457
Clinica Chimica Acta, 69 (1976) 457-462 0 Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CCA 7788
QUANTITATIVE DETERMINATION OF UNCONJUGATED PTERINS URINE BY GAS CHROMATOGRAPHY/MASS FRAGMENTOGRAPHY
FELIX
ReTHLER
and MANFRED
KAROBATH
Psychiatrische Universitir’tsklinik, Department A-I 090 Vienna (Austria) (Received
January
IN *
of Experimental
Psychiatry, Lazarettgasse 14,
23,1976)
Summary A gas chromatographic/mass fragmentographic method is described which permits the determination of unconjugated pterins in urine. After the addition of 6,7-dimethylpterin as an internal standard, the acidified urine samples are purified by liquid chromatography on Dowex-50 and Dowex-1 columns. The pterins are then converted to their corresponding trimethylsilyl derivatives and the base peaks of biopterin (m/e 409), neopterin (m/e 409) and 6,7-dimethylpterin (m/e 320) are determined. The method is sensitive and specific and permits the processing of large numbers of samples. By means of this method, the urinary excretion of biopterin and neopterin from 9 healthy subjects has been determined.
Introduction Biopterin (2-amino4-hydroxy-6-( L-erythro-1,2-dihydroxypropyl) pteridin) and other unconjugated pterins (2-amino-4-hydroxy-pteridins) occur in a variety of species [l] and only recently a functional role for them in mammalians has been shown by Kaufman [2]. 5,6,7,$-Tetrahydrobiopterin, a reduced unconjugated pterin, is the coenzyme in the enzymatic conversion of phenylalanine to tyrosine by phenylalanine hydroxylase (EC 1.14.16.1) [2]. While patients with phenylketonuria usually have a deficiency of phenylalanine hydroxylase, evidence has been presented recently that this disease can also be caused by a decreased availability of the reduced pt.&din cofactor [3]. Furthermore, tryptophan hydroxylase (EC 1.14.16.4) and tyrosine hydroxylase (EC 1.14.16.2) appear to require reduced pterins such as tetrahydrobiopterin as cofactor [ 2,4-61, and its concentration may be the limiting factor in tyrosine * Supported
by: Fonds ZUI Fiirderung
der wissenschaftlichen
Forschung. Projekt
1606.
458
hydroxylation in vivo [ 71. Although this role of biopterin in the synthesis of serotonin, tyrosine and ca~chol~ines has been known for several years, little is known about the metabolism, tissue levels and excretion of unconjugated pterins, since present analytical methods are not satisfactory, For instance, biopterin or other similar pterins are required for the growth of Crithidia fuscicul&u and this dependency has been used for a growth test [ 81. Pterins in human urine have also been analyzed by measuring their native fluorescence after purification by laborious ion-exchange chromatography, and essentially biopterin and neopterin (2-amino-4-hydroxy-6-(D-erythro-l,2,3-trihydroxy-propyl)pteridin) have been found [ 91. Recently, synthetic pterins have been separated by gas chromato~aphy as trimethylsilyl (TMS-) derivatives and their mass spectra have been described [lo]. Based on these observations we developed a gas chromatographic/mass fragmentographic method for the assay of biopterin and neopterin in urine which allows the specific determination of these compounds in a large number of samples. Materials and methods
Chemi~uls and reagents Biopterin was generously supplied by Dr. Wilfon, Smith, Kline and French Laboratories, Philadelphia, Pa., U.S.A. Xanthopterin, isoxanthopterin, pterin6-carboxylic acid, 6,7-dimethylpterin (6,7-DMP), and the silylation reagent bis(t~methyls~yl)trifluoacet~ide (BSTFA)~t~me~ylchlorosil~e ~TMCS)/t~methylsilylimid~ol (TSIM) (3 : 2 : 3, v/v/v) were purchased from Regis Chemical Co., Morton Grove, Ill., U.S.A. 3% Dexsil 300 on 100/120 Supelco Port was obtained from Supelco Inc., Bellefonte, Pa., US.A. Neopterin was synthesized and purified by published methods [ 111. All other reagents were obtained from commercial sources in their highest available purity and used without further purification.
