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Simultaneous determination of aromatic amino acids in human blood plasma by capillary electrophoresis with UV-absorption detection
Mauro Forteschi1,*, Salvatore Sotgia1, Stefano Assaretti1, Dionigia Arru1, Debora Cambedda1, Elisabetta Sotgiu1, Angelo Zinellu1, Ciriaco Carru1,2,*
1
Department of Biomedical Sciences, University of Sassari – Italy
2
Quality Control Unit, Hospital University of Sassari (AOU), Sassari, Italy
running title: Aromatic amino acids by CZE-UV detection
*Correspondence: Mauro Forteschi and Ciriaco Carru Dept. of Biomedical Sciences University of Sassari, Viale San Pietro 43/B - 07100 SASSARI – ITALY E-mail: [email protected] [email protected] Fax +39-079228275 - Phone +39-079229775
Keywords: Capillary Electrophoresis, methyl tryptophan, Phenylalanine, Tryptophan, Tyrosine.
Received: 10-Jan-2015; Revised: 21-Feb-2015; Accepted: 23-Feb-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201500038. This article is protected by copyright. All rights reserved.
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Abbreviations: AAA aromatic amino acid; CKD Chronic Kidney Disease; HBP Human Blood Plasma; IS internal standard; MPA metaphosphoric acid; mTrp methyl tryptophan; PCA perchloric acid; SSA sulfosalicylic acid; TCA trichloroacetic acid Abstract Phenylalanine, tyrosine and tryptophan, also known as aromatic amino acids, are involved in many physiological and pathophysiological conditions and are indicative of the liver and kidney function. In this work we describe a simple and accurate method for their simultaneous quantification, in a single capillary electrophoresis run. This method requires minimal sample manipulation, no derivatization procedures, and methyl tryptophan as internal standard. The human blood plasma sample was precipitated using sulfosalicylic acid and the supernatant was used for the analysis. All the analytes were baseline resolved within 16 min and detected at 200 nm using Tris phosphate 80 mmol/L at pH 1.4 as background electrolyte. The proposed method showed good linearity (r=0.998) and repeatability (intraassay RSD20 mL/min/1.73 m2 combined with a urinary protein excretion rate >0.3 g/24 h, without evidence of urinary tract infection or overt heart failure (New York Heart Association class III or more). Patients were classified as CKD at stage 3 and 4, and were not on dialysis. Exclusion criteria were represented by previous or concomitant treatment with steroids, anti-inflammatory and immunosuppressive agents, vitamin B6, B12, folate or statin, evidence or suspicion of renovascular disease, obstructive uropathy, type I diabetes mellitus and vasculitis. All patients were in stable treatment for at least six months with benazepril and valsartan, an inhibitor of the angiotensin-converting enzyme (ACE) and an angiotensin II receptor antagonist, respectively. Enrolled patients were randomized to receive 20 mg/day of sinvastatin and 10 mg/day of ezetimibe. Patients were treated for 12 months and evaluated at baseline and after four, eight and 12 months of therapy. The healthy controls’ group included 23 subjects (mean age, 58.2±11 years), recruited from accompanying relatives or friends of patients or from hospital personnel. Exclusion criteria for the control subjects were a history of diabetes, systemic hypertension, cardiovascular or cerebrovascular disease, renal failure, blood dyscrasias, tumors, retinal vascular disorders, age under 18 years old and current medication with vitamin B6, B12, or folic acid. The control subjects were recruited concurrently during the patients’ recruitment period. An informed consent was obtained from each patient and each control, and the study was approved by local Institution’s Ethics Committee.
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2.5 Statistical analysis All results are expressed as mean values (mean ± SD) or median values (median and range). The distribution of variables in the study group was assessed by the Kolmogorov–Simirnov test. The differences between cases and controls for quantitative variables were analyzed by Student’s t test or the Mann–Whitney test, when appropriate. The effect of drug treatment was evaluated by one-way repeated measures of analysis of variance (ANOVA). Calculations were performed using the software packages MedCalc for Windows, version 12.5 64 bit (MedCalc Software, Ostend, Belgium). 3 Results and Discussion 3.1 Electrophoretic conditions and BGE optimization The BGE features of the method were optimized testing a range of BGE concentration from 70 to 100 mmol/L and pH from 1.2 to 1.6. All of the experiments were performed in triplicate on HBP samples. The best separation performances were obtained using Tris phosphate 80 mmol/L at pH 1.4. As shown in fig 1A-B, this BGE allows to resolve all the analytes (Rs 1.35 between Trp and Phe Rs 1.56 between Tyr and IS) and to provide the highest separation efficiency (N > 15000 for all the analytes) and peak area (7.69, 11.41 15.7, and 21.99 Au.s for Phe, Trp, IS and Tyr respectively) fig 1 C-D. Migration times for all the analytes raise when BGE concentration increases (data not shown). In the selected electrophoretic condition all the analytes were resolved within 16 min (fig 2). All the following experiments were then performed using this BGE. 3.2 Sample preparation optimization The sample was prepared starting from 50 µL of HBP subjected to acidic precipitation. To optimize this process TCA, PCA, MPA, and SSA were tested at 3.2% m/v final concentration. While the samples precipitated with TCA, PCA and MPA showed current instability or disturbed baseline, the sample prepared using SSA showed the best overall electrophoretic behavior other than the highest peak area values for all the analytes. However, when acidic deproteinization is performed, a loss of analytes is often observed, especially in protein rich matrix, as HBP. In fact, during our preliminary experiments we observed an estimated 7% loss of analytes due to acidic precipitation (probably due to the analytes inclusion in the pellet). With the aim to improve the protein precipitation efficiency, and
