Peter N. Herbert

A specific apoprotein activator for lipoprotein lipase

Abstract These studies were designed to determine which of the apoproteins of high density lipoprotein (HDL) function as the cofactor for lipoprotein lipase (LPL). ApoLP-gln and apoLP-thr, the major HDL apoproteins, as well as apoLP-val, a minor apoprotein constituent, are inactive as cofactors even in the presence of phospholipid. ApoLP-ala, another minor constituent, is inactive alone but in the presence of phospholipid stimulates lipase activity twofold. Only apoLP-glu is able to stimulate LPL activity in the absence of phospholipid and, in the presence of phospholipid, increases activity twelvefold over baseline levels. It is possible that apoLP-glu and perhaps apoLP-ala are obligatory “co-factors” for the hydrolytic step required for normal clearing of triglyceride from the plasma.

Dietary and Drug Treatment of Primary Hyperlipoproteinemia

Abstract The first step in the management of primary hyperlipidemia is its translation into hyperlipoproteinemia, which can be done by measuring the plasma cholesterol and triglyceride concentratio...

Further Observations on the Activation and Inhibition of Lipoprotein Lipase by Apolipoproteins

ApoC-II was the only apolipoprotein from human very low density lipoprotein that activated rat adipose tissue lipoprotein lipase. Activation was blocked by antiserum against apoC-II. Addition of increasing amounts of activator did not alter the apparent Km of lipoprotein lipase (0.32 mM triolein), but it did produce a progressive increase in the apparent Vmax from 0.8 to 2.2 μmoles free fatty acid/mg hour−1. Substrate concentrations above 1.27 mM triolein diminished activation by 0.25–5.0 μg/ml of apoC-II as much as 20%. Reversal of this apparent substrate inhibition was achieved by increasing the activator concentration to 50.0 μg/ml. Each of five nonactivating apolipoproteins-apoC-I, C-III-1, C-III-2, A-I, and A-II-inhibited lipoprotein lipase up to 85–100%. ApoC-II also produced less inhibition under appropriate conditions. Inhibition was dependent on apoprotein concentration, inversely related to substrate triglyceride concentration, and unobserved with nonlipoprotein proteins. The inhibitory capacity of the nonactivating apolipoproteins was about the same, was independent of apoC-II concentration, and occurred when the ratio of nonactivator apoprotein to triglyceride exceeded 3% (w/w). It is possible that these apoproteins function partly to modulate the hydrolysis of very low density lipoprotein triglyceride by lipoprotein lipase.

Isolation and Characterization of an Abnormal High Density Lipoprotein in Tangier Disease

The nature of the high density lipoproteins has been investigated in five patients homozygous for Tangier disease (familial high density lipoprotein deficiency). It has been established that Tangier high density lipoproteins, as isolated by ultracentrifugation, are morphologically heterogenous and contain several proteins (Apo B, albumin, and Apo A-II). An abnormal lipoprotein has been isolated from the d = 1.063-1.21 g/ml ultracentrifugal fraction by agarose-column chromatography which contains apoprotein A-II as the sole protein constituent. In negative-stain electron microscopy, these lipoproteins appeared as spherical particles 55-75 A in diameter. By a variety of criteria (immunochemical, polyacrylamide electrophoresis, amino acid composition, and fluorescence measurements), apoprotein A-I the major apoprotein of normal high density lipoproteins and the C apoproteins were absent from this lipoprotein. As demonstrated by (125)I very low density lipoprotein incubation experiments with Tangier plasma, C apoproteins did not associate with lipoproteins of d = 1.063-1.21 g/ml. Tangier apoprotein A-II, isolated to homogeneity by delipidation of the apoprotein A-II-containing lipoprotein or Sephadex G-200 guanidine-HCl chromatography of the d = 1.063-1.21 g/ml fraction, was indistinguishable from control apoprotein A-II with respect to amino acid composition and migration of tryptic peptides in urea-polyacrylamide electrophoresis. The ability of Tangier apoprotein A-II to bind phospholipid was demonstrated by in vitro reconstitution experiments and morphological and chemical analysis of lipid-protein complexes. It is concluded that normal high density lipoproteins, as defined by polypeptide composition and morphological appearance, are absent from Tangier plasma and that as a consequence, the impairment of C apoprotein metabolism contributes to the hypertriglyceridemia and fasting chylomicronemia observed in these patients.

