Biomedical subjects

G Lazzarino

Publications and source records attributed to G Lazzarino.

13 recordsLinked to original sources

PubMed1992

Malondialdehyde is a biochemical marker of peroxidative damage in the isolated reperfused rat heart.

Concentration of MDA in isolated control, ischemic, and reperfused rat hearts was determined by using a new sensitive and reproducible HPLC method on the perchloric acid extract of the freeze-clamped tissues. By means of this HPLC assay for the direct measurements of MDA, concentrations of adenine nucleotide derivatives were also obtained in the same chromatographic run. Under the present experimental conditions, no detectable amount of MDA could be observed in control hearts while ischemic hearts showed 0.009 mumoles/g d.w. of MDA (s.d. = 0.001), this value representing the sensitivity limit of the method employed. On the contrary, reperfused hearts showed 0.118 mumoles/d d.w. of MDA (s.d. = 0.036), thereby indicating that this compound originates from an oxygen free radical-mediated breakdown of phospholipids and demonstrating the existence of quantifiable molecular damage occurring upon reperfusion. On the whole, our data demonstrate that MDA, if properly assayed, is a reliable index of peroxidative injury to biological systems.

Animals↗

PubMed1992

MDA, oxypurines, and nucleosides relate to reperfusion in short-term incomplete cerebral ischemia in the rat.

Short-term incomplete cerebral ischemia (5 min) was induced in the rat by the bilateral clamping of the common carotid arteries. Reperfusion was obtained by removing carotid clamping and was carried out for the following 10 min. Animals were sacrificed either at the end of ischemia or reperfusion. Controls were represented by a group of sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. Neutralized perchloric acid extracts of brain tissue were analyzed by a highly sensitive high-performance liquid chromatography (HPLC) method for the direct determination of malondialdehyde, oxypurines, nucleosides, nicotinic coenzymes, and high-energy phosphates. In addition, plasma concentrations of malondialdehyde, hypoxanthine, xanthine, inosine, uric acid, and adenosine were determined by the same HPLC technique. Incomplete cerebral ischemia induced the appearance of a significant amount (8.05 nmol/g w.w.; SD = 2.82) of cerebral malondialdehyde (which was undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde (53.30 nmol/g w.w.; SD = 17.77) and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia (0.050 mumol/L; SD = 0.015), significantly increased after 5 min of ischemia (0.277 mumol/L; SD = 0.056) and was strikingly augmented after 10 min of reperfusion (0.682 mumol/L; SD = 0.094). A similar trend was observed for xanthine, uric acid, inosine, and adenosine, while hypoxanthine reached its maximal concentration after 5 min of incomplete ischemia, being significantly decreased after reperfusion. From the data obtained, it can be concluded that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat. In particular, these results suggest the possibility of using the variation of malondialdehyde, oxypurines, and nucleosides in peripheral blood as a potential biochemical indicator of reperfusion damage occurring to postischemic tissues.

Adenine Nucleotides↗

PubMed1992

Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart.

Isolated Langendorff-perfused rat hearts after 20 min of normoxic perfusion in the presence of 2.5 mM Ca++ and 11 mM glucose were subjected to 30 min of global normothermic ischemia followed by 30 min of normoxic reperfusion with the starting buffer. At the end of each perfusion condition, hearts were freeze-clamped and deproteinized by 0.6 M HClO4. Two-hundred microL of the neutralized tissue extracts were analyzed by a recently developed high-performance liquid chromatography (HPLC) method for the simultaneous determination of malondialdehyde (MDA), ascorbic acid, and adenine nucleotides. By means of this analytical technique, it was possible to demonstrate that MDA is undetectable in control hearts. In contrast, 30 min of ischemia induced a modest production of MDA (0.012 mumol/g dw), while a large amount of MDA (0.103 mumol/g dw) was observed in reperfused hearts. Values referring to ascorbic acid showed that the concentration of this antioxidant progressively decreased from 1.190 (control hearts) to 0.837 (ischemic hearts) and to 0.595 mumol/g dw (reperfused hearts). The overall conclusions of this study are that reperfusion induces an oxidative stress to the isolated myocardium, a decrease of ascorbate, and an increase of lipid peroxidation. Therefore, by means of a proper analytical method, MDA may represent a valid biochemical parameter to demonstrate the relationship between myocardial reperfusion and a detectable tissue damage.

