Hepatic 3 alpha-dehydrogenation and 7 alpha-hydroxylation of deoxycholic acid in the guinea-pig

The metabolic fate of exogenous deoxycholate administered either intraperitoneally or intragastrically to male Hartley guinea-pigs was investigated. Two animals received a constant infusion of [24-14C]deoxycholate through an intraperitoneal catheter for 2 hr. Bile was quantitatively collected in 30 min samples during infusion and for 2 additional hours. Each bile sample was analyzed for composition and radioactivity. Five animals received for 15 days, through an intragastric catheter, 35 mg/kg/day of deoxycholate. The biliary bile acid composition was compared with that of a sham-operated control group. The studies with both animal models indicated that guinea-pigs, as the only species so far known, extensively oxidize deoxycholate to form 3-oxo,12 alpha-hydroxy-cholanic acid, which is secreted in bile mostly conjugated with glycine. In addition a small fraction (approx. 7%) of the administered deoxycholate is 7 alpha hydroxylated to form cholic acid. The metabolites being more hydrophilic than administered deoxycholate, it is suggested that guinea-pig liver counteracts the adverse increase in bile acids detergency, which follows deoxycholate administration, by converting most of the latter into less detergent compounds.     

7 alpha-hydroxylation of 27-hydroxycholesterol in human liver microsomes.

Human liver microsomes were found to catalyze 7 alpha-hydroxylation of 27-hydroxycholesterol at a rate of up to about 0.2 nmol/mg protein per min. The product of the reaction, 5-cholestene-3 beta, 7 alpha, 27-triol, was identified by means of combined gas chromatography-mass spectrometry. Liver microsomes from two patients with an upregulated cholesterol 7 alpha-hydroxylase, did not have higher 7 alpha-hydroxylase activity towards 27-hydroxycholesterol than those from untreated patients, suggesting that the 7 alpha-hydroxylase active towards 27-hydroxycholesterol is not the same as that active towards cholesterol. The mitochondrial fraction of liver from untreated patients and patients treated with cholestyramine, had negligible 7 alpha-hydroxylase activity towards 27-hydroxycholesterol less than 0.01 nmol/mg protein per min). The results are in accord with the possibility that there is a pathway to bile acids in human liver in which the first step is a 27-hydroxylation of cholesterol.     

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients

BACKGROUND/AIM: Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN: Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS: Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS: Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.     

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

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.     

Effect of different dietary triglycerides on 7alpha-hydroxylation of cholesterol and other mixed-function oxidations

The effect of a diet containing triglycerides of different fatty acid composition on hepatic 7alpha-hydroxylation of cholesterol was studied. 7alpha-Hydroxylation of exogenous as well as endogenous cholesterol was significantly lower in the liver of rats fed trilinolein and triolein than in those fed tripalmitin and trierucin. The concentration of cytochrome P-450 in liver microsomes was significantly lower in the rats fed tripalmitin and trierucin than in those fed triolein and trilinolein. The inhibitory effect of triolein and trilinolein on 7alpha-hydroxylation of cholesterol and the stimulatory effect of these triglycerides on the concentration of cytochrome P-450 was not due to the small amounts of peroxides present in the unsaturated triglycerides. Thus addition of the antioxidant butylated hydroxyanisol did not change the general pattern with respect to 7alpha-hydroxylation and concentration of cytochrome P-450. However, a diet consisting of peroxidized linoleic acid further decreased 7alpha-hydroxylation of cholesterol. The difference between the effect obtained with triolein and trilinolein on the one hand and trierucin and tripalmitin on the other was observed also in experiments with lower concentrations of fat in the diet and in experiments with different lighting conditions and feeding patterns. The inverse relation between cytochrome P-450 and 7alpha-hydroxylation of cholesterol, as well as results obtained with substrates for mixed-function oxidation other than cholesterol suggest that most of the changes observed due to the different diets are specific for 7alpha-hydroxylation of cholesterol. The level of cholesterol 7alpha-hydroxylase activity was found to be better related to the degree of absorption of fat than to total amount of absorbed fat or degree of unsaturation of the fat. The results are discussed in relation to previous knowledge concerning mechanisms regulating biosynthesis of bile acid.     

