Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.     

Dual metabolic pathways of 12-HETE in rat aortic smooth muscle cells.

12(S)-HETE, a major lipoxygenase-derived compound from arachidonic acid is incorporated and metabolized by vascular smooth muscle cells via beta-oxidation. We have now identified for the first time in this cell type 12(S)-HETE metabolites formed by a combination of reductase and oxidation pathways. HPLC and GC-MS analysis of time-course experiments allow us to characterize two different metabolic pathways: a direct peroxisomal beta-oxidation of 12(S)-HETE leading to the formation of 16:3 (8-OH) which accumulates first and a reduction of one of the conjugated double bonds of 12(S)-HETE giving the dihydro-intermediate 20:3(12-OH) that transiently accumulates before being converted itself by peroxisomal beta-oxidation to 16:2(8-OH). Taken together these results may suggest that the transient accumulation of 20:3(12-OH) through transcellular metabolism of 12(S)-HETE may represent a part of the modulatory effect of 12(S)-HETE on vascular function.     

12-Hydroxyeicosatetraenoic acid is metabolized by beta-oxidation in mouse peritoneal macrophages. Identification of products and proposed pathway

The products derived from the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by mouse peritoneal macrophages were characterized by high performance liquid chromatography (HPLC) and GC-mass spectrometry. HPLC analysis demonstrated two predominant polar products and several minor ones. The proportion and amounts of these products were dependent on the concentration of 12-HETE, the number of macrophages incubated with the monohydroxy fatty acid, and the time of incubation. The products identified by GC-mass spectrometry suggested that 12-HETE had undergone beta-oxidation. The intermediates identified were: 3,12-dihydroxy-5,8,10,14, 20:4; 10-hydroxy-3,6,8,12, 18:4; 3,10-dihydroxy-6,8,12, 18:3; 8-hydroxy-4,6,10, 16:3; 6-hydroxy-4,8, 14:2; and 4-hydroxy, 12:1. The major products, as identified by HPLC and GC-mass spectrometry, were 8-hydroxy-4,6,10, 16:3 and 4-hydroxy, 12:1. A minor product, 10-hydroxy-6,8,12, 18:3 was postulated to arise from either the isomerization and reduction of 10-hydroxy-3,6,8,12, 18:4 or from chain elongation of 8-hydroxy-4,6,10, 16:3. Inhibiting cyclooxygenase and lipoxygenase activities by ibuprofen and nordihydroguaiaretic acid, respectively, did not inhibit the formation of these products. 82% to 98% of 12-HETE was converted and released into the medium as products of beta-oxidation. The remainder was taken up into cellular lipids. beta-Oxidation of 12-HETE was decreased by only 12 and 21% after inhibiting mitochondrial fatty acid oxidation by 89 and 93% by 5 and 100 microM concentrations of the mitochondrial fatty acid oxidation inhibitor, methyl palmoxirate, respectively. It is thus postulated that the beta-oxidation of 12-HETE by mouse peritoneal macrophages occurs in peroxisomes.     

Differential effects of freezing on total hepatic peroxisomal fatty acid oxidation and on the rate-limiting enzyme, acyl-CoA oxidase

Fatty acid oxidation defects can be acutely fatal, leading to the collection of tissues which are frozen for future analysis. Since peroxisomes can also oxidize long-chain fatty acids, differentiation of the contributions from the peroxisome as opposed to the mitochondria is important. We studied the effects of freezing and storage of rat livers on peroxisomal and mitochondrial beta-oxidation as measured by cyanide sensitivity of the oxidation of [1-14C]oleoyl-CoA to 14CO2 and acid-soluble labeled products. In addition, we examined the effects of freezing and storage on the rate-limiting enzyme for peroxisomal beta-oxidation, acyl-CoA oxidase, by the H2O2 generation method. Marked reduction in the oxidation of [1-14C]oleoyl-CoA was found for both peroxisomal and mitochondrial systems upon freezing at -18 or -70 degrees C for 2 days which declined further on storage at these temperatures for 12 weeks. Loss of activity after freezing was greater for the mitochondrial than the peroxisomal beta-oxidation system. By contrast, acyl-CoA oxidase activity was resistant to these changes, maintaining prefrozen activities despite storage for 12 weeks. The contribution of the peroxisomal system to beta-oxidation was 32% of the total rate of oxidation of [1-14C]oleoyl-CoA in the rat liver. These findings indicate that the contributions of the peroxisomal system to total fatty acid oxidation may be considerable, that freezing of the liver results in drastic reduction in enzyme activities of both peroxisomal as well as mitochondrial beta-oxidation, but that the rate-limiting enzyme of the peroxisomal system, acyl-CoA oxidase, retains full activity despite freezing and storage.     

