Lipoprotein abnormalities in primary biliary cirrhosis: Information concerning control of plasma density lipoprotein levels

GASTKOENTEKOLOGY

1985;89:1426-35

EDITORIALS

Lipoprotein Abnormalities in Primary Biliary Cirrhosis: Information Concerning Control of Plasma Densitv LiDotwotein Levels J

High density lipoproteins (HDLs) are a collective term for spherical lipid-protein particles found in human plasma (for a recent review see Reference 1). High density lipoprotein particles have a molecular mass of 200-400 X 10" daltohs and range in diameter between 70 and 100 A. They are rich in protein [apolipoprotein (apo) A-I and apo A-II] and phospholipids, with minor amounts of cholesterol (mostly esterified) and triglyceride. On the basis of circulating particle number, HDL is the predominant plasma lipoprotein class. The other lipoprotein classes found in normal fasting plasma are very low density lipoproteins (VLDLs) and low density lipoproteins (LDLs). Very low density lipoproteins and LDLs are triglycerideand cholesterol-rich lipoproteins, respectively, that play important roles in plasma triglyceride and cholesterol transport. The role of HDL in lipid transport is more complicated. High density lipoprotein functions as a site for plasma cholesterol esterification through the action of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT), which requires apo A-I for activity (2). High density lipoprotein is also thought to function in the transport of cholesterol from peripheral tissues to the liver (“reverse cholesterol transport”). Associations have been demonstrated between low levels of HDL and the risk of developing coronary artery atherosclerosis (3). For these reasons, the investigation of the regulation of plasma HDL levels is an important research area. In the rat, and probably in humans, the intestine and liver contribute equally to plasma levels of apo A-I. Whether apo A-I is secreted by either organ as the free apoprotein that then combines with lipids in lymph or plasma to form HDL or is secreted as a lipoprotein is controversial. Both discoidal and spherical HDLs are found in rat mesenteric lymph (4).Both types of particles are apo A-I rich and phospholipid rich. Rat liver perfusate contains a discoidal HDL when the liver is perfused in the presence of an LCAT inhibitor. This “nascent HDL” is an apo E (arginine-rich apolipoprotein)-rich and phospholipid-rich particle. Another source of plasma apo A-I comes from intestinal chylomicrons or VLDLs, or both. The triglyceride content of these particles, as well as VLDL of hepatic origin, is

1

I

depleted in the capillary endothelium through the action of lipoprotein lipase (LPL), an enzyme that requires a specific apolipoprotein (apo C-II) for activity (5). During lipolysis, the apo A-I in these particles is released and combines with plasma HDL. It has been shown that phospholipid and unesterified cholesterol (“surface lipids”) from these particles also move to plasma HDL. Another pathway for the formation of HDL precursors is via apo A-I-phospholipid associations. The source of this phospholipid is thought to be cells, lipoproteins, or lipolyses of triglyceride-rich lipoproteins. These discoidal complexes then combine with unesterified cholesterol (derived from similar sources) to yield discoidal HDL. The action of LCAT on this discoidal HDL converts it to spherical, esterified cholesterol-rich HDL. Thus, LCAT plays a critical role in the formation of plasma HDL. Most of the esterified cholesterol formed in HDL through the action of LCAT does not remain in HDL. Through the action of the core-lipid transfer protein, it is transferred to lower density lipoproteins (VLDLs), and triglyceride is transferred from these lipoproteins to HDLs. Some investigators have suggested, but have not proved, that both LCAT and the core-lipid transfer protein are complexed together in an HDL subfraction. In addition to the exchange of esterihed cholesterol and triglyceride between VLDLs and HDLs, there is also exchange of phospholipid and unesterified cholesterol between lipoproteins and cell membranes. Thus, the lipid composition of plasma HDL is in a constant state of flux. Even the apoproteins in HDLs are known to exchange readily between themselves, and some (apo Cs) with other lipoprotein types. The “mobility” of HDL components has greatly complicated studies of catabolism. The use of animal models in which some of these complexities are eliminated has aided our understanding of HDL catabolism. These studies, using ligands attached to HDLs that are taken up by cells, and remain in the cell, have indicated that the liver is the major site of HDL cholesterol ester and apo A-I uptake (on an organ basis) (6).On a per gram basis, the adrenal gland and the gonads take up more HDL than the liver. In the rat, HDL serves as a source of cholesterol

