Peroxisomal Straight-chain Acyl-CoA Oxidase and D-bifunctional Protein Are Essential for the Retroconversion Step in Docosahexaenoic Acid Synthesis

Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Δ4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and “retroconverted” to DHA via peroxisomal β-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid β-oxidation pathway, exhibited impaired (5–20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2–5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal β-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts. Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Δ4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and “retroconverted” to DHA via peroxisomal β-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid β-oxidation pathway, exhibited impaired (5–20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2–5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal β-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts. docosahexaenoic acid (C22:6n-3) peroxisome biogenesis disorder peroxisomal straight-chain acyl-CoA oxidase peroxisomal D-bifunctional protein X-linked adrenoleukodystrophy sterol carrier protein X rhizomelic chondrodysplasia punctata very long-chain fatty acid medium- and very long-chain acyl-CoA dehydrogenase, respectively Docosahexaenoic acid (C22:6n-3, DHA1) plays an important role in normal neurological development, especially in brain and retina (1Innis S.M. Prog. Lipid Res. 1991; 30: 39-103Crossref PubMed Scopus (796) Google Scholar,2Uauy R. J. Pediatr. Gastroenterol. Nutr. 1990; 11: 296-302Crossref PubMed Scopus (32) Google Scholar), where it occurs in high concentration (3Fliesler S.J. Anderson R.E. Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (847) Google Scholar, 4Sastry P.S. Prog. Lipid Res. 1985; 24: 69-176Crossref PubMed Scopus (614) Google Scholar). A deficiency of brain DHA is associated with reduced learning ability in rats (5Lamptey M.S. Walker B.L. J. Nutr. 1976; 106: 86-93Crossref PubMed Google Scholar, 6Greiner R.S. Moriguchi T. Hutton A. Slotnick B.M. Salem Jr., N. Lipids. 1999; 34: S239-S243Crossref PubMed Google Scholar), impaired visual acuity in infant monkeys (7Neuringer M. Connor W.E. Lin D.S. Barstad L. Luck S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4021-4025Crossref PubMed Scopus (776) Google Scholar), and abnormal brain development in human infants with the Zellweger syndrome, a peroxisome biogenesis disorder (PBD) (8Moser H.W. Adv. Hum. Genet. 1993; 21: 1-106PubMed Google Scholar, 9Martinez M. J. Inherit. Metab. Dis. 1995; 18: 61-75Crossref PubMed Scopus (52) Google Scholar). DHA is derived from its parent precursor, the dietary essential fatty acid linolenic acid (C18:3n-3), via a series of alternating desaturation and elongation steps (see Fig. 1) (10Brenner R.R. Lipids. 1971; 6: 567-575Crossref PubMed Scopus (200) Google Scholar, 11Sprecher H. Bracco U. Deckelbaum R.J. Polyunsaturated Fatty Acids in Human Nutrition. Nestec Ltd., Vevey/Raven Press, Ltd., New York1992: 13-24Google Scholar). The primary products of C18:3n-3 are eicosapentaenoic acid (C20:5n-3), docosapentaenoic acid (C22:5n-3), and DHA. Until 1991, it had been postulated that conversion of C18:3n-3 to DHA was carried out entirely in microsomes. However, the last step in the biosynthetic pathway required conversion of C22:5n-3 to DHA via Δ4-desaturase, an enzyme that may not exist in higher animals (12Voss A. Reinhart M. Sankarappa S. Sprecher H. J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar). A modified pathway was therefore proposed in which C22:5n-3 is elongated to tetracosapentaenoic acid (C24:5n-3), desaturated to tetracosahexaenoic acid (C24:6n-3) in microsomes, and then retroconverted to DHA in peroxisomes (see Fig. 1) (12Voss A. Reinhart M. Sankarappa S. Sprecher H. J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar, 13Sprecher H. Luthria D.L. Mohammed B.S. Baykousheva S.P. J. Lipid Res. 1995; 36: 2471-2477Abstract Full