Date of Award
Doctor of Philosophy
John T. Smith
Ada Marie Campbell, Gail Disney, Jane R. Savage
This investigation was undertaken to study the effect of varying levels of dietary sulfate on the relative conjugation of 14C-cholic acid with taurine and glycine in rat liver microsomal preparations with a view to evaluating the factors controlling glycocholic:taurocholic acid (G:T) ratio at the cellular level. Pure microsomal preparations, obtained after centrifuging at high speed, were incubated with the optimum concentrations of 14C-cholic acid, glycine, taurine, coenzyme A, magnesium sulfate, ATP and sodium fluoride for 90 minutes at 37°. The radioactive cholic acid conjugates fonned were separated with thin layer chromatography and visualized by spraying with phosphomolybdic acid. The G:T ratios were computed by comparing the counts in glycocholate and taurocholate bands.
No diet related differences were observed in the G:T ratio except in rats fed 0. 0002% SO=4 + 0. 4% cysteine diet. Omission of coenzyme A from the reaction mixture resulted in alterations of the G:T ratio which were related to the level of sulfate in diets. A deficiency of inorganic sulfate or cysteine (0. 0002% SO=4 + 0. 4% cysteine diet and 0. 42% SO=4 diet) caused an increase in the G:T ratio as compared to the G:T ratio observed in rats fed 0. 1% SO=4 + 0.4% cysteine diet and 0. 42% SO=4 + 0. 4% cysteine diet. The overall increase in the G:T ratio in reaction mixtures not containing coenzyme A resulted from a decrease of approximately 85% in taurocholate synthesis and only 55% in glycocholate synthesis. iii The synthesis of taurocholate was more dependent upon the level of coenzyme A.
Further, the effect of these diets on the level of free coenzyme A in both the mitochondrial and nuclei free fraction of liver homogenates was studied. There was a decrease in the level of free coenzyme A in the rats fed 0. 0002% SO=4 + 0. 4% cysteine diet and 0. 42% SO=4 diet. These are the same diets which caused an increase in the G:T ratio. This showed that an increase in the G:T ratio observed after feeding these diets was caused by a deficiency of hepatic coenzyme A and any diet which changes taurine concentration and/or coenzyme A concentration would also tend to change the G:T ratio.
Michaelis constants (Km) of enzyme bile acyl transferase for glycine and taurine and glycine in the presence of small amounts of taurine were determined. Km of the enzyme for glycine was 15 times higher than its Km for taurine. The Km for glycine in the presence of taurine was further increased, thereby showing that taurine acts as a competitive inhibitor of glycine conjugation. From this investigation it is concluded that there is only one nonspecific enzyme, bile acyl transferase, for which taurine is a preferred substrate due to its lower Km, but this enzyme would also catalyze acyl transfer of other structurally similar compounds like glycine and B-alanine. These data have demonstrated that any dietary treatment which alters the level of taurine and/or coenzyme A in the tissue, will also change the G:T ratio; and may be important in the dietary management of serum cholesterol levels.
Verma, Ramesh S., "An Investigation of the Effect of Dietary Sulfate on Bile Acid Conjugation and Kinetic Studies of Bile Acyl Transferase in Rat Liver Microsomes. " PhD diss., University of Tennessee, 1974.