Chromatography A Hewlett-Packard 5982A gas chromatograph/mass spectrometer equipped with a multiple-ion accessory for the simult~eous destination of up to four fragments was used. Chromatographic conditions were as follows: for quantitative determination an isothermal 6 ft. 3% Dexsil 300 glass column (2 mm i.d.) with helium as carrier gas at 30 ml/min was used. Temperatures were: injection port, column, membrane separator and transfer lines 25O”C, ion source 2OO”C, analyzer 100°C. Ion source pressure was 2 X 10d torr and electron energy 70 eV. The injection port, gas-chromatographic column and transfer lines were of glass or had their inner surface lined with glass. In initial experiments 6 ft. columns packed with 3% OV-17 on 100/120 WAW Chromosorb G [lo] or with 3% Dexsil 400 on 100/120 Supelco Port were used. The columns were either interfaced by means of a membrane separator or directly coupled to the mass spectrometer. When the columns were connected directly, a helium flow of 12 ml/min was used which provided a pressure of 5 X 10e5 torr in the ion source. Essentially the same results were obtained with or without the separator.
459
Procedure 1 ml of urine was acidified with 7 N formic acid to about pH 2. 3 pg of 6,7DMP was added as an internal standard, mixed and the urine was poured over a Dowex 50 WX 8 column (H+-form, 100-200 mesh, 7 X 15 mm). The column was first washed with 2 X 5 ml water and the pterins were then eluted with 2 ml 1N NH40H on top of a Dowex 1-X 8 column (OH--form, 200-400 mesh, 5 X 7 mm). The Dowex-1 column was first washed with 2 X 3 ml of an aqueous NH3 solution (pH 8.6-9) and then with 0.5 ml water. Biopterin, neopterin and 6,7-DMP were eluted together with 1 ml 1 N formic acid. 0.5 ml of the formic acid eluate was transferred into 2-ml reaction vials and dried in a vacuum in a Buchler Vortex Evaporator at 70°C. 50 ~1 of the silylation reagent (BSTFA/TMCS/TSIM (3 : 2 : 3, v/v/v)) were then added to the dry residue. The vials were capped immediately and allowed to stand at room temperature for at least 3 hours. Normally they were kept overnight and analyzed the next day. 1 ~1 of the sample was injected which contained about 40 ng of endogenous compounds. Since after the injection of 0.05 ng TMS-biopterin or TMSneopterin an S/N ratio of about 2 : 1 is observed, the present assay can be adapted for the determination of unconjugated pterins in a few /.11of urine. The silylated derivatives remained stable for at least 2 weeks if the vials were kept dry at room temperature in a desiccator. In the routine assay of biopterin and neopterin the mass spectrometer recorded alternatively the base peak of TMS-6,7-DMP (m/e 320) and the base peaks of TMS-biopterin and TMS-neopterin both at m/e 409. Although both TMS-biopterin and TMS-neopterin have their base peak at m/e 409, simultaneous and independent determination of both compounds can be performed by measuring this fragment since they are eluted at different retention times. The peak heights of the derivatives were measured manually, a ratio of peak heights was formed and compared with a standard curve (Fig. 2). For the examination of other pterins the MID unit was adjusted to the following fragments: isoxanthopterin and xanthopterin (m/e 380), pterin-6-carboxylic acid (m/e 423). Although isoxanthopterin and xanthopterin were detected in urine samples, no attempts were made for their quantitative determination. Results and discussion In initial studies we separated silylated pterins, derivatized with bis(trimethylsilyl)acetamide (BSA) by means of a 6 ft. 3% OV-17 column [lo]. The retention times on this column and the mass spectra of the TMSderivatives of synthetic isoxanthopterin, xanthopterin, biopterin, pterin-6carboxylic acid and neopterin confirmed previously published data [lo]. Although a useful separation of the TMS-pterins could be obtained with the 3% OV-17 column, we found that a 3% Dexsil 300 column permits a separation of silylated 6,7-DMP, biopterin and neopterin under isothermal conditions with less bleeding. A combination of BSTFA, TMCS and TSIM (3 : 2 : 3, v/v/v) was chosen as the silylating agent since TMS-pterins derivatized and stored in this mixture were found to be stable for 2 weeks when kept dry. For the analysis of unconjugated pterins in urine samples it was necessary to
460 1
‘I ii
Fig. 1. Mass fragmentography of unconjugated pterins. (A) A mixture of synthetic &&ted 6.7-DMP, bioterin and neopterin was injected. (B) Endogenous pterins in human urine. (C) 6.7-DMP (3 pg) was added to urine before processing. (D) 6,7-DMP (3 /.&!) and biopterin (1 ug) were added to urine before processing. The peaks, their characteristic mass numbers (m/e) and retention times are: (1) 6.7-DMP, m/e 320, 53 s (seconds). (2) Biopterin. m/e 409, 100 s. (3) Neopterin, m/e 409,142 s. (4) ‘7-Biopterin (2-amino-4hydroxy-7-(L-erythro-l.2-dihydroxy-propyl)-pteridin) is an impurity of the synthetic biopterin, m/e 409, 75 s. A 6 ft. 3% Dexsil 300 column coupled by means of a membrane separator was used. For further details see text.