December 1985

for steroidogenesis. Because lipoprotein metabolism in the rat differs from that in humans, it is not presently known whether these results also apply to humans. There is also some evidence of a specific apo A-I membrane receptor on animal cells. The subject of HDL catabolism is under extensive study at present; thus our knowledge of this very important aspect of lipoprotein metabolism should increase greatly in the near future. As indicated above, the liver is the central organ in lipoprotein metabolism. Contributions of the liver include (a) the synthesis of apoproteins, which are constituents of lipoproteins and are essential cofactors in the activation of key enzymes involved in lipoprotein metabolism; (b) synthesis and secretion of LCAT; (c) uptake and degradation of triglyceridedepleted chylomicrons (remnants), LDLs, and HDLs; and (d) regulation of cholesterol homeosfasis by synthesis of bile acids from cholesterol and excretion of cholesterol in bile. The importance of these hepatic contributions to lipoprotein metabolism is dramatically demonstrated by the profound derangements in lipoprotein concentration and composition that occur as a consequence of hepatocellular or cholestatic liver disease (7,8). These changes are largely secondary to abnormalities in lipoprotein synthesis and catabolic functions of the liver. Recent studies of patients with alcoholic hepatitis (9-15) provide evidence that hepatocellular injury is associated with the following alterations in plasma lipoprotein composition: (a) VLDL is relatively‘norma1 in lipid composition but deficient in apoproteins E and C; (b) LDL contains mainly apoprotein (apo) B, but is heterogeneous in size and is enriched in triglyceride and deficient in esterified cholesterol; and (c) HDL is diminished in concentration, enriched in apo E and phospholipid, and deficient in apo A-I and esterified cholesterol. Using density gradient ultracentrifugal techniques, the abnocfnal HDL has been resolved into discoidal (apo E rich) and spherical particles (apo A-I rich). Several studies (7) documented low plasma LCAT activity in alcoholic liver disease associated with impaired cholesterol esterification. Lecithin-cholesterol acyltransferase deficiency also occurs in nonalcoholic hepatocellular injury. Associated with LCAT deficiency is decreased plasma apo A-I, an activator of LCAT. The catabolism of apo A-I is increased in alcoholic hepatitis, providing a rationale for reduced plasma levels. Hepatic triglyceride lipase (HTGL) is found on the endothelial cell plasma membrane and is heparinreleasable. Hepatic triglyceride lipase appears to be important in the intermediary catabolism of triglyceride-depleted VLDL and HDL metabolism (see below). This enzyme may be deficient in patients with liver disease and may partially account for accumu-

