Mohammad Perwaiz Iqbal

Polymorphisms in MTHFR, MS and CBS genes and homocysteine levels in a Pakistani population.

Background Hyperhomocysteinemia (>15 µmol/L) is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3 enzymes - methylenetetrahydrofolate reductase (MTHFR; C677T; A1298C), methionine synthase (MS; A2756G), cystathionine-β-synthase (CBS; T833C/844ins68, G919A) involved in homocysteine metabolism and investigated their interactions with nutritional and environmental factors in a Pakistani population. Methodology/Principal Findings In a cross-sectional survey, 872 healthy adults (355 males and 517 females; age 18–60 years) were recruited from a low-income urban population in Karachi. Fasting venous blood was obtained and assessed for plasma/serum homocysteine; folate, vitamin B12, pyridoxal phosphate and blood lead. DNA was isolated and genotyping was performed by PCR-RFLP (restriction-fragment-length- polymorphism) based assays. The average changes in homocysteine levels for MTHFR 677CT and TT genotypes were positive [β(SE β), 2.01(0.63) and 16.19(1.8) µmol/L, respectively]. Contrary to MTHFR C677T polymorphism, the average changes in plasma homocysteine levels for MS 2756AG and GG variants were negative [β(SE β), −0.56(0.58) and −0.83(0.99) µmol/L, respectively]. The average change occurring for CBS 844ins68 heterozygous genotype (ancestral/insertion) was −1.88(0.81) µmol/L. The combined effect of MTHFR C677T, MS A2756G and CBS 844ins68 genotypes for plasma homocysteine levels was additive (p value <0.001). Odds of having hyperhomocysteinemia with MTHFR 677TT genotype was 10-fold compared to MTHFR 677CC genotype [OR (95%CI); 10.17(3.6–28.67)]. Protective effect towards hyperhomocysteinemia was observed with heterozygous (ancestral/insertion) genotype of CBS 844ins68 compared to homozygous ancestral type [OR (95% CI); 0.58 (0.34–0.99)]. Individuals with MTHFR 677CT or TT genotypes were at a greater risk of hyperhomocysteinemia in folate and vitamin B12 deficiencies and high blood lead (p value <0.05) level. Conclusions Gene polymorphism (especially MTHFR C677T transition), folate and vitamin B12 deficiencies, male gender and high blood lead level appear to be contributing towards the development of hyperhomocysteinemia in a Pakistani population.

Association of Blood Lead (Pb) and Plasma Homocysteine: A Cross Sectional Survey in Karachi, Pakistan

Background High blood lead (Pb) and hyperhomocysteinemia have been found to be associated with cardiovascular disease (CVD). Mean blood Pb and mean plasma homocysteine levels have been reported to be high in Pakistani population. The objective of the present study was to assess the relationship of blood Pb to the risk of hyperhomocysteinemia in a low income urban population of Karachi, Pakistan. Methodology/Principal Findings In a cross sectional survey, 872 healthy adults (355 males, 517 females; age 18–60 years) were recruited from a low income urban population of Karachi. Fasting venous blood was obtained and assessed for blood Pb and plasma/serum homocysteine, folate, pyridoxal phosphate (PLP, a coenzymic form of vitamin B6) and vitamin B12. The study population had median (IQR) blood Pb of 10.82 µg/dL (8.29–13.60). Prevalence of high blood Pb (levels >10 µg/dL) was higher in males compared to females (62.5% males vs 56% females; p value = 0.05). Mean ± SD/median (IQR) value of plasma homocysteine was significantly higher in the highest quartile of blood Pb compared to the lowest quartile 16.13±11.2 µmol/L vs 13.28±9.7µmol/L/13.15 (10.33–17.81) µmol/L vs 11.09 (8.65 14.31) µmol/L (p value<0.001). Daily consumption of fruit juice had a positive influence on both levels of plasma homocysteine and blood Pb. Compared with the lowest quartile of blood Pb, the OR for hyperhomocysteinemia was 1.69 (95% CI, 1.00 to 2.85) for the fourth quartile when the model was adjusted for age, gender, folate and vitamin B12. Conclusions/Significance This study showed a relationship between blood Pb and hyperhomocysteinemia in a general population of Karachi, Pakistan. The harmful effect of Pb on cardiovascular system could be due to its association with hyperhomocysteinemia.

