CLB-08527; No. of pages: 4; 4C: Clinical Biochemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection Gabriele Neurauter a, Sabine Scholl-Bürgi b, Astrid Haara a, Simon Geisler a, Peter Mayersbach c, Harald Schennach c, Dietmar Fuchs a,⁎ a b c
Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria Department of Pediatrics, Medical University, Innsbruck, Austria Central Institute of Blood Transfusion and Immunology, University Clinics, Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 11 September 2013 Accepted 19 October 2013 Available online xxxx Keywords: Phenylalanine Tyrosine HPLC Phe/Tyr Fluorescence monitoring
a b s t r a c t Objectives: An HPLC method was developed to quantify serum concentrations of phenylalanine and tyrosine simultaneously using fluorescence detection without derivatization. Methods: Serum protein is precipitated with trichloroacetic acid, 0.015 mM dihydrogen-phosphate solution is used for separation on reversed-phase C18 material, and acetonitrile is avoided. Both amino acids are monitored utilizing their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths. Results: One analytical run is completed within 7 min. Lower detection limit for Phe and Tyr is 0.3 μM. Comparison of the new method with a classical HPLC method for total amino acids and using UV-absorption detection reveals a highly significant relationship for Phe and Tyr. Conclusion: The new HPLC method allows rapid and very sensitive measurement of phenylalanine and tyrosine concentrations. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Patients suffering from inflammatory conditions are at an increased risk for neuropsychiatric symptoms. Thereby the disturbed metabolism of essential aromatic amino acid phenylalanine (Phe) appears to play a major role [1–3]. Phe metabolism begins with its enzymatic conversion to tyrosine (Tyr), which is a precursor for the biosynthesis of L-dihydroxyphenylalanine (L-DOPA) and of important neurotransmitters dopamine, noradrenaline (norepinephrine) and adrenaline (epinephrine) [1]. Recently it has been observed that patients suffering from inflammatory diseases like infections with HIV-1 and hepatitis C virus (HCV), trauma, sepsis and burns but also healthy older-aged persons may present with moderate elevation of Phe concentrations in the blood, and also the Phe to Tyr ratio (Phe/Tyr) is increased [1,2]. Data imply a role of inflammation and immune activation in disturbing the activity of enzyme phenylalanine 4-hydroxylase (PAH) [1–3]. This view is further supported by the fact that in patients with HCV infection or malignant melanoma, therapy with interferon-α is accompanied by an increase of blood Phe and Phe/Tyr concentrations [3]. Moreover, the increase of Phe and/or Phe/Tyr concentrations under
IFN-α/ribavirin therapy is associated with reduced dopamine levels in the brain [3]. Serum or plasma concentrations of Phe and Tyr are usually measured by HPLC methods for total amino acids. Disadvantages of these methods are that derivatization is required and one run could last up to 85 min [4]. Recently several methods were created which are based on liquid chromatography combined with mass spectrometry (LC/MS) and UV detection [5,6]. Some of these methods are specifically devoted to the use of dried blood spots suitable for neonatal screening for phenylketonuria (PKU) [5,7]. However for specific studies of neuropsychiatric diseases, often only concentrations of Phe and Tyr, probably in addition to tryptophan but not of all the other proteinogenic amino acids, are of interest. Therefore we developed a sensitive reversedphase HPLC method for the simultaneous determination of Phe and Tyr concentrations that is based on an earlier created method for tryptophan and kynurenine analysis [8], 3-nitro-L-tyrosine (3NT) serves as an internal standard, and both, Phe and Tyr, are monitored by their natural fluorescence [9], which guarantees high sensitivity. Material and methods Chemicals and reagents
⁎ Corresponding author at: Division of Biological Chemistry, Biocenter, Medical University, Innrain 80, Innsbruck, Austria. fax: +43 512 9003 73110. E-mail address: [email protected] (D. Fuchs).
