O. David Taunton

Effects of Dietary Polyunsaturated and Saturated Fat on the Properties of High Density Lipoproteins and the Metabolism of Apolipoprotein A-I

In this study we have investigated, in four normal males the effects of dietary saturated and polyunsaturated fat on the chemical composition and thermotropic properties of human high density lipoproteins (HDL) and have measured the influence of the diets on the metabolism of that fraction of HDL apolipoprotein A-I (apoA-I) that undergoes exchange in vitro and accounts for approximately two-thirds of the lipoproteins apoA-I complement. When compared with the saturated fat diet, the polyunsaturated diet reduced plasma cholesterol (24%, P < 0.01) by affecting the cholesterol content in the very low density lipoprotein ( downward arrow25%, P < 0.02), low density lipoprotein ( downward arrow20%, P < 0.01), and high density lipoprotein fractions ( downward arrow33%, P < 0.01). Plasma triglyceride was also lowered (by 13%, P < 0.01). Furthermore, polyunsaturated fat ingestion caused a significant fall in the palmitate and stearate content of HDL triglyceride (41 and 37%, respectively), cholesteryl esters (29 and 35%), and phospholipids (17 and 9%) with a concomitant increase in the linoleate content of these moieties (157, 28, and 29%, respectively). The polyunsaturated diet also produced reciprocal changes in the percentage protein ( downward arrow9%, P < 0.02) and phospholipid ( downward arrow11.5%, P < 0.01) in HDl. These compositional changes were associated with an increase in the microscopic fluidity of the polyunsaturated HDL, although both diets had little effect on the fluidity parameters of HDL at body temperature. Rate zonal ultracentrifugation indicated that the HDL(2)/HDL(3) ratio fell by 28% (P < 0.05) on the polyunsaturated fat diet. In addition to the above, this diet reduced plasma apoA-I by 21% (P < 0.01). No change was seen in the fractional catabolic rate or the distribution of the apoprotein between intravascular and extravascular compartments on the two diets. However, when compared with the saturated diet, the synthetic rate of apoA-I was reduced by 26% during polyunsaturated fat feeding. The results show that polyunsaturated fat alters the chemical composition, thermotropic properties, and subfraction distribution of HDL without changing the fractional rate of catabolism of their major protein, apoA-I.These findings deserve careful consideration in determining the applicability and efficacy of polyunsaturated fat diet therapy in the prevention of atherosclerosis in man.

Effects of Nicotinic Acid Therapy on Plasma High Density Lipoprotein Subfraction Distribution and Composition and on Apolipoprotein A Metabolism

This report describes the effects of pharmacologic doses (3 g/d) of nicotinic acid on the plasma distribution and chemical composition of the high density lipoprotein (HDL) subfractions HDL(2) and HDL(3) and examines the influence of the drug on the metabolism of the major HDL apoproteins, apolipoproteins A-I (ApoA-I) and A-II (Apo-II). The drug lowered plasma cholesterol (15%, P < 0.05) and triglyceride (27%, P < 0.01); the former effect a result of a fall in the amount of cholesterol associated with very low density lipoproteins (31%, P < 0.02) and low density lipoproteins (36%, P < 0.02). Conversely, it raised plasma HDL cholesterol (23%, P < 0.05) and increased (by 345%) the plasma HDL(2):HDL(3) ratio. The latter derived from an absolute increment (646%) in circulating HDL(2), coupled with a fall (47%) in HDL(3). This change was not associated with major alterations in the overall cholesterol (free and esterified), triglyceride, phospholipid, or protein content of the subfractions; however, it was accompanied by substantial changes in their protein composition. In particular, the molar ratio of ApoA-I:ApoA-II in HDL(3) declined from 2.7:1 to 2.1:1 during nicotinic acid treatment.Significant perturbations of ApoA-I and ApoA-II metabolism accompanied the drug-induced HDL subfraction redistribution. Specifically, the plasma concentration of ApoA-I rose by 7% (P < 0.05) because of a decrease in its fractional catabolic rate. Moreover, whereas before treatment 6 and 94% of the plasma ApoA-I circulated with HDL(2) and HDL(3), after commencement of nicotinic acid therapy this distribution became 49 and 51% in HDL(2) and HDL(3), respectively. ApoA-II was found mainly in HDL(3), both before and during nicotinic acid treatment. Administration of the drug caused a 14% reduction in its plasma concentration (P < 0.05), which derived principally from a fall (22%, P < 0.01) in its synthetic rate. These data suggest that the effects of nicotinic acid on the HDL subfraction distribution may be mediated via (a) net transfer of ApoA-I from HDL(3) to HDL(2) and (b) a reduction in ApoA-II synthesis. Our present understanding of the association between HDL and atherosclerosis indicates that such changes may have prophylactic value in the prevention of coronary artery disease.

