Measurement of cholesterol biosynthesis in vivo with tritiated water.

The determination of the rate of cholesterogenesis has usually involved either the measurement of the incorporation of a radiolabelled substrate, such as acetate, or the assay of the enzyme /I-hydroxy-p-methylglutaryl-CoA reductase (EC 1.1.1.34), commonly believed to be rate-limiting. However, the use of acetate is of doubtful physiological relevance, and the size and rate of turnover of the free acetate pool depends on the nutritional and hormonal status. Consequently, data obtained from the incorporation of substrates in uitro and those obtained from the measurement of enzyme activities may be contradictory (Dugan et a/., 1972; Nervi et al., 1976). The use of H-labelled water does not suffer from such disadvantages. Rittenberg & Schoenheimer (1937) were the first to use 2Hz0 to demonstrate the synthesis de nouo of cholesterol. More recently 3Hz0 has been used to measure the rate of synthesis of lipid, in particular, fatty acid (Jungas, 1968). By using 3H20 it is possible to determine the actual overall flux of substrate into lipid, providing that the contributions from various substrates do not appreciably change in proportion to one another. For example, if fatty acids are oxidized to produce acetyl-CoA for cholesterogenesis, the /I-oxoacyl-CoA intermediates will non-enzymically incorporate 3H from 3Hz0 because of oxo(keto)*nol tautomerism (Gibbons & Pullinger, 1977). Thus acetyl-CoA substrate produced from fatty acids will tend to contain proportionately more 3H than that produced, for example, from glycolysis. The accuracy of the measurement of the rate of cholesterogenesis would also be decreased if a significant isotope effect existed, but this does not seem to be a problem (Lakshmanan & Veech, 1977). To calculate the actual rate of cholesterogenesis, it is necessary to ascertain the number of H atoms incorporated per molecule of cholesterol synthesized. Lakshmanan & Veech (1977) have shown that cholesterol biosynthesis incorporates 7protonsfromthemediumand 15 HatomsfromNADPH,which is approx. SO%labelled with 3H from 3H20, giving a net incorporation of 14.5 3H atoms (Fig. 1). For the experimental determination of the number of 3H atoms incorporated, we have compared the uptake of 3H from 3Hz0 with the simultaneous incorporation of I4C from [l-14C]acetate at a saturating concentration of the latter, in rat liver slices in vitro. We have also used the indirect method of Brunengraber et al. (1972), whereby the generally accepted 3H/14C ratio for fatty acid synthesis is used to correct for the dilution of 14C-radiolabelled substrate ([l-'4C]octanoate in the present experiment) administered together with 3H20 in uiuo. In such experiments, approx. 18 3H atoms were incorporated per molecule of cholesterol synthesized ; the excess over the theoretical 14.5 jHatomsmay indicate partial labelling of acetyl-CoA. We have validated the use of 3H20 by confirming known variations in hepatic cholesterogenesis, for example, the decrease and increase in rate after dietary cholesterol and cholestyramine respectively (Fears & Morgan, 1976). More recently the response to food deprivation was examined. Groups of eight male CFY rats were fed ad libirurn on a stock pelleted diet (Oxoid Breeding Diet) or were deprived of food for 12 or 24h. Rats were given 3H20 intraperitoneally 1 h before being killed and the incorporation of j H into digitonin-precipitable sterols in the liver was measured. In addition, [l-14C]acetate incorporation into digitonin-precipitable sterols was measured in liver