Dataset Information

Analysis of Total Thiols in the Urine of a Cystathionine ?-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 ?-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.

SUBMITTER: Chang CF

PROVIDER: S-EPMC7181027 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Chang Chun-Fang CF Hamase Kenji K Tsunoda Makoto M

Molecules (Basel, Switzerland) 20200409 7

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, cystei ...[more]

PMID: 32283791

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Project description:Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)‑induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild‑type (WT) mice and in mice with reduced expression of one of the three H2S‑producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S‑producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue‑dependent upregulation of some of the H2S‑producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE‑/‑ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/‑ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/‑, CSE‑/‑ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/‑ and CSE‑/‑ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)‑1β, IL‑6, IL‑10, IL‑12 and interferon (IFN)γ] in plasma; TNFα, IL‑10 and IL‑12 levels tended to be lower in all three groups of animals expressing lower levels of H2S‑producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

Dataset Information

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

Publications

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

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