Experimental Evaluation of ABCB1 C3435T (rs1045642) Gene Polymorphism and Its Association with Postoperative Analgesia in Hip Replacement Surgery

Summary

This study investigated whether a common genetic variant affects postoperative pain control following hip replacement. Patients with different genotypes showed clear differences in pain intensity, analgesic use, and pain-related biological responses.

Abstract

This study investigated the association between the ABCB1 (MDR1) C3435T (rs1045642) gene polymorphism and postoperative analgesia in patients undergoing hip replacement surgery. A total of 100 patients who underwent hip replacement in our hospital between January 2023 and January 2024 received postoperative patient-controlled intravenous analgesia with sufentanil. Preoperative serum samples were collected, and genomic detection was performed using the TaqMan SNP genotyping assay. According to the C3435T genotype, patients were classified into wild-type homozygotes (CC group), mutant heterozygotes (CT group), and mutant homozygotes (TT group). Clinical parameters, including baseline characteristics, Visual Analogue Scale (VAS) pain scores, sufentanil consumption, serum pain mediators (prostaglandin E2 (PGE2), substance P (SP), and β-endorphin (β-EP)), Hamilton anxiety and depression scores, sleep quality, and the incidence of adverse events within 24 h after surgery were compared among groups. Baseline characteristics were similar across groups (p > 0.05). VAS scores, sufentanil dosage, and serum PGE2, SP, and β-EP levels at 24 h were significantly lower in CT and TT groups compared with the CC group (p < 0.05). The TT group also exhibited reduced Hamilton anxiety and depression scores relative to both CC and CT groups (p < 0.05), whereas the CT group showed higher anxiety and depression scores than the CC group (p < 0.05). Sleep quality and adverse event rates were not significantly different among groups (p > 0.05). These findings suggest a potential association between the ABCB1 (MDR1) C3435T (rs1045642) polymorphism and variability in postoperative analgesic efficacy. Patients carrying the TT genotype tended to experience better analgesia, lower sufentanil requirements, and improved psychological outcomes. Although these results are promising, they should be interpreted with caution due to the small sample size and single-center design. Further large-scale studies are warranted to confirm these observations and evaluate their clinical applicability.

Introduction

Hip replacement refers to the replacement of the damaged hip joint with an artificial hip prosthesis through surgery. For early femoral head necrosis, hip osteoarthritis, and other diseases, the symptoms can be relieved, and the condition can be delayed through conservative treatment of integrated traditional Chinese and Western medicine¹. When the pain and deformity of the hip joint affect walking, it cannot be relieved by conservative treatment such as drugs, which seriously affects the quality of life. Hip replacement surgery can be chosen to relieve joint pain, rebuild hip function, and improve the quality of life. A series of pathophysiological changes caused by pain will increase the risk of cardiovascular and cerebrovascular diseases in elderly patients^2,3. Postoperative pain seriously affects the recovery of hip joint function, prolongs the bed rest time of patients, and increases the risk of thrombosis of the lower limbs⁴. Effective analgesia can not only alleviate stress reactions after an operation but also reduce the hospitalization time of patients. Epigenetic regulation means that the DNA sequence does not change, but the gene expression is affected by environmental factors, and so on⁵. Recent studies have shown^6,7 that there is a correlation between epigenetic regulation and the occurrence and development of chronic pain, and chronic pain or acute pain is often the result of the interaction between environmental factors and genes. Understanding the relationship between epigenetic regulatory components and chronic pain and identifying biomarkers (such as those related to epigenetic regulation) closely linked to pain occurrence and development, especially chronic pain, can help diagnose chronic pain susceptibility, evaluate drug treatment effects, and ultimately promote individualized and precise analgesia. The C3435T polymorphism (rs1045642) in the ABCB1 gene (multidrug resistance gene 1, MDR1) is a well-characterized single-nucleotide variant located in exon 26. This polymorphism encodes a variant of P-glycoprotein (P-gp), a transmembrane efflux transporter that regulates the cellular uptake and efflux of various drugs, including opioids, thereby influencing their pharmacokinetics and therapeutic response⁸. Among the genetic variants of ABCB1, the synonymous polymorphism C3435T (rs1045642) has been extensively studied in relation to interindividual variability in drug response. ABCB1 C3435T (rs1045642) is a synonymous substitution located in exon 26 of the MDR1 gene⁹. Although it does not alter the amino acid sequence, accumulating evidence suggests that this variant may influence mRNA stability and translation kinetics, thereby affecting P-glycoprotein folding, expression levels, or transport efficiency. While findings across studies are not entirely consistent, several reports have indicated that the T allele is associated with reduced P-gp expression or altered substrate specificity¹⁰. Because P-gp expressed at the blood-brain barrier limits the central nervous system penetration of many drugs, including certain opioids, the ABCB1 C3435T polymorphism may contribute to individual differences in sufentanil sensitivity and opioid-related adverse effects¹¹.

