Katsuyuki Nakajima

Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gels

We have developed a simple, rapid assay method for apo E-rich lipoproteins (d < 1.006 g/ml), using an immunoaffinity gel mixture of anti apo B-100 and apo A-I antibodies coupled to Sepharose 4B. The immunoaffinity mixed gels adsorb normal lipoproteins containing apo A-I quantitatively as well as most lipoproteins containing apo B-100. Unbound lipoproteins are quantified by assay of cholesterol. Characterization of the unbound lipoproteins of d < 1.006 g/ml (J Lipid Res 1992; 33: 369-380) has shown that they represent chylomicron and VLDL remnant-like particles (RLP). RLP-Cholesterol(C) levels in plasma have been determined in 363 male and female normolipidemic subjects (mean +/- S.D.: 72 +/- 16 mg/l) and have been found to be higher in patients with coronary heart disease and familial dysbetalipoproteinemia. Triglyceride-rich lipoproteins may well contain both atherogenic and non-atherogenic particles that can be separated by this simple immunoadsorption assay.

Remnant lipoprotein levels in fasting serum predict coronary events in patients with coronary artery disease

BACKGROUND Remnant lipoproteins are atherogenic, but assays of remnants have not been available in routine clinical laboratories because of the lack of practical and validated methods. A simple and reliable method for such an assay, using an immunochemical approach, has recently been developed. This study prospectively examined whether remnant lipoprotein levels in fasting serum, measured by our method, may have prognostic value in patients with coronary artery disease (CAD). METHODS AND RESULTS Remnant lipoprotein levels in fasting serum were measured in 135 patients with CAD by an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. Patients were followed up for </=36 months until occurrence of 1 of the following clinical coronary events: recurrent or refractory angina pectoris requiring coronary revascularization, nonfatal myocardial infarction, or cardiac death. Kaplan-Meier analysis demonstrated a significantly higher probability of developing coronary events in patients with the highest tertile of remnant levels (>5.1 mg cholesterol/dL; 75th percentile of distribution of remnant levels) than in those with the lowest tertile of remnant levels (</=3.3 mg cholesterol/dL; 50th percentile of the distribution). Higher levels of remnants were a significant and independent predictor of developing coronary events in multivariate Cox hazard analysis including the following covariates: extent of coronary artery stenosis, age, sex, smoking, hypertension, diabetes mellitus, hypercholesterolemia, low HDL cholesterol, and hypertriglyceridemia. CONCLUSIONS Higher levels of remnant lipoproteins in fasting serum predict future coronary events in patients with CAD independently of other risk factors. Thus, measurement of fasting remnant levels, assessed by the current immunoseparation method, may be helpful in assessment of CAD risk.

Association of Remnant Lipoprotein Levels With Impairment of Endothelium-Dependent Vasomotor Function in Human Coronary Arteries

BACKGROUND It remains undetermined whether triglyceride-rich lipoproteins are an independent risk factor for atherosclerosis. METHODS AND RESULTS The correlation of responses of coronary arterial diameter (quantitative coronary angiography) and coronary blood flow (intracoronary flow wire technique) to intracoronary infusion of acetylcholine (10 and 50 microg/min) with coronary risk factors including remnant lipoprotein levels was statistically analyzed in 106 consecutive subjects with normal coronary angiograms. Remnant lipoproteins were isolated from fasting blood with an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. In multivariate stepwise regression analysis, remnant lipoprotein levels had the most significant correlation with abnormal epicardial coronary vasomotor responses to acetylcholine infusion, reflected by impaired dilation or constriction of the epicardial coronary arteries, and the levels also had an inverse and independent correlation with the coronary blood flow increase in response to acetylcholine. In a subgroup of 53 consecutive subjects, constrictor responses of epicardial coronary diameters to intracoronary infusion of NG-monomethyl-L-arginine (50 micromol/min for 4 minutes) at baseline, reflecting the presence of coronary nitric oxide bioactivity, had an inverse and independent correlation with remnant lipoprotein levels by use of multivariate analysis. CONCLUSIONS Remnant lipoprotein levels were independently associated with abnormal endothelium-dependent vasomotor function in large and resistance coronary arteries in humans, indicating that remnant lipoproteins may impair endothelial vasomotor function in human coronary arteries. The decrease in coronary nitric oxide bioactivity may be responsible in part for the inhibitory effects of remnant lipoproteins.

