High density lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile.

Unesterified cholesterol (UC) that is taken up by the liver from lipoproteins is rapidly mixed by exchange with liver UC. Thus, it is not possible to quantitate the transport of UC from different lipoproteins into bile using radiolabeled UC. However, plant sterols do not exchange with UC and are secreted in bile with the same kinetics as UC. To compare the contribution to bile of sterols from different lipoproteins, we perfused isolated rat livers with VLDL, LDL, and HDL that were obtained from patients with hereditary phytosterolemia and were rich in plant sterols. After 30-min recirculating perfusions, hepatic concentrations of plant sterols were not different after different lipoproteins were perfused. However, biliary plant sterol secretion was markedly different: with the perfusion of either VLDL or LDL there was no increase in plant sterols in bile, but with perfusion of HDL, the secretion of plant sterols was increased two- to threefold (P = 0.0005). The increase in biliary plant sterols was detected 5-10 min after HDL was added to perfusates and was similarly large for each of three individual plant sterols that was tracked. Results show that when sterol transport from lipoproteins into bile can be determined, only HDL provides a vehicle for UC elimination in bile that is consistent with its putative function in reverse cholesterol transport.

Utilization of individual lecithins in intestinal lipoprotein formation in the rat.

To determine the molecular species composition of lecithins of different nascent lipoproteins, high density lipoproteins (HDL), very low density lipoproteins (VLDL), and chylomicrons (CM) were isolated from the mesenteric lymph of rats. Lymph was collected at 0 degrees C with 5,5'-dithiobis-2-dinitrobenzoic acid added to inhibit lecithin-cholesterol acyl transferase. CM were separated by ultracentrifugation and HDL from VLDL by dextran SO4-MG+2 precipitation. Molecular species of lecithin were directly isolated by reverse phase high performance liquid chromatography. In fasted animals, the lecithin compositions of lymph HDL and VLDL were virtually the same and closely resembled the lecithin composition of intestinal mucosa. When bile lecithin was eliminated (by bile diversion), there was a marked change in lecithin composition of all lipoprotein and mucosal samples, which was most notable for a reduction in 16:0-species (which are predominant in bile) and a relative increase in the corresponding 18:0-species. Feeding unsaturated triglycerides (triolein, trilinolein, or a combination of triolein and trilinolein) also resulted in a change in HDL and VLDL lecithin composition. The effect was similar whether bile lecithin was present or eliminated and was notable for a reduction in 16:0-species, an increase in 18:0-species, and the emergence of large amounts of diunsaturated lecithins that corresponded to the fatty acid composition of the triglycerides fed (i.e., 18:1-18:1, 18:2-18:2, and 18:1-18:2 lecithins). When bile-diverted rats were infused via the duodenum with a mix of [14C]choline-labeled lecithins (isolated from the bile of other rats), the incorporation of infused lecithins into different lymph lipoproteins was distinctly different. Individual lecithins were incorporated to a variable extent into each lipoprotein. In fasted rats the specific activities of all major molecular species of lecithin were relatively greater in VLDL than HDL, indicating that HDL derived proportionately more of its lecithins from an endogenous pool than did VLDL. Feeding triolein changed the specific activities of more of the lecithin species of VLDL than of HDL. The specific activities of lecithins in CM were more similar to VLDL than to HDL after triolein feeding. Results thus indicate that, although the lecithins of different mesenteric lymph lipoproteins are similar and may be derived from membrane sites with the same lecithin composition, lecithins incorporated into different lipoproteins originate from different metabolic pools and/or by different mechanisms.

Lipids of pigment gallstones.

The lipids of pigment gallstones were analyzed. In contrast to previous reports, pigment stones were found to contain a wide variety of free fatty acids. In addition, pigment stones contained unhydrolyzed phospholipids. Both free fatty acids and phospholipids were present in much higher concentrations in a brown stone obtained from a patient with a biliary tract infection than in a black stone obtained from a patient with sterile bile and a long-standing hemolytic anemia. However, the phospholipids in both kinds of stone consisted primarily of phosphatidylcholine. Separation of stone and bile phosphatidylcholines into their individual molecular species by high performance liquid chromatography indicated that the phosphatidylcholines in stones closely resemble those in bile. The data suggest that both the free fatty acids and the phosphatidylcholine of pigment stones derive from bile phosphatidylcholine, but that the extent of bile phosphatidylcholine participation in pigment stone formation may be variable.

The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal.

