Frank Petrat

Hypothermia injury/cold-induced apoptosis—evidence of an increase in chelatable iron causing oxidative injury in spite of low O2−/H2O2 formation

When incubated at 4°C, cultured rat hepatocytes or liver endothelial cells exhibit pronounced injury and, during earlier rewarming, marked apoptosis. Both processes are mediated by reactive oxygen species, and marked protective effects of iron chelators as well as the protection provided by various other antioxidants suggest that hydroxyl radicals, formed by classical Fenton chemistry, are involved. However, when we measured the Fenton chemistry educt hydrogen peroxide and its precursor, the superoxide anion radical, formation of both had markedly decreased and steady‐state levels of hydrogen peroxide did not alter during cold incubation of either liver endothelial cells or hepatocytes. Similarly, there was no evidence of an increase in O2−/H2O2 release contributing to cold‐induced apoptosis oc‐curring on rewarming. In contrast to the release/ level of O2− and H2O2, cellular homeostasis of the transition metal iron is likely to play a key role during cold incubation of cultured hepatocytes: the hepatocellular pool of chelatable iron, measured on a single‐cell level using laser scanning microscopy and the fluorescent indicator phen green, increased from 3.1 ± 2.3 μM (before cold incubation) to 7.7 ± 2.4 μM within 90 min after initiation of cold incubation. This increase in the cellular chelatable iron pool was reversible on rewarming after short periods of cold incubation. The cold‐induced increase in the hepatocellular chelatable iron pool was confirmed using the calcein method. These data suggest that free radicalmediated hypothermia injury/cold‐induced apoptosis is primarily evoked by alterations in the cellular iron homeostasis/a rapid increase in the cellular chelatable iron pool and not by increased formation of O2−/H2O2.—Rauen, U., Petrat, F., Li, T., de Groot, H. Hypothermia injury/coldinduced apoptosis—evidence of an increase in chelatable iron causing oxidative injury in spite of low O2−2/H2O2 formation. FASEB J. 14, 1953–1964 (2000)

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these “loosely bound” iron ions (presumably mainly FeII) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4‐[(1,10‐phenanthrolin‐5‐yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron‐binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4‐[(2,2′‐bipyridin‐4‐yl)aminocarbonyl]benzyl ester (RDA), with 2,2′‐bipyridine as chelating unit (log β3=17.5), essentially the same iron concentration (16.0±1.9 μM) was determined as with RPA (log β3=21.1), despite the four orders of magnitude difference in FeII‐binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron‐storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4‐[bis(pyridin‐2‐ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N‐bis(pyridin‐2‐ylmethyl)amine group as chelating component (log β2=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 μM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log Kapp) of between 9 and 14.

NAD(P)H, a Primary Target of 1O2 in Mitochondria of Intact Cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen (1O2) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2′,4′,5′,7′-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of 1O2, which readily oxidized NAD(P)H to NAD(P)+. In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating1O2 within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5–10 s, Rho 123 for 60 s) led to a significant (29.6 ± 8.2 and 30.2 ± 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow reincrease. Prolonged photoactivation (≥15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for 1O2 in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.

Free hemoglobin concentration in severe sepsis: methods of measurement and prediction of outcome

IntroductionHemolysis can be induced in sepsis via various mechanisms, its pathophysiological importance has been demonstrated in experimental sepsis. However, no data on free hemoglobin concentrations in human sepsis are available. In the present study we measured free hemoglobin in patients with severe sepsis as well as in postoperative patients using four methods. It was our aim to determine the potential value of free hemoglobin as a biomarker for diagnosis and outcome of severe sepsis in critical illness.MethodsPlasma concentration of free hemoglobin was determined in patients with severe sepsis (n = 161) and postoperative patients (n = 136) on day 1 of diagnosis and surgery. For the measurement of free hemoglobin, an enzyme linked immunosorbent assay and three spectrophotometric algorithms were used. Moreover, SAPS II- and SOFA scores as well as procalcitonin concentration and outcome were determined. Kaplan-Meier analysis was performed and odds ratios were determined after classification of free hemoglobin concentrations in a high and low concentration group according to the median. For statistical evaluation the Mann-Whitney test and logistic regression analysis were used.ResultsIn non-survivors of severe sepsis, free hemoglobin concentration was twice the concentration compared to survivors. Thirty-day survival of patients, as evidenced by Kaplan-Meier analysis, was markedly lower in patients with high free hemoglobin concentration than in patients with low free hemoglobin concentration. Best discrimination of outcome was achieved with the spectrophotometric method of Harboe (51.3% vs. 86.4% survival, p < 0.001; odds ratio 6.1). Multivariate analysis including free hemoglobin, age, SAPS II- and SOFA-score and procalcitonin demonstrated that free hemoglobin, as determined by all 4 methods, was the best and an independent predictor for death in severe sepsis (p = 0.022 to p < 0.001). Free hemoglobin concentrations were not significantly different in postoperative and septic patients in three of four assays. Thus, free hemoglobin can not be used to diagnose severe sepsis in critical illness.ConclusionsFree hemoglobin is an important new predictor of survival in severe sepsis.

Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4+/-1.3 microM.

Protection by Pyruvate Infusion in a Rat Model of Severe Intestinal Ischemia-Reperfusion Injury

BACKGROUND Several lines of evidence suggest a strong protective potential of pyruvate against ischemia-reperfusion injury. Here, we studied the effect of pyruvate infusion on injury of the small intestine and on systemic parameters in a rat model of severe mesenteric ischemia-reperfusion injury. MATERIALS AND METHODS Mesenteric ischemia-reperfusion was induced by occlusion/reopening of the superior mesenteric artery of male Wistar rats (90 min ischemia, 120 min reperfusion). Sodium pyruvate was infused at overall doses of 50, 250, and 1,000 mg/kg during two time windows: 30 min before until the induction of ischemia and 30 min before reperfusion until 60 min after beginning of reperfusion. RESULTS Pyruvate infusion attenuated ischemia-reperfusion injury of the small intestine between 25% and 55% as indicated by macroscopic and microscopic evaluation, intestinal hemorrhages, and myeloperoxidase activity (neutrophil invasion). There were no significant differences in the local protective effects exerted by the three doses of sodium pyruvate applied. At 250 mg sodium pyruvate/kg and 1,000 mg sodium pyruvate/kg, however, blood pH values were less acidotic, and at 250 mg sodium pyruvate/kg the mean arterial blood pressure remained at higher values during the reperfusion phase. A significant increase in the blood plasma sodium concentration only occurred at 1,000 mg sodium pyruvate/kg. CONCLUSIONS Pyruvate infusion clearly protects the small intestine against ischemia-reperfusion injury. Protection can already be achieved at doses where sodium overload is negligible. Protection primarily results from local effects on the small intestine. Only at a dose of 250 mg sodium pyruvate/kg and above, systemic effects may additionally contribute.

Protection Against Severe Intestinal Ischemia/Reperfusion Injury in Rats by Intravenous Resveratrol

BACKGROUND Repetitive enteral or intraperitoneal administration of resveratrol at high doses has recently been found to protect the small intestine against acute ischemia/reperfusion (I/R) injury. In the present work, the protective potential of solvent-free continuous intravenous infusions of small amounts of resveratrol was studied in a model of severe intestinal I/R injury. MATERIALS AND METHODS Mesenteric ischemia was induced in male Wistar rats (six animals/group) by superior mesenteric artery occlusion (SMAO, 90 min) and reperfusion (120 min) by reopening of the microvascular clamp. Resveratrol (0.056 or 0.28 mg/kg) was continuously perfused into the jugular vein (0.014 or 0.07 mg/kg × h) starting 30 min before SMAO; an SMAO control group and sham groups (no SMAO) receiving either 0.9% NaCl solution or resveratrol (0.28 mg/kg) were included. During the experimental procedure, isotonic saline was given at a systolic blood pressure below 90 mmHg, and several parameters including those of biomonitoring and blood gas analysis were measured. Small intestine injury was assessed macroscopically, from released plasma enzyme activities, from the tissue contents of thiobarbituric acid-reactive substances and hemoglobin, from the tissue myeloperoxidase activity, and histopathologically. RESULTS Resveratrol at only 0.056 mg/kg significantly decreased the macroscopic damage score, the tissue myeloperoxidase activity, the hemoglobin content, the histopathologic score, and the plasma glutamate-pyruvate transaminase activity, but it did not improve the systemic and metabolic parameters. Instead, during reperfusion, significantly higher volumes of saline were administered to animals receiving the polyphenol, although resveratrol did not significantly affect any parameters in sham-operated animals. CONCLUSIONS Low doses of intravenously administered resveratrol considerably protected the rat small intestine against severe I/R injury, despite some adverse effects on blood pressure under these conditions.

