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Measurement of urinary tryptophan metabolites by reverse-phase high-pressure liquid chromatography
BIOCHEMICAL
MEDICINE
26, 330-338 (1981)
Measurement of Urinary Tryptophan Metabolites by ReversePhase High-Pressure Liquid Chromatography JOHN B. TARR’ Department
of Developmental and Cell Biology, University Irvine, California 92717
of California.
Received March 18, 1981
The analysis of tryptophan and its metabolites in urine and other biological materials is of considerable clinical and experimental importance. Urinary excretion of tryptophan metabolites is elevated in human females during pregnancy (1) and use of oral contraceptive agents (2), particularly after a loading dose (2-10 g) of L-tryptophan. Large amounts of kynurenine and 3-hydroxykynurenine are excreted in most patients with myeloid or lymphoid leukemia, Hodgkin’s disease, and multiple myeloma (3). High levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid occur in the urine of many patients with bladder cancer (4); these metabolites have been implicated in the etiology of the disease (5). Elevated urinary tryptophan with low kynurenine following tryptophan loading has been reported in cases of Hartnup disease (6) and congenital tryptophanuria with dwarfism (7). Because the metabolism of tryptophan is heavily dependent on vitamin B-6 coenzymes, abnormalities in the metabolism of vitamin B-6 often lead to elevated urinary levels of tryptophan metabolites (8,9). Increased excretion of kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid has been observed in patients with infantile spasm due to vitamin B-6 deficiency or dependence (10). A regulatory role of kynurenine and 3-hydroxykynurenine in the synthesis of neurotransmitters in rat brain has been suggested (11). Several methods for the analysis of tryptophan and its metabolites in biological materials have been developed. The most comprehensive procedure (9) involves many steps of ion-exchange chromatography and ’ Present address: Analytical Development Corporation, Box 744, Monument, Cola. 80132. 330 0006-2944/81/060330-09$02.00/O Copyright All rights
8 1981 by Academic Press. Inc. of reproduction in any form reserved.
1875 Willow
Park Way, P.O.
ANALYSIS
OF
URINARY
TRYPTOPHAN
METABOLITES
331
spectrophotometric and calorimetric assays, and requires a great deal of effort, time, and laboratory space. A variety of methods using ionexchange (12-15), thin-layer (14,16,17), and gel permeation (18) chromatography, as well as spectroscopic (19,20) and fluorometric (21,22) methods, have been described; these are difficult to use quantitatively, require large samples, or are applicable to only a few compounds. A separation of four compounds by reverse-phase high-pressure liquid chromatography (HPLC), suitable for determination of tryptophan and kynurenine in urine, has been reported (23). A method for determination of nicotinic acid in urine by reverse-phase HPLC has been developed as well (24). Quantitative analysis of tryptophan, kynurenine, 3-hydroxykynurenine, and kynuramine in rat brain by a combination of ion-exchange HPLC and electron-capture gas-liquid chromatography (GLC) has been described (11). This procedure requires two HPLC steps followed by derivatization and GLC of each effluent fraction. The present report describes a rapid, sensitive analysis of tryptophan and nine of its metabolites using reverse-phase HPLC, and its use in the measurement of a normal urinary response to tryptophan loading. METHODS Apparatus. The Waters liquid chromatograph consisted of a Model 6000A pumping system, Model 440 ultraviolet absorbance detector set at 254 nm, and Model R401 differential refractometer. Injections were made via a Waters U6K injector. Monitor outputs were recorded on a Houston Omni-Scribe dual-pen chart recorder. The column was a Waters p.Bondapak C,,. 