Radio High-performance Liquid Chromatography Research Articles

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol.

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.

High-performance liquid radiochromatography: Its application for stability study of [3H] stobadin

An HPLC method was developed for studying the stability of [3H] stobadin stored in physiological saline or buffers at various pH. Optimum conditions for the separation of degradation products were elaborated. A high-performance liquid chromatograph equipped with a radioactivity detector was used.

Transfer of Added [18-14C]Octatriacontane - Cigarette Smoke Formation Studies

Abstract Radiolabelled [18-14C]octatriacontane (C38H78) was used as a model compound to study the transfer of compounds from tobacco into smoke. Non-filter cigarettes were made from Kentucky reference 2R1 cigarette tobacco filler oversprayed with alkane and were smoked on a single-port total recovery smoking machine. Of the recovered activity nearly 60 % was found in the mainstream and sidestream particulate phases of smoke, 5 % in the gaseous phase and 35 % in the butt. Reverse-phase high-performance liquid radiochromatography and thin-layer radiochromatography were used effectively to analyse the cyclohexane extracts of the filler, mainstream and sidestream total particulate matter, and butt. No degradation of the alkane was observed prior to smoking. The radioactivity in the mainstream total particulate matter extracts consisted mostly of intact [14C]octatriacontane. Sidestream total particulate matter extracts contained both intact octatriacontane and breakdown products. The radioactivity in butt extracts consisted almost entirely of intact octatriacontane, indicating that excess radioactivity in the butt is due to filtration of the intact alkane by the filler rod. The transfer of octatriacontane into mainstream smoke without pyrolytic degradation parallels that of the tobacco alkane dotriacontane.

Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with K m = 37 μM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.

Metabolism of ethyl eicosapentaenoate (EPA-E) in rats and effect of its metabolites on ellagic acid-induced thrombus formation in the stenosed femoral artery of rabbits.

The metabolism of ethyl eicosapentaenoate (EPA-E) was investigated in rats in vivo and in vitro with uniformly labelled 14C-EPA-E, and the antithrombotic properties of major metabolites of EPA-E in rabbits were examined.When 14C-EPA-E was administered orally to rats, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were identified in the liver as metabolites of EPA-E. These metabolites were incorporated mainly into phospholipids and triglycerides, and were found at the C-2 position of phospholipids and the C-1 and/or C-3 positions of triglycerides.When 14C-EPA coenzyme A (14C-EPA-CoA) was incubated with intact mitochondria, 14CO2 was released and chain-shortened metabolites were detected by radio-high performance liquid chromatography. When 14C-EPA-CoA was incubated with intact peroxisomes, chain-shortened metabolites were detected as in the case of mitochondria.When potassium eicosapentaenoate (14C-EPA·K) was incubated with microsomes, DPA and DHA were produced as a result of chain elongation and subsequent desaturation reactions. When 14C-EPA-CoA was incubated with intact peroxisomes, DPA was produced in the presence of malonyl-CoA as a cofactor.EPA-E significantly decreased the incidence of thrombus formation and also decreased the length of thrombi in rabbits. Major metabolites of EPA-E, such as EPA, DPA and DHA, moderately decreased the incidence of thrombus formation.

Comparison between condensation and overall chain elongation of arachidoyl-CoA and arachidonoyl-CoA in swine cerebral microsomes

The condensation and overall elongation products of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in swine cerebral microsomes were detected by radio gas chromatography. In addition, the condensation products with malonyl-CoA as substrate were analyzed by radio high-performance liquid chromatography. Three main condensation products were detected; the overall elongation products of exogenous 20:0-CoA were 22:0 and 24:0, and those of endogenous substrates were 18:0, 22:4, and 24: 4. The yield was estimated for the conversion of 3-ketoacyl-CoAs to the corresponding saponification products (methyl ketones or R-2-one; e.g., 2-heptadecanone = 17:0-2-one); these products were identified in the preceding paper ( S. Yoshida and M. Takeshita (1987) Arch. Biochem. Biophys. 254, 170–179). The extraction of R-2-one by hexane depended on the acyl chain length. The yield of 2-heneicosanone (21:0-2-one) detected by radio gas chromatography was 80% whereas the yields of 17:0-2-one and 2-heneicosatetraenone (21:4-2-one) from the corresponding 3-ketoacyl-CoAs were 56 and 48%, respectively. A quantitative comparison was performed for the condensation and overall elongation activity; it was noticed that the condensation activity for the system which simultaneously produced two elongation products was nearly the same as that of the corresponding overall elongation activity. This result suggests that the condensation step may be at least one of the rate-limiting steps in the overall elongation of very-long-chain fatty acyl-CoA.

Biotransformation of primary nicotine metabolites. II. Metabolism of [3H]-S-(-)-cotinine in the guinea pig: determination of in vivo urinary metabolites by high-performance liquid-radiochromatography.

