Cheryl Lalonde

Systemic lipid peroxidation and inflammation induced by thermal injury persists into the post-resuscitation period

We determined the time course of the oxidant-induced systemic lipid peroxidation seen after burn injury. Twelve sheep were given a 15% of total body surface third-degree burn and monitored for 3 or 5 days. Circulating lipid peroxides were monitored by both malondialdehyde (MDA) and conjugated dienes (CD). Lung and liver tissue MDA was also measured and compared to controls. A significant but transient increase in circulating MDA and CD was noted several hours after burn. Venous plasma levels increased again 3-5 days postburn with onset of wound inflammation. Oxygen consumption, VO2, also increased by 35 +/- 12% at this time. Lung MDA, which increased to 64 +/- 5 from a control of 45 +/- 4 nMol/gm, at 12 hours after burn was still increased 3 days after injury. Marked lung inflammation was present early after injury and persisted for the 5-day study period. Liver MDA also increased from control value of 110 +/- 20 to 252 +/- 25 at 12 hours and remained increased over the 5-day period. Serum alkaline phosphatase was also increased. Burn biopsies revealed no infection to explain the ongoing lipid peroxidation process, i.e., bacterial content was less than 10(5) organisms/gram burn tissue. We conclude that an initial system lipid peroxidation occurs immediately after burn injury, and that this process continues well into the post-resuscitation period, corresponding in time with increased VO2, lung inflammation, and evidence of liver dysfunction. The ongoing oxidant changes with the presence of a burn may explain the accentuated organ dysfunction seen with an additional septic insult in burned patients.

Oxidants and the pathophysiology of burn and smoke inhalation injury

A skin burn is a common traumatic injury that results in both local tissue damage and a systemic mediator-induced response. There is evidence of both local and systemic oxidant changes manifested by lipid peroxidation in animal burn models and also in burned man. Both increased xanthine oxidase and neutrophil activation appear to be the oxidant sources. Animal studies have also demonstrated decreased burn edema, and also decreased distant organ dysfunction with the use of antioxidants, suggesting a cause-and-effect relationship, which needs to be tested in man. Smoke inhalation injury, a chemical injury to the airways caused by incomplete products of combustion, is frequently seen in conjunction with a body burn. Lipid peroxidation, both in lung and in distant organs, is also seen with this injury. The combined body burn and smoke inhalation injury lead to a marked increase in mortality rate and also an increase in the degree of generalized oxidant release and lipid peroxidation. Although data in man are limited, the available information, along with that from animal research on burns and smoke inhalation, indicates oxidants may well play a key role, and antioxidants may be of clinical therapeutic use.

Excessive liver oxidant stress causes mortality in response to burn injury combined with endotoxin and is prevented with antioxidants

We studied the effect of the oral administration of a water-soluble antioxidant solution containing ascorbic acid, glutathione, and a precursor for glutathione synthesis, N-Acetyl-L-cysteine, on liver antioxidant activity, liver cell energetics, and mortality in rats in response to a 20% third-degree burn injury challenged 5 days later with an intraperitoneal injection of 30 mg/kg endotoxin. Rats with burns were fluid-resuscitated with subcutaneous Ringers lactate solution according to the Parkland formula (4 cc/kg/%burn). Rats challenged with endotoxin 5 days after burn were given an additional 100 ml/kg of subcutaneous Ringers lactate solution immediately after the injection of endotoxin. A group of rats with burns challenged with endotoxin 5 days after burn were given an oral antioxidant solution beginning after burn injury. Liver cell energetics were measured as tissue energy charge potential (ECP), adenosine triphosphate (ATP) content, and total adenine nucleotides. The levels of endogenous liver glutathione, catalase, vitamin C, and vitamin E were measured to monitor antioxidant status. We found that burn injury alone did not produce any mortality over the 6-day period despite a 35% decrease in liver energy charge potential resulting from a decrease in ATP, a 34% decrease in liver catalase activity, and a 20% decrease in liver vitamin C. It was interesting that glutathione increased and vitamin E remained unchanged. We found that endotoxin injury combined with burn injury produced a 61% mortality rate with a 63% decrease in liver energy charge potential, again resulting from a decrease in ATP, a 74% decrease in liver catalase activity, a 16% decrease in vitamin C, and a 29% decrease in vitamin E. Glutathione was significantly decreased compared with burn alone. We compared the liver antioxidant status of survivors with that of nonsurvivors who were killed when appearing moribund and found that glutathione was decreased by 51% and vitamin C by 73% in nonsurvivors over that in survivors, whereas catalase and vitamin E levels were comparable between the two groups. The oral administration of the antioxidants prevented mortality and the decrease in antioxidant activity and attenuated the decrease in energy charge potential. We conclude that a 20% burn produces a modest decrease in liver energy charge potential and antioxidant defenses without producing mortality. The addition of endotoxin further decreases liver antioxidant defenses, liver energy charge potential, and markedly increases mortality. Antioxidants, given post-burn, restored antioxidant defenses, attenuated the altered cell energetics, and prevented mortality, indicating oxidants to be the cause of mortality. This data also suggests that a critical value of decreases in antioxidant defenses and ATP exists, resulting in mortality.

