Association between B vitamins and schizophrenia: A population-based case-control study

Author’s Accepted Manuscript Association between B Vitamins and Schizophrenia: a Population-based Case-Control Study Bing Cao, Xiao-Yu Sun, Chuan-Bo Zhang, JingJing Yan, Qian-Qian Zhao, Si-Yu Yang, Lai-Lai Yan, Ning-hua Huang, Jing Zeng, Jie-Ying Liao, Jing-Yu Wang

PII: DOI: Reference:

www.elsevier.com/locate/psychres

S0165-1781(17)31236-2 https://doi.org/10.1016/j.psychres.2017.11.006 PSY10953

To appear in: Psychiatry Research Received date: 5 July 2017 Revised date: 11 October 2017 Accepted date: 2 November 2017 Cite this article as: Bing Cao, Xiao-Yu Sun, Chuan-Bo Zhang, Jing-Jing Yan, Qian-Qian Zhao, Si-Yu Yang, Lai-Lai Yan, Ning-hua Huang, Jing Zeng, JieYing Liao and Jing-Yu Wang, Association between B Vitamins and Schizophrenia: a Population-based Case-Control Study, Psychiatry Research, https://doi.org/10.1016/j.psychres.2017.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Association between B Vitamins and Schizophrenia: a Population-based Case-Control Study Bing Caoa, Xiao-Yu Suna, Chuan-Bo Zhangb, Jing-Jing Yana, Qian-Qian Zhaoa, Si-Yu Yanga, Lai-Lai Yana, Ning-hua Huanga, Jing Zenga, Jie-Ying Liaoc**, Jing-Yu Wanga* a

Department of Laboratorial Science and Technology, School of Public Health, Peking

University, Beijing 100191, P. R. China b

c

Mental Health Center of Weifang, Shandong 262400, P. R. China

Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences, Xiamen

361021, P.R. China [email protected] [email protected] *

Correspondence to: Jing-Yu Wang, PhD, Department of Laboratorial Science and

Technology, School of Public Health, Peking University, 38 Xue-Yuan Road, Haidian District, Beijing 100191, P.R. China. Tel./fax: +86 10 82801107. **

Correspondence to: Jie-Ying Liao, PhD, Xiamen Institute of Rare Earth Materials,

Chinese Academy of Sciences, Xiamen 361021, P.R. China. Tel./fax: +86-592-6081739.

Abstract To explore the association between schizophrenia and six types of B vitamins, including choline, biotin, riboflavin, pyridoxamine, pyridoxine and nicotinamide, based on the hydrophilic interaction liquid chromatography column (HILIC) Liquid Chromatography-Mass Spectrometry (LC-MS) platform. We conducted the case-control study between November 2015 and September 2016 in Weifang, Shandong Province, China. Blood samples from 128 cases of schizophrenia and 101 controls were collected, and B vitamin were measured by LC-MS coupled with HILIC. The HILIC UPLC-MS based analysis of serum B vitamins levels from 128 cases (30 cases with first-episode, 0

98 cases with relapse) and 101 controls were performed. The results indicated that lower pyridoxine level and schizophrenia was related. (total cases versus controls: β= -0.215, 95% CI: -0.271, -0.125, p <0.001; first-episode cases versus controls: β= -0.190, 95% CI: -0.277, -0.103, p <0.001). Higher nicotinamide level was also associated with schizophrenia after adjusting confounders (β= 0.343, 95% CI: 0.022, 0.664, p =0.036). Other four B vitamins, including biotin, riboflavin, pridoxamine and choline, were showed no statistically difference in cases versus controls, first episode cases versus relapse cases. Two types of B Vitamins, pyridoxine and nicotinamide, show significant association with the schizophrenia. Keywords Schizophrenia; vitamin; HILIC; UPLC-MS

