Some effect of source and level of nitrogen on the incorporation of radioactivity from C14 glucose to rumen volatile fatty acids.
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Animals↗
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Results for “FATTY ACIDS, RADIOACTIVE”
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1-14C-linoleic acid distribution in various tissue lipids of guinea pigs following an oral dose.
A recent study on the metabolism of 1-14C-alpha-linolenic acid in the guinea pig revealed that the fur had the highest specific activity of all tissues examined, 48 h after dosing. The present study investigated the pattern of tissue lipid labeling following an oral dose of 1-14C-linoleic acid after the animals had been dosed for the same time as above. Guinea pigs were fed one of two diets with a constant linoleic acid content (18% total fatty acids) and a different content of alpha-linolenic acid (0.3 or 17.3%) from weaning for 3 wk and 1-14C-linoleic acid was given orally to each animal for 48 h prior to sacrifice. The most highly labeled tissues (dpm/mg of linoleic acid) were liver, followed by brain, lung and spleen, heart, kidney and adrenal and intestines, in both diet groups. The liver had almost a three-fold higher specific activity than skin and fur which was more extensively labeled than the adipose and carcass. Approximately two-thirds of the label in skin plus fur was found in the fur which, because of a low lipid mass, would indicate that the fur was highly labeled. All tissues derived from animals on the diet with the low alpha-linolenic acid level were significantly more labeled than the tissues from the animals on the high alpha-linolenic acid diet, by a factor of 1.5 to 3. The phospholipid fraction was the most highly labeled fraction in the liver, free fatty acids were the most labeled fraction in skin & fur, while triacyglycerols were the most labeled in the carcass and adipose tissue. In these tissues, more than 90% of the radioactivity was found in fatty acids with 2-double bonds in the tissue lipids. These data indicate that the majority of label found in guinea pig tissues 48 h after dosing was still associated with a fatty acid fraction with 2-double bonds, which suggests there was little metabolism of linoleic acid to more highly unsaturated fatty acids in this time frame. In this study, the labeling of guinea pig tissues with linoleic acid, 48 h after dosing, was quite different from the labeling with alpha-linolenic acid reported previously. The retention of the administered radioactivity from 14C-linoleic acid in the whole body lipids was 1.6 times higher in the group fed the low alpha-linolenic acid diet (diet contained a total of 1.8 g PUFA/100 g diet) compared with the group fed the high alpha-linolenic acid diet (diet contained 3.6 g PUFA/100 g diet). The lack of retention of 14C-labeled lipids in the whole body would be consistent with an increased rate of beta-oxidation of the labeled fatty acid on the diet rich in PUFA, a result supported by other studies using direct measurement of labeled carbon dioxide.
Adrenal Glands↗
Effect of diet and substrate on the in vitro measurement of cholesterol and fatty-acid synthesis in hepatic tissue of Japanese quail.
A comparison of acetate, glucose, mevalonate, or water as radioactive substrates for the hepatic synthesis of cholesterol, fatty acids, and glyceride-glycerol was made in Japanese quail fed diets containing either beef fat or tuna oil. The quail fed a diet containing beef fat were fatter and had a significantly higher (P less than .01) concentration of serum cholesterol (5.6 mM per L) than that measured in the serum of quail given tuna oil (4.1 mM per L). Both in vitro cholesterol and fatty-acid synthesis were greater in the quail fed a diet of beef fat than in those fed a diet containing tuna oil. The results showed that mevalonate was the most-suitable radioactive substrate for measuring cholesterol synthesis, whereas glucose was the most-suitable radioactive substrate for measuring fatty-acid and glyceride-glycerol synthesis.
Animals↗
Fatty acid metabolism in the calanoid copepod Paracalanus parvus: 1. Polyunsaturated fatty acids.
The metabolic fate of radioactive linoleate and alpha-linolenate administered to the South Atlantic copepod Paracalanus parvus was studied. The wild copepod was able to incorporate the labeled acids dissolved in seawater. The radioactive linoleate was elongated to 20:2omega6 and 22:2omega6 and desaturated by a delta6 desaturase to 18:3omega6. alpha-Linolenate was also desaturated by a delta6 desaturase to 18:4omega3 and elongated to 20:3omega3. The copepod was able to convert alpha-18:3 to 20:5omega3 and 22:6omega3.
