NT-proBNP changes, oxidative stress, and energy status of hypertrophic myocardium following ischemia/reperfusion injury.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a sensitive functional marker in heart disease, including left ventricular hypertrophy (LVH) secondary to valvular aortic stenosis (AS). We evaluated the association between NT-proBNP changes, oxidative stress, energy status and severity of LVH in patients with AS. Ten patients undergoing aortic valve replacement for AS were studied. Plasma NT-proBNP concentrations were performed by electroluminescence immunoassay 15min after the induction of anesthesia (t0), before aortic cross-clamping (t1), before clamp removal (t2), 15min after myocardial reperfusion (t3), and 24h after surgery (t4). Heart biopsies were obtained and high energy phosphates (ATP, ADP, AMP) were analyzed by capillary electrophoresis (CE). In plasma samples from the coronary sinus, nitrate plus nitrite (NOx) concentrations were also analyzed by CE. Echocardiographic measurements were acquired and correlations between biochemical markers and severity of AS were assessed. NT-proBNP peaked significantly at t4 (p<0.001). A linear correlation between NT-proBNP values measured at t0 and t4 was found (R(2)=0.89; p<0.001). A negative correlation between NT-proBNP production and phosphorylation potential (ATP/ADP ratio) was observed (R(2)=0.62; p<0.01). NOx values positively correlated with NT-proBNP levels (p<0.01). NT-proBNP inversely correlated with aortic valvular area (r=81, p<0.01), positively correlated with mean (r=0.82, p<0.01) and maximum left ventricle-to-aortic gradients (r=0.80, p<0.01), and with left ventricular mass (r=0.69, p<0.01). NT-proBNP is a useful marker of LVH and severity of AS. It may complement echocardiographic evaluation of patients with AS in identifying the optimum time for surgery.

Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

[Preliminary results of the effects of electromagnetic fields ELF and TAMMEF on human lymphocytes: evaluation of some biological parameters]. / Studio preliminare sugli effetti di campi elettromagnetici, ELF e TAMMEF, nei linfociti umani: valutazione di alcuni parametri biologici.

AIMS: The Authors have evaluated the effects of low frequency electromagnetic fields on human peripheral blood lymphocytes metabolism. MATERIALS AND METHODS: It has been assayed total proteins and the activities of some purine metabolism enzymes after exposure of the cells to sinusoidal ELF fields (100 Hz frequency) or to the new TAMMEF system fields, characterized by variable frequency, intensity and shape of wave. RESULTS: The protein lymphocytes content, increased after both treatments. Instead, the enzyme activities did not vary, with the exception of Myokinase activity, which, respect to the control, increased after ELF field treatment, and slightly decreased after TAMMEF treatment. This preliminary result was interpreted as a variation of the cellular electric charge stability, stimulated by ELF fields, which induce the Myokinase to increase its rate, to rearrange the correct equilibrium, while the TAMMEF field were useless to maintain the cellular electric charge at normal levels. CONCLUSIONS: We interpreted these preliminary results as follow: the ELF fields influence the cellular electric charge stability, so that the cells must increase MK activity to restore the correct equilibrium, while TAMMEF fields are useful to maintain and regulate cellular electric charge. The results obtained by this preliminary study opens interesting prospective to be further investigated.

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.

Synthesis of adenine and guanine nucleotides at the 'inosinic branch point' in lymphocytes of leukemia patients.

The synthesis of purine nucleotides has been studied in human peripheral blood lymphocytes from healthy subjects and patients affected by B-cell chronic lymphocytic leukemia (B-CLL). The rate of the synthesis was measured by following the incorporation of 14C-formate into the nucleotides of lymphocyte suspensions. The whole sequence AMP-->ADP-->ATP was found reduced in B-CLL lymphocytes; in the case of guanylates only the last step of the sequence GMP-->GDP-->GTP was significantly lower in the same cells. From the analysis of these results, combined with previous data, we conclude that purine metabolism undergoes an imbalancement during CLL, which is partially compensated, and point out the importance of studying concomitantly purine metabolism and nucleic acid synthesis in leukemia cells.

