were no significant differences in hippocampal volumes between the two groups (left hippocampus: 3976 ± 472 mm3 versus 4031 ± 369 mm3, p = 0.571; right hippocampus: 4088 ± 401 mm3 versus 4195 ± 336 mm3, p = 0.214) (SZ group versus control group). Within the SZ group, there were significant positive relationships between serum levels of BDNF and both left and right hippocampal volumes (p = 0.026, p = 0.022 respectively, Fig. 1); these relationships remain to be significant after controlling for age, gender, education, smoking status, and disease duration (patients only) (Table 3). In contrast, such relationships did not exist in the control group (ps N 0.1) — both before and after controlling for potential confounding variables (Table 3). Within the SZ group, there was a trend of negative relationship between serum levels of folate and Hcy (r = 0.290, p = 0.051). There were significant negative relationships between serum levels of folate and PANSS-total scores and PANSS-negative symptom scores (p = 0.031, p = 0.030 respectively, Fig. 2). In addition, there was a positive relationship between serum levels of Hcy and PANSS-total scores (r = 0.312, p = 0.035). These relationships remain significant after
Fig. 1. Relationship between serum levels of BDNF and hippocampal volumes in patients with schizophrenia.
Table 3 Relationship between serum levels of BDNF, folate, homocysteine and hippocampal volumes after controlling for age, gender, education, smoking status, and disease duration (patients only). Characteristics
Schizophrenia group (N = 46)
Control group (N = 30)
Left HV
Hcy Folate BDNF
Right HV
Left HV
Right HV
r
p
r
p
r
p
r
p
−0.183 0.247 0.315
0.240 0.111 0.040
−0.235 0.193 0.329
0.130 0.214 0.031
−0.100 −0.155 0.231
0.620 0.440 0.246
−0.211 −0.115 0.262
0.292 0.567 0.186
Hcy, homocysteine; BDNF, brain derived neurotrophic factor; HV, hippocampal volume.
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X. Song et al. / Schizophrenia Research 159 (2014) 51–55
Fig. 2. Relationship between serum levels of folate and PANSS scores in patients with schizophrenia.
controlling for gender, age, education, smoking status and disease duration (Table 4).
4. Discussion To our knowledge, the present study was the first to examine serum levels of BDNF, folate, Hcy, and their correlations with hippocampal volume and psychopathology in single study in drug naïve, first episode schizophrenia patients. We found that the patient group had significantly lower serum levels of folate and BDNF and higher serum levels of Hcy compared to the healthy control group. Our results are consistent with the findings from some of previous studies (Kale et al., 2010; Kim and Moon, 2011; Rizos et al., 2011). However, some studies reported elevated blood levels of BDNF in patients with schizophrenia (Rizos et al., 2008; Jindal et al., 2010; Sotiropoulou et al., 2013). Other studies suggested elevated blood levels of Hcy but not folate in patients with schizophrenia compared with healthy controls (Haidemenos et al., 2007; Petronijevic et al., 2008). The discrepant findings across different studies might be explained by potential confounding factors such as the biological heterogeneity of study samples, disease duration, and antipsychotic treatment. Consistent with earlier reports in the literature (Erickson et al., 2010; Rizos et al., 2011; Martinotti et al., 2012), our study found a significant relationship between decreased serum levels of BDNF and reduced bilateral hippocampal volumes in the patient group. Animal studies suggested that BDNF can cross the blood brain barrier (Pan et al., 1998); blood levels of BDNF correlate with its levels in the brain (Karege et al., 2002; Sartorius et al., 2009). BDNF is widely distributed throughout the central nervous system; hippocampus is an area of
Table 4 Relationship between serum levels of BDNF, folate, homocysteine and the PANSS scores after controlling for age, gender, education, disease duration and smoking status in patients with schizophrenia (N = 46). Folate
PANSS-total PANSS-positive PANSS-negative PANSS-general
Hcy
BDNF
R
p
R
p
R
p
−0.322 −0.106 −0.321 −0.158
0.038 0.504 0.034 0.317
0.314 0.235 0.241 0.014
0.040 0.125 0.125 0.931
−0.209 −0.198 0.016 −0.087
0.185 0.208 0.922 0.584
Hcy, homocysteine; BDNF, brain derived neurotrophic factor; PANSS, the Positive and Negative Syndrome Scale, including positive symptoms, negative symptoms, and general psychopathology subscales.
