Metabolic studies in isolated rat liver cells: II. Biosynthesis of serum low density lipoproteins and its regulation

Dispersed rat liver cells were used for study of protein and serum lipoprotein synthesis. Cellular leucine incorporatein was tested in the presence of various cofactors, buffers and inhibitors.¹⁴C-leucine was incorporated into cellular protein at an active rate for 1 hr. Incorporation was more rapid in the presence of succinate, MgCl₂, phosphate and nicotinamide, but these cofactors were not absolutely required. The liver cells also incorporated labeled leucine into lower density lipoproteins (1DL) and released the newly labeled 1DL into the incubation medium. Puromycin strongly inhibited the production of cellular protein and 1DL. The mode of cellular regulation of 1DL synthesis was examined. The rate of 1DL production was selectively enhanced by short term alimentation with dietary triglyceride or by the addition of a mixture of lipogenic cofactors to incubated liver cells. The utility of isolated liver cells as a preparation for metabolic control studies is discussed.     

Lipid composition and erucic acid in rat liver cells in culture

Erucic acid (Δ¹³-docosenoic acid) was added to fetal calf serum, then fed to rat liver epithelial cells in culture, and uptake measured at intervals over 24 hr. During the first 6 hr. of incubation, uptake of the docosenoic acid was 21 nmoles/hr/mg protein in 7-day cells, and 15 mmoles/hr/mg protein in 14-day cells. Of¹⁴C-labeled erucic acid taken up by the cells in 24 hr, radioactivity measurements showed 60% of the total lipid¹⁴C activity derived from [1-¹⁴C] 22∶1 in neutral lipid (NL) and 40% in phospholipid (PL); whereas 55% of lipid¹⁴C activity was in NL and 45% in PL when the substrate was [14-¹⁴C] 22∶1. Within the NL fraction, 75% of¹⁴C activity derived from [1-¹⁴C] 22∶1 was in triglyceride (TG) and 11% in cholesterol (CHL), while 79% was in TG and 6.5% in CHL when the substrate was [14-¹⁴C] 22∶1. Triglycerides and cholesteryl esters accumulated in the cells during incubation with erucic acid. Among phospholipids separated by thin layer chromatography, 75% of¹⁴C activity was in lecithin (PC), 10% in phosphatidylethanolamine (PE), 5% in sphingomyelin (SPH), and 1% or less in cardiolipin (DPG). The highest specific activity (SA) was in PC, followed by SPH and PE. Incubation with erucic acid altered fatty acid composition of PC, PE, and SPH, although amounts of phospholipids were unaffected. Gas liquid chromatography analyses detected 18% erucic acid in PC, 2% in PE, and 4–5% in SPH.     

Bile lipid secretion in isolated perfused rat liver. A model for metabolic studies

Isolated perfused rat liver was used to study the effects of constant taurocholate perfusion, with or without the addition of phosphatidylcholine unilamellar vesicles, upon both the bile salt-dependent and bile salt-independent secretion of bile. Taurocholate introduction increased bile flow and normalized the bile lipid secretion by restoring the bile salt-dependent secretion. At a flow rate of 30 ml/min, the liver was perfused by a single-pass method. The perfusion medium contained 17.5 μM taurocholate with or without 5.83 μM phosphatidylcholine. In light of a recent quantitative dynamic concept on the interphase partition of lipids, it was calculated that more than99% of the taurocholate reaches the liver as monomers and/or dimers. It was also deduced that the lipids were secreted in bile as small discoidal lipoprotein structures rather than unilamellar lipoproteic vesicles. During the course of the experiments (2 hr), the excellent criteria of viability of this model make it highly suitable for the investigation of hepatic metabolism. Furthermore, the addition of phosphatidylcholine unilamellar vesicles to the perfusate consitutes a potential vector for various liposoluble molecular species.     

