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H Sprecher

Publications and source records attributed to H Sprecher.

At least 55 records · Page 3Linked to original sources
PubMed1994

Arachidonic acid formed by peroxisomal beta-oxidation of 7,10,13,16-docosatetraenoic acid is esterified into 1-acyl-sn-glycero-3-phosphocholine by microsomes.

Peroxisomal beta-oxidation of linoleic acid and arachidonic acid was depressed when 1-palmitoyl-sn-glycero-3-phosphocholine and microsomes were included in incubations. This reduction was due to the esterification of the substrate into the acceptor by microsomal 1-acyl-sn-glycero-3- phosphocholine acyltransferase. The first cycle of the beta-oxidation of 7,10,13,16-docosatetraenoic acid was independent of 1-acyl-sn-glycero-3-phosphocholine and microsomes. However, when arachidonate was produced it was esterified rather than serving as a substrate for continued beta-oxidation. When arachidonate and linoleate were incubated with peroxisomes alone, 2-trans-4,7,10-hexadecatetraenoic acid and 2-trans-4-decadienoic acid were the respective end products of beta-oxidation. 2-Oxo-8,11-heptadecadienone, a catabolite produced from linoleate, was most likely a nonenzymatic decarboxylation product of 3-oxo-9,12-octadecadienoic acid. In addition to the termination of beta-oxidation by microsomal-peroxisomal communication, our results with linoleate and arachidonate suggest that the reaction catalyzed by 2-trans-4-cis-dienoyl-CoA reductase is the control step in double bond removal. In addition, the beta-ketothiolase step may play a regulatory role in the peroxisomal beta-oxidation of linoleate but not arachidonate or 7,10,13,16-docosatetraenoic acid.

Animals↗
PubMed1994

A comparison of the specific activities of linoleate and arachidonate in liver, heart and kidney phospholipids after feeding rats ethyl linoleate-9,10,12,13-d4.

In order to determine how dietary linoleate is metabolized, rats were maintained on a chemically defined diet containing 1.6% ethyl linoleate. After 5 weeks the linoleate was replaced by an equal amount of ethyl 9,10,12,13-d4-linoleate. The animals were killed 3 days later and the molar percentage of d4-linoleate and d4-arachidonate were quantified in liver, heart and kidney phospholipids. In liver, 54 and 22.8 mol% respectively of the esterified linoleate and arachidonate was deuteriated. The lower specific activity of arachidonate versus linoleate suggests that desaturation of linoleate, by a 6-desaturase, is not only rate limiting for synthesis of arachidonate but that the amount of newly synthesized arachidonate is insufficient by itself to maintain steady state levels of esterified arachidonate. The molar fraction of deuteriated linoleate in heart and kidney phospholipids was respectively 35 and 37.4%. These values are lower than for liver phospholipids but it appears there is adequate dietary linoleate available in these tissues for the synthesis of arachidonate. However, of the esterified arachidonate in heart and kidney phospholipids only 4.2 and 8.6 mol% respectively was deuteriated. Our results suggest that arachidonate is made in liver and transported to heart and kidney.

Animals↗
PubMed1993

Studies to determine if rat liver contains chain-length-specific acyl-CoA 6-desaturases.

According to the revised pathway for 22:6(n - 3) biosynthesis in liver (Voss et al. (1991) J. Biol. Chem. 266, 19995-20000) both 18:3(n - 3) and 24:5(n - 3) serve as substrates for desaturation at position-6. The present study was undertaken to determine whether microsomes contain chain-length-specific 6-desaturases. Addition of [1-14C]20:3(n - 6), a substrate for desaturation at position-5, did not depress desaturation of either [1-14C]18:3(n - 3) or [3-14C]24:5(n - 3). An unexplained observation was that both 18:3(n - 3) and 24:5(n - 3) inhibited the metabolism of 20:3(n - 6) to 20:4(n - 6). When an enzyme-saturating level of [3-14C]24:5(n - 3) was now incubated alone and with 40, 80 and 120 nmol of [1-14C]18:3(n - 3), the production of 24:6(n - 3) was inhibited by 43, 67 and 81%. Conversely, when [1-14C]18:3(n - 3) was incubated with 40, 80 or 120 nmol of [3-14C]24:5(n - 3), the synthesis of 18:4(n - 3) was inhibited by only 15, 20 and 27%. These and other competitive studies showed that there was always preferential desaturation of 18:3(n - 3) rather than 24:5(n - 3). In addition, competitive studies between 18:2(n - 6) and 18:3(n - 3), as well as with 24:4(n - 6) and 24:5(n - 3) showed that there was always preferential desaturation of the (n - 3) acid. Although our results are consistent with a single 6-desaturase, further studies, including the isolation of the 6-desaturases(s), is obviously required to determine whether multiple forms of the 6-desaturase exist.

