Sepiapterin reductase in human amniotic and skin fibroblasts, chorionic villi, and various blood fractions

Clbtica C&mica Acta, 271 (1988) 271-282 Elsevier CCA 04165

Sepiapterin reductase in human amniotic and skin fibrob~asts, chorionic villi, and various blood fractions Juan Ferre a and Edwin W. Naylor b a Department of Genetics, Faculy of Biological Sciences, University of Valencia, Burjasot, Valencia (Spain) and b Department of Medical Genetics, West Penn Hospital, Pittsburgh, PA (USA) (Received 10 September 1987; revision received 12 February 1988; accepted after revision 16 February 1988) Key words: Sepiapterin reductase; Biopterin; Amniocytes; Chorionic vi& N-acetylserotonin

inhibition

Sepiapterin reductase activity has been measured in amniotic fibroblasts by two procedures: one photometric and the other HPLC-~uo~et~c. Both can be used for quantitative measurements, but the latter has considerable advantages including smaller standard deviation, much lower detection limit, and less volume of sample required. Sepiapterin reductase activity was also assayed in skin fibroblasts, chorionic villi and various blood fractions including stimulated mononuclear blood cells. Red blood cells have a low specific activity compared to unstimulated mononuclear blood cells, although the latter have a mean value with a high standard deviation. When the mononuclear blood cells were cultured for 5 days, the mean specific activity increased and the range became tighter. Enzyme stability and Nacetylserotonin inhibition were also studied.

Introduction

The enzymes phenyl~~ne hydroxylase, tyrosine hydroxylase, and t~tophan hydroxylase have significant metabolic roles in mammals including neurotr~s~tter biosynthesis. These three enzymes require tetrahydrobiopterin (BH,) as a cofactor in vivo [l], and a deficiency in its biosynthesis leads to impaired hydroxylation of the aromatic amino acids. In humans, a deficiency of BH, gives rise to a variant

Correspondence to: Dr. J. Ferre, Department of Genetics, Faculty of Biological Sciences, University of Valencia, Av. Dr. Moliner SO, 46100 Buqjasot, Valencia, Spain. OOOS-8981/88/$03.S0 Q 1988 Elsevier Science Publishers B.V. (Biomedical Division)

272 Guanosine Triphosphate 0 I Dihydron~pterin Tdp~sphate

4 Pyruvol-tetrahydropterin

1‘-H~roxy-~~x~ropyltet~hydropterin

Laces-tetrahydropterjn

Phenylalanine Tyrosine Tryptophan

Tyrosine / DOPA SOti-Tryptophan I

Tetrahydrobiopterin

kquinoid

Dihydrobiopterin/

Fig. 1. Proposed pathway for the biosynthesis of tetr~ydrobiopte~ in vivo showing its participation in the hydroxyIation of the aromatic amino acids. 1, GTP cyclohy~ol~; 2, p~voyl-tetr~ydropte~n synthase; 3, sepiapterin reductase; 4, pyNvoyl-tetr~y~opte~n reductase; 5, dihydropteridine reductase; 6, 7 and 8, phenylalanine, tyrosine and tryptophan hydroxylases.

form of phenylketonuria with somewhat more severe symptoms, which cannot be prevented by a low phenyl~~ne diet [Z]. Since 1975, several inborn errors of BH, metabolism have been discovered which initially presented with hyperphenylalaninemia. These have included cases with dihydropteridine reductase (EC 1.6.99.7) deficiency [3]; systemic, peripheral and partial BH, biosynthesis defects due to 6-pyruvoyl-tetrahydropterin synthase deficiency [4-61; and GTP cyclohydrolase (EC 354.16) deficiency [7,8] (Fig. 1). Sepiapte~ reductase (EC 1.1.1.153) is one of the enzymes involved in the biosynthesis of BH, from dihydroneopterin triphosphate, in both the de novo and the salvage pathways [9-111. Although a deficiency in sepiapterin reductase has not as yet been reported in a clinically affected patient, it seems likely that a defect in this enzyme will be found in the future. For this reason we have determined the activity of sepiapterin reductase in several tissues suitable for diagnosis, including amniotic fibroblasts and chorionic villi for prenatal diagnosis. Materials and methods

