Effects of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients: a double-blind randomized controlled trial

Etomidate has been advocated to be used in anesthesia for the elderly and the critically ill patients due to its faint effect on cardiovascular system. But the dose-dependent suppression of etomidate on adrenal cortex function leads to the limitation of its clinical application. Clinical research showed that dexmedetomidine could reduce the dose requirements for intravenous or inhalation anesthetics and opioids, and the hemodynamics was more stable during the operation. The objective was to observe the effect of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients. 180 elderly patients scheduled for elective ureteroscopic holmium laser lithotripsy were randomly allocated to PR group anesthetized with propofol-remifentanil, ER group anesthetized with etomidate-remifentanil, and ERD group anesthetized with dexmedetomidine combined with etomidate-remifentanil. Patients in each group whose operation time was less than or equal to 1 h were incorporated into short time surgery group (PR1 group, ER1 group and ERD1 group), and whose surgical procedure time was more than 1 h were incorporated into long time surgery group (PR2 group, ER2 group and ERD2 group). The primary outcome was the serum cortisol and ACTH concentration. The secondary outcomes were the values of SBP, DBP, HR and SpO2, the time of surgical procedure, the dosage of etomidate and remifentanil administered during surgery, the time to spontaneous respiration, recovery and extubation, and the duration of stay in the PACU. The Serum cortisol concentration was higher at t1~2   in ERD1 group compared to ER1 group (P < 0.05). The Serum cortisol concentration at t1~3   was higher in ERD2 group than in ER2 group (P < 0.05). The Serum ACTH concentration was lower at t1~2  in ERD1 group compared to ER1 group (P < 0.05). The Serum ACTH concentration at t1~3  was lower in ERD2 group compared to ER2 group (P < 0.05). The SBP at T1 and T3 were higher in ER2 and ERD2 group than in PR2 group (P < 0.05). The DBP in ER1 and ERD1 group were higher at T1 compared to PR1 group (P < 0.05). The dosage of etomidate was significantly lower in ERD1 group and ERD2 group than in ER1 group and ER2 group (P < 0.05), respectively. The administration of dexmedetomidine combined with etomidate can attenuate the inhibition of etomidate on adrenocortical function in elderly patients and maintain intraoperative hemodynamic stability.

σ stands for standard deviation and δ represents the difference of the means. Quantitative variables were expressed as mean ± standard deviation (SD), enumeration data was presented as frequencies. Comparison of the demographic data and clinical characteristics of the six groups were made using the Student's t-test, Mann-Whitney U test and x 2 test as appropriate. Repeated measures analysis of variance was used for comparisons of SBP, DBP, HR, serum cortisol and ACTH concentration levels among groups at each time point, if comparison between groups was positive, the SNK post hoc test was performed. The statistical significance was determined as p < 0.05.

Statement of ethics.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Following the approval by the Ethics Committee of the affiliated hospital of north Sichuan Medical College (Ref. 2018ER(R)008), we obtained the written informed consent from all the participants for this randomized prospective clinical trial conducted at the affiliated hospital of north sichuan medical college, on patients with kidney or ureteral calculi.

Results
One hundred and eighty patients were screened for eligibility, and subsequently allocated to three groups. No patient dropped out of the trial. A total of one hundred and eighty patients completed the study (shown in Fig. 1).
There were no differences in age, weight, ASA grade and sex ratio among patients in each group (shown in Tables 1 and 2).