desalt and purify urine with a two-step ion-exchange method, since omission of one of the columns yielded a tar-like precipitate after silylation. The purification method by ion-exchange chromatography is simple and at least 40 samples can be processed in a day. 6,7-DMP, which is commercially available, was selected as an internal standard, since it is a synthetic pterin which does not occur in the body and is not formed by degradation or metabolism of biopterin or neopterin [ 11. Furthermore, the chromatographic properties of 6,7-DMP on ion-exchange resins [ 121 and its gas-chromatographic retention time after silylation are comparable to those of biopterin and neopterin. The recoveries of biopterin and neopterin were 80% and 70%, respectively. Evidence that the elution of the ion m/e 409 in biological samples represents biopterin and neopterin, was obtained by direct and indirect identification methods. The retention times of synthetic or endogenous biopterin or synthetic or endogenous neopterin were identical on 3% Dexsil 300 (Fig. l), 3% Dexsil 400 and 3% OV-17 columns (not shown here). No elution of the fragment m/e 409 could be detected in urine after adsorption on charcoal, a treatment which is known to remove pterins quantitatively [ 9,131. Further identification of the endogenous compounds was possible by measuring the coelution of specific fragments derived from TMS-biopterin or TMS-neopterin. Thus,
461
ng
blopterln or neopterin
added
to
lml
urine
sample
Fig. 2. Standard curves for biopterin and neopterin derivatives. Increase of the ratio of peak heights of biopterin (a) or neopterin (0) and 6,7-DMP versus amounts of biopterin or neopterin added to 1 ml urine before processing.
biopterin was identified by the simultaneous elution of its parent ion m/e 525, its fragment m/e 320, and its base peak m/e 409. Neopterin was identified by the specific coelution of its parent ion m/e 613, its fragments m/e 598, m/e 320 and its base peak m/e 409. Since both TMS-biopterin and TMS-neopterin form the ion m/e 409 with high intensity, this fragment was used for the quantitative determination of both compounds. As shown in Fig. 2 a ratio between the peak height of the base peak of biopterin or neopterin at m/e 409 and of the TABLE
I
UNCONJUGATED
PTERINS
IN THE URINE
OF HEALTHY
HUMAN
SUBJECTS
The results of two separate determinations from the same urine sample are given. Excretion is expressed as ng unconjugated pterin per mg creatinine, which was determined by the method of Jaffa [141. Subjects
Biopterin
F.R. M.K. W.S. H. L. K.B. A.L. R.S. Ch.G.
1000 1507 757 752 1250 1527 900 608 520
Ch.H. Mean + S.D.
