Radioimmunoassay for biopterin in body fluids and tissues

ANALYTICAL

BIOCHEMISTRY

110,

182- 189 (1981)

Radioimmunoassay

for Biopterin

in Body Fluids and Tissues

TOSHIHARU NAGATSU, TOKIO YAMAGUCHI, TAKESHI KATO, TAKASHI SUGIMOTO,* SADAO MATSUURA,* MIKI AKINO,~ SHOICHIRO TSUSHIMA,$ NOBUHIKO NAKAZAWA, AND HIROSHI OGAWA~ Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227, Japan; *Department of Chemistry, College of General Education, Nagoya University, Nagoya 464, Japan; tDepartment of Biology, Tokyo Metropolitan University, Tokyo 158, Japan; and SDaiichi Radioisotope Laboratories, Ltd., Tokyo 103, Japan Received June 25, 1980 Specific antibodies against r-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against L-erythro-biopterin distinguished among L-erythro-tetrahydroor 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, D-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against L-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of L-eryrhro-biopterin with tyramine, 4-hydroxy-2-[2-(4hydroxyphenyl)ethylamino]-6-(L-eryrhro-l,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with lpsI as the labeled ligand for the radioimmunoassay. BPlz51-TYRA had similar binding affinity as the natural L-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP- ‘%TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro-, and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 ~1 of urine, but a pretreatment with a small Dowex SO-H+ column was required for serum, cerebrospinal fluid, and brain tissues.

L-eryrhro-Tetrahydrobiopterin [~-erythro2 - amino - 4 - hydroxy - 6 - (1,2 - dihydroxypropyl)-5,6,7,8-tetrahydropteridine] is the cofactor for three aromatic amino acid monooxygenases: phenylalanine hydroxylase (l), tyrosine hydroxylase (2,3), and tryptophan hydroxylase (4-6). These hydroxylases play an important role in the metabolism of aromatic amino acids into biogenic monoamines such as catecholamines (dopamine, norepinephrine, and epinephrine) and serotonin. Since these biogenie monoamines belong to the family of important intercellular messengers such as neurotransmitters or hormones, changes in biopterin concentrations may have sig0003-2697/81/010182-08$02.00/O Copyright AU tights

0 1981 by Academic Ress, Inc. of reproduction in any form reserved.

182

nificant physiological and pathological effects: Thus, hyperphenylalaninemia type III due to a deficiency of biopterin has recently been reported as a variant of phenylketonuria (7,8). The patients are supposed to have a defect in dihydrobiopterin synthesis and have such symptoms connected with the deficiency of catecholamines and serotonin. Therefore, assay of the biopterin cofactor in tissues and body fluids is now required in both basic and clinical studies. For this purpose biopt’erin has mainly been determined by a bioassay using Crithidia fuscicufuta (9). Although the method is sensitive and specifici’it is time

BIOPTERIN

183

RADIOIMMUNOASSAY

with 1251-tyramine, and have developed a highly sensitive radioimunoassay for Lerythro-biopterin in biological fluids including urine, blood, and cerebrospinal fluid, and in tissues such as the brain. MATERIALS

e -NHKH*)s-WOH f -NHlaC&CObWl#-*mi

w-w BP-CAPBSA

FIG. 1. Structures of L-eryrhro-5,6,7,8-tetrahydrobiopterin (l), quinonoid dihydrobiopterin (2), Lerythro-7,8-dihydrobiopterin (3), 4-hydroxy-6-(Lerythro-l,2-dihydroxypropyl)-2-methylthiopteridine (4a), L-eryrhro-biopterin (4b), 4-hydroxy-2-[2-(4hydroxyphenyl)ethylamino]-6-(L-eryrhro1,2dihydroxypropyl)pteridine (BP-TYRA) (4c) for lzJI labeling (BPJe51-TYRA) (4d), 6-[[4-hydroxy-6-(L-erythro-1,2dihydroxypropyl)-2-pteridinyllaminolhexanoic acid (BP-CAP) (4e), and t-eryrhro-biopterinyl-caproylbovine serum albumin (BP-CAP-BSA) (4f) for antiserum production.

consuming, and thus unsuitable to the assays of many samples. Several newly devised methods for the assay of biopterin or tetrahydrobiopterin by enzyme-radioassay (lo), gas chromatography-mass fragmentography (11)) or high-performance luquid chromatography (12,13), are still time consuming, and thus inapplicable to the screening of many samples. We prepared specific antibodies against biopterin, neopterin, and 6.7~dimethylpterin, and developed radioimmunoassay for these pterins (14). This new radioimmunoassay was applied to the determination of biopterin in urine (14). Although this new radioimmunoassay was simple and had high sensitivity (limit of detection, 5 pmol biopterin/tube) comparable to those of the above cited method, the sensitivity was not sufficient to measure biopterin in blood. We have synthesized a new radiolabeled ligand, a conjugate of L-erythro-biopterin

AND METHODS

Materials. All the pterins except folic acid were synthesized in our laboratory according to the known methods: the four isomers of biopterin (IS) [L-erythru-biopterm, D-eryythro-biopterin, L-three-biopterin, and D-three-biopterin], D-eryfhro-neopterin (16), 6-methylpterin (17), 6-hydroxymethylpterin (18)) and L-dihydroxyethylpterin (19). L-eryrhro-Tetrahydrobiopterin (1 in Fig. 1) and L-erythro-7,8dihydrobiopterin (3 in Fig. 1) were prepared by the chemical reduction of L-erythro-biopterin (4b in Fig. 1) (20). Folic acid was obtained from Sigma. The purity of the pterins was confirmed by their ultraviolet spectra, by paper chromatography, and by permanganate oxidation to the known pterind-carboxylic acid. The conjugate of L-erythro-biopterin with tyramine, 4-hydroxy-2-[2-#-hydroxyphenyl)ethylamino] - 6 - ( L -erythro - 1,2 - dihydroxyprolybpteridine (BP-TY RA;’ Fig. 1, 4c), was newly synthesized (21). A solution of 1.O g of 4-hydroxy-6-(L-erythro1,Zdihydroxypropyl)-2-methylthiopteridine (Fig. 1, 4a) (21), 3.0 g of tyramine, and 0.8 g of acetic acid in 10 ml of 50% aqueous 2methoxyethanol was heated at 100°C for 6 h. The solution, after being adjusted to pH l-2 with HCl, was fractionated on a Florisil column (3.5 x 40 cm): the column was washed first with 500 ml of 1 M formic acid and 300 ml of water, and then eluted with O-2% ammonia (1 liter). The intensely fluorescent fraction was evaporated to dryness under reduced pressure. The ’ Abbreviations used: BP-TYRA, conjugate of Leryrhro-biopterin with tyramine, 4-hydroxy-2-{2-(4-hydroxyphenyl)ethylamino]-6-(r-eryrhro-l,2-dihydroxyprolyl)pteridine; BP-CAP-TYRA, biopterinyl-caproyltyramide; BSA, bovine serum albumin.

184

NAGATSU

residue was extracted with 200 ml of 1% ammonia, and the extract was concentrated to about 80 ml. The concentrate was adjusted to pH 3 with formic acid to give 350 mg of bhydroxy-2-[2+hydroxyphenyl)ethylamino]-6(L-erythro-1,2dihydroxypropyl)pteridine (BP-TYRA; Fig. 1,4c) as yellow needles, which decomposed at 165°C when crystalhzed ‘from water. Elementary analysis: Calcd for C,7H19N50,.HzO: C, 54.39; H, 5.64; N, 18.66%. Found: C, 54.56; H, 5.62; N, 18.38%. Preparation

of the radiolabeled &and,

BP-[1251]TYRA. The conjugate of L-erythrobiopterin with tyramine (BP-TYRA; Fig. 1, 4 c) was iodinated, as described previously on iodination of biopterinyl-caproyl-tyramide (BP-CAP-TYRA) (14). Ten micrograms of BP-TYRA in 10 ~1 of NJV-dimethylformamide was added to 20 ~1 of 0.5 M phosphate buffer, pH 7.4. To this solution, 10 pg of Na1251 (1.2 mCi) and then 20 pg of chloramine-T in 10 ~1 of 0.05 M phosphate buffer, pH 7.4, were added. The solution as allowed to react at 25°C for 30 s and then the reaction was terminated by the addition of 40 pug of sodium metabisullite in 10 ~1 of 0.05 M phosphate buffer, pH 7.4. BP-[1251]TYRA (Fig. 1, 4d) formed was purified by preparative electrophoresis on cellulose acetate in 0.05 M phosphate buffer (pH 7.6). The iodinated compound was extracted by the phosphate buffer containing 5% bovine serum albumin. Preparation of immunogenic L-erythrobiopterinyl-caproyl -bovine serum albumin conjugate and immunization. L-erythro-

Biopterinyl-caproyl-bovine serum albumin conjugate [BP-CAP-BSA, Fig. 1, 4f] was synthesized, and using the conjugate in complete Freund’s adjuvant, the antiserum against biopterin was obtained in rabbits, by a similar method as described previously (14). Sixty-five microliters of ethylchloroformate was added to a solution of 6-[[4hydroxy&(L-erythro-1,2-dihydroxypropyl)2-pteridinyl]amino[hexanoic acid (BP-CAP; Fig 1, 4e) (22) (74 mg) and triethylamine (126 ~1) in 2 ml of NJV-dimethylformamide

ET AL.

at -5°C. After stirring for 15 min, the mixture was added to a solution of bovine serum albumin (114 mg) in 2.5 ml of 0.25 M NaOH at 0°C. The mixture was stirred at 0°C for 1 h and then at 25°C for 30 min, during which periods the solution was maintained at pH 9- 10 by addition of 1 M NaOH. Finally the solution was made strongly alkaline by addition of 1 M NaOH (2 ml) and kept at 25°C for 30 min. The alkaline solution was placed in a Celophane tube and dialyzed against running water for 24 h. The tube was immersed in aqueous 3% sodium acetate (300 ml) whose pH was brought to 4.5 to cause precipitation inside the tube. The precipitate was collected by centrifugation and lyophilized to give the 6-(bioopterinylamino)hexanoic acid- bovine serum albumin conjugate (BP-CAP-BSA; Fig. 1,4f) as colorless powder (95 mg). Quantitation of the moles of biopterin per mole of protein was determined to be 32 by uv spectrometry. The lyophilized conjugate was used for immunization of five New Zealand albino rabbits. The conjugate (1 mghabbit) in complete Freund’s adjuvant was injected intradermally, and after 1 month the animals received booster injections (0.4 mghabbit) four times in the same manner as above once a week, and the antiserum was collected 10 days after the last booster injection. Preparation of samples of urine, blood, cerebrospinal fluid, and tissues for radioimmunoassay of biopterin. Since human

urine contains biopterin, dihydrobiopterin, and tetrahydrobiopterin (23), the reduced biopterins were oxidized to biopterin before the assay either in 0.2 M HCl for the assay of total biopterin and in 0.2 M NaOH for measuring only biopterin and 7,8-dihydrobiopterin. To 100-500 ~1 of urine, a l/l0 volume of either 2 M HCl (or 2 M NaOH) and a l/10 volume of 2% 12-4% KI solution were added, and the solution was left for 1 h at room temperature in the dark. Oxidation was terminated by adding a l/l0 volume of a 2% ascorbic acid solution, and the mixture was lyophilized. The residue was dis-