EDITORIALS 1427

lation of triglyceride-rich LDL (13). Recently, it was proposed that the apo E-enriched HDL and apo Benriched VLDL, which accumulate in the plasma in alcoholic hepatitis, represent nascent HDL and VLDL and may be the principal pathway through which apo E and apo B are secreted by the liver (11). In this issue of GASTROENTEROLOGY, Jahn et al. (16) report on the nature and etiology of plasma lipoprotein abnormalities in patients in varying stages of primary biliary cirrhosis (PBC), a chronic form of cholestatic liver disease. Patients with advanced disease (group 2) had marked elevations in LDL with the presence of lipoprotein-X (a vesicular, abnormal lipoprotein) and a significant decrease in HDL. The ratio of unesterified cholesterol to total cholesterol was also elevated in these patients, which was consistent with their decreased plasma LCAT mass and activity. Lipoproteins of abnormal morphology (bilayered disks) could be seen in LDL and HDL by electron microscopy after negative staining. The subspecies HDL, was the only HDL subspecies present in the plasma; and apo A-I and apo A-II, the major protein components of HDL, were decreased. As was indicated above, similar findings have been reported for other forms of liver disease. The major findings of Jahn et al. concern those of patients with early and intermediate histologic stages of PBC (group 1). These patients had mild elevations of VLDL and LDL, but also had marked increases in plasma HDL. Parameters of cholesterol eqterification were normal and lipoproteins of normal (spherical) morphology were observed after negative staining. The subspecies of HDL elevated in these patients was HDL2, the larger and lighter of the two major HDL subspecies (see below). Apo A-I and apo A-II were also increased in this patient group. Mean postheparin hepatic HTGL activity was decreased in this group of patients with PBC and the group with advanced disease. The authors presented evidence that the decreased HTGL activity was caused by the presence of a heat-labile inhibitor in the patients’ plasma. That the patients with PBC in the early and intermediate stages of the disease had abnormally elevated HDLz levels is a surprising finding. This finding, along with the observation of decreased HTGL activity in patients in all stages of PBC, highlights the role of HTGL in HDL metabolism. HDi2 and HDL3 are the major HDL subspecies present in the plasma of r)ormal humans (1).HDL2 has about a 50% larger core diameter than HDL3, and therefore contains threefold to fourfold more esterified cholesterol and triglyceride molecules. The surface area of HDLz is double that of HDL3. Hence, more “surface lipids” (phospholipids and unesterified cholesterol) and apo A-I are found per particle of HDL2. Our current knowledge indicates that these

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GASTROENTEROLOGY

EDITORIALS

two HDL subspecies are interconvertible in plasma. Lipolysis of triglyceride-rich lipoproteins, catalyzed by LPL, the surface coat of other lipoproteins, and cell membranes supply phospholipids, unesterified cholesterol, and apolipoproteins to HDL,,. Conversion of HDL2 is catalyzed by LCAT. The transfoymation of HDL, to HDL, depends on a lipid transfer protein that transfers cholesteryl esters in HDL, to triglyceride-rich lipoproteins and transfers triglyceride from these particles to HDL2, thereby enriching HDL2 in triglyceride. Hydrolysis of this transferred predominantly by triglyceride in HDL2, catalyzed HTGL, results in decreased size and density of the lipoprotein. Continuation of this process leads ultimately to HDL3. The following factors favor a predominance of HDbZ: (a) the LPL and LCAT enzyme activities and a proper supply of phospholipids, (b) unesterified cholesterol, and (c) apolipoproteins. Conversely, the following factors favor a predominance of HDL3: (a) HTGL and lipid transfer protein activities and (b) the presence of a large mass of triglyceride-rich lipoproteins. Therefore, the distribution of HDL2 and HDL:, in plasma reflects the equilibrium of these pathways. the lowered HTGL activiIn group 1 PBC patients, ty along with the higher unesterified and esterified cholesterol concentrations in plasma (resulting from partial regurgitation of bile into plasma and the subsequent conversion of unesterified cholesterol to esterified cholesterol by the action of LCAT) favor an increased HDLz subspecies. Thus, the data of Jahn et a!. provide a confirmation of the above theories of HDL subspecies distribution. They postulate that the decreased HDL concentrations observed in the advanced disease, despite decreased HTGL activity, were probably due to the concomitant LCAT deficiency. Formation of normal HDL is not possible in the’ absence of LCAT activity. Previous workers have shown that in male alcoholics without severe liver disease (cirrhosis or alcoholic hepatitis), HDL levels are elevated, particularly in the HDL, subspecies, upon the start of abstention and decrease to normal levels within 2 wk (17-19). Alcoholics with severe liver disease failed to show this ethanol-induced rise in HDL. Several years ago we reported long-term (6-10 mo) follow-up studies on 4 alcoholic hepatitis patients (9). These studies indicated a substantial elevation of HDLz cholesterol and apo A-I in 3 of these patients, all of whom had continued to drink alcohol after discharge. The other patient, who had a normal HDL cholesterol level, had stopped drinking. The mechanism of the remarkable elevation of HDL,-like material in the plasma of patients who continued drinking may be related to the heat-labile plasma HTGL inhibitor reported by Jahn et al. It is conceivable that

Vol.