Behavior of L-threonine-degrading enzymes during liver regeneration.

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.

Nitrogen metabolism during liver regeneration.

Nitrogen metabolism was investigated in regenerating liver-bearing rats through the following parameters: (1) liver aminoacid content, (2) plasma and urinary urea and creatinine, (3) plasma and urinary oxypurines, uric acid and allantoin. Two groups of aminoacids were considered: (1) the essential aminoacids (phenylalanine, tyrosine, isoleucine, lysine, leucine, valine, arginine, histidine and methionine); (2) the non-essential aminoacids (aspartic acid, asparagine, glutamic acid, glutamine, alanine, glycine, serine, threonine and proline). Some of the first group tended to decrease, and those of the second group to increase, immediately after partial hepatectomy. Few ketogenic aminoacids are probably oxidized to provide energy. The flux of aminoacids for gluconeogenesis is minutely controlled, therefore, those of the second group being spared at first and set aside for protein synthesis, which increases on the second and third days after partial hepatectomy. Plasma and urinary urea, oxypurines, uric acid and allantoin did not show any significant variations after partial hepatectomy. The conclusion emerging from the present research is that, although variations in aminoacid composition and metabolism and in purine nucleotide metabolism have been demonstrated to occur in the regenerating liver, the overall nitrogen catabolism, as reflected by the principal end products, does not undergo substantial variations. The remaining liver is able to fulfil this function.

Purine nucleotide metabolism: specific aspects in chronic lymphocytic leukemia lymphocytes.

The metabolism of purine nucleotides was studied in human peripheral blood lymphocytes from healthy subjects and patients with B-cell chronic lymphocytic leukemia. Nucleotide content was determined by HPLC. The rate of de novo synthesis of purine nucleotides was measured kinetically by following the incorporation of 14C-formate into the nucleotides of a lymphocyte suspension. The patterns of the main enzymes involved in purine nucleotide metabolism (those of the salvage pathway and catabolism) were estimated by a radiochemical method. Although the data expressed in relation to cells and protein showed some discrepancies, several common differences were evident in both cases. The main differences were an increase in NAD and IMP, a sharp decrease in 5'-nucleotidase activities and in total guanylate content and synthesis, and an increase in the A/G ratio in lymphocytes of patients with respect to controls. The changes in these parameters in CLL indicate an imbalance in purine metabolism and may play a specific role in the biology of the leukemia cell. They are also potential biochemical markers of lymphoid malignancies and may be useful in chemotherapic applications.

The behavior of free purine nucleotides in lymphocytes infected with HIV-1 virus.

The purine nucleotide content was examined in various cells before and after HIV-1 virus infection: healthy peripheral blood lymphocytes (PBL) and cultured PBL after infection; the PBL of asymptomatic, ARC and AIDS patients. In all cases, changes in purine nucleotide concentrations were observed. The pattern of purine nucleosides and nucleobases was also evaluated by HPLC in PBL of controls and patients. The analysis was integrated by following the incorporation of a labelled precursor ([14C]formate) into purine nucleotides, which was investigated as an indication of the rate of purine metabolism in these cells. Many interesting variations in the catabolic and of anabolic pathways were observed, demonstrating that the viral penetration affects purine nucleotide metabolism. These results suggest interesting perspectives in AIDS research.

Determination of p185 and adenylosuccinate lyase (ASL) activity in preneoplastic colon lesions and intestinal mucosa of human subjects.