Studies were undertaken to assess the contribution of lipoprotein cholesterol to bile and to determine whether already-existent hepatic free cholesterol and the free cholesterol which is newly generated from the hydrolysis of hepatic cholesteryl esters are equally available for secretion into bile or constitute metabolically separate pools. Rats with a bile fistula were injected with an intravenous bolus of high-density lipoprotein recombinants containing free [14C]cholesterol and [3H]cholesteryl esters. Results showed (1) that bile free [14C]cholesterol secretion was a constant and linear proportion of the whole liver free [14C]cholesterol pool, (2) that secretion into bile of free [3H]cholesterol was in direct proportion to the rate of hydrolysis of hepatic [3H]cholesteryl esters, and (3) that rates of biliary cholesterol secretion were very similar when secretion was calculated using the specific activity of free [14C]cholesterol and free [3H]cholesterol in the entire liver to 'label' the precursor free cholesterol pool. Furthermore, rates of secretion that were calculated using either isotope closely approximated the mass of free cholesterol that was directly measured in bile. Results thus indicate that because of equilibration and extensive dilution by the large pool of already-existent free cholesterol, the transport of isotopic cholesterol from lipoproteins cannot be used to directly assess the contribution of lipoprotein cholesterol to the cholesterol that is secreted in bile. These studies further suggest that the totality of preformed free cholesterol in the liver is in metabolic equilibrium in one single kinetic pool and that all hepatic free cholesterol is potentially available for secretion into bile.

Gender differences in the development of hyperlipemia and atherosclerosis in hybrid hamsters.

In response to a diet enriched in saturated fat and cholesterol (CH), male Syrian hamsters develop hyperlipemia and changes of early atherosclerosis. However, it has not been determined if female hamsters are equally susceptible to an atherogenic diet. Male and female hamsters of the F1B hybrid strain (Bio Breeders, Fitchburg, MA) were fed either a chow diet or this diet (HiFat) with added saturated fat (10% coconut oil) and CH (0.05%) for up to 12 weeks. Female hamsters ate significantly more than males, and with the HiFat diet gained threefold more weight than males. However, with the HiFat diet, serum triglycerides (TGs) and CH were markedly increased only in male hamsters. Furthermore, only in males was there a significant increase in stainable fat in the aorta that corresponded to an increase in subintimal foam cells. In freely feeding males, the largest percentage increase in serum CH was in the TG-rich fraction of lipoproteins. After females were castrated, serum TG and CH levels increased to the same extent as in males. These studies demonstrate a profound gender difference in response to an atherogenic diet in these hamsters that has parallels to the lipid patterns of humans and their susceptibility to atherosclerosis.

Delineation of a novel hepatic route for the selective transfer of unesterified sterols from high-density lipoproteins to bile: studies using the perfused rat liver.

Cholesterol is principally excreted from the body in bile as unesterified cholesterol (UC). Using the unesterified plant sterol, sitostanol (SIT), as a nonexchangeable analog for UC, we have found that high-density lipoproteins (HDL), but not low-density lipoproteins, provide a vehicle for the direct delivery of cholesterol to bile. To determine the mechanism for preferential cholesterol transport from HDL to bile, isolated rat livers were perfused with a reconstituted HDL, made with radiolabeled unesterified SIT, UC, and cholesteryl esters (CE). Total biliary sterol secretion was independent of the concentration of HDL added to perfusions, but with increasing HDL-SIT perfused, the proportion of SIT to cholesterol in bile was linearly increased. Biliary SIT secretion was rapid (detected within 2 to 4 minutes after reconstituted HDL was added to perfusions) and was dependent on the immediate presence of SIT in the perfusate, but independent of the amount of SIT that had accumulated in the liver. The ratio of SIT to UC was seven- to ninefold greater in bile than in the liver, consistent with preferential mobilization of membrane sterols delivered from HDL. Although radiolabeled UC as well as SIT was taken up from HDL by the liver and secreted in bile, net UC uptake could not be quantitated because of both UC exchange and a sizable enrichment of HDL with UC mass that approximated the SIT removed during the passage of HDL through the liver. These results are consistent with sterol transport to bile from HDL by a direct plasma membrane pathway and by a mechanism that appears to involve substitution of unesterified (exogenous) sterol from HDL for plasma membrane UC during transport. By this process, HDL can promote reverse cholesterol transport from peripheral tissues to bile, even without an increase in biliary cholesterol secretion.

Effect of different molecular species of phosphatidylcholine on the clearance of emulsion particle lipids.