The Serum cortisol concentration was lower at t 1~2 in ER 1 group and t 1 in ERD 1 group compared to t 0 and PR 1 group (P < 0.05). The Serum cortisol concentration at t 1~2 was higher in ERD 1 group than in ER 1 group (P < 0.05), (shown in Fig. 2). The Serum cortisol concentration was lower at t 1~3 in ER 2 group and t 1~2 in ERD 2 n = z α + z β 2 * 2σ 2 www.nature.com/scientificreports/ group compared to t 0 and PR 2 group (P < 0.05). The Serum cortisol concentration at t 1~3 was higher in ERD 2 group than in ER 2 group (P < 0.05), (shown in Fig. 3). The Serum ACTH concentration was higher at t 1~2 in ER 1 group and t 1 in ERD 1 group compared to t 0 and PR 1 group (P < 0.05). The Serum ACTH concentration at t 1~2 was lower in ERD 1 group than in ER 1 group (P < 0.05), (shown in Fig. 4). The Serum ACTH concentration was higher at t 1~3 in ER 2 group and t 1~2 in ERD 2 group compared to t 0 and PR 2 group (P < 0.05). The Serum ACTH concentration at t 1~3 was lower in ERD 2 group than in ER 2 group (P < 0.05), (shown in Fig. 5).  The Serum cortisol concentration were lower at t 1~2 in ER1 group and t 1 in ERD 1 group compared to t 0 and PR 1 group (P < 0.05). The Serum cortisol concentration were higher at t 1~2 in ERD 1 group compared to ER 1 group (P < 0.05), (in this figure). www.nature.com/scientificreports/ The SBP was lower at T 1 compared to T 0 in short time surgery groups (P < 0.05). The SBP in ER 1 and ERD 1 group was higher at T 1 and T 3 compared to PR 1 group (P < 0.05). The SBP at T 4 was lower in ERD 1 group than in ER 1 group (P < 0.05), (shown in Table 3). The SBP was lower at T 1 compared to T 0 in long time surgery groups (P < 0.05). The SBP in ER 2 and ERD 2 group were higher at T 1 and T 3 compared to PR 2 group (P < 0.05). The SBP at T 4 were lower in ERD 2 group than in ER 2 group (P < 0.05), (shown in Table 4). The Serum cortisol concentration were lower at t 1~3 in ER 2 group and t 1~2 in ERD 2 group compared to t 0 and PR 2 group (P < 0.05). The Serum cortisol concentration were higher at t 1~3 in ERD 2 group compared to ER 2 group (P < 0.05), (in this figure). The Serum ACTH concentration were higher at t 1~2 in ER 1 group and t 1 in ERD 1 group compared to t 0 and PR 1 group (P < 0.05). The Serum ACTH concentration were lower at t 1~2 in ERD 1 group compared to ER 2 group (P < 0.05), (in this figure). www.nature.com/scientificreports/ The DBP was lower at T 1 compared to T 0 in short time surgery groups (P < 0.05). The DBP in ER 1 and ERD 1 group was higher at T 1 compared to PR 1 group (P < 0.05). The DBP at T 4 was lower in ERD 1 group than in ER 1 group (P < 0.05), (shown in Table 5). The DBP was lower at T 1 compared to T 0 in long time surgery groups (P < 0.05). The DBP in ER 1 and ERD 1 group was higher at T 1 compared to PR 1 group (P < 0.05). The DBP at T 4 was lower in ERD 1 group than in ER 1 group (P < 0.05), (shown in Table 6).
The HR was lower at T 1 and higher at T 4 compared to T 0 in short time surgery groups (P < 0.05). The HR in ERD 1 group was lower at T 3 and T 4 compared to PR 1 group and ER 1 group (P < 0.05), shown in (shown in Table 7). The HR was lower at T 1 compared to T 0 in long time surgery groups (P < 0.05). The HR was higher at T 4 compared to T 0 in PR 2 group and ER 2 group, and lower at T 4 compared to T 0 in ERD 2 group (P < 0.05). The HR in ERD 2 group was lower at T 3 and T 4 compared to PR 2 group and ER 2 group (P < 0.05), (shown in Table 8).
