Katharina M.
Rund
a,
Fabian
Nolte
a,
Julian
Doricic
b,
Robert
Greite
b,
Sebastian
Schott
b,
Ralf
Lichtinghagen
c,
Faikah
Gueler
b and
Nils Helge
Schebb
*a
aChair of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Wuppertal, Germany. E-mail: schebb@uni-wuppertal.de; Tel: +49 202-439-3457
bNephrology, Hannover Medical School, Hannover, Germany
cInstitute of Laboratory Medicine, Hannover Medical School, Hannover, Germany
First published on 3rd February 2020
Quantitative analysis of oxylipins in blood samples is of increasing interest in clinical studies. However, storage after sampling and transport of blood might induce artificial changes in the apparent oxylipin profile due to ex vivo formation/degradation by autoxidation or enzymatic activity. In the present study we investigated the stability of free (i.e. non-esterified) and total oxylipins in EDTA-plasma and serum generated under clinical conditions assessing delays in sample processing and automated transportation: Free cytochrome P450 monooxygenase and 5-lipoxygenase (LOX) formed oxylipins as well as autoxidation products were marginally affected by storage of whole blood up to 4 h at 4 °C, while total (i.e. the sum of free and esterified) levels of these oxylipins were stable up to 24 h and following transport. Cyclooxygenase (COX) products (TxB2, 12-HHT) and 12-LOX derived hydroxy-fatty acids were prone to storage and transport induced changes due to platelet activation. Total oxylipin patterns were generally more stable than the concentration of free oxylipins. In serum, coagulation induced higher levels of COX and 12-LOX products showing a high inter-individual variability. Overall, our results indicate that total EDTA-plasma oxylipins are the most stable blood oxylipin marker for clinical samples. Here, storage of blood before further processing is acceptable for a period up to 24 hours at 4 °C. However, levels of platelet derived oxylipins should be interpreted with caution regarding potential ex vivo formation.
Oxylipins are formed endogenously by conversion of PUFA via three major enzymatic pathways as well as non-enzymatic autoxidation leading to a multitude of oxygenated products from different PUFA precursors such as arachidonic acid (C20:4 n6), eicosapentaenoic acid (EPA, C20:5 n3) and docosahexaenoic acid (DHA, C22:6 n3): (I) conversion by cyclooxygenases (COX) results in formation of prostanoids and thromboxanes; (II) lipoxygenases (LOX) convert PUFA to regio- and stereoselective hydroperoxy-PUFA which can be reduced to hydroxy-PUFA or further converted to leukotrienes or multiple hydroxylated PUFA; and (III) action of cytochrome P450 monooxygenases (CYP) leads to formation of hydroxylated PUFA and epoxy-PUFA whereby the latter can be further hydrolyzed to vicinal dihydroxy-PUFA by soluble epoxide hydrolases.1,5,6 Besides regio- and stereoselective conversion by these enzymes, non-enzymatic radical mediated autoxidation gives rise to structurally similar oxygenated products with diverse regio- and stereochemistry such as isoprostanoids (IsoP), hydroxy-PUFA and epoxy-PUFA which also possess biological activity.7–10
Dysregulation of oxylipin formation is implicated among others in cardiovascular, immune or metabolic diseases11,12 and several pharmaceuticals act by modulating the oxylipin profile (e.g. non-steroidal anti-inflammatory drugs, anti-thrombotic drugs, the asthma drug zileuton).13–15 Considering the multitude and diversity of oxylipins as well as the different pathways involved in oxylipin formation the overall physiological effect might result rather from the general profile than from the levels of a single oxylipin.16,17 Therefore, comprehensive and reliable quantification of the oxylipin pattern may be valuable to evaluate the disease state or monitor the action of drugs.
