P-450 Level and Taurochenodeoxycholate 6 -Hydroxylase System of Rat Liver Microsomes

Abstract

The 6P-hydroxylation of taurochenodeoxycholate by rat liver microsomes has been previously reported ( 1 ) . The participation of cytochrome P-450 in this reaction was subsequently demonstrated by the photochemical action spectrum method ( 2 ) . In this respect, the enzyme is similar to a number of other mixed function oxidases involved in steroid and drug hydroxylations. These are complex enzymes which introduce one atom of molecular oxygen into the substrate molecule while reducing the other oxygen atom to water. In the process, electrons flow from NADPH through an electron transport system consisting of a flavoprotein at one end and cytochrome P-450 at the other. The steroid 1 lp-hydroxylase system of bovine adrenal mitochondria has been solubilized and resolved into three components of flavoprotein, nonheme-iron protein, and cytochrome P-450 (3). Mixed function oxidases of liver microsomes have, however, resisted similar attempts at resolution and purification, mainly because of instability of the P-450 towards the various solubilizing agents.Recently, Lu et al. (4) have reported the solubilization of fatty acid o-hydroxylase system from rabbit liver. Fractionation of the soluble preparation yielded a P-450 containing fraction and a TPNH-cytochrome c reductase fraction. The ratio of P-450 to protein in the former fraction was, however, the same as that of the intact microsomes.Using our previous method ( l ) , we have prepared an extract of liver microsomes in 1.0 M phosphate, active in 6 P-hydroxylation after removal of the 105,OOOg pellet. The ratio of P-450 to protein indicated a partial purification of P-450 over that in the original microsomes. The apparent K , for tauro-chenodeoxycholate was estimated at 1.2 )( Since the involvement of P-450 in the 6P-hydroxylase system was established, it became of interest to assess its influence in the overall rate of hydroxylation. We treated the experimental animals with various agents which were expected to alter either the levels of P-450 in the liver, the bile acid pool, or both, and measured in each case the 6P-hydroxylase activity and the concentration of P-450. The agents used were (a) phenobarbital, a known inducer of P-450 (S), (b) cholestyramine, a bile acid sequestering resin which causes an increased production of bile acids (6), (c) thyroxine, which enhances the production of chenodeoxycholic acid (7), and (d) chenodeoxycholate to increase the bile acid pool.Methods. Four groups of five male Sprague-Dawley rats (2 months old), weighing 200-250 g each, were kept in individual cages during a 5-day treatment period: (a) Control group, the animals were fed the regular Purina rat chow. (b) Cholestyramine group, the regular Purina pellets were pulverized and mixed with cholestyramine (Merck, Sharp and Dohme) in proportion of 1 g of resin to 19 g of chow. Pellets of approximately the same size as the original were then formed. Each rat ingested an average of 1 g of resin/day. (c) Phenobarbital group, each rat was injected intraperitoneally 25 mg of sodium pheno...

Partial characterization of 5.ALPHA.-reductase in the human epididymis.

“…These values are similar to the optimum pH value for the 5a-reductase in the rat epididymis (Monsalve and Blaquier, 1977), rat seminal vesicle (Suzuki and Tamaoki, 1974), and human neonatal foreskin (Voigt et al, 1970 …”

“…These results are consistent with reports on the rat epididymis (Monsalve and Blaquier, 1977) and the rat prostate (Shimazaki et al, 1965 ;Frederiksen and Wilson, 1971 ;Nozu and Tamaoki, 1974). On the other hand, it was reported that 5a-reductase activity in the human neonatal foreskin (Voigt et al, 1970) and immature rat testis (Oshima et al, 1970) is confined exclusively to the microsome.…”

“…NADPH is essential to microsomal 5a-reductase as a coenzyme in the human epididymis as well as in the rat epididymis (Monsalve and Blaquier, 1977), rat prostate (Shimazaki et al, 1965 ;Frederiksen and Wilson, 1971) and human neonatal foreskin (Voigt et al, 1970).…”

Engineered Three-Dimensional Liver Mimics Recapitulate Critical Rat-Specific Bile Acid Pathways

“…It is synthesized from CDCA in vivo by phenotypically stable hepatocytes. [41][42][43][44] The production of TbMCA was monitored to determine the fate of metabolized CDCA reported in the previous section (Fig. 6).…”

“…In the rat, beta-muricholic acid (bMCA) is considered an additional primary BA since it is directly synthesized by rat hepatocytes from CDCA. [41][42][43][44] The final step before functional BA secretion from the liver is conjugation to either glycine or taurine to increase their solubility in vivo. 8,12,45 In this study, we exploited the 1 ng/mL detection limit of our HPLC/MS methodology to quantify the following primary rat BAs: CA, glycocholic acid (GCA), taurocholic acid (TCA), CDCA, glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), bMCA, and tauro-beta-muricholic acid (TbMCA).…”