Studies were performed to assess the effect of changes in the molecular species composition of phosphatidylcholine (PC) on the clearance of emulsion particles that were made to approximate chylomicrons in size and lipid composition. Emulsions were prepared with free [14C]cholesterol, [3H]cholesteryl oleate, triolein, and one of four single PCs that differed in hydrophilic strength (as assessed by the relative rate of elution of these PCs from a reverse phase column). Emulsions were injected as an intravenous bolus into unanesthetized rats and the clearance of lipids was determined at 2-min intervals for 10 min. All emulsion lipids were cleared from the serum in parallel and in an order that closely corresponded to the relative hydrophilic strength of the particular PC that was administered. Fractional rates of clearance, calculated from log-linear plots, were 2- to 10-fold greater for all lipids for the emulsion that was made with the most hydrophilic PC compared to the least hydrophilic PC. Although hepatectomy, performed in acutely anesthetized animals, generally slowed the clearance of lipids, hepatectomy did not abolish differences in the clearance of triolein or specific PCs from emulsions prepared with the most and least hydrophilic PCs. These results indicate that a change in the composition of emulsion particle PCs, independent of any other change in the lipid composition of these particles, can significantly change the metabolism of the whole emulsion particle in the live animal. More specifically, these studies show that the rate of clearance of all emulsion lipids closely corresponds to the hydrophilic strength of the PCs that occupy the emulsion particle surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of bile salt on phosphatidylcholine composition and secretion of hepatic high-density lipoproteins.

Bile salts are necessary for the secretion of phosphatidylcholines (PCs) in bile and result in the selective secretion of highly hydrophilic molecular species of PC that contain a 16:0 acyl group. To determine the effect of bile salt on the secretion of PCs in lipoproteins, isolated rat livers were perfused with and without taurocholate. The PC composition of very-low-density lipoproteins (VLDL), newly synthesized by the liver, precisely mirrored the composition of PCs in the whole liver and was not changed with the administration of taurocholate. In contrast, both the composition of PCs in high-density lipoproteins (HDL) and the absolute amount of newly synthesized HDL were markedly affected by the administration of taurocholate. With taurocholate the PC content of HDL was increased, HDL was enriched, like bile, in 16:0 molecular species of PC, and the amount of HDL that was recovered in the perfusate was 2.5-fold greater than without taurocholate (P less than 0.001). These findings suggest that VLDL and HDL are differently derived from within the liver, that the PCs of HDL and bile originate from the same hepatic pool or by the same mechanism, and that both the secretion of bile and HDL from the liver are susceptible to regulation by bile salt.

Evidence that hepatic triglycerides provide acylglycerides for synthesis of bile phosphatidylcholines.

To determine the biochemical origin of bile phosphatidylcholines (PCs), rat liver perfusions with 16:1 fatty acid (FA) and [3H]glycerol were performed to generate novel radiolabeled bile and liver PCs and their hepatic glyceride precursors. Results showed total equilibration of bile and liver 16:1-16:1 PC when the specific activity of precursor glycerol-3-phosphate was kept constant. However, when the specific activity of glycerol-3-phosphate decreased during the labeling period and during a prolonged chase period with 17:1 FA and nonradiolabeled glycerol, the specific activity of bile 16:1-16:1 PC was appreciably higher than this same PC in the liver and during the chase period was even higher than its hepatic 16:1-16:1 acylglycerol precursors, phosphatidic acid and diglyceride. During the chase period with 17:1 FA, new radiolabeled 16:1-17:1 PC was formed, and again the specific activity of this PC in bile was greater than this PC and 16:1-17:1 phosphatidic acid and diglyceride in the liver. Only the specific activity of liver 16:1-16:1-(FA) triglyceride equaled or was high enough to support the formation of new bile 16:1-16:1 PC. These studies indicate that bile PCs do not directly derive from preexisting hepatic PCs or by de novo synthesis through phosphatidic acids and diglycerides, but likely originate by remodeling from a pool of hepatic triglycerides.

Hepatic phosphatidylcholines: evidence for synthesis in the rat by extensive reutilization of endogenous acylglycerides.