Antioxidants Prevent the Cellular Deficit Produced in Response to Burn Injury

We studied the effect of water-soluble antioxidants on the cell energetics in multiple organs in rats in response to a 20% total body surface area third-degree burn injury. Liver, lung, and heart tissue were studied. Cell energetics were measured as tissue energy charge potential (ECP), adenosine triphosphate (ATP) content, and total adenine nucleotides. The enzymatic antioxidant catalase was used as a marker of endogenous cell antioxidant activity, especially to hydrogen peroxide. The water-soluble antioxidants glutathione, N-acetylcysteine, and vitamin C were given orally beginning at the time of burn injury and for the 6-day study period. All rats were fluid resuscitated according to the Parkland formula. The mortality rate was 0% for this size burn. The ECP in lung, liver, and heart, was normal on day 1 after the burn injury. However, the ECP was significantly decreased from the controls in the liver by day 3, with a peak decrease at day 6 as inflammation increased. A decrease in the heart ECP occurred between day 3 and day 6. Total adenine nucleotides did not decrease, indicating the decrease in ECP to be the result of a decrease in ATP. ECP remained normal in the lung. Catalase was also decreased in the liver and the heart and remained at normal levels in the lung. The decrease in the liver and heart ECP and ATP was eliminated with the oral antioxidant administration after the burn injury. We conclude that a modest burn injury decreases cellular energy charge in the heart and liver not immediately after burn but 3 to 6 days later. The decrease in antioxidant activity precedes the decrease in ECP. The lung appears to be protected. Water-soluble antioxidants, given after burn injury, prevent the altered cell energetics-strongly suggesting a cause-and-effect relationship between increased oxidant release with inflammation, decreased antioxidant activity, and altered cell energetics.

Increased Lipid Peroxidation and Decreased Antioxidant Activity Correspond With Death After Smoke Exposure in the Rat

Oxidants are released directly from smoke and also as a result of the airways inflammation that occurs after smoke injury. We determined the relation between the degree of tissue oxidant change with the use of malondialdehyde content to measure lipid peroxidation and the degree of lung and systemic organ damage and resulting damage mortality 24 hours after a controlled smoke exposure in a rat model. We also monitored changes in the key tissue antioxidant catalase. We found that the degree of lung lipid peroxidation and the decrease in catalase activity directly correlated with mortality caused by respiratory failure and with the degree of lung inflammation but that they did not correlate with the peak carboxyhemoglobin level, a marker of smoke gas phase exposure. The lung oxidant changes also directly correlated with increased systemic lipid peroxidation and decreased catalase in liver and kidney tissue. We conclude that the initial smoke insult causes lung and in turn systemic inflammation with resulting release of oxidants, which leads to tissue oxidant injury. The degree of lung oxidant change significantly correlates with the degree of lung tissue injury, respiratory failure, and mortality, and the major source of the oxidant changes is tissue inflammation rather than oxidants in the smoke itself.

