![Figure 1. Excretion of 15d-PGJ2 in human urine and its in vitro conjugation with glutathione, L-cysteine and N-acetylcysteine. (A) Reaction of 30μM 15d-PGJ2 with each 1110μM glutathione (GSH), L-cysteine (Cys) or N-acetylcysteine (NAC) in 100mM phosphate buffer (pH 7.4) resulted in formation of the corresponding conjugates and concomitant decrease of 15d-PGJ2 as measured by high-performance liquid chromatography (HPLC). Retention time was 12.7, 3.6, 2.8 and 1.2 minutes for 15d-PGJ2 and the 15d-PGJ2-NAC, 15d-PGJ2-Cys, and 15d-PGJ2-GSH conjugates, respectively. Reaction of 15d-PGJ2 with Cys was accompanied by a shift of the maximum wavelength from 318 nm to 312 nm and an increase in absorbance at 230 nm. (B,C) The HPLC fractions of the above mentioned conjugates were collected and subjected to catalytical hydrogenation/desulfurization as described elsewhere for the cysteinyl leukotriene E4.5 After derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br) followed by N-methyl-N-trimethylsilyl-trifluoroacetamide (MTSFA) in the presence of NH4I and 2-mercaptoethanol (ME), gas chromatography-mass spectrometry (GC-MS) spectra were generated in the electron-capture negative-ion chemical ionization mode (B). The precursor ion at m/z 397 [M-PFB]− was subjected to collision-induced dissociation (CID) to generate GC-tandem MS (GC-MS/MS) spectra (C). Expectedly, virtually identical GC-MS and GC-MS/MS mass spectra were obtained from all thiol (RSH) conjugates of 15d-PGJ2. Inserts in panels B and C indicate schematically part of the analytical procedure used and the proposed structures for the ions obtained. (D) Excretion of 15d-PGJ2 and the isoprostane 15(S)-8-iso-PGF2α (iso-PGF2α) was measured in fresh spot urine samples of 12 healthy volunteers (4 females) by GC-MS/MS using 2H4-15d-PGJ2 and 2H4-15(S)-8-iso-PGF2α as internal standards. 15(S)-8-iso-PGF2α was extracted from urine (1 mL) by immunoaffinity column chromatography.6 15d-PGJ2 was extracted from acidified (pH 4.5) urine samples by solid-phase extraction and purified by isocratic reverse phase HPLC. In the urine samples no 15d-PGJ3 was detectable. 15(S)-8-iso-PGF2α was measured because it is considered a COX-independent metabolite, analogous to 15d-PGJ2 and 15d-PGJ3.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/119/12/10.1182_blood-2012-01-403105/4/zh89991288600001.jpeg?Expires=1735516089&Signature=abTA5BalCbEYZIMi1XSXylnNZ1Gu99KGAp2F28OWcBmze2H~aIn8gJRhvlw5nGoAYAV3Q8VspwAwGP0M~yAikz6cEJA~LeQlbQYBf2Jo~MVmrF0lIKE7AJAYdZ4AHSB914fgt1K5Uva-1~6xmsyQ9FDKff2H2xo-BlTieJfk6iAniA7mAjlXLu-kY0CVkZY93dj0VIeTwfLRZ8rEjPzWF9NNVt4-4nkXyGhD-4ImhHtf0cehV6Km~sS89TWbXrYfEtlAd2hX2EGkkEQXWv0lpMerYVi-vly0rQURagrZR44wMt-heo2MRNlIcl1Fx1bXwtYMrr-kdb4Dgp1trezExw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Excretion of 15d-PGJ2 in human urine and its in vitro conjugation with glutathione, L-cysteine and N-acetylcysteine. (A) Reaction of 30μM 15d-PGJ2 with each 1110μM glutathione (GSH), L-cysteine (Cys) or N-acetylcysteine (NAC) in 100mM phosphate buffer (pH 7.4) resulted in formation of the corresponding conjugates and concomitant decrease of 15d-PGJ2 as measured by high-performance liquid chromatography (HPLC). Retention time was 12.7, 3.6, 2.8 and 1.2 minutes for 15d-PGJ2 and the 15d-PGJ2-NAC, 15d-PGJ2-Cys, and 15d-PGJ2-GSH conjugates, respectively. Reaction of 15d-PGJ2 with Cys was accompanied by a shift of the maximum wavelength from 318 nm to 312 nm and an increase in absorbance at 230 nm. (B,C) The HPLC fractions of the above mentioned conjugates were collected and subjected to catalytical hydrogenation/desulfurization as described elsewhere for the cysteinyl leukotriene E4.5 After derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br) followed by N-methyl-N-trimethylsilyl-trifluoroacetamide (MTSFA) in the presence of NH4I and 2-mercaptoethanol (ME), gas chromatography-mass spectrometry (GC-MS) spectra were generated in the electron-capture negative-ion chemical ionization mode (B). The precursor ion at m/z 397 [M-PFB]− was subjected to collision-induced dissociation (CID) to generate GC-tandem MS (GC-MS/MS) spectra (C). Expectedly, virtually identical GC-MS and GC-MS/MS mass spectra were obtained from all thiol (RSH) conjugates of 15d-PGJ2. Inserts in panels B and C indicate schematically part of the analytical procedure used and the proposed structures for the ions obtained. (D) Excretion of 15d-PGJ2 and the isoprostane 15(S)-8-iso-PGF2α (iso-PGF2α) was measured in fresh spot urine samples of 12 healthy volunteers (4 females) by GC-MS/MS using 2H4-15d-PGJ2 and 2H4-15(S)-8-iso-PGF2α as internal standards. 15(S)-8-iso-PGF2α was extracted from urine (1 mL) by immunoaffinity column chromatography.6 15d-PGJ2 was extracted from acidified (pH 4.5) urine samples by solid-phase extraction and purified by isocratic reverse phase HPLC. In the urine samples no 15d-PGJ3 was detectable. 15(S)-8-iso-PGF2α was measured because it is considered a COX-independent metabolite, analogous to 15d-PGJ2 and 15d-PGJ3.