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considering the negative effect caused by acid on electrophoretic separation, we evaluated the effect of sample dilution. Moreover, we evaluated the effect of diluting sample before or after the acidic precipitation. Samples dilution results in an improvement of current stability, regardless if sample was diluted before or after acidic precipitation; while the recovery of analytes was effectively improved when sample was diluted before acidic precipitation with SSA. We, then, optimized the sample pre-precipitation dilution testing several dilution ratios (5:1, 5:2.5, and 1:1 by adding 10, 20 and 50 µL of water to the HBP samples respectively). When 1:1 dilution was applied, we reported a general improvement of the peaks resolution (Rs from 0.7 to 1.52 for Trp) and separation efficiency (N from 132233 to 193699 for Phe). Moreover, in this condition, the recovery was quantitative. Samples were then prepared as described on materials and method. 3.3 Recovery LOD and LOQ determination The Phe, Trp and Tyr recovery ratio in HBP was evaluated by adding a spike of different concentrations (5, 50 and 100 µmol/L) of the analytes to the sample and comparing it with the sample without any spike. The average recovery was 101.0% for Phe 101.9% for Trp and 100.1% for Tyr. The LOD, evaluated as S/N=3, was 4 µmol/L for Phe and 1.5 µmol/L for Tyr and Trp; while the LOQ, evaluated as S/N=10, was 13 µmol/L for Phe and 5µmol/L for Tyr and Trp. 3.4 Method validation The calibration curves, obtained as the ratio of Phe, Trp and Tyr peak areas to that of IS versus concentration, were linear in the range of concentration tested: Phe and Tyr between 120 and 15 µmol/L (Phe calibration curve Y=0.0052X+0.0063 R2=0.9982; Tyr calibration curve Y=0.0127X+0.0216, R2 0.9989) while Trp between 80 and 10 µmol/L (Trp calibration curve Y=0.0115X+0.0549 R2=0.9949). The RSD of injection repeatability test was 0.37% for Phe, 1.04 for Trp, 0.94% for IS and 0.68% for Tyr. The within-run precision (intra-assay) of the method was evaluated by injecting the same biological sample ten times consecutively, while the between-run (inter-assay) precision was determined by injecting the same biological sample in ten consecutive days. Precision tests indicate a good repeatability of the method, intra-assay 2.49, 3.01, 2.12 and 2.78% for Phe, Trp, IS and Tyr respectively while the inter-assay precision of the method was 4.83, 5.40, 4.76 and 4.74% for Phe, Trp, IS and Tyr respectively. As reported in Table 1, data in controls, obtained by the present method, are comparable with the values already reported in literature.
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3.5 Method application The applicability of the method was tested by measuring the AAAs concentration in 23 healthy volunteers (mean age 58.2 ± 11years) and 20 CKD subjects (mean age 58.8± 9.9 years). Enrolled subjects criteria were earlier described [25] and the results are reported in table 2. A significant difference was found between healthy subjects and CKD patients regarding the Trp HBP concentration (p= 0.020), while no significant difference was observed regarding the HBP Tyr, Phe concentration and the Tyr/Phe ratio. 20 CKD patients were subjected to simvastatin/ezetimibe treatment for 12 months and evaluated at baseline, 4, 8 and 12 months. As shown in table 3, no significant variations were found in the concentration of Phe, Trp and Tyr during the hypolipidemic treatment, while a weak increase (even if not statistically significant) was found for the Tyr/Phe ratio (p=0.103). 4 Concluding remarks The analysis of HBP amino acids is important in understanding mechanisms of disease in critical illnesses. Many HPLC methods are able to measure AAAs in assorted matrices, but most of them need derivatization procedures and are able to measure only one or two compounds [8,9]. Regarding CE, in 2010 a method was described that was able to determinate AAAs in decomposition fluid [34], and in urine by CE–MS/MS in 2013 [35]. Here we present a simple and accurate method for simultaneous quantification of Tyr, Trp and Phe without any derivatization procedure. Our method needs only 50 µL of HBP and a minimal sample manipulation so that the variability due to the operator is negligible. AAAs are detected within 16 min and good linearity and repeatability is obtained for all the analytes. Impaired AAAs metabolism is known to take place in CKD patients [1–4, 36] so we successfully applied the presented method on this kind of patients. A significant difference between healthy controls and CKD patients was found in the HBP Trp level while an indicative increasing trend was found about Tyr/Phe ratio during the hypolipidemic treatment with simvastatin/ezetimibe. The lowering in Trp level found in CKD patients, even if it may be explained as an alteration in the kidneys Kynurenine pathway, is a topic that needs to be further investigated. Regarding the Tyr/Phe ratio, kidney plays a pivotal role in maintain the circulating Tyr level in humans contributing for at least 50% of whole body conversion of Phe to Tyr [2]. Previous reports suggested that uremic patients have lower Tyr/Phe ratio [4,7,37] and this is due to the impairment of the kidney’s ability to convert Phe to Tyr [37– 39]. Therefore a slight but indicative trend that shows a raising of Tyr/Phe ratio may indicate the kidney's restoring function during the simvastatin/ezetimibe treatment.