high expression of BDNF and its TrkB receptor (Murer et al., 2001). Extensive previous work suggests the role of BDNF in impaired hippocampus-dependent forms of learning and memory and in behavioral disturbances related to psychiatric disorders including schizophrenia (Pardon, 2010). Our study also found that lower serum levels of folate and higher serum levels of Hcy are associated with worse clinical symptoms of schizophrenia, especially in the negative symptom domain. Fetal exposure to folate deficiency and elevation of maternal Hcy have been identified as risk factors for schizophrenia in offspring (Lewis et al., 2005). Goff et al. reported that there was a negative correlation between serum levels of folate and the severity of negative symptoms in individuals with schizophrenia (Goff et al., 2004). Hcy is formed by demethylation of methionine and acts as a neurotoxin via actions at NMDA receptors and by increasing oxidative stress (Mattson and Shea, 2003); folate and B12 are cofactors in its metabolism. Folate deficiency leads to elevated levels of Hcy, which likely contributes to the development of schizophrenia. The strengths of the present study include the use of a relatively homogeneous group of drug naïve, first episode schizophrenia patients. The selection of the study sample in our study minimized potential confounding factors such as previous exposure to antipsychotic medications, and variations in disease state (acute versus chronic) and disease duration. Another strength is that we were able to examine the relationship between serum levels of BDNF, folate, Hcy and hippocampal volume and psychopathology in single study. The present study also has some limitations: 1) Causal relationships relating serum levels of BDNF, folate and Hcy to hippocampal volume and psychopathology cannot be drawn given the cross-sectional study design. 2) The sample size was relatively small; and 3) multiple testing was not corrected. In summary, our study provided evidence that drug naïve, first episode schizophrenia presents decreased serum levels of BDNF and folate and increased serum levels of Hcy, which may play an important role in the neurodevelopmental process and clinical manifestation of schizophrenia. Future research should also explore the relationship between these serum measures and other brain regions such as frontal lobe, which is known to be affected in schizophrenia. Future intervention studies to normalize the levels of these biomarkers with the potential benefit of ameliorating brain volume loss and improving schizophrenia symptoms are warranted.
Role of funding source Funding for this study was provided by the National Natural Science Foundation of China (No. 30971058 to X-QS; No. 81071090 to L-XL), the Natural Science Foundation of Henan Province (No. 102300413208, 112300413226 to L-XL).
X. Song et al. / Schizophrenia Research 159 (2014) 51–55 Contributors Dr. Song and Dr. Fan were responsible for the analysis and interpretation of the data for this paper. All authors contributed to the writing of the paper. Conflict of interest Dr. Fan has received research support or honoraria from Eli Lilly, AstraZeneca, BristolMyer-Squibb, Janssen, and Pfizer. Other authors report no competing interests. Acknowledgments None.