Trans fatty acids - A risk factor for cardiovascular disease

Trans fatty acids (TFA) are produced either by hydrogenation of unsaturated oils or by biohydrogenation in the stomach of ruminant animals. Vanaspati ghee and margarine have high contents of TFA. A number of studies have shown an association of TFA consumption and increased risk of cardiovascular disease (CVD). This increased risk is because TFA increase the ratio of LDL cholesterol to HDL cholesterol. Food and Agriculture Organization of the United Nations and World Health Organization have come up with the recommendation that the contents of TFA in human dietary fat should be reduced to less than 4%. There is high prevalence of CVD in Pakistan. High consumption of vanaspati ghee which contains 14.2-34.3% of TFA could be one of the factors for this increased burden of CVD in Pakistan. Consumption of dietary fat low in TFA would be helpful in reducing the risk of CVD in South Asia. Denmark by banning the sale of food items with TFA has brought down the number of deaths due to coronary heart disease by nearly 50% over a period of 20 years. Public awareness about the adverse effects of TFA on human health would be extremely important. Media can play a very effective role in educating the masses and advocating the policy for the sale of only low TFA food items. Literature sources: Google and US National Library of Medicine, National Institute of Health were the sources of papers cited in this review article.

Relationship between vitamin B12, folate and homocysteine levels and H. Pylori infection in patients with functional dyspepsia: A cross-section study

BackgroundH. pylori infection has been associated with many micronutrient deficiencies. There is a dearth of data from communities with nutritional deficiencies and high prevalence of H. pylori infection. The aim of this study was to determine the impact of H. pylori infection on serum levels of vitamin B12, folate and homocysteine in patients with functional dyspepsia (FD).MethodsOne hundred and thirty-two patients with FD undergoing gastroscopy were enrolled. The serum was analyzed for B12, folate and homocysteine levels before gastroscopy. H. pylori infection was diagnosed by histopathological examination of gastric biopsies and urea breath test. An independent sample t-test and the Mann–Whitney test were used to compare mean serum concentrations of biomarkers between H. pylori- positive and H. pylori- negative groups of patients. A Chi-square test was performed to assess the differences among proportions, while Spearman’s rho was used for correlation analysis between levels of B12 and homocysteine.ResultsThe mean age of the group was 40.3 ± 11.5 (19–72) years. Folate deficiency was seen in 43 (34.6%), B12 deficiency in 30 (23.1%) and hyperhomocysteinemia in 60 (46.2%) patients. H. pylori was present in 80 (61.5%) patients with FD while it was absent in 50 (38.5%). Mean serum levels of B12, folate and homocysteine in the H. pylori- positive group of patients were not significantly different from the levels in the H. pylori- negative group (357 ± 170 vs. 313 ± 136 pg/mL; p = 0.13), (4.35 ± 1.89 vs. 4.42 ± 1.93 ng/mL; p = 0.84); (15.88 ± 8.97 vs. 16.62 ± 7.82 μmol/L; p = 0.24); respectively.B12 deficiency (≤200 pg/mL) was 23.8% in the H. pylori- positive patients versus 22.0% in the H. pylori- negative patients. Folate deficiency (≤3.5 ng/mL) was 33.8% in the H. pylori- positive group versus 36% in the H. pylori- negative group. Hyperhomocysteinemia (>15 μmol/L) was present in 46.2% of H. pylori- positive patients compared to 44% in the H. pylori- negative group. Correlation analysis indicated that serum B12 levels were inversely associated with serum levels of homocysteine in patients with FD (rho = −0.192; p = 0.028).ConclusionsThis study demonstrated an inverse relationship between serum levels of B12 and homocysteine in patients with FD. Moreover, no impact of the presence of H. pylori was found on B12, folate and homocysteine levels in such patients.

