Biotinylated phosphatidylinositol 3,4,5-trisphosphate as affinity ligand.

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a primary output signal of phosphoinositide (PI) 3-kinase, plays a crucial role in diverse cellular processes. Evidence indicates that PIP(3) exerts downstream signaling, in part, by recruiting effector proteins to plasma membranes. Consequently, identification of signaling enzymes with PIP(3)-binding motifs represents a viable approach to understand the mechanism by which specificity of the PI 3-kinase-mediated signaling network is maintained. To address this issue, we have developed biotinylated derivatives of PIP(3) as affinity probes for the purification and characterization of PIP(3)-binding proteins. Considering the relaxed requirement for the acyl moiety in PIP(3) recognition, these biotinylated PIP(3) analogues display two structural features. First, they contain short acyl side chains (C(4) and C(8)), allowing them to be soluble in aqueous milieu. This desirable feature avoids the formation of lipid aggregates, which minimizes nonspecific hydrophobic interactions with proteins. Second, the appended biotin is located at the terminus of the sn-1 acyl side chain, thereby maintaining the integrity of the phosphoinositol head group essential for selective recognition. The utility of these affinity ligands is validated by the purification of recombinant PIP(3)-binding proteins, expressed as GST fusion proteins, to homogeneity from bacterial lysates. These include the C-terminal SH2 domain of the p85 subunit of PI 3-kinase and the N-terminal PH domain of PLCgamma1. The efficiency of biotinylated PIP(3) analogues in the purification of these recombinant proteins was approximately 20% of that of glutathione beads

Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase).

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine-2,4- dinitrophenyl, where P(2), P(2)', and P(3) residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P(1) and P(1)' residues, and adjacent residues are designated P(2), P(3), P(2)', P(3)', etc.) There was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P(2) residues having the greatest effect. The S(3), S(2), and S(2)' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P(2) position, were among the best P(2)' residues. The specificity at P(3) was generally opposite that of P(2). Residues at P(2), and to a lesser extent at P(3), influenced the cleavage site. At the P(2) position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P(2) Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P(1)' or P(1) position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.

Hydroxyl radical generation in rat brain is initiated by iron but not aluminum, as determined by microdialysis with salicylate trapping and GC-MS analysis.

Free radical-induced brain oxidative injury is thought to play a role in the etiology and pathogenesis of neurodegenerative disorders. Iron (Fe) can catalyze the Fenton reaction and mediate hydroxyl radical (HO.) generation to initiate lipid peroxidation (LP). The neurotoxin aluminum (Al) can facilitate Fe-mediated LP. However, the mechanism of Al-facilitated LP has not been determined. In this study, microdialysis (MD) of the ventral hippocampus was performed in rats exposed to Fe and/or Al sulfate via the MD probe. Salicylate (SA) was included in the dialysate to trap the HO. by forming 2,3-dihydroxybenzoic acid (2,3-DHBA). 2,3-DHBA was quantified by capillary column gas chromatography-mass spectrometry (GC-MS) as a trimethylsilyl (TMS) derivative with 2,4-DHBA as an internal standard. Fe-catalyzed HO. generation was shown in brain interstitial fluid. Aluminum alone did not initiate HO. generation. Al did not facilitate Fe-catalyzed HO. generation. Therefore, the previously reported Al facilitation of Fe-mediated oxidative injury must be due to mechanisms other than increased extracellular HO. generation.

Increased plasma level of cholesterol-5 beta,6 beta-epoxide in endometrial cancer patients.

Plasma levels of alpha- and beta-isomers of cholesterol-5, 6-epoxides were quantitated in a pilot study of 9 women with endometrial cancer and 9 race- and age-matched control women by isotope dilution gas chromatography/mass spectrometry. Endometrial cancer cases had significantly higher cholesterol-5 beta,6 beta-epoxide levels than the controls (P = 0.01). The mean plasma beta-epoxide level was 0.71 +/- 0.46 ng/ml for the cases and 0.35 +/- 0.25 ng/ml for the controls. No significant differences were found between the plasma alpha-epoxide levels of the cases and the controls. Plasma levels of various carotenoids, tocopherols, and ascorbic acid were also quantitated by high performance liquid chromatography. An inverse correlation was present between the plasma levels of cholesterol-5 beta,6 beta-epoxide and cis-lutein/zeaxanthin (P = 0.01). These data suggest a possible role for cholesterol-5 beta,6 beta-epoxide as a biomarker of endometrial cancer risk. Further studies are warranted to investigate the possible association between the oxidation products of cholesterol and endometrial cancer and to determine the interactive role of antioxidants.

Hepatic metabolism of 3-oxoandrost-4-ene-17 beta-carboxylic acid in the adult rat: formation of carboxyl-linked glucuronides both in vivo and in vitro.

