Studies on liver phosphatidyl cholines: I. Effects of fatty liver induction on phosphatidyl cholines from liver mitochondria and microsomes

Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl₄ (0.3 ml of CCl₄ suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl₄. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.     

Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes

The effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities, the two enzymes involved in phosphatidyl choline biosynthesis in liver microsomes, has been investigated. Female rats were fed a 5% ethanol-liquid diet containing amino acids, minerals, vitamins, with and without choline, for 2, 6, and 10 weeks. Control animals were pair-fed the same isocaloric diet with 5% sucrose with and without choline. Ethanol administration with or without dietary choline stimulated significantly (P<0.001) the specific activities of phosphatidyl ethanolamine methyltransferase in liver microsomes in the animals fed 5% ethanol for 2, 6, and 10 weeks, when compared to those control animals pairfed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P<0.02) the specific activities of choline phosphotransferase. The specific activities of phosphatidyl ethanolamine methyltransferase continued to increase in the liver microsomes from the animals in which dietary choline was omitted for 2, 6, and 10 weeks in the sucrose controls and alcohol-fed animals. Ethanol administration stimulates significantly (P<0.001) the phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of animals fed the liquid diet with dietary omission of choline and methionine for 2 weeks.     

Effects of diet and type IIa hyperlipoproteinemia upon structure of triacylglycerols and phosphatidyl cholines from human plasma lipoproteins

Four normal and two individuals with Type IIa hyperlipoproteinemia were placed on the National Heart and Lung Institute Type IIa diet (low cholesterol, smaller than 300 mg/day, high polyunsaturated, low saturated fat diet) for 1 week and on a normal diet the following week. Plasma samples were obtained and the triacylglycerols, phospholipids, and cholesterol contents of plasma and of very low density lipoproteins, low density lipoproteins, and high density lipoproteins determined. Triacyglycerol fatty acid composition was determined and stereospecific analyses of triacglycerols and phosphatidyl cholines performed. Structural determinations were limited to one normal and one Type IIa individual. In normal and Type IIa individuals, chylomicrons contained twice the amount of 18:0 as did the very low density lipoproteins, low density lipoproteins, or high density lipoproteins. The structure of the triacyglycerols from the very low density lipoproteins and low density lipoproteins was asymmetric with at least 50M% 16:0 in the sn-1 position and mostly 18:1 in positions sn-2 and 3. There was a marked difference in the distribution of 18:2 in low density lipoproteins of the normal and Type IIa individuals. The control contained equal amounts of 18:2 in the sn-1 and sn-3 positions, whereas IIa low density lipoprotein was asymmetric with 26% of the 18:2 in position sn-1 and 3% in the sn-3 position. Very low density lipoprotein was asymmetric with regard to 18:2 in control and IIa samples with an average of 5% of the 18:2 in position sn-1 and 40% in position sn-3. The phosphatidyl cholines contained predominantly 16:0 and 18:0 in position sn-1, whereas the acids in position sn-2 were unsaturated with very little difference between lipoprotein classes. Neither the short dietary periods nor source of plasma affected the structure of the phosphatidyl cholines.     

Lipid composition of liver mitochondria and microsomes in hyperthyroid rats

Triiodothyronine-induced alteration of the lipid pattern in rat-liver mitochondria and microsomes has been investigated. In mitochondria, a 25% total cholesterol decrease and a 14% phospholipid increase have been detected. In these hyperthyroid rat liver organelles, a strong decrease in the total cholesterol/phospholipid molar ratio occurs. On the contrary, in microsomes from the same animals, a decrease of about 23% has been measured for both total cholesterol and phospholipids; hence, in this fraction, the total cholesterol/phospholipid molar ratio is unaffected by hyperthyroidism. The liver mitochondrial phospholipid composition, unlike the microsomal composition, is altered significantly in hyperthyroid rats; a 7.4% phosphatidylcholine decrease is accompanied by a similar additive percentage increase of both phosphatidylethanolamine and cardiolipin. In regard to total phospholipid fatty acid composition in liver microsomes from hyperthyroid rats, no variation has been observed compared with the control rats, whereas in mitochondria from the same animals, a meaningful linoleic acid decrease with a similar arachidonic acid increase has been found. In addition to fatty acid alteration, the separated mitochondrial phospholipid classes also exhibit some increase in stearic acid. Among phospholipids, cardiolipin changes the most of the esterified fatty acids in hyperthyroid rat liver. In this compound, a strong increase in the percentage of both palmitic and stearic acid and a 32.4% decrease of linoleic acid have been found.     

