Influence of preservation solutions on lipid peroxidation and mitochondrial respiration in rat kidneys

The role of hypothermic storage of rat kidneys on lipid peroxidation compared to its effect on mitochondrial respiratory parameters has been investigated. Rat kidneys were flushed with cold solutions of isotonic sodium chloride; Euro-Collins'; preservation solution containing histidine buffer, tryptophane, and alpha-ketoglutarate (HTK); or hypertonic citrate and then stored for 20 hr at 4 degrees C. After storage, the endogenous contents of malondialdehyde as well as the chemically (by Fe2+/ascorbic acid) and enzymatically (by Fe3+/ADP/NADPH) induced generation of malondialdehyde were measured in cortical homogenates and partly in mitochondria and microsomes by the thiobarbituric-acid reaction. Compared to the values measured in fresh, unstored kidneys, the levels of malondialdehyde were significantly higher in kidneys preserved in solutions of isotonic sodium chloride or HTK. This stimulating effect of the HTK solution on the generation of lipid peroxidation products could also be established when homogenate was incubated with this solution. Euro-Collins' and hypertonic citrate solution did not change the endogenous contents of malondialdehyde in kidneys during hypothermic storage. Both the chemically and enzymatically induced lipid peroxidation increased after hypothermic storage of kidneys in all solutions investigated. No direct relationship between the contents of malondialdehyde and respiratory mitochondrial parameters was detectable. The results demonstrate that the extent of lipid peroxidation does not correlate with preservation effectiveness.     

Expression of GMP-140 (P-selectin) correlates with graft viability in cold-preserved rat livers

BACKGROUND: Ischemia/reperfusion injury is an inflammatory process involving cytokine release, Kupffer cell activation, and sinusoidal endothelial cell activation. GMP-140 is synthesized by endothelial cells. METHODS: We analyzed by Western blotting the expression of GMP-140 in a syngeneic rat liver transplantation model using grafts preserved for different periods of time. RESULTS: Compared with prereperfusion samples, expression did not change significantly in freshly harvested and 4-hr preserved livers. In grafts preserved for 24 hr (100% survival), GMP-140 levels increased dramatically at 1 hr, then returned to baseline at 24 hr after transplantation. Forty-eight hour preserved grafts (0% survival) showed a decreasing expression. To identify possible mediators, the effects of tumor necrosis factor-alpha and interleukin-1beta on GMP-140 expression in primary sinusoidal endothelial cells were analyzed. These cytokines increased both the percentage of stained cells as well as their mean staining fluorescence. CONCLUSIONS: The absence of increase in 48-hr grafts suggests that GMP-140 may distinguish viable from nonviable livers.     

Effect of vitamin E on human sperm motility and lipid peroxidation in vitro

Aim: To assess the protective efficacy of vitamin E to counteract the reactive oxygen species (ROS) mediated damage onsperm motility, viability and lipid peroxidation. Melhods: Human semen samplns were obtained from the local hospi-tal. The split seminal fractions freed of seminal plasma v, ere reeonstimted in Ringer-Tymde and subjected to varied vita-min E concentrations (0.1-2 mmol/L), Results: Dose-dependent improvement in both motility and viability accom-panied by concomitant decrease in malondialdehyde (MDA an end product of lipid peroxidation) following vitamin Esuppllementation was noticed. Conclusion: Vitamin E protects against the ROS mediated damage on spermatozoa.Vimmth E supplementation could be of clinical importance for prolonged spermatozoal storage whenever needed.     

The role of alpha-tocopherol and allopurinol in lipid peroxidation and mitochondrial respiration in the ischemic rat liver

In the present study, the effects of alpha-tocopherol and allopurinol in liver ischemia and reperfusion injury on lipid peroxidation and mitochondrial respiratory function were investigated in rats. Ischemia was induced in the left and median liver lobes clamping the vessels for 90 minutes. After declamping reperfusion was continued for 60 minutes. Liver tissue was taken before and 90 minutes after ischemia and 60 minutes after reperfusion to measure lipid peroxides and mitochondrial respiratory function. In one group of rats alpha-tocopherol (10mg/kg) was given intraperitoneally for three consecutive days preoperatively and in the other group allopurinol (50mg/kg) was given intravenously 10 minutes before ischemia. alpha-Tocopherol caused inhibition of increase in lipid peroxides at reperfusion and improvement in lowering of mitochondrial respiratory function. This improvement was less than previously reported, probably due to not only reperfusion injury but also ischemic injury. Allopurinol, on the other hand, caused neither such inhibition nor such improvement, suggesting the other source of oxygen-derived free radicals than xanthine oxidase system.     