The purpose of this study was to investigate whether the ABCB1 C3435T polymorphism is associated with interindividual variability in postoperative analgesic efficacy and pain-related outcomes in patients undergoing hip replacement surgery. Hip replacement surgery is commonly associated with moderate to severe postoperative pain, and opioids such as sufentanil are routinely used for postoperative analgesia. However, substantial interindividual differences exist in analgesic response and opioid-related adverse effects, which cannot be fully explained by clinical factors alone. Because P-glycoprotein encoded by the ABCB1 gene plays a key role in regulating drug transport across biological membranes, including the blood-brain barrier, genetic variation in ABCB1 may influence opioid disposition and central analgesic effects. Therefore, examining the relationship between the ABCB1 C3435T polymorphism and postoperative analgesia may help clarify the genetic contribution to variability in pain control after hip replacement surgery. By discussing differences in postoperative analgesic effects among patients with different genotypes, this study aims to reveal individual variations in postoperative pain and provide a theoretical basis for clinical individualized analgesia. Simultaneously, it expands understanding of the mechanism by which the C3435T gene polymorphism influences postoperative analgesia, providing a foundation for subsequent research.

Protocol

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Fuzhou Hospital of Traditional Chinese Medicine (Approval No.: LC-2023-048). Written informed consent was obtained from all participants prior to enrollment.

Data and methods
Based on the research hypothesis, we developed a prospective cohort analysis study protocol. A total of 100 patients undergoing elective hip replacement surgery were prospectively enrolled, with a mean age of 58.41 ± 6.97 years. Figure 1 illustrates the technical roadmap of this protocol, with detailed descriptions of the study design provided in the following sections.

Study design and participants
This was a prospective cohort study enrolling consecutive adults scheduled for primary hip arthroplasty under general anesthesia between January 2023 and January 2024. Inclusion criteria were age 18-60 years, American Society of Anesthesiologists (ASA) class I-II, and surgery performed by the same orthopedic team. Exclusion criteria included significant cardiopulmonary, hepatic, or renal dysfunction (defined as serum creatinine >1.5 mg/dL or ALT >3x upper limit of normal); history of chronic pain (e.g., fibromyalgia, neuropathic pain); abuse of sedatives or opioids; known allergy to analgesics; exposure to other opioids during the current illness; and long-term preoperative use of analgesics or steroids (>1 week within 3 months prior to surgery). From the participants, 5 mL of EDTA-anticoagulated venous blood was collected preoperatively for genotyping. Genomic DNA was extracted from peripheral blood using a commercial DNA extraction kit according to the manufacturer's instructions. The ABCB1 C3435T (rs1045642) polymorphism was genotyped using polymerase chain reaction-based methods, and genotype determination was performed according to established protocols¹⁰. After genotyping the ABCB1 C3435T (rs1045642) polymorphism, participants were stratified into CC, CT, and TT groups. No external healthy control group was included; all comparisons were made among genotype groups (CC, CT, TT). Laboratory personnel analyzing biomarkers and genotypes were blinded to clinical outcomes.

Anesthesia and intraoperative management
Standard monitoring included electrocardiography, non-invasive blood pressure (measured every 5 min), pulse oximetry, and end-tidal carbon dioxide (EtCO₂). An intravenous line was established using an 18G catheter, and patients were preoxygenated with 100% O₂ via facemask for 3 min. Anesthesia was induced with midazolam 0.05 mg/kg, cisatracurium 0.1 mg/kg, sufentanil 0.4 µg/kg, and propofol titrated at 1-2 mg/kg until loss of consciousness and adequate jaw relaxation. Tracheal intubation was performed after confirming sufficient neuromuscular blockade (train-of-four count = 0). Mechanical ventilation was set in volume-controlled mode with a respiratory rate of 15 breaths/min, tidal volume of 6-8 mL/kg predicted body weight, and PEEP of 5 cm H₂O, with adjustments to maintain EtCO₂ at 35-45 mmHg. Anesthesia was maintained with propofol 0.08-0.12 mg/kg/min and sufentanil 0.15-0.30 µg/kg/h. Intravenous anesthetics were discontinued at wound closure. Hemodynamics were managed with fluid boluses or vasopressors as needed to maintain MAP within 20% of baseline.