0.39 x 30 cm, consisting of octadecyl groups covalently attached to a silica gel matrix of lo-pm particle size. Solvents and samples were filtered through 0.22~pm Millipore filters. Sample injections were made with Hamilton Syringes 801 (10 pl), 802 (25 Al), and 725 (250 ~1). Chemicals. L-Tryptophan, L-kynurenine, 3-hydroxy-or-kynurenine, 3hydroxyanthranilic acid, quinolinic acid, nicotinamide, trigonelline hydrochloride, kynurenic acid, and xanthurenic acid were purchased from Sigma. N’-Formyl-L-kynurenine and nicotinic acid were purchased from Calbiochem. Acetic acid, anhydrous sodium acetate and methanol were Mallinckrodt (AR) products. All water was deionized and glass-distilled. Analytical methods. All standards were prepared as 1.0 mM solutions, 3-hydroxykynurenine and 3-hydroxyanthranilic acid in 0.01 N HCl, kynurenic acid and xanthurenic acid in 10 mM sodium acetate, pH 4.84, in 20% methanol, and all others in 10 mM sodium acetate, pH 4.84. The concentration of each solution was standardized by measurements of ultraviolet absorbance. Solutions were mixed in the desired combinations in the injector. The column was stored in 50% methanol when not in use. It was
332
JOHN B. TARR
equilibrated to new solvents for at least 30 min before use. The eluant was 10 mM sodium acetate, pH 4.84,’ degassed in vacua for at least 10 min, and filtered. The flow rate was 1.0 ml/min, maintained by a pressure of 900 psi. After 42 min of sample elution, the flow rate was increased to 2.0 ml/min (1800 psi). All chromatograms were obtained at ambient temperature (ca. 20°C). Quantitative analysis of samples was provided by preparation of standard curves of peak area (peak height x width at half of peak height, or by integration) vs amount of each compound injected. Urine samples were collected from a human male, age 28 years, body weight 69.5 kg, before and at hourly intervals after a 6.95-g (100 mg/kg) oral dose of L-tryptophan. Extracts were prepared as described by Bieleski and Turner (25); unextracted samples were frozen in a solid CO,-ethanol mixture, thawed, filtered, and injected within 1 hr of collection. RESULTS Several chromatographic eluants were tested before adequate separation of all of the compounds was achieved. Distilled deionized water (23) was unsuitable because most of the peaks were too broad for complete resolution of mixtures. Peaks were much sharper in all buffered eluants tested, but 10 mM sodium phosphate, pH 7.2, and sodium 2morpholinoethanesulfonate, pH 6.0 and 5.5, were found to give inadequate separation of the compounds eluting in the first few minutes. The elution time of 3-hydroxyanthranilic acid is strongly affected by the pH of the eluant in the range tested, being 4.3 min at pH 7.2, 5.7 min at pH 6.0, 9.2 min at pH 5.5, and 20 min at pH 4.84. The elution times of tryptophan, kynurenine, and derivatives of kynurenine decrease below pH 5.5, and the elution time of nicotinic acid increases. The separation of a test mixture of tryptophan and nine metabolites with 10 mM sodium acetate, pH 4.84, as eluant is shown in Fig. 1. Analysis of seven compounds is complete after about 42 min; determination of xanthurenic acid and kynurenic acid is achieved after about 54 min when the flow rate is increased at 42 min, but complete analysis requires over 70 min if it is kept constant. Quinolinic acid was not resolved under the conditions used in this analysis; its elution was too broad to be of analytical use. All of the other compounds except xanthurenic acid and kynurenic acid are almost completely resolved. Retention times of all compounds showed daily variations of up to 5% due to changes in column state, buffer composition, temperature, and unknown factors. The system was routinely tested with a standard mix’ A 1 M stock solution, containing 2.500 g glacial acetic acid and 4.800 g annydrous sodium acetate in 100 ml water, was diluted l/100 immediately before use.
