The ischemic preconditioning paradox in deceased donor liver transplantation-evidence from a prospective randomized single blind clinical trial.

While animal studies show that ischemic preconditioning (IPC) is beneficial in liver transplantation (LT), evidence from few smaller clinical trials is conflicting. From October 2003 to July 2006, 101 deceased donors (DD) were randomized to 10 min IPC (n = 50) or No IPC (n = 51). Primary objective was efficacy of IPC to decrease reperfusion (RP) injury. Both groups had similar donor risk index (DRI) (1.54 vs. 1.57). Aminotransferases on days 1 and 2 were significantly greater (p < 0.05) in IPC recipients. In multivariate analyses, IPC had an independent effect only on day 2 aspartate transferase. Prothrombin time, bilirubin and histological injury were similar in both groups. IPC had no significant effect on plasma TNF-alpha, IL-6 and IL-10 in the donor and TNF-alpha and IL-6 in the recipient. In contrast, IPC recipients had a significant rise in systemic IL-10 levels after RP (p < 0.05) and had fewer moderate/severe rejections within 30 days (p = 0.09). Hospital stay was similar in both groups. One-year patient and graft survival in IPC versus No IPC were 88% versus 78% (p = 0.1) and 86 versus 76% (p = 0.25), respectively. IPC increases RP injury after DDLT, an 'IPC paradox'. Other potential benefits of IPC are limited. IPC may be more effective in combination with other preconditioning regimens.

Oxidative stress in fatty livers of obese Zucker rats: rapid amelioration and improved tolerance to warm ischemia with tocopherol.

Fatty livers in humans and rats are less tolerant of ischemia, endotoxin, and alcohol. We hypothesized that fatty livers of obese (Ob) Zucker rats are oxidatively stressed and oxidative stress could be relieved by antioxidant treatment, leading to improved tolerance to ischemia. Total glutathione (GSH), tocopherol (TOC), ascorbic acid (AA), catalase (CAT), superoxide dismutase (SOD), and selenium-glutathione peroxidase (Se-GPx) were measured in the livers of Ob and lean (Ln) Zucker rats before and after treatment with high-dose TOC and ascorbate. Also, survival in treated Ob rats following a lethal 90 minutes of partial in vivo warm ischemia was examined. Fatty livers of Ob rats contained significantly less GSH, TOC, and CAT, in comparison with livers of Ln rats. Immunoblotting showed significantly decreased CAT protein without changes in mRNA in fatty livers. There were no significant differences in AA, SOD, and Se-GPx between the 2 groups. Pretreatment with TOC and ascorbate over 48 hours completely corrected the decreases in GSH, TOC, and CAT. Most importantly, TOC with or without ascorbate pretreatment significantly improved survival in Ob rats following ischemia in a dose-dependent manner. In conclusion, TOC administration corrected the oxidative stress in fatty livers of Ob Zucker rats and improved survival following lethal ischemia. Additional studies are needed to determine the efficacy of TOC-a relatively inexpensive agent-in treating patients with fatty livers in a variety of clinical conditions, possibly including liver transplantation.

The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF.

PURPOSE: Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics. METHODS: Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA). RESULTS: 12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors. CONCLUSIONS: N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.

Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions.

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Correlation between red blood cell deformability and changes in hemodynamic function.