Animals↗
Fatty acid acylation of the 67K envelope glycoprotein of a baculovirus: Autographa californica nuclear polyhedrosis virus.
Modification of Autographa californica multicapsid nuclear polyhedrosis virus extracellular virion polypeptides with fatty acid was investigated. Fatty acid-derived radioactivity was incorporated into gp67, the predominant virion envelope glycoprotein, during metabolic labeling using [3H]palmitic acid or [3H]myristic acid. The acyl moiety resisted extraction with chloroform-methanol but was released by mild alkali or hydroxylamine treatment, features characteristic of an ester-type linkage. Palmitic acid was identified as the fatty acid liberated during acid hydrolysis of purified [3H]palmitic acid and [3H]myristic acid-labeled gp67. Acyl peptides were protected during proteinase K digestion of intact virions but they were larger (Mr 34.3K and 31.8K) than expected on the basis of membrane protection alone. Carbohydrate may have played a role in determining the protease resistance of the observed fragments. Potential acylation sites on gp67 within or adjacent to the C-terminal hydrophobic region are discussed with respect to the predicted amino acid sequence.
Acylation↗
Phospholipid metabolism in Plasmodium-infected erythrocytes: guidelines for further studies using radioactive precursor incorporation.
The biosynthesis of phospholipids is extensive in Plasmodium knowlesi-infected simian erythrocytes due to the synthesis of membranes by this single-cell eukaryote in a host erythrocyte devoid of any pathway for lipid biosynthesis. In the present paper, we show that the incorporation of [3H]glycerol, which reflects de novo biosynthesis, is better studied at 300 microM-1 mM than at the trace doses, since this non-physiological precursor does not modify the amount of phosphatidylcholine biosynthesis from [3H]choline. Time-course incorporation of radioactive glycerol, oleate, lysophosphatidylcholine, choline, and inositol in RPMI 1640 medium containing nutrients for lipid synthesis showed that the optimum incubation time for phospholipid studies is 60-90 min, after which radioactive incorporation slows considerably. On the other hand, studies with [14C]serine revealed that incubation for 2-3 h is necessary for isotopic labelling of phosphatidylcholine via phosphatidylserine decarboxylation and phosphatidylethanolamine N-methylation. Incorporation of the various fatty acids into individual lipids was related to the molecular species composing each of them. Studies with [14C palmitoyl] lysophosphatidylcholine showed a very fast intracellular release of radioactive fatty acids, which indicates a potent lysophospholipase activity. Taken together, these data define the indispensable conditions for an experimental system suitable for further studies.
Animals↗
Lipids associated with bovine kidney glomerular basement membranes.
1. After incubation of bovine glomeruli with D-[U-14C]glucose, about 21% of the total radioactivity is found in lipid extracts of glomerular basement membranes. 2. The concentration of lipids in glomerular basement membranes (4.3% of dry wt.) is lower than in the residual glomerular particles (10.8% of dry wt.). The concentrations of neutral lipids (13.9%), phospholipids (46.7%) and cholesterol (37.9%) in the total lipid extract of the glomerular basement membranes, however, differ from those in the residual glomerular particles (15.6, 54.0 and 30.9% respectively). Though residual glomerular particles show a higher lipid content, the radioactivity in this fraction only amounts to 38% of that found in the glomerular basement membranes. 3. The specific radioactivity of total glomerular basement-membrane lipids (12 600 d.p.m./mg) is about 4 times as high as that of the glomerular basement membranes. The specific radioactivities of the individual lipid components, however, differ. The highest values are found for phosphatidylcholine and triacylglycerols. The largest proportion of the radioactivity is found in the glycerol of the glycerides. The radioactivity in the fatty acids is much less and does not differ significantly in the various classes of lipids. 4. G.1.c. of methyl esters of the fatty acids does not reveal a clear difference between the fatty acid compositions of glomerular basement membranes and residual glomerular particles. 5. Treatment of glomerular basement-membrane preparations with ultrasound, the generally used procedure for glomerular basement-membrane preparations, drastically decreases the lipid content of glomerular basement membranes. 6. It is concluded that lipids are associated with the basement membranes. Further, the comparatively high radioactive labelling suggests that glomerular basement-membrane lipids may be an interesting class of substances for further pathological studies.