Association of Body Iron Status with the Risk of Premature Acute Myocardial Infarction in a Pakistani Population

Background Coronary artery disease is very common in Pakistani population. Some of the studies carried out on Western populations have shown a relationship between body iron status as determined by the ratio of concentrations of serum soluble transferrin receptor (sTfR) to ferritin and the risk of acute myocardial infarction (AMI). In order to investigate whether increased body iron status has any relationship with the risk of premature AMI in Pakistani population, a case-control study was carried out. Methodology/Principal findings In this case-control study, 203 consecutive AMI patients [146 males and 57 females; age range 18–45 years] admitted to the National Institute for Cardiovascular Diseases, Karachi, were enrolled with informed consent. In addition, 205 healthy controls whose gender and age (within 3 years) matched the patients, and who had a similar socio-economic background were recruited. Fasting venous blood was obtained and assessed for plasma/serum folate, vitamin B12, homocysteine, total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, sTfR and ferritin and blood lead. It was found that serum concentration of ferritin and blood lead levels were significantly higher in AMI patients compared to their age and gender-matched healthy controls (p value <0.05), while the concentrations of vitamin B12 and HDL-cholesterol were significantly lower in AMI patients compared to controls (p value <0.01). The ratio of sTfR to ferritin was significantly lower in AMI patients compared to controls [mean±SD/median (IQR) values 84.7±295/28.9 (38.4) vs 255±836/49.4 (83.8), respectively; p value <0.001]. Compared with the highest quartile of sTfR/ferritin (low body iron status), the OR for the risk of AMI was 3.29(95% CI, 1.54–7.03) for the lowest quartile (quartile 1) when the model was adjusted for vitamin B12 and HDL-cholesterol (p value for trend <0.01). Conclusions/Significance This study shows a positive association between total body iron status and risk of premature AMI in a Pakistani population.

Effect of methotrexate and folinic acid on skeletal growth in mice.

Aim: To investigate whether chronic administration of medium doses of methotrexate (MTX) causes suppression of skeletal growth in young mice and to determine whether folinic acid supplementation could reverse this effect. Methods: Four equal groups of Balb/c young male mice (6 animals in each group; mean body weight 11.9 ± 0.25g, in their rapid growth phase) were subjected to the following drug treatment for a period of 3 wk. Group 1 was given intraperitoneal MTX (3.5 mg kg‐1 body weight) every second day. Group 2 received folinic acid (7.0 mg kg‐1 body weight) intraperitoneally every second day. Group 3 was given both drugs (MTX every second day and folinic acid 8 h post‐MTX injection). Group 4 was injected with physiological saline every other day to serve as a control group. Total body weight of the animals in each group was monitored every second day for the entire study period. The animals were sacrificed, the bilateral femurs and tibias of each animal were harvested and X‐rays of the bones were taken. The length of each femur and tibia was measured using a micrometer. Measurements from the radiographs were also recorded using image analysis software. The MTX concentrations in the plasma and the folate levels in erythrocytes were determined. The heights of the distal femoral and the proximal tibial growth plate for each animal were measured on histological tissue sections. Results: Mean lengths of both the tibia and femur of animals were compared in the four treatment groups. A significant decrease in the mean lengths (one‐way ANOVA, p < 0.005) was observed in the group receiving MTX alone. Similarly, there was a significant decrease (p < 0.001) in the height of the femoral and tibial growth plate in this group when compared with the other groups. The main effect of MTX seemed to be on the hypertrophic proliferative zone of chondrocytes in the growth plate. Furthermore, animals in this MTX‐treated group also showed increased levels of MTX in plasma and low levels of erythrocyte folate.

Association of alkaline phosphatase with acute myocardial infarction in a population with high prevalence of hypovitaminosis D.

