Shinichi Mashiba

Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalent hemodialysis patients

BACKGROUND AND OBJECTIVES Here, we assessed the impact of oxidized high-density lipoprotein (oxHDL), dysfunctional HDL, on mortality and cardiovascular disease (CVD) events in prevalent HD patients and compared oxHDL to interleukin-6 (IL-6), a strong predictor of CVD events in HD patients. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS This prospective study examined a cohort of prevalent HD patients (n=412). Blood samples were obtained at baseline to measure lipids, high-sensitive C-reactive protein (hsCRP), IL-6, oxidized low-density lipoprotein, N-terminal pro B-type natriuretic peptide, intercellular adhesion molecule 1 (ICAM-1), myeloperoxidase, adiponectin, and oxHDL. Carotid intima-media thickness (CIMT) was assessed at baseline and 3-year follow-up. Nutritional status was assessed by subjective global assessment (SGA), body mass index, and geriatric nutritional risk index (GNRI). After the baseline assessment, study patients were prospectively followed up (mean observational period, 40 months). RESULTS At baseline, patients with high oxHDL had a worse nutritional state and higher HDL-cholesterol (HDL-chol), ICAM-1, and adiponectin levels and a higher oxHDL/HDL-chol ratio than low oxHDL patients. A combination of high oxHDL and high IL-6 was significantly associated with increased CIMT at baseline and a larger increase in CIMT at 3-year follow-up. High oxHDL did not predict all-cause mortality; however, it was significantly associated with CVD-related mortality and composite CVD events, particularly with concomitant high IL-6. These associations were confirmed in multivariate Cox hazard models adjusted with confounding variables. CONCLUSIONS High oxHDL, particularly with concomitant high IL-6, may be associated with an increased risk of CVD events and CVD-related mortality in prevalent HD patients.

Evaluation of oxidized alpha-1-antitrypsin in blood as an oxidative stress marker using anti-oxidative α1-AT monoclonal antibody

BACKGROUND alpha1-AT is a 52-kDa acute-phase protein and a typical serine proteinase inhibitor, which is present in human serum. In vivo, the inhibitor prevents tissue damage by inactivating proteinases, such as elastase, that are released from activated neutrophils in the presence of inflammation. METHODS We obtained a monoclonal antibody against oxidized alpha1-AT(3F4) using chloramine T-oxidized alpha1-AT as the antigen. RESULTS This antibody did not react with either the native alpha1-AT or the elastase-alpha1-AT complex. However, it reacted with alpha1-AT oxidized by various oxidants and peroxide lipid. The oxidized alpha1-AT is a polymer with a molecular mass of 100-200 kDa in addition to the 52-kDa protein that corresponds to the native alpha1-AT in sera. In vitro evaluations reveal that fatty acids are involved in the polymerization. Furthermore, the concentrations of oxidized alpha1-AT in the sera of patients with inflammatory and rheumatoid diseases were higher than those in healthy subjects. CONCLUSIONS We considered that 3F4 is an effective antibody that can specifically recognize oxidized alpha1-AT, a marker of oxidative stress.

Assessment of Myeloperoxidase and Oxidative α1-Antitrypsin in Patients on Hemodialysis

BACKGROUND AND OBJECTIVES The present study assesses the effects of the oxidative stress marker, myeloperoxidase (MPO), and the possible MPO-related oxidative stress marker, oxidative alpha(1)-antitrypsin (oxAT), on carotid intima-media thickness (CIMT) and protein-energy wasting (PEW) in patients on hemodialysis (HD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Blood samples were obtained from 383 patients before HD to measure WBC count, serum albumin, lipids, high-sensitivity C-reactive protein (CRP), alpha(1)-antitrypsin (AT), interleukin-6, oxidative LDL-C, MPO, and oxAT. We assessed both CIMT and the geriatric nutritional risk index (GNRI) in this cross-sectional competitive study. RESULTS Levels of MPO and oxAT correlated. Myeloperoxidase was associated with max-CIMT, and oxAT correlated with max-CIMT and GNRI. Multivariate linear regression models showed that MPO and oxAT were independent predictors of increasing max-CIMT, whereas oxAT, but not MPO, independently correlated with GNRI. In four combined MPO and oxAT groups classified according to median values, a multinomial logistic regression model showed that high MPO together with high oxAT was independently associated with increased max-CIMT. Moreover, the OR for max-CIMT with positive PEW and high MPO was significantly increased in the four groups with combined MPO and PEW. CONCLUSIONS High MPO with high oxAT and high MPO with PEW seem to contribute to plaque formation in patients on HD, whereas elevated MPO or oxAT alone might not predict increasing CIMT. In contrast, a high oxAT value seems to be an independent predictor of PEW in patients on HD.

