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Metabolism of postprandial lipoproteins
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pools of VLDL and HDL should not be affected by the in vitro incubation, Sv, = Svn
and
SHn = SHn = 0
The SA in the exchangeable pools of VLDL and HDL will reach a new equilibrium value which reflects fPv, the fraction of the exchangeable mass of apoC-III which is associated with VLDL, i.e., SHe =
ave
= fpvSve
The overall apoC-III SA in each lipoprotein class is a weighted average of the SA in the equilibrating and nonequilibrating pool. Sv = Svefv + Svn(1 - f v )
where fv =
mass of equilibrating VLDL apoC-III total mass of apoC-III in VLDL S . = SIaefH
where fH =
mass of equilibrating HDL apoC-III total mass of apoC-III in HDL
The ratio R of the apoC-III SA between VLDL and HDL as obtained from the decay curves is defined as: R = Sv/SH
[28] M e t a b o l i s m o f P o s t p r a n d i a l L i p o p r o t e i n s By ALAN R. TALL
Overview Although a significant portion of each day is spent in the postprandial state, there have been relatively few studies of the effects of a meal on lipoprotein metabolism. The absorption and transport of dietary fat lead to significant perturbations of the plasma lipoproteins. The changes reflect both the addition of newly synthesized, triglyceride-rich particles (chylomicrons) to the plasma, and also changes in the other lipoproteins, especially high-density lipoproteins (HDL), probably resulting from the meMETHODS IN ENZYMOLOGY, VOL. 129
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
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tabolism of chylomicrons. The changes in the d < 1.006 fraction (containing chylomicrons) and in the HDL after a fatty meal may be regarded as an important physiological model of the transfer of lipid and apolipoprotein components between the plasma lipoproteins, especially the transfer or exchange of surface and core components between triglyceride-rich lipoproteins and HDL. Studies of the metabolism of postprandial lipoproteins may shed light on the importance of different particles in the process of atherogenesis. The changes in the plasma lipoproteins that occur in the postprandial state are quite different in different individuals. Otherwise subtle abnormalities of plasma lipoprotein metabolism may be highlighted in the postprandial state. Changes in the postprandial lipoprotein profile in individuals susceptible to atherosclerosis may provide insights into the mechanisms of involvement of apoB-containing particles of HDL in the process of atherogenesis. This chapter will focus on the metabolism of postprandial lipoproteins in humans, especially the acute response to dietary fat or alcohol. The emphasis Will be on the metabolism of chylomicrons and HDL. Studies in animals or in vitro will be referred to when they are thought to elucidate human physiology. Methodological aspects of studying postprandial lipoproteins will be discussed. Methodological problems, especially the paucity of nonexchangeable markers of newly synthesized lipoproteins or apolipoproteins, have been a major factor limiting a detailed understanding of the genesis of changes noted in the postprandial plasma lipoprotein profiles. Study Design The majority of studies of postprandial lipoproteins in humans have measured sequential changes in the plasma following single or multiple oral fat loads. Triglyceride may be given as corn oil, olive oil, safflower oil, cream, or butter or incorporated into a meal. There are few studies which have compared the acute effects of different types of dietary fat, such as saturated versus polyunsaturated fats. Significant changes in the plasma lipids or apolipoproteins may be observed with doses of 0.75-3.0 g triglycerides/kg body weight. Some subjects experience nausea and diarrhea after ingesting fat alone. When fat is given as part of a meal it is possible that the presence of carbohydrate or protein will alter the subsequent metabolism of triglyceride. There have been few systematic studies of the effects of other dietary components on the response to a triglyceride load. Alcohol has a marked effect on the subsequent metabolism of
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dietary fat. 1 Alcohol has been given in several forms; there is no information on the effects of the nonalcoholic moieties of different types of alcoholic beverages on postprandial lipoprotein metabolism. One of the difficulties of studying changes in the lipoproteins after an oral fat load is the variable time between the ingestion of fat and the peak response of the plasma lipoproteins. This variability results primarily from differences in gastric emptying rate. In order to catch the peak changes in plasma lipoproteins it is necessary to take multiple blood samples. The peak in plasma triglycerides usually occurs about 5 to 6 hr after ingestion of 1.5 g/kg of oral fat, and the major alterations in the plasma lipoproteins (such as the HDL) may be observed at 5 to 10 hr. The variability in response necessitates the study of several individuals. More controlled methods for studying the metabolism of postprandial lipoproteins have involved the intravenous infusion of radiolabeled chylomicrons 2 or intraduodenal fat infusion. 3 These approaches have the advantage that they provide kinetic data for chylomicron removal. Human chylomicrons may be radiolabeled in the protein moiety by the IC1 method. 4 Optimally, human chylomicrons can be obtained from thoracic duct lymph. Unfortunately, thoracic duct cannulation is not frequently performed for therapeutic purposes. Human chylomicrons may also be prepared from other sources such as chylous pleural effusions or from the urine of subjects with chyluria. However, the supply of chylomicrons from these sources is irregular and the chylomicrons have probably already undergone extensive alteration due to contact with plasma components. Grundy and Mok 3 have studied the metabolism of chylomicrons during a constant intraduodenal fat infusion. This method results in a steady rate of fat absorption and constant plasma triglyceride levels and allows the kinetics of triglyceride removal to be determined. The subjects are intubated with a nasogastric tube the night before the study. The next morning the tube is positioned in the region of the ampulla of Vater, under X-ray guidance, then a constant infusion of triglyceride or other lipid (e.g., a monoglyceride/lecithin mixture) is begun. A triple lumen tube may be used to determine the amount of lipid absorbed across a segment of small intestine. In normal subjects, small intestinal triglyceride absorption should be more than 95%. After a 5-hr infusion of triglycerides, the i D. E. Wilson, P. H. Schreibman, A. C. Brewster, and R. A. Arky, J. Lab. Clin. Med. 75, 264 (1970). 2 p. j. Nestel, J. Clin. Invest. 43, 943 (1964). 3 S. M. Grundy and H. Y. I. Mok, Metabolism 25, 1225 (1976). 4 D. W. Bilheimer, S. Eisenberg, and R. I. Levy, Biochim. Biophys. Acta 260, 212 (1972).
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plasma triglyceride level becomes constant, and the study is begun. With steady plasma levels the removal rate of triglyceride is equal to the infusion rate (mg/kg/hr), assuming 100% absorption. From the turnover rate and the increment of chylomicron triglyceride, the tv2 of chylomicron triglyceride can be determined as follows: tv2 = 0.693/K, where K = input of chylomicron TG (mg/kg/hr)/plasma pool of chylomicron TG. The latter is determined from the plasma triglyceride concentration and the estimated plasma volume ( = 927 + 31.47 × body wt). The prior state of the patient is an important variable in determining the metabolism of postprandial lipoproteins. In normal subjects plasma triglycerides may continue to fall significantly during the morning after a fast from 11 PM; fasting for a full 14 hr before the onset of the study is advisable. The patient's prior metabolic state has an important influence on the response to a meal. Weight loss, alcohol intake, ~ exercise: and probably a variety of other variables may influence the individual's response to a fatty meal. Ideally, studies should be undertaken in the metabolic ward setting where caloric intake, diet composition, and exercise are all well-controlled variables. However, valuable information has been obtained under less rigorously controlled conditions. Preexisting hyperlipidemia has a large influence on the changes in plasma lipoproteins after ingestion of a fatty meal. Although several studies have contrasted the responses of broad groups of hyperlipidemic patients with those of normal controls, there is a paucity of information on the response of patients with genetically or otherwise well-defined hyperlipidemias. Even patients who are defined as normolipidemic, on the basis of a plasma triglyceride level of less than 200 mg/dl, have quite different responses depending on their preexisting plasma triglyceride level. This will be discussed in detail below. Preparation of Lipoproteins Chylomicrons are generally isolated as plasma lipoproteins of Sf > 400, as described. 6 There is evidence that triglyceride-rich lipoproteins of Sf < 400 may make a lesser contribution to the hypertriglyceridemia following a fatty meal, 6 especially during infusion of lecithin. 7 Plasma lipoproteins in the postprandial state may be isolated by procedures reviewed elsewhere in this volume. It is worth noting that during alimentary 5 j. R. Patsch, J. B. Karlin, L. W. Scott, L. C. Smith, and A. M. Gono, Proc. Natl. Acad. Sci. U.S.A. 80, 1449 (1983). 6 T. G. Redgrave and L. A. Carlson, J. Lipid Res. 20, 217 (1979). F. U. Beil and S. M. Grundy, J. Lipid Res. 21, 525 (1980).