BACKGROUND: In sepsis red blood cells (RBCs) have been shown to be less deformable (i.e., more rigid) and have been implicated in decreasing nutrient blood supply and possibly leading to organ dysfunction. However, no studies have demonstrated an association between organ dysfunction and rigid RBCs. This study examined cardiovascular physiologic and histologic changes in two different models to determine whether a relationship may exist between RBC deformability and organ function. METHODS: In the following two experiments, cardiac index (CI) was continuously measured, whereas both deformability index and histology were examined at the end of the experimental periods. The first experiment studied nonanesthetized, hydrated rats after a cecal ligation and puncture (CLP), a slow-developing means of inducing RBC rigidity. In a second experiment animals were anesthetized and received a 20% total blood volume transfusion of either diamide-treated (rigid) RBCs or normal RBCs. RESULTS: CLP-treated animals' CI gradually decreased during 18 hours (232 +/- 60 ml/min/kg to 123 +/- 90 ml/min/kg; p = 0.05), with an increase in systemic vascular resistance (1459 +/- 517 dyne.sec/cm5.m2 to 2337 +/- 1213 dyne.sec/cm5.m2; p = 0.02). Diamide-treated animals had a rapid decrease in CI (86 +/- 7.0 ml/min/kg to 58 +/- 13 ml/min/kg; p = 0.05) and increase in SVR (2269 +/- 373 dyne.sec/cm5.m2 to 3897 +/- 988 dyne.sec/cm5.m2; p = 0.05) from baseline to 120 minutes after treatment respectively. The DI was significantly lower in both CLP and diamide groups (p < 0.03) when compared with control animals. Histologic evidence of subendocardial necrosis was shown in both the CLP- and Diamide-treated animals. CONCLUSIONS: These data suggest an association with RBC deformability and organ function in both septic and nonseptic animal models.

The influence of arachidonic acid metabolites on leukocyte activation and skeletal muscle injury after ischemia and reperfusion.

Derivatives of arachidonic acid have been found to play a role in the reperfusion injury of various tissues. These compounds have a broad spectrum of activity, including modulation of white blood cell response to injured tissue. This study was designed to determine the effect of thromboxane and lipoxygenase derivatives on the local and systemic response to ischemia and reperfusion of skeletal muscle. Fifteen dogs were separated into three groups and subjected to gracilis muscle ischemia followed by 2 hours of reperfusion. One group served as controls, one group was treated with OKY-046 (a thromboxane synthetase inhibitor), and one group was treated with diethylcarbamazine (a lipoxygenase inhibitor). White blood cell activation as measured by superoxide anion production, and eicosanoid levels were measured both in the gracilis venous effluent and central venous circulation. These results were compared to infarct size in the gracilis muscle. OKY-046 significantly reduced thromboxane production in both the central venous (102 +/- 30 to 31 +/- 9 pg/ml, p less than 0.05) and gracilis samples (107 +/- 22 to 25 +/- 6 pg/ml, p less than 0.005). This was accompanied by a reduced white cell activation in the central venous blood (15 +/- 1 to 10 +/- 1 nmol O2-, p less than 0.05), but did not affect infarct size or white cell activation in the gracilis. Conversely, diethylcarbamazine significantly reduced both white cell activation (16 +/- 1 to 10 +/- 1 nmol O2-, p less than 0.005) and infarct size in the gracilis muscles (61.6% +/- 4.5% to 28.5% +/- 8.6%, p less than 0.01), as well as reduced systemic white blood cell activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte activation in ischemia-reperfusion injury of skeletal muscle.

Polymorphonuclear leukocyte (PMN) participation in the pathophysiology of the reperfusion injury following skeletal muscle ischemia has become recognized. We measured the activation of PMNs as evidenced by production of superoxide anion (O2-) in the isolated canine gracilis muscle preparation of ischemia-reperfusion injury. PMNs were isolated from the gracilis muscle venous effluent and central venous blood after 6 hr of bilateral gracilis ischemia and 1 hr of reperfusion in five dogs. Baseline samples were obtained prior to ischemia from the central venous circulation. Liberation of O2- from PMNs and from PMNs stimulated by opsonized zymosan was determined by measuring ferricytochrome reduction. Results are expressed as nanomoles of O2- produced/2 x 10(6) PMN +/- SEM. O2- production by unstimulated cells was increased from 0.33 +/- 0.15 nmole in the baseline samples to 0.96 +/- 0.08 nmole in the central venous sample (P less than 0.01). With stimulation by zymosan, production increased from 10.3 +/- 1.4 nmole in the baseline samples to 15.2 +/- 1.1 nmole in the central venous sample (P less than 0.05) and to 15.5 +/- 0.9 nmole in the gracilis venous sample (P less than 0.01). These increases in superoxide production were not seen in the three sham-operated animals. Mean infarct size determined by planimetry was 55%. O2- produced by PMNs from central venous blood correlated with infarct size (r = 0.934, P = 0.02). These data imply that PMNs are activated by muscular ischemia, and the degree of activation is directly related to the extent of the muscle infarction.