The hepatic metabolism of 3-oxoandrost-4-ene-17 beta-carboxylic acid (etienic acid), a probable acidic catabolite of deoxycorticosterone, was investigated using rats prepared with an external biliary fistula. Metabolic products were identified by GC-MS after hydrolysis with beta-glucuronidase and by proton nuclear magnetic resonance after chromatographic purification of protected glucuronides. About 80% of the injected dose was secreted into bile in 20 hours. Three fully reduced etianic acids (3 alpha-hydroxy-5 alpha-, 3 beta-hydroxy-5 alpha-, 3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acids) were identified as were several of their di- and trihydroxylated congeners. Glucuronides of these reduced and/or hydroxylated metabolites constituted over half of the recovered dose, with carboxyl-linked glucuronides predominating over 3-hydroxyl-linked glucuronides. The mode of glucuronidation correlated well with the ability of liver microsomes to form the corresponding compounds in vitro from the set of four 3,5-diastereomeric etianic acids.

Bile acids of marsupials. 2. Hepatic formation of vulpecholic acid (1 alpha,3 alpha,7 alpha-trihydroxy-5 beta-cholan-24-oic acid) from chenodeoxycholic acid in a marsupial, Trichosurus vulpecula (Lesson).

Free vulpecholic acid (1 alpha,3 alpha,7 alpha-trihydroxy-5 beta-cholan-24-oic) is the major biliary component of the Australian opossum (Trichosurus vulpecula), accompanied only by a few percent of its taurine conjugate. In order to exclude a microbial involvement in its formation (i.e., secondary origin) four sets of experiments were performed. It was found that a) the level of vulpecholic acid remained unchanged in the bile of opossums fed with neomycin and kanamycin for 7 days prior to bile collection; b) it also remained unchanged after long bile drainage; c) in opossums prepared with biliary cannula, intraportally injected [24-14C]chenodeoxycholic acid was transformed to [24-14C]vulpecholic acid; and d) in a similar experiment, the detectable transformation of [1 alpha,2 alpha-3H2]cholesterol to vulpecholic acid was observed. In experiment c) 28-66% of the administered radioactivity was secreted in 2 h in the form of free biliary vulpecholic and chenodeoxycholic acids. Only a trace amount of the corresponding taurine conjugates (approximately 0.4%) was formed. Moreover, rapidly declining specific radioactivity of the unconjugated chenodeoxycholic acid indicated its probable participation in the native formation of vulpecholic acid.

Inhibitors of sterol synthesis. Characterization of trimethylsilyl dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one. Applications in the analysis of mitochondrial metabolites of the 15-ketosterol by gas chromatography-mass spectrometry.

Derivatization of delta 8(14)-15-ketosterols as bis-TMS dienol ethers facilitates their analysis by gas chromatography-mass spectrometry (GC-MS). Conditions are presented for the preparation of each of the three possible bis-TMS dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism. Treatment of 1 with N,O-bis(tri-methylsilyl)-trifluoroacetamide (BSTFA) and pyridine for 20 h at 100 degrees C produced primarily 3 beta,15-bis(trimethylsilyloxy)-5 alpha-cholesta-7,14-diene. Treatment of 1 with BSTFA-pyridine in the presence of 0.1% perchloric acid for 20 h at 22 degrees C gave mainly the delta 8(14)15, dienol ether. Heating this reaction mixture at 100 degrees C for 4 days gave mainly the delta 8,14 ether. The structures of the three dienol ethers were established by GC-MS and 1H and 13C nuclear magnetic resonance spectroscopy. The three TMS dienol ethers of 1 were resolved by capillary GC and gave very simple mass spectra upon electron impact with fragmentation limited almost exclusively to the elimination of trimethylsilanol, methyl, and the side chain. The TMS dienol ethers showed reduced artefact formation, improved chromatographic resolution, and increased sensitivity relative to the delta 8(14)-15-ketosterols, properties that improve the detection and identification of minor components in analyses of complex biological mixtures. The utility of this derivatization is illustrated for the delta 7,14 TMS dienol ethers of the 3-deoxy, 3-keto, 3 alpha-hydroxy, and 3 beta-methoxy analogs of 1 and for delta 8(14)-15-ketosterols in mixtures obtained from incubations of 1 with rat liver mitochondria in the presence of NADPH.

Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols.

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.

5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent regulator of cholesterol metabolism: occurrence in rat skin.

