Due to the limitations of the conventional refinery methods, development of a new method such as oxidative denitrogenation (ODN) is highly desirable. This study described a novel ODN to remove organo-nitrogenous compounds (ONCs) in liquid fuel by ascorbic acid (AscH2) and H2O2 redox system under ambient conditions. Seven ONCs including pyridine, quinoline, acridine, 7,8-benzoquinoline, indole, N-methylpyrrolidone (NMP), and N,N-dimethylformamide (DMF) were chosen to assess the fuel-denitrified ability of the AscH2/H2O2 system. The results showed that the basic group of ONCs (pyridine, quinoline, and acridine) can be effectively removed (removal ratio > 95 %) while the removal efficiency of water-soluble compounds (7,8-benzoquinoline, NMP, and DMF) was moderate (61-68 %) under a mild temperature (30 °C) and atmospheric pressure. Free radical quenching and electron paramagnetic resonance experiments confirmed that hydroxyl and AscH2 radicals played a major role in the degradation of ONCs. The degraded products of quinoline were analyzed by gas chromatography-mass spectroscopy and ion chromatography. Based on the identified intermediate products, a putative reaction pathway majorly involving three steps of N-onium formation, transfer hydrogenation, and free radical oxidative ring-opening was suggested for the quinoline degradation. The presented approach can be performed at a normal temperature and pressure and will live up to expectations in the pre-denitrogenation and selective removal of basic ONCs in fuel oils.

Lactoferrin (Lf) possess a protective potential to liver, but whether it can prevent alcoholic liver injury (ALI) remains unclear.

Four groups of male C57BL/6J mice are fed with different diets, namely, AIN-93G diet for control (CON) and ethanol (EtOH) groups, and AIN-93G diet with 0.4% and 4% casein replaced by Lf for low-dose Lf (LLf) and high-dose Lf (HLf) groups, respectively. ALI is induced by giving 20% ethanol ad libitum combined with four "binges". Lf can remarkably decrease EtOH-induced mortality. Lf promotes aldehyde dehydrogenase-2 (ALDH2) expression and suppressing cytochrome P450 2E1 (CYP2E1) overexpression, resulting in the reduced hepatic superoxide and inflammation levels, which ultimately leads to the hepatic injury alleviation. However, HLf increases acetyl-CoA carboxylase and fatty acid synthase protein levels, which suggests that excessive intake may weaken the beneficial effects of Lf. Moreover, LLf increases the relative abundances of Akkermansia and Lactobacillus. Additionally, the study shows that Lf likely exerts action in its digestive product forms rather than intact Lf molecular in normal condition.

LLf can ameliorate ALI, which is associated with the regulation of hepatic alcohol metabolism and the modulation of gut microbiota. However, excessive Lf intake may result in a diminished benefit.

Reactive oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide play an important role in the maintenance of life. However, production of excessive ROS and/or deficiency of the antioxidant system lead to oxidative stress and cause a variety of diseases. In the present study, we used electron spin resonance (ESR) to detect ROS in vivo to clarify its roles in redox dynamics and organ damage. However, the limited permeability of microwaves and low anatomic resolution of ESR equipment made it difficult to apply clinically. Nitroxide is widely used as a sensitive redox sensor for in vivo ESR analysis. The unpaired electrons of nitroxide are known to cause the T1 relaxation time-shortening effect of water protons, creating magnetic resonance imaging (MRI) effects. The remarkable development of MRI has facilitated the spatiotemporal analysis of nitroxide, which was previously impossible. In a rat model, we have been able to image and analyze the process of nitroxide reduction using MRI. MRI using nitroxide as a contrast medium is considered to be clinically applicable for evaluation of organ redox, imaging of ROS (which cause organ damage), and evaluation of therapeutic effects. In this review, we describe current advances in the analysis of in vivo redox capacity in animals using ESR and MRI equipment. We consider that redox evaluation using MRI can contribute to advances in clinical medicine.

Chromolena odorata is a tropical species of flowering shrub in the family Asteraceae, leaves of it have been reported to be widely used as herbal remedy for the treatment of various ailments. It is particularly reported to be useful in the healing of wounds.

We investigated the possibility of amelioration of intestinal ischemia-reperfusion (IR) injury in rats treated with methanolic extract of C. odorata (MECO). Wistar albino rats were divided randomly into five groups of six animals each as control, IR-treated, IR+200 mg/kg MECO, IR+400 mg/kg MECO, and IR+200 mg/kg vitamin C. Pre-treatment with MECO or vitamin C was for 7 days.

The contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were significantly reduced by MECO and vitamin C, while there were significant enhancements of the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), as well as the content of reduced glutathione (GSH) in pre-treated rats compared to IR-treated rats. Glutathione S-transferase (GST) activity was not significantly affected in all the groups. Histopathological examination of small intestinal mucosa revealed significant attenuation of intestinal pathology in animals pre-treated with MECO, while IR injury produced severe villi erosion, necrosis, and inflammatory cell infiltrations.

The present study highlights the antioxidant activities of MECO and its ability to inhibit inflammatory cell infiltration as mechanisms involved in its protection against IR injury in the intestine of rats, an effect that was largely comparable to that of vitamin C.

Three urate chelations were obtained when uric acid was reacted with UO2(CH3COO)2H2O, VOSO4·XH2O and ZrOCl2·XH2O salts with neutralized with 0.1 M NaOH aqueous media. The 1:2 metal-to-ligand complexes [(UO2)2(C5H2N4O3)2](H2O), [(ZrO)2(H2O)2(C5H2N4O3)2] and [VO((C5H3N4O3)2] were characterized by elemental analyses, molar conductivity, (infrared, Raman and UV-vis) spectra, effective magnetic moment in Bohr magnetons, and thermal analysis (TG/DTG). The urate ligand coordinates as mononegative bidentate donor towards the mononuclear central vanadium atom and coordinated as binegative tetradentate mode towards the binuclear dioxouranium and zirconyl centers. The antibacterial activity of the metal complexes were tested against some kind of bacteria and fungi strains and compared with uric acid. The ligand, ZrO(II) and UO2(II) complex showed a week potential degradation on calf thymus DNA, whereas VO(II) complex slightly degraded the DNA.

Vibrational frequency analysis was performed for the complexes of alkali metal cations (Li+, Na+ and K+) with urate in the gas phase. The geometries of all possible metal cation-urate complexes were optimized at the B3LYP/6-311++G(d,p) level. The most stable complex corresponding to the each cation was used for the vibrational frequency analysis including the computation of % potential energy distribution (%PED). For comparison, the vibrational frequency analysis was also performed for the uric acid. The computed results are discussed in terms of the available experimental data. It was revealed that the characteristic stretching vibrational modes corresponding to the metal cation and the interacting nucleophilic sites of urate can be used to identify metals involved in the stone formation in the living system. Changes in different vibrational frequencies of uric acid consequent to the metal cation interactions are discussed.

The geometries and energetics of complexes of Li(+), Na(+), K(+), Be(2+), Mg(2+), and Ca(2+)metal cations with different possible uric acid anions (urate) were studied. The complexes were optimized at the B3LYP level and the 6-311++G(d,p) basis set. Complexes of urate with Mg(2+), and Ca(2+)metal cations were also optimized at the MP2/6-31+G(d) level. Single point energy calculations were performed at the MP2/6-311++G(d,p) level. The interactions of the metal cations at different nucleophilic sites of various possible urate were considered. It was revealed that metal cations would interact with urate in a bi-coordinate manner. In the gas phase, the most preferred position for the interaction of Li(+), Na(+), and K(+) cations is between the N(3) and O(2) sites, while all divalent cations Be(2+), Mg(2+), and Ca(2+) prefer binding between the N(7) and O(6) sites of the corresponding urate. The influence of aqueous solvent on the relative stability of different complexes has been examined using the Tomasi's polarized continuum model. The basis set superposition error (BSSE) corrected interaction energy was also computed for complexes. The AIM theory has been applied to analyze the properties of the bond critical points (electron densities and their Laplacians) involved in the coordination between urate and the metal cations. It was revealed that aqueous solvation would have significant effect on the relative stability of complexes obtained by the interaction of urate with Mg(2+) and Ca(2+)cations. Consequently, several complexes were found to exist in the water solution. The effect of metal cations on different NH and CO stretching vibrational modes of uric acid has also been discussed.

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.

