Aberrant pathways in the late stages of cholesterol biosynthesis in the rat: origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome

Abstract

Minor aberrant pathways of cholesterol biosynthesis normally produce only trace levels of abnormal sterol metabolites but may assume major importance when an essential biosynthetic step is blocked. Cholesta-5,8-dien-3 ␤ -ol, its ⌬ 5,7 isomer, and other noncholesterol sterols accumulate in subjects with the Smith-Lemli-Opitz syndrome (SLOS), a severe developmental disorder caused by a defective ⌬ 7 sterol reductase gene. We have explored the formation and metabolism of unsaturated sterols relevant to SLOS by incubating tritium-labeled ⌬ 5,8 , ⌬ 6,8 , ⌬ 6,8(14) , ⌬ 5,8( 14) , and ⌬ 8 sterols with rat liver preparations. More than 60 different incubations were carried out with washed microsomes or the 10,000 g supernatant under aerobic or anaerobic conditions; some experiments included addition of cofactors, fenpropimorph (a ⌬ 8 -⌬ 7 isomerase inhibitor), and/or AY-9944 (a ⌬ 7 reductase inhibitor). The tritium-labeled metabolites from each incubation were identified by silver ion high performance liquid chromatography on the basis of their coelution with unlabeled authentic standards, as free sterols and/ or acetate derivatives. The ⌬ 5,8 sterol was converted slowly to cholesterol via the ⌬ 5,7 sterol, which also slowly isomerized back to the ⌬ 5,8 sterol. The ⌬ 6,8 sterol was metabolized rapidly to cholesterol by an oxygen-requiring pathway via the ⌬ 7,9(11) , ⌬ 8 , ⌬ 7 , and ⌬ 5,7 sterols as well as by an oxygen-independent route involving initial isomerization to the ⌬ 5,7 sterol. The ⌬ 8 sterol was partially metabolized to ⌬ 5,8 , ⌬ 6,8 , ⌬ 7,9(11) , and ⌬ 5,7,9(11) sterols when isomerization to ⌬ 7 was blocked. The combined results were used to formulate a scheme of normal and aberrant biosynthetic pathways that illuminate the origin and metabolic fate of abnormal sterols observed in SLOS and chondrodysplasia punctata. -Ruan, B., J. Tsai, W. K. Wilson, and G. J. Schroepfer, Jr. Aberrant pathways in the late stages of cholesterol biosynthesis in the rat: origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome.

Sterols in blood of normal and Smith-Lemli-Opitz subjects

“…Blood from a severely affected SLOS subject contained substantially elevated levels of sitosterol and other plant sterols from intravenous feeding of a plant lipid emulsion. Micromolar levels of numerous unidentified components in this and other samples suggest a greater complexity of aberrant pathways in sterol synthesis than recognized previously (32).…”

“…The ⌬ 5,8 sterol was metabolized slowly to cholesterol via the ⌬ 5,7 sterol, and the ⌬ 6,8 sterol was rapidly converted to cholesterol under aerobic conditions, a process that cannot involve the slow-metabolizing ⌬ 5,8 sterol. Rat liver incubations of the ⌬ 6,8 sterol with a DHCR7 inhibitor (1 M AY-9944) produced only the ⌬ 5,7,9 (11) sterol (32). Thus, a significant metabolic flux through the ⌬ 6,8 sterol could account for the elevated ⌬ 5,7,9 (11) levels in SLOS.…”

“…Many, but not all (56), experimental results suggest that the ⌬ 8 (14) sterol is an intermediate of cholesterol synthesis (55), and our detection of the ⌬ 8 (14) , ⌬ 5,8 (14) , and ⌬ 6,8 (14) sterols supports this proposal. In a study of aberrant pathways of cholesterol biosynthesis in rat liver preparations (32), a small portion of the ⌬ 8 sterol was desaturated to ⌬ 5,8 and ⌬ 6,8 sterols. The ⌬ 5,8 sterol was metabolized slowly to cholesterol via the ⌬ 5,7 sterol, and the ⌬ 6,8 sterol was rapidly converted to cholesterol under aerobic conditions, a process that cannot involve the slow-metabolizing ⌬ 5,8 sterol.…”

“…11 min), ⌬ 5 sterol by GC-MS and Ag ϩ -HPLC; zone II-3 (t R 14 -15 min), ⌬ 5,24 sterol by GC-MS and Ag ϩ -HPLC; zone II-4 (t R 22 -24 min), unidentified sterol, GC-MS, t R 0.91 relative to the ⌬ 8 (14) TMS ether; m/z 456 (69), 441 (31), 366 (100), 351 (82), 327 (11), 253 (37), 129 (36); zone II-5 (t R 26 -28 min), ⌬ 5,8 (14) sterol by GC-MS, NMR, and Ag ϩ -HPLC; zone II-6 (t R 31 -34 min), ⌬ 5,8 sterol by GC-MS, accompanied by the 19-nor-⌬ 5,7,9 artifact. Zone III (t R 42 -44 min), a 97:3 mixture of ⌬ 5,8 and ⌬ 7 sterols by NMR, was subfractionated on Ag ϩ -HPLC as follows: zone III-1 (t R 8 -9 min), ⌬ 7 sterol by GC-MS, NMR, and Ag ϩ -HPLC); zone III-2 (t R 29 -35 min), mixture including a major component with GC-MS showing t R 1.09 relative to cholesterol (TMS ethers), m/z 456 (100), 441 (32), 366 (10), 351 (14), 343 (6), 255 (9), 229 (21), 107 (46); possibly the ⌬ 8(14),24 sterol; zone III-3 (t R 29 -35 min), ⌬ 5,8 sterol by GC-MS, accompanied by the 19-nor-⌬ 5,7,9 artifact. Zone IV (t R 45 -47 min) contained the ⌬ 5,7 sterol (Ͼ99% purity) by GC-MS, NMR, and Ag ϩ -HPLC.…”

“…Herein is described the application of practical analytical methods to the quantitation and identification of sterols in 17 SLOS and 6 normal blood samples. Comparisons between normal and SLOS samples, together with studies of the aberrant metabolic pathways of cholesterol biosynthesis (32), provide insight into the origin and fate of sterol intermediates.…”