Animals↗
PubMed1993

Purification and characterization of the trifunctional beta-oxidation complex from pig heart mitochondria.

The presence of a trifunctional beta-oxidation complex in pig heart and its relationship to the known long-chain enoyl-CoA hydratase (EC 4.2.1.74) from pig heart mitochondria were investigated. For this study, the complex was partially purified by chromatography on DEAE-cellulose in the absence of detergents and was purified to near homogeneity in the presence of Triton X-100. Both enzyme preparations contained long-chain specific activities of enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase but were virtually inactive toward short-chain (C4) substrates. Both preparations exhibited very low or no activities of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) and 2,4-dienoyl-CoA reductase (EC 1.3.1.34). The molecular weights of the two subunits of the pig heart complex were estimated to be 81,000 and 45,000, respectively. The partially purified preparation, obtained in the absence of detergent, was identified as a membranous fraction enriched with respect to the inner mitochondrial membrane. It is concluded that long-chain enoyl-CoA hydratase is a component enzyme of the trifunctional beta-oxidation complex which is associated with the inner membrane of pig heart mitochondria.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1993

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin endoperoxide synthase and by cytochrome P450 monooxygenase: structure and mechanism of formation of major metabolites.

Incubation of 5,8,11-[1-14C]eicosatrienoic acid with prostaglandin endoperoxide synthase of ram vesicular gland microsomes led to formation of a number of polar metabolites. Four major compounds were characterized by chemical and physical methods and found to be: (11R)-hydroxy-5,8,12-eicosatrienoic acid, 8,9,11-trihydroxy-5,12-eicosadienoic acid (two diastereoisomers), and 8,9-epoxy-11-hydroxy-5,12-eicosadienoic acid. On the basis of previous studies on the mechanism of prostaglandin biosynthesis it seemed likely that the initial step of conversion of 5,8,11-eicosatrienoic acid consisted of removal of the pro-S hydrogen from C-13. The resulting carbon-centered radical was apparently attacked by dioxygen at C-13 to provide a (13R)-(hydro)peroxy derivative, which served as the precursor of (13R)-hydroxyeicosatrienoic acid. Alternatively, attack by dioxygen occurred at C-11 to produce an (11R)-peroxy radical. This intermediate was further converted to (11R)-hydroxyeicosatrienoic acid by reduction, into two 8,9,11-trihydroxy-5,12-eicosadienoic acids by successive cyclization, oxygenation, and reduction, and into the epoxy-hydroxy acid by cyclization and intramolecular epoxidation. The relative abundance of (13R)-hydroxy-5,8,11-eicosatrienoic acid, (11R)-hydroxy-5,8,12-eicosatrienoic acid, and the epoxy alcohol plus the two 8,9,11-triols was 51, 9, and 40%, respectively. The oxygenation at C-13 and C-11 of 5,8,11-eicosatrienoic acid was inhibited by 90% in the presence of diclofenac, an inhibitor of prostaglandin endoperoxide synthase. The two diastereomeric 8,9,11-trihydroxy acids and the epoxy-hydroxy acid are novel oxylipins and their formation provides independent chemical evidence for the existence of an 11-peroxy radical intermediate in prostaglandin endoperoxide synthase catalysis. Oxygenation of 5,8,11-eicosatrienoic acid by cytochrome P450 from liver microsomes of cynomolgus monkeys and phenobarbital-treated rats was also investigated. The metabolites formed included 19- and 20-hydroxyeicosatrienoic acid, 8,9- and 11,12-dihydroxyeicosadienoic acids (formed by enzymatic hydrolysis of the corresponding epoxides), and (12R)-hydroxy-5,8,10-hydroxyeicosatrienoic acid.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

2-Alkenyl-4,4-dimethyloxazolines as derivatives for the structural elucidation of isomeric unsaturated fatty acids.

