Article Type: Original article
Pharmacogenetics, Plasma Concentrations, Clinical Signs and EEG during Propofol Treatment
Muhammad Suleman Khan1, Eva-Lena Zetterlund2, Henrik Gréen1,3,4, Anna Oscarsson2, Anna-Lena Zackrisson4, Eva Svanborg5, Maj-Lis Lindholm6, Harald Persson6 and Christina Eintrei2 1
Clinical Pharmacology, Division of Drug Research, Faculty of Health Sciences, Linköping University, Linköping, Sweden 2 Anesthesiology, Division of Drug Research, Faculty of Health Sciences, Linköping University, Linköping, Sweden 3 Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, KTH Royal Institute of Technology, Solna, Sweden 4 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden 5 Department of Clinical Neurophysiology, Linköping University Hospital and IKE; Linköping University, Linköping, Sweden 6 Department of Anaesthesia & Intensive Care, Kalmar Hospital, Sweden Author for correspondence: Christina Eintrei, Anesthesiology, Division of Drug Research Department of Medical and Health Sciences Faculty of Health Sciences, Linköping University, SE-581 85, Linköping, Sweden (fax:, e-mail: [email protected]). Running title: Propofol and depth of anaesthesia
Abstract: A variety of techniques have been developed to monitor the depth of anaesthesia. Propofol's pharmacokinetics and response vary greatly, which might be explained by genetic polymorphisms. We investigated the impact of genetic variations on dosage, anaesthetic depth, and recovery after total intravenous anaesthesia with propofol. A total of 101 patients were enrolled in the study. The plasma concentration of propofol during anaesthesia was measured using high-performance liquid chromatography. EEG was monitored during the surgical procedure as a measure of anaesthetic depth. Pyrosequencing This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12276 This article is protected by copyright. All rights reserved.
was used to determine genetic polymorphisms in CYP2B6, CYP2C9, the UGTIA9-promotor and the GABRE gene. The correlation between genotype and to plasma concentration at the time of loss of consciousness (LOC), the total induction dose, the time to anaesthesia, eye opening and clearance were investigated. EEG monitoring showed that the majority of the patients had not reached a sufficient level of anaesthetic depth (sub-delta) at the time of loss of consciousness despite a high induction dose of propofol. Patients with UGT1A9-331C/T had a higher propofol clearance than those without (p=0.03) and required a higher induction dose (p=0.03). The patients with UGT1A9 -1818T/C required a longer time to LOC (p=0.03). The patients with CYP2C9*2 had a higher concentration of propofol at the time of LOC (p=0.02). The polymorphisms in the metabolizing enzymes and the receptor could not explain the large variation seen in the pharmacokinetics of propofol and the clinical response seen. At LOC, the patients showed a large difference in EEG pattern.
The clinical signs of loss of consciousness (LOC) during induction of anaesthesia include hypoventilation and the loss of verbal response and eyelash reflex [1]. The transition from consciousness to unconsciousness is a complex phenomenon involving working memory, motor control, respiration and cardio-vascular performance. It is well known that the time as well as the anaesthetic dose needed for LOC (measured as loss of eyelash reflex) varies widely between patients. Additionally, anaesthesia-induced LOC can be detected by typical electroencephalogram (EEG) patterns consisting of low frequency and high-amplitude activity [2]. In the last 10–15 years, a variety of EEG-based techniques have been developed for monitoring the depth of anaesthesia. None of these techniques have been proven to distinguish between consciousness and unconsciousness in individual patients in a validated
This article is protected by copyright. All rights reserved.