Thyroid control over biomembranes: VI. Lipids in liver mitochondria and microsomes of hypothyroid rats

The lipids of liver mitochondria prepared from normal rats and from rats made hypothyroid by thyroidectomy and injection with¹³¹INa contained similar amounts, per mg protein, of total lipids, phospholipids, neutral lipids and lipid phosphorus. Hypothyroidism caused a doubling of the relative amounts of mitochondrial cardiolipins (CL; to 20.5% of the phospholipid P) and an accompanying trend (although statistically not significant) toward decreased amounts of both phosphatidylcholines (PC) and phosphatidylserines (PS), with phosphatidylethanolamines (PE) remaining unchanged. The pattern of elevated 18∶2 fatty acyl content and depleted 20∶4 acyl groups of the mitochondrial phospholipids of hypothyroid preparations was reflected to varying degrees in the resolved phospholipids, with PC showing greater degrees of abnormality than PE, and CL showing none. Hypothyroidism produced the same abnormal pattern of fatty acyl distributions in liver microsomal total lipids as was found in the mitochondria. Hypothyroid rats, when killed 6 hr after injection of [1-¹⁴C] labeled linoleate, showed the following abnormalities: the liver incorporated less label into lipids, and converted 18∶2 not exclusively to 20∶4 (as normals do) but instead incorporated the label mainly into saturated fatty acids. These data, together with the known decrease in β-oxidation, suggest that hypothyroidism involves possible defective step(s) in the conversion of 18∶2 to 20∶4. These studies were initiated during a leave at the University of California, Los Angeles.     

In vitro conversion of erucic acid by microsomes and mitochondria from liver,kidneys and heart of rats

Microsomes and mitochondria of liver, kidneys, and heart were incubated with [14-¹⁴C]erucic acid in three assay media: one favorable for chain elongation (NADPH+KCN), another favorable for β-oxidation and the last one for shortening (NADP+KCN). Elongating reactions occurred mainly in microsomes, those of kidneys being very active; the mitochondria also showed some activity, heart mitochondria being, however, more active than the microsomes, when considering the amount of erucic acid activated. In the medium for β-oxidation, practically no shortened fatty acids were found. On the contrary, when β-oxidation was inhibited, and in the presence of NADP, the formation of shorter monoenes, probably in the outer membrane of the mitochondria, was observed, namely eicosenoic acid in high amount, oleic acid and hexadecenoic acid. Mitochondria from liver were very active as were those of heart, when compared with the quantity of activated erucic acid. In heart, the mitochondria shortened erucic acid into oleic acid and hexadecenoic acid, which were then probably used as energy substrates. With carnitine and without NADP, shortened fatty acids were formed in the mitochondria of liver, probably by the first reactions of β-oxidation. In this case, the proportions of oleic acid and hexadecenoic acid were higher than with NADP alone. In the presence of carnitine and NADP, the level of the chain-shortening reaction did not differ from that observed with NADP alone. It appears, therefore, that the activated erucic acid is mainly directed towards shortening reactions and not towards transfer reactions across the mitochondrial membranes.     

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.     

