Antioxidant defense and free radical production in a rabbit model of kidney ischemia-reperfusion.
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Animals↗
Biomedical subjects
J Pincemail
Publications and source records attributed to J Pincemail.
Modifications of the synaptosomal dopamine uptake and release by two systems generating free radicals: ascorbic acid/Fe2+ and L-arginine/NADPH.
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Analysis of Variance↗
Are similar inflammatory factors involved in strenuous exercise and sepsis?
An increasing body of data suggest that strenuous exercise triggers an inflammatory response having some similarity with those occurring in sepsis. Indices of this inflammatory response to exercise (IRE) especially include leukocytosis, release of inflammatory mediators and acute phase reactants, tissue damage, priming of various white blood cell lines, production of free radicals; activation of complement, coagulation and fibrinolytic cascades. Inflammatory responses to strenuous exercise and sepsis could in part be due to the release of endotoxin in blood as common triggering factor, but it seems that tissue damage and/or contact system activation are more important triggering mechanisms in exercising subjects. While the magnitude and duration of cellular and humoral changes associated with IRE are quite different from those observed in sepsis, recent human studies suggested that chronic and/or excessive IRE could have adverse effects. Among the possible consequences of acute and chronic IRE are delayed onset muscular soreness and loss of force, cardiovascular complications, intravascular hemolysis, hypoferraemia and increased susceptibility to infection.
Blood Coagulation↗
Preservation of cortical microcirculation after kidney ischemia-reperfusion: value of an iron chelator.
Treatment of suprarenal aneurysms and renal artery reconstructions are both responsible for normothermic ischemia of the kidney (during clamping) followed by reperfusion (declamping). During reflow through an organ undergoing ischemia the production of free radicals can be associated with cell injury and a no-reflow phenomenon characterized by perfusion defects after a period of transient hyperemia. The objectives of this study were to demonstrate the existence of this phenomenon in the kidney undergoing ischemia followed by reperfusion and to test the potential protection afforded by an iron chelator (desferrioxamine) since free radical reactions are catalyzed by iron. Adult New Zealand white rabbits were divided into the following three groups: group A, 15 minutes of ischemia plus 10 minutes of reperfusion; group B, 60 minutes of ischemia plus 10 minutes of reperfusion; and group C, 60 minutes of ischemia plus 10 minutes of reperfusion combined with infusion of desferrioxamine (50 mg/kg). Cortical microcirculation in the kidney was measured by laser Doppler flowmeter before ischemia and 1, 5, and 10 minutes after reperfusion. Vitamin E content was determined in the cortex of the left kidney after 10 minutes of reperfusion and compared with that of the right (control) kidney. After 1 minute of reperfusion the cortical microcirculatory flow was significantly increased in all three groups (reactive hyperemia). In groups A and C blood flow returned to preclamping values after 10 minutes of reperfusion; however, blood flow in group B remained significantly reduced (29.2% +/- 10.5%) after 5 minutes of reperfusion with a further reduction to 48.5% +/- 5.7% after 10 minutes. These findings were correlated with the dosage of vitamin E since the vitamin E content was greatly reduced by 46.7% +/- 7.8% in group B but did not change significantly in groups A and C. This study shows that 60 minutes of normothermic ischemia is followed by a significant reduction in cortical microcirculatory flow (no-reflow phenomenon). Infusion of an iron chelator (desferrioxamine), however, which decreases the intensity of lipid peroxidation induced by the free radicals, preserves the microcirculatory flow.
Animals↗
Direct evidence of free radical production after ischaemia and reperfusion and protective effect of desferrioxamine: ESR and vitamin E studies.
After surgical renal revascularisation, warm renal ischaemia due to renal artery cross-clamping contributes to postoperative renal dysfunction. After reperfusion, free radicals are thought to be a significant cause of injury. Nevertheless, indisputable proof of free radical production is scarce, partly because of their transient nature. In this study, electron paramagnetic resonance and vitamin E levels were used to demonstrate the free radical production after renal ischaemia and reperfusion. Rabbit kidneys were submitted either to 15 or 60 minutes of ischaemia followed by reperfusion. A spin trap agent (alpha-phenyl-N-tert-butyl nitrone (PBN), 20mg/ml, 1 ml/min) was infused during reperfusion directly into the left renal artery via an aortic catheter before declamping. Blood samples were selectively drawn from the left renal vein for ESR analysis (Varian spectrometer E109) of lipidic residues extracted from blood samples. The vitamin E content of the left renal cortex was determined by HPLC procedure. The right renal cortex was used as a control for the vitamin E values. In the venous effluent, ESR analysis revealed the formation of a spectrum consisting of a triplet of asymmetric doublets. This signal resulted from the spin trapping by PBN of a mixture of both oxygen- and carbon- centred lipidic radicals. The amplitude of the signal which is proportional to the amount of free radicals was significantly higher after 60 minutes ischaemia than after 15 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Anaphylatoxin C5a production during short-term submaximal dynamic exercise in man.
