A new method for measuring cholesterol efflux capacity uses stable isotope-labeled, not radioactive-labeled, cholesterol.

The incidence of cardiovascular events correlates inversely with cholesterol efflux capacity (CEC) more than with HDL-cholesterol level. The measurement of CEC is used to qualify cardiovascular disease risk and is conventionally performed with radioisotope (RI)-labeled cholesterol. Here, we established a CEC measurement technique using stable isotope-labeled cholesterol as an alternative, and we compared the new method with RI and fluorescence (boron dipyrromethene difluoride-cholesterol) methods in cells and in patient serum. We incubated J774 cells labeled with [d7]cholesterol ([d7]C) with patient serum depleted of apoB, and [d7]C extracted from the culture medium was quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry. [d7]C efflux increased with greater apoB-depleted serum concentration and longer incubation time. The assay coefficient of variation (CV) of five consecutive measurements of three sets of samples ranged from 7.3% to 9.5%, and the interassay CV determined by measuring three samples four times ranged from 4.1% to 8.5%, both indicating good precision. We then measured CEC levels of 41 outpatients with serum HDL-cholesterol levels between 36 and 94 mg/dl (mean: 61.7 ± 18.0 mg/dl); in the presence of cAMP, we observed a significant, positive correlation between CEC levels determined with the stable isotope and RI methods that was stronger than the correlation between measurements obtained by the fluorescence and RI methods (r = 0.73, P < 0.0001 vs. r = 0.55, P < 0.001). Therefore, our stable isotope method can be considered useful as a non-RI method and thus deserves evaluation in future clinical studies.

Effect of Angiotensin II on Matrix Metalloproteinase-2 Secretion in Human Umbilical Vein Endothelial Cells.

Matrix metalloproteinase (MMP), which is secreted from vascular cells, is an enzyme-degrading extracellular matrix protein. MMP molecules, including MMP-2, are involved in the destabilization of atherosclerotic plaque and plaque rupture during the development of cardiovascular disease. Angiotensin II (Ang-II), a vascular stimulant associated with cardiovascular disease progression, has been demonstrated to be mainly involved in cardiovascular remodeling of atherosclerosis and cardiac hypertrophy. This study was performed to investigate the regulation of MMP-2 by Ang-II in human umbilical vein endothelial cells (HUVECs). Ang-II significantly increased MMP-2 secretion and MMP-2 messenger RNA expression in HUVECs. The effects of Ang-II were suppressed by the coexistence of telmisartan, a blocker of the Ang-II receptor type 1 (AT1 receptor), or PD123319, a blocker of Ang-II receptor type 2 (AT2 receptor). Especially, PD123319 showed marked suppression of the effect of Ang-II on MMP-2. Therefore, Ang-II-induced upregulation of MMP-2 in HUVECs was considered to be mainly achieved through AT2 receptors, although AT1 and AT2 receptors were expressed in HUVECs, but the detailed mechanisms remain undefined. These findings suggest that Ang-II can enhance MMP-2 mainly through AT2 receptors in endothelial cells, but the significance of circulating MMP-2 as a cardiovascular biomarker requires confirmation in further clinical studies.

Association of Renal Function and Statin Therapy with Lipoprotein(a) in Patients with Type 2 Diabetes.

AIM: A high level of serum lipoprotein(a) [Lp(a)] is associated with kidney disease development in patients with type 2 diabetes (T2DM). Recent studies have suggested that statins may affect serum levels of Lp(a). However, the statin effect is not well-defined in patients with T2DM with kidney dysfunction. This retrospective study aimed to investigate the relevance of kidney dysfunction and statin therapy to Lp(a) in patients with T2DM. METHODS: Japanese patients with T2DM (n=149, 96 men and 53 women) were divided into two groups: statin users (n=79) and non-statin users (n=70). Multiple logistic regression analyses were performed with Lp(a) as the objective variable and estimated glomerular filtration rate (eGFR), hemoglobin A1c, age, gender, and body mass index as the explanatory variables. RESULTS: Lp(a) serum levels were higher in statin users than in non-statin users (P=0.022). Multivariate regression analysis results showed an inverse correlation of eGFR to log Lp(a) in all patients (P=0.009) and in non-statin users (P=0.025), but not in statin users. In a multiple logistic regression analysis for median Lp(a), there was an inverse association between eGFR and Lp(a) level (odds ratio, 0.965; 95% confidence interval, 0.935-0.997; P=0.030) in non-statin users as well as in all participants, but not in statin users. CONCLUSIONS: The present study suggests that a high Lp(a) level in patients with T2DM, except in statin users, is significantly associated with decreased eGFR, indicating that the increased Lp(a) levels under statin therapy might diminish the relationship between Lp(a) and eGFR.

