Identification of the peroxisomal β-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid
2001; Elsevier BV; Volume: 42; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)31527-3
ISSN1539-7262
AutoresSacha Ferdinandusse, Simone Denis, Petra A.W. Mooijer, Zhongyi Zhang, Janardan K. Reddy, Arthur A. Spector, Ronald J. A. Wanders,
Tópico(s)Fatty Acid Research and Health
ResumoDHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by β-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the β-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in β-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal β-oxidation.—Ferdinandusse, S., S. Denis, P. A. W. Mooijer, Z. Zhang, J. K. Reddy, A. A. Spector, and R. J. A. Wanders. Identification of the peroxisomal β-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid. J. Lipid Res. 2001. 42: 1987–1995. DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by β-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the β-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in β-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal β-oxidation. —Ferdinandusse, S., S. Denis, P. A. W. Mooijer, Z. Zhang, J. K. Reddy, A. A. Spector, and R. J. A. Wanders. Identification of the peroxisomal β-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid. J. Lipid Res. 2001. 42: 1987–1995. For years, it was generally assumed that the biosynthesis of PUFAs takes place in the endoplasmic reticulum, which is also the main site for phospholipid biosynthesis (1Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Google Scholar). DHA (C22:6n-3), the major PUFA in adult mammalian brain and retina, was believed to be synthesized from dietary linolenic acid (C18:3n-3) in a pathway consisting of a series of elongation and desaturation reactions. This pathway required that n-3 docosapentaenoic acid (C22:5n-3) become desaturated at position 4 by a microsomal acyl-CoA-dependent Δ4-desaturase to form C22:6n-3. Several studies, however, have indicated that such a Δ4-desaturase does not appear to exist (2Voss A. Reinhart M. Sankarappa S. Sprecher H. 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.J. Biol. Chem. 1991; 266: 19995-20000Google Scholar, 3Wang N. Anderson R.E. Synthesis of docosahexaenoic acid by retina and retinal pigment epithelium.Biochemistry. 1993; 32: 13703-13709Google Scholar, 4Mohammed B.S. Sankarappa S. Geiger M. Sprecher H. 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.Arch. Biochem. Biophys. 1995; 317: 179-184Google Scholar, 5Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.J. Lipid Res. 1995; 36: 2433-2443Google Scholar). Instead, it was found that a 24-carbon n-3 fatty acid is synthesized, which is desaturated at position 6 to produce tetracosahexaenoic acid (C24:6n-3), followed by one round of β-oxidation with C22:6n-3 as final product. Although still disputed, the peroxisome is the likely site of C24:6n-3 β-oxidation. After its formation, DHA is transported back to the endoplasmic reticulum, where it is esterified into membrane lipids (6Baykousheva S.P. Luthria D.L. Sprecher H. Peroxisomal-microsomal communication in unsaturated fatty acid metabolism.FEBS Lett. 1995; 367: 198-200Google Scholar, 7Luthria D.L. Mohammed B.S. Sprecher H. Regulation of the biosynthesis of 4,7,10,13,16,19-docosahexaenoic acid.J. Biol. Chem. 1996; 271: 16020-16025Google Scholar). Figure 1 shows the revised pathway for the biosynthesis of DHA. The synthesis of arachidonic acid (C20:4n-6) and n-6 docosapentaenoic acid (C22:5n-6) from dietary linoleic acid (C18:2n-6) follows a similar pathway (1Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Google Scholar). The β-oxidation step in the revised pathway of PUFA biosynthesis requires a