Oxygen free radicals: effect on red cell deformability in sepsis.

OBJECTIVE: To examine the effect of alpha-tocopherol, a free radical scavenger, on RBC deformability, mixed venous hemoglobin saturation (SvO2), arterial-venous oxygen content difference (C[a-v]O2), pHv, and survival during sepsis. DESIGN: Randomized controlled study. INTERVENTIONS: Sprague-Dawley rats were randomized to three groups: sham, cecal ligation and puncture, or alpha-tocopherol/cecal ligation and puncture (pretreatment with alpha-tocopherol before cecal ligation and puncture). MEASUREMENTS AND MAIN RESULTS: The cecal ligation and puncture group had a significantly (p less than .05) higher SvO2 and lower C (a-v)O2, pHv, and survival rate when compared with alpha-tocopherol/cecal ligation and puncture and sham groups. No difference in pHa existed between groups. CONCLUSIONS: The alpha-tocopherol treatment improves survival in sepsis. RBC deformability during sepsis is prevented by alpha-tocopherol, suggesting that free radicals may cause the decrease in RBC deformability. This study provides indirect evidence that decreased RBC deformability may play a role in the physiologic peripheral shunting and decreased microcirculatory flow that occurs during sepsis.

Effect of oxygen-free radical scavengers on survival in sepsis.

UNLABELLED: Sepsis remains a leading cause of death in the surgical intensive care unit (SICU) patient following major surgery or trauma. Recent work has demonstrated that oxygen-free radicals (OFR) generated during sepsis contribute to the pathogenesis of this syndrome. The purpose of this study was to evaluate the effect of various new free radical scavengers on survival in sepsis. A total of 85 male Sprague-Dawley rats were placed into one of the following treatment groups. CONTROL: cecal ligation and puncture (CLP); PRE-AT: pretreatment with alpha-tocopherol (AT) 10 mg/100 gm SC x 3 days, and 5 mg/100 gm IV prior to CLP; AT: 20 mg/100 gm at time of CLP and 4 hours following CLP; U74006F: (21-aminosteroid which inhibits lipid peroxidation) 3 mg/kg IV at the time of and 4 hours following CLP; U78517F: (alpha-tocopherol analogue) 3 mg/kg at the time of and 4 hours following CLP. Survival was determined at various time points up to 72 hours. Pretreatment with AT resulted in improved survival, whereas the novel OFR scavengers U78517F and U74006F significantly improved survival and were efficacious without pretreatment. It was concluded that OFR scavengers can improve survival in sepsis.

Thromboxane synthetase inhibition decreases polymorphonuclear leukocyte activation following hindlimb ischemia.

Ischemia of the lower extremity has been shown to cause pulmonary leukostasis and increased pulmonary artery pressure. Thromboxane (TX) has been implicated as a mediator in this process. The effect of OKY-046, a TX synthetase inhibitor, on polymorphonuclear leukocyte (PMN) production of superoxide anion (O2-) as determined by ferricytochrome reduction was examined. Fourteen dogs were subjected to 6 hours of bilateral gracilis muscle ischemia followed by 1 hour of reperfusion. O2- production from resting PMNs and PMNs stimulated with opsonized zymosan (OZ, 0.1 mg/ml) was measured prior to ischemia or drug treatment (baseline), and following reperfusion in both treated (n = 7) and control groups (n = 7). Serum TX levels were measured using a radioimmunoassay. Following reperfusion, TX levels in the treated group were decreased as compared with the control group (18 +/- 2 pg/ml vs. 72 +/- 26 pg/ml, P less than 0.05). Superoxide production by both resting and stimulated PMNs was also decreased in the treated group; from 0.98 +/- 0.16 nmol to 0.43 +/- 0.12 nmol O2- in the resting state (P less than 0.05) and from 13.3 +/- 1.5 nmol to 9.0 +/- 1.1 nmol O2- after stimulation (P less than 0.005). O2- production was increased in the control group following reperfusion as compared with baseline samples, and this increase was attenuated by treatment with OKY-046. TX synthetase inhibition decreases activation of PMNs following hindlimb ischemia.