Studies were performed to compare the extent of fatty acid incorporation into liver phosphatidylcholines (PCs) by acyl remodeling and by de novo synthesis. To this end, isolated rat livers were first perfused with palmitoleic acid (16:1) and [2-3H]glycerol and then with 17:1 fatty acid and nonradiolabeled glycerol that resulted in the formation of new molecular species of radiolabeled PCs containing 16:1 and 17:1 acyl groups. The specific activities of newly formed molecular species of the de novo precursors of acylglyceride synthesis, phosphatidic acids (PAs) and diglycerides (DGs), and the products of synthesis, PCs and triglycerides (TGs), were measured at periods during both the labeling period of perfusion with 16:1 (first 15 min) and the more prolonged chase period with 17:1 (up to 120 min). At the end of the labeling period, the specific activity of all the 16:1-containing PAs, DGs, and 16:1-16:1-16:1 and 16:1-16:1-18:2 TGs were the same and were much higher than any molecular species that did not contain 16:1. The specific activities of these molecular species are indicative of the specific activity of molecular species synthesized exclusively by de novo synthesis (i.e., by acylation of glycerol 3-phosphate) during the labeling period. In contrast, the specific activity of 16:1-16:1 PC was only 2/3 that of the other 16:1-16:1 glycerides, and the specific activities of the other 16:1-containing PCs were only about 1/3 that of the corresponding 16:1-containing PAs, DGs, and 16:1-16:1 TGs. After the labeling period and during the chase period with perfusion of 17:1 and nonradiolabeled glycerol, the specific activities of major 16:1 PCs exceeded the specific activities of their corresponding PAs and DGs and remained considerably higher than these precursors of de novo synthesis for the duration of perfusions. However, during this period, the specific activities of major 16:1 PCs were less than their corresponding molecular species of TGs. During the chase period, new 17:1 molecular species of PCs were formed that were also radiolabeled. The specific activity of 16:1-17:1 PC, the 17:1 PC with the highest specific activity, always exceeded its corresponding PA and DG precursors. During the chase period, non-16:1 and non-17:1 molecular species of PCs that comprised the bulk of hepatic PCs were also radiolabeled and the specific activities of these molecular species progressively increased during this period.(ABSTRACT TRUNCATED AT 400 WORDS)

Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography methods are described for separating the major classes of phospholipids and for isolating the individual molecular species of phospholipids. Fractionation of a total lipid extract of rat liver on a silica column resulted in quantitative recoveries of all major phospholipids with preservation of their fatty acid composition. Rat liver phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were then each chromatographed on a C18 reverse phase column to isolate individual molecular species. Component peaks were identified by their fatty acid composition and quantitated by phosphorus determination. Using this method we found that for each of these phospholipids from 30 to 35 different molecular species can be routinely identified and reproducibly quantitated. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on a reverse phase column.

Response of serum triglycerides of endogenous origin to the administration of triglyceride-rich lipid particles.

Studies were performed in rats to quantitate the changes in the concentration of serum triglycerides (TGs) of endogenous and exogenous origin after the acute intravenous injection of TG-rich emulsion particles. Emulsions were prepared to approximate chylomicrons and to contain a TG with a single fatty acid that could be traced during its clearance from the serum. After injection of emulsions, there was a rapid increase of not only the emulsion TG but TGs that contained a variety of other fatty acids of endogenous origin. Endogenous TGs were cleared from the serum at a slower rate than the emulsion TG and accounted for the major increase in serum TGs, especially during the latter phase of the clearance period. The increase in endogenous TGs was completely abolished by hepatectomy, which had no effect on the clearance of the emulsion TG. Results thus show that TGs of hepatic origin accumulate in the serum in response to the introduction of new TG-rich lipoproteins. Feeding rats a specific TG produced a similar result, with a pronounced rise in endogenous TGs that, like the changes after emulsion administration, was particularly evident once the TG that was fed was largely cleared from the serum. These findings are consistent with a process in which the preferential clearance of chylomicron TGs interrupts the clearance of very low density lipoprotein TGs that are produced by the liver. Consequently, the composition of serum TGs that accumulate after a meal may not reflect the composition of the meal itself.

Utilization of different fatty acids for hepatic and biliary phosphatidylcholine formation and the effect of changes in phosphatidylcholine molecular species on biliary lipid secretion.

