Systemic Inflammatory Response After Open, Laparoscopic and Robotic Surgery in Endometrial Cancer Patients

Materials and Methods

Patients. This prospective study was undertaken at the University Hospital Olomouc between October 2012 and June 2015. The study was approved by the hospital's ethical committee and informed, written consent was obtained from all patients. The study group included 109 consecutive patients with histologically confirmed endometrial cancer, aged 65±10 (range=33-88) years who underwent hysterectomy with bilateral salpingo-ophorectomy, pelvic and para-aortic lymphadenectomy. These procedures were performed by LT, RS or LS approaches. Twenty four patients, aged 55±12 (range=36-80) years, who were scheduled for elective total hysterectomy for non-malignant disease, constituted a control group. The patients were randomly allocated to receive either an abdominal hysterectomy or laparoscopic assisted hysterectomy for their benign disease. Venous blood samples were collected from each patient at the following times: a baseline sample the day before surgery (day -1), before skin incision (day 0), 24 h after operation (day 1), second, third, fourth and fifth day after surgery (day 2-5). The samples were separated and the sera were all stored at −80°C until analysis for CRP, interleukin-6 (IL-6), citrulline, vitamin D, alpha-tocopherol, retinol, kynurenine and tryptophan. Urinary samples, with the same timing, were collected and stored at −20°C until analysis for urinary neopterin determination. The changes during the postoperative period were assessed by calculation of the area under curve (AUC). Other parameters evaluated were: patient age, body mass index (BMI), blood loss, decline of hemoglobin, increase of white blood cell count, increase of platelet count, number of lymph nodes obtained and visual analogue scale (VAS) scores. BMI was calculated using the formula: body weight/height². Preoperative and postoperative hemoglobin, white blood cells and platelets levels were analyzed on the same days (day-1 to day 5) as the venous blood samples were collected. The difference was calculated by subtracting postoperative levels from the preoperative levels. Postoperative pain was evaluated at the time of admission, before surgery and during the postoperative period (day-1 till day-5) using a 10-point VAS score (0, absence of pain; 10, worst possible pain).

Neopterin, kynurenine, tryptophan and creatinine in human serum. The blood samples were centrifuged (1,600 × g, 10 min, +4°C) and serum was separated. Then, 200 μl of serum was diluted with 100 μl phosphate buffer (15 mmol/l, pH 6.5) and deproteinized by 100 μl cooled ethanol (10 min, −25°C). After centrifugation (14,000 × g, 10 min), the supernatant was filtered using 0.2-μm micro-titration plate filters and vacuum manifold. Filtered solution was applied into the HPLC column. The analyses were performed using a high performance liquid chromatography (HPLC) set Prominence LC 20 Shimadzu (Kyoto, Japan) composed by degasser DGU 20A5, pump LC20-AB, special auto sampler SIL/20 AC for micro-titration plates (rack changer), column oven CTO-20 AC, diode array detector SPD-M20A, fluorescence detector RF10 AXL and communication bus module CBM-20A. Sample preparation technique was developed using micro-titration plates with filters AcroPrep 96 filter Plate 0.2 μm/350 μl Pall Corporation (Ann Arbor, MI, USA), vacuum manifold Phenomenex (Aschaffenburg, Germany), vacuum pump VAC Space-50 Chromservis (Prague, Czech Republic) and centrifuge Minispin Eppendorf (Hamburg, Germany). Analysis was performed at HPLC set Prominence LC 20 (Shimadzu) equipped by special auto sampler for micro-titration plates. As stationary phase, two monolithic columns RP-18e (4.6×50 mm, 3.0×100 mm) were connected together with monolithic security guard (4.6×10 mm). As mobile phase, 15 mmol/l phosphate buffer (KH₂PO₄ +K₂HPO₄·3H₂O) was used at pH 4.51 and flow rate 1 ml/min (0–3.09) and 2.3 ml/min (3.10–8.20). Injection volume was 1 μl. Kynurenine and creatinine were detected using diode array detection (230 and 235 nm), neopterin and tryptophan were detected using fluorescent detection (excitation and emission wavelengths of 353 nm and 458 nm for neopterin, respectively, and excitation and emission wavelengths of 254 nm and 404 nm for tryptophan, respectively). Separation was held at ambient temperature in 8.2 min.

Retinol and alpha-tocopherol in human serum. The method used in this study for the analysis of retinol and alpha-tocopherol in the serum was modified from the method previously published by Urbanek et al. and is briefly described below (6). In the liquid-liquid extraction (LLE) procedure, 500 μl of serum was deproteinized using cold ethanol with 5% of methanol. Then, 2,500 μl n-hexane was added to this mixture and extracted using a vortex apparatus. After centrifugation, an aliquot of the clean extract was separated and evaporated in a concentrator. The residue was dissolved in 400 μl of methanol and analyzed using a Prominence HPLC system (Shimadzu) (7). The separation of retinol and alpha-tocopherol was performed using the Chromolith Performance RP-18e monolithic column (100x4.6 mm; Merck, Darmstadt, Germany). The detection of retinol and alpha-tocopherol was carried out at 325 and 295 nm, respectively, using a diode array detector.

