Non-thyroidal illness syndrome in chronic diseases: role of irisin as modulator of antioxidants.

OBJECTIVE: Non-thyroidal-illness syndrome (NTIS) refers to condition found in chronic diseases that is an adaptive mechanism. However, oxidative stress is related to NTIS in a vicious circle, due to deiodinases alteration and negative effects of low T3 on antioxidant levels or activity. Muscle is one of the main targets of thyroid hormones and it can secrete a myokine named irisin, which is able to induce the browning of white adipose tissue, energy expenditure and protect against insulin resistance. Inconclusive data have been reported about irisin role in chronic diseases. Moreover, no correlation with antioxidants has been investigated. Therefore, we performed a case-control study with the primary endpoint to evaluate irisin levels in two models of NTIS, such as chronic heart failure (CHF) and chronic kidney disease (CKD) during haemodialytic treatment. The secondary endpoint was the correlation with total antioxidant capacity (TAC) to establish a possible role of irisin in the modulation of antioxidant systems. PATIENTS AND METHODS: Three groups of subjects were enrolled. Group A included CHF patients (n=18; aged 70.22 ± 2.78 ys; BMI ± 27.75 ± 1.28 kg/m2); Group B included CKD patients (n=29; aged 67.03 ± 2.64; BMI 24.53 ± 1.01); finally, 11 normal subjects (Group C) have been enrolled as controls. Irisin has been evaluated by ELISA method and Total Antioxidant Capacity (TAC) by spectrophotometric method. RESULTS: Irisin was significantly higher in Group B vs. A and C groups (Mean ± SEM: 20.18 ± 0.61 ng/ml vs. 2.77 ± 0.77 and 13.06 ± 0.56, respectively; p<0.05); a significant correlation between irisin and TAC was observed in group B. CONCLUSIONS: These preliminary data suggest a possible role of irisin in the modulation of antioxidants in two chronic syndromes with low T3 (i.e., CHF and CKD) with differential pattern in these two models studied. Further insights are needed to confirm this pilot study, which could be the basis for a longitudinal investigation, to assess a prognostic role of irisin with possible therapeutic implications.

Lycopene and cardiovascular diseases: an update.

Cardiovascular disease (CVD) is the leading cause of death in Western societies and accounts for up to a third of all deaths worldwide. In comparison to the Northern European or other Western countries, the Mediterranean area has lower rates of mortality from cardiovascular diseases and cancer, and this is attributed, at least in part, to the so-called Mediterranean diet, which is rich in plantderived bioactive phytochemicals. Identification of the active constituents of the Mediterranean diet is therefore crucial to the formulation of appropriate dietary guidelines. Lycopene is a natural carotenoid found in tomato, an essential component of the Mediterranean diet, which, although belonging to the carotenoid family, does not have pro-vitamin A activity but many other biochemical functions as an antioxidant scavenger, hypolipaemic agent, inhibitor of pro-inflammatory and pro-thrombotic factors, thus potentially of benefit in CVD. In particular, the review intends to conduct a systematic analysis of the literature (epidemiological studies and interventional trials) in order to critically evaluate the association between lycopene (or tomato products) supplementation and cardiovascular diseases and/or cardiovascular disease risk factors progression, and to prepare provision of evidence-based guidelines for patients and clinicians. Several reports have appeared in support of the role of lycopene in the prevention of CVD, mostly based on epidemiological studies showing a dose-response relationship between lycopene and CVD. A less clear and more complex picture emerges from the interventional trials, where several works have reported conflicting results. Although many aspects of lycopene in vivo metabolism, functions and clinical indications remain to be clarified, supplementation of low doses of lycopene has been already suggested as a preventive measure for contrasting and ameliorating many aspects of CVD.

New developments in anthracycline-induced cardiotoxicity.

Anthracyclines are among the most effective anticancer drugs ever developed. Unfortunately, their clinical use is severely limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure, usually refractory to conventional therapy. The pathophysiology of anthracycline-induced cardiomyopathy remains controversial and incompletely understood. The current thinking is that anthracyclines are toxic per se but gain further cardiotoxicity after one-electron reduction with ROS overproduction or two-electron reduction with conversion to C-13 alcohol metabolites. ROS overproduction can probably be held responsible for anthracycline acute cardiotoxicity, but not for all the aspects of progressive cardiomyopathy. Intramyocardial formation of secondary alcohol metabolites might play a key role in promoting the progression of cardiotoxicity toward end-stage cardiomyopathy and congestive heart failure. In this review we also discuss recent developments in: a) the molecular mechanisms underlying anthracycline-induced cardiotoxicity; b) the role of cytosolic NADPH-dependent reductases in anthracycline metabolism; c) the influence of genetic polymorphisms on cardiotoxicity outcome; d) the perspectives on the most promising strategies for limiting or preventing anthracycline-induced cardiotoxicity, focusing on controversial aspects and on recent data regarding analogues of the natural compounds, tumor-targeted formulations and cardioprotective agents.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes.