Abnormal concentration and anomalous distribution of apolipoprotein A-I in Tangier disease

A double-antibody radioimmunoassay was used to determine the concentration and distribution of apolipoprotein A-I (apoA-I) in the plasma of patients with Tangier disease. Obligate heterozygotes for Tangier disease had apoA-I levels that were 50% or less of controls, even when estimates of high-density lipoprotein cholesterol concentration were normal. Plasma apoA-I levels in Tangier homozygotes were at most 3% of normal. Most of the apoA-I in the plasma of homozygotes sedimented in the ultracentrifuge at density 1.21 g/ml, and no more than 20% was recovered in the plasma lipoprotein fraction. Radioimmunoassay demonstrated no immunochemical differences between Tangier and control apoA-I.

Structural and compositional changes attending the ultracentrifugation of very low density lipoproteins.

The effects of repetitive ultracentrifugation on the physical and chemical properties of very low density lipoproteins (VLDL) were investigated. VLDL recentrifuged one to seven times were characterized by chemical analyses, analytical ultracentrifugation and electron microscopy. The VLDL content of triglyceride was increased and the proportion of phospholipid decreased by ultracentrifugation. Recentrifugation of VLDL decreased the number of Sf-o 20-100 particles and generated particles of Sf-o greater than 400. The bulk of the material removed from VLDL by ultracentrifugation was lipoprotein having pre-beta mobility on paper electrophoresis, flotation rates of Sf-o 10-100 and a particle size of 300-400 A-O. Two ultracentrifugations separated an average of 14% of the starting VLDL protein. Characterization of the apoproteins in this material by polyacrylamide gel electrophoresis, gel chromatography, immunoprecipitation and amino acid analysis demonstrated a relatively high proportion of beta-apoprotein and relatively little C-apoproteins.

Properties of the plasma very low and low density lipoproteins in Tangier disease.

The absence of normal high density lipoproteins (HDL) in Tangier disease is well established, but the properties of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in this disorder have not been well defined. The profiles obtained by analytic ultracentrifugation and the chemical composition, morphology, and electrophoretic mobility of Tangier and normal VLDL and LDL were compared. Apolipoproteins were fractionated by gel chromatography and characterized by amino acid analysis, polyacrylamide-gel electrophoresis, and immunochemical reactivity. Concentrations of low density lipoproteins of S(f) (o) 0-12 were reduced in three of six Tangier plasmas studied by analytic ultracentrifugation. Accumulation of intermediate density lipoproteins (S(f) (o) 12-20) was not observed. Two subjects with hypertriglyceridemia had normal VLDL (S(f) (o) 20-400) levels, suggesting that abnormalities of chylomicron metabolism probably account for the hypertriglyceridemia frequently observed in this disorder. Tangier VLDL migrate more slowly than normal VLDL on paper electrophoresis, yet their morphology, gross chemical composition, and qualitative apolipoprotein content are similar. Quantitative abnormalities in C-apolipoproteins, however, were observed in Tangier VLDL. When patients were consuming unrestricted diets, C apoproteins accounted for 19-49% of the protein in lipoproteins of d < 1.006 g/ml. Ingestion of low-fat, high-carbohydrate diets reduced the VLDL-C-apoprotein content in all Tangier patients (mean = 17% of VLDL protein vs. 43% in controls). These findings suggested that a major proportion of the C apoproteins in Tangier plasma is associated with chylomicrons or their remnants, perhaps because the C-apoprotein reservoir normally provided by HDL is absent. This secondary mechanism for C-apoprotein conservation is lost when dietary fat is withdrawn.LDL-2 (1.035 < d < 1.063) from Tangier and control plasma had identical electrophoretic mobilities. Tangier LDL-2 had slightly smaller median diameters (210-225 A vs. 230-240 A in controls) and a quite different composition than normal LDL-2. Triglyceride accounted for a mean of 29% of Tangier LDL-2 mass (control = 6%) and the cholesteryl ester content was reduced by about 50%. Thus, HDL may be required for the generation of chemically normal LDL. Alternatively, the fundamental defect in Tangier disease may involve all lipoprotein classes.

