Synergistic Effect of Vitamin E & D in Reducing the Risk of Effects Associated With Atypical Antipsychotics
Phase: NA
Conditions: Antipsychotics and Neuroleptics Toxicity
Sponsor: Dr Rabia Arshad

 
 
 
SYNOPSIS 
SYNERGISTIC EFFECT OF VITAMIN-E AND VITAMIN-D IN 
REDUCING RISK OF SIDE EFFECTS ASSOCIATED WITH 
ATYPICAL ANTIPSYCHOTICS AND IMPROVEMENT OF 
PSYCHIATRIC ILLNESS 
 
 
 
Candidate: 
 
 
Dr Mohammad Abid 
MBBS, M.Phil. 
 
 
Supervisor:  
 
Professor Dr Kausar Aamir 
MBBS, M.Phil., Ph.D. 
Professor of Pharmacology 
 
 
 
 
Basic Medical Science Institute 
Jinnah Post Graduate Medical 
Center Karachi, 
University Of Karachi 
 
12th December 2019

 
List of abbreviations 
 
 
HDL 
High density lipoprotein 
LDL 
Low density lipoprotein 
VLDL 
Very low density lipoprotein 
SOD 
Superoxide dismutase 
GPx 
Glutathione peroxidase 
INF γ 
Interferon-γ 
S. L P 
Serum lipid profile 
PCR 
Polymerase chain reaction 
TNF-α 
Tumor necrosis factor alpha 

 
ABSTRACT 
Introduction 
 
Depressive disorders and schizophrenia are the serious mental illnesses. Many 
etiological factors like oxidative stress, socio-economic status, genetic involvement 
and environmental changes are responsible. Wide range of treatment including anti- 
depressants and anti-psychotics are available. These drugs especially atypical anti- 
psychotics causes serious side effects including obesity, changes in serum lipids and 
blood pressure. In our study we will emphasize the synergistic effects of Vit D and 
Vit E along with atypical anti psychotics not only in reducing the side effects but also 
to see the improvement in the disease. 
We will be dividing the patients into seven groups. First group will serve as 
control and other six as treatment groups. Group 2, 3 and 4 will be treated with 
atypical anti psychotics (olanzapine 10mg/day, risperidone2mg/day, quetiapine 
100mg/day respectively) for two months and then same patients will be given Vit D 
500- 1000 I.U and Vit E 1000 mg/day along with atypical anti psychotics. 
Serum glutathione peroxidase (GPx) and super oxide dismutase (SOD) will be 
examined by colorimetric method. Tumor necrosis factor alpha (TNF-α) and 
interferon-γ levels will be analyzed by PCR. Serum lipid profile will be assessed by 
commercially available kits. Improvement in illness will be assessed through Positive 
and Negative Syndrome Scale score (PANSS) 
OBJECTIVES: 
1. The objective of this study is to examine the safety and efficacy of vitamin D 
and vitamin E in the treatment of psychiatric disorders. 
2. To determine the improvement and reversibility of side effects of psychiatric 
disorders. 
MATERIAL AND METHODS: 
Study design and setting: It will be an interventional prospective case control study 
and it will be conducted in the Department of Pharmacology, in collaboration with 
Psychiatry Department JPMC, Karachi. 
Study period: Two years after approval of synopsis. 
Study sample size: Based on prevalence with confidence interval 99% and bond of 
error 1% using online software www.openepi.com, my sample size is 240 
Sampling technique: Non probability purposive sampling will be used. 
Ethical Approval: Ethical approval will be taken from IRB, JPMC, Karachi 
Result: Results will be evaluated and compared statistically between and within 
groups. 
Keywords: Anti- Psychotics, Olanzapine, Quetiapine, risperidone, tumor necrosing 
factor alpha, interferon gamma, positive and negative syndrome scale 

 
INTRODUCTION: 
Mental illness is defined as any disturbance of emotional equilibrium, as manifested 
in maladaptive behavior and impaired functioning, caused by genetic, physical, 
chemical, biologic, psychologic, or social and cultural factors (Singh et al., 2017). 
Mental illness is a major problem all over the world. Studies from developed 
countries have reported a prevalence rate of 15 per 1000 population (Jayanthi et al., 
2013). In Pakistan the reported rate is 73 per 1000 population (Sarumathy et al., 
2014). Organic psychosis (0.4%), schizophrenia (2.7%) and affective disorders 
(12.3%) contribute to a rate of 15.4% for psychosis. The prevalence rate for mental 
retardation (6.9%), epilepsy (4.4%), neurotic disorders (20.7%), alcohol/drug 
addiction (6.9%) and miscellaneous group (3.9%) were estimated (Pakpoor et al., 
2014). Different treatment modalities such as drugs, electro-convulsive therapy, 
counselling, psycho-surgery and psychotherapy are used in the treatment of mental 
illness. (Sarumathy et al., 2014) 
An adverse drug reaction (ADR) is defined by the World Health Organization 
(WHO) as “any response to a drug which is noxious and unintended, and which 
occurs at doses normally used in man for prophylaxis, diagnosis, or therapy of 
disease, or for the modification of physiological function” (Singh et al., 2017). 
Patients with psychiatric illness needs lifelong therapy with psychotropic 
drugs which predisposes them to an array of ADRs.(Sarumathy et al., 2014). The 
common adverse effects associated with psychotropic drugs are weight gain, 
somnolence, tremors, and tardive dyskinesia. These adverse effects tend to deteriorate 
the mental and physical well-being of the patient and thus lead to patient non- 
adherence to therapy (Sarumathy et al., 2014) 
Antipsychotics are a class of drugs used in the treatment of psychiatric 
disorders. Since the introduction of antipsychotic medication for the treatment of 
psychosis, a wide range of different types of drugs have been developed under this 
genre. The first generation of antipsychotic medication is known as the ‘typical 
antipsychotics’ and these were first discovered in the 1950s. Soon following their 
clinical use it was recognized that they caused extrapyramidal symptoms (EPS) in 
patients including parkinsonism, tardive dyskinesia, akathesia and dystonia. (Pakpoor 
et al., 2014). 

