Gender Influences Cold Preservation-Reoxygenation Injury in the Liver A. Colantoni, N. De Maria, P. Caraceni, M. Bernardi, and D.H. Van Thiel E ACH LIVER GRAFT sustains injury during the time it is removed from the donor (warm ischemia), stored (cold ischemia), and transplanted into the recipient (reper- fusion). Reactive oxygen species are produced massively during post-hypoxic reoxygenation and are implicated in the pathogenesis of ischemia/reoxygenation injury in the liver. Reactive oxygen species attack lipids, proteins, and nucleic acids causing irreversible structural and functional damage. 1 The glutathione (GSH) system is an important endoge- nous antioxidant. More than 95% of the intracellular GSH is maintained in the reduced form. Under conditions of oxidative stress, the formation of intracellular glutathione disulfide (GSSG) and, subsequently, GSSG efflux increase. GSSG formation has been used to detect and quantify oxidative stress during hypoxia/reoxygenation in models of isolated perfused rat liver. 2 Donor gender influences the outcome after liver trans- plantation. Livers from female donors show a poorer out- come in several series. In particular, Kahn et al reported the occurrence of graft failure within 60 days from transplant in 11% to 12% of the cases when a male graft was used, while 19% to 22% graft failures were found when a female organ was used. 3 In another more recent series, a decrease in graft survival of all the recipients of female allograft had been reported. 4 The reason for this finding still remains un- known. Among the determinants of early graft failure are intra- cellular alterations caused by cold preservation and reoxy- genation. Therefore, the goal of the present study was to investigate if gender influences the sensitivity of the liver to cold preservation/reoxygenation injury. MATERIALS AND METHODS Adult Wistar rats (260 to 280 g), 8 males and 8 females, were used. Under anesthesia, the abdomen was opened and the liver dissected free. The portal vein and the superior vena cava were cannulated. The livers were rinsed with cold University of Wisconsin preserva- tion solution (UW) then stored in UW at 4°C for 24 hours (cold preservation phase). After preservation, the livers were perfused with Krebs-Henseleit bicarbonate buffer (37°C, pH 7.4) saturated with oxygen for 60 minutes (reoxygenation phase). Samples of perfusate were collected every 5 minutes during reoxygenation. Liver tissue samples were obtained at the end of preservation and reoxygenation. The rate of lactic dehydrogenase (LDH) release in the perfusate was taken as an index of hepatocellular injury. LDH release was measured using a spectrophotometric technique (Sigma, St. Louis, MO). Total GSH and GSSG were evaluated in the perfusate according to Tietze. 5 Hepatic malonaldehyde (MDA) levels were taken as index of lipid peroxidation and were measured using the thiobarbituric acid reaction. 6 Liver protein oxidation was assessed as protein carbonyl group content (PCC) according to the method of Levine. 7 RESULTS AND DISCUSSION The time course of LDH release in the perfusate was obtained during the 60 minutes of reoxygenation that followed 24 hours of cold preservation. Female livers showed a significantly more increased rate of LDH release throughout the study period with respect to male livers (Fig 1). Both male and female livers showed significantly greater MDA levels at the end of the reoxygenation phase than at the end of the preservation phase. When the results ob- From the Liver Transplant Program, Loyola University Chicago (A.C., N.D.M., H.V.T.), Maywood, Illinois; and Departmente Me- dicina Interna, Cardioangiologia ed Epatologia, University of Bologna (P.C., M.B.), Bologna, Italy. Address reprint requests to Dr Colantoni, Liver Transplant Program, Loyola University Medical Center, Build 114, Room 48, 2160 South First Ave, Maywood, IL 60153. Fig 1. Rate of LDH release in the perfusate during 60 minutes of reoxygenation that follows 24 hours of cold preservation in male and female isolated rat livers. 0041-1345/99/$–see front matter © 1999 by Elsevier Science Inc. PII S0041-1345(98)01900-9 655 Avenue of the Americas, New York, NY 10010 1052 Transplantation Proceedings, 31, 1052–1053 (1999)