For clinical diagnostics blood specimen, i.e. plasma or serum, are the most widely used biofluids reflecting the systemic condition of the donor. In blood, oxylipins occur in their free form, are associated with proteins, such as albumin,18,19 however the major part (>90%) – particularly hydroxy- and epoxy-PUFA as well as isoprostanoids – is esterified in cellular lipids (i.e. in blood cells) and in lipoproteins.20–22
Endogenous factors, such as sex,23 age,24 physical exercise,25,26 health status,27 intake of drugs13,15,28 or diet17,29 as well as variation in expression or genetic polymorphisms in enzymes30–33 impact the oxylipin profile of an individual. Moreover exogenous, pre-analytical factors relevant during collection, processing and storage of samples until analysis affect the oxylipin pattern,5 thus complicating its reliable interpretation.
In hospitals clinical blood samples are usually collected on a ward or in the emergency unit and are afterwards transported from the phlebotomy site to the clinical chemistry laboratory via transport systems such as pneumatic tube system transport (PTS) where centrifugation and generation of plasma or serum takes place.
The oxylipin pattern and its information about the physiology of the patient strongly depend upon the choice between serum or plasma during blood withdrawal. During serum generation the blood coagulation cascade is triggered which includes activation of platelets resulting in a massive increase of platelet derived oxylipins such as 12-LOX metabolites and thromboxanes.34,35 For plasma different anticoagulants during blood collection, such as EDTA, heparin or citrate are used. The type of anticoagulant affects the formation of oxylipins, particularly hydroxy-PUFA and platelet derived oxylipins (12-LOX and COX derived products).34,36 Therefore, blood sampling, i.e. the choice of the blood specimen, has to be carefully considered in clinical studies with respect to the analytes of interest and standardized within studies.
Following blood withdrawal, temperature and transitory storage time as well as transport to the clinical chemistry laboratory such as pneumatic tube system transport (PTS), are relevant pre-analytical factors which are hard to control in a clinical setting. Few studies investigated the influence of duration and temperature during whole blood storage prior further processing and revealed changes for some free, i.e. non-esterified oxylipins in the generated plasma especially after longer times (>30 min) at elevated temperatures (room temperature, RT).16,34,37 However, no data about the influence of typical sample handling in the clinic on the oxylipin pattern exist. This is especially necessary to evaluate the suitability and reliability of the oxylipin pattern in blood samples from clinical studies, as well as in samples collected from patients in emergency or intensive care units or from biobanks. Moreover, these data are an essential prerequisite to deduce the physiological role of changes in the oxylipin pattern in clinical studies as well as their suitability as biomarkers for disease.
In the present study we therefore investigated the impact of storage of blood samples for different time periods at 4 °C after collection, as well as the transfer from the ward to the clinical chemistry laboratory within a large hospital using PTS prior to the removal of cells (plasma or serum generation). For all conditions, levels of both, free and total oxylipins, i.e. after alkaline hydrolysis, were determined in EDTA-plasma and serum to evaluate if free or total oxylipins are more stable in plasma or serum. In order to deduce pathway specific effects a comprehensive set of oxylipins covering representative analytes derived from ARA, EPA and DHA from COX, LOX, CYP as well as non-enzymatic conversion was analyzed.
From each individual plasma and serum samples were generated by centrifugation (4 °C, 10 min, 1844g, Thermo Scientific, Heraeus, Multifuge 3SR+) at 4 time points as described below and illustrated in Fig. 1: immediate, after 4 hours at 4 °C, after 24 hours at 4 °C and after pneumatic tube system transport (PTS) from the hospital ward to the clinical chemistry laboratory. For basal levels (t0) one plasma and serum monovette from each individual was centrifuged about 15 min after blood sampling. The supernatant, i.e. plasma or serum, was transferred to a cryotube and stored at −80 °C until analysis. To investigate the influence of storage on oxylipins, blood samples were maintained for about 30 min at room temperature reflecting the time blood sampling takes on a hospital ward. Afterwards monovettes from each individual were stored at 4 °C in the fridge for 4 hours or 24 hours until further processing to generate plasma or serum as described above. After 30 min of storage at room temperature an additional monovette was transferred by the PTS to the clinical chemistry laboratory where plasma and serum were generated in an analogous manner (ESI Fig. S1†). The pneumatic tube transport system of the Hannover Medical School comprises about 50 km pipe distance and works in suction operation. PTS transport of the samples ranged between 2 to 17 min. Overall samples sent via the PTS stayed approx. 1.5 to 3 h at room temperature until storage at −80 °C.