Effects of glycine, pyruvate, resveratrol, and nitrite on tissue injury and cytokine response in endotoxemic rats

BACKGROUND Glycine, pyruvate, resveratrol, and nitrite are well-known protective compounds among others in ischemic tissue injury. Here, we compared their effects in acute lipopolysaccharide (LPS)-induced shock in rats to assess whether inhibition of the proinflammatory cytokine response is a prerequisite for their protective actions. MATERIALS AND METHODS Rats (six or eight per group) were anesthetized, received LPS as an intravenous bolus (2.5 mg/kg), and were observed for 5 h. Glycine, sodium pyruvate, resveratrol, and sodium nitrite were continuously infused starting 30 min before LPS administration. Parameters included histopathologic changes, organ-specific cytokine levels, plasma nitrite and nitrate concentrations, and time courses of biomonitoring parameters, marker enzyme activities, and plasma cytokine concentrations. RESULTS Glycine, pyruvate, resveratrol, and nitrite enhanced arterial blood pressure after LPS-induced shock. Also, parameters reflecting tissue ischemia were significantly improved and plasma markers of organ injury ameliorated by all substances. Of the plasma cytokine concentrations increased by LPS, some were differently decreased or even further increased by the substances. None of them reduced the elevated plasma nitrite and nitrate concentration. Glycine diminished the increases in tissue cytokine levels organ specifically, pyruvate decreased some cytokine concentrations in all organs, and nitrite significantly affected only a few cytokine concentrations in some organs, whereas the levels of many cytokines were raised by resveratrol. All substances except resveratrol decreased granulocyte infiltrates in the liver. CONCLUSIONS The present results demonstrate that glycine, pyruvate, resveratrol, and nitrite protect against LPS-induced shock and tissue injury (cell death) in rats and suggest that inhibition of the proinflammatory cytokine response is not mandatory for their protective actions.

ADVERSE EFFECTS OF RESUSCITATION WITH LACTATED RINGER COMPARED WITH RINGER SOLUTION AFTER SEVERE HEMORRHAGIC SHOCK IN RATS

Lactated Ringer (LR) is a widely used resuscitation fluid that is known to mediate beneficial effects on acid-base balance when compared with normal saline. We here compared LR with the more physiological Ringer solution (RS) regarding acid-base status, hemodynamics, survival, and organ injury following fluid resuscitation subsequent to severe hemorrhagic shock. Anesthetized rats were hemorrhaged to a mean arterial blood pressure of 25 to 30 mmHg within 30 min. After 60 min, they were resuscitated with either RS or LR (three times the shed blood volume) or with RS or LR plus blood (shed blood plus twice its volume) within 30 min. Subsequently, the animals were observed for further 150 min. When the rats were resuscitated with pure LR or RS, all animals of the shock/LR group, but only three of eight shock/RS group rats were dead 100 min later (median survival, 50 ± 13.1 vs. 120 ± 14.1 min; P < 0.05). Coadministration of the shed blood with RS or LR increased the survival rates to 100%. In these blood-resuscitated groups, organ injury, especially of the kidney, was diminished by the use of RS compared with LR. Time-matched acid-base parameters were not different in all shock groups until death of the animals or euthanasia at the end of experimental time. We conclude that, in severe hemorrhagic shock, resuscitation with RS leads to an improved outcome compared with resuscitation with LR, regardless whether blood is coadministered or not. ABBREVIATIONS HEPES — 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid BE — base excess BW — body weight CK — creatine kinase LDH — lactate dehydrogenase LR — lactated Ringer MAP — mean arterial blood pressure MPO — myeloperoxidase NS — normal saline PCO2 — carbon dioxide partial pressure PO2 — oxygen partial pressure RS — Ringer solution bpm — beats per min

Quantification of ischemia-reperfusion injury to the small intestine using a macroscopic score.

ABSTRACT Introduction: Classical methods do not allow to rapidly quantify the heterogeneously distributed ischemia-reperfusion injury along the small intestine. We therefore established a suitable macroscopic score. Methods: A rat model based on superior mesenteric artery occlusion (45 or 90 min ischemia; 0, 120, or 180 min reperfusion) was used on 42 rats. Results: Subsequent to ischemia-reperfusion or pure ischemia, macroscopic phenotypes of the small intestine were defined and differentiated based on the severity of existing petechiae and hemorrhages (macroscopic types 0-1-2-3). Analysis of the interobserver variability verified the reliability of this macroscopic differentiation. Macroscopic types were directly correlated with their histological alterations (Chiu score). On the basis of the sample hemoglobin content, a nonlinear macroscopic score (0-1-3-9) was derived from the linearly related macroscopic types. Mean macroscopic scores after 45 min ischemia/180 min reperfusion were lower than those after 90 min ischemia/120 min reperfusion. Nevertheless, all scores correlated well with their respective hemoglobin contents (R2 = 0.87). Heterogeneous patterns of macroscopic scores that depended on the ischemic period and largely differed between individual animals were found to be distributed along the small intestine with the highest injury score at the last half of the jejunum. The iron chelator deferoxamine mesylate clearly decreased the overall tissue hemoglobin content and macroscopic score of the small intestine but local protection was restricted to the proximal and middle part of the jejunum. Conclusion: The local as well as overall ischemia-reperfusion injury in the rat small intestine can be rapidly and reliably assessed macroscopically.