This randomized prospective clinical study was performed after approval from the institutional review board of Inonu University (Malatya Clinical Research Ethics Committee, 2017/128) that the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration was obtained. Written consent was obtained from all donors, and the study was registered prior to patient enrolment at ClinicalTrials.gov (NCT04232904, https://classic.clinicaltrials.gov/ct2/show/NCT04232904, date of registration: 18 January 2020). The clinic trial was carried out at Inonu University Liver Transplant Institute, Malatya, Turkey. Seventy-two LLDs aged between 18–65 years, with American Society of Anesthesiologists (ASA) score I-II scheduled for right hepatectomy were included in the study. The donors participating in our study were relatives with their recipients and the relationship degree was a first- or second-degree relationship. Donors with systemic disease (such as diabetes and hypertension), recent (one week prior to the donor hepatectomy) opioid or nonsteroidal anti-inflammatory drug use, disturbaces in coagulation, history of allergy to the study drugs, previous chronic use of anticonvulsants such as pregabalin and/or gabapentin, and refusal to participate were excluded.
Donors were randomly assigned based on a web-based randomization generation to Group 1(the control group) received standard general anesthesia or the intervention group with subcostal TAP blocks (Group 2). The present study was designed as a single-blind trial in which the researchers who administered the intervention and collected the data blinded for the treatment groups.
No sedatives or premedications were administered. Donors were monitored with bispectral index (BIS) and electrocardiography, pulse oximeter, noninvasive blood pressure, and body temperature. Peripheral venous access was performed, and blood samples were obtained for preoperative cytokine and bupivacaine measurements. After preoxygenation, anesthesia was induced with 2–3 mg kg− 1 propofol and 1 mcg kg− 1 fentanyl. Neuromuscular blockage was achieved with 0.6 mg kg− 1 rocuronium. Anesthesia was maintained with desflurane, 0.25–0.5 mcg kg− 1 min− 1 remifentanil infusion, and an O2/air mixture. Desflurane at 0.6 and 1 MAC (minimum alveolar concentration) was used to keep the BIS value in a range of 40 to 60. After anesthesia induction, an arterial line (radial artery) was established all donors to collect blood samples during the study and monitor invasive arterial blood pressure during surgery.
All subcostal TAP blocks were performed following induction and immediately before the surgical incision by the same experienced anesthesiologist. The subcostal TAP block was applied bilaterally with real-time ultrasonography (LOGIQ S8, GE Ultrasound, Korea) guidance with a linear 6–13 MHz probe with a 22-G 80-mm needle (Stimuplex Ultra 360 B. Braun Aesculao Japan Co. Ltd. Japan) using the in-plane technique in the subcostal region as previously described [3, 4]. A total of 1.5 mg/kg 0.5% bupivacaine (in 2 syringes with 20 mL bupivacaine + saline) was applied to the abdominal wall bilaterally.
In both groups, blood samples were obtained preoperatively, 30 minutes and 2, 6, and 24 hours following injection for cytokine measurement. In Group 1, to examine the plasma bupivacaine level, blood samples were obtained preoperatively as well as 30 minutes and 1, 2, 6, 12, and 24 hours following injection.
Signs of toxicity of the central nervous and cardiovascular systems, including perioral numbness, metallic taste, dizziness, tingling sensation, muscle twitch, disorders of speech and convulsions were regularly checked in the post-operative period. Postoperative analgesia management of the all patients was provided by a patient-controlled analgesia device with morphine (90 mL of SF with 10 mL of morphine sulfate [100 mg]), the locking time of the device was adjusted for 15 minutes and an intermittent bolus of 2 mg.
The primary outcome was to examine the efficacy of subcostal TAP block in reducing the inflammatory response in the treatment arm of the study. Secondary outcomes included plasma bupivacaine concentrations and the correlation between plasma bupivacaine levels and cytokine levels in just the intervention group.
Whole blood samples drawn from the patients were centrifuged at 4000 rpm for 7 minutes, and their serum was separated. The samples were separated in to aliquots and kept in a -80°C until the analyses.
