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.     

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.     

Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life

Cholesterol catabolism to bile acids was stimulated in neonatal guinea pigs by feeding 1,11% cholestyramine (CT)-containing diet for 8 weeks. The animals were then switched to standard laboratory diet for an additional 4 weeks. At the end of the laboratory diet period: a) CT-pre-treated guinea pigs continued to excrete significantly higher (p<0.05) amounts of bile acids, b) the activity of hepatic 7α-hydroxylase was significantly elevated (p<0.01) in CT-pre-treated animals, and c) isolated hepatocytes from CT-pre-treated guinea pigs secreted significantly higher (p<0.05) amounts of bile acid when compared to controls during a 4-hour incubation. These data provide biochemical support for our contention that stimulation of cholesterol catabolism during neonatal life can have effects that persist into adult life.     

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.     

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.     

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).     

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%),     

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     

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.     

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.     

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.     

Myrianthic acid: a triterpene acid from the rootwood of Myrianthus arboreus

Arjunolic acid and a new triterpene acid, myrianthic acid, were isolated from the rootwood of Myrianthus arboreus. The structures of the two compounds were elucidated by IR, ¹H NMR and mass spectroscopy.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Successful prediction of the hydrogen bond network of the 3-oxo-12alpha-hydroxy-5beta-cholan-24-oic acid crystal from resolution of the crystal structure of deoxycholic acid and its three 3,12-dihydroxy epimers

Crystal structures of p-xylene-crystallized deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholan-24-oic acid) and its three epimers (3beta,12alpha-; 3alpha,12beta-; and 3beta,12beta-) have been solved. Deoxycholic acid forms a crystalline (P21) complex with the solvent with a 2:1 stoichiometry whereas crystals of the three epimers do not form inclusion compounds. Crystals of the 3beta,12beta-epimer are hexagonal, whereas the 3alpha,12beta-and 3beta,12alpha-epimers crystallize in the P2(1)2(1)2(1) orthorhombic space group. The three hydrogen bond sites (two hydroxy groups, i. e. O3-H, and O12-H, and the carboxylic acid group of the side chain, O24bO24a-H) simultaneously act as hydrogen bond donors and acceptors. The hydrogen bond network in the crystals was analyzed and the following sequences have been observed: two chains (abcabc... or acbacb... ) and two rings (abc or acb), which constitute a complete set of all the possible sequences which can be drawn for an intermolecular hydrogen bond network formed by three hydrogen bond donor/acceptor sites forming crossing hydrogen bonds. The orientation of O3-H (alpha or beta) determines the sequence of the acceptor and the donor groups involved in the pattern: O24a --> O12 --> O3 --> O24b when it is alpha and O24a --> O3 --> O12--> O24B when it is beta. These observations were used to predict the hydrogen bond network of p-xylene-crystallized 3-oxo,12alpha-hydroxy-5beta-cholan-24-oic acid. This compound has two hydrogen bond donor and three potential hydrogen bond acceptor sites. According to the previous sequence set, this compound should crystallize in the monoclinic P21 system, should form a complex with the solvent, O24b should not participate in the hydrogen bond network, and the chain sequence O24a --> O12 --> O3 would be followed. All predictions were confirmed experimentally.     

Oligomeric proanthocyanidins mitigate cholesterol and cholic acid diet–induced hepatic dysfunction in male Sprague Dawley rats

Dysregulated synthesis of hepatic cholesterol is a critical determinant of atherosclerosis. The combination of cholesterol and cholic acid (CC) diet supplementation to animal models is associated with hepatic dysfunction‐mediated atherosclerosis. The current study was designed to investigate the hepatic cholesterol–lowering effects of oligomeric proanthocyanidins (OPC) in CC diet fed rats. CC diet–induced group exhibited significant increase in the hepatic lipid profile, activities of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase (HMGR), PON‐1, LCAT, LPL, and LPO levels, and messenger RNA expression of HMGR, low‐density lipoprotein receptor (LDLr), and HNF‐4α. Administration of OPC (100 mg/kg/bwt) resulted in the significant reduction of lipid profile and HMGR levels, with concomitant increase in the levels of cholesterol‐regulating enzymes and upregulated expression of LDLr and HNF‐4α, which was similar to atorvastatin. Molecular docking studies also revealed that proanthocyanidins had a strong binding affinity to HMGR, similar to atorvastatin. Our findings suggest that OPC regulate the impaired cholesterol metabolism–associated atherosclerosis through hepatic cholesterol–lowering effect.     

Bile acids. LXIV. Synthesis of 5α-cholestane-3α,7α,25-triol and esters of new 5α-bile acids

Interest in the structural requirements of a sterol or bile acid for maximal activity by an hepatic microsomal steroid 12α-hydroxylase prompted the preparation of 5α-cholestane-3α, 7α, 25-triol and 5α-analogs of 3α, 7α-dihydroxy-5β-cholane-24-carboxylic acid. Methyl 3α, 7α-dihydroxy-5β-cholane-24-carboxylate derived from methyl chenodeoxycholate via the Arndt-Eistert reaction was allomerized with Raney nickel in boiling p-cymene to provide a number of products of which methyl 3,7-dioxo-5β- and 5α-cholane-24-carboxylates, methyl 3-oxo-7α-hydroxy-5β-and 5α-cholane-24-carboxylates, were identified. Reduction with K-Selectride of methyl 3-oxo-7α-hydroxy-5β-cholane-24-carboxylate, provided a high yield of methyl 3α, 7α-dihydroxy-5α-cholane-24-carboxylate. Treatment of this ester with an excess of methyl magnesium iodide afforded 5α-cholestane-3α, 7α, 25-triol. The products were characterized by thin-layer and gas liquid chromatography, proton resonance, infrared and mass spectrometry.     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2 weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.