Animals↗

PubMed1992

Fructose-1,6-bisphosphate improves efficiency of work in isolated perfused rat hearts.

The purpose of this study was to determine whether exogenous fructose-1,6-bisphosphate (F-1,6-P2) directly affects myocardial hemodynamics and certain metabolic parameters. Isolated working rat hearts were perfused for 30 min with 10 mM glucose (+insulin) as the exclusive exogenous substrate followed by 15 min with glucose plus one of the following concentrations (in mM) of F-1,6-P2: 1.25, 2.5, 5, or 10, and finally returned to the glucose only buffer. Additions of 2.5 and 5 mM F-1,6-P2 decreased (P less than 0.01) oxygen consumption (VO2) by 10.8 and 17.0% and coronary flow by 8.3 and 10.3%, respectively. No changes were observed in lactate release, cardiac output (CO), peak systolic pressure, heart rate, or pressure work (PW). Efficiency, expressed as PW divided by VO2, increased with F-1,6-P2 by 8.6% with 1.25 mM (P less than 0.05), 13.2% with 2.5 mM (P less than 0.01), and 16.9% with 5 mM (P less than 0.01). F-1,6-P2 at 10 mM produced no further improvements in VO2 or efficiency but was associated with declines (P less than 0.05) in CO and PW. Glucose plus 10 mM fructose had no effects on any of the above parameters, indicating that the F-1,6-P2-induced changes were not due to changes in osmolarity or to end products of F-1,6-P2 hydrolysis. Some chelation of buffer calcium by F-1,6-P2 occurred, but when free calcium was equalized in glucose and glucose plus 5 mM F-1,6-P2 buffers, the decline in VO2 (11.5%) was still far greater than could be explained by exogenous F-1,6-P2 metabolism in the glycolytic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Ischemia and reperfusion: effect of fructose-1,6-bisphosphate.

Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the "oxygen paradox" in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5 mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.

Animals↗

PubMed1992

[Ischemia and reperfusion: the production and release of malondialdehyde, oxypurines and nucleosides in the isolated rat heart].

Ischemia and reperfusion damage has been evaluated by determining the sum of adenine nucleotides, nucleosides, oxypurines and the concentration of malondialdehyde, ascorbic acid, lactate and pyruvate in the isolated rat heart subjected to global normothermic ischemia and subsequent reperfusion. In addition, the sum of oxypurines and nucleosides, and the concentration of malondialdehyde has been determined in the perfusate collected during the reperfusion. Data indicate that ischemia and reperfusion induce an oxidative stress to myocardial tissue (increase of tissue malondialdehyde and decrease of ascorbic acid, and release of malondialdehyde during reperfusion) that, due to the output of relevant amount of oxypurines (congruent to 6.7 mumol/g dry weight) and of nucleosides (congruent to 7.0 mumol/30/min/g dry weight), it is not even able to restore its energy metabolism after reperfusion.

Animals↗

PubMed1992

[L-propionylcarnitine taurine amide induces the metabolic recovery of the isolated postischemic rat heart].

The effect of reperfusion with L-propionyl-carnitine-taurinammide 1 mM was evaluated on the metabolic recovery of the isolated postischemic rat heart. Data referring to the tissue concentration of the high-energy phosphates, oxypurines, nucleosides, nicotinic coenzymes, lactate and pyruvate indicate that L-propionyl-carnitine-taurinammide significantly improves the metabolism of the reperfused myocardium. In particular, ATP, creatinphosphate, GTP, sum of adenine nucleotides and the energy charge resulted 1.80, 1.83, 3.47, 1.47 and 1.20 times higher respectively than the corresponding values recorded in control reperfused heart (p < 0.01 all). These data, out of supplying the necessary biochemical support to the beneficial effects of L-propionyl-carnitine-taurinammide on hemodynamics obtained in previous studies, suggest that L-propionyl-carnitine-taurinammide might represent a useful tool for the pharmacological treatment of myocardial infarction.

Animals↗

PubMed1991

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.

Adenine Nucleotides↗

PubMed1991

Effect of exogenous fructose-1,6-bisphosphate on glycolysis in the isolated perfused rat heart.