Animals↗
Influence of diet on conversion of 14C1-linolenic acid to docosahexaenoic acid in the rat.
14C1-Linolenic acid was incorporated into lipids of hearts, livers, and carcasses of male rats. We studied the influence of diet composition on extent and distribution of radioactivity. A CHOW diet, a purified, essential fatty acid (EFA)-deficient diet, a purified control diet, and EFA-deficient diets with four fatty acid supplements were used. Supplements of 18:2n-6, 20:4n-6, 15:3n-3, and 22:6n-3 were given as single doses. Radioactivities in liver phosphatidyl ethanolamines (PE), phosphatidyl cholines, and neutral lipids were measured. The distribution of radioactivity among the fatty acids in liver phospholipids was determined. Rats on CHOW diet incorporated far less radioactivity than any other group into lipids of hearts and livers. Most of the activity in livers was recovered as 20:5n-3 and 22:6n-3 in all rats. In EFA-deficient rats, the radioactivity in 22:6n-3 of liver PE was still increasing 36 hr after 14C1-linolenic acid had been administered. The n-6 supplements (18:2n-6 and 20:4n-6) seemed to reduce the coversion of 20:4n-3 to 20:5n-3 (desaturation), whereas the n-3 supplements (18:3n-3 and 22:6n-3) reduced the conversion of 20:5n-3 to 22:5n-3 (elongation). Formation of 22:6n-3 may be controlled by 22:6n-3 itself at the elongation of 20:5n-3 to 22:5n-3.
Animals↗
Partial purification and properties of the fatty acid elongation systems in the outer and inner membranes of beef liver mitochondria.
Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.
Animals↗
[Conversion of erucic acid in subcellular fractions from liver, kidneys and heart of rat, 8 min after intravenous injection (author's transl)].
Rats were intravenously injected with albumin-bound [14 14C] erucic acid, and after 8 min the 14C radioactivity (RA) of the fatty acids were studied in mitochondria, microsomes and other fractions of liver, kidneys and heart cells. 1. In liver, which contained 15% of the injected RA, oleic acid (18 : 1) was the main fatty acid (FA) formed (26% of the 14C recovered) whereas in kidneys (0,53% of the injected RA) the proportion of nervonic acid (24 : 1) was higher (20%) than that of 18 : 1 (14%). No appreciable transformation was encountered in heart, which contained 0,53% of the injected RA. 2. In liver, the microsomes showed the higher converted 14C RA (45%), mainly as 18 : 1 (33%), much higher than in mitochondria (11%), whereas the amount of total 14C FA was a little higher in the latter fraction. 3. In kidneys, the mitochondrial and microsomal fractions contained the same percentage of 14C oleic acid (15%), whereas nervonic acid was recovered in higher proportion, 29 and 20%, in microsomes and in mitochondria respectively. 4. Results show that the same kind of metabolic activity, i.e., shortening or elongation, was observed in the whole cell as that previously shown with isolated fractions. However, mitochondrial oxidation and exchanges of transformed fatty acids between organelles can modify the extent of the phenomenon.
Animals↗
Regulation of membrane release in apoptosis.
Apoptosis is a fundamental process of cell regulation whereby cells execute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to play roles in the regulation of apoptosis, including the activation of phospholipase A2 (PLA2), usually measured as release of 3H-labelled arachidonic acid (AA) from prelabelled cells. The current study was aimed at examining the role of PLA2 in regulating apoptosis in response to several inducers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [3H]fatty acids and the released radioactivity was characterized. These studies indicated that the AA release assay did not reflect release of non-esterified fatty acid via activation of the PLA2 pathway. Rather, studies using TLC and electron microscopy showed that AA release reflected a previously unsuspected shedding of a heterogeneous population of membrane vesicles and fragments, probably as components of apoptotic bodies. Further studies demonstrated that this process is an integral part of apoptosis. Overexpression of Bcl-2 or the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethane prevented the characteristic morphological changes of cell death, and completely inhibited the release of membrane vesicles and fragments. On the other hand, release of membrane vesicles and fragments was caused by various inducers of apoptosis, as measured by release of either 3H-labelled AA or palmitic acid. Thus the present study demonstrates that the release of membrane lipids during apoptosis defines a new assay for apoptosis and has allowed the investigation of the mechanisms regulating formation of apoptotic bodies.