The duration of surgery and the length of stay in the PACU were similar among the three short time surgery groups. There was no difference in remifentanil dosage between the ER 1 group and ERD 1 group. The dosage of The Serum ACTH concentration were higher at t 1~3 in ER 2 group and t 1~2 in ERD 2 group compared to t 0 and PR 2 group (P < 0.05). The Serum ACTH concentration were lower at t 1~3 in ERD 2 group compared to ER 2 group (P < 0.05) (in this figure). Table 3. The SBP (mmHg) at T 0 , T 1 , T 2 , T 3 , T 4 , T 5, T 6 and T 7 in short operation time groups. The SBP at different time points in short operation time groups are shown in this table. The SBP were lower at T 1 compared to T 0 in three groups (P < 0.05). The SBP in ER 1 and ERD 1 group were higher at T 1 and T 3 compared to PR 1 group (P < 0.05). The SBP in ERD 1 group were lower at T 4 compared to ER 1 groups (P < 0.05), (in this table).
Values are mean ± SD. PR 1 Propofol-remifentanil, ER 1 Etomidate-remifentanil, ERD 1 Etomidate-remifentanil and dexmedetomidine, T 0 before the induction of anesthesia, T 1 5 min after induction of anesthesia, T 2 the beginning of operation, T 3 during operation, T 4 6 h after surgery, T 5 12 h after surgery. T 6 24 h after surgery, T 7 48 h after surgery.*p < 0.05 vs. T 0 ; # p < 0.05 vs. PR 1 group; $ p < 0.05 vs. ER 1 group. www.nature.com/scientificreports/ etomidate was significantly lower in ERD 1 group compared with ER 1 group (P < 0.05). The time to spontaneous respiration, tracheal extubation time and the time to recovery were longer in group ERD 1 compared with group ER 1 (P < 0.05), (shown in Table 9). The duration of surgery was similar among the three long time surgery groups. The dosages of remifentanil and etomidate were significantly lower in ERD 2 group compared with ER 2 group (P < 0.05). The time to spontaneous respiration, tracheal extubation time, the time to recovery and the PACU stay time were increased more significantly in group ERD 2 compared with group ER 2 (P < 0.05), (shown in Table 10).

Discussion
In this study, we found that the plasma concentration levels of cortisol and ACTH returned to preoperative levels 24 h and 48 h after surgery in short time surgery group and long time surgery group, respectively. However, after administration of dexmedetomidine 0.4 μg/kg.h, the serum cortisol and ACTH concentrations returned to the preoperative level 12 h after surgery in short time surgery group, and 24 h after surgery in long time surgery group. The blood pressure during both induction of anaesthesia and surgery was more stable when anesthetized with etomidate than propofol, indicating that the elderly patients performed good hemodynamic stability when anesthetized with etomidate. After intravenous administration of dexmedetomidine, the recovery time was increased significantly especially in such short surgeries. Table 4. The SBP (mmHg) at T 0 , T 1 , T 2 , T 3 , T 4 , T 5, T 6 and T 7 in long operation time groups. The SBP at different time points in long operation time groups are shown in this table. The SBP were lower at T 1 compared to T 0 in three groups (P < 0.05). The SBP in ER 2 and ERD 2 group were higher at T 1 and T 3 compared to PR 2 group (P < 0.05). The SBP in ERD 2 group were lower at T 4 compared to ER 2 groups (P < 0.05), (in this table).