1. Introduction Schizophrenia is a severe mental disorder with psychopathological manifestations of thought and perception, along with distortion of emotion or reduced affect (Freedman, 2003). It more likely affects young-adulthood(Adachi et al., 2008) without certain pathogeny. Because of the persistency and refractory, the burden of schizophrenia is at the forefront of the total disease(Carpenter and Buchanan, 1994; Karimkhani et al., 2016). At present, diagnosis and severity evaluation of schizophrenia primarily relies on the subjective identification of symptom cluster with PANSS scale (Tamminga and Holcomb, 2005) by psychiatrist. However, it is difficult to distinguish schizophrenia from other mental disorders that have similar symptoms with schizophrenia (such as emotional disorders)(Ishii, 2007) only with the subjective diagnostic modality results of PANSS scale. It is greatly urgent to explore the etiology, pathogenesis and disease markers of schizophrenia. B vitamins play an essential role in cellular metabolism process, including homocysteine

toxicity(Moustafa

et

al.,

2014),

transmethylation

and

oxidation/reduction reactions(Roffman et al., 2013). Researchers have pointed out that some B vitamins may associate with the occurrence and development of schizophrenia, such as vitamin B6 (Brown and Roffman, 2014), vitamin B12(Tanner et al., 2015; Zhang et al., 2016), folic acid (Tanner et al., 2015), choline (Bustillo et al., 2002). Researches 1

indicated that serious folic acid deficiency increase the risk of neurodevelopmental disorders, psychiatric disorders and dementia(Czeizel et al., 2013; Mitchell et al., 2014); and changes of vitamin B12 levels lead to neuropsychiatric disorders, such as mental disorders, cognitive slowness, mood disorders, violent behavior, fatigue and paranoid psychosis (Bottiglieri, 1996). Metabolic dysregulation of elevated levels of homocysteine in first-episode schizophrenia patients was found associated with deficiency of folic acid and vitamin B12(Misiak et al., 2016; Moustafa et al., 2014). A number of RCTs of adjunct treatment with B vitamins illustrated clinical improvements in schizophrenic patients (Firth et al., 2017). It is helpful to clarify the mechanism of schizophrenia by exploring the alteration in schizophrenic patients, and that reflect the pathophysiology of the disorder. It is difficult to separate multiple chromatographic peaks of B vitamins by ordinary high performance liquid chromatography (HPLC), which hindered the process of B vitamins researches in schizophrenia. For better retention and separation of these polar metabolites, this study was performed based on the liquid chromatography column (HILIC) Liquid Chromatography-Mass Spectrometry (LC-MS) platform. This method used minimal sample cleanup to reduce sample loss during extraction, chemical derivatization to enhance ionization. Massive evidence have been showing the clear relationship of folic acid or vitamin B12 with development of schizophrenia, however, it remains unclear whether other B vitamins were involved in this process. In this study, we focus on six B vitamins including choline, biotin, riboflavin, pyridoxamine, pyridoxine and nicotinamide. Through comparison of blood B vitamins levels between schizophrenia patients and the controls, the potential association of the six B vitamins levels with schizophrenia was investigated. This study could provide new clues of the B vitamins research in schizophrenia, further our understanding of pathogenesis of schizophrenia, and provide potential diagnostic and therapeutic targets for schizophrenia. 2. Materials and Methods 2.1 Study population We conducted the case-control study between November 2015 and September 2016 in Weifang, Shandong Province, China. Cases were recruited consecutively from Mental Health Center of Weifang, who were screened by trained psychiatrists with the 2