Measurement of glycated albumin by the nitroblue tetrazolium colorimetric method

A method has been developed for the measurement of glycated albumin (albumin-fructosamine) by the nitroblue tetrazolium (NBT) colorimetric method. In this method, polyethylene glycol was added to the serum and then the mixture was centrifuged to separate globulin proteins from albumin proteins. This made it possible to measure the glycated albumin in the supernatant by the NBT colorimetric method, without the interference of globulin proteins. This measurement method correlated with the measurement of glycated albumin using boronate affinity chromatography with an r value of 0.942 (P < 0.001). Our method using polyethylene glycol permits easy measurement of albumin fructosamine and is therefore useful as an index of diabetic control and for diabetic screening.

Oxidized High-Density Lipoprotein Is Associated with Protein-Energy Wasting in Maintenance Hemodialysis Patients

BACKGROUND AND OBJECTIVES Oxidized HDL (oxHDL) may behave as proinflammatory HDL because of reduced anti-inflammatory capacity and is considered a risk factor for mortality in patients on maintenance hemodialysis (MHD). The study presented here assessed the effect of oxHDL on protein-energy wasting (PEW) in MHD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This prospective study examined a cohort of MHD patients (n = 176) who were not taking lipid-lowering drugs. Blood samples were obtained to measure albumin, lipids, high-sensitivity C-reactive protein (hsCRP), oxidized LDL (oxLDL), and oxHDL. PEW was assessed by subjective global assessment (SGA) and geriatric nutritional risk index (GNRI). Measurements and assessment of nutritional status were followed up 1 year later. RESULTS OxHDL was significantly increased in patients with PEW at baseline. High oxHDL and high hsCRP were significantly associated with PEW, and receiver operating characteristic curves for oxHDL and hsCRP showed statistically similar accuracy for predicting SGA-positive status. According to multivariate regression models, high oxHDL had a significant influence on PEW in patients, particularly those with high hsCRP. Decreased changes in GNRI and high prevalence of SGA-positive status at 1 year were more common in patients with high oxHDL at baseline and 1 year later than in patients with low oxHDL at both time points. CONCLUSIONS A high oxHDL state may be associated with PEW estimated by GNRI and SGA, particularly concomitant with inflammation in MHD patients.

Low-density lipoprotein (LDL), which includes apolipoprotein A-I (apoAI-LDL) as a novel marker of coronary artery disease

BACKGROUND Serum low-density lipoprotein (LDL), which includes apolipoprotein A-I (apoAI-LDL) may be generated by oxidization in the serum of patients with coronary artery disease (CAD). We determine the utility of the serum apoAI-LDL level as a novel coronary risk factor. METHODS We measured serum apoAI-LDL in 473 consecutive patients who underwent diagnostic coronary angiography. Serum levels of apoAI-LDL were assayed by a newly developed ELISA. RESULTS The patients consisted of 84 with unstable angina (UA), 259 with stable CAD, and 130 without CAD (control). The serum level of apoAI-LDL was higher in CAD patients than in the control group (31.4 (22.1-41.4) microg/ml vs. 24.6 (18.4-29.2) microg/ml, respectively, p<0.001), as well as in patients with UA compared to those with stable CAD 44.5 (35.8-51.9) microg/ml vs. 27.1 (19.5-35.6) microg/ml, respectively, p<0.0001) (data are expressed as the median (25th-75th percentiles)). By logistic regression analysis, only apoAI-LDL was independent, being significantly able to predict CAD (odds ratio: 1.50, 95% CI: 1.23-1.82, p<0.001), and differentiate unstable angina (odds ratio: 1.80, 95% CI: 1.48-2.17, p<0.001) after controlling for classical risk factors. CONCLUSION The serum level of apoAI-LDL, a newly identified component of oxidized LDL, may be a more sensitive marker of CAD and acute coronary syndrome than CRP.