Comparison between lung and liver lipid peroxidation and mortality after zymosan peritonitis in the rat.

We compared the mortality rate and the lung and liver histologic injury with the degree of tissue lipid peroxidation after zymosan-induced peritonitis. Male Wistar rats were given .75 or 1 mg/g of zymosan intraperitoneally and monitored for 24 h. Tissue lipid peroxides were measured as conjugated dienes and malondialdehyde (MDA) as were the antioxidants, ascorbic acid and catalase. Mortality rates for the .75 and 1 mg/g groups were 15 and 50%, respectively. In lung, the degree of increase in conjugated dienes and MDA was significantly greater in nonsurvivors than survivors. Ascorbic acid and catalase levels were also significantly decreased to a greater degree in the sicker animals with ascorbic acid decreased to a greater degree in lung than liver. In liver, conjugated dienes were not increased at 24 h, but MDA was increased to a greater degree in the higher dose and sicker animals. The level of MDA corresponded with the degree of histologic change. Catalase decreased to a greater degree in liver than lung. We conclude that the degree of lung and liver lipid peroxidation correlates with the degree of inflammation induced tissue injury and mortality.

FLUID RESUSCITATION WITH DEFEROXAMINE PREVENTS SYSTEMIC BURN-INDUCED OXIDANT INJURY

We studied the effect of deferoxamine (DFO) infused after burns on hemodynamic stability as well as local and systemic inflammation and oxidant-induced lipid peroxidation. Eighteen anesthetized sheep were given a 40% of total body surface burn and fluid resuscitated to restore oxygen delivery (DO2) and filling pressures to baseline values. Animals were resuscitated with lactated Ringers (LR) alone or LR plus 1,500 ml of a 5% hetastarch complexed with DFO (8 mg/ml). Animals were killed 6 hours postburn. The sheep resuscitated with LR and LR plus hetastarch demonstrated significant lung inflammation and significant increases in lung and liver malondialdehyde (MDA) from controls of 47 +/- 6 and 110 +/- 7 nMol/gm to 63 +/- 13 and 202 +/- 59 for LR and 67 +/- 4 and 211 +/- 9 for LR + hetastarch, respectively. The group resuscitated with hetastarch alone required 15% less fluid. VO2 returned to baseline values in both groups by 2 hours. Resuscitation with the 5% hetastarch-DFO decreased total fluids by 30% over LR and prevented the increase in lung and liver MDA. In addition, postburn VO2 increased by 25% above baseline values. Burn tissue edema, measured as protein-rich lymph flow, was significantly increased with the administration of DFO compared with the other groups. We conclude that DFO used for burn resuscitation prevents systemic lipid peroxidation and decreases the vascular leak in nonburn tissues while also increasing O2 utilization. Resuscitation with hetastarch-DFO may accentuate burn tissue edema, possibly by increased perfusion of burn tissue.

Alpha-tocopherol attenuates lung edema and lipid peroxidation caused by acute zymosan-induced peritonitis.

BACKGROUND Inflammation-induced disease as seen with trauma and infection can lead to increased lung oxidant activity resulting in cell membrane lipid peroxidation. Acute zymosan-induced peritonitis in rats produces lung inflammation, edema, and lipid peroxidation. We determined whether administered alpha-tocopherol (vitamin E), the key antioxidant protection against cell membrane lipid peroxidation, would improve this process. METHODS Male Wistar rats were given 0.75 mg/kg of intraperitoneal zymosan, volume resuscitated, monitored, and killed at 4 or 24 hours. Lung histologic changes and levels of conjugated dienes, a marker of lipid peroxidation, were used to monitor injury. The levels of vitamin E, vitamin C, and catalase were used to monitor antioxidant defenses. The effect of administering alpha-tocopherol (50 mg/kg) by gavage immediately after zymosan on the degree of the lung injury was then determined. RESULTS Twenty-four hours after zymosan was administered, the vitamin E levels in plasma were significantly decreased, but lung tissue vitamin E levels were maintained, whereas tissue catalase and vitamin E levels decreased. Lung tissue-conjugated diene levels, alveolar edema, and neutrophil count were significantly increased. alpha-Tocopherol treatment increased the postzymosan plasma vitamin E levels by 50%. Lung tissue vitamin E levels did not increase; however, the degree of lung injury and lipid peroxidation was significantly attenuated. Tissue catalase levels were also maintained. CONCLUSIONS We conclude that alpha-tocopherol given at the onset of a progressing inflammatory injury can protect the lung from oxidant damage and attenuate the degree of lung injury.