ANALYSIS
OF URINARY
TRYPTOPHAN
333
METABOLITES
.I2
.08
A 254
.04
0
L 0
, 20
40 TIME
60
(min)
FIG. I, Separation of a mixture of tryptophan metabohtes, containing 10 nmole of each compound, on a pBondapak C,, column, with IO mM sodium acetate, pH 4.84, as eluant. Flow rate: 1.O mlimin, increased to 2.0 ml/min at 42 min. Compounds are identified as follows: (1) trigonelhne, (2) nicotinic acid, (3) 3-hydroxykynurenine, (4) kynurenine, (5) 3-hydroxyanthranihc acid, (6) nicotinamide, (7) N’-formylkynurenine, (8) tryptophan, (9) xanthurenic acid, (10) kynurenic acid. The low, broad peak from 5 to 20 min is quinolinic acid.
ture before analysis of samples. Detection by ultraviolet absorbance was found to be more sensitive than refractive index measurements in all cases. Standard curves of peak area vs amount injected were linear at least in the range of 0 to 50 nmole; measurements were not made with larger amounts. Chromatograms of urine under basal conditions and 8 hr after a tryptophan load are shown in Figs. 2 and 3, respectively. Tryptophan, N’formylkynurenine, kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid were all detected in basal urine. The concentrations of these metabolites were higher in the S-hr sample, and 3-hydroxyanthranilic acid was also detected. The identification of the compounds was confirmed by spiking the urine samples with standards. Positions and relative intensities of the peaks matched those obtained with standard mixtures. The amount of each compound in the spiked sample was used,
334
JOHN B. TARR
.08 A 254
.04
d.J , , , 4
0 0
20
40 TIME
( ( 60
(min)
FIG. 2. Chromatogram of 5 bl of basal urine from a human male. Chromatographic conditions are described and peaks are identified in Fig. 1.
after subtracting endogenous levels, to determine recovery of added standards in the extraction procedure. Mean recoveries ranged from 66 to 106% (Table 1). The urinary excretion patterns of tryptophan and several metabolites after tryptophan loading, as measured in unextracted urine samples, are shown in Fig. 4. Excretion of tryptophan rose within 3 hr to a sustained maximum. The other compounds were excreted at maximal rates 6 to 7 hr after administration of tryptophan. DISCUSSION
Previously developed methods (9) are suitable for the determination olites with high sensitivity (largely samples are analyzed). The recently is capable of detecting femtomolar
using ion-exchange chromatography of most urinary tryptophan metabbecause complete 8 to 24-hr urine developed HPLC-GLC method (11) amounts of four tryptophan metab-
ANALYSIS
OF URINARY
TRYPTOPHAN
335
METABOLITES
.08
A 254
.04
0 t 0
20
40
60
TIME (mid 3. Chromatogram of 5 )LI of urine from a human male 8 hr after administration of a 100 mg/kg oral dose of L-tryptophan. Chromatographic conditions are described and peaks are identified in Fig. 1. FIG.
olites. Although less sensitive, the method described in the present report is of potential value in clinical diagnosis and metabolic studies for several reasons. It is much simpler, faster, and more comprehensive. It is suitable for analysis of very small samples, and therefore may be useful with plasma or serum, culture fluids, tissue extracts, and other materials available in limited quantities. It is about as reliable as other methods currently in use. Standard curves indicate that the measurements are accurate to 5% or less. The method has been used successfully with samples containing 50 pmole of each compound. The method of extraction used in this study (25) is more effective than simple extraction by water-immiscible solvents. It almost quantitatively extracts all of the compounds to be separated, despite the wide range of structure and hydrophobic character. The extraction method also removes many tissue solutes that might interfere with the analysis and shorten the life of the column. Although extraction of the urine resulted
JOHN B. TARR
336
RECOVERY
OF
TABLE I 100 nmole EACH OF ADDED STANDARDS IN URINE EXTRACTION’
Compound Tryptophan N’-Formylkynurenine Kynurenine 3-Hydroxykynurenine 3-Hydroxyanthranilic Nicotinic acid Nicotinamide
acid
Amount in 200~ul aliquoP (nmole)
Total amount in extracP @mole)
9.1 9.2 8.6 7.4 6.6 8.6 10.6
91 92 86 74 66 86 106
Recovery (%I 91
92 86 74 66 86 106
* Mean of three to eight experiments. ’ Endogenous amount subtracted.