BIOPTERIN

RADIOIMMUNOASSAY

185

volume of 0.02 M phosphate buffer, pH 7.5, and this solution was used for the radioimmunoassay. Radioimmunoassay procedure. In the entire assay procedure, 0.02 M phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin was used. One hundred microliters of the buffer, 100 ~1 of the sample, or 100 ~1 of the buffer containing 20. standard biopterin (from 0 to 100pmol), and IO. . 100 ~1 of 5000-fold diluted antiserum were 10-1 mixed, and the mixture was incubated at 37°C for 30 min. After cooling the mixture FIG. 2. Biopterin standard curve in radioimmunoat 4°C for 5 min, a solution of the above assay. (A) BP-%TYRA as @and; (B) L-eryrhro-bie pterinyl-caproyl-‘IJI-tyramide (BP-CAP-‘%I-TYRA) as prepared BPJz51-TYRA (100 ~1, 20,000 @and. (B) (cpm), bound BP-‘%TYRA (in (A)) or bound cpm) was added to each tube and the mixBP-CAP-~951-TYRA (in (B)) in the presence of various ture was left at 4°C for l-2 h. Antigen-antiamounts of bioptetin; Bo (cpm), bound BP-‘%TYRA (in body complex was precipitated by a second (A)) or bound BP-CAP-lPJI-TYRA (in (B)) in the absence antibody method (24) as follows: 100 ~1 of of biopterin; and N (cpm), nonspecific binding of BP-‘=Ianti-rabbit IgG was added to each tube and TYRA or BP-CAP-L251-TYRA in the absence of antiserum and biopterin. the mixture was left at room temperature for 10 min, followed by the addition of 500 ~1 of 4% Dextran T-70 (Pharmacia). The solved in a small volume of 0.02 M phos- mixture was shaken vigorously and then phate buffer, pH 7.5, containing 0.3% centrifuged at 2500 rpm at 4°C for 15 min. bovine serum albumin and used for the The supematant solution was removed and radioimmunoassay. radioactivity of the precipitate was counted Human serum samples were obtained by to determine BP-lz51-TYRA bound to venipuncture, placed at room temperature antibody (%) . for 30 min, and then centrifuged at 3000rpm for 10 min, and the serum was removed. RESULTS Tissues, lo-200 mg, were homogenized Standard Curve with 4 vol of 0.1 M phosphate buffer, pH 7.5. To fresh serum (l-5 ml) or tissue homogeThe standard curves for L-erythronate (50 ~1-2 ml) a l/4 volume of 2 M biopterin radioimmunoassay using either trichloroacetic acid, a l/IO volume of 5 M BP-1251-TYRA or BP-CAP-‘*1-TYRA as a HCl or 5 M NaOH, and a l/10 volume of a labeled ligand are shown in Fig. 2. The sen2% 1*-40/o KI solution were added, and the sitivity of the assay using the present mixture was left for 1 h at room temperature prepared l&and, BP-1351-TYRA, was about in the dark. Oxidation was terminated by 10times higher than the previously reported adding a l/10 volume of 4% ascorbic acid radioimmunoassay using BP-CAPJ%TYRA solution, and the mixture was centrifuged (14), and the limit of the sensitivity was at 3000 rpm for 10 min. The supematant 0.5 pmol/tube. The control% ligand bound was passed through a column of Dowex- specifically (Bo-N/T, Bo = bound labeled 50-H+ (0.5 x 1 cm). The column was ligand in the absence of biopterin, N = nonwashed with water (10 ml), and biopterin specific binding of labeled ligand in the abwas eluted with 1 ml of 1 M NH,OH. The sence of antiserum and biopterin, and T eluate was lyophilized, dissolved in a small = total labeled ligand) was 30%.

186

NAGATSU

ET AL. TABLE

1

PERCENTAGE CROSS-REACTION L-ERYTHRO-BIOPTERIN

OF P~ERINS ANTISERUM

Cross-reaction (%o)

Pterins

FIG. 3. Competition of binding of BP-*Z51-TYRA by various pterins with L-etythro-biopterin antiserum. (B) (cpm), bound BP-iZ51-TYRA in the presence of various amounts of the pterin ligands; Bo (cpm), bound BP-?-TYRA in the absence of the pterin ligands; and N (cpm), nonspecific binding of BP-‘% TYRA in the absence of antiserum and the pterin ligands. L-e-BP, t-erythro-biopterin; BPH,, L-erythro5,6,7,8-tetrahydrobiopterin; BPH*, L-etythro-7,8-dihydrobiopterin; DHEP, L-dihydroxyethylpterin; D-thBP, D-threo-biopterin; L-th-BP, L-threo-biopterin; D-e-BP, D-erythro-biopterin; 6-HMP, 6-hydroxymethylpterin; AHP, 2-amino-4-hydroxypteridine (pterin), D-e-NP, D-erythro-neopterin; 6MP, 6-methylpterin; 6CP, 6-carboxypterin; XP, xanthopterin; and FA, folic acid.