1. The metabolism of [3H]-S-(-)-cotinine in vivo has been investigated in the guinea pig. The quantitive determination of urinary metabolites has been carried out using a high-performance liquid radiochromagraphic procedure. Metabolite analysis of C- and N-oxidation products, and N-methylcotininium ion, were carried out on two chromatographic systems: (a) Partisil-10 SCX cation-exchange chromatography, and (b) Partisil-10 ODS reverse-phase chromatography. 2. 3-Hydroxycotinine was the major urinary metabolite of cotinine in the guinea pig, accounting for 57% of the total radioactivity in the urine. Cotinine-N-oxide constituted 18% of total metabolites in the urine, whereas neither nicotine nor the N-methylcotininium ion were detected as urinary metabolites of [3H]-S-(-)cotinine. S-(-)-Cotinine was extensively metabolized in the guinea pig; total recovery of radioactivity in 24 h urine was very high (greater than 95%) and very little cotinine was detected (less than 1%) in the urine. 3. Two unidentified metabolites of [3H]-S-(-)-cotinine were detected which collectively constituted approximately 20% of total urinary tritium.

Application of New Radiochemical Methods to the Study of the Cure of Epoxies by Various Imidazoles

The reaction between phenylglycidyl ether and a number of substituted imidazoles has been investigated using a combination of tritium nuclear magnetic resonance spectroscopy and radio-high performance liquid chromatography, two methods that have not been used previously in this area. For more volatile catalysts such as aromatic amines radio-gas chromatography can be employed to good effect.

Studies on 18F-labeled pyrimidines. II. Metabolic investigation of 18F-5-fluorouracil, 18F-5-fluoro-2'-deoxyuridine and 18F-5-fluorouridine in rats.

18F-labeled 5-fluorouracil(FUra), 5-fluoro-2'-deoxyuridine(FdUrd), and 5-fluorouridine ( FUrd ) were synthesized with high radiochemical purities. The 18F-labeled pyrimidines were injected into rats. The metabolites in serum, bile, and urine were analyzed up to 2 h after administration by radio-high performance liquid chromatography (HPLC). The blood clearance of three pyrimidines was very rapid. In the serum the nucleosides and base disappeared very rapidly with a biological half-life of about 2 min and most of them had disappeared by 60 min. The metabolites in the urine were similar to those in the serum. In the bile pyrimidine nucleosides and base were not detected. 18F- was found in the metabolites. Our results explain the high uptakes in the kidney and liver in biodistribution studies of the 18F-labeled pyrimidines.

Combined action of acivicin and d-galactosamine on pyrimidine nucleotide metabolism in hepatoma cells

The glutamine antagonist acivicin, l-(αS,5S)-α- amino-3- chloro-4,5- dihydro-5- isoxazoleacetic acid, strongly reduced CTP and GTP contents in AS-30D rat hepatoma cells in suspension. UTP only dropped to 63% of the respective control after 4hr; however, by combining acivicin with the uridylate-trapping sugar analogue d-galactosamine, a synergistic decrease in UTP contents to 7% of control was induced. Incorporation of 14CO 2 into purine and pyrimidine nucleotides followed by radio-high performance liquid chromatography showed marked inhibition of purine and pyrimidine biosynthesis de novo; the latter was reduced to 35% of control. The inhibitory potency of acivicin on glutamine-dependent carbamoyl-phosphate synthetase and consequently on de novo uracil nucleotide formation was also reflected by the complete suppression of the d-galactosamine-induced rise in total uridylate. Induction of UTP deficiency by interference with the first and rate-limiting step in pyrimidine biosynthesis de novo together with a trapping of uridylate by d-galactosamine may provide a promising approach to the chemotherapy of hepatocellular carcinoma.

Urine pH and phencyclidine excretion.

Subeffective doses (0.5 mg) of 3H-phencyclidine (PCP) were given intravenously to three healthy men under two regimens designed to alkalinize or acidify their urine (oral sodium bicarbonate or ammonium chloride). The concentrations of PCP and its metabolites in saliva, plasma, and urine for 7 hr after injection were determined by high-performance liquid radiochromatography. A sample of perspiration from one subject was analyzed. The effects of physical exercise on the plasma concentration and urinary excretion of PCP were also studied. Multiple linear regression analysis showed the logarithm of renal clearance the renal clearance of PCP. PCP and its metabolites are also excreted in perspiration. Our results support clinical reports of the importance of vigorous acidification of urine and diuresis in treatment of PCP intoxication.

Chloramine‐t for “no‐carrier‐added” labelling of aromatic biomolecules with bromine‐75,77

AbstractThe applicability of chloramine‐T (CAT) was examined for “no‐carrier‐added” labelling of aromatic compounds with the short‐lived isotopes bromine‐77 (T1/2 = 56 h) and bromine‐75 (T1/2 = 98 min). In aqueous solution the optimum reaction conditions with respect to pH, concentration of CAT, reaction time and added brominecarrier were determined using tyrosine as a model substrate. Highest radiochemical yields (>85%) were obtained at pH 1 within a few seconds at a CAT concentration of 10−3 M. At pH 7 a maximum radiochemical yield of 65% was achieved with 10−2 M after 2.5 minutes. Studies with some model peptides showed that only the tyrosyl residues were brominated and similar yields were obtained as in free tyrosine. Some aromatic molecules such as α‐methyl‐tyrosine, phenylalanine, uracil, cytosine, and phenol were radiobrominated by the CAT method with yields up to 90%. Separation and identification of the labelled products were achieved by means of radio high performance liquid chromatography.