Biomedical subjects

H Sprecher

Publications and source records attributed to H Sprecher.

At least 37 records · Page 2Linked to original sources

PubMed1997

Studies to determine if rat liver contains multiple chain elongating enzymes.

According to the revised pathways of polyunsaturated fatty acid biosynthesis three, rather than two acids, must be chain elongated for converting linoleate and linolenate, respectively, to 22:5(n-6) and 22:6(n-3) (Sprecher et al. (1995) J. Lipid Res. 36, 2471-2477). The present study was undertaken to determine whether microsomes contained chain-length specific chain-elongating enzymes and, secondly, whether reaction rates for any of these reactions might be rate limiting in the synthesis of 24:5(n-6) and 24:6(n-3), which are the immediate precursors of 22:5(n-6) and 22:6(n-3). Rates of total chain elongation products produced from both 18:4(n-3) and 20:5(n-3) were about 3 nmol/min/mg of microsomal protein while only about 0.5 nmol/min/mg of 24:5(n-3) plus 24:6(n-3) was synthesized from 22:5(n-3). The rate of 24:5(n-3) synthesis was similar to that for the desaturation of 24:5(n-3), at position 6, to yield 24:6(n-3) (Geiger et al. (1993) Biochim. Biophys. Acta 1170, 137-142). The results suggest that the last chain elongation step in unsaturated fatty acid biosynthesis may be equally regulatory in governing the synthesis of fatty acids as is desaturation at position 6. When an enzyme saturating level of [1-(14)C]18:4(n-3) was incubated with increasing amounts of 18:3(n-6) there was a decrease in the production [1-(14)C]20:4(n-3). In a similar way it was observed that 18:4(n-3) inhibited the chain elongation of [1-(14)C]18:3(n-6). Identical cross-over inhibitory studies, using 20:4(n-6) and 20:5(n-3), as well as 22:4(n-6) and 22:5(n-3) also suggested that microsomes contain chain length specific chain-elongating enzymes. This conclusion was further supported by the finding that neither 20:5(n-3) or 22:5(n-3) inhibited the chain elongation of [1-(14)C]18:4(n-3). However, 18:4(n-3), and to a lesser degree, 22:5(n-3) did inhibit the chain elongation of [1-(14)C]20:5(n-3). This latter finding suggests that 18:4(n-3) and 20:5(n-3) might interact with the enzyme that chain elongates 20:5(n-3) to depress its ability to synthesize 22:5(n-3). Our results are most consistent with the presence of multiple chain-elongating enzymes, but a more definitive answer requires the purification of these membrane-bound proteins. In addition our results suggest that the channeling of acids between enzymes in the endoplasmic reticulum may play an important role in regulating the biosynthesis of unsaturated fatty acids.

Animals↗

PubMed1997

Metabolites produced during the peroxisomal beta-oxidation of linoleate and arachidonate move to microsomes for conversion back to linoleate.

When [1-(14)C]4,7,10-16:3, a product produced after two cycles of arachidonate beta-oxidation, was incubated with rat liver peroxisomes and microsomes it was metabolized to 2-trans-4,7,10-16:4, a catabolic product; 6,9,12-18:3 and 8,11,14-20:3, anabolic products made via microsomal chain elongation of the substrate; and 7,10-16:2 and 9,12-18:2. Analysis of the acyl-CoAs produced when 6,9,12-18:3 and its catabolic product, 4,7,10-16:3, where incubated under the above conditions showed that the acyl-CoAs of all of the above compounds, as well as 5,8-14:2-CoA and 6:0-CoA accumulated. Our results show that when 5,8-14:2 and 4,7,10-16:3 are produced by peroxisomal beta-oxidation they can be further degraded to hexanoyl-CoA or move to microsomes for conversion back to linoleate, which is a precursor of arachidonate.

Adenosine Triphosphate↗

PubMed1997

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids.

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ratio. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-14C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.

8,11,14-Eicosatrienoic Acid↗

PubMed1997

Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells.

Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.

Adenosine↗

PubMed1996

Regulation of the biosynthesis of 4,7,10,13,16,19-docosahexaenoic acid.