All procedures were conducted according to the guidelines laid down in the Declaration of Helsinki and approved by the ethic committee of the Hannover Medical School (MHH ethical approval 6895) and all volunteers gave their written informed consent.
To characterize effects on the sample state levels of lactate dehydrogenase (LDH) were determined in plasma samples from an additional sample set generated in an analogous manner using an Olympus analyzer (AU400) in an automated fashion according to the manufacturer's instruction (ESI Fig. S2†).
Analysis of free oxylipins was carried out as described.39 In brief, to 500 μL plasma or serum 10 μL antioxidant solution (0.2 mg mL−1 BHT and EDTA, 100 μM cyclooxygenase inhibitor indomethacin, 100 μM of the soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) in MeOH/water (50/50, v/v)), 10 μL of 250 mM protease inhibitor PMSF in isopropanol, 10 μL of 100 μM LOX inhibitor 2-TEDC in MeOH and 10 μL of an internal standard (IS) solution (containing 100 nM of 2H4-6-keto-PGF1α, 2H4-15-F2t-IsoP, 2H11-5(R,S)-5-F2t-IsoP, 2H4-PGE2, 2H4-PGD2, 2H4-TxB2, 2H4-LTB4, 2H4-9,10-DiHOME, 2H11-14,15-DiHETrE, 2H4-9-HODE, 2H8-5-HETE, 2H8-12-HETE, 2H6-20-HETE, 2H4-9(10)-EpOME, 2H11-14(15)-EpETrE) were added. Samples were diluted with 1 mL 1 M sodium acetate (pH 6, water/MeOH 95/5 v/v) and loaded on the preconditioned SPE cartridge.
Total, i.e. free and esterified oxylipins were extracted from 100 μL plasma or serum as described with slight modifications.40 After addition of 10 μL antioxidant solution and 10 μL IS solution, 400 μL isopropanol was added and samples were stored for 30 min at −80 °C for protein precipitation. After centrifugation the supernatant was hydrolyzed (300 μL 1.5 M KOH (75/25, MeOH/water, v/v)), immediately neutralized with 55 μL 50% acetic acid, diluted with 2 mL sodium phosphate buffer (pH 5.5) and extracted by SPE.
The extracts were analyzed in scheduled selected reaction monitoring mode following negative electrospray ionization by LC-MS/MS (QTRAP 6500, Sciex, Darmstadt, Germany).38,41
In parallel to the samples aliquots of pooled human quality control plasma (QC) were extracted using the same sample preparation procedure for free (n = 6) and total oxylipins (n = 7), respectively.
In order to evaluate the influence of storage or transport of blood on oxylipin levels in plasma and serum % differences vs. t0 (immediate sample processing) were calculated for each time point (tx) for each individual using the following formula: 100 × (conctx − conct0)/conct0. The relevance of change in the analyte concentration was evaluated by comparison with the acceptable change limit (ACL) calculated using ACL = 2.77 × RSDQC according to DIN ISO 5725-6.42 The factor 2.77 is based on 1.96 × , where 1.96 is used to cover the 95% confidence interval for bi-directional changes and is used as the difference of two values is compared (at tx and t0). The relative standard deviation (RSD) was based on the RSD of quality control plasma samples (n = 6–7) analyzed in parallel with the samples (Table 1).
