Rat Liver Microsomal Fraction Research Articles

Antiperoxidative, anti-inflammatory, and antimutagenic activities of ethanol extract of the mycelium of Ganoderma lucidum occurring in South India.

Free radical mediated genetic instability is widely thought to be a major etiological factor for initiation of carcinogenesis. Mushrooms represent a largely untapped source of powerful new pharmaceutical products. In the present study, we examined the antiperoxidative, anti-inflammatory, and antimutagenic activities of the ethanol extract of the mycelium of a medicinal mushroom, Ganoderma lucidum, occurring in south India. Antiperoxidative activity was evaluated using Fe(2+)-ascorbate-induced lipid peroxidation in rat liver homogenate and a phorbol ester (croton oil)-induced lipid peroxidation in mouse skin. Antiinflammatory activity was evaluated against carrageenan-induced acute and formalin-induced chronic inflammatory paw edema in mouse and phorbol ester-induced mouse skin inflammation. Antimutagenic activity was determined by the Ames mutagenicity assay using histidine mutant of Salmonella typhimurium strains TA 98, TA100, and TA102. Sodium azide (NaN(3)), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NPD), and benzo[a]pyrene (B[a]P) were used as the mutagens. The extract showed significant inhibition of Fe(2+)-induced peroxidation of lipid in rat liver (IC(50) 510 +/- 22 microg/ml) and 37% inhibition of croton oil-induced peroxidation on the mouse skin at 20 mg/0.1 ml/skin. Carrageenan-induced acute and formalin-induced chronic inflammatory edema were inhibited by 56 and 60%, respectively, by the extract at 1,000 mg/kg body wt (i.p). The extract at a concentration of 5 mg/plate showed inhibition of mutagenicity elicited by direct acting mutagens, NaN(3) (55.5 and 75.7%) and MNNG (50.0 and 57.5%) for S. typhymurium strains TA100 and TA102, respectively. The extract at the same concentration also inhibited mutagenicity elicited by NPD (52.4 and 64.2%) and B[a]P (60.7 and 59.6%) for TA98 and TA100 strains, respectively. The B[a]P was activated in the presence of rat liver microsomal (S9) fraction. The results of our study revealed that ethanol extract of Ganoderma lucidum mycelium possessed significant antiperoxidative, antiinflammatory, and antimutagenic activities. The findings suggest a medicinal use for the ethanol extract of the mycelium of G. lucidum occurring in South India.

Enantioselective analysis of thiobencarb sulfoxide produced by metabolism of thiobencarb by hydroxypropyl-γ-cyclodextrin modified micellar electrokinetic chromatography

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of thiobencarb sulfoxide, which is produced by S-oxygenation of thiobencarb, a herbicide, using γ-cyclodextrin together with sodium dodecyl sulfate. Factors affecting chiral resolution and migration time of thiobencarb sulfoxide were studied. The optimum running conditions were found to be 20 mM phosphate-5 mM borate buffer (pH 8.5) containing 60 mM hydroxypropyl-γ-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of +20 kV at 20°C using direct detection at 220 nm. Under these conditions, the resolution (Rs) of racemic thiobencarb sulfoxide was approximately 1.7. Thiobencarb was treated with a rat liver microsomal fraction containing cofactors (known as S9mix). The ratio between (+)- and (-)-thiobencarb sulfoxide was found to be 15:85. It was also found that the ratio between (+)-and (-)-thiobencarb sulfoxide produced in soil spiked with thiobencarb was 3:7. These results indicated marked enantioselectivities for these metabolisms. The activities of thiobencarb, (+)- and (-)-thiobencarb sulfoxides on 5α-dihydrotestosterone-and 17β-estradiol-induced transcriptions were also investigated. Whereas thiobencarb and (+)-thiobencarb sulfoxide did not show any activities, (-)-thiobencarb sulfoxide showed significant anti-estrogenic and anti-androgenic activities, suggesting that thiobencarb sulfoxide can act as both an enantioselective anti-estrogen and an enantioselective anti-androgen.

