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Analysis of urinary eicosanoids by LC-MS/MS revealed alterations in the metabolic profile after smoking cessation Michael Goettel, Reinhard Niessner, Max Scherer, Gerhard Scherer, and Nikola Pluym Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00276 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Chemical Research in Toxicology

Analysis of urinary eicosanoids by LC-MS/MS revealed alterations in the metabolic profile after smoking cessation Michael Goettel‡, †, Reinhard Niessner‡, Max Scherer†, Gerhard Scherer†, Nikola Pluym†,* ‡Chair for Analytical Chemistry, Technische Universität München, Marchioninistraße 17, 81377 Munich, Germany †ABF, Analytisch-Biologisches Forschungslabor GmbH, Semmelweisstraße 5, 82152 Planegg, Germany KEYWORDS: urinary eicosanoids, smoking cessation, metabolomics.

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ABSTRACT: A preceding untargeted metabolic fingerprinting approach in our lab followed by a targeted fatty acid analysis revealed alterations in the arachidonic acid metabolism in samples derived from a diet-controlled smoking cessation study in which compliant subjects (N=39) quit smoking at baseline and were followed over the course of three months. Consequently, urinary eicosanoids were determined by means of a validated LC-MS/MS method. A significant decrease was obtained for the prostaglandins PGF2α, 8-iso-PGF2α, thromboxane 2,3-d-TXB2 and leukotriene E4 upon quitting smoking. These findings indicate a partial recovery of smoking-induced alterations in the eicosanoid profile due to a reduced oxidative stress and inflammatory response.


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INTRODUCTION Untargeted metabolic fingerprinting in combination with targeted analysis is a powerful tool to elucidate alterations between distinctive groups. Such a combined approach of untargeted profiling and targeted quantification has been performed recently in our laboratory in a first clinical study1,

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to identify

differences in the metabolome of smokers and non-smokers. We found several major metabolic pathways which differ significantly between these groups, especially lipid and fatty acid metabolism.1-3 The findings from the first clinical study comparing smokers and non-smokers encouraged us to conduct a second diet-controlled clinical study, in which we investigated metabolic alterations in smokers after cessation. The untargeted fingerprinting analysis using a validated GC-TOF-MS method1,

2

showed

changes in the fatty acid (FA), amino acid and energy metabolism, indicating a partial recovery of the smokers’ metabolome towards the non-smoker profile upon three months of cessation.4 Subsequently, we developed and validated a targeted GC-TOF-MS method for the quantification of FAs.5 Several FA species were significantly altered after quitting including saturated, as well as mono- and polyunsaturated FAs. Amongst other FAs, arachidonic acid showed a statistically significant increase after one month of smoking cessation which prompted us to investigate the eicosanoid profile in this second clinical study as arachidonic acid (FA 20:4 5Z, 8Z, 11Z, 14Z) is regarded as a key precursor towards the formation of various eicosanoids. Eicosanoids are signaling molecules produced through both enzymatic and non-enzymatic free radical catalyzed reactions.6 They are secreted from different human cell types under normal and pathophysiological conditions. Alterations in eicosanoid levels may be caused by various disease states like asthma7, 8, diabetes9, cancer9-11 and exogenous sources like smoking.9, 12, 13 Hence, eicosanoids are often investigated in plasma and urine as biomarkers of effect indicating oxidative stress or inflammation.6,

10, 12, 14, 15

We, therefore, decided to analyze urinary eicosanoids using a validated,

targeted LC-MS/MS method.12 In terms of smoking cessation only few analytes were investigated simultaneously in previous studies.16,

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Thus, we decided to extend our method to a total of nine

eicosanoids by inclusion of two additional analytes, namely the prostaglandins (PGs) tetranor-PG Dmetabolite (t-PGDM) and PGF2ɑ, to allow for the investigation of a more comprehensive eicosanoid profile (Figure 1).


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Figure 1 Structures of the nine eicosanoids analyzed by means of LC-MS/MS.

