Jonas Niemann

Divergence in urinary 8-iso-PGF2α (iPF2α-III, 15-F2t-IsoP) levels from gas chromatography–tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF2α: Possible methodological, mechanistic and clinical implications

Abstract Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F2-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC–tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF2α (iPF2α-III, 15-F2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF2α (i.e. 15(S)-8-iso-PGF2α) has been shown by others to be highly selective and specific for this 8-iso-PGF2α isomer when quantified by GC–MS. In the present study we established IAC for urinary 8-iso-PGF2α for subsequent quantification by GC–tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF2α. 8-iso-PGF2α was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF2α was determined to be 291±102 pg/mg creatinine by method A and 141±41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF2α at 128±55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF2α is heterogenous, with 15(S)-8-iso-PGF2α contributing by ∼50%. PGF2α and other 8-iso-PGF2α isomers including 15(R)-8-iso-PGF2α are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF2α. We assume that ent-15(S)-8-iso-PGF2α is also contributing by ∼50% to urinary 8-iso-PGF2α. This finding may have methodological, mechanistic and clinical implications.

Glutathione promotes prostaglandin H synthase (cyclooxygenase)-dependent formation of malondialdehyde and 15(S)-8-iso-prostaglandin F2α

Prostaglandin (PG) H synthases (PGHS) or cyclooxygenases (COX) catalyse the peroxidation of arachidonic acid (AA) to PGG2 and PGH2 which are further converted to a series of prostaglandins and thromboxane A2. Here, we report that GSH promotes concomitant formation of the current oxidative stress biomarkers malondialdehyde (MDA) and 15(S)‐8‐iso‐prostaglandin F2α from AA via PGHS. This illustrates an uncommon interplay of enzymatic and chemical reactions to produce species that are considered to be exclusively produced by free‐radical‐catalysed reactions. We propose mechanisms for the PGHS/AA/GSH‐dependent formation of MDA, 15(S)‐8‐iso‐prostaglandin F2α and other F2‐isoprostanes. These mechanisms are supported by clinical observations.

Production of prostaglandins, isoprostanes and thromboxane by Aspergillus fumigatus: identification by gas chromatography-tandem mass spectrometry and quantification by enzyme immunoassay.

Aspergillus fumigatus has been reported to produce prostaglandin (PG)-like substances. The molecular structure of these fungal eicosanoids is however still unknown. By using the gas chromatography-tandem mass spectrometry (GC-MS/MS) methodology we identified a number of eicosanoids that were formed upon incubation of the precursor arachidonic acid ethyl ester (AAE, 10 μM) with three strains of A. fumigatus. The eicosanoids identified include the prostaglandins (PG) PGE(2), 6-keto-PGF(1α) (the stable hydrolysis product of prostacyclin PGI(2)) and PGF(2α), the isoprostanes 15(S)-8-iso-PGF(2α) and 15(S)-8-iso-PGE(2), and thromboxane B(2) (TxB(2), the stable hydrolysis product of TxA(2)). These eicosanoids are identical with those produced by cyclooxygenases (COX) in humans. The biosynthesis of all of these eicosanoids could not be inhibited by the human COX inhibitors indomethacin (100 μM), acetylsalicylic acid (100 μM) or the non-selective human lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (100 μM). By contrast, the selen-containing antioxidant ebselen (100 μM) was found to inhibit their synthesis. Our results suggest that mammals and fungi employ different eicosanoid biosynthesis pathways. As some of the detected eicosanoids are potent immunomodulators and bronchoconstrictors, they could play a possible role in pulmonary diseases associated with A. fumigatus infection.

Extra-platelet NO and NO+-containing drugs are potent inhibitors of platelet aggregation in humans by cGMP-dependent and cGMP-independent mechanisms

To the editor: Zhang and colleagues stated in their recent article[1][1] that it was unknown until their study whether NO donors inhibit platelet function by cGMP-dependent and cGMP-independent mechanisms. Yet we[2][2],[3][3] and others[4][4] reported several years ago that S -nitrosocysteine (CSNO

Nitric oxide, peroxynitrite, S-nitrosothiols and thiols are unlikely to exert their effects on recombinant cyclooxygenase-1 and cyclooxygenase-2 activity in vitro by modifying cysteine moieties.

To the Editor, Nitric oxide (NO), prostacyclin (PGI2) and other prostaglandins (PGs), and thromboxane A2 (TxA2) are important short-lived signaling molecules involved in many physiological and pathological processes. NO is synthesized from L-arginine by NO synthase (NOS) isoforms. Prostaglandin synthase or cyclooxygenase (COX) isoforms convert arachidonic acid to the collectively named prostanoids. The L-arginine/NO pathway is generally accepted to interact with the prostanoids pathway and to modulate its activity and reversely [1–4]. For instance, the inducible NOS isoform (iNOS) has been shown to bind to the inducible COX isoform (COX-2), to S-nitrosylate and activate COX-2 [2]. The role of NO in prostaglandin biology has been recently updated by Kim in this Journal [4]. Potential mechanisms of direct NOS–COX cross-talk may include (1) binding of the NO radical ( NO) to the iron atom of the heme group of COX; (2) reaction of the nitrosyl cation (NO) with sulfhydryl (SH) groups of cysteine (Cys) moieties of COX; and (3) reaction of peroxynitrite (ONOO ), i.e., the reaction product of NO and superoxide radical anion ðO 2 Þ produced either by NOS itself or by other enzymes [5], with SH groups of Cys residues or with tyrosine (Tyr) residues of COX being involved in the catalytic process [4]. S-Nitrosylation of COX-Cys moieties by higher oxides of NO, notably dinitrogen trioxide (N2O3), and by ONOO , and S-transnitrosylation of COX-Cys moieties by S-nitrosothiols have been shown both to enhance and inhibit COX activity; in contrast, nitration of Tyr residues located in the catalytic domain of COX is assumed to inhibit COX activity [4,6–8]. On the other hand, ONOO has been reported to activate COX activity presumably by increasing the peroxide tonus that is required for the peroxidase activity of COX [9]. However, in this and in other studies very high lM-concentrations of peroxynitrite have been used. We found only a weak inhibition (25–50%) of isolated COX-1 and COX-2 activity by nM-concentrations of synthetic ONOO , i.e., at ONOO /COX molar ratios of 16:1–160:1 [10,11]. The divergent results obtained from the use of ONOO , as well as the contradicting results concerning the S-(trans)nitrosylation mechanism suggest that the interaction between the NOS and COX pathways is incompletely understood. One possible explanation for the discrepancies may be that the effects seen by NO, ONOO , and NO/NO /NO-donors are secondary because most investigations had been performed on cells under conditions that are likely to have influenced additional pathways, finally leading to activation or inhibition of COX activity and/or expression. Direct effects of NO-, ONOO -, and NO-donors, including physiological and non-physiological S-nitrosothiols, on COX activity are best studied by using isolated COX enzymes. An additional important issue that has been obviously overseen in

Antioxidants and Endothelial Dysfunction in Young and Elderly People: Is Flow-Mediated Dilation Useful to Assess Acute Effects?

To the Editor: Wray et al1 reported that acute consumption of antioxidants by healthy subjects had diametrically different effects: flow-mediated dilation increased in elderly people but decreased in young people. The opposite effects are difficult to interpret and reconcile with the measured biomarkers of oxidative stress and NO synthesis/bioavailability. Although not representative, an example for the strong limitation of acute changes is the enhancing effect of certain diuretics on the renal excretion of …