Direct laryngoscopy and endotracheal intubation are considered among the most invasive stimuli of the practice of general anaesthesia. Their physiopathological effects are as important as their traumatic complications. The cardiovascular response to these procedures arises from sympathoadrenal reflexes evoked by the stimulation of laryngeal and tracheal tissues during the procedure [1-4]. Coughing and bucking that may be caused during the procedure may add to the increases in venous, intracranial and intraocular pressures. Haemodynamic responses to induction and intubation, although short-lived and with various measures available to reduce them, can cause serious problems in patients with cardiovascular and cerebral diseases. In this study, we aimed to examine some of the putative mechanisms causing haemodynamic changes (the sympathetic, the vasopressin and the renin-angiotensin systems) in order to identify future areas of work in terms of preventing these changes.
Patients and methods
This study was approved by the Research and Ethics Committees of Başkent University (Project No. KA03/134) and supported by the Başkent University Research Fund.
One hundred ASA I patients, 50 male, 50 female aged 20-50 yr (mean ± SEM; 35.59 ± 0.99) were enrolled into this study. The same number of male and female were included to obtain a homogeneous study group with respect to gender. Informed consent was obtained from all patients. Individuals with chronic disease and/or under drug treatment (oral contraceptives, antidepressants and antihypertensives) were excluded. All patients were scheduled for elective surgery requiring general anaesthesia with orotracheal intubation.
All patients received diazepam 5 mg and famotidine 40 mg orally the night before surgery and midazolam 0.1 mg kg−1 orally 60 min prior to induction of anaesthesia. Electrocardiogram (ECG), non-invasive blood pressure, arterial oxygen saturation by pulse oximetry and end-tidal CO2 concentration by capnography were monitored. Blood pressure measurements and heart rates were recorded at the following times; following 10 min rest after arrival to the operating theatre, immediately before laryngoscopy, every minute for 5 min following intubation and at intervals as necessary afterwards.
All patients received standard induction and maintenance of anaesthesia. Anaesthesia was induced with fentanyl 1 μg kg−1 and thiopental sodium 5-7 mg kg−1 followed by vecuronium 0.1 mg kg−1. The same senior resident (HM) performed laryngoscopy and intubation 3 min later at one attempt using a standard Macintosh laryngoscope blade and conventional tracheal tubes with high-volume, low-pressure cuffs. The patients in whom more than one attempt to intubation was required were excluded from the study. Anaesthesia was maintained with nitrous oxide (60%) in oxygen (40%) and isoflurane (0.5-2%). All measurements and blood samples were completed before the surgical skin incision.
For determination of plasma epinephrine, norepinephrine and vasopressin concentrations, and angiotensin converting enzyme (ACE) activity, 4 mL of venous blood was drawn from a separate intravenous cannula. Prior to the first sampling, a steady state was achieved in all patients by allowing them rest for 10 min. All measurements were taken and recorded at four time points: 5 min prior to induction (baseline value, T1), immediately before laryngoscopy (T2) and 2 and 5 min after tracheal intubation (T3 and T4, respectively). Each blood specimen was collected into three different tubes for the following analysis; chilled ethylene diamide tetraacetic acid (EDTA) tubes for catecholamine, chilled serum tubes containing aprotinin (500 KIU mL−1) for arginine-vasopressin and serum tubes for ACE determinations. Samples were immediately centrifuged at 1600g for 15 min at +4°C and plasma and serum specimens were stored at −80°C until analysis.
