Rojeet Shrestha

Product of serum calcium and phosphorus (Ca × PO4) as predictor of cardiovascular disease risk in predialysis patients.

OBJECTIVES The mortality rate of chronic kidney disease (CKD) patients is very high due to cardiovascular diseases (CVD) which cannot be fully justified by traditional CVD markers. Since, mineral bone disorder is common in CKD, product of serum calcium and phosphorus (Ca × PO4) can be a predictor of future CVD. So, our study aims to assess the utility of higher Ca × PO4 in prediction of CVD risk in predialysis CKD patients. DESIGN AND METHODS 150 CKD patients defined by NKF-KDOQI guideline not undergoing dialysis were recruited. Anthropometric and electrocardiographic parameters were recorded. We evaluated CVD risk by: i) Biochemical CVD markers, ii) NCEP ATP III guideline postulated risk factors and iii) Framingham risk scores. RESULTS Higher Ca × PO4 is associated with presence of Left Ventricular Hypertrophy, oxidative stress, microinflammation, hyperhomocysteinemia, hypercholesterolemia, hypertriglyceridemia and increased LDLc. Compared to cases with Ca × PO4 <55 mg2/dL2, cases with ≥55 mg2/dL2 had relative risk (RR) of 1.82 (95% CI 1.25-2.64) for CVD, 3.24 (95% CI 2.37-4.41) for stroke and 2.43 (95% CI 1.37-4.31) for coronary heart disease (CHD). Moreover, compared to lowest quartile of Ca x PO4, the highest quartile group had RR of 2.13 (95% CI 1.06-4.28) for CVD, 2.61(95% CI 1.80-3.75) for stroke and 2.84 (95% CI 1.15-7.0) for CHD. CONCLUSION In predialysis patients, higher Ca × PO4 is independent predictor of CVD risk.

Development of homogeneous assay for simultaneous measurement of apoE-deficient, apoE-containing, and total HDL-cholesterol

BACKGROUND Pathophysiological role for high-density lipoprotein (HDL) subclasses remains to be elucidated. Homogeneous assay for simultaneous measurements of apoE-deficient HDL-cholesterol (HDL-C), apoE-containing HDL-C, and total HDL-C is desired, because apoE plays important roles in lipid metabolism. METHODS The proposed assay consists of a primary reaction to remove non-HDL-C, a secondary reaction to measure apoE-deficient HDL-C, and a tertiary reaction to measure apoE-containing HDL-C. The assay is completed within 10 min. For control study, 13% polyethylene glycol precipitation assay and phosphotungstate-dextran sulfate-magnesium precipitation assay were carried out. RESULTS Good correlations between the control assays and the proposed assay was obtained in serum samples from patients without liver disease (n=33): r=0.987, 0.957, and 0.991 for apoE-deficient, apoE-containing, and total HDL-C, respectively. ApoE-containing HDL-C by the proposed method in healthy individuals (n=12) and patients with hyper-HDL-cholesterolemia (n=5) were 0.11±0.03 and 0.26±0.05 mmol/l (4.1±1.3 and 10.1±2.0 mg/dl), respectively. ApoE-containing HDL-C increased rapidly at >2.59 mmol/l (100 mg/dl) of total HDL-C, suggesting a unique regulating mechanism of apoE-containing HDL-C. CONCLUSIONS The established homogeneous assay might be useful for clinical and epidemiological studies on apoE-deficient and apoE-containing HDL subclasses.

Plasma capric acid concentrations in healthy subjects determined by high-performance liquid chromatography

Background Capric acid (FA10:0, decanoic acid) is a medium-chain fatty acid abundant in tropical oils such as coconut oil, whereas small amounts are present in milk of goat, cow, and human. Orally ingested FA10:0 is transported to the liver and quickly burnt within it. Only few reports are available for FA10:0 concentrations in human plasma. Methods Fasting (n = 5, male/female = 3/2, age 31 ± 9.3 years old) and non-fasting (n = 106, male/female = 44/62, age 21.9 ± 3.2 years old) blood samples were collected from apparently healthy Japanese volunteers. The total FA10:0 in the plasma were measured by high-performance liquid chromatography after derivatization with 2-nitrophenylhydrazine followed by UV detection. Results Inter and intra-assay coefficient of variation of FA10:0 assay at three different concentrations ranged in 1.7–3.9 and 1.3–5.4%, respectively, with an analytical recovery of 95.2–104.0%. FA10:0 concentration was below detection limit (0.1 µmol/L) in each fasting human plasma. FA10:0 was not detected in 50 (47.2%) of 106 non-fasting blood samples, while 29 (27.4%) plasma samples contained FA10:0 less than or equal to 0.5 µmol/L (0.4 ± 0.1), and 27 (25.5%) contained it at more than 0.5 µmol/L (0.9 ± 0.3). Conclusion A half of the non-fasting plasma samples contained detectable FA10:0. This simple, precise, and accurate high-performance liquid chromatography method might be useful for monitoring plasma FA10:0 during medium-chain triglycerides therapy.