and reliable way [3]. The gold standard to assess cerebral activity and anaesthetic depth therefore remains the traditional EEG. Propofol (2,6-diisopropylphenol) exerts its pharmacological action through the activation of the GABAA receptor [4] by inhibiting the nerve impulse in the neural network in the brain. Although the major molecular target for propofol effect in the brain, like the GABAA-R is known, we still lack the knowledge as to how propofol disrupts neural transmission and stops the nerve impulse from travelling between neurons. It is possible that gene polymorphism in the GABAA receptor gene might have an impact on the pharmacological response to propofol anaesthesia. Propofol is used because of its rapid induction of anaesthesia (30-60 sec.) and fast recovery (4 to 6 min.) [5, 6]. The majority of anaesthetic agents, including propofol, are metabolized in the liver by the cytochrome P450 superfamily enzymes (CYPs) and phase II drug-metabolizing enzymes: glutathione S-transferases (GSTs), sulphotransferases (SULTs), UDP-glucuronosyl transferases (UGTs) and NAD(P) quione oxido-reductase (NQO1) [7]. Important genes associated with metabolism for propofol are: GABRG2, UGT1A9, CYP2B6, CYP2C9, GSTP1, SULT1A1 and NQO1 [7]. Over 90 % of the propofol dose is metabolized in the liver and the metabolites are eliminated through urinary excretion. About 70% of propofol is metabolized into propofol glucuronide, by UDP-glucuronosyl transferase encoded by UGT1A9 gene. Another pathway of propofol biotransformation (approx. 29%) is by the enzymes coded CYP2B6, CYP2C9 and SULT1A [7]. Other enzymes contribute minimally to the elimination of propofol. Clinically, it is well known that here is a marked inter-individual variability in the hypnotic dose of propofol. This could be due to several factors such as age, gender, genetics, ethnicity, stage of anxiety, and the use and/or abuse of drugs [5, 8-13]. As the pharmacokinetics of propofol seem to be dependent on a large number of enzymes encoded by polymorphic genes, we decided to identify whether the pharmacokinetics and the clinical response to propofol could be explained by genetic polymorphism. Prior studies have
This article is protected by copyright. All rights reserved.
shown a good correlation between the pharmacokinetics and the pharmacodynamics of propofol and clinical parameters such as age and gender [8, 9, 13]. In order to understand the reason for this inter-individual variability in the clinical response to propofol, we decided to investigate the impact of single nucleotide polymorphisms (SNPs) in CYP2B6, CYP2C9, UGT1A9 and GABRE on variations seen in the clinical response to propofol. Additionally, we wanted to compare the signs of depth of anaesthesia with EEG monitoring [14].
Methods The Regional Ethical Review Board, University Hospital, Linköping, approved this study (M184-07), and written informed consent was obtained from all participants. The study was registered in a Clinical Trials database (NCT 01029379). One hundred and three patients scheduled for elective surgery (minor orthopaedic-, plastic and hand-, back-, gynaecological-, endocrinological- and ENT-surgery) under general anaesthesia at the University Hospital in Linköping and the Regional County Hospital in Kalmar, Sweden, were enrolled in this study. Inclusion criteria were age 18–50 years, BMI 20–30, American Society of Anesthesiology (ASA) classification I, Caucasian origin, and planned surgery >30 min. Exclusion criteria were allergy to propofol, soya beans or peanuts; smoking; pregnancy; a history of alcohol and/or drug addiction, and the use of analgesics within 12 hr prior to surgery. No anxiolytic pre-medication was given, and the patients were monitored with three-lead ECG, noninvasive blood pressure, and pulsoximetry and end-tidal carbon dioxide. Intravenous access was achieved and one catheter was used for administering anaesthetics, and the second was used for blood sampling. Blood samples for measurement of s-albumin and for DNA extraction were obtained prior to induction of anaesthesia.
This article is protected by copyright. All rights reserved.
Anaesthetic method An infusion pump (AlarisTM TIVA care fusion, Rolle, Switzerland) with propofol (Propofol LipuroTM 10 mg/ml B. Braun AG, Melsungen, Germany) was connected to a peripheral IV catheter. Following pre-oxygenation and during continuous EEG monitoring, propofol infusion was commenced with an infusion rate of 300 ml/h until the patient lost consciousness, measured as loss of eyelash reflex. After loss of consciousness, the infusion rate of propofol was reduced to the pre-determined infusion rate based on weight and age. Continuous EEG recording was obtained until 10 min. after the clinical criterion for adequate anaesthetic depth, no response to a verbal command, was fulfilled. The time from the start of propofol infusion until LOC (loss of eyelash reflex) was documented i.e. time to anaesthesia, and blood samples to determine the plasma concentration of propofol were subsequently taken. A laryngeal mask or an endotracheal tube was inserted at this time point and the surgery commenced. Following the end of EEG monitoring, the patients received remifentanil 25 μg/kg/min. Twenty minutes after LOC, a second blood sample for measurement of plasma concentration of propofol was obtained. This was repeated at 30 min. The clearance of propofol was then calculated as the infusion rate divided by the mean plasma concentration at 20 and 30 min. At the end of surgery, morphine was administered in a dose depending on the severity of the surgery. Paracetamol and, in some cases, NSAIDs were also given as analgesics for postoperative pain. The time from termination of the propofol infusion until recovery of consciousness (ROC), defined as eye opening on command, was documented. Blood samples for plasma concentrations of propofol and genetic analysis were frozen to 70°C until the time of analyses. Serum albumin in the blood sample was analysed directly after surgery.
This article is protected by copyright. All rights reserved.
Electroencephalographic monitoring & analysis EEG recordings were performed on a NicoletOne Neurodiagnostic system (Viasys, CareFusion Inc., San Diego, USA). A bipolar montage of the electrodes F3-T3 and F4-T4, 6 electrodes in all, was used. All recordings were later analysed by the same clinical neurophysiologist who was blinded to the events at the time of surgery (e.g., when the injection of the anaesthetic was given and when signs of clinical anaesthesia were reached). The EEG was manually scored in 10-sec. epochs and classified into five different stages: The EEG changes as a patient passes into deeper planes of general anaesthesia [15]. EEG slowing generally accompanies loss of consciousness. Some anaesthetics among which propofol is one will induce CNS excitation during the phase of initiation of anaesthesia, with increased oscillatory activity in the higher beta-frequency bands (12.5–25 Hz) and decreased activity in slower frequency bands (3.5 – 12.5 Hz) [16]. This state is marked by disinhibition and loss of both motor and affective control [17]. With deeper sedation, the EEG will become slower with increased delta activity (1-3 Hz) and also subdelta activity (
Pharmacogenetics, Plasma Concentrations, Clinical Signs and EEG during Propofol Treatment
Muhammad Suleman Khan1, Eva-Lena Zetterlund2, Henrik Gréen1,3,4, Anna Oscarsson2, Anna-Lena Zackrisson4, Eva Svanborg5, Maj-Lis Lindholm6, Harald Persson6 and Christina Eintrei2 1
Clinical Pharmacology, Division of Drug Research, Faculty of Health Sciences, Linköping University, Linköping, Sweden 2 Anesthesiology, Division of Drug Research, Faculty of Health Sciences, Linköping University, Linköping, Sweden 3 Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, KTH Royal Institute of Technology, Solna, Sweden 4 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden 5 Department of Clinical Neurophysiology, Linköping University Hospital and IKE; Linköping University, Linköping, Sweden 6 Department of Anaesthesia & Intensive Care, Kalmar Hospital, Sweden Author for correspondence: Christina Eintrei, Anesthesiology, Division of Drug Research Department of Medical and Health Sciences Faculty of Health Sciences, Linköping University, SE-581 85, Linköping, Sweden (fax:, e-mail: [email protected]). Running title: Propofol and depth of anaesthesia
Abstract: A variety of techniques have been developed to monitor the depth of anaesthesia. Propofol's pharmacokinetics and response vary greatly, which might be explained by genetic polymorphisms. We investigated the impact of genetic variations on dosage, anaesthetic depth, and recovery after total intravenous anaesthesia with propofol. A total of 101 patients were enrolled in the study. The plasma concentration of propofol during anaesthesia was measured using high-performance liquid chromatography. EEG was monitored during the surgical procedure as a measure of anaesthetic depth. Pyrosequencing This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12276 This article is protected by copyright. All rights reserved.
