β 2 Adrenoceptor (ADRB2) Pharmacogenetics and Cardiovascular Phenotypes during Laryngoscopy and Tracheal Intubation

Abstract

Pharmacogenetics is the study of genetic variations that cause a variable drug response characterized by alteration in drug metabolism or in pharmacodynamics. The polymorphisms in genes encoding receptors relevant to treatment cause variation in sensitivity to many drugs. β2 Adrenoceptor genetic variation contributes to regulation of blood pressure and hemodynamic changes by mediating peripheral vasodilatation. Laryngoscopy and tracheal intubation associated with hemodynamic changes. Although there are four nonsynomic single-nucleotide polymorphisms (SNPs) of β2 adrenoceptor gene, codon 16 (Arg16Gly) and codon 27 (Gln27Glu) SNPs are both common and functionally important. In this paper, the authors investigated the β2 adrenoceptor Gly16 and Glu27 SNPs in response to drugs relevant to anesthesia and how these SNPs impacted upon the cardiovascular phenotypes. The authors measured arterial systolic and diastolic blood pressure, heart rate, and rate-pressure product before induction of anesthesia and 1 min following laryngoscopy and tracheal intubation. Genomic DNA was amplified and genotyped using allele-specific polymerase chain reaction (ASPCR) and restriction fragment length polymorphism (RFLP) assays, respectively. When the authors compared hemodynamic results according to genotypes, the patients with Gln homozygote allele at codon 27 exhibited significant increase of heart rate than patients with Glu allele after laryngoscope and tracheal intubation.

The promise of pharmacogenetics (pharmacogenomics) is that by examining the single-nucleotide polymorphism (SNP) in genes that determine drug behavior, an appealing alternative can be offered to the patient who could suffer a severe adverse reaction to medication (Galley, Mahdy, and Lowe 2005). This science can be used to predict whether a patient will have a good or a bad or no response at all to a drug. Pharmacogenomics offers tailor-made drugs for individuals (right, efficient, and safe drugs) depending on their genetic makeup (www.nchi.nlm.nih.gov), so as to optimize therapeutic benefit and minimize the potential for toxicity (Anderson et al. 2003). The realization of personalized medicine, or the fine tailoring of the practice of medicine to an individual, is being fostered through numerous efforts aimed at characterizing individual differences in the molecular processes underlying disease pathogenesis, disease progression, and the response to therapeutics (McLeod and Evans 2001; Meyer and Ginsburg 2002). Early insights into clinical impact of pharmacogenetics were provided by investigations into adverse effects of anesthetic substance such as succinylcholine apnea and thiopental-induced porphyria (Iohom, Fitzgerald, and Cunningham 2004).

Laryngoscopy and tracheal intubation by direct vision using a laryngoscope is associated with hemodynamic changes including hypertension, tachycardia, and dysrhythmias mediated by noxious stimulus that provokes sympathoadrenal responses. Mechanical stimulation of the epipharyngeal and laryngopharyngeal regions increases cervical sympathetic activity (Hung 2001; Stone and Gal 2000). The circulatory responses to direct laryngoscopy and tracheal intubation associated with changes in plasma catecholamine are well recognized. Measurements of the plasma concentrations of catecholamine have consistently demonstrated increases in noradrenaline following laryngoscopy. Although a reflex response to a noxious stimulus due to tracheal intubation is mediated at the subcortical levels of the brain, peripheral stimuli reaches the brain through the ascending reticular-activating systems of the brain. It is well recognized that laryngoscopic tracheal intubation is associated with hypertension and tachycardia mediated by sympathetic stress responses (Fox, Sklar, and Hill 1977; Derbyshire et al. 1983; Low et al. 1986; Shribman, Smith, and Achola 1987; Edwards et al. 1994; Kayhan et al. 2005).