Text PDF PubMed Google Scholar). Evidence for this new revised pathway was obtained when the intermediates C24:5n-3 and C24:6n-3 were detected in cultured cell studies (14Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. J. Lipid Res. 1995; 36: 2433-2443Abstract Full Text PDF PubMed Google Scholar, 15Delton-Vandenbrouke I. Grammas P. Anderson R.E. J. Lipid Res. 1997; 38: 147-159Abstract Full Text PDF PubMed Google Scholar) and tissue homogenates (16Yin F.Q. Chen Q. Sprecher H. Biochim. Biophys. Acta. 1999; 1438: 63-72Crossref PubMed Scopus (12) Google Scholar, 17Retterstol K. Haugen T.B. Christophersen B.O. Biochim. Biophys. Acta. 2000; 1483: 119-131Crossref PubMed Scopus (33) Google Scholar). That the retroconversion step for DHA synthesis, a chain shortening of C24:6n-3 to DHA, takes place only in peroxisomes has been called into question, and a mitochondrial contribution has also been suggested (18Infante J.P. Huszagh V.A. Mol. Genet. Metab. 2001; 72: 1-7Crossref PubMed Scopus (22) Google Scholar, 19Infante J.P. Huszagh V.A. Mol. Genet. Metab. 2001; 72: 185-198Crossref PubMed Scopus (49) Google Scholar, 20Infante J.P. Huszagh V.A. FEBS Lett. 2000; 468: 1-5Crossref PubMed Scopus (80) Google Scholar, 21Infante J.P. Huszagh V.A. FEBS Lett. 1998; 431: 1-6Crossref PubMed Scopus (56) Google Scholar, 22Infante J.P. Huszagh V.A. Mol. Cell. Biochem. 1997; 168: 101-115Crossref PubMed Scopus (51) Google Scholar). The identities of specific enzymes or mechanisms involved in the retroconversion of C24:6n-3 to DHA is not known. Both mitochondria and peroxisomes carry out fatty acid β-oxidation. This cyclic process is similar in both organelles with each cycle containing dehydrogenation/oxidation, hydration, dehydrogenation, and thiolytic cleavage steps. However, the functions of mitochondrial and peroxisomal β-oxidation pathways are significantly different (23Wanders R.J. Tager J.M. Mol. Asp. Med. 1998; 19: 69-154Crossref PubMed Google Scholar,24Wanders R.J.A. Barth P.C. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3219-3256Google Scholar). In general, one cycle of β-oxidation shortens an acyl chain by two carbons, releasing one molecule of acetyl-CoA, in a process mediated by a sequence of enzymes, each of which is specific for its substrate. In mitochondrial β-oxidation, the preferred substrates are fatty acids with a chain length of less than 20 carbons. These fatty acids enter the organelle by the carnitine transport system and are usually degraded completely to acetyl-CoA via several β-oxidation cycles. Peroxisomal fatty acid β-oxidation is capable of oxidizing longer chain length substrates, the very long-chain fatty acids (VLCFA), e.g. hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0). Entry of these substrates does not require carnitine but may involve ATP-binding cassette transporters such as the ALD protein. Peroxisomal β-oxidation does not proceed to completion but rather only through a few cycles in which the acyl chain is shortened. In this study, the mechanism and enzymes involved in the retroconversion of C24:6n-3 to DHA, a two-carbon shortening process, were investigated. We compared the rate of radiolabeled DHA synthesis from [1-14C]18:3n-3, the parent precursor, and [1-14C]22:5n-3, a more direct precursor, in human skin fibroblasts from normal controls with DHA synthesis rates in cells from patients with disorders of peroxisomal or mitochondrial fatty acid β-oxidation. Our results confirm the existence of the new revised DHA synthetic pathway in which C22:5n-3 is elongated to C24:5n-3, desaturated to C24:6n-3 in microsomes, and then retroconverted to C22:6n-3 in peroxisomes. Furthermore, we demonstrate that the peroxisomal β-oxidation enzymes straight-chain acyl-CoA oxidase (AOx) and D-bifunctional protein (DBP) are essential for the retroconversion process. One or both of the known peroxisomal thiolases, 3-oxoacyl-CoA thiolase and sterol carrier protein X (SCPx) thiolase, may participate in retroconversion, but the