Effect of modification of thyroid function on cholesterol 7 alpha-hydroxylation in rat liver.

Hepatic activities of cholesterol synthesis and cholesterol 7 alpha-hydroxylation were determined in hyper- and hypo-thyroid rats after oral administration of glucose or cholesterol. Increases in activities of both cholesterol synthesis and cholesterol 7 alpha-hydroxylation induced by glucose administration were enhanced by pretreatment with thyroid powder but suppressed by pretreatment with thiouracil. The enhancement of 7 alpha-hydroxylation was produced by a relatively small amount of thyroid powder, but high doses were required to increase cholesterol synthesis. On the other hand, the suppression of 7 alpha-hydroxylation was brought about by a low dose of thiouracil, but high doses were required to decrease cholesterol synthesis. Although cholesterol synthesis increased similarly in both hypo- and hyper-thyroid rats after glucose administration, hydroxylase activity in hypothyroid rats began to increase more slowly and was always lower than that in hyperthyroid rats. Thus it is concluded that cholesterol 7 alpha-hydroxylase activity is more sensitive to thyroid function than are activities of cholesterol-synthetic enzymes. When exogenous cholesterol was given, hypothyroid rats showed a larger increase in serum cholesterol concentration than hyperthyroid rats, and there was an inverse relationship between serum cholesterol concentrations and hepatic cholesterol 7 alpha-hydroxylase activities.     

Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7 alpha-hydroxylation.

Rat hepatic cytochrome P-450 form 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) and P-450 form RLM2 (testosterone 15 alpha-hydroxylase; P-450 gene IIA2) are 88% identical in primary structure, yet they hydroxylate testosterone with distinct and apparently unrelated regioselectivities. In this study, androstenedione and progesterone were used to assess the regioselectivity and stereospecificity of these two P-450 enzymes towards other steroid substrates. Although P-450 RLM2 exhibited low 7 alpha-hydroxylase activity with testosterone or progesterone as substrate (turnover number less than or equal to 1-2 nmol of metabolite/min per nmol of P-450), it did catalyse androstenedione 7 alpha-hydroxylation at a high rate (21 min-1) which exceeded that of P-450 3 (7 min-1). However, whereas P-450 3 exhibited a high specificity for hydroxylation of these steroids at the 7 alpha position (95-97% of total activity), P-450 RLM2 actively metabolized these compounds at four or more major sites including the nearby C-15 position, which dominated in the case of testosterone and progesterone. The observation that androstenedione is actively 7 alpha-hydroxylated by purified P-450 RLM2 suggested that this P-450 enzyme might make significant contributions to microsomal androstenedione 7 alpha-hydroxylation, an activity that was previously reported to be associated with immunoreactive P-450 3. Antibody inhibition experiments were therefore carried out in liver microsomes using polyclonal anti-(P-450 3) antibodies which cross-react with P-450 RLM2, and using a monoclonal antibody that is reactive with and inhibitory towards P-450 3 but not P-450 RLM2. P-450 3 was thus shown to catalyse only around 35% of the total androstenedione 7 alpha-hydroxylase activity in uninduced adult male rat liver microsomes, with the balance attributed to P-450 RLM2. The P-450-3-dependent 7 alpha-hydroxylase activity was increased to approximately 65% of the total in phenobarbital-induced adult male microsomes, and to greater than 90% of the total in untreated adult female rat liver microsomes. These observations are consistent with the inducibility of P-450 3 by phenobarbital and with the absence of P-450 RLM2 from adult female rat liver respectively. These findings establish that P-450 RLM2 and P-450 3 can both contribute significantly to microsomal androstenedione 7 alpha-hydroxylation, thus demonstrating that the 7 alpha-hydroxylation of this androgen does not serve as a specific catalytic monitor for microsomal P-450 3.     

