Analysis of Total Thiols in the Urine of a Cystathionine β-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography

Abstract

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine β-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

“…Good linearity was obtained, with correlation coefficients exceeding 0.999. The thiol sensitivity was lower than that obtained with HILIC methods [14][15][16][17] and was similar to those obtained in other studies [23,24], except for GSH. This was because the sensitivity of the fluorescence detector for GSH was lowered.…”

“…In this study, we developed an analytical method for detecting and quantifying intracellular thiols that can be applied to K562 cell samples. Thiols were first reduced by TCEP and then fluorescently derivatized by SBD-F. Six different SBD-thiols were successfully separated within 30 min on an ODS column, which can perform more stable analyses than HILIC columns [14][15][16][17]. By adjusting the sensitivity of the fluorescence detector, Cys, CysGly, γGC, and GSH in K562 cells could be quantified simultaneously.…”

“…Currently available separation-based analytical methods for thiols include HPLCfluorescence detection, LC-mass spectrometry, and capillary electrophoresis [10][11][12][13]. We have previously developed analytical methods for thiol detection based on fluorescence derivatization using ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) (Figure 1b) and hydrophilic interaction chromatography (HILIC) [14][15][16][17]. Although eight types of thiols could be separated within 10 min with high sensitivity, these methods were difficult to apply in routine analyses, owing to the low stability of the HILIC column, the long time required for column equilibrium, and limitations as regards the injection sample Molecules 2021, 26, 2365 2 of 7 volume [18][19][20].…”

Simple and fast detection of homocysteine by cucurbit[7]uril fluorescent probe based on competitive strategy

“…So far, a great number of methods have been reported for detecting Hcy, including radioenzyme immunoassay (RIA) [7], high performance liquid chromatography (HPLC) [8][9][10][11][12][13], electrochemistry [2,14], capillary electrophoresis (CE) [15,16], colorimetry [1,[17][18][19][20], liquid chromatography-mass spectrometry (LC-MS) [21][22][23], etc. [24].…”