MATERIAL AND METHODS Reagents and Standards Acetic acid (≥ 99 %), ammonium hydroxide (28% in water), creatinine (anhydrous), formic acid (≥ 95%), hydrochloric acid (~37%), sodium hydroxide (≥ 97%, pellets) were purchased from SigmaAldrich (Munich, Germany). Chloroform (picograde), ethyl acetate (optigrade), methanol (optigrade) and water (optigrade) were obtained from LGC Standards (Wesel, Germany). Tetranor PGE-M (tPGEM), tetranor PGD-M (t-PGDM), 2,3-dinor-8-iso-PGF2α, (2,3-d-8-iso-PGF2α) 8-iso-PGF2α, 2,3dinor-TXB2 (2,3-d-TXB2), 11-dehydro-TXB2 (11-dh-TXB2), LTE4, 12(S)-HETE, D6-tetranor-PGDM, D6-tetranor-PGEM, D4-8-iso-PGF2α, D4-11-dehydro-TXB2, D5-LTE4 and D8-12(S)-HETE were with purities higher than 97% and purchased from Biomol (Hamburg, Germany). 0.1% formic acid in water (ULC-MS grade) and 0.1% formic acid in acetonitrile (ULC-MS grade) were from Biosolve BV (Valkenswaard, Netherlands). Smoking Cessation Study and Sample Collection The clinical study was carried out as described in detail in Goettel et al.4 The protocol was approved in accordance with the Helsinki declaration18 by the ethical commission of the Medical Chamber of North ACS Paragon Plus Environment

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Rhine-Westphalia, Germany. Briefly, 39 smokers (15 to 24 cigarettes/day) were followed over the course of three months of smoking cessation. Subjects were healthy male individuals, still smoking at study start (TP0). Sampling was conducted during the in-patient stays at study start (TP0), after one week (TP1), one month (TP2) and three months (TP3) of smoking cessation. A controlled diet with 72% carbohydrates, 14% fat and 14% protein was necessary in order to reduce nutritional effects on eicosanoid synthesis and metabolism. Hence, twelve hours of fasting prior to the in-patient stays were mandatory. Meals were served at defined points in time during confinement (in-patient stays at TP0 – TP3). Moreover, the caloric intake was adjusted according to the individuals’ body weight (55-65 kg: ~7500 kJ, 66-75 kg: ~8500 kJ, 76-85 kg: ~9400 kJ, 86-95 kg: ~10200 kJ, and 96-105 kg: 10700 kJ). Compliance was verified by determination of carbon monoxide in exhaled breath and cotinine in saliva and urine in several ambulatory visits interspersed throughout the entire clinical study. Subjects with COex levels above 6 ppm and/or concentrations for cotinine over 15 ng/mL in saliva and 50 ng/mL in urine, respectively, were regarded as non-compliant and excluded from the clinical study. In total, 39 participants of the initially 60 subjects were compliant over the complete course of 3 months and selected for urinary bioanalysis of eicosanoids and subsequent data evaluation. Urine samples were collected in three fractions at the in-patient stays (TP0 to TP3) under strictly controlled conditions during a period of 24h of controlled diet (8 am first day to 8 am second day). For the analysis of eicosanoids, the urine fractions were combined proportionally to yield a 24h urine. Samples were stored at -20°C until analysis. Quality Control Samples In addition to the set of study samples, nine quality control samples in low, medium and high concentration levels (3 per level) were interspersed throughout the entire analytical batch. Therefore, urine samples were pooled and partly spiked or diluted to yield concentrations in the low, medium, and high calibration range. The acceptance criteria for the accuracy of QC samples were set according to FDA guidelines19 at ± 20% for the low level and ± 15% for the medium and high level, respectively. At least two-thirds of all QC samples and 50% per level had to meet the acceptance criteria, otherwise the batch had to be repeated. Method validation In addition to the seven analytes implemented initially, tetranor PG E-metabolite (t-PGEM), 8-iso- and 2,3-dinor-8-iso-PGF2α (8-iso-PGF2ɑ; 2,3-d-8-iso-PGF2α), the thromboxanes (TX) 11-dehydro- and 2,3dinor-TXB2 (11-dh-TXB2, 2,3-d-TXB2), leukotriene (LT) E4 (LTE4) and 12-hydroxyeicosatetraenoic acid (12-HETE), two further analytes were introduced, namely PG D-metabolite (t-PGDM) and PGF2ɑ, ACS Paragon Plus Environment