To search a regimen for prevention of post-ischemic reperfusional (I/R) injuries, I/R in the liver was induced by 30-min clamping and subsequent unfastening of the portal vein of a rat, which underwent previous i.v. administration with ascorbic acid (Asc) of 1 mg/kg or the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated Asc (Asc2G) or 2-O-phosphorylated Asc (Asc2P) of 1 mg Asc equivalent/kg from the viewpoint of utilization of antioxidants that can promptly scavenge I/R-derived reactive oxygen species. The administration with Asc, Asc2P or Asc2G prevented some features of hepatic I/R injuries such as release of hepatic marker enzymes GOT and GPT into the blood vessel, cellular degenerative symptoms including vacuolation and cell fragmentation, and nuclear DNA strand cleavage as detected by TUNEL staining. The preventive effects on I/R injuries were in the order: Asc2G > Asc2P >> Asc. This order of preventive degrees of three anti-oxidants is partly attributable to proper efficiency of conversion to vitamin C and stability in blood stream; Asc2P was moderately converted to a free monoanion form of Asc in human serum, but, in rat serum, so efficiently converted to Asc as to undergo the resultant oxidative decomposition before reaching the liver, whereas Asc2G underwent scarce conversion to Asc in human serum but moderate conversion in rat serum, suggesting that Asc2P might be less cytoprotective against I/R injury than Asc2G in the rat liver in a way different from the human liver. In contrast Asc was so susceptible to autooxidation as to be rapidly decomposed in either rat or human serum. The concentrations of ascorbyl radicals (AscR) in serum were unchanged during I/R for sham-operated rats, but appreciably diminished time-dependently for I/R-operated rats as shown by ESR spectra. A marked increase in serum AscR occurred in rats receiving Asc, Asc2G or Asc2P, but it was time-dependently restored down to the pre-ischemic level of AscR in I/R-operated rats more rapidly than in sham-operated rats. Thus, hepatic I/R injuries were shown to be prevented more markedly by Asc2G or Asc2P than by Asc, which is attributable to efficiencies of both vitamin C conversion and subsequent AscR retention.

The relationship between hepatic ischemia-reperfusion (I-R) and subsequent injury through neutrophil accumulation is well described. Although alterations in reticuloendothelial system (RES) function (specifically Kupffer cell function) after I-R have been delineated, the degree to which discrete components of RES function (phagocytosis and killing) are independently modulated under these conditions has not been quantified. A hepatic segmental I-R model was established in mice, in which blood supply to the left lateral lobe of the liver was occluded for 45 minutes, the liver was reperfused, and the laparotomy incision was closed. Experimental animals were pretreated with either vinblastin (1.5 mg/kg) to induce neutropenia or anti-P-selectin monoclonal antibody (mAb; 50 microg/mice) 4 days and 5 minutes before ischemia, respectively. We previously reported that after intravenous injection of chromium 51 ((51)Cr) and iodine 125 ((125)I) double-labeled Escherichia coli, hepatic (51)Cr levels could be used to reliably quantify hepatic phagocytic clearance (HPC) of bacteria from blood, whereas the subsequent release of (125)I from the liver accurately paralleled hepatic bacterial killing efficiency (HKE). Using this double-label bacteria clearance assay, HPC and HKE were depressed after I-R, in association with hepatic neutrophil accumulation. Segmental I-R resulted in decreased HPC and HKE activity in both ischemic and nonischemic hepatic lobes. Depressions in HPC and HKE were attenuated by either vinblastin-induced neutropenia or blocking neutrophil adhesion to the hepatic endothelium with anti-P-selectin mAb. These findings support the hypothesis that I-R induces hepatic RES dysfunction, at least in part, through P-selectin-mediated neutrophil accumulation.

Myocardial and endothelial damage is still a widely debated problem during the ischemia-reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed.

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Intermittent hepatic pedicle clamping is thought to cause less hepatic reperfusion injury compared with continuous clamping. The mechanisms underlying this difference are unknown. We examined the relationship between intermittent ischemia/reperfusion and the production of free radicals using electron spin resonance spectrometry.

Alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone was administered to rats as a spin trap agent. Continuous clamping (15, 30, or 60 minutes) or intermittent clamping (four cycles of 15-minute ischemia and 5 or 15 minutes of reperfusion) of hepatic pedicle was carried out. After reperfusion, free radical production in the liver was measured by an electron spin resonance spectrometer, and the level of hepatic injury was evaluated by measuring liver enzyme.

Longer periods of ischemia increased free radical production after reperfusion. There was no significant increase in free radical production or liver enzymes when the duration of ischemia was 15 minutes. Free radical production and liver damage were significantly less severe in intermittent pedicle clamping than in continuous clamping for 60 minutes, especially when the duration of the reperfusion between four cycles of ischemia was 15 minutes.