BACKGROUND Since Pakistanis have high prevalence of hypovitaminosis-D as well as acute myocardial infarction (AMI), the objective of the study was to investigate the relationship between vitamin-D deficiency and risk of AMI in a hospital-based population and to identify major risk factors for this disease. METHODS Fasting serum samples from 66 consecutive AMI patients [age 30-70 y] and 132 gender and age-matched (within 5 y) healthy controls were analyzed for concentrations of glucose, total-cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, calcium, inorganic phosphate, alkaline phosphatase (ALP), bone-ALP, parathyroid hormone (PTH), 25(OH) vitamin-D (25(OH)D) and alanine aminotransferase. RESULTS Mean concentrations of serum 25(OH)D, PTH, total-ALP, bone-ALP, LDL-cholesterol, HDL-cholesterol and glucose were significantly different compared to healthy controls (p<0.05). Percent vitamin-D deficiency/insufficiency (levels<30 ng/ml) was significantly greater in AMI patients compared to controls (93.9% vs.75.8%; p=0.001). Multiple conditional logistic regression analysis revealed that increased levels of 25(OH)D were associated with decreased risk of AMI [MAOR (95% CI)=0.821 (0.718, 0.940); p=0.004]. Hypertension and smoking were positively associated with AMI. CONCLUSIONS Increased vitamin-D levels were associated with decreased risk of AMI, while serum glucose, bone-ALP, hypertension and smoking were positively associated with it. Association of bone-ALP with AMI in hypovitaminosis-D is a novel finding of this study.

Vitamin E Supplementation Enhances Hemoglobin and Erythropoietin Levels in Mildly Anemic Adults

adults. A cohort of 60 apparently healthy adult volunteers, aged 18–45 years, with no known history of any major systemic illness during the previous 2 years, were randomly selected from general practitioners’ clinics at various localities in Karachi. All subjects met the criterion for borderline levels of hemoglobin [13] previously established by the Aga Khan University Clinical Laboratory (11.6–13.5 g/dl in males and 9.0–10.9 g/dl in females). Pregnant females, subjects using vitamins and/or iron supplements or EPO for the previous 6 months, and those having a history of tobacco smoking or iron deficiency were excluded. Those who had received blood transfusion during the past 6 months were also excluded. None of the subjects included in this study was found to be dehydrated. The study was approved by the Aga Khan University Ethics Committee. Age-, sexand body mass index-matched subjects were placed in 1 of 4 groups. Group A (control male) and group C (control female), comprising 15 subjects each, were given a placebo (olive oil) capsule once daily for a period of 3 months. Group B (test male) and group D (test female), comprising 15 subjects each, orally took a capsule of -tocopherol 400 mg (Evion; Merck Pharmaceuticals, Pakistan) every day for 3 months. Pre-and postsupplemental blood hemoglobin concentrations and serum levels of EPO, vitamin E and iron in each subject were determined using chemical analysis and kit methods. Paired sample t test was used Increased synthesis of erythropoietin (EPO) occurs mainly in response to relative tissue hypoxia [1] . However, studies in the last few years have suggested many biological factors other than hypoxia, such as thyroid hormone, androgenic steroids, insulin, catalase, progesterone and angiotensin II, which could influence the synthesis and/or secretion of EPO in humans [2–7] . It has been shown in vitro that a combination of antioxidant vitamins (A, C and E) provided to isolated, serum-free, hypoxically perfused rat kidneys significantly increased EPO production [8] . Studies of vitamin E supplementation in hemodialysis patients and children have shown enhanced erythropoiesis and improved levels of hemoglobin [9, 10] . Vitamin E, in combination with recombinant human EPO, reduced EPO dosage requirements and enhanced multiplication of erythroid precursor cells [9, 11] . Conflicting results have been reported in children with anemia of prematurity [12] , however, a possible role for vitamin E as a human erythropoietic agent in adults with mild anemia has never been reported. Apparently healthy, but mildly anemic, adults with borderline hemoglobin levels are common in the general population of Pakistan. Therefore, we carried out a casecontrol, randomized, prospective, single blind study in Karachi from June 2006 to April 2007. The goal was to study the effect of vitamin E supplementation on serum EPO and blood hemoglobin levels in mildly anemic Received: September 18, 2007 Accepted after revision: December 3, 2007 Published online: February 6, 2008

Smokeless tobacco use: a risk factor for hyperhomocysteinemia in a Pakistani population.

