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T L Kaduce

Publications and source records attributed to T L Kaduce.

49 records · Page 3Linked to original sources
PubMed1983

A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography.

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.

Animals↗
PubMed1981

Effect of essential polyunsaturated fatty acid modifications on prostaglandin production by MDCK canine kidney cells.

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE2 release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9- times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglyceride fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE2 release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE2 accounting for 55 to 68% of the total output from [1-14C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE2 formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. It is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids become highly enriched in arachidonate.

Animals↗
PubMed1981

Utilization of arachidonic and linoleic acids by cultured human endothelial cells.

When cultured human umbilical vein endothelial cells are supplemented with linoleic acid, the arachidonic acid content of the cellular phospholipids is reduced approximately 35%. Most of the fatty acid compositional change occurs during the first 24 h. One factor responsible for this effect is the inability of the endothelial cells to convert appreciable amounts of linoleic to arachidonic acid, due to a fatty acid delta 6-desaturase deficiency. By contrast, these endothelial cultures contain delta 5- and delta 9-desaturase activity and are able to elongate long-chain polyunsaturated fatty acids. The other factor that contributes to the decrease in arachidonic acid is that high concentrations of linoleic acid reduce the incorporation of arachidonate into cellular phospholipids. Stearic acid, a long-chain saturate, does not produce any reduction, whereas eicosatrienoic acid is an even more effective inhibitor than linoleic acid. In spite of the fact that high concentrations of these polyunsaturates produced inhibition, the endothelial cells were found to efficiently incorporate exogenous arachidonic acid into cellular phospholipids and triglycerides. This may serve to compensate for the inability of these cells to synthesize arachidonic acid from linoleic acid. These findings suggest that the endothelium obtains arachidonic acid from an extracellular source, that this cannot be provided in the form of linoleic acid and, in fact, that high concentrations of linoleic acid actually may interfere with the ability of the endothelium to maintain an adequate supply of intracellular arachidonic acid.

Arachidonic Acid↗
PubMed1980

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes.

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.

Acyltransferases↗
PubMedDate not supplied

Role of peroxisomal oxidation in the conversion of arachidonic acid to eicosatrienoic acid in human skin fibroblasts.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1-(14)C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [3H]20:4 to [3H]20:3. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [3H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [3H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [3H]16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal beta-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.

8,11,14-Eicosatrienoic Acid↗
PubMedDate not supplied

Comparison of 20-, 22-, and 24-carbon n-3 and n-6 polyunsaturated fatty acid utilization in differentiated rat brain astrocytes.

Astrocytes convert n-6 fatty acids primarily to arachidonic acid (20:4n-6), whereas n-3 fatty acids are converted to docosapentaenoic (22:5n-3) and docosahexaenoic (22:6n-3) acids. The utilization of 20-, 22- and 24-carbon n-3 and n-6 fatty acids was compared in differentiated rat astrocytes to determine the metabolic basis for this difference. The astrocytes retained 81% of the arachidonic acid ([(3)H]20:4n-6) uptake and retroconverted 57% of the docosatetraenoic acid ([3-(14)C]22:4n-6) uptake to 20:4n-6. By contrast, 68% of the eicosapentaenoic acid ([(3)H]20:5n-3) uptake was elongated, and only 9% of the [3-(14)C]22:5n-3 uptake was retroconverted to 20:5n-3. Both tetracosapentaenoic acid ([3-(14)C]24:5n-3) and tetracosatetraenoic acid ([3-(14)C]24:4n-6) were converted to docosahexaenoic acid (22:6n-3) and 22:5n-6, respectively. Therefore, the difference in the n-3 and n-6 fatty acid products formed is due primarily to differences in the utilization of their 20- and 22-carbon intermediates. This metabolic difference probably contributes to the preferential accumulation of docosahexaenoic acid in the brain.

Animals↗
PubMedDate not supplied

Linoleic acid metabolism and prostaglandin production by cultured bovine pulmonary artery endothelial cells.

When bovine pulmonary artery endothelial cells are cultured in a medium supplemented with linoleic acid, their capacity to produce prostacyclin (PGI2) is reduced by about 60%. This reduction occurs when PGI2 formation is stimulated by the addition of either the calcium ionophore A23187 or arachidonic acid. In addition, supplementation with linoleic acid reduced the production of prostaglandin E2 and F2 alpha from 1-14C-arachidonic acid by more than 50%. The capacity of cultured bovine pulmonary vein and aortic endothelial cells to convert extracellular arachidonic acid into PGI2 also was reduced by about 50% when the growth medium was supplemented with linoleic acid. Although bovine pulmonary artery endothelial cells incorporated large amounts of 1-14C-linoleic acid into cellular phospholipids and triglycerides, a maximum of only 2.3% of the radioactivity was converted to arachidonic acid in 24 hours. The most prevalent radioactive metabolite was eicosadienoic acid, the elongation product of linoleic acid. As compared with linoleic acid, the bovine endothelial cells incorporated 30% more 1-14C-arachidonic acid into phospholipids and 60% more into triglycerides. When the growth medium was supplemented with linoleic acid, the percentage of this fatty acid in cellular lipids increased 3- to 4.5-fold and eicosadienoic acid accumulated, accounting for up to 9% of the cellular fatty acids. This increase was accompanied by a 30% to 45% reduction in arachidonic acid. These findings, together with our previous results with human umbilical vein endothelium, suggest that an inability to convert large amounts of linoleic to arachidonic acid and a suppressive effect of linoleic acid enrichment on prostaglandin production may be general properties of endothelial cells.

Animals↗
PubMedDate not supplied

Arachidonic acid availability and prostacyclin production by cultured human endothelial cells.

When human umbilical vein endothelial cultures were grown in the presence of supplemental arachidonic acid, the cell phospholipids became enriched with arachidonic acid. Prostacyclin (PGI2) accumulated in the medium during supplementation with arachidonic acid. The capacity of these enriched cultures to produce PGI2 when subsequently incubated with either arachidonic acid or thrombin was reduced by as much as 90%, but release of arachidonic acid from the cell lipids in response to thrombin stimulation was not inhibited. Refractory cultures completely recovered the capacity to form PGI2 within 18 hours after removal of the medium containing supplemental arachidonic acid. However, recovery was prevented by cycloheximide. When enrichment with arachidonic acid was done in the presence of ibuprofen, a reversible cyclooxygenase inhibitor, PGI2 did not accumulate in the medium during supplementation, and the subsequent capacity of the cultures to produce PGI2 in response to thrombin increased by 70% to 240%. By contrast, the capacity of these supplemented cultures to convert added arachidonic acid to PGI2 did not increase. Therefore, the enhancement in thrombin-stimulated PGI2 production when the cultures are supplemented with arachidonic acid probably is due to the larger amount of arachidonic acid available in the intracellular lipid substrate pools, rather than to an activation of the PGI2 synthetic pathway. These findings suggest that changes in the arachidonic acid content of the endothelial cell lipids may modulate the capacity of the endothelium to produce PGI2 in response to stimulation.

Arachidonic Acid↗