considerable exchange of unsaturated fatty acids between different subcellular compartments (6Baykousheva S.P. Luthria D.L. Sprecher H. Peroxisomal-microsomal communication in unsaturated fatty acid metabolism.FEBS Lett. 1995; 367: 198-200Google Scholar). Several lines of evidence suggest that peroxisomes are the intracellular site of this β-oxidation step. First, patients with Zellweger syndrome (a peroxisome biogenesis disorder), who lack functional peroxisomes, have clearly reduced levels of DHA, especially in brain and retina but also in liver, kidney (8Martinez M. Abnormal profiles of polyunsaturated fatty acids in the brain, liver, kidney and retina of patients with peroxisomal disorders.Brain Res. 1992; 583: 171-182Google Scholar), and blood (9Martinez M. Mougan I. Roig M. Ballabriga A. Blood polyunsaturated fatty acids in patients with peroxisomal disorders. A multicenter study.Lipids. 1994; 29: 273-280Google Scholar). Inaddition, in newborn PEX5 knockout mice, a mouse model for Zellweger syndrome, the DHA concentration in the brain is also strongly reduced (40% as compared with levels in normal littermates) (10Janssen A. Baes M. Gressens P. Mannaerts G.P. Declercq P. Van Veldhoven P.P. Docosahexaenoic acid deficit is not a major pathogenic factor in peroxisome-deficient mice.Lab. Invest. 2000; 80: 31-35Scopus (47) Google Scholar). In an extensive study of n-3 fatty acid metabolism, Moore et al. (5Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.J. Lipid Res. 1995; 36: 2433-2443Google Scholar) reported that control human fibroblasts metabolized [1-14C]18:3n-3 to labeled tetracosapentaenoic acid (C24:5n-3), C24:6n-3, and C22:6n-3. In contrast, fibroblasts from patients with Zellweger syndrome metabolized [1-14C]18:3n-3 to C24:5n-3 and C24:6n-3, but not to C22:6n-3. Likewise, [3-14C]22:5n-3, [3-14C]24:5n-3, and [3-14C]24:6n-3 were all metabolized to C22:6n-3 in control fibroblasts, but not in Zellweger fibroblasts. Similar results were obtained by Petroni et al. (11Petroni A. Bertagnolio B. La Spada P. Blasevich M. Papini N. Govoni S. Rimoldi M. Galli C. The beta-oxidation of arachidonic acid and the synthesis of docosahexaenoic acid are selectively and consistently altered in skin fibroblasts from three Zellweger patients versus X-adrenoleukodystrophy, Alzheimer and control subjects.Neurosci. Lett. 1998; 250: 145-148Google Scholar), who incubated control and Zellweger fibroblasts with [14C]eicosapentaenoic acid ([1-14C]20:5n-3). In a more recent article, it was demonstrated that peroxisomes are required for biosynthesis of DHA from linolenic acid in livers from neonatal piglets (12Li Z. Kaplan M.L. Hachey D.L. Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development.Lipids. 2000; 35: 1325-1333Google Scholar). This was concluded from the observation that isotope-labeled DHA, and all the intermediates of the pathway, were formed only when combined microsomal and peroxisomal fractions were incubated with [U-13C]18:3n-3. In spite of the many experiments that show that peroxisomes are involved in the biosynthesis of PUFAs, Infante and Huszagh (13Infante J.P. Huszagh V.A. On the molecular etiology of decreased arachidonic (20:4n-6), docosapentaenoic (22:5n-6) and docosahexaenoic (22:6n-3) acids in Zellweger syndrome and other peroxisomal disorders.Mol. Cell. Biochem. 