Relationship of sociodemographic factors with serum levels of vitamin D in a healthy population of Pakistan

Background: High prevalence of vitamin D deficiency has been reported from Pakistan. Association of sociodemographic factors with vitamin D status has received little attention in this region. Therefore, we embarked on investigating the relationship of sociodemographic factors with vitamin D levels in a healthy Pakistani population. Venous blood from 226 healthy participants [age range 19-69 years] was collected and analyzed for serum concentrations of 25[OH] vitamin D [25[OH]D] and other related biomarkers. Demographic characteristics of the study participants were collected. Vitamin D deficiency [25[OH]D levels less than 20 ng/ml] was found to be 75% in this cohort. Gender, sunlight exposure and monthly household income emerged as predictors of hypovitaminosis D. Mean serum 25[OH]D levels in the groups with monthly household income less than Pakistani Rupees [PKR] 20,000, between PKR 20,000-50,000 and above PKR 50,000 were found to be 11.0+/-7.5, 13.9+/-9.6 and16.9+/-11.7 ng/ml, respectively. Using logistic regression the odds of having vitamin D deficiency was 3.22 [95% CI, 1.65-6.28] in the group with household income less than PKR 50,000 per month compared to the group with household income more than PKR 50,000 per month when the model was adjusted for gender and exposure to sunlight. There is an association between household income and hypovitaminosis D in a healthy Pakistani population

Lack of association of statin use with vitamin D levels in a hospital based population of type 2 diabetes mellitus patients

Objective: To investigate the relationship of statins [drug given to reduce serum levels of LDL-cholesterol] on vitamin D levels of Pakistani type 2 diabetes mellitus [DM] patients in a hospital in Karachi

Methods: In a cross-sectional survey, 312 consecutive patients with type 2 DM [219 males and 93 females, age 22-70 years] were recruited with informed consent. A questionnaire was administered to find out whether they were statin users or non-users. Serum was analyzed for concentrations of 25[OH] vitamin D [25[OH]D] and other related biomarkers such as serum cholesterol, triglycerides, HDL-cholesterol, LDLcholesterol, phosphate and calcium using kit methods. Multiple Linear Regression was used to evaluate association of statin use with serum levels of vitamin D while adjusting for related covariates including duration of statin use, duration of type 2 DM and smoking

Results: Mean concentrations of serum cholesterol, and LDL-cholesterol were lower among statin users compared to statin non-users [P < 0.01], while HDL-cholesterol levels were higher [P<0.01]. No relationship was observed between statin use and serum levels of vitamin D [P=0.768], when adjusted for age, gender, BMI, duration of type 2 DM, smoking, serum cholesterol and LDL-cholesterol. The adjusted regression coefficient [beta] and standard error [SE[beta]] for statin use duration were 0.012 [0.042], when serum levels of vitamin D was taken as an outcome

Conclusion: Lack of association was found between statin use and vitamin D levels in a hospital-based population of Pakistani patients with type 2 DM

Association of Vitamin D deficiency and VDBP gene polymorphism with the risk of AMI in a Pakistani population

Objective: To investigate the relationship of vitamin D deficiency and risk of AMI in a Pakistani population, and to find out any association between vitamin D binding protein [VDBP] genotypes and risk of AMI in this population

Methods: In a comparative cross-sectional study, 246 patients [age: 20-70 years; 171 males and 75 females] with first AMI were enrolled with informed consent. Similarly, 345 healthy adults [230 males and 115 females] were enrolled as controls. Their fasting serum samples were analyzed for 25 [OH] vitamin D, lipids and other biomarkers using kit methods, while DNA was analyzed for VDBP genotypes using PCR-RFLP based methods. Chi-squared test and logistic regression were used for association of vitamin D deficiency and VDBP genotypes with AMI

Results: Mean serum concentration of 25[OH] vitamin D was significantly lower in AMI patients compared to healthy subjects [p=0.015] and percent vitamin D deficiency was higher in AMI patients compared to healthy subjects [p=0.003]. VDBP IF-IF genotype was positively associated with the risk of AMI in subject above 45 years after adjusting for potential confounders [OR = 9.86; 95% CI=1.16 to 83.43]

Conclusion: Vitamin D deficiency and VDBP IF-IF genotype are associated with AMI in Pakistani adults

Polymorphisms in MTHFR, MS and CBS genes and premature acute myocardial infarction in a Pakistani population