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37 C) of intact human red blood cells to doxorubicinol (40 M) and to aglycone derivatives of doxorubicin (40 M) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells...

Inhibition of salivary enzymes by cigarette smoke and the protective role of glutathione.

Tobacco smoke is involved in the pathogenesis of several diseases regarding different body systems, mainly cardiovascular and respiratory in addition to its local toxic effect in the oral cavity. The noxious effects of smoke compounds justify the high incidence of periodontal diseases, caries, and neoplastic diseases of oral tissues in smokers. Some toxic components of tobacco smoke, unsaturated and saturated aldehydes, could interact with thiol rich compounds, leading to structural and functional modification of these molecules. Previous papers have demonstrated an in vitro significant decrease of some enzymatic activities, both in plasma and in saliva, following external addition of aldehydes or exposure to cigarette smoke (CS). Furthermore, the same studies underlined the protective effect exerted by the addition of glutathione (GSH) against the damaging role of smoke aldehydes. In this study some salivary enzymes (lactic dehydrogenase [LDH], aspartate aminotransferase [AST] and amylase), and total GSH were measured in 20 volunteers smokers, before and just after smoking a single cigarette. All enzymatic activities showed a significant inhibition following a single cigarette, probably due to the interaction between smoke aldehydes and -SH groups of the enzyme molecules. Moreover, the percentage of the enzymatic inhibition showed a negative correlation with the basal level of salivary GSH. Our results emphasize that not only one cigarette is sufficient to impair the salivary enzymatic activities but also strengthen the proposed protective role of GSH against the noxious biochemical effects of CS.

Human heart cytosolic reductases and anthracycline cardiotoxicity.

Anthracyclines are a class of antitumor drugs widely used for the treatment of a variety of malignancy, including leukemias, lymphomas, sarcomas, and carcinomas. Different mechanisms have been proposed for anthracycline antitumor effects including free-radical generation, DNA intercalation/binding, activation of signaling pathways, inhibition of topoisomerase II and apoptosis. A life-threatening form of cardiomyopathy hampers the clinical use of anthracyclines. According to the prevailing hypothesis, anthracyclines injure the heart by generating damaging free radicals through iron-catalyzed redox cycling. Although the "iron and free-radical hypothesis" can explain some aspects of anthracycline acute toxicity, it is nonetheless disappointing when referred to chronic cardiomyopathy. An alternative hypothesis implicates C-13 alcohol metabolites of anthracyclines as mediators of myocardial contractile dysfunction ("metabolite hypothesis"). Hydroxy metabolites are formed upon two-electron reduction of the C-13 carbonyl group in the side chain of anthracyclines by cytosolic NADPH-dependent reductases. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+ movements, ultimately impairing cardiac contraction and relaxation. In addition, alcohol metabolites can impair cardiac intracellular iron handling and homeostasis, by delocalizing iron from the [4Fe-4S] cluster of cytoplasmic aconitase. Chronic cardiotoxicity induced by C-13 alcohol metabolite might be primed by oxidative stress generated by anthracycline redox cycling ("unifying hypothesis"). Putative cardioprotective strategies should be aimed at decreasing C-13 alcohol metabolite production by means of efficient inhibitors of anthracycline reductases, as short-chain coenzyme Q analogs and chalcones that compete with anthracyclines for the enzyme active site, or by developing novel anthracyclines less susceptible to reductive metabolism.

Is homocysteine a pro-oxidant?

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

Effect of homocysteine on polymorphonuclear leukocyte activity and luminol-dependent chemiluminescence.

Homocysteine is a non-protein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H(2)O(2), enhanced platelet activity and increased production of macrophage-derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine-generated H(2)O(2) has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol-dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol-hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at micromol/L concentrations (10-100 micromol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol-hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H(2)O(2)-mediated neutrophil activity is not supported and, probably, the common opinion that views the H(2)O(2) generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered.

Oxidative stress in ischemia-reperfusion injury: assessment by three independent biochemical markers.

