Microsomal Synthesis Research Articles

Luminal accumulation of newly synthesized morphine-3-glucuronide in rat liver microsomal vesicles

Morphine is converted to morphine 3-β-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 μM). Therefore, the build-up of high (about 20 μM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 μM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion.

Metabolic N- and alpha-C-oxidation of norephedrine by rabbit liver microsomal fractions and synthesis of the metabolic pdocuts.

Abstract The products of metabolism of norephedrine by 10 000 g fractions of rabbit liver microsomes have been shown to be norephedrine hydroxylamine, l-hydroxy-2-oxo-l-phenylpropane oxime, 1-hydroxy-2-oxo-1-phenylpropane, 1,2-dihydroxy-l-phenylpropane (erythro). N-Oxidation was at least as important as α-C-oxidation. The synthesis and properties of the metabolic products are described.

Use of Human Microsomes and Deuterated Substrates: An Alternative Approach for the Identification of Novel Metabolites of Ketamine by Mass Spectrometry

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Cellular retinol-binding protein functions in the regulation of retinoid metabolism.

A microsomal retinyl ester hydrolase from rat tissues is activated by apo-cellular retinol-binding protein, which thus regulates the availability of retinol either for binding to serum retinol-binding protein or for metabolic oxidation. Microsomal retinal synthesis was found to occur in rat tissues with holo-cellular retinol-binding protein as substrate. Retinal could be further oxidized to retinoic acid by a cytosolic fraction.

How Does Fish Oil Lower Plasma Triglycerides?

Studies using dietary fish oil, safflower oil, and palm oil suggest that the decrease in plasma triacylglycerol due to dietary fish oils is somehow related to a decrease in the capacity of the liver to hydrolyze the phosphatidate, which then affects microsomal synthesis of triacylglycerol from diacylglycerol.

Book Review

Book Review

Inhibition of androgen synthesis in human testicular and prostatic microsomes and in male rats by novel steroidal compounds.

The C(17,20)-lyase and 5alpha-reductase are key enzymes in the biosynthesis of androgens. The effects of novel steroidal compounds were evaluated as inhibitors against both human C(17,20)-lyase and 5alpha-reductase in vitro. The concentrations of testosterone (T) and dihydrotestosterone (DHT) in the prostate, testis and serum and changes in the tissue weights were also determined in rats treated with the novel inhibitors. L-12 and L-26 showed potent inhibition of human testicular C(17,20)-lyase with IC50 values of 50 and 25 nM, respectively. L-12, L-38, and I-47 showed moderate inhibition of human testicular C(17,20)-lyase with IC50 values of 75, 108, and 70 nM, respectively similar to ketoconazole (78 nM). Interestingly, L-6, L-26, and L-38 also showed some inhibitory activity against 5alpha-reductase with IC50 values of 75, 125, and 377 nM, respectively. Finasteride, an inhibitor of 5alpha-reductase had an IC50 value of 33 nM. However, ketoconazole did not inhibit 5alpha-reductase nor did finasteride inhibit C(17,20)-lyase. Treatment of normal male rats with several of these novel inhibitors (50 mg/kg x day, s.c., for 14 consecutive days) caused about 45-91% decrease in serum, testicular and prostatic T concentration. Similarly, serum and prostatic DHT concentration were significantly decreased in rats treated with these novel compounds by 50-90% compared with controls. Surgical castration caused almost complete elimination of circulating T and DHT concentration in rat tissues. L-6 and L-12 were the most effective and reduced the wet weight of the prostate by 50%. Although future improvements in their bioavailability are necessary, these novel steroidal compounds show promise as potential agents for reducing T and DHT levels in patients with androgen dependent diseases.