TIME
(h)
FIG. 4. Urinary excretion of tryptophan and its metabolites in a human male after administration of a 100 m&g oral dose of L-tryptophan. Compounds are identified in Fig. 1.
ANALYSIS
OF URINARY
TRYPTOPHAN
METABOLITES
337
in an improvement in resolution, all of the compounds listed in Fig. 4 could be detected and measured in unextracted samples. Most of the compounds were easily detected in samples of basal urine (Fig. 2), but the presence of interfering peaks made measurement of trigonelline impossible in most cases. A normal response to tryptophan loading was easily measured in spite of interfering urinary solutes. The excretion patterns of tryptophan and kynurenine matched those observed previously in a similar study (23). The characteristic patterns of metabolic abnormalities should be even more readily detected. In most laboratories it has been the practice to collect 24-hr urine samples in tryptophan loading tests. Some investigators (2627) have analyzed urine collected during the first 6 to 8 hr after administration of tryptophan and found that the largest portion of the metabolites is excreted during this period. The present results indicate that maximum excretion of each metabolite occurs between 6 and 7 hr after tryptophan loading and that excretion is essentially complete after 10 hr. This suggests that shorter collection periods, or brief timed collections between 5 and 8 hr, may be sufficient when the situation dictates the need for a more rapid analysis. SUMMARY Rapid, sensitive separation and determination of tryptophan, W-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, nicotinic acid, nicotinamide, trigonelline, kynurenic acid, and xanthurenic acid in untreated and extracted urine were achieved using reverse-phase high-pressure liquid chromatography. Accuracy of the measurements is 5% or better and 50 pmole of each compound can be reliably measured. Determination of quinolinic acid was not possible with the conditions used. The method was found to be suitable for the analysis of urinary metabolites in a normal human subject under basal conditions and after tryptophan loading. ACKNOWLEDGMENTS This work was supported in part by Grant PCM76-21483 from the National Science Foundation to J. Arditti. I thank Dr. E. Rodriguez for the use of his liquid chromatograph, G. Reynolds and C. Wisdom for helpful discussions, and Dr. K. J. Tarr for reviewing the manuscript.
REFERENCES 1. Brown, R. R., Thornton, M. J., and Price, J. M., J. C/in. Invest. 40, 617 (1961). 2. Miller, L. T., Benson, E. M., Edwards, M. A., and Young, J., Amer. J. C/in. Nutr. 27, 797 (1974). 3. Musajo, L.. Benassi, C. A., and Parbajola, A., Clin. Chim. Acta 1, 229 (1956). 4. Boyland, E., Wallace, D. M., and Williams, D. C., &it. J. Cancer 9, 62 (1955). 5. Boyland, E., and Williams, D. C., Biochem. J. 64. 578 (1956).
