Association between plasma concentrations of linoleic acid-derived oxylipins and the perceived pain scores in an exploratory study in women with chronic neck pain

Female participants of age range 20–65 years were consecutively recruited during the period June 2011–March 2012. Twenty-seven healthy controls and 36 subjects with NP (of whom data from 35 are reported here) were recruited from a randomized controlled trial (Current Controlled Trials registration ISRCTN49348025; the trial comprised 120 subjects with NP and 40 healthy controls) [18]. Seventeen subjects with CWP (of whom data from 15 are reported here) were recruited via contact with the local patient organisation and/or advertising in local newspapers. Ethical approval for this project was granted by the ethical committee of Uppsala University (registration number 2011/081). All subjects participated voluntarily after informed written consent. For the NP group, inclusion criteria were more than 6 weeks of non-specific neck-shoulder pain (indicated as dominant pain area in a pain drawing), more than “no disability” but less than “complete disability” according to the Neck Disability Index (NDI), and self-reported impaired productivity to work the preceding month (for details see [18]). The CWP group had obtained their diagnosis using the American College of Rheumatology criteria for fibromyalgia [2] albeit without the requirement of at least 11/18 trigger points. Subjects with rheumatoid arthritis, systemic lupus erythematosus, Bechterew’s disease, multiple sclerosis, epilepsy or Parkinson’s disease, type I-diabetes, cardiovascular disease or endocrine diseases were excluded, as were subjects who did not eat either fish or meat on a regular basis.

All participants were asked not to use any pain medications except for paracetamol preparations three days before the blood sampling and to avoid intake of caffeine, nicotine and wholegrain during the 12 h prior to blood sampling. The subjects were allowed water alone during the last two hours of this period and were also asked not to perform any shoulder or neck-straining exercises for the last two days prior to blood sampling, except for ordinary daily work and/or leisure activities. All subjects rated the current pain (“NRS_day”) and the average pain during the last week (“NRS_week”) on a numerical rating scale [19] (0–10, where 0 is no pain and 10 is the worst pain imaginable) at the time of blood sample collection. NRS is adjudged to have a higher validity than several other commonly used pain scores [20].

Blood was sampled either during the morning (between 07:30 and 11:30) or afternoon (between 12:00 and 15:00). After arrival at the laboratory, subjects were allowed to rest for 15 min before venous blood was drawn into Li-Heparin prepared Vacutainer tubes from the bend of the arm while the subjects were in a sitting position. Blood for plasma analysis were treated according to a standardised protocol, whereby the Vacutainer tube was turned 10 times during a 30 s period at room temperature before being immediately centrifuged at 4 °C for 5 min at 2500 rpm. Supernatants were aliquoted (500 μL) into pre- labelled homopolymer tubes and frozen at −80 °C until analysis. Thus, the lipid analyses conducted here were undertaken on previously unthawed samples.

The following native and deuterated standards were purchased from Cayman Chemical (Ann Arbor, MI, USA): 2-AG, AEA, PEA, SEA, OEA, LEA, 9-HODE, 13-HODE, 9(10)-DiHOME (9(10)-dihydroxy-12Z-octadecenoic acid), 12(13)-DiHOME (12(13)-dihydroxy-9Z-octadecenoic acid), 13-oxo-ODE (13-oxo-9Z,11E-octadecadienoic acid), 5-HETE (5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid), 8,9-DiHETrE (8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid), 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA), 2-AG-d₈, AEA-d₈, PEA-d₄, SEA-d₃, OEA-d₄, TXB₂-d₄, 12(13)-DiHOME-d₄, 9(S)-HODE-d₄, 20-HETE-d₄, 5(S)-HETE-d₈ and 12(13)-EpOME-d₄. 9,10,13-TriHOME (9,10,13-trihydroxy-11-octadecenoic acid) and 9,12,13-TriHOME (9,12,13-trihydroxy-10E-octadecenoic acid) were obtained from Larodan (Sweden, Malmö). Butylhydroxytoluene (BHT) was obtained from the Cayman Chemical Co. All solvents and chemicals used were of HPLC grade or higher. A Milli-Q Gradient system (Millipore, Milford, MA, USA) was used to purify water.

The deuterated compounds were used as internal standards and added prior to extraction to mimic the isolation of the endogenous compounds from the plasma samples. For each native compound, a suitable internal standard was selected based on structural similarities for quantification purposes. Recovery rates of each internal standard were calculated by adding a known amount of the recovery standard CUDA in the last step before injection. The recovery rates (%, means ± SD) of the internal standards in the present study were: 2-AG-d₈ (92.3 ± 13.3, N = 74), AEA-d₈ (70.7 ± 11.3, N = 74), PEA-d₄ (70.0 ± 21.8, N = 74), SEA-d₃ (71.2 ± 6.2, N = 74), OEA-d₄ (62.1 ± 13.7, N = 74), TXB₂-d₄ (71.7 ± 16.6, N = 74), 12(13)-DiHOME-d₄ (77.2 ± 10.2, N = 74), 9(S)-HODE-d₄ (67.5 ± 16.4, N = 74), 20-HETE-d₄ (101.3 ± 12.2, N = 74), 5(S)-HETE-d₈ (81.1 ± 14.7, N = 74) and 12(13)-EpOME-d₄ (103.7 ± 13.7, N = 74).