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lipemia lipid enrichment of HDL species results in significant alterations of the density of the major subclasses of HDL. 8 It appears that a significant portion of lipid-enriched HDL-3 particles float at d 1.125 g/ml.8 Thus, the changes in HDL may in general be best defined by methods which make no assumptions about traditional HDL-2 or HDL-3 designations, such as isopycnic density gradient techniques. In the future it is likely that valuable information about changes in HDL subclasses will be obtained by noncentrifugal methods, such as those employing antibody affinity chromatography for isolation of apoA-I-containing particles from plasma. 9 A major problem in the study of the metabolism of postprandial lipoproteins has been the paucity of nonexchangeable lipid or protein markers to denote the fate of individual lipoprotein particles during lipemia. Although lipid core markers have been employed as a means of following chylomicron metabolism, triglycerides, cholesteryl esters, and retinyl esters are all susceptible to cholesteryl ester transfer protein-mediated exchange with other lipoprotein core lipids.l° Although these lipids may be used to follow the fate of chylomicron particles in animals which have low lipid transfer protein activity (dogs, pigs, and rats), they are not reliable particle markers in species which have significant cholesteryl ester transfer protein activity (humans, rabbits, and monkeys). Estimates of the extent of core lipid exchange based on incubations of plasma may underestimate physiological exchange rates, since lipolysis enhances the activity of the cholesteryl ester transfer protein.11 Thus, the redistribution of chylomicron retinyl ester radioactivity into Sf < 400 lipoprotein classes in intact animals cannot be taken as evidence that chylomicron remnants contribute to Sf < 400 particles. The recent recognition that the lowmolecular-weight form of apoB is a nonexchangeable marker for intestinally derived particles ~2has great potential in allowing intestinally derived particles to be traced. For example, this has allowed identification of an intestinally derived subfraction of/3-VLDL which is particularly efficacious in delivering cholesterol to macrophages. 13 In future studies mea8 A. R. Tall, C. B. Blum, G. P. Forester, and C. Nelson, J. Biol. Chem. 257, 198 (1982). 9 j. p. McVicar, S. T. Kunitake, R. L. Hamilton, and J. P. Kane, Proc. Natl. Acad. Sci. U.S.A. 81, 1356 (1984). 10 R. E. Morton and D. B. Zilversmit, J. Lipid Res. 23, 1058 (1982). H A. R. Tall, D. Sammett, G. Vita, R. J. Deckelbaum, and T. Olivecrona, J. Biol. Chem. 259, 9587 (1984). 12 j. p. Kane, D. A. Hardman, and H. E. Paulus, Proc. Natl. Acad. Sci. U.S.A. 77, 2465 (1980). i3 M. R. Fainaru, R. W. Mahley, R. L. Hamilton, and T. L. Innerarity, J. LipidRes. 23, 702 (1982).
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surements of the low-molecular-weight apoB (particularly as specific immunochemical methods become available) should serve to determine if accumulating particles after a fatty meal are intestinally derived or hepatogenous. Also, the characterization of intestinally derived particles should provide insights into their role in atherogenesis. The lack of a marker for newly synthesized apoA-I has limited the understanding of how apoA-I becomes incorporated into the HDL fraction during lipemia. In humans plasma and HDL apoA-I rise after a fatty meal, but the incremental apoA-I has not been distinguishable from the preexisting apoA-I. A potentially useful marker of newly synthesized apoA-I might be its isoprotein-2, which is the secretory proprotein of apoA-I.14 The isoprotein-2 has a half-life of several hours in the plasma before being converted to the more basic isoproteins 4 and 5. J5Analysis of apoA-I isoforms in the HDL fraction after a fatty meal might provide some clue as to how newly synthesized apoA-I becomes incorporated into plasma HDL particles. Results of Studies Overview o f Chylomicron Metabolism Based on in Vitro Studies and Studies in Experimental Animals
Absorbed dietary fat is packaged into chylomicron particles containing a core of triglyceride and a surface film of phospholipids, cholesterol, apoB, and soluble apolipoproteins (apoA-I, apoA-II, apoA-IV, and apoCIII). The chylomicron triglyceride is rapidly hydrolyzed in peripheral tissues due to the action of lipoprotein lipase, a triglyceride hydrolase located on the surface of the capillary endothelium. Fatty acids and partial glycerides enter the tissues or are bound to albumin. During the process of lipolysis of chylomicrons certain surface materials (phospholipids, apoA-I, and apoA-II) are transferred into the HDL fraction. Conversely, apoE, apoC peptides, and cholesteryl esters are transferred from HDL to chylomicron remnants. Thus, relative to nascent chylomicrons, chylomicron remnants are enriched in cholesteryl esters and apoE and apoC peptides (especially apoC-III), and depleted in apoA-I and apoA-IV; recently we have found that chylomicron and VLDL remnants also bind cholesteryl ester transfer protein. The chylomicron remnant is rapidly 14 V. I. Zannis, D. M. Kurnit, and J. L. Breslow, J. Biol. Chem. 257, 536 (1982). ~5G. Ghiselli, E. J. Schaefer, S. Law, J. A. Light, and H. B. Brewer, Clin. Res. 31, 500A (1983).
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removed from the circulation owing to the action of a hepatic chylomicron remnant or apoE receptor.~6
Studies of Postprandial Lipoproteins in HumanshChylomicron Metabolism Using intravenously injected thoracic duct chylomicrons, Nestel showed that the half-life of chylomicron triglyceride in the circulation was about 5 min. 2 Grundy and Mok 3 obtained similar results using an intraduodenal infusion method. Nestel z showed a strong correlation between fasting triglyceride levels and the height of the response of plasma triglyceride to a fatty meal. There was an inverse correlation between the level of fasting plasma triglycerides and the rate of chylomicron metabolism both in hypertriglyceridemia (>200 mg/dl) and also within the normal range of plasma triglycerides (<200 mg/dl). Grundy and Mok 3 speculated that the slower removal of chylomicron triglyceride in subjects with fasting hypertriglyceridemia reflected competition of the chylomicrons with VLDL for removal mechanisms rather than a basic defect in the ability to remove chylomicrons, because with weight loss and a fall in the plasma triglyceride level, most subjects showed a marked improvement in chylomicron clearance. Thus, in many patients with hypertriglyceridemia a marked alimentary lipemic response may reflect a tendency to overproduce VLDL, rather than a primary defect in clearance of chylomicrons. Exceptions to this statement are patients with lipoprotein lipase or apoCII deficiency and perhaps a minority of other patients who have a predominant clearance defect. 3 Redgrave and Carlson 6 contrasted the response to lipemia of hypertriglyceridemic and normal subjects. They found that the more pronounced lipemia of hypertriglyceridemic subjects occurred in Se > 400 particles, and was largely due to presence of particles of increased size in these subjects. It is notable that in several studies increases in number or size of triglyceride-rich lipoproteins of both Sf > 400 and Sf < 400 have been noted during alimentary lipemia. 3,6 It is uncertain if this represents continued degradation of chylomicron remnants to smaller particle size, or competition between chylomicron remnants and hepatogenous VLDL for removal sites, resulting in accumulation of the latter, or increased hepatic VLDL secretion due to the load of dietary fat returning to the liver. It should be possible to determine if the increase in Sf < 400 parti16 R. W. Mahley, D. Y. Hui, T. L. Innerarity, and K. H. Weisgraber, J. Clin. Invest. 68,
1197 (1981).
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cles is due to hepatic or intestinal particles by determining the distribution of the low-molecular-weight apoB particle in the postprandial VLDL fraction. In patients infused with lecithin the major increase in triglyceridecarrying particles in the plasma occurred in the Sf 20-400 fraction, possibly due to absorption of lecithin over a longer length of small intestine than triglyceride,7 (distal small intestinal fat absorption tends to result in formation of smaller chylomicrons). Another possibility is that lecithin fatty acid was predominantly absorbed via the portal vein and that the Sf 20-400 particles were, in fact, hepatogenous VLDL. Thus, both the mode of intestinal absorption and the subject's baseline lipid metabolism influence the response of triglyceride-rich lipoproteins during lipemia. Changes in HDL. Havel et al.17 noted an increase in HDL phospholipids and protein during alimentary lipemia and speculated that these changes might reflect transfer of chylomicron surface materials into the HDL fraction. These workers also showed that during lipemia there is transfer of apoC peptides, including the activator peptide of lipoprotein lipase, apoC-II, from HDL to the chylomicron remnant fraction. More recently, we have performed detailed studies of the changes in HDL subfractions during alimentary lipemia, using primarily density gradient methods to separate HDL subclasses. These studies showed an increase in HDL phospholipids, cholesteryl esters, apoA-I, and apoA-II during lipemia; these changes were thought to reflect at least in part the transfer of these lipids and proteins from chylomicrons into HDL, as demonstrated in the r a t : Another potential mechanism could be increased intestinal HDL secretion during lipemia. Analysis of HDL by equilibrium density gradient ultracentrifugation showed the presence of a major and a lesser peak of HDL in fasting plasma, corresponding to the traditional HDL-3 and HDL-2 subclasses, respectively. During lipemia the most pronounced change was an increase in mass and a shift to lower density of the major peak of HDL-3, with similar but less marked changes in the HDL-2 peak. Analyses of molecular compositions suggested that the lipid transferred into HDL was added to preexisting particles, since the particles retained the same basic complement of apoA-I and apoA-II molecules but had additional lipid molecules (especially phospholipids). Although there was an increase in mass of HDL apoA-I and apoA-II the particle contents of apoA-I and apoA-lI were unchanged during lipemia, suggesting that apolipoprotein transfer causes interconversion of existing HDL species, or formation of new particles with the same content of apoA-I and apoA-II as existing species. An interesting recent report 5 has shown that in a group of normolipi17 R. J. Havel, J. P. Kane, and M. L. Kashyap, J. Clin. Invest. 52, 32 (1973).
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demic healthy young adults there was an inverse relationship between the magnitude of the plasma triglyceridemic response and the levels of fasting HDL-2 (r = -0.86), HDL-cholesterol (r = -0.61), and apoA-I (r = -0.46). In two subjects followed for 3 years, when HDL-2 levels rose or fell in response to exercise or the lack of it, the triglyceridemic response decreased or increased, respectively.
Potential Mechanisms of Lipid Transfer Processes during Lipemia In Vitro Studies of Lipid Exchange Processes. Our recent in vitro studies suggest that lipid exchange processes may be stimulated during lipemia.11 These studies show that lipolysis enhances the cholesteryl ester transfer protein-mediated movement of cholesteryl esters from H D L to triglyceride-rich lipoproteins. The latter were isolated from both fasting and lipemic plasma. 18The stimulation of cholesteryl ester transfer activity was related to the accumulation of lipolytic products, especially fatty acids in the remnant particle. It appears that small amounts of negatively charged fatty acids enhance the binding of the cholesteryl ester transfer protein to the remnant surface. Under several different circumstances enhancement or reduction of cholesteryl ester transfer activity was related to increased or decreased binding of cholesteryl ester transfer protein to VLDL or VLDL remnants, suggesting that the increased binding resulted in increased cholesteryl ester transfer into the triglyceride-rich particles. There is also theoretical evidence to support the concept that increased affinity of cholesteryl ester transfer protein for acceptor particles relative to H D L should increase cholesteryl ester transfer from H D L to the acceptor particles.19 The enhancement of cholesteryl ester transfer processes by lipolysis was greatly favored by high V L D L / H D L ratios. Thus, cholesteryl ester transfer processes should be more pronounced in subjects who experience higher levels of plasma triglyceride during lipemia. In summary, these in vitro studies suggest that cholesteryl ester transfer from H D L to triglyceride-rich particles during lipemia may be related to activities of lipoprotein or hepatic lipases, and to the magnitude of the rise in plasma triglyceride levels. In other studies we have shown that the transfer of phospholipids from triglyceride-rich lipoproteins to H D L is enhanced by a partially purified plasma phospholipid transfer protein, m This phospholipid transfer protein appears to be a different protein than the lipid transfer protein referred to a b o v e , z° However, the phospholipid transfer protein-mediated transfer of t8 A. R. Tall, unpublished results, 1984. 19 p. j. Barter and M. E. Jones, J. LipidRes. 21, 238 (1980). 2o A. R. Tall, E. Abreu, and J. Shuman, J. Biol. Chem. 258, 2174 (1983).
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phospholipids from VLDL to HDL is also stimulated by lipolysis, suggesting common principles of action. Mechanisms of Lipid Changes during Lipemia in Vivo. The changes in HDL are thought to result from influx of chylomicron phospholipids, apoA-I, and apoA-II. In incubations of plasma it has been shown that enrichment of HDL with phospholipids is followed by influx of cholesterol from other lipoproteins, z~ The accumulation of phospholipids and cholesterol probably leads to cholesteryl ester formation as a result of activity of lecithin:cholesterol acyltransferase. Within HDL there is a major redistribution of cholesteryl ester mass toward particles of lower density, reflecting in large part the increase in HDL phospholipids. Different studies have reported that total HDL cholesteryl ester may be increased, 8 unchanged,~7 or lowered 22 during alimentary lipemia. It is possible that the net change in HDL cholesteryl esters is determined by the magnitude of the rise in the plasma triglycerides. Thus, individuals who experience the most profound hypertriglyceridemia may also display the greatest fall in HDL cholesterol during lipemia, owing to lipid transfer protein-mediated exchange of HDL cholesteryl esters with the triglycerides of chylomicron remnants and VLDL. Consistent with this lipid exchange hypothesis is the finding that changes in HDL-2 and HDL-3 cholesteryl esters (which varied from about +15 to -20%) showed a strong negative correlation with changes in triglycerides in the same lipoproteins (r = -0.85, -0.88, respectively)23; the changes in HDL triglycerides tended to parallel those of plasma triglycerides. During lipemia there is an approximate 40% increase in lecithin : cholesterol acyltransferase activity, as revealed by incubation of lipemic plasma. The increase in LCAT activity was correlated with the magnitude of rise in the plasma triglycerides.23 The increase in LCAT activity could reflect the greater availability of substrate (phospholipids and cholesterol) owing to influx of materials into HDL, or the removal of the inhibitory product lipid, cholesteryl esters, owing to lipid exchange processes; however, changes in LCAT activity did not seem to correlate with these processes. 23 An alternative explanation may be related to the recent observations of Barter, which provide evidence that LCAT may act directly on non-HDL lipoproteins. 24 It is conceivable that LCAT binds directly to triglyceride-rich lipoproteins in lipemic plasma, resulting in an increase in LCAT activity. Further support for this concept is derived from the fact that LCAT circulates in a complex with cholesteryl ester transfer pro2i A. R. Tall and P. H. R. Green, J. Biol. Chem. 256, 2035 (1981). 22 R. M. Kay, S. Rao, C. Arnott, N. E. Miller, and B. Lewis, Atherosclerosis 36, 567 (1980). z3 H. G. Rose and J. Juliano, J. Lipid Res. 20, 399 (1979). 24 p. j. Barter, Biochim. Biophys. Acta 751, 261 (1983).
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tein. 25 The latter has been shown to bind to triglyceride-rich particles as they undergo lipolysis. TM The explanation of the inverse relationship between the magnitude of alimentary lipemia and the levels of fasting HDL-2 and HDL cholesterol levels 5 could be related to some of the above suggestions about lipid transfer and exchange mechanisms. First, the efficiency of clearance of both chylomicrons and VLDL could be related to the quantities of chylomicron phospholipid, cholesterol, and apoA-I transferred into HDL. A second and perhaps more likely explanation is related to the magnitude of cholesteryl ester exchange processes that occur during alimentary hypertriglyceridemia. We have suggested that transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins may be more pronounced the more plasma triglycerides rise. Since lipemia occurs several times a day, this could have an impact on fasting HDL-cholesterol levels. Also, the transfer of HDL cholesteryl esters into fasting VLDL could be more marked in individuals experiencing greater lipemia, since those who show larger rises in plasma triglycerides during lipemia also have higher fasting plasma triglycerides. 2 A third possibility might be related to the distribution of LCAT activity in lipemic plasma. Individuals with more pronounced hypertriglyceridemia might tend to form cholesteryl esters at the surface of triglyceride-rich particles as they undergo lipolysis, rather than in HDL, with subsequent lipid transfer protein-mediated transfer of cholesteryl ester into the core of the triglyceride-rich lipoprotein. It is evident that these several putative mechanisms may be interrelated. The inverse correlation between the magnitude of alimentary lipemia and fasting HDL-2 levels may also have its origin in cholesteryl estertriglyceride exchange processes, or in the distribution of LCAT activity. Thus, exchange of HDL cholesteryl esters with chylomicron triglycerides may result in a triglyceride-enriched HDL-2 particle. In vitro the triglyceride-rich HDL-2 can be remodeled into a more dense HDL-3 as a result of lipoprotein lipase activity. 26 Thus, the triglyceride enrichment of HDL2 that occurs during alimentary lipemia may be an important step in the remodeling of HDL-2 to HDL-3. This remodeling into smaller HDL particles may be more pronounced in individuals who have more marked alimentary hypertriglyceridemia, accounting for their lower mass of HDL-2. Since HDL-2 formation may be dependent on LCAT activity, it is evident that a competitive distribution of LCAT activity between triglyceride-rich lipoproteins and HDL could also affect HDL-2 levels. 25 p. E. Fielding and C. J. Fielding, Proc. Natl. Acad. Sci. U.S.A. 77, 3327 (1980). 26 R. J. Deckelbaum, S. Eisenberg, Y. Oschry, M. Cooper, and C. Blum, J. Lipid Res. 23, 1274 (1982).