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to include further established inflammation and oxidative stress markers and expand the method profile to a total of nine eicosanoids. The method was re-validated with respect to the novel analytes t-PGDM and PGF2ɑ in analogy to Sterz et al.12 and according to FDA guidelines.19 Calibration Quantification was performed with the internal standard method. Separate calibrations were performed with a set of 8 to 10 calibrators for each analyte, depending on the target eicosanoid and the calibration range as described in Sterz et al.12 (for data of the additional analytes cf. Table 2). Calibrators were prepared by spiking non-smoker urine samples with increasing amounts of analytes. For the calibration of the new analytes [D6]-t-PGDM was used as internal standard (IS) for t-PGDM and [D4]-11-dh-TXB2 for PGF2α. Evaluation of the chromatographic data was performed using Analyst 1.6.3 (AB Sciex, Darmstadt, Germany) and Excel 2013 (Microsoft, Redmond, USA). Calculation of the calibration curves was done by linear regression with 1/x weighting. Sample Preparation Urine samples from the clinical study4 were prepared and measured according to Sterz et al.12 All samples were randomized prior to LC-MS/MS analysis. Aliquots of 3 mL 24h-urine were used for analysis. According to Sterz et al.12 20 µL of acetic acid and 30 µL of an IS-mixture, containing 6 ng [D6]-t-PGEM, 6 ng [D6]-t-PGDM, 6 ng [D4]-8-iso- PGF2α, 6 ng [D4]-11-dh-TXB2, 1.5 ng [D5]-LTE4, and 1.5 ng [D8]-12-HETE, were added to each sample prior to extraction. Extraction was performed according to Bligh & Dyer20 with modifications. For liquid-liquid extraction, 11.25 mL B&D solution (methanol:chloroform 2:1 v/v) was added to each sample. After vortexing, samples were incubated for 1 hour at ambient temperature. Subsequently, 3.75 mL chloroform and 3.75 mL water were added. The samples were vortexed again and centrifuged for 10 min at 2500 rpm. The bottom layer chloroform phase was transferred into a vial and evaporated to dryness in a SpeedVac centrifuge (Thermo Scientific, Dreieich, Germany). Finally, the residue was reconstituted in 100 µL methanol for injection into the LC-MS/MS system. LC-MS/MS Analysis An API 5000 triple quadrupole mass spectrometer (AB Sciex, Darmstadt, Germany) LC-MS/MS system, equipped with a 1200 series binary pump (G1312B), a degasser (G1379B) and a column oven (G1316B) (Agilent, Waldbronn, Germany) connected to an HTC Pal autosampler (CTC Analytics, Zwingen, Switzerland) was used for chromatographic separation. The negative electrospray ionization (ESI-) was performed on the Turbo V ion spray source (AB Sciex, Darmstadt, Germany). Nitrogen in 7 ACS Paragon Plus Environment


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high purity was provided by a nitrogen generator NGM 22-LC/MS (cmc Instruments, Eschborn, Germany). For chromatographic separation a Waters (Eschborn, Germany) Acquity ultra performance liquid chromatography (UPLC) BEH C18 column (2.1 x 50 mm) with 1.7 µm particle size was utilized. The column oven was operated at 30°C. 5 µL sample were injected into a constant flow of 600µL/min. Eluents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Separation of the analytes was achieved with a gradient elution starting with 5% B for 1 min followed by a linear increase to 53% B until 9.5 min and a second linear increase to 76% B until 11 min. Third, a step to 100% B until 11.1 min was performed and held for 1 min. Finally, re-equilibration of the analytical system was achieved by rinsing from 12.1 min to 14 min with 5% B. The turbo ion spray source operated with the following settings: ion spray voltage = -4 kV, heater temperature = 600°C, source gas 1 = 20 psi, source gas 2 = 5 psi, CAD gas = 5 psi and curtain gas = 40 psi. Data acquisition was performed with quadrupoles working at unit resolution and multiple reaction monitoring (MRM). Data Analysis Different statistical tests were performed applying R (Version 3.3.0) and RStudio (Version 0.99.902) in order to follow alterations in the eicosanoid profile over the course of three months of smoking cessation. The Quade test was utilized in order to identify significant (ɑ = 0.05) changes across all points in time. The Wilcoxon signed rank test was applied for cessation-relevant comparisons between two points in time (e.g. TP0/TP1) in order to verify the results of the Quade test. To counteract the problem of multiple statistical comparisons, the significance levels were adjusted by the Bonferroni correction.21, 22

RESULTS AND DISCUSSION The determination of 9 individual eicosanoid species was performed by means of LC-MS/MS operating in ESI-negative ionization mode. Baseline separation of the isobaric species PGF2α/8-iso-PGF2α and tPGDM/t-PGEM, respectively, was achieved within a total run time of 14 minutes (Figure 2). Gradient elution yielded satisfactory peak resolution and sharpness which allowed the sensitive detection of the nine eicosanoid species. The quality control samples fulfilled the acceptance criteria set according to FDA guidelines19,

23

throughout all batches proving the robustness, accuracy and precision of the

method.


