Lipoproteins--a current perspective of methods and concepts.

171

A B S T R A C T We have developed a double antibody radioimmunoassay (RIA) for human apolipoprotein B (ApoB). The assay measures not only the ApoB content of 8-lipoproteins (low density lipoproteins [LDL]) but also that contained in the other lipoproteins in plasma.Purified lymph and plasma chylomicrons and plasma very low density lipoproteins (VLDL) produced displacement curves in the assay system which paralleled those produced by pure LDL. Thus, the ApoB found in chylomicrons, VLDL, and LDL were immunologically identical. ApoB accounted for about 25 and 35%, respectively, of the total protein of chylomicrons and VLDL by RIA. VLDL and LDL preparations from normal and hyperlipoproteinemic subjects also produced parallel displacement curves, suggesting that the ApoB of normal and hyperlipoproteinemic subjects were immunologically identical. High density lipoproteins and abetalipoproteinemic plasma displaced no counts, nor did the sera of several animal species produce any useful displacement curves in this system.The fasting total plasma ApoB concentration of normal subjects was 83±16 mg/dl (mean+SD). ApoB levels were high in Type II (162±16), and less so in Type IV (112±24) and Type V (105±17). When plasma ApoB concentration in Type IV patients was graphed against plasma glycerides, two subpopulations, which may represent different genetic or biochemical subgroups, were apparent. ApoB concentration in individuals on constant diet and drug regimen was stable over weeks to months. Greater than 90% of ApoB of normal and Type II subjects was in the d> 1.006 plasma fraction. By contrast, only 50-80% of ApoB was in the d > 1.006 fraction in Types IV and V. Thus, hypertriglyceridemia was associated primarily with a redistribution of ApoB to the lighter density fractions; by contrast, in hypercholesterolemia absolute ApoB concentration was markedly increased.

Section: Discussionmentioning

confidence: 91%

Assay of Total Plasma Apolipoprotein B Concentration in Human Subjects

Schonfeld¹,

Lees²,

George³

et al. 1974

171

“…This suggests a smaller mean HDL particle size because available data indicate that HDL particles are spherical and are composed of an amiiphiphilic surface of protein, phospholipid, an(l uniesterified cholesterol, and a hydrophobic core of cholesteryl ester andl triglyceride (42)(43)(44)(45)(46)(47). Mlost of the cholesterol in HDL is esterified (48). Furthermore, anialytical ultracentrifugation in the studies of S. NI.…”

Section: 6(hdl3)mentioning

confidence: 99%

High Density Lipoprotein Metabolism in Man

Blum¹,

Levy²,

Eisenberg³

et al. 1977

383

115

A B S T R A C T The turnover of 125I-high density lipoprotein (HDL) was examined in a total of 14 studies in eight normal volunteers in an attempt to determiine the metabolic relationship between apolipoproteins A-I (apoA-I) and A-II (apoA-II) of HDL and to define further some of the determinants of HDL metabolism. All subjects were first studied unider conditions of an isocaloric balanced diet (40% fat, 40% carbohydrate). Four were then studied with an 80% carbohydrate diet, and two were studied while receiving nicotinic acid (1 g three times daily) anid ingesting the same isocaloric balanced diet. The dlecay of autologous 1251-HDL and the appearance of urinary radioactivity were followed for at least 2 wk in each study. ApoA-I and apoA-II were isolated by Sephadex G-200 chromatography from serial plasma samples in each study. The specific activities of these peptides were then measured directly.It was found that the decay of specific activity of apoA-I and apoA-II were parallel to one another in all studies. The mean half-life of the terminal portion of decay was 5.8 days during the studies with a balanced diet.Mathematical modeling of the decay of plasma radioactivity and appearance of urinary radioactivity was most consistent with a two-compartment model. One compartment is within the plasma and exchanges with a nonplasma component. Catabolism occurs from both of these compartments.With a balanced isocaloric diet, the mean synthetic rate for HDL protein was 8.51 mg/kg per day. HDL synthesis was not altered by the high carbohydrate Dr. Blum's current address is the Department of Medicine, College ofPhysicians & Surgeons, Columbia University, New York 10032. Dr. Eisenberg's current address is the Department of Medicine B, Hadassah Hospital, Jerusalem, Israel.Reprint requests should be addressed to Robert I. Levy, M.D., National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, 20014. Receivedfor publication 7January 1977 given beginning 3 days before the injection of '25I-HDL, and ferrous sulfate, 300 mg, was given three times daily. The patients received no other medications. Informed consent was obtained.Collectiot of plasmrla. Blood was obtained in 0.1% EDTA, usually after an overnight fast (12-14 h), and the plasma was separated at 40C in a refrigerated centrifuge. In three instances during each turnover study, nonfastinig blood was obtained; these were at 6, 12, and 36 h after the 9:00 a.m. initiation of the studies.Isolation and labeling of HDL. Autologous HDL was isolated at 4°C over the density range 1.09-1.21 g/ml according to the method of Havel et al. (17). Successive ultracentrifugation was carried out in a Beckman 60 Ti rotor (Beckman Instruments, Inc., Fullerton, Calif.) at 59,000 rpm for 18 h at d 1.09 g/ml, and for 24 h at d 1.21 g/ml. The HDL thus obtained was resuspended in an NaCl-KBr solution of d 1.21 g/ml and washed by ultracentrifugation for 24 h at 64,000 rpm in a Beckman 65 rotor. KBr was then removed by at least four successive 30-min dialyses ...