 
Antipsychotics 
Antipsychotics are associated with a range of side effects. It is well recognized 
that many people stop taking these drugs (around two thirds even in controlled drug 
trials) due in part to adverse effects.(Tiwari et al., 2012). The typical antipsychotics 
has adverse effects like extrapyramidal reactions including acute dystonias, akethesia, 
parkinsonism (rigidity and tremor), tardive dyskinesia, tachycardia, hypotension, 
impotence, lethargy, seizures, intense dreams or nightmares, and hyper-prolactinemia 
(Tiwari et al., 2012). On the other hand, the atypical antipsychotics causes obesity, 
alter lipid profile and subsequent metabolic syndrome specially by Olanzapine, 
Risperidone and Quetiapine (ketzang 2004). Side effects from antipsychotics can be 
managed by a number of different drugs. Continuous use of neuroleptics has been 
shown to decrease total brain volume by 10% in macaque monkeys.(Dorph-Petersen 
et al., 2005 
Role of Vitamin D 
Vitamin D deficiency is prevalent throughout the world, particularly in high- 
risk groups including pregnant woman, infants, dark-skinned migrants and the elderly 
(Lips et al., 2010). In Australia, a recent study showed that 31% of the population has 
vitamin D deficiency, which is defined as a serum concentration of 25- 
hydroxyvitamin D [25(OH)D] below 50 nmol/L (Daly et al., 2012). Although vitamin 
D is essential for calcium homeostasis and bone metabolism, it also has a role in other 
physiological functions, such as an immune modulator (Baeke et al., 2010) and in cell 
proliferation and differentiation (Bikle et al., 2009). Research over the past 15 years 
has revealed many functions of vitamin D in brain development and adult brain 
function (Harms et al., 2011). More recently, evidence has accumulated to suggest 
that low vitamin D levels during adulthood may also be associated with adverse brain- 
related outcome. A growing body of evidence from epidemiology and neuroscience 
links vitamin D deficiency with a range of neuropsychiatric disorders and 
neurodegenerative diseases (Eyles et al., 2013). Low vitamin D would have utmost 
public health implications and treatment is safe, cheap and publicly acceptable. 
Adequate vitamin D status is also needed for optimal muscular strength in youth 
(HELENA study) and the elderly.(Hirani et al., 2013). In addition to these diseases, 
vitamin D has also been linked to mental illnesses including Alzheimer's (Balion et 

 
al., 2012) premenstrual mood disorder, (Eskandari et al., 2007) major depression and 
psychosis. (Gracious et al., 2012). However, the relationship of vitamin D status to the 
functions of the brain is also in its infancy (Gracious et al., 2012) with some evidence 
of negative, cognitive( Annweiler et al., 2010) and mood(Graham et al., 2015) effects 
in people with insufficient vitamin D levels. Neuro-protective qualities of vitamin D 
have been observed in many studies, and recent reviews have suggested that vitamin 
D could play an important role in the central nervous system for healthy neural 
development and function. (Wrzosek et al.,2013; Deluca et al., 2013). A correlation 
between schizophrenia and low prenatal and early life vitamin D status has been well 
demonstrated (McGrath et al., 2010) and supplementation could be important for 
disease prevention. (Wrzosek et al., 2013) 
Although several studies have shown metformin to be effective, this still hasn’t been 
included in clinical guidelines on managing antipsychotic side effects. Several RCT 
which evaluated the efficacy of metformin in treating antipsychotic induced weight 
gain have been published recently (de Silva et al., 2015; Jarskog et al., 2013). 
Therefore it is important that the evidence regarding metformin is synthesized. 
Study conducted in New Zealand, in which they observed severe vitamin D 
deficiency in schizophrenia and other psychiatric illnesses.(Menkes et al., 2012). 
Vitamin D deficiency and associated psychosis has been noted in dark-skinned 
immigrant populations. In a retrospective chart review of 18 first generation 
immigrants from Africa and Haiti with acute psychosis, all patients had vitamin D 
levels in the insufficient range. (Dealberto et al., 2013), however many of the clinical 
descriptions of the episodes were not consistent with a schizophrenia diagnosis, and 
only seven of these patients went on to be diagnosed with schizophrenia. Similarly, in 
a cross-sectional study of Norwegian patients, vitamin D deficiency was present in 
80% of the psychotic immigrant population with dark complexions. Among native- 
born Norwegians with psychosis, 43% had vitamin D deficiency and had lower serum 
vitamin D levels compared to reference sample (Berg et al., 2010). In a cross- 
sectional study of 35 adolescent inpatients, 33.7% patients were vitamin D deficient; 
of those deficient 40% exhibited psychotic features compared to 16% of patients who 
were not vitamin D deficient. (Gracious et al., 2012). In a case-control study, Crews 