The β2 adrenoceptor (ADRB2) is a G protein–coupled protein family comprised of seven-transmembrane-domain receptors that is widely distributed across the tissues, including the heart, kidney, and vascular smooth muscle cells. ADRB2 contributes to the regulation of blood pressure by mediating cardiac chronotropy, renal sodium excretion, vascular tone, and actions of catecholamine in various tissues and organs (Johnson 1998; Busjahn et al. 2000). ADRB2 agonists induce vasodilatation following the activation of receptors, which are present on both endothelium and vascular smooth muscle (Littlejohn et al. 2002). The ADRB2 genes illustrate a link between genetic polymorphisms in drug targets and clinical responses. Genetic polymorphism of the β-adrenoreceptor can alter the process of signal transduction by these receptors (Evans and McLeod 2003). The ADRB2 is encoded by an intronless single gene product of just over 1200 bp. This gene is located on chromosome 5q31–32 (Kobilka et al. 1987). At least 13 distinct SNPs have been identified in ADRB2. This finding has led to evaluation of the importance of haplotype structure as compared with individual SNPs in determining receptor function and pharmacologic response (Evans and McLeod 2003). Although four amino acid polymorphisms have been reported within the open reading frame, only two of them are both functionally important (Hall 1996). These are two common coding SNPs within the amino-terminal extracellular domain of the ADRB2 gene. One of which leads to the substitution of glycine (Gly) for arginine (Arg) at amino acid position 16 (Arg16Gly); the other results in substitution of glutamate (Glu) for glutamine (Gln) at amino acid position 27 (Gln27Glu) (Littlejohn et al. 2002; Hall 1996). Both of them have been found to alter the receptor function significantly. It has been shown that the Gly16 mutant allele displays enhanced agonist-promoted down-regulation, whereas the Glu27 mutant allele is resistant to down-regulation compared with alleles of ADRB2 gene (Gratze et al. 1999). Racial differences in allele and genotype frequencies of ADRB2 polymorphisms can potentially explain the difference in ADRB2-mediated vasodilatation and impact hemodynamics underlying blood pressure regulation. The SNPs of ADRB2 have been studied extensively and certain polymorphisms are associated with hypertension or blood level (Busjahn et al. 2000; Gratze et al. 1999; Candy et al. 2000; Snieder et al. 2002; Timmermann et al. 1998; Rosmond et al. 2000; Hoit et al. 2000). However, overall the findings are inconsistent.

To our knowledge, there is only one study on the association of ADRB2 polymorphism with pressure response to laryngoscopy and tracheal intubation. In that study, it was suggested that this genetic variability in the human ADRB2 gene polymorphisms might be associated with this interaction (Kim et al. 2002). The main purpose of this study was to examine the effects of the Arg16Gly and Gln27Glu polymorphisms on hemodynamic response in patients scheduled for elective surgery and required tracheal intubation.

MATERIAL AND METHODS

Study Groups

After receiving the institutional approval and informed consent, 103 adult patients (ASA physical status I–II) undergoing elective surgery and required tracheal intubation were enrolled. Patients with cardiovascular and neurological diseases, cerebral pathology, altered conscience, history of hypertension, and epilepsy or muscle disorder, were not included. All patients received 0.07 mg kg⁻¹ intramuscular (IM) midazolam as premedicant approximately 30 min before induction of anesthesia. Routine monitoring, including blood pressure (BP), electrocardiogram (ECG), and SpO₂ (oxygen saturation), was conducted. Hypertension was defined as systolic blood pressure >160 mm Hg and/or diastolic blood pressure >95 mm Hg. Baseline readings of arterial pressure and heart rate were recorded after a stabilization period of 5 min. Anesthesia was induced with 1.5 μg kg⁻¹ intravenous (IV) fentanyl as a bolus, followed by thiopental (5 to 7 mg kg⁻¹ IV) titrated with additional boluses of 50 mg sufficient to abolish the corneal reflex and apnea. Manual ventilation via face mask (O₂, 100%) was established to the patients. Vecuronium 0.1 mg kg⁻¹ was injected in 5 s to facilitate tracheal intubation. The lungs were ventilated for 3 min with oxygen 100%, by means of a face mask. Three minutes after induction, direct laryngoscopy with a standard Macintosh laryngoscope blade was performed and tracheal intubation was undertaken. All laryngoscopic tracheal intubations were performed by the same investigator and accomplished within 30 s. Intubation conditions were evaluated according to Good Clinical Research Practice (GCRP). Acceptable intubating conditions, as argued in GCRP, are obviously an appropriate end point and represent the goal of an adequate anesthetic induction (Viby-Mogensen et al. 1996). The systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR), and rate-pressure product (RPP) were measured before endotracheal intubation (T₀) and 1 min following intubation (T₁). The RPP was calculated by multiplying systolic arterial pressure by HR. The percentage change expressed as (T₁ – T₀) × 100/T₀.

DNA Extraction and Genotype Analysis

Whole blood samples were collected from study population. All participants completed a questionnaire assisted by physician included information on age, sex, height, weight, occupation, history of cigarette smoking, and alcohol consumption.

