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PubMed1975

Adaptive changes in delta 9 desaturase activity in rat liver.

The delta 9 desaturase activity and the 14-C radioactivity of the de novo synthesized fatty acids incorporated into microsomal lipids and serum triglycerides were measured under different nutritional conditions. The results obtained indicate a correlation between the values of the three parameters studied after starvation or after refeeding Purina chow or either a high carbohydrate or a high protein diet. These data suggest that liver lipogenesis and delta 9 desaturase activities respond to the same regulatory factors.

Acetates↗
PubMed1978

Effect of pentadecan-2-one on lipid metabolism in HeLa cells.

HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1(-14)C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased 14C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.

Fatty Acids, Nonesterified↗
PubMed1975

Differential labeling of glycerol moieties of phospholipids and triacylglycerols of cultured mammalian cells by [U-14 C] glucose.

Rabbit liver cells, in which fatty acid synthesis was suppressed by the rabbit serum component of the medium, were grown through 8- to 120-fold increases in cell numbers and mass of cell lipid in the presence of [U-14 C]-glucose. Triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were isolated from the total cell lipid and deacylated. Carbons 1 and 3 of the glycerol from the triacylglycerols and the no. 1 glycerol carbons of the two deacylated phospholids were oxidized by periodate and isolated as the dimedon derivative of formaldehyde. The specific activities of the glycerol carbons indicated that 58, 44, and 37 percent of the glycerol of the triacylglycerols. phosphatidylcholine, and phosphatidylethanolamine, respectively, were derived from the glucose of the medium. An additional 8 percent and 1-2 percent of the glycerol of each lipid was derived, respectively, from [U-14 C] glycerol and U14C-labeled amino acids added to the medium. In agreement with an experiment with albumin-bound [9,10- minus 3H]-oleic acid, and with smilar earlier experiments, it appears likely that appriacylglycerols originated from serum lipoproteins, or their partial hydrolysis products. An appreciable part of the ethanolamine of the cells' phosphatidylethanolamine originated from exogenous U- minus 14 C-labeled amino acids. Phosphatidyl-ethanolamine, however, was not a primary source of phosphatidylcholine. Labeling of the fatty acids of triacylglycerols and phospholipids by radioactive glucose, glycerol and amino acids was negligible.

Amino Acids↗
PubMed1989

Expression and secretion of chicken apolipoprotein AI in transfected COS cells.

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.

Acylation↗
PubMed1999

Radiolabelling of parenteral O/W emulsions by means of neutron activation.

Parenteral O/W emulsions containing lanthanide fatty acid derivatives were prepared. With regard to enhancing the incorporation efficiency of the neutron activatable excipients, the addition of the non-ionic co-emulsifier Solutol HS 15 proved to be most suitable. Comparing the different chain lengths of the fatty acids, the long chain fatty acid derivative lanthanide(tri)stearate seemed to be superior in strengthening the interfacial layer. After neutron activation, the physical and chemical stability of the irradiated formulations was evaluated. The chemical stability, indicated by the concentration of lyso phosphatidylcholine as the degradation product of the main emulsifier, was shown to be dependent on the irradiation time. By applying a neutron flux of 2.1 x 10(13) neutrons/cm2 per s, the maximum should not rise above 60 s. The physical stability indicated by the particle size distribution was affected by the presence of the non-ionic co-emulsifier. Concerning the amount of radiation necessary for in vivo biodistribution studies the maximum load of Samarium fatty acid derivatives did not yield sufficient radioactivity levels. However, Europium derivatives could be shown to be suitable for in vivo studies.

Emulsions↗
PubMed1975

Relationship between sterol synthesis and DNA synthesis in phytohemagglutinin-stimulated mouse lymphocytes.

Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.

Animals↗
PubMed1994

Gastrointestinal effects and energy value of polydextrose in healthy nonobese men.