Analyte | Free oxylipins | Total, i.e. free and esterified oxylipins | ||||||
---|---|---|---|---|---|---|---|---|
LLOQ [nM] | Mean ± SD [nM] | RSD [%] | ACL | LLOQ [nM] | Mean ± SD [nM] | RSD [%] | ACL | |
a PGE2 and TxB2 are degraded during alkaline hydrolysis. | ||||||||
PGE2 | 0.01 | 0.03 ± 0.004 | 17 | 48 | ||||
TxB2 | 0.05 | 0.52 ± 0.06 | 12 | 32 | ||||
12-HHT | 0.05 | 0.53 ± 0.07 | 14 | 39 | 0.25 | 1.25 ± 0.13 | 9 | 26 |
12-HETE | 0.05 | 5.63 ± 0.89 | 16 | 44 | 0.25 | 8.48 ± 0.90 | 11 | 30 |
12-HEPE | 0.06 | 1.51 ± 0.28 | 18 | 51 | 0.31 | 1.14 ± 0.10 | 9 | 25 |
14-HDHA | 0.10 | 3.63 ± 0.55 | 15 | 42 | 0.50 | 2.98 ± 0.53 | 18 | 49 |
15-HETE | 0.13 | 1.14 ± 0.10 | 8 | 23 | 0.63 | 10.42 ± 1.50 | 14 | 40 |
15-HEPE | 0.06 | 0.20 ± 0.03 | 14 | 39 | 0.31 | 1.05 ± 0.15 | 14 | 39 |
17-HDHA | 0.20 | 0.89 ± 0.12 | 13 | 36 | 1.00 | 4.54 ± 0.75 | 17 | 46 |
5-HETE | 0.05 | 0.86 ± 0.09 | 11 | 29 | 0.25 | 17.77 ± 1.55 | 9 | 24 |
5-HEPE | 0.05 | 0.26 ± 0.03 | 13 | 36 | 0.25 | 2.69 ± 0.16 | 6 | 16 |
4-HDHA | 0.03 | 0.35 ± 0.03 | 7 | 21 | 0.13 | 3.62 ± 0.45 | 12 | 34 |
7-HDHA | 0.05 | 0.11 ± 0.02 | 21 | 58 | 0.50 | 2.47 ± 0.39 | 16 | 43 |
14(15)-EpETrE | 0.03 | 0.10 ± 0.01 | 9 | 24 | 0.25 | 54.46 ± 11.78 | 22 | 60 |
17(18)-EpETE | 0.10 | <LLOQ | 0.50 | 5.11 ± 0.88 | 17 | 48 | ||
19(20)-EpDPE | 0.05 | 0.31 ± 0.03 | 9 | 26 | 0.25 | 11.60 ± 2.06 | 18 | 49 |
14,15-DiHETrE | 0.01 | 0.64 ± 0.04 | 6 | 16 | 0.05 | 1.53 ± 0.16 | 11 | 29 |
17,18-DiHETE | 0.03 | 0.55 ± 0.06 | 11 | 31 | 0.13 | 0.80 ± 0.07 | 9 | 25 |
19,20-DiHDPE | 0.05 | 3.09 ± 0.17 | 5 | 15 | 0.25 | 3.77 ± 0.16 | 4 | 12 |
5(R,S)-5-F2t-IsoP | 0.05 | 0.11 ± 0.01 | 10 | 28 | 0.25 | 0.47 ± 0.05 | 11 | 31 |
11-HETE | 0.05 | 0.31 ± 0.02 | 7 | 20 | 0.25 | 6.42 ± 0.74 | 11 | 32 |
9-HETE | 0.25 | <LLOQ | 1.25 | 6.37 ± 0.77 | 12 | 34 | ||
18-HEPE | 0.10 | 0.42 ± 0.02 | 4 | 10 | 0.50 | 1.30 ± 0.15 | 12 | 33 |
Median concentrations of representative oxylipins with the interquartile range (25% percentil, 75% percentil) are given in the ESI (Tables S1 and S2†).