Biliary cholesterol secretion is ordinarily tightly coupled to phosphatidylcholine (PC) secretion. Bile PCs are distinct in composition and predominantly composed of molecular species with 16:0 in the sn-1 position and 18:2 and 18:1 in the sn-2 position. In an attempt to acutely change the composition of biliary PCs and to assess the effect of a change in PCs on biliary cholesterol secretion, isolated livers were perfused with a variety of single free fatty acids. Rat livers with bile duct cannulas were perfused with a recirculating medium, taurocholate (40 mumol/h), and albumin-bound 16:1, 17:1, 18:1, 20:1, 18:2, 20:4, or 20:5 fatty acids (90 mumol/h) for 2 h. Biliary lipid secretion was measured and bile and liver PC compositions were compared at the start and end of perfusion. Results showed 1) greater utilization of shorter chain than longer chain fatty acids for bile PC formation (16:1 greater than 17:1 greater than 18:2 or 18:1 greater than 20:5, 20:4 or 20:1); 2) no similar pattern of FA utilization for liver PC formation; 3) preferentially greater incorporation of fatty acids into bile PCs compared to liver PCs when perfused fatty acids were used for esterification at both sn-1 and sn-2 positions of PC (to form diunsaturated PCs); and 4) increased biliary secretion of cholesterol relative to PC only when the population of PCs that was newly formed included more hydrophilic molecular species of PC than are present in native bile (that was observed only with perfusion of 16:1). Changes in biliary PC secretion or cholesterol/PC secretion occurred independently of any change in bile salt secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol exchange and synthesis in the live rat.

The turnover of plasma cholesterol and de novo cholesterol synthesis were measured simultaneously in the live rat, immediately after administration of [3H]water together with a large volume exchange transfusion of whole blood prelabeled with [14C]cholesterol. It was possible to separate the exchange of unesterified cholesterol from the uptake and secretion of lipoprotein cholesteryl ester, and also to assess the impact of plasma cholesterol exchange on the measurement of in vivo rates of cholesterolgenesis by individual tissues. Cholesterol was measured by an HPLC procedure that effectively separated cholesterol from other structurally similar sterols, and synthesis was determined by the incorporation of [3H]water into cholesterol. Plasma unesterified cholesterol turnover was multiphasic and exceedingly rapid (initial T1/2, 4.1 min) in contrast to the near linear and much slower turnover of plasma cholesteryl ester (initial T1/2, 59.4 min). Plasma unesterified cholesterol equilibrated with different tissues at different rates, with the liver and adrenal equilibrating most rapidly. Full equilibration of plasma unesterified cholesterol was not achieved with any tissue during the course of this study. For rapidly exchanging tissues like the liver, which was responsible for about 60% of plasma unesterified cholesterol exchange, unesterified cholesterol appeared to be kinetically compartmentalized into rapidly, and much less rapidly, exchangeable pools. After [3H]water administration, the content of newly synthesized cholesterol was greatest in the liver, adrenal, and intestine, and appreciably lower in all other tissues studied. Hepatectomy and intestinal resection resulted in a profound reduction of newly synthesized cholesterol in the plasma and adrenal, but no certain change in the already low amounts at other sites. Thus, while it is clear that appreciable amounts of newly synthesized cholesterol in the adrenal were derived from the plasma by exchange, it was not possible to make this assessment for other selected individual tissues. When, however, newly synthesized cholesterol was determined in the total mass of all extrahepatic and extraintestinal tissues together, exchange could be calculated to account for close to 50% of the new cholesterol recovered in the carcass (in studies of 60 min duration). After correcting for exchange, the liver accounted for 82% of all newly synthesized cholesterol, the intestine for about 10%, and the remaining tissues of the body for just 9%. These results are in marked contrast to recent findings of others and demonstrate that in the live rat cholesterol synthesis is principally confined to the liver.

Influence of lecithin acyl chain composition on the kinetics of exchange between chylomicrons and high density lipoproteins.

The kinetics of lecithin exchange between native lipoproteins was characterized for individual molecular species of lecithins of rat mesenteric lymph chylomicrons and rat plasma HDL. Studies were performed in the absence of lipid transfer proteins. Donor (chylomicrons) and acceptor (HDL) particles were present in ratios of 1:1 and 1:10 with respect to total phospholipid. Biphasic exchange kinetics were observed for all major lecithins common to chylomicrons and HDL at both proportions of donor to acceptor particles. During the early rapid phase of exchange, complete in about 30 min, 40-60% of the total lecithin pool was exchanged. Initial exchange rates were most rapid for the more hydrophilic species of the major lecithins normally present in both lipoproteins. Calculated activation energies correspondingly were least for a diunsaturated lecithin (18:1-20:4), intermediate for lecithins were 16:0 in position-1 (16:0-18:2 and 16:0-20:4), and highest for analogous lecithins with 18:0 in position-1. A 10-fold increase in the ratio of acceptor to donor particles affected neither the biphasic nature of the exchange nor the rates of exchange of individual molecular species (consistent with exchange by diffusion rather than by particle collisions). Total equilibration of individual molecular lecithin species was achieved by 24 hr (37 degrees C, donor to acceptor ratio of 1:1) with only a small change in the relative mass of lecithins in chylomicrons and HDL. Novel lecithins containing 18:3, incorporated into chylomicrons, were found to exchange exceedingly rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for separate pathways of transport of newly synthesized and preformed cholesterol into bile.