Background Smokeless tobacco (ST) use is highly prevalent in the South Asian populations. While there have been a number of reports on association of ST consumption with cancer, very few studies have been conducted to investigate its relationship with cardiovascular disease. Hyperhomocysteinemia is a well-recognized risk factor for cardiovascular disease; however, its association with ST use has never been investigated. The objective of this study was to evaluate the relationship of ST use with hyperhomocysteinemia in an urban Pakistani population. Methodology/Principal Findings In a cross-sectional study for assessment of risks of hyperhomocysteinemia, 872 healthy adults (355 males and 517 females of age range 18–60 years) were recruited from a low-income population in Karachi, Pakistan. A detailed questionnaire was administered which included information about smoking, non-smoking, use of ST alone (chewing as well as sniffing) and use of ST with betel nuts. Fasting serum/plasma levels of homocysteine, folate, vitamin B12 and pyridoxal phosphate (PLP; a coenzymic form of vitamin B6) were analyzed. In this population, 43.4% males and 15.5% females were found to be regular users of ST products. Laborers and vendors were the major ST consumers. Smoking was not found to be associated with plasma/serum concentrations of homocysteine, folate, vitamin B12 and PLP. However, homocysteine concentrations in the group which consumed ST alone and the group which consumed ST along with betel nut were significantly higher compared to the non-user group (17.7±7.5 µmol/L, 25.48 µmol/L vs. 11.95 µmol/L, respectively; p<0.01). Odds ratio for the association of hyperhomocysteinemia (>15 µmol/L) was 11-fold higher in the ST-consumer group compared to the non-user group, [OR (95%CI)  = 11.34 (7.58–16.96); p<0.001], when the model was adjusted for age, gender, folate and vitamin B12 status. Conclusion This study shows a positive association between ST consumption and hyperhomocysteinemia in a low-income urban Pakistani population.

Effects of betel nut on cardiovascular risk factors in a rat model

BackgroundAreca nut (commonly known as betel nut) chewing has been shown to be associated with metabolic syndrome and cardiovascular disease (CVD). The mechanism by which betel nut ingestion could lead to development of CVD is not precisely known; however, dyslipidemia, hyperhomocysteinemia, hypertriglyceridemia and inflammation could be some of the potential risk factors. This study was undertaken to investigate the effects of two dosages of betel nut on homocysteinemia, inflammation and some of the components of metabolic syndrome, such as hypertriglyceridemia, low HDL-cholesterol, obesity and fasting hyperglycemia in a rat model.MethodsThirty-six adult female Sprague Dawley rats, aged 10–12 weeks were divided into three equal groups. Group-1 served as the control group (n = 12) and received water, whereas groups 2 and 3 were given water suspension of betel nut orally in two dosages, 30 mg and 60 mg, respectively for a period of 5 weeks. At the end of the fifth week, the animals were weighed and sacrificed, blood was collected and liver, kidney, spleen and stomach were removed for histological examination.Plasma/serum was analyzed for glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, homocysteine, folate, vitamin B12 and N-acetyl-β-D-glucosaminidase (NAG) – a marker of inflammation.ResultsWhen the mean concentration values of 3 groups were compared using one way ANOVA followed by Tukey’s HSD-test, there was a significant increase in the concentration of total cholesterol (p = 0.04) in the group receiving 30 mg/day betel nut compared to the control group. However, administration of a higher dose of betel nut (60 mg/day) had no significant effect on the serum concentrations of glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, and NAG. Histological examination of spleen revealed a dose-dependent extramedullary hematopoiesis. No other remarkable change in the tissues (liver, kidney and stomach) was observed.Mean serum/plasma levels of folate, vitamin B12 and homocysteine were not found to be significantly different in all the groups. Betel nut ingestion had no effect on the mean body weights of rats.ConclusionsLow dosage of betel nut is found to be associated with hypercholesterolemia. However, betel nut ingestion is not associated with hyperhomocysteinemia, hypertriglyceridemia, hyperglycemia, inflammation and increase in body weight in a rat model.