Optimized Gas Chromatography-Tandem Mass Spectrometry for 1,1'-sulfonylbis[2-(methylthio) ethane] Quantification in Human Urine.

Sulfur mustard (SM) which is a bifunctional alkylating vesicant is one of the mostly used chemical warfare agent in First World War and the Iran-Iraq War. ß-Lyase metabolites of SM especially 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) is an unequivocal biomarker of the exposure. An optimized gas chromatography-tandem mass spectrometry method was developed and validated for the retrospective detection of SBMTE in human urine. Urine samples were treated with acidic titanium trichloride to reduce ß-lyase metabolites to the single analyte SBMTE. After neutralization and precipitation, SBMTE was extracted from urine by C8 solid-phase extraction cartridge and analyzed in the multiple-reaction monitoring mode. The lower limit of quantification was 1 ng/mL with relative standard deviation of <10%. Acceptable intra-day and inter-day precisions and accuracies were obtained. The developed method was successfully measured various levels of SBMTE which could be used as the forensic evidence of such a chemical attack.

Historical control data on urinary and renal tissue biomarkers in naive male Wistar rats.

Total proteins, angiotensin-converting enzyme, N-acetyl-beta-D-glucosaminidase, glutamine transaminase K and glutamine synthetase were determined in urine collected overnight (14 h: 6:00 p.m.-8:00 a.m.) from naive male Wistar rats; glutamine transaminase K and glutamine synthetase in the kidney 10,000 g supernatant and p-aminohippurate uptake in renal cortical slices also were measured. Urinary parameters were related both to urinary creatinine concentration and urinary flow rate; kidney parameters were related to protein concentration (enzymes) or slice/medium (S/M) ratio (p-aminohippurate uptake). The following reference ranges (1.0 and 99.0 percentiles) were obtained: urine: total urinary proteins (195 samples) 0.03-0.29 g mmol(-1) creatinine and 0.13-1.77 mg h(-1); angiotensin-converting enzyme (115 samples) 8.9-63.7 micromol mmol(-1) creatinine and 59.4-282.7 nmol h(-1); glutamine transaminase K (115 samples) 0-1.7 micromol mmol(-1) creatinine and 0-8.5 nmol h(-1); N-acetyl-beta-D-glucosaminidase (72 samples) 0.7-5.0 micromol mmol(-1) creatinine and 4.9-28.4 nmol h(-1) (naive male rats did not excrete glutamine synthetase); kidney: glutamine transaminase K (36 samples) 14.5-32.8 nmol mg(-1) protein; glutamine synthetase (22 samples) 13.9-48.6 nmol mg(-1) protein and p-aminohippurate (54 samples) 4.77-17.89 S/M. Urinary creatinine (r = -0.780), total urinary proteins (r = -0.521), angiotensin-converting enzyme (r = -0.650) and N-acetyl-beta-D-glucosaminidase (r = -0.796) but not glutamine transaminase K were well correlated with diuresis. In addition, the same parameters, but not glutamine transaminase K, were well correlated with creatinine (r = 0.604,0.701 and 0.747, respectively). Significant correlation also was observed between urinary indices adjusted to creatinine or urinary flow rate (total urinary proteins: r = 0.813; angiotensin-converting enzyme: r = 0.677; glutamine transaminase K: r = 0.939; N-acetyl-beta-D-glucosaminidase: r = 0.657). Finally, a low but significant correlation was found between total urinary proteins and angiotensin-converting enzyme (r = 0.293) and N-acetyl-beta-D-glucosaminidase (r = 0.471).

Glutamine transaminase K intranephron localization in rats determined by urinary excretion after treatment with segment-specific nephrotoxicants.

Glutamine transaminase K(GTK) excretion assessed in urine and by kidney histology was evaluated in rats after single treatment with 1.0 mg/kg i.p. of mercuric chloride, 100 mg/kg i.p. of hexachloro-1:3-butadiene (both S3, pars recta, segment-specific nephrotoxicants) and 25 mg/kg s.c. of potassium dichromate (S1-S2, pars convoluta, segment-specific nephrotoxicant). The aim was to correlate segment-specific injury and enzyme excretion in order to assess, using non-vasive methods, localization of GTK along the proximal tubule. Mercuric chloride and hexachloro-1:3-butadiene produced early focal damage in the pars recta (focal necrosis was shown 10 h after treatment, and diffuse necrosis appeared later at 34 and 24 h after treatment). Changes of the pars convoluta were occasional and delayed (72 h after treatment for both substances). On the contrary, potassium dichromate induced damage of the pars convoluta (vacuolar degeneration and focal necrosis were evident 24 h and 48 h after treatment, respectively), whereas the pars recta was affected later (focal vacuolar degeneration was observed 72 h after treatment). Increase urinary GTK excretion was early after treatment with mercuric chloride and hexachloro-1:3-butadiene (significant increase was observed within 10 h), with a peak for both substances 24 h after treatment, in agreement with the necrosis of the pars recta. Potassium dichromate induced a significant increase of enzyme excretion in urine also 24 h after injection, according to histological features showing vacuolar degeneration of the pars convoluta; the peak of excretion was reached 48 h after treatment (delay was due, probably, to s.c. administration). The results show that GTK increased in urine after treatment with S3 and S1-S2 specific nephrotoxicants; the combination of histological examination and urinary enzyme supports the evidence that the enzyme is distributed along the whole of the proximal tubule.

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): identification of beta-lyase metabolites and hydrolysis products in human urine.

1. Samples of urine from two human subjects accidentally exposed to sulphur mustard were analysed for metabolites derived from hydrolysis (thiodiglycol, thiodiglycol sulphoxide), conjugation with glutathione (1,1'-sulphonylbis [2-S-(N-acetylcysteinyl)ethane]) and from further metabolism of glutathione conjugates by the beta-lyase pathway (1,1-sulphonylbis[2-(methylsulphinyl)ethane], 1-methylsulphinyl-2-[2-(methylthio)ethylsulphonyl]ethane). 2. Thiodiglycol sulphoxide was excreted in much higher concentrations than thiodiglycol, as was observed previously in rat exposed to sulphur mustard. However, the use of thiodiglycol sulphoxide as a biological marker for sulphur mustard poisoning is limited by its presence at low concentrations in normal human urine. 3. beta-lyase metabolites were detected at concentrations comparable with those of thiodiglycol sulphoxide. No background levels of beta-lyase metabolites have been detected in normal human urine, and they are proposed as unequivocal diagnostic and forensic indicators of sulphur mustard poisoning in man.