Polarization fluoroimmunoassay of biopterin and neopterin in human urine

Clinica Chimica Acta, 138 (1984) 275-282 Elsevier

275

CCA 02825

Polarization fluoroimmunoassay of biopterin and neopterin in human urine Makoto

Sawada a, Tokio Yamaguchi a, Takashi Sugimoto Sadao Matsuura b and Toshiharu Nagatsu a,*

b,

a L.aboratoty of Cell Physiology, Department of Life Chemistry Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227 (Japan); b Department of Chemistry College of General Education, Nagoya University, Nagoya 464 (Japan) (Received

September

lst, 1983; revision January

Key words: Fluorescence polarization;

Fluorescein

3rd. 1984)

-labeled;Biopterin; Neopterin

A polarization fluoroimmunoassay has been developed for the routine determination of biopterin and neopterin levels in human urine. The method employs fluorescein-labeled biopterin and neopterin. The assay is fast (incubation time: 2 min) and no separation step is required prior to measurement of fluorescence polarization. Linearity, recovery and precision were satisfactory. Estimations of biopterin and neopterin levels in human urine samples closely correlated with those obtained by radioimmunoassay.

Introduction

Three aromatic amino acid monooxygenases, phenylalanine hydroxylase [ 1,2], tyrosine hydroxylase [3,4], and tryptophan hydroxylase [5,6] are pterin-dependent, and play an important role for biosynthesis of biogenic amines, such as catecholamines and indolamines. Recent reports indicate that hyperphenylalaninemia as a variant of phenylketonuria is due to a deficiency of biopterin [7-91, and that the level of biopterin in the cerebrospinal fluid [lo] and brain [ll] of parkinsonian patients is lower than that of controls. On the other hand, 7JGdihydroneopterin triphosphate is an intermediate in the biopsynthesis of tetrahydrobiopterin [12-U], and the level of neopterin in patients with a deficiency of biopterin [8,9] and in cancer patients [16,17] has been reported to be higher than that of controls.

* To whom correspondence 0009-8981/84/$03.00

should be addressed.

0 1984 Elsevier Science Publishers

B.V.

276

A simple and rapid assay of biopterin and neopterin in human urine is required. High-performance liquid chromatography with fluorometry [18], bioassay by Crithidiu fmciculata [19] and radioimmunoassay [20,21] have been used for this purpose. High-performance liquid chromatography is most frequently used. It is a sensitive technique, and biopterin and neopterin can be measured at the same time. However, it is rather time-consuming for screening many samples. Bioassay is sensitive and specific, but also time-consuming. Radioimmunoassay is sensitive, specific and convenient for screening, but requires expensive reagents and involves the handling of radioactive materials. A relatively rapid method which has never been tried for the assay of biopterin and neopterin, is fluoroimmunoassay [22,23] in which a fluorescent molecule is substituted for a radioactive ligand used in radioimmunoassay. The advantages of fluoroimmunoassay are avoidance of radioactivity, a much longer lifetime of the assay kit, and less expensive equipment. In particular, recent reports (22,241 have indicated the advantages of polarization fluoroimmunoassay. Polarization of fluorescence results from the relative orientation of the excitation and emission dipoles which is affected by changes in size, rotational restrictions, conformation of the fluorescent molecules, solvent viscosity and temperature [22]. Polarization fluoroimmunoassay does not suffer from decreased sensitivity due to the variation and magnitude of the sample’s own fluorescence since it is based on measurement of changes in molecular size by antigen-antibody binding. We have developed a polarization fluoroimmunoassay technique for biopterin and neopterin, and applied this method to the determination of these pterins in human urine. Materials and methods Preparation of fluorescein-labeled biopterin and neopterin (Fig. 1) A solution of 4-hydroxy-6-L-erythro-1,2-dihydroxypropyl)-2-methylthiopteridine [25] (1.0 g) and 1,3-diaminopropane (1.0 g) in 50% aqueous 2-methoxyethanol

-!-L”, I

FITC-bloptrln

AHAH

CH2-w2-rH f=S NH

Fig. 1. Structure of fluorescein-labeled

FF -f: H

F--OH H

FIX-noptrrin

L-etyrhro-biopterin and meythro-neopterin.

