Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: An implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

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.