Genotoxic evaluation of the furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene in cultured human lymphocytes

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-βNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15μg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-βNF) were observed mainly in the cultures treated without the S9 fraction.

Isolation of peroxisomal subpopulations from mouse liver by immune free-flow electrophoresis

Peroxisomes (PO) are a heterogeneous population of cell organelles which in mammals are most abundant in liver and kidney. Commonly, differential and density gradient centrifugation are used for their isolation which, however, give only rise to the so-called "heavy" PO with a buoyant density of 1.22-1.24 g/cm(3). Subpopulations other than the heavy PO which are also present in both of these tissues have escaped adequate purification because of their sedimentation characteristics which are close to those of other major organelles, in particular microsomes. Since the purification of these subpopulations has become an essential task in view of the putative importance of peroxisomal subpopulations in the biogenesis of this organelle, alternatives to density gradient centrifugation are required. Recently, we have introduced such a novel approach, named immune free flow electrophoresis (IFFE). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of immunoglobulin G (IgG) molecules. pH-values other than 8.0 proved to be less efficient, probably because IgG molecules only focus at pH 8.0 but are scattered at any other. Applying IFFE to heavy and light mitochondrial as well as microsomal fractions of rat liver not only regular PO (rho = 1.22-1.24 g/cm(3)) but also other subpopulations could be isolated. To substantiate the validity of this approach, we now have subfractionated mouse liver homogenates accordingly. Of the PO subpopulations collected, mainly that obtained from the heavy mitochondrial fraction differed in its composition of matrix and membrane proteins as revealed by immunoblotting. This is in line with the data reported on rat liver thus confirming the potential of IFFE in the isolation of distinct subpopulations of hepatic PO.

Formation of retinoyl-CoA in rat tissues.

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro and in tissues in vivo. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and subsequent transfer and covalent binding of the retinoyl moiety to protein. However, such reactions are not well known. To gain further insight into retinoylation, we studied the synthesis of retinoyl-CoA, the first step in this multi-stage process. The formation of [(3)H]-retinoyl-CoA was determined in incubation mixtures containing rat liver extract, [(3)H]-RA, ATP, CoA, and MgCl(2). No retinoyl-CoA was formed in the presence of boiled extract, or in the absence of ATP, CoA, or MgCl(2) (a divalent cation). A greater amount of retinoyl-CoA was obtained from microsomal fractions of rat liver than from other subfractions. The presence of retinoyl-CoA was also detected in extracts prepared from rat testis, kidney, brain, spleen, and pancreas. The level of retinoylation in various tissue extracts was related directly to the amount of retinoyl-CoA formed. V(max) and K(m) values for RA in the formation of liver retinoyl-CoA were estimated to be 1.0 x 10(-4) micromol/min/mg protein and 24 nM, respectively. Synthesis of retinoyl-CoA was suppressed by fatty acids and fatty acyl-CoAs. These results indicate that ATP-dependent generation of retinoyl-CoA occurs in rat tissues and may play a significant physiological role in RA actions mediated by retinoylation.

Simultaneous Detection of Cholyl Adenylate and Coenzyme A Thioester Utilizing Liquid Chromatography/Electrospray Ionization Mass Spectrometry

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 A particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.

The in vitro effect of Helichrysi flos on microsomal lipid peroxidation

Helichrysum arenarium (L.) Moench has long been known as a medicinal plant in Europe for its cholagogue, choleretic, hepatoprotective and detoxifying activities. Antioxidant properties of its main phenolics, flavonoids may be supposed to be responsible for these effects. The aim of this study was to verify the antioxidant properties of lyophilized water extracts with different polyphenol and flavonoid contents from inflorescences. The effects of natural extracts on microsomal fraction of rat liver were examined. Enzymatically induced lipid peroxidation and NADPH cytochrome c reductase activity in liver microsomes were measured by spectrophotometric methods. Results were compared with the activity of silibinin flavonoid, the main agent of well-known milk thistle ( Silybum marianum L.). The natural plant extracts diminish the enzymatically induced lipid peroxidation in a concentration-dependent manner and reduce the cytochrome c dose dependently. The sample with higher polyphenol and flavonoid contents showed more stimulation of NADPH cytochrome c reductase. The lyophilized Helichrysi flos extracts proved to be more effective compared to silibinin in examined concentrations.

Mutagenicity and antioxidant assessment of Stachitarpheta jamaicensis (L.) Vahl.

Stachitarpheta jamaicensis (L.) Vahl. is a member of the Verbenaceae commonly used in Cuba, mainly as vermifugue and against diarrhoea. The mutagenic potential of a hydroalcohol extract of its aerial parts was assessed in vitro using the Salmonella/microsome assay and in vivo in the mouse bone marrow micronucleus test. No positive response was observed in a battery of four Salmonella typhimurium strains employed: TA 1535, TA 1537, TA 98 and TA 100, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation. In the same way, no increase in the micronucleus frequency in mitotic erythropoietic tissue was observed when animals were administered the extract orally in doses of 500, 1000 and 2000 mg/kg. The extract inhibited lipid peroxidation in the rat liver microsomal fraction (IC(50) = 3.6 microg/mL) but it does not seem to be an effective.OH radical scavenger (IC(50) = 76.7 microg/mL). Noteworthy, it increased in a dose dependent way the level of revertant colonies in E. coli IC 203, a strain sensitive to oxidative mutagenesis, when assayed together with hydrogen peroxide and ferrous sulphate, which suggests a pro-oxidant action.

Metabolism of DHEA by Cytochromes P450 in Rat and Human Liver Microsomal Fractions

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16α-hydroxy-DHEA, 7α-hydroxy-DHEA, and 7-oxo-DHEA. While 16α-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.

Ethanol Enhances Retinoic Acid Metabolism Into Polar Metabolites in Rat Liver via Induction of Cytochrome P4502E1

Long-term and excessive ethanol intake results in decreased plasma and hepatic levels of retinoic acid (RA), the most active derivative of vitamin A. The decrease of RA by ethanol treatment has been proposed to be a cytochrome P450 enzyme (CYP)-dependent process. However, the role of the major ethanol-induced CYP, CYP2E1, in the metabolism of RA has not been defined. In vitro incubations of RA with microsomal fractions of liver tissue containing CYPs from either ethanol-exposed or non-ethanol-exposed rats were carried out using chemical inhibitors and antibodies against various CYPs. In vivo, both ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole, a specific CYP2E1 inhibitor, for 1 month. RA and its catabolic metabolites were analyzed by high-performance liquid chromatography and spectral analysis. Incubation of RA with the liver microsomal fraction from ethanol-exposed rats resulted in greater disappearance of RA and increased appearance of 18-hydroxy-RA and 4-oxo-RA compared with control rat liver microsomal fractions. The enhancement of RA catabolism by ethanol was inhibited by both CYP2E1 antibody and specific inhibitors (allyl sulfide and chlormethiazole) in a dose-dependent fashion, whereas the metabolism of RA into polar metabolites was abolished completely by nonspecific CYP inhibitors (disulfiram and liarozole). Furthermore, treatment with chlormethiazole in ethanol-fed rats in vivo restored both hepatic and plasma RA concentrations to normal levels. Ethanol-induced CYP2E1 plays a major role in the degradation of RA, which may provide a possible biochemical mechanism for chronic and excessive ethanol intake as a risk for both hepatic and extrahepatic cell proliferation and carcinogenesis.

Antimutagens in gaiyou (Artemisia argyi levl. et vant.).

