Henry Danielsson

Relationship between diurnal variations in biosynthesis cholesterol and bile acids

Abstract The rate of incorporation of acetate into cholesterol by rat liver slices and the rates of 7α-hydroxylation of cholesterol, of 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and of 7α-hydroxylation of taurodeoxycholic acid by rat liver microsomes were assayed at six different times of the day. The diurnal variations in rate of incorporation of acetate into cholesterol (maximum between 8 p.m. and midnight and minimum at noon) and in rate of 7α-hydroxylation of cholesterol (maximum at 8 p.m. and minimum at noon) followed each other closely. 12α-Hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid remained at about the same level during the day. The pattern of diurnal variations in incorporation of acetate into cholesterol and in 7α-hydroxylation of cholesterol was changed by reversing the daily rhythm of the rats. The results indicate that changes in rates of biosynthesis of cholesterol and bile acids occur practically concomitantly.

Mechanisms of Bile Acid Biosynthesis

In this chapter, an attempt is made to summarize present knowledge concerning the mechanisms of the conversion of cholesterol into the primary bile acids. In addition, a section on the formation of bile salts in “primitive” animals is included. Emphasis in discussion and documentation of references will be placed on more recent developments. The early work will be briefly reviewed in the introductory section.

On serum-cholesterol levels and neutral fecal sterols in germ-free rats; bile acids and steroids 59.

Abstract On a semisynthetic diet without significant amounts of cholesterol, germ-free rats had significantly higher serum-cholesterol values than control rats, the mean value for germ-free animals being 110 mg.% and for controls 77 mg.%. On a diet with completely steroid-free synthetic fat, germ-free animals excreted daily 2.6 mg. of neutral sterols in feces. At least 82% of these consisted of cholesterol.

Effect of biliary obstruction on formation and metabolism of bile acids in rat

Abstract The effect of biliary obstruction in the rat on several hydroxylations involved in the formation and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The rate of 7α-hydroxylation of cholesterol increased two- to threefold between 24 and 48 hours after ligation of the bile duct and remained at this level the next 48 hours. During the first 24 hours of obstruction the rates of 1 2α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid decreased but returned to control levels between 24 and 48 hours after operation. The rate of 6β-hydroxylation of lithocholic acid and taurochenodeoxycholic acid increased gradually and reached a plateau between 24 and 48 hours at which time the rate was two to three times faster than in the controls. The increase in 6β-hydroxylase activity was reflected in the pattern of the bile acids excreted in urine. After 48 hours of obstruction β-muricholic acid accounted for 50% or more of the bile acids in urine.

Hydroxylations in biosynthesis and metabolism of bile acids

SummaryRecent development in research concerning hydroxylations in biosynthesis and metabolism of bile acids is reviewed. In the conversion of cholesterol into bile acids, hydroxyl groups may be introduced into the following positions: C-6α, C-6β, C-7α, C-12α, C-16α, C-23, C-24, C-25, and C-26. With the exception of the 26-hydroxylation and probably the 24-hydroxylation, which are also catalyzed by the mitochondrial fraction, the different hydroxylations are catalyzed by the microsomal fraction of liver homogenate and require NADPH and molecular oxygen. With the possible exception of the 12α-hydroxylase, all the microsomal hydroxylations appear to involve participation of cytochrome P-450 and NADPH-cytochrome P-450 reductase. Several of the hydroxylations have been demonstrated in reconstituted systems consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase and a phospholipid. The specificity of the hydroxylation is determined mainly by the cytochrome P-450 fraction. The cholesterol 7α-hydroxylase catalyzes the major rate-limiting step in the overall conversion of cholesterol into bile acids. The combined activities of the microsomal 26-hydroxylase and the 12α-hydroxylase play a major role in determining the ratio between cholic acid and chenodeoxycholic acid formed from cholesterol. There is a close relationship between the cholesterol 7α-hydroxylase activity and the rate of cholesterol biosynthesis. The regulatory role of the cholesterol 7α-hydroxylase, the 12α-hydroxylase and the microsomal 26-hydroxylase may be correlated with some specific properties of the enzyme systems which differ markedly from the properties of the other hydroxylases involved in biosynthesis and metabolism of bile acids. The influence of different factors on the hydroxylations has been studied and the physiological implications of the different effects are discussed.

Assay of liver microsomal cholesterol 7α-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

Abstract The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-2H3]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at m e 456 (M-90) and m e 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-14C]cholesterol into 5-cholestene-3β,7α-diol.

Influence of dietary bile acids on formation of bile acids in rat.

Abstract Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.

Effects of thyroidectomy and thyroxine treatment on the activity of 12α-hydroxylase and of some components of microsomal electron transfer chains in rat liver

Thyroid hormone increases the rate of formation of total bile acids in rats [ 1 ] . This effect is due mainly to increased production of chenodeoxycholic acid (3a,7adihydroxy-Sfl-cholanoic acid), with a slight inhibitory effect [2,3], or no effect [l] , on the production of cholic acid (3&,7a,l2ar-trihydroxy-5+3choIanoic acid). In this communication we show that the microsomall2or-hydroxylation of 7ol-hydroxycholest-4en-3-one, an intermediate in the biosynthesis of cholic acid from cholesterol [4,5,6] , is decreased by thyroxine treatment and is increased by thyroidectomy. In an attempt to explain the effect of thyroid hormone on the 12~hydroxylation required for cholic acid formation we have measured NADPH-cytochrome c reductase, cytochrome P-450 and cytochrome b, in the liver microsomes of our experimental animals, since all three factors have been shown to participate in the transfer of electrons from reduced pyridine nucleotides in microsomes.

12α-Hydroxylation of 7α-hydroxy-4-cholesten-3-one by a reconstituted system from rat liver microsomes

Abstract A preparation of partially purified cytochrome P-450 from rat liver microsomes was found to catalyze 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one in the presence of NADPH and phosphatidyl choline. The reaction was stimulated two- to four-fold by addition of a preparation of cytochrome P-450 reductase. The reaction was inhibited by carbon monoxide to a considerably less extent than other hydroxylations catalyzed by the reconstituted system. In the presence of optimal concentrations of cytochrome P-450 reductase, cytochrome P-450 prepared from livers of starved rats catalyzed the 12α-hydroxylation more efficiently than cytochrome P-450 prepared from livers of normal rats or rats treated with phenobarbital.

7α-hydroxylation of cholesterol by reconstituted systems from rat liver microsomes

Abstract A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4- 14 C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.