Chang Medium was obtained from Hana Media, Inc. {Berkeley, CA, USA). N-Acetylserotonin and RPM1 1640 culture media from Sigma (St. Louis, MO,

213

USA). Sepiapterin was obtained from Schircks Laboratories (Jona, Switzerland), phytohemagglutinin (PHA) from Wellcome Reag. Ltd. (Dartford, Kent, UK), antibiotic-antimycotic mixture (100 x ) from Gibco (Chagrin Falls, OH, USA), fetal bovine serum from HyClone Lab. Inc. (Logan, UT, USA), Ficoll-Paque and Sephadex G-25 from Pharmacia (Uppsala, Sweden), and C,, Sep-Pak cartridges from Waters Assoc. (Milford, MA, USA). Preparation of enzyme extracts from amniotic fibroblasts, skin fibroblasts and chorionic villi

Amniotic fibroblasts were obtained by culturing amniocytes (obtained at 14-18 wk gestation) in Chang Medium. When cultures were confluent (in 75 cm* flasks), cells were trypsinized, washed with 0.1 mol/l KH,PO,/O.lS mol/l KC1 (pH 7.4) and suspended in 2 ml of 0.1 mol/l Tris-HCl buffer (pH 7.8) containing 0.15 mol/l KCl, 0.68 mol/l glycerol and 2.5 mmol/l EDTA. Cell membranes were disrupted by freezing and thawing three times in an acetone-dry ice bath. After centrifugation at 15 000 x g for 2 min at room temperature, the supernatant was stored at -40°C until analyzed. For inhibition studies with N-acetylserotonin, the supematant was passed through a small Sephadex G-25 column (1.5 ml bed volume) equilibrated in the above Tris buffer using the procedure of Neal and Florini [12]. Skin fibroblasts were prepared in the same way as described for amniotic fibroblasts but starting from cells obtained from skin biopsies. Chorionic villi were obtained from spontaneous abortions at 10 and 18 weeks gestation. Samples were stored frozen at -40°C until used. Approximately lo-20 mg of villi was dissected, washed in 154 mmol/l NaCl and suspended in 0.5 ml of the Tris buffer. Disruption of the cell membranes was carried out in a glass homogenizer. The homogenate was centrifuged at 15 000 X g for 2 min at room temperature and the supematant loaded on a Sephadex G-25 column (1.5~ml bed volume) equilibrated in the same buffer. The eluate was immediately used for the enzyme assay. Preparation of blood fractions

Heparinized venous blood (4-8 ml) was centrifuged at 500 x g for 10 min at room temperature. Portions (1 ml each) of plasma and red blood cells were saved and the remaining blood fractions were mixed again. Mononuclear blood cells were separated from whole blood (4-8 ml) in Ficoll-Paque. Portions of 2 ml of whole blood were mixed with 2 ml of 154 mmol/l NaCl and the mixture layered on top of 3 ml of Ficoll-Paque in a centrifuge tube. After centrifugation at 400 X g for 30 min at room temperature, the layer containing the separated mononuclear blood cells was collected and washed twice with 3 vol of 154 mmol/l NaCl. Hemolysis of red blood cells was performed as described by Katoh et al [13]. Lysis of mononuclear blood cells was carried out as follows: cells were suspended in 0.5 ml of 0.1 mol/l Tris-HCl buffer (pH 7.8) containing 0.15 mol/l KCl, 0.68 mol/l glycerol and 2.5 mmol/l EDTA, and freeze-thawed three times in an acetone-dry ice bath. Cell debris was eliminated by centrifugation at 15 000 X g for 2 min at room temperature. Prior to analysis, plasma, red cell hemolysate and

274

mononuclear cell lysate samples were passed through small Sephadex G-25 columns (1.5 ml bed volume) equilibrated in the above buffer.

