Biomedical subjects

J B Marsh

Publications and source records attributed to J B Marsh.

At least 55 records · Page 3Linked to original sources

PubMed1981

Metabolic heterogeneity of apolipoprotein B in the rat.

Triglyceride-rich lipoprotein apoprotein catabolism was studied in rats from 5 to 60 min after intravenous injection of 125I-labeled lipoproteins. The plasma and liver labeled apoprotein content was analyzed by gel filtration column chromatography using an elution buffer containing 1% sodium dodecyl sulfate. The method resolved two B apoproteins of lower (apo B1) and higher (apo Bh) molecular weight. Total apoprotein B disappeared from plasma faster than either apo E or apo C and the smaller sized apo B1 had the most rapid disappearance, with 90% being lost after 60 min. The larger sized apo Bh disappeared rapidly from the plasma in the first 15 min but between 15 and 60 min 40% of the apo Bh remained in the plasma, associated with low density lipoprotein. Apoprotein analysis of liver homogenates was consistent with the plasma results. There was 28% of apo B1 compared to 16% of apo Bh present in the liver 5 min after injection, expressed as percent of initial injected radioactivity in each fraction. Apo B1 and apo Bh were the predominant liver apoproteins up to 30 min but by 60 min there was little of either apo B in the liver. In contrast to apo B, there was a relatively constant amount of apo E and apo C, about 10%, associated with the liver over 60 min. Plasma apo E declined progressively to 68% and apo C to 86% of initial concentration by 60 min. These findings suggest that there is differential hepatic catabolism of a subpopulation of triglyceride-rich lipoproteins containing apo B1. A population of triglyceride-rich lipoproteins containing apo Bh preferentially enters the low density lipoprotein pool with a slower catabolism. The results are consistent with an hypothesis that apo B1 mediates binding and rapid hepatic catabolism of its associated lipoproteins. Metabolic heterogeneity of the triglyceride-rich lipoproteins may be explained by the molecular heterogeneity of apoprotein B.

Animals↗

PubMed1979

Lipoproteins in experimental nephrosis: plasma levels and composition.

Experimental nephrosis was induced in rats by administration of puromycin aminonucleoside and the levels of plasma lipoproteins were examined 7 days later and compared to controls. As determined by density ultracentrifugation, VLDL, IDL, LDL, and HDL protein levels were increased by 8, 4, 5, and 5 times, respectively. These increases were accompanied by changes in lipid and apoprotein composition. The VLDL, IDL, and LDL fractions contained less triglyceride and more phospholipid and cholesterol, while HDL lipid composition was not altered. The apoprotein composition of VLDL and IDL were not measurably altered, but LDL contained less apoE. HDL had a markedly abnormal composition characterized by an almost complete absence of apoA-IV and apoE, increased apoA-1, and decreased apoC. While increased hepatic synthesis can account for much of the observed hyperlipoproteinemia in nephrosis, the changes in lipoprotein composition suggest impaired catabolism as a contributory factor.

Animals↗

PubMed1979

Hepatic secretion of lipoproteins in the rat and the effect of experimental nephrosis.

Livers from normal and nephrotic rats were perfused by the nonrecirculating technique. Nephrosis was studied on the 7th d after the injection of puromycin animonucleoside. Amino acid-labeled lipoproteins (d < 1.21) were isolated from the perfusion medium by agarose column chromatography or by sequential density ultracentrifugation. In both groups of animals, in addition to very low density lipoproteins and nascent high density lipoproteins, column chromatography revealed the presence of a peak of 2-3 x 10(6) daltons. This peak contained lipoproteins of densities corresponding to <1.006, 1.006 < d < 1.02, and 1.02 < d < 1.06, which indicated that rat liver secretes a heterogeneous mixture of triglyceride-rich lipoproteins. The amount of these lipoprotein density classes was measured and their lipid and apoprotein composition and their apoprotein specific activity were determined. In both groups of rats there was a progressive rise in phospholipid and decrease in triglyceride content as the isolation density increased from 1.006 and 1.06. The lipoproteins from the nephrotics had higher amounts of cholesterol. The livers from the nephrotic rats secreted two to three times as much lipoprotein as controls in all density classes in the first 20 min, but during the next 40 min only the 1.02 < d < 1.06 and nascent high density lipoproteins remained at this high level compared to controls. A larger total liver pool of apolipoproteins in nephrotic livers was inferred from their lower specific activities during the first 20 min. The apoprotein composition of liver perfusate lipoproteins from nephrotics differed from controls. There was a 40% decrease in the amount of low molecular weight apoproteins in all density classes, with corresponding increases in apo B and apo E in the triglyceride-rich fractions. The apo A-1 content of nascent HDL was increased from 16% in controls to 52% in nephrotics, with corresponding decreases in apo C and apo E. When these results were combined with specific activity measurements of the individual apoproteins and the net secretion rate of total protein in each lipoprotein class, it was possible to estimate the total amount of each apoprotein secreted and the total incorporation of labeled amino acids into each. The incorporation of label gave results similar to those obtained by direct measurement of the amounts of apoproteins. Apo E secretion was increased by a factor of 1.8, apo B by 2.8, and apo A-1 by 8.4, whereas the secretion of apo C was not significantly altered. We explain these results by postulating that the primary stimulus to hepatic plasma protein synthesis in response to proteinuria is general and that subsequent negative feedback regulation affects individual apolipoprotein synthesis rates. A corollary of this hypothesis is that the biosynthesis and secretion of an apoprotein may be regulated independently of the lipoprotein density class in which it is found.

Animals↗

PubMed1978

Proteolysis of very low density lipoprotein in perfused lung.