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Further support for a metabolic relationship between triglyceride-rich lipoproteins and HDL-2 levels has been derived from studies of patients with abetalipoproteinemia. In patients with classical abetalipoproteinemia, who lack chylomicrons and VLDL, the major H D L particle is a cholesteryl ester-enriched HDL-2. 26 However, in a patient with normotriglyceridemic abetalipoproteinemia, the major H D L was HDL-3. 27 This patient appeared to make intestinal but not hepatic apoB and thus experienced alimentary lipemia, but lacked hepatogenous VLDL. The difference in H D L in the two types of abetalipoproteinemia implies that the alimentary lipemic process is important in the remodeling of HDL-2 into HDL-3. Effects of Alcohol In different studies the acute administration of a single dose of alcohol has led to a variable, small increase in plasma VLDL-triglyceride levels. The changes seem to be more pronounced with larger doses of alcohol and in subjects with higher fasting triglyceride levels.l,28,29 In a study of normal subjects with mean fasting plasma triglycerides of 40 to 70 mg/dl we found no consistent change in VLDL triglycerides after acute ingestion of 100 g alcohol (as whiskey). 29 However, VLDL phospholipids were markedly increased. Also, HDL-3 and HDL-2a phospholipid levels were increased. These changes in the lipoproteins were associated with a mean 33% decrease in hepatic lipase activity, as determined in postheparin plasma. Given the evidence in experimental animals that hepatic lipase may be involved in the catabolism of H D L and possibly VLDL phospholipids, it appears likely that the changes in the plasma lipoproteins may have reflected in part the inhibition of hepatic iipase activity. It is also possible that increased VLDL flux, without a rise in plasma triglycerides, could lead to increased transfer of VLDL phospholipids into HDL. Several studies have shown that ingestion of alcohol prior to or with a fat load results in a more pronounced increase in plasma triglyceride level than occurs after ingestion of either alcohol alone or fat alone. 1 The magnitude of this synergistic effect is more pronounced the higher the subject's baseline plasma triglyceride levels. Compared to the effects of fat alone, the synergistic effects of fat and alcohol result in a later and more 27 M. J. Malloy, J. P. Kane, D. A. Hardman, R. L. Hamilton, and K. B. Dalai, J. Clin. Invest. 67, 1441 (1981). 28 p. Avogaro and G. Cazzolato, Metabolism 24, 1231 (1975). 29 C. S. Goldberg, A. R. Tail, and S. Krumholz, J. Lipid Res. 25, 714 (1984).
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sustained rise in plasma triglycerides; also, the increment is due to greater levels of Sf < 400 particles. It is not known if the increment in Sf < 400 triglycerides represents delayed clearance of chylomicron remnants, or greater incorporation of dietary fat and fatty acids into hepatogenous VLDL. Experimental evidence in rats has suggested both greater hepatic secretion of V L D L 3° and also impaired clearance of chylomicron remnants when a fatty meal is given to alcohol-treated animals. 3~ Studies in humans in which the low-molecular-weight apoB is quantitated in the d < 1.006 fraction should be able to resolve this question. The combined effects of alcohol and fat on H D L and LDL lipid levels have not been reported. Significance of the Changes in Postprandial Lipoproteins The metabolism of chylomicron remnants could have direct relevance to atherogenesis. Zilversmit 32postulated that chylomicron remnants bearing dietary cholesterol might deposit cholesterol in arterial tissues. Lipoprotein lipase present in arterial tissues could lead to in situ remnant formation and movement of remnants or remnant lipids into arterial tissues.32 There is evidence that perfusion of cholesterol-enriched chylomicrons through the rat heart leads to nonendocytotic transfer of cholesteryl esters into the coronary artery tissue. 33 Tissue culture studies indicate that cholesterol-enriched fl-VLDL of intestinal origin (presumably chylomicron remnants) are particularly active in causing cholesterol deposition in macrophages, promoting foam cell formation. Internalization of these particles seems to depend on receptor-mediated endocytosis mediated by the/3-VLDL receptor.~3 Since subjects with low HDL-cholesterol and low HDL-2 levels have a more pronounced lipemia, it is possible that the longer circulation of remnants leads to greater arterial deposition of atherogenic chylomicron remnants) Thus, low fasting HDL-cholesterol levels could be a marker of a propensity to form atherogenic chylomicron remnants. 5 A more generalized interpretation of the potential significance of the inverse relationship between levels of alimentary lipemia and H D L levels is suggested in this chapter. Subjects with a more pronounced lipemic response may have a tendency to drop their HDL-cholesterol levels dur3o E. 3t T. 32 D. 33 C.
Baraona and C. S. Lieber, J. Clin. Invest. 49, 769 (1970). G. Redgrave and G. Marton, Atherosclerosis 28, 69 (1977). B. Zilversmit, Circulation 60, 473 (1979). J. Fielding, J. Clin. Invest. 62, 141 (1978).
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OF PLASMA LIPOPROTEINS
[29]
ing lipemia and to have lower fasting HDL-cholesterol levels; the more pronounced lipemic response may result from increased VLDL apoB or triglyceride production, on a genetic basis (e.g., in familial combined hyperlipidemia) or on an acquired basis (e.g., in obesity). Thus, the lipemic changes in H D L cholesterol may mark the tendency to overproduce apoB-containing particles. The resulting chylomicron or VLDL remnants or L D L may all be atherogenic. It should be noted that this hypothesis is based on an opposite interpretation of the significance of the HDL-cholesterol to that made in the reverse cholesterol transport theory. Thus, subjects who show greatest transfer of H D L cholesteryl esters to remnant particles would be at greatest risk for atherogenesis. By contrast, one version of the reverse cholesterol transport theory suggests that individuals with high HDL-cholesterol levels would transport more cholesterol to VLDL or chylomicron remnants, because this step allows a path for exit of cholesterol from the plasma. Further experiments aimed at measuring the transfer of cholesteryl esters between H D L and lipemic particles may be helpful in evaluating these different theories of the relationship between H D L and atherogenesis. Acknowledgments Supported by NIH Grants HL 22682 and 21006. Author is an Established Investigator of the A.H.A.
[29] In Vivo M e t a b o l i s m o f A p o l i p o p r o t e i n E in H u m a n s B y RICHARD E . GREGG and H. BRYAN BREWER, JR.
Apolipoprotein E (apoE), a glycoprotein of Mr 34,000, is associated predominantly with very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) in human plasma.l,2 Human apoE is a polymorphic protein 3 with multiple alleles inherited in a codominant fashion at a 1 C. B. Blum, L. Aron, and R. Sciacca, J. Clin. Invest. 66, 1240 (1980). 2 R. J. Havel, L. Kotite, J. L. Vigne, J. P. Kane, P. Tun, N. Phillips, and G. C. Chen, J. Clin. Invest. 66, 1351 (1980). 3 G. Utermann, M. Jaeschke, and J. Menzel, FEBS Lett. 56, 352 (1975).