Probucol markedly reduces HDL phospholipids and elevated preβ1-HDL without delayed conversion into α-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling

Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma prebeta1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate prebeta1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 microg/L by week 4 (p<0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5-15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p<0.01) and 53% (p<0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r=0.455, p<0.05). The absolute and relative concentrations of prebeta1-HDL increased by 14% (p<0.01) and 60% (p<0.001), respectively. The conversion rate of prebeta1-HDL into alpha-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway.

Thujaplicin-copper chelates inhibit replication of human influenza viruses.

The effects of alpha-, beta- and gamma-thujaplicins and six of their metal chelates on human influenza virus-induced apoptosis in Madin-Darby canine kidney (MDCK) cells were examined by DNA fragmentation and flow cytometry. Among the compounds tested, thujaplicin copper chelates inhibited apoptosis induced in the infected MDCK cells with influenza A/PR/8/34(H1N1), A/Shingapol/1/57(H2N2), A/Aichi/2/68(H3N2) and B/Lee/40 viruses, at concentrations of more than 5 microM. These results indicate that the copper chelates inhibit influenza virus-induced apoptosis and that the inhibitory effects may be independent of influenza virus subtype or types. Furthermore, the copper chelates also inhibited the release of the viruses from the infected MDCK cells during apoptosis. The anti-apoptotic effects of the copper chelates may occur 2 4 h postinfection, suggesting that the copper chelates affect MDCK cells directly in the early stage of influenza virus-induced apoptosis. In this study, we demonstrated that thujaplicin-copper chelates inhibit influenza virus-induced apoptosis of MDCK cells and also inhibit virus replication and release from the infected cells.

The possible role of remnant-like particles as a risk factor for sudden cardiac death.

Abstract Postmortem plasma lipid and lipoprotein levels were analyzed in two groups of Japanese subjects who died suddenly and unexpectedly due to cardiac (n = 93) or non-cardiac (n = 26) causes. No individuals in either group had a significant medical or cardiac history. In this study, we measured plasma total cholesterol, triglycerides, VLDL-cholesterol, LDL-cholesterol, HDL-cholesterol, and especially triglyceride-rich lipoprotein remnants. Triglyceride and apo E-rich remnant-like particles (RLP) were studied as a possible risk factor for sudden cardiac death in relation to the progression of coronary atherosclerosis. The receiver-operating characteristic curve (ROC) analysis showed that RLP-TG was the most significant risk factor for sudden cardiac death among the lipids and lipoproteins and RLP-C was the best predictor for coronary atherosclerosis. HDL-C and LDL-C levels were within normal limits in the majority of the cases and did not appear to relate to the sudden cardiac death. Apo E phenotyping was performed for the detection of the genetic background in the lipid metabolism. The frequency of the Apo E3/3 (wild type) phenotype, which closely relates with the remnant metabolism, was significantly reduced in the sudden cardiac death group. Our study on the postmortem plasma lipid analysis suggested that RLP-C and RLP-TG are the best risk predictor for coronary atherosclerosis and sudden cardiac death, respectively.

Remnant-like lipoproteins stimulate whole blood platelet aggregation in vitro.