1997; 168: 101-115Google Scholar, 14Infante J.P. Huszagh V.A. Analysis of the putative role of 24-carbon polyunsaturated fatty acids in the biosynthesis of docosapentaenoic (22:5n-6) and docosahexaenoic (22:6n-3) acids.FEBS Lett. 1998; 431: 1-6Google Scholar) propose that synthesis of these fatty acids occurs in the outer mitochondrial membrane via a channeled carnitine-dependent pathway. Although there is not much direct experimental evidence to support the existence of such a mitochondrial pathway, a role for the mitochondrion in the biosynthesis of DHA cannot be ruled out with absolute certainty. We therefore set out to study the role of peroxisomes and mitochondria, and their fatty acid oxidation systems, in DHA synthesis in more detail. Figure 2 shows a schematic representation of the peroxisomal β-oxidation system. There are two complete sets of β-oxidation enzymes present in the peroxisome (15Wanders R.J. Vreken P. Ferdinandusse S. Jansen G.A. Waterham H.R. Van Roermund C.W. Van Grunsven E.G. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases.Biochem. Soc. Trans. 2001; 29: 250-267Google Scholar). Straight-chain acyl-CoA oxidase (SCOX) is responsible for the initial oxidation of very long-chain fatty acyl-CoAs, whereas branched-chain acyl-CoA oxidase (BCOX) oxidizes branched-chain fatty acyl-CoA. The enoyl-CoA esters of both straight- and branched-chain fatty acids are then hydrated and subsequently dehydrogenated by the same enzyme: d-bifunctional protein (DBP). The function of the second multifunctional protein present in the peroxisome, l-bifunctional protein (LBP), is still unknown. The last step of the β-oxidation process, the thiolytic cleavage, is performed by sterol carrier protein X (SCPx) in the case of the branched-chain substrates, whereas straight-chain substrates most likely are handled by both SCPx and the classic 3-ketoacyl-CoA thiolase. Until now, only patients with an isolated defect of SCOX and DBP have been identified. In addition, patients with rhizomelic chondrodysplasia punctata (RCDP) type 1 lack 3-ketoacyl-CoA thiolase in their peroxisomes. This is, however, not the only deficiency in these patients. Because of a defect in PEX7, the gene encoding the peroxisome targeting signal 2 (PTS2) receptor, their peroxisomes lack all proteins imported via this receptor, including alkyldihydroxyacetonephosphate synthase, an enzyme of the plasmalogen biosynthetic pathway, and phytanoyl-CoA hydroxylase, the first enzyme of the peroxisomal α-oxidation pathway (16Purdue P.E. Zhang J.W. Skoneczny M. Lazarow P.B. Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor.Nat. Genet. 1997; 15: 381-384Google Scholar, 17Motley A.M. Hettema E.H. Hogenhout E.M. Brites P. ten Asbroek A.L. Wijburg F.A. Baas F. Heijmans H.S. Tabak H.F. Wanders R.J. Distel B. Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a nonfunctional PTS2 receptor.Nat. Genet. 1997; 15: 377-380Google Scholar, 18Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata.Nat. Genet. 1997; 15: 369-376Google Scholar). No patients have been identified with a deficiency of BCOX, LBP, and SCPx, but knockout mice have been created for the latter two enzymes (19Qi C. Zhu Y. Pan J. Usuda N. Maeda N. Yeldandi A.V. Rao M.S. Hashimoto T. Reddy J.K. Absence of spontaneous peroxisome proliferation in enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase-deficient mouse liver. Further support for the role of fatty acyl CoA oxidase in PPARalpha ligand metabolism.J. Biol. Chem. 1999; 274: 15775-15780Google Scholar, 20Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W. Wanders R.J. Maeda N. Assmann G. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-X gene function.Genes Dev. 1998; 12: 1189-1201Google Scholar). To elucidate the role of both the peroxisome and mitochondrion,we studied the biosynthesis of DHA from [1-14C]-18:3n-3, [1-14C]20:5n-3, and [3-14C]24:6n-3 in fibroblasts from patients with a peroxisome biogenesis disorder and from patients with a deficiency of one of the following mitochondrial enzymes: carnitine palmitoyltransferase 1 (CPT1), carnitine acylcarnitine translocase (CACT), carnitine palmitoyltransferase 2 (CPT2), and very long-chain acyl-CoA dehydrogenase (VLCAD). The first three enzymes are necessary for the transport of activated fatty acids across the inner mitochondrial membrane (21McGarry J.D. Brown N.F. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.Eur. J. Biochem. 1997; 244: 1-14Google Scholar) and the last enzyme is part of the mitochondrial β-oxidation system (22Wanders R.J. Vreken P. den Boer M.E. Wijburg F.A. van Gennip A.H. IJlst L. Disorders of mitochondrial fatty acyl-CoA beta-oxidation.J. Inherit. Metab. Dis. 1999; 22: 442-487Google Scholar). In addition, we investigated the role of the various peroxisomal β-oxidation enzymes in DHA biosynthesis by incubating fibroblasts from patients with a deficiency of SCOX and DBP, patients with RCDP type 1, and from LBP and SCPx knockout mice with 14C-labeled precursors. We also studied DHA synthesis in fibroblasts from a patient with X-linked adrenoleukodystrophy (X-ALD). These patients accumulate very long-chain fatty acids because of impaired peroxisomal β-oxidation of these fatty acids. However, this is not caused by a deficiency of one of the enzymes of the β-oxidation system, but by a defect of the peroxisomal membrane protein ALDP (adrenoleukodystrophy protein) (23Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.Nature. 1993; 361: 726-730Google Scholar, 24Mosser J. Lutz Y. Stoeckel M.E. Sarde C.O. Kretz C. Douar A.M. Lopez J. Aubourg P. Mandel J.L. The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein.Hum. Mol. Genet. 1994; 3: 265-271Scopus (229) Google Scholar). Radiolabeled [1-14C]18:3n-3, [1-14C]20:5n-3, and [1-14C]22:6n-3 were purchased from New England Nuclear (DuPont, Boston, MA). [3-14C]24:6n-3 was synthesized as described previously (2Voss A. Reinhart M. Sankarappa S. Sprecher H. 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.J. Biol. Chem. 1991; 266: 19995-20000Google Scholar). Each radiolabeled fatty acid had a specific activity between 50 and 55 mCi/mmol. Patient cell lines. Cell lines were used from several patients with various peroxisomal and mitochondrial fatty acid β-oxidation disorders. The fibroblasts from patients with a peroxisome biogenesis disorder studied in this article were from four patients with Zellweger syndrome and one patient with neonatal adrenoleukodystrophy (NALD), which is a less severe form of a peroxisome biogenesis defect. These patients all had the clinical and biochemical abnormalities described for patients with a peroxisome biogenesis disorder, including deficient hexacosanoic acid (C26:0) and pristanic acid β-oxidation and phytanic acid β-oxidation (25Wanders R.J. Schutgens R.B. Barth P.G. Peroxisomal disorders: a review.J. Neuropathol. Exp. Neurol. 1995; 54: 726-739Google Scholar). The fibroblasts from the X-ALD patient had impaired C26:0 β-oxidation, which is caused by a mutation in the gene encoding the peroxisomal membrane protein ALDP (23Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.Nature. 1993; 361: 726-730Google Scholar, 24Mosser J. Lutz Y. Stoeckel M.E. Sarde C.O. Kretz C. Douar A.M. Lopez J. Aubourg P. Mandel J.L. The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein.Hum. Mol. Genet. 1994; 3: 265-271Scopus (229) Google Scholar). The SCOX- and DBP-deficient patients all had mutations in the encoding gene and no enzyme activity could be measured in fibroblasts of these patients (26Fournier B. Saudubray J.M. Benichou B. Lyonnet S. Munnich A. Clevers H. Poll-The B.T. Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy.J. Clin. Invest. 