structured Clinical Interview for International Classification of Diseases and Related Health Problems -10th edition. Patients with brain damage, serious complications, alcohol dependence, and substance-related psychotic disorders were excluded. The volunteers from health examination population who were willing to attend an anonymous survey and didn’t have any history of mental diseases, brain damage, serious complications, and alcohol dependence were enrolled as the controls. All subjects were aged from 18 to 40 years old. The ethical committee of the Peking University Health Science Health Science Center (Beijing, China) reviewed and approved the protocol of this study and the procedures for sample collection and analysis (IRB00001052-14071). All the subjects signed an informed consent form and agreed to serve as serum donors for the experiments. 2.2 Serum samples preparation Fasting blood samples (approximately 5ml) of cases were collected before clinical treatment, which were obtained from forearm vein and drawn in serum separation hoses for biomarker studies. Samples were maintained at 4℃ for 20-30 min and resulted serum aliquoted into Eppendorf tubes, which were subsequently stored at -80℃. The serum samples of control group were collected with the same methods of the case group. Before analysis, all serum samples were thawed in ice, vortexed well and centrifuged at 12,000 rpm for 10 min. From the remaining supernatant of each sample, 100 µl was used for HILIC analysis. Briefly, 300 µl of cold acetonitrile was added to a labeled 1.5 ml micro-centrifuge tube with100 µl of each sample. Samples were vortexed and maintained at 4 °C for 10min and centrifuged operating at 4 °C 12,000 rpm for 10 min and then transferred 100 µl supernatant to a 200 µl vial insert for HILIC analysis. 2.3 Quality Control Samples Equal volumes of the supernatant from all samples were pooled to prepare quality control samples, which were used to monitor the stability and repeatability of the actual samples.

3

2.4 Reagent The reference standard substances of B vitamins and HPLC-grade ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA); HPLC-grade Formic acid and Acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA); pure water was purchased from Wahaha Co., Ltd. (Hangzhou, China). 2.5 HILIC mode and UPLC-MS analysis Thermo Scientific™ Dionex™ UltiMate™ 3000 Rapid Separation LC system (Thermo Fisher Scientific, Waltham, MA, USA) performed UPLC separations with HILIC using the gradient conditions. For HILIC separation, mobile phase A was acetonitrile and mobile phase B was water; both A and B contained 0.1% formic acid and 10mmol/L ammonium acetate. The column was a BEH Amide column (2.1 x 100 mm, 1.7 µm, Waters, Milford, MA, USA) operated at 40 °C. The analysis was carried out using ESI+ mode. The flow rate was 300 µL/min and the injection volume was 1 µL. The gradient was optimized to get maximum separation (0-1 min: 5% B, 1-10 min: 5-50% B, 10-12 min: 50% B, 12-12.1 min: 50-5% B, 12.1-16 min: 5% B). A Thermo Scientific™ Q Exactive™ hybrid quadrupole Orbitrap mass spectrometer equipped with a HESI-II probe was employed (Thermo Fisher Scientific, Waltham, MA, USA). The pos HESI-II spray voltages were 3.7 kV, the heated capillary temperature was 320°C, the sheath gas pressure was 30 psi, the auxiliary gas setting was 10 psi, and the heated vaporizer temperature was 300°C. Both the sheath gas and the auxiliary gas were nitrogen. The collision gas was also nitrogen at a pressure of 1.5 mTorr. The parameters of the full mass scan were as follows: a resolution of 70,000, an auto gain control target under 1 × 106, a maximum isolation time of 50 ms, and an m/z range 100–1000. The calibration was customized for the analysis of Q Exactive to keep the mass tolerance of 5 ppm. The parameters of the dd-MS2 scan were as follows: a resolution of 17,500, an auto gain control target under 1.0 × 105, a maximum isolation time of 50 ms, a loop count of top 10 peaks, an isolation window of m/z, a normalized collision energy of 30v and an intensity threshold under 1.0 × 105. The LC-MS system was controlled using Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific, Waltham, MA, USA), and raw data was collected and processed with 4

the same software. Skyline (64-bit, 3.5.0.9319, MacCoss Lab, UW, USA) was used to create metabolite transitions, visualize results, integrate observed signals, and quantify all metabolites that were detected by MS. 2.6 Statistical analysis Each data set of B vitamins detected by LC-MS was subjected to log transformation (decimal base) before univariate and multiple analysis. Descriptive statistics were performed, with continuous variables summarized as the mean and standard deviation (SD) or median and interquartile range (IQR), and categorical variables summarized as frequencies and proportions. Statistical significance between various groups was tested using the χ2 test for categorical variables and t-test or Mann-Whitney U-test for continuous variables. Generalized linear models (GLM) were used to explore the association between schizophrenia and the logarithm values of B vitamins. The variables of age, sex, BMI, FBG, TG and TC were adjusted in the GLM models. β-values and their 95% confidence intervals (CIs) were estimated using maximum likelihood methods. A two-sided p <0.05 was statistically significant. All statistical analysis was performed using Stata 12.0 (Stata Corp LP, College Station, TX, USA).