Values are mean ± SD. PR 1 Propofol-remifentanil, ER 1 Etomidate-remifentanil, ERD 1 Etomidate-remifentanil and dexmedetomidine, T 0 before the induction of anesthesia, T 1 5 min after induction of anesthesia, T 2 the beginning of operation, T 3 during operation, T 4 6 h after surgery, T 5 Table 5. The DBP (mmHg) at T 0 , T 1 , T 2 , T 3 , T 4 , T 5, T 6 and T 7 in short operation time groups. The DBP at different time points in short operation time groups are shown in this table. The DBP were lower at T 1 compared to T 0 in three groups (P < 0.05). The DBP in ER 1 and ERD 1 group were higher at T 1 compared to PR 1 group (P < 0.05). The DBP in ERD 1 group were lower at T 4 compared to ER 1 groups (P < 0.05), (in this table). Values are mean ± SD. PR 1 Propofol-remifentanil, ER 1 Etomidate-remifentanil, ERD 1 Etomidateremifentanil and dexmedetomidine, T 0 before the induction of anesthesia, T 1 5 min after induction of anesthesia, T 2 the beginning of operation, T 3 during operation, T 4 6 h after surgery, T 5  www.nature.com/scientificreports/ Although etomidate provides rapid onset, rapid recovery and reliable cardiovascular stability 2 , the suppressive effects of etomidate on adrenocortical function limits its clinical application by anesthetists, especially the increased mortality in critically ill patients was potentially due to the adrenal suppressive effects of etomidate 8 . A specific and reversible blockade of the 11α-hydroxylation and 11β-hydroxylation of adrenal steroid synthesis caused by etomidate lead to the decrease of cortisol, corticosterone and aldosterone synthesis 9 . It was found that the serum corticosterone concentration decreased significantly and lasted for more than 3 h after 120 min of etomidate infusion in rats 9 . Clinical studies found that when intravenous infusion of etomidate was used for anesthesia induction, the levels of plasma cortisol were suppressed in the first 6 h after induction by intravenous infusion of etomidate, and returned to pre anesthesia levels 24 h later 10 . The serum cortisol concentration of patients anesthetized with etomidate for electroconvulsive therapy for several times was decreased significantly at 24 h after each anesthesia, and returned to the preoperative level 48 h after anesthesia 11 . These studies suggested that the suppression of etomidate on adrenal cortical function was dose-dependent。In this study, etomidate 0.3 mg/kg was used for anesthesia induction, and the anesthesia was maintained with intravenous target concentration of etomidate 4 to 6 μg/ml. In the short time surgery group, the serum cortisol level was significantly lower compared to preoperative level at 6 to 12 h after surgery, and there was no significant difference in serum cortisol level between the baseline and 24 h after surgery. The plasma cortisol concentration was decreased more Table 6. The DBP (mmHg) at T 0 , T 1 , T 2 , T 3 , T 4 , T 5, T 6 and T 7 in long operation time groups. The DBP at different time points in long operation time groups are shown in this table. The DBP were lower at T 1 compared to T 0 in three groups (P < 0.05). The DBP in ER 1 and ERD 1 group were higher at T 1 compared to PR 1 group (P < 0.05). The DBP in ERD 1 group were lower at T 4 compared to ER 1 groups (P < 0.05), (in this table). Values are mean ± SD. PR 2 Propofol-remifentanil, ER 2 Etomidate-remifentanil, ERD 2 Etomidate-remifentanil and dexmedetomidine, T 0 before the induction of anesthesia, T 1 5 min after induction of anesthesia, T 2 the beginning of operation, T 3 during operation, T 4 6 h after surgery, T 5 12 h after surgery, T 6 24 h after surgery, T 7 48 h after surgery.*p < 0.05 vs. T 0 ; # p < 0.05 vs. PR 2 group; $ p < 0.05 vs. ER 2 group.  Table 7. The HR (beats per minute) at T 0 , T 1 , T 2 , T 3 , T 4 , T 5, T 6 and T 7 in short operation time groups. The HR at different time points in short operation time groups are shown in this table. The HR were lower at T 1 and higher at T 4 compared to T 0 in three groups (P < 0.05). The HR in ERD 1 group were lower at T 3 and T 4 compared to PR 1 group and ER 1 group (P < 0.05), (in this table). Values are mean ± SD. PR 2 Propofolremifentanil, ER 2 Etomidate-remifentanil, ERD 2 Etomidate-remifentanil and dexmedetomidine, T 0 before the induction of anesthesia, T 1 5 min after induction of anesthesia, T 2 the beginning of operation, T 3 during operation, T 4 6 h after surgery, T 5 12 h after surgery, T 6  www.nature.com/scientificreports/  www.nature.com/scientificreports/ significantly at 6 to 24 h after surgery, and