We studied seven healthy volunteers before and during acute (PD1) and chronic (PD2) ingestion of 30 g polydextrose (PD)/d. The energy value of PD was assessed after [U-14C]PD was added to the 10-g morning dose of PD during PD1 and at the end of PD2. Thirty-one +/- five percent (mean +/- SD) (PD1) and 29 +/- 4% (PD2) of the dose appeared in breath within 48 h. A small fraction of the ingested radioactivity was recovered in urine (4 +/- 1%) and excreted in flatus (< or = 1%) and in feces as volatile fatty acids (VFAs) (< 1%) and bacteria (3-4%); the remaining radioactivity in stools, 33 +/- 3% (PD1) and 32 +/- 4% (PD2), was assumed to be intact PD. Breath excretion of the label was 49 +/- 5% after intracolonic infusion of [U-14C] acetate. The energy value of PD, calculated by means of Miller and Wolin's stoichiometric equation of colonic fermentation, was similar during PD1 and PD2: 4.0 and 6.1 kJ/g, respectively, when breath 14CO2 and VFA production from PD were used for calculation.

Adult↗
PubMed1969

Lipid synthesis by isolated Chlamydia psittaci.

The isolated cells of the host-dependent meningopneumonitis agent, Chlamydia psittaci, were shown to incorporate radioactive carbon from aspartate, isoleucine, and glucose-6-phosphate into cell lipids. The nature of this incorporation was investigated. Radioactivity was found only in the fatty acids and primarily in the phosphatidyl ethanolamine, and, to a lesser extent, in the phosphatidyl choline fractions. Branched-chain fatty acids, not found in host lipid, were shown to constitute a large proportion of the fatty acid content of phosphatidyl ethanolamine. The reasons why only fatty acid synthesis took place under the conditions of our experiments with isolated meningopneumonitis agent cells remain obscure.

Aspartic Acid↗
PubMedDate not supplied

Endogenous lipids of the earthworm Lumbricus terrestris.

Earthworms (Lumbricus terrestris) were given [1-14C]-labeled palmitic acid by gavage on days 0 and 3, and sacrificed on day 7. The distribution of label among lipid classes indicated that glycerides, sterol esters, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylserine and (or) phosphatidylinositol, phosphatidylcholine, and sphingomyelin turn over in, or are synthesized by, the earthworm. Free fatty acids still had the highest specific radioactivity of any lipid class at the end of the experiment. Incorporation of label into sterol and hydrocarbon fractions was insignificant and there was no detectable label incorporated into gangliosides. Phosphatidylethanolamine apparently turned over quite slowly compared with other lipid classes, while the cerebroside fraction became highly labeled. Elongation of palmitic acid to stearate and oxidation to CO2 occurred extensively, but there was no evidence for desaturation.

Animals↗
PubMed1977

[In vivo incorporation of labeled palmitic and oleic acids into skeletal muscle lipids of normal and thyroidectomized rats during swimming].

The purpose of this work was to study the incorporation of [1-14C] palmitic and and [9,10(-3)H] oleic acids, after intravenous administration in the lipids of rat hind leg muscles. The animals were fasting or fed, at rest or swimming during 10 min before test, euthyroid or thyroidectomised. All these animals have been taking the same daily swimming training, during 15 days before the injection of labelled molecules. They were killed 10 min (+/-1)later. The lipidic muscle composition, the incorporation rate of labelled fatty acids in these lipids and the radioactivity distribution among the different lipids in the various cases have been determined. Moreover the plasmatic non-esterified acid radioactivity has been measured. These various values are affected by nutritional, hormonal state, and by physical activity of the animals. Particularly, it seems that supplementary energy spent during swimming test will be covered by the oxidation of different nutriments, according to the nutritional and hormonal state of animals.

Animal Nutritional Physiological Phenomena↗
PubMed1992

Transport of n-3 fatty acids from the intestine to the retina in rats.