OBJECTIVES: The HER2 gene has been found amplified in a number of human adenocarcinoma leading to elevated levels of expression of its encoded product, p185 protein. Because little information is available on the tissue and tumor specificity of this gene product, we studied the expression of p185 protein in preneoplastic colon lesions. Adenylosuccinate lyase (ASL, EC 4.3.2.2) is known to increase in malignancies such as colorectal, breast, and prostate cancer. In order to evaluate the potential of ASL as a tumor marker, its activity was determined and compared with the expression of p185. DESIGN AND METHODS: p185 was determined by an immunohistochemical procedure in patients with the preneoplastic lesions. ASL activity was evaluated in intestinal mucosa adjacent to colorectal cancers (patient group A) and in preneoplastic colorectal lesions (group B). The enzyme activity was evaluated in dialyzed supernatants, following the disappearance of substrate (adenylosuccinate AMP-S) and the formation of product (adenosine 5'-monophosphate-AMP), separated by high performance liquid chromatography. RESULTS AND CONCLUSIONS: Increased expression of p185 and elevated ASL activity were observed in tubular and tubulo-villous adenoma and may, therefore, be associated with the early stages of colorectal cancer.

Myocardial ischemic injury and purine metabolism in patients undergoing coronary artery bypass.

OBJECTIVES: High-energy phosphates and their catabolic products were determined in myocardium during coronary artery bypass surgery with blood cardioplegic reperfusion in order to evaluate the effects of aortic cross-clamping and reoxygenation on myocardial purine metabolism. DESIGN AND METHODS: Transmural left ventricular biopsy specimens were taken with ITu-Cut biopsy needles, before aortic cross-clamping, before cross-clamp removal and after 30' of reperfusion; perchloric extracts of the material were analyzed for nucleotide content by capillary zone electrophoresis (CZE). The CZE procedure used separates the complete spectrum of purine metabolites in myocardial extracts obtained from 0.6-8.6 mg biopsy material. RESULTS: The basal values of ATP/ADP ratio and energy charge were low, IMP content was high. After the ischemic period, ATP levels further decreased and IMP, nucleosides and bases accumulated. After reperfusion, nucleoside and base basal levels, but not energy charge, were restored to some extent. CONCLUSIONS: The study arises the problem of myocardial preservation during heart surgery. In this investigation, capillary electrophoresis was an extremely adaptable technique for the evaluation of ischemic injury and could be useful in studying the effects of cardioplegic solutions.

5'-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic leukemia.

OBJECTIVES: The activity of membrane-bound ecto-5'-nucleotidase and soluble e-Ns and c-N-II 5'-nucleotidases was evaluated on lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL). A statistically significative decrease in ecto-5'-nucleotidase, e-Ns, and c-N-II activities was observed in peripheral blood lymphocytes and in B and T populations from affected individuals. DESIGN AND METHODS: For the assay of ecto-5'-nucleotidase, e-Ns, and c-N-II activity we used a radioactive procedure coupled to HPLC. Since the ecto-5'-nucleotidase is identified as CD73 antigen, we performed immunofluorescence analysis using a specific monoclonal antibody. We analyzed ecto-5'-nucleotidase mRNA by RT-PCR to ascertain the possibility of an alteration in the transcription of its gene. RESULTS: A decrease in ecto-5'-nucleotidase activity was correlated to reduction in ecto-5'-nucleotidase positive cells (CD73+) in leukemia patients. RT-PCR produced a fragment of the expected size and the specific mRNA was found expressed in both healthy subjects and leukemia patients. CONCLUSIONS: The decrease in ecto-5'-nucleotidase activity in patients with B-CLL is not due to loss of transcription of the specific mRNA. The presence of point mutations, splicing alteration, or posttranslational modifications must be investigated. If a defect at DNA or RNA level will be detected, the molecular analysis will be considered for diagnosis of B-cell chronic lymphocytic leukemia.

The regulation of aminotransferase activity by carbamoyl-phosphate.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

Behaviour of human lymphocytic isoenzymes of 5'-nucleotidase.

