Ultraviolet-absorbing Product Research Articles

Citrate in urine determined with a new citrate lyase method

An enzyme-spectrophotometric method to determine citrate in biological fluids is proposed, based on citrate lyase-catalyzed and phenylhydrazine reactions. The enzyme converts citrate into oxaloacetate, which, in the presence of phenylhydrazine, is transformed into the corresponding phenylhydrazone. The ultraviolet-absorbing product is determined by absorbance measurement at 330 nm. The method is more precise and twice as sensitive as the traditional citrate lyase method and, because it does not require the use of additional enzymes and coenzymes, is cheaper and simpler. Mean analytical recovery of citrate averaged 100.7% +/- 2.2%, imprecision (CV) of the assay for citrate at 0.96 mmol/L (urine) was 2.0%, and the lower limit of quantification was 0.08 mmol/L. Results correlated well with those by both ion-chromatographic and traditional citrate lyase methods.

An optimized micromethod for determining the catalytic activity of serum ribonuclease.

An optimized assay is described for the catalytic activity determination of serum ribonuclease, using polycytidylic acid as substrate and measuring the released acid-soluble ultra-violet absorbing products. Recommended final reaction concentrations are 0.3 mmol/l polycytidylic acid, 200 mmol/l imidazole/HCl buffer, pH 7.0, and 50 mmol/l NaCl. Optimal concentrations for the precipitation procedure, guaranteeing sufficient precipitation and minimal decomposition of unreacted substrate, are 160 mmol/l perchloric acid and 4 mmol/l lanthanum nitrate. Coefficients of variation for the method (within series and between days) ranged from 2.2 to 7.9%. No sex-related differences of catalytic activity were observed. In 63 blood donors with normal values of serum creatinine, the upper limit of the reference intervals (99th percentile) was 33.7 kU/l.

Studies on testosterone hydroxylation and pentobarbital oxidation by rat liver microsomes: Selective effects of age, sex, castration and testosterone propionate treatment

The effect of sex, age, castration and testosterone propionate treatment on the metabolism of testosterone and pentobarbital by rat liver microsomes have been studied. The adult male rat metabolized testosterone primarily to a polar ultraviolet absorbing fraction composed of hydroxylated products. The rates of 6β-, 7α- and 16α-hydroxylation of testosterone by liver microsomes of male rats varied independently as a function of age, castration and testosterone propionate treatment. Changes in the rate of pentobarbital oxidation most closely paralleled changes in the rate of the 16α-hydroxylation reaction. These data suggest that a single component in liver microsomes cannot alone determine the specificity in the 6β-, 7α- and 16α-hydroxylation of testosterone. In the female rat, little metabolism of testosterone to polar ultraviolet absorbing products occurred and castration and/or testosterone propionate treatment had little or no effect on this pathway.

Chemical analysis of DNA alterations: II. Alteration and liberation of bases of deoxynucleotides and deoxynucleosides induced by hydrogen peroxide and hydroxylamine

During the reaction of H 2O 2 with deoxyribonucleotides or their components, the ultraviolet absorption decreased at rates which were for cytosine > thymine > adenine > guanine . The chromatographic separation of the reaction mixtures revealed that all four bases were partially removed from both deoxynucleotides and deoxynucleosides, the reaction rates being for thymine > adenine > cytosine > guanine . Since no deoxynucleosides were formed from deoxynucleotides, H 2O 2 apparently cleaved only the N- glycosidic linkage, the phosphate group not being involved. dpA, dA, and adenine gave rise to an additional ultraviolet-absorbing base, A 1. Kinetic arguments showed that dpA was first altered to dpA 1 and then A 1 was liberated. A quantitative analysis of the ultraviolet-absorbing products showed that the base liberation was only partially responsible for the loss of ultraviolet absorption: some of the purine and the majority of the pyrimidine bases must have been converted to non-ultraviolet-absorbing products, the relative effectiveness of alteration being for cytosine > thymine > adenine > guanine . Experiments with nucleotides labeled in the base moiety showed that most of the bases which lost their ultraviolet absorption remained attached to the deoxyribose phosphate. The relative frequency with which the doexynucleotide molecules were decomposed was for adenine > thymine > cytosine > guanine . 0.1 M NH 2OH also liberated and destroyed bases, similar to H 2O 2, but it produced a different altered adenine: A 2.