was used to determine genetic polymorphisms in CYP2B6, CYP2C9, the UGTIA9-promotor and the GABRE gene. The correlation between genotype and to plasma concentration at the time of loss of consciousness (LOC), the total induction dose, the time to anaesthesia, eye opening and clearance were investigated. EEG monitoring showed that the majority of the patients had not reached a sufficient level of anaesthetic depth (sub-delta) at the time of loss of consciousness despite a high induction dose of propofol. Patients with UGT1A9-331C/T had a higher propofol clearance than those without (p=0.03) and required a higher induction dose (p=0.03). The patients with UGT1A9 -1818T/C required a longer time to LOC (p=0.03). The patients with CYP2C9*2 had a higher concentration of propofol at the time of LOC (p=0.02). The polymorphisms in the metabolizing enzymes and the receptor could not explain the large variation seen in the pharmacokinetics of propofol and the clinical response seen. At LOC, the patients showed a large difference in EEG pattern.
The clinical signs of loss of consciousness (LOC) during induction of anaesthesia include hypoventilation and the loss of verbal response and eyelash reflex [1]. The transition from consciousness to unconsciousness is a complex phenomenon involving working memory, motor control, respiration and cardio-vascular performance. It is well known that the time as well as the anaesthetic dose needed for LOC (measured as loss of eyelash reflex) varies widely between patients. Additionally, anaesthesia-induced LOC can be detected by typical electroencephalogram (EEG) patterns consisting of low frequency and high-amplitude activity [2]. In the last 10–15 years, a variety of EEG-based techniques have been developed for monitoring the depth of anaesthesia. None of these techniques have been proven to distinguish between consciousness and unconsciousness in individual patients in a validated
This article is protected by copyright. All rights reserved.
and reliable way [3]. The gold standard to assess cerebral activity and anaesthetic depth therefore remains the traditional EEG. Propofol (2,6-diisopropylphenol) exerts its pharmacological action through the activation of the GABAA receptor [4] by inhibiting the nerve impulse in the neural network in the brain. Although the major molecular target for propofol effect in the brain, like the GABAA-R is known, we still lack the knowledge as to how propofol disrupts neural transmission and stops the nerve impulse from travelling between neurons. It is possible that gene polymorphism in the GABAA receptor gene might have an impact on the pharmacological response to propofol anaesthesia. Propofol is used because of its rapid induction of anaesthesia (30-60 sec.) and fast recovery (4 to 6 min.) [5, 6]. The majority of anaesthetic agents, including propofol, are metabolized in the liver by the cytochrome P450 superfamily enzymes (CYPs) and phase II drug-metabolizing enzymes: glutathione S-transferases (GSTs), sulphotransferases (SULTs), UDP-glucuronosyl transferases (UGTs) and NAD(P) quione oxido-reductase (NQO1) [7]. Important genes associated with metabolism for propofol are: GABRG2, UGT1A9, CYP2B6, CYP2C9, GSTP1, SULT1A1 and NQO1 [7]. Over 90 % of the propofol dose is metabolized in the liver and the metabolites are eliminated through urinary excretion. About 70% of propofol is metabolized into propofol glucuronide, by UDP-glucuronosyl transferase encoded by UGT1A9 gene. Another pathway of propofol biotransformation (approx. 29%) is by the enzymes coded CYP2B6, CYP2C9 and SULT1A [7]. Other enzymes contribute minimally to the elimination of propofol. Clinically, it is well known that here is a marked inter-individual variability in the hypnotic dose of propofol. This could be due to several factors such as age, gender, genetics, ethnicity, stage of anxiety, and the use and/or abuse of drugs [5, 8-13]. As the pharmacokinetics of propofol seem to be dependent on a large number of enzymes encoded by polymorphic genes, we decided to identify whether the pharmacokinetics and the clinical response to propofol could be explained by genetic polymorphism. Prior studies have
This article is protected by copyright. All rights reserved.
shown a good correlation between the pharmacokinetics and the pharmacodynamics of propofol and clinical parameters such as age and gender [8, 9, 13]. In order to understand the reason for this inter-individual variability in the clinical response to propofol, we decided to investigate the impact of single nucleotide polymorphisms (SNPs) in CYP2B6, CYP2C9, UGT1A9 and GABRE on variations seen in the clinical response to propofol. Additionally, we wanted to compare the signs of depth of anaesthesia with EEG monitoring [14].
Methods The Regional Ethical Review Board, University Hospital, Linköping, approved this study (M184-07), and written informed consent was obtained from all participants. The study was registered in a Clinical Trials database (NCT 01029379). One hundred and three patients scheduled for elective surgery (minor orthopaedic-, plastic and hand-, back-, gynaecological-, endocrinological- and ENT-surgery) under general anaesthesia at the University Hospital in Linköping and the Regional County Hospital in Kalmar, Sweden, were enrolled in this study. Inclusion criteria were age 18–50 years, BMI 20–30, American Society of Anesthesiology (ASA) classification I, Caucasian origin, and planned surgery >30 min. Exclusion criteria were allergy to propofol, soya beans or peanuts; smoking; pregnancy; a history of alcohol and/or drug addiction, and the use of analgesics within 12 hr prior to surgery. No anxiolytic pre-medication was given, and the patients were monitored with three-lead ECG, noninvasive blood pressure, and pulsoximetry and end-tidal carbon dioxide. Intravenous access was achieved and one catheter was used for administering anaesthetics, and the second was used for blood sampling. Blood samples for measurement of s-albumin and for DNA extraction were obtained prior to induction of anaesthesia.
This article is protected by copyright. All rights reserved.
Anaesthetic method An infusion pump (AlarisTM TIVA care fusion, Rolle, Switzerland) with propofol (Propofol LipuroTM 10 mg/ml B. Braun AG, Melsungen, Germany) was connected to a peripheral IV catheter. Following pre-oxygenation and during continuous EEG monitoring, propofol infusion was commenced with an infusion rate of 300 ml/h until the patient lost consciousness, measured as loss of eyelash reflex. After loss of consciousness, the infusion rate of propofol was reduced to the pre-determined infusion rate based on weight and age. Continuous EEG recording was obtained until 10 min. after the clinical criterion for adequate anaesthetic depth, no response to a verbal command, was fulfilled. The time from the start of propofol infusion until LOC (loss of eyelash reflex) was documented i.e. time to anaesthesia, and blood samples to determine the plasma concentration of propofol were subsequently taken. A laryngeal mask or an endotracheal tube was inserted at this time point and the surgery commenced. Following the end of EEG monitoring, the patients received remifentanil 25 μg/kg/min. Twenty minutes after LOC, a second blood sample for measurement of plasma concentration of propofol was obtained. This was repeated at 30 min. The clearance of propofol was then calculated as the infusion rate divided by the mean plasma concentration at 20 and 30 min. At the end of surgery, morphine was administered in a dose depending on the severity of the surgery. Paracetamol and, in some cases, NSAIDs were also given as analgesics for postoperative pain. The time from termination of the propofol infusion until recovery of consciousness (ROC), defined as eye opening on command, was documented. Blood samples for plasma concentrations of propofol and genetic analysis were frozen to 70°C until the time of analyses. Serum albumin in the blood sample was analysed directly after surgery.
This article is protected by copyright. All rights reserved.
Electroencephalographic monitoring & analysis EEG recordings were performed on a NicoletOne Neurodiagnostic system (Viasys, CareFusion Inc., San Diego, USA). A bipolar montage of the electrodes F3-T3 and F4-T4, 6 electrodes in all, was used. All recordings were later analysed by the same clinical neurophysiologist who was blinded to the events at the time of surgery (e.g., when the injection of the anaesthetic was given and when signs of clinical anaesthesia were reached). The EEG was manually scored in 10-sec. epochs and classified into five different stages: The EEG changes as a patient passes into deeper planes of general anaesthesia [15]. EEG slowing generally accompanies loss of consciousness. Some anaesthetics among which propofol is one will induce CNS excitation during the phase of initiation of anaesthesia, with increased oscillatory activity in the higher beta-frequency bands (12.5–25 Hz) and decreased activity in slower frequency bands (3.5 – 12.5 Hz) [16]. This state is marked by disinhibition and loss of both motor and affective control [17]. With deeper sedation, the EEG will become slower with increased delta activity (1-3 Hz) and also subdelta activity (
