Anesthetic Methods and Gene Expression Profile

By using a computer-generated randomization table, hospitalized patients undergoing elective hip arthroplasty will be randomly consecutively allocated to receive general (GA group), regional (RA group), or integrated (IA group) anesthesia. Patients with contraindication to spinal anesthesia or lumbar catheter placement, as well as obese patients, with arterial hypertension not controlled by oral medication, severe pulmonary, cardiovascular, renal, hepatic, cerebrovascular, or psychiatric diseases will be excluded from the study.

Whole blood samples (10 mL) will be obtained from all enrolled patients at three time points: early morning on the operation day (T0), after surgery (T1) and third day (T2) after surgery. The samples will be collected in heparin tubes and PBMCs will be isolated and used for gene expression analysis. Serum obtained after blood centrifugation will be used for hematological and biochemical analysis such as glutamate oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), bilirubin (BIL), creatinine (CREA), creatine phosphokinase (CPK), hemoglobin (HB).

The sample size was determined according to Lee- Whitmore * and G*Power Ftest for ANOVA Fixed effects, omnibus, one-way (Lee ML, Whitmore GA. Stat Med. 2002;21: 3543-3570). Assuming a Poisson distribution for the expected value of the false-positive gene expression of the 9 chosen genes and fixing at 1 the maximum expected value for false positives E(R0) and considering all the 84 genes as not differentially expressed (G0=G=9), the probability α for any single gene among the G genes that are not differentially expressed is given by α=E(R_0 )/G=1/9=0.011, with the Bonferroni correction it becomes α_c=(0.011)/3=0.037. This is the type I error of a false positive expression. Moreover, considering as primary endpoint the fold increase of the gene expression, assuming a log-normal distribution with standard deviation of 0.7 which is typical of moderate-high gene expression and therefore a conservative one for the sample size determination and imposing a minimum difference on logarithmic scale among the 3 groups of 0.5 with a power of at least 0.8 and a Bonferroni corrected type I error α=0.05/3=0.0167 (which is smaller and then conservative, than the previous α_c) the minimum sample size(G*POWER)^ for each group is 30 patients, by considering a 10% of drop-out , the chosen sample size was 33 patients per group which leads to a total sample size of 99 patients.

^F tests - ANOVA: Fixed effects, omnibus, one-way Analysis: A priori: Compute required sample size Input: Effect size f = 0.7 α err prob = 0.0167 Power (1-β err prob) = 0.8 Number of groups = 3 Output: Noncentrality parameter λ = 14.7000000 Critical F = 4.7803455 Numerator df = 2 Denominator df = 27 Total sample size = 30 Actual power = 0.8009945

The normal distribution of continuous variables will be evaluated by Kolmogorov-Smirnov test. The Chi-square test will be used to evaluate categorical variables. The differences between groups will be evaluated by means of parametric ANOVA tests followed by Tukey test. Multiple regression analysis will be performed to evaluate the influence of biochemical parameters on gene expression in response to anesthetics considering confounding factors such as age, gender, BMI, smoking. Values of p <0.05 will be considered statistically significant. All tests will be performed using software (SPSS, Chicago, USA).

Inclusion criteria

• American Society of Anesthesiologists Classification (ASA Class) = I-II.

Exclusion criteria

• American Society of Anesthesiologists Classification (ASA Class) > III
• contraindication to spinal anaesthesia or lumbar catheter placement
• arterial hypertension not controlled by oral medication
• severe pulmonary
• cardiovascular disease
• renal disease
• hepatic disease
• cerebrovascular disease
• psychiatric diseases