3 Results 3.1 Demographic and Clinical Characteristics of subjects A total of 128 cases (30 cases with first-episode, 98 cases with relapse) and 101 controls were included in the analysis. The characteristics of the subjects were listed in Table1. The distribution of age, gender, body mass index (BMI), fasting blood glucose (FBG), triglyceride (TG) and serum total cholesterol (TC) were similar between cases and controls, but the differences were not statistically significant (all p > 0.05). 3.2 The comparison of B vitamins between schizophrenia cases and controls We determined six types of B vitamins in this study (the molecular structure were shown in Supplemental Figure 1). After the log conversion of determination data, the results were analyzed by logistic regression models. Distribution of the six types of B vitamins were shown in Figure 1. Lower level of pyridoxine was significantly 5

associated with schizophrenia, both before and after controlling for potential confounders including age, sex, BMI, FBG, TG and TC (total cases versus controls β= -0.215, 95% CI: -0.271, -0.125, p <0.001; first-episode cases versus controls: β= -0.190, 95% CI: -0.277, -0.103, p <0.001). Higher level of nicotinamide and schizophrenia were significantly associated after adjusting the above potential confounders (β= 0.343, 95% CI: 0.022, 0.664, p =0.036). All the details were shown in Table2 and Table 3. 3.3 The comparison of B vitamins between first-episode cases and relapse cases As shown in Table 4, compared to first-episode schizophrenia patients, the nicotinamide level was lower in the relapse patients (β= 0.339, 95% CI: 0.005, 0.674, p =0.048), and the difference was statistically significant. But after adjusting the confounding factors, the statistical difference disappeared.

4 Discussion In this population-based case-control study, differences of FBG, TG and TC were not found between cases and controls (p >0.05). The results indicated that lower pyridoxine and higher nicotinamide associated with schizophrenia after adjusting the confounders. And the nicotinamide level was found lower in first episode cases than relapse cases (p ＜0.05) from the univariate analysis results. Other four types of B vitamins, including biotin, riboflavin, pridoxamine and choline, were showed no difference in cases versus controls, first episode cases versus relapse cases. Pyridoxine is one form of Vitamin B6 which is an essential water-soluble vitamin working as a coenzyme of cysteic acid decarboxylase and cystathionine-β-synthetase, and a cofactor that involves in many decarboxylation and transamination reactions (Fujii et al., 2017). Lack of pyridoxine could result in raising central free radicals and hydrogen peroxide, which would deepen the damage to hippocampal neurons and involved

in

the

pathophysiological

process

of

schizophrenia

cognitive

impairment(Geng et al., 1995; Yoo et al., 2011). Our study verified that lower pyridoxine and schizophrenia were significantly associated, which was consistent with the recent research results(Arai et al., 2010). But in this study, we did not find the difference of pridoxamine between cases and controls, which is also one form of 6