Hip arthroplasty procedure¹²
Patients were positioned in the lateral decubitus position with the operative side up. A posterolateral approach was used: a 15-20 cm skin incision was made centered over the greater trochanter. Subcutaneous tissue and fascia lata were incised, and the gluteus maximus was split bluntly. The posterior capsule was exposed, and a T-shaped capsulotomy was made along the acetabular rim. The hip was dislocated by external rotation and adduction. The femoral head was resected, and the femoral neck was trimmed to 1.0-1.5 cm above the lesser trochanter. For hemiarthroplasty, the femoral canal was prepared with sequential broaches (size increase: 1 mm steps), and the femoral stem was implanted with approximately 15° of anteversion. For total hip arthroplasty, the acetabulum was reamed until punctate bleeding was observed, targeting a cup abduction angle of 45° and an anteversion angle of 15°. Components were cemented or press-fit based on intraoperative assessment of bone quality and implant stability. After reduction, hip stability and range of motion were assessed intraoperatively by the operating surgeon through passive flexion, extension, internal and external rotation, and abduction, with particular attention to the presence of impingement or dislocation. A closed-suction drain was placed if necessary, and the wound was closed in layers using absorbable sutures for the deep fascia and subcutaneous tissue, and non-absorbable sutures or skin staples for skin closure.

Postoperative patient-controlled intravenous analgesia (PCIA)
A standardized sufentanil PCIA regimen was used for all participants. The analgesic solution consisted of sufentanil at 1.5 µg/mL, diluted in 0.9% saline, and loaded into a programmable infusion pump. Pump settings were background infusion 1 mL/h, bolus dose 1 mL, and lockout interval 10 min, without a loading dose unless clinically indicated. Patients received standardized instruction on PCIA use in the recovery area. Cumulative sufentanil consumption was recorded from the pump log at 24 h postoperatively. All pump models, disposables, and consumables are listed in the Table of Materials.

Blood sampling and processing
Preoperative genotyping samples consisted of 5 mL of EDTA-anticoagulated venous blood mixed by gentle inversion. For pain mediator analyses, 5 mL of venous blood was collected on postoperative days 1, 2, 3, and 7. For serum preparation, samples were allowed to clot for 30 min at room temperature; for plasma, the anticoagulant specified by the assay was used. Samples were centrifuged at approximately 1,000 x g for 15 min at room temperature to separate serum or plasma. The supernatant was aliquoted into labeled low-binding tubes and stored at −80 °C until analysis. To minimize freeze-thaw cycles, a minimum of two aliquots per time point were prepared.

Genomic DNA extraction and SNP genotyping
Genomic DNA was extracted from whole blood using a silica-membrane column-based genomic DNA extraction kit (commercially available) following the manufacturer's protocol. DNA concentration and purity were measured spectrophotometrically, with samples exhibiting A260/280 ratios of 1.8-2.0 considered acceptable. DNA was stored at −20 °C until use.

The ABCB1 C3435T (rs1045642) polymorphism was genotyped using a pre-designed TaqMan SNP Genotyping Assay on a real-time PCR platform. The assay included pre-optimized primers and hydrolysis probes. Thermal cycling conditions followed the manufacturer's recommendations (95 °C for enzyme activation, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s). No-template controls and duplicate samples (≥10% of total) were included for quality control. Genotype calling was performed using allelic discrimination analysis software, with a call rate ≥95% and 100% duplicate concordance required for acceptance.

Pain mediator assays
Serum prostaglandin E₂ (PGE₂), substance P (SP), and β-endorphin (β-EP) were measured using validated radioimmunoassay or sandwich immunoassay kits according to the manufacturers' instructions. Calibration curves were generated using at least five standards spanning the expected concentration range; unknowns and controls were run in duplicate. The lower limits of detection, intra-assay coefficients of variation, and inter-assay coefficients of variation were recorded; assays with intra-assay CV >10% or inter-assay CV >15% were repeated. All kit names and catalog numbers are listed in the Table of Materials.