METHODS IN ENZYMOLOGY, VOL. 129
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pools of VLDL and HDL should not be affected by the in vitro incubation, Sv, = Svn
and
SHn = SHn = 0
The SA in the exchangeable pools of VLDL and HDL will reach a new equilibrium value which reflects fPv, the fraction of the exchangeable mass of apoC-III which is associated with VLDL, i.e., SHe =
ave
= fpvSve
The overall apoC-III SA in each lipoprotein class is a weighted average of the SA in the equilibrating and nonequilibrating pool. Sv = Svefv + Svn(1 - f v )
where fv =
mass of equilibrating VLDL apoC-III total mass of apoC-III in VLDL S . = SIaefH
where fH =
mass of equilibrating HDL apoC-III total mass of apoC-III in HDL
The ratio R of the apoC-III SA between VLDL and HDL as obtained from the decay curves is defined as: R = Sv/SH
[28] M e t a b o l i s m o f P o s t p r a n d i a l L i p o p r o t e i n s By ALAN R. TALL
Overview Although a significant portion of each day is spent in the postprandial state, there have been relatively few studies of the effects of a meal on lipoprotein metabolism. The absorption and transport of dietary fat lead to significant perturbations of the plasma lipoproteins. The changes reflect both the addition of newly synthesized, triglyceride-rich particles (chylomicrons) to the plasma, and also changes in the other lipoproteins, especially high-density lipoproteins (HDL), probably resulting from the meMETHODS IN ENZYMOLOGY, VOL. 129
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
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tabolism of chylomicrons. The changes in the d < 1.006 fraction (containing chylomicrons) and in the HDL after a fatty meal may be regarded as an important physiological model of the transfer of lipid and apolipoprotein components between the plasma lipoproteins, especially the transfer or exchange of surface and core components between triglyceride-rich lipoproteins and HDL. Studies of the metabolism of postprandial lipoproteins may shed light on the importance of different particles in the process of atherogenesis. The changes in the plasma lipoproteins that occur in the postprandial state are quite different in different individuals. Otherwise subtle abnormalities of plasma lipoprotein metabolism may be highlighted in the postprandial state. Changes in the postprandial lipoprotein profile in individuals susceptible to atherosclerosis may provide insights into the mechanisms of involvement of apoB-containing particles of HDL in the process of atherogenesis. This chapter will focus on the metabolism of postprandial lipoproteins in humans, especially the acute response to dietary fat or alcohol. The emphasis Will be on the metabolism of chylomicrons and HDL. Studies in animals or in vitro will be referred to when they are thought to elucidate human physiology. Methodological aspects of studying postprandial lipoproteins will be discussed. Methodological problems, especially the paucity of nonexchangeable markers of newly synthesized lipoproteins or apolipoproteins, have been a major factor limiting a detailed understanding of the genesis of changes noted in the postprandial plasma lipoprotein profiles. Study Design The majority of studies of postprandial lipoproteins in humans have measured sequential changes in the plasma following single or multiple oral fat loads. Triglyceride may be given as corn oil, olive oil, safflower oil, cream, or butter or incorporated into a meal. There are few studies which have compared the acute effects of different types of dietary fat, such as saturated versus polyunsaturated fats. Significant changes in the plasma lipids or apolipoproteins may be observed with doses of 0.75-3.0 g triglycerides/kg body weight. Some subjects experience nausea and diarrhea after ingesting fat alone. When fat is given as part of a meal it is possible that the presence of carbohydrate or protein will alter the subsequent metabolism of triglyceride. There have been few systematic studies of the effects of other dietary components on the response to a triglyceride load. Alcohol has a marked effect on the subsequent metabolism of
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dietary fat. 1 Alcohol has been given in several forms; there is no information on the effects of the nonalcoholic moieties of different types of alcoholic beverages on postprandial lipoprotein metabolism. One of the difficulties of studying changes in the lipoproteins after an oral fat load is the variable time between the ingestion of fat and the peak response of the plasma lipoproteins. This variability results primarily from differences in gastric emptying rate. In order to catch the peak changes in plasma lipoproteins it is necessary to take multiple blood samples. The peak in plasma triglycerides usually occurs about 5 to 6 hr after ingestion of 1.5 g/kg of oral fat, and the major alterations in the plasma lipoproteins (such as the HDL) may be observed at 5 to 10 hr. The variability in response necessitates the study of several individuals. More controlled methods for studying the metabolism of postprandial lipoproteins have involved the intravenous infusion of radiolabeled chylomicrons 2 or intraduodenal fat infusion. 3 These approaches have the advantage that they provide kinetic data for chylomicron removal. Human chylomicrons may be radiolabeled in the protein moiety by the IC1 method. 4 Optimally, human chylomicrons can be obtained from thoracic duct lymph. Unfortunately, thoracic duct cannulation is not frequently performed for therapeutic purposes. Human chylomicrons may also be prepared from other sources such as chylous pleural effusions or from the urine of subjects with chyluria. However, the supply of chylomicrons from these sources is irregular and the chylomicrons have probably already undergone extensive alteration due to contact with plasma components. Grundy and Mok 3 have studied the metabolism of chylomicrons during a constant intraduodenal fat infusion. This method results in a steady rate of fat absorption and constant plasma triglyceride levels and allows the kinetics of triglyceride removal to be determined. The subjects are intubated with a nasogastric tube the night before the study. The next morning the tube is positioned in the region of the ampulla of Vater, under X-ray guidance, then a constant infusion of triglyceride or other lipid (e.g., a monoglyceride/lecithin mixture) is begun. A triple lumen tube may be used to determine the amount of lipid absorbed across a segment of small intestine. In normal subjects, small intestinal triglyceride absorption should be more than 95%. After a 5-hr infusion of triglycerides, the i D. E. Wilson, P. H. Schreibman, A. C. Brewster, and R. A. Arky, J. Lab. Clin. Med. 75, 264 (1970). 2 p. j. Nestel, J. Clin. Invest. 43, 943 (1964). 3 S. M. Grundy and H. Y. I. Mok, Metabolism 25, 1225 (1976). 4 D. W. Bilheimer, S. Eisenberg, and R. I. Levy, Biochim. Biophys. Acta 260, 212 (1972).
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plasma triglyceride level becomes constant, and the study is begun. With steady plasma levels the removal rate of triglyceride is equal to the infusion rate (mg/kg/hr), assuming 100% absorption. From the turnover rate and the increment of chylomicron triglyceride, the tv2 of chylomicron triglyceride can be determined as follows: tv2 = 0.693/K, where K = input of chylomicron TG (mg/kg/hr)/plasma pool of chylomicron TG. The latter is determined from the plasma triglyceride concentration and the estimated plasma volume ( = 927 + 31.47 × body wt). The prior state of the patient is an important variable in determining the metabolism of postprandial lipoproteins. In normal subjects plasma triglycerides may continue to fall significantly during the morning after a fast from 11 PM; fasting for a full 14 hr before the onset of the study is advisable. The patient's prior metabolic state has an important influence on the response to a meal. Weight loss, alcohol intake, ~ exercise: and probably a variety of other variables may influence the individual's response to a fatty meal. Ideally, studies should be undertaken in the metabolic ward setting where caloric intake, diet composition, and exercise are all well-controlled variables. However, valuable information has been obtained under less rigorously controlled conditions. Preexisting hyperlipidemia has a large influence on the changes in plasma lipoproteins after ingestion of a fatty meal. Although several studies have contrasted the responses of broad groups of hyperlipidemic patients with those of normal controls, there is a paucity of information on the response of patients with genetically or otherwise well-defined hyperlipidemias. Even patients who are defined as normolipidemic, on the basis of a plasma triglyceride level of less than 200 mg/dl, have quite different responses depending on their preexisting plasma triglyceride level. This will be discussed in detail below. Preparation of Lipoproteins Chylomicrons are generally isolated as plasma lipoproteins of Sf > 400, as described. 6 There is evidence that triglyceride-rich lipoproteins of Sf < 400 may make a lesser contribution to the hypertriglyceridemia following a fatty meal, 6 especially during infusion of lecithin. 7 Plasma lipoproteins in the postprandial state may be isolated by procedures reviewed elsewhere in this volume. It is worth noting that during alimentary 5 j. R. Patsch, J. B. Karlin, L. W. Scott, L. C. Smith, and A. M. Gono, Proc. Natl. Acad. Sci. U.S.A. 80, 1449 (1983). 6 T. G. Redgrave and L. A. Carlson, J. Lipid Res. 20, 217 (1979). F. U. Beil and S. M. Grundy, J. Lipid Res. 21, 525 (1980).
