Longitudinal analysis of serum oxylipin profile as a novel descriptor of the inflammatory response to surgery

Complications following colorectal surgery, such as surgical site infection (2–25% [1, 2]) and anastomotic leak (3–15% [3]), frequently induce the systemic inflammatory response syndrome (SIRS) or sepsis which are the major causes of post-operative morbidity and mortality [3‐6]. To detect such complications, clinicians are generally reliant on the longitudinal assessment of physiological parameters (e.g. respiratory rate, heart rate, temperature) and biomarkers of inflammation, such as C-reactive protein (CRP) and white cell count (WCC). However, pertubations in these parameters are the final common pathway of many distinct pathologies and are therefore relatively late indicators and are extremely non-specific. A more detailed understanding of the inflammatory response and the ability to quantify it with higher resolution may enhance the early detection of acute post-operative complications.

Eicosanoids, and more generally oxylipins, are potent, locally-acting lipid mediators derived from the metabolism of polyunsaturated fatty acid (PUFA) precursors such as linoleic acid (C18:2, LA), dihomo-γ-linolenic acid (C20:3, DGLA), arachidonic acid (C20:4, AA), eicosapentaenoic acid (C20:5, EPA) and docosahexaenoic acid (C22:6, DHA). These PUFAs are principally converted to biologically active downstream mediators by three enzymatic pathways. Cyclooxygenase (COX) produces prostaglandins (PG) and thromboxanes (Tx), while lipoxygenase (LOX) produces leukotrienes (LT). Finally, cytochrome P450 (CYP450), together with LOX, is responsible for generating the less well-characterised “non-classical” eicosanoids including AA-derived hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), LA-derived hydroxyoctadecadienoic acids (HODEs) and DHA-derived hydroxy-docosahexaenoic acids (HDoHEs). Oxylipins form a complex of interlinked cascades with a variety of precursors and metabolites that may or may not have biological activity. The biological effects of oxylipins are diverse, pleiotropic, antagonistic and, in some cases, still unclear. However, their ability to regulate the propagation and resolution of inflammation via effects on smooth muscle, cytokine release, the coagulation cascade and leukocyte chemotaxis is well-described [7‐9]. Despite the acknowledged importance of oxylipins, there remains an incomplete knowledge of their systemic profiles during acute inflammation in humans due to a lack, until recently, of suitable quantitative bioanalytical platforms.

We have previously described a novel, highly sensitive assay for the detection in biofluids of a panel of oxylipins and PUFAs spanning the major PUFA precursors and the three principal biosynthetic enzyme pathways (CYP450, COX and LOX) [10]. In the present study, we applied this assay to longitudinally quantify the serum levels of key oxylipins in patients undergoing colorectal surgery. The aims of our investigation were, (1) to provide proof-of-principle that multiple oxylipin concentrations can be quantified simultaneously in human biofluids in the clinical environment and; (2) to better characterise the evolution of the oxylipin profile in response to surgical trauma. We hypothesise that the serum oxylipin profile may provide novel insights into the inflammatory response to surgery and perhaps ultimately show potential as an early biomarker of inflammatory dysregulation.

Oxylipins, such as prostaglandins and leukotrienes, have been individually studied for over fifty years but technological limitations restricting the number of oxylipins that could be simultaneously assayed have necessarily focussed work on allocating distinct regulatory functions to individual mediators [23, 24]. Recent developments in analytical platforms allowed us to develop an assay for the simultaneous analysis of numerous lipid mediators in biofluids [10]. In the present investigation we applied this oxylipin assay to adult patients undergoing elective colorectal surgery to demonstrate that oxylipin profiling is feasible in a clinical context and that it has the potential to provide novel insights into the systemic inflammatory response.

PCA of oxylipin profiles demonstrated that surgery induces profound time-dependent shifts. We observed significant variability amongst pre-operative oxylipin profiles that may reflect the heterogeneity in the demographics (e.g. age, gender, physical condition), underlying aetiologies, attendant comorbidities and pre-operative preparations of the patient population. However, it should be noted that previous studies have shown that even when individuals were well-matched with regard to gender, health and physical condition, their basal oxylipin levels still differed significantly [25]. Despite this underlying heterogeneity, surgery induced a more uniform oxylipin profile at the early post-operative phase, perhaps reflecting a coherent response to surgical trauma. By the late post-operative phase, significant variability in oxylipin profile was again noted, perhaps reflecting unique recovery trajectories between patients. Visual differences between the PCA scores plots were confirmed by supervised multivariate analysis which was able to discriminate between each time-point on the basis of the oxylipin profile.