Prostate Biomarkers with Reference to Body Mass Index and Duration of Prostate Cancer

OBJECTIVE This study was performed to assess prostate biomarkers with reference to body mass index and duration of prostate cancer. MATERIALS AND METHODS A hospital based retrospective study was undertaken using data retrieved from the register maintained in the Department of Biochemistry of Manipal Teaching Hospital, Pokhara, Nepal between 1st January, 2009 and 28th February, 2012. Biomarkers studied were prostate specific antigen (PSA), acid phosphatase (ACP) and prostatic acid phosphatase (PAP), alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (γGT). Demographic data including age, duration of disease, body weight, height and body mass index (BMI) were also collected. Duration of disease was categorized into three groups: <1 year, 1-2 years and >2 years. Similarly, BMI (kg/m2) was categorized into three groups: <23 kg/m2, 23-25 kg/ m2 and >25 kg/m2. Descriptive statistics and testing of hypothesis were used for the analysis using EPI INFO and SPSS 16 software. RESULTS Out of 57 prostate cancers, serum level of PSA, ACP and PAP were increased above the cut-off point in 50 (87.5%), 30 (52.63%) and 40 (70.18%) respectively. Serum levels of PSA, ACP and PAP significantly declined with the duration of disease after diagnosis. We observed significant and inverse relation between PSA and BMI. Similar non-signficiant tendencies were apparent for ACP and PAP. CONCLUSIONS Decreasing levels of prostate biomarkers were found with the duration of prostate cancer and with increased BMI. Out of prostate biomarkers, PSA was found to be significantly decreased with the duration of disease and BMI.

Effects of Long-term Use of Depo-medroxyprogesterone Acetate on Lipid Metabolism in Nepalese Women

Various synthetic progestogens that are used as contraceptives have been reported to influence lipid and lipoprotein fractions differently. Depo-medroxyprogesterone acetate (DMPA), a synthetic progestogen, is used by Nepalese women as a contraceptive agent. Our study aims to determine the effects of long-term use of DMPA on lipid metabolism. We performed this study on 60 healthy Nepalese women who had been using DMPA for more than 2 yr and age- and weight-matched control subjects who were not using hormonal contraceptives. Fasting blood samples were collected from the subjects for the estimation of total cholesterol (TC) and triglyceride (TG) levels, and the levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were estimated using the Friedewalds equation. TC and LDL-C levels in DMPA users were significantly higher than those in non-users. Our study concluded that DMPA use induces lipid metabolism changes that can increase the risk of cardiovascular diseases.

Association of High Sensitivity C-Reactive Protein with the Components of Metabolic Syndrome in Diabetic and Non-Diabetic Individuals

BACKGROUND AND OBJECTIVES High sensitivity C-reactive protein (hsCRP) has been associated with metabolic syndrome (MetS) and its components. Several studies have suggested hsCRP to be used as a marker for the primary prevention of cardiovascular diseases. So, we aimed to evaluate the association between hsCRP levels and the components of MetS in diabetic and non-diabetic population. MATERIALS AND METHODS Type II diabetic patients (T2DM) (n= 121) and healthy controls (n= 121) were enrolled for the study. Anthropometric measurements were taken along with blood pressure from the arm. Ten ml of blood was collected after overnight fasting for the measurement of lipid profile, hsCRP, C-peptide and glucose levels. Insulin resistance (HOMA2-IR) was estimated by HOMA2 calculator utilizing glucose and C-peptide values. All participants were classified into two groups on the basis of the presence or absence of MetS. Data were analysed through SPSS 14 software. RESULTS hsCRP, C-peptide and HOMA2-IR were significantly higher in T2DM subjects when compared with controls. As the number of the components of MetS increased, there was a linear increase in hsCRP levels in whole study population (p trend <.001), diabetic subjects (p trend <.001), as well as in controls (p trend <.001). HOMA2-IR and hsCRP levels were found to be better than LDL cholesterol and waist circumference for predicting the presence of MetS. CONCLUSION hsCRP was found to be better than LDL cholesterol and waist circumference for the prediction of MetS. Hence, hsCRP could be used as a defining marker of MetS in the near future.