former enzyme does not appear to be essential for this process. Thus, we conclude that the retroconversion step in DHA synthesis from C24:6n-3 proceeds via the peroxisomal straight-chain fatty acid β-oxidation pathway. We find no evidence for involvement of mitochondrial enzymes in this process. Radiolabeled [1-14C]18:3n-3 (52 mCi/mmol) and [1-14C]22:5n-3 (55 mCi/mmol) were purchased from PerkinElmer Life Sciences and American Radiolabeled Chemicals (St. Louis, MO), respectively. Standards of fatty acids and fatty acids methyl esters were purchased from Matreya (Pleasant Gap, PA). The free fatty acid of C24:5n-3 and C24:6n-3 were generous gifts from A. Spector and H. Sprecher, respectively. Human skin fibroblasts from normal controls and patients with peroxisomal or mitochondrial fatty acid oxidation disorders were obtained through the Kennedy Krieger Institute's Mental Retardation Research Center. The peroxisomal disorders included Zellweger syndrome patients with different genotypes (25Moser H.W. Mol. Genet. Metab. 1999; PubMed Scopus Google Scholar), deficiency D.S. R.J. J. Lipid Res. Full Text PDF PubMed Google Scholar), deficiency Chen S. Jr., A. H.W. J. 83: PubMed Scopus Google Scholar, E. H.W. T. J. R.J. J. Hum. Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), rhizomelic chondrodysplasia punctata M. Res. 1999; 24: PubMed Scopus Google Scholar), Refsum R.J.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar), and X-linked adrenoleukodystrophy H.W. J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). The disorders of mitochondrial fatty acid β-oxidation included deficiencies of very long-chain acyl-CoA and acyl-CoA Biochem. Biophys. Res. PubMed Scopus Google Scholar, C.R. J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). Cells were in essential with and The radiolabeled substrates [1-14C]18:3n-3 and [1-14C]22:5n-3 were by a of with a of Biochem. 1998; PubMed Scopus (33) Google Scholar). were in of modified containing and of [1-14C]18:3n-3 or of [1-14C]22:5n-3 in a in the were for this the was the cells were with and then cells were The cells were and the were with and the with were in of and a for of an for protein were from the cell C22:5n-3, and were and fatty were to methyl esters as D.S. H.W. Res. 1999; 24: PubMed Scopus (80) Google Scholar). Radiolabeled fatty acid methyl esters were by high by a of the of (14Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. J. Lipid Res. 1995; 36: 2433-2443Abstract Full Text PDF PubMed Google Scholar). were to a and with a of and The of for by a to and this concentration for an This methyl esters of C22:5n-3, C24:5n-3, C24:6n-3, and DHA. products were a The was a of and was by products were by times with fatty acid methyl The of the fatty acid methyl was by D.S. H.W. Res. 1999; 24: PubMed Scopus (80) Google Scholar). as and DHA methyl esters were as C22:5n-3, C24:5n-3 and C22:5n-3 methyl esters and fatty The C22:5n-3 acid the C24:5n-3 and and the C24:6n-3 These associated fatty acids not to the of the concentration was by the of A.L. R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) with as are as of the specific Until the terminal steps in the pathway for synthesis of DHA from dietary essential fatty acids required a no enzyme with this had been (12Voss A. Reinhart M. Sankarappa S. Sprecher H. J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar) proposed the retroconversion pathway in Fig. Furthermore, (14Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. J. Lipid Res. 1995; 36: 2433-2443Abstract Full Text PDF PubMed Google Scholar) that retroconversion in peroxisomes. and Huszagh (18Infante J.P. Huszagh V.A. Mol. Genet. Metab. 2001; 72: 1-7Crossref PubMed Scopus (22) Google Scholar, 22Infante J.P. Huszagh V.A. Mol. Cell. Biochem. 