Univariate analysis of individual oxylipin concentrations revealed that, compared to pre-operative levels, there was a generalised dampening of several anti-inflammatory (15-HETE, 8-HETE and degradation products of EET) and anti-thrombotic (9-HODE and 13-HODE) mediators, combined with an increase in the pro-inflammatory 11-HETE. In the late post-operative phase, the levels of anti-inflammatory mediators were typically elevated compared to the early post-operative time-point suggesting a return towards baseline and indeed, no significant differences were observed for these mediators between the pre-operative and late post-operative samples. In contrast, the pro-inflammatory 11-HETE and anti-thrombotic 9-HODE and 13-HODE did not appear to return to baseline, although the differences between pre-operative and late post-operative measurements were non-significant. Interestingly, levels of 12-oxo-LTB4 (the stable degradation product of pro-inflammatory leukotriene B4) showed a trend towards reduction at the early post-operative point and were significantly reduced by the late post-operative phase. We attempted to correlate oxylipin concentrations with with CRP concentration and WCC as known references of inflammation. However, no consistent correlations we found, perhaps reflecting temporal differences in the dynamics of oxylipin, CRP and WCC levels [26, 27]. When interpreting our results it is important to bear in mind that, due to the lipid mediator’s bioactivity as inflammatory signalling compounds, sample collection and handling could result in in vivo and ex vivo alterations [28]. For example, the stimulation of platelets and leukocytes following venipuncture and serum formation could potentially cause activation of the oxylipin biosynthetic cascade [29]. Equally, anticoagulants used for the collection of plasma (such as heparin) can stimulate oxylipin production via activation of phospholipase A₂ [30, 31]. However, the practicalities of collecting samples within a clinical environment limit the extent to which such potential effects can be prevented.

Although it is interesting to postulate on how changes in the oxylipin profile fit with the current understanding of the inflammatory response to surgery, we urge caution to avoid overstating and oversimplifying the results of our single study. That said, the early reduction in the concentration of mediators with anti-inflammatory and anti-thrombotic effects could be viewed as “releasing the brake” on inflammation, consistent with the pro-inflammatory, pro-thrombotic phenotype that is thought to dominate the early post- operative phase [32, 33]. Early elevation of pro-inflammatory 11-HETE would also support this hypothesis. The subsequent return of the anti-inflammatory mediators towards basal levels may reflect a reinstatement of normal inflammatory homeostasis. Evidence of early and persistent reduction in the pro-inflammatory leukotriene B4 may seem contrary to this hypothesis but other investigations have demonstrated that leukotriene B4 release by neutrophils peaks extremely rapidly following surgery (e.g. within 6 h) and that levels remain low thereafter [34, 35]. The early post-operative sample in the present study was taken at 24 h, and may well have missed an initial peak in pro-inflammatory oxylipin concentration, leaving only the later nadir. Strassburg et al. recently investigated the effects of on-pump cardiac surgery on serum oxylipin levels in five patients and demonstrated increases in other oxylipins with pro-inflammatory effects at 24 h post-operatively, including 12-HETE and 5-HETE which were quantified in our assay [36]. However, the large physiological insult of on-pump cardiac surgery, small sample size and discrepancies in their sampling protocol (venous blood pre-operatively and arterial blood post-operatively) makes direct comparison between our investigations difficult.

The immunophysiological impact of laparoscopic versus open colorectal surgery is not well understood with conflicting evidence [15‐21]. Our investigation was not powered to detect differences in oxylipin profile by outcome (e.g. the development of complications) or by surgical approach as patients were not randomised. With these caveats, we did find that length of stay was shorter in the laparoscopic group and demonstrated interesting differences in the post-operative oxylipin profiles between the two approaches. Patients undergoing open surgery had lower levels of some anti-inflammatory oxylipins (8,9-DHET and 17-HDoHE) in addition to reduced concentrations of anti-thrombotic mediators (9-HODE and 13-HODE) with increased concentration of their pro-thrombotic counterpart (TxB2). Higher levels of anti-inflammatory 17-HDoHE were found amongst the laparoscopic cohort. Whilst almost certainly an oversimplification, these results could be suggestive of a more pro-inflammatory, pro-thrombotic phenotype amongst patients undergoing open surgery.

This is the first investigation to quantify systemic oxylipin profile longitudinally in a cohort of patients undergoing major surgery. We have demonstrated that surgery induces a common shift in lipid mediators, marked at 24 h post-operatively by a combination of elevation in pro-inflammatory oxylipin concentration with suppression of specific oxylipins thought to have anti-inflammatory and/or anti-thrombotic effects. Furthermore, differences between patients undergoing open versus laparoscopic surgery suggests that the oxylipin profile may be sensitive to the degree of surgical trauma. Based on this evidence, the stage is set to more completely define the “normal” oxylipin dynamics in response to surgery using a more comprehensive sampling strategy and a larger patient cohort. Deviations from this “normal” profile may then offer a useful prognostic tool for the early identification of patients at higher risk of post-operative complications.