These results indicate that intermittent pedicle clamping lessens free radical production when compared with continuous clamping, although many free radicals are produced.

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Sodium salicylate can be used as a chemical trap for hydroxyl radicals, the most damaging reactive oxygen species. Because reactive oxygen species are involved in the pathogenesis of hepatic hypoxia/reoxygenation injury, the goal of this study was to determine if trapping hydroxyl radicals with salicylate would prevent or at least ameliorate such injury. Isolated rat livers, continuously perfused with Krebs-Henseleit bicarbonate buffer in the presence or absence of salicylate (2 mM), were exposed, after 30 min of recovery, to 60 min of hypoxia, followed by 30 min of reoxygenation. During reoxygenation, control livers experienced a sharp increase in the rate of lactic dehydrogenase release, taken as index of cell injury, protein carbonyl content, and malondialdehyde, taken as index of protein oxidation and lipid peroxidation, respectively. The presence of salicylate in the solution perfusion significantly reduced the rate of lactic dehydrogenase release, protein carbonyl content, and malondialdehyde production during reoxygenation. Hepatic histology documented a significantly reduced cell injury in salicylate-perfused livers compared to control livers. These data suggest that the hydroxyl radical chemical trap sodium salicylate, acting as an antioxidant, may represents an effective agent to reduce liver injury due to hypoxia/reoxygenation in a model of isolated-perfused rat liver.

Chronic production of reactive oxygen species (ROS) and/or deficiency in the antioxidant defense system are observed in non-insulin-dependent diabetes mellitus (NIDDM) patients. As an adjunct to the usual indirect parameters for evaluating oxidative stress, we assessed the feasibility of oxyradicals detection in venous blood by electron spin resonance spectroscopy (ESR). Detection of the ascorbate pool was also performed using the validated ESR analysis of the ascorbyl free radial (AFR)-dimethyl sulfoxide (DMSO) complex.

Plasma lipoperoxidation was characterized by higher levels of total MDA (1.50 +/- 0.08 nmol/L), lower levels of GSH (0.54 +/- 0.02 mmol/L) and of vitamin A (2.13 +/- 0.52 mumol/L) in the NIDDM group than in the controls (0.75 +/- 0.05 nmol/L, 0.90 +/- 0.05 mmol/L, 3.52 +/- 1.04 mumol/L, respectively). Improvement of the ESR measurement of oxyradical adducts has been previously obtained by addition of a new sensitive nitrone (DEPMPO), which acts as a spin-trap. However, in our experiment the ESR signal-to-noise ratio was too low to detect significative oxyradicals adducts in total venous blood of NIDDM patients having a weak production of ROS. A significant difference (p < 0.002) was observed in DMSO/AFR index between controls (24.00 +/- 4.10 nmol/L) and NIDDM patients (7.28 +/- 2.36 nmol/L) suggesting ascorbate depletion related to the free radical production.

The DMSO/AFR index could be an interesting additional marker of oxidative stress during a chronic production of ROS.

Metallothioneins (MTs) are low molecular weight, zinc-binding proteins that by activating zinc metalloenzymes participate in the regulation of growth and development. The present study was designed to examine the roles of MTs in cell proliferation using an in vivo model of liver regeneration following partial hepatectomy (PH) in rats. The levels of MT-I and MT-II were studied with respect to regulation of proliferative potential, cell cycle checkpoint activity, and oxidative stress in the rat PH model. We synthesized a 17-mer antisense phosphorothioate oligodeoxynucleotide (S-ODN), named aMT, complimentary to the start site of the MT-I mRNA sequence and an appropriate control. Both S-ODNs were administered intraperitoneally at the dose of 5 mg/kg following 70% PH. MT became induced 57.4 +/- 9.8-fold following PH and the said effect became attenuated dramatically following administration of aMT. In addition, PH rats treated with aMT exhibited decreased rate of liver regeneration as measured by expression of proliferating cell nuclear antigen and elevated cell cycle checkpoint activity as determined by expression of p53. The results of these studies suggest that MT isoforms with their high thiol contents do play an important role in cellular functions and especially during stressful states induced by a broad range of mediators generating free radicals.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.