L-Phe and albumin from Serva (Heidelberg, Germany), L-Tyr and 3NT from Sigma-Aldrich (Vienna, Austria), trichloroacetic acid from Merck
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
2
G. Neurauter et al. / Clinical Biochemistry xxx (2013) xxx–xxx
(Darmstadt, Germany) and potassium dihydrogen phosphate (KH2PO4) from Carl Roth GmbH (Vienna, Austria) were used. Stock solutions of Phe, Tyr (200 μM) and 3NT (500 μM) were prepared in aqua dest., and aliquots were stored at −18 °C. Calibrators were prepared freshly for each run by mixing 15 μl Tyr, 15 μl Phe and 30 μl albumin stock solutions. Instruments and chromatography The HPLC system consisted of a Prostar 210 solvent delivery system (Varian, Palo Alto, CA). Sample injection was controlled by an autosampler Prostar 400, Phe and Tyr concentrations were detected by a fluorescence detector (ProStar 360) set at 210 nm excitation and 302 nm emission wavelengths. The internal standard 3NT was monitored by an ultraviolet detector (ProStar 325) at 360 nm wavelength [8]. Separation was accomplished at room temperature using a reversed-phase LiChroCART 55-4 mm cartridge (Merck), filled with Purosphere STAR RP18 (3 μm grain size, Merck) together with a reversed-phase C18 pre-column (Merck). Mobile phase was aqueous 15 mM KH2PO4, injection volume was 30 μl, and the flow rate was 0.9 mL/min. One single chromatographic run was completed within 10 min. Concentrations of components were calculated according to peak heights using Prostar and MicroSoft Excel software.
P b 0.05. Data analysis was performed using statistical software package PASW Statistics 18.0. Results Using 0.015 mM dihydrogen-phosphate solution for separation at a flow rate of 0.9 mL/min, retention times at room temperature were around 2.2 min for Tyr and 3.8 min for Phe (Fig. 1). Retention time for the internal standard 500 μM 3NT detected by UV absorption was around 8.5 min (not shown). One single chromatographic run was completed within 7 min. The measurement of Tyr in calibrator dilutions was linear from 1.25 μM to 80 μM with an abrupt end; the highest 87.7 μM concentration of Tyr was above the upper detection limit. Phe was linear from 1.25 μM to 200 μM. Lower detection limit for Phe and Tyr was 0.31 μM. After measuring 20 aliquots of one reference pool on 20 consecutive days, Phe and Tyr concentrations resulted as 68.6 ± 6.89 μM (mean ± SD; CV = 10.0%) and 104 ± 5.28 μM (CV = 5.1%). Comparing the new method with fluorescence detection with a classical HPLC method for total amino acids using UV-absorption detection [4], measurement of 19 serum specimens revealed highly significant correlations for Phe and Tyr (Fig. 2). Other performance characteristics were comparable to earlier described methods [6–8] and are therefore not presented in detail.
Sample preparation and quantitative analysis method Blood serum (30 μl) was diluted with 30 μl 15 mM KH2PO4 solution, calibration mixtures, pool sera and samples were diluted with 300 μl 3NT. After addition of 75 μl 2 M trichloroacetic acid the reaction vials were vortexed immediately to precipitate; to separate the protein, samples were centrifuged at 13,000 ∗ g for 6 min. at room temperature. 370 μl of each supernatant was diluted with 400 μl 0.015 M KH2PO4 and transferred to the autoinjector. When Phe and Tyr measurements are performed in cerebrospinal fluid (CSF) or cell culture supernatants, 60 μl of the undiluted sample is used. To study linearity of the method, a serum pool with previously measured concentrations of Phe and Tyr was diluted with H2O to achieve 1:2.5, 1:5, 1:10, 1:50 and 1:100 dilutions and measured in quadruplicate. In addition, duplicates of 10 different concentrations of calibrators over the range of 0–200 μM were analyzed. Recovery studies were performed by analyzing a pooled blood serum with known concentrations before and after addition of 50, 30, 10 μl of 25 μM Tyr and 100 μM Phe in quadruplicates. To test the reproducibility of the method, one serum pool was aliquoted into 20 samples and stored at −20 °C. Between-day variation was assessed by measuring these aliquots on 20 consecutive days using one freshly thawed sample every day. Another serum pool was aliquoted into 10 samples all of which were measured on 3 consecutive days. Between each of the series, samples were stored at 4 °C in the dark overnight.