returned to the preoperative level at 48 h after surgery in the long time surgery group. The results showed that the adrenocortical function of the elderly patients anesthetized with etomidate was suppressed, and the suppression was also prolonged with the increase of anesthesia time. Dexmedetomidine is a highly specific α 2 -adrenoreceptor agonist with short half-life period (about 2 h). It has a dose-dependent sedative and analgesic effect, and has no adverse effect on respiration 12 . The application of dexmedetomidine (0.5 g/kg) in pediatric patients anesthetized with sevoflurane could decrease the heart rate of children, but there were no significant changes in SBP, DBP or PETCO 2 4 . It was shown that dexmedetomidine as an adjunct for inhalation anesthetics could effectively maintain the stability of circulation and respiration during surgery. In recent clinical trials, the effect of dexmedetomidine on the requirement for propofol and remifentanil in total intravenous anesthesia was studied. It was found that the administration of dexmedetomidine significantly decreased both the requirements for propofol and remifentanil during anesthesia induction and the dosage of propofol administered during surgery 3,13 . In this study, when dexmedetomidine was added to intravenous anesthesia with etomidate, the intraoperative dosages of etomidate were reduced by 17% and 22% in the short time surgery group and long time surgery group respectively, and the dosage of remifentanil was reduced by 13% in long time surgery group, which was consistent with the above research results.
In this study, the administration of dexmedetomidine not only reduced the etomidate requirements for total intravenous anesthesia in elderly patients, but also attenuated and shortened the inhibitory effect of etomidate on adrenocortical function in elderly patients. This is mainly because the inhibition effects of etomidate on adrenal cortex function were dose-dependent 14 , the administration of dexmedetomidine significantly reduced the requirement for etomidate, and with the reduction of etomidate dose, the inhibitory effect of etomidate on adrenal cortex was correspondingly attenuated in this study. In vitro studies showed that dexmedetomidine combined with etomidate had a stronger inhibitory effect on human adrenocortical cells than etomidate alone 15 . However, some scholars studied the effect of dexmedetomidine and etomidate on adrenocortical function in children and found that 3 h after induction of anesthesia, the serum cortisol concentration of patients in the etomidate group was the lowest, while there was no difference between dexmedetomidine group and the control group, indicating that dexmedetomidine had little or no effect on adrenocortical function 16 . The researches above showed that the inhibitory effect of dexmedetomidine on adrenal function is controversial currently. The inhibitory effect of dexmedetomidine on adrenocortical function in elderly patients was not studied in our study, so the effect of dexmedetomidine on adrenocortical function in elderly patients is still unknown.
In clinic, the induction of anesthesia with propofol often leads to the decrease of arterial blood pressure 17 . Due to the degradation of organ function and the declination of physiological function, it is easier to induce hypotension in elderly patients anesthetized with propofol 18 . The blood pressure was decreased significantly compared to the baseline in patients by using propofol and etomidate for anesthesia induction, and the decrease was greater in the propofol group compared to etomidate group 19 . In this study, 5 min after induction of anesthesia with propofol, the systolic pressure, diastolic pressure and heart rate were decreased by 23.5%, 28.4% and 13%, respectively. This was mainly attributed to propofol reducing cardiac output and systemic vascular resistance, and inhibiting baroreceptor reflex 18 . However, in the etomidate group, systolic blood pressure was decreased by 14.6%, diastolic blood pressure was decreased by 12% and heart rate was decreased by 14% 5 min after induction of anesthesia. Although there was no difference in the decrease of heart rate, the decrease of systolic and diastolic blood pressure was more gently in etomidate group compared to propofol group. Meanwhile, the blood pressure of patients during surgery was significantly lower in propofol group compared to etomidate group. It was suggested that the hemodynamic stability in the elderly patients could be better maintained with etomidate anesthesia.