Several types of unsaturated fatty acid methyl esters were converted into 4,4-dimethyloxazoline (DMOX) derivatives and analyzed by mass spectrometry to further evaluate the feasibility of using this derivative for locating the positions of double bonds in isomeric fatty acids. Five isomeric 20-carbon tetraenoic acids were analyzed in which the four cis double bonds were systematically moved from the 4,7,10,13- to the 8,11,14,17-positions. It was possible to locate the positions of all four double bonds in the 7,10,13,16- and 8,11,14,17-isomers by appropriate ions differing by 12 atomic mass units. In a similar way the three terminal double bonds in the 4,7,10,13-, 5,8,11,14- and 6,9,12,15-isomers could be assigned. Odd-numbered ions at m/z 139, 153 and 167 which are accompanied by an even mass ion at 138, 152 and 166, respectively, are diagnostic for DMOX derivatives of acids with their first double bond, respectively, at positions 4, 5 and 6. It was thus possible to assign the location of all four double bonds in these three isomers. A comparison of the spectra of the DMOX derivatives of 17,17,18,18-d4 vs. 9,10,12,13-d4 linoleic acid suggests that double bonds preferentially migrate toward the polar end of the molecule prior to fragmentation. The merit of using DMOX derivatives to locate double-bond positions in mono- and dicarboxylic acids, produced during beta-oxidation of polyunsaturated fatty acids, was evaluated. The spectra of 3-cis- and 4-cis-decenoic acids differ as do the spectra of 8-carbon dicarboxylic acids with their double bonds at positions 3 and 4.

Alkenes↗
PubMed1993

Synthesis of ethyl arachidonate-19,19,20,20-d4 and ethyl dihomo-gamma-linolenate-19,19,20,20-d4.

Ethyl 5,8,11,14-eicosatetraenoate-19,19,20,20-d4 and ethyl 8,11,14-eicosatrienoate-19,19,20,20-d4 were synthesized by Grignard coupling of the methanesulfonyl ester of 2,5-undecadiyn-1-ol-10,10,11,11-d4 with 5,8-nonadiynoic acid and 8-nonynoic acid, respectively. The coupled products upon Lindlar reduction, followed by the preparation of their ethyl esters, yielded deuteriated ethyl arachidonate and ethyl dihomo-gamma-linolenate, which were completely characterized by 13C and 1H nuclear magnetic resonance and mass spectral analysis.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Stearidonic acid, an inhibitor of the 5-lipoxygenase pathway. A comparison with timnodonic and dihomogammalinolenic acid.

Leukotrienes have been shown to play an important role as mediators in various disease processes, including asthma and inflammation; thus, their synthesis is tightly regulated. The major precursor of leukotrienes is arachidonic acid (20:4n-6). Fatty acids which are structurally similar to 20:4n-6, such as eicosatrienoic acid (20:3n-6; dihomogammalinolenic acid) and eicosapentaenoic acid (20:5n-3; timnodonic acid) have been found to inhibit leukotriene biosynthesis. Because of the structural similarity of octadecatetraenoic acid (18:4n-3; stearidonic acid) with 20:4n-6, the present study was undertaken to determine whether stearidonic acid also exerts an inhibitory effect on the 5-lipoxygenase pathway. Human leukocytes were incubated with 18:4n-3 (20 microM or 10 microM), 20:5n-3 (20 microM) or 20:3n-6 (20 microM) and subsequently stimulated with 1 microM ionophore A23187 and 20:4n-6 (20 microM or 10 microM). The 5-lipoxygenase products were then measured by high-performance liquid chromatography. Leukotriene synthesis was reduced by 50% with 20 microM 18:4n-3 and by 35% with 10 microM 18:4n-3. Formation of 5S,12S-di-hydroxy-eicosatetraenoic acid and of 5-hydroxy-eicosatetraenoic acid was decreased by 25% with 20 microM 18:4n-3 and by 3% with 10 microM 18:4n-3. The inhibition observed with 20 microM 18:4n-3 appeared to be of the same order as that observed with 20 microM 20:5n-3; the inhibition observed with 18:4n-3 was shown to be dose-dependent. The inhibition produced by 20 microM 20:3n-6 was greater than that observed with either 20 microM 18:4n-3 or with 20 microM 20:5n-3.(ABSTRACT TRUNCATED AT 250 WORDS)

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Peroxisomal beta-oxidation of polyunsaturated long chain fatty acids in human fibroblasts. The polyunsaturated and the saturated long chain fatty acids are retroconverted by the same acyl-CoA oxidase.

