In an attempt to provide immunological tools for subfractionation of high-density lipoproteins (HDL), monoclonal antibodies were raised against HDL complexes. Two clones identified a peptide, provisionally named K-45 (PI 4.5-4.9; molecular mass 45 kDa, range 42-48 kDa), whose plasma distribution and lipoprotein association were fully characterised. Gel filtration localised the peptide to the HDL region of human plasma where it co-eluted with apolipoprotein (apo) A-I, the structural protein of HDL. Complementary studies employing immunoabsorption with anti-(apo A-I) antibodies removed 90 % of K-45 from plasma: conversely, anti-(apo A-11) antibodies eliminated only 10 % of K-45. Immunoaffinity chromatography on an anti-(K-45) column revealed that the peptide was present in a distinct HDL subspecies containing three major proteins: K-45, apo A-I and clusterin or apo J. The lipoprotein nature of the bound fraction was indicated by electron microscopy (diameter 9.6 ? 3.3 nm) and quantification of lipids, the latter showing an unusually high triacyglycerol concentration. Plasma concentrations of K-45 were positively correlated with apo A-I and HDL-cholesterol and negatively correlated with apo B and total cholesterol. Thus, the peptide appears to be linked, directly or indirectly, to processes which give rise to an anti-atherogenic lipid profile. After completion of the present studies, an N-terminal sequence identical to that of K-45 was reported in recently isolated cDNA clones. These clones encode paraoxonase.Lipoprotein complexes are the principal transport vehicles for plasma lipids. As such, they are the focus of particular attention which derives from the designation of blood lipids as primary cardiovascular risk factors [l, 21. The rationale behind these studies is that a dysfunctional lipid transport system will be a major cause of the dyslipidaemias associated with premature cardiovascular disease [3]. In this context, an obvious pre-requisite is a comprehensive understanding of the normal functioning of the lipoprotein metabolic system ; unfortunately, this is not currently the case. It is due, in part, to the highly dynamic nature of this metabolic process, rendered even more intricate by extensive interactions between the major subclasses, very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoproteins [4]. The latter are of particular interest as they afford a measure of protection against cardiovascular disease [5, 61. Yet many aspects of HDL metabolism are poorly understood. Neither the origins nor the sites of catabolism of this lipoprotein species have been convincingly demonstrated. Additionally, the mechanisms by which HDL assure their postuCorrespondence to R.

No abstract

Paraoxonase is a serum protein bound to high-density lipoproteins (HDLs). The physiological function of the enzyme is unknown, but a role in lipid metabolism has been postulated. To date, studies of the protein have had to rely on measurements of enzyme activity with various substrates. We have developed a highly specific, competitive e.l.i.s.a. using a previously characterized monoclonal antibody. The assay can detect 20 ng of paraoxonase with a working range of 75-600 ng. Intra- and interassay coefficients of variation were 6.5 and 7.9% respectively. Serum concentrations of paraoxonase in healthy subjects from Geneva and Manchester ranged from 25 to 118 micrograms/ml. There were significant differences in mean concentrations between the two groups (Geneva, 79.3 +/- 18.7 micrograms/ml; Manchester, 59.9 +/- 24.1 micrograms/ml: P < 0.001), differences also apparent when subjects were compared according to paraoxonase phenotype. These appeared to be largely a consequence of differences in apolipoprotein A-I concentrations between the two populations, suggesting that HDL particle number may be important in determining serum levels of paraoxonase. Paraoxonase specific activities were also significantly different between the two groups of subjects (Geneva, 2.08 +/- 0.96 units/mg; Manchester, 3.08 +/- 1.73 units/mg: P < 0.001), which may reflect differences in HDL particle composition. The e.l.i.s.a. should furnish the necessary complement to studies of paraoxonase enzymic activity and has already provided evidence for differences with respect to serum levels of the protein both between populations and between phenotypes within populations.