Vitamin B6. To some extent, this study further provided a molecular basis for the clinical practice and dietary supplement of vitamin B6 in schizophrenia patients. Excess nicotinamide can increase plasma serotonin and histamine levels and cause an abnormal metabolism of monoamine neurotransmitters (Tian et al., 2013). One nicotinamide intervention study indicated that five-hour postload plasma serotonin and histamine levels were significantly increased after oral loading with 100 mg nicotinamide (Tian et al., 2013).. Schizophrenia is one of the monoamine-related mental diseases (Richard and Brahm, 2012), which also exhibits metabolic disturbance of monoamine neurotransmitters(Cai et al., 2011; Richard and Brahm, 2012). The present study showed high nicotinamide was found in patients with schizophrenia, which further our understanding of pathogenesis of this disease. Genetic factor is one of the main causes of schizophrenia (Harrison, 2015), a genetically-determined deficiency in alpha-7 nicotinic receptors as a genetic factor in the onset and development of schizophrenia(Ross et al., 2010; Stefansson et al., 2008). Researches performed that alpha-7 nicotinic receptors can be activated by choline (Li and Buccafusco, 2004) in pregnancy. Choline do not impact cholinergic neurotransmission in adults, because choline is no longer a significant source of activation of alpha-7 nicotinic receptors after cholinergic synapses form (late in pregnancy) (Freedman and Ross, 2015).. Our results didn’t find that the choline is associated with schizophrenia. In addition, biotin is the coenzyme of many enzymes in human body, which is involved in metabolism of fatty acid and carbohydrate, vitamin B12, folic acid and pantothenic acid(Chen et al., 2012; Kennedy, 2016). Riboflavin has antioxidant activity in the metabolism of vitamin B6 and niacin, and maybe is related to the Flavin-glutathione reductase(Batchelor et al., 2017; van der Kooi et al., 2016). To our knowledge, there was no previous research concerned about the association between schizophrenia and riboflavin or biotin. It is well worth further comprehensive and systematic studies to elucidate the mechanisms of the associations between B vitamins and the schizophrenia. There were some limitations to our study. First of all, this study is a cross-sectional study, we cannot conclude about the causality between schizophrenia and B vitamins. Secondly, we did not control the dietary of all the study subjects and antipsychotic treatment therapy of patients, which might affect the concentration of B vitamins in samples. Also, another important limitation of this study was the lack of data for some potential confounders, such as cigarette smoking, alcohol drinking. With the 7

continuous update of instruments and methods, more metabolic biomarkers that are closely related to schizophrenia will be discovered in the future. Thus, the material basis will be found for the mechanism researches of schizophrenia. In a conclusion, this study measured six types of different B vitamins in schizophrenia patients and controls by HILIC UPLC-MS method. We concluded that the pyridoxine and nicotinamide levels may be associated with schizophrenia. The study not only provided clues to the more comprehensive researches of the mechanisms of B vitamins and schizophrenia but also provide potential diagnostic and therapeutic targets for schizophrenia.

Author Contributions Wang JY, Zhang CB and Cao B conceived and designed the study; Cao B, Sun XY and Yan JJ collected the data; Cao B and Sun XY performed the statistical analysis; Cao B, Huang NH, Zeng J and Liao JY contributed to the discussion; Yan LL, Yang SY, Zhao QQ, Wang JY and Liao JY revised the paper. All authors have read and approved the final version of this article.

Acknowledgements The authors alone are responsible for the content and writing of the paper. We thank team members for their support and contributions to this study.

Conflict of interest Regarding this report, the authors do not have any commercial or other association that would be considered a conflict of interest.

Institutional review board statement This study was reviewed and approved by the Ethics Review Committee of Public Health at Peking University Health Science Center (IRB00001052-14071).

8

Funding This work was supported by the Medicine Interdisciplinary Seed Fund (BMU20140435) by Health Science Center, Peking University. The funding agents had no role in the design and conduct of the study; collection, management, analysis, interpretation of the data; preparation, review, or approval of the manuscript.