Whole blood samples (≅5 ml) were removed via venepuncture into tubes containing EDTA and samples were stored at −20°C until DNA extraction. Genomic DNA was extracted from whole blood using sodium perchlorate/chloroform extraction.

The ADRB2 alleles were detected by modification of the method described by Martinez et al. (1997). Allele-specific polymerase chain reaction (ASPCR) and restriction fragment length polymorphism (RFLP) assays were used to determine the genotypes, respectively. The sequences of the PCR primers were 5′-GCCTTCTTGCTGGCACCC C AT-3′ for the forward and 5′-CAGAGCACATCAATGGAAGTC-3′ for the reverse primer. The underlined base was modified from the reported sequence to create NcoI restriction sites on the polymerase chain reaction (PCR) product generated from the Gly16 allele but not from the Arg16. A 36-μl PCR reaction was performed using approximately 0.2 to 0.5 μg genomic DNA, 30 ng each primer, 200 μM of each dNTP, 2.5 mM MgCl₂, and 3.75 U Taq polymerase (Promega, Madison, WI) in the 1 × PCR buffer supplied by the manufacturer (50 mM KCl, 10 mM Tris-HCl, pH 9, 0.1% Triton X-100). Amplification was carried out for 1 cycle by first denaturing at 96°C for 2 min, and then for 35 cycles by denaturing at 96°C for 25 s, annealing at 58°C for 25 s, extending at 72°C for 35 s, and final extention at 72°C for 10 min for 1 cycle. A PCR product of 318 bp was obtained. The same PCR aliquots were used for different restriction enzyme digests to identify the Arg16 and Glu27 SNPs. To detect the Arg16 allele, 10 μl PCR products were digested with 2U NcoI (New England Biolabs) and 2U BtsI (NEBiolabs) in 2 μl 1 × NEB4 buffer at 37°C, and analyzed by electrophoresis on 3% Nusieve agarose gels, followed by visualization of the bands with ethidium bromide. NcoI cut 18 bp from the 5′ end, and BtsI cuts 236 bp from the 5′ end of the Gly16 allele. Glu27 allele was detected by restriction digestion with 0.9 U BbvI (NEBiolabs) in 2 μl 1 × NEB2 buffer at 37°C. BbvI digests only Gln27 allele to produce 105-bp and 63-bp fragments, which are separated from the uncut Glu27 allele on 3% Nusieve agarose gels.

Statistical Analysis

The physical variables were expressed as mean ± standard deviation (SD). The differences of SBP, DBP, MAP, HR, and RPP were compared using the Wilcoxon test. The difference of percentage change according to Arg16Gly and Gln27Glu genotypes was compared by Kruskal-Wallis analysis of variance (ANOVA). The relationship between Arg16Gly and Gln27Glu genotypes and percentage change of MAP, HR, and RPP was expressed as β coefficients and its associated 95% confidence interval. Multiple linear regression was used to evaluate independence of Arg16Gly and Gln27Glu with respect to changes in MAP, HR, and RPP. Analyses were performed by SPSS version 11.5 software (Chicago, USA). A p value of <.05 was considered significant.

RESULT

Our study population included 103 patients (43 male, 60 female), 77 (68%) of whom were nonsmoking individuals. The mean of ages was 38 (± 12 SD; range 18 to 65 years). Physical variables of the study population are given in Table 1. The allelic frequencies of mutant Gly16 and Glu27 alleles were found to be 66% and 51%, respectively. The frequencies of Arg/Arg (wild-type), Arg/Gly (heterozygote), Gly/Gly (mutant) genotypes at codon 16 were found to be 0.49, 0.34, 0.18, whereas Gln/Gln (wild-type), Gln/Glu (heterozygote), Glu/Glu (mutant) genotypes at codon 27 were 0.32, 0.38, 0.30 in the study population. The genotype frequencies of ADRB2 16 and 27 SNPs are shown in Table 2.

We detected significant increases in SBP, DBP, MAP, HR and RPP following laryngoscopy and tracheal intubation (Table 3). When we examined the percent change of SBP, DBP, MAP, HR, and RPP according to genotypes, only in cases of ADRB2 27 (Gln/Gln, Gln/Glu, Glu/Glu) that we observed significantly high percentage change of HR in Gln/Gln genotypes (26.66) in comparison to Glu/Glu and Gln/Glu genotypes (15.85, 15.00, respectively) (Table 4).

