Simultaneous determination of total homocysteine, cysteine, glutathione, and N‐acetylcysteine in brain homogenates by HPLC

Abstract

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10 min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20 μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 μmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.

Alterations in the Antioxidant Enzyme Activities in the Neurodevelopmental Rat Model of Schizophrenia Induced by Glutathione Deficiency during Early Postnatal Life

“…The high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was used for determination of total Cys and GSH levels in the striatal samples. The HPLV-UV method is based on the reduction disulfide bonds by tris(2-carboxyethyl) phosphine (TCEP) prior to precolumn derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) [ 49 , 50 ].…”

Micronutrient Status of Recreational Runners with Vegetarian or Non-Vegetarian Dietary Patterns

“…Liquid chromatography with mass spectrometry coupling (LC-MS/MS) was applied to assess methylmalonic acid (MMA) in serum [25]. Plasma homocysteine (tHcy) was determined by HPLC with a fluorescence detector in accordance with [26]. The four marker combined vitamin B-12 indicator (4cB12) was computed from concentrations of vitamin B 12 in serum, Holo-TC, MMA, and homocysteine according to published formula [27]: 4cB12=log10(HoloTC∗B12MMA∗tHcy)−(age factor).…”

Glutathione Deficiency and Alterations in the Sulfur Amino Acid Homeostasis during Early Postnatal Development as Potential Triggering Factors for Schizophrenia-Like Behavior in Adult Rats

“…Total GSH and Cys levels in the brain samples (PFC and HIP) were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection according to the method described by Bald et al [102] with a modification developed by Kamińska et al [103]. Simultaneous determination of thiols was carried out using HPLC after precolumn derivatization with CMQT [100].…”