Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine

Abstract

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ∼50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 μmol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Reversed‐phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1‐benzyl‐2‐chloropyridinium bromide

“…The method was successfully applied to total thiols measurement in the authentic samples. Our plasma values of total thiols fall within the ranges for healthy men reported in the literature (Pastore et al, 1998;Ueland, 1995;Mudd et al, 2000). The described procedure is simple, low-cost and reliable for the analysis of plasma for cysteine, glutathione, homocysteine and cysteinylglycine.…”

“…Moreover, thanks to the large bathochromic shift accompanying the reaction, the excess derivatization reagent peak does not appear on the chromatogram. The above performance of the BPCB reagent compares well with some other florescence and ultraviolet tagging reagents (Fahey et al, 1981;Pastore et al, 1998;Chassaing et al, 1999;Winters et al, 1995;Ercal et al, 1996Ercal et al, , 2001Bald et al, 2000Bald et al, , 2001Bald et al, , 2004, which give much more crowded chromatograms. Among plasma thiol derivatization reagents only halogenosulfonylbenzofurazans (Ercal et al, 2001;Toyo'oka and Imai, 1983;Nolin et al, 2007) and 1,1′-thiocarbonyldiimidazole (Amarnath et al, 2003) give equally clean chromatograms, although with longer overall analysis time caused by longer derivatization time (Toyo'oka and Imai et al, 1983;Nolin et al, 2007) or inclusion of solid-phase extraction in the assay procedure (Amarnath et al, 2003).…”

“…Since thiols lack strong chromophores or fluorophores, the analytical methods, except those based on mass spectrometry or electrochemical detection, require derivatization for ultraviolet or fluorescence detection. The most popular and sensitive detection reagents are the fluorimetric bimanes (Fahey et al, 1981;Pastore et al, 1998), maleimides [fluorescein-5-maleimide (Chassaing et al, 1999), N-(1-pirenyl)maleimide (Winters et al, 1995;Ercal et al, 1996), ThioGlo TM (Ercal et al, 2001)], halogenosulfonylbenzofurazans [SBD-F (Toyo'oka and Imai, 1983;Imai et al, 1983;Nolin et al, 2007), ABD-F (Ling et al, 1991)] and o-phthalaldehyde (Paroni et al, 1995;Tcherkas and Denisenko, 2001). In the recent years, several methods utilizing precolumn derivatization with UV labeling reagents, such as 2-chloro-1-methylpyridinium iodide (CMPI) (Bald et al, 2001) and 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) (Bald et al, 2001(Bald et al, , 2004, followed by HPLC separation, have also been described.…”

The proteome of cblC defect: in vivo elucidation of altered cellular pathways in humans

“…To determine GS-Pro, the protein pellet was resuspended in 150 μL of NaOH 0.1 mol/L and derivatized as described below. Plasma total homocysteine and the levels of the different forms of glutathione were determined using the derivatization and chromatography procedures reported previously (Pastore et al 1998). Total glutathione (tGSH) amounts were calculated by the sum of free GSH, GSSG and GS-Pro.…”

Glutathione metabolism in cobalamin deficiency type C (cblC)

“…For GS-Pro determinations, protein pellet was resuspended in 150 μL of NaOH 0.1 mol/L and derivatized as described below. Cysteine, total homocysteine and the levels of the different forms of glutathione were determined by using the derivatization and chromatography procedures performed as previously reported (Pastore et al 1998). Total glutathione (TotGSH) amounts were calculated by the sum of free GSH, GSSG and GS-Pro.…”