High prevalence of premature coronary heart disease in Pakistanis compared to other populations points towards the genetic predisposition of this population to develop this disease. Since no investigations have been carried out in Pakistan to study the relationship of polymorphisms in genes involved in homocysteine cycle, the objective of the present study was to find out if there is any association of methylenetetrahydrofolate reductase [MTHFR] C677T, A1298C; methionine synthase [MS] A2756G; cystathionine-beta-synthase [CBS] 844ins68, G919A polymorphisms with premature acute myocardial infarction [AMI] in a population of Pakistani patients with this disease. In a cross-sectional study, DNA samples of 143 AMI patients [age <45 years] and 153 healthy controls were genotyped for the above mentioned polymorphisms using PCR-RFLP methods. Plasma/serum samples of both patients and healthy controls were screened for homocysteine, folate and vitamin B12. One way ANOVA and chi-squared test were used for analysis of data. Mean plasma homocysteine levels in premature AMI patients and healthy controls were found to be 23 +/- 17.2 and 23 +/- 13.4 micro mol/l, respectively which are higher than the upper normal limit of this biomarker [15micro mol/l]. MTHFR 677 CT genotype in healthy controls and MTHFR 677 TT genotype in AMI patients were found to have significantly increased levels of plasma homocysteine [p value <0.05], while all other polymorphisms did not show any significant difference in mean levels of homocysteine between AMI patients and healthy controls. Moreover, no association was observed between MTHFR C677T, A1298C; MS A2756C; CBS844ins68 polymorphisms and premature AMI in this population. This indicates that common polymorphisms in MTHFR, MS and CBS genes have no role in premature AMI in Pakistani population

Lack of association between MTHFR gene polymorphisms and response to methotrexate treatment in Pakistani patients with rheumatoid arthritis

Methylenetetrahydrofolate reductase [MTHFR] gene polymorphisms have been reported to be associated with response to methotrexate [MTX] in certain populations of patients with rheumatoid arthritis [RA]. This study aims at investigating any relationship of two single nucleotide polymorphisms [SNPs] in MTHFR gene, C677T and A1298C with response to therapy with MTX in Pakistani RA patients. Allelic frequencies of the two polymorphisms [C677T and A1298C] were determined in 67 RA patients [9 males and 58 females; mean age 42.87 +/- 13.5 years] who had previously participated in a prospective clinical trial. Fifty-one patients had received MTX and were followed up for response up to 6 months. Genotyping of the two MTHFR polymorphisms was carried out using PCR-RFLP, while fasting concentration of plasma homocysteine was determined using a kit method. Twenty-eight patients were found to be "good responders", while twenty-three were [poor responders]. MTHFR 1298C and MTHFR 677T alleles' frequencies in [good responders] were not different from frequencies in [poor responders] [0.574 vs. 0.521; p=0.6 and 0.197 vs. 0.196; p=0.75, respectively]. Plasma homocysteine levels in female RA patients were significantly higher compared to general population in Karachi [13.1 +/- 6.7 micromol/1 vs. 11.4 +/- 5.3 micromol/1; p<0.00l]. MTHFR C677T and A1298C polymorphisms are not associated with response to MTX in a population of Pakistani RA patients

High prevalence of mild hyperhomocysteinemia and folate, B12 and B6 deficiencies in an urban population in Karachi, Pakistan

To find out the prevalence of hyperhomocysteinemia, and deficiencies of folate, vitamin B6 and vitamin B12 in an urban population in Karachi, Pakistan. In a pre and post experimental study, eight hundred and seventy-two apparently healthy adults [aged 18-60 years; 355 males and 517 females] were recruited from a low-income urban locality in East of Karachi from February 2006 to March 2007. Fasting venous blood was obtained. Serum was analyzed for folate and vitamin B12. Plasma was analyzed for pyridoxal phosphate [PLP, coenzymic form of B6] and total homocysteine. A group of vitamin-deficient individuals [n=194] was given 3-week supplementation with folic acid [5mg/day], methycobalamin [0.5mg/day] and pyridoxine hydrochloride [vitamin B6, 50 mg/day]. After supplementation, serum/plasma levels of folate, vitamin B12, PLP and homocysteine were again determined. Prevalence of hyperhomocysteinemia [>15micromol/l] was 32%. Similarly percent values of folate deficiency [<3.5ng/ml], vitamin B6 deficiency [PLP<20 nmol/l] and vitamin B12 deficiency [<200pg/ml] in the study population were 27.5%, 33.7% and 9.74%, respectively. Hyperhomocysteinemia was associated with male sex, folate deficiency, vitamin B12 deficiency [OR [95%CI], 8.3[5.7-12.1]; 2.5[1.76-3.58]; 2.6[1.5-4.5], respectively]. A 3-week supplementation with folic acid, methycobalamin and pyridoxine hydrochloride in vitamin-deficient subjects decreased plasma homocysteine levels by 37%. High prevalence estimates of folate, vitamin B12, and vitamin B6 deficiencies appear to be the major determinants of hyperhomocysteinemia in a low income general population in Karachi