We performed a multiple linear regression model with the independent variables of ADRB2 16 and ADRB2 27 SNPs and dependent variables of the percentage changes in MAP, HR, and RPP. According to this model, there was no association between these variables in terms of MAP. However, when we used the percentage change of HR as a dependent variable, HR was significantly greater in patients with GlyGly genotype in ADRB2 16 SNPs than those with the ArgArg genotype. On contrary, in the case of ADRB2 27, the significant correlation was found in patients with GlnGln genotype. When we used the percent change of RPP as a dependent variable, RPP was significantly greater in patients with GlnGln genotype in ADRB2 27 SNPs than those with the GluGlu genotype. In addition, RPP was significantly greater in patients with GlnGlu genotype than those with the GluGlu genotype (Table 5).

When we performed power analysis depending on the multiple linear regression of percent change of HR, there was 92% power in case of alpha = 0.05 and 78% power in control of alpha = 0.01.

DISCUSSION

Pharmacogenetics will have its application in clinical research. Clinical investigations in various populations will help clarify interethnic and interindividual differences in response to given anesthesia. The differences can be explained by genetically determined ones in drug receptors (Iohom, Fitzgerald, and Cunningham 2004).

The ADRB2 gene locus is linked to the quantitative traits of SBP, DBP, and cardiac size, indicating that this gene locus is a quantitative trait locus for blood pressure and heart size in normotensive individuals (Snieder et al. 2002). In consideration of physiological importance of the ADRB2 gene, it was shown that functional molecular variations of the gene cause attenuated vasodilatation and influences catecholamine production, leading to increased total peripheral resistance and hence ultimately resulting in hypertension (Johnson 1998; Busjahn et al. 2000; Liitlejohn et al. 2002).

ADRB2 polymorphisms with Gly or Arg at codon 16 (Arg16Gly) and Glu or Gln at codon 27 can alter physiological and pharmacological responses to ADRB2-mediated stimulation (Gren et al. 1994). The Gly16 polymorphism appears to be highly labile in recombinant and endogenously expressing cells and compared with the Arg16 receptor, displays a marked decrease in expression in the presence of catecholamine. Gly16 allele also indicates an increased propensity for down-regulation of the receptor. Such a down-regulation pattern could lead to impaired vasodilatation in peripheral arteries in response to peripheral vasoconstriction. Normal individuals with the Gly16 polymorphisms, compared with the Arg16 genotype have attenuated vasodilator responses (plethysmographic blood flow and vascular resistance) to β ₂-agonist stimulation (Snieder et al. 2002; Hoit et al. 2000; Li et al. 2001). According to in vitro studies, the Glu27 allele of the ADRB2 exhibits a strong resistance towards agonist-promoted down-regulation, such that the responsiveness in individuals with the Glu27 polymorphism should be greater than in those with the Gln27 allele (Leineweber et al. 2004). In vitro and ex vivo studies had shown that the functional properties of the Arg16Gly and Gln27Glu alleles of the ADRB2 did not differ from those of the wild-type receptor; the existing in vivo data regarding ADRB2-mediated changes in heart rate, contractility, and blood pressure in general confirmed these findings. The conditional analysis supports the notion that the Arg16Gly polymorphism is responsible for the effects on blood pressure and heart size rather than the Gln27Glu polymorphism (Snieder et al. 2002). In the recent study, after screening many candidate SNPs of postural tachycardia syndrome, Nickander et al. (2005) showed that a significant association of HR and DBP with Gly16 or Glu27 SNPs could aggravate orthostatic tachycardia by excessive venous pooling.

The interpretation of cardiovascular responses to anesthetic drugs becomes even more difficult when a noxious intervention, such as a laryngoscopic tracheal intubation, introduces confounding variables. These include not only the magnitude and duration of the noxious intervention, but also the variables introduced by various medical conditions, such as cardiovascular diseases and diabetes (Hung 2001). Low et al. (1986) demonstrated that following laryngoscopy there was an increase in systolic pressure in normotensive patients. To determine the stress response to laryngoscopy, in that study, the diastolic pressures were also high following laryngoscopy. The maximum pressures were recorded 1 min following laryngoscopy, and returned to their control values 5 min after laryngoscopy. McCoy, Mirakhur, and McCloskey (1995) compared HR, MAP, and plasma nor-adrenaline and adrenaline concentrations before and at laryngoscopy, and 1, 3, and 5 min later. They found a significant increase in both HR (33%) and MAP (27%) after laryngoscopy using the Macintosh blade.

