The present study has validated kinetically a convenient method to measure tolbutamide hydroxylation capacity in human beings by use of urinary metabolic ratios. The known in vivo and in vitro inhibitory properties of sulfaphenazole were used to convert control phase subjects to phenotypically "poor" metabolizers of tolbutamide. Six healthy subjects were given a single 500 mg oral dose of tolbutamide with and without sulfaphenazole, 500 mg every 12 hours. Tolbutamide, hydroxytolbutamide, and carboxytolbutamide in urine were determined by newly developed HPLC procedures. Plasma tolbutamide clearance and half-life were measured, as were the metabolic ratio (hydroxytolbutamide + carboxytolbutamide/tolbutamide) in successive 6-hour urine collections. The mean tolbutamide plasma clearance decreased from 0.196 +/- 0.026 ml/min/kg without sulfaphenazole to 0.039 +/- 0.009 ml/min kg with sulfaphenazole, and the mean half-life of tolbutamide increased from 7.28 +/- 0.89 hours to 38.76 +/- 13.30 hours. The metabolic ratio determined in the 6 to 12 hour urine collection period decreased from 794.0 +/- 86.6 to 126.0 +/- 79.3, and this collection period also gave the best separation of subjects between phases. There was a good correlation between tolbutamide plasma clearance and metabolic ratio (rs = 0.853, p less than 0.01, n = 12) and between the percentage decrease in plasma tolbutamide clearance and the percentage decrease in metabolic ratio (r = 0.932, p less than 0.01, n = 6). The tolbutamide urinary metabolic ratio therefore effectively distinguishes tolbutamide hydroxylase activity in "normal" subjects and in those converted to model phenotypically "poor" metabolizers by sulfaphenazole.

Theaerobicdegradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed byperoxidase(EC 1.1 1.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3 H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(hH)pterin, and this finally rearranges t o 7,g-dihydroxanthopterin.Spectroscopic evidence (ultraviolet, ' H NMR and I3C N MR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3 H)pterin.The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a K,,, of 16.3 pM and k,,, of 22.5 s-I.The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6 R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural ( 6 s ) isomer.By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (hR)-'I,X-dihydro(6H)biopterin K, = 1.3 pM and k,,, = 22.8 s -' ; with (6S)-7,8-dihydro(6H)biopterin K,,, = 13.5pM and k,,, = 51.6s-'; and with (hRS)-7,%dihydro(6H)neopterin K, = 19.2pM and k,,, = 1 2 6 s y ' . (1) is the natural cofactor for the enzymes that hydroxylatc phenylalanine, tyrosine and tryptophan [l] in the presence of oxygen. The cofactor is oxidised to qui~onoid-7,8-BH~ ( 2 ) which is reduced enzymically by dihydropteridine reductase (EC 1.6.99.7) in the presence of NADH back to BH4 and restores the cofactor for further hydroxylation reactions. Whereas simple pterins, e.g. quinonoid-7,8-6-MPHz, which are effective substrates for dihydropteridine reductase, behave normally during kinetic rneasuremcnts, the oxidised natural cofactor ( 2 ) does not. Simple pterins give linear initial rate plots in the standard assay procedure for the uncoupled rcaction (i.e. without hydroxylase) when the quinonoid-7,8-dihydro(6H)-pterin is gencrated from the 5,6,7,8-tetrahydropterin by peroxidase together with hydrogen peroxide [2] or oxygen [3]. When using the same procedure, quinonoid-7,8-BH~ (2) gives biphasic initial rate plots consisting of a fast linear trace followed by a slower one from which two sets of kinetic parameters ( K , and V) can be derived. This is true also [or 5,6,7,8-Tetrahydrobiopterin (BH,)

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The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8‐dihydro(6H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8‐trimethyl‐5,6,7,8‐tetrahydro(3H)pterin was synthesised and the quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin derived from it is extremely stable with a half‐life in 0.1 M Tris/HCl (pH 7.6, 25°C) of 33 h. Quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin is not a substrate for dihydropteridine reductase but it is reduced non‐enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 μM, pH 7.6, 25°C when using quinonoid 6‐methyl‐7,8‐dihydro(6H)pterin as substrate. 6,6,8‐Trimethyl‐5,6,7,8‐tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.