-desaturase deficiency in human skin fibroblasts

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85– 95% less [114 C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [314 C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [114 C]linolenic acid (18:3n-3) and [314 C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [114 C]tetradecadienoic acid (14:2n-6) was 18:2n-6. However, they produced 50–90% as much 20:4n-6 as the NF cultures from [114 C]hexadecatrienoic acid (16:3n-6), [114 C] g -linolenic acid (18:3n-6), and [114 C]dihomog -linolenic acid (20:3n-6), PUFA substrates that contain D 6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in D 6 desaturation. This was confirmed by Northern blots demonstrating an 81–94% decrease in D 6 desaturase mRNA content in the DF cultures, whereas the D 5 -desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a D 6 -desaturase deficiency. Furthermore, the finding that the 18and 24-carbon substrates are equally affected suggests that a single enzyme carries out both D 6 desaturation reactions in human PUFA metabolism. — Williard, D. E., J. O. Nwankwo, T. L. Kaduce, S. D. Harmon, M. Irons, H. W. Moser, G. V. Raymond, and A. A. Spector. Identification of a fatty acid D 6 -desaturase deficiency in human skin fibroblasts. J. Lipid Res. 2001. 42: 501–508. Supplementary key words polyunsaturated fatty acids • fatty acid desaturation • D 5 -desaturase • linoleic acid • a -linolenic acid • arachidonic acid • docosahexaenoic acid • gene expression • genetic defect The mammalian polyunsaturated fatty acid (PUFA) metabolic pathway consists of a series of elongation and desaturation reactions that convert the 18-carbon essential fatty acids to 24-carbon intermediates, followed by a peroxisomal retroconversion reaction that forms the final 22-carbon products (1–5). Two fatty acid desaturase enzymes operate in this pathway, the D 6 and D 5 -desaturases. The human, mouse, and rat D 6 -desaturase (6, 7), and the human D 5 -desaturase (8), have been cloned. D 5 -Desaturase acts once in the pathway, inserting a double bond into the 20-carbon intermediates formed during n-3 and n-6 PUFA metabolism. D 6 -Desaturase acts twice, once on the 18-carbon PUFA substrates and again after they are converted to 24-carbon derivatives. The first D 6 -desaturase reaction is the rate-limiting step in the conversion of linoleic acid (18:2n-6) and a -linolenic acids (18:3n-3) to the longer, more highly unsaturated members of the n-6 and n-3 PUFA families. Both of these fatty acids can go through the entire PUFA metabolic pathway to form the final 22-carbon products (2–4). However, under most conditions, this occurs only with n-3 PUFA. The main product formed from 18:2n-6 ordinarily is arachidonic acid (20:4n-6), the principal substrate for eicosanoid synthesis. This conversion requires only the initial segment of the PUFA metabolic pathway, D 6 desaturation of 18:2n-6 followed by chain elongation to a 20-carbon intermediate and then D 5 desaturation (1). Thus, D 6 -desaturase usually acts only once in n-6 PUFA metabolism. On the other hand, 18:3n-3 ordinarily goes through the complete pathway because the main n-3 PUFA produced by mammalian tissues is docosahexaenoic acid (22:6n-3, DHA), the product formed by Abbreviations: 14:2n-6, tetradecadienoic acid; 16:0, palmitic acid; 16:3n-6, hexadecatrienoic acid; 18:2n-6, linoleic acid; 18:3n-3, a -linolenic acid; 18:3n-6, g -linolenic acid; 18:4n-3, stearidonic acid; 20:3n-6, dihomog -linolenic acid; 20:4n-6, arachidonic acid; 22:4n-6, docosatetraenoic acid; 22:5n-6 or n-3, docosapentaenoic acid; 22:6n-3 or DHA, docosahexaenoic acid; 24:4n-6, tetracosatetraenoic acid; 24:5n-3 or n-6, tetracosapentaenoic acid; 24:6n-3, tetracosahexaenoic acid; DF, deficient human skin fibroblasts; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLC, gas-liquid chromatography; HPLC, high performance liquid chromatography; NF, normal human skin fibroblasts; PUFA, polyunsaturated fatty acid. 1 To whom correspondence should be addressed at the Department of Biochemistry, 4-403 BSB, University of Iowa, Iowa City, IA 52242. e-mail: [email protected] at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom