Appropriate questionnaire was developed as to obtain personal information and to identify factors responsible for the onset of Schizophrenia. All patients selected for sampling were first asked to read and understand the consent form with the help of their attendant and sign before taking the samples. Total diagnosed Schizophrenic subjects were 35 with the age range between 15 - 50 years for males and 12 - 59 years for females with 20 siblings as controls. All the subjects were Pakistanis. Schizophrenic patients were divided into two groups. First group contain 23 patients who were on antipsychotic drugs (old diagnosed) and second group contain 12 newly diagnosed (ND) patients. The ND patients are also schizophrenics but they were being just diagnosed and were not on medication. However they were addicted to drugs such as cannabis, tobacco, hashish, alcohol and betel nut. The study was approved by the Human Ethics Committee of Jinnah postgraduate medical center (JPMC), Karachi, Pakistan with the ethic number of F.2-81/2008-GENL/2128/JPMC.

Appropriate self made questionnaire was developed as to obtain personal information and to identify many socio-economic factors that could be responsible for the onset of schizophrenia. All patients were clinically evaluated by expert psychiatrists by taking the history and clinical examinations. Patients self assessment survey was done by government hospitals and some private hospitals situated in Karachi, Pakistan.

Blood samples were collected from Schizophrenic patients and their control (sibling). These patients were diagnosed according to the DSM-IV and ICD-10 criteria and were admitted to the psychiatric wards of Jinnah postgraduate medical center and Civil Government Hospital, Karachi, Pakistan. 5 ml blood samples were collected from each subject (patients and controls) into metal free plastic tubes containing heparin.

For digestion of biological samples of patients and controlled subjects of human whole blood were directly taken into Pyrex flasks separately, added 5 ml whole blood was mixed into digestion acid of a freshly prepared mixture of concentrated HNO₃-H₂O₂ (2:1, v/v) digested at 60˚C - 70˚C for 1 - 2 h. The digests were then treated with 2 ml nitric acid and few drops of H₂O₂, heated on hot plate about 80˚C until the clear digested solution was obtained, the excess acid mixture was evaporated to semidried mass, cooled and diluted with 0.1 mol/l nitric acid. Once the beaker was cooled then the content was diluted with de-ionized water up to 50 ml volume.

It is a very sensitive technique of mass spectrometry which is capable of detecting metals and several non-metals at concentrations as low as one part in 10⁶ (part per million) (Table 1). ICP-OES has greater speed, precision, and sensitivity than Atomic Absorption (AA) Technique. We have used inductively coupled plasma optical emission spectrometer (ICP-710-OES version, that is UKAS approved QC material ordered from (MERCK Germany) for the determination of trace metals of interest in the Central Laboratories Unit, Research Affairs, at the UAE University. Element standards (0.01, 0.1, 1.0, 10.0, 50.0 mg/L) are commercially available or prepared per instrument manufacture recommendations. Standard Argon and compressed Nitrogen gas was used supplied by Emirates Gas Company, Dubai.

Standard solutions of heavy metals were provided by Merck (Darmstadt, Germany). The standards were prepared from the individual 1000 mg/l standards (Merck), in 0.1M HNO₃. Working standards were prepared from the stock solutions. Quality assurance (QA) and quality control (QC) procedures were strictly followed through- out the present study. The level of accuracy and QC for the determination of heavy metal concentrations was comprised of measurements of natural matrix Certified Reference Material (CRM) and the measurements of duplicates for each batch of samples. NIST standard 1573 was used for the evaluation of procedures. Reproducibility of the measurements for the metals ranged between 92.3 and 113 percentage relative standard deviation (%RSDs). RSD is generally better than 5% if an analyte concentration is about 10 times higher than the relevant limit of detection. The mean recovery for certified reference material (CRM 1573) was below 8.7% whereas the percent recovery of RSD for duplicate samples were less than 9%. Student’s t-test and chi square was carried out for statistical analysis with the program SPSS version 17 for windows (Table 1).

SPSS software version 17 was used for the analysis of the data. The results were expressed as Means ± SD by using SPSS software. Students paired t test were performed for the determination of difference between group means. The significance level was set as p < 0.05. Chi square was also carried out to determine the association of socio-economic factors such as marital status, education, age, and residential area to the levels of trace metals in Schizophrenic patients.

According to our questionnaire all participants were living in the nearby vicinity of the same city i.e. Karachi, though they were from different ethnic background. The socio-demographic and clinical characteristics were compared between schizophrenic male and female patients (Table 2). Out of 140 participants 80 were females and 60 were male patients. All groups were matched for mean age, age at contact, gender and clinical symptoms.