Antimutagens from gaiyou (Artemisia argyi Levl. et Vant., Compositae) were examined. The methanol extract prepared from aerial parts of this plant strongly reduced the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), when Salmonella typhimurium TA98 was used in the presence of the rat liver microsomal fraction. The antimutagens were purified chromatographically while monitoring the antimutagenic activity against Trp-P-2 with a modified Ames test employing a plate method. This purification resulted in the isolation of four strong antimutagens, 5,7-dihydroxy-6,3',4'-trimethoxyflavone (eupatilin), 5, 7,4'-trihydroxy-6,3'-dimethoxyflavone (jaceosidin), 5,7, 4'-trihydroxyflavone (apigenin) and 5,7, 4'-trihydroxy-3'-methoxyflavone (chrysoeriol) from the methanol extract. These antimutagenic flavones exhibited strong antimutagenic activity against not only Trp-P-2 but also against other heterocyclic amines, such as 3-amino-1,4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA(alpha)C) in S. typhimurium TA98. In contrast, they did not exhibit antimutagenic activity against benzo[a]pyrene (B[a]P), 4-nitroquinoline-1-oxide (4-NQO), 2-aminofluorene (2-AF), 2-nitrofluorene (2-NF) or furylfuramide (AF-2) in S. typhimurium TA98, or B[a]P, 4-NQO, 2-NF, AF-2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium azide (SA) in Salmonella typhimurium TA100, whereas they decreased the mutagenicity caused by aflatoxin B(1) (AFB(1)) and 2-aminoanthracene (2-AA) in both of these tester strains. Regarding the structure-activity relationship, the tested flavones had distinct differences in the intensities of their antimutagenic activities according to the differences of their substitution patterns. Namely, the intensity of antimutagenic activities against Trp-P-2 decreased in the order of: 5,7,3',4'-tetrasubstituted flavones (IC(50): <0.1 mmol/plate), 5,7,4'-trisubstituted flavones (IC(50): 0.120-0.260 mmol/plate), 5,6,7,3',4'-pentasubstituted flavones (IC(50): 0.440-0. 772 mmol/plate). The four isolated flavones were also studied regarding their antimutagenic mechanisms with preincubation methods of the modified Ames test and emission spectroscopic analysis. The results suggested that all isolated flavones were desmutagens which directly inactivated Trp-P-2 or inhibited its metabolic activation.

Detection of Triazolam and Its Hydroxy Metabolites in Rat Hair by Reversed-Phase Liquid Chromatography with Electrospray Ionization Mass Spectrometry

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) for simultaneous determination of triazolam (TZ) and its hydroxy metabolites in hair has been developed. After the addition of deuterium-labeled 1 -hydroxymethyltriazolam (1-HT-d4) as an internal standard, analytes in hair shaft and hair root samples were extracted with a basic medium, CH2Cl2/MeOH/28% NH4OH (20:80:2), at room temperature overnight. The chromatographic separation of the analytes was achieved using a 3-microm micro HPLC column (100 x 2.0-mm i.d.) with a gradient of acetonitrile in water containing 1% acetic acid as the mobile phase at a flow rate of 0.15 mL/min. The mass spectrometer was operated in selected-ion monitoring mode at quasi molecular ions [M+H]+ of TZ and its metabolites. Under the proposed conditions, the ranges of quantitation of TZ, 1-HT, and 4-HT were 0.1-10 ng/0.2 mL. The method has been applied to determine the hair shaft and hair root incorporation of TZ and its metabolites into Dark Agouti rats administered with 3 mg/kg or 6 mg/kg intraperitoneally twice a day for five days. Judging from the retention behavior by the chromatography and the mass spectra of the peaks detected, TZ, 1-HT, and 4-HT were incorporated in the hair shaft and the hair root. The concentration of 4-HT was the highest of all compounds detected. An unknown substance thought to be 1,4-diHT also appeared in both hair shaft and hair root samples. This substance was obtained from in vitro metabolic studies of TZ using rat liver microsome fraction and was accompanied by the other two metabolites, 1-HT and 4-HT. Structural elucidation was performed with online high-performance liquid chromatography-MS after acetylation of the substance with acetic anhydride and pyridine. This is the first report of the detection of the hydroxy metabolites of TZ in hair. The method has been found to be useful as a screening procedure of TZ intake in humans.

The in vitro effect of dandelions antioxidants on microsomal lipid peroxidation.