Analysis of blood samples
Serum IL-1β, IL-6 and TNF-α levels were evaluated by enzyme-linked immunosorbent assay (ELISA) kits (YLBiont, Shanghai, China). The ELISA kit was evaluated spectrophotometric analysis using 595 nm UV in microplate reader (BioTek Synergy H1, BioTek Instruments) in accordance with the kit procedure. The concentration values of the samples were determined from the standard curve based on absorbances using the data analysis program (Gen5, BioTek Instruments), and the values were drawn with the help of known standards.
The determination of bupivacaine in serum can be performed by several methods including high-performance liquid chromatography (HPLC), GC-MS, capillary electrophoresis and LC-MS [9, 10].
We chose HPLC foe determination of serum bupivacaine because post-elimination doses of the drug required measurement with a highly sensitive test that was minimally affected by other factors such drugs that are administered concomitantly.
Reagents and materials
Standard for bupivacaine was obtained from Sigma-Aldrich (Germany). The pure bupivacaine standard was weighed accurately, and a 1.00 mg L− 1 stock standard solution was prepared by dissolving in 10 mL acetonitrile and water (75:25 v/v). A calibration curve was prepared with five points in the range of 0.1 to 100 mg mL− 1 by diluting the stock standard solution with acetonitrile and water (75:25, v/v). The calibration curve is used to calculate the samples of the treatment and the control arm of the study.
Instrumentation
Chromatographic qualitative and quantitative analyses of bupivacaine were performed on a Shimadzu chromatography system. This system contains a photodiode array (PDA) detector. All analyses were performed via a Shimadzu HPLC equipped with a Shimadzu DGU-20 A5 model vacuum degasser and a Shimadzu 20 ADXR solvent pump. Separations were performed through an ODS-2, C18 (150 mm × 4.6 mm, 3 µm) column at room temperature and at 207 nm (Fig. 1).
Bupivacaine measurements were performed using the optimum separation conditions by HPLC with gradient elution and a flow rate of 1.0 ml min− 1.
The mobile phase included of solvent A (0.03 M sodium dihydrogen phosphate solution) and solvent B (acetonitrile 100%). The following linear gradient was used: at 0 min, 85% solvent A and 15% solvent B; between 3–15 min, 40% solvent A and 60% solvent B; in 15 minutes, 85% solvent A and 15% solvent B; and in 20 minutes, return to initial conditions.
Sample preparation procedure
Plasma bupivacaine concentrations were determined using HPLC using the methodology described by Purvis et al. and Faura et al. [11–13].
Acetonitrile (500 µL) was added to the plasma sample (1 ml) in the tube, and tubes were screwed on and mixed by vortexing for 5 min. The tubes were centrifuged for approximately 10 min at 5000 rpm. The extract was injected into the HPLC system after it was filtered through a membrane filter (0.22-µm PVDF).
Discrete chromatograms were observed. Peaks were spate from one and other without any interference. The bupivacaine chromatography results are summarized in Fig. 1.
Statistical analyses
All statistical analyses were performed using Statistical Software Package for Social Sciences software package version 22 (SPSS v22) (IBM, New York, ABD. Shapiro Wilk test was used to determine the normal distribution of the continuous data. Continuous data were analyzed using independent samples t test or Mann-Whitney U test. Categorical data were analyzed using the chi-square or Yates corrected chi-square test, depending on the situation. Differences between repeated measurements were analyzed using the Friedman test. Correlations between different variables were analyzed with the Spearman correlation coefficient. Data are presented as medians ± quartiles or means and standard deviations, and categorical data are presented as numbers and percentages. Α p-value < 0.05 was considered significant for all analyses. Based on IL-6 values in a previous study, when the amount of type I error (alpha) is 0.05, the power of the test (1-beta) is 0.8 [14]. Thus, it was determined that a minimum sample size of 34 individuals with 17 in each group is required to observe a 20% difference in the IL-6 mean values with an effect size of 1.01 (large). The power analysis was performed using Web-Based Sample Size & Power Analysis Software (WSSPAS) [15].