“…On paper electrophoresis, VLDL1 occupy a pre-Pposition (1,2) and on starch block (3) they migrate as a fairly homogeneous band in the position of a2-globulins. The composition of VLDL isolated by electrophoresis or preparative ultracentrifugation is usually quite reproducible, consisting of about 10, 12, and 65% by weight of protein, cholesterol, and triglyceride, respectively (4,5).…”

Section: Introductionmentioning

confidence: 99%

On the lipoprotein abnormality in type III hyperlipoproteinemia

Quarfordt¹,

Levy²,

Fredrickson³

1971

A B S T R A C T Two lipoprotein species were isolated by starch block electrophoresis from the very low density lipoproteins (VLDL) (St 20-400) of patients with type III hyperlipoproteinemia. One of these, a2-VLDL, had a content of lipid and protein and physical characteristics similar to VLDL from normal subjects or patients with other forms of hyperglyceridemia. The other species, 9-VLDL, contained more cholesterol and less triglyceride in relation to the protein, than normal VLDL. Only the apoprotein of low density lipoprotein was immunochemically detectable in the P-VLDL; the proteins in the a2-VLDL reacted with antisera specific for low density lipoprotein and high density lipoprotein. The electrophoretic mobility of P-VLDL was similar to that of low density lipoprotein and significantly less than that of a2-VLDL. Isolated P-VLDL had a lesser mean flotation rate than a2-VLDL, but both a2-and 8-VLDL were found throughout the Si 20-400 flotation range.The relative quantities of a2-and f-VLDL could be varied by changing the diet or by heparin administration. Most of the VLDL from type III patients on a high carbohydrate diet was in the a2-VLDL form. During fasting, caz-VLDL fell and P-VLDL increased becoming the predominant species of VLDL. Heparininduced acceleration of triglyceride clearance also increased p-VLDL and decreased a2-VLDL. These findings suggest a precursor-product relationship between the a2-and P-forms of VLDL. in most electrophoretic systems. On paper electrophoresis, VLDL1 occupy a pre-Pposition (1, 2) and on starch block (3) they migrate as a fairly homogeneous band in the position of a2-globulins. The composition of VLDL isolated by electrophoresis or preparative ultracentrifugation is usually quite reproducible, consisting of about 10, 12, and 65% by weight of protein, cholesterol, and triglyceride, respectively (4, 5). INTRODUCTIONA group of patients has recently been identified with an aberrant form of VLDL. This VLDL has an unusual electrophoretic mobility, extending from the jS-to the a2-zone on paper electrophoresis (2), and an abnormal composition, including significantly more cholesterol in proportion to triglyceride. The presence of such abnormal VLDL appears to be genetically determined and is associated with unique clinical features leading to its classification as type III hyperlipoproteinemia (2). The isolation from patients with this disorder of two distinct forms of VLDL, characterized by their electrophoretic mobility as p-VLDL and a2-VLDL, is described in this report. METHODS Patients12 patients with type III, 5 with type IV hyperlipoproteinemia, and 5 normal volunteers (aged 19-26), classified according to previously published criteria (2), were studied as in-patients at the National Institutes of Health Clinical Center. Each was fed two different diets. The "balanced low cholesterol diet" contained approximately 20, 40, and 40% of calories, respectively as protein, carbohydrate, and fat (P/S ratio of about 2.0), and less than 300 mg cholesterol per day. The "high carbohy...