Plasma levels of epinephrine and norepinephrine were determined by using a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle (IBL, RE59251 and RE59261, Hamburg, Germany). These assays use monoclonal antibodies directed against N-acyl-metanephrine and N-acyl-normetanephrine for the determination of epinephrine and norepinephrine, respectively. Quantitations were achieved spectrophotometrically using a microwell plate reader at 490 nm with a reference filter of 630 nm (Bio-Tek Instruments, INC. ELX 800, USA). Assay values were calculated from a four-parameter logistics calibration curve. Results were expressed as nmol L−1. The analytical sensitivity was 0.11 nmol L−1 for epinephrine and 0.12 nmol L−1 for norepinephrine. The highest detectable limits were 819 nmol L−1 for epinephrine and 2955 nmol L−1 for norepinephrine. Epinephrine to norepinephrine ratio was also calculated since it was considered as a reliable index of plasma catecholamine response in this study.
Serum vasopressin concentrations were assayed using a competitive immunoassay technique (Assay Designs' Correlate-EIA, MI, USA). This method uses a polyclonal antibody against vasopressin in the sample to bind, in a competitive manner with an alkaline phosphatase molecule, which has covalently attached to vasopressin. The optical density of the samples was measured spectrophotometrically at 405 nm using a microplate reader. Assay values were calculated from a four-parameter logistic calibration curve. Results were expressed as ng L−1. The analytical sensitivity was 3.14 ng L−1 and the highest detectable limit of the method was 926 ng L−1.
Determination of ACE activity in serum samples was performed spectrophotometrically with a commercially available kit (Bühlman, ACE colorimetric, Schönenbuch, Switzerland). The principle of this method is based on the cleavage of a synthetic substrate (N-hippuryl-histidyl-L-leucine) by ACE in the sample to yield hippuric acid. The released hippuric acid is then complexed with cyanuric chloride. The absorbance of the complexed hippuric acid was measured spectrophotometrically at 382 nm (Shimadzu uv-1600, Japan). Results were expressed as ACE units. One unit of ACE activity is defined as the amount of enzyme required to release 1 μmol of hippuric acid per minute per litre of serum at 37°C. The analytical sensitivity of the procedure was 2.6 ACE units.
Data were expressed as mean ± SEM. Statistical evaluations were performed using the SPSS 12.0 statistical package programme. Repeated measured design and Pearson's correlation coefficient tests were used to analyze data. Logarithmic transformations were also made for parameters showing non-normal distribution. Statistical significance was set at P < 0.05.
Five patients in the epinephrine group, four patients in the norepinephrine group, three in the vasopressin group and one in the ACE activity group were excluded because there was insufficient amount of specimen to allow duplicate analysis of each biochemical parameter. Laryngoscopy and endotracheal intubation were easily performed in all patients in <1 min. Arterial oxygen saturation did not fall below 95% in any patient during the procedure.
After the induction of anaesthesia heart rate increased and continued to increase after intubation (P < 0.05) and then decreased significantly at T4 (P < 0.05) (Table 1).
Systolic blood pressure decreased significantly (P < 0.05) after induction and increased slightly after intubation decreasing to below baseline value (P < 0.05) at T4. Diastolic blood pressure increased slightly after intubation and decreased significantly (P < 0.05) at T4 (Table 1).
Plasma epinephrine and norepinephrine concentrations decreased after induction and increased at T3 and T4 without reaching significance. Epinephrine to norepinephrine ratio also showed a similar pattern (Table 2; Figs 1-3). Vasopressin concentrations increased slightly at T2 and T3 and decreased significantly at T4 (P < 0.05) (Table 2; Fig. 4). ACE activity was unaffected when compared with baseline values (Fig. 5).
The main findings of this study indicate that in normotensive patients with standard anaesthesia induction and maintenance:
- heart rate increased after induction and intubation (P < 0.05) and decreased significantly at T4 (P < 0.05);
- systolic blood pressure decreased significantly (P < 0.05) after induction and increased slightly after intubation decreasing to below baseline (P < 0.05) at T4;
- diastolic blood pressure increased slightly after intubation and decreased significantly (P < 0.05) at T4;
- plasma epinephrine and norepinephrine concentrations decreased after induction and increased at T3 and T4;
- vasopressin concentrations increased slightly at T1 and T3 decreasing significantly at T4;
- ACE activity was unaffected when compared with baseline values.
