The extraction-chromatographic method for determining of sulfur containing amino acids in biological fluids
Abstract
Sulfur-containing amino acids (SCAA) cysteine (Cys), homocysteine (Hcy) and tripeptide glutathione (GSH) are involved in many biochemical processes of the body. SCAA and glutathione are present in the body in two forms: in the form of oxidized derivatives (disulfides) and in free (reduced) form, the free forms being rapidly oxidized. To determine the total content of analytes, they must first be recovered from disulfides and protein conjugates. There are many ways to determine SCAA and glutathione by various methods including chromatographic ones. SCAA have a low molar absorption coefficient in the UV range, and therefore, for the spectrophotometric detection SCAA is subjected to pre-columnar derivatization. And the SCAA derivatives are detected after reaction with thiol-specific and nonspecific reagents. The use of a number of reagents (reducing agents, deproteinizers, antioxidants and derivatizing reagent) creates a problem for UV detection which high demands are placed on the purity of the extracts. In addition, the sequential addition of reagents results in a dilution of the bioassay.
In this study the extraction-chromatographic method is suggested for determining of sulfur- containing amino acids and gluthatione in biological fluids using HPLC with UV detection at 330 nm. Reduction from disulfide bonds to thiol groups was carried out with 1,4-dithioerythritol. The derivatization of analytes was carried out with Ellman's reagent [5,5'-dithiobis (2-nitrobenzoic acid)]. Cartridges with hyper-crosslinked polystyrene Purosep-200 were used for purification and concentration of samples. The results of HPLC determination of SCAA and gluthatione showed what purposed methods is characterized by simplicity of sample preparation, good reproducibility and sensitivity. This is makes this method suitable for routine clinical analysis, as well as for biochemical studies plasma and serum of the experimental animals - mice and rats, with uses small sample volumes of biological fluids.
Downloads
References
2. Ueland P.M., Refsum H., Stabler S.P., Malinow R.M., Andersson A. and Allen R.H. // Clinical Chemistry, 1993, Vol. 39, No 9, pp. 1764-1779.
3. Shevchenko O.P., Olefirenko G.A., Laboratoriya, 2002, No 1, pp. 3-6 (in Russian).
4. Melnyk S., Pogribna M., Pogribny I.P., Yi P. et al., Clinical Chemistry, 2000, Vol. 46, pp. 265-272.
5. Severin E.S. Biokhimiya [Biochemistry]. М., GEOTAR - Media, 2005. 784 p. (in Russian).
6. Cacciatore I., Cornacchia C., Pinnen F., Mollica A. et al., Molecules, 2010, Vol. 15, pp. 1242-1264.
7. Wu G., Fang Y-Z., Yang S., Lupton J.R et al., Journal of Nutrition, 2004, Vol. 134, pp. 489-492.
8. Townsend D.M., Tew K. D., Tapiero H., Biomedicine & Pharmacotherapy, 2003, Vol. 57, pp. 145-155.
9. Dawson P, Elliott D Elliott Y, Jones K. Spravochnik biokhimika [Handbook of biochemist]. Мoscow, Мir, 1991. 544 p (in Russian).
10. Jones D.P., Methods Enzymol, 2002, Vol. 348, pp. 93-112.
11. Rudakov O.B., Selemenev V.F., Rudakova L.V., HPLC. Sorbaty, sorbenty i elyuyenty [Sorbate, sorbent and eluent]. Voronezh, VGASU, 2016, 205 с. (in Russian).
12. Rudakov O.B., Selemenev V.F., Vostrov I.A. Sputnik khromatografista. Metody zhidkostnoy khromatografii [Handbook of chromatography. Liquid chromatography methods]. Voronezh: Vodoley, 2004, 526 p. (in Russian).
13. Rudakov O.B., Khorokhordina E.A., Rudakov L.V. Groshev E.N., Vestnik VGU. Seriia: Khimiia. Biologiia. Farmatsiia, 2015, Vol. 3, pp. 42-47 (in Russian).
14. Winters R.A., Zukowski J., Ercal N., Matthews R.H. et al., Anal. Biochem, 1995, Vol. 227, pp. 14-21.
15. Lynn G., Hellwig L.C. Handbook of derivatization reactions for HPLC. John Wiley & Sons, Inc., 1998, 1795 р.
16. Amores-Sanchez M.I., Medina M.A., Clin. Chem. Lab. Med, 2000, Vol. 38(3), pp. 199-204.
17. Zhloba A., Blashko E., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2004, Vol. 800(1-2), pp. 275-280.
18. Gilfix B.M. Novel reductant for determination of total plasma homocysteine. Clin. Chem., 1997, Vol. 43, pp. 687-688.
19. Hyland K., Bottiglieri T., J. Chromatogr, 1992, Vol. 579, pp. 55-62.
20. Waters Oasis Sample preparation product (Application Notebook), (Water Corporations www.waters.com).
21. Tcherkas Y.V., Denisenko A.D., Journal of Chromatography A, 2001, Vol. 913, pp. 309-313.
22. Andersson A., Isaksson A., Brattstrom L., Hultberg B., Clinical Chemistry, 1993, Vol. 39, pp. 1590-1597.
23. Tsyurupa M.P., Blinnikova Z.K., Proskurina N.A., Pastukhov A.V et al., Rossiyskiye nanotekhnologii [Russian nanotechnologies], 2009, Vol. 4, No 9-10, pp. 109-117 (in Russian).
24. Dutov A.A., Nikitin D.A., Tereshkov P.P., Martynova A.V. et al., Problems of biological, medical and pharmaceutical chemistry, 2015, No 6, pp. 12-15 (in Russian).