Table shows highly associated risk factors of Schizophrenia between male and female patients. The data did not find significant differences at the time of onset between males and females. Similarly, no significant correlation of the disease was found with the level of education and awareness and symptoms type. Effects of education, monthly income and drug abuse were found significantly associated with schizophrenia. SD = Standard Deviation; df = Degree of Freedom; ^*p = 0.03, ^**p = 0.01, ^***p = 0.000001; NS = Not Significant; t/x² = Chi Square; n = Number of patients male or female.

Our data shows no significant difference in the onset of disease between the genders and the mean age of the groups i.e. from 12 - 59 years. There were no significant differences found to be present between the ages of onset and contact among the groups. This shows that males and females are equally prone to have schizophrenia. Moreover men and women of ages between 12 - 59 years were equally susceptible and can develop schizophrenic symptoms. Interestingly there was a slight non-significant difference (x² = 3.472, p = 0.06) found between positive and negative symptoms only in between the genders. It was found that 78% of men shows a negative and 36.25% women shows positive symptoms of schizophrenia (Table 2). Similarly, no significant correlation of the disease was found with the level of education and awareness.

According to our survey all Pakistani Schizophrenic subjects have completed their primary or secondary education except for 2.5% of the effected population that has reached up to the university level. In Pakistani population we have observed that single men and married or divorced women have significantly higher tendency to develop schizophrenia (Table 2). Whereas in men with lower income and females with higher incomes shows a significant (X² = 7.47; p < 0.05) correlation with the disease. Present data recommends a major role of drugs and other addictives such as Cannabis, Charas, Alcohol, Tobacco and Betel nut in the onset of schizophrenia. According to our survey 16 % men uses cannabis and 36.6% men are addictive to tobacco and alcohol, in contrast to this 25% of women are addictive to betel nut. Our results demonstrate that male schizophrenic patients are using more illicit drugs as compare to their female counterparts.

Trace elements are directly involved in human metabolism and there is a growing interest in the role of certain elements in physiological and psychological processes. The level of trace metals such as Cr, Pb, Zn, Cu, Fe, Se, As and Mn have been detected in ppm (mg/L) in blood samples of Pakistani Schizophrenic patients (Table 2) by using inductive couple plasma-optical emission spectrometry (ICP-OES). ICP-OES is the most reliable and the only technique available which can provide the correct levels of these metals even if they are present in trace amounts (<5 ppm). We have used this method not only to obtain the trace metal levels in both male and female patients but also to compare the iron levels between both genders (Figure 1). ICP-OES used in a clinical laboratory for a variety of specimens, including whole blood, serum, hair and urine [17] . Trace level values are typically defined <5 mg/dL [18] . Current study did not find any significant differences between the levels of trace metals in any group (Control, Newly diagnosed and Old diagnosed). We also did not observed significant any decrease or increase in the levels of any trace metals in newly diagnosed and medicated-schizophrenic patients when compared to controls (Table 3). The present results showed that these toxic metals were not significantly involved in the progression and onset of schizophrenia at least in Pakistani population.

When we compared individual values of trace metals in male and female Schizophrenic patients, we found higher values of Cr, Pb, Fe and Mn in males while Zn and Cu in females (Table 4(a) and Table 4(b)). While comparing male patients with their controls we found that in newly diagnosed patient the levels of all trace metals were higher except for Fe. Similarly, for females all trace metal values were higher in newly diagnosed patients except for Fe and Mn.

Clearly demonstrates that the levels of trace metals were not significantly different in both groups of schizophrenic patients when compared with the controls. ^aControl when compared to newly diagnosed , ^bControl when compared to old diagnosed, ^cNewly diagnosed when compared to old diagnosed, SD = Standard Deviation, n = number of patients, Ppm = part per million.

Data compared individual values of trace metals in female and found higher values of Zn, and Cu in female patients. SD = Standard Deviation, n = number of patients, ppm = part per million.

(b)

Data showing the comparison of individual values of trace metals levels in male patients among three groups. We found higher values of Cr, Pb, and Mn in male patients. SD = Standard Deviation, n = number of patients, ppm = part per million.

In Pakistan 1.5% of the population is suffering from Schizophrenia and its prevalence is higher because of the poor care and un-acceptance of disorder. Their families believed in Shamans (Aamil/Peer) for the treatment which usually makes their conditions worse. Major factors like unemployment, living standard, education, marital status, economic status and cost of treatment play significant roles for the onset of schizophrenia in Pakistan. In this study we identified factors such as drug abuse, marital status, education, monthly income that are related with Schizophrenia. Likewise the levels of trace metals observed in the controls and patients were directing some clues for the disease.

