Tetrahydrobiopterin deficiency and brain nitric oxide metabolism

Tetrahydrobiopterin is an essential cofactor for the aromatic amino acid monooxygenase group of enzymes. Inborn errors of tetrahydrobiopterin metabolism result in hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. Tetrahydrobiopterin is also a cofactor for all known isoforms of nitric oxide synthase (NOS). The effect of tetrahydrobiopterin deficiency on brain nitric oxide metabolism has to date been given little consideration. In this thesis the effect of tetrahydrobiopterin deficiency on brain nitric oxide metabolism has been studied using a mouse model of tetrahydrobiopterin deficiency, the hph-1 mouse. Tetrahydrobiopterin was measured in 10 and 30 day old mice in whole brain and cerebellum. At both age points there was a significant -50% reduction in tetrahydrobiopterin content for whole brain and cerebellum in the hph-1 mouse compared to corresponding control mice. NOS activity was measured in whole brain from 10 and 30 day old hph-1 and control mice. No difference was observed in enzyme activity when tetrahydrobiopterin was included in the incubation medium. However, omission of tetrahydrobiopterin from the reaction buffer resulted in significantly lower NOS activity in the hph-1 mouse group compared to controls. Tetrahy-drobiopterin was also shown to have a potent effect on the affinity of brain NOS for arginine. The Km for arginine was virtually identical for the control and hph-1 mouse when tetrahydrobiopterin was included in the reaction buffer. In the absence of cofactor, the for arginine was three fold greater for control and five fold higher for hph-1 preparations. The accumulation of cGMP from slices prepared from the cerebellum was measured in both groups of mice at both 10 and 30 days using the glutamate analogue, kainate. In the 10 day old hph-1 mouse there was a significant 50% reduction in cGMP levels under basal and stimulated conditions. In the 30 day old hph-1 mouse there was a significant 30% reduction in cGMP accumulation in both basal and activated states. Whole brain amino acid levels were measured. In the 10 day old hph-1 mouse confounding hyperphenylalaninaemia may affect the availability of the NOS substrate, arginine. In the 30 day old hph-1 mouse, which has normal phenylalanine levels, reduced citrulline levels may indicate reduced NOS activity. Reduced levels of tetrahydrobiopterin and also arginine, have been shown to lead to superoxide formation by nitric oxide synthase. Superoxide can react with nitric oxide to form the oxidising species, peroxynitrite, which has been shown to damage of the mitochondrial electron transport chain. Mitochondrial function together with the antioxidant, glutathione, were analysed in both hph-1 and control mice at 10 and 30 days to measure oxidative stress. However, no differences were observed between the two groups. In summary, partial deficiency of tetrahydrobiopterin appears to lead to impaired brain NOS function leading to an impairment of the nitric oxide/cGMP pathway