Nitin Bhardwaj

Bio: Nitin Bhardwaj is an academic researcher from Institute of Genomics and Integrative Biology. The author has contributed to research in topics: Bisulfite sequencing & DNA methylation. The author has an hindex of 4, co-authored 5 publications receiving 36 citations.

Plasma proteomic analysis of stable coronary artery disease indicates impairment of reverse cholesterol pathway.

Abstract: Coronary artery disease (CAD) has remained as one of the most important causes of mortality and morbidity worldwide. According to WHO, almost seven million deaths occur yearly due to this disease1. It is estimated that global cardiovascular death would increase from 17.1 million (in 2004) to 23.4 million by 2030 with CAD contributing a significant proportion2. Furthermore, the burden of this disease has increased rapidly in the developing countries3. In India alone the mortality due to CAD has increased from 1.17 million to 1.59 million from 1990 to early 20004. Since CAD is a complex disorder where both genetic and lifestyle (including dietary habits) contribute significantly, finding new potential markers holds its own clinical importance specifically in early detection and efficient management of the disease5. In recent years, several studies focussed to identify genetic markers that could be associated with CAD. To this end several Genome Wide Association Studies (GWAS) have been undertaken by various groups to identify single nucleotide polymorphisms (SNPs) that are associated with CAD6,7,8,9. However, other than a few SNPs most of them could not be replicated in different populations. Further, most of the SNPs have low discriminative accuracy and the common variants account for about 10% of predicted genetic heritability of CAD5. Thus, even now, the classical risk factors in blood like total cholesterol, HDL, LDL etc. are routinely determined to assess the risk of CAD1. These traditional risk factors are useful in identifying people at high risk of developing CAD. Wang et al. showed a predictive accuracy of 0.7 area under the curve with classical risk factors for major cardiovascular events in Framingham heart study. However, identification of newer markers are necessary to increase the predictive accuracy especially since various prospective studies using the classical markers of CAD did not show a high predictive significance for the disease10,11. With the advent of mass spectrometry based high throughput proteomic technologies, protein markers have gained attention as it is feasible to compare the proteome of diseased and healthy individuals and identify differentially expressed proteins that could potentially act as disease markers. Proteomics has helped in identifying markers for several diseases like cancer, neurological diseases etc12,13,14. Although several groups have also attempted to identify markers for various cardiovascular diseases including acute coronary syndrome, unstable and stable angina, myocardial infarction etc15,16,17, surprisingly, studies on identifying markers for stable coronary artery disease are limited. A couple of studies have been done based on peptide profiling in urine and a few peptide signatures were identified as potential biomarkers18,19. However the use of peptide signatures has an inherent problem since they can be detected only using mass spectrometer thereby limiting their utility as biomarkers in clinical practice as mass spectrometers are till date not routinely used as a tool for biomarker profiling20,21. Donahue et al. reported a qualitative proteomic analysis using pooled plasma from 53 CAD cases and 53 controls22. However, qualitative proteomic measurement will have limited application in terms of biomarkers. Thus, a panel of defined quantifiable proteins (and not peptide signatures) from an easily accessible biological fluid (like urine or plasma) is necessary for it to be considered as potential biomarkers23. In the present study using high throughput iTRAQ (Isobaric tag for relative and absolute quantitation) based relative quantitation of plasma proteome we have identified proteins that are differentially expressed in stable CAD patients. The differentially expressed proteins in the discovery phase were further validated in two phases in more than 500 samples. After multivariate logistic regression we report four proteins involved in the reverse cholesterol pathway to be significantly associated with CAD.

Low holo-transcobalamin levels are prevalent in vegetarians and is associated with coronary artery disease in Indian population.

Abstract: Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B12 deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B12 is internalised in the cells by the proteins transcobalamin II. Vitamin B12-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B12. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p = 2.57E-4) in CAD (26.81 pmol/l) cases as compared to controls (29.97 pmol/l).

