We conducted a single-center, randomized, controlled trial with two parallel groups. The trial was approved by the Ethics committee of the Hospital Clínico Universidad de Chile, José Joaquin Aguirre and was conducted according to the principles of the Helsinki Declaration under monitoring by the Good Clinical Practice unit of our institution. Written informed consent was obtained from all patients before they were included in the trial. The outcome assessors were blinded to the group allocation of the patients. This study adhered to CONSORT guidelines for reporting randomized trials .
We included 16 patients scheduled to undergo elective knee-ligament reconstruction surgery with at least 60 min of tourniquet-induced ischemia of one of the lower extremities at the Hospital Clínico de la Universidad de Chile. The inclusion criteria were age between 18 and 60 years and an American Society of Anesthesiologists (ASA) classification I and II. We excluded patients with allergies to egg or soya, previous history of critical events during surgery and the perioperative period, those at risk of malignant hyperthermia, and patients with 3 or more predictors of difficult airway management.
Blood samples were collected with the informed consent of the patients. All patients entered the operating room and after standard ASA monitorization and placement of an intravenous line (IV), a 50 mL·h− 1 Ringer lactate infusion was started. All additional drugs were bolus administered and pushed with 10 mL saline solution. Spinal anesthesia was performed under 1 mg midazolam and 1 μg kg− 1 fentanyl sedation. After analgesia and motor block establishment, another mg of midazolam was administered, and according to the study arm allocation, an intravenous or inhalation hypnosis-induction was performed. The airway was secured by laryngeal mask placement before the surgery started. A femoral tourniquet was installed and inflated by the surgeon 120 mmHg above patient systolic blood pressure. Surgery and tourniquet duration, total fluid administration, and the use of vasoactive agents were registered. The sevoflurane group hypnotic anesthetic target was 0.8–1.0 age-corrected minimum alveolar concentration (MAC) and the propofol hypnotic anesthetic target was set to a site-effect target-controlled infusion of 2–2.5 μg·mL− 1 (Marsh, keO 1.21 min− 1). Both targets allow spontaneous ventilation or pressure support ventilation during surgery. Finally, a femoral nerve block for postoperative analgesia was performed in the post-anesthesia care unit for all patients.
To measure endothelial damage biomarkers, five different venous blood samples were collected: at the IV placement (baseline value, T1); during surgery before tourniquet release (T2); and 10 (T3), 60 (T4), and 90 (T5) minutes after tourniquet release. At the end of surgery, all patients were transferred to a post-operative care unit where the blood samples were collected. All blood samples were collected by the anesthesiologist team and coded before delivering them to the processing laboratory. Blood samples were incubated for one hour at 37 °C and centrifuged at 1500 rpm for 10 min. Blood serum was stored in a − 80 °C freezer for final analysis.
To characterize the endothelial damage, we decided to measure two superficial biomarkers (syndecan-1 and heparan sulfate) and one deep biomarker (thrombomodulin). To evaluate the biomarker levels in blood serum, we used the following assays: syndecan-1 (CD138) Human ELISA Kit (Catalog #ab46506, Abcam, Cambridge, MA, USA), Heparan Sulfate BioAssay™ ELISA kit (Catalog #356350, USBiological, Salem, MA, USA), and human Thrombomodulin ELISA kit (Catalog #CSB-E07937h, CUSABIO, Houston, TX, USA). All measurements were performed in duplicate. Assays and analyses were conducted according to the manufacturer’s instructions. Standard solutions of syndecan-1, heparan sulfate, and thrombomodulin were provided in each test, with a detection range of 8 ng/mL to 256 ng/mL for syndecan-1, 78 ng/mL to 5 μg/mL for heparan sulfate, and 0.312 ng/mL to 20 ng/mL for thrombomodulin.
The primary outcome was the difference in syndecan-1 concentrations between sevoflurane and propofol groups 90 min after tourniquet release (T5). Heparan sulfate and thrombomodulin blood serum concentrations were compared between both groups as secondary outcomes. All the comparisons were assessed at the pre-established five points during the perioperative period.
We based our sample size calculation on the normal range values of syndecan-1 reported by Rahbar et al. and on the increase in syndecan-1 levels reported in cardiac surgery patients, septic patients, and trauma injured patients [11, 12, 22]. These findings describe an increase of two or three times to several folds from baseline values. Considering a normal syndecan-1 value of 31.6 ng/mL, a standard deviation of 15.3 ng/mL, and a three-fold increase from the normal range values, a sample size calculation was performed to detect a 25% change in the mean concentration of syndecan-1 with an alpha of 0.05 and a power of 80%. Considering a 20% loss of patients, 16 patients (8 patients per arm) were needed for the two-sided test analysis.
Computer-generated permuted eight-block randomization was performed. With the results, sixteen consecutively sealed and numbered envelopes were generated. Each envelope was opened consecutively after each patient signed the informed consent form. Blood tubes were then labelled with unique patient and sample codes. Sample collectors carried each tube from the operating room to the processing laboratory for analysis.
Sample collectors, laboratory processing investigators, and the outcome assessor were blinded to patients’ allocations. After study termination, the outcome assessor had access to the patient’s sample codes and performed the final analysis.
Categorical variables were summarized as relative frequencies. Continuous variables for primary and secondary variables were expressed as the mean and standard deviation (SD) or interquartile rank (IQR). Non-paired results were compared with the Mann-Whitney test. For comparison of repeated assessments, we used a two-way ANOVA. Post-hoc comparisons with baseline values were performed with a Bonferroni correction. For paired data, a Wilcoxon test was performed. Two-tailed P values less than 0.05 were considered significant. Data were analyzed with GraphPad Prism software, version 8.0 (La Jolla, CA, USA).