Adenosine kinase gene expression in human colorectal cancer.

Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate gene expression of adenosine kinase, a key enzyme in adenosine metabolism, in human intestinal biopsy specimens of 10 colorectal cancer patients. Quantitative mRNA expression levels were normalized against the reference gene beta-actin. The results showed that adenosine kinase gene expression was significantly higher in cancer than in normal-appearing tissue, in line with our previous measurements of adenosine kinase enzyme activities in colorectal tumor samples.

A kinetic study of the rat liver adenosine kinase reverse reaction.

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.

Circulating gastrin and ghrelin levels in patients with colorectal cancer: correlation with tumour stage, Helicobacter pylori infection and BMI.

Many studies have pointed out a possible role of gut peptides, including gastrin and ghrelin, in the pathogenesis and natural history of gastrointestinal malignancies, one of the most common death cause in the Western world. The objective of this work is to check gastrin and ghrelin serum levels in patients with colorectal cancer according to tumour's location, stage, Helicobacter pylori infection and BMI, in order to understand the two peptides' behaviour through the tumour's natural history and evaluate their assay's use in research and clinical practice. Twenty-nine subjects affected by colorectal cancer and 50 healthy controls were studied. Circulating gastrin and ghrelin levels and H. pylori serum antibodies were assessed by radioimmunologic assay and ELISA method. Gastrin and ghrelin serum levels were respectively slightly higher and significantly lower in colon cancer patients than in controls. Gastrin levels were higher in patients carrying left colon cancer and H. pylori infection while ghrelin levels were lower in both these groups. Both hormones' serum levels decreased from tumour earlier to later stages. Significant differences persisted in the correlation between BMI and ghrelin levels in controls but not in patients. Additional studies are necessary to ascertain the significance of gastrin and ghrelin opposite behaviour in colon cancer probably linked with interferences in endocrine pathways involving other gut peptides in this compromised condition.

Adenosine kinase from rat liver: new biochemical properties.

Adenosine kinase is a well-known enzyme which catalyzes the phosphorylation of adenosine to AMP: Its metabolic and kinetic properties are well studied. Here, we report new properties of rat liver enzyme, demonstrating a new reaction: ADP can be a phosphate donor instead ATP, according to the reaction: adenosine + ADP --> 2AMP) demonstrating the efficiency of AdK to phosphorylate adenosine, also starting from ADP. Cells could exploited this property in situations in which ATP levels are strongly decreased and ADP decreases slowly.

Serum folate and vitamin B12 levels in children from Mozambique.

In order to investigate the behaviour of biochemical parameters in children from Mozambique, we have determined the serum levels of folic acid and vitamin B12, two well known markers of nutritional anemia. We have correlated their values with other blood parameters and have evidenced potential interesting relationship between folate content and platelets count.

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.

Effect of testosterone on purine nucleotide metabolism in rat liver.

In previous studies, we found that castration induced interesting morphological and biochemical changes in rat liver. For the present study, we have examined the effects of testosterone on the kinetics of purine nucleotide metabolism with the aim of determining the steps affected by testosterone deficiency. A biomathematical model of purine nucleotide metabolism was used to analyze the many reactions involved. The model simplifies purine nucleotide metabolism to four main steps: 1) de novo synthesis from PRPP to IMP; 2) the inosinic branch point from IMP to GMP or AMP; 3) catabolism of IMP, AMP and GMP to uric acid; 4) RNA and DNA formation from AMP and GMP. We evaluated rate constants from each step from variations in specific radioactivity of metabolites labelled with (14)C-formate, a precursor of de novo synthesis. The model was applied to the liver of normal and castrated rats before and after testosterone treatment. All four steps were slowed after castration, and were not completely restored by androgen administration. The model can give a clear representation of the kinetics of the reactions involved in the liver nucleotide metabolism investigated here, and we propose that a similar approach could be useful whenever a quantitative evaluation of the results obtained in vivo after administration of labelled precursors is required.

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.

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives.

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.

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.

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.

Some chemical properties and biological role of thiazolidine compounds.

In this study we have investigated some chemical properties and the biological role of thiazolidine compounds, obtained by condensation of aminothiols (L- or D-cysteine, cysteamine) with pyridoxal-5'-phosphate. These products have been tested in presence of rat liver extracts (supernatant and mitochondria); bacterial suspensions and enzymes (L- or D-aminoacid oxidase, xanthine oxidase) with interesting results which gives evidence to a biological role. Their formation in vivo may represent the regulation of intracellular levels of pyridoxal-5'-phosphate and aminothiols. Moreover, we have analysed the two diastereoisomers of the thiazolidine compounds derived from L-cysteine and D-cysteine: we have succeeded to distinguish by NMR analysis the cis and the trans forms, concluding that the interconversion of the free forms is extremely rapid at pH 7: thus, it may be relevant for the protein bound forms.

Biological role of carbamoyl pyridoxal 5'-phosphate.

A new compound, carbamoyl-pyridoxal 5'-phosphate (C-PLP), was synthetized by condensation of pyridoxal 5'-phosphate (PLP) with KCNO. It may be obtained under certain physiological conditions of pH, temperature and concentration of reagents. Formation and degradation of C-PLP are readily reversible chemical reactions, not involving enzymes, at least in rat tissues. However, different considerations suggest that synthesis and breakdown of C-PLP play a biological role in the cell, providing 'protective synthesis' and a 'variable reservoir' of PLP and KCNO, which can be trapped by other proteins, apoenzymes and metabolites, to regulate many cell metabolic functions.

Effect of testosterone on purine metabolism and morphometric parameters in the rat liver.

The effect of testosterone on the morphology and biochemistry of adult castrated rat liver is described. Castration decreases mean weight and volume of hepatocytes, volume and surface area of sinusoidal lumen, and apparently increases cell number per g of tissue. These variations indicate cell distress. Testosterone administration restored sinusoidal volume and surface area, indicating a true hyperplastic response and improved trophic conditions. Acid soluble nucleotides, RNA and DNA content were lower after castration, being partially restored after testosterone treatment. This restoration, however, was only statistically significant for total guanylate. We concluded that testosterone deficiency and administration exerts a specific effect on the liver in terms of morphological and biochemical changes. Purine nucleotide metabolism is probably the first target of hormonal action, since its changes are the most significant and useful to explain all the other observations.