Vitamin D in human serum. A total of 50 μl of 4 % monohydrate zinc sulfate as the precipitation reagent and 400 μl of methanol were added to 200 μl of human serum in an Eppendorf tube. After 20 s of vortexing, the sample was incubated for 10 min at 4°C. The sample was then centrifuged (14,000 × g, 5 min, 21°C) and 300 μl of supernatant was filtered using 0.2-μm well filter plates and a vacuum manifold. The filtered solution was injected into the UHPLC system. Analysis was performed by using an UHPLC Nexera set (Shimadzu) coupled with an LCMS-8030 triple-quadrupole mass spectrometer operating in positive ESI mode. Chromatographic separation was achieved on a Kinetex C18 analytical column (1.7 μm, 3×100 mm) connected to a security guard ultra cartridge C18, both purchased from Phenomenex (Aschaffenburg, Germany). The column oven, CTO-20 AC, was used to set the temperature of the analytical column at 50°C. Acetonitrile and water, both with the addition of FA (c=0.01 mol/l) were used as the mobile phase at a flow rate of 0.5 ml/min. The separation of metabolites 25-OHD₂ and 25-OHD₃ was realized in 6 min and under isocratic conditions, 72:28 v/v of acetonitrile/ water. After analyte separation, the switching valve was activated and the pre-column and the analytical column were washed and equilibrated for 3min into the waste by increasing the flow rate to 0.8 ml/min with 90:10 v/v of acetonitrile/water. The sample injection volume was 20 μl. The total analysis time was 9 min. System operation, data acquisition and data processing were controlled using the LabSolutions 5.41 SP1 software. MS conditions were investigated using automated optimization of software LabSolution as the first step of the analytical method development. Voltages of Q1, Q3, collision cell and five of the most intensive multiple reaction monitoring (MRM) transitions for identification and quantification of 25-OHD₃ and 25-OHD₂ were found. Other optimized conditions of ion source were as following: interface ESI-positive polarity; interface temperature 350°C; desolvation line (DL) temperature 250°C; nebulising gas flow 3ml/min; heat block temperature 400°C and drying gas flow 15 l/min.

Citrulline in human serum. For citruline analysis, 20 μl of serum samples were mixed with 100 μl of methanol solution containing 0.1% FA and deuterated internal standards (amino acids and acylcarnitines, non-derivatised reagent kit; Chromsystems, Munich, Germany). The final solutions were vortexed and centrifuged (24,400 × g, 10 min at 24°C, 400 × g at 4°C) and 80 μl of supernatant was transferred to a 96-well plate and used for direct injection mass spectrometry analysis. The remaining supernatant was pooled and used for quality control purposes (8). The samples were measured on 4000 (AB Sciex, Framingham, MA, USA) by flow injection mass spectrometry direct analysis infusion with settings as follows: polarity was set to positive mode with IonSpray voltage of 5500 V, capillary temperature of 450°C, Curtain curtain Gas gas of - 20 arbpsi, Ion ion Source source Gas gas (GS1/GS2) of - 40 arbpsi. Methanol containing 0.1 % FA was chosen as a mobile phase. Flow rate was set at 0.03 ml/min (0.1-0.4 min) and 0.30 ml/min (0.0-0.1 and 0.4-0.5 min). Citrulline was measured in MRM mode under optimized parameters of declustering potential and collision energy for each mass transition. Unit resolution was set for isolation ions in mass analyzer. Data were processed by software Chemoview 2.0 (AB Sciex).

CRP levels in human serum. Cobas 8000 imunoturbidimetry analyzer (Hitachi, Japan) was used.

IL-6 levels in human serum. Detection was performed by electrochemiluminescence immunoassay (ECLIA) using a Cobas 8000 analyzer.

Urinary neopterin concentrations. Urine sample were collected and stored at −20°C until analysis. After centrifugation (5 min, 1,300 × g) and diluting 100 μl of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium-EDTA per liter, samples were injected onto a column and neopterin was determined using HPLC, Prominence LC20 (Shimadzu). Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission) and quantified by external standard method. Creatinine was determined by the Jaffe reaction after 1:50 dilution of the sample on a Modular analyzer (Roche) using a commercial kit according to the manufacturer's instructions. Neopterin concentrations were expressed as neopterin/creatinine ratio (μmol/mol creatinine).