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[Establishment of a method for detection of thiols in seminal plasma and its application in patients with
Tong Cui1, Ya-Wen Gong1, Jing Ma2
1School of Chemistry and Materials Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China.
Objective:
To establish a method of reversed-phase ultra-performance liquid chromatography with fluorescence (UPLC-FL) detection for simultaneous determination of homocysteine (Hcy), cysteine (Cys), cysteine glycine (CysGly) and glutathione (GSH) and analysis of the contents of the four thiols in the seminal plasma of normal men and patients with hyperuricemia (HUA).
Methods:
Seminal plasma samples were collected from 30 normal sperm donors and 30 HUA patients and reduced with tris (2-carboxyethyl) phosphine hydrochloride. Then, the samples were subjected to protein precipitation with trichloroacetic acid solution, derivative reaction with 7-fluorobenzofuran-4-sulfate and isolation with a C18 column, with 0.025 mol/L KH2PO4 (pH 3.0) for mobile phase A and pure methanol for B, followed by equal gradient elution with an excitation wavelength of 380 nm and an emission wavelength of 510 nm.
Results:
The correlation coefficients (R2) of the four thiols all exceeded 0.999, with an average recovery rate of 94.23－107.87%. Compared with the normal sperm donors, the HUA patients showed significantly increased contents of Cys (［108.01 ± 48.03］ vs ［250.10 ± 55.87］ μmol/L, P < 0.05), Hcy (［113.97 ± 6.32］ vs ［48.35 ± 15.07］ μmol/L, P < 0.05), and GSH (［3.15 ± 1.48］ vs ［4.63 ± 1.17］ μmol/L, P < 0.05), but a decreased level of CysGly (［12.79 ± 3.18］ vs ［5.94 ± 0.99］ μmol/L, P < 0.05).
Conclusions:
The method of reversed-phase UPLC-FL detection established in this study has made it possible simultaneous detection of Hcy, Cys, CysGly and GSH in the seminal plasma, which is applicable to laboratory research and clinical routine examination. Patients with hyperuricemia may incur oxidative damage to the reproductive system.