Neopterin 933 1515 853 827 1228 1627 872 672 568
995 i- 359
777 582 339 393
828 596 -
598 499 680 328 318
660 583 660 328 340
513 f 170
462
base peak of the internal standard (m/e 320) was determined and used for quantitation. The standard curve was linear for biop~~n ‘and neopterin in the examined concentration range (Fig. 2). In all 9 persons, more biopterin than neopterin was excreted and the values show that the ratio between biopterin and neopterin varies considerably (Table I). The values closely resemble those of Fukushima and Shiota [9], who measured the urinary output of biopterin and neopterin in the urine of three persons. The present method is the first rapid, specific and sensitive assay of unconjugated pterins in biological samples. Work is in progress to study factors which influence the excretion of unconjuga~d pterins in urine and to adapt the method for the determination of these compounds in tissue samples. Acknowledgements We thank Dr. M. Pailer for his advice and encou~gement stad for her help during the preparation of this manuscript,
and Ms. B. Bjijrn-
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Remboid, H. and Gyure, W.L. (1972) Angew. Chem. 84,1088-1099 Kaufman, S. (1967) Annu. Rev. Biochem. 36,171-184 Butler, I.J., Holtzman. N.A., Kaufman, S.H. (1975) Pediatr. Res. 9, 348 Jequier, E.. Lovenberg, W. and Sjoerdsma, A. (1967) Mol. Pharmacol. 3, 247-278 Nagatsu, T., Levitt, M. and Udenfriend, S. (1964) J. Biol. Chem. 239, 2910-2917 Karobath, M. and Baldessarini, R. (1972) Nature 236.206-208 Kettler, R., Bartholini, G. and Pletscher, A. (1974) Nature 249, 476478 Dewey, V.C. and Kidder, G.W. (1971) in Methods of Enzymology (Colowick, S.P. and Kaplan, N.O., eds.) Vol. 18 B pp. 618624, Academic Press, New York Fukushima, T. and Shiota, T. (1972) J. Biol. Chem. 247, 46494556 Lloyd. T., Markey. S. and Weiner, N. (1971) Anal. Biochem. 42.108-112 Rembold, H. and Buschmann, L. (1963) Chem. Ber. 96,1406-1410 Rembofd, H. and Buschmann, L. (1962) Hoppe-Seyler’s 2. Physiol. Chem. 330,132-139 Asatoor, A. and Dalgliesh, C.E. (1956) J. Chem. See. 445,2291-2299 Jaffa, M. (1886) Hoppe-Seyler’s 2. Physiol. Chem. 10, 399400
Clinica Chimica Acta, 69 (1976) 457-462 0 Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CCA 7788
QUANTITATIVE DETERMINATION OF UNCONJUGATED PTERINS URINE BY GAS CHROMATOGRAPHY/MASS FRAGMENTOGRAPHY
FELIX
ReTHLER
and MANFRED
KAROBATH
Psychiatrische Universitir’tsklinik, Department A-I 090 Vienna (Austria) (Received
January
IN *
of Experimental
Psychiatry, Lazarettgasse 14,
23,1976)
Summary A gas chromatographic/mass fragmentographic method is described which permits the determination of unconjugated pterins in urine. After the addition of 6,7-dimethylpterin as an internal standard, the acidified urine samples are purified by liquid chromatography on Dowex-50 and Dowex-1 columns. The pterins are then converted to their corresponding trimethylsilyl derivatives and the base peaks of biopterin (m/e 409), neopterin (m/e 409) and 6,7-dimethylpterin (m/e 320) are determined. The method is sensitive and specific and permits the processing of large numbers of samples. By means of this method, the urinary excretion of biopterin and neopterin from 9 healthy subjects has been determined.
Introduction Biopterin (2-amino4-hydroxy-6-( L-erythro-1,2-dihydroxypropyl) pteridin) and other unconjugated pterins (2-amino-4-hydroxy-pteridins) occur in a variety of species [l] and only recently a functional role for them in mammalians has been shown by Kaufman [2]. 5,6,7,$-Tetrahydrobiopterin, a reduced unconjugated pterin, is the coenzyme in the enzymatic conversion of phenylalanine to tyrosine by phenylalanine hydroxylase (EC 1.14.16.1) [2]. While patients with phenylketonuria usually have a deficiency of phenylalanine hydroxylase, evidence has been presented recently that this disease can also be caused by a decreased availability of the reduced pt.&din cofactor [3]. Furthermore, tryptophan hydroxylase (EC 1.14.16.4) and tyrosine hydroxylase (EC 1.14.16.2) appear to require reduced pterins such as tetrahydrobiopterin as cofactor [ 2,4-61, and its concentration may be the limiting factor in tyrosine * Supported
by: Fonds ZUI Fiirderung
der wissenschaftlichen
Forschung. Projekt
1606.