WITH

L-etythro-Biopterin L-etythro-5,6,7,8Tetrahydrobiopterin L-erythro-7,8Dihydrobiopterin L-Dihydroxyethylpterin D-threo-Biopterin L-threo-Biopterin D-erythro-Biopterin 6-Hydroxymethylpterin pterin D-erythro-Neopterin 6Methylpterin 6Carboxypterin Xanthopterin Folic acid

100 2.1 1.9 1.1 0.5 0.3 0.2 0.2 0.03 0.01 0.009 0.006 0.005 0.003

stereochemical structures of the side chain at the B-position.

SpeciJicity

Linearity,

The cross-reactivity of various ligands for the anti-r.-erythro-biopterin antibody was determined by preparing serial dilutions of all the pterin ligands in the radioimmunoassay system (Fig. 3). The L-erythrobiopterin equivalent was read off the standard curve and this value was divided by the amount of each pterin originally added and multiplied by 100 to give percentage cross-reaction (Table 1). The antiserum against L-erythro-biopterin distinguished among the stereochemical isomers of biopterin (D-erythro-, L-threo- and Dthree-biopterins), L-erythro-5,6,7,&tetrahydrobiopterin, L-eryrhro-7,kiihydrobiopterin), Derythro-neopterin, xanthopterin, folk acid, pterin, and other natural and unnatural pterins including 6-hydroxyethyl-, 6-hydroxymethyl-, 6-methyl-, and 6-carboxypterin. Therefore, the antiserum toward L-erythro-biopterin was highly specific; it distinguished aromatic pterin ring from the reduced pterin, and recognized the size and

Linearity of assay for biopterin in human urine is shown in Fig. 4. The total amount of biopterin was completely linearly proportional to the amount of urine used for the assay. The recovery of L-erythro-biopterin, Lerythro-7,8,-dihydrobiopterin, or L-erythro5,6,7,8-tetrahydrobiopterin added into human urine, serum, cerebrospinal fluid, rat brain, and human brain was nearly 100%

Recovery, and Reproducibility

Oo

I

2 Urlm

3 4 ( rl/tubm

5 I

FIG. 4. Linearity studies on the radioimmunoassay of total biopterin in human urine.

BIOPTERIN TABLE DISTRIBUTION

OFTOTAL ANDRAT

2 BIOP~ERIN IN HUMAN TISSUES Biopterin (mean k SEM)

Tissue

PmoYg creatinine 7.89 c 0.85

Human urine (13)

pmol/ml Human serum (7) Human cerebrospinal fluid (8)

25.5 +- 2.9 10.2 2 1.3 nmoYg tissue

Human brain (7) (caudate nucleus) Rat brain (6) (brain stem)

1.25 + 0.25 0.99 c 0.04

a Autopsy brains from control patients without a history of neurological disorders.

in this radioimmunoassay system, when oxidation was carried out in acid. Thus, tetrahydrobiopterin in test fluids was oxidized to biopterin by iodine in an acidic solution nearly quantitatively. In contrast, oxidation of tetrahydrobiopterin in an alkaline solution yielded biopterin at about 1%. The interassay reproducibility with replicates of the same urine sample containing 5.4 and 33.6 pmol of biopterin was 100 + 6.1% and 100 + 7.9% (SD for four determinations), respectively, and the precision was satisfactory. Physiological