89, No. 6

this inhibitor could be a cellular HTGL regulator released to plasma by hepatocellular necrosis. The identity of this inhibitor is a fertile area for further research and could provide further insight into the regulation of plasma HDL levels. SEYMOUR M. SABESIN, STUART W. WEIDMAN. Department

Section

of Internal

of Digestive

Hush-Presbyterian-St.

Chicago,

M.D. Ph.D. Medicine Diseases

Luke’s

Medical

Center

Illinois

References 1. Eisenberg 2. 3.

4.

5.

6.

7. 8. 9.

10.

11.

12.

13. 14.

S. High density lipoprotein metabolism. J Lipid Kes 1984;25:1017-58, Marcel YL. Lecithin:cholesterol acyltransferase and intravascular cholesterol transport. Adv Lipid Res 1982;19:85-135. Miller GJ, Miller NE. Plasma high-density lipoprotein concentration and development of ischaemic heart-disease. Lancet 1975;i:16-9. Bisgaier CL, Glickman RM. Intestinal synthesis, secretion, and transport of lipoproteins. Ann Rev Physiol 1983;45:62536. Jackson RL. Lipoprotein lipase and hepatic lipase. In: Boyer PD. ed. The enzymes. 3rd ed. Vol. 16. New York: Academic, 1983:141-81. Pittman RC, Steinberg D. Sites and mechanisms of uptake and degradation of high density and low density lipoproteins. J Lipid Res 1984;25:1577-85. Sabesin SM, B&tram PD, Freeman MR. Lipoprotein metabolism in liver disease. Adv Intern Med 1980;25:117-46. Sabesin SM. Cholestatic lipoproteins-their pathogenesis and significance. Gastroenterology 1982;83:704-9. Weidman SW, Ragland JB, Sabesin SM. Plasma lipoprotein composition in alcoholic hepatitis: accumulation of apolipoprotein E-rich high density lipoprotein and preferential reappearance of “light” HDL during partial recovery. J Lipid Res 1982;23:556-69. Sabesin SM, Hawkins HL, Kuiken L, et al. Abnormal plasma lipoproteins and lecithin: cholesterol acyltransferase deliciency in alcoholic liver disease. Gastroenterology 1977;72: 510-8. Ragland JB. Bertram PD. Sabesin SM. Identification of nascent high density lipoproteins containing arginineirich protein in human plasma. Biochem Biophys Res Commun 1978;80:818. Ragland JB, Heppner C, Sabesin SM. The lole of lecithin: cholesterol acyltransferase deficiency in the apoprotein metabolism of alcoholic hepatitis. Stand J Clin Lab Invest 1978; 38(Supp1 150):208-13. Freeman M, Kuiken L, Ragland JB, et al. Hepatic triglyceride lipase deficiency in liver disease. Lipids 1976;12:443-5. Sabesin.SM, Ragland JB, Freeman MR. Lipoprotein disturbances in liver disease. In: Popper H, Schaffner F, eds. Progress in’ liver disease. New York: Grune & Stratton, 1979:243-62.’

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Seidel D, Greten H, Geisen H, et al. Further aspects on the characterization of high and low density lipoproteins in patients with liver disease. Eur J Clin Invest 1972;2:359-64. 16. Jahp CE, Schaefer EJ, Taam LA, et al. Lipoprotein abnormalities in primary biliary cirrhosis. Association with hepatic lipase inhibition as well as altered cholesterol esterification. Gastroenterology 1985;89:1266-78.