Existence of acyl-CoA hydrolase-mediated pathway supplying arachidonic acid for prostaglandin synthesis in microsomes from rabbit kidney medulla

We have previously shown that acyl-coenzyme A (CoA) hydrolase that hydrolyzes arachidonoyl-CoA (AA-CoA) to arachidonic acid (AA) and CoA is present in the cytosol of rabbit kidney medulla and that this enzyme can supply AA for prostaglandin (PG) synthesis in this region. In the present study, the existence of the acyl-CoA hydrolase-mediated pathway that supplies AA available for PG synthesis in microsomes from the kidney medulla was examined. AA-CoA (20 μM) was preincubated with the 105 000 g pellet (microsomes, 0.5 mg of protein) from the medulla for 5 min at 37°C followed by incubation with the medulla microsomes (0.5 mg of protein) (the source of PG synthesizing enzymes) in the presence of hydroquinone and reduced glutathione for 5 min at 37°C. The PGs formed were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of the microsomal fraction from the medulla in the preincubation mixture increased total PG formation from 3.86 to 8.70 nmol, and this stimulatory effect was somewhat weaker than that of the cytosolic fraction. On the other hand, the microsomal fraction in the kidney cortex has an extremely lower capacity to supply AA for PG synthesis than do medulla microsomes. These results suggest that, in kidney medulla, the microsomes as well as the cytosol have the potential route that supplies AA from AA-CoA for PG synthesis and that this pathway is mediated by acyl-CoA hydrolase.

1,25-Dihydroxyvitamin D Synthesis in Rat Liver Microsomes

Previous studies from our laboratory have shown 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by rat liver homogenates and a low affinity of the hepatic enzyme for 25-hydroxyvitamin D3. Because the liver microsomal vitamin D-25-hydroxylase is the main source of systemic 25(OH)D3, we examined the subcellular location and the kinetics of liver 1,25(OH)2D3 production. Unlike the renal 1 alpha-hydroxylase activity which was assayed simultaneously, 1,25(OH)2D3 synthesis was undetectable in rat liver mitochondria, whereas in microsomes, 1,25(OH)2D3 production followed typical Michaelis Menten kinetics with a Km for 25(OH)D3 of 13.4 microM and a Vmax of 109.8 pg/min per mg protein accounting for most of the 1,25(OH)2D3 synthesized by rat liver cytosol free homogenates. Thus, microsomes are the site for 1,25(OH)2D3 synthesis in the rat liver. This microsomal compartmentalization of the two major steps in the activation of vitamin D to 1,25(OH)2D3 suggests a role for the liver as an autocrine/paracrine organ for 1,25(OH)2D3.

Sources of triacylglycerol accumulation in livers of rats fed a cholesterol‐supplemented diet

The source of free fatty acids (FFA) and the pathways contributing to the accumulation of neutral fats in livers of rats fed a cholesterol-enriched diet were investigated in this report. Supplementation with 1% cholesterol in the diet for four weeks resulted in hepatomegaly in the rats. The contents of cholesterol and triacylglycerols (TG) per gram liver measured in rats fasted overnight increased by 48 mg (approximately tenfold) and 66 mg (approximately fourfold), respectively. The activities of glycerophosphate acyltransferase and diacylglycerol acyltransferase, the two key enzymes for TG synthesis in liver microsomes, were found to increase by 23 and 19%, respectively, in the cholesterol-fed rats. The secretion of plasma TG present predominantly in very low density lipoprotein was found to decrease by approximately 30%. The incorporation of tritium from tritiated water in liver FFA increased by twofold in rats fed the cholesterol-supplemented diet, whereas the activity of CPT I in liver mitochondria decreased by 23%. The uptake of plasma FFA in vivo in livers of fasted rats maintained on the cholesterol-supplemented diet decreased by 60%. Our data thus indicate that the excess TG accumulated in livers of rats fed the cholesterol-enriched diet resulted from increased synthesis and decreased secretion of TG. To meet the demand of fatty acids for this purpose, de novo lipogenesis increased, whereas beta-oxidation decreased. Although difference in the uptake of extrahepatic FFA may be discounted, a difference in the uptake of chylomicron remnants between the control and cholesterol-fed rats may not be ruled out.

Morpholinoalkylindenes as antinociceptive agents: Novel cannabinoid receptor agonists

Indence analogs of pravadoline exhibited antinociceptive activity in several animal models. The inhibition of prostaglandin (PG) synthesis in mouse brain microsomes was diminished in these pravadoline analogs, but they were potent in inhibiting electrically stimulated contractions in the mouse vas deferens (MVD) preparations. Binding studies with ligand WIN 55212-2 have aided to demonstrate that the morpholinoalkyl-indene binding site is functionally equivalent with cannabinoid binding site. The antinociceptive activity of the indene derivatives appears to be mediated by increased affinity for the cannabinoid receptor.