458
hydroxylation in vivo [ 71. Although this role of biopterin in the synthesis of serotonin, tyrosine and ca~chol~ines has been known for several years, little is known about the metabolism, tissue levels and excretion of unconjugated pterins, since present analytical methods are not satisfactory, For instance, biopterin or other similar pterins are required for the growth of Crithidia fuscicul&u and this dependency has been used for a growth test [ 81. Pterins in human urine have also been analyzed by measuring their native fluorescence after purification by laborious ion-exchange chromatography, and essentially biopterin and neopterin (2-amino-4-hydroxy-6-(D-erythro-l,2,3-trihydroxy-propyl)pteridin) have been found [ 91. Recently, synthetic pterins have been separated by gas chromato~aphy as trimethylsilyl (TMS-) derivatives and their mass spectra have been described [lo]. Based on these observations we developed a gas chromatographic/mass fragmentographic method for the assay of biopterin and neopterin in urine which allows the specific determination of these compounds in a large number of samples. Materials and methods
Chemi~uls and reagents Biopterin was generously supplied by Dr. Wilfon, Smith, Kline and French Laboratories, Philadelphia, Pa., U.S.A. Xanthopterin, isoxanthopterin, pterin6-carboxylic acid, 6,7-dimethylpterin (6,7-DMP), and the silylation reagent bis(t~methyls~yl)trifluoacet~ide (BSTFA)~t~me~ylchlorosil~e ~TMCS)/t~methylsilylimid~ol (TSIM) (3 : 2 : 3, v/v/v) were purchased from Regis Chemical Co., Morton Grove, Ill., U.S.A. 3% Dexsil 300 on 100/120 Supelco Port was obtained from Supelco Inc., Bellefonte, Pa., US.A. Neopterin was synthesized and purified by published methods [ 111. All other reagents were obtained from commercial sources in their highest available purity and used without further purification.
Chromatography A Hewlett-Packard 5982A gas chromatograph/mass spectrometer equipped with a multiple-ion accessory for the simult~eous destination of up to four fragments was used. Chromatographic conditions were as follows: for quantitative determination an isothermal 6 ft. 3% Dexsil 300 glass column (2 mm i.d.) with helium as carrier gas at 30 ml/min was used. Temperatures were: injection port, column, membrane separator and transfer lines 25O”C, ion source 2OO”C, analyzer 100°C. Ion source pressure was 2 X 10d torr and electron energy 70 eV. The injection port, gas-chromatographic column and transfer lines were of glass or had their inner surface lined with glass. In initial experiments 6 ft. columns packed with 3% OV-17 on 100/120 WAW Chromosorb G [lo] or with 3% Dexsil 400 on 100/120 Supelco Port were used. The columns were either interfaced by means of a membrane separator or directly coupled to the mass spectrometer. When the columns were connected directly, a helium flow of 12 ml/min was used which provided a pressure of 5 X 10e5 torr in the ion source. Essentially the same results were obtained with or without the separator.
