Shlomo Eisenberg

The metabolism of very low density lipoprotein proteins I. Preliminary in vitro and in vivo observations

1. n1. The metabolic fate of the apoproteins of very low density lipoprotein was studied by labeling the protein moiety of very low density lipoprotein with 125I. 46–57% of the label bound to low density lipoprotein (d 1.019–1.063) apoprotein and 31–44% attached to apolipoprotein glutamic acid, apolipopotein alanine1 and apolipropotein alanine2. Following in vitro incubation of labeled very low density lipoprotein in plasma, or 10 min after intravenous injection into humans, labeled apolipoprotein glutamic acid, apolipoprotein alanine1 and apolipropotein alanine2 transferred to other lipoproteins, mainly high density lipoprotein. Low density lipoprotein apoprotein, in contrast, was confined to the very low density lipoprotein density range under these conditions. n n2. n2. During the first 24 h after injection of 125I-labeled very low density lipoprotein to humans, the decay rate of low density lipoprotein apoprotein from very low density lipoprotein was faster than that of apolipoprotein glutamic acid, apolipoprotein alanine1 and apolipoprotein alanine2. During this interval, radioactivity accumalated first in a lipoprotein of intermediate density (d 1.006–1.019) and later in low density lipoprotein (d 1.019–1.063). It is suggested that apolipoprotein glutamic acid, apolipoprotein alanine1 and apolipoprotein alanine2 exchange readily between lipoproteins, but the low density lipoprotein apoprotein moiety of very low density lipoprotein moves unidirectionally from very low density lipoprotein to low density lipoprotein through a lipoprotein of intermediate density (d 1.006–1.019).

The metabolism of very low density lipoprotein proteins: II. Studies on the transfer of apoproteins between plasma lipoproteins

Abstract 1. 1. Apolipoprotein-glutamic acid (apoLP-Glu) and apolipoprotein-alanine (apoLP-Ala), small molecular weight apolipoproteins, readily transfer in vitro from very low density lipoprotein to other lipoproteins. Their transfer to high density lipoprotein always exceeds that to low density lipoproteins, and is proportional to the concentration of lipoproteins present in the incubation mixture. A similar transfer of radioactivity occurs in vivo , and is proportional to both plasma triglyceride and high density lipoprotein cholesterol levels. The transfer of apoLP-Glu and apoLP-Ala between very low density and high density lipoproteins is bidirectional, and thus represents, at least in part, an exchange phenomenon. In contrast, the apoprotein moiety of low density lipoprotein does not participate in this type of transfer. 2. 2. Apolipoproteins can be separated into groups following their reassociation properties with lipids and lipoproteins. ApoLP-Glu and apoLP-Ala reassociate with all plasma lipoproteins, predominantly very low density and high density lipoprotein. Apolipoprotein-glutamine 1 (apoLP-Gln 1 ) and apolipoprotein-glutamine 2 (apoLP-Gln 2 ) reassociate primarily with their parent lipoprotein, high density lipoprotein. Representative proteins of both groups however, reassociate with lipid (lecithin or triglyceride). The recombination of apoproteins with lipoproteins thus may be specific and involve a process of “recognition” of the lipoprotein by the apoprotein. This specificity may not be involved in the simple recombination of apolipoproteins and lipids. These observations may explain the distribution of apoproteins among plasma lipoproteins and provide insight into their metabolic fate.

Dietary fat clearance in normal subjects is regulated by genetic variation in apolipoprotein E.

Apolipoprotein E (apo E) plays an important role in receptor mediated clearance of lipoprotein particles from plasma. Common genetic variation in apo E exists with three alleles coding for proteins called E2, E3, and E4. In in vitro receptor binding assays, E2 binds poorly, whereas E3 and E4 function normally. Recently, the apo E phenotype has been shown to have an effect on low density lipoprotein (LDL) cholesterol levels with levels in subjects with E2 lower and E4 higher than E3. We have examined the effect of the apo E polymorphism on dietary fat clearance using the vitamin A-fat loading test, which specifically labels intestinally derived lipoproteins with retinyl palmitate (RP). 27 normal subjects were studied, 10 with E3/3, 9 with E3/2, 7 with E4/3, and 1 with E4/4. After a vitamin A-containing fatty meal, postprandial RP concentrations were measured in chylomicron (Sf greater than 1,000) and nonchylomicron (Sf less than 1,000) fractions for 14 h. Compared with E3/3 subjects, E3/2 subjects had a significantly higher nonchylomicron RP concentration (P less than 0.05) (peak heights and areas below the curves) indicating slower clearance and the E4/3, E4/4 group had a significantly lower nonchylomicron RP concentration (P less than 0.05) indicating faster clearance. The clearance in the latter group was twice that of E3/2 subjects (P less than 0.01). Thus, heterozygosity for the defective form of apo E, E2, delays, and the surprising presence of a functionally normal allele, E4, increases clearance. This apo E effect on exogenous fat clearance may explain the recently described effect of the apo E phenotypes on LDL cholesterol levels.

