Chromatographic characterization of amino acid profiles in urinary samples of patients suffering from prostate carcinoma
Authors:
N. Cernei 1,2; Z. Heger 1,2; Š. Veselý 3; O. Zítka 1,2; V. Adam 1,2; R. Kizek 1,2
Authors‘ workplace:
Ústav chemie a biochemie, Mendelova univerzita v Brně, Zemědělská 1, CZ-61 00 Brno, Česká republika
1; Středoevropský technologický institut, VUT v Brně, Technická 058/10, CZ-616 00 Brno, Česká republika
2; Urologická klinika, 2. Lékařská fakulta Univerzity Karlovy a FN V Motole, V Úvalu 84, CZ-150 06 Praha 5, Česká republika
3
Published in:
Klin. Biochem. Metab., 22 (43), 2014, No. 4, p. 196-202
Overview
Objective:
The aim of our study was to prepare a protocol for pre-treatment of clinical urinary samples, subsequently employed for acquirement of amino acid profiles via ion exchange chromatography with detection in visible spectrum (IEC/Vis). Further for clinically interesting biomolecules as sarcosine and taurine the optimization was carried out to shorten the analysis time.
Design:
Methodological
Material and methods:
Urine specimens from patients diagnosed with prostate cancer (n = 500) were collected within 1 year and stored in -80°C. Samples were processed for analysis of amino acid profiles through acidic hydrolysis in a microwave reactor. Using optimized conditions (80 W, 120°C, 25 bar, 105 min), 500 µL sample, mixed with 500 µL of 35% hydrochloric acid hydrolyzed. That resulted in product, which was subsequently diluted in buffer of sodium cycle (0.2 mol/L NaCl, 60 C6H807 mmol/L, 1.5 mmol/L and 0.4% N3Na S(CH2CH2OH)2). After centrifugation (25000 g at 4°C, 20 min), the samples were neutralized (0.6 mol/l NaOH) and analyzed using ion-exchange liquid chromatography with post-column derivatization with ninhydrin, using the detection wavelength λ = 440 and 570 nm (IEC/Vis). For analysis of sarcosine and taurine, the samples were prepared by evaporation of 250 µL of sample employing a nitrogen evaporator (40 min, temperature 60°C. Nitrogen pressure of 1 bar) and resuspended with buffer of sodium cycle (250 µL).
Results and conclusion:
The analyses of amino acid profiles offer interesting clinical information not only in non-invasively collected urinary samples, but also in other organic matrices. Employment of nitrogen evaporator for sample pre-treatment leads to reduction of manipulation with sample and its combination with shortened analyses times of sarcosine and taurine may serve as a sensitive and low-cost method for analysis of clinical specimens.
Keywords:
Ion-exchange chromatography, Non-invasive biomarker, Prostate carcinoma, Sarcosine, Taurine.
Sources
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