Role of eicosanoids and white blood cells in the beneficial effects of limited reperfusion after ischemia-reperfusion injury in skeletal muscle.

Limiting the rate of reperfusion blood flow has been shown to be beneficial locally in models of ischemia-reperfusion injury. We investigated the effects of this on eicosanoids (thromboxane B2, 6-keto-PGF1 alpha, and leukotriene B4), white blood cell activation, and skeletal muscle injury as quantitated by triphenyltetrazolium chloride and technetium-99m pyrophosphate after ischemia-reperfusion injury in an isolated gracilis muscle model in 16 anesthetized dogs. One gracilis muscle in each dog was subjected to 6 hours of ischemia followed by 1 hour of limited reperfusion and then by a second hour of normal reperfusion. The other muscle was subjected to 6 hours of ischemia followed by 2 hours of normal reperfusion. Six dogs each were used as normal reperfusion controls (NR) and limited reperfusion controls (LR), with 5 dogs being treated with a thromboxane synthetase inhibitor (LR/TSI) and another five with a leukotriene inhibitor (LR/LI). LR in all three groups (LR, LR/TSI, and LR/LI) showed a benefit in skeletal muscle injury as measured by triphenyltetrazolim chloride and technetium-99m pyrophosphate when compared with NR. However, there was no significant difference between the groups with LR regarding eicosanoid levels and white blood cell activation when compared with NR. These results demonstrate that LR produces benefits by mechanisms other than those dependent upon thromboxane A2, prostacyclin, or white blood cell activation.

Organ distribution of radiolabeled enteric Escherichia coli during and after hemorrhagic shock.

Translocation of intestinal bacteria to the blood during hemorrhagic shock (HS) has been confirmed in rats and humans. The current study was designed to trace the path of translocated intestinal bacteria in a murine HS model. Thirty-one rats were gavaged with 1,000,000 counts of viable 14C oleic acid-labeled Escherichia coli. Forty-eight hours later the animals were bled to 30 mmHg until either 80% of their maximal shed blood was returned or 5 hours of shock had elapsed and they were resuscitated with Ringer's lactate as previously described. Control animals were cannulated but not shocked. Eight rats immediately after shock and resuscitation, 6 rats 24 hours after shock, 3 rats 48 hours after shock, and 4 animals that died in shock had their heart, lung, liver, spleen, kidney, and serum harvested, cultured, and radioactive content measured. Translocated enteric bacteria are found primarily in the lung immediately after shock with redistribution to the liver and kidney 24 hours later. Animals surviving to 48 hours were capable of eliminating the majority of the bacteria from their major organ systems. Positive cultures for E. coli were also found in the blood, lung, liver, and kidney. We speculate that the inflammatory response stimulated by the bacteria in these organs may contribute to the multiple-organ failure syndrome seen after HS.

The incidence of decreased red blood cell deformability in sepsis and the association with oxygen free radical damage and multiple-system organ failure.