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lipemia lipid enrichment of HDL species results in significant alterations of the density of the major subclasses of HDL. 8 It appears that a significant portion of lipid-enriched HDL-3 particles float at d 1.125 g/ml.8 Thus, the changes in HDL may in general be best defined by methods which make no assumptions about traditional HDL-2 or HDL-3 designations, such as isopycnic density gradient techniques. In the future it is likely that valuable information about changes in HDL subclasses will be obtained by noncentrifugal methods, such as those employing antibody affinity chromatography for isolation of apoA-I-containing particles from plasma. 9 A major problem in the study of the metabolism of postprandial lipoproteins has been the paucity of nonexchangeable lipid or protein markers to denote the fate of individual lipoprotein particles during lipemia. Although lipid core markers have been employed as a means of following chylomicron metabolism, triglycerides, cholesteryl esters, and retinyl esters are all susceptible to cholesteryl ester transfer protein-mediated exchange with other lipoprotein core lipids.l° Although these lipids may be used to follow the fate of chylomicron particles in animals which have low lipid transfer protein activity (dogs, pigs, and rats), they are not reliable particle markers in species which have significant cholesteryl ester transfer protein activity (humans, rabbits, and monkeys). Estimates of the extent of core lipid exchange based on incubations of plasma may underestimate physiological exchange rates, since lipolysis enhances the activity of the cholesteryl ester transfer protein.11 Thus, the redistribution of chylomicron retinyl ester radioactivity into Sf < 400 lipoprotein classes in intact animals cannot be taken as evidence that chylomicron remnants contribute to Sf < 400 particles. The recent recognition that the lowmolecular-weight form of apoB is a nonexchangeable marker for intestinally derived particles ~2has great potential in allowing intestinally derived particles to be traced. For example, this has allowed identification of an intestinally derived subfraction of/3-VLDL which is particularly efficacious in delivering cholesterol to macrophages. 13 In future studies mea8 A. R. Tall, C. B. Blum, G. P. Forester, and C. Nelson, J. Biol. Chem. 257, 198 (1982). 9 j. p. McVicar, S. T. Kunitake, R. L. Hamilton, and J. P. Kane, Proc. Natl. Acad. Sci. U.S.A. 81, 1356 (1984). 10 R. E. Morton and D. B. Zilversmit, J. Lipid Res. 23, 1058 (1982). H A. R. Tall, D. Sammett, G. Vita, R. J. Deckelbaum, and T. Olivecrona, J. Biol. Chem. 259, 9587 (1984). 12 j. p. Kane, D. A. Hardman, and H. E. Paulus, Proc. Natl. Acad. Sci. U.S.A. 77, 2465 (1980). i3 M. R. Fainaru, R. W. Mahley, R. L. Hamilton, and T. L. Innerarity, J. LipidRes. 23, 702 (1982).
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METABOLISM OF PLASMA LIPOPROTEINS
[28]
surements of the low-molecular-weight apoB (particularly as specific immunochemical methods become available) should serve to determine if accumulating particles after a fatty meal are intestinally derived or hepatogenous. Also, the characterization of intestinally derived particles should provide insights into their role in atherogenesis. The lack of a marker for newly synthesized apoA-I has limited the understanding of how apoA-I becomes incorporated into the HDL fraction during lipemia. In humans plasma and HDL apoA-I rise after a fatty meal, but the incremental apoA-I has not been distinguishable from the preexisting apoA-I. A potentially useful marker of newly synthesized apoA-I might be its isoprotein-2, which is the secretory proprotein of apoA-I.14 The isoprotein-2 has a half-life of several hours in the plasma before being converted to the more basic isoproteins 4 and 5. J5Analysis of apoA-I isoforms in the HDL fraction after a fatty meal might provide some clue as to how newly synthesized apoA-I becomes incorporated into plasma HDL particles. Results of Studies Overview o f Chylomicron Metabolism Based on in Vitro Studies and Studies in Experimental Animals
Absorbed dietary fat is packaged into chylomicron particles containing a core of triglyceride and a surface film of phospholipids, cholesterol, apoB, and soluble apolipoproteins (apoA-I, apoA-II, apoA-IV, and apoCIII). The chylomicron triglyceride is rapidly hydrolyzed in peripheral tissues due to the action of lipoprotein lipase, a triglyceride hydrolase located on the surface of the capillary endothelium. Fatty acids and partial glycerides enter the tissues or are bound to albumin. During the process of lipolysis of chylomicrons certain surface materials (phospholipids, apoA-I, and apoA-II) are transferred into the HDL fraction. Conversely, apoE, apoC peptides, and cholesteryl esters are transferred from HDL to chylomicron remnants. Thus, relative to nascent chylomicrons, chylomicron remnants are enriched in cholesteryl esters and apoE and apoC peptides (especially apoC-III), and depleted in apoA-I and apoA-IV; recently we have found that chylomicron and VLDL remnants also bind cholesteryl ester transfer protein. The chylomicron remnant is rapidly 14 V. I. Zannis, D. M. Kurnit, and J. L. Breslow, J. Biol. Chem. 257, 536 (1982). ~5G. Ghiselli, E. J. Schaefer, S. Law, J. A. Light, and H. B. Brewer, Clin. Res. 31, 500A (1983).
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removed from the circulation owing to the action of a hepatic chylomicron remnant or apoE receptor.~6
Studies of Postprandial Lipoproteins in HumanshChylomicron Metabolism Using intravenously injected thoracic duct chylomicrons, Nestel showed that the half-life of chylomicron triglyceride in the circulation was about 5 min. 2 Grundy and Mok 3 obtained similar results using an intraduodenal infusion method. Nestel z showed a strong correlation between fasting triglyceride levels and the height of the response of plasma triglyceride to a fatty meal. There was an inverse correlation between the level of fasting plasma triglycerides and the rate of chylomicron metabolism both in hypertriglyceridemia (>200 mg/dl) and also within the normal range of plasma triglycerides (<200 mg/dl). Grundy and Mok 3 speculated that the slower removal of chylomicron triglyceride in subjects with fasting hypertriglyceridemia reflected competition of the chylomicrons with VLDL for removal mechanisms rather than a basic defect in the ability to remove chylomicrons, because with weight loss and a fall in the plasma triglyceride level, most subjects showed a marked improvement in chylomicron clearance. Thus, in many patients with hypertriglyceridemia a marked alimentary lipemic response may reflect a tendency to overproduce VLDL, rather than a primary defect in clearance of chylomicrons. Exceptions to this statement are patients with lipoprotein lipase or apoCII deficiency and perhaps a minority of other patients who have a predominant clearance defect. 3 Redgrave and Carlson 6 contrasted the response to lipemia of hypertriglyceridemic and normal subjects. They found that the more pronounced lipemia of hypertriglyceridemic subjects occurred in Se > 400 particles, and was largely due to presence of particles of increased size in these subjects. It is notable that in several studies increases in number or size of triglyceride-rich lipoproteins of both Sf > 400 and Sf < 400 have been noted during alimentary lipemia. 3,6 It is uncertain if this represents continued degradation of chylomicron remnants to smaller particle size, or competition between chylomicron remnants and hepatogenous VLDL for removal sites, resulting in accumulation of the latter, or increased hepatic VLDL secretion due to the load of dietary fat returning to the liver. It should be possible to determine if the increase in Sf < 400 parti16 R. W. Mahley, D. Y. Hui, T. L. Innerarity, and K. H. Weisgraber, J. Clin. Invest. 68,
1197 (1981).
