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\u3c0.01). Odds ratio for the association of hyperhomocysteinemia (\u3e15 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\u3c0.001], 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

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 \u3c0.05), while the concentrations of vitamin B12 and HDL-cholesterol were significantly lower in AMI patients compared to controls (p value \u3c0.01). 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 \u3c0.001]. 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 \u3c0.01). Conclusions/Significance This study shows a positive association between total body iron status and risk of premature AMI 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 microg/dL (8.29-13.60). Prevalence of high blood Pb (levels\u3e10 microg/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 micromol/L vs 13.28+/-9.7micromol/L/13.15 (10.33-17.81) micromol/L vs 11.09 (8.65 14.31) micromol/L (p valu

Polymorphisms in MTHFR, MS and CBS Genes and Homocysteine Levels in a Pakistani Population

Background: Hyperhomocysteinemia (\u3e15 mol/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-beta-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 [beta(SE beta), 2.01(0.63) and 16.19(1.8) mol/L, respectively]. Contrary to MTHFR C677T polymorphism, the average changes in plasma homocysteine levels for MS 2756AG and GG variants were negative [beta(SE beta), -0.56(0.58) and -0.83(0.99) mol/L, respectively]. The average change occurring for CBS 844ins68 heterozygous genotype (ancestral/insertion) was -1.88(0.81) mol/L. The combined effect of MTHFR C677T, MS A2756G and CBS 844ins68 genotypes for plasma homocysteine levels was additive (p valu

High Plasma Homocysteine Increases Risk of Metabolic Syndrome in 6 to 8 Year Old Children in Rural Nepal

Little attention has been given to the association of plasma homocysteine (Hcy) and metabolic syndrome (MetS) in children. We have evaluated the risk of MetS with plasma Hcy in a cohort of 6 to 8 year old rural Nepalese children, born to mothers who had participated in an antenatal micronutrient supplementation trial. We assessed Hcy in plasma from a random selection of n = 1000 children and determined the relationship of elevated Hcy (>12.0 μmol/L) to MetS (defined as the presence of any three of the following: abdominal adiposity (waist circumference ≥ 85th percentile of the study population), high plasma glucose (≥85th percentile), high systolic or diastolic blood pressure (≥90th percentile of reference population), triglyceride ≥ 1.7 mmol/L and high density lipoprotein < 0.9 mmol/L.) and its components. There was an increased risk of low high-density lipoproteins (HDL), [odds ratios (OR) = 1.77, 95% confidence intervals (CI) = 1.08–2.88; p = 0.020], high blood pressure [OR = 1.60, 95% CI = 1.10–2.46; p = 0.015] and high body mass index (BMI) [OR = 1.98, 95% CI = 1.33–2.96; p = 0.001] with elevated Hcy. We observed an increased risk of MetS (OR = 1.75, 95% CI = 1.06–2.90; p = 0.029) with elevated Hcy in age and gender-adjusted logistic regression models. High plasma Hcy is associated with increased risk of MetS and may have implications for chronic disease later in life

Nutritional environmental and genetic causes of hyperhomocysteinemia in a Pakistan population