Marked kinetic differences were observed when hepatic newly synthesized cholesterol and preformed cholesterol were separately radiolabeled and separately traced into bile. Whereas newly synthesized cholesterol was not evenly distributed throughout the liver but was preferentially secreted into bile, preformed cholesterol was in near-complete equilibrium in the whole liver and bile. Furthermore, whereas newly synthesized cholesterol in bile originated from the interior of the hepatocyte, results suggest that biliary preformed cholesterol may be transported directly from the blood through the plasma membrane of the hepatocyte and secreted from the canaliculus without first entering the interior of the cell and mixing with newly synthesized cholesterol.

Hepatic transport and secretion of unesterified cholesterol in the rat is traced by the plant sterol, sitostanol.

The hepatic uptake, transport, and secretion into bile of unesterified cholesterol cannot be directly quantitated because of extensive exchange and equilibration between different pools of unesterified cholesterol. Plant sterols are structurally similar to cholesterol but because of poor intestinal absorption are ordinarily not present in the liver. To quantitate hepatic sterol uptake and transport in the absence of exchange with endogenous sterols, isolated rat livers were perfused with the plant sterol, sitostanol, incorporated in phosphatidylcholine liposomes. Appreciable amounts of sitostanol were taken up by the liver and uptake was independent of the presence of bile salt. In contrast, like unesterified cholesterol, the secretion of sitostanol in bile required bile salt. Sitostanol was detected in bile within 5 min after a perfusion was begun and reached a plateau by about 20 min. The rate of appearance of sitostanol in bile was precisely the same as unesterified cholesterol when both sterols were perfused together. Furthermore, the output of sitostanol in bile was directly proportional to the output of cholesterol. At the peak of biliary sitostanol secretion, the amount of sitostanol relative to unesterified cholesterol was much greater in bile (40-50% of sterols) than in the whole liver (11% of sterols). Selective biliary secretion of sitostanol was associated with much greater concentrations of sitostanol in canalicular membranes than in the interior membranes of the hepatocyte and in newly secreted high density lipoproteins compared to newly secreted very low density lipoproteins. These results indicate that sitostanol parallels the secretion from and distribution of unesterified cholesterol in the liver and suggest that sitostanol can be used as a physiologic analog of unesterified cholesterol to trace the transport of sterols through the liver.

Hepatic cholesterol synthesis and the secretion of newly synthesized cholesterol in bile.

To determine the effect of increased hepatic cholesterol synthesis on the secretion of newly synthesized cholesterol in bile, rats were fed with cholestyramine, a bile-acid-binding resin that increases the number of hepatocytes that synthesize cholesterol. Cholesterol synthesis was measured 15 min after [3H]water injection to avoid appreciable exchange between the liver and serum of newly synthesized cholesterol that accumulates in the serum in studies of several hours duration. At 15 min after [3H]water injection, the specific radioactivity of cholesterol in the liver and hepatic microsomes was greatly increased in resin-fed animals compared with controls. However, with resin, the specific radioactivity of newly synthesized cholesterol that was secreted in bile was the same as for controls. At 15 min after [3H]water injection the specific radioactivity of serum cholesterol was minimally increased and not different in resin and control groups. In contrast, in studies that were longer than 60 min, newly synthesized cholesterol in serum was appreciably increased in resin-fed animals, and newly synthesized cholesterol in bile was also greatly increased compared with controls. Thus, when appreciable cholesterol exchange is avoided, an increase in hepatic cholesterol synthesis and the number of hepatocytes that synthesized cholesterol does not result in an increase in newly synthesized cholesterol in bile. Our results suggest that newly synthesized cholesterol is secreted in bile from a fixed subpopulation of hepatocytes. From a comparison of the specific radioactivity of newly synthesized cholesterol in whole liver and bile, it can be estimated that this subpopulation of hepatocytes represents about 20% of the total hepatocyte mass.