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METABOLISM OF PLASMA LIPOPROTEINS
[28]
cles is due to hepatic or intestinal particles by determining the distribution of the low-molecular-weight apoB particle in the postprandial VLDL fraction. In patients infused with lecithin the major increase in triglyceridecarrying particles in the plasma occurred in the Sf 20-400 fraction, possibly due to absorption of lecithin over a longer length of small intestine than triglyceride,7 (distal small intestinal fat absorption tends to result in formation of smaller chylomicrons). Another possibility is that lecithin fatty acid was predominantly absorbed via the portal vein and that the Sf 20-400 particles were, in fact, hepatogenous VLDL. Thus, both the mode of intestinal absorption and the subject's baseline lipid metabolism influence the response of triglyceride-rich lipoproteins during lipemia. Changes in HDL. Havel et al.17 noted an increase in HDL phospholipids and protein during alimentary lipemia and speculated that these changes might reflect transfer of chylomicron surface materials into the HDL fraction. These workers also showed that during lipemia there is transfer of apoC peptides, including the activator peptide of lipoprotein lipase, apoC-II, from HDL to the chylomicron remnant fraction. More recently, we have performed detailed studies of the changes in HDL subfractions during alimentary lipemia, using primarily density gradient methods to separate HDL subclasses. These studies showed an increase in HDL phospholipids, cholesteryl esters, apoA-I, and apoA-II during lipemia; these changes were thought to reflect at least in part the transfer of these lipids and proteins from chylomicrons into HDL, as demonstrated in the r a t : Another potential mechanism could be increased intestinal HDL secretion during lipemia. Analysis of HDL by equilibrium density gradient ultracentrifugation showed the presence of a major and a lesser peak of HDL in fasting plasma, corresponding to the traditional HDL-3 and HDL-2 subclasses, respectively. During lipemia the most pronounced change was an increase in mass and a shift to lower density of the major peak of HDL-3, with similar but less marked changes in the HDL-2 peak. Analyses of molecular compositions suggested that the lipid transferred into HDL was added to preexisting particles, since the particles retained the same basic complement of apoA-I and apoA-II molecules but had additional lipid molecules (especially phospholipids). Although there was an increase in mass of HDL apoA-I and apoA-II the particle contents of apoA-I and apoA-lI were unchanged during lipemia, suggesting that apolipoprotein transfer causes interconversion of existing HDL species, or formation of new particles with the same content of apoA-I and apoA-II as existing species. An interesting recent report 5 has shown that in a group of normolipi17 R. J. Havel, J. P. Kane, and M. L. Kashyap, J. Clin. Invest. 52, 32 (1973).
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METABOLISM OF POSTPRANDIAL LIPOPROTEINS
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demic healthy young adults there was an inverse relationship between the magnitude of the plasma triglyceridemic response and the levels of fasting HDL-2 (r = -0.86), HDL-cholesterol (r = -0.61), and apoA-I (r = -0.46). In two subjects followed for 3 years, when HDL-2 levels rose or fell in response to exercise or the lack of it, the triglyceridemic response decreased or increased, respectively.
Potential Mechanisms of Lipid Transfer Processes during Lipemia In Vitro Studies of Lipid Exchange Processes. Our recent in vitro studies suggest that lipid exchange processes may be stimulated during lipemia.11 These studies show that lipolysis enhances the cholesteryl ester transfer protein-mediated movement of cholesteryl esters from H D L to triglyceride-rich lipoproteins. The latter were isolated from both fasting and lipemic plasma. 18The stimulation of cholesteryl ester transfer activity was related to the accumulation of lipolytic products, especially fatty acids in the remnant particle. It appears that small amounts of negatively charged fatty acids enhance the binding of the cholesteryl ester transfer protein to the remnant surface. Under several different circumstances enhancement or reduction of cholesteryl ester transfer activity was related to increased or decreased binding of cholesteryl ester transfer protein to VLDL or VLDL remnants, suggesting that the increased binding resulted in increased cholesteryl ester transfer into the triglyceride-rich particles. There is also theoretical evidence to support the concept that increased affinity of cholesteryl ester transfer protein for acceptor particles relative to H D L should increase cholesteryl ester transfer from H D L to the acceptor particles.19 The enhancement of cholesteryl ester transfer processes by lipolysis was greatly favored by high V L D L / H D L ratios. Thus, cholesteryl ester transfer processes should be more pronounced in subjects who experience higher levels of plasma triglyceride during lipemia. In summary, these in vitro studies suggest that cholesteryl ester transfer from H D L to triglyceride-rich particles during lipemia may be related to activities of lipoprotein or hepatic lipases, and to the magnitude of the rise in plasma triglyceride levels. In other studies we have shown that the transfer of phospholipids from triglyceride-rich lipoproteins to H D L is enhanced by a partially purified plasma phospholipid transfer protein, m This phospholipid transfer protein appears to be a different protein than the lipid transfer protein referred to a b o v e , z° However, the phospholipid transfer protein-mediated transfer of t8 A. R. Tall, unpublished results, 1984. 19 p. j. Barter and M. E. Jones, J. LipidRes. 21, 238 (1980). 2o A. R. Tall, E. Abreu, and J. Shuman, J. Biol. Chem. 258, 2174 (1983).
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METABOLISM OF PLASMA LIPOPROTEINS
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phospholipids from VLDL to HDL is also stimulated by lipolysis, suggesting common principles of action. Mechanisms of Lipid Changes during Lipemia in Vivo. The changes in HDL are thought to result from influx of chylomicron phospholipids, apoA-I, and apoA-II. In incubations of plasma it has been shown that enrichment of HDL with phospholipids is followed by influx of cholesterol from other lipoproteins, z~ The accumulation of phospholipids and cholesterol probably leads to cholesteryl ester formation as a result of activity of lecithin:cholesterol acyltransferase. Within HDL there is a major redistribution of cholesteryl ester mass toward particles of lower density, reflecting in large part the increase in HDL phospholipids. Different studies have reported that total HDL cholesteryl ester may be increased, 8 unchanged,~7 or lowered 22 during alimentary lipemia. It is possible that the net change in HDL cholesteryl esters is determined by the magnitude of the rise in the plasma triglycerides. Thus, individuals who experience the most profound hypertriglyceridemia may also display the greatest fall in HDL cholesterol during lipemia, owing to lipid transfer protein-mediated exchange of HDL cholesteryl esters with the triglycerides of chylomicron remnants and VLDL. Consistent with this lipid exchange hypothesis is the finding that changes in HDL-2 and HDL-3 cholesteryl esters (which varied from about +15 to -20%) showed a strong negative correlation with changes in triglycerides in the same lipoproteins (r = -0.85, -0.88, respectively)23; the changes in HDL triglycerides tended to parallel those of plasma triglycerides. During lipemia there is an approximate 40% increase in lecithin : cholesterol acyltransferase activity, as revealed by incubation of lipemic plasma. The increase in LCAT activity was correlated with the magnitude of rise in the plasma triglycerides.23 The increase in LCAT activity could reflect the greater availability of substrate (phospholipids and cholesterol) owing to influx of materials into HDL, or the removal of the inhibitory product lipid, cholesteryl esters, owing to lipid exchange processes; however, changes in LCAT activity did not seem to correlate with these processes. 23 An alternative explanation may be related to the recent observations of Barter, which provide evidence that LCAT may act directly on non-HDL lipoproteins. 24 It is conceivable that LCAT binds directly to triglyceride-rich lipoproteins in lipemic plasma, resulting in an increase in LCAT activity. Further support for this concept is derived from the fact that LCAT circulates in a complex with cholesteryl ester transfer pro2i A. R. Tall and P. H. R. Green, J. Biol. Chem. 256, 2035 (1981). 22 R. M. Kay, S. Rao, C. Arnott, N. E. Miller, and B. Lewis, Atherosclerosis 36, 567 (1980). z3 H. G. Rose and J. Juliano, J. Lipid Res. 20, 399 (1979). 24 p. j. Barter, Biochim. Biophys. Acta 751, 261 (1983).