Identification of molecular species of cholesteryl ester hydroperoxides in very low-density and intermediate-density lipoproteins

Background Oxidation of lipoproteins is thought to play a crucial role in atherogenesis. Role for triglyceride-rich lipoproteins in atherogenesis is unclear. Thus, we aimed to investigate whether cholesteryl ester hydroperoxides (CEOOH) are present in very low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) by using highly sensitive liquid chromatography/mass spectrometry. Methods Total lipids were extracted from the plasma of healthy donors (n = 6) and their fractions of VLDL and IDL. Additional three plasma samples were analysed freshly for CEOOH. Detection and identification of CEOOH was conducted by liquid chromatography/LTQ ion trap mass spectrometry/Orbitrap high mass accuracy mass spectrometry. Authentic standards of CEOOH were used for unequivocal identification on the basis of their mass spectra. Results We identified six molecular CEOOH species overall, namely, Ch18:1-OOH, Ch18:2-OOH, Ch18:3-OOH, Ch20:4-OOH, Ch20:5-OOH and Ch22:6-OOH. Of them, Ch18:2-OOH, Ch20:5-OOH, Ch20:4-OOH and Ch22:6-OOH were detected in all IDL samples, while only Ch20:4-OOH was detected in all VLDL samples. All of CEOOH species except for Ch18:3-OOH were detected in plasma, with constant detection of Ch20:5-OOH, and Ch22:6-OOH in all plasma samples. Conclusion The presence of CEOOH species in VLDL and IDL was confirmed with the analytical sensitivity of 0.1 pmol, showing the constant appearance of more CEOOH species in IDL than VLDL. This finding might add biochemical evidences of atherogenicity of these lipoproteins. Clinical utility of measuring CEOOH level in these lipoproteins need to be investigated for the risk assessment of the cardiovascular disease.

Liver Involvement in Multiple Myeloma: A Hospital Based Retrospective Study

OBJECTIVE This study was to assess liver involvement in multiple myeloma with the aid of liver function tests. MATERIALS AND METHODS A hospital based retrospective study was undertaken using data retrieved of multiple myeloma from the register maintained in the Department of Biochemistry of the Manipal Teaching Hospital, Pokhara, Nepal between 1st January, 2007 and 28th February, 2012. We collected biomarkers of liver profiles including bilirubin (Total, Direct and Indirect), total protein, albumin, AG ratio, SGOT, SGPT, ALP, γGT, LDH, ferritin, renal profile and hematological profile. Descriptive statistics and testing of hypothesis were used for the analysis using EPI INFO and SPSS 16 software. RESULTS Out of 37 cases of multiple myeloma, serum level of AST, ALT, ALP, γGT and LDH were increased above the cut-off point in 22 (59.5%), 24 (64.86%), 13 (35.13%), 9 (24.3%) and 11 (29.7%) respectively. The mean values of AST (65.5±28.18 U/L), ALT (68.37±29.74 U/L), ALP (328.0±148.4 U/L), γGT (44.5±29.6 U/L) and LDH (361.7±116.5 U/L), total protein (9.79±1.03 gm/ dl) were significantly increased when compared with controls. In contrast, albumin (3.68±0.43 gm/dl) and the AG ratio (0.62±0.15) were significantly decreased. Similarly, anemia, hyperuricemia, azotemia, hypercalcaemia and Bence Jones proteinuria were found in 30 (78.9%), 27 (71.1%), 19 (51.5%), 15 (39.5%) and 16 (42.1%) respectively, in cases of multiple myeloma. CONCLUSIONS While clinical manifestation of liver disease among the multiple myeloma was not common, abnormalities in liver function were characteristic.

Elastic modulus of low-density lipoprotein as potential indicator of its oxidation

Background Evaluation of low-density lipoprotein oxidation is important in the risk assessment of cardiovascular disease. Atomic force microscope is widely used to evaluate the physical properties including stiffness on a single-particle scale. In this study, the effect of low-density lipoprotein oxidation on the low-density lipoprotein stiffness was investigated using an atomic force microscope. Methods Isolated low-density lipoprotein particles with or without oxidation were densely bound to an Au substrate on mica, and then pressed and deformed by the atomic force microscope tip. The stiffness of each low-density lipoprotein particle was estimated as the elastic modulus obtained by the force curve analysis. Biochemical change of low-density lipoprotein due to oxidation was studied by electrophoresis. Results and conclusion The elastic modulus of low-density lipoprotein particles ranged between 0.1 and 2 MPa. The oxidation of low-density lipoprotein increased the number of low-density lipoprotein particles with smaller elastic moduli, indicating the decrease in low-density lipoprotein stiffness. The elastic modulus of low-density lipoprotein might be potentially useful to evaluate low-density lipoprotein oxidation.

Mass Spectrometric Quantification of Amphipathic, Polyphenolic Antioxidant of the Pacific Oyster (Crassostrea gigas).

A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography-tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 - 16 pmol (r(2) = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 μg g(-1) whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.