The synthesis of 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) requires that when 6,9,12,15,18,21-tetracosahexaenoic acid (24:6(n-3)) is produced in the endoplasmic reticulum, it preferentially moves to peroxisomes for one cycle of beta-oxidation rather than serving as a substrate for membrane lipid synthesis. Both 24:6(n-3) and its precursor, 9,12,15,18,21-tetracosapentaenoic acid (24:5(n-3)), were poor substrates for acylation into 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) by rat liver microsomes. When peroxisomes were incubated with 1-14C- or 3-14C-labeled 7,10,13,16,19-docosapentaenoic acid (22:5(n-3)), [1-14C]22:6(n-3), [3-14C]24:5(n-3), or [3-14C]24:6(n-3), only small amounts of acid-soluble radioactivity were produced when double bond removal at positions 4 and 5 was required. When microsomes and 1-acyl-GPC were included in incubations, the preferred metabolic fate of acids, with their first double bond at either positions 4 or 5, was to move out of peroxisomes for esterification into the acceptor rather than serving as substrates for continued beta-oxidation. When [1-14C]22:6(n-3) or [3-14C]24:6(n-3) was incubated with peroxisomes, 2-trans-4,7,10,13,16,19-22:7 accumulated. The first cycle of 20:5(n-3) beta-oxidation proceeds through 2-trans-4,8,11,14,17-20:6 and thus requires both Delta3,5,Delta2, 4-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. The accumulation of the substrate for 2,4-dienoyl-CoA reductase, as generated from 22:6(n-3), but not from 20:5(n-3), suggests that this enzyme distinguishes between subtle structural differences. When 22:6(n-3) is produced from 24:6(n-3), its continued degradation is impaired because of low 2,4-dienoyl-CoA reductase activity. This slow reaction rate likely contributes to the transport of 22:6(n-3) out of peroxisomes for rapid acylation into 1-acyl-GPC by microsomes.

Acyl Coenzyme A↗

PubMed1996

Active synthesis of C24:5, n-3 fatty acid in retina.

The formation of 14C-labelled long-chain and very-long-chain (n-3) pentaenoic and hexaenoic fatty acids was studied in bovine retina by following the metabolism of. [14C]-docosapentaenoate [C22:5, n-3 fatty acid (22:5 n-3)], [14C]-docosahexaenoate (22:6 n-3), and [14C]acetate. With similar amounts of 22:5 n-3 and 22:6 n-3 as substrates, the former was actively transformed into 24:5 n-3, whereas the latter was virtually unmodified. Labelled 24:5, 26:5, 24:6 and 22:6 were formed from [1-14C]22:5 n-3, showing that pentaenoic fatty acids including 24:5 n-3 can be elongated and desaturated within the retina. When retinal microsomes were incubated with [1-14C]22:5 n-3, 24:5 n-3 was the only fatty acid formed. In retinas incubated with [14C]acetate, 24:5 n-3 was the most highly labelled fatty acid among the polyenes synthesized, 24:6 n-3 being a minor product. Such selectivity in the elongation of two fatty acids identical in length, 22:5 n-3 and 22:6 n-3, despite the fact that 22:5 is a minor and 22:6 a major fatty acid constituent of retina, suggests that the active formation of 24:5 n-3 plays a key role in n-3 polyunsaturated fatty acid (PUFA) metabolism. This compound might give rise to even longer pentaenes via elongation, and to the major PUFAs of retina, 22:6 n-3, by 6-desaturation and chain shortening. Of all retinal lipids, a minor component, triacylglycerol (TG), incorporated the largest amounts of [14C]22:5 and 22:6. TG also concentrated most of the [14C]24:5 formed in retina, whether from [14C]22:5 n-3 or from [14C]acetate, suggesting an important role for this lipid in supporting PUFA metabolism and the synthesis of 22:6 n-3.

Acetates↗

PubMed1996

Very long chain acyl-CoA dehydrogenase deficiency: successful treatment of acute cardiomyopathy.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a severe defect of mitochondrial fatty acid oxidation characterized by hypertrophic cardiomyopathy, pericardial effusion, steatosis, and hypoglycemia, often resulting in death by 4-5 months of age. The onset of cardiomyopathy and pericardial effusion is insidious and sudden, necessitating early diagnosis and intervention to prevent death. A family affected with this defect is described in which dietary therapy with medium-chain triglycerides (MCT) was associated with rapid reversal of these severe clinical symptoms. Diagnosis by acylcarnitine analysis in the neonatal period can provide the opportunity for early clinical intervention. Prenatal diagnosis from amniocytes by enzymology or in vitro analysis of the fat oxidation pathway with deuterated fatty acid precursors has also been successful and permits intervention at birth. Of 10 affected children, 7 untreated cases died within the first several months while the remaining 3 cases survived when treated with medium-chain triglycerides as the major source of dietary fat.