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tein. 25 The latter has been shown to bind to triglyceride-rich particles as they undergo lipolysis. TM The explanation of the inverse relationship between the magnitude of alimentary lipemia and the levels of fasting HDL-2 and HDL cholesterol levels 5 could be related to some of the above suggestions about lipid transfer and exchange mechanisms. First, the efficiency of clearance of both chylomicrons and VLDL could be related to the quantities of chylomicron phospholipid, cholesterol, and apoA-I transferred into HDL. A second and perhaps more likely explanation is related to the magnitude of cholesteryl ester exchange processes that occur during alimentary hypertriglyceridemia. We have suggested that transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins may be more pronounced the more plasma triglycerides rise. Since lipemia occurs several times a day, this could have an impact on fasting HDL-cholesterol levels. Also, the transfer of HDL cholesteryl esters into fasting VLDL could be more marked in individuals experiencing greater lipemia, since those who show larger rises in plasma triglycerides during lipemia also have higher fasting plasma triglycerides. 2 A third possibility might be related to the distribution of LCAT activity in lipemic plasma. Individuals with more pronounced hypertriglyceridemia might tend to form cholesteryl esters at the surface of triglyceride-rich particles as they undergo lipolysis, rather than in HDL, with subsequent lipid transfer protein-mediated transfer of cholesteryl ester into the core of the triglyceride-rich lipoprotein. It is evident that these several putative mechanisms may be interrelated. The inverse correlation between the magnitude of alimentary lipemia and fasting HDL-2 levels may also have its origin in cholesteryl estertriglyceride exchange processes, or in the distribution of LCAT activity. Thus, exchange of HDL cholesteryl esters with chylomicron triglycerides may result in a triglyceride-enriched HDL-2 particle. In vitro the triglyceride-rich HDL-2 can be remodeled into a more dense HDL-3 as a result of lipoprotein lipase activity. 26 Thus, the triglyceride enrichment of HDL2 that occurs during alimentary lipemia may be an important step in the remodeling of HDL-2 to HDL-3. This remodeling into smaller HDL particles may be more pronounced in individuals who have more marked alimentary hypertriglyceridemia, accounting for their lower mass of HDL-2. Since HDL-2 formation may be dependent on LCAT activity, it is evident that a competitive distribution of LCAT activity between triglyceride-rich lipoproteins and HDL could also affect HDL-2 levels. 25 p. E. Fielding and C. J. Fielding, Proc. Natl. Acad. Sci. U.S.A. 77, 3327 (1980). 26 R. J. Deckelbaum, S. Eisenberg, Y. Oschry, M. Cooper, and C. Blum, J. Lipid Res. 23, 1274 (1982).
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METABOLISM OF PLASMA LIPOPROTEINS
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Further support for a metabolic relationship between triglyceride-rich lipoproteins and HDL-2 levels has been derived from studies of patients with abetalipoproteinemia. In patients with classical abetalipoproteinemia, who lack chylomicrons and VLDL, the major H D L particle is a cholesteryl ester-enriched HDL-2. 26 However, in a patient with normotriglyceridemic abetalipoproteinemia, the major H D L was HDL-3. 27 This patient appeared to make intestinal but not hepatic apoB and thus experienced alimentary lipemia, but lacked hepatogenous VLDL. The difference in H D L in the two types of abetalipoproteinemia implies that the alimentary lipemic process is important in the remodeling of HDL-2 into HDL-3. Effects of Alcohol In different studies the acute administration of a single dose of alcohol has led to a variable, small increase in plasma VLDL-triglyceride levels. The changes seem to be more pronounced with larger doses of alcohol and in subjects with higher fasting triglyceride levels.l,28,29 In a study of normal subjects with mean fasting plasma triglycerides of 40 to 70 mg/dl we found no consistent change in VLDL triglycerides after acute ingestion of 100 g alcohol (as whiskey). 29 However, VLDL phospholipids were markedly increased. Also, HDL-3 and HDL-2a phospholipid levels were increased. These changes in the lipoproteins were associated with a mean 33% decrease in hepatic lipase activity, as determined in postheparin plasma. Given the evidence in experimental animals that hepatic lipase may be involved in the catabolism of H D L and possibly VLDL phospholipids, it appears likely that the changes in the plasma lipoproteins may have reflected in part the inhibition of hepatic iipase activity. It is also possible that increased VLDL flux, without a rise in plasma triglycerides, could lead to increased transfer of VLDL phospholipids into HDL. Several studies have shown that ingestion of alcohol prior to or with a fat load results in a more pronounced increase in plasma triglyceride level than occurs after ingestion of either alcohol alone or fat alone. 1 The magnitude of this synergistic effect is more pronounced the higher the subject's baseline plasma triglyceride levels. Compared to the effects of fat alone, the synergistic effects of fat and alcohol result in a later and more 27 M. J. Malloy, J. P. Kane, D. A. Hardman, R. L. Hamilton, and K. B. Dalai, J. Clin. Invest. 67, 1441 (1981). 28 p. Avogaro and G. Cazzolato, Metabolism 24, 1231 (1975). 29 C. S. Goldberg, A. R. Tail, and S. Krumholz, J. Lipid Res. 25, 714 (1984).
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481
sustained rise in plasma triglycerides; also, the increment is due to greater levels of Sf < 400 particles. It is not known if the increment in Sf < 400 triglycerides represents delayed clearance of chylomicron remnants, or greater incorporation of dietary fat and fatty acids into hepatogenous VLDL. Experimental evidence in rats has suggested both greater hepatic secretion of V L D L 3° and also impaired clearance of chylomicron remnants when a fatty meal is given to alcohol-treated animals. 3~ Studies in humans in which the low-molecular-weight apoB is quantitated in the d < 1.006 fraction should be able to resolve this question. The combined effects of alcohol and fat on H D L and LDL lipid levels have not been reported. Significance of the Changes in Postprandial Lipoproteins The metabolism of chylomicron remnants could have direct relevance to atherogenesis. Zilversmit 32postulated that chylomicron remnants bearing dietary cholesterol might deposit cholesterol in arterial tissues. Lipoprotein lipase present in arterial tissues could lead to in situ remnant formation and movement of remnants or remnant lipids into arterial tissues.32 There is evidence that perfusion of cholesterol-enriched chylomicrons through the rat heart leads to nonendocytotic transfer of cholesteryl esters into the coronary artery tissue. 33 Tissue culture studies indicate that cholesterol-enriched fl-VLDL of intestinal origin (presumably chylomicron remnants) are particularly active in causing cholesterol deposition in macrophages, promoting foam cell formation. Internalization of these particles seems to depend on receptor-mediated endocytosis mediated by the/3-VLDL receptor.~3 Since subjects with low HDL-cholesterol and low HDL-2 levels have a more pronounced lipemia, it is possible that the longer circulation of remnants leads to greater arterial deposition of atherogenic chylomicron remnants) Thus, low fasting HDL-cholesterol levels could be a marker of a propensity to form atherogenic chylomicron remnants. 5 A more generalized interpretation of the potential significance of the inverse relationship between levels of alimentary lipemia and H D L levels is suggested in this chapter. Subjects with a more pronounced lipemic response may have a tendency to drop their HDL-cholesterol levels dur3o E. 3t T. 32 D. 33 C.
Baraona and C. S. Lieber, J. Clin. Invest. 49, 769 (1970). G. Redgrave and G. Marton, Atherosclerosis 28, 69 (1977). B. Zilversmit, Circulation 60, 473 (1979). J. Fielding, J. Clin. Invest. 62, 141 (1978).
482
METABOLISM
OF PLASMA LIPOPROTEINS
[29]
ing lipemia and to have lower fasting HDL-cholesterol levels; the more pronounced lipemic response may result from increased VLDL apoB or triglyceride production, on a genetic basis (e.g., in familial combined hyperlipidemia) or on an acquired basis (e.g., in obesity). Thus, the lipemic changes in H D L cholesterol may mark the tendency to overproduce apoB-containing particles. The resulting chylomicron or VLDL remnants or L D L may all be atherogenic. It should be noted that this hypothesis is based on an opposite interpretation of the significance of the HDL-cholesterol to that made in the reverse cholesterol transport theory. Thus, subjects who show greatest transfer of H D L cholesteryl esters to remnant particles would be at greatest risk for atherogenesis. By contrast, one version of the reverse cholesterol transport theory suggests that individuals with high HDL-cholesterol levels would transport more cholesterol to VLDL or chylomicron remnants, because this step allows a path for exit of cholesterol from the plasma. Further experiments aimed at measuring the transfer of cholesteryl esters between H D L and lipemic particles may be helpful in evaluating these different theories of the relationship between H D L and atherogenesis. Acknowledgments Supported by NIH Grants HL 22682 and 21006. Author is an Established Investigator of the A.H.A.
[29] In Vivo M e t a b o l i s m o f A p o l i p o p r o t e i n E in H u m a n s B y RICHARD E . GREGG and H. BRYAN BREWER, JR.
Apolipoprotein E (apoE), a glycoprotein of Mr 34,000, is associated predominantly with very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) in human plasma.l,2 Human apoE is a polymorphic protein 3 with multiple alleles inherited in a codominant fashion at a 1 C. B. Blum, L. Aron, and R. Sciacca, J. Clin. Invest. 66, 1240 (1980). 2 R. J. Havel, L. Kotite, J. L. Vigne, J. P. Kane, P. Tun, N. Phillips, and G. C. Chen, J. Clin. Invest. 66, 1351 (1980). 3 G. Utermann, M. Jaeschke, and J. Menzel, FEBS Lett. 56, 352 (1975).
METHODS IN ENZYMOLOGY, VOL. 129
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