To test the mechanism of action of fructose-1,6-bisphosphate (F-1,6-P2), experiments were conducted on isolated perfused rat hearts to measure the glycolytic rate supported by exogenous glucose with simultaneous measurement of oxygen consumption and the release of lactate and pyruvate. Glycolysis was assayed in terms of the release of tritiated water from [5-3H] glucose, a measure of the rate through the aldolase step. It was found that 5 mmol/L F-1,6-P2 reduced the glycolytic rate parallel to the decrease in oxygen consumption. The results suggest that the cardioprotective action of F-1,6-P2 is related to a substrate effect and a decrease in adenosine triphosphate consumption as indicated by a decrease in oxygen consumption in accordance with the recent demonstration of Ca2+ binding by F-1,6-P2.

Animals↗

PubMed1991

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.

Adenine Nucleotides↗

PubMed1991

Mechanism of the effect of exogenous fructose 1,6-bisphosphate on myocardial energy metabolism.

The effects of fructose 1,6-bisphosphate (F-1,6-P2) on the isolated Langendorff-perfused heart were studied by monitoring flavoprotein fluorescence, oxygen consumption (MVO2), coronary flow (Fc), systolic intraventricular pressure (Psys), diastolic intraventricular pressure, and contraction frequency. The cellular energy state and cytosolic pH were determined by means of 31P nuclear magnetic resonance. Infusion of 5 mM F-1,6-P2 caused a rapid shift toward reduction in the flavoprotein redox state and initial 50% and 44% decreases in Psys and MVO2, respectively. After a partial recovery, these measures remained 11% and 25% below the basal value. Concomitantly, after an initial transient increase of 13%, Fc remained 17% lower than in the basal state. When the F-1,6-P2 concentration was subsequently increased to 10 mM, psys and MVO2 dropped temporarily to 31% and 29% of the basal value and then remained at 50% and 53%, respectively. Simultaneously, a brief increase was observed in Fc, which then fell 34% below the basal value. Rapid reoxidation of the flavoproteins and increases in MVO2, Psys, and Fc occurred on discontinuation of the F-1,6-P2 infusion. 31P nuclear magnetic resonance during infusions of both 5 and 10 mM F-1,6-P2 revealed a decrease in cytosolic inorganic phosphate and a tendency to increase creatine phosphate, suggesting elevation in the cellular energy state. No changes in intracellular pH occurred as estimated from the chemical shift of the nuclear magnetic resonance of inorganic phosphate. F-1,6-P2 (5 mM and 10 mM) lowered the free Ca2+ concentration in the Krebs-Henseleit bicarbonate buffer (by 32% and 47%, respectively). This probably explains the effects of F-1,6-P2 on mechanical work performance and cellular respiration. A direct metabolic effect also exists, however, because flavoprotein reduction by F-1,6-P2 could be observed in the K(+)-arrested heart, where its effects on MVO2 were minimal. This redox effect may not be caused by changes in free Ca2+ concentration because it could not be reproduced by infusion of EGTA.

Animals↗

PubMed1990

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.

Animals↗

PubMedDate not supplied

Exogenous fructose-1,6-bisphosphate is a metabolizable substrate for the isolated normoxic rat heart.

Isolated rat hearts were perfused by the recirculating Langendorff mode under normoxic conditions for 60 min. The Krebs-Ringer buffer was supplemented with 10 mM glucose + 12 IU/l insulin and either [U-14C]-fructose-1,6-bisphosphate (together with 5 mM cold fructose-1,6-bisphosphate) or [U-14C]-fructose (together with 5 mM cold fructose). At the end of perfusion, gaseous 14CO2, 14CO2 trapped in the perfusates, 14C-lactate output and tissue 14C-lactate were assayed in both groups of hearts. Analysis of high-energy compounds, glycogen, lactate, and pyruvate was also performed on the neutralized perchloric acid extracts of the freeze-clamped hearts. Data obtained from the 14C catabolites, originating from the metabolism of the radiolabeled substrates, indicated that the isolated normoxic rat heart metabolizes an 8.5 times higher amount of fructose-1,6-bisphosphate (7.07 mumoles/min/g d.w.) than of fructose (0.83 mumoles/min/g d.w.). CrP, CrP/Cr, glycogen, and total lactate in both tissue and perfusate were significantly higher in fructose-1,6-bisphosphate-perfused hearts. The overall indication is that fructose-1,6-bisphosphate can be taken up in its intact form by myocytes and successively metabolized to support their energy demand, and that its effects on myocardial performance and metabolism should be attributed to the molecule itself rather than to its eventual degradation products.

Animals↗