Acute Disease↗

PubMed1996

New advances in fatty-acid biosynthesis.

When [1-14C]7,10,13,16,19-22:5 was incubated with microsomes, it was not desaturated to 4,7,10,13,16,19-22:6 by an acyl-CoA-dependent 4-desaturase. Subsequent studies with rat hepatocytes showed that 7,10,13,16, 19-22:5 was the precursor of 4,7,10,13,16,19-22:6, but the pathway proceeded through 24-carbon fatty acids. The implication of this finding is that 24-carbon acids, when produced in microsomes, must move to a site for partial beta-oxidation. Subsequent studies using a number of labeled acids showed that peroxisomes chain-shorten fatty acids, which then move back to the endoplasmic reticulum for esterification. The biosynthesis of both 22:5(n-6) and 22:6(n-3) thus requires the extensive movement of fatty acids between subcellular compartments.

Animals↗

PubMed1995

Peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase and thus possess all enzymes required for the beta-oxidation of unsaturated fatty acids by a novel reductase-dependent pathway.

The presence of delta 3,5,delta 2,4-dienoyl-CoA isomerase in peroxisomes was demonstrated by determining the subcellular distribution of this enzyme in rat liver. The peroxisomal and mitochondrial forms of the isomerase exhibit similar chain length specificities and they are homologous as indicated by the recognition of the peroxisomal 66-kDa enzyme by an antiserum raised against the mitochondrial 32-kDa isomerase. This report demonstrates that peroxisomes contain all enzymes required for the beta oxidation of unsaturated fatty acids with odd-numbered double bonds by a novel pathway in which double bonds are reductively removed by the NADPH-dependent 2,4-dienoyl-CoA reductase.

Animals↗

PubMed1995

Alteration of mitochondrial gene expression and disruption of respiratory function by the lipophilic antifolate pyrimethamine in mammalian cells.

To clone the mammalian gene(s) associated with a novel lipophilic antifolate resistance provoked by the antiparasitic drug pyrimethamine (Assaraf, Y. G., and Slotky, J. I. (1993) J. Biol. Chem. 268, 4556-4566), differential screening of a cDNA library from pyrimethamine-resistant (PyrR100) cells was used. This library was screened with total cDNA from wild-type and PyrR100 cells. Surprisingly, several differentially overexpressed cDNA clones were isolated from PyrR100 cells, many of which mapped to the mitochondrial genome. Several lines of evidence establish mitochondria as a new target for the cytotoxic activity of pyrimethamine. (a) At > or = 10 microM, pyrimethamine inhibited mitochondrial respiration in viable wild-type cells. (b) Electron microscopy revealed degenerated mitochondrial membrane cristae in PyrR100 cells. (c) Some mitochondrially encoded transcripts were prominently elevated, whereas the normally stable 12 S/16 S rRNA was decreased in PyrR100 cells. (d) Metabolic pulse-chase labeling suggested an increased turnover rate of mitochondrially synthesized proteins in PyrR100 cells. (e) The specific activity of the key respiratory enzymatic complex cytochrome c oxidase was reduced by 6-fold in PyrR100 cells. (f) Consequently, the rate of respiration in intact PyrR100 cells was reduced by 3-fold. We conclude that pyrimethamine and possibly lipophilic analogues of methotrexate possess a folinic acid nonrescuable toxicity involving disruption of mitochondrial inner membrane structure and respiratory function, thereby establishing a new organellar target for the cytotoxic effect elicited by lipid-soluble antifolates.

Adenosine Triphosphatases↗

PubMed1995

Peroxisomal-microsomal communication in unsaturated fatty acid metabolism.

