Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

7β,12β-dihydroxy-5β-cholan-24-oic acid as an internal standard for quantitative determination of bile acids by gas chromatography

In order to find an artificial internal standard compound for quantitative determination of bile acids by gas chromatography, 7α,12α-,7α, 12β-, 7β,12α- and 7β,12β-dihydroxy-5β-cholan-24-oic acids were chemically synthesized with cholic acid (1) as the first starting material. The gas chromatographie retention time of 7β,12β-dihydroxy-5β-cholan-24-oic acid (ββ-isomer) was more different from that of natural bile acids than the other isomers. Moreover, ββ-isomer was extracted in the same fraction as the bile acids from urine, and no urinary substance had the same retention time as ββ-isomer. No artifact was produced from ββ-isomer during the analysis procedure. It was concluded that the ββ-isomer is an internal standard compound with certain advantages for the quantitative determination of bile acids in urine by gas chromatography, irrespective of the recovery rate during the analysis procedure.     

Synthesis, intestinal absorption and metabolism of sarcosine conjugated ursodeoxycholic acid

Sarcosine conjugated ursodeoxycholic acid (SUDC) was synthesized and its intestinal absorption and metabolism were studied in rat and hamster. Intestinal absorption study using bile fistula rat shows that more than 90% of SUDC administered intraduodenally was excreted in the bile within 24 hr. No change of the administered bile acid was seen during the absorption from the intestine, the passage of the liver, and the excretion into the bile. When [24-14C]SUDC and [11,12-3H2]-ursodeoxycholic acid were administered orally to a hamster, more than 95% of both the administered 14C and 3H were recovered from the feces within 6 days. Most (77%) of the fecal 14C-labeled compound was SUDC, whereas 95% of the fecal 3H-labeled compound was unconjugated lithocholic acid. These results indicate that SUDC, unlike taurine or glycine conjugated bile acid, resists bacterial deconjugation and 7-dehydroxylation.     

Methoxylation of 3β,7α-dihydroxychol-5-en-24-oic acid,a key intermediate of chenodeoxycholic acid biogenesis,compared with that of its 7β-epimer

The conventional methods of gas liquid chromatography or mass spectrometry failed to be useful for the identification of the biliary 3β, 7α-hydroxychol--en-24-oic acid, a key intermediate of chenodeoxycholic acid biogenesis. It has been preliminarily reported that this acid in human bile was successfully identified by gas chromatography-mass spectrometry, after the methoxylation of its allyl alcohol group. Physical as well as spectral properties of the methoxylation products derived from the acid were reported, compared with those from its 7β-epimer.     

Antibacterial properties of bile acid oxazoline compounds

Chenooxazoline³ (50–100 μM) inhibited (>50%) both 7α and 7β-dehydroxylase activities in whole cells and cell extracts of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (>50 μM) and methylchenooxazoline (>25 μM) but not lithooxazoline (≤100 μM) inhibited growing cultures of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (100 μM) also inhibited the growth of certain members of the genera $\text{Eubacterium}$, $\text{Clostridium}$, $\text{Bacteroides}$ and $\text{Staphylococcus}$ but not $\text{Pseudomonas}$, $\text{Escherichia}$, $\text{Salmonella}$ or the eucaryotic microorganism, $\text{Saccharomyces cerevisiae}$ (_< 400 μM).     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

Assays for cholesterol 7 alpha-hydroxylase and 12 alpha-hydroxylase using high performance liquid chromatography

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7 alpha-hydroxylating) and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (enzyme not yet registered) are described. 7 alpha-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12 alpha-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.     

Methoxylation of methyl 3α,7α-dihydroxychol-4-en-24-oate and its 3β-epimer. —a contribution to chenodeoxycholic acid biogenesis

By the conventional methods of gas liquid chromatography (GLC) as well as mass spectrometry, 3β,7α-dihydroxychol-5-en-24-oic acid (Δ⁵-acid), a key intermediate of chenodeoxycholic acid biogenesis and its metabolic by-product, 3α,7α-dihydroxychol-4-en-24-oic acid (Δ⁴-acid) have not yet been identified as such probably due to thermal decomposition. However, taking advantage of the observation that they are readily methoxylated in methanoi containing a trace of acids, their individual methoxy-compounds were easily prepared and proved to be useful for their identification, even though they are present in minimal amounts as was the case with the human or hen bile. The present paper reported physical as well as spectral properties of the methoxy-compounds derived from methyl 3α,7α-dihydroxychol-4-en-24-oate, compared with those of its 3β-epimer     