Lipid synthesis by rat lung in vitro

Oxidation and lipogenesis in isolated rat lung tissue were studied in vitro. The minced tissue was incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) with 1-¹⁴C-acetate, 2-¹⁴C-pyruvate, U-¹⁴C-D-glucose, 1,5-¹⁴C-citrate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, 1-¹⁴C-linoleate. The lung tissue readily oxidized all of these substrates to¹⁴CO₂ and incorporated them into¹⁴C-lipids with the exception of 1,5-¹⁴C-citrate, for which there was no significant incorporation into¹⁴C-lipids. Most of the lipid¹⁴C was recovered in phospholipids, more specifically phosphatidyl choline. Twenty-eight per cent of glucose carbons was incorporated into the fatty acid moiety of phospholipids, while more than 90% of the carbons of other substrates was found in phospholipid fatty acids. The main fatty acid of the phospholipid fraction synthesized from acetate, pyruvate or glucose was palmitic acid. The oxidation of fatty acids was apparently influenced both by the carbon chain length and number of double bonds. Accumulation of¹⁴C-fatty acids in the tissue was observed when fatty acids were used as substrates; this finding suggests that the rate limiting step was not in the uptake of fatty acids. Chemical degradation of¹⁴C-myristic and palmitic acids obtained by hydrolysis of phospholipids biosynthesized from 1-¹⁴C-laurate indicated that the phospholipid fatty acids were synthesized via the de novo synthesis pathway. Presented at the AOCS-ISF World Congress, Chicago, September 1970.     

Metabolic control in isolated brown fat cells

Experiments with brown fat cell preparations from the adult hamsters are described. The mitochondria of brown adipose tissue were shown to have a classical electron transport system. The basal respiration of brown fat cells was demonstrated to be coupled to oxidative phosphorylation. Evidence is presented for partial uncoupling of oxidative phosphorylation as a mechanism for controlling respiration during norepinephrine stimulation. Exogenously added fatty acids were found to mimick the norepinephrine stimulation of respiration. Norepinephrine and cyclic AMP were shown to have no effect on brown fat mitochondria. Experiments with labeled oleate showed that the triglyceride reesterification cycle does not control respiration in brown adipose tissue. One of nine papers to be published from the Symposium “Brown Adipose Tissue,” presented at the AOCS-AACC Joint Meeting, Washington, D.C., March 1968.     

Lipoprotein synthesis. I. rat plasma lipoprotein composition and synthesis from radioactive precursors

The in vivo synthesis of rat plasma lipoproteins was studied by the use of isotopic protein and lipid precursors. Labelled amino acids, palmitic acid and tripalmitin were administered by stomach tube and the radioactivity in the plasma lipoproteins was determined following preparative ultracentrifugal isolation at densities of 1.006, 1.019, 1.063 and 1.21 g/ml. In response to triglyceride feeding, amino acid composition of the high density lipoprotein changed little, but in the low density lipoproteins proportionality in the amino acid pattern was changed as reflected by increases and decreases in certain amino acids. Isotopic amino acids were not incorporated in proportion to the relative abundance with which they occurred in the lipoproteins. Triglyceride feeding markedly stimulated isotope utilization, especially in the low density fractions. Methionine, though only present in small amounts, was extensively utilized and it is suggested that this amino acid may play a significant role in the synthesis of lipoproteins, other than the role of a methyl donor for phosphatidylcholine.     

Chemically regenerative redox fuel cells. I Membrane studies

Three different types of membrane have been tested in a chemically regenerative redox fuel cell. It was found that a Nafion membrane gave the best polarization curves, but also that a much cheaper silica-filled polyethylene membrane could be used. A polysulphone membrane ranked number three     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis: II. Perfused rat liver

