Technical note: simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection

A reproducible and robust ultra-high performance liquid chromatography method was evaluated for the simultaneous determination of cysteine, cysteinylglycine, homocysteine, gamma-glutamylcysteine, and glutathione in solid tissue. The method will facilitate novel insights into tissue-specific and whole-body sulfur amino acid metabolism in livestock animals. Lay Summary The synthesis of glutathione, a major intracellular antioxidant, in animal tissue accounts for a considerable fraction of the intake of the sulfur amino acids methionine and cysteine. Animal scientists accordingly need methods suitable for measuring the abundance of metabolites related to sulfur amino acid metabolism in solid tissue. However, methods currently available are either validated for measuring these metabolites in plasma, serum, or urine, do not fully describe all procedures needed to prepare tissue samples for analysis, or are validated for measuring only cysteine and glutathione in tissue. The focus of this work was to describe the sample preparation and analysis methods needed to measure these metabolites in solid tissue. Sample preparation time is less than 2 h and sample analysis time is less than 5 min. The method is robust and reproducible and is applied to identify weaning-induced differences in sulfur amino acid metabolism in liver and small intestine in pigs. The method will also help evaluate the impact of diet, stress, or inflammation on cysteine and glutathione metabolism on a tissue-by-tissue basis to help optimize levels of sulfur amino acids in swine diets. Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), gamma-glutamylcysteine (gamma-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 mu m, 2.1 mm x 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and gamma-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r(2) > 0.99) and excellent recovery of amino thiols from spiked samples (mean +/- SD across tissues; Cys, 100.0 +/- 2.2%; CysGly, 95.4 +/- 5.1%; Hcys, 96.6 +/- 2.0%; gamma-GluCys, 102.2 +/- 2.7%; and GSH, 100.6 +/- 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both gamma-GluCys and GSH decline in jejunum of weaned pigs.

Automated summary

A reproducible and robust ultra-high performance liquid chromatography method was evaluated for the simultaneous determination of cysteine, cysteinylglycine, homocysteine, gamma-glutamylcysteine, and glutathione in solid tissue. The method will facilitate novel insights into tissue-specific and whole-body sulfur amino acid metabolism in livestock animals.