Under nearly all physiologic conditions, the rate of cellular cholesterol synthesis is determined by the activity of the microsomal enzyme HMG CoA reductase. Accurate assessment of changes in the catalytic activity and/or total quantity of this enzyme in man should, in theory, provide insight into fluctuations in endogenous rates of cholesterolgenesis under conditions of health or disease. To date, studies of the regulation of reductase have primarily been restricted to the use of either whole animals, animal tissues or cultured human cells. Because of certain species-specific controlling influences on reductase, however, it is often difficult to extrapolate results from animal experiments to the in vivo regulation of the enzyme in man. In addition, prior studies of the human-derived reductase have relied on estimation of enzyme activity in suspensions of mitochondria free cell homogenates, rather than from either microsomal preparations or purified enzyme. Furthermore, it has so far been impossible to identify a phosphorylation-dephosphorylation control mechanism for the human enzyme, despite the well documented existence of this important modulating feature for reductase in animal cells. We propose to characterize human reductase, viz. its physicochemical and regulatory properties and to utilize the measurement of human leukocyte reductase in vivo as a probe to investigate cholesterol metabolism in man. The enzyme will be isolated from cultured human IM-9 lymphoid cells, freshly isolated peripheral mononuclear cells and human liver. Methods have already been developed by us for isolation of both the microsomally-bound and solubilized, partially purified enzyme, and a phosphorylation-dephosphorylation mechanism of control has been discovered. The kinetic features of the solubilized enzyme will be explored. In order to provide a means of quantitating changes in enzyme protein, anti-human reductase antibodies will be generated. Using this technology, the in vivo regulation of human leukocyte reductase will be studied with the use of select nutritional and pharmacologic perturbants of cholesterol metabolism. Quantitative and qualitative changes in cata-lytic enzyme activity and total enzyme content under varying in vivo conditions will thus be investigated. Ultimately, we will test the hypothesis that the activity of peripheral leukocyte reductase accurately reflects endogenous rates of cholesterol-genesis in both healthy subjects and patients with various forms of abnormal lipid metabolism
| Stacpoole, P W; Alig, J; Ammon, L et al. (1989) Dose-response effects of dietary marine oil on carbohydrate and lipid metabolism in normal subjects and patients with hypertriglyceridemia. Metabolism 38:946-56 |