A previously reported and validated SPE protocol was used for isolation of the lipids [21]. In brief, plasma samples (400 μL) were thawed on ice and spiked with 10 μL internal standards solutions and 10 μL antioxidant (0.2 mg/mL BHT/EDTA in methanol:water (1:1)) solution before being extracted using Waters Oasis HLB cartridges (60 mg of sorbent, 30 μm particle size). Eluates were reconstituted in 100 μL of MeOH spiked with 10 μL CUDA (0.05 μg/mL) and transferred to vials before analysis using an ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method.

Lipid analyses were undertaken on an Agilent UPLC (Infinity 1290) coupled with an electrospray ionization (ESI) source to an Agilent 6490 Triple Quadrupole system equipped with the iFunnel Technology (Agilent Technologies, Santa Clara, CA, USA). Analyte separation was achieved using a Waters BEH C₁₈ column (2.1 mm x 150 mm, 130 Å, 1.7 μm particle size) according to a previously validated analytical protocol [21]. Injection volumes of 10 μL were employed, and the mobile phases consisted of (A) 0.1 % acetic acid in MilliQ water and (B) acetonitrile:isopropanol (90:10). For the endocannabinoids and related NAEs, the system operated in positive mode (ESI+) with the following gradient: 0.0–2.0 min 30–45 % B, 2.0–2.5 min 45–79 % B, 2.5–11.5 min 79 % B, 11.5–12 min 79–90 % B, 12–14 min 90 % B, 14–14.5 min 90–79 % B, 14.5–15.5 min 79 % B, 15.6–19 min 30 % B. For the linoleic acid- and arachidonic acid-derived oxylipins, the negative ionisation mode was used (ESI-), with the following gradient: 0.0–3.5 min 10–35 % B, 3.5–5.5 min 40 % B, 5.5–7.0 min 42% B, 7.0–9.0 min 50 % B, 9.0–15.0 min 65 % B, 15.0–17.0 min 75 % B, 17.0–18.5 min 85 % B, 18.5–19.5 min 95 % B, 19.5–21 min 95-10 % B, 21.0–25.0 min 10 % B. The dynamic multiple reaction monitoring (dMRM) option was performed for the lipids with optimized transitions and collision energies [21]. All peaks were integrated manually using the MassHunter Workstation software, and the stable isotope dilution method was used to quantify the peaks using calibration curves of peak areas of native compounds divided by the corresponding IS peak areas. Accuracy and precision for the method is described elsewhere [21], but in general at the levels detected in the current study the precision ranged from 4.6–9.8 % (endocannabinoids and related NAEs) and 2.1–11.2 % (oxylipins) and accuracies ranged from 98–119 % (endocannabinoids and related NAEs) to 100–111 % (oxylipins).

Groups (both patient characteristics and outcome variables) were tested for normal distribution using the D’Agostino and Pearson normality test and differences between groups were tested using Kruskal-Wallis test, Mann Whintney U-test or Chi squared test, all using GraphPad Prism v. 6 for the Macintosh (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis of the effects of sampling time and pain group upon the observed lipid concentrations were undertaken using two-way robust Wilcoxon analyses (available with the function raov in the Rfit package version 0.22 of the R computer programme [22, 23]). Correlations between AEA, 2-AG, related NAEs and linoleic acid-derived oxylipins and self rated pain scores were conducted with Spearman’s correlation coefficients (GraphPad Software). For comparison between independent correlation coefficients, the standard Fisher’s transformation was used with an on-line calculator (http://​vassarstats.​net/​index.​html, URL checked on 19 February 2016). The false discovery rate (FDR) by Benjamin and Hochberg [24] was used to control for the expected proportion (5 %) of incorrectly rejected null hypotheses (i.e. false discoveries) due to multiple comparisons.

Subject characteristics are presented in Table 1. There were no significant differences in the distribution of the time of day of sampling (morning vs. afternoon), the body weight, BMI and number of nicotine users among the subjects. A significant group difference for age was seen, due to a difference between the CWP and NP groups (CWP > NP, P < 0.05, Dunn’s multiple comparisons post-hoc test). The scores for the average pain during the last week (“NRS_week”) were greater for the CWP group than the NP group, whereas the current pain scores (“NRS_day”) were not significantly different between the two groups.