The procedure of Blau et al [14] was followed with minor modifications. The culture medium was RPM1 1640 containing 24 mmol/l sodium bicarbonate, antibiotic-antimycotic mixture (5 ml/l) (the mixture consisted of 10000 U penicillin, 10000 pg streptomycin and 25 pg Fungizone/ml), 0.1 mmol/l mercaptoethanol, fetal bovine serum (200 ml/l), PI-IA (20 mg/l) and HCI to bring the pH to 7.2. Mononucle~ blood cells separated as described above, were suspended (0.5 x lo6 cells/ml) in the medium in conical bottom sterile tubes (Coming, 15 ml). Culture tubes, containing 5 ml of culture medium, were kept horizontal in an incubator at 37°C and 5% CO2 for 5 days. Culture tubes were then centrifuged and the cells pooled and washed with 154 mmol/l NaCl. Cells were suspended in 0.5 ml of the above Tris buffer and the same procedure was followed as for non-stimulated mononuclear blood cells. Sepiapterin reductase assays Sepiapterin was purified in Cis Sep-Pak prior to use [15]. This step eliminates degradation products that could inhibit sepiapterin reductase, especially 6-pterincarboxylic acid 1161.Care was also taken to avoid exposure of sepiapterin solutions to direct light. Protein determination was carried out using the method of Bradford v71. Photometric assay This assay is a modification of the procedure of Katoh [16]. The reaction mixture contained 0.1 mol/l KH,PO, buffer (pH 6.8), 50 pmol/l sepiapterin, 100 ,umol/l NADPH and 435 ~1 of sample, in a final volume of 750 yl (this dilutes the buffer in which the sample was prepared to a final con~ntration of 58 mmol/l Tris, 87 mmol/l KCl, 0.39 mol/l glycerol and 1.45 mmol/l EDTA). Controls without NADPH were run simultaneously with each sample. The reaction was started with the addition of sepiapterin and incubation was carried out at 37 o C for 2-4 h in the dark. The reaction was stopped by boiling the mixture for 1 min. After removing the precipitated proteins by cent~fugation at 15 000 X g for 4 min at room temperature, 100 ,el of 1 mol/l KOH was added to the supematant and the absorbance read at 440 nm in a Varian model DMS 70 spectrophotometer. The difference in absorbance between the complete reaction mixtures and their respective controls is a measure of the sepiapterin reductase activity. High-performance liquid chromatography (HPLC)-fluorimetric assay The reaction mixture was the same as in the photomet~c assay described above but with 29 ~1 of sample and a final volume of 50 ~1. Incubation was performed at 37OC for 30 min in the dark. The reaction was stopped by adding 25 JLI of a mixture of 33 rmnol/l I,/100 mmol/l KI in 1 mol/l HCl. After 10 min at room temperature, the precipitated proteins were removed by centrifugation at 15 000 x g for 3 min at room temperature. The excess of iodine was reduced by addition of 25 ~1 of 57 mmol/l ascorbic acid. The fluorescence of the resulting biopterin was measured after separation by HPLC. The separation was carried out in a reversed phase C,,

215

Partisil-10 ODS column (25 x 0.46 cm, Whatm~) with an IBM LC/934 liquid chromatograph using 5% methanol in water as the mobile phase. Fluorescence was monitored at 362 nm for excitation and 435 nm for emission with a McPherson fluorescence detector model FL-749. The flow rate was kept at 1 ml/mm with a pressure of 90 atm. The chromatographic peaks were integrated using an IBM 9001 computer. Results Sepiapterin reductase activity in amniotic fibrobhsts

Incubation of an extract of amniotic fibroblasts with sepiapterin and NADPH gave rise to a decrease in absorbance at 440 nm due to the cons~ption of sepiapterin. Controls lacking enzyme extract or sepiapterin showed very little decrease in absorbance ( < 5%). The product of the reaction was identified as dihydrobiopterin (BH,) by its retention time in HPLC (monitoring for absorbance at 280 nm) and by the identification of biopterin as its oxidation product (Fig. 2). The conversion of sepiapterin into biopterin was found to be appro~ately 95%, calculated assuming an extinction coefficient for sepiapterin at 420 nm of 10400 (mol/l-’ per cm [18}. Sepiapterin reductase activity was linear up to 4 h using the photometric method and up to 45 min with the HPLC-fluorimetric method (Fig, 3). The precision of both methods was estimated from 6 replicates of the same amniotic fibroblast homogenate. For the photometric method the reaction mixture was incubated for 2 h and a specific activity of 200 f 25 pmol BH,/min per mg

RETENTION TIME (min)

Fig. 2. HPLC analysis of the sepiapterin reductase product in amniotic fibroblasts. (A), Complete reaction mixture without iodine oxidation; (B), complete reaction mixture after iodine oxidation; (C) and (D) reaction mixtnre without NADPH, with and witbout iodine oxidation respectively. Bp = biopterin.