Discussion Recent findings on the interrelationship of Phe and Tyr metabolism with immune response and inflammation stimulated new interest in the determination of the two amino acids in body fluids [1–3]. Disturbed biochemistry of neurotransmitters may influence the mental state and may relate to specific neuropsychiatric symptoms like fatigue and depression in patients with chronic inflammation [1,3]. Conventional methods for Phe and Tyr measurements rely on the determination of total proteinogenic amino acids concentrations by, e.g., ion exchange chromatography and ninhydrin derivatization, and the completion of one analytical run may require 1 h or even longer [4]. Although fluorescence dyes such as o-phthalaldehyde are available and increase sensitivity of the conventional ninhydrin method [10], the focus of specific studies in the neuropsychiatric field often relies solely on PAH activity, and therefore only the concentrations of Phe and Tyr are of interest. Our described HPLC method for the simultaneous measurement of Phe and Tyr allows considerably shorter separation times with high sensitivity. The presented method achieves both aims: The method works fast as one single run is completed within 7 min and more than 100 specimens can be analyzed with one chromatographic system per day. High sensitivity is guaranteed by fluorescence detection which is applicable for Phe and Tyr although it has to be mentioned that the
Method comparison In sera obtained from 50 healthy blood donors (25 males and 25 females), Phe and Tyr concentrations were measured and compared to those measured with a conventional ion exchange chromatography method and ninhydrin derivatization [4]. All specimens were obtained from the Central Institute of Blood Transfusion and Immunology at the University Hospital Innsbruck, and informed consent was obtained from donors that their residual blood might be used for scientific purposes in the case when it was not used for transfusion. Statistical analysis Results were expressed as mean ± standard deviation (SD). To test for associations between results obtained with two different methods, linear regression analysis was applied. Test significance was set at
Fig. 1. Typical chromatogram of the simultaneous measurement of 103 μM phenylalanine (Phe) and 30 μM tyrosine (Tyr): both amino acids were detected by fluorescence detection at 210 nm excitation and 302 nm emission wavelengths, retention times were 1.8 min for tyrosine and 3.4 min for phenylalanine.
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
G. Neurauter et al. / Clinical Biochemistry xxx (2013) xxx–xxx
3
specimens without any other extra pre-analytical treatments required. Only changing buffer solution and wavelengths at the fluorescence detector is required. Thus, the new method might provide an easy tool for the decision whether the individual patient suffering from neuropsychiatric disturbances is more likely to respond to either serotonergic or adrenergic antidepressant medication [1]. This approach seems very reasonable because in older-aged individuals already significant relationships between specific neuropsychiatric deviations and alterations of Phe/Tyr vs. Kyn/Trp have been described [2]. Similarly, in patients with HCV infection under IFN-α/ribavirin treatment only the higher risk of fatigue development was associated with the increase of Phe/Tyr concentrations [3]. To allow the easy switch between the HPLC methods for Phe and Tyr and Kyn and Trp determinations is the only reason why 3NT is applied as an internal standard in this newly described method. In case when only measurements of Phe and Tyr are in the focus of interest, N-methyl phenylalanine instead of 3NT can be applied as an internal standard [5]. Then all three analytes can be monitored by their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths and a UV-detector is no longer required. Abbreviations
Fig. 2. Correlation between phenylalanine (upper) and tyrosine (lower) concentrations of the newly described HPLC method with fluorescence detection (ordinates) and the conventional ion exchange chromatography method and ninhydrin detection (abscissa); n = 19. Linear regression analyses reveal the following equations for phenylalanine: y = 0.9122 × − 0.1123; R2 = 0.996 and tyrosine y = 1.1224 × − 5.4226; R2 = 0.968.