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Serum levels of BDNF, folate and homocysteine: In relation to hippocampal volume and psychopathology in drug naïve, first episode schizophrenia Xueqin Song a,b,c,⁎,1, Xiaoduo Fan c,1, Xue Li a, David Kennedy c, Lijuan Pang a, Meina Quan c, Xumei Chen a, Jinsong Gao a, Wei Zhang a, Jianjiang Zhang a, Luxian Lv b,d,⁎⁎ a
The First Affiliated Hospital/Zhengzhou University, Zhengzhou, China Henan Province Biological Psychiatry Key Laboratory, Xinxiang Medical University, Xinxiang, China c Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA, USA d Henan Province Mental Hospital, The Second Affiliated Hospital/Xinxiang Medical University, Xinxiang, China b
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 15 July 2014 Accepted 25 July 2014 Available online 12 August 2014 Keywords: Schizophrenia Folate Homocysteine BDNF Hippocampus
a b s t r a c t Objective: The present study was to examine serum levels of brain-derived neurotrophic factor (BDNF), folate, homocysteine (Hcy), and their relationships with hippocampal volume and psychopathology in drug naïve, first episode schizophrenia. Method: Drug naïve, first episode schizophrenia patients and healthy controls were enrolled in the study. Serum levels of BDNF, folate and Hcy were measured using enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECLIA), and enzymatic cycling method respectively. Hippocampus was parcellated and bilateral hippocampal volumes were measured using FreeSurfer. Results: Forty-six patients with drug naïve, first episode schizophrenia (SZ group) and 30 healthy controls (control group) were enrolled. The SZ group had significantly lower serum levels of BDNF and folate, and significantly higher serum levels of Hcy compared with the control group (p = 0.013, p b 0.001, p = 0.003 respectively). There were no significant differences in hippocampal volumes between the two groups (ps N 0.2). Within the SZ group, there were significant positive relationships between serum levels of BDNF and both left and right hippocampal volumes (r = 0.327, p = 0.026; r = 0.338, p = 0.022 respectively). In contrast, such relationships did not exist in the control group. Within the SZ group, there were significant negative relationships between serum levels of folate and PANSS-total scores and PANSS-negative symptom scores (r = 0.319, p = 0.031; r = 0.321, p = 0.030 respectively); and there was a positive relationship between serum levels of Hcy and PANSS-total scores (r = 0.312, p = 0.035). Controlling for potential confounding variables resulted in similar findings. Conclusions: Drug naïve, first episode schizophrenia presents decreased serum levels of BDNF, folate and increased serum levels of Hcy, which may play an important role in the neurodevelopmental process and clinical manifestation of schizophrenia. © 2014 Published by Elsevier B.V.
1. Introduction Schizophrenia is a severe and common psychiatric disorder characterized by disturbed brain development; abnormalities in brain morphology have been found in various brain regions in patients with schizophrenia (Wright et al., 2000; Shenton et al., 2001; Honea et al., 2005; Ellison-Wright et al., 2008; Glahn et al., 2008). Temporal
⁎ Correspondence to: X. Song, The First Affiliated Hospital/Zhengzhou University, Zhengzhou, China, 450052. Tel.: +86 371 6796 6822. ⁎⁎ Correspondence to: L. Lv, Henan Mental Hospital, The Second Affiliated Hospital/ Xinxiang Medical University, Xinxiang, China, 453003. Tel.: +86 373 337 3969. E-mail addresses: [email protected] (X. Song), [email protected] (L. Lv). 1 Xueqin Song, M.D., PH.D. and Xiaoduo Fan M.D., M.P.H., M.S. share co-first authorship.
http://dx.doi.org/10.1016/j.schres.2014.07.033 0920-9964/© 2014 Published by Elsevier B.V.