20

40

60

INcUBATK)I\I

80 TIME

100

120

imn.)

Fig. 3. Time course of the sepiapterin reductase reaction using the photometric (A) and the HPLC-fluorimetric method (B). For the latter the values are the means of two measures. Amniotic fibroblasts were used as the source of the enzyme.

protein (mean A-SD) was found. For the HPLC-fluorimetric method the incubation was carried out for 30 min and the specific activity was 213 f 7 pmol BH,/min per mg protein (mean rt SD). The detection limit of the assay using the photome~c method is imposed by the extinction coefficient of sepiapterin and the minimum measurable change in the spectrophotometer. If a change of 0.001 absorbance units is considered significant, the amount of sepiapterin to be reduced to produce this change in a volume of 850 ~1 is 64 pmol (the extinction coefficient of sepiapterin at 440 nm in alkali is approximately 1.33 X lo4 (mol/l)-’ per cm). Using the HPLC-fluorimetric method, the detection limit is imposed by the sensitivity of the fluorescence detector. In our conditions we could integrate peaks corresponding to < 2 pmol of biopterin.

TABLE I Sepiapterin reductase activity in fibroblasts, chorionic villi and blood fractions Sample

Amniotic fibroblasts Skin fibroblasts Chorionic villi Plasma Red blood cells Mononuclear blood cells Stimulated mononuclear blood cells

Number of samples analyzed

Spec act (pm01 BH, /min per mg protein) Mean

SD

Range

21 6 7 10 26 30

170 201 137 0.08 0.92 27

76 48 98 0.04 0.39 28

95-315 120-263 43-334 0.04-0.16 0.33-1.86 o-91

26

115

29

67-177

211 2

E w Ii

0 5 u_ 0 z

015

010

0.05

g C CONCENTRATION OF PROTEIN Wn-4 Fig. 4. Sepiapterin reductase activity in amniocytes vs. concentra&on of protein in the reaction mixture.

The results presented in the rest of this paper were obtained with the HPLC-fluorimetric method (unless otherwise specified). A summary of the values obtained for 21 different amniotic fibroblast samples is shown in Table I. Dependence of sepiupterin reductase activity on the co~po~en?~ of the reaction mixture The enzyme activity was proportional to the concentration of protein in the assay in the range of protein used (Fig. 4). The dependence of sepiapterin reductase activity on the NADPH and sepiapterin concentration at the standard assay concentration of sepiapterin and NADPH respectively is shown in Fig. 5. The concentration of NADPH in the standard assay (100 pmol/l) is practically saturating, and the standard con~ntration of sepiapterin (SO ,umol/l), although not saturating, falls close to the asymptotic part of the curve.

40

.

t 50

I

,

100

150

[NADPH~ m4

I 200

20

40

60

[Sepiipterin] Wit

Fig. 5. Dependence of the sepiapterin reductase activity from amniocytes on the concentration of NADPH and sepiapterin in the assay. The reaction mixture was incubated for 10 min using (A) different concentrations of NADPH and 50 pmol/l sepiapterin, and (B) different concentrations of sepiaptexin and 100 pmol/l NADPH. BH, = dihydrobiopterin.