Effect of pentoxifylline on veno-occlusive priapism-induced corporeal tissule lipid peroxidation in a rat model

Objective: To investigate whether pentoxifylline could play a role in attenuation of the hazardous effects of ischemia/reperfusion on corporeal tissue in a rat model of veno-occlusive priapism (VOP). Materials and methods: Placebo and pentoxifylline were given to eight groups of rats prior to priapism being induced by a vacuum constrictive device for durations of 6 and 12 h, respectively. Half of the groups of rats that underwent the same duration of priapism (ischemic) were subjected to 1 h of detumescence after band removal (reperfusion). One group underwent no manipulation and no drug administration and served as a baseline determination (control). Corporeal homogenates were examined for lipid peroxidation (LP) derived malondialdehyde (MDA) accumulation via thiobarbituric acid assay. Results: MDA concentration differed significantly between VOP rats and controls (P < 0.001) but did not differ significantly between ischemic-only groups and reperfused groups (P > 0.05). In the pentoxifyllinepretreated groups, although MDA accumulation tended to be slightly lower than in the placebo groups, the difference was not statistically significant (P > 0.05) either in the 6- or 12-h duration priapic groups. Conclusions: LP, an indicator of radical oxygen metabolite (ROM) induced injury, occurs in rat corporeal tissue during and after abolishment of VOP. Single-dose pentoxifylline pretreatment failed to exert a protective effect on corporeal tissue in a rat model of VOP in terms of attenuation of LP.     

异丙酚对心肌缺血再灌注损伤大鼠离体心脏脂质代谢的影响

目的观察异丙酚对心肌缺血再灌注损伤大鼠离体心脏脂质代谢的影响。方法雄性Wistar大鼠68只，体重350～450g，随机分为2组（n=34）：正常对照组（C组）和心肌缺血再灌注组（I／R组）。I／R组阻断左冠状动脉前降支，缺血30min，再灌注2h制备心肌缺血再灌注模型，取心脏在Langendorff灌注模型上用Krebs-Hemseleit（KBH）缓冲液灌注，将用放射性物质标记的（TAG）0．4mol/L加入循环回路中。分别用含不同浓度异丙酚[0（生理盐水）、0．1、1．0、5．0、10．0、500μmol／L]的灌注液进行灌注，监测平均动脉压、心率、主动脉流量（AFR）、冠状动脉血流（CFR），计算Hvdraulic work反映心肌作功情况。测定灌注液中TAG浓度，计算脂肪酸（FFA）氧化率，并测定心肌脂蛋白酶（LPL）活性。结果心肌缺血再灌可导致Hydraulic work降低，FFA氧化率和心肌LPL活性升高；与C组异丙酚浓度为0时比较，C组异丙酚浓度为10．0、50．0μmol／L时Hydraulic work降低，异丙酚浓度为5．0～50．0μmol／L时灌注液中TAG浓度升高，I／R组异丙酚浓度为10．0、50．0μmol／L时Hydraulic work降低，异丙酚浓度为5．0—50．0μmol／L时灌注液中TAG浓度升高，异丙酚浓度为10．0～50．0μmol／L时FFA氧化率和心肌LPL活性降低（P〈0．05）；与I／R组异丙酚浓度为0时比较，I／R组异丙酚浓度为0．1～10．0μmol／L时Hydraulic work升高，异丙酚浓度为5．0～50．0μmol／L时灌注液中TAG浓度升高，FFA氧化率及心肌LPL活性降低（P〈0．05）。结论异丙酚可减轻心肌缺血再灌注损伤大鼠心脏功能的降低，其机制与改善心肌脂质代谢有关。     

Investigation of lipid peroxidation in regenerating rat liver]

The purpose of this study was to estimate the effects of lipid peroxidation in regenerating rat liver. Partial 70% hepatectomy was performed in rat according to Higgings and Anderson. EPC (alpha Toc: Ascorbic acid = 6:4, radical scavenger) was injected intravenously (5mg/kg weight) one hour before operation. Lipid peroxidation in regenerating liver reached a peak at 24 hours after operation. The administration of EPC markedly suppressed the increase of lipid peroxide in the plasma and remnant liver and that of GPT after hepatectomy, with subsequent good liver regeneration ratio. Moreover, the pretreatment with EPC remarkably elevated the activity of thymidine kinase (index of DAN synthesis). The EPC administration had not notable effects on the level of plasma ketone body ratio in animal but pathologically caused early advent of glycogen granule in the remnant liver tissue after partial hepatectomy, which reflected restoration of mitochondrial energy level. The results of the present study suggest that scavenger may be useful not only for impairment of liver dysfunction but also for recovery of mitochondrial energy level and DNA synthesis after liver resection.     

含吡那地尔的心脏保存液对大鼠离体心脏线粒体呼吸功能的影响

目的 探讨含吡那地尔的心脏保存液(HTK液)对大鼠离体心脏线粒体呼吸功能的影响.方法 健康清洁级SD大鼠120只,建立离体心脏Langendorff灌注模型,随机分为5组,HTK组(Ⅰ组)、吡那地尔+HTK液组(Ⅱ组)、吡那地尔+HTK液+5-羟葵酸(5-HD)组(Ⅲ组)、吡那地尔+HTK液+HMR-1098组(Ⅳ组)、吡那地尔+HTK液+HMR-1098+5-HD组(Ⅴ组).K-H液平衡灌注10 min后灌注心脏停搏液:Ⅰ组灌注HTK液;Ⅱ组灌注含吡那地尔0.5 mmol/L的HTK液;Ⅲ组灌注含吡那地尔0.5 mmol/L和5-HD 100μmol/L的HTK液;Ⅳ组灌注含吡那地尔0.5 mmol/L和HMR-1098 100μmol/L的HTK液;Ⅴ组灌注含吡那地尔0.5 mmol/L、5-HD 100μmol/L和HMR-1098 100 μmol/L的HTK液.停搏后取心脏保存于4℃各组相应液体中8 h,然后用37 ℃含氧K-H液再灌注60 min.于平衡灌注10 min(T1)、保存8 h(T2)、复灌60 min(T3)时测定线粒体呼吸功能[3态呼吸(S3)和4态呼吸(S4)的耗氧速率、呼吸控制率(RCR)及磷氧比(P/O)].于T1和T3时收集冠脉流出液测定心肌肌钙蛋白I(cTnI)浓度、肌酸激酶同功酶(CK-MB)及乳酸脱氢酶(LDH)的活性.电镜下观察心肌细胞超微结构.结果 与Ⅰ组比较,Ⅱ组～Ⅳ组RCR、P/O、S3耗氧速率升高,cTnI、CK-MB和LDH的水平降低(P＜0.05).与Ⅱ组比较,Ⅲ组～Ⅴ组RCR、P/O、S3耗氧速率降低,cTnI、CK-MB和LDH的水平升高(P＜0.05).与Ⅲ组比较,Ⅳ组RCR、P/O、S3耗氧速率升高,cTnI、CK-MB和LDH的水平降低,Ⅴ组RCR、P/O、S3耗氧速率降低,cTnI、CK-MB和LDH的水平升高(P＜0.05).与Ⅳ组比较,V组RCR、P/O、S3耗氧速率降低,cTnI、CK-MB和LDH水平升高(P＜0.05).各时点S4耗氧速率组间比较差异无统计学意义(P＞0.05).Ⅱ组心肌细胞损伤较轻,Ⅲ组和Ⅳ组损伤程度相近但重于Ⅱ组,Ⅰ组和Ⅴ组损伤最重.结论 含吡那地尔的HTK液可改善心脏线粒体呼吸功能,减轻心肌损伤,提高心脏保存效果,线粒体ATP敏感性钾通道和细胞膜ATP敏感性钾通道均参与了吡那地尔的心肌保护效应,且线粒体ATP敏感性钾通道发挥主导作用.     