lobe structure abnormalities, including amygdala, hippocampus and parahippocampal gyrus, are among the most frequently reported findings in this patient population (Shenton et al., 2001; Honea et al., 2005; Buehlmann et al., 2010; Ebdrup et al., 2010). Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, mediates differentiation and survival of neurons as well as synaptic plasticity during the brain development (Lu and Chow, 1999; Poo, 2001; Lu, 2003). Several lines of evidence suggest an important role of BDNF in schizophrenia. Post-mortem studies have found reductions in BDNF mRNA expression and proteins in the prefrontal cortex and hippocampus of schizophrenia brain (Durany et al., 2001; Weickert et al., 2003; Hashimoto et al., 2005; Weickert et al., 2005). Studies have shown decreased serum levels of BDNF in chronic, first episode, and drug naïve schizophrenia patients
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(Buckley et al., 2007; Ikeda et al., 2008; Chen da et al., 2009; Xiu et al., 2009); however, the relationship between serum levels of BDNF and schizophrenia psychopathology is less clear (Martinotti et al., 2012). Folate provides the substrate for intracellular methylation reactions that are essential to normal brain development and function. Methylation is important for DNA synthesis and repair, gene expression, neurotransmission synthesis and degradation, and homocysteine (Hcy) metabolism (Frankenburg, 2007). Abnormal folate metabolism has been implicated in schizophrenia. For example, reduced maternal folate intake (St Clair et al., 2005) and increased maternal blood levels of Hcy (Brown et al., 2007) are both associated with an increased risk for schizophrenia. Low blood levels of folate have been reported in patients with schizophrenia, and are associated with clinical manifestation especially in the negative symptom domain (Goff et al., 2004; Roffman et al., 2008). Among various brain regions, hippocampus contains the highest levels of neurotrophic factors, which play an important role in the brain development (Rizos et al., 2011). Hippocampus is an area of high expression of BDNF and its TrkB receptor (Murer et al., 2001). Previous studies have found a positive relationship between hippocampal volume and serum levels of BDNF in both chronic and first episode schizophrenia (Erickson et al., 2010; Rizos et al., 2011). In addition, a negative relationship between hippocampal volume and symptom severity has been found in chronic schizophrenia (Flaum et al., 1995; Rajarethinam et al., 2001). The present study was to examine serum levels of BDNF, folate, homocysteine (Hcy), and their relationships with hippocampal volume and psychopathology in drug naïve, first episode schizophrenia. We hypothesized that higher serum levels of BDNF and folate, and lower serum levels of Hcy are associated with larger hippocampal volume and less severe psychopathology in this patient population. 2. Methods 2.1. Subjects All subjects provided written informed consent to participate in the study, which was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Inpatients 18 and 45 years old diagnosed with first-episode schizophrenia were recruited. The study sample was composed of a consecutive series of admissions between November 2011 and December 2012. Patients were diagnosed with first-episode schizophrenia according to the criteria of DSM-IV (Runeson and Rich, 1994), and were never previously treated with antipsychotic medications or other psychotropics. The diagnosis of schizophrenia was further determined by a research psychiatrist (X.S.) using the Structured Clinical Interview for DSM-IV Axis I Disorders. Exclusion criteria included ongoing infections or allergies, autoimmune disorders, pregnancy, use of folate supplement, being medically unstable, history of alcohol or other substance use, and history of significant neurological illness. Healthy controls were recruited from the local community through advertisement. A complete medical history, physical examination, and routine laboratory tests were obtained from all subjects to rule out possible medical conditions. None of them had a history of any psychiatric conditions, or history of alcohol or substance use. 2.2. Clinical symptoms Symptoms of schizophrenia were assessed for all patient subjects using the Positive and Negative Syndrome Scale (PANSS). The PANSS was administered by the same rater (J.G.) throughout the study. 2.3. Bioassay Venous blood (5 mL) was collected between 7:00 and 8:00 AM to avoid circadian fluctuation of the parameters to be measured. The