fluorescence intensity of Phe is considerably lower as compared to Tyr (Fig. 1) still being approximately 5–10 times more sensitive than UV detection at 210 nm wavelength [6]. Because of the natural fluorescence of Phe and Tyr, no derivatization step is required. No acetonitrile or methanol is used within the chromatographic procedure, and in contrast to UV-detection [6], the fluorescence monitoring reveals clean chromatograms with only rarely appearing peaks related to other substances. Serum or plasma are the superior sample types for the analysis of Phe and Tyr but this method can also be applied for the measurement of concentrations in cerebrospinal fluid (CSF) and cell culture supernatants (not shown). Whereas blood sera are diluted 1:1 to stay within linearity range of the method, CSF specimens are used undiluted because of their comparably lower Phe and Tyr contents. The new method allows rapid detection of Phe, Tyr and Phe/Tyr concentrations and is thus able to support new strategies in research and care of patients which focus on the association between Phe and Tyr metabolism in chronic inflammatory conditions and neuropsychiatric deviations [1–3]. The method presented here is based on the standard equipment and methodology used for the earlier described determination of tryptophan and kynurenine concentrations in human specimens [8]. Immune activation and inflammation are not only accompanied by disturbed Phe metabolism but also by tryptophan breakdown that is activated by pro-inflammatory cytokines [1,3]. IDO activity can be estimated by the kynurenine to tryptophan ratio (Kyn/Trp) and was recognized as another important link between the immunological and neuroendocrine circuits. Since only analysis buffers and fluorescence wavelengths differ between the two analytical methods for Phe/Tyr and Kyn/Trp measurements, the new method can easily be adapted for the measurement of tryptophan and kynurenine concentrations. This offers the opportunity of a sequential measurement of Phe/Tyr and Kyn/Trp utilizing the same deproteinized
BH4 CSF L-DOPA IDO 3NT PHA Phe Phe/Tyr PKU SSRI TDO Tyr
5,6,7,8-Tetrahydrobiopterin cerebrospinal fluid L-Dihydroxyphenylalanine indoleamine 2,3-dioxygenase 3-Nitro-L-tyrosine phenylalanine (4)-hydroxylase phenylalanine phenylalanine to tyrosine ratio phenylketonuria selective serotonin reuptake inhibitor tryptophan 2,3-dioxygenase tyrosine
Conflict of interest statement There are no conflicts of interest regarding the publication of this article. Acknowledgments The authors thank Mrs. Maria Pfurtscheller and Mr. Thomas Nuener for their excellent technical assistance. References [1] Neurauter G, Schröcksnadel K, Scholl-Bürgi S, Sperner-Unterweger B, Schubert C, Ledochowski M, et al. Chronic immune stimulation correlates with reduced phenylalanine turnover. Curr Drug Metab 2008;9:622–7. [2] Capuron L, Geisler S, Kurz K, Leblhuber F, Sperner-Unterweger B, Fuchs D. Activated immune system and inflammation in healthy ageing: relevance for tryptophan and neopterin metabolism. Curr Pharm Des 2013 [in press]. [3] Felger JC, Li L, Marvar PJ, Woolwine BJ, Harrison DG, Raison CL, et al. Tyrosine metabolism during interferon-alpha administration: association with fatigue and CSF dopamine concentrations. Brain Behav Immun 2013;31:153–60. [4] Scholl-Bürgi S, Haberlandt E, Heinz-Erian P, Deisenhammer F, Albrecht U, Sigl SB, et al. Amino acid cerebrospinal fluid/plasma ratios in children: influence of age, gender, and antiepileptic medication. Pediatrics 2008;121:e920–6. [5] Kand'ár R, Záková P. Determination of phenylalanine and tyrosine in plasma and dried blood samples using HPLC with fluorescence detection. J Chromatogr B Anal Technol Biomed Life Sci 2009;877:3926–9. [6] Mo XM, Li Y, Tang AG, Ren YP. Simultaneous determination of phenylalanine and tyrosine in peripheral capillary blood by HPLC with ultraviolet detection. Clin Biochem 2013;46:1074–8.
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
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[7] Prinsen HC, Holwerda-Loof NE, de Sain-van der Velden MG, Visser G, Verhoeven-Duif NM. Reliable analysis of phenylalanine and tyrosine in a minimal volume of blood. Clin Biochem 2013;46:1272–5. [8] Widner B, Werner ER, Schennach H, Wachter H, Fuchs D. Simultaneous measurement of serum tryptophan and kynurenine by HPLC. Clin Chem 1997;43:2424–6.