Estimation of dolichol and cholesterol synthesis in microsomes and peroxisomes isolated from rat liver

The participation of peroxisomal and microsomal fractions from rat liver in dolichol and cholesterol synthesis was investigated using marker enzymes. Recovery was 8% for peroxisomes and 33% for microsomes, with virtually no cross-contamination between these fractions. Using these data, it was calculated that the peroxisomal branch-point enzyme activities for dolichol and cholesterol biosynthesis, i.e. cis-prenyltransferase and squalene synthase, were 25% and 12%, respectively, of the total homogenate activity. Treatment with mevinolin increased the peroxisomal contribution in the case of both enzymes, to levels almost equal to that of their microsomal counterparts. These results indicate that peroxisomes play a role in the biosynthesis of isoprenoid lipids and that the extent of this participation is increased extensively when peroxisomes are induced by various treatments.

Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for β-oxidation and glycerolipid synthesis

The dissociation constants for octanoyl-CoA, dodecanoyl-CoA and hexadecanoyl-CoA binding to acyl-CoA-binding protein (ACBP) were determined by using titration microcalorimetry. The KD values obtained, (0.24 +/- 0.02) x 10(-6) M, (0.65 +/- 0.2) x 10(-8) M and (0.45 +/- 0.2) x 10(-13) M respectively, were much lower than expected. ACBP was able to extract hexadecanoyl-CoA from phosphatidylcholine membranes immobilized on a nitrocellulose membrane. The acyl-CoA/ACBP complex formed was able to transport acyl-CoA to mitochondria or microsomes in suspension, or to microsomes immobilized on a nitrocellulose membrane, and to donate them to beta-oxidation or glycerolipid synthesis in mitochondria or microsomes, respectively.

Interaction of the di-catechols rooperol and nordihydroguaiaretic acid with oxidative systems in the human blood: A structure-activity relationship

The effects of the di-catechols rooperol [( E)-1,5-bis(3',4'-dihydroxyphenyl)pent-4-en-1-yne; P2] and nordihydroguaiaretic acid (NDGA) on oxidative systems in the human blood were investigated. P2 and NDGA gave comparable results in the inhibition of leukotriene synthesis in the polymorphonuclear leukocyte and prostaglandin synthesis in platelet microsomes. The oxidation states of myeloperoxidase in the presence of H 2O 2 were also similarly affected by both P2 and NDGA. In these systems, the 4'4'-β- d-diglucopyranoside of rooperol, hypoxoside [( E)-1,5-bis(4'β- d-glucopyranosyloxy-3'-hydroxyphenyl) pent-4-en-1-yne; P2A] had no effect. The only system which showed significant differences in the effects of the catechols was the red blood cell. NDGA in the presence of H 2O 2 had a pronounced haemolytic effect, which did not correlate with its ability to induce methaemoglobin formation, while P2 had a much lower haemolytic effect, but stimulated the oxidation of haemoglobin to a greater extent than NDGA. NDGA is more hydrophobic than P2 which would result in a greater membrane effect. The pent-4-en-1-yne chain which links the catechol moieties in P2 can take part in the resonance structures of the semiquinone free radical, thus assisting in its stabilization and leading to increased methaemoglobin formation. This stabilization is also demonstrated by the fact that P2A affected the oxidation of haemoglobin to nearly the same extent as NDGA.

O-226 - Effects of fatty acyl-CoA esters on prostaglandin synthesis in rabbit kidney medulla microsomes.

O-226 - Effects of fatty acyl-CoA esters on prostaglandin synthesis in rabbit kidney medulla microsomes.

Effects of endogenous and exogenous n-3 and n-6 fatty acids on microsomal synthesis of docosahexaenoic acid in vitro.

