Correlation between serum concentrations of homocysteine, folate and vitamin B12 in patients with schizophrenia

Introduction : The role of hyperhomocysteinemia in psychotic disorder can be explained by partial antagonism of homocysteine on NMDA-glycine receptor. Plasma concentration of homocysteine is an indicator of the status of the B-vitamins (folate, B12, B6). Folate de ﬁ ciency may have different effects on the neurochemical processes of schizophrenia. This suggests that the association between elevated levels of homocysteine and schizophrenia is biologically very likely. Methods : The study was consisted of 20 patients with schizophrenia and 20 healthy controls. We investigated the levels of serum homocysteine concentration using AxSYM (Abbott), levels of folate assay is two-step immunoassay to determine the presence folate in human serum using CMIA (chemiluminescent microparticle immunoassay) technology and Axsym Holo Tc is microparticle enzyme immunoassay (MEIA) for the quantitative determination of human holo TC in serum and determination de ﬁ cit of vitamin B 12. Results : The patients group has higher levels of homocysteine in compare with controls group for 3.85 μ mol/L while the concentration of folate in the group of patients was lower for 9.17 ng/mL. The mean level of vitamin B-12 in investigation groups were in reference range 19.1-119 pmol/L, but patient group have lower average concentration of vitamin B-12 lower for 24.81 pmol/L compared to the control group. Conclusion : Our results showed that homocysteine concentration is inversely proportionate to folate concentration, i.e. as homocysteine concentration in serum increases, folate concentration falls. Shizophrenic patients with elevated tHcy level and low folate levels should have vitamin supplementation with folic acid.


INTRODUCTION
Conceptualization of glutaminergic system in the last fi fty years follows the historical development of models phencyclidine (PCP) in patients who suff ering from schizophrenia, a description of its psyhomimetic eff ect, through the discovery of Nmethyl-D-aspartate glutamat receptor (NMDAR), and fi nally the nineties years set new treatment strategies to base on these receptors (1,2). NMDAR antagonists induce an increase of the positive and negative symptoms of schizophrenia. In this connection we now have several hypotheses that explain the mechanism by which NMDAR dysfunction leads to abnormalities in thinking, perception disorders, bizarre behavior and cognitive damage, which schizophrenia remains of intrigue when it comes to the status of reality as the notion of mental deterioration, as well as several theories in the new concept of treatment. Oxidation of homocysteine leads to the formation homocysteine sulfuric acid and homocysteine acid; both products have expressed excitotoxic action of the diff erent subtypes of the N-methyl-Daspartate (NMDA) glutamat receptor. Excitotoxic cell type was observed when human cells in diff erent parts of the brain exposed to homocystein excitotoxic acids and amino acids. Recent studies in rats have shown that homocysteine causes DNA damage to neurons inducing apoptosis of hypersensitivity to excitotoxicity. In, the brain, there is a limited capacity for homocysteine methabolism. In addition, folate metabolism play a critical roles in the methabolism of homocysteine (4). Th e level of 5-methyltetrahydrofolate in cerebrospinal fl uid is at least three times that found in plasma and an active transport process exists at the blood-brain-barrier. Th e folates have important role in the brain and the necessity for protection agains the eff ects of defi ciency (5). Tests on rats have shown that elevated homocysteine leads to apoptosis of neurons in the hippocampus. Relatively large storage of folate in CSF provides the brain with folate and thereby protects nerve cells from the toxic eff ects of increased concentrations of homocysteine. Besides folate defi ciency may have diff erent eff ects on the neurochemical processes of schizophrenia, because it works as a carbon donor in the synthesis of glycine from serine. Folate is also involved in the synthesis of dopamine, norepinephrine and serotonin through pathways the methylation of S-adenosyl-methionine and conversion of methionine to homocysteine (6). Homocysteine is an amino acid produced by demethylation of methionine, an essential amino acid that in the body intake of food. Homocysteine was fi rst described in 1932 in the works by Butz and Vigneauda that homocysteine synthesis activity concentrated strong acid on methionine. In the body comes from food or produced during degradation of endogenous proteins. Methyl group of methionine is activated by switching to S-adenosyl-methionine (SAM). Th e reaction catalyzes of L-methionine-S-adenoziltransferaza the presence of ATP. After the transfer of the methyl group exceeds S-adenosyl-methionine in tioester S-adenosyl-homocysteine (SAH). S-adenosyl-methionine is a donor of numerous reactions in the brain, including many that are involved in the synthesis and metabolism of monoamines such as dopamine, norepinephrine and serotonin (6). Th is suggests that the association between elevated levels of homocysteine and schizophrenia is biologically very likely. Numerous epidemiological studies have linked increased levels of homocysteine with vascular disease, dementia, defects of fetal development, increasing the level of mortality and with neuropsychiatric disorders (2) such as schizophrenia, bipolar aff ective disorder and depression (6,7,8). Homocysteine is at about 45% of elderly patients increased and its concentration correlates well with the severity of the memory functions. Th erefore homocysteine it is better for diagnosis of cognition (cognitive impairment) than the concentration of folate and vitamin B12 in serum (9). Today there is interest in measuring the biologically active form of cobalamin. Th e vitamin B 12 (cobalamin) in serum is bound to two proteins, transcobalamin (TC) and haptocorrin (HC). About 20% of cobalamin is related to transcobalamin and it is biologically active, while 80% is related to haptocorrin is biologically inactive. Th e transcobalaminvitamin B-12 complex is called holotranscobalamin (Holo Tc) and it is used in vitro diagnostic assay for the quantitative determination of cobalamin. Th e reduced concentration of Holo Tc, it is an indicator of defi cit vitamin B 12. Notably, low values have been reported in vegetarians and populations with low intake of vitamin B 12. Th e tests for measuring total vitamin B 12 are based on the measurement of active and inactive fractions, but very often do not correlate with existing symptoms of the defi cit. Th e reference area of vitamin B 12 is diffi cult to determine because it is too close to «normal» samples. Holo Tc levels refl ect vitamin B 12 status, independent of recent absorption of the vitamin (10,11). Homocysteinaemia is a newly defi ned term connected to the increased risk of atherothrombotic and atherosclerotic systemic and retinal vascular occlusive diseases, and its role in the course of many anatomical or functional abnormalities of the vessels of the optic nerve head such as arteriosclerosis or vascular dysregulation, homocistein might be the causative factor (12,13). Some studies showed that elevated Hcy may increase the risk of retinal vascular diseases, such as retinal artery and vein occlusion and non-artheritic ischemic optic neuropathy (14,15). Hcy-induced vascular problem may be a multifactorial case, including direct toxic damage to the endothelium, stimulation of proliferation of smooth muscle cells, enhanced low density lipoprotein peroxidation, increased platelet aggregation, and eff ects upon the coagulation system (16). Maybe, this reason of much work in the cognitive neuroscience of schizophrenia has focused on attention, memory, and executive functioning. To date, less work has focused on perceptual processing. However, perceptual functions are frequently disrupted in schizophrenia, and thus this domain has been included available ophthalmology tests in this article, how we can describe the basic science presentation and the breakout group discussion on the topic of perception when we use some test for the assessment of cognitive function in schizophrenia. Th e importance of perceptual dysfunction in schizophrenia, the nature of perceptual abnormalities in this disorder, and the critical need to develop perceptual ophthalmology tests appropriate for future clinical trials and validity of some tests for the assessment of cognitive function in schizophrenia were discussed.

