OP03 Absorption behaviour and blood baseline concentrations of sulfide monitored with gas chromatography/mass spectrometry

Abstract

The reported sulfide absorption values and baseline blood concentrations are still controversial [1] . We modified an existing gas chromatographic/mass spectrometric method of routine sulfide quantification using a bis-pentafluorobenzyl derivative [2] . This approach allows sensitive determination of sulfide in small sample volumes under mild chemical conditions. In addition, in in vitro experiments, it allows to distinguish between endogenous and exogenous sulfide by administration of the stable isotope 34 S 2− . A standard protocol utilises 100 μl of blood, but can be scaled down to 25 μl for rodent samples. In brief, a mixture, consisting of 400 μl internal standard (5 μg/ml tribromobenzene in isooctane), 200 μl of alkylation reagent (10 μ l/ml pentafluorobenzylbromide in isooctane) and 400 μl of phase transfer catalyst (2 mg/ml tetradecyldimethylbenzylammonium chloride in sodium tetraborate saturated water) was prepared. After addition of 100 μl sample, the mixture was vigorously shaken for 1 min, 400 μl of saturated potassium dihydrogenphosphate solution were added and the cup was shaken for another 10 s. 2 μl of the isooctane phase were injected into an Agilent 5890/5970 gas chromatography/mass spectrometry instrument, housing a 12 m Macherey–Nagel Optima-5MS capillary column. In sim mode ions m/z 313.7 for the internal standard and 394.0 for the sulfide derivative (or 396.0 for 34 S 2− ) were recorded. The method was used to determine basal sulfide blood concentrations in pigs and to measure the sulfide release from the sulfide releasing drug GYY4137 over 24 h in cell culture medium. In addition this method reproducibly was used to determine the absorption of a spike of 100 μM 34 S 2− at pH 7.4 in buffer, blood, plasma, cysteine, glutathione and albumine. Baseline blood sulfide concentrations in pigs were between 0.2 and 2.1 μM. Sulfide concentrations of a 300 μ M solution of GYY4137 in cell culture medium (in cell culture flask, gas exchange enabled) were 1.02 μM (10 min after GYY addition), 1.32 μM (2 h), 1.37 μM (4 h), 1.60 μM (8 h), 1.58 μM (12 h) and 1.81 μM (24 h). The sulfide concentration of the corresponding medium without GYY4137 ranged between 0.25 and 0.50 μM. An initial 100 μM spike of 34 S 2− in blood decreased to 23 μM after 1 min, 12 μM after 10 and 1 μM after 60 min. The corresponding values for plasma were 60, 41 and 3 μM, for phosphate buffer (pH 7.4) 103, 96 and 85 μM. In a 50 mg/ml solution of albumine the 34 S 2− concentration decreased to 22, 15, 3 μM, in 5 mg/ml oxidized glutathione to 32, 18, 3 μM, in 5 mg/ml reduced glutathione to 79, 76, 75 μM, in 120 μg/ml cystine to 91, 64, 12 μM and in 1 mg/ml cysteine to 110, 102, 99 μM. Our findings show, that sulfide baseline concentrations in blood are in the low micromolar range. Added sulfide is rapidly absorbed by blood, plasma and disulfide bridge containing peptides and proteins, but much lower by the reduced species. Adding the sulfide releasing drug GYY4137, increased the sulfide concentration of cell culture media for 24 h.