Enrico Marinello

The protective effect of Mucuna pruriens seeds against snake venom poisoning.

ETHNOPHARMACOLOGICAL RELEVANCE The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite. AIM OF THE STUDY To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms. MATERIALS AND METHODS Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization. RESULTS In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested. CONCLUSIONS The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.

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.

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: Characterization and cross-reactions

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivoin mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatusvenom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A2, the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins ofM. pruriens extract. Proteins of E. carinatusvenom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.

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.

Comparative determination of purine compounds in carotid plaque by capillary zone electrophoresis and high-performance liquid chromatography

Allantoin, uric acid (UA), hypoxanthine (Hx) and xanthine (X) were determined on carotid plaque by capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). Comparison of the results showed that capillary zone electrophoresis may have similar or even superior analytical performance to HPLC, especially for the determination of allantoin in biological samples.

Effects of testosterone on cholesterol levels and fatty acid composition in the rat.

The effects of testosterone treatment on cholesterol levels and its fatty acid components were studied in adult rats. Cholesterol levels increased both in the liver and in the serum of castrated rats. Androgen administration restored the normal values only in the serum. A general decrease in unsaturated and essential fatty acids in cholesterol esters was evident after testosterone administration. In the liver, only the C16:O/C16:1 ratio clearly increased after testosterone administration, which inhibited the delta 9 unsaturation of palmitic acid, but not of stearic acid. In the serum the C16:O/C16:1, C18:O/C18:1, and C18:2/C2O:4 ratios decreased after castration and were restored by testosterone. The results indicate a clear inhibition of delta 9 unsaturation of palmitic and stearic acids, of delta 5 unsaturation and elongation in organs other than the liver. No effect was evident on delta 6 unsaturation. This suggests that fatty acid unsaturations are regulated differently by testosterone in different tissues. For delta 9 unsaturation in the liver, the effect also seems to be substrate-dependent.

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.

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.

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short‐ (1 injection) and long‐term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short‐term and one long‐term MPE treatment. One group received EV alone. Two test groups received EV with either a short‐term or long‐term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2‐DE/MALDI‐TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV‐induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.