Cholinesterase activity in serum during general anesthesia in health and disease

Atherosclerotic patients challenge the anesthesiologist as they display a signicant instability between the two parts of the autonomic nervous system. We aimed to assess the effect of general anesthesia (GA) and surgery on serum cholinesterase activity. Prospective study of 57 patients undergoing ambulatory or vascular surgery under GA. Cholinesterase levels were measured before induction of anesthesia, 15 minutes thereafter and at the end of surgery by measuring the capacity of serum Acetylcholinesterase (AChE) and Butyrylcholinesterase to hydrolyze AcetylThioCholine. Data of atherosclerotic disease, anesthesia management were analyzed. Both AChE and total Cholinergic Status (CS) decreased signicantly after GA induction at 15 minutes and furthermore by the end of surgery. Vascular surgery patients, presented lower baseline cholinesterase activity compared to patients for ambulatory surgery. In patients requiring intraoperative administration of phenylephrine for hemodynamic support (21.1%), a signicant lower level of AChE and CS was observed compared to untreated patients. A positive correlation was found between the lowest temperatures measured and the AChE and CS change from the baseline values. Our ndings serve as a mirror to the sympathetic/parasympathetic disbalance during GA with a marked decrease in the parasympathetic tone. Our data show that low cholinesterase activity increase the need for hemodynamic support.

heart rate recovery in healthy adults. 6,11−14 Repetitive measurements of BChE activity levels during 5 years show little variation in healthy adults. 15 In contrast, following acute events, such as myocardial infarction or stroke, decreased level of both cholinesterase were observed which were associated with worse prognosis. 16 Data regarding anesthesia and surgery effects on cholinesterase activity levels are limited. During general anesthesia, the anesthesiologist uses different drugs that inhibit competitively the activity of cholinesterase, for example neostigmine. 10,[17][18][19][20]21 Also, a recent study found that cholinesterase activity could be related to post-operative delirium. 22 In the present study we aimed to assess the effect of general anesthesia and surgery on cholinesterase activity levels 10,23 and to evaluate whether patients with atherosclerotic disease demonstrate altered activity levels when compared to healthy adults.
Additionally, we aimed to examine in patients undergoing general anesthesia whether there is an association between enzyme activity levels and hemodynamic stability.

Materials And Methods:
Study population This is a comparative prospective study. The study was approved by the Tel Aviv Sourasky Medical 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 re ects 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 spectro uorometry (Spectra uor 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 speci c 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 re ected 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 coe cients 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 oneway 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 signi cant for all-analyses. We used IBM SPSS Statistics 24 statistical package (IBM Corporation, Armonk, New York, USA) for all statistical analysis.

Results:
Patient's demographics During the study period 57 patients were enrolled, of whom 17 (29.8%) underwent vascular surgery (peripheral arterial bypass surgery or carotid endarterectomy) and 40 (70.2%) underwent ambulatory surgery, mainly lumpectomy or laparoscopic cholecystectomy. Their demographics are presented in Table   1. Third of the cohort suffered from essential hypertension, almost all treated with angiotensin receptor blocker/ angiotensin converting enzyme inhibitor (ACEI/ARB) while some with more than one medication (beta blockers or calcium channel blockers). Anemia 28 was found in 9 patients (15.7%). Comorbidities associated with atherosclerotic disease such as myocardial infraction, carotid artery stenosis, peripheral vascular disease, cerebrovascular disease affected 14%, 7%, 17.5%, 3.5% of patients, respectively, and were prevalent only in the vascular surgery group. Comorbidities and patients' medications are listed in Table 1.  Table 1 Population demographics, Comorbidities & medications Table 2 Cholinesterase activity at baseline, 15 minutes after induction of anesthesia and at the end of anesthesia Table 3 Drug and anesthesia methods and their effect on Cholinesterase activity at baseline, 15 minutes after induction of anesthesia and at the end of anesthesia  Both AChE and total cholinergic status (CS) decreased signi cantly after general anesthesia induction at 15 minutes and furthermore by the end of surgery (Table 2). anesthesia were observed only in the ambulatory surgery group (patients without atherosclerotic disease), with further signi cant reduction by the end of anesthesia compared to baseline measurement (Fig. 1a, 1b).

