Studies on the Biosynthesis of Cholesterol

We have recently presented evidence indicating that during biosynthesis of squalene from farnesyl pyrophosphate by rat liver microsomes, 1 hydrogen atom attached to carbon 1 of 1 of the 2 precursor molecules condensing to squalene was exchanged for a hydride ion derived from reduced triphosphopyridine nucleotide (1). This conclusion was drawn from the observations, (a) that during synthesis of squalene by microsomes from biosynthetically prepared C14-farnesyl pyrophosphate in the presence of H3-labeled reduced triphosphopyridine nucleotide, each micromole of squalene formed contained nearly 1 microgram atom of labeled hydrogen; and (b) that squalene biosynthesized from mevalonate-2-Cl4-5Dz contained 11 atoms of deuterium, 3 of these being arranged asymmetrically around the 2 central carbon atoms thus: -CHD . CD2-. Uptake of a proton from the water of the incubation medium into squalene during its synthesis from farnesyl pyrophosphate with washed liver microsomes did not occur. Since it is known that the conversion of mevalonate to squalene proceeds through the intermediate formation of farnesyl pyrophosphate (2, 3), and that mevalonate-2-C14-5-Dt yields, in all probability, 4,8, 12-C&l-D2-5-Dz-9-Dp-farnesyl pyrophosphate, our conclusions were well founded. Nevertheless, further formal proof for the exchange of the 1 hydrogen atom, referred to, was required. We have therefore synthesized chemically l-H&2-@ and 1-D2-2-C%rans-trans-farnesyl pyrophosphate and examined these as substrates for squalene synthesis. We found, in confirmation of our previous data, that only 3 atoms of the hydrogen isotope were contained in squalene biosynthesized from either of these synthetic substrates. We have also analyzed, by mass spectrometry, a specimen of farnesol, obtained from the hydrolysis with intestinal alkaline phosphatase of farnesyl pyrophosphate biosynthesized from mevalonate-2-Cl4-5-Dz, and found it to contain 6 atoms of deuterium, 2 of these being attached to carbon 1, in confirmation of our former assumptions (1). Some of these observations have already been reported briefly (5).