blood was put into a glass tube and allowed to clot at room temperature. Serum was obtained through centrifugation at 3000 rpm for 10 min, and was then divided into 3 Eppendorf tubes, and stored at −70 °C for assay. Serum levels of BDNF were measured by enzyme linked immunosorbent assay (ELISA) (R&D Systems, USA). Serum levels of folate were measured by electrochemiluminescence immunoassay (ECLIA) (Abbott Laboratories, USA). Serum levels of Hcy were measured by enzymatic cycling method (Kangte Bio-Tech, China). All assays were performed according to manufacturers' instructions. The sensitivity for BDNF was 20 pg/mL with inter-assay variation coefficient of 9.0% and intra-assay variation coefficient of 5.0%. Standard curve concentrations were calculated in triplicate for each plate. Absorbencies were measured by a microtiter plate reader (absorbency at 492 nm). All assays were carried out at the same time, and were conducted blinded to the subjects' group status. 2.4. Image acquisition and processing T1-weighted Spoiled Gradient Echo (SPGR) images were collected for all subjects on a 3.0 Tesla Scanner (Signa HDxt 3 T GEHCGEHC) at the Magnetic Resonance Center of the First Affiliated Hospital of Zhengzhou University. The acquisition protocol included the following pulse sequence and parameters: repetition time (TR) = 12 ms, echo time (TE) = 4.5 ms, inversion time (TI) = 1100 ms, flip angle: 7°, field of view (FOV) = 256 × 256 × 220 mm3, matrix size: 256 × 256, slice thickness: 1 mm without spacing, scan piles: 192, and scan time: 17 min. All data acquisitions were performed in the coronal plane, which was perpendicular to the anterior commissure–posterior commissure (AC–PC) line. Scan results were optimized by high contrast between gray matter and white matter, and between gray matter and cerebral spinal fluid (CSF) to obtain the best structure and surface images. MRI data were coded and cataloged, and transferred to the Center for Morphometric Analysis (CMA), Department of Psychiatry at University of Massachusetts Medical School (UMMS) for analysis with subject's identity and clinical information blinded. Images were realigned using the line between the anterior and posterior commissures and the sagittal sulcus to correct for head tilt. The FreeSurfer software (http://surfer. nmr.mgh.harvard.edu/) was used to parcellate T1-weighted SPGR images into cortical and subcortical gray and white matter regions for each subject, based on probabilistic information automatically
Table 1 Demographic and clinical characteristics of the study sample. Characteristics
Schizophrenia group (N = 46) (Mean ± SD)
Control group (N = 30) (Mean ± SD)
t
p
Age (years) Education (years) Age of illness onset (years) Disease duration (months) PANSS-positive PANSS-negative PANSS-general PANSS-total
22.54 12.63 21.57 7.79 21.85 18.13 38.78 78.72
24.33 ± 7.42 13.73 ± 3.51
−1.118 −1.550
0.269 0.129
N (%)
χ2
p
± ± ± ± ± ± ± ±
N (%) Gender Male Female Smoking status Yes No
5.79 2.09 5.14 7.54 3.17 2.28 2.30 4.74
28 (61%) 18 (39%)
17 (57%) 13 (43%)
9 (20%) 37 (80%)
7 (23%) 23 (77%)
0.133
0.716
0.155
0.694
PANSS, the Positive and Negative Syndrome Scale, including positive symptoms, negative symptoms, general psychopathology subscales.
X. Song et al. / Schizophrenia Research 159 (2014) 51–55 Table 2 Comparison of serum levels of BDNF, folate and homocysteine between the schizophrenia group and the control group. Characteristics
Hcy (μmol/L) Folate (ng/mL) BDNF (pg/mL)
Schizophrenia group (N = 46)
Control group (N = 30)
t
p
Effect size (Cohen d)
(Mean ± SD)
(Mean ± SD)
25.31 ± 9.30
19.12 ± 7.02
3.109
0.003
0.75
5.62 ± 2.61
7.66 ± 1.70
−3.784
b0.001
−0.93
746.31 ± 171.89
828.67 ± 109.30
−2.553
0.013
−0.57
Hcy, homocysteine; BDNF, brain derived neurotrophic factor.
estimated from a manually labeled training set (Fischl et al., 2002). Hippocampus is one of the regions that have shown comparable accuracy as with manual labeling.
2.5. Statistical analysis The data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL). Demographics, clinical measures and laboratory values were reported using descriptive statistics. Group comparisons were performed using the Student's t-test for continuous variables and χ2 test for categorical variables. Pearson correlation was used to examine the relationships between variables of interest. Further, partial correlation was used to examine the relationships between variables of interest after controlling for potential confounding variables. A p value of less than 0.05 (2-tailed) was used for statistical significance.
