The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.

Section: Experimental Methodsmentioning

confidence: 98%

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from [2-14C]mevalonic acid in vitro

Gibbons

Mitropoulos

1973

“…T'he materials and methods were those described by Wilton et al (1968) Preparation of4,4'-[3fl-l80H]dimethylcholest-7-enol 4,4'-Dimethylcholest-7-enone was prepared from 4,4-dimethylcholesta-5,7-dienone by reduction with Raney nickel (Watkinson et al, 1971) followed by oxidation with Jones reagent (Bowden et al, 1946).…”

Section: Methodsmentioning

confidence: 99%

The mechanism of C-4 demethylation during cholesterol biosynthesis. Evidence for a decarboxylation mechanism not involving a Schiff-base intermediate

Wilton

Akhtar

1975

Self Cite

“…Preparation of 4,4-dimethylcholesta-8,14-dien-3fl-of and 4,4-dimethylkholesta-7,14-dien-3fl-of These two compounds and their corresponding acetates were prepared as described previously (Akhtar et al, 1969b;Watkinson et al, 1971) by methods adapted from those of Gautschi & Bloch (1958) and Knight et al (1966).…”

Section: Analysis Of Incubations For Radioactive Formic Acid Water Amentioning

confidence: 99%

“…Additional background information pertinent in this connexion includes the knowledge that the removal of a 15ax-hydrogen atom from lanosterol (V, Scheme 4) is intimately linked to the loss of C-32 (Canonica et al, 1968a,b;Gibbons et al 1968;Akhtar et al, 1968Akhtar et al, , 1969aGoodwin, 1971, and references cited therein), and also that a 8,14-diene system of type (VIII) is one of the early products of demethylation Watkinson et al, 1971;Fiecchi et al, 1969). These features have been reconciled in terms of two alternative mechanistic proposals (Fiecchi et al, 1972;Akhtar et al, 1972;Watkinson et al, 1971 ;Spike et al, 1974, and references cited therein;Schroepfer et al, 1972;Goodwin, 1971), which in the light of the new information that the C-32 is removed as formic acid and not as CO2 may be presented in modified forms as shown in Scheme 4. Pathway A assumes that activation for the cleavage of the C-14-C-32 bond of the compound (VI) is provided through protonation of the double bond by a group on the enzyme, to give an electron-deficient centre at C-8 which facilitates deformylation to produce the A81"4)-steroid (VII).…”

Section: H (% Of Original Radioactivity) Associated Withmentioning

confidence: 99%

Chemical and enzymic studies on the characterization of intermediates during the removal of the 14α-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives

Akhtar¹,

Alexander²,

Boar³

et al. 1978

By using cell-free preparations of rat liver it was shown that the removal of the 14alpha-methyl group (C-32) of steroids containing either a delta7(8) or a delta8(9) double bond is attended exclusively by the formation of the corresponding 7,14- and 8,14-dienes respectively (structures of types III and VIII). Cumulative evidence from a variety of experimental approaches had led to the deduction that delta8(14)-steroids are not involved as intermediates on the major pathway of cholesterol biosynthesis. The metabolism of [32-3H]lanost-7-ene-3beta,32-diol (structure of type I) results in the formation of radioactive formic acid, no labelled formaldehyde being formed. By using appropriately labelled species of the compound (I) it was found that the release of formic acid and the formation of 4,4-dimethylcholesta-7,14-dien-3beta-ol (strurcture of type III) were closely linked processes, and that in the conversion of compound (I) into compound (III), 3-beta-hydroxylanost-7-en-32-al (II) is an obligatory intermediate. Both the conversion of lanost-7-ene-3beta,32-diol (I) into 3beta-hydroxylanost-7-en-32-al (II) and the further metabolism of the latter (II) to 4,4-dimethylcholesta-7,14-dien-3beta-ol (III) exhibited a requirement for NADPH and O2. This suggests that the oxidation of the 32-hydroxy group of compound (I) to the aldehyde group of compound (II) does not occur by the conventional alcohol dehydrogenase type of reaction, but may proceed by a novel mechanism involving the intermediacy of a gem-diol. A detailed overall pathway for the 14alpha-demethylation in cholesterol biosynthesis is considered, and proposals about the mechanism of individual steps in the pathway are made.