 
found significantly lower levels of serum vitamin D among 69 first-episode psychosis 
inpatients compared to matched healthy controls ( Crews et al., 2013). In another 
small study of first-episode patients, more severe negative symptoms and cognitive 
impairment were correlated with lower vitamin D levels. These patients had not 
received more than a total of four months of antipsychotic treatment (Graham et al., 
2015) Finally, in a recent meta-analysis, Belvederi Murri et al. found that patients 
with psychotic disorders (mainly schizophrenia) had consistently lower vitamin D 
levels compared to healthy controls (Murri et al., 2013) The authors examined 7 
studies overall for a total of 523 patients and 7545 controls. Of note there was 
heterogeneity of effect size, and most studies were case-control or cross-sectional. As 
with all observational studies, we emphasize that the results have to be taken with 
caution, given there may be many potential confounding variables that are present in 
patients with schizophrenia and also affect vitamin D levels such as insufficient 
nutrient intake or little sunlight exposure. 
Vitamin D deficiency early in life may also contribute to schizophrenia risk. 
McGrath et al. examined a Danish cohort of over 400 patients with schizophrenia and 
carefully matched controls in which neonatal dried blood spots had been collected 
(McGrath et al., 2010). Among individuals in the lowest quintiles of neonatal vitamin 
D there was a two-fold elevated risk of schizophrenia compared to those in higher 
quintiles. Interestingly, those neonates with the highest vitamin D levels also had an 
increased risk of schizophrenia. In a birth cohort of over 2000 people in the UK, 
Sullivan et al. did not find an association between maternal vitamin D levels and risk 
of psychotic illness at age 18 (Sullivan et al., 2013). However, it is likely that some 
individuals at this age have not yet experienced onset of the illness; it will be 
important to follow the cohort over the subsequent years. 
Intervention studies have had mixed results. In a Finnish birth cohort of over 
9000 people, vitamin D supplementation with at least 2000 IU/day in males in the 
first year of life reduced the risk of schizophrenia by 77% (RR .23) compared to those 
receiving less than 2000 IU/day; these findings did not hold true in females(McGrath 
et al., 2004). Dealberto et al., 2013 hypothesized that early vitamin D supplementation 
is crucial for pro-differentiating signals in the developing brain, and potentially also 

 
for normal brain recovery after insult. In the study of immigrant population discussed 
above, provided daily vitamin D supplementation of 1000 IU/day in addition to 
ongoing antipsychotic treatment, and concluded there were no changes in psychiatric 
symptoms after vitamin supplementation. 

 
Role of Vitamin E 
Vitamins E is an antioxidants that protects against cellular damage due to 
inflammation or highly reactive oxygen-containing molecules. In another small study 
among 14 in patients with schizophrenia in India, plasma vitamin E and C levels were 
significantly lower when compared to control subjects (D’Souza et al., 2003) 
One study reported a decrease in the Brief Psychiatric Rating Scale (BPRS) 
and Positive and Negative Syndrome Scale (PANSS) scores among patients on 
antipsychotic treatment after supplementation with vitamin E (Avindakshan et al., 
2003); however given the combination treatment it is difficult to conclude what 
effects are due to vitamin supplementation alone. A randomized placebo-control study 
by Bentsen et al 2013 also supplemented vitamin E (1000 mg/day) to patients with 
schizophrenia on antipsychotic medication; among those patients with low red blood 
cell polyunsaturated fatty acids (PUFAs), the vitamin supplementation actually 
impaired recovery from acute psychosis compared to placebo. The authors 
hypothesize that vitamin E at a high enough dose can act as a pro-oxidant if there is 
inadequate antioxidant activity, thus increasing oxidative stress; it could also inhibit 
the beneficial γ- and δ tocopherols. Ten of the thirteen patients showed improvement 
on CGI scores and plasma vitamin C levels increased over the eight weeks of 
treatment. The authors report that most of the symptomatic improvement occurred in 
the positive symptom domain. Notably, there was no control group in this study, and 
the weekly study visits with medical staff may also contribute to improvement in 
psychotic symptoms. 

 
Need for the Study 
Patients suffering from physical illness are given specific treatment because 
the causes are specific and the signs and symptoms are also specific. In a psychiatric 
setting the treatment may not be so specific and most patients are given more than one 
treatment. These treatment methods vary from patient to patient. Some patients do not 
want treatment and may not co-operate with the doctors because of hazards of side 
effects. Some do not realize that they are ill and may actively resist all forms of 
treatment. To minimize side effects, doses should be increased slowly until there is a 
therapeutic effect, side effects emerge, or the maximum recommended dose is 
reached. It may take 6 to 8 weeks for effective results, or as determined by the 
practitioner. Each drug has its own set of actions and side effects, some more serious 
than others; these should be evaluated in terms of each user’s medical status, 
including interaction with other medications. Consider the effect of one psychotropic 
medication at a time. 
A study was conducted on “psychotropic side effects of commonly prescribed 
medications in the elderly” are prevalent and, in most instances, predictable and 
preventable. They are also associated with considerable morbidity and mortality. 
Delirium, mood changes, and psychotic symptoms are the most serious categories of 
psychotropic side effects. Although any drug that crosses the blood-brain barrier has 
the potential for causing psychotropic side effects, certain commonly prescribed 
classes of drugs, such as anticholinergics, psychotropics, antihistaminics, and many 
over-the-counter medications, are particularly suspect. A variety of interventions, 
such as decreasing the inappropriate prescription and performing a routine evaluation 
of the older patient’s drug regimen, can prevent or minimize psychotropic side effects. 
Practitioners must be vigilant in monitoring patients for psychotropic side effects of 
all commonly prescribed drugs. 
A study was conducted on “Educating and informing patients receiving 
psychopharmacological medications. Are family physicians in Pakistan up to the 
Task? A total of 354 adult patients were interviewed during 3 days. Among them, 73 
(20.6%) were prescribed psychopharmacological medications. Among patients 
receiving psychopharmacological medicines, 37 (50.7%) did not know their 
diagnosis; 50 (68.5%) were unaware of the disease process; 52 (71.2%) were unaware 

 
of alternative treatments; 63 (86.3%) were not cautioned about the potential adverse 
effects of the drugs; 24 (32.9%) were unaware of the duration of treatment and in 60 
(82.2%) of the participants an appropriate referral had not been discussed. For all 
aspects of education, patients prescribed psychopharmacological medications knew 
less as compared to those patients that were prescribed other medications. 
When a person knows the side effects of the medication he/she is taking, 
he/she is better equipped to make a decision about whether this is an appropriate 
medical treatment for her. The side effects of some treatments may make some people 
decide not to take the medication or to pursue another treatment option. If a medical 
emergency or serious complication arises as a result of medicinal treatment, a person 
who is aware that this is a possible side effect of the medication can more quickly 
seek appropriate medical treatment. People who are taking medications that have 
chronic but not necessarily serious side effects can learn strategies to manage the 
side effects in our population. Vitamin D and Vitamin E are pro-hormone with 
skeletal and extra skeletal properties that could potentially reduce the severity of these 
side effects. Their role as an adjunctive therapy for the management of side effects of 
drug agents has not been adequately studied. Effective strategies to curb these side 
effects will improve the overall health of patients with psychiatric illnesses who 
receive antipsychotics drugs. This is the reason the researcher selected this topic for 
the study. 
OBJECTIVES: 
The objectives of this study are: 
1. The objective of this study is to examine the safety and efficacy of vitamin D and 
vitamin E in the treatment of psychiatric disorders. 
2. To determine the improvement and reversibility of side effects of psychiatric 
disorders. 

 
HYPOTHESIS OF THE STUDY: 
 
Null Hypothesis 
 
There is no statistically significant difference of Synergistic effect of Vitamin E 
and Vitamin D in reducing risk of side effects associated with atypical anti 
psychotics 
 
Alternate Hypothesis 
 
There is statistically significant difference of Synergistic effect of Vitamin E 
and Vitamin D in reducing risk of side effects associated with atypical anti psychotics 
 
OPERATIONAL DEFINITIONS: 
Vitamin D level: It will be Based on laboratory reference values, Vitamin D 
severe deficiency, deficiency, insufficiency, and sufficiency were defined as a 25- 
hydroxy Vitamin D (25(OH)D) levels <10 ng/mL, 10–20 ng/mL, 21–30 ng/mL, and 
>30 ng/mL, respectively. 
 
Vitamin E level: It will be based on below Reference Values: 
 
 
0-17 years: 3.8-18.4 mg/L 
 
or =18 years: 5.5-17.0 mg/L 
 
Significant deficiency: <3.0 mg/L 
 
Significant excess: >40 mg/L 
Atypical Antipsychotic drugs: 
In this study it refers to, a class of drugs used to control psychotic symptoms 
in patients with psychotic disorders such as schizophrenia, severe depression, and 
delusional disorder that is, atypical [Risperidone, Olanzapine, quetiapine] 
 
MATERIAL AND METHODS: 
Study design: 
It will be interventional prospective case control study. 
Study period: 
Two year after approval of synopsis. 

 
Study setting: 
Study will be conducted in the Department of Pharmacology 
with 
collaboration of Psychiatry department JPMC, Karachi 
Study sample size: 
Based on the prevalence of psychiatric illnesses10.0%a total of 240 patients 
will be selected based on 99% confidence interval and 1% margin of error using 
online software www.openepi.com The sample size estimated is 240 (Goswami et al., 
2014) 
Sampling technique: 
Non probability purposive sampling technique will be used. 
SAMPLE SELECTION: 
Inclusion Criteria: 
1. Participants who were under antipsychotic therapy such as olanzapine, 
risperidone and quetiapine. 
2. Participants who were between the ages 20 – 70 years both sex male and 
female. 
3. Participants who were taking a combination of one or two antipsychotics. 
4. Participants who were under antipsychotic therapy 
5. Patients having vitamin-D and vitamin-E levels below normal 
Exclusion Criteria: 
1. Psychiatric patients those who were pregnant and nursing mothers. 
2. Participants who have renal and hepatic failure or parathyroid disorders. 
3. Patients who use phosphorous, calcium or vitamin D supplements, or had co- 
administered medications of anticonvulsants, anti-fungal and corticosteroids 
with history of other psychiatric and neurologic disorders. 
4. Any metabolic syndrome 
Data collection procedure: 
During the study period, 240 were will be registered into the study who selected 
according to the inclusion and exclusion criteria for psychiatric disorders with 
baseline serum vitamin D and vitamin E concentrations. All patients will be on 
antipsychotics for at least two months and a stable dose for at least 2 month will be 

 
included in the study and the antipsychotics dose remained unchanged during the 
study period. 
Patients groups: 
All the patients will be equally divided (n = 35) into seven groups as: 
Group 1: Treatment group with Resperidone 
The patients who will receive 2 mg/day Risperidone (n =35 ) at baseline along 
and will be followed for 2 months will be known as the intervention group. 
Group 2: Treatment group with Olanzapine 
The patients who will receive 10 mg/day Olanzapine (n =35 ) at baseline along 
and will be followed for 2 months will be known as the intervention group. 
Group 3: Treatment group with Quetiapine 
The patients who will receive 50-100 mg/day Quetiapine (n =35 ) at baseline 
along and will be followed for 2 months will be known as the intervention group. 
Group 4: Treatment group with Vitamin D + Vitamin E + Risperidone 
The patients who will receive 5000 IU per week plus vitamin E 1000 mg per 
day plus 2mg Risperidone (n = 35) at baseline along with their antipsychotic regimen 
and followed for 2 months will be known as the intervention group. 
Group 5: Treatment group with Vitamin D + Vitamin E + Olanzapine 
The patients who will receive from 5000 IU per week plus vitamin E 1000 mg 
per day plus 10 mg Olanzapine (n = 35) at baseline along with their antipsychotic 
regimen and followed for 2 months will be known as the intervention group. 
 