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Acknowledgements: This study was supported by the “FondazioneBanco di Sardegna – Sassari – Italy” and by the “Ministero dell’Università e della Ricerca” Italy. The manuscript language revision by Mrs Maria Antonietta Meloni is greatly appreciated.
The authors have declared no conflict of interest
5 References [1] Kopple, J. D., J. Nutr. 2007,137, 1586S–1590S. [2] Møller, N., Meek, S., Bigelow, M., Andrews, J., Nair, K.S., PNAS 2000, 97, 1242–1246. [3] Laidlaw, S. A., Berg, R. L., Kopple, J. D., Naito, H., Walker, W. G., Walser, M., Am. J. Kidney Dis. 1994, 23, 504–13. [4] Pawlak, D., Tankiewicz, A., Mysliwiec, P., Buczko, W., Nephron 2002, 90, 328–335. [5] Fernstrom, J. D., J. Nutr. Biochem. 1990, 1, 508–17. [6] Fernstrom, J. D., Fernstrom, M. H., J. Nutr. 2007, 137, 1539S–1547S. [7] Neurauter, G., Schröcksnadel, K., Scholl-Bürgi, S., Sperner-Unterweger, B., Schubert, C., Ledochowski, M., Fuchs, D., Curr. Drug Metab. 2008, 7, 622–7. [8] Neckers, L. M., Delisi, L. E., Wyatt, R. J., Clin. Chem. 1981, 27, 146–8. [9] Kalghatgi, K., Ambrus, C. M., Horvath, C., Karakousis, C. P., Sharma, S. D., Barren, E., Res. Commun. Chem. Pathol. Pharmacol. 1984, 45, 253–9. [10] Bayle, C., Siri, N., Poinsot, V., Treilhou, M., Caussé, E., Couderc, F., J. Chromatogr. A, 2003, 1013, 123–30. [11] Poinsot, V., Ong-Meang, V., Gavard, P., Couderc, F., Electrophoresis 2014, 35, 50–68. [12] Poinsot, V., Carpéné, M.A., Bouajila, J., Gavard, P., Feurer, B., Couderc, F., Electrophoresis 2012, 33, 14–35.
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[13] Viglio, S., Fumagalli, M., Ferrari, F., Bardoni, A.,Salvini, R., Giuliano, S., Iadarola, P., Electrophoresis 2012, 33,36–47 [14] Kašička, V., Electrophoresis 2014, 35, 69–95 [15] Kašička, V., Electrophoresis 2012, 33, 48–73 [16] Kandàr, R., Záková, P., J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2009, 877, 3926–9. [17] Tang, A. G., Mo, X. M., Luo, X. B., Ao, X., Clin. Biochem. 2009, 42, 420–5. [18] Uchikura K., Chem. Pharm. Bull. 2003, 51(9), 1092–4. [19] Kand'ár, R., Záková, P., Jirosová, J., Sladká, M., Clin. Chem. Lab. Med. 2009, 47(5), 565–72. [20] Deng, C., Deng, Y., Wang, B., Yang, X., J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2002, 780(2), 407–13. [21] Chace, D. H., Sherwin, J. E., Hillman, S. L., Lorey, F., Cunningham, G. C., Clin. Chem. 1998, 44(12), 2405–9. [22] Ramautar, R., Mayboroda, O. A., Derks, R. J., Van Nieuwkoop, C., Van Dissel, J. T., Somsen, G. W., Deelder, A. M., De Jong, G. J., Electrophoresis 2008, 29(12), 2714–22. [23] Jeong, J. S., Kim, S. K., Park, S. R, Anal. Bioanal. Chem. 2013, 405(25), 8063–72. [24] Desiderio, C., Iavarone, F., Rossetti, D.V., Messana, I., Castagnola, M., J. Sep. Sci. 2010, 33, 2385–93 [25] Forteschi, M., Sotgia, S., Pintus, G., Zinellu, A., Carru, C., J. Sep. Sci. 2014, 37(17), 2418–23 [26] Sa, M., Ying, L., Tang, A.G., Xiao, L.D., Ren, Y.P., Clin. Chim. Acta 2012, 14, 413, 973–7. [27] Mayoral-Mariles, A., Cruz-Revilla, C., Vega-Manriquez, X., Aguirre-Hernández, R., Severiano-Pérez, P., Aburto-Arciniega, E., Jiménez-Mendoza, A., Guevara-Guzmán, R., Arch. Med. Res. 2012, 43, 375 382. [28] Ehrlich, ,S., Franke, L., Schneider, N., Salbach-Andrae, H., Schott, R., Craciun, E.M., Pfeiffer, E., Uebelhack, R., Lehmkuhl, U., Int. J. Eat. Disord. 2009, 42, 166–72. [29] Li, Y., Tang, A.G., Mu, S., Clin. Chim. Acta 2011, 12, 412, 1032–5
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[30] Psychogios, N., Hau, D.D., Peng, J., Guo, A.C., Mandal, R., Bouatra, S., Sinelnikov, I., Krishnamurthy, R., Eisner, R., Gautam, B., Young, N., Xia, J., Knox, C., Dong, E., Huang, P., Hollander, Z., Pedersen, T.L., Smith, S.R., Bamforth, F., Greiner, R., Mc Manus, B., Newman, J.W., Goodfriend, T., Wishart. D.S., PLoS One 2011, 16, 6 16957. [31] Domingues, D.S., Crevelin, E.J., De Moraes, L.A., Cecilio Hallak, J.E., De Souza Crippa, J.A., Costa Queiroz, M.E., J. Sep. Sci. 2014 26, 1–8. [32] Slocum, R.H., Cummings, J.G,. Amino acids analysis of physiological samples. In: Hommes FA, editor. Techniques in Diagnostic Human Biochemical Genetics – A Laboratory Manual. New York: Wiley-Liss, 1991, pp 87–126 [33] Borum, P.R., Manual for amino acid analysis of physiological samples. Proceedings of American Association for Clinical Chemistry and Canadian Society of Clinical Chemists 37th National Meeting, Chicago (A-TB-127) 1986, pp 1–12. [34] Swann, L. M., Forbes, S. L., Lewis,. S. W., Talanta 2010, 81, 4–5. [35] Alberice, J. V., Amaral, A. F., Armitage, E. G., Lorente, J. A., Algaba, F., Carrilho, E., Márquez, M., García, A., Malats, N., Barbas, C., J. Chromatogr. A 2013, 1318, 163–70. [36] Viljoen, M., Bipath, P., Govender, C., Potgieter, C. D., Clin. Nephrol. 