We studied the effects of short-term submaximal exercise on the plasma levels of myeloperoxidase ([MPO]) and C5a anaphylatoxin ([C5a]), taken as specific markers of polymorphonuclear neutrophil (PMN) and complement activation, respectively. Eleven young, healthy male volunteers were subjected to a constant-load concentric exercise on a cycle ergometer (20 min at 80% maximal oxygen uptake). Mean resting MPO and C5a concentrations were 437 +/- 113 and 0.47 +/- 0.21 ng/ml, respectively. During exercise, [MPO] and [C5a] increased significantly (p < 0.001) towards respective peak values of 649 +/- 131 and 1.3 +/- 0.6 ng/ml. A rapid decrease of both [MPO] and [C5a] was observed during recovery. The similar time course of [MPO] and [C5a] changes and the highly significant relationship between these two variables (r = 0.651; p < 0.001) argues for the possible involvement of the complement anaphylatoxin C5a in the process of PMN degranulation. During exercise, the number of circulating PMN increased (+80%; p < 0.001) and remained practically unchanged up to 20 minutes of recovery. As [MPO] and PMN count were not significantly related (r = 0.2; p < 0.1), we concluded that the activation of PMN was independent of their mobilization.
Adult↗
[Direct approach for demonstrating free radical phenomena during equine postanesthetic myopathy: preliminary study].
This preliminary study demonstrated the existence of a free radical generation during an experimental postischemic muscular reperfusion in a halothane anesthetized horse. The authors used alpha-phényl-N-tert-butylnitrone as a spin trap agent and the electronic paramagnetic resonance method to observe in vivo a free radical generation.
Anesthesia, Inhalation↗
Evidence for free radical formation during human kidney transplantation.
Fourteen patients undergoing kidney transplantation were studied for evidence of the production of free radicals as assessed by the measurement of vitamin E (an index of lipid peroxidation) and of myeloperoxidase (a marker of neutrophil activation) in the systemic blood. Early (2 min) and late revascularization (30 min) of the kidney were respectively associated with a significant decrease of 35.5 and 40% of the initial level of plasma vitamin E. This consumption paralleled to the decrease of the vitamin E/total lipids ratio, a better indicator of vitamin E status. Heparin administration preceding renal artery clamping resulted in a twofold significant increase of baseline plasma myeloperoxidase (MPO) level (523 +/- 214 ng/ml). At kidney reperfusion, MPO concentration rose again and reached a maximum value of 1,653 +/- 882 ng/ml, indicating the presence of considerable neutrophil activation. A return to the baseline value was observed after 30 min of reperfusion. A short discussion about the possible origin of this MPO increase is given. Taken together, these data strongly suggest that free radical production, leading to lipid peroxidation phenomena, can occur within the early phase of kidney revascularization. Preliminary data using electron spin resonance with the spin-trapping technique strengthen this hypothesis.
Adult↗
In vivo free radical production after cross-clamping and reperfusion of the renal artery in the rabbit.
Postischaemic reperfusion injury is often attributed to the generation of oxygenated free radicals which may subsequently promote lipid peroxidation in cell membranes. Electron paramagnetic resonance spectroscopy in association with the spin trap molecule alpha-phenyl-N-tert-butyl-nitrone allowed direct confirmation of lipid free radical production after renal ischaemia-reperfusion in an in vivo rabbit model. A 60-min period of ischaemia followed by reperfusion caused free radical production twofold greater than after 15 min of ischaemia. Glutathione and alpha-tocopherol have been measured in renal tissue, as indirect markers of lipid peroxidation. After 15 min of ischaemia followed by 10 min of reperfusion, the mean(s.e.m.) glutathione content of the ischaemic kidney was slightly but significantly reduced by 11.9(2.5)% (P < 0.003). The content of alpha-tocopherol was unchanged. However, 10 min of reperfusion following 60 min of ischaemia led to significant decrease in mean(s.e.m.) content of both glutathione (30.4(3.7)%) (2.23(0.2) versus 3.14(0.18) mumol/g wet tissue, P < 0.001) and alpha-tocopherol (46.1(7.8)%) (0.57(0.10) versus 1.09(0.14) micrograms/g wet tissue, P < 0.001) when compared to the control kidney. Under these experimental conditions, desferrioxamine (15 mg/kg administered intravenously before inducing ischaemia), a drug known to limit free radical production, significantly limited the decrease of alpha-tocopherol to 20.8(6.4)% (0.83(0.08) versus 1.05(0.04) micrograms/g wet tissue, P < 0.05), but did not prevent glutathione consumption in the reperfused kidney.