459
Procedure 1 ml of urine was acidified with 7 N formic acid to about pH 2. 3 pg of 6,7DMP was added as an internal standard, mixed and the urine was poured over a Dowex 50 WX 8 column (H+-form, 100-200 mesh, 7 X 15 mm). The column was first washed with 2 X 5 ml water and the pterins were then eluted with 2 ml 1N NH40H on top of a Dowex 1-X 8 column (OH--form, 200-400 mesh, 5 X 7 mm). The Dowex-1 column was first washed with 2 X 3 ml of an aqueous NH3 solution (pH 8.6-9) and then with 0.5 ml water. Biopterin, neopterin and 6,7-DMP were eluted together with 1 ml 1 N formic acid. 0.5 ml of the formic acid eluate was transferred into 2-ml reaction vials and dried in a vacuum in a Buchler Vortex Evaporator at 70°C. 50 ~1 of the silylation reagent (BSTFA/TMCS/TSIM (3 : 2 : 3, v/v/v)) were then added to the dry residue. The vials were capped immediately and allowed to stand at room temperature for at least 3 hours. Normally they were kept overnight and analyzed the next day. 1 ~1 of the sample was injected which contained about 40 ng of endogenous compounds. Since after the injection of 0.05 ng TMS-biopterin or TMSneopterin an S/N ratio of about 2 : 1 is observed, the present assay can be adapted for the determination of unconjugated pterins in a few /.11of urine. The silylated derivatives remained stable for at least 2 weeks if the vials were kept dry at room temperature in a desiccator. In the routine assay of biopterin and neopterin the mass spectrometer recorded alternatively the base peak of TMS-6,7-DMP (m/e 320) and the base peaks of TMS-biopterin and TMS-neopterin both at m/e 409. Although both TMS-biopterin and TMS-neopterin have their base peak at m/e 409, simultaneous and independent determination of both compounds can be performed by measuring this fragment since they are eluted at different retention times. The peak heights of the derivatives were measured manually, a ratio of peak heights was formed and compared with a standard curve (Fig. 2). For the examination of other pterins the MID unit was adjusted to the following fragments: isoxanthopterin and xanthopterin (m/e 380), pterin-6-carboxylic acid (m/e 423). Although isoxanthopterin and xanthopterin were detected in urine samples, no attempts were made for their quantitative determination. Results and discussion In initial studies we separated silylated pterins, derivatized with bis(trimethylsilyl)acetamide (BSA) by means of a 6 ft. 3% OV-17 column [lo]. The retention times on this column and the mass spectra of the TMSderivatives of synthetic isoxanthopterin, xanthopterin, biopterin, pterin-6carboxylic acid and neopterin confirmed previously published data [lo]. Although a useful separation of the TMS-pterins could be obtained with the 3% OV-17 column, we found that a 3% Dexsil 300 column permits a separation of silylated 6,7-DMP, biopterin and neopterin under isothermal conditions with less bleeding. A combination of BSTFA, TMCS and TSIM (3 : 2 : 3, v/v/v) was chosen as the silylating agent since TMS-pterins derivatized and stored in this mixture were found to be stable for 2 weeks when kept dry. For the analysis of unconjugated pterins in urine samples it was necessary to
460 1
‘I ii
Fig. 1. Mass fragmentography of unconjugated pterins. (A) A mixture of synthetic &&ted 6.7-DMP, bioterin and neopterin was injected. (B) Endogenous pterins in human urine. (C) 6.7-DMP (3 pg) was added to urine before processing. (D) 6,7-DMP (3 /.&!) and biopterin (1 ug) were added to urine before processing. The peaks, their characteristic mass numbers (m/e) and retention times are: (1) 6.7-DMP, m/e 320, 53 s (seconds). (2) Biopterin. m/e 409, 100 s. (3) Neopterin, m/e 409,142 s. (4) ‘7-Biopterin (2-amino-4hydroxy-7-(L-erythro-l.2-dihydroxy-propyl)-pteridin) is an impurity of the synthetic biopterin, m/e 409, 75 s. A 6 ft. 3% Dexsil 300 column coupled by means of a membrane separator was used. For further details see text.
desalt and purify urine with a two-step ion-exchange method, since omission of one of the columns yielded a tar-like precipitate after silylation. The purification method by ion-exchange chromatography is simple and at least 40 samples can be processed in a day. 6,7-DMP, which is commercially available, was selected as an internal standard, since it is a synthetic pterin which does not occur in the body and is not formed by degradation or metabolism of biopterin or neopterin [ 11. Furthermore, the chromatographic properties of 6,7-DMP on ion-exchange resins [ 121 and its gas-chromatographic retention time after silylation are comparable to those of biopterin and neopterin. The recoveries of biopterin and neopterin were 80% and 70%, respectively. Evidence that the elution of the ion m/e 409 in biological samples represents biopterin and neopterin, was obtained by direct and indirect identification methods. The retention times of synthetic or endogenous biopterin or synthetic or endogenous neopterin were identical on 3% Dexsil 300 (Fig. l), 3% Dexsil 400 and 3% OV-17 columns (not shown here). No elution of the fragment m/e 409 could be detected in urine after adsorption on charcoal, a treatment which is known to remove pterins quantitatively [ 9,131. Further identification of the endogenous compounds was possible by measuring the coelution of specific fragments derived from TMS-biopterin or TMS-neopterin. Thus,
461
ng
blopterln or neopterin
added
to
lml
urine
sample
Fig. 2. Standard curves for biopterin and neopterin derivatives. Increase of the ratio of peak heights of biopterin (a) or neopterin (0) and 6,7-DMP versus amounts of biopterin or neopterin added to 1 ml urine before processing.
