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Technical Briefs |
1 Departement de Biologie, Pharmacologie, Centre Hospitalier de Versailles, Faculte de Medecine Paris Ile de France Ouest, Le Chesnay, France;2 Service de Neurovirologie, Commissariat à l’Energie Atomique (CEA), Centre de Recherches du Service de Santé des Armées, Universite Paris XI, Ecole Pratique des Haute Etudes, Institut Paris Sud sur les Cytokines, Fontenay-aux-Roses, France;3 Unite de Biologie du Développement et de la Reproduction, Departement de Physiologie Animale, Institut National de Recherche Agronomique, Jouy-en-Josas, France;4 SPI-BIO, c/o Service de Neurovirologie, CEA, Fontenay-aux-Roses, France;
aaddress correspondence to this author at: Departement de Biologie, Pharmacologie, Centre Hospitalier de Versailles–Hopital Andre Mignot, 177 rue de Versailles, 78157 Le Chesnay, France; fax 33-1-3963-9598, e-mail bmaneglier{at}ch-versailles.fr;
L-Tryptophan (Trp) is an essential amino acid and an abundant protein component. Trp is metabolized in mammals via two pathways: biosynthesis of the neurotransmitter serotonin and the kynurenine (Kyn) pathway (1)(2). Trp is the substrate for the first step in both of these pathways. In the Kyn pathway, the indole ring of Trp can be opened by the enzymes tryptophan pyrrolase [tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO)] (3). The reactions catalyzed by both of these enzymes constitute the rate-limiting step in the Kyn metabolic pathway (4). The organ distribution of the two enzymes differs, allowing them to be clearly distinguished (5)(6). Tryptophan 2,3-dioxygenase is located primarily in the liver, and its activity is up-regulated in response to Trp and metabolic steroids. IDO can be found in various cells, but it is not active in healthy humans and its activity is induced only during immune responses mediated by proinflammatory cytokines such interferon-
(7)(8).
The enzymatic reactions of the Kyn pathway produce NAD and other intermediates, including Kyn and quinolinic acid (5)(9). The removal of Trp from the microenvironment via this pathway protects the organism by limiting the growth of intracellular pathogens and malignant cells and may also prevent maternal immune reactions against the fetus during pregnancy (10)(11)(12)(13)(14). The Kyn-to-Trp ratio provides an estimate of IDO activity that is independent of baseline Trp concentrations (15)(16)(17). In plasma, Kyn/Trp may be correlated with the concentrations of inflammatory markers, such as neopterin, to ensure that Trp degradation is induced by interferon- and involves IDO (18). Kyn/Trp is higher in individuals with diseases that involve an activated cellular immune response or those undergoing cytokine immunotherapy (16)(17)(19).
Here we describe a procedure for the determination of Kyn and Trp concentrations in human plasma or in supernatants of human cell cultures. This method involves HPLC coupled to coulometric detection.
L-Kyn and L-Trp were purchased from Sigma. We obtained potassium dihydrogen phosphate (KH2PO4) and sodium monohydrogen phosphate (Na2HPO4) from Prolabo. Acetonitrile, perchloric acid (HClO4), and L-ascorbic acid were from Merck. The mobile phase was a 94:6 mixture (by volume) of 16.2 mmol/L KH2PO4 and acetonitrile. The mobile phase was used at ambient temperature at a flow rate of 0.9 mL/min.
Chromatography was performed with a Thermofinnigan solvent delivery SpectraSeries P100 pump. Sample injection was controlled by a Spark Holland Triathlon autosampler, with a partial loop feed of 100 µL. A Merck LiChrospher 100 RP-18 column [250 x 4 mm (i.d.); 5 µm bead size] was used. The coulometric detection system consisted of a thin-layer flow-through electrochemical ESA Coulochem II detector connected to an ESA Model 5011 analytical cell containing two working electrodes made of porous graphite. The analytical cell voltage was set at +0.45 V for the first detector and +0.60 V for the second detector. Kyn and Trp were detected at +0.60 V. Each chromatographic run took 14 min. The samples were treated as follows: 100 µL of the appropriate specimen was deproteinized by incubation with 200 µL of 0.5 mol/L HClO4 supplemented with 0.06 mmol/L ascorbic acid. Samples were vortex-mixed and centrifuged at 12 000g for 5 min. We then mixed 50 µL of the supernatant with 250 µL of 0.1 mol/L Na2HPO4 in a microsampling vial to buffer the sample after acid precipitation of the proteins. We injected 50 µL of the resulting mixture into the HPLC system. We used the same method to generate calibration curves with Trp and Kyn calibration solutions. Individual stock solutions of Trp and Kyn were prepared in methanol and stored at –80 °C for a maximum of 3 months. Immediately before each HPLC analysis, these stock solutions were used to prepare a calibration mixture containing Trp and Kyn. Calibration curves were constructed with seven concentrations of the two compounds, including a zero point. The concentrations were calculated from peak heights. Retention times were 6.8 min for Kyn and 11.6 min for Trp (Fig. 1A
). It was not necessary to replace the precolumn or the analytical cell during the 4-month study period, during which 
3000 specimens were analyzed.