The metabolism of cholest-4-en-3-one-7α-ol by rat-liver cell fractions

1. 1. The metabolism of cholest-4-en-3-one-7α-ol was studied by incubating the tritium-labelled substance with the different cell fractions of rat-liver. After incubation, a lipid extract was made, the constituents of which were crudely separated on small alumina columns. The metabolites were further separated and identified chemically and by radioactive means, by thin-layer chromatography on fluorescent plates. 2. 2. In mitochondria, a more-polar ultraviolet-absorbing product, suggested to be cholest-4-en-3-one-7α,26-diol, was formed. The reaction was not stimulated by NADPH or NADH. Supernatant factor (boiled supernatant fraction) possibly stimulated the reaction. Disruption of the mitochondria either sonically or osmotically did not destroy the enzymic activity, which was found to lie in the debris sedimented after disruption. An acetone powder was found to be inactive. 3. 3. No metabolism of cholest-4-en-3-one-7α-ol occurred in the microsomal fraction. However, on addition of either active supernatant or supernatant factor, the polar ultraviolet-absorbing material was formed. Thus, a thermostable co-factor found in the supernatant fraction gives microsomes the power to hydroxylate. This cofactor was inactivated by treatment with alkali at 100°. 4. 4. The supernatant fraction was partially purified by ammonium sulphate precipitation. Cholest-4-en-3-one-7α-ol was reduced to cholest-4-en-3α,7α-diol and 3α,7α-dihydroxycoprostane. The production of both substances was found to depend on substrate concentration and NADPH concentration. Cholest-4-en-3-one inhibited the production of 3α,7α-dihydroxycoprostane only. Cholest-4-en-3α,7α-diol was found to be not readily converted to 3α,7α-dihydroxycoprostane. The enzyme system was not specific, and readily reduced many α,β-unsaturated ketones. Coprostan-3-one-7α-ol was converted readily to 3α,7α-dihydroxycoprostane, with either NADPH or NADH as co-factor, thus suggesting two possible routes for the formation of 3α,7α-dihydroxycoprostane by reduction of cholest-4-en-3-one-7α-ol. Biochim. Biophys. Acta, 116 (1966) 362–378

A sensitive assay for pancreatic ribonuclease

A convenient, rapid assay for pancreatic ribonuclease is based on the release of acid-soluble ultraviolet-absorbing products from polycytidylate. The sensitivity (ca. 10 μμg of ribonuclease) is comparable to the most sensitive assays available.

A new method for determination of hepatic glutathione reductase activity.

SummaryA reaction between sodium nitrite and reduced glutathione (GSH) yielded a product having an ultraviolet absorption maximum at 334 mμ. No ultraviolet absorbing product was formed when oxidized glutathione was reacted with sodium nitrite. Optimal conditions and stoichiometry of the reaction were described. The reaction was not specific for GSH; ultraviolet absorbing products were formed with a variety of sulfhydryl compounds. The reaction was used to determine glutathione reductase activity of rat liver, and yielded data which compared favorably with that obtained by an accepted method.

Effect of X-Radiation on Aqueous Solutions of Adenosine Diphosphate

The absorbancy at 260 m mu of a dilute solution of adenosine diphosphate decreases linearly with x ray dose up to 3 x 10/sup 5/ r. The principal ultraviolet-absorbing product is adenine. The concentrations of ADP and adenine in the irradiated solution were measured. Adenosine monophosphate is present only in trace amounts equal to that in control nonirradiated solutions. With combined paper chromatographic and autoradiograph techniques alter the irradiation of uniformly labeled C/sup 14/-ADP, at least seven irradiation products were formed. Irradiation of adenosine mono- and triphosphate under similar conditions also yielded adenine as the principal ultraviolet-absorbing product. (auth)

Effect of ionizing radiation on aqueous solutions of guanylic acid and guanosine.

Aqueous solutions of guanylic acid irradiated wth high-energy electrons were analyzed by paper chromatography using an acid solvent. Two ultra-violet absorbing products were found, one of which had values identical wth granine in three solvent systems, and the other having spectra sinlilar to those for products obtained by the alkylation of guanylic acid under mildly alkaline conditions. The radiation degradation of granylic acid and guanosine are compared wth that of corresponding adenine derivatives, where adenine is the sole ultra-violet absorbing product. (C.H.)