BACKGROUND: Oxidative stress plays a key role in ischemia-reperfusion injury, causing peroxidation of tissue lipids and proteins. However, it is debated whether brief ischemic episodes are sufficient to cause detectable oxidative stress in humans, since biochemical markers used so far in the setting of percutaneous transluminal coronary angioplasty (PTCA) gave conflicting results. METHODS: We determined lipid hydroperoxides (ROOHs), conjugated dienes (CD) and total radical-trapping antioxidant capacity (TRAP), three different independent markers of oxidative stress, in aortic and great cardiac vein blood of 5 patients undergoing PTCA before a single balloon inflation lasting 115 +/- 38 s (t0), and 1 min (t1), 5 min (t5), 15 min (t15) after balloon deflation (Group 1). ROOHs and CD were also determined in aortic and great cardiac vein blood of 5 patients with mitral valve disease (Group 2). RESULTS: In Group 1, great cardiac vein levels of ROOHs and CD at t1 increased by 219% and 79%, respectively, compared to t0 (p < 0.01); this sharp and consistent increase persisted up to t15 (+189% and +63%, respectively, compared to t0; p < 0.01). Great cardiac vein levels of TRAP were significantly lower than aortic levels at t0, and exhibited a further decrease at tl. No significant differences in aortic and great cardiac vein levels of ROOHs and CD at t0 were observed between Group 1 and Group 2. CONCLUSIONS: The three methods we used showed a remarkable sensitivity for the detection of post-ischemic reperfusion injury in cardiac venous blood and may be useful for detecting small foci of ischemia-reperfusion injury in microvascular angina.

Doxorubicin metabolism and toxicity in human myocardium: role of cytoplasmic deglycosidation and carbonyl reduction.

The anthracycline doxorubicin (DOX) is an exceptionally good antineoplastic agent, but its use is limited by formation of metabolites which induce acute and chronic cardiac toxicities. Whereas the acute toxicity is mild, the chronic toxicity can produce a life-threatening cardiomyopathy. Studies in laboratory animals are of limited value in predicting the structure and reactivity of toxic metabolites in humans; therefore, we used an ethically acceptable system which is suitable for exploring DOX metabolism in human myocardium. The system involves cytosolic fractions from myocardial samples obtained during aorto-coronary bypass grafting. After reconstitution with NADPH and DOX, these fractions generate the alcohol metabolite doxorubicinol (DOXol) as well as DOX deoxyaglycone and DOXol hydroxyaglycone, reflecting reduction of the side chain carbonyl group, reductase-type deglycosidation of the anthracycline, and hydrolase-type deglycosidation followed by carbonyl reduction, respectively. The efficiency of each metabolic route has been evaluated at low and high DOX:protein ratios, reproducing acute, single-dose and chronic, multiple-dose regimens, respectively. Low DOX:protein ratios increase the efficiency of formation of DOX deoxyaglycone and DOXol hydroxyaglycone but decrease that of DOXol. Conversely, high DOX:protein ratios facilitate the formation of DOXol but impair reductase- or hydrolase-type deglycosidation and uncouple hydrolysis from carbonyl reduction, making DOXol accumulate at levels higher than those of DOX deoxyaglycone and DOXol hydroxyaglycone. Structure-activity considerations have suggested that aglycones and DOXol may inflict cardiac damage by inducing oxidative stress or by perturbing iron homeostasis, respectively. Having characterized the influence of DOX:protein ratios on deglycosidation or carbonyl reduction, we propose that the benign acute toxicity should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity should be attributed to alterations of iron homeostasis by DOXol. This picture rationalizes the limited protective efficacy of antioxidants against chronic cardiomyopathy vis-à-vis the better protection offered by iron chelators, and forms the basis for developing analogues which produce less DOXol.

High total antioxidant activity and uric acid in tracheobronchial aspirate fluid of preterm infants during oxidative stress: an adaptive response to hyperoxia?

The effect of O2 exposure, expressed by mean daily fractional inspired oxygen concentration (FiO2), was evaluated during the first 6 d of life in the tracheobronchial aspirate fluid of 16 mechanically ventilated preterm infants in terms of both antioxidant response and oxidative damage, by measuring total antioxidant activity, uric acid concentrations and protein carbonyl content. Each day linear regression analysis was performed and a positive correlation was found between total antioxidant activity and FiO2 during the study period, especially on day 2 of life (r = 0.91, p < 0.0001), but uric acid correlated only in the first 3 d, especially on the 2nd day (r = 0.83, p < 0.0001). No correlation was found between carbonyl content and FiO2. The highest values of total antioxidant activity (416 and 790 micromol l(-1)) were found in 2 babies ventilated with highest FiO2: 1 and 0.80, respectively. Total antioxidant activity was not detectable or was very low in the babies not requiring O2 therapy. The highest value of uric acid (270 micromol l(-1)) was found in the baby ventilated with 100% oxygen. Uric acid concentrations obtained in these babies were much higher then those reported in the bronchoalveolar lavage fluid of adults. Preterm babies seem to have an antioxidant response in the tracheobronchial aspirate fluid following an oxidative stress and uric acid may be physiologically important as an antioxidant of the respiratory tract, especially during the first days of life.

Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers.

Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically significant difference between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were significantly (p < 0.05) higher in smokers. Smoking of a single cigarette induced a significant reduction in glutathione concentration (p < 0.05). Salivary antioxidant power may affect individual sensitivity toward tobacco stress.