Transient hyperthermia during oxygenated rewarming of isolated rat livers

Pretransplant machine perfusion of donor grafts has gained clinical appreciation to improve graft function and survival after transplantation. This study was aimed as pilot investigation to evaluate the additive potential of a transient ex vivo heat shock treatment of the isolated organ during machine perfusion to further protect the graft from subsequent reperfusion injury. Rat livers were retrieved after 20 min of cardiac arrest and preserved for 18 h by cold storage in HTK solution. Prior to reperfusion, livers were subjected to 2 h of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35 °C. In half of the livers (n = 7), a brief hyperthermic impulse (10 min perfusion at 42 °C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Induction of heat shock protein 70 was followed on the mRNA and protein level. Chaperone induction by transient hyperthermia was associated with a significant improvement of bile production upon reperfusion and significantly reduced enzyme loss of mitochondrial GLDH. Heat shock treatment further affected pro-inflammatory upregulation in the graft in significantly reducing gene expression as wells as protein release of TNF-alpha. It is concluded, that graft conditioning by controlled hyperthermia ex vivo may represent a feasible and useful tool to improve liver recovery after preservation.     

Interactions between mitochondrial proteins and lipid peroxidation products in the maintenance of the glomerular filtration barrier in the in vitro perfused kidney

BACKGROUND: The fourth complex of the mitochondrial respiratory chain, cytochrome-c oxidase (CytC) consists of thirteen both mitochondrially and nuclearly encoded subunits, which are differently regulated in proteinuric kidneys. The effect of mitochondrial involvement on proteinuria is not known. METHODS: We set up an in vitro kidney perfusion model to study the direct effect of inhibitors of the mitochondrial respiratory chain, rotenone and antimycin A, on the glomerular filtration barrier by using immunohistochemistry and Northern blotting and quantitating the resulting proteinuria. RESULTS: Rapid onset of proteinuria and characteristic changes in CytC subunits were seen in the perfused kidneys. Urinary protein excretion increased significantly in the rotenone- and antimycin-A-treated groups during perfusion. Downregulation of CytC subunits I and IV was similarly found in the groups treated with rotenone and antimycin A, while increases in the lipid peroxidation (LPO) products malondialdehyde and 4-hydroxynonenal which reflect mitochondrial damage, were observed. CONCLUSIONS: These data show rapid changes in mitochondrial proteins and induction of proteinuria in response to exposure to mitochondrial inhibitors. Together with the concomitant increase in local LPO products, these results suggest that continuous maintenance of a proper energy balance is important to maintain the glomerular filtration barrier.     

Short-term treatment with mycophenolic acid increases bile flow in continuously perfused and cold-preserved rat livers and does not affect hepatic ischemia-reperfusion injury

Mycophenolate mofetil (MMF) is a new immunosuppressive agent which has been used successfully after kidney and heart transplantation. Experience with MMF after liver transplantation is still limited. In particular, there is no information about influence on ischemia-reperfusion injury (IRI). Therefore, the aim of this investigation was to assess the effects of mycophenolic acid (MPA), the pharmacologically active metabolite of MMF, in the cold-preserved or normal rat liver. Livers of male Sprague-Dawley rats were subjected to cold ischemia in University of Wisconsin (UW) solution (24 h, 4 degrees C) and reperfused for 2 h in the absence or presence of MPA (100 microg/ml, n=5-6 each). Another group received MPA pretreatment for 20 min prior to ischemia ( n=7). In further experiments, livers were perfused with a bile salt-free Krebs-Henseleit buffer in a continuous fashion (controls, n=5). MPA was infused from 20-40 min after starting perfusion in therapeutic concentrations (5 microg/ml, 10 microg/ml, 40 microg/ml, and 100 microg/ml; n=3-6 each). There was no significant influence of MPA on portal pressure nor on postischemic efflux rates of LDH. MPA pretreatment resulted in a significant improvement of bile flow during reperfusion (0.32+/-0.05 microl/min x g liver) compared with controls (0.17+/-0.04 microl/min x g liver, mean+/-SEM). In contrast, postischemic bile flow was not influenced by continuous administration of MPA during the reperfusion period only (0.18+/-0.07 microl/min x g liver). In continuously perfused livers, MPA increased bile salt-independent bile flow (1.00+/-0.06 microl/min x g liver) in a dose-dependent manner, reaching half-maximal effects around 5 microg/ml (1.66+/-0.15 microl/min x g liver) and maximal effects at 40 microg/ml (2.61+/-0.28 microl/min x g liver). In conclusion, neither preischemic nor postischemic administration of MPA influences IRI to hepatocytes significantly after hypothermic liver preservation in UW solution. In contrast to other immunosuppressive agents, MPA exhibits strong choleretic effects, which are related to a stimulation of bile salt-independent bile formation.     

Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo

Nitric oxide, a highly reactive radical, was recently identified as an intermediate of L-arginine metabolism in mammalian cells. We have shown that nitric oxide synthesis is induced in vitro in cultured hepatocytes by supernatants from activated Kupffer cells or in vivo by injecting rats with nonviable Corynebacterium parvum. In both cases, nitric oxide biosynthesis in hepatocytes was associated with suppression of total protein synthesis. This study attempts to determine the effect of nitric oxide biosynthesis on the activity of specific hepatocytic mitochondrial enzymes and to determine whether inhibition of protein synthesis is caused by suppression of energy metabolism. Exposure of hepatocytes to supernatants from activated Kupffer cells led to a 30% decrease of aconitase (Krebs cycle) and complex I (mitochondrial electron transport chain) activity. Using NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, we demonstrated that the inhibition of mitochondrial aconitase activity was due, in part, to the action of nitric oxide. In contrast, in vivo nitric oxide synthesis of hepatocytes from Corynebacterium parvum-treated animals had no effect on mitochondrial respiration. This suggests that inhibition of protein synthesis by nitric oxide is not likely to be mediated by inhibition of energy metabolism.     

Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa

We demonstrated previously that pentoxifylline in millimolar concentrations can inhibit superoxide anion production by human spermatozoa. In the present study we have examined the effects of the same concentrations of pentoxifjrlline on experimentally induced lipid peroxidation, as measured by malondialdehyde formation in the thiobarbituric (TBA) assay. Under the experimental conditions used, preincubation of spermatozoa with pentoxifjdline led to a significant dose-dependent stimulation (p<0.005) of malondialdehyde production amounting to 10.77 ± 2.35%, 13.45 ±2.99% and 17.4 ± 1.99% (mean ± SEM) for 1.9, 3.7 and 11.2 mmol/l pentoxifylline, respectively. In the presence of 11.2 mmol/l pentoxifylline, an increase in iron-catalysed lipid peroxidation potential was detected in samples of spermatozoa from 29 infertile men, regardless of their initial levels of malondialdehyde. The results of this study indicate that pentoxifylline might further augment the ferrous ion-stimulated decomposition of pre-accumulation lipid hydroperoxides in the sperm plasma membrane and thus promote malondialdehyde generation in the TBA assay.
It is concluded that the stimulatory effect of pentoxifjdline on iron-induced lipid peroxidation may have an adverse effect on the quality of sperm suspensions prepared for in vitro fertilization, a possibility which should be investigated further.     

Effects of melatonin on lipid peroxidation and antioxidant enzymes in streptozotocin-induced diabetic rat testis

Aim:To examine the effects of melatonin treatment on lipid peroxidation(LPO)and the activities of antioxidantenzymes in the testicular tissue of streptozotocin(STZ)-induced diabetic rats.Methods:Twenty-six male rats wererandomly divided into three groups as follows:group Ⅰ,control,non-diabetic rats(n=9);group Ⅱ,STZ-induced,untreated diabetic rats(n = 8);group Ⅲ,STZ-induced,melatonin-treated(dose of 10 mg/kg-day)diabetic rats(n=9).Following 8-week melatonin treatment,all rats were anaesthetized and then were killed to remove testes from thescrotum.Results:As compared to group Ⅰ,in rat testicular tissues of group Ⅱ,increased levels of malondialdehyde(MDA)(P<0.01)and superoxide dismutase(SOD)(P<0.01)as well as decreased levels of catalase(CAT)(P<0.01)and glutathione peroxidase(GSH-Px)(P>0.05)were found.In centrast,as compared to group Ⅱ,in rat testiculartissues of group Ⅲ,levels of MDA decreased(but this decrease was not significant,P>0.05)and SOD(P<0.01)aswell as CAT(P<0.05)increased.GSH-Px was not influenced by any of the treatment.Melatonin did not signifi-cantly affect the elevated glucose concentration of diabetic group.At the end of the study,there was no significantdifference between the melatonin-treated group and the untreated group by means of body and testicular weight.Conclusion:Diabetes mellitus increases oxidative stress and melatonin inhibits lipid peroxidation and might regulatethe activities of antioxidant enzymes of diabetic rat testes.(Asian J Androl 2006 Sep;8:595-600)     