1994; 94: 526-531Google Scholar, 27Van Grunsven E.G. vanw Berkel E. Lemonde H. Clayton P.T. Wanders R.J. Bifunctional protein deficiency: complementation within the same group suggesting differential enzyme defects and clues to the underlying basis.J. Inherit. Metab. Dis. 1998; 21: 298-301Google Scholar, 28Van Grunsven E.G. van Berkel E. IJlst L. Vreken P. de Klerk J.B. Adamski J. Lemonde H. Clayton P.T. Cuebas D.A. Wanders R.J. Peroxisomal d-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency.Proc. Natl. Acad. Sci. USA. 1998; 95: 2128-2133Google Scholar). Peroxisomes from the patients with RCDP type 1 under study lack 3-ketoacyl-CoA thiolase because of a mutation in the PEX7 gene encoding the PTS2 receptor. Immunoblot studies performed with an antibody raised against 3-ketoacyl-CoA thiolase revealed that only the unprocessed protein of 44 kDa is present in fibroblast homogenates. It is known that 3-ketoacyl-CoA thiolase is synthesized as a precursor protein and is proteolytically cleaved to its mature form of 41 kDa inside the peroxisome (29Purdue P.E. Skoneczny M. Yang X. Zhang J.W. Lazarow P.B. Rhizomelic chondrodysplasia punctata, a peroxisomal biogenesis disorder caused by defects in Pex7p, a peroxisomal protein import receptor: a minireview.Neurochem. Res. 1999; 24: 581-586Google Scholar). Cultured skin fibroblasts from an SCPx knockout mouse were obtained from U. Seedorf (Westphalian Wilhelms-University, Münster, Germany) (20Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W. Wanders R.J. Maeda N. Assmann G. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-X gene function.Genes Dev. 1998; 12: 1189-1201Google Scholar) and fibroblasts of an LBP knockout mouse were generated by Qi et al. (19Qi C. Zhu Y. Pan J. Usuda N. Maeda N. Yeldandi A.V. Rao M.S. Hashimoto T. Reddy J.K. Absence of spontaneous peroxisome proliferation in enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase-deficient mouse liver. Further support for the role of fatty acyl CoA oxidase in PPARalpha ligand metabolism.J. Biol. Chem. 1999; 274: 15775-15780Google Scholar). Both knockout mice have been fully characterized and completely lack SCPx and LBP gene function, respectively. The fibroblasts used in this study were from patients with a mitochondrial β-oxidation disorder, that is, a confirmed deficiency of CPT1, CACT, CPT2, or VLCAD due to mutations in the encoding genes [see ref. (22Wanders R.J. Vreken P. den Boer M.E. Wijburg F.A. van Gennip A.H. IJlst L. Disorders of mitochondrial fatty acyl-CoA beta-oxidation.J. Inherit. Metab. Dis. 1999; 22: 442-487Google Scholar) for review]. These mutations result in a deficiency of mitochondrial fatty acid oxidation as established by individual enzyme activity measurements in cultured skin fibroblasts. All patient cell lines used in this study were taken from the cell repository of the Laboratory for Genetic Metabolic Diseases (Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands) and were derived from patients diagnosed in this center. Informed consent was obtained from parents or guardians of the patients whose fibroblasts were studied in this article and the studies were approved by the Institutional Review Board of the Academic Medical Center, University of Amsterdam. Experimental protocol. DHA synthesis from [1-14C]18:3n-3, [1-14C]-20:5n-3, and [3-14C]24:6n-3 was studied in cultures of fibroblasts grown in tissue culture flasks (25 cm2). Incubations were carriedout in MEM supplemented with penicillin-streptomycin, and containing 10% fetal calf serum (fatty acid free), 20 mM HEPES, and 14C-labeled fatty acid. In the case of [1-14C]18:3n-3 and [1-14C]20:5n-3 the incubation was carried out with 2 μM labeled fatty acid, whereas [3-14C]24:6n-3 was used at a concentration of 0.2 μM. The fibroblasts were kept in an incubator at 37°C for 96 h except for the incubations with [3-14C]24:6n-3, which were