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The main aim of this study was to improve post-ischemic ATP recovery by (i) increasing the availability of PRPP and/or by (ii) increasing the availability of the free purine bases hypoxanthine and adenine. This was achieved with simple manipulations, which are known to be tolerated in humans. Therefore, results of this study may provide important information for development of new treatments for stroke patients.

1.5.1 D-Ribose to increase the available PRPP pool

As in brain, brief periods of ischemia produces a rapid depletion in myocardial ATP levels (Zimmer, 1992). Likewise, the restoration of post-ischemic ATP levels is slow, due to the heart’s dependence on the purine salvage pathway and the loss of salvageable purine metabolites to the systemic circulation (Zimmer, 1992, 1998; Pauly et al., 2003). It has been shown that due to the weak capacity of the oxidative PPP, the available pool of PRPP is small and limits post-ischemic cardiac adenine nucleotide synthesis via de novo and salvage pathways (Zimmer, 1998). Therefore, elevating the PRPP pool by bypassing the rate limiting step of the oxidative PPP is a

34 possibility to enhance adenine nucleotide synthesis. This can be achieved by administration of D-ribose, a simple pentose sugar, which is directly phosphorylated to ribose-5-phosphate by ribokinase (Park et al., 2007), and subsequently phosphoribosylated to PRPP by PRPP synthetase.

In fact, addition of D-ribose increases myocardial PRPP levels and efficiently improves postischemic recovery of the myocardial ATP in rat hearts by enhanced purine salvage as well as de novo synthesis (Zimmer and Gerlach, 1978; Zimmer, 1992, 1996, 1998). Accordingly this metabolic approach has been utilized in different experimental in vivo models: for example during myocardial infarction in rats (Zimmer, 1982; Zimmer et al., 1989) or after global myocardial ischemia in dogs (St Cyr et al., 1989), where it has been shown to enhance the functional recovery of the heart.

Ribose is known to be tolerated in humans (Gross and Zollner, 1991; Pauly et al., 2003) and administration of 10 mmol/kg per day has has no adverse effects in humans (Salerno et al., 1999). In patients with coronary artery disease D-Ribose improved cardiac function, exercise tolerance and general quality of life (Pliml et al., 1992). Ribose has also been given to a patient with fibromyalgia, a disease which is correlated with reduced ATP levels in muscles, where it decreased certain symptoms such as muscle pain, weakness or sleeping disturbances (Gebhart and Jorgenson, 2004).

In rat brain extracts ribose is phosphorylated to ribose-5-phosphate and subsequently PRPP, thereby implying the presence of ribokinase (Mascia et al., 2000; Barsotti and Ipata, 2002). Therefore – similar to the heart - increasing the available PRPP pool for APRT and HGPRT might help brain cells to recover their ATP pools faster after periods of ischemia and may improve functional recovery as well as cell viability. Therefore the D-Ribose administration will be tested as a potential neuroprotective intervention for the post-ischemic in vitro brain tissue.

35 1.5.2 Xanthine oxidase inhibition to increase available hypoxanthine levels

As explained above, the conversion of hypoxanthine to xanthine and uric acid, catalysed by xanthine oxidase, also contributes to a reduction of the TAN pool, since neither of these metabolites can be used in the purine salvage pathway. In rat forebrain following focal cerebral ischemia (Kanemitsu et al., 1988), as well as in cerebrospinal fluids of human stroke patients (Stover et al., 1997) xanthine and uric acid levels are increased, suggesting a role of xanthine oxidase in the degradation of hypoxanthine. Therefore, another possibility to enhance the purine salvage pathway could be to inhibit xanthine oxidase, and thereby increase the available hypoxanthine pool for HGPRT. The xanthine oxidase inhibitor allopurinol is known to be well tolerated in humans, since it is a commonly used drug to treat gout (Pacher et al., 2006).

This approach of inhibiting xanthine oxidase has already been studied in in vivo

models of cerebral ischemia and pre-treatment of rodents with oxypurinol, the active inhibitor, has been shown to enhance the postischemic recovery of adenine nucleotide levels in the brain (Phillis et al., 1995). In other experiments pretreatment with allopurinol increased adenosine and inosine levels in cerebral cortex of newborn piglets, which was believed to be responsible for the protective effects of allopurinol during ischemia (Marro et al., 2006). Therefore, allopurinol will be used for comparative studies to D-ribose and its effect on postischemic recovery of cellular ATP levels and synaptic transmission will be studied.

1.5.3 Administration of the purine base adenine to enhance APRT activity

I further tested whether addition of the free purine base adenine can help to improve post-ischemic ATP recovery by enhancing the activity of APRT. In rat heart it has been shown that adenine is effectively converted to ATP and can further enhance the D-ribose mediated purine salvage (Brown et al., 1985; Zimmer, 1996; Kalsi et al., 1998). Likewise high concentrations of adenine can improve the post-ischemic recovery of ATP levels in brain slices (Newman et al., 1998).

36 Adenine has been given to humans suffering from Lesh-Nyhan syndrome (Schulman et al., 1971). However, adenine has to be administered in combination with allopurinol, to avoid its degradation by xanthine oxidase to an insoluble metabolite, 2,8-dihydroxy-adenine, that can cause the formation of kidney stones (Greenwood et al., 1982). Therefore it will be tested, whether adenine and adenine/allopurinol administration is an effective strategy to improve post-ischemic recovery of ATP and synaptic transmission, as well as cell viability.

Other work in this field attempted to maintain nucleotide levels by pre-incubation with creatine, to increase phospho-creatine levels, and thereby delay the degradation of ATP during ischemia (Balestrino et al., 1999; Balestrino et al., 2002). Therefore, experiments with creatine will be used for comparative purposes, but the main focus of this study will be on D-Ribose, allopurinol and adenine supplementation.