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Figure 2. Representative UPLC-MS/MS chromatograms of a smoker’s urine after three months of cessation (TP3). Quantifier MRM transitions are given in the respective chromatograms. Numbers are assigned according to Table 3. Validation data for the additional analytes are shown in Table 1. All acceptance criteria for precision and accuracy according to FDA guidelines were met proving the suitability of the method for bioanalysis of t-PGDM and PGF2α together with seven further eicosanoids. Recovery rates of 20 to 24 % were found for t-PGDM and 66% to 73% for PGF2α. A broad linear range and excellent sensitivity with an LOQ of 0.10 ng/mL allow to determine the analytes at physiologically relevant concentrations in healthy subjects and patients as well (Table 2). It is noteworthy that robust results were obtained for PGF2α, 2,3d-TXB2, and 2,3-d-8-i-PGF2α throughout the whole method validation although specific stable isotopelabeled analogs of these analytes were not used as internal standards ([D4]-11-dh-TXB2 for PGF2α, 2,3d-TXB2 and [D4]-8-i-PGF2α in case of 2,3-d-8-i-PGF2α). ACS Paragon Plus Environment

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As expected, t-PGDM showed a similar fragmentation to t-PGEM with a loss of water as most intense fragment (Table 2; Figure 3). Clearly, high peak resolution of the isobaric compounds t-PGDM and tPGEM which was achieved using a 1.7 µm UPLC column was imperative for robust quantification. For PGF2α, ion 193 m/z was identified as the most abundant fragment (quantifier MRM transition) without interfering signals resulting from a complex fragmentation including a break-up of the cyclopentane ring, the dispatch of a C9-side chain, water and 2 protons similarly to 8-iso-PGF2α described by Sterz et al.12 Fragmentation of the other analytes was already discussed extensively in Sterz et al.12 Stability of the analytes was regarded as given in case of accuracies between 85 and 115 % (80 to 120 % for low concentrations up to 3x LOQ). The stability of the analytes in matrix was confirmed for 24 h at ambient temperature. The freeze/thaw (F/T) stability of the analytes in urine was determined for six F/T cycles. Post-preparative stability at 10°C was confirmed for 14 days (data not shown). Finally, longterm stability was proven for 10.5 months at < -78°C, both for t-PGDM and PGF2α (Table 1). Matrix effects were assessed by comparing three different matrix samples to water in two levels. As for the previously validated analytes, no significant matrix effects were observed (differences between urine and matrix-free samples of -9.9 to 16.2%). Table 1. Precision, accuracy, recovery and stability investigations for t-PGDM and PGF2α. Recovery

Conc.

Short-term stability r.t. after 24 h

Freeze-thaw stability after 6 cycles

Long-term stability at < - 78 °C after 10.5 months

[%]

[%]

[ng/mL]

[%]

[%]

[%]

0.25

93.5

23.6

0.40

89.8

89.7

96.3

7.2

1.00

94.3

20.5

3.8

2.8

10.00

92.7

20.3

10.90

91.0

101.0

105.4

0.40

4.5

6.0

0.25

94.2

66.7

0.80

112.0

98.3

112.1

1.10

6.7

5.4

1.00

96.5

72.7

9.20

3.8

3.6

10.00

93.4

68.5

9.00

106.0

106.0

110.5

Conc.

Intraday precision (N=5)

Interday precision (N=6)

Conc.