Perfusion of homologous 125I-labeled rat very low density lipoprotein through isolated rat lungs in the presence of heparin resulted in apoprotein proteolysis. At least the apoprotein C was degraded into two peptides smaller than 7500 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lung uptake of radioactivity was small and due mainly to the presence of the larger of the two peptides. The lung protease was not active against an 125-I-labeled albumin substrate and was not released into the medium by heparin.

Animals↗

PubMed1978

Labeling of high density lipoproteins with [3H] acetic anhydride.

Rat serum HDL was labeled by reaction with [3H] acetic anhydride at pH 7.2 for 30 min at room temperature by a modification of the method of Montelaro and Rueckert (1975. J. Biol. Chem. 250: 1413). Protein specific activities of 60 dpm/ng were achieved. Seven percent of the label was in lipid, of which 92 percent was recovered in phospholipid. The labeled HDL migrated as a single band as seen by electrophoretic or column chromatographic analysis. When the labeled HDL was injected into rats without re-isolation, the biological half-life was not significantly different from HDL labeled in vitro with 125I or in vivo with amino acids. All of the apoproteins were labeled; their specific activities were closer to one another than those obtained with 125I. For some applications, acetylation may provide a useful alternative to the 125I labeling procedure.

Acetates↗

PubMed1976

Metabolism of 125I-labeled lipoproteins by the isolated rat lung.

The capacity of the isolated perfused rat lung to metabolize the protein moieties of serum lipoproteins was assessed using homologous (rat) and heterologous (human) plasma lipoproteins. The protein and lipid moieties of the plasma lipoproteins were labeled in vivo with Na[125I]. In selected cases the lipoprotein peptides were labeled in vivo with 14C- or 3H-labeled amino acids. Uptake of lipoprotein label during perfusion was monitored by measure of losses in perfusate label and by rises in pulmonary tissue labeling as shown by radioassay and by light and electron microscope radioautography. Lipoprotein degradation was assessed by fractionation of perfusate and lung tissue radioactive material into trichloroacetic acid (TCA)-isoluble, TCA-soluble, and ether-ethanol-soluble fractions. When heparin was included in the perfusion medium, there was selective degradation of the protein portion of very low density lipoprotein (VLDL) in the perfusate and concomitant uptake of radioactive label by the lungs. Low density lipoprotein (LDL)) was neither taken up nor catabolized by the isolated rat lung in the absence or presence of heparin. By light and electron microscopy, the label was localized over the interalveolar septa, predominantly the capillary endothelium. Disappearance of TCA-insoluble radioactivity from the perfusate was associated with the generation of both TCA-soluble iodide and noniodide radioactivity. Greater than 50% of the radioactive label taken up by the lungs was found in the delipidated TCA-insoluble fraction. This study provides in vitro evidence for pulmonary catabolism of VLDL apolipoproteins and uptake of peptide catabolic products of VLDL by the lung.

Animals↗

PubMed1976

Apoproteins of the lipoproteins in a nonrecirculating perfusate of rat liver.

The apoproteins of serum lipoproteins and of lipoproteins present in a nonrecirculating perfusate of rat liver were compared by immunochemical, gel electrophoretic, and solubility techniques. Serum and perfusate very low density lipoprotein apoprotein composition were not different. No evidence for the presence of a lipoprotein resembling serum low density lipoprotein was obtained. However, the apoprotein composition of circulatory high density lipoprotein was quantitatively different from the secretory product in the density 1.06-1.21 range. As measured by stained sodium dodecyl sulfate gel electrophoretic patterns, the arginine-rich protein was the major secretory apoprotein while the A-I protein was the major apoprotein in circulating high density lipoprotein. A very similar pattern was seen in perfusates of orotic acid-fatty livers. It was concluded that although the liver secrets lipoproteins in the high density class, circulatory high density lipoprotein is largely a product of catabolic processes.

Amino Acids↗

PubMed1974

Lipoproteins in a nonrecirculating perfusate of rat liver.

Rat livers were perfused in a nonrecirculating system for 30-40 min with Krebs-Ringer bicarbonate-0.1% glucose solution gassed with 95% O(2)-5% CO(2) at 37 degrees C at a flow rate of 3 ml/g/min. The livers appeared normal as judged by O(2) uptake, bile flow, transaminase release, and net protein output (2.5 mg/g/hr). The perfusate was concentrated by ultrafiltration using Amicon PM-10 or PM-30 membranes. The concentrated perfusate was subjected to sequential ultracentrifugation at solution densities of 1.006, 1.04, 1.06, and 1.21, and the top fractions were analyzed for protein and lipid. The net release of protein in the four density classes, suitably corrected, averaged 39, 10, 5, and 20 micro g/g/hr. The lipid composition of the perfusate lipoprotein fractions differed from that of serum mainly in the high percentage of free cholesterol, reflecting the lack of exposure to lecithin:cholesterol acyltransferase. When rat serum was fractionated in the same way, most of the lipoprotein in the d 1.006-1.06 range had a density greater than 1.04. It was concluded from these experiments that the liver secretes very low density lipoprotein (VLDL), high density lipoprotein (HDL), and a modified form of VLDL containing less lipid. Comparison of secretion rates and serum lipoprotein levels leads to the conclusion that the latter are largely determined by catabolic rates. When labeled amino acids were present, the perfusate HDL had a higher specific activity than VLDL. Addition of carrier whole serum did not alter recovery of labeled lipoproteins, but when these were isolated from Golgi membranes after a 40-min perfusion, more than twice as much label was recovered in HDL, suggesting the presence of precursors within the Golgi. The main advantages of the nonrecirculation perfusion technique are the avoidance of catabolic reactions, simplicity, and complete control over the composition of the perfusing medium.

Animals↗

PubMed1973