Effect of clofibric acid on the molecular species composition of diacyl glycerophosphocholine of rat liver microsomes

The effect of administeringp-chlorophenoxyisobutyric (clofibric) acid to rats on the molecular species composition of diacyl-glycerophosphocholine (GPC) of rat liver microsomes was studied. Microsomal choline glycerophospholipids were converted to 1,2-diradyl-3-acetylglycerol and were separated into molecular species by reverse-phase high performance liquid chromatography. Diacyl-GPC consisted of 17 different molecular species. The predominant species were arachidonoyl derivatives, such as 18∶0–20∶4 (22.2% of the total) and 16∶0–20∶4 (22.0%). Administration of clofibric acid to rats caused a marked increase in 16∶0–18∶1 species of diacyl-GPC from 8% to 30%, making these the predominant species of diacyl-GPC in clofibric acid-fed rats. Also, a significant decrease (50% of controls) in 18∶0–18∶2 and 18∶0–20∶4 species was observed, whereas the decrease in molecular species containing 16∶0 at the 1-position such as 16∶0–18∶2 and 16∶0–20∶4 was small (approximately 85% of control). The results show that clofibric acid caused marked changes in the molecular species composition of diacyl-GPC. The participation of 1-acyl-GPC acyltransferase and stearoyl-CoA desaturase in the regulation of the molecular species composition of diacyl-GPC is discussed.     

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes

The biosynthesis of [¹⁴C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [¹⁴C] phosphatidic acid by preincubation of unlabeled microsomes withsn-[¹⁴C]glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgC1₂. The newly formed [¹⁴C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [¹⁴C] phosphatidate and [¹⁴C]CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP:phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation ofsn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol. Presented in part at the Annual Meeting of the Canadian Federation of Biological Societies, Winnipeg, June 1975.     

Existence of cholinephosphotransferase in mitochondria and microsomes of liver and lung of guinea pig and rat

We reported earlier on the occurrence of cholinephosphotransferase in the mitochondria of guinea pig lung. In order to determine whether organ and/or species specificities exist in regard to the cholinephosphotransferase activity in mitochondria, we have compared the subcellular distribution of the enzyme in the liver and lungs of rats and guinea pigs. Even though the activity of the enzyme was higher in microsomes than it was in mitochondria, the mitochondrial activity was authentic in both tissues of both species. The authenticity of mitochondrial activity was established by marker enzyme studies and ultrastructural examination of mitochondrial preparations. Presented in part at the Seventy-third Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, LA, March 1989.     

Effect of diet on choline phosphotransferase,phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in liver microsomes

Phosphatidylcholine (PC) biosynthesis has been investigated in female rats fed a liquid amino acid, choline-methionine-free diet by assaying in liver microsomes the specific and total activities of choline phosphotransferase, phosphatidyldimethylethanolamine methyltransferase and phosphatidylethanolamine methyltransferase. There was a significant decrease in the specific activity (sp act) of choline phosphotransferase in the liver of rats fed a choline-methionine-free diet. The dietary omission of methionine for 2 wk resulted in a significant decrease in the sp act of choline phosphotransferase. The dietary omission of choline, methionine, B₁₂, folic acid and the addition of a methyl group acceptor, guanidoacetic acid, decreased further the sp act of choline phosphotransferase. The phosphatidyl-ethanolamine methyltransferase sp act increased with the dietary omission of choline and methionine. The dietary omission of choline, methionine, B₁₂, folic acid and the addition of a methyl group acceptor, guanidoacetic acid, resulted in a decrease in the sp act of phosphatidyldimethylethanolamine methyltransferase and an increase in phosphatidylethanolamine methyltransferase. The dietary omission of choline, methionine, B₁₂, folic acid and the addition of a methylation inhibitor, 2-amino-2-methyl-1-propanol, did not result in a significant decrease in the sp act of choline phosphotransferase; however, it did significantly decrease the sp act of phosphatidylethanolamine methyltransferase. The addition of dietary methionine with the inhibitor resulted in a significant decrease in the sp act of the choline phosphotransferase and phosphatidylethanolamine methyltransferase when compared to control and/or when compared to deficient with or without inhibitor. The dietary supply of methionine, as a source of choline, did affect the activity of the enzymes that synthesize PC. The ratio of the substrate, S-adenosylmethionine, and the inhibitory product, S-adenosylhomocysteine, affected the enzymatic activity of phosphatidylethanolamine methyltransferase. It is suggested that the concentrations of these 2 compounds may be important in regulating the methylation of phosphatidylethanolamine in the liver cell.     