We studied the incidence of decreased red blood cell deformability (RBCD) in sepsis and the association between decreased RBCD and oxygen free radical generation (as measured by malonyldialdehyde) and the occurrence of multiple-system organ failure (MSOF). Patients were divided into three groups: septic (n = 28), nonseptic (n = 15), and control (n = 5). Serial measurements of deformability index, malonyldialdehyde, and MSOF were made. The deformability index for the septic group (0.23 +/- 0.17) was significantly less than that for the nonseptic (1.12 +/- 0.48) and control (1.11 +/- 0.25) groups. The malonyldialdehyde levels for the septic group (4.5 +/- 1.0 nmol/mL) were significantly greater than those for the nonseptic (2.7 +/- 0.9 nmol/mL) and control (2.6 +/- 0.8 nmol/mL) groups. The MSOF index for the septic group (10.1 +/- 2.5) was significantly greater than that for the nonseptic (7.6 +/- 1.7) and control (6.0 +/- 0.0) groups. An inverse correlation existed between malonyldialdehyde and deformability index ( = .501, less than .001, n = 40) and between deformability index and MSOF index (= .350, less than .05, n = 61). We conclude that RBCD is decreased during human sepsis; free radicals generated during sepsis may play a role in the decrease in RBCD. Decreased RBCD may contribute to the MSOF that occurs during sepsis.

Influence of pH on the modification of thiols by carbamoylating agents and effects on glutathione levels in normal and neoplastic cells.

In previous studies, we have suggested that the selective inhibitory effect of sodium cyanate (NaOCN) on hepatoma metabolism may be due to the lower pH observed in tumors relative to normal tissues. Lower pH might enhance the action of NaOCN by increasing the formation of isocyanic acid and carbamoylation of sulfhydryl groups. In the present work, studies were conducted on the effect of pH on the carbamoylation of sulfhydryl groups. The data indicated that carbamoylation of the sulfhydryl group of glutathione by NaOCN was enhanced by decreasing the pH from 7.4 to 6.6. A less pH-dependent response was observed with organic isocyanates. However, all reactions were reversible after the pH was increased by the addition of base. Kinetic studies showed that the rate of the reaction is very rapid, a maximal effect occurring within the first 10 min. Dose-dependent modifications of cellular glutathione by NaOCN and organic isocyanates were observed in human HT29 colon tumor cells, rat HTC hepatoma cells, and rat hepatocytes. The rate of carbamoylation of the glutathione sulfhydryl group in cells was similar to that of pure glutathione (GSH). The effect of buthionine sulfoxamine on GSH levels in cells was at least as great as that of sodium cyanate, but only the latter showed inhibitory effects on macromolecular synthesis; these were very rapid, pH-dependent, and reversible in tumor cells. Our results suggest that cellular sulfhydryl group(s) other than that of GSH might be involved in the effect of NaOCN on macromolecular synthesis.

Temporal relationship of hepatocellular dysfunction and ischemia in sepsis.

To determine whether hepatic dysfunction in sepsis results from hypoperfusion or direct cellular injury, Sprague-Dawley rats underwent either cecal ligation and puncture or sham operation. After either two or six hours, effective hepatic blood flow was measured using the galactose clearance method. Hepatocytes were isolated and intracellular sodium and potassium and glucose production were measured. Hepatic blood flow in septic rats decreased as early as two hours after sepsis when compared with sham-operated rats (3.8 +/- 1.4 vs 8.7 +/- 3.1 mL/min/100 g body weight). Intracellular sodium and potassium levels and glucose production in septic rats were not significantly different when compared with controls at two hours. After six hours, hepatic blood flow remained depressed and intracellular sodium level was increased compared with sham-operated rats (41.7 +/- 10.4 vs 31.4 +/- 5.9 mmol/L [41.7 +/- 10.4 vs 31.4 +/- 5.9 mEq/L]) and potassium decreased compared with controls (90.7 +/- 7.9 vs 111.5 +/- 6.7 mmol/L [90.7 +/- 7.9 vs 111.5 +/- 6.7 mEq/L]). Glucose production was decreased in septic rats after six hours when compared with controls (4.7 +/- 1.5 vs 15.4 +/- 6.4 mumol/g hepatocytes). These data suggest that hepatic blood flow is decreased before alterations in intracellular sodium and potassium as well as glucose production.

Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.