[ng/mL]

CV [%]

CV [%]

[ng/mL]

0.40

11.0

5.2

1.00

5.8

10.00

Analyte

t-PGDM

PGF2α

Accuracy (N=5)

Table 2. MRM parameters, limits, and calibration data for t-PGDM and PGF2α. MRM

MRM IS

Dwell time

DP

CE

RT

LOD

LOQ

Calibration range

Slope

Correlation coefficient R'

[m/z]

[m/z]

[ms]

[V]

[V]

[min]

[ng/mL]

[ng/mL]

[ng/mL]

50

-85

-18 4.1

0.040

0.10

50

-80

-32

0.10 – 25.0

0.605

0.9996

50

-150

-36 8.0

0.045

0.10

50

-140

-28

0.10 – 25.0

0.210

0.9999

Analyte

t-PGDM

PGF2α

327.0 → 309.0 (Quantifier) 327.0 → 143.0 (Qualifier) 353.1 → 193.0 (Quantifier) 353.1 → 309.1

332.8 → 315.2 [D6]-t-PGDM

371.1 → 309.1 [D4]-11-dh-TXB2


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Figure 3. MS2 scans of reference compounds for t-PGDM (1) and PGF2α (2).

Table 3 summarizes the results for all nine eicosanoid species over the four points in time of the study (TP0 - TP3), given as total amount excreted within 24 hours, as well as minimum and maximum values and the trend of the concentration after three months of smoking cessation. Table 3. Results (concentration as median (Med.), minimum (Min.) and maximum (Max.) range) of the LC-MS/MS analysis and trend during the period of three months of smoking cessation. Numbering of the analytes is according to Figure 2. No.

Analyte

Trend

TP0 (start)

TP1 (1 week)

TP2 (1 month)

TP3 (3 month)

Excretion (ng/24 h)

Excretion (ng/24 h)

Excretion (ng/24 h)

Excretion (ng/24 h)

Med.

Min-Max

Med.

Min-Max

Med.

Min-Max

Med.

Min-Max

1

t-PGEM

-

4346

1200 - 37596

5099

2554 - 17100

4845

1392 - 16562

4636

1039 - 21638

2

t-PGDM

-

2524

1358 - 4680

2595

1479 - 5197

2532

1106 - 5221

2670

1505 - 5078

3

8-iso-PGF2α

▼

595

197 - 1098

510

245 - 1576

486

180 - 1439

473

219 1820

4

11-dh-TXB2

-

596

153 - 1358

509

185 - 1653

513

155 - 863

526

76 - 5454

5

LTE4

▼

130

39 - 355

77

25 - 149

61

24 - 164

67

32 - 157

6

12-HETE

-

20

6 - 320

15

5 - 418

13

3 - 190

16

4 - 185

7

2,3-d-8-iso-PGF2α

-

5139

2057 - 9031

4845

2760 - 9142

4702

1566 - 9999

5211

1798 - 15743

8

2,3-d-TXB2

▼

1450

236 - 3050

997

216 - 3655

702

167 - 2828

928

177 - 4120

9

PGF2ɑ

▼

3235

1199 - 6748

2718

1057 - 21821

2852

1242 - 6487

2984

1481 - 17225

▼ = decrease; Only trends of in both tests significantly altered analytes are given.


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Table 4. P-values of the statistical evaluation using Quade test and Wilcoxon signed rank test (analytes with significant alterations are highlighted in bold). Analyte