Group 6: Treatment group with Vitamin D + Vitamin E + Quetiapine 
The patients who will receive from 5000 IU per week plus vitamin E 1000 mg 
per day plus 100 mg Quetiapine (n = 35) at baseline along with their antipsychotic 
regimen and followed for 2 months will be known as the intervention group. 
Group 7: Control group 
Twenty apparently age and sex matched controls that will come for routine 
checkup, will be included from outpatient department. 
All the studied groups received mentioned treatment for 2 months. All psychiatric 
patients will be managed based on the guidelines of the American Psychiatric 

 
Association (18). All patients will beexamined clinically, and their demographic and 
menstrual and medical history will be recorded by a trained nurse using a check list. 
Sample collection and measurement 
A sample of 10 mL of venous blood will be collected into EDTA tube that will 
contain anticoagulant at the baseline by the end of the 2nd month (Graham et al., 
2015). Each sample will be centrifuged (3500×g) for 15 min. 
ASSESEMENT OF VIT D3 AND VIT E 
Then the serum will be separated and the serum Vitamin D and Vit E 
concentration will be analyzed using a commercially available 25-OH Vitamin D and 
Vit E ELISA kit. 
ASSESMENT OF PSYCHIATRIC IMPROVEMENT 
For each patient, a set of variables will be collected including demographics 
(age, gender, marital and smoking status, duration of disease, and initial Positive and 
Negative Syndrome Scale score (PANSS) (20).The PANSS assessment will be blind 
and made by two trained and expert psychologists independently. The whole study 
will be consisted of 2 visits: a baseline, and a follow up visit after 2 months of 
treatment. At baseline visit, blood will be collected for MDA, vitamin D, and vitamin 
E levels assessment. All patients will receive standard routine medical care 
throughout the study. Regularity of drug intake will be ensured over telephone, and 
with patient’s compliance sheet. 
Assessment of lipid profile 
The metabolic panel includes a lipid profile for the measurement of total 
cholesterol (TC), triglycerides (TAG), high density lipoproteins (HDL), low density 
lipoproteins (LDL), and very low-density lipoproteins (VLDL). 
 
Determination of Glutathione Peroxidase and Superoxide Dismutase Activity 
 
As the oxidative stress of tissue generally involves the Glutathione Peroxidase and 
Superoxide Dismutase system, the levels of serum Glutathione Per-oxidase and 
Superoxide Dismutase will be measured in each group by colorimetric method using 
commercially available kits. 

 
Anti-inflammatory Markers 
TNF α and interferon gamma expression levels will be done though PCR 
Appropriate primers for TNF-α and IFN-γ will be synthesized and used for the 
production of copies by PCR 
Primer for TNF-α (Wang et al., 2003) 
 
Forward 
5-CAGGCGGTGCCTATGTCTC-3 
 
Reverse 
5-CGATCACCCCGAAGTTCAGTAG-3 
 
Primer for interferon-γ (Harris et al., 2005) 
 
Forward 
5-CCATCGGCTGACCTAGA-3 
 
Reverse 
5-GCCACTTGAGTTAAAATAGTTATTCAGAC-3 
 
INSTRUMENTS 
Brief Psychiatric Rating Scale (BPRS). 
This instrument has 16 items each scored on a seven points scale. For 
example, absent, minimal, mild, moderate, moderate-severe, severe, and extreme. 
The items include anxiety, emotional withdrawal (autism), depressive mood, guilt 
feeling etc. There are criteria to define the symptomatology items but not the severity 
ratings. It is suitable for rating of sever psychiatric illness but not minor disorders. 
Positive and negative syndrome rating scale (PANSS) 
This scale rates blunted effect, poor rapport, social withdraw difficulty in 
abstract and stereo typed thinking and lack of spontaneity. A thirty minutes interview 
is required. It has seven items for positive symptoms and seven items for negative 
symptom and scoring is the same as in BPRS. 
Extra pyramidal symptom rating scale (EPSRS) 
On which quantitative rating of the symptoms of parkinsonism, dystonia and 
dyskinesia is done. It has twelve items each scored on four points scale. 
Quality of life questionnaire (WHO QOL-BREF) 
Quality of life is a general term applied to the totality of physical, 
psychological and social functioning. It is determined by physical impairment, 

 
emotional reaction, personality, illness etc. QOL scale requires great deal of 
concentration and responsibility and assessed very carefully as involves many diverse 
aspects and is laborious. It has 26 items each scored on five-point scale. Increase in 
total count means an improvement in the symptoms and hence in the quality of life. 
Ethical considerations: 
 
The study will be undertaken after an ethical approval for the current study 
will be taken by the Institutional Review Board (IRB) of Basic Medical Sciences 
Institute, JPMC, Karachi. Data which will be obtained during the study will be kept 
extremely confidential. 
In a psychiatric study it would always be necessary to ask and discuss about 
some sensitive issues, regarding personal and family affairs. But extreme precaution 
would be taken not to break the limits of ethical issues and not to harm physically, 
psychologically socially and spiritually to the participants, during the course of thesis 
work. 
Points to preserve the ethical issues- 
1. This study will not involve one’s body organ or fetal tissues. 
2. Precaution will be taken to ensure one’s anonymity. 
3. Subject or key relatives will be clearly informed about the scope and 
limitation of the study. 
4. A written consent will be taken from the subject and / or from the key relatives 
if subjects are minor or unable to give reliable information (and for that purpose 
a consent form was supplied). 
5. The patient and or relatives who will be unwilling to participate will be 
excluded from the study. 
6. Precautions will be taken to maintain ones confidentiality of personal data and 
obscurity. 
7. No financial involvement of the client or respondents will be encouraged. 
8. Basic human rights to refuse, or to accept will be maintained. 
9. During the study period no medication will be given to the patient for trial 
excepting therapeutic measures provide by a psychiatrist. 

 
During the course of this study Precautions will be taken about not to produce any 
environmental hazards or breach. 

 
Statistical analysis: 
 
Data will be processed and analyzed using computer software SPSS (Statistical 
Package for Social Sciences) version 22.0. Data will be assessed for test of normality 
in SPSS.Qualitative variables will be recorded according to frequency and percentage 
and quantitative variables in terms of Mean ± SD (Standard Deviation). To compare 
continuous variables in five groups, independent sample t-test will be applied and to 
compare categorical variables Chi- Square or Fisher›s exact test will be used. 
Kruskal-Wallis test will be used, for comparing The correlation between serum 
vitamin D and vitamin E level and negative and positive symptoms at baseline will be 
made by Pearson correlation test. All the data will be calculated on 99% confidence 
interval. P value < 0.05 will be considered significant level for all the comparisons. 

