OPRM1 A118G НА ТЕЧЕНИЕ ТОТАЛЬНОЙ ВНУТРИВЕННОЙ АНЕСТЕЗИИ У ПАЦИЕНТОК ГИНЕКОЛОГИЧЕСКОГО ПРОФИЛЯ

Subjects and methods. A sample consisted of 161 gynecological patients who had under(cid:26) gone elective surgery under conventional total intravenous anesthesia. Heart rate, noninvasive mean blood pressure, peripheral oxygen saturation, bispectral index, and somatosensory evoked potentials were monitored in all the examinees before and after administration of the induction dose of an anesthetic, in the intraoperative and early postoperative period. The polymorphic variants of the gene in question were determined by allele(cid:26)specific PCR. Results. According to the iden(cid:26) tified genotype, the patients were divided into 3 groups: 1) 118A/A genotype carriers ( n =101); 2) 118A/G genotype carri(cid:26) ers ( n =48); 3) 118G/G genotype carriers ( n =12). It was intraoperatively found that the 118G/G genotype carriers tended to have hypertension and to consume higher quantities of fentanyl and droperidol than the 118A/A and 118A/G carriers. The intergroup difference in the bispectral index was statistically insignificant during surgery. In the early postoperative period, the 118G(cid:26)allele homozygotes showed a deeper level of sedation, which correlated with the significantly lower val(cid:26) ues of the bispectral index ( p <0.01) and the higher incidence of adverse reactions ( p <0.01). At the same time, the latency and amplitude of somatosensory evoked potentials in the 118G/G genotype carriers showed the least variations as com(cid:26) pared to the A(cid:26)allele homozygotes and heterozygotes ( p <0.01). Conclusion. The A118G polymorphism of the μ(cid:26)opioid receptor gene (OPRM1) affects the course of total intravenous anesthesia. The 118G/G genotype patients needed larger the


Introduction
The improving pain relief strafegies in surgery is a challenging problem in contemporain anesthesiology reanimatology [1][2][3][4]. The perception and response to the sample nociceptive stimulus in patients occurs in different ways [5][6][7]. Genetic characteristics of an individual might play important role in this process [8][9][10][11][12]. For example, gene polymorphism of glutaminase leads to the develop ment of cognitive dysfunction and formation of chronic pain syndrome [13][14]. Genetic polymorphism of sero tonin receptors significantly affects the perception of pain associated with thermal irritation. Low expression of the serotonin transporter leads to the formation of hypoalgesia to a threshold heat pain and above threshold thermal noci ceptive stimuli [15][16][17]. Patients with reduced activity of catechol O methyltranspherase possess higher pain threshold to repeated nociceptive stimuli [18][19][20]. In rela tion to the antinociceptive system the native isoforms of opioid receptor MOR 1K significantly reduce the effec tiveness of narcotic analgesics [21,22]. Carriers of geno types of 118A/G, 118G/G of μ opioid receptor gene OPRM1 are characterized by reduced responses to the fen tanyl and alfentanil resulted in a need in higher doses of narcotic analgesics in early postoperative period [23][24][25]. Side effects such as nausea and vomiting associated with the administration of narcotic analgesics were reported more frequently in post surgery homozygous carriers of the minor alleles [26,27]. However, other authors did not reveal any statistically significant differences in the con sumption of narcotic analgesics among the different carri ers of the OPRM1 genotypes [28].
The aim of our study was to evaluate the effect of the A118G polymorphism in μ opioid receptor OPRM1 gene on total intravenous anesthesia.

Materials and Methods
The study was approved by the local independent ethics committee of the Rostov state medical university (Protocol No.20112 from 20.12.2012). The work was performed as two ran domized, double blind clinical studies.
Study enrolled 161 patients (European and Asian popula tion) of 20 to 45 years old (mean age 33.25±6.26 years) who were residents of the Rostov region, hospitalized in the Department of gynecology, City Hospital №6, Rostov on Don. All patients received total intravenous anesthesia during gyne cological operations.
Criteria for inclusion in the study: 1.

2.
No comorbidity in patients on addmittance.

3.
Patients with chronic bronchitis, chronic pharyngitis, chronic gastritis with remission for at least 3 months and more (patients with diseases of central nervous system, liver and kid neys without failure were excluded from the study).
The body mass index less than 27.5 kg/m 2 . Criteria for exclusion from the study: 1.
A history of pathology of the central nervous system including vegetative vascular dystonia.