Apoptosis↗
Prepaid medical care plans in relation to medical education.
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Education, Medical↗
A microtubule-binding protein of Trypanosoma brucei which contains covalently bound fatty acid.
Covalently linked fatty acids are increasingly recognized as an important type of post-translational protein modification. Many of such acylated proteins are found associated with cellular membranes. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a regular array of microtubules which are tightly bound to the overlying cell membrane. A microtubule-binding protein (p41) has been identified within this structure which carries covalently bound fatty acid. The fatty acid linkage is sensitive to hydroxylamine treatment. After chemical transesterification, the released radioactivity co-migrates with fatty acid methyl esters in thin-layer chromatograms, establishing that the fatty acid was covalently bound to p41 via an ester (thioester) linkage. Upon detergent extraction of trypanosomes, p41 remains tightly bound to the cytoskeleton as long as Ca2+ ions are present. It can selectively be released from this structure by the addition of excess EGTA. Conversely, p41 binds to isolated cytoskeletons and to purified microtubules in vitro, the reaction again being entirely Ca2+-dependent.
Animals↗
Erucic acid metabolism in rat liver. A combined biochemical and radioautographical study.
Metabolism of erucic acid was studied in rat liver in comparison with oleic acid in relation with diet lipids. Rats were fed for 3 or 60 days a balanced diet containing 30% of the calories of either rapeseed oil rich in erucic acid or sunflower seed oil rich in linoleic acid. They were intravenously injected with tritiated erucic or oleic acid. After 1 or 15 min, the radioactivity recovered in liver lipids was 9 to 26% whatever the diet or the acid injected. One minute after injection of erucic acid a high part of radioactivity was recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. The autoradiography did not show any marked difference between the labelling of peroxisomes and mitochondria when tritiated erucic or oleic acid was injected. These data do not bring about arguments for a peroxisomal nor for a mitochondrial location of erucic acid shortening in liver.
Animals↗
Release of lysosomal phospholipase A1 and A2 into cytosol and rapid turnover of newly-formed lysophosphatidylcholine in FL cells during fusion-from-within induced by measles virus.
The subcellular distribution of the phospholipase activities and metabolism of lysophosphatidylcholine in cultured human cell line (FL cells) during "fusion-from-within" induced by measles virus were studied. During cell fusion, fairly high activities of phospholipase A1 and A2, the optimal pHs of which were acidic, appeared in the cytosol. These phospholipases were confirmed to be of lysosomal origin by the following facts: (1) the properties of these phospholipases were the same as those of lysosomal phospholipase A1 and A2; (2) a decrease in the total activity of phospholipases A1, A2 and acid phosphatase in the lysosomal fraction of the infected cells coincided with an increase in their activity in the cytosol. The release of lysosomal phospholipase A1 and A2 into the cytosol of the infected cells appeared to be related to the extent of cell fusion-from-within. Phospholipase A1 and A2 released to the cytosol hydrolyzed the cell membrane-bound phospholipids to form lysophospholipids. 1-[1-14C]Palmitoyl-GPC, incorporated into the cells from the culture medium, was rapidly converted into phosphatidylcholine, triacylglycerol and phosphatidylethanolamine in the cells during fusion-from-within, but the radioactive lysophosphatidylcholine and fatty acids were hardly detectable, indicating a rapid turnover of cellular lysophosphatidylcholine during cell fusion. The subcellular lysophospholipid acyl-hydrolase activities of the infected cells were higher than those of normal cells. Possible relation between the membrane fusion induced by measles virus and the phospholipid metabolism in the infected cells were discussed.
Cell Fusion↗
Lipids of cultured hepatoma cells. VI. Glycerolipid and monoenoic fatty acid biosynthesis in minimal deviation hepatoma 7288C-1.