The metabolism of the C22 unsaturated fatty acids erucic acid (22:1(n-9)), adrenic acid (22:4(n-6)), docosapentaenoic acid (22:5(n-3)) and docosahexaenoic acid (22:6(n-3)) was studied in cultured fibroblasts from patients with acyl-CoA oxidase deficiency, the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. [3-14C] 22:4 (n-6) and [3-14C] 22:5 (n-3) were shortened (retroconverted) to [1-14C] 20:4 (n-6) and [1-14C] 20:5 (n-3), respectively, in normal and X-ALD fibroblasts. In Zellweger and acyl-CoA oxidase deficient fibroblasts these reactions were deficient. Since the retroconversion is normal in X-ALD fibroblasts peroxisomal very long chain (lignoceryl) CoA ligase is probably not required for the activation of C22 unsaturated fatty acids. The present work with fibroblasts from patients with a specific acyl-CoA oxidase deficiency, previously shown to have a deficient peroxisomal clofibrate-inducible acyl-CoA oxidase, and which accumulate 24:0 and 26:0 fatty acids, supports the view that this enzyme is responsible for the chain-shortening of docosahexaenoic acid (22:6(n-3)), erucic acid (22:1(n-9)), docosapentaenoic acid (22:5(n-3)), and adrenic acid (22:4(n-6)) as well.

Acyl-CoA Oxidase↗
PubMed1992

Differences in the interconversion between 20- and 22-carbon (n - 3) and (n - 6) polyunsaturated fatty acids in rat liver.

When male weanling rats were fed diets containing either 5% corn oil or a diet in which half of the corn oil was replaced by fish oil, the 20:5(n - 3) in liver choline and ethanolamine phosphoglycerides, not only partially replaced arachidonate but also paired with palmitic and stearic acids in the same molar ratio as did arachidonate. The 22:5(n - 3)/22:6(n - 3) ratio in the liver phospholipids of corn oil fed rats was similar to that found when the esterified levels of these two acids were increased 5-fold by feeding fish oil. Moreover, the pairing of both 22:5(n - 3) and 22:6(n - 3) with palmitic and stearic acids, on a molar ratio basis, was relatively independent of the total amount of esterified 22:5(n - 3) and 22:6(n - 3). When (3-14C)-labeled 22:4(n - 6) was injected into rats raised on a chow diet or incubated with hepatocytes from these animals, its primary metabolic fate was retroconversion to arachidonate followed by esterification. Conversely, [3-14C]22:5(n - 3) was a poorer substrate for retroconversion with a larger amount being esterified directly into phospholipids and, in addition, this acid served as a precursor for 22:6(n - 3). The enhanced metabolism of both [3-14C]22:4(n - 6) to 22:5(n - 6) and of [3-14C]22:5(n - 3) to 22:6(n - 3) in animals raised on a diet devoid of fat or in their hepatocytes may possibly be due to elevated 6-desaturase activity and/or the level of this enzyme or enzymes. This hypothesis is based on studies showing that the synthesis of 22:6(n - 3) proceeds via a pathway independent of a 4-desaturase but requires the use of a 6-desaturase at two steps (Voss, A., Reinhart, M., Sankarappa, S. and Sprecher, H. (1991) J. Biol. Chem. 266, 19995-20000).

Animals↗
PubMed1992

Beta-oxidation of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid by MOLT-4 lymphocytes.

MOLT-4 lymphocytes metabolize 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE via beta-oxidation with retention of the hydroxyl group at the omega 9 carbon atom. The isolation of 6-hydroxy-4,8-tetradecadienoic acid documents that these cells have the capacity to catabolize the conjugated diene system. 12(S)-HETE was also metabolized to 3,12-dihydroxy-8,10,14-eicosatrienoic acid and 1,9-dihydroxy-5,7,11-heptadecatriene as well as to 17- and 19-carbon aldehydes. When MOLT-4 cells were incubated with the beta-oxidation product, 10-hydroxy-6,8,12-octadecatrienoic acid, it was in part further catabolized but in addition it served as an anabolic precursor as defined by the accumulation 3,12-dihydroxy-8,10,14-eicosatrienoic acid as well as 1,11-dihydroxy-7,9,13-nonadecatriene. Neither 10-hydroxy-6,8,12-octadecatrienoic acid nor 13-hydroxy-5,8,11-octadecatrienic acid was as potent in inhibiting phytohemagglutin-induced lymphocyte mitogenesis as were their parent compounds--i.e., 12(S)- and 15(S)-HETE. These findings argue against the hypothesis that beta-oxidation products of 12(S)- and 15(S)-HETE are the potential modulators of lymphocyte function. However, neither the pathway for synthesis, nor the role of odd chain aldehydes and diols as potential lipid mediators was determined in this study.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Binding of carbamyl-platelet-activating factor to the Raji lymphoblast platelet-activating factor receptor.