Identification of differentially expressed proteins in vitamin B 12

Abstract: Background: Vitamin B 12 (cobalamin) is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC) (Holo-TC [HoloTC]) enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD) in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

Investigating Coronary Artery Disease methylome through targeted bisulfite sequencing

Abstract: BackgroundGene environment interactions leading to epigenetic alterations play pivotal role in the pathogenesis of Coronary Artery Disease (CAD). Altered DNA methylation is one such epigenetic factor that could lead to altered disease etiology. In this study, we comprehensively identified methylation sites in several genes that have been previously associated with young CAD patients.nnMethodsThe study population consisted of 42 healthy controls and 33 young CAD patients (age group < 50 years). We performed targeted bisulfite sequencing of promoter as well as genic regions of several genes in various pathways like cholesterol synthesis and metabolism, endothelial dysfunction, apoptosis, which are implicated in the development of CAD.nnResultsWe observed that the genes like GALNT2, HMGCR were hypermethylated in the promoter whereas LDLR gene promoter was hypomethylated indicating that intracellular LDL uptake was higher in CAD patients. Although APOA1 did not show significant change in methylation but APOC3 and APOA5 showed variation in methylation in promoter and exonic regions. Glucokinase (GCK) and endothelial nitric oxide synthase 3 (NOS3) were hyper methylated in the promoter. Genes involved in apoptosis (BAX/BCL2/AKT2) and inflammation (PHACTR1/LCK) also showed differential methylation between controls and CAD patients.nnConclusionsThis study is unique because it highlights important gene methylation alterations which might predict the risk of young CAD in Indian population. Large scale studies in different populations would be important for validating our findings and understanding the epigenetic events associated with CAD.

Homocysteine and holotranscobalamin and the risk of Alzheimer disease A longitudinal study

Abstract: Objective: To examine the relation between serum levels of homocysteine (tHcy) and holotranscobalamin (holoTC), the active fraction of vitamin B12, and risk of incident Alzheimer disease (AD) in a sample of Finnish community-dwelling elderly. Methods: A dementia-free sample of 271 subjects aged 65–79 years derived from the Cardiovascular Risk Factors, Aging, and Dementia (CAIDE) study was followed up for 7 years to detect incident AD. The association between serum tHcy and holoTC with AD was analyzed with multiple logistic regression after adjusting for several potential confounders, including common vascular risk factors. Results: The odds ratios (ORs) (95% confidence interval [CI]) for AD were 1.16 (1.04–1.31) per increase of 1 μmol/L of tHcy at baseline and 0.980 (0.965–0.995) for each increase of 1 pmol/L baseline holoTC. Adjustment for several potential confounders including age, sex, education, APOE ϵ4 allele, body mass index, Mini-Mental State Examination, smoking, stroke, and blood pressure did not alter the associations: ORs (95% CI) for AD became 1.19 (1.01–1.39) for tHcy and 0.977 (0.958–0.997) for holoTC. Adjusting for holoTC attenuated the tHcy–AD link (OR changed from 1.16 to 1.10, 95% CI 0.96–1.25). The holoTC–AD relationship was less influenced by controlling for tHcy (OR changed from 0.980 to 0.984, 95% CI 0.968–1.000). Addition of folate did not change any of the results. Conclusions: This study suggests that both tHcy and holoTC may be involved in the development of AD. The tHcy–AD link may be partly explained by serum holoTC. The role of holoTC in AD should be further investigated.

Identification of differentially expressed proteins in fresh and frozen-thawed boar spermatozoa by iTRAQ-coupled 2D LC-MS/MS.

Abstract: Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa In this study, protein samples prepared from fresh and frozen-thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC-MS/MS analysis A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen-thawed spermatozoa by at least a mean of 179-fold (P<005) On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm-oocyte binding and fusion The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity This is the first study to compare protein levels in fresh and frozen-thawed spermatozoa using the iTRAQ technology Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen-thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease

Abstract: Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD.

Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet—A Randomized Controlled Trial in Healthy Participants

Abstract: Vegans are at an increased risk for certain micronutrient deficiencies, foremost of vitamin B12. Little is known about the short-term effects of dietary change to plant-based nutrition on vitamin B12 metabolism. Systemic biomarkers of vitamin B12 status, namely, serum vitamin B12 and holotranscobalamin, may respond quickly to a reduced intake of vitamin B12. To test this hypothesis, 53 healthy omnivore subjects were randomized to a controlled unsupplemented vegan diet (VD, n = 26) or meat-rich diet (MD, n = 27) for 4 weeks. Vitamin B12 status was examined by measurement of serum vitamin B12, holotranscobalamin (holo-TC), methylmalonic acid (MMA) and total plasma homocysteine (tHcy). Holo-TC decreased significantly in the VD compared to the MD group after four weeks of intervention, whereas metabolites MMA and tHcy were unaffected. Body weight remained stable in both groups. VD intervention led to a significant reduction of cholesterol intake, and adequate profiles of nutrient and micronutrient status. Lower intake of vitamin B12 was observed in VD, which was mirrored by a lower concentration of serum vitamin B12 and reduced holo-TC after 4 weeks. Plasma holo-TC may be a fast-responding biomarker to monitor adequate supply of vitamin B12 in plant-based individuals.