Plasma levels of fifteen lipids (AEA and 2-AG, four related NAEs, and seven linoleic acid-derived oxylipins including 9- and 13-HODE, and two arachidonic acid-derived oxylipins, 5-HETE and 8,9- DiHETrE) were quantified in serum samples from 27 controls, 15 women with CWP and 35 women with NP. The plasma concentrations of 9- and 13-HODE were highly correlated for all three groups (P for Spearman rank correlation coefficients <0.001, Fig. 1a) and a similar pattern was seen for the correlation between the two NAEs PEA and SEA (Fig. 1b). In contrast, the plasma concentrations of the two endocannabinoids were not significantly correlated (Fig. 1c). This is not merely a reflection of potential quantification issues for the low abundant AEA, since 9,10,13-TriHOME, which has a similar abundance to AEA, is very highly correlated to 9,12-13-TriHOME, which has a similar abundance to 2-AG (Fig. 1d).

The median values and interquartile ranges are shown for all fifteen lipids in Table 2, stratified both on the basis of the condition (control, NP, CWP) and of the time of sampling (morning and afternoon). The P values for two-way robust Wilcoxon analyses [22, 23] are also given in the Table. In no case did the ANOVA P values for the condition as main effect reach significance. Significant interactions were seen for the condition × time of sampling for LEA (P = 0.024) and 13-oxo-ODE (P = 0.047), but these levels were below the threshold for significance upon implementation of the false discovery rate approach of Benjamini and Hochberg [24]. A similar result was seen when the cases were stratified into two BMI groups: BMI in the normal range (18.5–24.9 kg/m²; N = 57 in total) and overweight/obese (BMI ≥25 kg/m²; N = 22 in total; only 6 individuals were obese [BMI ≥30 kg/m²] and this was adjudged to be a too small sample size). The main effect of condition again did not reach significance for any of the lipids (P > 0.27) and the only significant finding was an interaction condition × BMI for AEA (P = 0.027, otherwise P > 0.14 for the interactions, two-way robust Wilcoxon analyses).

The patients were asked to rate their pain on the day of blood sampling (“NRS_day”) and for the week prior to the blood sampling (“NRS_week”). The NRS_day and NRS_week scores (median, with range in brackets) for the NP group were 2 (1–6) and 4 (1–8), respectively. The corresponding scores for the CWP group were 3 (1–8) and 6 (2–7), respectively (Table 1).

Bivariate (zero-order) Spearman rank correlation coefficients were calculated for the correlations between the lipid concentrations and the NRS_day scores for the CWP and NP patients. An example of the raw data is shown for 9-HODE in Fig. 2a, and the correlation coefficients are given in Table 3 and presented visually in Fig. 2b and c. The data points are colour coded on the basis of their group (orange for the linoleic acid-derived oxylipins, red for the endocannabinoids, blue for the related NAE’s and yellow for the two arachidonic acid-derived oxylipins). Given that there are multiple comparisons, we have presented the significance levels “as is”, showing two vertical lines: one at P = 0.05, and one at P = 0.0033, which represents the limit using the false discovery rate approach of Benjamini and Hochberg [24] for the 30 comparisons. For the NP cases, five of the linoleic acid-derived oxylipins (from left to right in Fig. 2b: 13-HODE, 9-HODE, 13-oxo-ODE, 12,13-DiHOME and 9,10-DiHOME) reached significance at the P < 0.05 level, and the P values for the two HODEs were smaller than the 5 % Benjamini and Hochberg [24] cut-off value. For the CWP cases, none of the correlations reached significance (Fig. 2b). However, it is important to consider the significance of the difference rather than the difference in the significance [25]. Using the Fisher r-to-z transformation, the significance of the differences for the correlation coefficients for 9- and 13-HODE, 9,10- and 12,13-DiHOME and 13-oxo-ODE were determined. In no case was the difference significant (P > 0.13). In consequence, we have also presented the data for the combined NP + CWP groups in Table 3.

Table 3 also presents the first-order Spearman rank correlation coefficients for the NP cases taking into account the exact time of sampling (N = 31; in the remaining four cases, the exact time of sampling was not available in the dataset) and the BMI (N = 34). The correlation coefficients were very similar to the bivariate coefficients between the lipids and the NRS_day scores, indicating that the findings described above are retained when controlled for time of sampling or for the BMI of the individuals. Further, first-order Spearman rank correlation coefficients for the NP cases were not changed when controlling for either age (0.52 and 0.56, N = 35) or pain duration (0.52 and 0.53, N = 34; data for 9- and 13-HODE, respectively).

Spearman rank correlation coefficients were also calculated for the NP, CWP and NP + CWP groups for the correlation between all the lipid concentrations and the NRS_week scores and pain duration. In no case was a significant correlation found (see Table 3 for the combined NP + CWP groups).