Oxidation of fatty acids by Leishmania braziliensis panamensis

Heating cultures of Leishmania braziliensis panamensis (grown at 26 degrees C) to 34 degrees C for 1.5-12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-14C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26 degrees C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of 14CO2 release from [1-14C]laurate to that from [12-14C]laurate was generally larger than four, whereas this ratio from [1-14C]oleate relative to [10-14C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the omega-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

Studies on the biosynthesis of phenols in fungi. Conversion of [14C]orsellinic acid and [14C]orcinol into fumigatol by Aspergillus fumigatus I.M.I. 89353

1. Sodium [1-(14)C]acetate was incorporated into orsellinic acid and fumigatol by Aspergillus fumigatus. 2. [(14)C]Orsellinic acid was prepared biosynthetically. It was converted almost entirely into fumigatol and fumigatin within 2 days of supplementation of the medium. The apparent decrease in incorporation after a longer period of growth was due to decomposition of radioactive fumigatol and the production of relatively unlabelled material. The addition of orcinol to these cultures decreased the conversion of [(14)C]orsellinic acid into fumigatol. [(14)C]Orsellinic acid was incorporated into 3,4-dihydroxytoluquinol in both sets of cultures. 3. [(14)C]Orcinol was prepared from [(14)C]orsellinic acid after acid hydrolysis. It was also very effective as a precursor of fumigatol (60% incorporation). 4. The specific activity of fumigatin was lower than that of fumigatol at early stages of growth (4-5 days after inoculation) with all the labelled substrates that were tested. This indicated that fumigatin arose from fumigatol after oxidation in the medium. 5. The presence of orcinol in the medium greatly stimulated the incorporation of radioactivity (presumably derived from the (14)CO(2)H of orsellinic acid) into the isoprenoid compounds, ergosterol and ubiquinone, in the mycelium.     

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.     

Contribution of omega-oxidation to fatty acid oxidation by liver of rat and monkey.

Contributions of omega-oxidation to overall fatty acid oxidation in slices from livers of ketotic alloxan diabetic rats and of fasted monkeys are estimated. Estimates are made from a comparison of the distribution of 14C in glucose formed by the slices from omega-14C-labeled compared to 2-14C-labeled fatty acids of even numbers of carbon atoms and from [1-14C]acetate compared to [2-14C]acetate. These estimates are based on the fact that 1) the dicarboxylic acid formed via omega-oxidation of a omega-14C-labeled fatty acid will yield [1-14C]acetate and [1-14C]succinate on subsequent beta-oxidation, if beta-oxidation is assumed to proceed to completion; 2) only [2-14C]acetate will be formed if the fatty acid is metabolized solely via beta-oxidation; and 3) 14C from [1-14C]acetate and [1-14C]succinate is incorporated into carbons 3 and 4 of glucose and 14C from [2-14C]acetate is incorporated into all six carbons of glucose. From the distributions found, the contribution of omega-oxidation to the initial oxidation of palmitate by liver slices is estimated to between 8% and 11%, and the oxidation of laurate between 17% and 21%. Distributions of 14C in glucose formed from 14C-labeled palmitate infused into fasted and diabetic rats do not permit quantitative estimation of the contribution of omega-oxidation to fatty acid oxidation in vivo. However, the distributions found also indicate that, of the fatty acid metabolized by the whole animal in the environment of glucose formation, at most, only a minor portion is initially oxidized via omega-oxidation. As such, omega-oxidation cannot contribute more than a small extent to the formation of glucose.     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Effects of culture age and hexoses on fatty acid oxidation by Leishmania major

The effect of culture age on the rate of oxidation of short-, medium, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10-14C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-14C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-14C]palmitate and of [1-14C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in beta-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.     

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

Radiometric detection of formate 14C and formaldehyde 14C oxidation in folic acid deficiency]

An ionization chamber method was used in vivo to demonstrate a delayed oxidation of [14C] formaldehyde and [14C] formate to 14CO2 in folic acid-deficient rats as compared to control rats or folic-acid-deficient rats treated by folic acid. Results obtained showed that oxidation of these two molecules required the presence of folic acid.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Differences in the conversion of the polyunsaturated fatty acids [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3) in isolated rat hepatocytes

The reasons why most cellular lipids preferentially accumulate 22:6(n-3) rather than 22:5(n-6) are poorly understood. In the present work the metabolisms of the precursor fatty acids, [1-(14)C]20:4(n-6), [1-(14)C]22:4(n-6) versus [1-(14)C]20:5(n-3), [1-(14)C]22:5(n-3) in isolated rat hepatocytes were compared. The addition of lactate and L-decanoylcarnitine increased the formation of [(14)C]24 fatty acid intermediates and the final products, [(14)C]22:5(n-6) and [(14)C]22:6(n-3). In the absence of lactate and L-decanoylcarnitine, no [(14)C]24 fatty acids and [(14)C]22:5(n-6) were detected when [1-(14)C]22:4(n-6) was the substrate, whereas small amounts of the added [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). Lactate reduced the oxidation of [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) while L-decanoylcarnitine did not. No significant differences between the total oxidation or esterification of the two substrates were observed. By fasting and fructose refeeding the amounts of [(14)C]24:4(n-6) and [(14)C]24:5(n-3) were increased by 2.5- and 4-fold, respectively. However, the levels of [(14)C]22:5(n-6) and [(14)C]22:6(n-3) were similar in hepatocytes from fasted and refed versus fed rats. With hepatocytes from rats fed a fat free diet the levels of [(14)C]24 fatty acid intermediates were low while the further conversion of the n-6 and n-3 substrates was high and more equal, approx. 33% of [1-(14)C]22:4(n-6) was converted to [(14)C]22:5(n-6) and 43% of [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). The moderate differences found in the conversion of [1-(14)C]22:4(n-6) versus [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3), respectively, and the equal rates of oxidation of the two substrates could thus not explain the abundance of 22:6(n-3) versus the near absence of 22:5(n-6) in cellular membranes.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.     

Studies on the transport of acetyl groups from peroxisomes to mitochondria in isolated liver cells oxidizing the polyunsaturated fatty acid 22:4n-6.

The oxidation of the fatty acid [1-(14)C]22:4n-6 was studied in isolated hepatocytes. Labeled acetate was the main acid soluble product identified by HPLC after short incubation periods. At low substrate concentrations and longer incubations [(14)C]acetate was gradually replaced by labeled beta-hydroxybutyrate, acetoacetate and oxaloacetate/malate. Preincubation with 2-tetradecylglycidic acid (TDGA), an inhibitor of mitochondrial fatty acid oxidation, did not reduce the oxidation but acetate was the only product recovered. TDGA also strongly inhibited the metabolism of added [1-(14)C]acetate to mitochondrial oxidation products. During the preparation procedure of hepatocytes the cellular L-carnitine concentration was decreased but it was restored after preincubation with L-carnitine. With low [1-(14)C]22:4n-6, concentrating a low level of [(14)C]acetate and high levels of labeled mitochondrial oxidation products were recovered after preincubation with L-carnitine. A small amount of [(14)C]acetylcarnitine was also detected under this incubation condition. The results suggest that a significant part of labeled acetyl groups from the peroxisomal oxidation of [1-(14)C]22:4n-6 is transported to the mitochondria as free acetate. Moreover, the results also suggest that L-carnitine at physiological concentrations may facilitate the transport of part of the acetyl groups from peroxisomes to mitochondria as acetylcarnitine. However, the possibility that an increased cellular L-carnitine concentration may stimulate oxidation of [1-(14)C]22:4n-6 in mitochondria could not be excluded.