High Density Lipoprotein Metabolism in Man

The turnover of (125)I-high density lipoprotein (HDL) was examined in a total of 14 studies in eight normal volunteers in an attempt to determine 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 under 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) and ingesting the same isocaloric balanced diet. The decay of autologous (125)I-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 diet and was only slightly decreased by nicotinic acid treatment. These perturbations had effects on HDL catabolic pathways that were reciprocal in many respects. With an 80% carbohydrate diet, the rate of catabolism from the plasma compartment rose by a mean of 39.1%; with nicotinic acid treatment, it fell by 42.2%. Changes in the rate of catabolism from the second compartment were generally opposite those in the rate of catabolism from the plasma compartment, suggesting that these two catabolic pathways may be reciprocally regulated.

On the metabolic conversion of human plasma very low density lipoprotein to low density lipoprotein.

Abstract The relationship of 125I-labelled apoproteins of very low density lipoprotein to that of other lipoproteins was studied in humans during steady-state conditions and following heparin injection. Heterogeneous metabolism of very low density lipo-protein apoproteins in normal individuals was apparent during steady-state conditions. Radioactivity transferred to high density lipoprotein immediately in vivo. With time radioactivity also transferred to an intermediate density lipoprotein (d = 1.006−1.019) and thereafter to low density lipoprotein (d = 1.019−1.063). Labelled apoLP-glu and apoLP-ala, but not labelled apoprotein of low density lipoprotein (apoLDL), disappeared initially from very low density lipoprotein (10 min after the injection). At later time intervals, the rate of disappearance of labelled apoLDL from very low density lipoprotein ( t 1 2 = 2−4 h ), far exceeded that of labelled apoLP-glu and apoLP-ala ( t 1 2 = 17–18 h ). Heparin affected primarily the distribution of apoprotein radioactivity between very low density lipoprotein and high density lipoprotein and among very low density lipoprotein subfractions. 45 min after heparin injection, a net transfer of more than 50 % of labelled apoLP-glu and apoLP-ala from very low density lipoprotein to high density lipoprotein occurred. Almost no change in content of labelled apoLDL in very low density lipoprotein occurred during this interval. Both very low density lipoprotein mass and radioactivity accumulated in a very low density lipoprotein subtraction of Sf 20–60, which contained about twice as much 125I-labelled apoLDL as 125-labelled apoLP-glu and 125I-labelled apoLP-ala. Further decay of labelled apoLDL to lipoproteins of density 1.006–1.019 and then to low density lipoprotein, occurred later, with peak activities 6 and 24 h, respectively, after heparin injection. 6 h after heparin, when plasma triglyceride had increased toward pre-heparin levels, labelled apoLP-glu and apoLP-ala retransferred to very low density lipoprotein (mainly to triglyceride rich, newly synthesized Sf > 100 molecules). During the conversion of very low density lipoprotein molecules of Sf 100–400 (mol. wt 20 · 106-130 · 106) to low density lipoprotein (mol. wt 2.2 · 106), all apoLDL moiety of very low density lipoprotein is preserved. In contrast, more than 95% of apoLP-ser, apoLP-glu and apoLP-ala, more than 99% of triglyceride, and more than 85% of the very low density lipoprotein cholesterol and phospholipids are removed. These observations suggest that concomitant with continuous triglyceride hydrolysis, apoLP-glu and apoLP-ala leave the very low density lipoprotein density range resulting in molecules relatively poor in triglyceride and relatively rich in apoLDL. These molecules occupy a notation rate range of Sf 12–60 and are trans-formed ultimately to low density lipoprotein, presumably by a different mechanism.

Dietary polyunsaturated fats of the W-6 and W-3 series reduce postprandial lipoprotein levels. Chronic and acute effects of fat saturation on postprandial lipoprotein metabolism.