53
3. Results Fifty-four patients were approached for the study, 5 refused to participate in the study, and 3 didn't fit the inclusion/exclusion criteria. Forty-six first episode, drug naïve schizophrenia patients (SZ group) and 30 healthy controls (control group) were enrolled in the study. The age range for patients enrolled in the study was 18–36 years old. There were no significant differences in age, gender, education, and smoking status between the two groups (ps N 0.1, Table 1). The SZ group had significantly lower serum levels of BDNF and folate and higher serum levels of Hcy and was compared with the control group (p = 0.013, p b 0.001, p = 0.003 respectively, Table 2). However, there were no significant differences in hippocampal volumes between the two groups (left hippocampus: 3976 ± 472 mm3 versus 4031 ± 369 mm3, p = 0.571; right hippocampus: 4088 ± 401 mm3 versus 4195 ± 336 mm3, p = 0.214) (SZ group versus control group). Within the SZ group, there were significant positive relationships between serum levels of BDNF and both left and right hippocampal volumes (p = 0.026, p = 0.022 respectively, Fig. 1); these relationships remain to be significant after controlling for age, gender, education, smoking status, and disease duration (patients only) (Table 3). In contrast, such relationships did not exist in the control group (ps N 0.1) — both before and after controlling for potential confounding variables (Table 3). Within the SZ group, there was a trend of negative relationship between serum levels of folate and Hcy (r = 0.290, p = 0.051). There were significant negative relationships between serum levels of folate and PANSS-total scores and PANSS-negative symptom scores (p = 0.031, p = 0.030 respectively, Fig. 2). In addition, there was a positive relationship between serum levels of Hcy and PANSS-total scores (r = 0.312, p = 0.035). These relationships remain significant after
Fig. 1. Relationship between serum levels of BDNF and hippocampal volumes in patients with schizophrenia.
Table 3 Relationship between serum levels of BDNF, folate, homocysteine and hippocampal volumes after controlling for age, gender, education, smoking status, and disease duration (patients only). Characteristics
Schizophrenia group (N = 46)
Control group (N = 30)
Left HV
Hcy Folate BDNF
Right HV
Left HV
Right HV
r
p
r
p
r
p
r
p
−0.183 0.247 0.315
0.240 0.111 0.040
−0.235 0.193 0.329
0.130 0.214 0.031
−0.100 −0.155 0.231
0.620 0.440 0.246
−0.211 −0.115 0.262
0.292 0.567 0.186
Hcy, homocysteine; BDNF, brain derived neurotrophic factor; HV, hippocampal volume.
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X. Song et al. / Schizophrenia Research 159 (2014) 51–55
Fig. 2. Relationship between serum levels of folate and PANSS scores in patients with schizophrenia.
controlling for gender, age, education, smoking status and disease duration (Table 4).
