The HPLC analysis of salivary cysteine, homocysteine and glutathione

А.А. Дутов; Д.А. Никитин; Е.В. Фефелова; М.Н. Мищенко; А.Н. Семенова; А.В. Сверкунова; А.В. Мартынова; Ю.Л. Лукьянова; А.В. Ермолина

The HPLC analysis of salivary cysteine, homocysteine and glutathione

А.А. Дутов¹, Д.А. Никитин², Е.В. Фефелова³, М.Н. Мищенко⁴, А.Н. Семенова⁵, А.В. Сверкунова⁶, А.В. Мартынова⁷, Ю.Л. Лукьянова⁸, А.В. Ермолина⁹

research article Pages: 29-32

¹ д.м.н., ст. науч. сотрудник, лаборатория экспериментальной и клинической биохимии и иммунологии НИИ молекулярной медицины, Читинская государственная медицинская академия
² зав. лабораторией химического анализа, Забайкальский государственный университет (г. Чита)
³ к.м.н., доцент, кафедра патологической физиологии, Читинская государственная медицинская академия
⁴ к.м.н., врач-стоматолог, стоматологическая клиника, Читинская государственная медицинская академия
⁵ врач-стоматолог, стоматологическая клиника, Читинская государственная медицинская академия
^6,7 аспирант, кафедра химии, Забайкальский государственный университет (г. Чита)
⁸ врач-ординатор, кафедра неврологии, Читинская государственная медицинская академия
⁹ врач-офтальмолог, Краевая больница № 2 (г. Чита)

Keywords: HPLC UV detection cysteine homocysteine glutathione saliva hyper cross-linked polystyrene

Abstract

An HPLC method for measuring cysteine, homocysteine and glutathione in saliva was validated. Reduction from disulfides was achieved with 1,4-dithioerythritol. Aminothiols derivatization was made with 5,5'-dithio-bis-(2-nitrobenzoic acid). The derivatives were cleaned on a cartridges, packed hyper cross-linked polystyrene (Purosep-200) and separated with reversed phase (C18) HPLC and UV detection at 330 nm. Simplicity and reproducibility of a method, allow its use in a clinical practice for an estimation of biochemical changes at stomatologic diseases and also for non-invasive diagnostic hyperhomocysteinemia.

How to cite

Дутов А.А., Никитин Д.А., Фефелова Е.В., Мищенко М.Н., Семенова А.Н., Сверкунова А.В., Мартынова А.В., Лукьянова Ю.Л., Ермолина А.В. ВЭЖХ-анализ цистеина, гомоцистеина и глутатиона в слюне // Вопросы биологической, медицинской и фармацевтической химии. 2014. № 7. С. 29–32.

References

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Shevchenko O.P., Olefirenko G. A. Gipergomeczisteinemiya i ee klinicheskoe znachenie // Laboratoriya. 2002. № 1. 3-6.
Melnyk S., Pogribna M., Pogribny I.P.,, Yi P. and James S.J. Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations // Clinical Chemistry. 2000. V. 46. P. 265-272.
Bioximiya / pod red. E.S. Severina. M.: GE'OTAR-Media. 2005. 784 s.
Winters R.A., Zukowski J., Ercal N., Matthews R.H., Spitz D.R. Analysis of glutathione, glutathione disulfide, cysteine, homocysteine and other biological thiols by high-performance liquid chromatography following derivatization by N-(1-pyrenyl)maleimide // Anal. Biochem. 1995. V. 227. P. 14-21.
Lynn G., Hellwig L.C. Handbook of derivatization reactions for HPLC // John Wiley & Sons, Inc. 1998.
Amores-Sanchez M. I., Medina M. A. Methods for the determination of plasma total homocysteine: a review // Clin. Chem. Lab. Med. 2000. V. 38(3). P. 199-204.
Zhloba A., Blashko E. Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection // J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2004. V. 800. № 1-2. P. 275-280.
Battino M., Ferreiro M.S., Gallardo I., Newman H.N., Bullon P. The antioxidant capacity of saliva // J. Clin. Periodontol. 2002. V. 29. № 3. P. 189-194.
Chapple I.L., Brock G., Eftimiadi C., Matthews J.B. Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease // Mol. Pathol. 2002. V. 55. № 6. P. 367-373.
Benitez-Sillero Jde D., Perez-Navero J.L., Tasset I., Guillen-Del Castillo M., Gil-Campos M., Tunez I. Influence of intense exercise on saliva glutathione in prepubescent and pubescent boys. Eur // J. Appl Physiol. 2009. V. 106. № 2. P. 181-186.
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Dutov A.A., Nikitin D.A., Fedotova A.A. Opredelenie gomoczisteina i czisteina v plazme/sy'vorotke krovi VE'ZhX metodom s UF detekcziej i tverdofaznoj e'kstrakcziej na polimernom sorbente // Biomediczinskaya ximiya. 2010. T. 56. № 5. S. 609-615.
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[1] Ueland P.M., Refsum H., Stabler S.P., Malinow R.M., Andersson A. and Allen R.H. Total homocysteine in plasma or serum: methods and clinical applications (review) // Clinical Chemistry. 1993. V. 39. № 9. P. 1764-1779.