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We used a pool of EDTA-plasma samples from healthy blood donors, aliquoted into 10 samples, for evaluating the stability of Trp and Kyn on 3 consecutive days. Between analyses, samples were stored protected from light at 4 °C. The CVs were 2.3% and 1.3% for mean (SD) Trp concentrations of 73.2 (1.7) µmol/L (day 2; n = 10) and 72.1 (0.9) µmol/L (day 3; n = 10), respectively. The CVs were 2.0% and 1.2% for Kyn concentrations of 2.4 (0.05) µmol/L (day 1; n = 10) and 2.5 (0.03) µmol/L (day 2; n = 10), respectively. We evaluated the stability of these compounds over 24 h in Na2HPO4, stored in microsampling vials protected from light at 4 °C. The CVs were 3.6% and 3.1% for a Trp concentration of 70.4 (2.51) µmol/L (n = 10) and a Kyn concentration of 2.7 (0.08) µmol/L (n = 10), respectively. Stability during storage in microsampling vials was therefore satisfactory for up to 24 h at 4 °C.
We also tested the reproducibility of this method over 20 consecutive days. For this purpose, two pools of plasma samples were each aliquoted into 20 samples and stored at –20 °C. Each day, one sample from each pool was assessed in duplicate. The between-day variations were 4.2% for a Trp concentration of 63.3 (2.7) µmol/L (n = 20) and 4.4% for a Kyn concentration of 2.7 (0.1) µmol/L (n = 20) for the first serum pool and 3.4% for a Trp concentration of 44.4 (1.5) µmol/L (n = 20) and 4.1% for a Kyn concentration of 1.6 (0.06) µmol/L (n = 20) for the second serum pool. Seven-point calibration curves were constructed in duplicate for the 20-day period, and on 3 days, we plotted six independent calibration curves. The mean (SD) Trp and Kyn concentrations were calculated and compared with theoretical values. Peak heights for Trp were correlated over a range of concentrations from 0.30 to 100 µmol/L (r2 = 0.9997) and for Kyn over a range of concentrations from 0.07 to 10 µmol/L (r2 = 0.9998). The lowest quantifiable concentrations of Trp and Kyn were determined by comparing the measured concentrations from six samples of phosphate-buffered saline with no added analyte with those obtained from six samples to which target concentrations of Trp or Kyn had been added. The lowest quantifiable concentrations for Trp and Kyn were 0.24 and 0.06 µmol/L, respectively. The actual values observed were 0.27 (0.03) µmol/L (n = 6) for Trp, with a within-day CV of 13%, and 0.07 (0.01) µmol/L (n = 6) for Kyn, with a within-day CV of 9.5%.
Recovery from the column was evaluated by use of a 100-µL control sample containing 44.4 µmol/L Trp and 1.61 µmol/L Kyn. In additional test series, this control sample was supplemented with 100 µL volumes of a phosphate buffer solution containing 8 and 10 nmol of Trp (final concentrations, 62.2 and 72.2 µmol/L) or 0.8 and 1.0 nmol of Kyn (final concentrations, 4.81 and 5.81 µmol/L). The recoveries were 99.1% for Trp and 101.4% for Kyn. The mean (SD) Trp and Kyn concentrations obtained for 40 EDTA-plasma samples from healthy blood donors [20 women; mean (SD) age, 40.4 (13) years; and 20 men; mean (SD) age, 47.6 (10) years] were 64.2 (10.8) and 2.0 (0.4) µmol/L, respectively. These concentration ranges are consistent with those reported previously for a HPLC method that used fluorescence detection for Trp and ultraviolet detection for Kyn (20)(21).
The main advantage of our method lies in the use of a coulometric detection system, which is devoid of the quenching problems associated with fluorescence detection and provides high specificity. In contrast to the methods of Laich et al. (21) and Vaarmann et al. (22), we used only one detector with one analytical cell. The choice of 0.06 mmol/L ascorbic acid is important. When the ascorbic acid concentration was high (0.6 mmol/L), Trp concentrations in samples left for 8 h at ambient temperature increased by 25%, and when no ascorbic acid was added, the observed Kyn concentrations were threefold lower.
Trp and Kyn concentrations were determined in the supernatants from cultures of human primary macrophages in serum-free medium with this HPLC method (Fig. 1B
). Between-day reproducibility was assessed on the supernatants of macrophages, which contained Trp and were supplemented with Kyn and aliquoted into 20 samples. We analyzed one sample per day for 20 days. The CV were 4.7% for a Trp concentration of 42.6 (2.0) µmol/L and 7.6% for a Kyn concentration of 0.73 (0.06) µmol/L. The lowest quantifiable concentrations of Trp and Kyn determined in supernatants of cell cultures with this HPLC method were similar to those for plasma.
Acknowledgments
We thank Julie Sappa for excellent help in the preparation of this manuscript, Sylvia Cantaluppi for assistance, and the Centre de Transfusion Sanguine (Le Chesnay, France). This work was supported by the Faculte de Medecine Paris-Ile-de-France-Ouest (Bonus Qualite Recherche–Soutien Specifique) and by the Commissariat à l’Energie Atomique (CEA). This work is dedicated to Prof. Dominique Dormont, who died in November 2003.
Footnotes
References
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