2008, 70, 561–2. [37] Boirie, Y., Albright. R., Bigelow, M., Nair, K. S., Kidney Int. 2004, 66(2), 591–6. [38] Tessari, P., De Ferrari, G., Robaudo, C., Vettore, M., Pastorino, N., De Biasi, L., Garibotto, G., Kidney Int. 1999, 56(6), 2168–72. [39] Letteri, J. M., Scipione, R. A., Nephron 1974, 13, 365–371.
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Figure 1. A) resolution of Trp calculated respect to Phe; and B) in resolution of Tyr respect to IS (mTrp) in relation to BGE concentration and pH; C) Phe Trp IS (mTrp) and Tyr area values (Au.s); and D) separation efficiency (N) in relation to BGE concentration at pH 1.4
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Figure 2. A) Electropherogram of Phe Trp IS and Tyr standards (Phe and Tyr 120 µmol/L, Trp 80 µmol/L, and IS 200 µmol/L); and B) Electropherogram of HBP sample (Phe 95.32 µmol/L, Trp 92.42 µmol/L, IS 200 µmol/L, and Tyr 114.35 µmol/L ), in the same run are also detectable Arg at 8.7 min, creatinine at 9 min, His at 9.5 min, Ala at 11 min, Val at 11.3 min and Cys at 14.5 min and two other unknown peaks at 12.2 and 14.3 min respectively.
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Table 1. Reported HBP levels of AAAs in controls (CTRLs) Phe Trp Tyr Method (mol/L) (mol/L) (mol/L) CE HPLC
60.3 ± 18.9 --
60.4 ± 13.4 42.4 ± 1.1
68.5 ± 13.3 63.6 ± 2.2
HPLC
51.4 ± 1.2
46.5 ± 1.3
61.8 ± 2.1
HPLC 58.7 ± 9.6 63.8 ± 8.9 HPLC 44.2 ± 4.6 63.5 ± 4.6 Direct Flow Injection MS/MS 85.2 ± 23 78.4 ± 15.5 LC MS/MS -53 ± 17.4 Beckman 6300 Amino Acid 35 – 85* -Analyzer. Beckman 6300 Amino Acid 36-88 -Analyzer. (55)** All data are expressed as mean ± SD. *range; **range (mean)
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Reference
54.5 ± 17.5 63.5 ± 4.6 143 ± 35 57 ± 16
Our study Mu Sa et al [26] Mayoral-Mariles et al [27] Ehrlich et al [28] Y. Li et al. [29] Psychogios et al [30] Domingues et al [31]
34 – 112*
Slocum et al.[32]
30 – 97 (58)**
Borumet al. [33]
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Table 2. Comparison of Phe, Trp, Tyr and Tyr/Phe HBP concentrations (µmol/L ± SD) between Controls and CKD patients
Phe
Trp
Tyr
Tyr/Phe
AGE
CTRLs
60.3 ± 18.9
60.4 ± 13.4
68.5 ± 13.3
1.18 ± 0.26
58.3 ± 11.1
CKDs
56.5 ± 12
46.2 ± 11.4
57.3 ± 16.4
1.09 ± 0.2
58.8 ± 9.9
t-test
P = 0.443
P = 0.020
P = 0.102
P = 0.242
P = 0.872
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Table 3. Variations on Phe, Trp, Tyr (µmol/L ± SD) and Tyr/Phe ratio during statine/ezetimibe treatment, I basal level time 0, II after four months treatment, III after eight months treatment, IV after one year treatment
I
II
III
IV
P value
Phe
56.5 ± 12
62.3 ± 12.9
56.8 ± 11.8
55.2 ± 13.8
P = 0.475
Trp
46.1 ± 11.4
50.0 ± 14.0
49.2 ± 12.0
49.0 ± 13.5
P = 0.496
Tyr
57.3 ± 16.4
62.5 ± 18.3
59.6 ± 14.7
60.1 ± 20.6
P = 0.712
Tyr/Phe
1.02 ± 0.2
1.00 ± 0.2
1.06 ± 0.2
1.08 ± 0.2
P = 0.103
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Simultaneous determination of aromatic amino acids in human blood plasma by capillary electrophoresis with UV-absorption detection
Mauro Forteschi1,*, Salvatore Sotgia1, Stefano Assaretti1, Dionigia Arru1, Debora Cambedda1, Elisabetta Sotgiu1, Angelo Zinellu1, Ciriaco Carru1,2,*
1
Department of Biomedical Sciences, University of Sassari – Italy
2
Quality Control Unit, Hospital University of Sassari (AOU), Sassari, Italy
running title: Aromatic amino acids by CZE-UV detection
*Correspondence: Mauro Forteschi and Ciriaco Carru Dept. of Biomedical Sciences University of Sassari, Viale San Pietro 43/B - 07100 SASSARI – ITALY E-mail: [email protected] [email protected] Fax +39-079228275 - Phone +39-079229775
Keywords: Capillary Electrophoresis, methyl tryptophan, Phenylalanine, Tryptophan, Tyrosine.