Human paraoxonase and HDL-cholesterol in Pakistani patients with acute myocardial infarction and normal healthy adults

Human serum paraoxonase is a high density lipoprotein [HDL]-bound enzyme exhibiting antiatherogenic properties. The aim of this study was to investigate any relationship between serum paraoxonase activity and serum levels of HDL-cholesterol in Pakistani patients with acute myocardial infarction [AMI] compared to normal healthy subjects and to examine possible association between serum paraoxonase activity and AMI in Pakistani population. In a case-control study, serum paraoxonase activity and serum levels of HDL-cholesterol and LDL-cholesterol were monitored in 164 Pakistani patients with AMI and 106 normal healthy adults matched for gender, BMI and age within 10 years. Mean serum concentration of HDL-cholesterol and mean serum paraoxonase activity in AMI patients were not significantly different from the corresponding values in normal healthy subjects. Mean serum paraoxonase activity value was significantly lower in normal healthy subjects with low HDL-cholesterol [serum levels<40mg/dl] compared to the value in those with normal levels of HDL-cholesterol [P=0.04]. In AMI patients, paraoxonase activity was lower in subjects with low HDL-cholesterol compared to those with normal levels of HDL-cholesterol, however, the decrease was not statistically significant. Correlation analyses of the data revealed a moderate association of paraoxonase activity with HDL-cholesterol [Pearson's r=0.225, P<0.01 for AMI patients and r=0.281, P<0.01 for normal healthy controls]. Seventy three percent of normal healthy subjects and 65% of AMI patients in this study had low HDL-cholesterol. Low serum paraoxonase activity and high prevalence of low HDL-cholesterol in Pakistani population could be contributing to the high rates of coronary heart disease in this population

Plaque formation reduction with glutathione monoester in mice fed on atherogenic diet

To determine the role of glutathione monoester on reducing the development of plaque formation in an animal model. Laboratory control trial. Aga Khan University, Karachi, from January 2004 to December, 2004. Twenty-four Balb/c mice were divided into 3 equal groups. First group was fed on atherogenic diet alone, while the second group received atherogenic diet plus twice weekly injections of glutathione monoester. The third group was fed on normal diet for mice. After one year, the animals were sacrificed. Blood was analyzed for lipid levels, while liver, kidney, spleen, heart and aorta were removed to study morphological changes. In the groups of mice receiving atherogenic diet [with and without glutathione monoesters], there was significant increase in levels of total cholesterol [p=0.011] and LDL cholesterol [p=0.001] compared to levels of these lipids in mice on normal diet. However, a significant decrease in levels of triglycerides [p=0.01] was observed in the group receiving atherogenic diet along with glutathione monoester. Supplementation with glutathione monoester had the most pronounced effect only on triglyceride levels. Atherosclerotic plaques were seen in heart and/or aorta of mice receiving atherogenic diet. However, such plaques were either totally absent or if seen in an animal, were extremely small and diffuse in the group receiving glutathione monoester along with atherogenic diet. Mice on normal diet had no evidence of any plaque formation. Cholesterol granuloma was seen in liver of mice on atherogenic diet alone. In mice receiving atherogenic diet plus glutathione monoester, no cholesterol granuloma was found in liver. There were no remarkable morphological changes in spleen and kidney in the three groups of mice. Glutathione monoester appears to inhibit or reduce the development of plaque formation in mice