Carbamyl-platelet-activating factor (1-hexadecyl-2-N-methylcarbamyl-glycero-3-phosphocholine; CPAF) is an analog of platelet-activating factor (PAF) containing an N-methylcarbamyl moiety at the sn-2 position. CPAF was tested for effects on the Raji lymphoblast PAF receptor. Binding studies conducted at 4 degrees C demonstrated specific binding that reached saturation within 60-80 min. Scatchard analysis of CPAF binding data revealed a single class of CPAF binding sites (14,800/cell) with a K = 2.9 +/- 0.9 nM. Competition binding studies with PAF indicated that CPAF has about one-third the potency of native PAF. Unlike PAF, however, CPAF was not significantly metabolized by Raji lymphoblasts at 37 degrees C. CPAF was shown to have PAF-agonistic qualities, since 100 pM to 1 microM CPAF increased free intracellular calcium in a dose-dependent manner. The structurally dissimilar PAF receptor antagonists CV-6209 and alprazolam inhibited the CPAF-induced calcium changes at doses that competed with CPAF binding. Treatment of Raji lymphoblasts with PAF or CPAF (10 pM-1 microM) did not affect spontaneous proliferation, suggesting that the PAF receptor is not involved in the proliferative process in this cell line. These studies demonstrate that CPAF is a metabolically stable lymphoblast PAF receptor agonist that may provide a useful tool in the further elucidation of the role of PAF in lymphocyte function.

Burkitt Lymphoma↗
PubMed1992

An in vitro model for essential fatty acid deficiency: HepG2 cells permanently maintained in lipid-free medium.

A stable essential fatty acid-deficient cell type, known as HepG2-EFD, was derived from the lipoprotein-producing human hepatoma cell line HepG2. These cells are particularly useful for quantitative studies involving essential fatty acids (n-6 and n-3 fatty acids) in secreted lipoproteins. Radiolabeled essential fatty acids can be delivered to these cells without altering the specific activity of the fatty acids, since the deficient cells contain no endogenous essential fatty acids. Using these cells, radioactivity data (dpm) from metabolic studies can be converted directly to mass, and masses as low as a few pmoles can be accurately measured. HepG2-EFD cell cultures were established by growing HepG2 cells in medium containing delipidated serum. After 10 days of growth in delipidated medium, HepG2 cells were completely depleted of all essential fatty acids. Compensatory increases in nonessential fatty acids (n-9 and n-7 fatty acids) including 20:3n-9 (the Mead acid), which is the hallmark fatty acid of essential fatty acid deficiency, were also observed in HepG2-EFD cells. Despite the lack of exogenous fatty acids in the medium and the lack of essential fatty acids in the cells, export of very low density lipoprotein (VLDL)-associated apolipoprotein B by HepG2-EFD was the same as observed for parent HepG2 cells. However, the activity of beta-oxidation of fatty acids in HepG2-EFD cells was much lower than in the parent cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins B↗
PubMed1991

The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase.

The hypothesis that the last step in the biosynthesis of 4,7,10,13,16,19-22:6 from linolenate is catalyzed by an acyl-CoA-dependent 4-desaturase has never been evaluated by direct experimentation. When rat liver microsomes were incubated with [1-14C]7,10,13,16,19-22:5, under conditions where linoleate was readily desaturated to 6,9,12-18:3, it was never possible to detect the product of the putative 4-desaturase. In the presence of malonyl-CoA, 7,10,13,16,19-22:5 was sequentially chain-elongated to 9,12,15,18,21-24:5, followed by its desaturation at position 6 to give 6,9,12,15,18,21-24:6. Microsomes desaturated 9,12,15,18,21-24:5 at rates similar to those observed for metabolizing linoleate to 6,9,12-18:3. Rat hepatocytes metabolize [1-14C]7,10,13,16,19-22:5 to 22:6(n-3), but in addition, it was possible to detect small amounts of esterified 24:5(n-3) and 24:6(n-3) in phospholipids, which is a finding consistent with their role as obligatory intermediates in 22:6(n-3) biosynthesis. When 3-14C-labeled 24:5(n-3) or 24:6(n-3) were incubated with hepatocytes, only a small amount of either substrate was esterified. [3-14C] 24:5(n-3) was metabolized both by beta-oxidation to 22:5(n-3) and by serving as a precursor for the biosynthesis of 24:6(n-3) and 22:6(n-3). The primary metabolic fate of [3-14C]24:6(n-3) was beta-oxidation to 22:6(n-3), followed by its acylation into membrane lipids. Our results thus document that 22:5(n-3) is the precursor for 22:6(n-3) but via a pathway that is independent of a 4-desaturase. This pathway involves the microsomal chain elongation of 22:5(n-3) to 24:5(n-3), followed by its desaturation to 24:6(n-3). This microsomal product is then metabolized, via beta-oxidation, to 22:6(n-3).

Animals↗