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.

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%).

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.

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).

Glucagon stimulation of hepatic Na+, K+-ATPase.

In the perfused rat liver administration of glucagon was shown to result in a transiently increased uptake of K+, indicating the possible involvement of the Na+, K+-ATPase. Direct measurement of the activity of Na+, K+-ATPase revealed a two-fold stimulation of the enzyme by glucagon. The effect of glucagon on the activity of the enzyme was immediate. Simultaneously with the increase in the activity of the Na+, K+-ATPase, the activity of Mg2+-ATPase decreased. In order to evaluate whether the activation of the Na+, K+-ATPase by glucagon is related to the metabolic effects of the hormone, experimental conditions known to interfere with the activity of the enzyme were employed and glucagon stimulation of Ca2+-efflux, mitochondrial metabolism and gluconeogenesis were measured. K+-free perfusate, high K+ perfusate or ouabain interfered to varying degrees with the glucagon stimulation of these responses. The combination of K+-free perfusate and ouabain almost completely abolished the glucagon stimulation of all three parameters. These results demonstrate the glucagon stimulation of Na+, K+-ATPase and raise the possibility that the activation of the enzyme by glucagon might be a necessary link for the manifestation of its metabolic effects.

Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli.

The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector. The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo. The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E. coli. The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism. The isolated hybrid plasmids have been mapped by restriction endonuclease analysis. The presence and location of other genetic markers have also been established. The above data, along with analysis of deletion derivatives of these plasmids and subcloning of appropriate restriction fragments, have established the position of the pgsA locus on the hybrid plasmids. From this data, the position of the pgsA locus has been determined to le between flaI and uvrC on the E. coli genetic map.

Induction of an attachment and spreading on glass of Ehrlich ascites tumour cells by alpha-saturated polyprenols.

It has been observed that a dolichol, alpha dihydro-undecaprenol, induces phenotypic changes in Ehrlich ascites tumour [EAT] cells. In the presence of this dolichol in the medium. EAT cells attach to glass and spread on it but grow in an overlapping pattern. This spreading takes place after a lag of between 72-96 hours at continuous exposure to the dolichol. When cells, once induced to spread, are trypsinized, they can spread once more within 3-6 hours in the dolichol-free medium. It is suggested that dolichols, which act as lipid intermediates in protein glycosylation, may represent a class of compounds which by interference with the biosynthesis of plasma membrane constituents influence surface properties of EAT cells and induce spreading. The results presented in this paper support the view that sugar moieties of plasma membrane constituents play a role in controlling cell attachment, spreading, and intercellular communication.

Synthesis and content of ether-linked glycerophospholipids in the harderian gland of rabbits.

Although harderian glands are rich in neutral glycerolipids with ether bonds, less than 20% of the choline glycerophospholipids have ether bonds in the white and pink portions of the adult rabbit harderian gland. Only 6% of these are plasmalogens while 94% are alkylacyl glycerophosphocholines. The ethanolamine glycerophospholipids include 37% with ether bonds in both white and pink portions. In the white portion 96% are plasmalogens but only 19% are plasmalogens in the pink portion. The microsomal ethanolaminephosphotransferase (EC 2.7.8.1) is more active with diacylglycerols than with alkylacylglycerols. The microsomal cholinephosphotransferase (EC 2.7.8.2) is equally active with both diradylglycerols. Particularly with microsomes from the pink portion, the apparent Km values for CDPethanolamine and CDPcholine are ower in the presence of alkylacylglycerols than in the presence of diacylglycerols. The incorporation of radioactivity from CDP[14C]ethanolamine and CDP[14C]choline into ethanolamine and choline plasmalogens was increased several-fold by addition of alkylacylglycerols but was not increased substantially by addition of diacylglycerols.

Choline and ethanolamine glycerophospholipid synthesis in isolated synaptosomes of rat brain.

