Micronutrient Deficiencies and Anemia in Urban India-Do We Need Food Fortification?

Prevalence of anemia in India is almost 40% with no significant change since 1998-99, whereas globally this prevalence has been reduced to < 15%. This could be because our national nutritional programs (mainly National Nutritional Anemia Control Program-NNACP) focus on supplementation with iron and folate but not with vitamin B12. Some Indian studies, including our study (2012), indicated high prevalence of B12 deficiency in North Indian urban population. Hence, we conducted a retrospective analysis of 3 years' data (2012-2014 including 48,317 subjects) and compared it with last year's retrospective data (April 2019-March 2020 including 4775 subjects) to ascertain prevalence of deficiencies of these micronutrient with special reference to patients of anemia, and improvement therein over the subsequent 5-year period. Our results indicate that amongst our subjects with anemia, iron deficiency has reduced from 66.73% (2012-2014) to 56.86% (2019), but prevalence of vitamin B12 deficiency is still the same (36.54% in 2012-2014; 37.04% in 2019). Folate deficiency was similar in both sets of data (2.95% in 2012-2014 and 2.55% in 2019). Thus, NNACP has reduced prevalence of iron deficiency by ~ 10%points and folate deficiency marginally; B12 deficiency has not been addressed. It would, therefore, follow that we need to add to our current national programs to effectively deal with these deficiencies. Food fortification (with iron, folate and B12) seems the most likely means to add value to the existing programs. In addition, food diversification needs to be included in regular school curriculum to bring about community awareness and change in food habits.

Interpreting HbA1c in Presence of Deficiency Anemias.

HbA1c is used extensively for the diagnosis and management of diabetes mellitus. It constitutes 80% of glycated HbA1(Glycated haemoglobin(GHb)A), and depends upon blood glucose and RBC life span. RBC life span varies with anemia, leading to a consequent alteration in the HbA1c value irrespective of the circulating blood glucose concentration. But to the best of our knowledge no Hb cut offs have been derived for appropriate interpretation of HbA1c. The prevalence of anemia in Indian population is nearly 40% as per its definition by WHO-Hb < 12 g/dL in females and < 13 g/dL in males-with most cases attributable to nutritional deficiencies. Hence, we aimed to identify Hb cut-off for accurate interpretation of HbA1c in presence of deficiency anemias. Partial correlation between random blood glucose (RBG) and HbA1c was studied in 1312 subjects, 470 of whom had deficiency-related anemia]. The data was adjusted for age, sex and Hb. Partial correlation between RBG and HbA1c was highly significant (p < 0.0001) till Hb of 8.1 gm/dL. Significance reduced to p = 0.003 and p = 0.006 as the cut off of Hb reduced to 7.1 gm/dL and 5.0 gm/dL, respectively, but was not lost. Hence, caution in interpretation of HbA1c is not required till an Hb of 5 g/dL.

The Hemolyzed Sample: To Analyse Or Not To Analyse.

Preanalytical errors constitute about 40-65% of laboratory errors, of which 60% are due to hemolysis. This leads to imprecise reporting and misinterpretation of the actual concentration of analytes. Hence the aim of this study was to estimate the extent of different degrees of interference by visible hemolysis. 25 hemolysed samples along with their fresh unhemolysed sample were studied. Hemolyzed serum was mixed with unhemolyzed serum in predefined serial ratios from 100%, 70%, 50%, 30% and 10% to achieve different grades of hemolysis. Each dilution was analysed for BUN, creatinine, uric acid, phosphorus, Na, K, total protein, amylase, lipase, LDH, tacrolimus and methotrexate. Percentage difference of each dilution of the hemolyzed sample as compared to the unhemolyzed sample was calculated and considered acceptable only if less than TEa. It was observed that Percentage difference of BUN, creatinine, amylase and lipase in all dilutions of hemolyzed samples were within TEa while phosphorus, Na, K, total protein and LDH were beyond the acceptance criteria. Hence It was concluded that it may be safe to analyse a hemolysed sample for BUN, creatinine, amylase, lipase, tacrolimus and methotrexate while uric acid may be estimated in a moderately hemolysed sample. Phosphorus, sodium, potassium, total protein and LDH must never be analyzed in any hemolysed sample.