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B. TARR
6. Milne, M. D., Crawford, M. A., Girao, C. B.. and Loughridge, L. W.. Quart. J. Med. 29, 407 (1960). 7. Tada, K., Ita, H., Wada, Y.. and Arakawa, T., Tohoku J. Exp. Med. 80, 118 (1963). 8. Allegri, G., and DeAntoni. A., Acta Vitaminol. Enzymol. 28, 233 (1974). 9. Price, J. M., Brown, R. R., and Yess, N., Advan. Merabol. Disorders 2, 159 (1965). 10. French, J. H.. Grueter, R. B., Druckman. R., and O’Brien, D.. Neurology 15, 101 (1965). 11. Gal, E. M., and Sherman, A. D.. J. Neurochem. 30, 607 (1978). 12. Chilcote, D. D., and Mrochek, J. E., Clin. Chem. 18, 778 (1972). 13. Chen, N. C., and Gholson, R. K., Anal. Biochem. 47, 139 (1972). 14. Morris, K. M., and Moon, R. J., Anal. Biochem. 61, 313 (1974). 15. Yeh, J. K., and Brown, R. R., Biochem. Med. 14, 12 (1975). 16. Anderson, G. M., J. Chromatogr. 105, 323 (1975). 17. Baumann, P., J. Chromatogr. 109, 313 (1975). 18. Stratakis, E., Insect Biochem. 6, 149 (1976). 19. Hood, L. V. S., and Winefordner. J. D., Anal. Biochem. 27, 523 (1969). 20. Buxto, J., and Guilbault, G. G., C’lin. Chem. 20, 765 (1974). 21. Nakken, K. F., Stand. J. Clin. Lab. Invest. 15, Suppl. 76, 78 (1963). 22. Gaitonde, M. K., Evans, G., and Hartmann, M. K., Anal. Biochem. 92, 338 (1979). 23. Grushka, E., Kikta, E. J., Jr., and Naylor, E. W.. J. Chromatogr. 143, 51 (1977). 24. Hengen, N.. Seiberth, V., and Hengen, M.. Clin. Chem. 24, 1740 (1978). 25. Bieleski, R. L., and Turner, N. A., Anal. Biochem. 17, 278 (1966). 26. Kowlessar, 0. D., Haeffner. L. J., and Benson, G. D.. J. C/in. Invest. 43, 894 (1964). 27. Michael, A. F.. Drummond, K. N., Doeden, D.. Anderson. J. A.. and Good, R. A., .I. Clin. Invest. 43, 1730 (1964).
MEDICINE
26, 330-338 (1981)
Measurement of Urinary Tryptophan Metabolites by ReversePhase High-Pressure Liquid Chromatography JOHN B. TARR’ Department
of Developmental and Cell Biology, University Irvine, California 92717
of California.
Received March 18, 1981
The analysis of tryptophan and its metabolites in urine and other biological materials is of considerable clinical and experimental importance. Urinary excretion of tryptophan metabolites is elevated in human females during pregnancy (1) and use of oral contraceptive agents (2), particularly after a loading dose (2-10 g) of L-tryptophan. Large amounts of kynurenine and 3-hydroxykynurenine are excreted in most patients with myeloid or lymphoid leukemia, Hodgkin’s disease, and multiple myeloma (3). High levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid occur in the urine of many patients with bladder cancer (4); these metabolites have been implicated in the etiology of the disease (5). Elevated urinary tryptophan with low kynurenine following tryptophan loading has been reported in cases of Hartnup disease (6) and congenital tryptophanuria with dwarfism (7). Because the metabolism of tryptophan is heavily dependent on vitamin B-6 coenzymes, abnormalities in the metabolism of vitamin B-6 often lead to elevated urinary levels of tryptophan metabolites (8,9). Increased excretion of kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid has been observed in patients with infantile spasm due to vitamin B-6 deficiency or dependence (10). A regulatory role of kynurenine and 3-hydroxykynurenine in the synthesis of neurotransmitters in rat brain has been suggested (11). Several methods for the analysis of tryptophan and its metabolites in biological materials have been developed. The most comprehensive procedure (9) involves many steps of ion-exchange chromatography and ’ Present address: Analytical Development Corporation, Box 744, Monument, Cola. 80132. 330 0006-2944/81/060330-09$02.00/O Copyright All rights
8 1981 by Academic Press. Inc. of reproduction in any form reserved.
1875 Willow
Park Way, P.O.
