Non-stereoselective formation of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid during cholic acid biosynthesis

Incubation of (25RS)-, (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA, 6, 6a, 6b) and (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (7) with rat liver mitochondria gave all four stereoisomers (9a,9b,9c,9d) of 3 alpha,7 alpha,12 alpha,24-Tetrahydroxy-5 beta-cholestan-26-oic acid (TeHCA). The corresponding 27-nor analogs (10,11) were also converted non-stereoselectively to a 1:1 mixture of the epimeric 24-hydroxy compounds (12).     

Formation of varanic acid, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in Bombina orientalis

Varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid; 24-OH-THCA) is almost the sole component of bile acids in the bile of Bombina orientalis. To examine in the mechanism of the formation of 24-OH-THCA, radiolabeled (25R)- and (25S)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholestanoic acids [(25R)- and (25S)-THCA] and (24E)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) were administered intraperitoneally to B. orientalis, gallbladder bile was collected after 24 h, and bile acids were subsequently extracted. Then the bile acids were analyzed by means of radio thin-layer chromatography and radio high-performance liquid chromatography after conversion to p-bromophenacyl ester derivatives. Although delta 24-THCA was not converted to 24-OH-THCA, (25R)-THCA and (25S)-THCA were transformed to (24R,25R)-24-OH-THCA and (24R,25S)-24-OH-THCA, respectively. These results strongly suggest that 24-OH-THCA was transformed via direct hydroxylation of the saturated side chain of THCA, not via hydration to an alpha, beta-unsaturated acid, delta 24-THCA, in B. orientalis.     

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.     

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.     

Sterol synthesis. Syntheses of 15-oxygenated 5alpha, 14beta-cholest-7-en-3beta-ol derivatives

Treatment of 3beta-benzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3alpha-benzoyloxy-5alpha, 14beta-cholest-7-en-15-one with the unnatural C-D ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15alpha, 14beta-cholest-7-en-3beta, 15alpha-diol and 5alpha, 14beta-cholest-7-en-3beta, 15beta-diol in 9% and 81% yields, respectively.     

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.     

Fate of etiproston, a synthetic analogue of PGF2 alpha, in cows

The pharmacokinetics and metabolic fate of labelled compounds were investigated after intramuscular administration of 3H-radiolabelled etiproston to nine cows. Elimination was rapid (t 1/2 beta = 2.8 h). Forty-eight h after administration 92.6% of the radioactivity had been eliminated, mainly via the urinary (66% at 48 h) and faecal routes (26% at 48 h). In comparison, little elimination in milk occurred (less than 0.034% dose/l by 24 h). Radioactivity at the injection site 48 h after administration was seen in one cow (< 4.68 x 10(-5%) dose/g). No radioactivity was detected in the tissues. Urinary metabolites were purified and isolated using XAD-2 extraction and preparative HPLC in reverse and normal phases. The main urinary metabolite, identified by mass spectrometry, was the tetranor acid derivative in equilibrium with its lactone form.     

Regulation of the epididymal synthesis of P26h, a hamster sperm protein

Erythromycin with or without additional zinc acetate is used topically in the treatment of acne vulgaris. A potential effect of zinc on the stratum corneum penetration of erythromycin was investigated in human volunteers. Skin surface washings and tape strippings from the skin of the back were collected after drug applications in 12 subjects for quantification of erythromycin levels. Zinc acetate increased the amount remaining on the back skin at 6 h after application from 40 +/- 19 to 56 +/- 15% of the dose and, vice versa, reduced the amount in stratum corneum strips from 22 +/- 7 to 18 +/- 7%, both with statistical significance. The effect varied with body region. Zinc acetate thus provided to prolong the residence time of erythromycin on the skin.     

The effect of idebenone, a coenzyme Q analogue, on hydrophobic bile acid toxicity to isolated rat hepatocytes and hepatic mitochondria

Oxidant stress induced by hydrophobic bile acids has been implicated in the pathogenesis of liver injury in cholestatic liver disorders. We evaluated the effect of idebenone, a coenzyme Q analogue, on taurochenodeoxycholic acid (TCDC)-induced cell injury and oxidant stress in isolated rat hepatocytes and on glycochenodeoxycholic acid (GCDC)-induced generation of hydroperoxides in fresh hepatic mitochondria. Isolated rat hepatocytes in suspension under 9% oxygen atmosphere were preincubated with 0, 50, and 100 micromol/l idebenone for 30 min and then exposed to 1000 micromol/l TCDC for 4 h. LDH release (cell injury) and thiobarbituric acid reactive substances (measure of lipid peroxidation) increased after TCDC exposure but were markedly suppressed by idebenone pretreatment. In a second set of experiments, the addition of 100 micromol/l idebenone up to 3 h after hepatocytes were exposed to 1000 micromol/l TCDC resulted in abrogation of subsequent cell injury and markedly reduced oxidant damage to hepatocytes. Chenodeoxycholic acid concentrations increased to 5.15 nmol/10(6) cells after 2 h and to 7.05 after 4 h of incubation of hepatocytes with 1000 micromol/l TCDC, and did not differ in the presence of idebenone. In freshly isolated rat hepatic mitochondria, when respiration was stimulated by succinate, 10 micromol/l idebenone abrogated the generation of hydroperoxides during a 90-minute exposure to 400 micromol/l GCDC. These data demonstrate that idebenone functions as a potent protective hepatocyte antioxidant during hydrophobic bile acid toxicity, perhaps by reducing generation of oxygen free radicals in mitochondria.     

Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.     

Localization of the gene encoding the alpha subunit of human interleukin-5 receptor (IL5RA) to chromosome region 3p24-3p26

The chromosomal location of the human gene for the alpha subunit of interleukin-5 receptor (IL5RA) has been determined. The human IL5RA gene was localized to the short arm of chromosome 3 by Southern blot analysis of DNA from a panel of mouse-human hybrid somatic cell lines. The IL5RA gene has been further localized to human chromosome region 3p24-3p26 by in situ hybridization of a molecularly cloned IL5RA cDNA fragment to metaphase chromosomes. The results suggest that the IL5RA locus is unlinked to other members of the hematopoietic receptor family. Assignment of the IL5RA gene to chromosome 3 at bands p26-p24 raises the possibility that it may be altered by certain nonrandom chromosomal abnormalities arising in human hematopoietic malignancies and solid tumors.     

Protective effect of beraprost sodium, a stable prostacyclin analogue, in development of monocrotaline-induced pulmonary hypertension

Commercial cheese products were surveyed for beta-casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection. beta-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for beta-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to beta-casomorphins. Therefore, beta-casomorphins were absent, or concentrations were below the HPLC detection threshold for beta-casomorphin of 2 micrograms/ml of cheese extract. The susceptibility of beta-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp. cremoris was investigated. beta-Casomorphin standards were incubated at 4 degrees C with bacterial cell lysate at pH 5.0, 5.2, 5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added beta-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of beta-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, beta-casomorphins may be degraded under conditions common for Cheddar cheese.     

Metabolism of 17 beta-hydroxy-2alpha, 3alpha-cyclopropano-5alpha-androstane in the rabbit

The neutral urinary excretion products of 17beta-hydroxy-2alpha, 3alpha-cyclopropano-5alpha-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4alpha-position and one in the 6alpha-position with the cyclopropane ring intact. The fifth substance, 17beta-hydroxy-3beta-methyl-5alpha-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4-C-6-C-2.