Livers from rats with experimental hypoproteinemia induced by aminonucleoside-nephrosis or plasmapheresis were perfused with a [¹⁴C]-labeled amino acid mixture at physiological concentration. Compared to control rats, a significantly increased incorporation of the amino acid label was found in the apolipoproteins of the ultracentrifugally separated very low and high density lipoproteins (VLDL, HDL), and into albumin secreted into the perfusate. However, no increase in the amino acid-derived label was detected in VLDL- or HDL-borne lipids in nephrosis or plasmapheresis. Perfusion with U-[¹⁴C] leucine as a lipogenesis precursor at >10 times higher than physiological concentration resulted in 5-fold increase in the label incorporation into perfusate proteins in nephrosis but only in a slightly significant increase in perfusate lipids. In contrast, the incorporation of a preformed fatty acid, 9,10-[³H] oleate into VLDL and HDL lipids increased 3- to 4-fold in nephrosis. Both with leucine and oleate as precursors, the increments in the label appearing in perfusate proteins or lipids, respectively, were markedly greater than the increases in hepatic tissue proteins or lipids. The results indicate that amino acids are preferentially directed by the liver into the synthesis of circulating apolipoproteins and albumin in hypoproteinemia and do not seem to constitute an important precursor of the lipoprotein lipids. The increased production of apolipoproteins is associated with an increased incorporation of preformed fatty acids into lipoprotein lipids in addition to the previously reported stimulation of hepatic de novo lipid synthesis from precursors other than amino acids.     

Desaturation and chain elongation of essential fatty acids in isolated liver cells from rat and rainbow trout

Isolated hepatocytes from rainbow trout and rat were incubated with¹⁴C-labeled linoleic acid, linolenic acid, dihomogammalinolenic acid or eicosapentaenoic acid. The most striking difference in the desaturase activity was the lower level of Δ5 desaturase in trout than in rat. No Δ4 desaturation of 22∶4(n−6) to 22∶5(n−6) was observed in either of the two species, while the conversion of 22∶5(n−3) to 22∶6(n−3) was significant in both groups and highest in rainbow trout. The chain-elongating activity was remarkably similar in the two species, except for the “dead-end” elongation which was distinctly more important in fish.     

Glycerylbisether sulfates. I: Improved synthesis

Sparse literature outlines previous syntheses for glycerylbisether sulfates, which, while giving rise to the desired molecules, are somewhat cumbersome and suffer from undersired by-product formation. We now report an improved synthesis of the title compounds that results in greater simplicity and reduced by-product formation. An hypothesis is advanced to explain the by-product formation.     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis. I: Studies in rat in vivo

The incorporation of L-4,5-[³H]leucine into the ultracentrifugally separated apolipoproteins of very low, low, and high density lipoproteins (VLDL, LDL, HDL) and into serum albumin was found three-to four-fold higher in nephrotic than in normal rats one hour after intravenous injection. Incorporation of leucine into the circulating lipids was negligible. Increases of similar magnitude were obtained in the incorporation of simultaneously injected 1,5[¹⁴C] citrate into the lipids of VLDL, LDL, and HDL of nephrotic rats. Of the citrate carbons incorporated into serum and liver lipids, the proportion in cholesterol was higher in nephrotic rats when compared to normal rats. The incorporation of both precursors into total proteins and lipids of the liver vs. the incorporation into the lipoproteins was relatively lower in nephrotic than in control rats, indicating a preferential channeling into secretable products. The occurrence of enhanced new lipid synthesis in nephrosis was corroborated by the finding of markedly enhanced synthesis of lipoprotein-borne fatty acids and cholesterol from³H₂O. These results point out that while leucine is not an efficient in vivo precursor of lipoprotein lipids in nephrosis, de novo lipogenesis proceeds from other precursors. Similar trend of changes, though of smaller magnitude, was elicited in rats after double plasmapheresis, 18 hr apart, when measured 3 hr after the second plasma withdrawal. This indicates that the loss of circulating proteins either by direct removal or through kidney lesion stimulates the compensatory hepatic response involving excessive lipoprotein synthesis. Time-course studies showed that peak incorporation of leucine and citrate into the protein and lipid components of lipoproteins, respectively, as well as into serum albumin, occurred coincidentally 3 hr after the second plasmapheresis, suggesting an interdependence of the enhanced protein and lipid synthesis.     

Studies on the lipids of sheep red blood cells. I. Lipid composition in low and high potassium red cells

The lipid composition of whole red blood cells was investigated in five sheep with red cells containing a low concentration of potassium (LK) and in five sheep with red cells containing a high concentration of potassium (HK). No apparent differences within the limit of error of the experiment were detected in the lipid class composition between the HK and LK red cells. Cholesterol, the only nonpolar lipid detected in the tissue, was present in oneto-one molar ratio to the total phospholipids. Phosphatidyl ethanolamine and sphingomyelin accounted for 85% of the total phospholipids; phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, and lysolecithin were present in lesser amounts. No lecithin was detected in any of the animals in this investigation. Plasmalogen compounds were found only in the ethanolamine lipids. The molar ratio of choline to noncholine phospholipids was also approximately one to one. It was concluded that the major lipid class distribution in the two types of red cells cannot be directly responsible for the differences observed in the cation concentrations in these cells in the two species of sheep.     