Effects of endogenous and exogenous polyunsaturated fatty acids (PUFA) on microsomal synthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA) in vitro were studied using rat livers. Liver microsomes prepared from three groups of rats which had been fed n-3/n-6 ratios of 0.01, 0.39, and 2.70, respectively, for 1 month, were incubated with [1-14C]EPA at 37 degrees C for 30 min. There were no significant differences between the formations of docosapentaenoic acid (DPA) or DHA in the groups. Liver microsomes from rats fed a diet with an n-3/n-6 ratio of 2.70 for 3 weeks were then incubated with [1-14C]EPA in the presence of unlabeled DHA or arachidonic acid (AA). The additional DHA did not have any effect. However, a significant inhibition of DHA synthesis was observed by addition of AA (EPA/AA = 5:2). These results suggest that the microsomal biosynthesis of DHA in rat liver is not susceptible to feedback inhibition.

Effects of fatty acyl-coenzyme A esters on prostaglandin synthesis in rabbit kidney medulla microsomes

The effects of fatty acyl-coenzyme A (CoA) esters (palmitoyl-, stearoyl-, oleoyl-, linoleoyl- and arachidonoyl-CoA) on the synthesis of prostaglandins (PGs) in rabbit kidney medulla microsomes were examined. Medulla microsomes were incubated with arachidonic acid in 0.1 M-Tris HCl buffer (pH 8.0) containing reduced glutathione and hydroquinone and the formed PGE 2, PGF 2α and PGD 2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. Under our incubation conditions rabbit kidney medulla was found to produce PGE 2 mainly. The addition of fatty acyl-CoA esters inhibited total PG formation (the sum of PGE 2, PGF 2α and PGD 2) in a dose-dependent manner. Palmitoyl-, stearoyl- and oleoyl-CoA were about 10 times more potent than linoleoyl- and arachidonoyl-CoA as inhibitors of total PG formation. Linoleic acid was slightly more effective than linoleoyl-CoA, while palmitic acid had no influence on PG formation. All the fatty acyl-CoA esters inhibited the formation of PGE 2. Simultaneously, the production of PGF 2α and PGD 2 was increased. These results suggest that the CoA derivatives of palmitic, stearic and oleic acids have the potential to modulate PGE 2, PGF 2α and PGD 2 synthesis by affecting the activities of bothcyclooxygenase and endoperoxide E 2 isomerase.

Fatty acid chain elongation synthesis in eel-liver Microsomes

1. 1. Fatty acid chain elongation synthesis, which is remarkably active in eel-liver microsomes, requires [2- 14C] malonyl-CoA as the two-carbon donor. NADPH is much more effective than NADH as reducing agent. 2. 2. Maximum elongation activity was obtained at an ATP concentration of 0.5 mM, although in its absence a noticeable incorporation of malony-CoA into fatty acids was observed. Coenzyme A had no effect up to concentration of 1μM, but strongly inhibited lipogenesis at higher concentrations. 3. 3. Thin layer chromatography (TLC) analysis showed that most radioactivity was associated with free fatty acids while nearly 30% of the labelled products were esterified into phospatidylcholine, phosphatidylethanolamine and neutral lipids.

Microsomal Triacylglycerol Synthesis and Diacylglycerol Concentration in the Liver of Rats Fed with Soybean and Egg Yolk Phospholipids.

Diets containing 10% soybean oil, soybean phospholipid and egg yolk phospholipid decreased rat liver microsomal diacylglycerol acyltransferase activity when measured with an endogenous diacylglycerol substrate relative to that in rats fed with a low-fat diet (0.5% soybean oil). Although no significant difference was detected in the enzyme activity among the rats fed with the diets containing 10% lipids, the extent of the decrease was more prominent with two kinds of phospholipids compared to soybean oil. When the enzyme activity was measured with an exogenous dioleoylglycerol substrate dispersed in ethanol, it was also significantly depressed by the diets containing 10% phospholipids and soybean oil, but to a much lesser extent. Dietary phospholipids reduced the microsomal concentration of diacylglycerol to a value less than one-third that in the animals fed with a low-fat diet. Soybean oil also decreased this parameter, but to a lesser extent. In addition, soybean and egg yolk phospholipids compared to soybean oil were more effective in reducing the triacylglycerol concentration in the liver. It seemed that dietary phospholipid compared to soybean oil exerted a more potent triacylglycerol lowering effect by modifying the concentration of microsomal diacylglycerol available for triacylglycerol synthesis in the liver.

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodeling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the 1-position of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.