1997; 168: 101-115Crossref PubMed Scopus (51) Google Scholar) have recently the involvement of peroxisomes in DHA synthesis, these we the synthesis of DHA in cultured human skin fibroblasts two [1-14C]18:3n-3 and Cells from normal controls with these substrates for into DHA a rate of and of respectively. confirm the of (14Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. J. Lipid Res. 1995; 36: 2433-2443Abstract Full Text PDF PubMed Google Scholar) that DHA synthesis required we cells from patients with Zellweger syndrome, a in which peroxisomal functions are are by in one of known (25Moser H.W. Mol. Genet. Metab. 1999; PubMed Scopus Google Scholar, Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar), and (14Moore S.A. Hurt E. Yoder E. Sprecher H. Spector A.A. J. Lipid Res. 1995; 36: 2433-2443Abstract Full Text PDF PubMed Google Scholar) two patients with the The of patients from to to Refsum and is no S.J. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). In the in cells from Zellweger syndrome patients with in and were The rate of radiolabeled DHA synthesis in fibroblasts from Zellweger syndrome with either [1-14C]18:3n-3 or [1-14C]22:5n-3 as was less than of that in Thus, the is not to a These data therefore and the that peroxisomes are essential for DHA radiolabeled C22:5n-3, C24:5n-3, and C24:6n-3 derived from both substrates were detected in Zellweger syndrome cell not These intermediates peroxisomal retroconversion of C24:6n-3 to DHA in the revised pathway. these results that peroxisomes are essential for DHA In to in peroxisomal β-oxidation, patients with such as the Zellweger syndrome have a rate of biosynthesis H. D. R.J. 1993; PubMed Scopus Google Scholar). and Huszagh (18Infante J.P. Huszagh V.A. Mol. Genet. Metab. 2001; 72: 1-7Crossref PubMed Scopus (22) Google Scholar) have that the in biosynthesis in patients to rate of DHA we DHA synthesis from [1-14C]18:3n-3 and [1-14C]22:5n-3 in fibroblasts from patients with deficiency of either or the first step in peroxisomal β-oxidation of straight-chain fatty and the and of this pathway 1) R.J.A. Barth P.C. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3219-3256Google Scholar). from patients with deficiency DHA from either [1-14C]18:3n-3 or [1-14C]22:5n-3 a rate less than of that of cell DHA synthesis in cells from patients with deficiency was less than of with [1-14C]18:3n-3 as and was of with [1-14C]22:5n-3 as with or deficiency have peroxisomal fatty acid β-oxidation of but normal synthesis R.J.A. Barth P.C. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3219-3256Google Scholar). these data that both and are involved in DHA We also DHA synthesis in fibroblasts from patients with Cells from patients to the peroxisomal β-oxidation enzyme 3-oxoacyl-CoA thiolase into the organelle and have biosynthesis M. Res. 1999; 24: PubMed Scopus Google Scholar). In to and cells from patients DHA normal rates from either [1-14C]18:3n-3 or [1-14C]22:5n-3 a thiolase derived from in to 3-oxoacyl-CoA thiolase U. P. T. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to the of thiolase, fibroblasts are to normal rates S. Chen A. J. J.M. L. T. H.W. J. Pediatr. Full Text PDF PubMed Scopus Google Scholar). These data that peroxisomal 3-oxoacyl-CoA thiolase is not required for DHA Furthermore, the results that thiolase this but not 3-oxoacyl-CoA thiolase is to be the enzyme in peroxisomal straight-chain fatty acid β-oxidation and is the first enzyme to the pathway T. M. T. S. T. Biochem. Biophys. Res. PubMed Scopus Google Scholar). we the of DHA synthesis intermediates in and fibroblasts for with either [1-14C]18:3n-3 or [1-14C]22:5n-3 in only a of the [1-14C]18:3n-3 accumulated in either normal or cell In cell the of radiolabeled DHA with to and to In contrast, DHA synthesis was significantly reduced in cell In both cell the radiolabeled C22:5n-3, C24:5n-3, and C24:6n-3, accumulated the that is not required for of the fatty acid synthetic pathway from C18:3n-3 through to the the two direct of DHA, C24:5n-3 and C24:6n-3, accumulated to a higher in cell than in fibroblasts the that peroxisomal straight-chain fatty acid β-oxidation is essential for DHA results were obtained [1-14C]22:5n-3 as of the in normal cells