 
Synergistic effect of Vitamin E and Vitamin D in reducing risk of side effects 
associated typical anti psychotics and improvement of psychiatric illness 
 
 
Questionnaire 
 
1. Identification Number:   
 
1.1 I.D Code:    
 
1.2 Name:  
 
1.3 Father’s/ Husband’s Name:  
 
1.4 Sex: 
1. Male 
2. Female 
1.5 Marital Status:  
 
1.6 Date of Birth (dd/mm/yy):   
 
1.7 Age (yr):     
 
1.8 Mailing address:     
 
 
 
1.9 Permanent address:   
 
 
 
1.10 Religion:   
 
1.11 Nationality:   
 
 
1.12 Duration of illness:  
 
 
2. Personal history: 
 
2.1 Area of residence: 
1. Rural 
2. Urban 
3. Sub-Urban 
 
2.2. Education: 
1. Illiterate 
2. Primary 
3. Middle 
 
4. Matriculation 
5. Intermediate 
6. Graduate 
 
7. Masters or above 

 
2.3.Occupation: 
1. Professional/Managerial/ Business 
2. Clerical 
3. Technical 
4. Skilled worker 
5. Unemployed/ Pensioner 
6. Housewife 
7. Labour 
8. Govt Job 
2.4. Socio economic status 
1. Lower 
2. Middle 
3. Upper 
 
 
 
 
3. Previous history of psychiatric disorder: 
1. Yes 
2. No 
4. Family history of psychiatric disorder: 
1. Yes 
2. No 
 
 
 
 
 
ANTIPSYCHOTICS 
 
At baseline 
Group 1: 
Control 
Group 2: 
Risperidone 
Group 3: 
Olanzapine 
Group 4: 
Quetiapine 
Group 5: 
Vitamin D + Vitamin 
E + Risperidone 
Group 6: 
Vitamin D + Vitamin 
E + Olanzapine 
Group 7: 
Vitamin D + Vitamin 
E + Quetiapine 
 
 
At 2 months 
Group 1: 
Control 
Group 2: 
Risperidone 
Group 3: 
Olanzapine 
Group 4: 
Quetiapine 
Group 5: 
Vitamin D + Vitamin 
E + Risperidone 
Group 6: 
Vitamin D + Vitamin 
E + Olanzapine 
Group 7: 
Vitamin D + Vitamin 
E + Quetiapine 

 
LIPID PROFILE 
Total cholesterol 
At baseline 
  
 
At 2 months 
  
 
LDL 
  
 
  
 
HDL 
  
 
  
 
Triglyceride 
  
 
  
 
 
 
 
 
Glutathione 
TNF alpha 
At baseline 
At 2 months 
Interferon gamma through PCR 
 
 
 
 
Weight: 
Gained 
Not weight gained 
Improved 
Not improved 
Brief Psychiatric Rating Scale (BPRS). 
Positive and negative syndrome rating scale (PANSS) 
Extra pyramidal symptom rating scale (EPSRS) 
Quality of life questionnaire (WHO QOL-BREF) 

 

 
INFORMED CONSENT FORM (ENGLISH) 
Reg. No:   
 
I, Mr./Ms./Mrs. 
do hereby state willingly that I have been 
briefly explained about the details of this clinical research project entitled as 
“Synergistic effect of Vitamin E and Vitamin D in reducing risk of side effects 
associated typical anti psychotics and improvement of psychiatric illness”, which is 
being undertaken by Dr.Mohammad Abid. I do hereby give my written consent to get 
myself registered in this study after considering the potential benefits that it will offer to 
the health care services for the general public without any monetary settlements. 
 
 
 
 
 
 
 
Participant’s Signature:   
 
 
 
Date:   
 
 
 
 
 
 
Researcher’s Signature:   
 
 
 
Date:   
 

 
INFORMED CONSENT FORM (URDU) 
 
 
یضرا ہامن 
تحقیقی کام میں شر
کت کرنے کے لئے ا ٓپ
کا بہت ش
کریہ اگر ا ٓپ اس کام میں ہورہے ہیں تو ا ٓپ
وک 5 یلم نوخریٹل ونہ مناک 
دینے کو کہا جائیگا جو کہ ایک اس کار ڈاک
رٹ جن سر کے  ذریعے ےل ۔اگ یہ نہومن ریٹرابیل ںیم حم
فوظ ک
یا شامل گا۔ خونکالنے 
سے ی کس سم ق اک یئوک ناصقن ںیہن اگوہ ۔ عد ب ںازا ہی ہنومن بہرجت  ےک  ےئل  المعت اس  ک
ای  ئےاج  ۔اگ  یہ  قحت
یقی  ماک  ی کس  
ریابوراک  صمق
د جائے ا ٓپ
کی شمولیت ا ٓپ
ک
ی مرضی پر ہے اور ا ٓپ ک
س
ی بھی وقت اس تح
قیق ہیں ا ٓپ سے 
حاص
ل ےنوہ یالو مامت تامولعم وک ص
یغہ کے
لئے نہیں ہے۔  یہ  معلومات  صرف  تح
قیق  نے کر  ںوالو  وک  میسر حت
قیق سے 
الگ ہو
س
ک
ےت ازر  ںیم  ر
ئےاجاھک  اگ  روا 
ہونگی
۔ اگر اس تح
قیق ےک جئاتن ےس تحص ےک قلعتم ئین ئاس
ی نس تامولعم 
حاص
ل ہوئیں تو وہ ا ٓپ
کے نام کو ظاہر ک
ےئ ریغب سئناس
ی جریدے یا ر
ےلاس یم شائع ں ک
ی ئیگی اج
۔ اس خون کے نمون
ے سے 
سا کے ہوالع یئوک ماک ںیہن ایل 
جائ
ے گا۔ 
 