[9] Bailey SW, Ayling JE. An assay for picomole levels of tyrosine and related phenols and its application to the measurement of phenylalanine hydroxylase activity. Anal Biochem 1980;107:156–64. [10] Teerlink T, van Leeuwen PA, Houdijk A. Plasma amino acids determined by liquid chromatography within 17 min. Clin Chem 1994;40:245–9.
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection Gabriele Neurauter a, Sabine Scholl-Bürgi b, Astrid Haara a, Simon Geisler a, Peter Mayersbach c, Harald Schennach c, Dietmar Fuchs a,⁎ a b c
Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria Department of Pediatrics, Medical University, Innsbruck, Austria Central Institute of Blood Transfusion and Immunology, University Clinics, Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 11 September 2013 Accepted 19 October 2013 Available online xxxx Keywords: Phenylalanine Tyrosine HPLC Phe/Tyr Fluorescence monitoring
a b s t r a c t Objectives: An HPLC method was developed to quantify serum concentrations of phenylalanine and tyrosine simultaneously using fluorescence detection without derivatization. Methods: Serum protein is precipitated with trichloroacetic acid, 0.015 mM dihydrogen-phosphate solution is used for separation on reversed-phase C18 material, and acetonitrile is avoided. Both amino acids are monitored utilizing their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths. Results: One analytical run is completed within 7 min. Lower detection limit for Phe and Tyr is 0.3 μM. Comparison of the new method with a classical HPLC method for total amino acids and using UV-absorption detection reveals a highly significant relationship for Phe and Tyr. Conclusion: The new HPLC method allows rapid and very sensitive measurement of phenylalanine and tyrosine concentrations. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Patients suffering from inflammatory conditions are at an increased risk for neuropsychiatric symptoms. Thereby the disturbed metabolism of essential aromatic amino acid phenylalanine (Phe) appears to play a major role [1–3]. Phe metabolism begins with its enzymatic conversion to tyrosine (Tyr), which is a precursor for the biosynthesis of L-dihydroxyphenylalanine (L-DOPA) and of important neurotransmitters dopamine, noradrenaline (norepinephrine) and adrenaline (epinephrine) [1]. Recently it has been observed that patients suffering from inflammatory diseases like infections with HIV-1 and hepatitis C virus (HCV), trauma, sepsis and burns but also healthy older-aged persons may present with moderate elevation of Phe concentrations in the blood, and also the Phe to Tyr ratio (Phe/Tyr) is increased [1,2]. Data imply a role of inflammation and immune activation in disturbing the activity of enzyme phenylalanine 4-hydroxylase (PAH) [1–3]. This view is further supported by the fact that in patients with HCV infection or malignant melanoma, therapy with interferon-α is accompanied by an increase of blood Phe and Phe/Tyr concentrations [3]. Moreover, the increase of Phe and/or Phe/Tyr concentrations under
IFN-α/ribavirin therapy is associated with reduced dopamine levels in the brain [3]. Serum or plasma concentrations of Phe and Tyr are usually measured by HPLC methods for total amino acids. Disadvantages of these methods are that derivatization is required and one run could last up to 85 min [4]. Recently several methods were created which are based on liquid chromatography combined with mass spectrometry (LC/MS) and UV detection [5,6]. Some of these methods are specifically devoted to the use of dried blood spots suitable for neonatal screening for phenylketonuria (PKU) [5,7]. However for specific studies of neuropsychiatric diseases, often only concentrations of Phe and Tyr, probably in addition to tryptophan but not of all the other proteinogenic amino acids, are of interest. Therefore we developed a sensitive reversedphase HPLC method for the simultaneous determination of Phe and Tyr concentrations that is based on an earlier created method for tryptophan and kynurenine analysis [8], 3-nitro-L-tyrosine (3NT) serves as an internal standard, and both, Phe and Tyr, are monitored by their natural fluorescence [9], which guarantees high sensitivity. Material and methods Chemicals and reagents
⁎ Corresponding author at: Division of Biological Chemistry, Biocenter, Medical University, Innrain 80, Innsbruck, Austria. fax: +43 512 9003 73110. E-mail address: [email protected] (D. Fuchs).