Patients
Th e patients group consisted of inpatients (n=20) with Schizophrenia (Sch) diagnosed according to the tenth revision of the International Classifi cation of Diseases (ICD-10), treated at the Psychiatric Clinic, Clinical Centre University of Sarajevo (CCUS). Th e patients were followed for eight weeks during 2012. All participants signed an informed consent and study was approved by the Ethics Committee of the Clinical Center of the University of Sarajevo. Th e study was, for the most part, conducted via the prospective method of clinical research and partly as a descriptively controlled study. Th e patients were treated with typical antipsychotics (haloperidol and fl uphenazine) (n = 14) and atypical antipsychotics (olanzapine and risperidone) (n = 6) for at least eight weeks. Th e inclusion criteria were female and male patients with a diagnosis of schizophrenia according to ICD-10 criteria (International Classifi cation of Diseases); Th e presence of positive and negative symptoms, cognitive defi cits and functioning on a social level; Antipsychotic therapy, typical or atypical; Subjects 18-65 years of age; One or more hospitalizations. Th e exclusion criteria were comorbidity; organic brain damage; other medical conditions (diabetes mellitus, hypertension, neurological disorders, infl ammatory disease or gastroenterological disease); alcohol and drug abuse.

Blood samples
Th e patient samples of blood were collected in serum separation Vacutainer test tubes (Beckton Dickinson, Rutherford, NJ 07,070 U.S.) in volume of 3.5 mL. Test tubes with gel were used. After collection, samples were placed in ice and after 30 to 60 minutes, samples were obtained by centrifugation at 3000 rpm using centrifuge (Sigma 4-10). After centrifuging, serum concentration of homocysteine, folate, active form of vitamin B-12 and creatinine were determined.