(20

27-J

ml) was heated under reflux for 2 h. The solution was adjusted to pH 2-3 with formic acid and fractionated on a Florisil column (3.5 x 40 cm), using 3% ammonia as solvent. The eluate was evaporated to dryness and the residue was chromatographed on a Dowex 50WX 8 column (2 x 8 cm), eluted gradiently by O-5% ammonia [25]. Evaporation of the eluate gave a yellow viscousoil (0.9 g) which was chromatographically pure but did not crystallize. A part of the oil (0.1 g) was treated with acetic anhydride and pyridine in a conventional way to give ivory needles (80% yield) of 2-(3-acetylaminopropyl)-amino-4-hydroxy-6-(L-e~f~~~-l,2-dihydroxypropyl)-pteridine, m.p. 232-234”C, decomp. (recrystallized from ethanol) (Found: C, 50.25; H, 6.10; N, 24.85%. Calculated for C,,H,,N,O,: C, 50.00; H, 5.95; N, 25.00%); X,,,(log 0 in methanol: 238 (shoulder, 4.01), 280 (4.30), 352 (3.74) nm. A solution of the above oil (0.5 g), fluorescein isothiocyanate (0.5 g), and potassium carbonate (0.2 g) in 20% aqueous methanol (30 ml) was kept at room temperature for 10 h. The solution was adjusted to pH 3 with formic acid and chromatographed on a silica gel column (Wako Gel C-100, 3 x 40 cm) using a mixture of acetic acid, water, and 1-butanol (1: 1 : 4, v/v/v) as solvent. The eluate was evaporated to dryness and the residue was crystallized from methanol to give orange needles (0.21 g) of the fluorescein-labeled biopterin, which did not melt below 300°C (Found: C, 57.60; H, 4.55; N, 14.65%. Calculated for C,,H,,N,O,S: C, 57.97; H, 4.28; N, 14.34%). Aminolysis of 4-hydroxy-6-(D-erythro-l,2,3-trihydroxypropyl)-2-methylthiopteridine with 1,3-diaminopropane gave the 2-(3-aminopropyl)-aminopteridine (75% yield), which was similarly characterized as an acetamide. The amide melted at 205-207°C decomp. (recrystallized from methanol) (Found: C, 47.52; H, 5.73; N, 23.81%. Calculated for C,,H,,N,OS: C, 47.73; H, 5.86; N, 23.86%) and exhibited x max (log e) in methanol at 237 (shoulder, 4.01), 280 (4.30) and 352 (3.74) nm. Condensation of the amine with fluorescein isothiocyanate and isolation of the product in-an analogous way as above gave the fluorescein-labeled neopterin as orange needles (30% yield), m.p. not below 300°C (Found: C, 56.86; H, 4.46; N, 14.34%. Calculated for C,,H,,N,O,S: C, 56.65; H, 4.18; N, 14.01%). Preparation of anti-serum Anti-sera of biopterin and neopterin previously described [20,21]. Standard Reference standards of biopterin previously reported [20,21].

were raised in rabbits

and neopterin

and characterized

were synthesized

and purified

as

as

Specimens Since human urine contains dihydro- and tetrahydro-derivatives of biopterin and neopterin [18], the reduced derivatives of both biopterin and neopterin were oxidized by iodine in 0.2 mol/l HCl to the parent pteridines before the assay. To 500 ~1 of urine, a l/10 vol. of 2 mol/l HCl and l/10 vol. of 2% I,-4% KI solution were added in this order and the solution was left for 1 h at room temperature in the dark.

278

Oxidation was terminated by adding a l/10 vol. of a 2% ascorbate solution, and the mixture was lyophilized. The residue was dissolved in 500 ~1 of 0.02 mol/l phosphate buffer (pH 7.5), and used for the fluoroimmunoassay. Fluorometers

Two fluorescence depolarization analyzers, JASCO SFP-3 (Japan Spectroscopic Co., Hachioji, Tokyo 192, Japan) and FS-501 (Union Giken Co., Hirakata, Osaka 573-01, Japan) were used. Assay procedure

Throughout the assay procedure, 0.02 mol/l phosphate buffer (pH 7.5) was used. For routine assay, 100 ~1 of the sample, or 100 ~1 of the buffer containing standard biopterin or neopterin (from 0.1 to 10 nmol), and 100 ~1 of a FITC-biopterin or FITC-neopterin solution (3 mg/l; the final pterin content: 100 ng/ml) were mixed with 2.7 ml of the buffer. Then 100 ~1 of lOO-fold diluted anti-serum were added to give a final dilution of 1 : 3000 in a final vol. of 3.0 ml. After incubation at 25°C for at least 2 n-tin, the fluorescence polarization was measured. The percent polarization was calculated by using the equation: percent polarization = (P - Pblank/PO P,,,,) X 100, where P = polarization of each sample, Pblank = polarization of a FITC-pterin alone, and P,, = polarization in the absence of a pterin. The percent polarization was plotted against the logarithm of the concentration of standard pterins, and the biopterin or neopterin levels in urine samples determined from this standard curve.

ReSUltS Fig. 2 shows the antibody dilution curves of biopterin and neopterin, where the concentrations of FITC-biopterin and FITC-neopterin were kept constant at 100

0.3 -

0.2 1

\

0.1 -

lo Fig. 2. Antibody anti-serum (W-

loo

dllutbn (xlO0) dilution curves showing binding of FITC-ptetins n) and neopterin anti-serum (O0).

to serial dilution

of biopterin

219

0

I

0.1 Crnertnl

I

(nmol/tube)

Fig. 3. Standard curves showing decreasing percent polarization (_m) and neopterin (am).

with increasing levels of added biopterin

ng/ml. The anti-sera of biopterin or neopterin showed about 30% binding of FITC-biopterin and FITC-neopterin, respectively, at a dilution of 1: 3000 in final concentrations. In Fig. 3 typical assay standard curves of biopterin and neopterin are indicated. In these conditions, sensitivities of biopterin and neopterin are 0.05 nmol/tube and 0.03 nmol/tube, respectively. The two FITC-pterins gave similar

Fig. 4. Effects of FITC-neopterin concentration on standard curve of neopterin. 0 0, anti-serum 1: 2.4 x 10 4, FITC-neoptexin 25 q/tube; n -& anti-serum 1: 3.2 X 104, FITC-neopterin 20 ng/tube; O-O, anti-serum 1: 3.2 x 105, FITC-neopterin 2.5 &tube.