Carbide phases on iron-based Fischer-Tropsch synthesis catalysts part I: Characterization studies

The characterization of iron carbide phases formed on iron-based Fischer-Tropsch catalysts is reviewed in this part with particular emphasis on Mössbauer and XPS studies of selected iron and supported iron catalysts, in combination with some specific results in activity and selectivity. Mössbauer studies have shown clearly the evolution of bulk carbide species on iron catalysts under synthesis reaction conditions; the relationship of these to the nature of near-surface species and their temporal evolution under synthesis conditions is indicated by XPS results. Comparisons among reduced, unreduced and carbided iron catalysts are given; XPS and reaction results suggest that Fe₃O₄ is active for synthesis even in the absence of carbide phases.     

The electrochemical synthesis of aminonitriles I. H-cell studies with adiponitrile and azelanitrile

Adiponitrile and azelanitrile were electrochemically hydrogenated to their corresponding aminonitriles in a divided H-cell using Raney nickel powder as the cathode material. The effects of current, temperature, and solvent/supporting electrolyte composition on product selectivities were investigated. Syntheses of the fully hydrogenated diamine by-product increased with increasing current and solution temperature. When a 0.8 M adiponitrile/alcohol/water/ammonium actetate electrolyte was hydrogenated at temperatures of 35–45°C, 6-aminocapronitrile selectivities in the range of 79–97% and current efficiencies of 50–60% were obtained. The optimum applied current was 60 mA for each 2.5 g of catalyst (an apparent current density of 4.8 mA cm^–2). For the case of azelanitrile, reaction selectivities for the partially hydrogenated 9-aminononanenitrile product ranged from 80–93%.     

Cholesteryl ester metabolism in tissue culture cells. I. Accumulation in Fu5AH rat hepatoma cells

Exposure of Fu5AH rat hepatoma tissue culture cells to hyperlipemic rabbit serum results in the accumulation of cellular cholesteryl esters. Accumulation is not a characteristic of all cells in culture, as evidenced by the lack of response of mouse and human fibroblasts. Fu5AH cells grown for 24 hr on 5% hyperlipemic rabbit serum have an 8- to 12-fold increase of cellular cholesteryl esters, small increases in free cholesterol and triglycerides, and no change in phospholipids, when compared to cells grown in normal rabbit serum. Rapid accumulation of cholesteryl esters occurs during the first 8–12 hr of incubation, and maximum cellular concentration is achieved within 24 hr. The maximum level of cellular cholesteryl esters obtained with individual samples of hyperlipemic rabbit serum is correlated with the cholesterol content of the original sera, even when the incubation medium is adjusted to a constant concentration of cholesterol. Heating hyperlipemic rabbit serum (60 C/30 min) does not destroy activity; however, no cholesteryl ester accumulation occurs in heated cells. The stimulatory activity of hyperlipemic rabbit serum primarily is associated with lipoproteins having densities <1.006. High levels of cellular cholesteryl ester are associated with the appearance of cytoplasmic vacuoles containing cholesteryl esters. The increase in cellular cholesteryl esters is accompanied by a decrease of the cholesteryl esters in the growth medium. Cellular cholesteryl esters are not rapidly hydrolyzed or released upon removal of hyperlipemic rabbit serum.     