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a potent inhibitor of cholesterol biosynthesis which has significant hypocholesterolemic activity upon oral administration to animals. Described herein are the results of experiments that indicate the presence of the 15-ketosterol in rat skin. The 15-ketosterol was, after purification by medium pressure liquid chromatography on Lichroprep RP-8 columns, thin-layer chromatography on silica gel G, and reverse phase high performance liquid chromatography, characterized by gas-liquid chromatography-mass spectrometry in the form of its trimethylsilyl ether derivative. The use of an internal standard containing both tritium and deuterium permitted the determination of the levels of the 15-ketosterol by mass fragmentography. The results of five separate analyses of portions of the skin of a male Sprague Dawley rat showed a mean value of 84.5 +/- 4.1 (SEM) ng per g. Analyses of hair samples of ten male Sprague Dawley rats indicated a mean level of 143 +/- 19 (SEM) ng per g of hair. Most (approximately 72%) of the 15-ketosterol in hair was esterified. This report constitutes the first isolation of the 15-ketosterol from animal tissues.

Enzymatic formation and chemical synthesis of an active metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, a potent regulator of cholesterol metabolism.

The enzymatic (rat liver mitochondria) conversion of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one to 5 alpha-cholest-8(14)-ene-3 beta,26-diol-15-one is described. The enzymatic product was judged, on the basis of IH and 13C NMR studies, to be a 4:1 mixture of its 25R and 25S isomers. (25R)-5 alpha-Cholest-8(14)-ene-3 beta,26-diol-15-one was prepared through a five-step synthesis from (25R)-26-hydroxycholesterol. The (25R) isomer of the new compound was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells and to inhibit the esterification of cholesterol in jejunal microsomes.

Inhibitors of sterol synthesis. Spectral characterization of derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one.

Described herein are the chemical syntheses of a number of deuterated derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These include the [2,2,3 alpha,4,4,7,7,9 alpha,16,16-2H10]-, [7 alpha,9 alpha,16,16-2H4]-, [7,7,9 alpha,16,16-2H5]-, and [2,2,3 alpha,4,4-2H5]-analogs of the delta 8(14)-15-ketosterol. Also included are the syntheses of the 3 beta-acetate derivatives of the latter three deuterated analogs and of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, and 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one. Low resolution mass spectral data on these compounds and on 5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, and the trimethylsilyl ethers of the free sterols have been presented. The results of these studies, supplemented with high resolution mass spectral data on five of these compounds, have been used to evaluate the electron impact mass spectral fragmentation of the delta 8(14)-15-ketosterols and their derivatives. Also presented herein are the results of 1H, 2H, and 13C nuclear magnetic resonance studies of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and its derivatives.

Inhibitors of sterol synthesis. Synthesis and spectral characterization of fatty acids esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one.

Reported herein are the chemical syntheses of 29 fatty acid esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These compounds were characterized by the results of ultraviolet, 1H nuclear magnetic resonance, and mass spectral studies.

Glucuronidation of 6 alpha-hydroxy bile acids by human liver microsomes.

The glucuronidation of 6-hydroxylated bile acids by human liver microsomes has been studied in vitro; for comparison, several major bile acids lacking a 6-hydroxyl group were also investigated. Glucuronidation rates for 6 alpha-hydroxylated bile acids were 10-20 times higher than those of substrates lacking a hydroxyl group in position 6. The highest rates measured were for hyodeoxy- and hyocholic acids, and kinetic analyses were carried out using these substrates. Rigorous product identification by high-field proton nuclear magnetic resonance and by electron impact mass spectrometry of methyl ester/peracetate derivatives revealed that 6-O-beta-D-glucuronides were the exclusive products formed in these enzymatic reactions. These results, together with literature data, indicate that 6 alpha-hydroxylation followed by 6-O-glucuronidation constitutes an alternative route of excretion of toxic hydrophobic bile acids.

Constituents of human meconium--III. Identification of 3,20-dihydroxypregnan-21-oic acids.

Seven 3,20-dihydroxypregnan-21-oic acids (C21) were detected by capillary gas chromatography-mass spectrometry in the acid fraction isolated from human meconium after solvolysis and mild alkaline hydrolysis. The two major acids were isolated and identified by nmr as 3 alpha, 20 alpha-dihydroxy-5 alpha-and 3 alpha, 20 alpha-dihydroxy-5 beta-pregnan-21-oic acids. Two minor C21 components were identified as 3 alpha, 20 beta-dihydroxy-5 beta-pregnan-21-oic and 3 beta, 20 alpha-dihydroxypregn-5-en-21-oic acids. A trace amount of 3 alpha, 20 beta-dihydroxy-5 alpha-, 3 beta, 20 alpha-dihydroxy-5 alpha-pregnan-21-oic, and 3 beta, 20 beta-dihydroxypregn-5-en-21-oic acids were also detected. The identification of these C21 acids was confirmed by mass spectral and chromatographic comparison with standards of eight saturated and two 5-unsaturated 3, 20-dihydroxylated C21 acids. The decisive predominance of 20 alpha isomers confirmed the native presence of C21 acids in meconium. Six acids, with an exception of 3 beta, 20 beta-dihydroxy-pregn-5-en-21-oic acid, were detected in the extract of meconium prepared in the presence of large excess of sodium borohydride; this minor acid, together with a part of its 20-epimer, was evidently formed as result of the artefactual oxidation of 21-hydroxypregnenolone, the known constituent of meconium. The co-occurrence of relatively high concentrations of C21 acids and monohydroxylated C20 acids, previously detected in human meconium, pointed to the significance of the oxidative catabolism of neutral C21 steroids in the case of feto-placental-maternal unit.