Quantitation of phosphatidyl N-methyl- and N,N-Dimethylaminoethanol in rat liver

The contents of phosphatidyl N-methyl-and N,N-dimethylaminoethanol were determined in the liver of rats injected with (Me-C¹⁴) methionine. Total phospholipids were extracted from aliquots of the liver and fractionated by two-dimensional thin layer chromatography after addition of carrier phosphatidyl-N-methyl-and N,N-dimethylaminoethanol. The radioactivity present in the two phosphatide spots was determined and used to calculated total disintegrations per min/100 g body wt. The remainder of the livers was pooled, and total phospholipids were isolated and subjected to acid hydrolysis. N-methyl- and N,N-dimethylaminoethanol were purified by thin layer chromatography, and their specific activity was determined after quantitation by gas liquid chromatography and radioactivity measurement. The liver contents of phosphatidyl N-methyl- and N,N-dimethylaminoethanol were determined by dividing disintegrations per min/100 g body wt by the specific activity of N-methyl- or N,N-dimethylaminoethanol.     

Studies on composites from wood and polypropylenes. II

Composites of polypropylene (PP) or maleic-anhydride-modified polypropylene (MPP) with refiner ground pulp (RGP) were prepared under various kneading conditions (mixing temperature, rate of rotation, and mixing time) and evaluated for their tensile strength (σ_max), break elongation (?_max), and Young's modulus (E). Particularly, for the rate of rotation, fiber length distribution and its fibrillation were investigated in connection with mechanical properties of the composites. Also evaluated in this study was the effect of modifiers of PP on the properties. Modified PP that can be grafted or can have affinity to RGP was proved to improve the tensile strength of the molded composites as the RGP content increases, while the strength was decreased for PP–RGP composite without a modifier. MPP was also evaluated as a compatibilizer for the PP–RGP composites, and a tremendous improvement of tensile strength was achieved with MPP addition of only 2.5%, indicating that MPP can act as a compatibilizer in its system. These lines of evidence is interpreted to be caused from the improvement of the adhesion between RGP and PP through localizing MPP at the interface of these two components.     

Effect of pyridoxine deficiency on phospholipid methylation in rat liver microsomes

The effect of altered methionine metabolism during pyridoxine deficiency on the activity of phosphatidyl-ethanolamine methyltransferase (EC 2.1.1.17) and the levels of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) has been evaluated in rat liver microsomes. Animals fed a pyridoxine-deficient diet for 7 wk displayed a fivefold increase in the hepatic tissue level of S-adenosylhomocysteine when compared to either control or pair-fed animal counterparts. When PE methyltransferase was assayed in vitro, a significant increase in specific activity was observed using enzyme preparations from either pair-fed or pyridoxine-deficient rats. On the other hand, phospholipid levels did not conform to the measured enzyme activity. The level of PC in microsomes from either pyridoxine-deficient or pair-fed animal groups was significantly lower than that determined for the control group of rodents. However, the level of PC was noticeably lower in microsomes from pyridoxine-deficient animals than that from pair-fed animals, which received 45% of the feed intake of the control animals. In addition, the level of PE in microsomes from pair-fed and pyridoxine-deficient animals was significantly higher than that analyzed from the control animals, further confirming decreased methylation of substrate to product. It is concluded that pyridoxine deficiency may alter the methylation of phospholipid in the endoplasmic reticulum above and beyond that produced by feed restriction alone.     

