Response to letter by Tsikas et al. 15-deoxy-Δ12,14-PGJ2: An interesting but unapproachable...
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Letter to the Editor Response to letter by Tsikas et al. 15-deoxy-Δ 12,14 -PGJ 2 : An interesting but unapproachable pharmacological target? Dimitrios Tsikas ⁎, Jonas Niemann, Bibiana Beckmann Center of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany article info Article history: Received 21 August 2014 Accepted 26 August 2014 Available online 30 August 2014 Keywords: Cardiomyocytes Cyclooxygenase Cyclopentenone prostaglandins Mass spectrometry Oxidative stress Thiols To the Editor: We read with great interest the article by Koyani et al. reporting that 15-deoxy-Δ 12,14 -prostaglandin (PG) J 2 (15d-PGJ 2 ) promotes apoptosis and inflammation in cardiomyocytes [1]. Although there is no question that 15d-PGJ 2 and relatives including Δ 12 -PGJ 3 have multiple biological activities [2–9], the most important question is still [10] whether 15d- PGJ 2 is formed in vivo at sufficiently high concentrations or can be ap- plied pharmacologically or nutritionally at such doses that are likely to provide 15d-PGJ 2 concentrations high enough to exert biologic activity. This question cannot be answered without addressing quantitative is- sues and the chemistry of 15d-PGJ 2 . Formally, PGJ 2 is the dehydrated product of PGD 2 which is formed in vivo from arachidonic acid by the catalytic action of cyclooxygenase (COX). PGJ 2 is further dehydrated and isomerized to produce 5d-PGJ 2 . From the chemical perspective, the most characteristic feature of 15d- PGJ 2 is the highly reactive cyclopentenone ring, which is readily attacked by ubiquitous intra- and extra-cellular thiols such as cysteine and glutathione (GSH) to form thioethers [10]. Rapid and exhausting thiolation of 15d-PGJ 2 will greatly reduce the bioavailability of authentic 15d-PGJ 2 . Yet, thiolation will also modify the inherent biological activity of 15d-PGJ 2 and produce a cocktail of bioactive lipids including cysteinyl leukotrienes-like thioethers. Indeed, by means of an accurate and precise gas chromatography–mass spectrometry–mass spectrometry (GC–MS/MS) method (Fig. 1) we measured 15d-PGJ 2 in the urine of healthy subjects at concentrations in the pM-range, equivalent to a few nmol 15d-PGJ 2 per mol creatinine (e.g., 67 ± 14 nmol/mol [10]). Thus, the excretion rate of 15d-PGJ 2 is comparable to other prostaglan- dins including PGD 2 [11] and suggests that plasma and tissue concentra- tions of 15d-PGJ 2 are likely to be in the pM-range. In fact, in heart tissue of rabbits basal 15d-PGJ 2 concentration was reported to be 117 pg/mL which increased to 224 pg/mL upon desflurane anesthesia [12]. These values correspond to 15d-PGJ 2 concentrations of 0.4 and 0.7 nM, respec- tively. Thus, in the study by Koyani et al. [1] and in studies from other groups [3–8], 15d-PGJ 2 was used in the majority of their experi- ments at a concentration of 15 μM, which is at least three orders of mag- nitude higher than expectable tissue 15d-PGJ 2 concentrations. Lower, more pathophysiologically relevant but still too high concentrations (e.g., 50 nM) were used only in very few experiments [1]. Many biological effects of 15d-PGJ 2 including apoptosis in cardiomyocytes are attributed to the intracellular production of reactive oxygen species (ROS) which are frequently measured by the DCF fluo- rescence assay [1]. Yet, the ROS studies by Koyani et al. [1] were per- formed at the very high 15d-PGJ 2 concentration of 15 μM, which renders the oxidative effects of 15d-PGJ 2 at least partially non- pathophysiologically relevant. Furthermore, the use of antioxidants such as N-acetylcysteine (NAC) in studies involving highly reactive elec- trophiles such as 15d-PGJ 2 is not very meaningful, because a consider- able fraction of the strong nucleophilic NAC adds to the double bond of the cyclopentenone ring 15d-PGJ 2 covalently but relatively slowly in a time-dependent manner [5]. In addition, thiols are known to alter COX activity and shift the prostaglandin spectrum. Thus, we found that NAC potently (IC 50 ≈ 10–20 μM NAC) inhibits COX-1- and COX- 2-catalyzed formation of PGD 2 and PGE 2 from arachidonic acid (Fig. 2). 