Metabolism of 24-norlithocholic acid in the rat: formation of hydroxyl- and carboxyl-linked glucuronides and effect on bile flow.

24-Norlithocholic (3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic) acid is the lower homologue of lithocholic acid, a potent cholestatic agent. In order to characterize its cholestatic potential and metabolic fate, 3 beta-tritiated 24-norlithocholate was infused intravenously into adult male Sprague-Dawley rats prepared with an external biliary fistula. The results demonstrate that 24-norlithocholate does not induce cholestasis in rats when administered in doses in excess of those necessary for lithocholate to produce cholestasis. Hydroxyl- and carboxyl-linked glucuronides were identified as major metabolites secreted in the bile. Especially noteworthy is the identification of carboxyl-linked glucuronides of mono-, di- and trihydroxylated C23 bile acids. Their total amount (25% of recovered radioactive products) is comparable to that of the hydroxyl-linked glucuronide of 24-norlithocholic acid (41%). In this study, for the first time, a bile acid diglucuronide, substituted both at 3-hydroxyl and carboxyl groups, was detected (11%).

Glucuronides of monohydroxylated bile acids: specificity of microsomal glucuronyltransferase for the glucuronidation site, C-3 configuration, and side chain length.

The ability of rat liver microsomes to catalyze UDP-glucuronic acid-dependent glucuronidation of monohydroxy-bile acids was examined. The following bile acids were used as substrates, each as the 3 alpha and 3 beta epimer: 3-hydroxy-5 beta-cholanoic acid (C24), 3-hydroxy-5 beta-norcholanoic acid (C23), 3-hydroxy-5 beta-bisnorcholanoic acid (C22), 3-hydroxy-5 beta-pregnan-21-oic acid (C21), and 3-hydroxy-5 beta-androstane-17 beta-carboxylic acid (C20). The corresponding glucuronides were chemically synthesized to serve as standards and were characterized by thin-layer and gas-liquid chromatography as well as by nuclear magnetic resonance. Enzymatic glucuronidation reactions were optimized with respect to pH for each product formed and the kinetic parameters for each reaction were measured. Analytical techniques necessary to separate products from unreacted substrates and to identify them included thin-layer chromatography, gas-liquid chromatography, and nuclear magnetic resonance. It was found that the 3 alpha epimers of the five bile acids listed above enzymatically formed 3-O-glucuronides, C24 being the best substrate, followed by C21 and C20; C22 and C23 gave rise to only small amounts of this product. The 3 beta epimers of all bile acids tested were poorer substrates, although by a factor that varied widely. In addition to the expected hydroxyl-linked glucuronide, three of the 3 alpha-bile acids (C23, C22, and C20) and at least one 3 beta-bile acid (C20), gave rise to a novel metabolite in which the 1-OH of glucuronic acid was esterified with the steroidal carboxyl group (carboxyl-linked glucuronide).

Preparation and characterization of 3-monohydroxylated bile acids of different side chain length and configuration at C-3. Novel approach to the synthesis of 24-norlithocholic acid.

A series of 3-monohydroxylated bile acids, in unlabeled and radioactive form, of varying side chain length and configuration at C-3 has been synthesized and rigorously characterized. They include: 3 alpha- and 3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acids (C20); 3 alpha- and 3 beta-hydroxy-5 beta-pregnan-21-oic acids (C21); 3 alpha- and 3 beta-hydroxy-23,24-bisnor-5 beta-cholan-22-oic acids (C22); 3 alpha- and 3 beta-hydroxy-24-nor-5 beta-cholan-23-oic acids (C23, norlithocholic and isonorlithocholic acids); and 3 beta-hydroxy-5 beta-cholan-24-oic acid (C24, isolithocholic acid). A novel approach to the degradation of lithocholic acid acetate to 24-norlithocholic acid is described. This degradation involves the photochemical modification of a Hunsdiecker reaction and Kornblum oxidation of the intermediate 23-bromide. The availability of these compounds makes it possible to study the metabolism and biological effects of short chain bile acids.

Detection of 3-hydroxy-etianic and 3-hydroxy-bisnorcholanoic acids in human serum.

Capillary GLC-MS analysis of the free/sulfate/glucuronide bile acid fraction obtained from human cholestatic serum demonstrated the presence of 3-hydroxyandrostan-17 beta-carboxylic (etianic) and 3-hydroxybisnorcholanoic acids.