According to our survey females are more affected than men (Table 2). Previous finding showed that many women became schizophrenics or acquired mood disorder illnesses after or during pregnancy, delivery or nursing [19] . This could be explained by considering possibilities of postpartum depression in which the level of amino acid tryptophan, the main precursor for serotonin, is decreased leading to low serotonin levels which leads which lead to depression [20] . Furthermore evidences have suggested estrogen hormone is involved in memory and cognition function so any imbalances in its levels might affect the mother or its unborn child [21] . Though, we didn’t find any significant differences between the onset of schizophrenia and these physical factors in Pakistani women. The negative symptoms precipitated more in men than in women, however the results remained slightly not significant (p = 0.06). We proposed that negative symptoms (such as reduction of emotional responsiveness, motivation, socialization, movement, and monosyllabic speech, few gestures, and inability to feel pleasure or act spontaneously) and positive symptoms (such as exaggerations and distortions of normal perception and thinking and hallucinations) are equally precipitated in males and females but more common in men. This could be explained perhaps by the fact that usually men do not like to share their true feelings with their friends or family members. Generally they tend to suppress their emotions which lead to stress and depression and may cause negative symptoms of schizophrenia. However poverty and huge difference between “social classes” are also major issues in Pakistan which induces frustrations leading to mental disorders.

We observed that patients were addicted to tobacco, cannabis, betel nut, and charas. We found that high percentages of males (16.6%) were using cannabis than tobacco and alcohol and cannabis increases the levels of dopamine in the brain, which can lead to Schizophrenic symptoms [22] . Similarly 25% of women are addictive to betel nut. Moreover those who were using cannabis under the age of 15 were three times more likely to develop schizophrenia [23] . This is consistent with our study that Pakistani men who use cannabis are having more schizophrenic symptoms as compare to their female counterparts.

There is increasing evidence suggesting that the transition metals are essential for many metabolic processes and their homeostasis is crucial for life [24] . In this study we found that Fe levels were decreased in pregnant female patients as compared to male patients leading to more schizophrenic symptoms in married females. This could be explained by the fact that the low availability of iron in plasma results in increased Fe deposition in the brain of the patients [25] . Klinker and colleagues (2011) demonstrated that the Iron deficiency may also decrease dopamine receptors in rats and mice, deregulating spontaneous motor activity and behavior performance during learning exercises [26] [27] . On the other hand excess Fe levels are neurotoxic in adults however results remained not significant [28] . Mn is an essential metal but also have toxic effects [29] . Our results showed an increase in the levels of Mn in newly and old diagnosed patients which are different from the previous finding that showed a reduced level of Mn in patients who were on medications [30] but similar to the research [31] showing an association between reduced levels of Mn in patients taking anti-psychotics medications. We highly recommend follow-ups of the patients using antipsychotic medications. Their Mn levels should be monitored after every three to four months while they are on medicine. On the other hand there is a slight increase in the levels of Zn and Cu in newly and old diagnosed male patients as compared to its control healthy siblings. Both of these metals are essential for the development of brain especially Zn which involves in the neurotransmission of glutamate in the limbic system [32] . Zn is also important for the normal function of enzymes involved in DNA synthesis and repairing such as super oxide dismutase (SOD) and thymidylate synthase [33] . However excess copper have significant neurological consequences such as Wilson disease a genetic disorder resembling schizophrenia [34] . There is non-significant increase in the levels of Pb in our case-control study, care should be taken as lead is a toxic trace metal and its elevated levels have been implicated in schizophrenia [35] . Guilarte et al. (2011) demonstrated that Pb poisoning of mice leads to the expression of mutant forms of DISC-1 gene in Schizophrenic animals. Thus a procedure for decreasing blood Pb level may be beneficial for the management of schizophrenia. In the present study, elevated levels of Cr were also observed in newly diagnosed drug-free male schizophrenic patients when compared to male controls, whereas the levels of Cr were reduced in old diagnosed patients who were on antipsychotic medications. Chromium is an essential trace element that potentiates insulin secretion and enhances the activity of neurotransmitters in the brain. Hence increased levels of Cr may lead to increased activity of neurotransmitters in the brain, suggesting its role in the pathogenesis of schizophrenia.

Caution should be taken even the levels of Cr, Zn, Mn and Pb is non-significantly elevated and may make the patients vulnerable to acquire more psychotic symptoms. Importantly it was reported [36] that patients had more depressed mood than controls and siblings but both patients and siblings exhibited a significant increased risk of depressive episodes compared to the controls. In other words siblings have higher chances of meeting lifetime depressive episodes than the controls [36] . Most of the subjects in our study were living in densely populated commercial areas of Karachi, which may increase their risk of having psychotic symptoms in controls as well as subjects with faulty genes.