Substantial activities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found with lysed synaptosomes but not with intact synaptosomes isolated from adult rat brains. Synaptosomal and non-synaptosomal microsomal transferases were similar in kinetic properties. Substantial activities of synaptosomal transferases have not been described previously. Part of the glycerophospholipids in synaptosomal membranes may be synthesized in the nerve ending in addition to the glycerophospholipids supplied by axonal transport. The synthesis of the alkylacyl type of choline and ethanolamine glycerophospholipids was moderately inhibited by 1 mM ATP and 1 microM cyclic AMP. This synthesis was also inhibited by more than 50% by 1 mM norepinephrine and to a lesser extent by 5 mM hydroxytryptamine and 1 mM acetylcholine. Cyclic AMP may mediate the effects of biogenic amines. The relative synthesis of different glycerophospholipid classes and the relative proportion of alkylacyl type (plasmalogen precursors) and diacyl type of glycerophospholipids may be influenced by the levels of adenine nucleotides and/or biogenic amines. Elevated cyclic AMP levels will decrease the synthesis of plasmalogen precursors.

Acyl esters of polyprenols: specificity of microsomal transacylase for polyprenols of different chain length and saturation.

Transfer of fatty acids from phospholipids to polyprenols, catalysed by the transacylase from rat liver microsomes, was investigated. The specificity of the enzyme for polyprenols of different chain length and different degree of saturation was studied using individual isoprenologues, the preparation of which in highly tritiated form is described. It was found that short-chain polyprenols are better substrates for the enzyme than long-chain polyprenols, and alpha-saturated better than unsaturated or multiply saturated polyprenols. Short-chain, alpha-saturated single isoprenologues were several-fold more active as acyl acceptors than natural dolichol.

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.

Effects of free fatty acids on the enzymic synthesis of diacyl and ether types of choline and ethanolamine phosphoglycerides.

Activities of ethanolaminephosphotransferases (EC 2.7.8.1) and choline phosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats are increased several fold by the addition of 1,2-diacyl-sn-glycerols or 1-alkyl-2-acyl-sn-clycerols. Oleic acid added with diacylglycerols stimulated further the synthesis of lecithins by liver microsomes, confirming the work of Sribney and Lyman (Can J. Biochem. 51: 1479-1486, 1973). With alkylacylglycerols, oleic and stearic acids were inhibitory and linoleic acid was even more inhibitory for the synthesis of both 1-alkyl-1-acyl-sn-glycero-3-phosphorylcholines and the corresponding ethanolamine compounds with microsomes from both tissues. Free fatty acids without added diglycerides had mixed effects. These results are best explained by postulating the presence of two isoenzymes each for ethanolaminephosphotransferase and cholinephosphotransferase of which only one is affected by free fatty acids. Regulation of the phosphotransferases by free fatty acids may determine the proportion of CDP-choline and CDP-ethanolamine used for synthesis of diacyl and alkylacyl types of these phosphoglycerides.

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines by the CDP-ethanolamine: 1-radyl-2-acyl-sn-glycerol ethanolaminephosphotransferase from microsomal fraction of rat brain.

The incorporation of radioactivity from cytidine-5'-phosphate-[(32)P]phosphorylethanolamine into 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines and 1,2-diacyl-sn-glycero-3-phosphorylethanolamines was stimulated more than fourfold by 1-alkyl-2-acyl-sn-glycerols and 1,2-diacyl-sn-glycerols, respectively, with an ethanolaminephosphotransferase (EC 2.7.8.1) present in the microsomal fraction from brains of mature rats. The K(m) values, 0.28 mm for CDP-ethanolamine and 1.9 mm for 1-alkyl-2-acyl-sn-glycerols, were similar to those obtained by other investigators with other 1-radyl-2-acyl-sn-glycerols. The formation of 1,2-diacyl-sn-glycero-3-phosphorylethanolamines from endogenous 1,2-diacyl-sn-glycerols was inhibited by 1-alkyl-2-acyl-sn-glycerols. These properties indicate that the ethanolaminephosphotransferase lacks specificity for the type of group at the 1-position of the lipid substrate. The synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines from 1-alkyl-2-acyl-sn-glycerols and CDP-ethanolamine by an enzyme from rat brain supports the inclusion of this reaction in the metabolic pathway for the synthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.