Restoration of rat liver L-threonine dehydratase activity by pyridoxamine 5'-phosphate: the half-transaminating activity of L-threonine dehydratase and its regulatory role.

When a highly purified preparation of rat liver l-threonine deaminase (l-TDH, EC 4.2.1.16) was 99% inactivated by dialysis, removing bound pyridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPLC, the commercial PMP used in the incubation mixture was found to contain only extremely small amounts of PLP, which could not account for restoration of l-threonine dehydratase activity. HPLC analysis of the assay mixtures showed that during incubation, sufficient PLP had been formed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, which either contaminated, or were formed by the minimal amount of PLP-holoenzyme always present even in the dialyzed preparation. When sufficient PLP was formed, the PLP-holoenzyme and the original 'true' l-threonine dehydratase activity were restored. When PMP was incubated with the apoenzyme in the presence of small quantities of keto acids (pyruvate or 2-oxobutyrate) small amounts of l-alanine or l-aminobutyrate were formed. The reaction was not reversible; l-alanine and l-aminobutyrate did not react with the PLP-holoenzyme. No transaminating activity occurred with other amino acids. These results show that l-threonine dehydratase exists in two forms: the well known stable apoenzyme-PLP (hydrolase deaminating) and the transient apoenzyme-PMP (non-reversible half-transaminating). Half-transamination has the biological role of keeping the activity of the 'true' l-TDH constant and of regulating intracellular levels of pyruvate, alanine, oxobutyric acid, l-aminobutyric acid, l-threonine and l-serine.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues.

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

Metabolism of adenosine in human colorectal tumour.

The aim of this work is to analyse the activities of the enzymes metabolising adenosine in fragments of neoplastic and normal-appearing mucosa, surrounding the tumour in 20 patients affected by colorectal cancer. The results show that the activities of the enzymes are markedly higher in tumour in comparison to normal mucosa to coope with the accelerated purine metabolism in cancerous tissues.

["Qualitative test of fecal fat" and "steatocrit", simple complementary methods for the evaluation of steatorrhea in childhood]. / L'"esame qualitativo dei grassi fecali" e lo "steatocrito", semplici metodi complementari di valutazione della steatorrea nell'infanzia.

The evaluation of fecal fat elimination (steatorrhoea) is of primary importance for diagnosis of gastroenterological disorders. In childhood it is quite difficult to apply the ordinary methods of evaluation, on the other hand it is necessary to make use of them to screen and diagnose maldigestion and/or malabsorption syndromes. In this work "Steatocrit" method by Phuapradit and "Fecal fat qualitative test" (FFQT) on glass, by Jacobson, have been used in a parallel study on stool samples from subjects with suspected gastrointestinal disease. While Steatocrit was determined on 200 samples, FFQT was determined on 1574 samples. Our data show that steatocrit is fully able to detect quantitative steatorrhoea with high significance when compared to controls. Likewise FFQT shows a sensibility of 100% compared to controls and it is able to predict coeliac disease and cystic fibrosis in 85.5% of cases and in 89.9% of cases respectively. We conclude that these two tests are sure and auxiliary each other. They allow, when performed on the same sample, to go toward diagnosis of both malabsorption and maldigestion, furthermore they allow to monitor steatorrhoea under therapy.

[Antigliadin antibodies (AGA) in the various stages of celiac disease in children]. / Gli anticorpi antigliadina (AGA) nelle varie fasi della malattia celiaca del bambino.

Antigliadin antibodies (AGA), both IgA and IgG, were studied in the serum of 84 coeliac children during the various stage (Diagnosis, GFD, Challenge) and in 29 healthy children, with a micro-ELISA technique. The results demonstrated the presence of AGA in the serum of coeliac children and a different behaviour between the two Ig-classes in the various stages of the disease. During acute phase both classes were present at high titre. When gluten was withdrawal from the diet, while the titre of IgA fell rapidly since the first month, the IgG titre decreased slowly and raised the normal limits after six months. If the children didn't observe a corrected GFD, the serum AGA titres remained at high levels. During challenge, while IgG raised since the early days, IgA titres raised later, when the intestinal damage became important. The explanation of this different behaviour could be that AGA-IgA are derived from gut mucosa, on the contrary AGA-IgG are not synthesised in the intestine. We believe that serum AGA seem to be good markers of the immune reaction in the intestine triggered by gluten. Furthermore we conclude that the assay of AGA in the serum of coeliac patients is: 1) high sensible and specific method; 2) the most important screening test for intestinal biopsy; 3) the most important test for diagnosis and follow-up of CD; 4) the test which could substitute 1 or 2 intestinal biopsies of the ESPGAN protocol.

Determination of urinary methylated purine pattern by high-performance liquid chromatography.

We describe the group selective separation and quantification of unmodified and modified purines in human urine by high-performance reverse phase liquid chromatography. The pattern of oxypurines and methylated purines: hypoxanthine (Hx), xanthine (X), 1-methyl hypoxanthine (1-MHx), 1-methyl guanine (1-MG), 3-methyl guanine (3-MG), 7-methyl guanine (7-MG), 1-methyl xanthine (1-MX), 3-methyl xanthine (3-MX), 7-methyl xanthine (7-MX), 1,7-dimethyl guanine (1,7-dMG), 1,3-dimethyl xanthine (1,3-dMX), 1,7-dimethyl xanthine (3,7-dMX) and 1,3,7-trimethyl xanthine (1,3,7-tMX) were determined in a single run in urine of a healthy subject and a gout patient before and after treatment with allopurinol. This method may be useful to investigate the urinary pattern of methylated bases in diseases involving purine metabolism.