Tetrahydrobiopterin is synthesized by separate pathways from dihydroneopterin triphosphate and from sepiapterin in adrenal medulla preparations.

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 40-60% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.