Introduction: Hyperhomocysteinemia have been found to be associated with cardiovascular disease. The objectives of this study were to find out the prevalence of hyperhomocysteinemia and deficiencies of folate, vitamin B6 and vitamin B12 in a population in Karachi. We also identified major dietary patterns and investigated their association with plasma homocysteine and assessed the relationship of blood lead (Pb) to the risk of hyperhomocysteinemia in this population. Study objectives also included: the determination of the prevalence of C677T and A1298C mutations in MTHFR gene, A2756G mutation in MS gene, and T833C/844ins68 and G919A mutations in CBS gene; investigation of any relationship between these mutations (and their haplotypes) and hyperhomocysteinemia in the study population; study of gene-gene, gene-nutrient and gene-environment interactions with hyperhomocysteinemia; and determination of the impact of vitamin(s) supplementation on plasma homocysteine levels in vitamin-deficient individuals. Methodology: 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 11111 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. DNA was isolated and single nucleotide polymorphisms of the genes (MTHFR, MS and 1111 CBS) were studied by polymerase chain reaction followed by restriction fragment length 111110 polymorphism assay. Dietary intake was assessed by a food frequency questionnaire and factor analysis was used to define major dietary patterns. Results: In the study population, prevalence of hyperhomocysteinemia (\u3e 15pmo1/1) was 32%. Similarly, percent folate (\u3c 3.5 ng/ml), vitamin B12 (\u3c 200 pg/ml) and vitamin B6 (\u3c 20 nmol/1) deficiencies in this population were found to be 27.5%, 9.74%, and 33.7%, respectively. Hyperhomocysteinemia was associated with male sex, folate deficiency and 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. Three major dietary patterns were identified and labeled as prudent diet , high animal-protein diet and high plant-protein diet . We observed protective effect of a prudent dietary pattern for the highest quartile of intake in comparison with the lowest quartile on hyperhomocysteinemia when the model was adjusted for age, gender, household income, body mass index (BMI), tobacco chewing and smoking; OR = 0.52 (95% CI = 0.30 - 0.90). The high plant-protein diet pattern was inversely related to hyperhomocysteinemia with a higher intake being protective. Compared with the first quartile, the adjusted OR was 0.42 (95% CI = 0.25 — 0.69) for the fourth quartile. The high animal-protein diet showed a positive association with hyperhomocysteinemia with participants in the highest quartile of intake having the highest increase in risk; OR = 2.10 (95% CI = 1.22 — 3.60). The study population had mean ± SD blood Pb of 11.65 ± 5.5 pg/d1. Prevalence of high blood Pb (levels \u3e 10 pg/d1) was higher in males compared to females (62.5% males vs 56% females; p value =0.05). Mean ± SD value of plasma homocysteine was significantly higher in the highest quartile of blood Pb compared to the lowest quartile (16.13 ± 11.2 pmo1/1 vs 13.28 ± 9.7,umo1/1; p value \u3c 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. The frequencies of MTHFR 677 and MTHFR 1298 mutant alleles (677T and 1298C) were found to be 0.15 and 0.55, respectively. The prevalence of heterozygote AG individuals of MS (A2756G) was 38.8% with mutant allele G frequency of 0.29. The mutant allele I frequency of CBS 844ins68 insertion was 0.07 with heterozygous WI insertion prevalence to be. 13.2%. CBS T833C polymorphism was present in all alleles carrying the CBS 844ins68 insertion. Genotyping for CBS G919A mutation showed that all the individuals had GG genotype. The mean homocysteine concentration was significantly higher in individuals who were either heterozygous or homozygous variant for MTHFR C677T mutation as compared to the wild type CC genotype (p=0.04 and p\u3c0.001, respectively). It has also been observed that serum folate levels were significantly less in MTHFR TT genotype with respect to other genotypes of MTHFR 677 (p=0.004). Compared to the wild type MTHFR 677 genotype CC , the odds of having hyperhomocysteinemia with homozygous TT variant was 10.17 (p\u3c 0.001) after adjustment for age, gender, folate and vitamin B 12 levels. We also observed protective effect towards hyperhomocysteinemia with heterozygous genotype of CBS 833ins64 when compared to wild type CBS 833ins64 [OR (95% CI); 0.58 (0.34-0.99)]. Regarding influence of gene-gene interaction on plasma homocysteine, mean plasma levels in individuals who were carriers of variants of CBS and MS genes (CBS 844ins68 WI; MS 2756 AG) were lower compared to those who were carriers of either variant (CBS 844ins68 WW, MS 2756 AG or CBS844ins68 WI, MS 2756 AA). We have also observed that individuals having MTHFR TT variant along with MTHFR 1298 AC variant had 8.78 times increased risk of hyperhomocysteinemia. MTHFR TT variant along with MS 2756 AA variant increased the risk for hyperhomocysteinemia by 19.1 fold, while MTHFR TT variant along with CBS 833ins64 WW variant had 14 times higher risk for hyperhomocysteinemia. Regarding geneenvironment interaction, the risk also increased significantly with combinations of MTHFR C677T variants (CT or TT) with folate and vitamin B12 deficiency states (OR 4.84 and 4.33, respectively). Individuals who had either heterozygous CT or homozygous TT mutant genotype of MTHFR 677 and also had blood Pb levels greater than 10 ug/dl, have increased risk for hyperhomocysteinemia [OR (95% CI) 2.4 (1.43- 4.24); p=0.001] compared with those having CC genotype of MTHFR 677 and blood Pb levels less than or equals to 10 ug/dl. A three-week supplementation with folic acid (5 mg/day), methycobalamin (0.5 mg/day) and pyridoxine hydrochloride (50 mg/day) in vitamin-deficient subjects (n=194) decreased plasma homocysteine levels by 37%. Conclusions: High prevalence of folate, vitamin B12, and vitamin B6 deficiencies appears to be the major determinant of hyperhomocysteinemia in a general population in Karachi. A diet rich in fruits and uncooked-vegetables decreased the risk of hyperhomocysteinemia, while diets rich in red meat, chicken and tea with milk were found to be positively associated with hyperhomocysteinemia. This study also showed a relationship between blood Pb and hyperhomocysteinemia suggesting that the harmful effect of Pb on cardiovascular system could be due to its association with hyperhomocysteinemia. MTHFR 677 TT genotypes is strongly associated with hyperhomocysteinemia in study population. Current data suggest that environmental factors (exposure to Pb), nutritional factors (diet and B vitamin status) and genetic factors (especially MTHFR 677 TT genotype) are main determinants of the mild hyperhomocysteinemia observed in study population. The overall contribution of blood Pb, folate and vitamin B12 status, male gender and genetic polymorphisms in 3 genes (MTHFR, MS, CBS) towards plasma homocysteine in this population turns out to be 30%. Since hyperhomocysteinemia is a pathogenic risk factor for atherosclerosis, B vitamin supplementation and increased consumption of high plant protein diet and fruit juices would be of extreme importance in reducing the risk of cardiovascular disease which is highly prevalent in Pakistani population