B. Frosi

Fast-atom bombardment mass spectrometry for mapping of endogenous methylated purine bases in urine extracts.

Fast-atom bombardment (FAB) mass spectrometry, linked with tandem mass spectrometry (MS/MS), was employed for the identification of methylated purine bases in four urinary extracts of healthy subjects and fourteen urinary extracts of patients bearing colorectal tumors. In order to obtain an easy structural identification of the species present in urinary extracts, the MS/MS spectra of MH+ species of twenty nine diagnostically relevant purine bases were studied. Even if definitive quantitative data cannot be obtained by this approach, FAB mass spectra of urine extracts lead to a readily reproducible mapping of endogenous purine bases, allowing a distinction between healthy and sick subjects. Bases such as 9-ethyladenine, N6-2-isopentenyladenine and N6-benzyladenine were detected only in urine samples of colorectal tumor bearing patients. The detection in urine of compounds such as 7-methylguanine and 1-methylguanine, and their increase in the urine of colorectal tumor bearing patients, has been justified either by a more rapid turnover of nucleic acids in tumor tissue or by an increase in the extent of their methylation. The obtained results indicate that the method can be employed for diagnostic purposes.

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.

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.

Immunohistochemical Analysis of P185 and ASL Activity in Pre-Neoplastic Lesions and Intestinal Mucosa of Human Subjects

Colorectal cancer is the second neoplasia in terms of incidence and mortality (after lung-cancer in men and breast cancer in women), with 20,000 new cases in Italy every year and 10,000 deathes. The starting point of the multiphasic process leading to colorectal cancer (somatic and inherited genetic alterations which involve activation of onco-genes and inactivation of oncosuppressor genes) seems to be adenomatous polyposis. Since mortality due to colorectal cancer is high in our country, new perspectives for early diagnosis and chemotherapy, based on molecular and biochemical patterns at different stages of the pathological process, are needed.