278

The effect of the Tris buffer (containing KCl, glycerol and EDTA) on the reaction was checked comparing the sepiapterin reductase activity in two samples from which an aliquot had been harvested in 0.1 mol/l potassium phosphate buffer (pH 6.8) and another aliquot in the Tris buffer as described in ‘Materials and Methods’. No difference in the specific activity of the aliquots of each sample was found (using the photometric method). Sepiapterin reductase in skin fibroblasts and in chorionic villi

Sepiapterin reductase activity was also found in skin fibroblasts and in chorionic villi. The specific activity in skin fibroblasts was comparable to that found in amniotic fibroblasts with a slightly higher mean value. The activity in chorionic villi showed considerable variation, ranging from 43 to 334 pmol BH,/min per mg protein (Table I). Sepiapterin reductase in blood fractions

Plasma, red blood cell hemolysates and mononuclear cell homogenates were assayed for sepiapterin reductase activity (Table I). The activity in plasma was around the detection limit of the assay. Red blood cell fractions, however, showed a measurable activity of 0.92 f 0.39 pmol BH,/min per mg protein (mean f SD).The sepiapterin reductase activity found in mononuclear blood cell homogenates was quite variable, ranging from O-91 pmol BH,/min per mg protein (27 f 28, mean f SD),with some samples showing almost no measurable activity. However, when cells from 26 different samples were cultured for 5 days with PHA, the sepiapterin reductase activity levels increased to a value of 115 f 29 pmol BH,/min per mg protein (mean f SD),with a much lower dispersion of the values. Stability of sepiapterin reductase in frozen samples

Sepiapterin reductase activity was stable -40’ C. Samples kept at this temperature when freshly prepared. With samples from pletely stable at least up to 15 days at sepiapterin reductase is not as stable with after 1 wk and 90% after 2 wk at - 40 ’ C. although a small percentage (approximately activity is lost upon freezing, the activity -20°C.

in amniotic fibroblast samples kept at for 1 month had the same activity as skin fibroblasts, the activity was com- 40° C. In chorionic villi, however, approximately 70% of the activity lost In stimulated mononuclear blood cells, 14%) of the initial sepiapterin reductase was stable for at least two weeks at

Inhibition of sepiapterin reductase by N-acetylserotonin

The effect of N-acetylserotonin on the reaction was checked since this compound has been reported to be a strong inhibitor of sepiapterin reductase from rat brain and rat erythrocytes [19]. The concentration of N-acetylserotonin that produced 50% inhibition (IC,,) was determined in Dixon plots (Fig. 6). The IC,, for amniotic fibroblasts was 1.5 pmol/l, 2.3 pmol/l for skin fibroblasts, 3.0 pmol/l for chorionic villi and 1.8 rmol/l for stimulated mononuclear blood cells. A complete inhibition of sepiapterin reductase activity was not achieved in any case, since at the highest

279

[N-acetylserotorin]

(@t)

Fig. 6. Dixon plots of sepiapterin reductase inhibition by N-acetylserotonin. The lines were adjusted by the least square method. The ICso value for chorionic villa was obtained using an appropriate ordinate scale. 0, Amniotic fibroblasts; l, stimulated mononuclear blood cells; 0, skin fibroblasts; a, chorionic villi.

concentration of N-acetylserotonin tested (200 pmol/l for stimulated mononuclear blood cells and 100 pmol/l for the other samples) there was still 3-1056 of the initial activity left. Discussion

Sepiapterin reductase was first discovered because of its involvement in the reduction of sepiapterin to BH, in rat liver extracts [20]. More recently it has been shown that the enzyme can catalyze the reduction of carbonyl moieties in a wide variety of compounds [21]. In fact, sepiapterin reductase is involved in the aromatic amino acid hydroxylating system catalyzing several steps in the biosynthetic pathway of BH, (Fig. 1). None of the intermediates in the biosynthesis of BH, are commercially available, and the three intermediates that are substrates for sepiapterin reductase are extremely unstable. For this reason, sepiapterin is normally used as the substrate of choice to assay sepiapterin reductase, even at the knowledge that probably the reduction of sepiapterin to BH, in vivo is of minor, if any, importance.