L-Phe and albumin from Serva (Heidelberg, Germany), L-Tyr and 3NT from Sigma-Aldrich (Vienna, Austria), trichloroacetic acid from Merck
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
2
G. Neurauter et al. / Clinical Biochemistry xxx (2013) xxx–xxx
(Darmstadt, Germany) and potassium dihydrogen phosphate (KH2PO4) from Carl Roth GmbH (Vienna, Austria) were used. Stock solutions of Phe, Tyr (200 μM) and 3NT (500 μM) were prepared in aqua dest., and aliquots were stored at −18 °C. Calibrators were prepared freshly for each run by mixing 15 μl Tyr, 15 μl Phe and 30 μl albumin stock solutions. Instruments and chromatography The HPLC system consisted of a Prostar 210 solvent delivery system (Varian, Palo Alto, CA). Sample injection was controlled by an autosampler Prostar 400, Phe and Tyr concentrations were detected by a fluorescence detector (ProStar 360) set at 210 nm excitation and 302 nm emission wavelengths. The internal standard 3NT was monitored by an ultraviolet detector (ProStar 325) at 360 nm wavelength [8]. Separation was accomplished at room temperature using a reversed-phase LiChroCART 55-4 mm cartridge (Merck), filled with Purosphere STAR RP18 (3 μm grain size, Merck) together with a reversed-phase C18 pre-column (Merck). Mobile phase was aqueous 15 mM KH2PO4, injection volume was 30 μl, and the flow rate was 0.9 mL/min. One single chromatographic run was completed within 10 min. Concentrations of components were calculated according to peak heights using Prostar and MicroSoft Excel software.
P b 0.05. Data analysis was performed using statistical software package PASW Statistics 18.0. Results Using 0.015 mM dihydrogen-phosphate solution for separation at a flow rate of 0.9 mL/min, retention times at room temperature were around 2.2 min for Tyr and 3.8 min for Phe (Fig. 1). Retention time for the internal standard 500 μM 3NT detected by UV absorption was around 8.5 min (not shown). One single chromatographic run was completed within 7 min. The measurement of Tyr in calibrator dilutions was linear from 1.25 μM to 80 μM with an abrupt end; the highest 87.7 μM concentration of Tyr was above the upper detection limit. Phe was linear from 1.25 μM to 200 μM. Lower detection limit for Phe and Tyr was 0.31 μM. After measuring 20 aliquots of one reference pool on 20 consecutive days, Phe and Tyr concentrations resulted as 68.6 ± 6.89 μM (mean ± SD; CV = 10.0%) and 104 ± 5.28 μM (CV = 5.1%). Comparing the new method with fluorescence detection with a classical HPLC method for total amino acids using UV-absorption detection [4], measurement of 19 serum specimens revealed highly significant correlations for Phe and Tyr (Fig. 2). Other performance characteristics were comparable to earlier described methods [6–8] and are therefore not presented in detail.
Sample preparation and quantitative analysis method Blood serum (30 μl) was diluted with 30 μl 15 mM KH2PO4 solution, calibration mixtures, pool sera and samples were diluted with 300 μl 3NT. After addition of 75 μl 2 M trichloroacetic acid the reaction vials were vortexed immediately to precipitate; to separate the protein, samples were centrifuged at 13,000 ∗ g for 6 min. at room temperature. 370 μl of each supernatant was diluted with 400 μl 0.015 M KH2PO4 and transferred to the autoinjector. When Phe and Tyr measurements are performed in cerebrospinal fluid (CSF) or cell culture supernatants, 60 μl of the undiluted sample is used. To study linearity of the method, a serum pool with previously measured concentrations of Phe and Tyr was diluted with H2O to achieve 1:2.5, 1:5, 1:10, 1:50 and 1:100 dilutions and measured in quadruplicate. In addition, duplicates of 10 different concentrations of calibrators over the range of 0–200 μM were analyzed. Recovery studies were performed by analyzing a pooled blood serum with known concentrations before and after addition of 50, 30, 10 μl of 25 μM Tyr and 100 μM Phe in quadruplicates. To test the reproducibility of the method, one serum pool was aliquoted into 20 samples and stored at −20 °C. Between-day variation was assessed by measuring these aliquots on 20 consecutive days using one freshly thawed sample every day. Another serum pool was aliquoted into 10 samples all of which were measured on 3 consecutive days. Between each of the series, samples were stored at 4 °C in the dark overnight.
