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Human livers can only be stored for a short time before transplantation, thus a better understanding of potential pharmacologic and nutritional strategies that might protect the liver against anoxic damage may be important clinically. Fructose has been shown recently to prevent liver damage induced by warm ischemia as assessed by lactate dehydrogenase (LDH) release.1 To determine whether fructose protects the liver from anoxic damage by production of glycolytic adenosine triphosphate (ATP), and whether an elevation of cytosolic free calcium (Cai2+) is involved in anoxic liver cell injury, the intracellular ATP level in real time assessed by 31P nuclear magnetic resonance (NMR) spectroscopy and intracellular free calcium determinations with aequorin using freshly isolated rat hepatocytes was studied. Lactate dehydrogenase release was measured to monitor liver cell damage.
Freshly isolated hepatocytes were prepared from adult male Sprague-Dawley rats weighing between 200 and 250 g. The cells were isolated from fed animals with collagenase (Sigma type IV, Sigma Chemicals, St Louis, Mo) using a previously described perfusion method.2 Cell viability assessed by trypan blue exclusion averaged 90% and was never less than 85%. The cells were imbedded in agarose gel threads and perfused at a rate of 0.6 mL/min with standard Krebs-Henseleit bicarbonate buffer (KHB) at 37°C. Daring the 1-hour control period, the KHB perfusate contained 1.3 mmol/L Ca2+, 5 mmol/L glucose, and it was exposed to a gas phase of 95% O2–5% carbon dioxide (CO2) Anoxia was induced by saturating the perfusate for 2 hours with 95% N2–5% CO2 During the period of anoxia the medium contained glucose or fructose. After 2 hours of anoxia, reoxygenation was initiated by perfusing the cells with KHB containing glucose ad 1.3 mmol/L Ca2+ saturated with 95% O2–5% CO2.
Intracellular ATP was measured by 31P-NMR spectroscopy in real time.3 The ATP content was expressed as the ratio of the βATP peak to the peak of the external standard methylene diphosphonic acid contained in a sealed spherical glass bulb positioned within the agarose threads.
Cai2+ was measured using the Ca2+-sensitive photoprotein aequorio4 incorporated into the hepatocytes by gravity-loading. The aequorin loaded cells were imbedded in agarose gel threads, placed in the cuvette of an aequorin luminescence photometer, and perfused at a rate of 0.6 mL/min with KHB at 37°C, as previously described.4
Cell injury was monitored by measuring LDH release from the cells into the effluent perfusate before, during, and after the anoxic period. Because the concentration of perfused cells imbedded in the gel threads varied slightly between individual experiments, LDH release was expressed as the percent increase over the control value measured during the 1-hour control period of each experiment.
During the first hour of anoxia, ATP decreased faster and to a lower level with fructose than with glucose in the medium. On the other hand, the intracellular ATP concentration of the fructose group progressively increased during the second hour of anoxia. In contrast, the ATP content of the glucose exposed group continuously declined. The major surge in Cai2+ that occurred during anoxia was depressed 52% when glucose was replaced by fructose: Cai2+ reached only 0.7 ± 0.2 μmol/L instead of 1.45 ± 0.42 μmol/L (P <.01). Fructose did not eliminate the early transient increase in Cai2+ which is released from the mitochondria. Fructose completely prevented the release of LDH that occurs with anoxia. Indeed, when the cells were perfused with KHB containing glucose, LDH release increased sixfold within 2 hours of anoxia. In contrast, in the presence of fructose, LDH release declined 30%.
These results confirm the earlier reports of other investigators who found that the presence of high concentrations of fructose during anoxia protects liver cells against anoxic injury.1 In the present experiments, fructose completely abolished the release of LDH in anoxic liver cells. However, the beneficial effect of fructose, during the first hour of anoxia, was not a result of a maintenance of higher ATP levels as proposed by several investigators. Indeed, with fructose the intracellular ATP concentration decreased faster and to a lower level than occurred in hepatocytes perfused with glucose. On the other hand, during the second hour of anoxia, the concentration of ATP within the fructose exposed cells increased to 40% of the control value, while it continued to decline in the glucose-exposed group. This increase in ATP concentration can only be explained by a glycolytic formation of ATP that contributes to the protection against anoxia provided by fructose during the second hour of the 2 hours of experimental anoxia. Furthermore, fructose abolished the massive increase in Cai2+ that occurred during the second hour of anoxia which arises from extracellular source and is responsible for the activation of Ca2+ -sensitive degradative enzymes that produce liver cell death.