Stable isotope gas chromatography–tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples

Abstract

Aminoethylcysteine ketimine decarboxylated dimer [AECK-DD; systematic name: 1,2–3,4–5,6–7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one] is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, cells in culture and vegetables, and to possess potent anti-oxidative properties. Here, we describe a stable-isotope GC-MS/MS method for specific and sensitive determination of AECK-DD in biological samples. 13C2-AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected-reaction monitoring of the mass transitions m/z 328 to m/z 268 for AECK-DD and m/z 330 to m/z 270 for 13C2-AECK-DD in the electron-capture negative-ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above ~ 4 nM, but was present in urine samples of healthy humans at a maximal concentration of 46 nM. AECK-DD was detectable in rat brain at very low levels of about 8 pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (~1 nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (~ 6.8 pmol/g fresh tissue).

Comment on Absorption of Aminoethyl Cysteine Ketimine Decarboxylated Dimer in Mice: Effect on Plasma Antioxidant Potential

“…We noted that urine also has very low AECKK-DD concentrations . However, we did show that AECK-DD is present at a maximal concentration of 46 nmol/L in urine samples obtained from the same cohort of human volunteers from which we obtained the plasma samples . This value, although considerably lower than that reported previously for human urine, may nevertheless suggest that, in humans, AECK-DD accumulates in urine.…”

“…Interestingly, Piazzon et al reported AECK-DD plasma concentrations of ≤10 ng/mL (≤43.9 nmol/L) in nonsupplemented mice (control group). We noted that urine also has very low AECKK-DD concentrations . However, we did show that AECK-DD is present at a maximal concentration of 46 nmol/L in urine samples obtained from the same cohort of human volunteers from which we obtained the plasma samples .…”

“…Here, we wish to compare these published results with our previous findings. We recently reported a highly specific and sensitive GC-MS/MS approach for the reliable quantitation of AECK-DD in plasma, urine, tissue, and dietary vegetables using a newly synthesized and structurally fully characterized 13 C-labeled AECK-DD ( 13 C 2 -AECK-DD) as internal standard (Figure C) . We performed quantitative analyses in the selected-reaction monitoring (SRM) mode using electron-capture negative-ion chemical ionization (ECNICI) and several different m / z transitions for physiological and 13 C 2 -AECK-DD, including those for the stable 34 S isotope (natural abundance, 4.22%).…”

Alternative functions of the brain transsulfuration pathway represent an underappreciated aspect of brain redox biochemistry with significant potential for therapeutic engagement

“…Cyclic thioether ketimines do themselves possess modest antioxidant activity, as one might expect from the nature of their thioether moiety, but certain of these (particularly aminoethylcysteine ketimine or AECK) can undergo spontaneous oxidative decarboxylation and dimerization to produce “decarboxylated dimers” that have greater antioxidant potential in vitro and in cell cultures [51–53] (Fig. 2).…”

“…2). Evidence has been presented for the presence of aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) in plasma and urine, but the extent to which AECK-DD forms through bimolecular reactions in their tissue of synthesis, and the biological relevance of the decarboxylated dimers to brain antioxidant balance, is a subject of some controversy [53]. …”