Received: 10-Jan-2015; Revised: 21-Feb-2015; Accepted: 23-Feb-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201500038. This article is protected by copyright. All rights reserved.
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Abbreviations: AAA aromatic amino acid; CKD Chronic Kidney Disease; HBP Human Blood Plasma; IS internal standard; MPA metaphosphoric acid; mTrp methyl tryptophan; PCA perchloric acid; SSA sulfosalicylic acid; TCA trichloroacetic acid Abstract Phenylalanine, tyrosine and tryptophan, also known as aromatic amino acids, are involved in many physiological and pathophysiological conditions and are indicative of the liver and kidney function. In this work we describe a simple and accurate method for their simultaneous quantification, in a single capillary electrophoresis run. This method requires minimal sample manipulation, no derivatization procedures, and methyl tryptophan as internal standard. The human blood plasma sample was precipitated using sulfosalicylic acid and the supernatant was used for the analysis. All the analytes were baseline resolved within 16 min and detected at 200 nm using Tris phosphate 80 mmol/L at pH 1.4 as background electrolyte. The proposed method showed good linearity (r=0.998) and repeatability (intraassay RSD20 mL/min/1.73 m2 combined with a urinary protein excretion rate >0.3 g/24 h, without evidence of urinary tract infection or overt heart failure (New York Heart Association class III or more). Patients were classified as CKD at stage 3 and 4, and were not on dialysis. Exclusion criteria were represented by previous or concomitant treatment with steroids, anti-inflammatory and immunosuppressive agents, vitamin B6, B12, folate or statin, evidence or suspicion of renovascular disease, obstructive uropathy, type I diabetes mellitus and vasculitis. All patients were in stable treatment for at least six months with benazepril and valsartan, an inhibitor of the angiotensin-converting enzyme (ACE) and an angiotensin II receptor antagonist, respectively. Enrolled patients were randomized to receive 20 mg/day of sinvastatin and 10 mg/day of ezetimibe. Patients were treated for 12 months and evaluated at baseline and after four, eight and 12 months of therapy. The healthy controls’ group included 23 subjects (mean age, 58.2±11 years), recruited from accompanying relatives or friends of patients or from hospital personnel. Exclusion criteria for the control subjects were a history of diabetes, systemic hypertension, cardiovascular or cerebrovascular disease, renal failure, blood dyscrasias, tumors, retinal vascular disorders, age under 18 years old and current medication with vitamin B6, B12, or folic acid. The control subjects were recruited concurrently during the patients’ recruitment period. An informed consent was obtained from each patient and each control, and the study was approved by local Institution’s Ethics Committee.
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2.5 Statistical analysis All results are expressed as mean values (mean ± SD) or median values (median and range). The distribution of variables in the study group was assessed by the Kolmogorov–Simirnov test. The differences between cases and controls for quantitative variables were analyzed by Student’s t test or the Mann–Whitney test, when appropriate. The effect of drug treatment was evaluated by one-way repeated measures of analysis of variance (ANOVA). Calculations were performed using the software packages MedCalc for Windows, version 12.5 64 bit (MedCalc Software, Ostend, Belgium). 3 Results and Discussion 3.1 Electrophoretic conditions and BGE optimization The BGE features of the method were optimized testing a range of BGE concentration from 70 to 100 mmol/L and pH from 1.2 to 1.6. All of the experiments were performed in triplicate on HBP samples. The best separation performances were obtained using Tris phosphate 80 mmol/L at pH 1.4. As shown in fig 1A-B, this BGE allows to resolve all the analytes (Rs 1.35 between Trp and Phe Rs 1.56 between Tyr and IS) and to provide the highest separation efficiency (N > 15000 for all the analytes) and peak area (7.69, 11.41 15.7, and 21.99 Au.s for Phe, Trp, IS and Tyr respectively) fig 1 C-D. Migration times for all the analytes raise when BGE concentration increases (data not shown). In the selected electrophoretic condition all the analytes were resolved within 16 min (fig 2). All the following experiments were then performed using this BGE. 3.2 Sample preparation optimization The sample was prepared starting from 50 µL of HBP subjected to acidic precipitation. To optimize this process TCA, PCA, MPA, and SSA were tested at 3.2% m/v final concentration. While the samples precipitated with TCA, PCA and MPA showed current instability or disturbed baseline, the sample prepared using SSA showed the best overall electrophoretic behavior other than the highest peak area values for all the analytes. However, when acidic deproteinization is performed, a loss of analytes is often observed, especially in protein rich matrix, as HBP. In fact, during our preliminary experiments we observed an estimated 7% loss of analytes due to acidic precipitation (probably due to the analytes inclusion in the pellet). With the aim to improve the protein precipitation efficiency, and