Dexmedetomidine could maintain intraoperative hemodynamic stability by inhibiting sympathetic nervous system and attenuating the stress response 20 . Davy A et al. 21 reported about 42% patients who were administered with dexmedetomidine developed various degree hypotension and bradycardia. Dexmedetomidine could decrease the heart rate and blood pressure in a dose-dependent manner 22 . A clinical study found that low dose dexmedetomidine (0.5u.kg-1.h-1) can effectively reduce the requirement of propofol and maintain the intraoperative hemodynamics of patients undergoing laparoscopic cholecystectomy 23 . Josephine et al. 24 pointed out in their review on hemodynamic response of high-and low-dose dexmedetomidine that compared with high-dose dexmedetomidine, low-dose dexmedetomidine had better hemodynamic stability and shorter recovery time. In our study, there was no difference in systolic and diastolic blood pressure between etomidate alone group and combined dexmedetomidine group at 5 min after anesthesia induction, the beginning of surgery and during surgery. But the addition of dexmedetomidine could decrease the intraoperative heart rate more significantly than etomidate alone, and no patient developed bradycardia. It showed that although the combination of dexmedetomidine (0.4 µ/kg/h) could decrease intraoperative heart rate, but had little effect on the intraoperative blood pressure in elderly patients undergoing general anesthesia, which was consistent with the results of recent studies 23,25 . There was little correlation between intraoperative dexmedetomidine and the recovery time after propofol anesthesia in common outpatient procedures, and the potential dose relationship was that the administration of per μg/kg dexmedetomidine would increase recovery time for about 15 min 26 . In present study, when etomidate was combined with dexmedetomidine, the time to spontaneous respiration, time to recovery and tracheal extubation time were prolonged. However, intraoperative intravenous infusion of dexmedetomidine (0.4 μ/kg/h) did not affect postoperative anesthesia recovery in patients undergoing thoracic surgery 27 , or provided faster recovery in patients undergoing tympanoplasty surgery 28 . It was suggested that the administeration of dexmedetomidine in long-term surgery rather than short-term surgery could provide faster recovery.
There are limitations in this study. Firstly, we didn't design a trial to identify the effect of dexmedetomidine alone on adrenocortical function in elderly patients. It is not clear whether the administration of dexmedetomidine suppress the adrenocortical function. Secondly, an enzymatic block of 11-b-hydroxylase was demonstrated in a patient who received a prolonged infusion of etomidate 29 . We didn't observe the enzymatic block www.nature.com/scientificreports/ of 11-b-hydroxylase both in short and long time surgery groups in this study, and the effects of intravenous infusion of dexmedetomidine combined with etomidate on the enzymatic block of 11-b-hydroxylase at different times need to be further studied. Thirdly, the principal adrenocortical products are cortisol, aldosterone and dehydroepiandrosterone sulphate 30 . We only observed changes in plasma concentrations of cortisol and adrenocorticotropin in present study, but the effect of administration of etomidate combined with dexmedetomidine on adrenocortical secretion of aldosterone was not clear. Fourthly, in our study, the surgery time of ureteral holmium laser lithotripsy was within 2.5 h, and the effect of etomidate combined with dexmedetomidine infusion for more than 2.5 h on adrenocortical function was unclear. We will apply the combination of dexmedetomidine and etomidate in long-term surgery to observe the changes of adrenal cortex function of elderly patients in future studies. Finally, the sample size of our study is too small. If this study had been performed on a larger sample size, there would probably have been more significant results in terms of the dose-and time-dependent effects of dexmedetomidine on etomidate-induced inhibition of adrenal cortical function.
In conclusion, the inhibitory effect of etomidate on adrenocortical function in elderly patients was prolonged with the duration of intravenous anesthesia with etomidate. The administration of dexmedetomidine combined with etomidate can attenuate the inhibition of etomidate on adrenocortical function in elderly patients and maintain intraoperative hemodynamic stability.