Glycine Protects Hepatocytes from Injury Caused by Anoxia, Cold Ischemia and Mitochondrial Inhibitors, But Not Injury Caused by Calcium Ionophores or Oxidative Stress

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Abstract

Isolated hepatocytes, suspended in an organ preservation solution, can be preserved at 4°C for up to 6 days. After preservation, normothermic-normoxic incubation causes loss of hepatocyte viability. The addition of 3 mmol/L glycine to the rewarming medium prevents the loss of viability. In this study we investigated the cytoprotective effects of glycine under many conditions known to cause hepatocellular injury to understand the mechanism of cold-induced injury in the liver. Hepatocytes were suspended in modified Krebs-Henseleit buffer with or without 3 mmol/L glycine and exposed to agents or conditions known to induce cell death. Hepatocyte viability was assessed by measuring the percentage of lactate dehydrogenase leakage from the cells and the concentration of ATP during incubation at 37°C under room air for up to 90 min. Mitochondrial inhibitors (potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone); calcium ionophores (ionomycin and A23187); an oxidizing agent, tert-butyl hydroperoxide; and anoxia were all used to cause cell injury. Hepatocytes were also isolated from fasted rats and hypothermically preserved as another model of cell death. Other amino acids were also tested in the hypothermic preservation model to study the specificity of the amino acid requirement for prevention of lactate dehydrogenase leakage. Of the amino acids tested, only alanine (10 mmol/L) and the combination of alanine (3 mmol/L) and serine (3 mmol/L) were as effective as glycine in preventing lactate dehydrogenase release in the hypothermic preservation model. Anoxia, potassium cyanide (3 and 5 mmol/L) and carbonyl cyanide m-chlorophenylhydrazone (5 and 10 μmol/L) all induced cell injury, as indicated by release by the cell membrane of lactate dehydrogenase (60% to 90% release) and severe depletion of ATP content (less than 10% of freshly isolated cells). In this model of cell death, glycine suppressed lactate dehydrogenase release (25% to 35% release) but had no effect on ATP content. A23187 (10 to 50 μmol/L) and ionomycin (5 to 15 μmol/L) (calcium-induced cell death) caused lactate dehydrogenase leakage that was not prevented by glycine. Nor was induction of cell death by tert-butylhydroperoxide (oxidative stress) suppressed by glycine. Fasting the rat before hepatocyte isolation sensitized the cells to hypothermic preservation injury (80% lactate dehydrogenase release in fasted cells vs. 35% in fed cells) after 1 day of cold storage followed by rewarming. Glycine suppressed lactate dehydrogenase release from hepatocytes in fasted rats. Glycine was effective in suppressing hepatocellular injury caused by cold storage, anoxia, fasting and mitochondrial poisons. However, neither calcium-induced nor oxidative stress–induced cell death was prevented by glycine. This study suggests that hepatocellular injury caused by hypothermia (and suppressed by glycine) may not, therefore, be caused by calcium or oxidative stress (injuries not suppressed by glycine). However, in this hepatocyte model, the methods used to induce calcium or oxidative stress–dependent cell death may have resulted in effects more severe than those obtained in hepatocytes exposed to hypothermia, and these methods may have overwhelmed the cytoprotective effects of glycine against calcium-induced or oxidative stress–induced cell death. The beneficial effect of some amino acids (glycine, alanine and serine) in hypothermic preservation of hepatocytes suggest that these agents may be useful in improving liver preservation for transplantation. (HEPATOLOGY 1993;17:91–98.)

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