Non-HDL as a Valid Surrogate Marker of Small Dense LDL in a Young Indian Population.

Small dense (sd) LDL is a significant independent risk factor for premature coronary artery disease (CAD). Unfortunately, its estimation is not popular, due to the limited availability of specialized equipment, high cost and time-consuming technique. Non-HDL is a calculated, single index measure of all atherogenic apolipoprotein-B containing lipoproteins. This study aimed at identifying non-HDL as a superior surrogate marker of sdLDL cholesterol in a young Indian population. 161 healthy subjects < 45 years were tested for lipid profile, apolipoproteins A1 and B, and sdLDL particle size. sdLDL particles showed negative correlation with non-HDL (r = - 0.283, p < 0.001), LDL (r = - 0.195, p = 0.013) and apoB/apo A1 (r = - 0.175, p = 0.026), the significance being greatest with non-HDL. ROC showed AUC for non-HDL, LDL and apoB/apo A1 as 0.704, 0.686, and 0.596 respectively. For LDL < 130 mg/dL, sdLDL showed a more significant negative correlation with non-HDL (r = - 0.291, p < 0.001) as compared with apoB/apoA1 (r = - 0.172, p = 0.037). For triglycerides < 200 mg/dL, sdLDL particle size showed higher significant negative correlation with non-HDL (r = - 0.213, p = 0.015) than with LDL (r = - 0.176, p = 0.045) while for triglycerides between 200 and 400 mg/dL, significant negative correlation was observed only with non-HDL (r = - 0.372, p = 0.043). Hence, our study suggested that non-HDL is a superior surrogate marker of sdLDL particle size as compared to LDL and apoB/A1 ratio in a young healthy Indian population and should be used for optimum assessment of dyslipidemias and CAD risk.

Lead as a Risk Factor for Osteoporosis in Post-menopausal Women.

Lead exposure is increasingly becoming an important risk factor for osteoporosis. In adults, approximately 80-90 % of absorbed lead is stored in the bones. These bone lead deposits are released into the blood during periods of enhanced bone resorption like menopause, forming a potential endogenous source of lead exposure. Postmenopausal women are at a higher risk for bone lead release because of hormonal and age related changes in bone metabolism. Estrogen deficiency is associated with increase in osteoclasts number and activity leading to both the early and late form of osteoporosis. Hence, high blood lead level coupled with concomitant environmental exposure exposes women in this age group to lead related adverse outcomes like hypertension, reduced kidney and neurocognitive functions as well as increased risk of atherosclerosis and cardiovascular mortality. A few studies have also identified coexisting variates like ethnicity, occupation, residence, education, smoking, alcohol medications, water etc. as significant determinants of bone and blood lead in women, thus increasing the magnitude of postmenopausal bone changes. Hence, interventions focused on reducing the intensity of bone resorption during menopause will help decrease exposure to endogenous lead. This would play a significant role in decreasing the morbidity and mortality associated with menopause. Also, identification of modifiable factors that prevent bone lead release will reduce the risk of chronic lead exposure and improve the health outcomes of post-menopausal women.

Lipoprotein (a): a Unique Independent Risk Factor for Coronary Artery Disease.

The current epidemic affecting Indians is coronary artery disease (CAD), and is currently one of the most common causes of mortality and morbidity in developed and developing countries. The higher rate of CAD in Indians, as compared to people of other ethnic origin, may indicate a possible genetic susceptibility. Hence, Lp(a), an independent genetic risk marker for atherosclerosis and cardiovascular disease assumes great importance. Lp(a), an atherogenic lipoprotein, contains a cholesterol rich LDL particle, one molecule of apolipoprotein B-100 and a unique protein, apolipoprotein (a) which distinguishes it from LDL. Apo(a) is highly polymorphic and an inverse relationship between Lp(a) concentration and apo(a) isoform size has been observed. This is genetically controlled suggesting a functional diversity among the apo(a) isoforms. The LPA gene codes for apo(a) whose genetic heterogeneity is due to variations in its number of kringles. The exact pathogenic mechanism of Lp(a) is still not completely elucidated, but the structural homology of Lp(a) with LDL and plasmin is possibly responsible for its acting as a link between atherosclerosis and thrombosis. Upper limits of normal Lp(a) levels have not been defined for the Indian population. A cut off limit of 20 mg/dL has been suggested while for the Caucasian population it is 30 mg/dL. Though a variety of assays are available for its measurement, standardization of the analytical method is highly complicated as a majority of the methods are affected by the heterogeneity in apo(a) size. No therapeutic drug selectively targets Lp(a) but recently, new modifiers of apo(a) synthesis are being considered.