Dandelions have long been used in herbal medicine for their choleretic, diuretic, antiinflammatory, appetite-stimulating and laxative properties. An antioxidant property can be supposed as a basis of their-therapeutic effects. To understand the mechanism of the drug's action, the effects of natural extracts on a microsomal fraction of rat liver were examined. The extracts diminished the enzymatically induced-lipid peroxidation and reduced the cytochrome c with and without NADPH in a concentration dependent manner.

The in vitro effect of dandelions antioxidants on microsomal lipid peroxidation

Dandelions have long been used in herbal medicine for their choleretic, diuretic, antiinflammatory, appetite-stimulating and laxative properties. An antioxidant property can be supposed as a basis of their­therapeutic effects. To understand the mechanism of the drug's action, the effects of natural extracts on a microsomal fraction of rat liver were examined. The extracts diminished the enzymatically induced­lipid peroxidation and reduced the cytochrome c with and without NADPH in a concentration dependent manner. Copyright © 2000 John Wiley & Sons, Ltd.

Genotoxicity of ochratoxin A by Salmonella mutagenicity test after bioactivation by mouse kidney microsomes

Ochratoxin A (OTA) was, up to now, believed to be non-mutagenic in the classical Salmonella typhimurium reverse mutation test. This was confirmed using rat liver microsomal fractions with the strains, TA1535, TA1538 and TA98, and up to 1210 μg/plate, utilizing an Ames microtest. However, using mice kidney microsomal fractions as metabolic activators, reverse mutations were obtained with the three strains used, in the presence of either NADP or arachidonic acid as cofactors. The mutagenicity was higher with arachidonic acid than with NADP using the TA1535 strain. This lends support to the results concerning the DNA or dGMP adducts obtained in vitro which were also higher in the presence of arachidonic acid, and indicate that several metabolic pathways of OTA can lead to genotoxic compounds. In addition, both base pair substitutions and frameshift mutations can be caused by OTA after metabolic activation.

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.

In vitro and in vivo glucuronidation of 24,25-dihydroxyvitamin D 3

Glucuronidation of 24,25-dihydroxyvitamin D 3 has been investigated in in vitro and in vivo experiments. Three positional isomers of 24,25-dihydroxyvitamin D 3 monoglucuronide were synthesized from 24,25-dihydroxyprovitamin D 3 derivatives with Koenigs-Knorr reaction and used as standard samples. In the presence of the rat liver microsomal fraction and uridine-5′-diphosphoglucuronic acid, 24,25-dihydroxyvitamin D 3 gave 3- and 24-glucuronides as the main products in almost equal amounts, but only a small amount of the corresponding 25-glucuronide was obtained. 24,25-Dihydroxyvitamin D 3 monoglucuronide was deconjugated with rat intestine homogenate, which indicated the entero-hepatic circulation of 24,25-dihydroxyvitamin D 3. After the administration of 24,25-dihydroxyvitamin D 3 to rats, its 3- and 24-glucuronides were identified from the bile as inferred from the in vitro experiment. However, the in vivo glucuronidation occurred at the 24-position in preference to the 3-position, and the corresponding 25-glucuronide was not detected. These glucuronides were identified in comparison with standard samples based on their chromatographic behavior during high-performance liquid chromatography and data obtained from liquid chromatography-electrospray ionization-mass spectrometry, which was helpful in identifying these compounds.

Pharmacokinetic interactions between HIV-protease inhibitors in rats.