Sterol and bile acid metabolism during development. 1. Studies on the gallbladder and intestinal bile acids of newborn and fetal rabbit

Bile acid composition and content in the intestine and gallbladder of newborn and fetal rabbits were investigated. Unlike the circumstances in adult rabbits, the bile acids were conjugated with both taurine and glycine. The major bile acids of the fetus and newborn rabbit were cholic acid, chenodeoxycholic acid, and deoxycholic acid. This is different from the known bile acid composition of adult rabbits, in which deoxycholic acid is the major bile acid (> 80%). The proportion of chenodeoxycholic acid was higher in the fetal than in the newborn tissues. The total bile acid pool in the newborn was higher than in the fetus. In the fetus, large proportions of bile acids (60.9%) were associated with the gallbladder fraction, whereas in the newborn the bulk of the bile acids were found with the intestinal fraction (64.4%),     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

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

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.     

Regio- and stereo-selective 1β-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1β-hydroxylation of lithocholic acid (LCA) to produce 1β-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1β-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1β-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp³-H activation by the developed P450 BM3 mutants.     

Intermediates in 16-androstene biosynthesis in neonatal porcine testis

The metabolism of pregnenolone has been studied in microsomal preparations of testes from 3-week old piglets. Metabolites identified after derivatization by capillary gas-chromatography and further by combined gas chromatography-mass spectrometry were: 17-hydroxy- and 16 alpha-hydroxypregnenolone, dehydroepiandrosterone, 5-androstenediols, 5-pregnenediol and andien-beta. Pregnenolone and 16-dehydro-pregnenolone, previously not separable by other methods, were successfully resolved as methyl oximetrimethylsilyl ethers using capillary column gas-chromatography.     

The conversion of testosterone to 7α-hydroxy-testosterone by incubated rat testes

Incubations of testes of adult rats with testosterone yield rather important amounts of a very polar metabolite which is identified as 7α-hydroxytestosterone. The identification of the metabolite is based on chromatography, spectrophotometry, fluorimetry, counter current distribution and NMR spectrometry.     

The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

Ontogeny of mitochondrial steroidogenesis in the guinea pig gonad

To determine whether steroidogenesis in the developing guinea pig may be limited by the formation of pregnenolone, cholesterol side chain cleavage activity was ascertained at various stages of development. The conversion of [1,2-³H]cholesterol to [1,2-³H]pregnenolone was detected in mitochondria isolated from fetal guinea pig ovaries and testes as early as day 35 of gestation, while no metabolism was noted in day 30 animals. Moreover, no [l,2-³H]progesterone was formed during the 60 minute incubation. From day 35 of gestation to the day of birth, the percentage of pregnenolone formed per testis (total activity) increased, while total activity in the ovary declined. In contrast, gonadal mitochondria from adult guinea pigs converted cholesterol to both pregnenolone and progesterone and total activity in these animals was substantially higher than in their fetal counterparts. In the three females examined, the rate of pregnenolone and progesterone synthesis varied according to the stage of the estrous cycle during which these animals were sacrificed. Conversion of pregnenolone to progesterone was most rapid in the early luteal phase animal, while conversion of cholesterol to pregnenolone occurred more rapidly in the periovulatory animals than in ovarian mitochondria from the late luteal phase of the cycle. The results indicate that during prenatal and postnatal development of the gonad, cholesterol side chain cleavage activity changes and that mitochondria may acquire a Δ⁵-3β-hydroxysteroid dehydrogenase.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Metabolism of 4-14 C-progesterone in the adult female chimpanzee.

The metabolism of 4- 14-C-progesterone was studied in two adult female chimpanzees (Pan troglodytes). After intravenous injection of 4- 14-C-progesterone, urine was collected for 5 days. The urinary conjugates were hydrolyzed with Glusulase and the extracts purified by chromatography on silica gel, paper and alumina and then crystallized to constant specific activity with or without carrier steroid. The major metabolite in both experiments was pregnanediol which accounted for 39.8% and 49.5% of the recovered dose respectively. Pregnanolone (0.6% and 2.1%) and pregnanetriol (0.1% and 1.5%) were also isolated in smaller quantities. These data suggest that the pattern of progesterone metabolism in the chimpanzee is similar to that of man in that pregnanediol is the major metabolite whereas androsterone is not.