酒精对大鼠前列腺抗氧化酶活性和脂质过氧化的影响

目的 探讨酒精对大鼠前列腺组织抗氧化酶活性和脂质过氧化水平的影响。方法 40只成年健康SD雄性大鼠随机均分为对照组、低剂量组、中剂量组和高剂量组，分别用50．0％、25．0％、12．5％的乙醇溶液和蒸馏水按10．0ml／kg每晚灌胃1次持续60d后，观察酒精毒染模型大鼠前列腺组织的形态结构的变化；用化学比色法测定前列腺组织中超氧化物岐化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）和前列腺酸性磷酸酶（PAP）的水平。结果 随着酒精剂量的增加，SOD、CAT、GSH-Px活力和PAP含量呈先升高后下降的趋势，MDA含量和前列腺相对重量分别呈现上升和下降趋势，前列腺组织馆牛棼缩。结论 长期过量饮洒可导致大鼠前列腺绸织结构和功能异常，氧化应激在其中发挥了重要作用。     

Effect of cyclosporine A on accumulation of tetraethylammonium and p-aminohippurate, and on lipid peroxidation in rat renal microsomes and cortical slices

The effect of cyclosporine A (CsA) on lipid peroxidation (LPO) was assessed in renal cortical slices and renal microsomes. Cortical slices were incubated with 1500 micrograms/ml CsA and microsomes with 0.5-20 micrograms/ml under identical conditions (pH 7.4, 37 degrees C) for 3 hours, and LPO monitored by the formation of malondialdehyde (MDA). CsA at concentrations of 3 micrograms/ml and higher caused a significant increase MDA in microsomes and renal cortical slices showed a time dependent release of MDA into the incubation medium. The influence of CsA on tetraethammonium (TEA) and p-aminohippurate (PAH) accumulation in renal cortical slices was investigated for up to 3 hours with concentration of CsA from 10 to 1000 micrograms/ml. CsA caused a time- and concentration-dependent decrease of TEA accumulation and higher concentrations of CsA decreased PAH accumulation in renal cortical slices. The results add further evidence to the suggestion that lipid peroxidation participate in CsA-induced impairment of kidney function.     

体外添加黄芪注射液对人精子膜拮抗脂质过氧化的作用

目的 :观察体外添加黄芪注射液对弱精子症患者精子膜脂质过氧化的拮抗作用。方法 :应用计算机辅助精子分析仪 (CASA)和丙二醛 (MDA)及超氧化物歧化酶 (SOD)检测试剂盒 ,分别分析体外添加黄芪注射液 (A组 )对 30例弱精子症患者精子运动参数的影响 ,检测精子悬液MDA含量及总SOD(TSOD)力 ,并进行相关分析 ,同时设立空白对照组进行对比观察。结果 :A组的精子活率 (Mot)、前向运动精子百分率、曲线速度 (VCL)、平均路径速度 (VAP)和精子头摆幅度 (ALH)较空白对照组显著提高 (P <0 .0 5或P <0 .0 1) ;A组精子悬液的MDA含量显著降低 (P <0 .0 5 ) ,TSOD活力与对照组比较差异无统计学意义 (P >0 .0 5 )。A组MDA含量与Mot、前向运动精子百分率、VAP和ALH间均有显著性负相关 (P <0 .0 5或P <0 .0 1)。结论 :体外添加黄芪注射液对弱精子症患者精子具有拮抗脂质过氧化作用 ,为临床应用黄芪减少递质对精子的损害提供理论基础。