Tangier disease: one explanation of lipid storage.

Normal high-density lipoproteins are absent from plasma in Tangier disease, and the disorder is characterized by accumulation of cholesteryl esters in several tissues, particularly those of the reticuloendothelial system. Electron microscopy of the abnormal high-density lipoproteins in the plasma of three patients with Tangier diseases revealed large (68-nm), flattened, translucent particles in all cases. These particles were most abundant in the plasma of the splenectomized patient. Restriction of dietary fat eliminated or drastically reduced the numbers of these particles among the Tangier high-density lipoproteins. Thus abnormal products of chylomicron metabolism that appear to occur in plasma in this disorder may be targets for phagocytosis and may be at least one source of the cholesteryl esters that accumulate in reticuloendothelial tissues in Tangier disease.

Lipodystrophic diabetes mellitus. Investigations of lipoprotein metabolism and the effects of omega-3 fatty acid administration in two patients☆

We investigated the metabolic effects of omega-6 (safflower oil) and omega-3 (fish oil) fatty acid-enriched diets (65% carbohydrate, 20% fat) in two patients with a syndrome of diabetes mellitus, lipodystrophy, acanthosis nigricans, chylomicronemia, and abdominal pain. 3H-glycerol was used to evaluate triglyceride-rich lipoprotein-triglyceride (TRLP-TG) metabolism, and changes in glucose and insulin dynamics were also studied. On the omega-6 diet, both subjects demonstrated four- to five-times normal rates of TRLP-TG production and glycerol biosynthesis, and striking decrements in the fractional catabolic rate (FCR) for TRLP-TG and TRLP-particles. Both subjects had elevations in nonesterified fatty acid (NEFA) concentrations. In one patient, the omega-3 diet markedly decreased serum triglycerides and newly synthesized triglyceride glycerol production, in association with a fall in NEFA. In both subjects, plasma glycerol reutilization for triglyceride synthesis, normal on the omega-6 diet, was abolished on the omega-3 regimen. Plasma postheparin lipolytic activity was normal on both diets. On the omega-3 diet, xanthomas and hepatomegaly decreased and, in the patient who had no reduction in serum triglycerides, pancreatitis attacks virtually ceased. Mean 24-hour serum glucose levels were higher, and both basal and peak C-peptide responses to a carbohydrate meal were blunted on the omega-3 diet. One patient became ketonuric. We conclude the cause of hypertriglyceridemia in these patients was due to increased lipid synthesis and hypothesize that this is secondary to high plasma concentrations of NEFA. In addition, an omega-3 diet in these subjects inhibited insulin secretion and worsened glucose tolerance.

Therapeutic failure in familial type II hyperlipoproteinemia

The extended use of diet and cholestyramine therapy in familial type II hyperlipoproteinemia was examined in patients who previously participated in a short-term, double-blind trial. A striking secondary failure in therapeutic response during 4 yr of use of this therapy was noted with plasma cholesterol rising an average of 15%. A 3 mo, out-patient, follow-up study designed to reinforce patient motivation and dietary and drug adherence resulted in a prompt but partial reversal of this therapeutic deterioration in 16 patients. Additional inpatient studies confirmed that patient noncompliance with the dietary regimen was the major factor responsible for the secondary failure. Cholestyramine together with a low cholesterol diet can be an effective agent in familial type II hyperlipoproteinemia, given a comprehensive program of out-patient follow-up with continued emphasis on dietary principles and drug adherence.