4. Discussion To our knowledge, the present study was the first to examine serum levels of BDNF, folate, Hcy, and their correlations with hippocampal volume and psychopathology in single study in drug naïve, first episode schizophrenia patients. We found that the patient group had significantly lower serum levels of folate and BDNF and higher serum levels of Hcy compared to the healthy control group. Our results are consistent with the findings from some of previous studies (Kale et al., 2010; Kim and Moon, 2011; Rizos et al., 2011). However, some studies reported elevated blood levels of BDNF in patients with schizophrenia (Rizos et al., 2008; Jindal et al., 2010; Sotiropoulou et al., 2013). Other studies suggested elevated blood levels of Hcy but not folate in patients with schizophrenia compared with healthy controls (Haidemenos et al., 2007; Petronijevic et al., 2008). The discrepant findings across different studies might be explained by potential confounding factors such as the biological heterogeneity of study samples, disease duration, and antipsychotic treatment. Consistent with earlier reports in the literature (Erickson et al., 2010; Rizos et al., 2011; Martinotti et al., 2012), our study found a significant relationship between decreased serum levels of BDNF and reduced bilateral hippocampal volumes in the patient group. Animal studies suggested that BDNF can cross the blood brain barrier (Pan et al., 1998); blood levels of BDNF correlate with its levels in the brain (Karege et al., 2002; Sartorius et al., 2009). BDNF is widely distributed throughout the central nervous system; hippocampus is an area of
Table 4 Relationship between serum levels of BDNF, folate, homocysteine and the PANSS scores after controlling for age, gender, education, disease duration and smoking status in patients with schizophrenia (N = 46). Folate
PANSS-total PANSS-positive PANSS-negative PANSS-general
Hcy
BDNF
R
p
R
p
R
p
−0.322 −0.106 −0.321 −0.158
0.038 0.504 0.034 0.317
0.314 0.235 0.241 0.014
0.040 0.125 0.125 0.931
−0.209 −0.198 0.016 −0.087
0.185 0.208 0.922 0.584
Hcy, homocysteine; BDNF, brain derived neurotrophic factor; PANSS, the Positive and Negative Syndrome Scale, including positive symptoms, negative symptoms, and general psychopathology subscales.
high expression of BDNF and its TrkB receptor (Murer et al., 2001). Extensive previous work suggests the role of BDNF in impaired hippocampus-dependent forms of learning and memory and in behavioral disturbances related to psychiatric disorders including schizophrenia (Pardon, 2010). Our study also found that lower serum levels of folate and higher serum levels of Hcy are associated with worse clinical symptoms of schizophrenia, especially in the negative symptom domain. Fetal exposure to folate deficiency and elevation of maternal Hcy have been identified as risk factors for schizophrenia in offspring (Lewis et al., 2005). Goff et al. reported that there was a negative correlation between serum levels of folate and the severity of negative symptoms in individuals with schizophrenia (Goff et al., 2004). Hcy is formed by demethylation of methionine and acts as a neurotoxin via actions at NMDA receptors and by increasing oxidative stress (Mattson and Shea, 2003); folate and B12 are cofactors in its metabolism. Folate deficiency leads to elevated levels of Hcy, which likely contributes to the development of schizophrenia. The strengths of the present study include the use of a relatively homogeneous group of drug naïve, first episode schizophrenia patients. The selection of the study sample in our study minimized potential confounding factors such as previous exposure to antipsychotic medications, and variations in disease state (acute versus chronic) and disease duration. Another strength is that we were able to examine the relationship between serum levels of BDNF, folate, Hcy and hippocampal volume and psychopathology in single study. The present study also has some limitations: 1) Causal relationships relating serum levels of BDNF, folate and Hcy to hippocampal volume and psychopathology cannot be drawn given the cross-sectional study design. 2) The sample size was relatively small; and 3) multiple testing was not corrected. In summary, our study provided evidence that drug naïve, first episode schizophrenia presents decreased serum levels of BDNF and folate and increased serum levels of Hcy, which may play an important role in the neurodevelopmental process and clinical manifestation of schizophrenia. Future research should also explore the relationship between these serum measures and other brain regions such as frontal lobe, which is known to be affected in schizophrenia. Future intervention studies to normalize the levels of these biomarkers with the potential benefit of ameliorating brain volume loss and improving schizophrenia symptoms are warranted.
Role of funding source Funding for this study was provided by the National Natural Science Foundation of China (No. 30971058 to X-QS; No. 81071090 to L-XL), the Natural Science Foundation of Henan Province (No. 102300413208, 112300413226 to L-XL).
X. Song et al. / Schizophrenia Research 159 (2014) 51–55 Contributors Dr. Song and Dr. Fan were responsible for the analysis and interpretation of the data for this paper. All authors contributed to the writing of the paper. Conflict of interest Dr. Fan has received research support or honoraria from Eli Lilly, AstraZeneca, BristolMyer-Squibb, Janssen, and Pfizer. Other authors report no competing interests. Acknowledgments None.
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