The behaviour of 5'-nucleotidase isoenzymes (ecto-5'-nucleotidase, e-Ns and c-N-II soluble 5'-nucleotidases) was studied in lymphocytes from patients with B-cell chronic lymphocytic leukemia. A strong reduction in ecto- and soluble activities was observed, although the pattern of the three 5'-nucleotidases did not always strictly overlap. A significant decrease (p<0.05) in ecto-5'-nucleotidase, e-Ns and c-N-II was found in B and T populations (B lymphocytes: 1.13, 0.88 and 1.26 nmol/h/10(6) cells versus 95.96, 9.64 and 13.73 nmol/h/10(6) cells in controls; T lymphocytes: 1.31, 0.23 and 0.06 nmol/h/10(6) cells versus 9.25, 1.31 and 2.10 nmol/h/10(6) cells in healthy subjects). The percentage of ecto-5'-nucleotidase-positive cells (CD73+) was reduced in leukemia patients, indicating a lower number of active molecules on the cell surface. The results of RT-PCR analysis showed that the ecto-5'-nucleotidase mRNA of leukemia patients was not defective.

Ecto-5'-nucleotidase in B-cell chronic lymphocytic leukemia.

Literature on the behaviour of ecto-5'-nucleotidase in the course of B-cell chronic lymphocytic leukemia is briefly reviewed and aims for further researches are highlighted.

Relationships between hemodynamic parameters and myocardial energy and antioxidant status in heart transplantation.

The relationships between high-energy phosphate levels, oxidative insult and mechanical function represent a key point in heart transplantation and related post-ischemic functional recovery. We evaluated myocardial purine compounds and glutathione antioxidant defence mechanism during 19 heart transplant operations. Heart biopsies were taken before harvesting on beating heart (t1), at the end of cold static preservation (t2) and 30 min after implantation and reperfusion (t3); perchloric extracts of the tissue were analyzed by capillary electrophoresis (CE). Correlation analyses were performed with hemodynamic parameters evaluated 90 min after aortic declamping (T90), 6 h following admission in intensive care unit (T6A) and 1 d post-operation (D1). We evidenced that AMP levels measured at T1 negatively correlate with both cardiac index (CI) and oxygen delivery index (DO2I) evaluated at T6A, respectively. The same behavior was evident plotting IMP levels measured at T3 with CI and DO2I evaluated at D1. After t2 the nucleotide/(nucleoside + base) ratio was in positive correlation with hemodynamic parameters at T6A. Energy charge and GSH/GSSG ratio measured before harvesting were in positive correlation with DO2I evaluated at T90. The present research shows that despite the complexity of the high-energy phosphate metabolism and that of the events associated to a clinical heart transplantation, there are some parameters that, besides reflecting the degree of myocardial preservation, also represents predictive parameters for the following organ functional recovery. It also suggests that heart preservation strategies should carefully take into account the sub-optimal nature of the donor heart at the time of procurement, through a broad spectrum of purine compound and glutathione antioxidant system measurements.

Determination, activity and biological role of adenylosuccinate lyase in blood cells.

Adenylosuccinate lyase deficiency, which is associated with severe mental retardation and autistic features, was discovered in 1984. Since then this enzyme has been analyzed in many human tissues and it is now generally agreed that screening for this enzyme defect should be performed in all unexplained neurological diseases. The aim of the present study was to analyze adenylosuccinate lyase activity in blood cells by a fast simple method adaptable to screening purposes. The activity was also analyzed in B-lymphocytes from patients with B-cell chronic lymphocytic leukemia. The biological role of adenylosuccinate lyase and its importance in regulating cellular levels of AMP is discussed.

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.

Analysis of purine nucleotides in lymphocytes from healthy subjects and AIDS patients.

The most important purine nucleotides (NAD, AMP, IMP, GMP, XMP, ADP, ATP, GDP, GTP) were analyzed by HPLC in the lymphocytes of healthy subjects and HIV-1 seropositive patients at different stages of the disease (ARC-AIDS). Several differences, which focus attention on the behaviour of purine nucleotide metabolism in the lymphocytes of these patients, were observed.

Levels and variability of purine nucleotides in normal human lymphocytes.

Anion-exchange, high performance liquid chromatography was used to determine purine nucleotides in lymphocytes of healthy males and females of various ages. We observed a considerable dispersion of values which were unrelated to age or sex, possibly linked to various states of activation of circulating lymphocytes and to other unknown factors.