[2] Shevchenko O.P., Olefirenko G. A. Gipergomeczisteinemiya i ee klinicheskoe znachenie // Laboratoriya. 2002. № 1. 3-6.

[3] Melnyk S., Pogribna M., Pogribny I.P.,, Yi P. and James S.J. Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations // Clinical Chemistry. 2000. V. 46. P. 265-272.

[4] Bioximiya / pod red. E.S. Severina. M.: GE'OTAR-Media. 2005. 784 s.

[5] Winters R.A., Zukowski J., Ercal N., Matthews R.H., Spitz D.R. Analysis of glutathione, glutathione disulfide, cysteine, homocysteine and other biological thiols by high-performance liquid chromatography following derivatization by N-(1-pyrenyl)maleimide // Anal. Biochem. 1995. V. 227. P. 14-21.

[6] Lynn G., Hellwig L.C. Handbook of derivatization reactions for HPLC // John Wiley & Sons, Inc. 1998.

[7] Amores-Sanchez M. I., Medina M. A. Methods for the determination of plasma total homocysteine: a review // Clin. Chem. Lab. Med. 2000. V. 38(3). P. 199-204.

[8] Zhloba A., Blashko E. Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection // J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2004. V. 800. № 1-2. P. 275-280.

[9] Battino M., Ferreiro M.S., Gallardo I., Newman H.N., Bullon P. The antioxidant capacity of saliva // J. Clin. Periodontol. 2002. V. 29. № 3. P. 189-194.

[10] Chapple I.L., Brock G., Eftimiadi C., Matthews J.B. Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease // Mol. Pathol. 2002. V. 55. № 6. P. 367-373.

[11] Benitez-Sillero Jde D., Perez-Navero J.L., Tasset I., Guillen-Del Castillo M., Gil-Campos M., Tunez I. Influence of intense exercise on saliva glutathione in prepubescent and pubescent boys. Eur // J. Appl Physiol. 2009. V. 106. № 2. P. 181-186.

[12] Zappacosta B., Manni A., Persichilli S., Boari A., Scribano D., Minucci A., Raffaelli L., Giardina B, De Sole P. Salivary thiols and enzyme markers of cell damage in periodontal disease // Clin Biochem. 2007. V. 40. № 9-10. P. 661-665.

[13] Dutov A.A., Nikitin D.A., Fedotova A.A. Opredelenie gomoczisteina i czisteina v plazme/sy'vorotke krovi VE'ZhX metodom s UF detekcziej i tverdofaznoj e'kstrakcziej na polimernom sorbente // Biomediczinskaya ximiya. 2010. T. 56. № 5. S. 609-615.

[14] Doson R, E'lliot D, E'lliot U, Dzhons K. Spravochnik bioximika: per. s angl. M.: Mir. 1991. 544 s.

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