The addition of 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) to peroxisomes decreased the production of acid-soluble radioactivity formed by beta-oxidation of [1-(14)C]arachidonate due to substrate removal by esterification into the acceptor. This peroxisomal-associated acyl-CoA:1-acyl-GPC acyltransferase activity was due to microsomal contamination. The production of acid-soluble radioactivity from [1-(14)C]7,10,13,16-22:4, but not from [3-(14)C]7,10,13,16-22:4 was independent of 1-acyl-GPC, with and without microsomes. By comparing rates of peroxisomal beta-oxidation with those for microsomal acylation, it was shown that the preferred metabolic fate of arachidonate, when added directly to incubations, or generated via beta-oxidation, was esterification by microsomal 1-acyl-GPC acyltransferase, rather than continued peroxisomal beta-oxidation.

1-Acylglycerophosphocholine O-Acyltransferase↗

PubMed1995

Double bond removal from odd-numbered carbons during peroxisomal beta-oxidation of arachidonic acid requires both 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase.

The pathway for the peroxisomal beta-oxidation of arachidonic acid (5,8,11,14-20:4) was elucidated by comparing its metabolism with 4,7,10-hexadecatrienoic acid (4,7,10-16:3) and 5,8-tetradecadienoic acid (5,8-14:2) which are formed, respectively, after two and three cycles of arachidonic acid degradation. When [1-14C]4,7,10-16:3 was incubated with peroxisomes in the presence of NAD+ and NADPH, it resulted in a time-dependent increase in the production of acid-soluble radioactivity which was accompanied by the synthesis of 2-trans-4,7,10-hexadecatetraenoic acid and two 3,5,7,10-hexadecatetraenoic acid isomers. The formation of conjugated trienoic acids suggests that peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase with the ability to convert 2-trans-4,7,10-hexadecatetraenoic acid to 3,5,7,10-hexadecatetraenoic acid. When 1-14C-labeled 6,9,12-octadecatrienoic acid or 7,10,13,16-docosatetraenoic acid was incubated without nucleotides, the 3-hydroxy metabolites accumulated, since further degradation requires NAD(+)-dependent 3-hydroxyacyl-CoA dehydrogenase. When [1-14C]5,8,11,14-20:4 was incubated under identical conditions, no polar metabolite was detected, but 2-trans-4,8,11,14-eicosapentaenoic acid accumulated. When NADPH was added to incubations, 3-hydroxy-8,11,14-eicosatrienoic, 2-trans-4,8,11,14-eicosapentaenoic, 2-trans-8,11,14-eicosatetraenoic, and 8,11,14-eicosatrienoic acids were produced. Analogous compounds were formed from [1-14C]5,8-14:2. Our results show that the removal of double bonds from odd-numbered carbons in arachidonic acid thus requires both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase. One complete cycle of 5,8-14:2 and 5,8,11,14-20:4 beta-oxidation yields, respectively, 6-dodecenoic and 6,9,12-octadecatrienoic acids.

Animals↗

PubMed1995

Reevaluation of the pathway for the metabolism of 7,10,13, 16-docosatetraenoic acid to 4,7,10,13,16-docosapentaenoic acid in rat liver.

When rat liver microsomes were incubated with [1-14C]22:4(n-6) under standard conditions for measuring acyl-CoA desaturases, it was not possible to detect the synthesis of any 22:5(n-6). When malonyl-CoA and NADPH were included in the incubation, 22:4(n-6) was chain elongated to 24:4(n-6), which was then desaturated to 24:5(n-6). Rat hepatocytes metabolized [1-14C]22:4(n-6), [3-14C]24:4(n-6), and [3-14C]24:5(n-6) to yield esterified radioactive 22:5(n-6). The results show that 22:4(n-6) is the precursor of 22:5(n-6) but the pathway is independent of an acyl-CoA-dependent 4-desaturase and probably requires intracellular communication between the endoplasmic reticulum and a site for beta-oxidation. Microsomal reaction rates for (n-6) versus (n-3) polyunsaturated fatty acid biosynthesis cannot per se be used to explain why in vivo most membrane lipids preferentially accumulate 22:6(n-3) rather than 22:5(n-6). Rates of desaturation of 24:4(n-6) and 24:5(n-3) at position 6 were similar (M. Geiger et al., Biochim. Biophys. Acta 1170, 137-142, 1993). We now show that 20:4(n-6) and 20:5(n-3) are chain elongated at the same rate as are 22:4(n-6) and 22:5(n-3). At present, no single reaction can be defined as being substrate specific or rate limiting to explain why there is an apparent selective synthesis and acylation of 22:6(n-3) rather than 22:5(n-6) into membrane lipids.