280

Two procedures have been used to measure sepiapterin reductase in amniotic fibroblasts. The photometric assay is based on the decrease of absorbance due to the consumption of sepiapterin. This change in absorbance is more intense and free of interferences when measured in alkali at 440 nm. The HPLC-fluorimetric method measures the product of the reaction after it has been oxidized to a highly fluorescent derivative (biopterin) and once separated from the other fluorescent components in the sample by HPLC. As a result, the latter method is much more sensitive and reliable since it measures the formation of the product in the reaction, instead of the disappearance of the substrate, which can be due to other causes not related to sepiapterin reductase activity. Furthermore, the HPLC-fluorimetric method has additional advantages over the photometric one, including smaller standard deviation, much lower detection limit and less volume of sample required. The presence of sepiapterin reductase has been reported in several mammalian tissues [16], including erythrocytes [22] and T-cells [23]. The activity has also been found in fetal erythrocytes [24] and we have demonstrated that it can also be measured in amniotic fibroblasts and chorionic villi for prenatal diagnosis [25,26]. For chorionic villi samples, a wide range has been obtained. Since chorionic villi sampling is becoming more and more used in prenatal screening for inborn defects, and sometimes replaces amniocentesis, the possibility of measuring sepiapterin reductase is of interest. The amniotic fibroblasts had a relatively high specific activity (Table I) that could be easily measured even using the photometric method. Screening for sepiapterin reductase deficiency can be performed either in skin biopsies or in blood samples. The specific activity of sepiapterin reductase in skin fibroblasts falls within the range obtained for amniotic fibroblasts, but with a somewhat higher mean value. The activity in plasma is extremely low (at the limit of detection with the HPLC-fluorimetric assay), and the possibility of contamination with enzyme from blood cells during the preparation procedure cannot be discarded. Red blood cells have a low specific activity compared to unstimulated mononuclear blood cells, but the latter have specific activities ranging from O-91 pmol BH,/min per mg protein, giving a mean value with a high SD (Table I). This wide range is probably due to the heterogeneity of the sample, containing different types of white cells in different proportions. When the mononuclear blood cell fraction is cultured in the presence of PHA, the cell population becomes more homogeneous (mainly T-lymphocytes), thus giving a mean value with a smaller standard deviation. Both types of cells, red blood cells and stimulated mononuclear blood cells, can be regarded as suitable sources for complementary assays for sepiapterin reductase. The advantage of using stimulated mononuclear blood cells is that the mean specific activity ‘is much higher, but on the other hand, the culturing procedure is more time consuming. Inhibition by N-acetylserotonin resulted in similar ICsO values for the enzyme from different sources. This suggests that the same enzyme (instead of a different isoenzyme) is produced in the different tissues. The IC,, values are slightly higher, although comparable, to that reported for sepiapterin reductase from rat erythrocytes (0.6 pmol/l) [21]. It is also interesting to note that even at the highest concentration of N-acetylserotonin used, the inhibition was not complete. In

281

contrast, it has be&n reported that this inhibitor can completely inhibit rat sepiapterin reductase, although in this case the authors used a much higher concentration of the inhibitor [19]. Acknowledgements