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1985

17. Ekman R, Fex G, Johansson BG, et al. Changes in plasma high density lipoprotein and lipolytic enzymes after long-term, heavy ethanol consumption. Stand J Clin Lab Invest 1981; 41:709-15. 18. Devenyi P, Robinson GM, Kapur BM. et al. High-density lipoprotein cholesterol in male alcoholics with and without severe liver disease. Am J Med 1981;71:589-94. 19. Weidman SW. Beard JD, Sabesin SM. Plasma lipoprotein

changes during abstinence rosis 1984;52:151-66.

alcoholics.

Atheroscle-

Address requests for reprints to: Seymour M. Sabesin, M.D., Department of Internal Medicine, Section of Digestive Diseases, Rush-Presbyterian-St. Luke’s Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612. 0 1985 by the American Gastroenterological Association

Extraher,atic Henadnavirus Does It &lean? A For many years the conventional view of hepatitis B virus (HBV) biology held that the virus was strictly hepatotropic and did not initiate the productive infection of nonhepatocytic cells. Recent rapid progress in hepatitis B virology has, however, reopened the question. The recognition that viruses structurally related to HBV exist naturally in ducks, ground squirrels, and woodchucks (I] has created new opportunities for examination of the issue in animal models; in addition, newer molecular techniques for virus detection are increasingly being applied to clinical specimens from human HBV carriers. Both lines of investigation have now documented the presence of hepadnaviral genomes in extrahepatic sites. Before considering the results generated by these new studies, however, a few comments are in order regarding the nature of the methods used and the interpretation of the kinds of data they generate. Because no cell culture system for growing hepadnaviruses exists as yet, the determination of viral tropism rests upon the demonstration of viral antigens or nucleic acids in various tissues rather than on the recovery of infectious virus. In earlier days this was accomplished by immunofluorescence (or immunohistochemistry) for viral antigens. Current approaches use nucleic acid hybridization to detect viral DNA directly, and properly performed and interpreted they can yield a great deal more information about the replicative state of viral genomes in specimens. This is particularly true for the hepadnaviruses, because in cells in which active viral replication is proceeding (in virologic parlance, productively infected cells) molecular forms of viral DNA are present that are not found in circulating mature virions (2). The seminal experiments of Summers and Mason (3) established that the basic replication cycle of these agents involves the synthesis of viral DNA by reverse transcription of an RNA template, a step previously thought to be unique to retroviruses. Productively infected cells harbor abundant amounts of RNA: DNA hybrid molecules that are the actual replicative forms of the genome, and that have charac-

in chronic:

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DNA: What

teristic structural features readily apparent in electrophoretic analyses. These forms are the precursors of the viral genomes that are packaged into virions and exported into serum, and can easily be distinguished from them. Thus, cells that are productively infected by hepadnaviruses can be readily differentiated from uninfected cells contaminated with serum-derived virus, and from cells in which virus has entered but in which productive infection has been aborted or is somehow aberrant. Such nonproductive infections are well known in virology and can, in principle, be either inconsequential or highly significant (latent herpes simplex infection of ganglionic cells would be an example of the latter). In 1983, Mason and coworkers (4) presented the first solid evidence that productive hepadnavirus infections can occur at extrahepatic sites. They detected not only viral antigens but also replicative intermediates in cells of exocrine and endocrine pancreas and in the proximal renal tubular epitheliurn of duck hepatitis B virus (DHBV)-infected ducks; these tissues were also shown to contain the normal complement of viral RNA species (4). In this issue of GASTROENTEROLOGY, Tagawa et al. (5) present confirmatory results using similar methods and additionally suggest the presence of transient splenic infection in this animal model. In general, replication in these extrahepatic sites is considerably less extensive than in the liver (steady-state levels of viral DNA are usually