280 TABLE

I

Pterin content in human urine samples Pterin content (nmol/ml) neopterin

biopterin

5.13 0.41 1.40 0.71 2.13 1.73 1.48 3.96 0.57

3.49 2.20 1.37 1.61 2.00 1.96 2.20 2.56 0.97

curves at the same concentrations of biopterin and neopterin. It was found that the standard curve shifted to lower concentrations when the concentration of FITCpterins was lowered, as illustrated in Fig. 4 in the case of neopterin. The contribution to the fluorescence polarization signal made by the intrinsic fluorescence of urine samples or standards was found negligible, therefore the blank value ( Pblank) was given by fluorescence polarization of FITC-pterins alone. When the minimum incubation time was assessed by adding anti-sera to FITCbiopterin and FITC-neopterin in the presence of various amounts of biopterin and neopterin, fluorescence polarization increased and stability was reached within 2 min at 25°C. Linearity of assay for the pterins in human urine was observed from 50-500 ~1 urine, and recoveries of authentic biopterin and neopterin added to urine samples were 96% and 95% in this polarization fluoroimmunoassay. The inter-assay variation

(A)

FIA

( nmol/ml)

FIA

hnol/ml)

Fig. 5. (A) Correlation between neopterin levels in human mine samples as determined by polarization fluoroimmunoassay (FIA) and those by radioimmunoassay @IA). r = 0.93, p < 0.01, n = 23. -, line of the best fit. (B) Correlation between biopterin levels in human urine samples as determined by , line of the best fit. polarization FIA and those by RIA. r = 0.96, p -C0.01, n = 20.

281

(CV) with replicates of the same urine sample was within 6.0% (n = 12), and precision was satisfactory. Table I shows the biopterin and neopterin contents of urine samples measured by polarization fluoroimmunoassay. Normal ranges of pterins in human urine samples are 0.5-5 nmol/ml urine in neopterin and 0.5-3 nmol/ml urine in biopterin. The correlation between fluoroimmunoassay and radioimmunoassay [20,21] was satisfactory. The correlation coefficients (r) between the two assays of neopterin and biopterin were 0.93 (p -z 0.01, n = 23) and 0.96 (p -c 0.01, n = 20) respectively, as illustrated in Fig. 5, A and B. Discussion The polarization fluoroimmunoassay of urinary biopterin and neopterin has several practical advantages over established assay methods. The assay is fast, takes only 2 min to read the fluorescence polarization after mixing the sample, FITC-pterins and anti-serum. It is economical, and the specificity and accuracy are satisfactory. Furthermore, it does not require a separation step. One drawback is its relatively low sensitivity, approximately 30 pmol/tube, as compared to the sensitivity of radioimmunoassay (0.1 pmol/tube) [20,21] or bioassay (0.05 pmol/ml) [19], but it is sensitive enough to measure biopterin and neopterin in human urine, and therefore, it is useful in clinical chemistry for the screening of the two pterins in urine samples from patients. The method is also ideally suited to total automation by the continuous-flow system. Acknowledgements The authors wish to thank Japan Spectroscopic Co. (JASCO) and Union G&ken Co. for permitting us to use the polarization fluorescence analyzer. This work was supported in part by a grant from the Ministry of Education, Science and Culture. References 1 Kaufman S. Studies on the mechanism of the enzymatic conversion of phenylalanine to tyrosine. J Biol Chem 1959; 234: 2677-2682. 2 Kaufman S. The structure of the phenylalanine-hydroxylation cofactor. Proc Nat1 Acad Sci USA 1963; 50: 1085-1093. 3 Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis, J Biol Chem 1964; 239: 2910-2917. 4 Brenneman AR, Kaufman S. The role of tetrahydropteridine in the enzymatic conversion of tyrosine to 3,4-dihydroxyphenylalanine. Biochem Biophys Res Commun 1964; 17: 177-183. 5 Lovenberg W, Jequier E, Sjoerdsma A. Tryptophan hydroxylation: measurement in pineal grand, brain stem, and carcinoid tumor. Science 1967; 155: 217-219. 6 Friedman PA, Kappelman AH, Kaufman S. Partial purification and characterization of tryptophan hydroxylase from rabbit hind brain. J Biol Chem 1972; 247: 4165-4173. 7 Kaufman S, Berlow S, Summer GK et al. Hyperphenylalaninemia due to a deficiency of biopterin. A variant form of phenylketonuria. New Engl J Med 1978; 299: 673-679.

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