ANALYSIS
OF
URINARY
TRYPTOPHAN
METABOLITES
331
spectrophotometric and calorimetric assays, and requires a great deal of effort, time, and laboratory space. A variety of methods using ionexchange (12-15), thin-layer (14,16,17), and gel permeation (18) chromatography, as well as spectroscopic (19,20) and fluorometric (21,22) methods, have been described; these are difficult to use quantitatively, require large samples, or are applicable to only a few compounds. A separation of four compounds by reverse-phase high-pressure liquid chromatography (HPLC), suitable for determination of tryptophan and kynurenine in urine, has been reported (23). A method for determination of nicotinic acid in urine by reverse-phase HPLC has been developed as well (24). Quantitative analysis of tryptophan, kynurenine, 3-hydroxykynurenine, and kynuramine in rat brain by a combination of ion-exchange HPLC and electron-capture gas-liquid chromatography (GLC) has been described (11). This procedure requires two HPLC steps followed by derivatization and GLC of each effluent fraction. The present report describes a rapid, sensitive analysis of tryptophan and nine of its metabolites using reverse-phase HPLC, and its use in the measurement of a normal urinary response to tryptophan loading. METHODS Apparatus. The Waters liquid chromatograph consisted of a Model 6000A pumping system, Model 440 ultraviolet absorbance detector set at 254 nm, and Model R401 differential refractometer. Injections were made via a Waters U6K injector. Monitor outputs were recorded on a Houston Omni-Scribe dual-pen chart recorder. The column was a Waters p.Bondapak C,,. 0.39 x 30 cm, consisting of octadecyl groups covalently attached to a silica gel matrix of lo-pm particle size. Solvents and samples were filtered through 0.22~pm Millipore filters. Sample injections were made with Hamilton Syringes 801 (10 pl), 802 (25 Al), and 725 (250 ~1). Chemicals. L-Tryptophan, L-kynurenine, 3-hydroxy-or-kynurenine, 3hydroxyanthranilic acid, quinolinic acid, nicotinamide, trigonelline hydrochloride, kynurenic acid, and xanthurenic acid were purchased from Sigma. N’-Formyl-L-kynurenine and nicotinic acid were purchased from Calbiochem. Acetic acid, anhydrous sodium acetate and methanol were Mallinckrodt (AR) products. All water was deionized and glass-distilled. Analytical methods. All standards were prepared as 1.0 mM solutions, 3-hydroxykynurenine and 3-hydroxyanthranilic acid in 0.01 N HCl, kynurenic acid and xanthurenic acid in 10 mM sodium acetate, pH 4.84, in 20% methanol, and all others in 10 mM sodium acetate, pH 4.84. The concentration of each solution was standardized by measurements of ultraviolet absorbance. Solutions were mixed in the desired combinations in the injector. The column was stored in 50% methanol when not in use. It was
332
JOHN B. TARR
equilibrated to new solvents for at least 30 min before use. The eluant was 10 mM sodium acetate, pH 4.84,’ degassed in vacua for at least 10 min, and filtered. The flow rate was 1.0 ml/min, maintained by a pressure of 900 psi. After 42 min of sample elution, the flow rate was increased to 2.0 ml/min (1800 psi). All chromatograms were obtained at ambient temperature (ca. 20°C). Quantitative analysis of samples was provided by preparation of standard curves of peak area (peak height x width at half of peak height, or by integration) vs amount of each compound injected. Urine samples were collected from a human male, age 28 years, body weight 69.5 kg, before and at hourly intervals after a 6.95-g (100 mg/kg) oral dose of L-tryptophan. Extracts were prepared as described by Bieleski and Turner (25); unextracted samples were frozen in a solid CO,-ethanol mixture, thawed, filtered, and injected within 1 hr of collection. RESULTS Several chromatographic eluants were tested before adequate separation of all of the compounds was achieved. Distilled deionized water (23) was unsuitable because most of the peaks were too broad for complete resolution of mixtures. Peaks were much sharper in all buffered eluants tested, but 10 mM sodium phosphate, pH 7.2, and sodium 2morpholinoethanesulfonate, pH 6.0 and 5.5, were found to give inadequate separation of the compounds eluting