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considering the negative effect caused by acid on electrophoretic separation, we evaluated the effect of sample dilution. Moreover, we evaluated the effect of diluting sample before or after the acidic precipitation. Samples dilution results in an improvement of current stability, regardless if sample was diluted before or after acidic precipitation; while the recovery of analytes was effectively improved when sample was diluted before acidic precipitation with SSA. We, then, optimized the sample pre-precipitation dilution testing several dilution ratios (5:1, 5:2.5, and 1:1 by adding 10, 20 and 50 µL of water to the HBP samples respectively). When 1:1 dilution was applied, we reported a general improvement of the peaks resolution (Rs from 0.7 to 1.52 for Trp) and separation efficiency (N from 132233 to 193699 for Phe). Moreover, in this condition, the recovery was quantitative. Samples were then prepared as described on materials and method. 3.3 Recovery LOD and LOQ determination The Phe, Trp and Tyr recovery ratio in HBP was evaluated by adding a spike of different concentrations (5, 50 and 100 µmol/L) of the analytes to the sample and comparing it with the sample without any spike. The average recovery was 101.0% for Phe 101.9% for Trp and 100.1% for Tyr. The LOD, evaluated as S/N=3, was 4 µmol/L for Phe and 1.5 µmol/L for Tyr and Trp; while the LOQ, evaluated as S/N=10, was 13 µmol/L for Phe and 5µmol/L for Tyr and Trp. 3.4 Method validation The calibration curves, obtained as the ratio of Phe, Trp and Tyr peak areas to that of IS versus concentration, were linear in the range of concentration tested: Phe and Tyr between 120 and 15 µmol/L (Phe calibration curve Y=0.0052X+0.0063 R2=0.9982; Tyr calibration curve Y=0.0127X+0.0216, R2 0.9989) while Trp between 80 and 10 µmol/L (Trp calibration curve Y=0.0115X+0.0549 R2=0.9949). The RSD of injection repeatability test was 0.37% for Phe, 1.04 for Trp, 0.94% for IS and 0.68% for Tyr. The within-run precision (intra-assay) of the method was evaluated by injecting the same biological sample ten times consecutively, while the between-run (inter-assay) precision was determined by injecting the same biological sample in ten consecutive days. Precision tests indicate a good repeatability of the method, intra-assay 2.49, 3.01, 2.12 and 2.78% for Phe, Trp, IS and Tyr respectively while the inter-assay precision of the method was 4.83, 5.40, 4.76 and 4.74% for Phe, Trp, IS and Tyr respectively. As reported in Table 1, data in controls, obtained by the present method, are comparable with the values already reported in literature.
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3.5 Method application The applicability of the method was tested by measuring the AAAs concentration in 23 healthy volunteers (mean age 58.2 ± 11years) and 20 CKD subjects (mean age 58.8± 9.9 years). Enrolled subjects criteria were earlier described [25] and the results are reported in table 2. A significant difference was found between healthy subjects and CKD patients regarding the Trp HBP concentration (p= 0.020), while no significant difference was observed regarding the HBP Tyr, Phe concentration and the Tyr/Phe ratio. 20 CKD patients were subjected to simvastatin/ezetimibe treatment for 12 months and evaluated at baseline, 4, 8 and 12 months. As shown in table 3, no significant variations were found in the concentration of Phe, Trp and Tyr during the hypolipidemic treatment, while a weak increase (even if not statistically significant) was found for the Tyr/Phe ratio (p=0.103). 4 Concluding remarks The analysis of HBP amino acids is important in understanding mechanisms of disease in critical illnesses. Many HPLC methods are able to measure AAAs in assorted matrices, but most of them need derivatization procedures and are able to measure only one or two compounds [8,9]. Regarding CE, in 2010 a method was described that was able to determinate AAAs in decomposition fluid [34], and in urine by CE–MS/MS in 2013 [35]. Here we present a simple and accurate method for simultaneous quantification of Tyr, Trp and Phe without any derivatization procedure. Our method needs only 50 µL of HBP and a minimal sample manipulation so that the variability due to the operator is negligible. AAAs are detected within 16 min and good linearity and repeatability is obtained for all the analytes. Impaired AAAs metabolism is known to take place in CKD patients [1–4, 36] so we successfully applied the presented method on this kind of patients. A significant difference between healthy controls and CKD patients was found in the HBP Trp level while an indicative increasing trend was found about Tyr/Phe ratio during the hypolipidemic treatment with simvastatin/ezetimibe. The lowering in Trp level found in CKD patients, even if it may be explained as an alteration in the kidneys Kynurenine pathway, is a topic that needs to be further investigated. Regarding the Tyr/Phe ratio, kidney plays a pivotal role in maintain the circulating Tyr level in humans contributing for at least 50% of whole body conversion of Phe to Tyr [2]. Previous reports suggested that uremic patients have lower Tyr/Phe ratio [4,7,37] and this is due to the impairment of the kidney’s ability to convert Phe to Tyr [37– 39]. Therefore a slight but indicative trend that shows a raising of Tyr/Phe ratio may indicate the kidney's restoring function during the simvastatin/ezetimibe treatment.