Determination of homocysteine (Hcy)
Th e value of serum homocysteine concentration was determined using AxSYM (Abbott), based on measurements of fl uorescence polarization immunoassay (FPIA) technology. Th e reaction principle is conversion of homocystine, mixed disulfi de and protein-bound forms of homocysteine in the sample to form of free homocysteine by the use of dithiothreitol (DTT). After that free homocysteine is converted to S-adenosyl-L-homocysteine (SAH). Under physiological conditions, SAH -hydrolases converts SAH to homocysteine. Existing methods is to determine L-homocysteine in human serum. Normal concentration of homocysteine in serum of women is 3.36-20.44 μmol/L and in men is 5.90-16 μmol/L (17). Th e creatinin was determined using automatic analyzer Dimension (Dade Behring). Method for determination of creatinine is a modifi cation of the kinetic reaction of Jaff ee. Th e reference value for serum creatinine concentration is 45-115 μmol/ L (18).

Determination of folate
All immunoassays require the use of labeled material in order to measure the amount of antigen or antibody. A label is a molecule that will react as a part of the assay, so a change in signal can be measured in the blood after added reagent solution. CMIA (chemiluminescent microparticle immunoassay) is noncompetitive sandwich assay technology to measure analytes. Th e amount of signal is directly proportional to the amount of analyte present in the sample. Architect folate asssay is two-step immunoassay to determine the presence folate in human serum using CMIA technology. Two pre-treatment steps mediate the release of folate from endogenous folate binding protein. In pre-treatment step 1 sample ans pre-treatment reagent 2 (dithiothreitol or DDT) are aspirated and dispensed into a reaction vessels (RV). In pre-treatment step 2, an aliquot of sample/ pre-treatment reagent 2 mixture is aspirated and dispensed into a second RV. Pre-treatment reagent (KOH) is then added. An alliquot of the pre-treated sample is transfered into a third RV, followed by the addition of folate binding protein (FBP) coated paramagnetic microparicles and assay specifi c diluent. Folate presented in the sample binds to the FBP coated microparticles. After washing, pteroic acidacridinium labeled conjugate is added and binds to unoccupied sites on the FBP-coated microparticles. Following another incubation and wash, pre-trigger and trigger solutions are then added to the reaction mixture. Th e pre-trigger solution (hydrogen peroxide) creates an acidic environment to prevent early release of energy (light emission), helps to keep microparticles from clumping and splits acridinium dye off the conjugate bound to the microparticle complex (this action prepares the acridinium dye for the next step). Th e trigger solution (sodium hydroxide) dispenses to the reaction mixture. Th e acridinium undergoes an oxidative reaction when is exposed to peroxide and an alkaline solution. Th is reaction causes the occurence of chemiluminescent reaction. N-methylacridone forms and releases energy (light emission) as it returns to its ground state. Th e resulting chemiluminescent reaction is measured as relative light units (RLU). A direct relationship exists between the amount of folate in the sample and RLU detected by Architect System optics. Th e reference value for serum folate concentration is 7.0-31.4 ng/mL (19).

Determination of active B-12
Axsym Holo Tc is microparticle enzyme immunoassay (MEIA) for the quantitative determination of human holo TC in serum and determination defi cit of vitamin B 12. Microparticles are coated with an Anti-Holo Tc monoclonal antibodies in the presence of human antigens on the Holo Tc microparticles arises immune complex. On Anti-Holo Tc antibodies present a conjugate of alkaline phosphatase in the next reaction, which reacts with the substrate 4-methylumbelliferyl phosphate (MUP). Th e resulting fl uorescent product is measured by MEIA optical system. Th e reference value for the healthy population Holo Tc is 19.1-119 pmol/L (20).

Statistical analysis
Th e results were statistically analysed using SPSS version 12 and Microsoft Offi ce Excel 2003. Average values, standard deviation (SD) and Pearson correlation coeffi cient (r) were calculated, as well Wilcoxon signed rang test with statistical signifi cance level of 0.05 P<0.05.