terminated after 24 h. Parallel incubations were performed to determine the amount of protein. Lipid analyses. Lipids were extracted from the incubation medium as described by Moore et al. (5Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.J. Lipid Res. 1995; 36: 2433-2443Google Scholar). Briefly, the lipids were extracted with a 2:1 (v/v) mixture of chloroform-methanol containing 1% acetic acid (v/v). The chloroform phase was dried under N2, and the residue was resuspended in 2 ml of 1.5 N HCl-methanol and heated to 90°C for 2 h to produce fatty acid methyl esters. After extraction of the methyl esters in heptane, the heptane phase was dried under N2 and the residue was resuspended in 150 μl of 70% acetonitrile, which was stored at −20°C until analysis. Seventy microliters of the sample were subjected to HPLC analysis as described below. To isolate cellular lipids, the incubation medium was removed and the fibroblasts were scraped into 1 ml of methanol and transferred to a screw-top glass vial. The tissue culture flasks were washed with 1 ml of 3 N HCl-methanol, which also was transferred to the glass vial. Finally, fatty acid methyl esters were produced and extracted as described above. HPLC analysis. Radioactive methyl esters prepared from the cell lipids or incubation medium were separated by reversed-phase HPLC. A reversed-phase C18 column (4.6 × 150 nm; Beckman, Fullerton, CA) with 5-μm spherical packing was used with a mobile phase of water and acetonitrile in a two-step isocratic elution (76% acetonitrile for 50 min, 90% acetonitrile for 10 min), followed by an equilibration period of 10 min at 76% acetonitrile. The effluent was mixed with scintillation solution at a 1:1 ratio, and the radioactivity was measured by passing the mixture through an online Radiomatic Instruments (Packard, Meriden, CT) Flo One-β radioactivity detector. The system was standardized with methyl esters of the following radiolabeled fatty acids: C18:3n-3, C20:5n-3, C24:6n-3, and C22:6n-3. Linolenic acid (C18:3n-3) utilization. After a 96-h incubation of control human skin fibroblasts with [1-14C]18:3n-3, substantial amounts of the radiolabeled fatty acids contained in the cells consisted of C22:6n-3 (mean value in five different control fibroblast cell lines was 15.5%) (see Table 1). In addition, radioactivity was detected in almost all intermediates of the proposed pathway of DHA biosynthesis (Fig. 1), including radiolabeled C24:5n-3 and C24:6n-3 (Fig. 3). Similar results were obtained with fibroblasts from patients with a deficiency of mitochondrial fatty acid oxidation. Fibroblasts from patients with a deficiency in one of the steps involved in the mitochondrial carnitine shuttle (CPT1, CACT, or CPT2) as well as from a patient with a defect of the first enzyme of the mitochondrial fatty acid oxidation system, VLCAD, revealed normal synthesis of DHA from radiolabeled linolenic acid compared with the synthesis observed in control fibroblasts (Table 1). In contrast, no radiolabeled C22:6n-3 was formed in fibroblasts from patientswith Zellweger syndrome, although the [1-14C]18:3n-3 was converted to other intermediates in the biosynthetic pathway. In addition, increased amounts of radiolabeled C24:6n-3, the precursor of DHA, were found. Fibroblasts from a patient with NALD, a milder variant of Zellweger syndrome characterized by a less severe peroxisomal deficiency, synthesized some radiolabeled DHA but only 1% of the radioactivity was converted to C22:6n-3 after the incubation period (Table 1).TABLE 1.Radiolabeled fatty acids produced by human and mouse skin fibroblasts after a 96-h incubation with [1-14C]18:3n-3Fibroblast TypeAmount of Radiolabeled Fatty Acid