Bile acid metabolism in mammals: VII. Studies on sex differences in deoxycholic acid metabolism in isolated perfused rat liver

The hepatic metabolism of deoxycholic acid was studied using the isolated perfused rat liver technique. In 20 perfusions, 10 involving the livers of male rats and 10 involving the livers of female rats, 30 μmoles deoxycholic acid was added to the perfusion medium. In 10 perfusions, 5 male and 5 female, 1 μmole deoxycholic acid was added to the perfusion medium. In 10 of the high dose studies and in the 10 low dose studies, 1 μCi deoxycholic acid-C-24-C¹⁴ also was added. The deoxycholic acid was added to 100 ml perfusion medium after 2 hr of baseline perfusion, and the studies were continued another 3 hr. Biliary bile acids were analyzed by combined thin layer and gas chromatography, and the radioactivity content of the perfusion medium and liver was documented. Although there was no sex difference in total bile acid secretion in the high dose studies, there were sex differences in the bile acid secretion rate and in the quantitative secretion of individual bile acids. The biliary secretion of deoxycholic acid and cholic acid was immediate in the female studies and delayed in the male, and the amounts of cholic acid and sulfated deoxycholyl-taurine secreted were considerably greater in the male studies. In the low dose studies the isolated perfused liver of the female rat converted more deoxycholic acid to cholyl-taurine than did that of the male rat. There are sex differences in the hepatic metabolism of deoxycholic acid. In contrast to those found in the case of chenodeoxycholic acid, these sex differences are not impressive when physiological amounts of deoxycholic acid are presented to the liver.     

Bile acid metabolism in mammals: IX. Conversion of chenodeoxycholic acid to cholic acid by isolated perfused rat liver

Current dogma of bile acid synthesis in mammals insists that hydroxylation of the ring structure at C-12 precedes side chain oxidation, and that chenodeoxycholic acid is not converted to cholic acid under normal conditions. This report concerns the conversion of chenodeoxycholic acid to cholic acid by isolated, perfused rat liver. Results indicate that isolated perfused rat liver has a definite, but limited, capacity for synthesis of cholic acid from chenodeoxycholic acid.     

The paired electrochemical synthesis of sorbitol and gluconic acid in undivided flow cells. I

The strategy of paired electrochemical synthesis for the production of organic chemicals, in which the reactions at both the anode and cathode simultaneously contribute to the formation of the final product(s), could result in as much as a 50% reduction in energy consumption as compared to conventional electro-organic syntheses. In order to evaluate this hypothesis the electrochemical oxidation of glucose to gluconic acid and the reduction of glucose to sorbitol were paired in undivided flow-through parallel plate and packed bed cells.To date, the optimum electrode materials and operating conditions for the paired synthesis are: an amalgamated zinc cathode, a graphite anode, an initial glucose concentration of 0.8 mol dm^–3, a 0.8 mol dm^–3 NaBr supporting electrolyte, an electrolyte flow rate of 0.81 min^–1 and an electrolyte pH of 7. Under these conditions the current efficiencies for sorbitol and gluconic acid were 26% and 68%, respectively at 0.25 F mole^–1. Current losses are believed to be due to hydrogen evolution and the reduction of-gluconolactone (an intermediate in the formation of gluconic acid) to glucose.     

Lipid and fatty acyl composition of rat brain capillary endothelia isolated by a new technique

A method is described for the isolation of pure capillary endothelia from rat brain and the phospholipid composition of these cells is reported. This method is rapid and requires only a small amount of starting material. It involves: (a) tissue disruption by high speed homogenization, (b) separation of the capillary endothelia from other brain structures using sucrose gradients, and (c) a final purification using a glass bead column. Choline and ethanolamine phosphoglycerides were found to be the predominant lipid classes of these cells amounting to 31.9% and 24.4%, respectively, of total phospholipids. The choline phosphoglycerides consisted almost exclusively of 1,2-diacyl glycerophosphorylcholine, whereas the ethanolamine phosphoglycerides consisted of approximately equal amounts of 1,2-diacyl and 1-alk-l’-enyl-2-acyl glycerophosphorylethanolamine. The composition of the constituent fatty acids of both choline and ethanolamine phosphoglycerides and the alk-1-enyl composition of ethanolamine phosphoglycerides is reported. Saturated fatty acids accounted for 45% of the total fatty acids in choline phosphoglycerides and for 53% in ethanolamine phosphoglycerides. Arachidonic acid accounted for approximately 48% of the total fatty acids in alk-1-enyl ethanolamine phosphoglyceride.