The effect of orlistat, a nonabsorbed inhibitor of gastric and pancreatic lipases, was examined in patients with primary hyperlipidaemia (serum cholesterol > or = 6.2 mmol.l-1 and triglycerides < or = 5.0 mmol.l-1) not responsive to dietary change alone. In a multicentre, randomised, double-blind study, 103 men and 70 women received 30, 90, 180, or 360 mg or orlistat or placebo for 8 weeks. Total and low-density lipoprotein cholesterol levels were reduced by 4% and 5% with 30 mg orlistat, by 7% and 8% with 90 mg orlistat, by 7% and 7% with 180 mg orlistat and by 11% and 10% with 360 mg orlistat compared to placebo. High density lipoprotein cholesterol levels significantly decreased in the 360 mg orlistat group. Triglyceride levels significantly increased in the placebo group but not in the drug groups. Body weight decreased by 1.2 kg with 360 mg orlistat, despite a weight maintenance diet. Decreases in vitamin E and D levels occurred, although both vitamins remained within the normal range. Adverse effects from the gastrointestinal tract were frequent, but led to discontinuation of therapy in only seven patients. Orlistat is a new therapeutic drug for the treatment of hyperlipidaemia that may be particularly useful among overweight patients. Its potential place in therapy will await long-term studies. Vitamin supplementation should be considered during treatment.

Low density lipoprotein (LDL), at concentrations high enough for receptor binding but not high enough to saturate the receptor, induces activation of phosphatidylinositot (PtdIns) turnover in a variety of cell types with various biological functions. Using both biochemical and electron microscopic studies, we have shown that blood platelets take up and degrade LDL in a manner reminiscent of phagocytic cell types. The activation of both PtdlIns turnover and LDL metabolism is inhibited by high density lipoprotein. Thus, LDL at hormonal concentrations causes general cellular activation.Since all cell types studied responded to LDL with increased PtdIns turnover and uptake of LDL cholesterol, the PtdIns cycle may also be involved in the cellular regulation of LDL cholesterol metabolism.It has recently been demonstrated that low density lipoprotein (LDL), at concentrations in the range of its dissociation constant (Kd) for receptor binding, =1 nM, rapidly affects human platelets in several ways: (i) their shape and ultrastructural morphology are transiently altered; (ii) phosphatidylinositol (PtdIns) turnover and the molar concentration of intracellular free calcium, [Ca2+]i, are increased; and (iii) thromboxane B2 formation is enhanced (1). All of these effects are inhibited by high density lipoprotein fraction 3 (HDL3), which is known to interfere with LDL binding in platelets (2).Studies with fibroblasts have shown that Ca2+-and phospholipid-dependent protein kinase C, which is activated by stimulation ofPtdIns turnover, controls the activity ofcertain enzymes involved in cellular LDL cholesterol (LDL-Chol) metabolism (3-5). This suggests an interrelationship between the PtdIns response and LDL-Chol metabolism, but it is not known whether low concentrations of LDL stimulate the PtdIns cycle in cells other than platelets. Furthermore, it remains to be clarified whether platelets, like other cells, are capable of metabolizing LDL-Chol. Although platelets, which freely exchange cholesterol with plasma under normocholesteremic conditions, are unable to synthesize cholesterol, there is an unexplained increase in the cholesterol-phospholipid ratio in familial and experimental hypercholesterolemia (6-9). Also HDL fractions are known to be internalized and degraded by platelets (10). These findings suggested to us that there might be a catabolic pathway for LDL-Chol in the platelets.We show here that the LDL-induced activation of the PtdIns cycle occurs not only in platelets but also in various cell types that metabolize LDL-Chol, including arterial smooth muscle cells, lung fibroblasts, lymphocytes, and vascular endothelial cells. We also establish that the catabolism of lipoproteins occurs not only in these cell types but also in platelets. Thus, LDL induces general cellular activation at concentrations near the Kd for receptor binding, which appears to be comparable to hormonal effects. MATERIALS AND METHODSLipoprotein Isolation. LDL (density, 1.019-1.063 g/ml) and HDL3 (density, 1.125-1.3 g/ml) were isolate...