Secondary outcome
Vasopressor support and cholinesterase activities The association between vasopressors use (re ecting the need to optimize hemodynamics) and cholinesterase activity before and following anesthesia was assessed. In patients requiring intraoperative administration of phenylephrine for hemodynamic support (n = 12, 21.1%), a signi cant lower levels of AChE and CS was observed at the beginning and at the end of anesthesia compared to patients not treated with phenylephrine (p = 0.008 and p = 0.013 for AChE; p = 0.011 and p = 0.011 respectively, Table

Remifentanil infusion
Remifentanil is an ultrashort opioid which metabolized by non-speci c blood and tissue esterase, but its effect on cholinesterase activity has not been studied to date. We found that patients receiving remifentanil continues infusion (n = 11, 19.3%) presented similar baseline measurements when compared to patients that did not receive remifentanil, but signi cant decrease of AChE and total CS 15 minutes after induction of anesthesia was observed (p = 0.013; p = 0.03, respectively) which remain signi cant also at the end of anesthesia (p = 0.029; p = 0.031, respectively, Table 3). The results remained signi cant even after adjustment for vasopressors use.

Type of anesthesia and cholinesterase activities
To further investigate the reason for decline of AChE and CS activity during general anesthesia we compared patients who were under total intra venous anesthesia (TIVA) (n = 8, 14%) vs. patients receiving volatile anesthesia (VA). We found that patients under TIVA had higher levels of CS at the baseline measurement compared to VA group (p = 0.044). All other comparisons of cholinesterase activities between TIVA to VA did not reach signi cance levels.

Discussion:
Knowledge regarding the effect of general anesthesia and surgery on cholinesterase activity levels is scarce. This is the rst study to show a decrease in cholinesterase activities during general anesthesia; signi cant decrease in AChE and total Cholinergic Status (CS) levels were found at 15 minutes post general anesthesia induction and at the end of surgery compared to baseline levels. Additionally, the present study demonstrated that vascular patients admitting for elective surgery show signi cantly lower levels of AChE and CS at baselined and during anesthesia when compared to non-vascular patients. The results might have clinical implications. Previous study by Arbel et al. found that patients arriving for cardiac catheterization with lower levels of cholinesterase had higher risk for major adverse cardiac events. 29 Goliash et al. report that low cholinesterase could be used as a biomarker for mortality prediction in stable coronary artery disease. 30 In the present study, lower levels of cholinesterase activities during surgery and anesthesia were associated with higher demand for drugs that support hemodynamics such as phenylephrine or ephedrine. As our department follows good clinical practice 31 of keeping mean arterial pressure 65 mmHg or more, this association is a re ection of the prevalence of hypotension with the anesthesiologists actively treating hypotension to prevent postoperative morbidities. To the best of our knowledge, this is also the rst time that direct plasma measurements of cholinergic activity were assessed in order to re ect peripheral sympathetic/parasympathetic balance during anesthesia. Current review of the anesthesia literature suggests the use of heart rate variability tracking as a surrogate marker for sympathetic response and for the analgesics management in clinical practice. 32,33 Our method offers a more precise measurement for evaluating the cholinergic tone, in addition to the HRV measurement.
Anesthetics and cholinesterase during anesthesia Our data did not nd difference between patients undergoing anesthesia using total intravenous anesthesia with propofol and patients anesthetized using volatile anesthetics suggesting that the type of agent used for general anesthesia did not explain the decrease in cholinesterase activity level we found.
Remifentanil, a synthetic ultrashort opioid analgesic has ester linkage which undergoes hydrolysis by non-speci c plasma esterase, 35 In vitro studies showed that remifentanil is not a good substrate for plasma cholinesterase. 10 Nevertheless we found signi cant lower levels of cholinesterase for patients that were anesthetized using remifentanil. A possible explanation is the known decreased sympathetic tone when using remifentanil which might extrapolate higher parasympathetic tone. Further studies will be needed to investigate on this nding.

Body temperature and Cholinesterase during anesthesia
We found a correlation between lowest body temperature measured during anesthesia to the level of AChE and CS. Furthermore, the decline in cholinesterase levels was correlated with lower body temperature. This nding is in line with the need for higher vasopressor use and the low level seen in the vascular group. Another possible explanation is low enzymatic activity in cold in-vivo environment, however, the lab measurement was done under temperature control.
Our study has few limitations; rst, this is a relatively small group size, thus further studies are needed in larger patient population, more so when assessing sub-populations. Second limitation is that baseline enzyme activity measurement was done at the entrance to the operating room when we might assume that the patient is anxious thus it might not re ect the true baseline of the patient, but rather anxious state before surgery. 36,37 However, this measurement was done in the same setting for all patients.
In summary, the main nding of our study is that anesthesia and surgery induced a signi cant reduction in serum cholinesterase activity associated with higher need for hemodynamic support. Correlation between minimal body temperature and the delta of AChE (a) and total cholinergic status (b) from the baseline to the end of anesthesia