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer's disease.

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimer's disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P < 0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P < 0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium.

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of -SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.

Antioxidant properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone).

Idebenone [2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone] is a synthetic analogue of coenzyme Q that is currently employed in the treatment of vascular and degenerative diseases of the central nervous system. There is some evidence to suggest that idebenone might function as an antioxidant; however, it has not been demonstrated whether this function pertains to the quinone or hydroquinone form of idebenone. Here we demonstrate that idebenone can scavenge a variety of free radical species, including organic radicals such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and diphenylpicrylhydrazyl, peroxyl and tyrosyl radicals, and peroxynitrite. Idebenone can also redox couple with hypervalent species of Mb or Hb, thus preventing lipid peroxidation promoted by these species. Likewise, idebenone inhibits microsomal lipid peroxidation induced by ADP-iron complexes or organic hydroperoxides. In so doing, idebenone prevents the destruction of cytochrome P450, which otherwise would accompany lipid peroxidation. Irrespective of the experimental system under investigation, idebenone functions by virtue of the electron-donating properties of the hydroquinone form. Redox coupling of this hydroquinone with free radicals generates the quinone compound, which per se lacks antioxidant activity. In many experiments, the antioxidant effects of idebenone become appreciable at approximately 2 microM, which is well in the range of plasma levels attainable in patients given oral doses of this drug. Moreover, comparative experiments have shown that the antioxidant efficiency of idebenone varies from no less than 50% to slightly more than 100% of that of vitamin E or Trolox. We would therefore propose that the neuroprotective effects of idebenone can be attributed, at least in part, to its ability to function as an antioxidant, involving redox cycling between hydroquinone and quinone.

Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM.

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 +/- 131 vs. 955 +/- 104 micromol/l, P < 0.001) and significantly increased levels of ROOHs (7.13 +/- 2.11 vs. 2.10 +/- 0.71 micromol/l, P < 0.001) and conjugated dienes (0.0368 +/- 0.0027 vs. 0.0328 +/- 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 +/- 0.0028 vs. 0.0259 +/- 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 +/- 0.0011 vs. 0.0069 +/- 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.

Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. Pharmacologic and molecular reappraisal of anthracycline cardiotoxicity.

Anticancer therapy with doxorubicin (DOX) and other quinone anthracyclines is limited by severe cardiotoxicity, reportedly because semiquinone metabolites delocalize Fe(II) from ferritin and generate hydrogen peroxide, thereby promoting hydroxyl radical formation and lipid peroxidation. Cardioprotective interventions with antioxidants or chelators have nevertheless produced conflicting results. To investigate the role and mechanism(s) of cardiac lipid peroxidation in a clinical setting, we measured lipid conjugated dienes (CD) and hydroperoxides in blood plasma samples from the coronary sinus and femoral artery of nine cancer patients undergoing intravenous treatments with DOX. Before treatment, CD were unexpectedly higher in coronary sinus than in femoral artery (342 +/- 131 vs 112 +/- 44 nmol/ml, mean +/- SD; P < 0.01), showing that cardiac tissues were spontaneously involved in lipid peroxidation. This was not observed in ten patients undergoing cardiac catheterization for the diagnosis of arrhythmias or valvular dysfunctions, indicating that myocardial lipid peroxidation was specifically increased by the presence of cancer. The infusion of a standard dose of 60 mg DOX/m(2) rapidly ( approximately 5 min) abolished the difference in CD levels between coronary sinus and femoral artery (134 +/- 95 vs 112 +/- 37 nmol/ml); moreover, dose fractionation studies showed that cardiac release of CD and hydroperoxides decreased by approximately 80% in response to the infusion of as little as 13 mg DOX/m(2). Thus, DOX appeared to inhibit cardiac lipid peroxidation in a rather potent manner. Corollary in vitro experiments were performed using myocardial biopsies from patients undergoing aortocoronary bypass grafting. These experiments suggested that the spontaneous exacerbation of lipid peroxidation probably involved preexisting Fe(II) complexes, which could not be sequestered adequately by cardiac isoferritins and became redox inactive when hydrogen peroxide was included to simulate DOX metabolism and hydroxyl radical formation. Collectively, these in vitro and in vivo studies provide novel evidence for a possible inhibition of cardiac lipid peroxidation in DOX-treated patients. Other processes might therefore contribute to the cardiotoxicity of DOX.

NA+/K+ ATPase impairment and experimental glycation: the role of glucose autoxidation.

Non enzymatic glycation could be involved in the early impairment of Na+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2 was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formation. No gross structural and conformational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements. Our results suggest that during the exposure of brain NA+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at lease in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.

Oxygen transport by fetal bovine hemoglobin.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20 degrees C. However, on going from 20 degrees C to 37 degrees C, the difference in O2 affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.