Studies on the 12α and 26-hydroxylation of bile alcohols by rabbit liver microsomes

12α-Hydroxylation of two C₂₇-steroids by rabbit liver microsomes was studied. Optimal assay conditions were determined with 7α-hydroxy-4-cholesten-3-one and 5β-cholestane-3α,7α-diol as substrates. The rate of 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was found to be greater than that of 5β-cholestane-3α,7α-diol by ca. 60%. Microsomal 26-hydroxylation of 5β-cholestane-3α7α-diol was also measured, and the ratio of 26-hydroxylation to 12α-hydroxylation of 5β-cholestane-3α,7α-diol was found to be ca. 0.4. Rabbit liver 12-αhydroxylase was more active than that of three other species (man, rat, monkey), explaining in part the predominance of cholic acid in rabbit bile.     

Studies on the hydrolysis and utilization of long chain acyl CoA thioesters by liver microsomes

Acyl CoA thioester “hydrolase” activity exhibited by liver microsomal preparations using a spectrophotometric assay for released CoA has been shown to be a composite of several enzymatic reactions. Pig liver microsomes form a considerable amount of triacylglycerol from endogenous precursors, with essentially all acyl groups being incorporated into the 1,3 positions. Rat and rabbit liver microsomes form very little triacylglycerol under the same conditions. The true acyl CoA hydrolase activity of pig liver microsomes is relatively insensitive to diisopropylfluorophosphate (DFP), whereas this compound strongly inhibits the rat and rabbit enzymes. The hydrolase remaining in the liver microsomes after DFP treatment is rapidly inactivated when incubated in the presence of substrate.     

Changes of fatty acid composition of phospholipids in liver mitochondria and microsomes of the rat during growth

The fatty acid patterns of rat liver mitochondrial and microsomal phospholipids were analyzed from term fetuses, 1 and 4 days old, and adult rats. The main fatty acids of phosphatidylethanolamine and-choline were stearic and palmitic acids, although the patterns differed slightly. The fatty acid composition of corresponding phospholipids in mitochondria and microsomes was similar. The fatty acid pattern of cardiolipin was dominated by linoleic acid. The most consistent feature of the developmental changes in the fatty acid patterns of all phospholipids studied was a decrease in the relative amount of monounsaturated fatty acids. The percentages of saturated fatty acids in phosphatidyl-ethanolamine and-choline increased during neonatal development. It is suggested that the high levels of fetal monounsaturated fatty acids were due to low availability of polyunsaturated fatty acids.     

Kinetic studies of detergency. II. Effect of age,temperature, and cleaning time on rates of soil removal

The effects of variation in temperature, flow rate, cleaning time, and age were studied in a model circulation system. The results are recorded as changes in the rate constants k₁ and k₂ and changes in the relative proportions of the two soil species. The removal of tristearin in this simple system was found to proceed by two independent mechanisms, acting simultaneously. The first, named the “flow mechanism,” is dependent on time, and its rate increases with flow rate. The second, which the authors term the “Dupré mechanism,” is not dependent upon time and arises from the air-detergent interface that moves over the surface. The Dupré effect is independent of flow rate at all rates of flow. In most of the experiments the Duprè mechanism accounted for about 90% of the removal of tristearin. A comparison is made between the removal of monomolecular films and thin polymolecular films. It is shown that the empirical finding of apparently simple first-order kinetics is not inconsistent with the complex processes that remove tristearin from the surface.     

Effect of malonyl-CoA on Δ6 desaturation activity of rat liver microsomes

The effect of malonyl-CoA on linoleic acid desaturation and elongation reactions of rat liver microsomes was studied. Under strict desaturation conditions, the in vitro microsomal conversion of linoleic acid to γ-linolenic acid is time-dependent. When malonyl-CoA was added to the aforementioned incubation medium, linoleic acid was desaturated to γ-linolenic acid and elongated to its higher homologues. Under these conditions, Δ6 desaturation activity, calculated by adding γ-18∶3, 20∶3 and 20∶4 acids, was neither inhibited nor activated by malonyl-CoA. These results indicate that the elongation of γ-linolenyl-CoA coupled to the desaturation of linoleic acid did not modify Δ6 desaturase activity.