Discussion Recent findings on the interrelationship of Phe and Tyr metabolism with immune response and inflammation stimulated new interest in the determination of the two amino acids in body fluids [1–3]. Disturbed biochemistry of neurotransmitters may influence the mental state and may relate to specific neuropsychiatric symptoms like fatigue and depression in patients with chronic inflammation [1,3]. Conventional methods for Phe and Tyr measurements rely on the determination of total proteinogenic amino acids concentrations by, e.g., ion exchange chromatography and ninhydrin derivatization, and the completion of one analytical run may require 1 h or even longer [4]. Although fluorescence dyes such as o-phthalaldehyde are available and increase sensitivity of the conventional ninhydrin method [10], the focus of specific studies in the neuropsychiatric field often relies solely on PAH activity, and therefore only the concentrations of Phe and Tyr are of interest. Our described HPLC method for the simultaneous measurement of Phe and Tyr allows considerably shorter separation times with high sensitivity. The presented method achieves both aims: The method works fast as one single run is completed within 7 min and more than 100 specimens can be analyzed with one chromatographic system per day. High sensitivity is guaranteed by fluorescence detection which is applicable for Phe and Tyr although it has to be mentioned that the
Method comparison In sera obtained from 50 healthy blood donors (25 males and 25 females), Phe and Tyr concentrations were measured and compared to those measured with a conventional ion exchange chromatography method and ninhydrin derivatization [4]. All specimens were obtained from the Central Institute of Blood Transfusion and Immunology at the University Hospital Innsbruck, and informed consent was obtained from donors that their residual blood might be used for scientific purposes in the case when it was not used for transfusion. Statistical analysis Results were expressed as mean ± standard deviation (SD). To test for associations between results obtained with two different methods, linear regression analysis was applied. Test significance was set at
Fig. 1. Typical chromatogram of the simultaneous measurement of 103 μM phenylalanine (Phe) and 30 μM tyrosine (Tyr): both amino acids were detected by fluorescence detection at 210 nm excitation and 302 nm emission wavelengths, retention times were 1.8 min for tyrosine and 3.4 min for phenylalanine.
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
G. Neurauter et al. / Clinical Biochemistry xxx (2013) xxx–xxx
3
specimens without any other extra pre-analytical treatments required. Only changing buffer solution and wavelengths at the fluorescence detector is required. Thus, the new method might provide an easy tool for the decision whether the individual patient suffering from neuropsychiatric disturbances is more likely to respond to either serotonergic or adrenergic antidepressant medication [1]. This approach seems very reasonable because in older-aged individuals already significant relationships between specific neuropsychiatric deviations and alterations of Phe/Tyr vs. Kyn/Trp have been described [2]. Similarly, in patients with HCV infection under IFN-α/ribavirin treatment only the higher risk of fatigue development was associated with the increase of Phe/Tyr concentrations [3]. To allow the easy switch between the HPLC methods for Phe and Tyr and Kyn and Trp determinations is the only reason why 3NT is applied as an internal standard in this newly described method. In case when only measurements of Phe and Tyr are in the focus of interest, N-methyl phenylalanine instead of 3NT can be applied as an internal standard [5]. Then all three analytes can be monitored by their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths and a UV-detector is no longer required. Abbreviations
Fig. 2. Correlation between phenylalanine (upper) and tyrosine (lower) concentrations of the newly described HPLC method with fluorescence detection (ordinates) and the conventional ion exchange chromatography method and ninhydrin detection (abscissa); n = 19. Linear regression analyses reveal the following equations for phenylalanine: y = 0.9122 × − 0.1123; R2 = 0.996 and tyrosine y = 1.1224 × − 5.4226; R2 = 0.968.