High-Density Lipoprotein Subfractions and Their Oxidized Subfraction Particles in Patients with Chronic Kidney Disease

AIM Chronic kidney disease (CKD) may lead to reduced concentrations of high-density lipoprotein (HDL) and its subfractions (HDL2 and HDL3), and damage them via inflammation and oxidative stress. The present study aimed to determine the contribution of such changes to cardiovascular disease (CVD) in patients with CKD. METHODS The levels of total cholesterol, low-density lipoprotein cholesterol, HDL-C, HDL2, HDL3, apolipoproteins, malondialdehyde-modified LDL (MDA-LDL), oxidized (ox) HDL, oxHDL2, and oxHDL3 were measured in blood samples from patients with CKD (stages 2-5, n=86) who were not on dialysis and from patients undergoing hemodialysis (CKD stage 5D, n=25). The patients were followed up for 28±9 months after baseline examinations and CVD events were recorded. RESULT The levels of HDL3 and ApoA1 in HDL3 fraction decreased according to CKD severity, whereas those of HDL2 and ApoA1 in HDL2 fraction did not differ. The levels of oxHDL were similar across CKD stages. The levels of oxHDL3 and MDA-LDL were decreased, whereas those of oxHDL2 increased according to CKD severity. Multivariate analyses using the Cox proportional hazards model selected high levels of oxHDL and its subfractions, and those adjusted with HDL-C and HDL subfractions or ApoA1 in HDL fractions respectively, compared with HDL-C and HDL subfractions or ApoA1 in HDL fractions alone as independent risk factors for CVD events. CONCLUSION The levels of HDL subfractions and their oxidized subfraction particles differed among patients with CKD. The increasing levels of oxHDL subfractions might cause a high frequency of CVD events in such patients.

Fenofibrate ameliorates insulin resistance, hypertension and novel oxidative stress markers in patients with metabolic syndrome.

SUMMARY OBJECTIVE The benefits of fenofibrate, a peroxisome proliferator-activated receptor α agonist, against cardiovascular risk factors have been established. To clarify the underlying mechanisms of these benefits, we examined the effects of fenofibrate on insulin resistance, hypertension, inflammation, oxidative stress and coagulation markers in patients with metabolic syndrome. METHODS Eleven Japanese patients with metabolic syndrome underwent physical examinations and blood tests before and after treatment with fenofibrate 200 mg daily for 8 weeks. RESULTS Fenofibrate significantly decreased systolic blood pressure, pulse wave velocity, serum insulin, insulin resistance (calculated from the homeostasis model assessment), total cholesterol, triglyceride, remnant-like particles cholesterol, uric acid, D-dimer, fibrinogen, serum amyloid A/low-density lipoprotein (LDL) and apoA1/LDL levels. It also significantly increased levels of high molecular weight adiponectin, thrombomodulin and high-density lipoprotein cholesterol in these patients. Plasminogen activator inhibitor-1, C-reactive protein, fasting plasma glucose and thrombin-antithrombin complex levels did not change. LIMITATION Small sample size. CONCLUSION Short-term fenofibrate administration not only improved lipid profiles, but also ameliorated insulin resistance, hypertension and oxidative stress markers in patients with metabolic syndrome, suggesting that fenofibrate can decrease the risk of arteriosclerosis through various pathways.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Oxidized high-density lipoprotein is associated with increased plasma glucose in non-diabetic dyslipidemic subjects

BACKGROUND Oxidized high-density lipoprotein (oxHDL) has reduced capacity for cholesterol efflux and some of other anti-atherogenic properties of HDL, but the role of oxHDL in the pathogenesis of cardiometabolic disease has not been fully demonstrated. This study investigated the association of oxHDL with plasma glucose (PG) and the other atherosclerotic risk variables in non-diabetic dyslipidemic subjects. METHODS Conventional atherosclerotic markers and LDL particle size (LDL-PS), as determined by gel electrophoresis, were measured in 155 non-diabetic subjects (mean age of 57 years) with dyslipidemia. Serum oxHDL levels were quantified using an antibody against oxidized human apoA-I in a sandwich ELISA format. RESULTS Multiple regression analysis adjusted for possible confounders revealed that HDL-cholesterol was independently, significantly and positively correlated with LDL-PS and oxHDL. By multiple regression analysis, oxHDL was independently, significantly and positively correlated with fasting PG (β=0.19, P=0.01). Subjects in the highest PG tertile group had approximately 30% higher oxHDL levels than the lowest PG tertile group. CONCLUSIONS These results suggest that high PG levels may contribute to the HDL oxidation, irrespective of HDL-cholesterol levels, even in non-diabetic subjects with dyslipidemia, and that the measurement of oxHDL may be a useful marker of dysfunctional HDL.