References Adachi, N., Hara, T., Oana, Y., Matsuura, M., Okubo, Y., Akanuma, N., et al., 2008. Difference in age of onset of psychosis between epilepsy and schizophrenia. Epilepsy Research 78 (2-3), 201-206. Arai, M., Yuzawa, H., Nohara, I., Ohnishi, T., Obata, N., Iwayama, Y., et al., 2010. Enhanced carbonyl stress in a subpopulation of schizophrenia. Arch Gen Psychiatry 67 (6), 589-597. Batchelor, R.R., Kwandou, G., Spicer, P.T., Stenzel, M.H., 2017. (-) - Riboflavin (vitamin B2) and flavin mononucleotide as visible light photo initiators in the thiol-ene polymerisation of PEG-based hydrogels. Polymer Chemistry 8 (6), 980-984. Bottiglieri, T., 1996. Folate, vitamin B12, and neuropsychiatric disorders. Nutr Rev 54 (12), 382-390. Brown, H.E., Roffman, J.L., 2014. Vitamin supplementation in the treatment of schizophrenia. CNS Drugs 28 (7), 611-622. Bustillo, J.R., Rowland, L.M., Lauriello, J., Petropoulos, H., Hammond, R., Hart, B., et al., 2002. High choline concentrations in the caudate nucleus in antipsychotic-naive patients with schizophrenia. Am J Psychiatry 159 (1), 130-133. Cai, H.L., Fang, P.F., Li, H.D., Zhang, X.H., Hu, L., Yang, W., et al., 2011. Abnormal plasma monoamine metabolism in schizophrenia and its correlation with clinical responses to risperidone treatment. Psychiatry Res 188 (2), 197-202. Carpenter, W.T., Jr., Buchanan, R.W., 1994. Schizophrenia. N Engl J Med 330 (10), 681-690. Chen, Y., Elizondo-Noriega, A., Cantu, D.C., Reilly, P.J., 2012. Structural classification of biotin carboxyl carrier proteins. Biotechnol Lett 34 (10), 1869-1875. Czeizel, A.E., Dudas, I., Vereczkey, A., Banhidy, F., 2013. Folate deficiency and folic acid supplementation: the prevention of neural-tube defects and congenital heart defects. Nutrients 5 (11), 4760-4775. Firth, J., Stubbs, B., Sarris, J., Rosenbaum, S., Teasdale, S., Berk, M., et al., 2017. The effects of vitamin and mineral supplementation on symptoms of schizophrenia: a systematic review and meta-analysis - CORRIGENDUM. Psychol Med, 1. Freedman, R., 2003. Schizophrenia. N Engl J Med 349 (18), 1738-1749. Freedman, R., Ross, R.G., 2015. Prenatal choline and the development of schizophrenia. Shanghai Arch Psychiatry 27 (2), 90-102. Fujii, T., Hattori, K., Miyakawa, T., Ohashi, Y., Sato, H., Kunugi, H., 2017. Metabolic profile alterations in the postmortem brains of patients with schizophrenia using capillary electrophoresis-mass spectrometry. Schizophr Res 183, 70-74. Geng, M.Y., Saito, H., Katsuki, H., 1995. Effects of vitamin B6 and its related compounds on survival of 9

