L. Arezzini

Determination and separation of allantoin, uric acid, hypoxanthine, and xanthine by capillary zone electrophoresis.

Hypoxanthine, xanthine, uric acid and allantoin are the main products of purine nucleotide catabolism and they are formed through the sequence: nucleotide→ hypoxanthine → xanthine→ uric acid→ allantoin. Under normal conditions, they represent the balance between synthesis and breakdown of nucleotides. Their levels change, for example, under oxidizing conditions and may be useful for quantifying oxidant generation in human extracts. Uric acid is oxidized to allantoin by variuos reactive oxygen species and is thought to act as an antioxidant in human bodily fluids. Allantoin concentrations may therefore indicate free radical damage in vivo.

Levels of folic acid in plasma and in red blood cells of colorectal cancer patients

The levels of folic acid have been determined by radioimmunological method in the plasma and in the red blood cells of normal subjects and colorectal cancer patients. A decrease was evident both in the plasma and erythrocytes of cancer patients. The possible reasons and applications of this observation are discussed.

Separation and determination of liver uric acid and allantoin.

We previously described the only satisfactory procedure yet achieved for separating uric acid and allantoin from rat liver. The procedure was based on trichloroacetic acid (TCA) extraction, acid hydrolysis, treatment with Hg-acetate, and cation- and anion-exchange chromatography. After separation, allantoin was quantified by a colorimetric method, and uric acid enzymatically using uricase. Since this procedure is too time-consuming, we propose an improved version which avoids the need for anion-exchange chromatography and the complex assay of catabolic compounds. The new method consists of a very fast and simple HPLC separation and direct determination of uric acid and allantoin at 220 nm. The method can be used for fresh tissue or after treatment of the tissue with labeled precursor.

EFFECT OF ESTRADIOL ON PHOSPHOLIPID LIPOPROTEIN LEVELS AND FATTY ACID COMPOSITION IN THE RAT

Phospholipid content and fatty acid composition in the different serum lipoproteins showed specific variations after castration and estradiol administration. Only the levels of phospholipids in HDL, the principal lipoprotein carrying phospholipids, increased after castration and were further enhanced by estradiol treatment, especially at low doses. Fatty acid composition showed many variations and an irregular pattern. The EFA/NEFA, EFA/ME and SAT/ME ratios were calculated. EFA/ME increased in VLDL after both doses of estradiol, while EFA/NEFA and EFA/ME of LDL enhanced at the low dose and decreased at the high one in a dose-dependent manner. HDL showed higher EFA/ME and SAT/ME ratios after castration and lower values of all ratios after both doses of estradiol. The correlation with diseases more frequent in men is discussed.

Labelling of uric acid and allantoin in different purine organs and urine of the rat.

We studied the incorporation of 14C-formate into uric acid and allantoin in different organs (liver, lung, kidney, spleen), isolated hepatocytes, perfused liver and urine of the rat. Allantoin had a higher specific radioactivity than uric acid after 14C-formate load in the liver in vivo. This was found to be a strictly hepatic phenomenon and not due to the influence of other tissues.

INFLUENCE OF ESTROGEN ON CHOLESTEROL ESTERIFICATION AND FATTY ACID COMPOSITION IN SERUM LIPOPROTEINS OF CASTRATED RATS

Total, free and esterified cholesterol and its fatty acid composition were measured in the serum lipoproteins of castrated rats after estradiol administration. In general, castration and treatment with estradiol led to a decrease in total esterified cholesterol content. However, estradiol induced an effect opposite to that of castration on the fatty acid composition in VLDL. The effects were variable in HDL and insignificant in LDL. Similarly, the ratios of essential fatty acid to non-essential fatty acid (EFA/NEFA) and that to monoenoic acid (EFA/ME) were affected differently in castration and estradiol treatment in VLDL, but not in HDL or LDL. The pattern of lipid metabolism in castrated and estradiol-treated rats thus appears opposite to that described in human pathology.

Separation and determination of allantoin, uric acid, hydantoin and urea

Abstract Hydantoin and urea, obtained by reducing the allantoin ring with hydroiodic acid or uric acid after treatment with uricase, were separated from each other and from their starting compounds by high-performance liquid chromatography and anion-exchange chromatography.

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors.

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.

Melting Temperature Analysis as quantitative method for detection of point mutations.

Abstract Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.

Uric Acid and Allantoin in Rat Liver after Oxonic Acid and 14C-Formate

Uric acid and allantoin are the key catabolites of purine degradation: their quantity and patterns of radioactive labelling from precursors (such as 14C-glycine, 14C-formate, 14C-adenine) are two parameters used in the evaluation of purine catabolism1-5. The correct evaluation of both compounds has always presented several difficulties, due to the fact that many interfering metabolites are present in tissues.