In the present study arterial cannulation was not considered ethical purely for research purposes as well as the concern of its possible effect on blood pressure and heart rate. Blood pressure was measured non-invasively at 1-min intervals during the study period and recordings were evaluated against the corresponding time points. Patients without anticipated difficulties in airway management were included in the study.
The influence of laryngoscopy and intubation on heart rate and blood pressure has long been recognized and many studies to modify it have been published . The magnitude of the haemodynamic changes observed may be dependent on various factors such as the depth of anaesthesia, whether any measure is taken prior to airway manipulation, the anaesthetic agent used , the duration of laryngoscopy and intubation . Cardiovascular responses may be attenuated by the fentanyl used for induction in our study.
To date, the exact mechanism of haemodynamic response to laryngoscopy and intubation has not been clarified. It has been reported to be associated with increased plasma concentrations of catecholamines, mainly norepinephrine and to a lesser extent to epinephrine in some studies [8-12]. But others did not find any increase in catecholamine levels after intubation and did not correlate it with the haemodynamic changes [13,14]. Adrenocorticotrophic hormone (ACTH) and dopamine have also been implicated .
Pernerstorfer and colleagues  found that serum epinephrine levels decreased slightly after induction and remained unaltered after intubation. On the other hand norepinephrine levels were significantly higher after laryngoscopy and intubation.
In contrast to epinephrine, norepinephrine is released in considerable amounts from peripheral nerve endings. Thus plasma norepinephrine concentrations represent adrenergic activity, whereas epinephrine concentrations represent epinephrine mainly derived from the adrenal medulla. For this reason, we also considered the epinephrine to norepinephrine ratio as a reliable index of plasma catecholamine response in this study. This ratio also decreased after induction, increasing after intubation but with no statistical significance.
Vasopressor systems regulating blood pressure to counteract hypotension are the sympathetic system, vasopressin system and renin-angiotensin system each acting on the vascular smooth muscle cell [17,18]. Therefore they may be responsible for the hyperdynamic response to airway manipulation. So we decided to measure their plasma levels to find out if any of them had a role.
Vasopressin, a vasoactive hormone, contributes to acute blood pressure regulation as well as catecholamines and the renin-angiotensin system. Increased plasma levels of vasopressin have been reported in some previous studies dealing with haemodynamic responses to thoracic surgery [19,20]. Höhne and colleagues also showed an elevation in vasopressin levels during spinal anaesthesia in patients on long-term treatment with ACE inhibitors. They related the increase in vasopressin levels to the compensatory mechanism against an inhibited renin-angiotensin system . In the present study, we observed a slight increase in vasopressin concentrations at 2 min after intubation (T3). This may be due to a response to counteract a decrease in blood pressure probably by replacing the angiotensin II concentrations since we observed unaffected ACE activity during defined time points, a finding which might result in unaltered angiotensin II levels.
On the other hand, the only significant change among neuroendocrine parameters in this study was the decline in vasopressin concentrations at T4 when compared to T3. Also both systolic and diastolic blood pressures showed a significant decrease at the respective time points. The significant reduction of blood pressures observed at 5 min after intubation might have probably resulted from the sharp decline in vasopressin levels.
Besides catecholamines, vasopressin and the renin-angiotensin system, also other regulators of circulatory homeostasis might have played a role in haemodynamic response to intubation. Among them, endothelin I is known to have a potent vasoconstrictor effect, even greater than that of vasopressin and norepinephrine. Also, interaction of endothelin I with other vasoactive substances such as inhibition of release of catecholamines and renin might have further influenced the plasma concentrations of vasoactive hormones and ACE activity in this study .
In conclusion, in normotensive ASA I patients following standard induction of anaesthesia including fentanyl, plasma levels of catecholamines and vasopressin, and ACE activity regarded as representing the vasopressor response did not show any significant increase following laryngoscopy and intubation.
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