Control of 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity in Cultured Human Fibroblasts by Very Low Density Lipoproteins of Subjects with Hypertriglyceridemia

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 mug of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 mug of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively different from the cellular defect found in familial hypercholesterolemia, since the regulation of HMG-CoA reductase activity is normal in Type III fibroblasts. The metabolic defect in hypertriglyceridemia is related to the triglyceriderich lipoproteins which, free of other lipoproteins, have an enhanced ability to interact with cultured fibroblasts to regulate HMG-CoA reductase activity. These studies suggest that, in hypertriglyceridemia, there is a mechanism for direct cellular catabolism of VLDL which is not functional for normal VLDL.

The in vitro interaction of human apolipoprotein A-I and high density lipoproteins☆

Abstract Radioiodinated human apolipoprotein A-I, when incubated with plasma lipoproteins, associates exclusively with high density lipoproteins. It does not interact with very low density lipoproteins or low density lipoproteins. Binding is rapid, being complete within 10 min, and is not affected by variation of pH within the range 6.0–9.5 or of temperature over the range 0°–37°C. At equimolar concentrations of apolipoprotein A-I and high density lipoproteins, 0.58 mol of the apoprotein bind per mol of high density lipoproteins. Binding increases progressively with apolipoprotein A-I concentration up to an apolipoprotein A-I : high density lipoprotein molar ratio of 20 : 1, when each mol of lipoprotein binds 7.62 mol of apoprotein. Interaction of apolipoprotein A-I with high density lipoproteins displaces endogenous apolipoprotein A-I from the particle to a maximum of 2 mol of apolipoprotein A-I per mol of high density lipoproteins. This displacement occurs at an apoprotein : lipoprotein molar ratio of 10 : 1. The rest of the endogenous apolipoprotein A-I is not displaceable, even when the exogenous apoprotein : lipoprotein ratio is increased to 20 : 1. Incubation of apolipoprotein A-I and high density lipoproteins at molar ratios of 0.3 or less results in a mol for mol exchange of exogenous with endogenous apolipoprotein A-I on the lipoprotein. The half-life of 131I-labeled apolipoprotein A-I/high density lipoproteins in rats and rabbits is 15 h and 1.40 days respectively; the material decays exponentially from the bodies of these animals. More than 94% of the radioactivity in their plasma is associated with a component of hydrated density 1.063–1.21 kg/l. When injected into humans, the radioiodinated apoprotein incorporated into high density lipoproteins by in vitro incubation is cleared from the plasma 20% faster than endogenous apolipoprotein A-I in high density lipoproteins.

Metabolism of apolipoproteins A‐I and A‐ll and its influence on the high density lipoprotein subfraction distribution in males and females

Abstract. Rate zonal ultracentrifugation of plasma samples from ten healthy age‐matched volunteers (five males, five females) indicated that the high density lipoprotein subfraction ratio (HDL2:HDL3) in females was significantly higher than in males. The cause of this phenomenon was investigated by simultaneous examination of the metabolism of the major HDL apoproteins (apoA‐I and apoA‐II) in both groups. The results show that there is no significant sex‐related difference in the plasma pool size, fractional catabolic rate, or synthetic rate of either apoprotein. We conclude that the increased HDL2:HDL3 ratio in females versus males does not derive from measurable differences in the metabolic handling of either apoprotein.

Apolipoprotein B metabolism in normal, type IV, and type V hyperlipoproteinemic subjects

Abstract Apolipoprotein B (apoB) metabolism was investigated in four normal, three type IV, and three type V hyperlipoproteinemic subjects. Following injection of autologous radioiodinated very low density lipoprotein (VLDL) the rate of clearance of the apoprotein from this particle and its subsequent appearance in low density lipoprotein (LDL) was measured by frequent apoB specific activity determinations over an 11-day period. The resultant data were analyzed using the SAAM 27 computer program. In the normal subjects, more than 95% of the injected VLDL apoB was rapidly transferred to the LDL density range and accounted for all LDL apoB synthesis in that group. The plasma VLDL apoB concentration in the type IV group was, on average, five times the normal level. This resulted primarily from a doubling of the VLDL apoB synthetic rate associated with a defective or saturated catabolic mechanism. Only 60% of this material subsequently appeared in LDL, while the remainder was catabolized via an LDL-independent pathway. The turnover parameters of LDL apoB were normal in the type IV patients. Type V hyperlipoproteinemic subjects exhibited a 12- to 35-fold increase in plasma VLDL apoB concentration over normal. This again derived from increased VLDL apoB synthesis in the presence of defective removal of the apoprotein; the fractional catabolic rate of VLDL apoB in this group was 14% of the normal value. However, in contrast to the type IV patient data, more than 85% of the apoB in type V VLDL eventually appeared in LDL whose turnover rate was raised as a result of an increase in its catabolism; the fractional catabolic rate of LDL apoB in type V patients was four-fold above normal. The plasma LDL apoB pool size was substantially reduced in these subjects. This study shows that in hyperlipoproteinemic pheno-types IV and V there exist multiple anomalies of apoB metabolism affecting both VLDL and LDL.

Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Chemical and physical properties.

Subfractions of CLDL (VLDL), Sf 100-400; CLDL2, Sf 60--100; VLDL3, Sf 20--60) and LDL (LDL), Sf 12--20; LDL2, Sf 6--12; LDL3, Sf 3--6) were isolated from the plasma of three normal, three type III and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies were of normal composition but were increased in concentration in the order VLDL1 greater than VLDL2 greater than VLDL3. In the same subjects, although LDL2 was lowered and LDL3 increased, the total plasma LDL concentration was normal. All VLDL subfractions were elevated in the type III group, but in this case VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL1 and was attributed to a substantial alteration in the cholesteryl ester : triglyceride ratio in the particle. A similar argument was used to explain thction in normal or type IV subjects. Particle diameters, determined by laser light-scattering spectroscopy were in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship between the physical and metabolic properties of VLDL and LDL subfractions in type III and IV hyperlipoproteinemia.

Comparative studies on plasma low density lipoproteins from pig and man.

Abstract 1. 1. Low density lipoproteins (LDL) were isolated from pig plasma by ultracentrifugal flotation in KBr between densities 1·020 and 1·060 g/ml (LDL 1 ) and between 1·060 and 1·090 g/ml (LDL 2 ). 2. 2. The chemical, immunological and physical properties of LDL 1 and LDL 2 were compared with each other and with human LDL. 3. 3. LDL 1 and LDL 2 were delipidated and fractionated by chromatography on Sephadex G-150 in 0·1 M Tris-HC1, pH 8·0, containing 2 mM sodium decyl sulfate. ApoLDL 1 and apoLDL 2 were immunochemically identical, had only minor differences of amino acid composition and had indistinguishable circular dichroic spectra. The apoproteins were very similar in amino acid composition and circular dichroic spectra to human apoLDL. 4. 4. Our findings strongly suggest that the apoproteins from pig LDL 1 and LDL 2 are identical and that this protein is similar but not identical to apoLDL from man.

Effects of dietary saturated and polyunsaturated fat on the metabolism of apolipoproteins A-I and B: Study of a patient with type IIb hyperlipoproteinaemia

The effects of dietary saturated and polyunsaturated fat on the metabolism of apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) were studied in a patient with type IIb hyperlipoproteinaemia. On the saturated fat diet, the rate of synthesis of very low density lipoprotein apoprotein B (VLDL-apoB) was approximately twice normal, accounting for the increased plasma VLDL pool in this subject. However, 54% of the synthesized VLDL-apoB was catabolized by a pathway independent of low density lipoproteins (LDL). The metabolic conversion rate of VLDL-apoB to LDL-apoB was normal in this subject and his expanded plasma LDL-apoB pool resulted, not from increased input of the apoprotein from VLDL, but from a decrease in its fractional clearance rate. On the polyunsaturated diet, there was a significant fall in the plasma cholesterol and triglyceride concentrations and a change in the fatty acid composition of all plasma lipoprotein fractions. These changes were accompanied by a decrease in the plasma concentrations of apoA-I and apoB which resulted from a reduction of apoprotein synthetic rate.

Effects of insulin, tolbutamide, and glucagon on activities of jejunal carbohydrate-metabolizing enzymes in humans

The activities of jejunal carbohydrate-metabolizing enzymes show adaptive drugs, and sex hormones. To learn whether insulin, tolbutamide, and glucagon had effects on these enzymes, we performed serial peroral jejunal biopsies in normal young men and in obese patients, before and after treatment with these agents. Jejunal mucosa was assayed for glycolytic enzyme activities, pyruvate kinase (PK), hexokinase (HK), and fructose-1,6-diphosphate aldolase (FDPA), and the nonglycolytic enzyme activity, fructose diphosphatase (FDPase). Insulin significantly increased the activity of jejunal PK (+48% change from control) and HK (+6%), decreased the activity of FDPase (-36%),and had no effect on FDPA. Glucagon had opposite effects; the activity of PK was decreased (-33%) and FDPase was increased (+50%). Tolbutamide significantly increased the activities of PK (+47%), HK (+14%), and FDPA (+7%), and decreased the activities of FDPase (-36%). The results of tolbutamide on glycolytic enzyme activities were independent of endogenous insulin. The data support the concept that jejunal carbohydrate-metabolizing enzymes in man respond to hormones and drugs similar to responses observed in rat liver. This is important because it now gives us a means of studying the actions of these hormones directly in human tissue.