4-Hydroxy-TEMPO at 1 mM had no appreciable effects of COX-1 and COX-2 activity when measured as PGD 2 and PGE 2 formation rate (data not shown). This inhibition is likely to result from reduction of COX-derived PGH 2 to PGF 2α [13]. Finally, by means of reliable GC– MS/MS methods for 15d-PGJ 2 [10] and 15(S)-8-iso-PGF 2α [14], we found no relationship between the lipid peroxidation biomarker 15(S)-8-iso-PGF 2α and 15d-PGJ 2 in the urine of healthy subjects (Fig. 1). This observation suggests that 15d-PGJ 2 is not associated with oxidative stress, presumably because of its very low quantity in biolog- ical samples. International Journal of Cardiology 177 (2014) 307–309 ⁎ Corresponding author at: Center of Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: +49 511 532 3984; fax: +49 511 532 2750. E-mail address: [email protected] (D. Tsikas). http://dx.doi.org/10.1016/j.ijcard.2014.08.145 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved. Contents lists available at ScienceDirect International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Transcript of Response to letter by Tsikas et al. 15-deoxy-Δ12,14-PGJ2: An interesting but unapproachable...
International Journal of Cardiology 177 (2014) 307–309
Contents lists available at ScienceDirect
International Journal of Cardiology
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Letter to the Editor
Response to letter by Tsikas et al. 15-deoxy-Δ12,14-PGJ2: An interestingbut unapproachable pharmacological target?
Dimitrios Tsikas ⁎, Jonas Niemann, Bibiana BeckmannCenter of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany
⁎ Corresponding author at: Center of Pharmacology andSchool, Carl-Neuberg-Str. 1, 30625Hannover, Germany. Te511 532 2750.
E-mail address: [email protected] (D. T
http://dx.doi.org/10.1016/j.ijcard.2014.08.1450167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved
a r t i c l e i n f o
Article history:
Received 21 August 2014Accepted 26 August 2014Available online 30 August 2014Keywords:CardiomyocytesCyclooxygenaseCyclopentenone prostaglandinsMass spectrometryOxidative stressThiols
leukotrienes-like thioethers. Indeed, by means of an accurate andprecise gas chromatography–mass spectrometry–mass spectrometry(GC–MS/MS) method (Fig. 1) we measured 15d-PGJ2 in the urine ofhealthy subjects at concentrations in the pM-range, equivalent to afew nmol 15d-PGJ2 per mol creatinine (e.g., 67 ± 14 nmol/mol [10]).Thus, the excretion rate of 15d-PGJ2 is comparable to other prostaglan-dins including PGD2 [11] and suggests that plasma and tissue concentra-tions of 15d-PGJ2 are likely to be in the pM-range. In fact, in heart tissueof rabbits basal 15d-PGJ2 concentration was reported to be 117 pg/mLwhich increased to 224 pg/mL upon desflurane anesthesia [12]. Thesevalues correspond to 15d-PGJ2 concentrations of 0.4 and 0.7 nM, respec-tively. Thus, in the study by Koyani et al. [1] and in studies from
To the Editor:
We readwith great interest the article by Koyani et al. reporting that15-deoxy-Δ12,14-prostaglandin (PG) J2 (15d-PGJ2) promotes apoptosisand inflammation in cardiomyocytes [1]. Although there is no questionthat 15d-PGJ2 and relatives including Δ12-PGJ3 have multiple biologicalactivities [2–9], the most important question is still [10] whether 15d-PGJ2 is formed in vivo at sufficiently high concentrations or can be ap-plied pharmacologically or nutritionally at such doses that are likely toprovide 15d-PGJ2 concentrations high enough to exert biologic activity.This question cannot be answered without addressing quantitative is-sues and the chemistry of 15d-PGJ2.