This study was undertaken to determine the mode of transport of the essential (n-3) fatty acids docosahexaenoic acid 22:6(n-3) and linolenic acid 18:3(n-3). Male weanling Sprague-Dawley rats received a mixture of corn oil and [14C]18:3(n-3) or [14C]22:6(n-3) by gavage. At periods of 1 to 4 days after the injection, four rats per time point were killed and samples of blood were taken via heart puncture and the livers and retinas were collected. Blood lipoproteins and plasma proteins were separated by ultracentrifugation and analyzed by HPLC. Lipids were extracted and saponified and the fatty acids were converted to phenacyl esters for separation of individual fatty acids. After 1 and 2 h, radioactivity from 18:3(n-3) and 22:6(n-3) was observed primarily in the chylomicron/very low density lipoprotein fraction. By 4 h, radioactivity in the lipoprotein fraction was greatly decreased, with a small amount of radioactivity associated with albumin in the soluble protein fraction. After 24 h, the total amount of radioactivity associated with lipoprotein was further reduced, with more than half of the remaining label occurring in association with albumin and another unidentified protein. In the liver, 22:6(n-3) was concentrated in triacylglycerols (40.7%) and phospholipids (51.1%), with a maximum specific activity at 4 h. In the rod outer segments (ROS), the specific activity of [14C]22:6(n-3) increased to a maximum at 24 h and maintained a high level even at 4 days. These data suggest that after injection, 18:3(n-3) and 22:6(n-3) are esterified to triglyceride and phospholipid by the intestinal absorptive cells and transported in chylomicrons to the liver. After conversion of 18:3(n-3) to 22:6(n-3) in the liver, the retina accumulates 22:6(n-3) which may be transported from the liver via albumin and another unidentified protein, and is retained by the rod outer segments.

Animals↗
PubMed1987

Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer.

Using deuterium-labeled glycerol as tracer and gas-liquid chromatography-mass spectrometry techniques for the determination of isotopic enrichment, we have developed a simple and ethically acceptable method of determining glycerol appearance rate in humans under steady-state and nonsteady-state conditions. In normal subjects, the appearance rate of glycerol in the post-absorptive state was 2.22 +/- 0.20 mumol X kg-1 X min-1, a value in agreement with those reported in studies with radioactively labeled tracers. The ratio nonesterified fatty acid (NEFA) appearance rate/glycerol appearance rate ranged from 1.95 to 3.40. In insulin-dependent diabetic patients with a mild degree of metabolic control, the appearance rate of glycerol was 2.48 +/- 0.29 mumol X kg-1 X min-1. The volume of distribution of glycerol, determined by the bolus injection technique, was (mean) 0.306 l X kg-1 in normal subjects and 0.308 l X kg-1 in insulin-independent diabetic patients. To evaluate the usefulness of the method for determination of glycerol kinetics in nonsteady-state conditions, we infused six normal subjects with natural glycerol and calculated the isotopically determined glycerol appearance rate using a single compartment model (volume of distribution 0.31 l X kg-1). During these tests, the expected glycerol appearance rates were successively 5.03 +/- 0.33, 7.48 +/- 0.39, 9.94 +/- 0.34, 7.48 +/- 0.39, and 5.03 +/- 0.33 mumol +/- kg-1 X min-1, whereas the corresponding isotopically determined appearance rates were 4.62 +/- 0.45, 6.95 +/- 0.56, 10.85 +/- 0.51, 7.35 +/- 0.34, and 5.28 +/- 0.12 mumol X kg-1 X min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1982

[Profile of the capacity for lipid synthesis in vivo in various rat tissues: effect of age].

Changes in 3H2O incorporation in vivo to lipid fractions have been studied in rats 1, 3 and 13 months old. The incorporation of tritiated water into non-saponifiable fraction showed and age-dependent decrease in liver. The values were 4.32, 2.91 and 2.56 mumol 3H2O incorporated/g wet weight of liver/hour, at 1, 3 and 13 months respectively. Similar variations could be observed in adipose tissue and in blood. The rates of lower lipogenesis increase markedly from 1 to 3 months (7.02 to 13.36 mumol 3H2O/g/h) and decrease sharply from the 3rd to 13th months (13.36 to 3.75 mumol 3H2O/g/h). The incorporation of radioactivity into blood and adipose tissue fatty acids, also decrease by age.

Adipose Tissue↗
PubMed1996

Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evidence from sterol regulation-defective cells.

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRD-6) levels of transcriptionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental CHO-7 cells were regulated by sterols. SRD-2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD-2 cells than in either SRD-6 or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These observations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are dependent on the availability of transcriptionally active SREBP. We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRD-6 cells. Finally, we demonstrate that the concentrations of cholesterol, cholesteryl ester, and triglyceride were all significantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride synthesis.

Acetates↗
PubMed1983

Extent of 14C incorporation into cholesterol of infant skin fibroblasts incubated with carboxyl-labeled oleic, -linoleic or -arachidonic acid.

In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.

Arachidonic Acid↗
PubMed2000

Selective inhibition of reactions involved in methanogenesis and fatty acid production on rice roots.

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.

Journal Article↗