Contribution of mitochondria and peroxisomes to palmitate oxidation in pea tissues

Total, mitochondrial and peroxisomal palmitate oxidation capacities were compared in pea, from the dry seed to 14 days after imbibition. Total beta-oxidation varied over the measured time period and showed four peaks of activity at day 2, days 5-6, day 10 and days 12-13. The contribution of peroxisomal and mitochondrial beta-oxidation to this overall beta-oxidation varied. Over the first 48 h of seed germination, peroxisomal beta-oxidation accounted for 80-100% of the total observed beta-oxidation. The larger peaks of beta-oxidation at days 5-6, day 10 and days 12-13 were due primarily to mitochondrial beta-oxidation activity, which accounted for 70-90% of the observed total beta-oxidation at these times. The peaks of activity are related to observed stages in seedling development.     

Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome

We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.     

Metabolism of 15-hydroxy-5,8,11,13-eicosatetraenoic acid by MOLT-4 cells and blood T-lymphocytes

MOLT-4 lymphocytes metabolize 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) via beta-oxidation with retention of the hydroxyl group at the omega 6-carbon atom. 15-HETE oxidation is accompanied by the time-dependent accumulation of both beta-hydroxy acids and metabolites produced by repetitive cycles of the beta-oxidation spiral. Detection of 7-hydroxy-5-dodecenoic acid shows that these cells continue to beta-oxidize the substrate when the conjugated diene is allylic to a hydroxyl group. When 15-HETE was the substrate, it was also possible to detect 12-hydroxy-5,8,10-heptadecatrien-1-al and 3,15-dihydroxy-8,11,13-eicosatrienoic acid. The former product may be produced by alpha-oxidation of 13-hydroxy-6,9,11-octadecatrienoic acid followed by its decarboxylation. Detection of a 20-carbon metabolite, lacking a double bond at position 5, suggests that an intermediate of beta-oxidation was used as a substrate for chain elongation. When 13-hydroxy-6,9,11-octadecatrienoic acid was used as a substrate, it was indeed possible to detect 3,15-dihydroxy-8,11,13-eicosatrienoic acid as well as 15-hydroxy-8,11,13-eicosatrienoic acid. In addition, 13-hydroxy-6,9,11-octadecatrienoic acid was a precursor for the biosynthesis of both 14-hydroxy-7,10,12-nonadecatrien-1-al and 1,14-dihydroxy-7,10,12-nonadecatriene. These studies with MOLT-4 cells as well as with T-lymphocytes isolated from blood show that products of the 15-lipoxygenase pathway are metabolized with the accumulation of a variety of compounds. Since 15-HETE has been implicated as a modulator of T-cell function, these findings raise the possibility that the newly described metabolites may be involved in regulating lymphocyte function.     

Myristic acid utilization in Chinese hamster ovary cells and peroxisome-deficient mutants.

Chinese hamster ovary (CHO) cells convert [9,10-3H]myristic acid ([3H]14:0) to several lipid-soluble, radioactive metabolites that are released into the medium. The main products are lauric (12:0) and decanoic (10:0) acids. Some of the 12:0 formed also is retained in cell lipids. Similar metabolites are not synthesized from palmitic (16:0), oleic (18:1), or arachidonic (20:4) acids, and the addition of these fatty acids does not reduce the conversion of [3H]14:0 to 12:0. Two peroxisome-deficient CHO cell lines do not convert [3H] 14:0 to any polar metabolites, but, they elongate, desaturate, and incorporate [3H]14:0 into intracellular lipids and proteins normally. While BC3H1 muscle cells convert some [3H]14:0 to 12:0, they also produce at least nine lipid-soluble polar products from [3H]12:0. These findings suggest that a previously unrecognized function of myristic acid is to serve as a substrate for the synthesis of 12:0, which can be either secreted into the medium or converted to other oxidized metabolites. The absence of this peroxisomal oxidation pathway, however, does not interfere with other aspects of myristic acid metabolism, including protein myristoylation.     

Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae

We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA beta-oxidation activity was markedly reduced in intact ypr128cDelta mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA beta-oxidation is impaired in ypr128cDelta cells. Since MCFA beta-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cDelta mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.     

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids.     

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.     