cultured brain neurons. Neurosci Res 24 (1), 61-65. Harrison, P.J., 2015. Recent genetic findings in schizophrenia and their therapeutic relevance. J Psychopharmacol 29 (2), 85-96. Ishii, T., 2007. [Differential diagnosis between pervasive developmental disorder and schizophrenia]. Nihon Rinsho 65 (3), 497-501. Karimkhani, C., Wanga, V., Coffeng, L.E., Naghavi, P., Dellavalle, R.P., Naghavi, M., 2016. Global burden of cutaneous leishmaniasis: a cross-sectional analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis 16 (5), 584-591. Kennedy, D.O., 2016. B Vitamins and the Brain: Mechanisms, Dose and Efficacy--A Review. Nutrients 8 (2), 68. Li, X.Y.D., Buccafusco, J.J., 2004. Role of alpha 7 nicotinic acetylcholine receptors in the pressor response to intracerebroventricular injection of choline: Blockade by amyloid peptide A beta 1-42. J Pharmacol Exp Ther 309 (3), 1206-1212. Misiak, B., Laczmanski, L., Sloka, N.K., Szmida, E., Piotrowski, P., Loska, O., et al., 2016. Metabolic dysregulation in first-episode schizophrenia patients with respect to genetic variation in one-carbon metabolism. Psychiatry Res 238, 60-67. Mitchell, E.S., Conus, N., Kaput, J., 2014. B vitamin polymorphisms and behavior: evidence of associations with neurodevelopment, depression, schizophrenia, bipolar disorder and cognitive decline. Neurosci Biobehav Rev 47, 307-320. Moustafa, A.A., Hewedi, D.H., Eissa, A.M., Frydecka, D., Misiak, B., 2014. Homocysteine levels in schizophrenia and affective disorders-focus on cognition. Front Behav Neurosci 8, 343. Richard, M.D., Brahm, N.C., 2012. Schizophrenia and the immune system: pathophysiology, prevention, and treatment. Am J Health Syst Pharm 69 (9), 757-766. Roffman, J.L., Lamberti, J.S., Achtyes, E., Macklin, E.A., Galendez, G.C., Raeke, L.H., et al., 2013. Randomized multicenter investigation of folate plus vitamin B12 supplementation in schizophrenia. JAMA Psychiatry 70 (5), 481-489. Ross, R.G., Stevens, K.E., Proctor, W.R., Leonard, S., Kisley, M.A., Hunter, S.K., et al., 2010. Research review: Cholinergic mechanisms, early brain development, and risk for schizophrenia. J Child Psychol Psychiatry 51 (5), 535-549. Stefansson, H., Rujescu, D., Cichon, S., Pietilainen, O.P., Ingason, A., Steinberg, S., et al., 2008. Large recurrent microdeletions associated with schizophrenia. Nature 455 (7210), 232-236. Tamminga, C.A., Holcomb, H.H., 2005. Phenotype of schizophrenia: a review and formulation. Mol Psychiatry 10 (1), 27-39. Tanner, A.S., Eryilmaz, H., Nitenson, A.Z., Ho, N.F., Manoach, D.S., Goff, D.C., et al., 2015. Effects of Supplemental Folic Acid and Vitamin B12 on Brain Activation During Working Memory and Related Functional Connectivity in Schizophrenia. Biol Psychiat 77 (9), 559. Tian, Y.J., Li, D., Ma, Q., Gu, X.Y., Guo, M., Lun, Y.Z., et al., 2013. Excess nicotinamide increases plasma serotonin and histamine levels. Sheng Li Xue Bao 65 (1), 33-38. van der Kooi, A., Jaeger, B., van Spaendonck, K., Bosch, A., 2016. Riboflavin transporter deficiency diagnosed 30 years after onset of symptoms. Neuromuscular Disorders 26, S201-S201. Yoo, D.Y., Kim, W., Kim, D.W., Yoo, K.Y., Chung, J.Y., Youn, H.Y., et al., 2011. Pyridoxine enhances cell proliferation and neuroblast differentiation by upregulating the GABAergic system in the mouse dentate gyrus. Neurochem Res 36 (5), 713-721. Zhang, Y., Hodgson, N.W., Trivedi, M.S., Abdolmaleky, H.M., Fournier, M., Cuenod, M., et al., 2016. 10

Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One 11 (1), e0146797.

Figure Legends Figure 1 The beanplots of six B vitamins among 3 groups. Green, red, and blue represent healthy controls, first-episode patients and relapse patients, respectively. * p <0.05 between healthy controls and all patients. patients and relapse patients.

+

#

p <0.05 between first-episode

p <0.05 between healthy controls and first-episode

patients.

Tables: Table 1 Demographic and Clinical Characteristics of subjects at the case-control study Case (n=128) Variables

Total

Age

Control

p

(n=101)

-value*

First-episode

Relapse

(n=30)

(n=98)

28.93±6.19

26.8±7.40

29.59±5.65

29.91±4.12

0.172

57 (44.53)

13 (43.33)

44 (44.90)

36

0.174

(year), mean ± SD Gender

Male

(n, %)

(35.64) Female

71 (55.47)

17 (56.66)

54 (55.10)

65 (64.36)

BMI

23.91± 4.04

22.33±3.24 11

24.39±4.15

23.61±4.34

0.585

(M±SD, kg/m2) Underweight

7 (5.56)

5 (17.24)

2 (2.06)

5(4.95)

Normal

54 (42.86)

12 (41.38)

42 (43.30)

55 (54.46)

Overweight

49 (38.89)

12 (41.38)

37 (38.14)

29 (28.71)

Obese

16 (12.70)

0 (0)

16 (16.49)

12 (11.88)