The interactions of four HIV-protease inhibitors, ritonavir (RIT), saquinavir (SAQ), indinavir (IND) and nelfinavir (NEL), were examined by in vitro metabolic studies using rat liver microsomal fractions. The substrate concentrations employed were 0.75 approximately 12 microM, and the inhibitor concentrations were 2.5 approximately 60 microM. The metabolic clearance rates of SAQ, NEL and IND as determined by V(max)/K(m) were 170.9+/-10.9, 126.0+/-4.4 and 73.0+/-2.0 microL/min/mg protein, respectively. RIT was a potent inhibitor of the other three protease inhibitors, and the inhibition constants (K(i)) were 1.64 microM for SAQ, 0.95 microM for IND and 1. 01 microM for NEL. NEL was the second strongest inhibitor with a K(i) for NEL inhibition of IND metabolism of 2.14 microM. IND was the third strongest inhibitor with K(i)s of 2.76 microM for inhibition of NEL and 3.55 microM for inhibition of SAQ. As SAQ has the highest metabolic clearance rate, the K(i) for the SAQ inhibition of IND metabolism was high, 9.50 microM. Based on these in vitro results, drug interactions between NEL and IND or RIT were studied after oral administration to rats where the dose of each drug was 20 mg/kg. The C(max) and AUC of NEL were increased 3.6- and 8.5-fold by the co-administration with RIT. However, in contrast to co-administration of NEL and RIT, the effect of IND on the pharmacokinetics of NEL was negligible and the t(1/2) of NEL was not significantly increased by IND. Therefore, the combination of NEL and IND is recommended as a combination therapy for AIDS patients.

In vivo and in vitro studies on the regulatory link between 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rat liver.

The activities of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase; EC 1.1.1.34), rate-limiting enzyme of cholesterol biosynthesis, and cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), key enzyme of the neutral bile acid synthesis pathway, were measured in the microsomal fraction of rat liver and in rat liver cells to investigate the coordinate regulation of the two pathways. Both enzyme activities exhibited the same diurnal rhythm and responded in a coordinate fashion to fasting or bile acid-feeding (decrease) and to cholestyramine-feeding (increase). Cholesterol-feeding decreased the activity of HMGCoA reductase, increased that of cholesterol 7 alpha-hydroxylase, and concomitantly increased free cholesterol in microsomes. In an ex vivo setting using primary hepatocytes from animals fed a high cholesterol diet the activity of HMGCoA reductase was initially low and that of cholesterol 7 alpha-hydroxylase was elevated. Release of cholesterol into the medium with ongoing incubation caused HMGCoA reductase activity to increase, and that of cholesterol 7 alpha-hydroxylase to decline. Incubation of hepatocytes with a cholesterol-containing lipoprotein fraction stimulated the activity of cholesterol 7 alpha-hydroxylase, but left HMGCoA reductase activity unaffected. The results confirm the idea of a joint regulation of the two key enzymes of cholesterol metabolism in response to the levels of substrate and metabolites, and support the notion that with respect to bile acid and cholesterol levels, respectively, regulation of HMGCoA reductase activity may be secondary to that of cholesterol 7 alpha-hydroxylase. The in vitro studies supply evidence that the effects of cholesterol and bile acid excess or deficiency are direct and do not involve accessory changes of hormone levels or mediators.

Characterization of Cholyl-Adenylate in Rat Liver Microsomes by Liquid Chromatography/Electrospray Ionization–Mass Spectrometry

The cholyl-adenylate, covalently bound 3α, 7α, 12α-trihydroxy-5β-cholanoic acid (cholic acid) with adenosine 5′-monophosphate having an acid anhydride linkage, has been characterized by means of liquid chromatography/mass spectrometry in an incubation mixture with a rat liver microsomal fraction. The authentic specimen of cholyl-adenylate was synthesized using the carbodiimide method and the structure was confirmed by MS and nuclear magnetic resonance spectroscopy. After incubation of cholic acid with a hepatic microsomal fraction in the presence of adenosine 5′-triphosphate, bile acids were extracted and purified by solid-phase extraction on a Sep-Pak C18cartridge and then subjected to a LC/MS analysis, where cholyl-adenylate and a CoA thioester of cholic acid (cholyl-CoA) were monitored with characteristic negative ions ofm/z736 and 577, respectively. Cholyl-adenylate was definitely characterized and preferential biotransformation into the acyl-adenylate prior to formation of cholyl-CoA was noted. The nonenzymatic formation of taurine-conjugated cholic acid by incubation of cholyl-adenylate with taurine in a buffer solution was also demonstrated.