Outcome measures
Baseline characteristics included age, sex, body mass index, surgical duration, surgical approach (anterior/posterior), prosthesis type (cemented/uncemented), preoperative pain VAS (0-10), preoperative anxiety and depression scores, and intraoperative sufentanil dose. Primary outcomes at 24 h were pain intensity at rest measured by the Visual Analogue Scale (VAS, 0-10) and cumulative sufentanil consumption recorded from the PCIA pump. Secondary outcomes at 24 h included sleep quality (score 0-3; higher scores indicate poorer sleep), incidence of postoperative nausea and vomiting (PONV), and Hamilton Anxiety and Hamilton Depression Scale scores. Exploratory outcomes included serum PGE₂, SP, and β-EP levels on postoperative days 1, 2, 3, and 7.

Statistical analysis
Continuous variables are presented as mean ± standard deviation (SD) or median (interquartile range) according to distribution; categorical variables as counts (percentages). Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene's test. For normally distributed variables with equal variances, one-way analysis of variance (ANOVA) with Bonferroni (or Tukey) post-hoc comparisons was applied; otherwise, the Kruskal-Wallis test with Dunn's post-hoc procedure was used. Categorical variables were analyzed using  χ² or Fisher's exact test, as appropriate. Genotype and allele frequencies were calculated, and the Hardy-Weinberg equilibrium was assessed by the χ² test. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated for associations between genotype (e.g., T-allele carriers vs CC) and dichotomized outcomes where applicable. Prior to applying parametric tests, data distributions were tested for normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test). In cases where assumptions were violated, non-parametric alternatives (Kruskal-Wallis test with post-hoc Dunn's test) were used. Perform Hardy-Weinberg equilibrium verification using the chi-square test. Calculate the odds ratio (OR) and 95% confidence interval (CI) to analyze the association between genotype and postoperative pain score, as well as sufentanil consumption. Two-sided p < 0.05 was considered statistically significant. Analyses were conducted with validated statistical software.

Results

Participant characteristics and genotype distribution
A total of 100 patients were enrolled and successfully genotyped for the ABCB1 C3435T polymorphism. The genotype distribution was 40.0% (40/100) for CC, 49.0% (49/100) for CT, and 11.0% (11/100) for TT. The allele frequencies were 64.5% for C (129/200) and 35.5% for T (71/200). The genotype distribution conformed to Hardy-Weinberg equilibrium (χ² =0.49, p=0.48; Table 1). Baseline characteristics, including age,...