Animals↗

PubMed1995

Evidence for intermediate channeling in mitochondrial beta-oxidation.

The accumulation of beta-oxidation intermediates was studied by incubating normal and beta-oxidation enzyme-deficient human fibroblasts with [2H4]linoleate and L-carnitine and analyzing the resultant acylcarnitines by tandem mass spectrometry. Labeled decenoyl-, octanoyl-, hexanoyl-, and butyrylcarnitines were the only intermediates observed with normal cells. Intermediates of longer chain length, corresponding to substrates for the beta-oxidation enzymes associated with the inner mitochondrial membrane, were not observed unless a cell line was deficient in one of these enzymes, such as very-long-chain acyl-CoA dehydrogenase, long-chain 3-hydroxyacyl-CoA dehydrogenase, or electron transfer flavoprotein dehydrogenase. Matrix enzyme deficiencies, such as medium- and short-chain acyl-CoA dehydrogenases, were characterized by elevated concentrations of intermediates corresponding to their respective substrates (octanoyl- and decenoylcarnitines in medium-chain acyl-CoA dehydrogenase deficiency and butyrylcarnitine in short-chain acyl-CoA dehydrogenase deficiency). These observations agree with the notion of intermediate channeling due to the organization of beta-oxidation enzymes in complexes. The only exception is the incomplete channeling from thiolase to acyl-CoA dehydrogenase in the matrix. This situation may be a consequence of only one 3-ketoacyl-CoA thiolase being unable to interact with the several acyl-CoA dehydrogenases in the matrix.

Acyl-CoA Dehydrogenase↗

PubMed1995

Investigation of beta-oxidation intermediates in normal and MCAD-deficient human fibroblasts using tandem mass spectrometry.

Mitochondrial fatty acid beta-oxidation was studied by incubating stable isotope-labeled fatty acid probes with human fibroblasts in the presence of L-carnitine. The acylcarnitine intermediates produced were analyzed by tandem mass spectrometry. Oxidation by normal fibroblasts produced specific acylcarnitine intermediates corresponding to acyl-CoA dehydrogenase substrates mainly of 10 or less carbons. These probes demonstrated that the pathway, involving all beta-oxidative steps, could be examined. Oxidation of the same precursors by cells with medium chain acyl-CoA dehydrogenase (EC 1.3.99.2) (MCAD) deficiency, which is caused by different DNA mutations, produced acylcarnitine profiles which appear to be specific to this enzyme defect, regardless of the DNA mutation. Increased amounts of octanoyl-, decanoyl-, or decenoylcarnitine were detected. The ratios of octanoylcarnitine to decanoyl- or decenoylcarnitine appear specific for MCAD deficiency. Even though the concentration of labeled decenoylcarnitine (C10:1) was elevated in incubations of MCAD-deficient cells with labeled linoleate or with a fatty acid mixture which included palmitate, oleate, and linoleate, the predominant intermediate was octanoylcarnitines. These results suggest that MCAD-deficient cells readily convert decanoyl-CoA into octanoyl-CoA. This in vitro system could be utilized to study fatty acid oxidation disorders and to study the origins of metabolic intermediates associated with them.

Acyl Coenzyme A↗

PubMed1995

Metabolism of deuterium-labeled linoleic, 6,9,12-octadecatrienoic, 8,11,14-eicosatrienoic, and arachidonic acids in the rat.