The chronic and acute effects of different types of dietary fat on postprandial lipoprotein metabolism were studied in eight normolipidemic subjects. Each person was placed for 25 d on each of three isocaloric diets: a saturated fat (SFA), a w-6 polyunsaturated fat (w-6 PUFA) and a w-3 polyunsaturated fat (w-3 PUFA) diet. Two vitamin A-fat loading tests were done on each diet. The concentrations in total plasma and chylomicron (Sf greater than 1,000) and nonchylomicron (Sf less than 1,000) fractions of retinyl palmitate (RP) were measured for 12 h postprandially. Compared with the SFA diet, the w-6 PUFA diet reduced chylomicron and nonchylomicron RP levels 56 and 38%, respectively, and the w-3 PUFA diet reduced these levels 67 and 53%, respectively. On further analysis, the main determinant of postprandial lipoprotein levels was the type of fat that was chronically fed, which appeared to mediate its effect by changing the concentration of the endogenous competitor for the system that catabolizes triglyeride-rich lipoproteins. However, there was a significant effect of the acute dietary fat load, which appeared to be due to a differential susceptibility to lipolysis of chylomicrons produced by SFA as opposed to PUFA fat loads. The levels of postprandial lipoproteins are determined by the interaction of these chronic and acute effects.

Increased apo A-I and apo A-II fractional catabolic rate in patients with low high density lipoprotein-cholesterol levels with or without hypertriglyceridemia.

Low HDL-cholesterol (HDL-C) levels may elevate atherosclerosis risk, and often associate with hypertriglyceridemia (HTG); however, the metabolic causes of low HDL-C levels with or without HTG are poorly understood. We studied the turnover of radioiodinated HDL apolipoproteins, apo A-I and apo A-II, in 15 human subjects with low HDL-C, six with normal plasma TG levels (group 1) and nine with high TG (group 2), and compared them to 13 control subjects with normal HDL-C and TG levels (group 3). The fractional catabolic rate (FCR) was equally elevated in groups 1 and 2 vs. group 3 for both apo A-I (0.313 +/- 0.052 and 0.323 +/- 0.063 vs. 0.245 +/- 0.043 pools/d, P = 0.003) and apo A-II (0.213 +/- 0.036 and 0.239 +/- 0.037 vs. 0.185 +/- 0.031 pools/d, P = 0.006). Thus, high FCR characterized low HDL-C regardless of the presence or absence of HTG. In contrast, transport rate (TR) of apo A-I did not differ significantly among the groups and the apo A-II TR differed only between groups 2 and 3 (2.15 +/- 0.57, 2.50 +/- 0.39, and 1.83 +/- 0.48 mg/kg per d for groups 1 to 3, respectively, P = 0.016). Several HDL-related factors were similar in groups 1 and 2 but differed in group 3, as with FCR, including the ratio of lipoprotein lipase to hepatic lipase activity (LPL/HL) in post-heparin plasma, the ratio of the HDL-C to apo A-I plus apo A-II levels, and the percent of tracer in the d greater than 1.21 fraction. In linear regression analysis HDL-C levels correlated inversely with the FCR of apo A-I and apo A-II (r = -0.74, P less than 0.0001 for both). Major correlates of FCR were HDL-C/apo A-I + apo A-II, LPL/HL, and plasma TG levels. We hypothesize that lipase activity and plasma TG affect HDL composition which modulates FCR, which in turn regulates HDL-C. Thus, HTG is only one of several factors which may contribute to elevated FCR and low HDL-C. Given the relationship of altered HDL composition with high FCR and low HDL-C levels, factors affecting HDL composition may increase atherosclerosis susceptibility.

A low-fat diet decreases high density lipoprotein (HDL) cholesterol levels by decreasing HDL apolipoprotein transport rates.

Diets that reduce atherosclerosis risk lower levels of HDL cholesterol (HDL-C), but the significance of this is unclear. To better understand the mechanism of this phenomenon we studied the turnover of HDL apolipoproteins A-I and A-II in 13 subjects on two contrasting metabolic diets. Upon changing from high to low intake of saturated fat and cholesterol the mean HDL-C decreased 29% from 56 +/- 13 (SD) to 40 +/- 10 mg/dl, while apo A-I levels fell 23% from 139 +/- 22 to 107 +/- 22 mg/dl (both P less than 0.001). Mean apo A-II levels did not change. The fractional catabolic rate (FCR) of apo A-I increased 11% from 0.228 +/- 0.048 to 0.254 +/- 0.063 pools/d, while its absolute transport rate (TR) decreased 14% from 12.0 +/- 2.7 to 10.3 +/- 3.4 mg/kg per d (both P = 0.005). The decrease in HDL-C and apo A-I levels correlated with the decrease in apo A-I TR (r = 0.79 and 0.83, respectively; P less than 0.001), but not with the increase in apo A-I FCR (r = -0.04 and -0.02, respectively). In contrast, within each diet the HDL-C and apo A-I levels were inversely correlated with apo A-I FCR both on the high-fat (r = -0.85 and -0.77, P less than 0.001 and = 0.002, respectively) and low-fat diets (r = -0.67 and -0.48, P = 0.012 and 0.098, respectively) but not with apo A-I TR. In summary, diet-induced changes in HDL-C levels correlate with and may result from changes in apo A-I TR. In contrast, differences in HDL-C levels between people on a given diet correlate with and may result from differences in apo A-I FCR. Therefore, the mechanism of dietary effects on HDL levels differs substantially from the mechanism explaining the differences in levels between individuals on a fixed diet. In assessing coronary heart disease risk, it may be inappropriate to conclude that diet-induced decreases in HDL are equivalent to low HDL within a given diet.