biopterin was identified by the simultaneous elution of its parent ion m/e 525, its fragment m/e 320, and its base peak m/e 409. Neopterin was identified by the specific coelution of its parent ion m/e 613, its fragments m/e 598, m/e 320 and its base peak m/e 409. Since both TMS-biopterin and TMS-neopterin form the ion m/e 409 with high intensity, this fragment was used for the quantitative determination of both compounds. As shown in Fig. 2 a ratio between the peak height of the base peak of biopterin or neopterin at m/e 409 and of the TABLE
I
UNCONJUGATED
PTERINS
IN THE URINE
OF HEALTHY
HUMAN
SUBJECTS
The results of two separate determinations from the same urine sample are given. Excretion is expressed as ng unconjugated pterin per mg creatinine, which was determined by the method of Jaffa [141. Subjects
Biopterin
F.R. M.K. W.S. H. L. K.B. A.L. R.S. Ch.G.
1000 1507 757 752 1250 1527 900 608 520
Ch.H. Mean + S.D.
Neopterin 933 1515 853 827 1228 1627 872 672 568
995 i- 359
777 582 339 393
828 596 -
598 499 680 328 318
660 583 660 328 340
513 f 170
462
base peak of the internal standard (m/e 320) was determined and used for quantitation. The standard curve was linear for biop~~n ‘and neopterin in the examined concentration range (Fig. 2). In all 9 persons, more biopterin than neopterin was excreted and the values show that the ratio between biopterin and neopterin varies considerably (Table I). The values closely resemble those of Fukushima and Shiota [9], who measured the urinary output of biopterin and neopterin in the urine of three persons. The present method is the first rapid, specific and sensitive assay of unconjugated pterins in biological samples. Work is in progress to study factors which influence the excretion of unconjuga~d pterins in urine and to adapt the method for the determination of these compounds in tissue samples. Acknowledgements We thank Dr. M. Pailer for his advice and encou~gement stad for her help during the preparation of this manuscript,
and Ms. B. Bjijrn-
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Remboid, H. and Gyure, W.L. (1972) Angew. Chem. 84,1088-1099 Kaufman, S. (1967) Annu. Rev. Biochem. 36,171-184 Butler, I.J., Holtzman. N.A., Kaufman, S.H. (1975) Pediatr. Res. 9, 348 Jequier, E.. Lovenberg, W. and Sjoerdsma, A. (1967) Mol. Pharmacol. 3, 247-278 Nagatsu, T., Levitt, M. and Udenfriend, S. (1964) J. Biol. Chem. 239, 2910-2917 Karobath, M. and Baldessarini, R. (1972) Nature 236.206-208 Kettler, R., Bartholini, G. and Pletscher, A. (1974) Nature 249, 476478 Dewey, V.C. and Kidder, G.W. (1971) in Methods of Enzymology (Colowick, S.P. and Kaplan, N.O., eds.) Vol. 18 B pp. 618624, Academic Press, New York Fukushima, T. and Shiota, T. (1972) J. Biol. Chem. 247, 46494556 Lloyd. T., Markey. S. and Weiner, N. (1971) Anal. Biochem. 42.108-112 Rembold, H. and Buschmann, L. (1963) Chem. Ber. 96,1406-1410 Rembofd, H. and Buschmann, L. (1962) Hoppe-Seyler’s 2. Physiol. Chem. 330,132-139 Asatoor, A. and Dalgliesh, C.E. (1956) J. Chem. See. 445,2291-2299 Jaffa, M. (1886) Hoppe-Seyler’s 2. Physiol. Chem. 10, 399400