DetectedC18:3n-3C20:4n-3C20:5n-3C22:5n-3C24:5n-3C24:6n-3C22:6n-3cpm/mg proteinControls (n = 5)3,470 ± 407aMean value ± standard deviation.1,269 ± 55441,264 ± 10,15939,346 ± 13,977803 ± 6041,057 ± 76216,265 ± 6,117Zellweger (n = 4)7,570 ± 2,055aMean value ± standard deviation.3,500 ± 77533,305 ± 5,61278,883 ± 20,3052,696 ± 3618,081 ± 2,1850 ± 0NALD3,0921,72713,25560,4047554,834917CPT18,3933,78674,21479,2152,5572,06815,488CACT (n = 3)8,696 ± 2,999aMean value ± standard deviation.4,215 ± 1,85260,030 ± 9,77699,987 ± 28,9473,814 ± 8182,624 ± 80018,419 ± 4,478CPT27,1992,84555,79884,1133,5812,04918,919VLCAD6,5993,31954,99860,4492,7812,02414,501X-ALD10,5602,21161,67858,8282,4821,63513,498SCOX (n = 3)7,861 ± 1,706aMean value ± standard deviation.4,625 ± 2,10454,654 ± 15,35094,991 ± 42,3003,783 ± 1,1246,310 ± 2,5051,791 ± 55DBP (n = 3)4,137 ± 2,590aMean value ± standard deviation.2,203 ± 1,19942,769 ± 21,36854,065 ± 21,8381,892 ± 8143,090 ± 1,0283,078 ± 1,518RCDP type 1 (n = 3)3,262 ± 1,093aMean value ± standard deviation.2,698 ± 1,01336,447 ± 10,01766,120 ± 23,7552,626 ± 2,6702,688 ± 13718,015 ± 2,724Control mouse1,67177356,85870,2288822,02410,050LBP−/−mouse2,6690122,89078,4761,1873,06518,273SCPX−/−mouse2,0051,91288,11548,9121,4391,67112,588The methyl esters of the radiolabeled fatty acids contained in the cells were separated by HPLC. n = number of different cell lines; all incubations were performed in triplicate.a Mean value ± standard deviation. Open table in a new tab The methyl esters of the radiolabeled fatty acids contained in the cells were separated by HPLC. n = number of different cell lines; all incubations were performed in triplicate. Analysis of the radiolabeled fatty acids excreted in themedium revealed a pattern similar to that of the fatty acids contained in the cells. This was true for both control skin fibroblasts as well as fibroblasts from patients with a mitochondrial or peroxisomal defect (data not shown). This is in agreement with the findings by Moore et al. (5Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.J. Lipid Res. 1995; 36: 2433-2443Google Scholar), who concluded that these mitochondrial and peroxisomal defects do not cause selective retention or release of certain radiolabeled fatty acids. Therefore, all results shown are obtained by analysis of the cells only. Eicosapentaenoic acid (C20:5n-3) utilization. Similar results were obtained after incubation of fibroblasts with [1-14C]-20:5n-3 (Table 2). After an incubation of 96 h, control fibroblasts produced radiolabeled C22:5n-3 and C22:6n-3, as well as small amounts of C24:5n-3 and C24:6n-3. Fibroblasts from patients with a deficiency of either CPT1, CACT, CPT2, or VLCAD revealed normal synthesis of DHA from [1-14C]20:5n-3. Fibroblasts from patients with Zellweger syndrome, however, produced no radiolabeled DHA. In contrast, they accumulated large amounts of C24:6n-3 (about 10 times more than observed in control fibroblasts). Incubations of fibroblasts from a patient with NALD resulted in intermediate values. These fibroblasts produced 10% of the amount of radiolabeled DHA formed in control fibroblasts and accumulated about six times the normal amount of C24:6n-3.TABLE 2.Radiolabeled fatty acids produced by human and mouse skin fibroblasts after a 96-h incubation with [1-14C]20:5n-3Fibroblast TypeAmount of Radiolabeled Fatty Acid DetectedC20:5n-3C22:5n-3C24:5n-3C24:6n-3C22:6n-3cpm/mg proteinControls (n = 5)52,198 ± 8,570aMean value ± standard deviation.57,325 ± 10,241113 ± 1791,782 ± 48925,740 ± 5,801Zellweger (n = 4)35,375 ± 5,460aMean value ± standard deviation.173,244 ± 47,838454 ± 11418,217 ± 6,3190 ± 0NALD12,434127,83425410,4242,709CPT175,871104,389622,39525,703CAC
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