Differential expression of lipid peroxidation and total antioxidant status in Indian patients with cardiovascular and cerebrovascular disease.

Data from studies examining lipid peroxidation as a mechanism involved with hyperhomocysteinemia (HHcy)-induced vascular remodeling in patients with occlusive vascular disease have been contradictory. It has not yet been studied in Indians within the context of atherogenesis. Therefore, we measured the levels of homocysteine (Hcy), malondialdehyde (MDA) as a measure of lipid peroxides (LPOs), and total antioxidant status (TAS) in the serum of 167 patients with occlusive vascular disease [coronary artery disease (CAD) = 43; cerebrovascular disease (CVD) = 82; peripheral vascular disease (PVD) = 42]. Each of these groups was further divided into groups of individuals with or without HHcy. In the case of CAD and CVD, patients with HHcy had significantly higher LPOs than those without HHcy (p = 0.009, 0.001, respectively). TAS was significantly lower in CVD patients with HHcy than in those without (p = 0.014). In patients with CAD or CVD, Hcy directly correlated with LPOs (p = 0.002, 0.001, respectively). Lipid peroxidation is a significant mechanism in HHcy-induced vascular remodeling in CAD and CVD, but not in PVD, probably because it is not relevant in thrombosis (38 of 42 patients of PVD had deep-vein thrombosis). To explain the significantly lower TAS in CVD, we hypothesized that CVD patients present very early with grave symptoms, whereas CAD and PVD occur over a longer period of time. Therefore, when CVD presents, TAS is still overwhelmed by HHcy-induced oxidative stress. Hence, adjuvant therapy with antioxidants would benefit patients with CVD.

The potential of Cystatin C and small dense LDL as biomarkers of coronary artery disease risk in a young Indian population.

Coronary artery disease (CAD) affects Indians 5-6 years earlier than in the west, is diffuse and malignant, and poses a heavy burden on India's developing economy. Traditional risk factors have failed to explain this high incidence of premature CAD and hence this study investigated the association of two novel risk biomarkers, cystatin C and small dense LDL (sdLDL) with the presence and severity of CAD. Cystatin C and sdLDL were estimated in 204 CAD patients ≤45 years of age and compared with 161 age-matched healthy controls. The traditional lipid profile parameters, i.e., cholesterol, LDL, HDL, triglycerides, apolipoproteins A1 and B, and Lp(a) were also measured in both groups. Cystatin C was significantly raised and mean LDL particle size significantly reduced in CAD patients as compared to controls. 62.7 % of CAD patients showed pattern B while 37.3 % patients showed pattern A. Of the traditional lipid tests, only HDL and apolipoprotein A1 showed a significant decrease in the CAD group. sdLDL was significantly associated with the severity of CAD, while cystatin C was not. Both cystatin C and sdLDL emerged as independent risk factors, however, of the two, sdLDL was a more sensitive predictor of CAD events. Cystatin C and mean LDL particle size are significantly and independently associated with the presence of CAD events in patients ≤45 years with normal kidney function. Hence, these novel risk biomarkers can be useful tools in reducing the morbidity and mortality associated with CAD in the productive Indian workforce.

Lowering homocysteine and modifying nutritional status with folic acid and vitamin B(12) in Indian patients of vascular disease.