Effect of graded increases in smoke inhalation injury on the early systemic response to a body burn

OBJECTIVE To study the early (first 24 hrs) effect of increasing lung exposure to smoke on the hemodynamic response to a modest body burn. DESIGN A prospective randomized study. SETTING Laboratory at a university medical center. SUBJECTS Thirty-two adult yearling female sheep. INTERVENTIONS Adult sheep (n = 32) were given an 18% of body surface burn; 24 sheep were then exposed to cotton toweling smoke using 12 breaths of a tidal volume of 5, 10, or 20 mL/kg. Animals were awakened, resuscitated to baseline oxygen delivery, and then killed at 24 hrs. MEASUREMENTS AND MAIN RESULTS Vascular pressure, cardiac output, and oxygen consumption and delivery were measured, as well as blood gases, lung and soft tissue lymph flow, and fluid balance. We found that a 5-mL/kg tidal volume smoke exposure x 12 breaths did not produce significant airway inflammation or alter the cardiopulmonary response to a burn alone. Oxygen consumption (VO2) remained at baseline and the net 24-hr positive fluid balance of 1.5 L was comparable to a burn alone. Increasing the smoke exposure to 10 mL/kg tidal volume, which produced a moderate airway injury, resulted in a significant increase in early fluid requirements, a 40% early increase in VO2, a doubling of positive fluid balance, as well as a marked increase in burn edema. However, gas exchange was not impaired. The 20-mL/kg tidal volume exposure resulted in an early 100% increase in VO2, a three-fold increase in fluid requirements at 1 to 4 hrs, compared with burn alone, in addition to a severe airway inflammation with mucosal slough and resulting impaired gas exchange. CONCLUSIONS The addition of a smoke exposure which produces airway inflammation and injury significantly increases early post burn systemic metabolic demands and fluid requirements, as well as the degree of burn edema and positive fluid balance compared with a burn alone. The magnitude of the accentuated response appears to correspond with the degree of airway inflammation and not with alveolar dysfunction.

Changes in lung and systemic oxidant and antioxidant activity after smoke inhalation.

We determined the oxidant activity in lung airways, parenchyma, and systemic tissues in response to smoke inhalation, comparing lipid peroxidation with physiologic and histologic change. Adult sheep were given a controlled amount of cooled smoke from burned cotton toweling, containing a uniform particle diameter of 3–4 μm. The mean peak carboxyhemoglobin was 45 ± 4%. Animals were monitored unanes-thetized for 24 h and killed. Severe respiratory failure was noted, as a result of airways mucosal ulceration, submucosal edema, and atelectasis, along with increased airways fluid, but minimal alveolar edema. Airway fluid malondialdehyde (MDA) content was threefold greater than plasma. However, airways mucosa and lung parenchymal tissue, lipid peroxidation, and oxidized glutathione were not increased, suggesting the only direct oxidant activity was present only at the airways surface. Other factors besides oxidants are likely to be involved in the lung injury. However, a marked systemic oxidant stress was noted as evidenced by a significant increase in liver tissue MDA and decrease in reduced glutathione and catalase activity. The tissue oxidant stress also corresponded with a 75% increase in systemic oxygen consumption and an increase in soft tissue vascular permeability. We conclude that: 1) the only direct lung oxidant stress after smoke was noted in airways fluid, while lung tissue lipid peroxidation was not seen despite severe airways injury and atelectasis, and 2) major systemic physiologic changes, as evidenced by increased systemic oxygen demands and systemic micro-vascular permeability are seen with smoke exposure in addition to evidence of systemic tissue oxidant stress. The likely source of the oxidant activity was a smoke-induced systemic inflammation.