fluorescence intensity of Phe is considerably lower as compared to Tyr (Fig. 1) still being approximately 5–10 times more sensitive than UV detection at 210 nm wavelength [6]. Because of the natural fluorescence of Phe and Tyr, no derivatization step is required. No acetonitrile or methanol is used within the chromatographic procedure, and in contrast to UV-detection [6], the fluorescence monitoring reveals clean chromatograms with only rarely appearing peaks related to other substances. Serum or plasma are the superior sample types for the analysis of Phe and Tyr but this method can also be applied for the measurement of concentrations in cerebrospinal fluid (CSF) and cell culture supernatants (not shown). Whereas blood sera are diluted 1:1 to stay within linearity range of the method, CSF specimens are used undiluted because of their comparably lower Phe and Tyr contents. The new method allows rapid detection of Phe, Tyr and Phe/Tyr concentrations and is thus able to support new strategies in research and care of patients which focus on the association between Phe and Tyr metabolism in chronic inflammatory conditions and neuropsychiatric deviations [1–3]. The method presented here is based on the standard equipment and methodology used for the earlier described determination of tryptophan and kynurenine concentrations in human specimens [8]. Immune activation and inflammation are not only accompanied by disturbed Phe metabolism but also by tryptophan breakdown that is activated by pro-inflammatory cytokines [1,3]. IDO activity can be estimated by the kynurenine to tryptophan ratio (Kyn/Trp) and was recognized as another important link between the immunological and neuroendocrine circuits. Since only analysis buffers and fluorescence wavelengths differ between the two analytical methods for Phe/Tyr and Kyn/Trp measurements, the new method can easily be adapted for the measurement of tryptophan and kynurenine concentrations. This offers the opportunity of a sequential measurement of Phe/Tyr and Kyn/Trp utilizing the same deproteinized
BH4 CSF L-DOPA IDO 3NT PHA Phe Phe/Tyr PKU SSRI TDO Tyr
5,6,7,8-Tetrahydrobiopterin cerebrospinal fluid L-Dihydroxyphenylalanine indoleamine 2,3-dioxygenase 3-Nitro-L-tyrosine phenylalanine (4)-hydroxylase phenylalanine phenylalanine to tyrosine ratio phenylketonuria selective serotonin reuptake inhibitor tryptophan 2,3-dioxygenase tyrosine
Conflict of interest statement There are no conflicts of interest regarding the publication of this article. Acknowledgments The authors thank Mrs. Maria Pfurtscheller and Mr. Thomas Nuener for their excellent technical assistance. References [1] Neurauter G, Schröcksnadel K, Scholl-Bürgi S, Sperner-Unterweger B, Schubert C, Ledochowski M, et al. Chronic immune stimulation correlates with reduced phenylalanine turnover. Curr Drug Metab 2008;9:622–7. [2] Capuron L, Geisler S, Kurz K, Leblhuber F, Sperner-Unterweger B, Fuchs D. Activated immune system and inflammation in healthy ageing: relevance for tryptophan and neopterin metabolism. Curr Pharm Des 2013 [in press]. [3] Felger JC, Li L, Marvar PJ, Woolwine BJ, Harrison DG, Raison CL, et al. Tyrosine metabolism during interferon-alpha administration: association with fatigue and CSF dopamine concentrations. Brain Behav Immun 2013;31:153–60. [4] Scholl-Bürgi S, Haberlandt E, Heinz-Erian P, Deisenhammer F, Albrecht U, Sigl SB, et al. Amino acid cerebrospinal fluid/plasma ratios in children: influence of age, gender, and antiepileptic medication. Pediatrics 2008;121:e920–6. [5] Kand'ár R, Záková P. Determination of phenylalanine and tyrosine in plasma and dried blood samples using HPLC with fluorescence detection. J Chromatogr B Anal Technol Biomed Life Sci 2009;877:3926–9. [6] Mo XM, Li Y, Tang AG, Ren YP. Simultaneous determination of phenylalanine and tyrosine in peripheral capillary blood by HPLC with ultraviolet detection. Clin Biochem 2013;46:1074–8.
Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
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Please cite this article as: Neurauter G, et al, Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.10.015