Hyperhomocysteinemia is more commonly associated with vascular disease in Indians than in the western populations. It is caused by genetic polymorphisms or dietary deficiencies of the B vitamins. We attempted to identify the association of hyperhomocysteinemia with vitamin B(12) and folate in Indian patients of vascular disease. Homocysteine, vitamin B(12) and folate levels were estimated in 100 controls and 100 patients of vascular disease. Homocysteine estimation was repeated in 73 patients on different vitamin supplements for 6 months. Homocysteine exhibited a significant negative correlation with B(12) only in cerebrovascular disease and peripheral vascular diseasepatients, and with folate in coronary artery disease and cerebrovascular disease patients as well as controls. Single daily dose of folate was as effective as a combination of folate and cobalamin in reducing plasma homocysteine concentrations. Low levels of B(12) contribute to the higher incidence of cerebrovascular disease and peripheral vascular disease, and low folate levels account for higher prevalence of hyperhomocysteinemia in coronary artery disease and cerebrovascular disease. Moreover, irrespective of the cause of hyperhomocysteinemia, folate is known to ameliorate it. Hence, large-scale corrective measures like food fortification or dietary supplementation with folate might benefit the Indian population and reduce the incidence and morbidity of vascular disease.

Homocysteine in occlusive vascular disease: a risk marker or risk factor.

Homocysteine has emerged as a significant marker for occlusive vascular disease, but there has been some debate as to whether it is just an association (risk marker) or actually a causative factor (risk factor). To elucidate this, a retrospective statistical analysis was done of data generated in the course of our study on homocysteine and vascular disease. Homocysteine, lipid profile components and lipoprotein(a) were estimated in fasting blood samples drawn from 252 controls and 536 patients of occlusive vascular disease. The data were analyzed by SPSS version 17. Mean homocysteine levels were significantly higher (p < 0.001) in all patients categories, as compared to controls. In fact, homocysteine level was the most significant biochemical risk factor for vascular disease. The odds ratios due to hyperhomocysteinemia varied from 3.170-4.153. When the cut-off was increased by 5 micromol/L, the odds ratio became almost three-fold. The prevalence of hyperhomocysteinemia increased by approximately equal to 20%, when the cut-off was reduced by 5 micromol/L. Statistical analysis of our data revealed that homocysteine conformed to Hill's criteria of causation. Moreover, hyperhomocysteinemia was treatable by the administration of B-vitamins, even if the cause was genetic. Hence morbidity due to vascular disease could be reduced by identification and treatment of hyperhomocysteinemia.

Prevalence of hyperhomocysteinemia in vascular disease: comparative study of thrombotic venous disease vis-à-vis occlusive arterial disease.

Studies on hyperhomocysteinemia in vascular occlusive disease have included mostly patients with arterial occlusion. However, more recent studies have included cases of venous occlusive disease as well. Our present study is aimed at comparing the prevalence of hyperhomocysteinemia in venous occlusive disease vis-à-vis arterial occlusive disease in the North Indian urban population. Homocysteine was estimated by chemiluminescent immunoassay in 205 normal controls and 536 patients, 244 presenting with arterial occlusion and 292 with venous thrombotic disease. The mean homocysteine in patients with arterial occlusion was 21.79 +/- 0.09 micromol/L (mean +/- standard error of measurement), in patients with venous thrombosis was 25.53 +/- 0.1 micromol/L, and in controls was 11.33 +/- 0.18 micromol/L. The prevalence of hyperhomocysteinemia (> 15 micromol/L) was 56.38% in arterial occlusive disease and 54.64% in venous thrombosis. In patients with peripheral vascular occlusive disease, patients with deep venous thrombosis (DVT) had the highest mean homocysteine level (25.51 micromol/L), which was even higher (32.14 micromol/L) when associated with pulmonary embolism (PE). There is a high prevalence of hyperhomocysteinemia in arterial and venous occlusive disease. Hence, in all patients with vascular occlusive disease, hyperhomocysteinemia should be elucidated and treated. In addition, long-term follow-up is required to ascertain whether the reduction in homocysteine decreases the thrombotic events and whether homocysteine levels can actually be of prognostic or predictive value in cases of DVT with PE.