Different patterns of postprandial lipoprotein metabolism in normal, type IIa, type III, and type IV hyperlipoproteinemic individuals. Effects of treatment with cholestyramine and gemfibrozil.

To study exogenous fat metabolism, we used the vitamin A-fat loading test, which specifically labels intestinally derived lipoproteins with retinyl palmitate (RP). Postprandial RP concentrations were followed in total plasma, and chylomicron (Sf greater than 1,000) and nonchylomicron (Sf less than 1,000) fractions. In normal subjects postprandial lipoproteins were present for more than 14 h, and chylomicron levels correlated inversely with lipoprotein lipase activity and fasting high density lipoprotein (HDL) cholesterol levels and nonchylomicron levels correlated inversely with hepatic triglyceride lipase activity. The main abnormality in type IV patients was a 5.6-fold increase in the chylomicron fraction, whereas in type III patients it was a 6.4-fold increase in nonchylomicrons. Type IIa patients had abnormally low chylomicron fractions. In type IV patients gemfibrozil decreased, whereas in type IIa patients cholestyramine increased the chylomicron fraction 66 and 88%, respectively. This study demonstrates an unexpectedly large magnitude and long duration of postprandial lipemia in normal subjects and patients. These particles are potentially atherogenic, and their role in human atherosclerosis warrants further study.

Very Low Density Lipoprotein: METABOLISM OF PHOSPHOLIPIDS, CHOLESTEROL, AND APOLIPOPROTEIN C IN THE ISOLATED PERFUSED RAT HEART

The fate of rat plasma very low density lipoprotein (VLDL) constituents was determined in the isolated perfused rat heart. VLDL was labeled with [(14)C]palmitate, (32)P-phospholipids, [(3)H] cholesterol, and (125)I-apolipoprotein C (apoC). Perfusions were performed with an albumin-containing buffer and without plasma. Radioactivity was followed in fractions of d < 1.019, d 1.019-1.04, d 1.04-1.21, and d > 1.21 g/ml, prepared by ultracentrifugation.VLDL triglycerides were progressively hydrolyzed to fatty acids (10-120-min perfusions). Concomitantly, phospholipids, cholesterol (predominantly unesterified), and apoC were removed from the VLDL to all other fractions. About 30-35% of the phosphatidylcholine was hydrolized to lysophosphatidylcholine and was recovered at d > 1.21 g/ml. The phosphatidylcholine-and triglyceride-hydrolyzing activities were confined to membrane supported enzyme(s). The other 60-65% of the phosphatidylcholine was removed unhydrolyzed and was found in fractions of d 1.019-1.04 (10-15%), d 1.04-1.21 (25-30%), and d > 1.21 g/ml (15-20%). [(32)P]Sphingomyelin accumulated at the fraction of d 1.04-1.21 g/ml. Unesterified cholesterol was found in the fraction of d 1.04-1.21 g/ml. ApoC was recovered predominantly in fractions of d 1.04-1.21 (50-60%) and d > 1.21 g/ml (30-40%). Cholesteryl esters were associated with VLDL during the hydrolysis of 50-70% of the triglycerides, but with advanced lipolysis, appeared in higher densities, mainly d 1.019-1.04 g/ml. The fraction of d 1.04-1.21 g/ml, (containing phosphatidylcholine, sphingomyelin, unesterified cholesterol, and apoC) contained by negative staining, many disk-like structures. The study demonstrated that removal of surface constituents (phospholipids, unesterified cholesterol, and apoC) during lipolysis of VLDL is an intrinsic feature of the lipolytic process, and is independent of the presence of plasma. It also indicated that surface constituents may be removed in a particulated form.