in the first few minutes. The elution time of 3-hydroxyanthranilic acid is strongly affected by the pH of the eluant in the range tested, being 4.3 min at pH 7.2, 5.7 min at pH 6.0, 9.2 min at pH 5.5, and 20 min at pH 4.84. The elution times of tryptophan, kynurenine, and derivatives of kynurenine decrease below pH 5.5, and the elution time of nicotinic acid increases. The separation of a test mixture of tryptophan and nine metabolites with 10 mM sodium acetate, pH 4.84, as eluant is shown in Fig. 1. Analysis of seven compounds is complete after about 42 min; determination of xanthurenic acid and kynurenic acid is achieved after about 54 min when the flow rate is increased at 42 min, but complete analysis requires over 70 min if it is kept constant. Quinolinic acid was not resolved under the conditions used in this analysis; its elution was too broad to be of analytical use. All of the other compounds except xanthurenic acid and kynurenic acid are almost completely resolved. Retention times of all compounds showed daily variations of up to 5% due to changes in column state, buffer composition, temperature, and unknown factors. The system was routinely tested with a standard mix’ A 1 M stock solution, containing 2.500 g glacial acetic acid and 4.800 g annydrous sodium acetate in 100 ml water, was diluted l/100 immediately before use.
ANALYSIS
OF URINARY
TRYPTOPHAN
333
METABOLITES
.I2
.08
A 254
.04
0
L 0
, 20
40 TIME
60
(min)
FIG. I, Separation of a mixture of tryptophan metabohtes, containing 10 nmole of each compound, on a pBondapak C,, column, with IO mM sodium acetate, pH 4.84, as eluant. Flow rate: 1.O mlimin, increased to 2.0 ml/min at 42 min. Compounds are identified as follows: (1) trigonelhne, (2) nicotinic acid, (3) 3-hydroxykynurenine, (4) kynurenine, (5) 3-hydroxyanthranihc acid, (6) nicotinamide, (7) N’-formylkynurenine, (8) tryptophan, (9) xanthurenic acid, (10) kynurenic acid. The low, broad peak from 5 to 20 min is quinolinic acid.
ture before analysis of samples. Detection by ultraviolet absorbance was found to be more sensitive than refractive index measurements in all cases. Standard curves of peak area vs amount injected were linear at least in the range of 0 to 50 nmole; measurements were not made with larger amounts. Chromatograms of urine under basal conditions and 8 hr after a tryptophan load are shown in Figs. 2 and 3, respectively. Tryptophan, N’formylkynurenine, kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid were all detected in basal urine. The concentrations of these metabolites were higher in the S-hr sample, and 3-hydroxyanthranilic acid was also detected. The identification of the compounds was confirmed by spiking the urine samples with standards. Positions and relative intensities of the peaks matched those obtained with standard mixtures. The amount of each compound in the spiked sample was used,
334
JOHN B. TARR
.08 A 254
.04
d.J , , , 4
0 0
20
40 TIME
( ( 60
(min)
FIG. 2. Chromatogram of 5 bl of basal urine from a human male. Chromatographic conditions are described and peaks are identified in Fig. 1.
after subtracting endogenous levels, to determine recovery of added standards in the extraction procedure. Mean recoveries ranged from 66 to 106% (Table 1). The urinary excretion patterns of tryptophan and several metabolites after tryptophan loading, as measured in unextracted urine samples, are shown in Fig. 4. Excretion of tryptophan rose within 3 hr to a sustained maximum. The other compounds were excreted at maximal rates 6 to 7 hr after administration of tryptophan. DISCUSSION
Previously developed methods (9) are suitable for the determination olites with high sensitivity (largely samples are analyzed). The recently is capable of detecting femtomolar
using ion-exchange chromatography of most urinary tryptophan metabbecause complete 8 to 24-hr urine developed HPLC-GLC method (11) amounts of four tryptophan metab-
ANALYSIS
OF URINARY
TRYPTOPHAN
335
METABOLITES
.08
A 254
.04
0 t 0
20
40
60
TIME (mid 3. Chromatogram of 5 )LI of urine from a human male 8 hr after administration of a 100 mg/kg oral dose of L-tryptophan. Chromatographic conditions are described and peaks are identified in Fig. 1. FIG.
