Sanne de Bruin

132 Chapter 5 on the PPP activity in SAGM stored RCCs have been published. Progressively decreasing activity but also increased oxidative PPP activity has been reported 40,42 . Higher flux through the PPP does not necessarily mean higher NADPH levels. The total NADPH and NADP+ pool gradually decreases during storage in AS-3 53 . These levels are similar in AS-7 stored RCCs even though PPP activationwas higher in AS-7 than in AS-3 47 . Furthermore, nicotinamide accumulates during storage, which is a breakdown product of NADPH and NADP+ 46 . The current hypothesis that this breakdown product has an inhibiting effect on G6PD activity and thus inhibits the oxidative PPP. Even when PPP activity is increased, RCCs are unable tomaintain their GSH levels during storage 40,53 . Total GSH and GSSG levels decrease over time independent of the additive solution 40,47,53 . GSH levels decrease rapidly during storage, and most studies reported decreased GSSG levels as well. However, one study showed increased GSSG levels 39 . Nevertheless, in literature there is consensus that the GSH/GSSG ratio decreases over time in RCCs 39,40,53 . This ratio is seen as the best parameter for antioxidant capacity. Until now, AS-7 stored RCCs seemed to maintain GSH levels best, resulting in a higher GSH/GSSG pool compared to AS-3 RCCs 47 . The breakdown of GSH seemed to be similar to that in other additive solutions because no difference in breakdown products were observed. Thus, in AS-7 GSH biosynthesis is upregulated or the reduction of GSSG to GSH is increased 47 . Decreased GSH levels are the result of a combination of increased turnover, increased reduction into GSSG or decreased GSH synthesis. One study showed that in AS-3 stored RCCs there is an increase in the GSH turnover product 5-oxproline and accumulation of glutathionyl-cysteine, implicating increased consumption of GSH 53 . However, there are also indications that GSH synthesis is impaired. Reduced NADPH levels result in less re- duction of GSSG into GSH. Besides NADPH dependent conversion of GSSG into GSH, also the novo synthesis of GSH takes place 55,56 . In this ATP-dependent process, GSH is formed by three amino acids; cysteine, glutamate and glycine (see figure 1). This synthesis is decreased by 45%when stored in SAGM 57 . However, when extra amino acids were added, the de novo synthesis remained stable 57 . This suggests that decreased GSH is not only caused by lower ATP levels but also by decreased levels of intracellular substrates for de novo synthesis of GSH. However, the availability of the three precursors amino acids does not decrease during storage. On the contrary, one study has shown that the levels of all three precursors for GSH are increased during storage 41 . Another study reported also increased glycine and cysteine levels during storage while the third GSH precursor glutamate decreased compared to baseline levels 36 .