Development of circulatory and metabolic shock following transient portal triad occlusion.

Liver ischemia is purposefully induced by portal triad occlusion (PTO) in several clinical situations including liver surgery for trauma, tumor, and transplantation. Despite significant morbidity from PTO, the hemodynamic and metabolic effects of PTO have not been evaluated relative to duration of ischemia. We investigated this using a total hepatic ischemia model. Rats received isoflurane anesthesia, carotid artery and jugular vein cannulation, and serial measurements of cardiac output (CO), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), stroke volume (SV), systemic vascular resistance (SVR), superior mesenteric artery blood flow (SMAF), intestinal vascular resistance (IVR), pH, pCO2, pO2, lactate, glucose, hematocrit (HCT), white blood cell count (WBC), and total neutrophils. Each group received 0, 15, 30, 45, or 60 min of PTO followed by 2 hr of reperfusion. All sham ischemia animals remained hemodynamically stable throughout the study. However, in the ischemic groups, there were significant time-dependent decreases in MAP, HR, CO, CVP, SV, SMAF, and pH, and increases in SVR, IVR, HCT, and lactate, while pCO2, pO2, glucose, and WBC remained stable. All of the ischemic animals survived except those that received 60 min of PTO. In this group, all of the animals survived the ischemic period; however, only one animal survived beyond 60 min of reperfusion. These data demonstrate a time-dependent circulatory and metabolic shock following PTO heralded by intestinal venous pooling and loss of intravascular fluid, and culminating in death. Careful hemodynamic monitoring and restoration of blood volume in the trauma patient may reduce morbidity and mortality.

A new model for inducing total hepatic ischemia while preventing circulatory collapse secondary to splanchnic vascular congestion.

Animal models used to study liver ischemia are limited by the lethal effect of splanchnic venous engorgement from portal triad occlusion (PTO). We compared a passive porto-systemic shunt (PSS) to a pump-driven PSS. The passive and pumped PSS groups (n = 6) received 60 min of PTO followed by 2 h of reperfusion. A control group received all interventions, but no PTO, and remained stable throughout. During PTO, severe circulatory shock with intestinal ischemia occurred in the passive group, while the pumped group remained stable. During reperfusion, both shunted groups experienced varying degrees of metabolic acidosis with decreases in cardiac index, stroke volume, superior mesenteric artery flow, and increases in systemic and intestinal vascular resistance. The mortality rate for the passive group was 83% vs. 0% for the pumped group. These results suggest that pumped PSS prevents splanchnic engorgement and allows for reproducible, isolated total hepatic ischemia in vivo.

Evidence that the large loss of glutathione observed in ischemia/reperfusion of the small intestine is not due to oxidation to glutathione disulfide.

Reperfusion injury following ischemia is thought to be the consequence of reactive oxygen species possibly generated either by xanthine oxidase activity or by processes associated with neutrophil activation in the affected organ or tissue. The conversion of xanthine dehydrogenase to the oxidase as well as the interactions between endothelium and neutrophils in the margination and activation of the latter are all considered to be results of conditions resulting from the ischemic episode. Determination of the redox status of glutathione in an ischemic/reperfused organ is frequently employed as an indicator of oxidative stress created by the production of oxygen free radicals during the reperfusion period. In this procedure, the ratio of oxidized glutathione (GSSG) to total glutathione (GSH + GSSG) is utilized to demonstrate the proportion of glutathione oxidized during reperfusion. We determined this ratio in the rat small intestine during ischemia and reperfusion and found that while the ratio of GSSG/(GSH + GSSG) does increase, this increase was the result of GSH disappearance rather than an increase in GSSG, and that essentially all of this loss occurred during the ischemic episode. We demonstrated that no oxidation of GSH occurred that was attributable to reperfusion per se; nor was there an increase of GSSG during this reoxygenation period.

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.

GSH-dependent inhibition of lipid peroxidation: properties of a potent cytosolic system which protects cell membranes.

Properties of a heat labile, nondialyzable cytosolic factor which prevents lipid peroxidation in membranous organelles are described. The factor is present in liver and other animal tissues, and its capacity to inhibit lipid peroxidation in membranes subjected to oxidative stress is greatly potentiated by glutathione (GSH), although GSH by itself has no inhibitory effect on lipid peroxidation. The data obtained thus far indicate that one or more sulfhydryl groups associated with the factor is required for the inhibition. The mechanism by which lipid peroxidation is inhibited must involve prevention of initiation of peroxidation in the membranes, presumably by a process requiring one or more sulfhydryl groups associated with the heat labile factor. The latter appears to be protected by GSH while the factor is exerting its inhibitory effect on lipid peroxidation. The factor is not one of the known GSH-dependent enzymes, and appears to be a potent and ubiquitous system for stabilizing cell membranes against oxidative damage.

Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor not glutathione peroxidase.

Both enzymic and nonenzymic lipid peroxidation in membranes are inhibited by a)certain chelating compounds, b)some metal ion (Mn2+, Co2+, and Ce3+), and c)lipid soluble antioxidants. The commonalities suggest that the processes of oxidative lipid degradation in the two types of systems may be similar, differing only in the mechanism of initiation. This is further borne out by studies with a glutathione-dependent, heat-labile cytosolic factor that inhibits malondialdehyde formation (a product of lipid peroxidation) in both systems. Studies in the authors' laboratory, however, have demonstrated that the cytosolic factor protects membranous organelles from oxidative damage to the lipids by preventing peroxidation from occurring at all. Analyses of the fatty acid composition of the membranes demonstrate that the polyunsaturated fatty acid content remains stable when the membranes are subjected to peroxidizing conditions in the presence of the cytosolic factor and GSH. Both the cytosolic factor and GSH are required for the protective action since neither can provide this marked stabilizing effects by itself. High concentrations of GSH reduce lipid peroxidation to some extent, but low concentrations are not effective without the addition of the cytosolic factor. The mechanism of this inhibition of peroxidative attack is unknown. Partial purification of rat liver cytosolic glutathione peroxidase demonstrated that the heat-labile cytosolic factor was not glutathione peroxidase. The cytosolic factor may be a glutathione transferase, but that is not known with certainty. Possibly more than one cytosolic protein possesses this GSH-dependent property for inhibiting lipid peroxidation under conditions of oxidative stress. The conditions for the functioning of this protective system in intact cells appear to be optimum and it may constitute a ubiquitous membrane-stabilizing system in that it is also present in other tissues (heart and lung, for example).

Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor in animal tissues.

A glutathione-dependent, cytosolic factor (previously thought to be glutathione peroxidase), inhibits lipid peroxidation in both microsomal and mitochondrial membranes. Studies in this laboratory had shown that the inhibition was due to prevention of peroxidative attack on the polyunsaturated fatty acids in the membrane lipids even under conditions that would otherwise promote rapid lipid peroxidation. A glutathione-dependent factor is also present in rat liver cytosol which can utilize peroxides of both free fatty acid salts in solution and free fatty acids in micellar suspension as substrates. It does not, however, utilize peroxidized lipids of microsomal and mitochondrial membranes as substrates. Whether or not this is the same factor which inhibits lipid peroxidation is not known with certainty, but current information indicates that they are not the same. Data presented in this report support the conclusion that neither glutathione peroxidase nor glutathione S-transferase activities appear to be responsible for the inhibition of lipid peroxidation in biological membranes. After partial purification of active preparations of both of these peroxidases, it was observed that neither preparation inhibited lipid peroxidation. The results of this study further support the conclusion that the glutathione-dependent cytosolic factor which inhibits lipid peroxidation in biological membranes does so by preventing the peroxidation rather than by reducing lipid peroxides.

Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes.

Results are presented indicating that, although glutathione peroxidase activity inhibits lipid peroxidation in membranes, it does not appear to do so by reducing membrane lipid peroxides to lipid alcohols, as has been shown by others to be the case for free fatty acid peroxides in solution. Lipid peroxidation was studied in an enzymic system (microsomal NADPH oxidase) and in a non-enzymic system (mitochondria plus ascorbate). A study of the fatty acids in the phospholipids of microsomes and mitochondria demonstrated that detectable amounts of hydroxy fatty acids were not formed in the membranes when the latter were incubated in the presence of the glutathione peroxidase system even under conditions known to have generated significant levels of lipid peroxides in the membrane. Fatty acid analyses of the microsomal and mitochondrial particles indicated that glutathione peroxidase activity inhibited loss of polyunsaturated fatty acids when these organelles were exposed to peroxidizing conditions. If glutathione peroxidase activity were inhibiting the formation of malondialdehyde (a product of lipid peroxidation) by converting peroxide groups to alcohols, the loss of the constitutive polyunsaturated fatty acids in the membrane should not have been appreciably affected by addition of the peroxidase system. The protective effect cannot be due to quenching of an autocatalytic type of lipid peroxidation (at least in the microsomal system) since it has been established that the microsomal enzyme system (NADPH oxidase) catalyzes a continuous attack on microsomal polyunsaturated fatty acyl groups during the reaction and that the peroxidative process is not autocatalytic in nature. It appears, therefore, that glutathione peroxidase activity must exert its effect on this system by preventing free radical attack on the polyunsaturated membrane lipids in the first place. A possible mechanism for the interruption of a free radical attack on the lipids is proposed.