It is well known that ischemia causes functional and structural damage to liver cells, and that the status of energy metabolism provides an important means of assessing the functional viability of the ischemic organ. However, the specific sequence leading to ischemic liver cell injury is not yet fully understood; therefore, it is clinically and pathophysiologically important to elucidate the mechanism of cellular injury during hepatic ischemia and subsequent reperfusion. Whereas the conventional view attributes this injury process to the ischemia itself, recent studies have demonstrated that a variable but often substantial proportion of this injury is caused by reactive oxygen metabolites that are generated at the time of reperfusion. This article presents an outline of the mechanism of cellular injury caused during hepatic ischemia and subsequent reperfusion resulting from certain types of surgery, with special reference to the xanthine-xanthine oxidase system and the activation of neutrophils and macrophages.

The significance of poor medullary reperfusion in the etiology of acute tubular necrosis during renal transplantation is poorly understood. Our objective was to determine the kinetics of renal hemoglobin oxygenation using near-infrared spectroscopy during renal transplantation, to provide a framework against which the timing of mitochondrial dysfunction could be considered. New Zealand White rabbit kidneys were flushed with hypertonic citrate solution (0-2 degrees C and autografted immediately (group 1) or stored at 0-2 degrees C for 72 hours before autografting (group 2). Changes in oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) were monitored by near-infrared spectroscopy for 3 hours of reperfusion. Intrarenal perfusion was evaluated separately by barium sulfate angiography. Reperfusion resulted in rapid increases in HbO2 within 1 minute in both groups. Group 1 HbO2 fell sharply to a minimum at 3 minutes but recovered by 20 minutes; group 2 changes were similar, but there was no recovery (P<0.05 by 10 minutes). Hb increased rapidly in both groups upon reperfusion but in group 2 was significantly greater after 10 minutes (P<0.05). Total hemoglobin levels were similar in both groups. Renal hemoglobin saturation was 69% at 1 minute in both groups; there was no significant change in group 1 but a profound desaturation in group 2 to 25% at 10 minute (P<0.005) and no recovery thereafter. Barium sulfate distribution was normal in all group 1 kidneys; cortical distribution was normal in all group 2 kidneys, but medullary perfusion was poor for the first 60 minutes. Renal hemoglobin oxygenation kinetics as determined here do not correlate with the timing of mitochondrial dysfunction previously reported (Thorniley et al., Kidney International, 1994; 45: 1489). We conclude that secondary ischemia during reflow is not the only mechanism leading to acute tubular necrosis.

The mitochondrial dysfunction in ischaemia-reperfusion is shortly reviewed. During ischaemia the ATP level and pH drops, phospholipids are degraded, membrane permeabilities increased and the cytosolic levels of Na+ and Ca2+ raised. During the following reperfusion the Ca2+ levels may further increase while pH is raised. The oxidative phosphorylation is resumed and the ATP used for membrane repair and ion pumping. The mitochondrial Ca2+ handling is important in removing Ca2+ from the cytosol since the mitochondria are able to take up substantial amounts of Ca2+. However, if a certain threshold is exceeded, mitochondria undergo a so-called permeability transition (MPT), release their Ca2+, undergo swelling and become uncoupled. MPT has been shown to be due to the opening of large pore allowing passage of substances with a M(R) < 1500. Data are presented showing by electron microscopy swelling of mitochondria in cells in perfused liver before other gross morphological changes have taken place. There are a number of factors lowering the threshold for Ca2+ in inducing the MPT: inorganic phosphate, pro-oxidants that oxidize membrane SH-groups, oxidation of NAD(P)H and GSH, while a protective effect is exerted by Mg2+, ADP (and ATP), some antioxidants, carnitine, decrease in pH, and cyclosporin A that binds to cyclophilin. The potential benefit of these in minimizing reperfusion-induced tissue damage is discussed.

The reduction of nitroxide free radicals was investigated in detail by Electron Paramagnetic Resonance (EPR) spectroscopy in perfused liver. The nitroxide free radical was rapidly reduced to the corresponding hydroxylamine more efficiently at the lower flow rate of 8 [ml/min], while at higher flow rates, the amount of reduced nitroxide showed a significant decrease. Oxidation of hydroxylamine using hydrogen peroxide provided dynamic information concerning the reduction of the free radical within the liver. In addition, liver homogenates were also investigated to determine the level of nitroxide uptake. The results suggested that a portion of the infused nitroxide was taken up by the liver and cleared from the circulation.