We previously proposed a novel disease entity, tetrahydrobiopterin (BH 4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH 4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375-378]. Subsequent reports indicate that the prevalence of BH 4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH 4, little is known about the mechanism of BH 4 responsiveness. An early report indicates that BH 4 concentration in rat liver was 5 μM where K m for BH 4 of rat PAH was estimated to be 25 μM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH 4 in liver in vivo. In the present study, we developed a breath test for mice using [1- 13C] phenylalanine in order to examine the BH 4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pah enu2. BH 4 supplementation significantly enhanced 13CO 2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH 4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH 4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH 4. Thus, we propose a possible hypothesis that the responsiveness to BH 4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH 4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.
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