 
میں ت
صدیق ک
تار /رک
یت ںوہ کہ ںیم نے سا دستاویز وک ھجمس ایل ہے ںیم پنیا 
یضمر ےس نےپا نوخ اک ہنومن سا حت
یقیق ماک ںیم ےنید ےک ےئل تیار ۔ںوہ 
 
 
 
 
 
نام یرم
ض:۔۔۔۔۔۔۔۔۔۔۔۔ ۔۔۔۔۔۔ 
                     دستخط:۔۔۔۔۔۔۔ ۔۔۔۔ 
 
 
 
ضی ار نامہ نیل
ے لےاو اک نام ۔۔۔۔۔۔۔۔۔۔۔۔ ۔۔۔۔۔ 
         یخرات:۔ ۔۔۔۔

REFERENCES: 
 
Annweiler, C., Schott, A. M., Allali, G., Bridenbaugh, S. A., Kressig, R. W., Allain, 
P., ... & Beauchet, O. (2010). Association of vitamin D deficiency with 
cognitive       impairment       in       older       women:        cross-sectional 
study. Neurology, 74(1), 27-32. 
Arvindakshan, M., Ghate, M., Ranjekar, P. K., Evans, D. R., & Mahadik, S. P. (2003). 
Supplementation with a combination of ω-3 fatty acids and antioxidants 
(vitamins E and C) improves the outcome of schizophrenia. Schizophrenia 
research, 62(3), 195-204. 
Baeke, F., Takiishi, T., Korf, H., Gysemans, C., & Mathieu, C. (2010). Vitamin D: 
modulator of the immune system. Current opinion in pharmacology, 10(4), 
482-496. 
Balion, C., Griffith, L. E., Strifler, L., Henderson, M., Patterson, C., Heckman, G., ... 
& Raina, P. (2012). Vitamin D, cognition, and dementia: a systematic review 
and meta-analysis. Neurology, 79(13), 1397-1405. 
Bentsen, H., Osnes, K., Refsum, H., Solberg, D. K., & Bøhmer, T. (2013). A 
randomized placebo-controlled trial of an omega-3 fatty acid and vitamins E+ 
C in schizophrenia. Translational psychiatry, 3(12), e335. 
Berg, A. O., Melle, I., Torjesen, P. A., Lien, L., Hauff, E., & Andreassen, O. A. 
(2010). A cross-sectional study of vitamin D deficiency among immigrants 
and Norwegians with psychosis compared to the general population. The 
Journal of clinical psychiatry, 71(12), 1598-1604. 
Bikle, D. (2009). Nonclassic actions of vitamin D. The Journal of Clinical 
Endocrinology & Metabolism, 94(1), 26-34. 
Crews, M., Lally, J., Gardner-Sood, P., Howes, O., Bonaccorso, S., Smith, S., ... & 
Gaughran, F. (2013). Vitamin D deficiency in first episode psychosis: a case– 
control study. Schizophrenia research, 150(2-3), 533-537. 

 
Daly, R. M., Gagnon, C., Lu, Z. X., Magliano, D. J., Dunstan, D. W., Sikaris, K. A., 
... & Shaw, J. E. (2012). Prevalence of vitamin D deficiency and its 
determinants in Australian adults aged 25 years and older: a national, 
population‐based study. Clinical endocrinology, 77(1), 26-35. 
de Silva, V. A., Dayabandara, M., Wijesundara, H., Henegama, T., Gunewardena, H., 
Suraweera, C., & Hanwella, R. (2015). Metformin for treatment of 
antipsychotic-induced weight gain in a South Asian population with 
schizophrenia or schizoaffective disorder: A double blind, randomized, 
placebo controlled study. Journal of Psychopharmacology, 29(12), 1255- 
1261. 
Dealberto, M. J. (2013). Clinical symptoms of psychotic episodes and 25‐hydroxy 
vitamin D serum levels in black first‐generation immigrants. Acta Psychiatrica 
Scandinavica, 128(6), 475-487. 
DeLuca, G. C., Kimball, S. M., Kolasinski, J., Ramagopalan, S. V., & Ebers, G. C. 
(2013).   the   role   of   vitamin   D   in   nervous   system   health    and 
disease. Neuropathology and applied neurobiology, 39(5), 458-484. 
Dorph-Petersen, K. A., Pierri, J. N., Perel, J. M., Sun, Z., Sampson, A. R., & Lewis, 
D. A. (2005). The influence of chronic exposure to antipsychotic medications 
on brain size before and after tissue fixation: a comparison of haloperidol and 
olanzapine in macaque monkeys. Neuropsychopharmacology, 30(9), 1649. 
D'souza, B., & D'souza, V. (2003). Oxidative injury and antioxidant vitamins E and C 
in Schizophrenia.Indian Journal of Clinical Biochemistry, 18(1), 87-90. 
Eskandari, F., Martinez, P. E., Torvik, S., Phillips, T. M., Sternberg, E. M., Mistry, S., 
... & Reynolds, J. C. (2007). Low bone mass in premenopausal women with 
depression. Archives of Internal Medicine, 167(21), 2329-2336. 
Eyles, D. W., Burne, T. H., & McGrath, J. J. (2013). Vitamin D, effects on brain 
development, adult brain function and the links between low levels of vitamin 
D and neuropsychiatric disease. Frontiers in neuroendocrinology, 34(1), 47- 
64 