We have developed a simple, rapid assay method to measure remnant-like lipoproteins by using an immunoaffinity gel mixture of anti apo B-100 and apoA-1 antibodies to Sepharose 4B. Characterization of the unbound lipoproteins has shown that they represent chylomicron and VLDL remnant particles (RLP). Preincubation of whole blood with RLP resulted in the enhanced activation of aggregation with ADP and collagen. Such enhancement was not observed in the presence of lipoprotein deficient serum or albumin preparation. The extent of enhancement was 2.78 times by 7.5 microM of ADP and 44 times by 0.5 microgram/ml of collagen in the presence of RLP-preparation 1 (RLP-1), respectively. In the presence of RLP-2, the enhancement was 5.37 times by 7.5 microM of ADP and 102 times by 0.5 microgram/ml of collagen, respectively. On the other hand RLP slightly inhibited PRP aggregation by these agonists. Inhibitions were 19% by 7.5 microM of ADP and 18% by 1.0 microgram/of collagen in the presence of RLP-1, respectively. Incubation of whole blood with RLP did not result in the release of factors to stimulate platelets or ADP- or collagen-induced platelet aggregation in vitro. The extents of enhanced aggregation in whole blood or inhibition in PRP were not correlated with RLP-cholesterol nor RLP-protein concentrations of RLP preparations used. These results may indicate that RLP not only interact with platelets but with erythrocytes or leukocytes. Our findings support the hypothesis that the postprandial increase in remnant lipoproteins is an atherosclerotic risk factor and may be a part of the reasons of thrombotic complications by stimulating platelets in patients with remnant hyperlipoproteinemia.

Inhibition of infection with human immunodeficiency virus type 1 by sulfated gangliosides

Four kinds of gangliosides, namely GM1a, GD1a, GD1b and GT1b and their sulfated derivatives were examined for antiviral activities against human immunodeficiency virus type 1 and abilities to modulate CD4 antigen on the cell surface. The infection of human T cells with the virus was markedly inhibited by treatment with the sulfated gangliosides at a concentration of 10 micrograms/ml, while the non-sulfated gangliosides had only weak antiviral activities. The sulfated gangliosides completely inhibited syncytium formation induced by HIV-1 at 30 micrograms/ml. The CD4 antigen on the surface of T cells became hardly detectable after treatment with them. They did not damage cells, nor prolong the activated partial thromboplastin time at concentrations of up to 100 micrograms/ml, suggesting that they may have little side effect in vivo.

A micro-scale affinity-purification of Fab'-horseradish peroxidase conjugates and its use for sandwich enzyme immunoassay of insulin in human serum.

We describe methods for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge of Fab’ using reaction with maleimide groups or the thiol-disulfide exchange reaction [l]. The Fab’-horseradish peroxidase conjugates prepared by these methods were demonstrated to be superior in both sandwich enzyme immunoassay and immunohistochemical staining to conjugates prepared by methods in which the enzyme was conjugated through Fab’ amino groups [l], with detection limits in the attomole range [2]. Hinge conjugation is, however, not suitable for small amounts of affinity-purified Fab’, due to loss of thiol groups during gel filtration and concentration. We describe a micro-scale method for affinity purification after Fab’-peroxidase conjugation and its application to a sandwich enzyme immunoassay of insulin in human serum. The non-specific binding of the affinity-purified conjugate was lowered by gel filtration, and the sensitivity for insulin was 1 nU/tube and 100 nU/ml of serum.

A Highly Sensitive Sandwich Enzyme Immunoassay of Human Growth Hormone in Serum Using Affinity-Purified Anti-Human Growth Hormone Fab′-Horseradish Peroxidase Conjugate

Abstract A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab1 -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were coated by physical adsorption with rabbit anti-hGH IgG which was not affinity-purified. Rabbit anti-hGH Fab1 which had been affinity-purified was coajugated with horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The sensitivity was 0.06 pg of hGH per tube or 6 pg/ml of serum when 0.01 ml of serum samples was used. No significant interference was observed in the presence of 8 ng/tube of human prolactin, 8 μU/tube of human thyroid-stimulating hormone, 50 mU/tube of human luteinizing hormone, 10 mU/tube of human follicle-stimulating hormone, or 5 U/tube of human c...