187

RADIOIMMUNOASSAY

Data

Biopterin concentrations obtained by this radioimmunoassay in human urine, human serum, human cerebrospinal fluid, rat brain, and human brain are shown in Table 2. About two-thirds of the total biopterin in human urine was alkaline labile, indicating that about two-thirds of the biopterin in human urine was tetrahydrobiopterin or quinonoid dihydrobiopterin. This agrees with the results of Fukushima and Nixon by high-performance liquid chromatography

(12). The level of biopterin in serum was much lower than that in urine. Biopterine concentration in rat brain was similar to that in human brain. In a preliminary study, when rat or human brain was left at 23”C, the total biopterin levels decreased only by 5-10% after 1 h. DISCUSSION

The quantitative analysis of biopterin has been tried by using bioassay (9), radioenzymatic assay based on the cofactor activity for phenylalanine hydroxylase (lo), gas chromatography-mass fragmentography (1 l), and high-performance liquid chromatography (12,13). Bioassay of biopterin for a growth factor of Crithidia fasciculata was the most frequently used, because of its high sensitivity. But it is time consuming and not exclusive of other pteridines possessing the growth factor activity such as L-erythro-neopterin. The radioenzymatic assay is also fairly sensitive, but detects various tetrahydropterins which are active as a cofactor for phenylalanine hydroxylase, and the values obtained using the radioenzymatic method are known to be higher than those obtained by the bioassay (12). The other chemical methods are also time consuming. A gas chromatography-mass fragmentography method requires the silylation of pterins and thus seems inapplicable for the assay of pterins in tissues. A recently reported procedure using highperformance liquid chromatography-fluorometry is sensitive and applicable for the analysis of pterins in urine, blood, and tissues (12). But it is difficult to analyze many samples simultaneously by the chemical methods. The radioimmunoassay reported here has many advantages. First, it is highly specific for the natural L-erythro-biopterin, as compared with other methods such as bioassay, radioenzymatic assay or high-performance liquid chromatography. This radioimmunoassay can distinguish even among the

188

NAGATSU

stereochemical isomers of biopterin, which cannot be distinguished by other methods. Although the values in Table 1 and Fig. 3 show slight but significant cross-reactivity with some isomers, the potential for interference with the assay depends on the concentrations of these isomers in test fluids and tissues. Second, it is highly sensitive, and the total biopterin as well as reduced forms of biopterin in any body fluids and tissues can be assayed. Third, it is very simple and rapid. It was not necessary to preincubate the antiserum with 6,7dimethylpterinylcaproyl-tyramide to remove antibodies against caproic acid moiety of the biopterin conjugate antigen to increase the sensitivity as in our previous method with BP-CAPJ2”I-TYRA as the radiolabeled ligand. This can further simplify the radioimmunoassay procedure. Samples as many as hundreds can be analyzed in a day, and this method is applicable to body fluids such as urine, serum, and cerebrospinal fluid and to any tissues. We have reported that the biopterin values obtained by our radioimmunoassay showed a, fairly good agreement with the values obtained by bioassay (14). Biopterin concentrations in human urine and blood and in rat brain, obtained by this radioimmunoassay are also in good agreement with those by high-performance liquid chromatography by Fukushima and Nixon (12). The concentrations of active biopterin cofactor, i.e., tetrahydrobiopterin plus quinonoid dihydrobiopterin, can be measured as the difference between the values obtained by oxidation in acid, and those in alkaline: the values obtained by oxidation in acid give total biopterin (biopterin, 7,8-dihydrobiopterin, quinonoid dihydrobiopterin, and tetrahydrobiopterin) (Fig. 1) and those by oxidation in alkaline give only biopterin and 7,8dihydrobiopterin, because tetrahydrobiopterin and quinonoid dihydrobiopterin convert to pterin but not to biopterin by alkaline oxidation (12). This radioimmunoassay is very useful not

ET AL.

