F. Rosi

Cardiac surgery: myocardial energy balance, antioxidant status and endothelial function after ischemia–reperfusion

Myocardial and endothelial damage is still a widely debated problem during the ischemia-reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed.

Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione.

An understanding of tissue energy metabolism and antioxidant status is of major interest in the field of organ preservation for transplantation. Nucleotide and glutathione are indicators of cell damage occurring during ischemia and reperfusion. A high performance capillary electrophoresis (HPCE) method with UV detection (185 nm) for the simultaneous analysis of intracellular free ribonucleotides, nucleosides, bases and glutathione (oxidized and reduced form) in myocardial tissues is described. The method does not involve thiol derivatization. The separations were carried out in an uncoated fused‐silica capillary, 60 cm long, 52.5 cm to detector, 75 μm ID, with 20 mM Na‐borate buffer, pH 10.00, at 20 kV voltage and reading at 185 nm. Injection was hydrostatic for 12 s and total analysis time was 20 min. The technique enables optimum separation of all the compounds examined and has a resolution similar to that of HPLC analysis, with the advantage of fast simultaneous measurement of cell nucleotide metabolism and redox state, not possible with HPLC.

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.

Fatty acid composition of phospholipids, triglycerides and cholesterol in serum of castrated and estradiol treated rats.

We have studied the levels of phospholipids, triglycerides, cholesterol esters, and their fatty acid composition in serum for normal, castrated and estradiol treated rats. The sex hormones did not greatly affect the levels of the various lipid fractions which did not undergo great significant variations, under the different treatments. More evident variations occurred in the percent composition of fatty acid and in the content of the various saturated (SAT), unsaturated (UNSAT), essential (EFA) and non essential fatty acids (NEFA). We studied the most important ratios: EFA/NEFA; UNS/SAT; 16:0/16:1; 18:0/18:1, 18:2/18:3; 18:2/20:4. 16:0/16:1; 18:0/18:1 represent the delta9 desaturase, one specific for palmitic, the other for stearic acid. 18:2/18:3 ratio is an index of the delta6 desaturase activity: 18:2/20:4 ratio of delta5 desaturase-elongase. Most changes were evident in triglycerides. We observed a different behaviour of the UNS/SAT and EFA/NEFA ratios in phospholipids and cholesterol esters, which may reflect either an effect of the sex hormones on the exchange of fatty acids between the same lipid fractions, or a redistribution of lipids among different tissues. Great variations were observed of the ratios 16:0/16:1; 18:0/18:1; 18:2/18:3; 18:2/20:4, which are ascribed a different effect of the sex hormones of delta9, delta6, delta5 desaturases.

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.

A 57-gene expression signature in B-cell chronic lymphocytic leukemia

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

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.

Purine metabolism in B-cell lymphocytic leukemia : A microarray approach

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Purine metabolism in HIV-1 virus-infected T lymphocyte population

Purine nucleotide metabolism has been studied in T-lymphocyte population in healthy subjects and in AIDS bearing patients. Nucleotide content was determined by HPLC. The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a T cell suspension. The authors discuss the results, which indicate interesting variations in nucleotide content and a lower nucleotide synthesis, determined by kinetic studies.

Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Undergoing Gene Therapy

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.