Associations of Homocysteine with B Vitamins and Zinc in Serum Levels of Patients with Type 2 Diabetes Mellitus: A Cross-Sectional Study.

The association of homocysteine metabolism-related nutrients along with renal function to homocysteine levels is not well known in patients with type 2 diabetes mellitus (T2DM). We investigated the relevance of kidney function, albuminuria, and nutritional factors to serum homocysteine in T2DM patients. This cross-sectional study enrolled 149 T2DM patients (96 men and 53 postmenopausal women), and patient characteristics and laboratory data including kidney-related data [glomerular filtration rate (eGFR), urinary albumin excretion (UACR), uric acid] and metabolism parameters (hemoglobin A1c and lipids) were collected from the medical record and serum levels of vitamin B12, folic acid, zinc, homocysteine and UACR were also acquired. In total subjects, serum levels of homocysteine, vitamin B12, and folic acid were within reference intervals, but zinc levels were close to lower limits of its reference interval. A multivariate-adjusted analysis showed that gender (ß=-0.259, p<0.001), uric acid (ß=0.267, p<0.001), eGFR (ß=-0.188, p=0.001), log UACR (ß=0.190, p=0.002), log folic acid (ß=-0.259, p<0.001), log vitamin B12 (ß=-0.224, p<0.001) and zinc (ß=-0.169, p=0.006) were correlated to log homocysteine. In multiple regression analysis by gender, these correlations were found similarly in men, but neither log folic acid nor zinc showed correlations with log homocysteine in women. The present study suggests that renal function parameters and the certain nutritional factors have a possible influence on serum homocysteine, in T2DM patients including diabetes kidney disease.

Clinical Significance of Intermediate-Density Lipoprotein Cholesterol Determination as a Predictor for Coronary Heart Disease Risk in Middle-Aged Men.

Background: Not only low-density lipoprotein (LDL) cholesterol but also non-high-density lipoprotein cholesterol (non-HDL-C), very low-density lipoprotein (VLDL) cholesterol (VLDL-C), and intermediate-density lipoprotein (IDL) cholesterol (IDL-C) are reported to be significant risk markers for coronary heart disease (CHD). We reported the relevance of IDL-C to Framingham risk score (F-score), but the present study addressed the relevance of IDL-C to Suita score (S-score), a risk score for coronary heart disease (CHD) developed for the Japanese individuals in addition to F-score. Methods: The cholesterol levels of lipoproteins, including triglyceride (TG)-rich lipoproteins (IDL and VLDL), were measured by an anion exchange high-performance liquid chromatography (AEX-HPLC). This study enrolled 476 men, aged mean 51 years and free of CHD and stroke. Results: Non-HDL-C, IDL-C, and VLDL-C significantly correlated with F-score and S-score. In the multiple stepwise regression analysis, IDL-C as well as body mass index (BMI) significantly correlated with both F-score and S-score in both the total subjects and the subjects without drug therapy. The multivariate logistic analysis with the model composed of BMI and IDL-C as the predictor variables demonstrated that 1 SD increase in IDL-C was an independent predictor for 10-year CHD risk >10% of F-score (OR 1.534, 95% CI 1.266-1.859, p < 0001) and that of S-score (OR 1.372, 95% CI 1.130-1.667, p = 0.0014) in the total subjects. Even in the subjects without the drug therapy, the increased IDL-C, as well as BMI, were significant predictors for 10-year CHD risk >10% of S-score as well as F-score. Conclusion: These results suggest the significant relevance of the increased IDL-C for CHD risk scores in middle-aged men free of CHD and stroke. Further investigations are needed in women and elderly subjects.

Age and sex differences in serum adiponectin and its association with lipoprotein fractions.

Objective The correlation of adiponectin with cholesterol concentration of fractionated lipoproteins has not been well investigated. Methods This study included 174 subjects (79 men and 95 women) without diabetes. The medical record data were investigated retrospectively. The study subjects with adiponectin <8.3, > 8.3 but less 13.9, and ≥ 13.9 were classified into tertile groups: Groups A ( n = 59), B ( n = 58) and C ( n = 57), respectively. Results In women, age and HDL-C were higher in Group C than in Groups A and B, but BMI, TG, IDL-C and VLDL-C were lower in Group C than in Groups A and B. In men, BMI was lower in Group C than in Groups A and B, and HDL-C was higher in Group C than in Groups A and B. In multiple stepwise regression analysis, BMI and HDL-C were significantly correlated with adiponectin in whole, male and female subjects, but TG-rich lipoprotein cholesterol concentrations were not independently correlated. Conclusions HDL-C and BMI were independently correlated with adiponectin in non-diabetic men and women. These results suggest that high adiponectin may play a role in the increased HDL-C concentrations, implicated in the reduction of cardiovascular disease risk, in non-diabetic subjects.