Dutkowski P, Southard J H, Junginger T
Klinik für Allgemein- und Abdominalchirurgie, Universität Mainz, Germany.
Langenbecks Arch Chir. 1997;382(6):343-8. doi: 10.1007/s004230050078.
Simple cold storage of livers for transplantation activates glycolysis due to lack of oxygen. Energy derived from glycolysis may be critical for cell survival and liver cell death may occur once glycolysis is inhibited in the liver due to accumulation of end products or lack of substrates (glycogen). The relationship between cell death (lactate dehydrogenase, LDH release), anaerobic glycolysis (lactate production), and glycogen content of liver tissue was studied during cold incubation of liver slices in UW solution. Rat livers slices from male Sprague Dawley rats were incubated at 4 degrees C in UW solution, with continuous gentle shaking, under conditions of chemical hypoxia (KCN, 5 mM). The rate of lactate production, LDH release-ATP and glycogen content were measured spectrophotometrically and by HPLC. Lactate increased nearly linearly for the first 48 h of incubation; total lactate which had accumulated after 48 h was 33.9 +/- 0.81 mumol/g and at 96 h nearly the same, 31.3 +/- 1.2 mumol/g. Glycolysis stopped, apparently, because of the depletion of liver slice glycogen which was initially 228.8 +/- 1.7 mumol/g wet wt. It decreased to 34.7 +/- 2.7 mumol/g at 48 h and to 18.7 +/- 1.1 mumol/g at 72 h and remained at this level for the next 24 h. An increased leakage of LDH occurred once glycogen metabolism (and accumulation) ceased. LDH release could be stimulated after only a few hours of cold incubation of liver tissue slices by adding glycolysis inhibitor (iodoacetic acid) to the medium. After 24 h. LDH release was 24.4 +/- 1.8% and increased to 52.8 +/- 5.2% (P < 0.05, Student's t-text) with iodoacetic acid. Adding a glycolytic substrate (fructose, 10 mM) to the medium maintained lactate production for 96 h. The stimulation of glycolysis by fructose also reduced cell death: LDH release was significantly lower at 72- and 96-h incubation (P < 0.001, two-way ANOVA). The ATP content was significantly higher with fructose (P < 0.001). Adding glucose (20 mM) and fructose (10 mM) in combination resulted in prolonged cell survival, significantly delayed glycogen depletion and significantly higher ATP content at 48 and 72 h (two-way ANOVA). Livers from rats who had fasted for 24 h demonstrated the same LDH release at 48 h when incubated with glucose (20 mM) and fructose (10 mM). In conclusion, LDH leakage from hypoxic cold-stored liver slices is related to anaerobic glycolysis. Anaerobic glycolysis appears to continue slowly under hypothermia and provides sufficient energy for maintenance of cell viability. A stimulation of glycolysis in the cold is possible by fructose and results in prolonged cell survival under hypothermic conditions. Glycogen depletion can be slowed down by combining glucose and fructose.
用于移植的肝脏简单冷藏会因缺氧而激活糖酵解。糖酵解产生的能量对于细胞存活可能至关重要，一旦肝脏中的糖酵解因终产物积累或底物（糖原）缺乏而受到抑制，肝细胞就可能发生死亡。在UW溶液中对肝切片进行冷孵育期间，研究了细胞死亡（乳酸脱氢酶，LDH释放）、无氧糖酵解（乳酸产生）与肝组织糖原含量之间的关系。将雄性Sprague Dawley大鼠的肝脏切片在4℃下于UW溶液中孵育，持续轻柔摇晃，处于化学性缺氧（5 mM KCN）条件下。通过分光光度法和高效液相色谱法测量乳酸产生速率、LDH释放-ATP和糖原含量。孵育的前48小时乳酸几乎呈线性增加；48小时后积累的总乳酸为33.9±0.81 μmol/g，96小时时几乎相同，为31.3±1.2 μmol/g。糖酵解显然停止了，这是因为肝切片糖原最初为228.8±1.7 μmol/g湿重，在48小时时降至34.7±2.7 μmol/g，72小时时降至18.7±1.1 μmol/g，并在接下来的24小时保持在该水平。一旦糖原代谢（和积累）停止，LDH的泄漏就会增加。通过向培养基中添加糖酵解抑制剂（碘乙酸），仅在肝组织切片冷孵育数小时后就能刺激LDH释放。24小时后，添加碘乙酸时LDH释放为24.4±1.8%，并增加至52.8±5.2%（P<0.05，学生t检验）。向培养基中添加糖酵解底物（10 mM果糖）可使乳酸产生维持96小时。果糖对糖酵解的刺激也减少了细胞死亡：在72小时和96小时孵育时LDH释放显著更低（P<0.001，双向方差分析）。添加果糖时ATP含量显著更高（P<0.001）。联合添加葡萄糖（20 mM）和果糖（10 mM）可延长细胞存活时间，显著延迟糖原消耗，并在48小时和72小时时使ATP含量显著更高（双向方差分析）。禁食24小时的大鼠肝脏在与葡萄糖（20 mM）和果糖（10 mM）一起孵育时，48小时时显示出相同的LDH释放。总之，缺氧冷保存肝切片的LDH泄漏与无氧糖酵解有关。无氧糖酵解在低温下似乎会缓慢持续，并为维持细胞活力提供足够的能量。在低温条件下，果糖可刺激糖酵解，从而延长细胞存活时间。联合使用葡萄糖和果糖可减缓糖原消耗。