2.
A history of pathology of the cardiovascular system.

3.
A history of respiratory failure.

4.
A history of liver disease and biliary tract.
A history of autoimmune diseases, allergies. 7.
A history of diabetes. 8.
A history of HIV infection. 9.
The body mass index more then 27.5 kg/m 2 . All patients were operated in a planned manner under total intravenous anesthesia (TIVA). The most common operations were: resection of ovarian cysts, laparoscopic conservative myomectomy, supracervical amputation of the uteri, hysterecto my. The operative time ranged from 35 to 98 minutes (average 62.48±11.27 minutes).
Scheme of total intravenous anesthesia was standard and included the following step. Premedication: administration of sibazon at a dose of 10 mg at night before surgery and two hours before entering the operating room.
Intraoperative period: after admittance at the operating room patients were administered with atropine intravenously at a dose of 0.5 mg, sibazon 10 mg («Moscow endocrine plant»), promedol 20 mg and ketamine 0.4 mg/kg («Moscow endocrine plant»). Induction was performed by intravenously bolus injec tion of propofol (Propofol Lipuro, B. Braun, Germany), 10 mg every 5 seconds, until the level of bispectral index decreased less than 60. Tracheal intubation was performed in 3-5 minutes after administration of succinylcholine (1.5-2 mg/kg), in means of bispectral index from 45 to 55. Mechanical lung ventilation was performed with a mixture of air and oxygen in the ratio 2:1 (DatexOhmedaAespire) in a normoventilation mode. In two minutes before the incision fentanyl at a dose of 0.005±0.0007 mg/kg was administered. Myorelaxation was carried out by the introduction of pipecuronii at a dose of 0.06 to 0.07 mg/kg. During the surgery propofol (100-150 ng/kg/min) and keta mine (0.2 mg/kg/hour) were administered for maintenance of anaesthesia. Analgesia was carried out by bolus administration of fentanyl at a dose of 0.003±0.00045 mg/kg if heart rate and/or mean arterial pressure increased more than 20% of initial values.
The assessment of the adequacy of general anesthesia was performed according to Harvard standards monitoring anesthesia (heart rate (HR), noninvasive mean arterial pressure (NIMBP), electrocardiography (I -first standard abstraction) and plethys mography were analyzed, the oxygen saturation (SpO 2 ) was esti matied by pulse oximetry and analysis of changes of bispectral and somatosensory evoked potentials (SSEP).
after the patient entered the operating room (start point); 2. after induction and intubation (in 2 minutes), but prior to skin incision; 3. during laparotomy (on a stage of the most significant somatic component of pain); 4. at the stage of removing, amputation of the uterus, enu cleation of the node (at the stage of most significant visceral com ponent of pain); 5. in early postoperative period (15 minutes after the surgery was completed).
The polymorphism of the μ opioid receptor gene OPRM1 was detected by polymerase chain reaction using specific forward and reverse primers (Biosune Biotechnology Co. Ltd, China) accord ing to the method described previously [27]. Depending on the identified genotype patients were divided into three groups (table 1): group I -101 carriers genotype 118A/A (carriers of the normal allele, the major variant), group II -48 carriers 118A/G genotype (heterozygous carriers of the polymorphic alleles), group III -12 carriers of genotype 118G/G (homozygous carriers of the poly morphic (minor) alleles) (P=0.073 demonstrating not significant diference from the Hardy Weinberg equilibrium). Genotyping was performed in the laboratory of human genetics, Institute of Biology, Southern Federal University, Rostov on Don).
Statistical processing of the obtained data was performed using analysis of variance (ANOVA) and discriminant analysis by SPSS 17,0 and Microsoft Excel. To evaluate the significance of intra group differences between the original and final values of variables nonparametric Wilcoxon criterion was employed. Statistically significant differences were considered at P 0,05.

