The metabolism of progesterone by animal tissues in vitro
The problem of progesterone metabolism thus provides biochemists with a subject of great interest and importance and one which will not be readily solved. Studies on excreta and on blood, especially investigations with isotopically labelled steroid; perfusion experiments and other in vitro techniques will all play their part in providing information. Until we have grained some insight into the biochemical mechanisms involved in progesterone metabolism we cannot hope to understand much about the causes of the variations which occur in the metabolism of this steroid in different species and in health and disease.GENERAL INTRODUCTION • • GENERAL METHODS AND MATERIALS a) Experimental animals • b) Liver preparations i) Slices ii) Homogenates • c) Suspension media • d) Diphosphopyridine nucleotide (DPN) • e) Measurement of pH • f) Addition of "co- factors" • g) Addition of steroid • h) The general arrangement of incubation experiments • i) Steroids • j) Melting points • k) Specific rotations • • SECTION I i) Introduction • ii) Incubation of aqueous suspensions of progesterone and pregnanolone with tissue preparations a) Incubation of progesterone with rabbit uterus b) Incubation of progesterone with rat liver c) Incubation of pregnanolone with rat liver • iii) Discussion • iv) Methods used for the dispersion of steroids and their possible application to in vitro studies with progesterone a) Water- soluble derivatives b) Solubilisation of progesterone in aqueous media • v) Determination of the solubility of progesterone in aqueous media a) Materials and methods b) Results c) Discussion • vi) Incubation of protein and detergent solutions of progesterone with rut and rabbit liver and rabbit uterus a) Incubation of pregnanediol in serum solution with rat liver homogenate b) Incubation of progesterone in ox serum solution with rat liver homogenate c) Incubation of progesterone in rabbit serum solution with rabbit uterus d) Incubation of progesterone in "Triton X 100" solution with rabbit uterus and rabbit liver • vii) Discussion • viii) Summary of Section I • • SECTION II I. INTRODUCTION • II. METHODS i) Development of a method for the determination of progesterone in tissue preparations a) Introduction b) Protein precipitation c) Extraction of aqueous residue after removal of acetone d) Application of extracts to partition columns • ii) Column partition chromatography a) Purification of celite b) Preparation of columns c) Transference of samples to columns d) Separation of progesterone from liver extract • iii) The accuracy of the method a) Recovery of progesterone from rabbit liver slices b) Recovery of progesterone from rat liver homogenate • iv) Specificity of the method a) The nature of the "blank" • v) Application of the method to the determination of other steroids • vi) Paper chromatography • vii) Ultraviolet spectroscopy • viii) Miscellaneous • III. THE DESTRUCTION OF PROGESTERONE BY RABBIT LIVER i) Incubation of progesterone with slices ii) Comparison of slices and homogenate; effect of citrate iii) Effect of added DPN and nicotinamide • IV. THE DESTRUCTION OF PROGESTERONE BY RAT LIVER i) Incubation of progesterone with slices and homogenate ii) The effect of added DPN und nicotinamide iii) The effect of pre -incubation of homogenate with and without added DPN and nicotinamide iv) The destruction of enzymic activity by heat v) The effect of varying DPN and nicotinamide concentrations vi) The effect of pH vii) Incorporation of acid hydrolysis into the extraction procedure viii) Varying incubation time ix) The effect of anaerobic conditions x) The effect of citrate xi) The effect of tricarboxylic acid cycle intermediates, adenylic acid (AMP) and adenosine triphosphoric acid (ATP) • V. PRODUCTION OF FORMALDEHYDOGENIC MATERIAL AFTER INCUBATION OF PROGESTERONE WITH RABBIT AND RAT LIVER a) Materials and methods • b) Results i) Rabbit liver 100 ii) Rat liver a) Progesterone added in propylene glycol b) Progesterone added in ethanol 45 min. incubation 60 min. incubation 60 and 120 min. incubation • VI. THE ISOLATION OF METABOLIC PRODUCTS AFTER INCUBATION OF PROGESTERONE WITH RAT LIVER HOMOGENATE i) Introduction • ii) a) Preparation of homogenate b) Incubation c) Extraction d) Counter-current distribution e) Adsorption chromatography f) Isolation of 5α(-pregnane-3:20-dione g) Isolation of 5α-pregnan-3α-ol-20-one h) Substance "Z" • • SECTION III GENERAL DISCUSSION i) Introduction • ii) The effect of DPN and nicotinamide on progesterone metabolism in vitro • iii) The significance of the formaldehydogenic material formed after incubation of progesterone with liver • iv) The nature of the metabolic products • v) The reduction of non-benzenoid steroid hormones • vi) Some general observations regarding the metabolism of progesterone • vii) Conclusion • • ÁPPENDICES I. Partition coefficients • II. Metabolism of progesterone by rat liver mitochondria • III. Purification of organic solvents • IV. numerical details of Figures • V. References • VI. Publications: • The influence of steroids on citrate metabolism. By J.K. Grant and W. Taylor. • Enzymic 11-ß-hydroxylation of 11-deoxycorticosterone (DOC). By A.C. Brownie, J.K. Grant and W. Taylor.