Formally, PGJ2 is the dehydrated product of PGD2 which is formedin vivo from arachidonic acid by the catalytic action of cyclooxygenase(COX). PGJ2 is further dehydrated and isomerized to produce 5d-PGJ2.From the chemical perspective, the most characteristic feature of 15d-PGJ2 is the highly reactive cyclopentenone ring, which is readilyattacked by ubiquitous intra- and extra-cellular thiols such as cysteineand glutathione (GSH) to form thioethers [10]. Rapid and exhaustingthiolation of 15d-PGJ2will greatly reduce the bioavailability of authentic15d-PGJ2. Yet, thiolationwill alsomodify the inherent biological activity
Toxicology, Hannover Medicall.:+49511 532 3984; fax:+49
sikas).
.
of 15d-PGJ2 and produce a cocktail of bioactive lipids including cysteinyl
other groups [3–8], 15d-PGJ2 was used in the majority of their experi-ments at a concentration of 15 μM,which is at least three orders ofmag-nitude higher than expectable tissue 15d-PGJ2 concentrations. Lower,more pathophysiologically relevant but still too high concentrations(e.g., 50 nM) were used only in very few experiments [1].
Many biological effects of 15d-PGJ2 including apoptosis incardiomyocytes are attributed to the intracellular production of reactiveoxygen species (ROS) which are frequently measured by the DCF fluo-rescence assay [1]. Yet, the ROS studies by Koyani et al. [1] were per-formed at the very high 15d-PGJ2 concentration of 15 μM, whichrenders the oxidative effects of 15d-PGJ2 at least partially non-pathophysiologically relevant. Furthermore, the use of antioxidantssuch asN-acetylcysteine (NAC) in studies involving highly reactive elec-trophiles such as 15d-PGJ2 is not very meaningful, because a consider-able fraction of the strong nucleophilic NAC adds to the double bondof the cyclopentenone ring 15d-PGJ2 covalently but relatively slowlyin a time-dependent manner [5]. In addition, thiols are known to alterCOX activity and shift the prostaglandin spectrum. Thus, we foundthat NAC potently (IC50 ≈ 10–20 µM NAC) inhibits COX-1- and COX-2-catalyzed formation of PGD2 and PGE2 from arachidonic acid(Fig. 2). 4-Hydroxy-TEMPO at 1 mM had no appreciable effects ofCOX-1 and COX-2 activity whenmeasured as PGD2 and PGE2 formationrate (data not shown). This inhibition is likely to result from reductionof COX-derived PGH2 to PGF2α [13]. Finally, by means of reliable GC–MS/MS methods for 15d-PGJ2 [10] and 15(S)-8-iso-PGF2α [14], wefound no relationship between the lipid peroxidation biomarker15(S)-8-iso-PGF2α and 15d-PGJ2 in the urine of healthy subjects(Fig. 1). This observation suggests that 15d-PGJ2 is not associated withoxidative stress, presumably because of its very low quantity in biolog-ical samples.