In our study, we showed that carriers of Gln homozygote allele of ADRB2 at codon 27 exhibited significantly greater in the increases of HR than patients with Glu allele after laryngoscopy and tracheal intubation. We also determined significant effects of ADRB2 mutant alleles versus wild-type alleles of each SNPs on HR (β coefficient: 14.371, 17.896, respectively). Moreover, the significant difference was found in ADRB2 27 alleles when comparing mutant and heterozygous genotypes to wild-type genotypes according to their RPP results (β coefficient: 29.240, 37.396, respectively) (Table 5).

The impact of the ADRB2 SNPs at codons 16 and 27 on cardiovascular phenotype has been investigated only in a very few studies in vivo, and the data were quite controversial. However, the effects of ADRB2 SNPs on pressure response during the laryngoscopy and tracheal intubation were investigated only in a previous study by Kim et al. (2002). They found an association between changes of MAP and RPP and Glu27Glu (mutant) as compared with the Gln27Gln (wild-type) genotype. Their results were consistent with our findings; however, this relation was reversed. Furthermore, we found a relation between ADRB2 16 and 27 SNPs. On the other hand, Martinez et al. (1997) showed that Glu27 allele was resistant to down-regulation when compared Gln27, but only when coexpressed with Arg16. They also emphasized a linkage disequilibrium between the two SNPs, with 97.8% of all chromosomes that carried Arg16 also carrying Gln27 allele. This disequilibrium also was observed in our study.

In contrast to our results, some studies determined an association between ADRB2 16 alleles and arterial blood pressure. Gratze at al. (1999) found that the young adult Caucasians with Gly16 genotype had significantly higher resting mean arterial pressure than subjects with the Arg16 genotype. According to their results, the Arg16 allele appeared to be associated with a greater degree of vasodilator response compared with the Gly16 allele. The Gly16 allele of ADRB2 was found to indicate a lesser propensity to vasodilatation in response to agonist infusion in normotensive subject. These effects were apparent despite the baroreceptor reflex counterregulation. When the effects of ADRB2 SNPs on hemodynamic responses to adrenaline were investigated, the results suggested that upon acute adrenaline infusion, the ADRB2 Arg16Arg genotype conferred reduced vasodilatation (Snapir et al. 2003). Timmermann et al. (1998) showed that the Gly16 variant rather than the Arg16 variant was associated with a propensity for hypertension and higher blood pressure values in the northern European population studied. When the alteration of cardiovascular responses to isometric exercise was investigated, Eisenach et al. (2004) observed a greater change in HR in the Gly16 homozygote. They suggested that the greater HR response to exercise in the Gly16 homozygotes might serve to maintain the pressure response (increased cardiac output) in the face of augmented peripheral vasodilatation (decreased total peripheral resistance) in that group.

Castellano et al. (2003) observed an association of the Arg16 allele with higher SBP at the age of below 50 years. The haplotype analysis showed that higher blood pressure values were more specifically associated with the presence of Arg16–Gln27. Though their results, in our study, any gene-gene interaction did not significantly effect to our pressure response results (data not shown).

The allelic frequency of Gly16 allele of the ADRB2 in Caucasians (50% in the homozygous mutant) is higher than the Arg16 allele (Gren et al. 1994). The allelic frequency of Glu27 SNP in Caucasians accounts about 25.35% in the homozygous wild-type genotype (Liggett 1995). We have found that the genotypic and allelic frequencies of Glu27Gln polymorphism of ADRB2 gene in our samples was significantly different from previous Turkish population study reported by Aynacioglu, Cascorbi, and Gungor (1999), but was similar to those in Caucasian and Asian population studies (Kim 2002). The allelic frequency of the Arg16Gly SNPs determined in our study group was the same as the frequencies of Caucasians and Turkish population, but differed from Asian populations.

In conclusion, we found an association between Glu27 homozygote allele of ADRB2 and increased mean HR and RPP after laryngoscope and tracheal intubation. Taking into account that ADRB2 is an important target of many drugs and endogenous substances, interethnic and individual differences in this receptor may explain individual hemodynamic response after laryngoscopy and tracheal intubation. The knowledge of pharmacogenetics may help reduce the adverse reactions of treatment and optimize efficacy.