Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates.

The biosynthetic pathway of tetrahydrobiopterin (BH(4)) from dihydroneopterin triphosphate (NH(2)P(3)) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH(2)P(3) was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH(4) to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH(4) biosynthesis under normal in vivo conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH(2)P(3) may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH(4) by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH(4) biosynthesis in vivo .

Effect of high-protein meal plus aspartame ingestion on plasma phenylalanine concentrations in obligate heterozygotes for phenylketonuria.

The effect of a protein-rich meal alone or in combination with 85 mumol/kg body weight aspartame (APM) on plasma phenylalanine and large neutral amino acids (LNAA) was evaluated in obligate heterozygotes for phenylketonuria (PKU) and normal subjects (controls). Thirteen PKU heterozygotes (seven women, six men) and 13 controls (five women, eight men) ingested a 12-noon meal providing approximately 303 mumol/kg Phe. In addition, 10 PKU heterozygotes (five women, five men) and 10 controls (five women, five men) ingested the same meal with 85 mumol/kg APM (providing 75 mumol/kg Phe). Plasma amino acids were analyzed at baseline (-4 and 0 hours) and at 1, 3, and 20 hours after the meal or meal plus APM. Compared with the meal alone, ingestion of the meal plus APM significantly increased plasma Phe concentrations in both controls and PKU heterozygotes. Mean plasma Phe values were higher for controls at 1 hour (95 +/- 7 mumol/L) and for PKU heterozygotes at 3 hours (153 +/- 21 mumol/L). After the addition of APM to the meal, the highest mean plasma Phe concentration was only slightly greater than the usual postprandial range for both controls and PKU heterozygotes. Ingestion of the meal did not increase the plasma Phe/LNAA ratio in either controls or PKU heterozygotes. Compared with baseline, the plasma Phe/LNAA ratio increased significantly 1 hour after combined ingestion of the meal plus APM in both groups (P = .020 and P = .008, respectively); however, the ratios were well below the range of Phe/LNAA values in individuals with mild hyperphenylalaninemia, who are clinically normal and do not require a Phe-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass fragmentographic determination of endogenous glycine and glutamic acid released in vivo from the pigeon optic tectum. Effect of electric stimulation of a midbrain nucleus.

Various investigations suggest glycine to be an inhibitory transmitter in the pigeon optic lobe in a pathway originating in the nucleus isthmi, pars parvocellularis (Ipc) terminating in the optic tectum. In order to obtain additional evidence for this hypothesis the in vivo release of endogenous glycine in the optic tectum upon electrical stimulation of Ipc was investigated. By perfusing the upper strata of the optic tectum with Ringer solution using a push-pull cannula endogenous amino acids released from the surrounding tissue were collected. Concentration of glycine and glutamic acid in the perfusates were determined by mass fragmentography of their N-pentafluoropropionyl hexafluoroisopropyl esters. Deuterium-labeled glycine and glutamic acid were used as internal standards for quantitative measurements. The resting release of glycine and glutamic acid was 2.9 pmol/min and 1.4 pmol/min, respectively. Electrical stimulation of Ipc was found to induce a 2--40-fold increase of the glycine efflux into the perfusate whereas the efflux of glutamic acid remained at a constant level. These findings strongly support the hypothesis that glycine is a transmitter in Ipc-tectal neurons.

Serotonin and dopamine synthesis in phenylketonuria.

Two regulation systems of the serotonin and dopamine biosynthesis in patients with classical and atypical PKU were investigated. In classical PKU, the serotonin and dopamine biosynthesis is inhibited by high L-phenylalanine in blood and tissues. The dopamine formation in vivo was inhibited by phenylalanine blood concentrations higher than 25 mg/dl: the serotonin formation was inhibited even at a phenylalanine blood concentration of only 8 mg/dl. In two patients with dihydrobiopterin synthetase deficiency, the dopamine, and even more pronounced the serotonin, excretions are considerably reduced. The dopamine excretion was reduced to about 50% and the serotonin excretion to only 10% compared to controls. Under BH4 therapy (16 mg daily), the dopamine values increased about twice, serotonin threefold and the phenylalanine blood concentration normalized to 1-1.5 mg/dl. On loading a patient with BH2 synthetase deficiency with 50 mg/kg deuterated tryptophan-d5 and 150 mg/kg deuterated tyrosine d2 (phenylalanine blood concentration of 16 mg/dl), deuterated dopamine d1 and serotonin d4 could only be formed in detectable amounts after BH4 administration. During BH4 therapy the amount of dopamine d1 and serotonin d4 formed was lower than but comparable to normal controls.

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver.

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.