Microbial 7α-Hydroxylation of 3-Ketobisnorcholenol

The transformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one to 7α-22-dihydroxy-23,24-bisnorchol-4-en-3-one by Botryodiploida theobromae, Lasiodiplodia theobromae, and various Botryosphaeria strains is described. Factors affecting the reaction were incubation temperature, sonication of the substrate, and addition of 2,2′-dipyridyl, extra carbohydrate, and Amberlite XAD-7. The enzyme responsible for the reaction appeared to be very specific and was not characteristic of all members of the genera listed above.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

Microbial degradation of 7-ketocholesterol

7-Ketocholesterol (7KC) is an oxidized derivative of cholesterol suspected to be involved in the pathogenesis of atherosclerosis and possibly Alzheimer’s disease. While some oxysterols are important biological mediators, 7KC is generally cytotoxic and interferes with cellular homeostasis. Despite recent interest in preventing the accumulation of 7KC in a variety of matrices to avoid adverse biological effects, its microbial degradation has not been previously addressed in the peer-reviewed literature. Thus, the rate and extent of biodegradation of this oxysterol was investigated to bridge this gap. A wide variety of bacteria isolated from soil or activated sludge, including Proteobacterium Y-134, Sphingomonas sp. JEM-1, Nocardia nova, Rhodococcus sp. RHA1, and Pseduomonas aeruginosa, utilized 7KC as a sole carbon and energy source, resulting in its mineralization. Nocardia nova, which is known to produce biosurfactants, was the fastest degrader. This study supports the notion that microbial catabolic enzymes could be exploited to control 7KC levels in potential biotechnological applications for agricultural, environmental, or medical use.     

The 1 alpha-hydroxylation of 24-hydroxyvitamin D3 by chick kidney homogenates

Tritium-labeled 24(R)-hydroxyvitamin D3 and 24(S)-hydroxyvitamin D3 were chemically synthesized and the 1 alpha-hydroxylation of these compounds by chick kidney homogenates was studied. A marked stereospecific preference with regard to the orientation of the hydroxyl functionality on carbon-24 was noted: while the 24(R)-epimer could be 1 alpha-hydroxylated in readily detectable amounts, the 24(S)-epimer was not hydroxylated. Thus, 1.2 micrograms of 1 alpha,24(R)-dihydroxyvitamin D3 was isolated and its structure confirmed by mass spectrometry. The relative rate of 1 alpha-hydroxylation of 125 nM substrate tritiated 24(R)-hydroxyvitamin D3 and 25-hydroxyvitamin D3 (the presumed natural substrate for the renal 1 alpha-hydroxylase) was 1:6.7.     

In vivo evaluation of cholesterol 7 alpha-hydroxylation in humans: effect of disease and drug treatment

7 alpha-Hydroxylation of cholesterol is a stereospecific reaction consisting of the replacement of the 7 alpha-hydrogen with a hydroxyl group. When cholesterol labeled with tritium at the 7 alpha position is administered, the hydroxylation of the substrate will result in the loss of tritium which in turn will label the body water. The rate of tritium enrichment of the body water could thus give a quantitative estimate of the hydroxylation rate. This study describes the validation of the procedure with some 21 studies performed on 15 subjects in different conditions. [7 alpha-3H]cholesterol was administered intravenously in 50 ml of plasma and thereafter blood was sampled at timed intervals for 4 to 5 days. The rate of the hydroxylation of cholesterol was calculated from the time course of the specific activities of plasma cholesterol and body water after tracer administration and was expressed as 7 alpha-hydroxycholesterol formed/24 hr. Calculated values of hydroxylation in three control subjects (493 +/- 206), five patients with hyperlipoproteinemia (539 +/- 168), and seven cirrhotic patients (153 +/- 136) are in good agreement with figures reported for bile acid synthesis determined with other techniques. Cholesterol 7 alpha-hydroxylation rate is reduced in patients with cirrhosis, the impairment being related to the severity of the disease. Cholestyramine administered to one subject for 4 weeks produced a threefold increase of the hydroxylation. Administration of chenodeoxycholic acid resulted in a 50% decrease, whereas that of ursodeoxycholic did not produce consistent changes of the hydroxylation rate. The results support the current view that 7 alpha-hydroxylation of cholesterol is rate-limiting in the synthesis of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbiological 1 alpha-hydroxylation of norethisterone.