Results and Discussion
Statistically significant differences between East Europians and ethnic Asian carriers of the same genotypes were not identified in consumption of drugs for TIVA, course of anesthesia and early postoperative period (P 0,05).
Comparison of doses of drugs for general anesthesia revealed increased consumption of fentanyl in heterozy gous and homozygous carriers of the minor G allele com pared to homozygous carriers of the major A alleles. Thus in the first hour of operation the dose of fentanyl in the first group (the carriers of the genotype 118A/A) was 0.0045±0.0003 mg/kg, in second group (118A/G) -0.0054±0.0004 mg/kg, and in the third group (118G/G) -0.0081±0.0008 mg/kg. The total dose of fentanyl in homozygous carriers of the a allele was 0.0059±0.0006 mg/kg, heterozygous carriers of the G allele 0.0073±0.007 mg/kg and in homozygous carriers of the G allele -
Differences in the consumption of other drugs for anesthesia were not significant ( Table 2).
Differences in changes between groups in values of bispectral index were minor and statistically insignificant on the first, second, third and fourth stages of the study. However, at the fifth stage of the research, values of bis pectral index were significantly lower (P 0,05) in carriers of the genotype 118G/G of μ opioid receptor OPRM1 (76.75±4.94) compared to the values of this parameter in carriers of a genotype 118A/A (82.32±3.48) and 118A/G (82.73±3.95) indicating a deeper level of sedation, and was associated with higher total dose of fentanyl and droperi dol during operation.
The dynamic of changes in the amplitude and laten cy of somatosensory evoked potentials at the second, third and fourth stages demostrated that the patients of group III had less variation of that parameters than in the I and II groups, and exhibited an increased sensitivity to noci ceptive stimuli despite the higher doses of narcotic anal gesics (table 3). This fact is presumably related to to altered μ opioid receptor signaling in brain compartments of carriers of 118G/G genotype [30]. Homozygous
In early postoperative period carriers of major geno type had increased latency of N19 (15.24±0.32%) with a simultaneous decrease of the amplitude of N19 (34.14±1.82%, P<0.01) in comparison with the original data (start point). Carriers of the genotype 118A/G had higher latency of N19 in comparison with original values (11.18±0.92%), and the amplitude was lower in compari son with original values (18.34±2.27%, P<0.05). Carriers of the minor genotype exhibited an increased latency of N19 (4.37±0.06%), with a simultaneous decrease of the amplitude of N19 (5.26±0.16%).Thus in homozygous car riers of the polymorphic alleles of μ opioid receptor gene OPRM1 the latency and amplitude of SSEP differed in comparison with the original data, which indicated almost complete recovery of conduction of nociceptive impulse, while the carriers of the genotype 118A/A genotype 118A/G were characterized by slower speed of the impulse conduction (residual analgesia).
Significant differences were not revealed among groups in heart rate at any stage of the study (Fig. 1-2).
At the same time, medium arterial pressure (NIMBP) was significantly higher in homozygous carriers of the minor G allele at 3-5 m phases of the study, com pared to homozygous carriers of the major A allele (P<0.05). At the third stage medium arterial pressure of homozygous carriers of the major allele was 77.29±3.43 mm Hg, in heterozygous carriers of the minor allele -84.31±5.38 mm Hg, and in homozygous carriers of the minor allele it was 91.25±8.37 mm Hg.
At the fourth stage in homozygous carriers of the A allele NIMBP value wwas 76.14±3.32 mm Hg., in het erozygous carriers of the G allele it was 81.35±6.12 mm Hg and in homozygous carries of the G allele OPRM1 -91.02±6.34 mm Hg.
In early postoperative period (the fifth stage) NIMBP in carriers of genotype 118A/A OPRM1 was 73.93±3.36 mm Hg, in carriers of the genotype 118A/G OPRM1 was 81.15±4.67 mm Hg, and in carriers of the genotype 118G/G was 85.33±5.52 mm Hg (Fig. 2).
Homozygous carriers of the major allele began exe cute verbal commands on 6.63±3.11 minutes, the extuba tion in that group was made on 9.74±3.46 minutes after surgery. Heterozygous carriers of the minor alleles began execute verbal commands on 9.27±3.69 minute, and extu bation was performed on 14.69±4.42 minute. In homozy gous carriers of the minor allele execution start time of ver bal commands were 27.75±8.23 minutes, and the time of extubation was 34.08±11.13 minutes after surgery, which was significantly higher (P 0.01) in comparison with the patients of first and second groups.
The application of discriminant analysis allowed to identify the most important criteria for predicting the course of anesthesia in carriers of the polymorphic variants of the gene OPRM1. Standardized coefficients that deter mine the value (weight) in the discrimination of the stud ied groups (Table. 4) were established. Analysis of the results showed that the important value in predicting the course of anesthesia and early postoperative period depending on the OPRM1 genotype were: latency and amplitude of somatosensory evoked potentials (primarily the amplitude N19 on 2 nd , 3 rd and 5 th phases of the study, the latency N19 on the 4 th stage, latency N19 and N32 on the 5 th stage) and extubation time.
All of the indicators included in the model have a high statistical significance (table 5).
The high statistical significance of variables allowed to applythis results to other patients.

Conclusion
1. The carrier genotypes 118A/G and 118G/G μ opioid receptor OPRM1, primarily 118G/G are more tol erant to narcotic analgesics (P 0.01) and required higher doses of narcotic analgesics to achieve adequate analgesia during surgery.
2. Homozygous carriers of the minor allele had deeper level of sedation, and they recovered the sponta neous breathing later demonstrating that the patients with 118A/G and 118G/G genotypes needed more attention in early postoperative period (P 0.01). This fact could be connected to higher doses of narcotic analgesics, which were administered during surgery.