from to and then to In of the [1-14C]22:5n-3 were times higher than in consistent with a DHA accumulated in cells with of but was than of in In contrast, the of C24:6n-3, the direct of DHA to retroconversion, to more than in cells with of of C24:5n-3, the intermediate C24:6n-3 in the DHA biosynthetic pathway, was times higher in cell as compared with controls. A similar in C24:6n-3 and C24:5n-3 was observed in fibroblasts from patients with Zellweger syndrome and deficiency not These are consistent with a in the DHA biosynthetic pathway C24:6n-3 and DHA. We DHA synthesis was in human disorders of peroxisomal fatty acid Peroxisomal β-oxidation of straight-chain is in to in the S. M. J. Res. 1999; 24: PubMed Scopus Google Scholar). This a peroxisomal protein to the ATP-binding cassette of the of which S. M. J. Res. 1999; 24: PubMed Scopus Google Scholar). of DHA from either [1-14C]18:3n-3 or [1-14C]22:5n-3 was not impaired in fibroblasts from patients This peroxisomal β-oxidation of C24:6n-3 to DHA does not require the of the of the with Refsum have a in the peroxisomal of a fatty acid R.J.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). the with fibroblasts from Refsum patients DHA from either radiolabeled a role for peroxisomal β-oxidation in the retroconversion of C24:6n-3 to DHA. that in mitochondrial β-oxidation not a similar in DHA synthesis, we fibroblasts from patients with deficiency of either or with [1-14C]18:3n-3 or in disorder exhibited DHA synthesis from either substrate. Furthermore, was no of the C24:6n-3 intermediate in cells from or These the hypothesis that not are the subcellular of retroconversion in DHA this is the first study that the peroxisomal β-oxidation enzymes and are essential for the retroconversion step in DHA synthesis that shortens C24:6n-3 by two carbons. The involvement of peroxisomal 3-oxoacyl-CoA thiolase or peroxisomal thiolase in this process is also Our data the existence of the new revised DHA synthetic pathway proposed by (12Voss A. Reinhart M. Sankarappa S. Sprecher H. J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar) in which C22:5n-3 is elongated to C24:5n-3 and desaturated to C24:6n-3 We conclude that in human skin retroconversion of C24:6n-3 to DHA proceeds via the peroxisomal straight-chain fatty acid β-oxidation pathway for the of The of two radiolabeled substrates, [1-14C]18:3n-3 and [1-14C]22:5n-3, to DHA synthesis from both its parent and from an intermediate in the pathway to the retroconversion The is the direct radiolabeled of DHA that is The radiolabeled intermediates C24:5n-3 and C24:6n-3, derived from either [1-14C]18:3n-3 or [1-14C]22:5n-3, were detected in normal human skin fibroblasts and in cells from patients with peroxisomal disorders or mitochondrial disorders in this higher of two were in Zellweger syndrome cell and in patients with either deficiency or deficiency but not in with mitochondrial β-oxidation of fatty acids in cells from to with substrates to that the proposed intermediates in the pathway were Our are consistent with the hypothesis that C18:3n-3 is to DHA via a series of alternating desaturation and elongation steps to C24:6n-3, that in H. Chen Q. Fatty 1999; Full Text PDF PubMed Scopus Google Scholar). Our studies that mitochondrial fatty acid β-oxidation is not essential for DHA The first step to mitochondrial fatty acid β-oxidation is by the acyl-CoA a of enzymes with distinct but chain length for C.R. J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). acyl-CoA have been and as very long-chain long-chain and Human patients with and acyl-CoA deficiency are but long-chain acyl-CoA deficiency has not been C.R. J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). Our that DHA biosynthesis from either [1-14C]18:3n-3 or [1-14C]22:5n-3 in cells from patients with and deficiency is consistent with the hypothesis that retroconversion of C24:6n-3 to DHA does not in In contrast, peroxisomes are essential for DHA The rate of DHA synthesis from either [1-14C]18:3n-3 or [1-14C]22:5n-3 in fibroblasts from Zellweger syndrome patients was less than of is in these in (8Moser H.W. Adv. Hum. Genet. 