Acremonium strictum and Acremonium kiliense converted norethisterone predominantly into its 1α-hydroxyl derivative. Chemical and spectroscopic /UV, IR, PMR, MS, ORD, CD/ methods were used in establishing the structure and stereochemistry of the product. The 1α-hydroxyl group is shown to be axially oriented in the preferred, normal half-chair ring A conformation.     

Microbial Preparation of Guanosine 7-N-Oxide

KS-504a inhibited bovine brain calmodulin-dependent cyclic nucleotide phosphodieaterase (CaM-PDE) with an IC₅₀ value of 122 μm. The inhibition was reversed by a high concentration of calmodulin. Cal modulin-independent activities of the enzyme were not affected by the compound at the same concentration ranges. Ca²⁺-dependent interaction of the compounds with calmodulin was shown using hydrophobic fluorescence probes. These data indicated that the compound exerted its effects on CaM-PDE by interacting with calmodulin. KS-504a also inhibited other calmodulin-dependent enzymes at different concentration ranges; myosin light chain kinase was inhibited at the lowest concentrations with an IC₅₀ value of 6.3 μm. The inhibition mechanism was competetive with respect to calmodulin and non-competetive to ATP.     

Dehydroepiandrosterone 7alpha-hydroxylation in human tissues: possible interference with type 1 11beta-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7alpha-hydroxy-DHEA that is a substrate for 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which exists in the same tissues and carries out the inter-conversion of 7alpha- and 7beta-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11beta-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7alpha-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11beta-HSD1. Human skin was selected then and used to test its ability to produce 7alpha-hydroxy-DHEA, and to test the interference of 7alpha- and 7beta-hydroxy-DHEA and 7-oxo-DHEA with the 11beta-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11beta-HSD1 in the liver, skin and tonsils. DHEA was readily 7alpha-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11beta-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA was competitive with a Ki at 1.85+/-0.495 and 0.255+/-0.005 microM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a Ki at 1.13+/-0.15 microM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7alpha-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Factors affecting the 16 alpha-hydroxylation of estrone 3-sulfate by guinea pig liver microsomes

The estrone 3-sulfate 16 alpha-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O2 ratio of 0.64 caused 50% inhibition of activity. Since inhibition was also obtained in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate . HCl it seems likely that the hydroxylase is a cytochrome P450 containing system. A fourfold increase in enzyme activity was brought about by 40 mM Mg2+ or Ca2+ while the same concentration of Mn2+ resulted in a twofold increase. Lesser increases were seen with Na+ or K+ and complete inhibition was obtained in the presence of Fe2+, Cu2+, or EDTA. When assayed in the presence of detergent concentrations sufficiently small to guard against cytochrome P450 destruction, it was found that Cutscum, Triton X-100, and Triton N-101 each caused greatest inhibition of enzyme activity. Lesser inhibition was apparent in the presence of Miranol H2M, cholate, or deoxycholate. The nonionic detergent, Brij 35, caused least inhibition of all and, when hepatic microsomes were treated higher concentrations of Brij 35, about 80% of protein and over 95% cytochrome P450 were to be found in the 100 000 X g supernatant. Microsomal activity was more stable when stored at -20 degrees C in buffer containing glycerol, EDTA, and dithiothreitol than in buffer alone. Under best conditions only 10% of the hydroxylase activity was lost in one week.     

Evidence for 16alpha-hydroxylation of estrone 3-sulphate by guinea pig liver slices.

[6,7-3H,35S]Estrone 3-sulfate (E13S) of 3H/35S = 3.57 was incubated with female guinea pig liver slices. Small amounts of free steroid and estrone-3-glucuronide, each containing 3H, were found. In addition, E13S, 17beta-estradiol 3-sulfate, and a 'disulfate' fraction, with 3H/35S = 4.4, 4.3, and 4.7, respectively, were also isolated from the incubated tissue. The latter fraction was a major metabolite and about 45% of it consisted of 'disulfates' of 16alpha-hydroxyestrone and estriol, thus providing strong evidence for 16alpha-hydroxylation in guinea pig liver slices.