olites. Although less sensitive, the method described in the present report is of potential value in clinical diagnosis and metabolic studies for several reasons. It is much simpler, faster, and more comprehensive. It is suitable for analysis of very small samples, and therefore may be useful with plasma or serum, culture fluids, tissue extracts, and other materials available in limited quantities. It is about as reliable as other methods currently in use. Standard curves indicate that the measurements are accurate to 5% or less. The method has been used successfully with samples containing 50 pmole of each compound. The method of extraction used in this study (25) is more effective than simple extraction by water-immiscible solvents. It almost quantitatively extracts all of the compounds to be separated, despite the wide range of structure and hydrophobic character. The extraction method also removes many tissue solutes that might interfere with the analysis and shorten the life of the column. Although extraction of the urine resulted
JOHN B. TARR
336
RECOVERY
OF
TABLE I 100 nmole EACH OF ADDED STANDARDS IN URINE EXTRACTION’
Compound Tryptophan N’-Formylkynurenine Kynurenine 3-Hydroxykynurenine 3-Hydroxyanthranilic Nicotinic acid Nicotinamide
acid
Amount in 200~ul aliquoP (nmole)
Total amount in extracP @mole)
9.1 9.2 8.6 7.4 6.6 8.6 10.6
91 92 86 74 66 86 106
Recovery (%I 91
92 86 74 66 86 106
* Mean of three to eight experiments. ’ Endogenous amount subtracted.
TIME
(h)
FIG. 4. Urinary excretion of tryptophan and its metabolites in a human male after administration of a 100 m&g oral dose of L-tryptophan. Compounds are identified in Fig. 1.
ANALYSIS
OF URINARY
TRYPTOPHAN
METABOLITES
337
in an improvement in resolution, all of the compounds listed in Fig. 4 could be detected and measured in unextracted samples. Most of the compounds were easily detected in samples of basal urine (Fig. 2), but the presence of interfering peaks made measurement of trigonelline impossible in most cases. A normal response to tryptophan loading was easily measured in spite of interfering urinary solutes. The excretion patterns of tryptophan and kynurenine matched those observed previously in a similar study (23). The characteristic patterns of metabolic abnormalities should be even more readily detected. In most laboratories it has been the practice to collect 24-hr urine samples in tryptophan loading tests. Some investigators (2627) have analyzed urine collected during the first 6 to 8 hr after administration of tryptophan and found that the largest portion of the metabolites is excreted during this period. The present results indicate that maximum excretion of each metabolite occurs between 6 and 7 hr after tryptophan loading and that excretion is essentially complete after 10 hr. This suggests that shorter collection periods, or brief timed collections between 5 and 8 hr, may be sufficient when the situation dictates the need for a more rapid analysis. SUMMARY Rapid, sensitive separation and determination of tryptophan, W-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, nicotinic acid, nicotinamide, trigonelline, kynurenic acid, and xanthurenic acid in untreated and extracted urine were achieved using reverse-phase high-pressure liquid chromatography. Accuracy of the measurements is 5% or better and 50 pmole of each compound can be reliably measured. Determination of quinolinic acid was not possible with the conditions used. The method was found to be suitable for the analysis of urinary metabolites in a normal human subject under basal conditions and after tryptophan loading. ACKNOWLEDGMENTS This work was supported in part by Grant PCM76-21483 from the National Science Foundation to J. Arditti. I thank Dr. E. Rodriguez for the use of his liquid chromatograph, G. Reynolds and C. Wisdom for helpful discussions, and Dr. K. J. Tarr for reviewing the manuscript.
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