Churchill T A, Fuller B J
Surgical Medical Research Institute, University of Alberta, Edmonton, Canada.
Transplantation. 1996 Aug 15;62(3):346-52. doi: 10.1097/00007890-199608150-00008.
This study examined the effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP) and okadaic acid (a specific inhibitor of protein phosphatases 1 and 2A) as additives to a cold storage solution. The effects on levels of glycogen phosphorylase, the resultant effects on flux through the glycolytic pathway, and the consequences of these changes on adenylate (ATP, ADP, and AMP) levels in rat liver during a 24-hr period of cold hypoxia were studied. The rapid transition to anaerobic metabolism was reflected in the increases in lactate levels for all groups. Total lactate accumulation in control livers (flushed and stored with a histidine-lactobionate-raffinose solution) was 9.8 micromol/g. The one notable difference between the control and experimental groups was the total lactate increase in one of the groups treated with db-cAMP; lactate accumulation was 16.0 micromol/g. There was a preferential maintenance of ATP that correlated with the increased flux through glycolysis observed with db-cAMP treatment; levels were 0.4-0.6 micromol/g higher than control group values between 2 and 10 hr of storage. In the control group, levels of glycogen phosphorylase in the active 'a' form began to decrease within 1 hr of exposure to cold hypoxic storage. Values dropped from 86% to 78% within the first 1 hr and by 10 hr, % 'a' was 57%. The separate addition of db-cAMP and okadaic acid resulted in a sustained maintenance of phosphorylase % 'a' throughout the entire cold hypoxic storage period; % 'a' values at 10 hr ranged from 75% to 81%. The major finding of this study was the clear and distinct correlation between phosphorylase % 'a' and total lactate accumulation (index of flux through glycolysis). This relationship was statistically significant after only 1 hr of storage, with a correlation coefficient of r=0.52 (P<0.025); however, the correlation became stronger as the time of storage progressed (by 10 hr, r=0.72; P<0.001). According to the relationship established, the maximum theoretical limit for lactate accumulation with 100% phosphorylase 'a' is approximately 30 micromol/g lactate. This finding suggests that glycogen phosphorylase and not necessarily glycogen content is one major determinant in maintaining anaerobic metabolism and energy production during cold liver storage. Hence, previous experiments that investigated the effects of nutritional status and glycogen content on tissue viability after experimental transplantation need to be reassessed.
本研究检测了二丁酰环磷酸腺苷（db-cAMP）和冈田酸（蛋白磷酸酶1和2A的特异性抑制剂）作为冷藏溶液添加剂的效果。研究了其对糖原磷酸化酶水平的影响、对糖酵解途径通量的后续影响，以及在24小时冷缺氧期间这些变化对大鼠肝脏中腺苷酸（ATP、ADP和AMP）水平的影响。所有组乳酸水平的升高反映了向无氧代谢的快速转变。对照肝脏（用组氨酸-乳糖酸盐-棉子糖溶液冲洗并储存）中的总乳酸积累量为9.8微摩尔/克。对照组和实验组之间的一个显著差异是用db-cAMP处理的一组中总乳酸增加；乳酸积累量为16.0微摩尔/克。ATP有优先维持现象，这与db-cAMP处理后观察到的糖酵解通量增加相关；在储存2至10小时期间，其水平比对照组高0.4 - 0.6微摩尔/克。在对照组中，活性“a”形式的糖原磷酸化酶水平在暴露于冷缺氧储存1小时内就开始下降。在最初1小时内，该值从86%降至78%，到10小时时，“a”形式的比例为57%。单独添加db-cAMP和冈田酸导致在整个冷缺氧储存期内糖原磷酸化酶“a”形式的比例持续维持；10小时时“a”形式的比例在75%至81%之间。本研究的主要发现是糖原磷酸化酶“a”形式的比例与总乳酸积累量（糖酵解通量指标）之间存在明确且显著的相关性。仅在储存1小时后，这种关系就具有统计学意义，相关系数r = 0.52（P < 0.025）；然而，随着储存时间的延长，相关性变得更强（到10小时时，r = 0.72；P < 0.001）。根据所建立的关系，100%糖原磷酸化酶“a”形式时乳酸积累的最大理论极限约为30微摩尔/克乳酸。这一发现表明，糖原磷酸化酶而非糖原含量是冷肝脏储存期间维持无氧代谢和能量产生的一个主要决定因素。因此，先前研究营养状况和糖原含量对实验性移植后组织活力影响的实验需要重新评估。