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Acknowledgements: This study was supported by the “FondazioneBanco di Sardegna – Sassari – Italy” and by the “Ministero dell’Università e della Ricerca” Italy. The manuscript language revision by Mrs Maria Antonietta Meloni is greatly appreciated.
The authors have declared no conflict of interest
5 References [1] Kopple, J. D., J. Nutr. 2007,137, 1586S–1590S. [2] Møller, N., Meek, S., Bigelow, M., Andrews, J., Nair, K.S., PNAS 2000, 97, 1242–1246. [3] Laidlaw, S. A., Berg, R. L., Kopple, J. D., Naito, H., Walker, W. G., Walser, M., Am. J. Kidney Dis. 1994, 23, 504–13. [4] Pawlak, D., Tankiewicz, A., Mysliwiec, P., Buczko, W., Nephron 2002, 90, 328–335. [5] Fernstrom, J. D., J. Nutr. Biochem. 1990, 1, 508–17. [6] Fernstrom, J. D., Fernstrom, M. H., J. Nutr. 2007, 137, 1539S–1547S. [7] Neurauter, G., Schröcksnadel, K., Scholl-Bürgi, S., Sperner-Unterweger, B., Schubert, C., Ledochowski, M., Fuchs, D., Curr. Drug Metab. 2008, 7, 622–7. [8] Neckers, L. M., Delisi, L. E., Wyatt, R. J., Clin. Chem. 1981, 27, 146–8. [9] Kalghatgi, K., Ambrus, C. M., Horvath, C., Karakousis, C. P., Sharma, S. D., Barren, E., Res. Commun. Chem. Pathol. Pharmacol. 1984, 45, 253–9. [10] Bayle, C., Siri, N., Poinsot, V., Treilhou, M., Caussé, E., Couderc, F., J. Chromatogr. A, 2003, 1013, 123–30. [11] Poinsot, V., Ong-Meang, V., Gavard, P., Couderc, F., Electrophoresis 2014, 35, 50–68. [12] Poinsot, V., Carpéné, M.A., Bouajila, J., Gavard, P., Feurer, B., Couderc, F., Electrophoresis 2012, 33, 14–35.
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[13] Viglio, S., Fumagalli, M., Ferrari, F., Bardoni, A.,Salvini, R., Giuliano, S., Iadarola, P., Electrophoresis 2012, 33,36–47 [14] Kašička, V., Electrophoresis 2014, 35, 69–95 [15] Kašička, V., Electrophoresis 2012, 33, 48–73 [16] Kandàr, R., Záková, P., J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2009, 877, 3926–9. [17] Tang, A. G., Mo, X. M., Luo, X. B., Ao, X., Clin. Biochem. 2009, 42, 420–5. [18] Uchikura K., Chem. Pharm. Bull. 2003, 51(9), 1092–4. [19] Kand'ár, R., Záková, P., Jirosová, J., Sladká, M., Clin. Chem. Lab. Med. 2009, 47(5), 565–72. [20] Deng, C., Deng, Y., Wang, B., Yang, X., J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2002, 780(2), 407–13. [21] Chace, D. H., Sherwin, J. E., Hillman, S. L., Lorey, F., Cunningham, G. C., Clin. Chem. 1998, 44(12), 2405–9. [22] Ramautar, R., Mayboroda, O. A., Derks, R. J., Van Nieuwkoop, C., Van Dissel, J. T., Somsen, G. W., Deelder, A. M., De Jong, G. J., Electrophoresis 2008, 29(12), 2714–22. [23] Jeong, J. S., Kim, S. K., Park, S. R, Anal. Bioanal. Chem. 2013, 405(25), 8063–72. [24] Desiderio, C., Iavarone, F., Rossetti, D.V., Messana, I., Castagnola, M., J. Sep. Sci. 2010, 33, 2385–93 [25] Forteschi, M., Sotgia, S., Pintus, G., Zinellu, A., Carru, C., J. Sep. Sci. 2014, 37(17), 2418–23 [26] Sa, M., Ying, L., Tang, A.G., Xiao, L.D., Ren, Y.P., Clin. Chim. Acta 2012, 14, 413, 973–7. [27] Mayoral-Mariles, A., Cruz-Revilla, C., Vega-Manriquez, X., Aguirre-Hernández, R., Severiano-Pérez, P., Aburto-Arciniega, E., Jiménez-Mendoza, A., Guevara-Guzmán, R., Arch. Med. Res. 2012, 43, 375 382. [28] Ehrlich, ,S., Franke, L., Schneider, N., Salbach-Andrae, H., Schott, R., Craciun, E.M., Pfeiffer, E., Uebelhack, R., Lehmkuhl, U., Int. J. Eat. Disord. 2009, 42, 166–72. [29] Li, Y., Tang, A.G., Mu, S., Clin. Chim. Acta 2011, 12, 412, 1032–5