1993; 21: 1-106PubMed Google Scholar, S. I. PubMed Scopus Google Scholar). peroxisomal have been Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar, R. S.J. J.M. J.M. H.W. T. A. S. T. Valle D. I. M. J. Biol. PubMed Scopus Google Scholar, S. 1998; PubMed Scopus Google Scholar). of are known to in (25Moser H.W. Mol. Genet. Metab. 1999; PubMed Scopus Google Scholar). In the study, the genotypes of the Zellweger syndrome patients included of and peroxisomal fatty acid β-oxidation of is observed in of these genotypes S.J. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). the rate of DHA synthesis was of for genotypes results from Zellweger syndrome fibroblasts are as a These data are consistent with the hypothesis that peroxisomal β-oxidation is for DHA deficiency and C24:6n-3 in from patients with the genotypes also have biosynthesis of S.J. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). and Huszagh (18Infante J.P. Huszagh V.A. Mol. Genet. Metab. 2001; 72: 1-7Crossref PubMed Scopus (22) Google Scholar) proposed that to the rate of DHA synthesis in Our that DHA synthesis was normal in fibroblasts from patients this results from in the the for to peroxisomes via S.J. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: Scholar). the β-oxidation enzyme 3-oxoacyl-CoA thiolase and a enzyme in synthesis, L. R.J. H. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). synthesis is in these β-oxidation proceeds to the of a thiolase, thiolase, which is to peroxisomes by a different (23Wanders R.J. Tager J.M. Mol. Asp. Med. 1998; 19: 69-154Crossref PubMed Google Scholar). in Fig. obtained from fibroblasts. We also that DHA synthesis from [1-14C]18:3n-3 was normal in fibroblasts from two patients with and one with not and patients have synthesis, normal β-oxidation, and normal of both 3-oxoacyl-CoA thiolase and thiolase to peroxisomes R.J.A. Barth P.C. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3219-3256Google Scholar). in synthesis a β-oxidation deficiency does not DHA studies that thiolase participate in peroxisomal retroconversion of C24:6n-3 to DHA, not out the that 3-oxoacyl-CoA thiolase is also capable of this last step in DHA Human peroxisomes two fatty acid β-oxidation one for straight-chain fatty acids and the for fatty acids T. Res. 1999; 24: PubMed Scopus Google Scholar, R.J. Res. 1999; 24: PubMed Scopus Google Scholar, R.J. Biochem. 2000; PubMed Scopus Google Scholar). Until it had been that human peroxisomal β-oxidation of fatty acids such as the required protein, and 3-oxoacyl-CoA it was that human β-oxidation of fatty acids such as acid involved acyl-CoA and In R.J.A. Barth P.C. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic & Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3219-3256Google Scholar) proposed that the human pathway for β-oxidation of be revised to not and either 3-oxoacyl-CoA the pathway Our that or both of which are essential for β-oxidation of are also required for the retroconversion step of DHA biosynthesis is consistent with proposed pathway. Our that DHA synthesis is normal in fibroblasts one important peroxisomal β-oxidation of and retroconversion of C24:6n-3 to DHA. The ALD protein, of the in is not required for DHA the ALD protein to the of ATP-binding cassette its is not known. DHA synthesis in Refsum fibroblasts is consistent with the that peroxisomal does not participate in DHA In results evidence that in human skin fibroblasts the retroconversion step of DHA synthesis, a two-carbon shortening system from C24:6n-3, peroxisomal enzymes and as as either 3-oxoacyl-CoA thiolase or We conclude that the retroconversion step in DHA synthesis proceeds via peroxisomal straight-chain fatty acid β-oxidation but not via peroxisomal fatty acid β-oxidation or mitochondrial fatty acid β-oxidation. Our data of the new revised DHA synthetic pathway.