only for biochemical, physiological, and pharmacological studies on the pterin cofactor of aromatic amino acid hydroxylases, but also for the screening of atypical phenylketonuria due to bipterin deficiency (7,8) and for the studies of pathogenesis of various diseases such as nervous and mental diseaseswhere some abnormality in biopterin metabolism may occur. In fact, we have found that in the striatum of parkinsonian human brain both biopterin concentrations and tyrosine hydroxylase activity were greatly reduced as compared with age-matched control human brains (25). REFERENCES 1. Kaufman, S. (1963) Proc. Nat. Acad. Sci. USA 50, 1085-1092. 2. Nagatsu, T., Levitt, M., and Udenfriend, S. (1964) J. Biol. Chem. 239, 2910-2917. 3. Brenneman, A. R., and Kaufman, S. (1964) Proc. Nat. Acad. Sci. USA 17, 177-183. 4. Jequir, E., Robinson, D. S., Lovenberg, W., and Sjoerdsma, A. (1969) Biochem. Pharmacol. 18, 1071-1081. 5. Ichiyama, A., Nakamura, S., Nishizuka, Y., and Hayaishi, 0. (1970) J. Biol. Chem. 245, 1699-1709. 6. Friedman, P. A., Kappelman, A. H., and Kaufman, S. (1972) J. Biol. Chem. 247, 41654173. 7. Kaufman, S., Berlow, S., Summer, G. K., Milstien, S., Schulman, J. D., Orloff, S., Speilberg, S., and Pueschel, S. (1978) New Engl. J. Med. 299, 673-679. 8. Niedenvieser, A., Curtius, H.-Ch., Bettoni, O., Bieri, J., Schircks, B., Viscontini, M., and Schaub, J. (1979) Lancer 1, 131-133. 9. Dewey, V. C. and Kidder, G. W. (1971) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 18B, pp. 618-624, Academic Press, New York. 10. Guroff, G., Rhoads, C. A., and Abramowitz, A. (1%7) Anal. Biochem. 21, 273-278. 11. Riithler, F., and Korobath, M. (1976) C/in. Chim. Acta 69, 457-462. 12. Fukushima, T., and Nixon, J. C. (1980) Anal. Biochem. 102, 176-188. 13. Stea, B., Halpem, R. M., kalpem, B. C., and Smith, R. A. (1980) J. Chromatogr. 188, 363-375.

14. Nagatsu, T., Yamaguchi, T., Kato, T., Sugimoto, T. Matsuura, S., Kobayashi, K., Akino, M.,

BIOPTERIN

15. 16. 17. 18. 19. 20.

RADIOIMMUNOASSAY

Tsushima, S., Nakazawa, N., and Ogawa, H. (1979) Proc. Japan Acad. 55, Ser. B., 317-322. Sugimoto, T., and Matsuura, S. (1975)BufI. Gem. Sot. Japn. (1975) 48, 3767-3768. Numata(Sudo), Y., Ikuta, K., Kato, T., Nagatsu, T., Sugimoto, T., and Matsuura, S. (1975) Biothem. Pharmacol. 24, 1998-2000. Armareg, W. L. F., and Shou, H. (1977) J. Chem. Sot. Parkin I 2529-2536. Viscontini, M., and Furuta, Y. (1973) Helv. Chim. Acta 56, 1710-1715. Sugimoto, T., and Matsura, S. (1979) BUN. Chem. Sot. Japn. 52, 181-183. Kaufman, S. (1%7)J. Biof. Chem. 242,3934-3943.

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21. Sugimoto, T., Matsuura, S., and Nagatsu, T. (1980) Bull. Chem. Sot. Japn. 53, 2344-2347. 22. Sugimoto, T., Shibata, K., Matsuura, S., and Nagatsu, T. (1979) Bull. Gem. Sot. Japn. 52, 2933-2937. 23. Fukushima, T., Kobayashi, K., Eto, I., and Shiota, T. (1978) Anal. Biochem. 89, 71-79. 24. Morgan, C. R., and Lazarow, A. (1%3) Diaberes 12, 115-126. 25. Nagatsu, T, Yamagushi, T., Kato, T., Sugimoto, T., Matsuura, S., Akino, M., Nagatsu, I., Iizuka, R., and Narabayashi, H. (1980) Abstract, 12th Collegium Intemationale Neuro-Psychopharmacologicum.