Male weanling rats were fed a diet that contained 2.1% ethyl oleate, 1% ethyl linoleate, and 0.2% ethyl linolenate. After 4 weeks all the linoleate was replaced by the deuterium-labeled analog and the animals were killed 4 days later. The molar fraction of total 20:4(n-6) in liver, heart, and kidney phospholipids containing deuterium was 33.9, 8.9, and 13.3%, respectively. Second, animals were preconditioned by incorporating either 0.2% of 18:3(n-6), 20:3(n-6), or 20:4(n-6) into the above diet and again after 4 weeks all the linoleate was replaced with the labeled analog. Now the molar fraction of labeled 20:4(n-6) in liver phospholipids from these three groups of animals was reduced from 33.9 to 27.1, 23.9, and 24.1% respectively. In contrast, there was little change in the specific activity of 20:4(n-6) in heart and kidney phospholipids. The third protocol was a direct crossover study in that again unlabeled linoleate was fed during the entire period. Four days prior to killing the unlabeled 18:3(n-6), 20:3(n-6), and 20:4(n-6) were replaced with the deuterium-labeled analogs. The mole % of total esterified 20:4(n-6) in liver phospholipids was now 24.6, 32.0, and 26.2%, respectively. Even though 18:3(n-6), 20:3(n-6), and 20:4(n-6) were all fed at only 20% of the level of 18:2(n-6), it can be calculated that the molar fraction of esterified 20:4(n-6) in liver phospholipids was between 65 to 77% of that found when 18:2(n-6) was the only dietary (n-6) acid as under these conditions 33.9 mol % of the 20:4(n-6) was labeled. Interestingly, when deuterium-labeled 18:3(n-6), 20:3(n-6), or 20:4(n-6) was fed, the specific activity of esterified 20:4(n-6) in kidney and heart phospholipids was always equal to or greater than what was derived from deuterium-labeled 18:2(n-6). The results show that under steady-state dietary conditions, (n-6) dietary fatty acids are processed in different ways by liver, heart, and kidney.

8,11,14-Eicosatrienoic Acid↗

PubMed1995

Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.

The purpose of this study was to determine whether the formation of docosahexaenoic acid in human cells occurs through a pathway that involves 24-carbon n-3 fatty acid intermediates and retroconversion. Normal human skin fibroblasts synthesized radiolabeled docosahexaenoic acid from [1-(14)C]18:3n-3, [3-(14)C]22:5n-3, [3-(14)C]24:5n-3, and [3-(14)C]24:6n-3. The amount of docosahexaenoate formed was reduced in fibroblasts defective in peroxisomal biogenesis, by 90-100% in Zellweger's syndrome and by 50-75% in infantile Refsum's disease. Fatty acid elongation and desaturation were intact in these mutant cells. No decrease in radiolabeled docosahexaenoic acid production occurred in mutant fibroblasts defective in peroxisomal alpha-oxidation or mitochondrial beta-oxidation, or in normal fibroblasts treated with methyl palmoxirate to inhibit mitochondrial beta-oxidation. Therefore, the retroconversion step in docosahexaenoic acid formation occurs through peroxisomal beta-oxidation in normal human cells. These results demonstrate that the pathway for docosahexaenoic acid synthesis in human cells involves 24-carbon intermediates. The limited ability to synthesize docosahexaenoic acid may underlie some of the pathology that occurs in genetic diseases involving peroxisomal beta-oxidation.

Cells, Cultured↗

PubMed1995

Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids.

Recent studies refute the commonly accepted, but untested, hypothesis that 7,10,13,16-22:4 and 7,10,13,16,19-22:5 are desaturated at position 4 by a microsomal acyl-CoA-dependent desaturase. The synthesis of 4,7,10,13,16,19-22:6 occurs via the following reaction sequence: 7,10,13,16,19-22:5-->9,12,15,18,21-24:5-->6,9,12,15,18,21-24:6 4,7,10,13,16,19-22:6. The synthesis of 4,7,10,13,16-22:5 from 7,10,13,16-22:4 takes place via an analogous pathway. According to these pathways the 24-carbon acids that are made in the endoplasmic reticulum move to a site for partial beta-oxidation, which is most likely peroxisomes. The products of partial beta-oxidation, 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6, then move back to the endoplasmic reticulum where they are used as substrates for membrane lipid biosynthesis. The ability of a fatty acid to serve as a substrate for continued peroxisomal beta-oxidation, versus its transfer out of peroxisomes for subsequent endoplasmic reticulum-associated esterification reactions, may be an important control for regulating membrane lipid fatty acid composition. Indeed, the revised pathways of polyunsaturated fatty acid biosynthesis imply that there is considerable intracellular movement and recycling of fatty acids between peroxisomes and the endoplasmic reticulum. In addition, these revised pathways require that two 18-carbon and two 24-carbon acids are substrates for desaturation at position 6. Also, as linoleate and linolenate are metabolized, respectively, to 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6, three n-6 acids and three n-3 acids are substrates for malonyl-CoA dependent chain elongation. It remains to be determined how many microsomal enzymes are required to carry out these reactions and whether other ancillary enzymes are expressed in tissues whose membrane lipids accumulate very long-chain polyunsaturated acids with up to 36 carbon atoms.

Acyl-CoA Dehydrogenase, Long-Chain↗