Animals↗
Plasma levels of polymorphonuclear elastase and myeloperoxidase after uphill walking and downhill running at similar energy cost.
This study was designed to compare the effects of eccentric and concentric exercises on blood polymorphonuclear neutrophil count (nPMN) and plasma levels of myeloperoxidase ([MPO]) and elastase ([EL]) used as markers of neutrophil (PMN) activation. Ten healthy male subjects underwent 2 periods of exercise of 20 min duration each at 60% VO2max on an inclined treadmill. They initially walked up a 5% grade (UW). Ten days later they ran downhill on a 20% grade (RD). Blood samples were collected 10 min before and immediately after exercise. A postexercise sample was drawn after 20 min recovery. Both exercise bouts were accompanied by a similar and significant (p < 0.01) increase of 33% in nPMN above resting values. Baseline nPMN values were reached after 20 min recovery. There were no significant changes in [MPO] and [EL] following UW. In contrast, RD was followed by significant increases (p < 0.001) in [MPO] (+97%) and [EL] (+70%) above resting levels. While [MPO] returned to its pre-exercise level after 20 min recovery, [EL] remained significantly elevated (p < 0.05). These results clearly demonstrate the importance of the eccentric component of muscle contraction in exercise-induced PMN activation.
Adolescent↗
Plasma myeloperoxidase and vitamin E levels in head injury: preliminary results related to outcome.
This preliminary study was designed to assess a possible role of neutrophil activation and to determine the prognostic value of plasma myeloperoxidase (MPO) and vitamin E (Vit. E) levels in severe head injury. Plasma MPO and Vit. E levels were measured in nine severely head-injured patients (Glasgow Coma Score </=8) (ages 12-80 years) 6, 12. 18, 24, and 30 h after trauma. Patients were classified into two groups according to outcome after discharge from the ICU: group D (death; n = 5) and group S (survival; n = 4). Plasma MPO levels were increased immediately after trauma and then decreased. The MPO peak observed after 6 h was significantly higher (p < 0.05) in group D (mean +/- SEM: 1,237 +/- 122 ng/ml) than in group S (mean +/- SEM: 543 +/- 148 ng/ml). Plasma Vit. E levels were lower than normal values and decreased over time. They were always significantly lower (p < 0.05) in group D than in group S, except for the first sample. These differences cannot be explained entirely by total plasma lipid (TL) values since no statistical difference in TL concentrations was found between the two groups during the course of study. The ratio of Vit. E to TL. considered as the best index of Vit. E status, was lower in group D than in group S. and the difference reached statistical significance (p < 0.05) 12 h after trauma. In conclusion, in spite of the limited number of patients included in this study, it appears that severe head injury is associated with an increase in MPO and a decrease in Vit. E levels. These biochemical changes are of greater magnitude in group D than in group S; they suggest neutrophil activation and lipoperoxidation processes. Finally, plasma MPO and Vit. E seem to be new discriminant factors of outcome in head-injured patients.
Journal Article↗
Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.
Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.
Animals↗
Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with 125iodine.
In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with 125Iodine:chloramine T, lactoperoxidase, and an original technique of 'self labeling' based on the ability of the enzyme to oxidize and bind 125I in the presence of H2O2. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 microCi/micrograms MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (less than or equal to 3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.
Chloramines↗
Fast double antibody radioimmunoassay of human granulocyte myeloperoxidase and its application to plasma.
The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml.
Animals↗
Plasma vitamin E, total lipids and myeloperoxidase levels during spinal surgery. A comparison between two anesthetic agents: propofol and isoflurane.
Plasma levels of vitamin E (Vit. E), total lipids (TL), Vit. E to TL ratio and myeloperoxidase (MPO) were studied in 20 patients undergoing lumbar spinal surgery and randomly allocated to two anesthetic groups: propofol (bolus dose + continuous infusion and thiopental/isoflurane. Peripheral blood samples were withdrawn prior to induction, each hour during anesthesia and 1 h after the end of surgery. Mean Vit. E and TL levels as well as mean Vit. E to TL ratios remained in the normal range over the entire period of study whatever the anesthetic regimen. MPO levels rose significantly in the post-operative period only, but without statistical difference between the two groups. Therefore, anesthesia with propofol or thiopental/isoflurane modifies neither total lipid concentrations nor plasma Vit. E, which is a potent endogenous inhibitor of lipid peroxidation bound to lipoproteins. The rise of plasma MPO suggests a moderate post-operative neutrophil activation which is not influenced by anesthetic techniques.
Adult↗
Myeloperoxidase and elastase as markers of leukocyte activation during cardiopulmonary bypass in humans.