RESULTS
Th e investigation included 20 patients with shizophrenia and 20 healty subjects. Th e study included 6 (30%) men and 14 (70%) women in control group. Th e group with shizophrenia have 5 (25 %) men and 15 (75%) women. Th e average age in the control group was 49.45 years and in shizophrenia patients was 44.5 years. Normal homocysteine concentration in serum is 3.36-20.44 μmol/L for women and 5.90-16 μmol/L for men. Th e reference folate concentration is 7.0-31.4 ng/mL and vitamin B-12 19.1-119 pmol/L. Th e mean concentration of homocysteine, folate and vitamin B-12 in shizophrenia patients and control group are shown in Table 1. Th e patients with shizophrenia have higher levels of homocysteine in compare with controls group for 3.85 μmol/L while the concentration of folate in the group of patients was lower for 9.17 ng/mL. Th e patients with shizophrenia have a folate defi ciency. Th e mean level of vitamin B-12 in investigation groups were in ref-erence range 19.1-119 pmol/L, but patient group have lower average concentration of vitamin B-12 lower for 24.81 pmol/L compared to the control group. Folate defi ciency signifi cantly changes the concentration of homocysteine, which could aff ect the diff erences between patients with shizophrenia and control group. It is indicated that high level of homocysteine follows low level of folate in serum at patients with shizophrenia. We have shown in Table 2. Z and P values mean serum homocysteine, folate and vitamin B-12 concentration in patients and control subjects. Using Wilcoxon signed ranks test we have concluded that average concentration of homocysteine in shizophrenia patients were signifi cantly diff erend (P<0.05) from serum concentration in control subjects. Th ere was a signifi cant diff erence in folate serum concentration between patient group and control group for P<0.05. According Wilcoxon signed ranks test mean vitamin B-12 concentration was signifi cant for P<0.05 in investigated groups. Th e correlation between homocystein and vitamin levels in patients serum are shown in Table 3. Th ere was a signifi cant diff erence between the mean serum homocysteine and folate (P <0.05) with a negative Pearson correlation coeffi cient (r = -0.52) in the group of patients. Using the same test, a signifi cant diff erence between the mean serum homocysteine and vitamin B-12 (p <0.05) with a negative Pearson correlation coeffi cient (r = -0.47).

DISCUSSION
Th e role of hyperhomocysteinemia in psychotic disorder can be explained by partial antagonism of homocysteine on NMDA-glycine receptor (21). Epidemiological studies show a correlation between total plasma homocysteine concentration and the risk of neurodegenerative diseases (22). Plasma concentration of homocysteine is an indicator of the status of the B-vitamins (folate, B12, B6). It was found that the values of homocysteine levels are significantly elevated in young male patients with schizophrenia and BP, especially those who show signs of cognitive deterioration, compared to the general population (23). However, there are studies whose results indicate no diff erence in homocysteine levels between bipolar patients and healthy controls (24). Increased levels of homocysteine can cause apoptosis and exocytosis as important mechanisms of neurodegeneration. Th e brain tissues utilize three mechanisms for maintaining low steady state concentrations of homocysteine: 1) effi cient recycling through cobalamin-dependent methionine synthase, given an adequate supply of cobalamin and folate; 2) catabolism throught CBS to cystathionine; 3) export homocysteine into the circulation (25). In our study group patients with shizophrenia have higher levels of homocysteine in compare with controls group for 3.85 μmol/L while the concentration of folate in the group of patients was lower for 9.17 ng/mL. Our results are compared with the results Mabrouk et al (26) have shown that the increased value of homocysteine and a lower value of folate in patients with schizophrenia compared with the control group. Th e low folate status in schizophrenics and folate-responsive schizophrenic-like behavior are connected with defi ciency of MTHFR of TT genotype (27). Eren et al (28)    It is known that a diet rich in animal protein may in-crease, and foods rich in beans and green vegetables to lower homocysteine levels. Diff erent groups of drugs, such as drugs for lowering fat (fi brates and niacin), oral hypoglycemics (metformin), insulin, drugs used to treat rheumatoid arthritis and anticonvulsants may cause an increase in serum levels of homocysteine (30), which could in the future be interesting in expanding the study introduce more detailed and complete test diet. With regard to gender diff erences in the prevalence of hyperhomocysteinemia results of numerous studies indicate that the values of homocysteine levels are generally higher in men (31). All of the above would be fully subject of our next study, given that they are now working on the preliminary results.

CONCLUSION
Th ese fi ndings support the hypothesis that altered levels of homocysteine, folate and vitamin B12 may coexist in patients with schizophrenia and contribute to pathophysiological aspects of this illness. Th e homocysteine could be an independent risk factor for neuropsychiatric disorders and risk factor for development of cerebrovascular disease. In our group of shizophrenia patients we have a limited number of patients and we have not informations about concentration of homocysteine, folate and vitamin B-12 during shizophrenia treatment. Th e hyperhomocysteinemia is presented in about 25 % and low folat leves in 60 % of shizophrenia patients. In patients group homocysteine level were elevated compared to the levels of controls when folate levels were low, these resuls indicate that enzymatic defect could be involved. Our results showed that homocysteine concentration is inversely proportionate to folate concentration, i.e. as homocysteine concentration in serum increases, folate concentration falls. Th e shizophrenia patients with elevated tHcy level and low folate levels should have vitamin supplementation with folic acid. Th is would be the view of the preliminary results of our research, which in the future demanded a larger sample, the relationship between these biochemical parameters related to the predominance of positive or negative symptoms of schizophrenia, with the inclusion of other diagnostic entities, depression and bipolar aff ective disorder, and complete processing of data related to the metabolic syndrome and nutrition, which will be planned.