5.22±1.01

5.08±0.57

5.27±0.11

5.34±0.54

0.287

1.22±0.89

0.96±0.08

1.30±0.97

1.08±0.75

0.208

4.59±1.08

4.25±0.99

4.69±1.09

4.33±0.91

0.056

Total score

88.85±18.61

89.32±22.45

88.72±17.58

——

——

Positive

21.82±8.10

22.88±9.32

21.54±7.77

——

——

20.47±8.19

18.92±7.59

20.88±8.34

——

——

43.39±12.58

44.56±15.00

43.09±11.93

——

——

(n, %)

FBG (M±SD, mmol/L) TG (M±SD, mmol/L) TC (M±SD, mmol/L) PANSS (M±SD) symptoms Negative symptoms General psychopathology

*p-values were the results of total cases compared with controls. BMI: body mass index; FBG: fasting blood glucose; TG: triglycerides; TC: total cholesterol; PANSS: positive and negative syndrome scale.

Table 2 The comparison of B vitamins between total schizophrenia cases and controls. Variables

Pyridoxamine Nicotinamide Biotin Riboflavin Pyridoxine Choline

Adjusted*

Crude β (95% CI)

p

β (95% CI)

p

-0.009 (-0.223, 0.205) 0.241 (-0.077, 0.558) -0.535 (-1.139, 0.068) -0.117 (-0.255, 0.022) -0.198 (-0.271, -0.125) -0.019 (-0.396, 0.358)

0.935 0.140 0.082 0.099 <0.001 0.920

0.018 (-0.200, 0.237) 0.343 (0.022, 0.664) -0.583 (-1.238, 0.072) -0.103 (-0.238, 0.033) -0.215 (-0.289, -0.140) -0.231 (-0.630, -0.167)

0.870 0.036 0.081 0.195 <0.001 0.254

*

Adjusted β and 95% CI were calculated by adjusting age, gender, BMI, FBG, TG and TC.

12

Table 3 The comparison of B vitamins between first-episode cases and controls Variables

Pyridoxamine Nicotinamide Biotin Riboflavin Pyridoxine Choline

Adjusted*

Crude β (95% CI) 0.122 (-0.113, 0.357) -0.076 (-0.432, 0.280) -0.174 (-0.885, 0.506) -0.116 (-0.274, 0.041) -0.196 (-0.283, -0.108) -0.219 (-0.695, 0.257)

p 0.309 0.675 0.615 0.148 <0.001 0.367

β (95% CI) 0.173 (-0.063, 0.409) -0.080 (-0.421, 0.260) -0.285 (-1.006, 0.435) -0.115 (-0.265, 0.036) -0.190 (-0.277, -0.103) -0.020 (-0.500, 0.461)

p 0.150 0.644 0.438 0.135 <0.001 0.936

*

Adjusted β and 95% CI were calculated by adjusting age, gender, BMI, FBG, TG and TC.

Table 4 The comparison of B vitamins between first-episode cases and relapse cases Variables

Pyridoxamine Nicotinamide Biotin Riboflavin Pyridoxine Choline

Adjusted*

Crude β (95% CI) -0.150 (-0.374, 0.075) 0.339 (0.005, 0.674) -0.027 (-0.996, 0.421) 0.036 (-0.118, 0.191) 0.025 (-0.053, 0.104) 0.161 (-0.216, 0.538)

p 0.191 0.048 0.426 0.645 0.526 0.402

β (95% CI) -0.136 (-0.383, 0.110) 0.242 (-0.177, 0.661) -0.614 (-1.442, 0.215) 0.110 (-0.058, 0.278) 0.032 (-0.053, 0.116) -0.110 (-0.555, 0.335)

p 0.278 0.257 0.146 0.200 0.462 0.629

*

Adjusted β and 95% CI were calculated by adjusting age, gender, BMI, FBG, TG and TC.

Highlights 1. The aim of this study is to investigate the potential association between B vitamins levels and schizophrenia. 2. For better retention and separation of B vitamins, this study was performed based on the HILIC LC-MS platform. 3. The study adjusted the confounding factors that might influence the results including age, sex, BMI, FBG, TG and TC.

13