Study population
This is a comparative prospective study. The study was approved by the Tel Aviv Sourasky Medical Center ethics committee, conformed to the principles outlined in the declaration of Helsinki (0342-19-TLV), and informed consent was obtained from all participants.
Two groups of patients undergoing surgery under general anesthesia of longer than one hour were enrolled. The first group of patients have known atherosclerotic disease (coronary, peripheral vascular or cerebral) arriving for vascular surgery. The second group are patients for ambulatory surgery without known atherosclerotic disease. Patients were enrolled consecutively from November 1st, 2019 to December 30th 2019.
Inclusion criteria –men and women, age > 18, who can sign informed consent, with either known atherosclerotic disease or no such background, scheduled for either a planned vascular surgery or ambulatory surgery, respectively. The surgery is assumed to last more than an hour of anesthesia time.
Exclusion criteria patients with medical history of Parkinson or Alzheimer disease, known muscle paralysis diseases with characteristics of muscle nerve synapse injury, acute kidney injury by KDIGO criteria.24
Cholinesterase activities
Blood samples were taken from all patients at three time points; prior to, and 15 minutes after induction of anesthesia, and at the end of surgery prior to neuro-muscular blockade reversal agent administration (Neostigmine).
We used the ACh analog Acetylthiocholine (ATCh) as a substrate that is hydrolyzed by both ACh degrading enzymes (Acetylcholinesterase and Butyrylcholinestersase) and reflects the total serum capacity for acetylcholine hydrolysis, referred to as Cholinergic Status (CS). (appendix 1)
Serum samples were frozen at − 80 °C until acetylcholine hydrolysis analysis. Acetylcholinesterase and total cholinesterase activity levels were assayed in triplicates in a microtiter plate using an adaptation of the Ellman assay.25 Hydrolysis of 1 mM acetylthiocholine (ATCh, Sigma) was followed by spectrofluorometry (Spectrafluor Plus, Tecan) at 405 nm.(appendix 2) Prior to read, we incubated the samples for 20 min in the dark with (for acetylcholinesterase activity) or without (for total cholinesterase activity) 50 μM tetra isopropyl pyrophosphoramide (iso-OMPA, Sigma) which is a specific ButyrylCholinestersase inhibitor. We calculated enzyme activity using 13,600 M/cm as the e405 for 5-thio-2-nitrobenzoate.26,27
Intra-operative measurements
Data of anesthesia management were taken from the Metavision with the aid of MDclone novel program: medications given during anesthesia including hypnotic, analgesics, sympathomimetics (Ephedrine, Phenylephrine) and Norepinephrine, type of anesthesia (total intravenous anesthesia versus volatile anesthesia), lowest temperature measured during anesthesia.
Primary outcome- to characterize the dynamics of cholinesterase activity in patients undergoing general anesthesia and surgery.
Secondary outcome- to assess the association between cholinesterase activity and hemodynamic instability during general anesthesia reflected by the intraoperative use of vasopressors.
Statistical analysis
All continuous variables are displayed as means (SD) for normally distributed variables or median [interquartile range] for variables with abnormal distribution. Categorical variables are displayed as numbers (%) of subjects within each group.
To test differences in continuous variables between 2 groups, the independent-samples t-test or the Mann–Whitney test were performed. For comparison of dichotomous or categorical variables, the Pearson Chi-Square test was performed. To compare continuous variables between 2 time-points the paired-samples t-test was performed or the sign-rank test when needed. The Pearson or Spearman correlation coefficients were used to assess correlations between continues variables. To compare continuous variables between 3 time points the repeated-measures general linear model was used. A one-way Analysis of Variance (ANOVA) with a linear contrast was used to compare the CS values between vascular and non-vascular groups for blood pressure measurements and In order to identify possible confounders, a multivariate regression with the use of pressor drugs (sympathomimetics and Norepinephrine) was used controlling for the age, and department as covariates. P < 0.05 was considered statistically significant for all-analyses. We used IBM SPSS Statistics 24 statistical package (IBM Corporation, Armonk, New York, USA) for all statistical analysis.