An assay has been established for the selective measurement of tamoxifen and its monohydroxy derivative, metabolite B, in human plasma. The assay was used to examine the concentrations of these compounds, relative to oestradiol-17 beta, in the plasma of patients undergoing tamoxifen therapy for advanced breast cancer. Oral administration of the drug (20 mg twice a day) raised the level of tamoxifen in plasma to approximately 200 ng/ml 20 days after the commencement of treatment. This level was 3000-fold higher than the corresponding concentration of oestradiol which remained within the range for post-menopausal women. Metabolite B was present in plasma at a much lower concentration than tamoxifen although in considerable excess over oestradiol. The overall results are discussed in relation to the possible mechanism of action of the drug.
The enzyme, CTP:phosphatidate cytidylyltransferase (EC2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer-detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleolphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60mM for MG2+, 6MM for CTP and 250 mug per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90,000 X g fraction (microsomal) with small but significant activity in the 12,000 X g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses); 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl-(3.85), 1-stearoyl-2-arachidonoyl-(3.31), 1-arachidonoyl-2-stearoyl-(3.16), 1,2-diarachidonoyl-(0.72), 1,2-dicaproyl-(0.67), 1,2-dipalmitoyl-(0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.
On the specificity of cytidine diphosphate diglycerides in monophosphoinositide biosynthesis by rat brain preparations. Can. J. Biochem. 48, 269-277 (1970).Some properties of the enzyme cytidine diphosphate diglyceride : inositol transferase which catalyzes the reaction CDP diglyceride + inositol -+-monophosphoinositide + CMP have been studied with various preparations from rat brain. The enzyme was found to be primarily located in the microsomal fraction (either the 12 808 x g supernatant or the 98 800 x g pellet). Optimal conditions were established for pH and concentrations of each of the reactants and Mn2+ ion. A number of cytidine diphosphate diglycerides with different fatty acids in the 1-and 2-positions were synthesized chemically and used as substrates for the above reaction. The substrates included those with the folIowing distribution of fatty acids : 1,2-dipalmitoyl ; 1 ,2-distearoyl ; 1-palmitoyl,2oleoyl; 1 -stearoy192-oleoyl ; 1-oleoyl,2-stearoyl, and 1,2-dioleoyl. It was observed that those substrates with oleic at the 2-position were more readily utilized than those containing palmitic or stearic acids at the Zposition or at both the 1-and 2-positions. This selectivity for cytidine diphosphate diglyceride with an unsaturated fatty acid at the 2-position was consistent with the pattern of fatty acid distribution found for isolated rat brain monophosphoinositide. In the latter, saturated fatty acids (stearic and palmitic acids) were predominantly located at the 1-position and unsaturated fatty acids (20:4, arachidonic =id) were predominantly located in the 2-position.
AsstractThe properties of diacylglycerol and monoacylglycerol kinase activities present in 90,000 x g pellet and 90,000 × g supernatant fractions from rat brain were examined and compared. Of the properties examined, time course (linear for 10 min), enzyme concentration, pH optimum (7.4–7.5), varying ATP (5 mM) and Mg2+ (10 mM) concentrations all showed similar optima for both activities. The optima for acylglycerol (5 mM for diacylglycerols; 3 mM for monoacylglycerols) and deoxycholate concentrations (0.1% for diacylglycerol kinase; 0.03% for monoacylglycerol kinase) differed slightly. Examination of the subcellular distribution for these activities also showed a similar pattern. All fractions showed significant activities, but the most was in the supernatant fraction. The similarities in properties and localization of the 2 kinase activities suggest a single enzyme may function. On this assumption, an extensive study using mono‐ and diacylglycerols of varying fatty acid composition gave the following results: (a) acylglycerols with the same fatty acid present showed increasing activity in the order: 1‐monoacylglycerol, 2‐monoacylglycerol and 1,2‐diacylglycerols; (b) when saturated fatty acids were present the order of decreasing activity varied directly with increasing chain length for C10→C20; (c) when one or more unsaturated fatty acids were present good activities resulted, but no clear pattern emerged, although acylglycerols with18∶1 and18∶3 fatty acids were more active than those with18∶2 and20∶4 fatty acids. These patterns do not support a role for this kinase in producing phosphatidic acids or lyso phosphatidic acids of the correct composition to act as precursors for the de novo synthesis of the predominant 1‐stearoyl, 2‐arachidonoyl molecular species of phosphatidylinositol.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.