Data evaluation was carried out using GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla California USA, http://www.graphpad.com).
Fig. 2 ARA derived free oxylipins in (A) plasma and (B) serum. Shown are individual concentrations (n = 6) of selected oxylipins from major formation pathways at t0 (immediate processing) and % difference vs. t0 after storage for 4 hours and 24 hours at 4 °C and after pneumatic tube system transport before centrifugation to generate plasma or serum. The different symbols represent samples from different individual human subjects. The grey lines indicate acceptable change limits calculated based on relative SD of quality control plasma (summarized in Table 1). |
Fig. 3 ARA derived total oxylipins in (A) plasma and (B) serum. Shown are individual concentrations (n = 6) of selected oxylipins from major formation pathways at t0 (immediate processing) and % difference vs. t0 after storage for 4 hours and 24 hours at 4 °C and after pneumatic tube system transport before centrifugation to generate plasma or serum. The different symbols represent samples from different individual human subjects. The grey lines indicate acceptable change limits calculated based on relative SD of quality control plasma (summarized in Table 1). |
Regarding concentrations after immediate processing following alkaline hydrolysis (Fig. 3, ESI Fig. S5, S6†), levels of total oxylipins showed the same trend as their free counterparts when comparing plasma and serum: levels of epoxy- and dihydroxy-PUFA, as well as 5(R,S)-5-F2t-IsoP were comparable in plasma and serum. Serum levels of total 12-HHT were elevated to a similar extent (51-441-fold) compared to free 12-HHT. For hydroxy-PUFA differences between plasma and serum were moderate: levels of 12-LOX derived 12-HETE, 12-HEPE, 14-HDHA were clearly elevated (5-50; 2-14; 2-16-fold, respectively) while 15-LOX derived 15-HETE, 15-HEPE, 17-HDHA (1-3; 1-2; 1-3-fold, respectively) were same or only slightly higher in serum and levels of 5-LOX derived 5-HETE, 5-HEPE and 4-HDHA and 7-HDHA were similar in plasma and serum after hydrolysis.
Comparing levels of free and total oxylipins revealed similar concentrations for dihydroxy-PUFA while levels of hydroxy-PUFA, epoxy-PUFA and 5(R,S)-5-F2t-IsoP were massively higher after hydrolysis.
In serum overall storage/transport-induced changes of free oxylipins were more pronounced. PGE2, TxB2 and 12-HHT showed a similar pattern in all stored and transported samples with high inter-individual variance being within the ACL for samples from 2–3 individuals, but massively higher for 2–4 individuals. For 12-LOX and 15-LOX metabolites changes were higher, though similar in 4 h and 24 h stored samples, but massively higher in PTS samples with high inter-individual variance. 5-LOX metabolites were within the ACL for most samples stored for 4 h, but exceeded the ACL in 24 h stored and PTS samples. While dihydroxy-PUFA were stable in all conditions, changes of epoxy-PUFA and 5(R,S)-5-F2t-IsoP were within the ACL in ≥50% of samples stored 4 h and after PTS, but were slightly higher than the ACL in 24 h stored blood samples.
Respective hydroxy-PUFA derived from EPA and DHA showed the same pattern as their ARA derived counterparts supporting the contribution of these enzymatic pathways to their formation during serum generation (ESI Fig. S3–S6†). However, these metabolites were elevated to a lesser extent reflecting the low levels of EPA and DHA in blood competing with ARA for their enzymatic conversion as human 12/15-LOX and 12-LOX enzymes have been shown to prefer DHA and EPA over ARA.53 The observation that levels of non-enzymatically formed 5(R,S)-5-F2t-IsoP were similar in plasma and serum suggests that autoxidative processes do not play a major role in freshly prepared serum. Similar only marginal alterations were seen for 9-HETE (ESI, Fig. S7†), which is discussed as autoxidation product.