Ascorbic acid contents of Pakistani fruits and vegetables

Fresh fruits and vegetables are good sources of vitamin C which is known for its antioxidant and immune-enhancing effects. The objective of this study was to determine ascorbic acid [vitamin C] contents of regularly consumed fruits and vegetables available in Pakistani markets. Most commonly used fresh fruits and vegetables were homogenized in 5% trichloroacetic acid, and ascorbic acid contents in the extracts were determined using a spectrophotometric method. Banana, custard apple, orange, lemon, guava and papaya were found to be very rich in ascorbic acid. Among vegetables, capsicum [green sweet pepper], cauliflower, bittergourd, roundgourd, beetroot, spinach, cabbage and radish contained high concentrations of ascorbic acid. Chikoo, grapes, pear, apricot, peach, carrot, cucumber, lettuce and 'kakri' were found to be poor sources of ascorbic acid. Several Pakistani fruits and vegetables [pear, melon, onion, sweet green pepper, spinach, cucumber] had ascorbic acid values similar to those reported by US Department of Agriculture in these fruits and vegetables in USA. However, wide differences in vitamin C contents were also observed in certain other fruits and vegetables from these two countries. This indicates that regional varieties of fruits and vegetables could vary in their ascorbic acid contents. Since subclinical deficiency of vitamin C appears to be quite common in developing countries like Pakistan, there is a need to develop awareness among masses to consume fresh fruits and vegetables with high contents of vitamin C

Variability in lipid profile before and after coronary artery bypass grafting [CCABG]

To investigate changes in total cholesterol, low density lipoprotein [LDL] cholesterol, high density lipoprotein [HDL] cholesterol, and triglycerides in serum of Pakistani patients before, immediately after and 5 days post CABG. Serum samples from 31 consecutive Pakistani angina patients undergoing CABG at the Aga Khan University Hospital were analyzed for total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides using kit methods. Immediately after CABG, there is a significant decline in the mean levels of serum cholesterol, LDL cholesterol, HDL cholesterol and triglycerides. However, 5 days post CABG, there is a significant increase in the concentrations of total cholesterol [P=0.01] and LDL cholesterol [P=0.001] in nondiabetic angina patients [n=13]. Among the diabetic group of patients [n=18], the levels of total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides went back to the pre-operative levels within 5 days post CABG. Compared to European patients, Pakistani patients tend to have very low levels of HDL cholesterol [24.9 +/- 7.1mg/dl] and high levels of triglycerides [185 +/- 50 mg/dl] on day 5 post CABG. Since risk of mortality following CABG increases with low level of HDL cholesterol and high level of triglycerides, close monitoring and treatment of high lipid levels of Pakistani patients following CABG is necessary to prevent further coronary events

Association study of the angiotensin-converting enzyme (ACE) gene G2350A dimorphism with myocardial infarction

The angiotensin converting enzyme (ACE) is a strong candidate gene for myocardial infarction (MI). Insertion-deletion dimorphism in intron 16 of this gene has been inconclusively found to be associated with it. Several new polymorphisms in the ACE gene have been identified and among these, a dimorphism in exon 17, ACE G2350A, has a significant effect on plasma ACE concentrations. To assess the value of genotyping the ACE G2350A dimorphism in a genetically homogeneous population, we carried out a case-control study of dimorphism G2350A for a putative association with MI among Pakistani nationals. We investigated a sample population of 370 Pakistanis, comprising 163 controls, and 207 patients with clinical diagnosis of acute MI (AMI). ACE G2350A alleles were visualized by assays based on polymerase chain reaction and restriction endonuclease analysis. Frequencies of G alleles were 0.68 among controls and 0.72 among AMI patients. The ACE G2350A dimorphism showed no significant association with MI (c2=0.90, 2 df, P=0.64), plasma levels of homocysteine (P=0.52) or with serum levels of folate (P=0.299). The results indicate that ACE G2350A polymorphism is not associated with risk of myocardial infarction in the Pakistani population investigated here.

N-Acetyl-beta-D-glucosaminidase in acute myocardial infarction

The objective of the study was to investigate whether the lysosomal enzyme, N-Acetyl-beta-D-glucosaminidase (NAG) activity is increased in plasma of patients with acute myocardial infarction (AMI) and to determine if there is any association between plasma levels of NAG and severity of myocardial infarction (MI). NAG activity in plasma was monitored in 69 patients with AMI and 135 normal healthy subjects using a spectrofluorimetric method. A modified Aldrich ST elevation score was used to gauge the severity of MI in terms of size of the infarct. Plasma NAG levels in AMI patients and normal healthy subjects were found to be 10.92+/-7.5 U/l and 6.8+/-2.2 U/l, respectively. These two mean value when compared by Student's t-test were significantly different P = 0.0001. No statistically significant differences in NAG activity were observed in patients in terms of gender, age, location of infarct, time from onset of chest pain to blood sampling in the hospital and size of the infarct.

Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: interim analysis

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.