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[30] Psychogios, N., Hau, D.D., Peng, J., Guo, A.C., Mandal, R., Bouatra, S., Sinelnikov, I., Krishnamurthy, R., Eisner, R., Gautam, B., Young, N., Xia, J., Knox, C., Dong, E., Huang, P., Hollander, Z., Pedersen, T.L., Smith, S.R., Bamforth, F., Greiner, R., Mc Manus, B., Newman, J.W., Goodfriend, T., Wishart. D.S., PLoS One 2011, 16, 6 16957. [31] Domingues, D.S., Crevelin, E.J., De Moraes, L.A., Cecilio Hallak, J.E., De Souza Crippa, J.A., Costa Queiroz, M.E., J. Sep. Sci. 2014 26, 1–8. [32] Slocum, R.H., Cummings, J.G,. Amino acids analysis of physiological samples. In: Hommes FA, editor. Techniques in Diagnostic Human Biochemical Genetics – A Laboratory Manual. New York: Wiley-Liss, 1991, pp 87–126 [33] Borum, P.R., Manual for amino acid analysis of physiological samples. Proceedings of American Association for Clinical Chemistry and Canadian Society of Clinical Chemists 37th National Meeting, Chicago (A-TB-127) 1986, pp 1–12. [34] Swann, L. M., Forbes, S. L., Lewis,. S. W., Talanta 2010, 81, 4–5. [35] Alberice, J. V., Amaral, A. F., Armitage, E. G., Lorente, J. A., Algaba, F., Carrilho, E., Márquez, M., García, A., Malats, N., Barbas, C., J. Chromatogr. A 2013, 1318, 163–70. [36] Viljoen, M., Bipath, P., Govender, C., Potgieter, C. D., Clin. Nephrol. 2008, 70, 561–2. [37] Boirie, Y., Albright. R., Bigelow, M., Nair, K. S., Kidney Int. 2004, 66(2), 591–6. [38] Tessari, P., De Ferrari, G., Robaudo, C., Vettore, M., Pastorino, N., De Biasi, L., Garibotto, G., Kidney Int. 1999, 56(6), 2168–72. [39] Letteri, J. M., Scipione, R. A., Nephron 1974, 13, 365–371.
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Figure 1. A) resolution of Trp calculated respect to Phe; and B) in resolution of Tyr respect to IS (mTrp) in relation to BGE concentration and pH; C) Phe Trp IS (mTrp) and Tyr area values (Au.s); and D) separation efficiency (N) in relation to BGE concentration at pH 1.4
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Figure 2. A) Electropherogram of Phe Trp IS and Tyr standards (Phe and Tyr 120 µmol/L, Trp 80 µmol/L, and IS 200 µmol/L); and B) Electropherogram of HBP sample (Phe 95.32 µmol/L, Trp 92.42 µmol/L, IS 200 µmol/L, and Tyr 114.35 µmol/L ), in the same run are also detectable Arg at 8.7 min, creatinine at 9 min, His at 9.5 min, Ala at 11 min, Val at 11.3 min and Cys at 14.5 min and two other unknown peaks at 12.2 and 14.3 min respectively.
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Table 1. Reported HBP levels of AAAs in controls (CTRLs) Phe Trp Tyr Method (mol/L) (mol/L) (mol/L) CE HPLC
60.3 ± 18.9 --
60.4 ± 13.4 42.4 ± 1.1
68.5 ± 13.3 63.6 ± 2.2
HPLC
51.4 ± 1.2
46.5 ± 1.3
61.8 ± 2.1
HPLC 58.7 ± 9.6 63.8 ± 8.9 HPLC 44.2 ± 4.6 63.5 ± 4.6 Direct Flow Injection MS/MS 85.2 ± 23 78.4 ± 15.5 LC MS/MS -53 ± 17.4 Beckman 6300 Amino Acid 35 – 85* -Analyzer. Beckman 6300 Amino Acid 36-88 -Analyzer. (55)** All data are expressed as mean ± SD. *range; **range (mean)
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Reference
54.5 ± 17.5 63.5 ± 4.6 143 ± 35 57 ± 16
Our study Mu Sa et al [26] Mayoral-Mariles et al [27] Ehrlich et al [28] Y. Li et al. [29] Psychogios et al [30] Domingues et al [31]
34 – 112*
Slocum et al.[32]
30 – 97 (58)**
Borumet al. [33]
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Table 2. Comparison of Phe, Trp, Tyr and Tyr/Phe HBP concentrations (µmol/L ± SD) between Controls and CKD patients
Phe
Trp
Tyr
Tyr/Phe
AGE
CTRLs
60.3 ± 18.9
60.4 ± 13.4
68.5 ± 13.3
1.18 ± 0.26
58.3 ± 11.1
CKDs
56.5 ± 12
46.2 ± 11.4
57.3 ± 16.4
1.09 ± 0.2
58.8 ± 9.9
t-test
P = 0.443
P = 0.020
P = 0.102
P = 0.242
P = 0.872
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Table 3. Variations on Phe, Trp, Tyr (µmol/L ± SD) and Tyr/Phe ratio during statine/ezetimibe treatment, I basal level time 0, II after four months treatment, III after eight months treatment, IV after one year treatment
I
II
III
IV
P value
Phe
56.5 ± 12
62.3 ± 12.9
56.8 ± 11.8
55.2 ± 13.8
P = 0.475
Trp
46.1 ± 11.4
50.0 ± 14.0
49.2 ± 12.0
49.0 ± 13.5
P = 0.496
Tyr
57.3 ± 16.4
62.5 ± 18.3
59.6 ± 14.7
60.1 ± 20.6
P = 0.712
Tyr/Phe
1.02 ± 0.2
1.00 ± 0.2
1.06 ± 0.2
1.08 ± 0.2
P = 0.103
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