In order to increase the tissue level of tetrahydrobiopterin (BH
4), supplementation with 6
R-tetrahydrobiopterin (6RBH
4) has been widely employed. In this work, the effectiveness of 6RBH
4 was compared with 7,8-dihydrobiopterin (7,8BH
2) and sepiapterin by administration to mice. Administration of 6RBH
4 was the least effective in elevating tissue BH
4 levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH
4 treatment suggested that systemic oxidation of the administered BH
4 had occurred before accumulation of BH
4 in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH
4 was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH
2 to BH
4. 2) When the unnatural diastereomer 6SBH
4 was administered to mice, a large proportion of the recovered BH
4 was in the form of the 6
R-diastereomer, suggesting that this BH
4 was the product of a dihydrofolate reductase process by which 7,8BH
2 converts to 6RBH
4. These results indicated that the exogenous BH
4 was oxidized and the resultant 7,8BH
2 circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH
2 in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by
de novo synthesis and by salvage of extracellular dihydrobiopterin.
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