 
Goswami, N. N., Gandhi, A. M., Patel, P. P., & Dikshit, R. K. (2014). An evaluation 
of the metabolic effects of antipsychotic medications in patients suffering from 
psychiatric illness. Journal of Applied Pharmaceutical Science, 4(4), 14. 
Gracious, B. L., Finucane, T. L., Friedman-Campbell, M., Messing, S., & Parkhurst, 
M. N. (2012). Vitamin D deficiency and psychotic features in mentally ill 
adolescents: a cross-sectional study. BMC psychiatry, 12(1), 38. 
Graham, K. A., Keefe, R. S., Lieberman, J. A., Calikoglu, A. S., Lansing, K. M., & 
Perkins, D. O. (2015). Relationship of low vitamin D status with positive, 
negative and cognitive symptom domains in people with first‐episode 
schizophrenia. Early intervention in psychiatry, 9(5), 397-405 
Harms, L. R., Burne, T. H., Eyles, D. W., & McGrath, J. J. (2011). Vitamin D and the 
brain. Best Practice & Research Clinical Endocrinology & Metabolism, 25(4), 
657-669. 
Harris, D.P., Goodrich,S., Gerth, A.J., Peng, S.L.,and Lund, F.E., 2005. Regulation of 
INF-γ Production by B Effector 1 Cells:Essential Roles for T-bet and the INF-γ 
Receptor. J. Immunol. 174: 6781-6790. 
 
Hirani, V., Naganathan, V., Cumming, R. G., Blyth, F., Le Couteur, D. G., 
Handelsman, D. J., ... & Seibel, M. J. (2013). Associations between frailty and 
serum 25-hydroxyvitamin D and 1, 25-dihydroxyvitamin D concentrations in 
older Australian men:   the   Concord   Health   and   Ageing   in   Men 
Project. Journals of Gerontology Series A: Biomedical Sciences and Medical 
Sciences, 68(9), 1112-1121. 
Jarskog, L. F., Hamer, R. M., Catellier, D. J., Stewart, D. D., LaVange, L., Ray, N., ... 
& METS Investigators. (2013). Metformin for weight loss and metabolic 
control in overweight outpatients with schizophrenia and schizoaffective 
disorder. American Journal of Psychiatry, 170(9), 1032-1040. 

 
Jayanthi, C. R., Divyashree, M., & Sushma, M. (2013). Adverse drug reactions in 
psychiatry outpatients: Clinical spectrum, causality and avoidability. J Chem 
Pharm Res, 5(8), 128-35. 
Lips, P. T. A. M. (2010). Worldwide status of vitamin D nutrition. The Journal of 
steroid biochemistry and molecular biology, 121(1-2), 297-300. 
McGrath, J. J., Burne, T. H., Féron, F., Mackay-Sim, A., & Eyles, D. W. (2010). 
Developmental vitamin D deficiency and risk of schizophrenia: a 10-year 
update. Schizophrenia bulletin, 36(6), 1073-1078. 
McGrath, J. J., Eyles, D. W., Pedersen, C. B., Anderson, C., Ko, P., Burne, T. H., ... & 
Mortensen, P. B. (2010). Neonatal vitamin D status and risk of schizophrenia: 
a population-based case-control study. Archives of general psychiatry, 67(9), 
889-894. 
McGrath, J., Saari, K., Hakko, H., Jokelainen, J., Jones, P., Järvelin, M. R., ... & 
Isohanni, M. (2004). Vitamin D supplementation during the first year of life 
and risk of schizophrenia: a Finnish birth cohort study. Schizophrenia 
research, 67(2-3), 237-245. 
Menkes, D. B., Lancaster, K., Grant, M., Marsh, R. W., Dean, P., & du Toit, S. A. 
(2012). Vitamin D status of psychiatric inpatients in New Zealand’s Waikato 
region. BMC psychiatry, 12(1), 68. 
Murri, M. B., Respino, M., Masotti, M., Innamorati, M., Mondelli, V., Pariante, C., & 
Amore,   M.   (2013).    Vitamin    D    and    psychosis:    mini    meta- 
analysis. Schizophrenia research, 150(1), 235-239. 
Pakpoor, J., & Agius, M. (2014). A review of the adverse side effects associated with 
antipsychotics as related to their efficacy. Psychiatr Danub, 26(Suppl 1), 273- 
284. 
Park, C. H., Park, T. W., Yang, J. C., Lee, K. H., Huang, G. B., Tong, Z., ... & Chung, 
Y. C. (2012). No negative symptoms in healthy volunteers after single doses 
of amisulpride, aripiprazole, and haloperidol: a double-blind placebo- 
controlled trial. International clinical psychopharmacology, 27(2), 114-120. 

 
Sarumathy, S., Menaka, K., George, P. S., & Ravichandiran, V. (2014). A study on 
drug use pattern and adverse drug reactions of anti-psychiatric medications in 
a psychiatry specialized hospital. Int J Pharm Pharm Sci, 6(6), 332-4. 
Singh, H., Yacob, M., Sabu, L., & Mamatha, K. (2017). Adverse drug reactions 
monitoring of psychotropic drugs&58; a tertiary care centre study. Open 
Journal of Psychiatry and Allied Sciences, 8(2), 136-140. 
Sullivan, S., Wills, A., Lawlor, D., McGrath, J., & Zammit, S. (2013). Prenatal 
vitamin D status and risk of psychotic experiences at age 18 years—a 
longitudinal birth cohort. Schizophrenia research, 148(1-3), 87-92. 
Tiwari, P., Panik, R., Bhattacharya, A., Ahirwar, D., & Chandy, A. (2012). Evidences 
of possible side effects of neuroleptic drugs: A systematic review. Asian 
Pacific Journal of Reproduction, 1(4), 330-336. 
Wang X, and Seed B., 2003. A PCR primer bank for quantitative gene expression 
analysis. Nucleic. Acid. Res.,31(21):1-8. 
 
Wrzosek, M., Łukaszkiewicz, J., Wrzosek, M., Jakubczyk, A., Matsumoto, H., 
Piątkiewicz, P., ... & Nowicka, G. (2013). Vitamin D and the central nervous 
system. Pharmacological Reports, 65(2), 271-278.