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

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine ß-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.

Optimization of tris(2-carboxyethyl) phosphine reduction conditions for fast analysis of total biothiols in mouse serum samples.

In this study, we investigated suitable conditions for the reduction of disulfides in mouse serum samples by tris(2-carboxyethyl) phosphine (TCEP) for fast analysis of total biothiols. Disulfides were reduced with TCEP, and then, thiols were derivatized with the fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Interference peaks on chromatograms of mouse serum samples disappeared when the TCEP reaction was conducted on ice instead of at room temperature, which is used classically. Low-molecular-weight disulfides, such as cystine and glutathione disulfide, were nearly completely reduced by TCEP on ice. Six SBD-biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and N-acetylcysteine) were separated within 7.5 min on a sulfoalkylbetain-type column (ZIC-HILIC: 150 × 2.1 mm i.d., 3.5 µm), without interference peaks. The developed method showed good linearity and reproducibility, with inter- and intra-day precisions of less than 3%.

Phylogenetic relationships and status quo of colonies for gayal based on analysis of cytochrome B gene partial sequences.

Thirty-three mutations and four different haplotypes were found when cytochrome b(Cytb) gene partial sequences of 12 gayals were analyzed. Together with sequences of Bos indicus, Bos taurus, Bos grunniens and Bos gaurus with Bubalus bubalis as the out group, the partial sequences of Cytb gene of gayals were aligned and base composition and nucleotide variation of Cytb gene were analyzed. The phylogenetic trees were constructed by the NJ method and the MP method respectively, both supporting almost the same topology. Gayal is an independent species of Bos from Bos indicus, Bos taurus and Bos gaurus. The results also indicate that a great proportion of gayal bloodline was invaded by other species, and the protection of gayal is facing a formidable situation.