A comparison of lipoprotein secretion, bile production and hepatic morphology in isolated rat livers perfused with a perfluorocarbon emulsion or rat erythrocytes.

Isolated rat livers were perfused with an oxygenated perfluorocarbon emulsion, FC-43 emulsion for 1 to 4 hr. FC-43 emulsion contained 20% FC-43 (wt/vol) perfluorotributylamine (the fluorocarbon component for the transport of oxygen and carbon dioxide) emulsified with 2.56% Pluronic F-68 (a nonionic surfactant) in Krebs-Ringer bicarbonate buffer. FC-43 emulsion also contained 3% hydroxyethyl starch as an oncotic agent and 1.8 mg/ml glucose. The viability (oxygen consumption), bile secretion, structural integrity and secretion of nascent lipoproteins by FC-43-perfused rat livers was compared with livers perfused with Krebs-Henseleit bicarbonate buffer that contained rat erythrocytes (25% hematocrit) and 1.5 mg/ml glucose (red blood cell medium). Oxygen consumption was somewhat higher in livers perfused with FC-43 emulsion. Bile secretion of livers perfused with FC-43 emulsion for 4 hr was reduced significantly to 40% of that by red blood cell medium. The structural integrity of livers perfused with FC-43 emulsion varied from normal to marked cellular damage. Light-microscopical examination of rat livers perfused with FC-43 emulsion showed ballooning of sinusoids, presence of vacuoles in sinusoidal lining cells in some hepatocytes and detachment of endothelium in sinusoids. The number of vacuoles progressively increased in longer perfusions. Electron-microscopical studies showed the presence of small (60 to 100 nm) vesicles of varying electron density, presumably fluorocarbon particles inside the vacuoles in sinusoidal lining cells (Kupffer and endothelial) and hepatocytes. After 4 hr of perfusion with FC-43 emulsion, most of the sinusoidal endothelia were denuded, and the microvilli of the hepatocytes all but disappeared. In contrast, the ultrastructure of rat livers perfused with red blood cell medium for 4 hr was unaltered. The accumulation of nascent lipoproteins in perfusates of FC-43-perfused livers was markedly reduced, and no normal very-low-density lipoprotein, low-density lipoprotein or high-density lipoprotein were isolated. Chemical analysis showed the presence of Pluronic F-68 in all lipoprotein fractions. Our data strongly suggest that, during recirculating liver perfusions with FC-43 emulsion (between 1 and 4 hr), the nonionic surfactant detergent Pluronic F-68 dissociated from the emulsion and markedly affected hepatic structure, lipoprotein secretion and the composition of lipoproteins isolated from perfusate. Therefore FC-43 emulsion is not a suitable liver-perfusion medium for studies of lipoprotein metabolism.

Colorimetric assay for pluronic F-68 as measured in isolated rat liver perfusion systems.

A colorimetric assay has been developed for the quantitation of Pluronic F-68, a nonionic detergent (surfactant) which is a polyoxypropylene-polyoxyethylene (POP-POE) block copolymer. We measured this substance in organic extracts of rat liver perfusates from livers which had been perfused with an oxygenated perfluorocarbon, FC-43 Emulsion (Oxypherol).

Nascent high density lipoproteins from liver perfusates of orotic acid-fed rats.

Uniformly fatty livers from orotic acid-fed rats secreted almost no very low density lipoproteins (VLDL) but normal amounts of nascent high density lipoproteins (HDL) accumulated in perfusates. When lecithin:cholesterol acyltransferase (LCAT) was inhibited, nascent HDL were uniformly discoidal and lacked cholesteryl esters. Lipid and apoprotein compositions of nascent HDL from normal and fatty livers were similar whether LCAT was inhibited or not. Apolipoprotein B-100 was not detected in perfusates of uniformly fatty livers, but small amounts of apolipoprotein B-48 were present in HDL2 fractions. Nascent lipoproteins were not seen in Golgi compartments, but lipid-rich particles were clearly evident in endoplasmic reticulum cisternae adjacent to the cis face of the Golgi complex, suggesting that orotic acid blocks VLDL secretion by preventing translocation of nascent particles from the endoplasmic reticulum to the cis Golgi compartment. The accumulation of normal amounts of discoidal HDL in liver perfusates despite virtual absence of triglyceride-rich lipoproteins in Golgi secretory compartments, the space of Disse, and the perfusate is inconsistent with the concept that nascent HDL are exclusively a product of surface remnants cast off during lipolysis of chylomicrons and VLDL.

Secretion of lipoprotein-X by perfused livers of rats with cholestasis.

The major abnormal plasma lipoprotein of cholestasis (LP-X) was isolated from blood plasma and from perfusates of isolated livers of rats with biliary obstruction. In both cases LP-X was composed mainly of about equimolar parts of phospholipids and unesterified cholesterol; the small protein component was primarily the arginine-rich apolipoprotein. By electron microscopy, LP-X appeared as a unilamellar liposome (690 A mean diameter, range 400-1000 A) with the trilaminar staining image typical of phospholipid bilayers. Extensive block staining of cholestatic livers for 48 hr with warmed uranyl acetate (37 degrees) permitted the visualization of vesicles indistinguishable from LP-X within hepatic parenchyma. These trilaminar-staining vesicles occurred predominantly within bile canaliculi. They also were seen in nearby cytoplasmic vacuoles or invaginations between hepatocytes and in the space of Disse. Similar vesicles were not seen in the endoplasmic reticulum or Golgi cisternae. These observations raise the possibility that the vesicles are formed within bile canaliculi and are transported from the canaliculi to the space of Disse within pinocytotic vacuoles.

Evidence that a separate particle containing B-apoprotein is present in high-density lipoproteins from perfused rat liver.

High-density lipoproteins (HDL) (1.075 less than d less than 1.175) from perfusates of rat liver, unlike those of blood plasma, contain protein with the properties of B-apolipoprotein. This protein remains near the origin upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, has beta electrophoretic mobility in agarose gel, is insoluble in tetramethylurea, and precipitates with antisera to the B-apoprotein isolated from low-density lipoprotein. B-apolipoprotein in HDL from perfusates binds to concanavalin-A Sepharose and can thus be separated from the characteristic HDL, the chemical and physical properties of which are otherwise preserved. These observations suggest that in addition to the discoidal lipoproteins, another particle that contains B-apoprotein exists in HDL of perfusates.

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.