A B
C
Fig. 1. (A) Relationship (Pearson correlation) between 15(S)-8-iso-PGF2α and 15d-PGJ2 in the urine of twelve healthy volunteers. (B) Linear regression analysis between the 15d-PGJ2 con-centration measured (y) in the urine and the 15d-PGJ2 concentration added (x) to a pooled urine sample of a healthy female donor. Data are shown as mean ± SD from a validation ex-periment performed on 5 days and in duplicate for each concentration point. The slope of the inserted regression equation of 1.02 indicates an average accuracy of 102%. The imprecision(RSD) ranged between 5 and 25%. 15d-PGJ2 and 15(S)-8-iso-PGF2α were measured by GC–MS/MS using 2H4-15d-PGJ2 (200 pg/mL) and 2H4-15(S)-8-iso-PGF2α (1000 pg/mL) as internalstandards as described previously for 15d-PGJ2 [10] and 15(S)-8-iso-PGF2α [14]. Creatinine wasmeasured by GC–MS using 2H3-creatinine (10mM) as internal standard as reported else-where [15]. (C) A representative chromatogram from the quantitative GC–MS/MS determination of 15d-PGJ2 in urine of a healthy volunteer using [3,3′,4,4′-2H4]15d-PGJ2 (200 pg/mL)as the internal standard. Quantification was performed by selected-reaction monitoring of the common product ions at m/z 203 produced by collision-induced dissociation (CID) of theprecursor anions atm/z 315 for endogenous 15d-PGJ2 (upper panel) andm/z 319 for [3,3′,4,4′-2H4]15d-PGJ2 (lower panel). 15d-PGJ2 and [3,3′,4,4′-2H4]15d-PGJ2 were analyzed after de-rivatization to their pentafluorobenzyl esters and electron-capture negative-ion chemical ionization. Insets show the structures of the precursor ions and the common product anion. AllGC–MS/MS analyses were performed on the triple-stage quadrupole mass spectrometer model TSQ 7000 (ThermoFisher, Dreieich, Germany). RT, retention time; PA, peak area; S/N,signal-to-noise.
308 D. Tsikas et al. / International Journal of Cardiology 177 (2014) 307–309
Prostaglandins, thromboxanes, leukotrienes and their derivativesare targets of pharmacological treatment in cardiovascular diseases formany decades. The PGD2-derived 15d-PGJ2 has been recently found toexertmultiple biological activities [2–9] and has been proposed to be in-volved inmany diseases. Induction of oxidative stress is assumed to be amechanism bywhich 15d-PGJ2 may exert (some of) its biological activ-ities such as damaging cardiomyocytes [1]. Yet, our present knowledgeabout the biological activities and the pharmacological potential of 15d-PGJ2 is mainly based on the use of very high concentrations/doses of15d-PGJ2 in vitro and in vivo experiments in animals. Itmust be empha-sized that the commonly used 15d-PGJ2 concentrations of 15 μM [1–9]
are of the same order of magnitude of the concentration of free arachi-donic acid, the precursor of PGD2 and all other eicosanoids, in biologicalfluids and tissues.
Non-consideration of quantitative issues in the research area of 15d-PGJ2 and other endogenous substances such as nitro-fatty acids [7,16],which are also electrophilic species and share with 15d-PGJ2 commonbiological activities, will certainly generate interesting scientific resultsand reveal basic mechanisms that would have been otherwise undis-covered. However, this practice is likely to raise unrealizable expecta-tions with regard to 15d-PGJ2, nitro-fatty acids and other endogenousbioactive substances in the cardiovascular and other systems.
Fig. 2. Inhibition of recombinant COX-1- andCOX-2-catalyzed formation of PGD2 and PGE2byN-acetylcysteine (NAC) at the indicated concentrations from arachidonic acid (10 μM).Recombinant ovine COX-1 and COX-2were obtained from CaymanChemicals (Ann Arbor,MI, USA). 5 units (equivalent to 90 ng COX-1 and 600 ng COX-2) were used and COXactivity was measured by GC–MS/MS in two independent experiments as describedelsewhere [13]. In the absence of NAC, mean COX-1 and COX-2 activities were 89 and46 pg/min × ng protein for PGD2, and 333 and 175 pg/min × ng protein for PGE2, respec-tively. The IC50 values are about 20 μMNAC for COX-1 and 10 μMNAC for COX-2 with re-spect both to PGD2 and PGE2. All GC–MS/MS analyses were performed on the triple-stagequadrupole mass spectrometer model TSQ 7000 (ThermoFisher, Dreieich, Germany).
309D. Tsikas et al. / International Journal of Cardiology 177 (2014) 307–309
Conflict of interest disclosure
The authors report no relationships that could be construed as a con-flict of interest.
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