The major portion of hydroxy- and epoxy-PUFA, as well as 5(R,S)-5-F2t-IsoP was found esterified in plasma and serum, because massive higher concentrations were observed after alkaline hydrolysis, consistent with earlier reports.20–22 Dihydroxy-PUFA were only present non-esterified with same apparent concentrations for free and total oxylipins. Since under alkaline conditions thromboxanes as well as β-hydroxy-keto-prostanoids, e.g. PGE2 and PGD2, are degraded,54 we used formation of 12-HHT as a surrogate for COX activity.47 The levels of 12-HHT were similar with and without hydrolysis yet massively higher in serum. Thus, based on 12-HHT also analysis of total oxylipins reflects increased COX activity due to platelet activation during serum generation.
Serum generation was also accompanied by an increase in total hydroxy-PUFA levels which was however less pronounced compared to the respective free hydroxy-PUFA. Even though it has been shown that eosinophilic 12/15-LOX directly form esterified 15-HETE in phospholipids49 and platelet 12-LOX derived hydroxy-PUFA are readily esterified contributing to the esterified pool,55 only a minor increase of 12- and 15-HETE (and respective EPA and DHA products) was observed in total oxylipins in comparison to their free levels. Esterified oxylipins in the cell free fraction of blood (i.e. serum and plasma) are thus hardly influenced by the activation of the coagulation cascade.
Free oxylipins in plasma from whole blood stored at 4 °C for 4 h remained almost stable (≥67% of the samples were within the ACL) with exception of samples from a few individuals showing higher deviations for 12-LOX and COX-1 derived products. After 24 h at 4 °C besides considerable increase in platelet-derived oxylipins also 15-LOX products were clearly increased. Though these results are consistent with a previous study reporting that free oxylipins in EDTA-whole blood are stable up to 2 h at 4 °C (ref. 34) or on wet ice,37 another study showed a clear decrease of some prostanoids (PGE2, PGF2α), hydroxy-PUFA (11- and 15-HETE) and epoxy-PUFA (14(15)- and 11(12)-EpETrE) in EDTA-whole blood already after 1 h on ice.16
In contrast to plasma, free oxylipins in serum of stored clotted whole blood were largely unstable. Especially platelet-derived (COX and 12-LOX) as well as 15-LOX products showed a strong increase with high inter-individual variance similar for 4 h and 24 h at 4 °C. 5-LOX derived products were stronger elevated after longer storage (24 h vs. 4 h). Consistently, La Frano et al. reported that a “freezing delay” of clotted whole blood dramatically elevated hydroxy-PUFA attributed to ongoing enzymatic processes in cooled whole blood.57
Apparent total oxylipin patterns were considerably more stable towards transitory storage of whole blood. However, the observed changes revealed a similar pattern as for free oxylipins: total plasma oxylipins were stable for 4 h at 4 °C and showed only elevation of platelet derived oxylipins after 24 h. In serum similarly to free oxylipins platelet derived oxylipins were massively increased after 4 h and 24 h, however increase in 15-LOX products was less pronounced and absent for 5-LOX products. These results suggest that transitory storage at 4 °C without removal of the blood cells or/and the blood clot led to enzymatic activity resulting from ex vivo activation of platelets. This leads to considerable artificial ex vivo formation of COX and 12-LOX products. While in plasma this can be ascribed to continuing enzymatic conversion related to the duration of storage, the high inter-individual variances in platelet-derived metabolites in serum which are similar at 4 h and 24 h indicate that these changes result rather from individual differences in the platelets and enzymatic activity of the blood coagulation cascade. One should note, that under all conditions the levels of free as well as total oxylipins of CYP derived dihydroxy-PUFA were stable and free levels of epoxy-PUFA showed only slight increases after 24 h. Moreover, the almost unchanged 5(R,S)-5-F2t-IsoP and 9-HETE levels indicate that only minor autoxidation takes place during the delay in sample processing.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9an01880h |
This journal is © The Royal Society of Chemistry 2020 |