This work was supported in part by a grant from the US Department of Health and Human Services, Bureau of Health Care Delivery and Assistance (Project MCJ 9049). Support for J. Ferre was provided by a fellowship from the ‘Conselleria de Cultura, Educacio i Ciencia de la Generalitat Valenciana’ (Spain). We also wish to acknowledge M. Guetthoff for carrying out the cell cultures and some of the enzyme assays and D.C. Ennis for his technical assistance with the liquid chromatograph. References 1 Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Ann Rev Biochem 1985;54:729-764. 2 Kaufman S. Phenylketonuria and its variants. In: Harris H, Hirschhorn K, eds. Advances in human genetics, Vol. 13. New York: Plenum Press, 1983;217-297. 3 Kaufman S, Holtzman NA, Milstien S, Butler IJ, Krumholz A. Phenylketonuria due to a deficiency of diiydropteridine reductase. N Engl J Med 1975;293:785-790. 4 Bartholome K, Byrd DJ, Kaufman S, Milstien S. Atypical phenylketonuria with normal phenylalanine hydroxylase and dihydropteridine reductase activity in vitro. Pediatrics 1977;59:757-761. 5 Niederwieser A, Leimbacher W, Curtius HCh, Ponzone A, Rey F, Leupold D. Atypical phenylketonuria with ‘dihydrobiopterin syntbetase’ deficiency: absence of phosphate-eliminating enzyme activity demonstrated in liver. Eur J Pediatr 1985;144:13-16. 6 Hoganson G, Berlow S, Kaufman S, et al. Biopterin synthesis defects: problems in diagnosis. Pediatrics 1984;74:1004-1011. 7 Niederwieser A, Blau N, Wang M, Joller P, Atares M, Cardesa-Garcia J. GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia. Eur J Pediatr 1984;141:208-214. 8 Naylor EW, Ennis D, Davidson AGF, Wong LTK, Applegarth DA, Niederwieser A. Guanosine triphosphate cyclohydrolase I deficiency: early diagnosis by routine urine pteridine screening. Pediatrics 1987;79:374-378. 9 Nichol CA, Lee CL, Edelstein MP, Chao JY, Duch DS. Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo. Proc Nat1 Acad Sci USA 1983;80:1546-1550. 10 Milstien S, Kaufman S. Tetrahydro-sepiapterin is an intermediate in tetrahydrobiopterin biosynthesis. Biochem Biophys Res Commun 1983;115:888-893. 11 Milstien S, Kaufman S. Biosynthesis of tetrahydrobiopterin: conversion of dihydroneopterin triphosphate to tetrahydropterin intermediates. Biochem Biophys Res Commun 1985;128:1099-1107. 12 Neal MW, Florini JR. A rapid method for desalting small volumes of solution. Anal Biochem 1973;55:328-330. 13 Katoh S, Arai Y, Taketani T, Yamada S. Sepiapterin reductase in blood of various animals and of leukemic rats. Biochim Biophys Acta 1974;370:378-388. 14 Blau N, Joller P, Atares M, Cardesa-Garcia J, Niederwieser A. Increase of GTP cyclohydrolase I activity in mononuclear blood cells by stimulation: detection of heterozygotes of GTP cyclohydrolase I deficiency. Clin Chim Acta 1985;148:47-52. 15 Ferre J, Jacobson KB. Use of reversed-phase C,, SEP-PAK cartridges for the purification and concentration of sepiapterin and other pteridines. J Chromatogr 1985;350:389-398.

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16 Katoh S. Sepiapterin reductase from horse Iiver: purification and properties of the enzyme. Arch Biochem Biophys 1971;146:202-214. 17 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utihzing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. 18 Tsusue M, Akin0 M. Yellow pterins in mutant lemon of silkworm and mutant sepia of D. melanogater. Zoo1 Mag 1965;74:91-94. 19 Katoh S, Sueoka T, Yamada S. Direct inhibition of brain sepiapterin reductase by a catecholamine and an indoleam;ne. Biochem Biophys Res Commun 1982;105:75-81. 20 Matsubara M, Katoh S, Akino M, Kaufman S. Sepiapterin reductase. Bicchim Biophys Acta 1966;122:202-212. 21 Sueoka T, Katoh S. Carbonyl reductase activity of sepiapterin in reductase from rat erythrocytes. Biochim Biophys Acta 1985;843:193-198. 22 Sueoka T, Katoh S. Purification and characterization of sepiapterin reductase from rat erythrocytes. Biochim Biophys Acta 1982;717:265-271. 23 Ziegler I, EIlwart J, Schwulera U. Pteridine synthesis during interaction of interhrkin 2 with T lymphocytes: modulator function in IL-2 signal transmission. In: Cooper BA, Whitehead VM, eds. Chemistry and biology of pteridines. Berlin/New York: Walter de Gruyter, 1986;209-212. 24 Niedenvieser A, Shintaku H, Hasler Th, et al. Prenatal diagnosis of ‘dihydrobiopterin synthetase’ deficiency, a variant form of phenyIketonuria. Eur J Pediatr 1986;145:176-178. 25 Ferre J, Naylor EW. Pteridine biosynthesis in human amniocytes and chorionic viIli. In: Cooper BA, Whitehead VM, eds. Chemistry and biology of pteridines. Berlin/New York: Walter de Gruyter, 1986;309-313. 26 Ferre J, Naylor EW. Sepiapterin reductase in human amniocytes, skin fibroblasts, chorionic vilIi and stimulated mononuclear blood cells. In: Curtius H-Ch, Blau N, Levine RA, eds. Unconjugated pterins and related biogenic amines. Berlin/New York: Walter the Gruyter, 1987;247-256.