Bruno Giardina

Myocardial release of malondialdehyde and purine compounds during coronary bypass surgery.

Free radicals and lipid peroxidation have been suggested to play an important role in the pathophysiology of myocardial reperfusion injury. The purpose of the present study was to monitor myocardial malondialdehyde (MDA) production as an index of lipid peroxidation during ischemiareperfusion sequences in patients undergoing elective coronary bypass grafting. There has been a lot of debate on the role of xanthine oxidase as a potential superoxide anion generator and thus lipid peroxidation in human myocardium. To evaluate the activity of xanthine oxidase pathway, we measured the changes in the transcardiac concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid. Methods and ResultsThe coronary sinus-aortic root differences (CS-Ao) of MDA, oxypurines, and nucleosides were measured by a recently developed ion-pairing high-performance liquid chromatographic (HPLC) method. Fifteen patients were included in the study, and 13 of them demonstrated a more than 10-fold increase in net myocardial production of MDA on intermittent reperfusion during the aortic crossclamp period. In 2 patients, MDA was not detectable in any of the CS or Ao samples. Before aortic cross-clamping, the CS-Ao concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid were 0.59±0.19, 0.23±0.05, 0.89±0.36, 0.58±0.32, and 11.4±4.9,μmol/L, respectively. After aortic cross-clamping, the sum of the transcardiac differences of these compounds increased up to 2.8-fold and then gradually decreased after declamping of the aorta. There was a weak positive correlation between transcardiac concentration differences of MDA and xanthine plus uric acid (r = .48, P < .01). The postoperative functional recovery or leakage of cardiac enzymes was not affected by the level of MDA net release during the aortic cross-clamp period, however. ConclusionsWe conclude that myocardial lipid peroxidation, estimated as MDA formation, is common during intermittent ischemia-reperfusion sequences in coronary bypass surgery, although some patients may be better protected. Xanthine oxidase appears to be operative in human myocardium, and free radicals generated in this reaction might also be involved in the observed lipid peroxidation process. Increased degradation of myocardial adenine nucleotides and concomitant lipid peroxidation may play a specific role in the development of reperfusion injury. In this study, however, more extensive lipid peroxidation was not associated with impaired functional recovery.

Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 microns particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.

Preserving effect of fructose-1,6-bisphosphate on high-energy phosphate compounds during anoxia and reperfusion in isolated langendorff-perfused rat hearts

Isolated Langendorff-perfused rat hearts after 10 min pre-perfusion, were subjected to a substrate-free anoxic perfusion (20 min) followed by 20 min reperfusion with a glucose-containing oxygen-balanced medium. A similar experimental protocol was repeated in the presence either of 5 mM fructose or of 5 mM fructose-1,6-bisphosphate throughout the different perfusion conditions. High-energy phosphate compounds (adenosine triphosphate, creatine phosphate), adenine nucleotides, nicotinic coenzymes, lactate, pyruvate and glycogen content in the tissue were determined at the end of each perfusion period, while coronary flow, heart rate and lactate and pyruvate output were monitored throughout the whole duration of the experiments. On the whole, the results indicate that exogenous fructose-1,6-bisphosphate preserves high-energy metabolites during anoxia and restores myocardial metabolism and contractility during reperfusion, that a prolonged period of substrate-free anoxic perfusion renders the heart unable to normalize its metabolism during re-oxygenation and that fructose is not utilized by the heart for its energy demand. A possible hypothesis concerning the mechanism of action of fructose-1,6-bisphosphate is presented.

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H2O2 (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO2 (1, 5, 10, 20, and 50 mm), for 15 min at 37 °C. AMP-deaminase tremendously increased by increasing H2O2 or NaNO2 at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5–100 mm), N-ethylmaleimide (0.1–10 mm), or p-hydroxymercuribenzoate (0.1–5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase ofV max, modification of the curve shape ofV o versus [S], Hill plots, and coefficients) were recorded with 4 mmH2O2- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe2++ H2O2) or –SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.

Oxygen transport in extreme environments

Evolution has adopted different strategies to solve the problem of transporting oxygen to respiring tissues, according to needs dictated by the environment. A thermodynamic analysis of haemoglobins of organisms living in extreme polar environments (mammals and fish) provides elegant examples of such adaptations.

Arctic adaptation in reindeer The energy saving of a hemoglobin

Previous results [(1988) Arct. Med. Res. 47, 83–88] have shown that hemoglobin from reindeer is characterized by a low overall heat of oxygenation. This particular aspect has been investigated further in a series of precise oxygen equilibrium experiments. The results obtained show a peculiar dependence of the temperature effect on the fractional saturation of hemoglobin with oxygen, which could be regarded as a very interesting case of molecular adaptation to extreme environmental conditions.

Oxygen radical injury and loss of high-energy compounds in anoxic and reperfused rat heart: Prevention by exogenous fructose-1,6-bisphosphate

Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1,6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and of TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1,6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1,6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release of lactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1,6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed. On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1,6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.

A method for preparing freeze clamped tissue samples for metabolite analyses

A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.

Arctic life adaptation—II. the function of musk ox (Ovibos muschatos) hemoglobin

1. The hemoglobin system from musk ox (Ovibos muschatos) has been characterized from the functional point of view with special regard to the effect of organic phosphates and temperature. 2. The results are similar to those previously obtained in the case of reindeer and confirm that hemoglobins from arctic animals may display very low enthalpy change for the reaction with oxygen. 3. This finding is considered an example of molecular adaptation of respiratory pigments to extreme environmental conditions.

Arctic life adaptation--I. The function of reindeer hemoglobin

1. The functional properties of hemoglobin from the reindeer (Rangifer tarandus tarandus L.) are characterized as a function of pH, temperature and organic phosphate concentration. 2. Alongside overall similarities shared with most vertebrate hemoglobins, hemoglobin from the reindeer shows significant differences with respect to the effect of both organic phosphates and chloride anions. 3. The limited effect of temperature on oxygen binding (delta H = -4 kcal/mol O2) could be regarded as an interesting case of molecular adaptation to extreme environmental conditions.