Quade test

Wilcoxon signed rank test

TP0-3

TP0/1

TP0/2

TP0/3

t-PGEM

0.0286

0.0459

0.6637

0.0459

t-PGDM

0.2136

0.3154

0.3721

0.1847

2,3-d-TXB2

6.7E-06

0.0009

1.2E-06

0.0093

2,3-d-8-iso-PGF2α

0.4070

0.7667

0.1311

0.4265

PGF2ɑ

0.0386

0.0132

0.0098

0.0197

11-dh-TXB2

0.1849

0.2041

0.0137

0.1275

8-iso-PGF2α

0.0153

0.1501

0.0400

0.0078

LTE4

2.5E-16

3.6E-12

6.9E-11

3.6E-11

12-HETE

0.2464

0.0222

0.0205

0.2248

The statistical analysis (Table 4) was performed applying the Quade test and Wilcoxon signed rank test in order to follow alterations in the eicosanoid levels during the three months of smoking cessation. We observed a decrease of 36% for 2,3-d-TXB2, 8% for PGF2ɑ, 21% for 8-iso-PGF2α and 49% for LTE4 after 3 months of smoking cessation which was found to be statistically significant in both tests applied (Quade test and Wilcoxon signed rank test). In addition, t-PGEM was identified as significantly altered in the Quade test, while showing no significance in the Wilcoxon signed rank test. In a previous study in our lab, Sterz et al.12 determined 80% lower levels for 2,3-d-TXB2 (Mann Whitney U test (MWU) < 0.000), 27% for 8-iso-PGF2α (MWU 0.042) and 32% for LTE4 (MWU 0.105; not significant) comparing smokers and non-smokers with the same LC-MS/MS method. Nowak et al.24 reported 36% lower values in non-smokers for 2,3-d-TXB2 levels. Both studies comparing smokers and non-smokers are in accordance with the differences we determined after smoking cessation. The reduction in urinary concentrations of the four eicosanoids was already observed after one week of smoking cessation (TP1, Figure 4). The most pronounced decrease was observed for LTE4 and 2,3-dTXB2 with a highly significant decline occurring already at TP1, while PGF2ɑ and 8-iso-PGF2ɑ showed significant decreases after 1 month (TP2) and 3 months (TP3) of cessation, respectively. Our findings are in line with other investigations that also show rather fast alterations in eicosanoid levels after quitting. For example, Riutta et al. described decreased levels of TXs, PGs and LTs in their 14-day cessation trail with 30 subjects25 while Benowitz et al. found a significant decrease of 11-dh-TXB2 in 12 quitters already after 5 days.26 In a long-term study, decreased levels of F2-isoprostanes were reported after one year of smoking cessation in 344 quitters.16


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In general, rather high inter-individual variations were observed for eicosanoids in our study in accordance with literature.12, 24, 27 Presumably, these variations result from endogenous formation and metabolic pathways. Variations from exogenous sources, other than smoking, seem less plausible here due to our study design including only healthy male subjects comprising a strict diet-control. In addition, we performed extensive and frequent compliance monitoring by means of COex and salivary/urinary cotinine measurements within several ambulatory visits in contrast to previous studies with less rigorous compliance measures.28-30 Hence, it is highly likely that the alterations found here are directly linked to smoking cessation.

Figure 4. Boxplots of the significantly altered eicosanoids up to three months of smoking cessation.

CONCLUSION Previous investigations mainly focused on the determination of single eicosanoid species like PGF2α and 8-iso-PGF2α or 2,3-d-TXB2 showing increased concentrations in smokers’ urine as compared to nonsmokers.12, 24, 31, 32 Elevated levels of LTE4 in smokers are also reported in literature by Riutta et al.25 Hence, the reduced levels of 2,3-d-TXB2, PGF2ɑ, 8-iso-PGF2ɑ and LTE4 indicate a partial recovery of eicosanoid levels after smoking cessation, which is mainly driven by the reduction of oxidative stress and inflammatory effects after quitting. Interestingly, reductions became already significant after one week of cessation indicating a rather fast response upon quitting especially for LTE4 and 2,3-d-TXB2.

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Corresponding Author *ABF, Analytisch-Biologisches Forschungslabor GmbH, Semmelweisstraße 5, 82152 Planegg Tel.: +49 89 535395; Fax: +49 89 5328039. E-mail address: [email protected] (N. Pluym). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was financially supported by the Imperial Tobacco Ltd and the Analytisch-biologisches Forschungslabor (ABF) GmbH (Planegg). There has not been any influence on the work or publication by the sponsor Imperial Tobacco Ltd. ABBREVIATIONS 11-dh-TXB2, 11-dehydro thromboxane B2; 12-HETE, 12-hydroxyeicosatetraenoic acid; 2,3-d-8-isoPGF2α, 2,3-dinor-8-iso prostaglandin F2α; 2,3-d-TXB2, 2,3-dinor thromboxane B2; 8-iso- PGF2α, 8-isoprostaglandin F2ɑ; FDA, Food and Drug Administration of the United States of America; LT, leukotriene; LTE4, leukotriene E4; MS, mass spectrometry; PG, prostaglandin; PGF2ɑ, Prostaglandin F2ɑ; PUFA, polyunsaturated fatty acid; SD, standard deviation; TP; point in time; t-PGDM, tetranor prostaglandin D metabolite; t-PGEM, tetranor prostaglandin E metabolite; TX, thromboxane; UPLC, ultra-performance liquid chromatography. REFERENCES

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