Discussion

Hip replacement is a common major orthopedic operation, and postoperative pain is one of the main problems faced by patients. The effect of postoperative analgesia is influenced by many factors, including individual factors such as the patient's age, gender, weight, and pain sensitivity, as well as external factors such as operation mode and postoperative rehabilitation exercise^13,14. In addition, the drug analgesia scheme is also an important factor affectin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Injection	Fresenius Kabi	19194	Diluent for sufentanil (PCIA solution) and other drugs; used for wound irrigation during surgery [document: "Postoperative PCIA", "Hip arthroplasty procedure"].
Allelic Discrimination Analysis Software (for real-time PCR)	Not specified (same as real-time PCR platform)	Not specified	Used for genotype calling of ABCB1 C3435T; requires call rate ≥95% and duplicate concordance 100% [document: "Genomic DNA extraction and SNP genotyping"].
Centrifuge (for serum/plasma separation)	Beckman Coulter	Avanti J - E	Operates at ~1000 × g (≈3000 rpm, 10 cm rotor radius); centrifuges blood samples for 10-20 min at room temperature to separate serum/plasma [document: "Blood sampling and processing"].
Cisatracurium Injection	Jiangsu Hengrui Pharmaceutical Co., Ltd	H20060869	Neuromuscular blocking agent for tracheal intubation, dose 0.1 mg/kg [document: "Anesthesia and intraoperative management"].
Clinical Chemistry Analyzer	Roche Diagnostics	Cobas c 501	Detects serum biomarkers (PGE2, SP, β-EP) [document: "Pain mediator assays"].
EDTA Anticoagulant	BD Vacutainer	366450	Used for preoperative venous blood collection (5 mL) for genomic DNA extraction and genotyping; samples mixed by gentle inversion to prevent coagulation [document: "Study design and participants", "Blood sampling and processing"].
Electrocardiograph (ECG)	Philips	CardioPerfect MX800	Part of standard intraoperative monitoring [document: "Anesthesia and intraoperative management"].
EndNote	Clarivate Analytics	X9	Assists in reference management and formatting (aligns with journal citation norms) [reference: "JoVE_Materials.xls"].
End-Tidal Carbon Dioxide (ETCO2) Monitor	Masimo	Rad - 97	Part of standard intraoperative monitoring; used to maintain normocapnia [document: "Anesthesia and intraoperative management"].
Low-Temperature Refrigerator (-80°C/-20°C)	Haier	DW - 86L626 (-80°C); DW - 25L262 (-20°C)	-80°C for storing aliquoted serum/plasma; -20°C for storing extracted genomic DNA [document: "Blood sampling and processing", "Genomic DNA extraction and SNP genotyping"].
Midazolam Injection	Jiangsu Enhua Pharmaceutical Co., Ltd	H10980025	Anesthesia induction agent, dose 0.05 mg/kg [document: "Anesthesia and intraoperative management"].
Non-Invasive Blood Pressure Monitor	Omron	HEM - 9072T	Part of standard intraoperative monitoring [document: "Anesthesia and intraoperative management"].
Programmable Intravenous Analgesia (PCIA) Pump	Phoenix Pharmaceuticals	EK-022-16	Used for postoperative sufentanil delivery; settings: background infusion 1 mL/h, bolus dose 1 mL, lockout interval 10 min; records cumulative sufentanil consumption [document: "Postoperative PCIA"].
Propofol Injection	AstraZeneca	H20120088	Anesthesia induction (titrated to loss of consciousness) and maintenance (0.08-0.12 mg·kg-1·min-1) [document: "Anesthesia and intraoperative management"].
Prostaglandin E2 (PGE2) Radioimmunoassay Kit	Cayman Chemical	514010	Validated kit for serum PGE2 detection; run in duplicate, with intra-assay CV ≤10% and inter-assay CV ≤15% (repeated if exceeding limits) [document: "Pain mediator assays"].
Pulse Oximeter			Part of standard intraoperative monitoring (measures SpO2) [document: "Anesthesia and intraoperative management"].
Real-Time PCR Platform	Phoenix Pharmaceuticals	EK-022-16	Used for ABCB1 C3435T genotyping; thermal cycling conditions: enzyme activation, 40 cycles of denaturation (95°C, ~15 s) and annealing/extension (60°C, ~60 s) [document: "Genomic DNA extraction and SNP genotyping"].
Silica-Membrane Column-Based Genomic DNA Extraction Kit	Qiagen	51104	Used for extracting genomic DNA from whole blood; procedures include cell lysis, protein digestion, DNA adsorption, sequential washes, and elution in 100 μL elution buffer [document: "Genomic DNA extraction and SNP genotyping"].
Spectrophotometer	Thermo Fisher Scientific	NanoDrop One	Measures DNA concentration and purity; samples with A260/280 ≈1.8-2.0 considered acceptable [document: "Genomic DNA extraction and SNP genotyping"].
SPSS	IBM Corporation	26	Used for statistical analysis: Shapiro-Wilk normality test, Levene’s test, ANOVA, Kruskal-Wallis test, χ² test, etc. [document: "Statistical analysis"; reference: "JoVE_Materials.xls"].
Substance P (SP) Radioimmunoassay Kit	Merck Millipore	RAB0456	Validated kit for serum SP detection; same quality control standards as PGE2 kit [document: "Pain mediator assays"].
Sufentanil Injection	Chia Tai Tianqing Pharmaceutical Group Co., Ltd.	NA	Used for anesthesia induction (0.4 μg/kg), intraoperative maintenance (0.15-0.30 μg·kg-1·h-1), and postoperative PCIA (1.5 μg/mL diluted in 0.9% saline) [document: "Anesthesia and intraoperative management", "Postoperative PCIA"].
TaqMan SNP Genotyping Assay (for ABCB1 C3435T, rs1045642)	Qiagen	51104	Pre-designed allele-specific hydrolysis-probe assay for real-time PCR genotyping of ABCB1 C3435T polymorphism [document: "Genomic DNA extraction and SNP genotyping"].
Tracheal Intubation Equipment (laryngoscope, endotracheal tube)	Draeger	Capnomac Ultima	Used for tracheal intubation after confirmation of neuromuscular blockade [document: "Anesthesia and intraoperative management"].
Volume-Controlled Ventilator	Dräger	Primus	Used for intraoperative mechanical ventilation; settings: respiratory rate ~15 breaths/min, tidal volume 6-8 mL/kg predicted body weight [document: "Anesthesia and intraoperative management"].
β-Endorphin (β-EP) Sandwich Immunoassay Kit	Phoenix Pharmaceuticals	EK-022-16	Validated kit for serum β-EP detection; calibration curves generated with ≥5 standards [document: "Pain mediator assays"].