To assess leukocyte activation during cardiopulmonary bypass, we measured white blood cell and neutrophil counts and lysosomal enzyme release, especially myeloperoxidase and elastase, throughout the operation and for 5 days postoperatively. A newly developed double antibody radioimmunoassay of myeloperoxidase and an enzyme-linked immunosorbent assay for detection of the polymorphonuclear elastase-alpha 1-proteinase inhibitor complex were used to determine their plasma levels in 15 patients undergoing elective aorta-coronary bypass grafting. Preoperatively white blood cell counts and plasmatic levels of myeloperoxidase and elastase-alpha 1-proteinase inhibitor were normal. Because no correlation has yet been established between levels of myeloperoxidase and elastase-alpha 1-proteinase inhibitor, the aim of this prospective study was to evaluate the use of these enzyme levels as markers for leukocyte activation in vivo. We addressed the clinical situation of cardiopulmonary bypass because it offered the possibility of monitoring the comparative evolution of blood levels of these enzymes in parallel to white blood cell counts through well-defined steps corresponding to known events. We document the advantages of myeloperoxidase blood levels over elastase measurement as reflecting more rapidly the in vivo activation of leukocytes. The time course kinetics of these three measurements were not parallel. White blood cell counts remained stable at the beginning of bypass, whereas myeloperoxidase levels increased sharply and continuously as soon as bypass was instituted until the end of bypass. Elastase levels also increased, but later than myeloperoxidase, beginning when the patients was rewarmed. High elastase plasma levels persisted later than myeloperoxidase after bypass, in parallel with white blood cell counts. It thus clearly appears that changes in myeloperoxidase levels more rapidly reflect the activation state of leukocytes induced by cardiopulmonary bypass and surgery, whereas peak levels of elastase were delayed and parallel to white blood cell counts. From this model, in which the evolution of leukocyte numbers could be followed in relation with known steps of stimulation, it appears that myeloperoxidase is a sensitive marker for monitoring in vivo activation of white blood cells.
Adult↗
[Mobilization of leukocytes during sub-maximal dynamic exercise].
Increase of blood leucocytes induced by a dynamic exercise has been studied in 10 adult male subjects (age: 22 +/- 2 years; body weight 73 +/- 10 kg; VO2max: 54 +/- 8 ml O2/kg.min. The exercise consisted of a 20 minutes test on a cycle ergometer at a work rate eliciting a total energy expenditure roughly equal to 80% of the maximal aerobic power (80% VO2max). During exercise the absolute number of circulating leucocytes (n Leu) increased significantly toward a maximal value reached in most cases at the end of exercise. The maximal values of n Leu--expressed as percentage of n Leu measured at rest--in the different blood leucocyte types (monocytes:n Mono; granulocytes:n GR; natural killer cells:n NK) and lymphocyte subsets (lymphocytes:n LY; lymphocytes T:n T; lymphocytes B:n B; lymphocytes T helper:n T4; lymphocytes T suppressor:n T8)--were as follows:n Mono:240%; n GR:170%; n NK:490%; n LY:220%; n T:200%; n B:190%; n T4:140%; n T8:190%. With the exception of n GR, n Leu decreased during recovery at a rate depending on the leucocyte type. The n T4/n T8 ratio, equal to 1 at rest, was reduced by about 40% during exercise. This ratio rose after exercise and reached a value equal to that measured at rest after 20 minutes recovery. In agreement with FERRY (1989), we concluded that exercise is accompanied by a selective mobilization of the different leucocyte types. The magnitude of this phenomenon and the kinetics of n Leu recuperation depend on cell types.
Adult↗
Exercise induces pentane production and neutrophil activation in humans. Effect of propranolol.
The effect of beta-adrenergic receptor blockade on exercise-induced lipid peroxidation in man has been examined by measuring the production of pentane in expired air. For this purpose, five healthy male subjects were subjected to dynamic exercise of graded intensity on a cycle ergometer (10 min at 45%, 5 min at 60% and 75% maximal oxygen uptake 1 h after ingestion of either a placebo or 40-mg propranolol. At rest, mean pentane concentration [( pent]) with placebo was 4.13 pmol.l-1, SD 2.14. After exercise, this value significantly increased by 310% (17.1 pmol.l-1, SD 7.73, P less than 0.01). Oral administration of 40-mg propranolol significantly lowered the mean resting [pent] to 1.75 pmol.l-1, SD 0.77, P less than 0.05. After exercise, the increase of [pent] was much smaller (240%) and was less significant (P less than 0.2) than with the placebo. The mechanism of this inhibitory effect of propranolol remains to be elucidated. However, as indicated by the measurement of plasma myeloperoxidase concentration, it can be concluded that the antioxidant property of propranolol cannot be attributed to the inhibition of neutrophil activation, a possible source of free radicals during exercise.
Adult↗