1-14C-Acetic, 1-14C-palmitic, or 1-14C-stearic acid was incubated with minimal deviation hepatoma 7288C cells grown in culture to assess: de novo fatty acid synthesis, oxidation, desaturation, and elongation of saturated fatty acids, as well as the ability of media fatty acids to serve as precursors of cellular glycerolipids. Distribution of radioactivity in the individual lipid classes and the various fatty acids of triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was determined. The radioactivity among the monoenoic acid isomers derived from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was analyzed by reductive ozonolysis. Only small amounts of the labeled substrates were oxidized to carbon dioxide. Except for labeled stearic acid, which also was incorporated heavily into phosphatidyl inositol and phosphatidyl serine, most radioactivity was recovered in triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine. Synthesis of cholesterol and long chain fatty acids from labeled acetic acid demonstrated that these cells can perform de novo synthesis of fatty acids and cholesterol. Both labeled palmitic and stearic acids were desaturated to the corresponding delta9 monoenes, and palmitic and palmitoleic acids were elongated. The nexadecenoic acid fraction isolated from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine, when acetic or palmitic acid was the labeled substrate, showed that greater than 70 percent of the labeled acids were the delta9 isomer. Radioactivity of the octadecenoic acid fraction derived from labeled acetic or palmitic acids was nearly equally divided between the delta9 isomer, oleic acid, and the delta11 isomer, vaccenic acid. Desaturation of labeled stearic acid produced only oleic acid. These data demonstrate that the biosynthesis of vaccenic acid in these cultured neoplastic cells proceeds via the elongation of palmitoleic acid. The relatively high level of vaccenic acid synthesis in these cells suggests that the reported elevation of "oleic acid" in many neoplasms may result from increased concentration of vaccenic acid.
Acetates↗
Metabolism of 14C-labelled sucrose esters of stearic acid in rats.
Rats were dosed by oral gavage (250 mg/kg) with compounds containing sucrose esterified in four, six or eight positions with stearic acid. For each compound, rats excreted greater than 95% of the dose in the faeces. The extent of absorption and metabolism of radioactivity was inversely related to the degree of esterification. For rats dosed with sucrose esters labelled in the fatty acid moieties, the degree of absorption of radioactivity was highest for the tetraesterified compound (5.9% of the dose). At 120 hr after dosing with this derivative, the highest concentrations of radioactivity, aside from tissues of the gastrointestinal tract, were found in fat (183 micrograms-equivalents/g tissue), lymph nodes (117 micrograms-equivalents/g tissue) and the liver (88 micrograms-equivalents/g tissue); appreciable radioactivity appeared in the blood (3.9 micrograms-equivalents/g tissue) and collected lymph (5.0-7.6% of the dose). For rats dosed with esters labelled in the sucrose moiety, the amounts of radioactivity absorbed were lower than after dosing with the corresponding sucrose derivatives labelled in the fatty acid moieties; the absorbed radioactivity was greatest following administration of the tetraesterified compound (3.0%). Relatively little radioactivity was found in tissue samples collected from these rats. These results are consistent with limited hydrolysis of the sucrose esters, presumably to sucrose and fatty acids, prior to intestinal absorption.
Administration, Oral↗
Liver and adipose tissue contributions to newly formed fatty acids in an ascites tumor.
We determined the contribution from host hepatic and extrahepatic tissues to newly synthesized fatty acids (FA) in the Ehrlich ascites tumor (EAT). We administered 3H2O (subcutaneously) and [14C]glucose (in a test meal) and measured the appearance of radioactivity in plasma triglyceride fatty acids (TGFA) and free fatty acids (FFA) and in tumor total lipid fatty acids (TLFA). Using [14 C]FFA, we selectively labeled epididymal fat TGFA to estimate the FA transport rate from intraperitoneal adipose tissue directly to the tumor. Contributions of four major pathways to newly synthesized FA in EAT were determined by multicompartmental analysis. De novo FA synthesis by EAT accounted for more than 93% of the TLFA radioactivity found in the tumor. Contributions from liver TGFA via plasma TGFA (less than 0.5%), adipose tissue TGFA via plasma FFA (less than 6%), and adipose tissue TGFA via direct intraperitoneal transport of FFA (less than 1%) accounted for less than 7% of all TLFA radioactivity measured in the EAT. Thus the present study establishes that practically all labeled esterified FA in the EAT is derived from de novo synthesis by tumor cells.
Adipose Tissue↗