Identification and characterization of Arabidopsis indole-3-butyric acid response mutants defective in novel peroxisomal enzymes

Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the active auxin indole-3-acetic acid (IAA) by removal of two side-chain methylene units in a process similar to fatty acid beta-oxidation. Previous studies implicate peroxisomes as the site of IBA metabolism, although the enzymes that act in this process are still being identified. Here, we describe two IBA-response mutants, ibr1 and ibr10. Like the previously described ibr3 mutant, which disrupts a putative peroxisomal acyl-CoA oxidase/dehydrogenase, ibr1 and ibr10 display normal IAA responses and defective IBA responses. These defects include reduced root elongation inhibition, decreased lateral root initiation, and reduced IBA-responsive gene expression. However, peroxisomal energy-generating pathways necessary during early seedling development are unaffected in the mutants. Positional cloning of the genes responsible for the mutant defects reveals that IBR1 encodes a member of the short-chain dehydrogenase/reductase family and that IBR10 resembles enoyl-CoA hydratases/isomerases. Both enzymes contain C-terminal peroxisomal-targeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA beta-oxidation to IAA.     

Physiological role of peroxisomal beta-oxidation in liver of fasted rats

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.     

酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。     

Effect of clofibrate on peroxisomal and mitochondrial beta-oxidation in chicken liver

The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial beta-oxidation in chicken liver was studied. The activities of antimycin antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal beta-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O2 consumption (mitochondrial beta-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal beta-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.     

Metabolism of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) in corneal tissues: formation of novel metabolites

12(R)-Hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a cytochrome P450 arachidonate metabolite, is metabolized by corneal tissues via three distinct metabolic pathways: beta-oxidation, omega-hydroxylation, and keto-reduction. The major metabolite released from the intact rabbit corneal epithelium or cultured cells was identified by mass spectrometric analysis as 8-hydroxy-4,6,10-hexadecatrienoic acid, the tetranor metabolite derived following two steps of beta-oxidation from the carboxy terminus. The beta-oxidation pathway was expressed in both microsomes and mitochondria isolated from bovine corneal epithelium and was dependent on the addition of oxidizing equivalents. The major metabolite of 12(R)-HETE in subcellular fractions of bovine corneal epithelial cells was a dihydro compound, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). This derivative is presumably formed by an oxidation of the hydroxyl group followed by two keto-reduction steps, since its formation was accompanied by the appearance of a keto metabolite identified as 12-oxo-5,8,14-eicosatrienoic acid. The omega-hydroxylation, in contrast to other cell types, was a minor route for 12(R)-HETE metabolism in these tissues. Since 12(R)-HETE has been implicated as a modulator of Na(+)-K(+)-ATPase activity and its related functions in ocular tissues, these findings raise the possibility that the newly described metabolites may be involved in regulating corneal functions. In addition, the presence of a keto reductase in the cornea may be of great importance following injury since 12(R)-HETrE resulting from 12(R)-HETE by this activity is a potent ocular proinflammatory compound.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells

The identification and polarity of release of the major metabolite of 12-HETE produced by cultured canine renal tubular epithelial cells was determined. When incubated with 1.0 microM [3H]12-HETE for 1 h, cultured Madin Darby Canine Kidney (MDCK) cells converted 35% of the radiolabeled 12-HETE to a more polar metabolite. Following high performance liquid chromatography isolation and chemical derivatization, gas-liquid chromatography combined with mass spectrometry was used to identify the compound as 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. The electron impact mass spectrum of the hydrogenated derivative contained major ions at m/z = 215 and 245, corresponding to cleavage on either side of the trimethylsilyl group, and chemical ionization with NH3 yielded a major ion at m/z = 359, corresponding to the protonated molecular weight of the methyl ester. Incubation with 25 mM alpha-naphthoflavone, 20 microM nordihydroguaiaretic acid, and 0.1 mM 4-pentenoic acid failed to inhibit the formation 16:3 (8-OH), suggesting that the formation of 16:3 (8-OH) is not mediated by the cytochrome P-450, lipoxygenase, or mitochondrial beta-oxidation pathways. When grown on fibronectin-treated polycarbonate filters, MDCK cells released the 16:3 (8-OH) in both the apical and basolateral directions, irrespective of which side the 12-HETE was encountered. These results demonstrate the conversion of 12-HETE to a 16-carbon monohydroxy derivative by renal tubular epithelium and suggest that this product can be released to either the potential urinary space or the kidney parenchyma and renal microcirculation.