TITLE: Oxidative stress markers in laparoscopic vs. open appendectomy for acute appendicitis: A double-blind randomized study

  ABSTRACT.BACKGROUND:
Oxidative stress is a complicated process, which was defined as an increase in prooxidants and decrease in antioxidants caused by various mechanisms, including inflammation and surgical trauma. The association between acute appendicitis and oxidative stress has been showed in previous studies. However, comparison of oxidative stress in laparoscopic or open appendectomy (OA) has not been established.

ABSTRACT.PATIENTS AND METHODS:
Patients who were diagnosed as acute appendicitis between October 2012 and January 2013 were randomized to open (OA, n = 50) and laparoscopic appendectomy (LA, n = 50). Blood samples for oxidative stress markers (total oxidant status [TOS] and total antioxidant status [TAS]), C-reactive protein (CRP) and white blood cells (WBC's) were collected just before the surgery and 24 h after surgery.

ABSTRACT.RESULTS:
There were no differences in preoperative values of WBC and CRP between LA and OA groups (P = 0.523 and 0.424), however, in postoperative 24th h, CRP was reduced in LA group (P = 0.031). There were no differences in preoperative levels of TOS, TAS, and oxidative stress index (OSI) between LA and OA groups. In the postoperative 24th h, TOS and OSI were found to be significantly higher in OA group when compared to LA group (P = 0.017 and 0.002) whereas no difference was detected in TAS level in the postoperative 24th h (P = 0.172).

ABSTRACT.CONCLUSIONS:
This double-blind, randomized clinical trial provides evidence that LA for uncomplicated appendicitis is associated with significantly lower oxidative stress compared with OA. Some of the advantages of LA may be attributed to the significant reduction of oxidative stress in these patients.

BODY.INTRODUCTION:
Oxidative stress is a complicated process, which was defined as an increase in prooxidants and decrease in antioxidants caused by various mechanisms, including inflammation and surgical trauma. Free radicals are highly active molecules with important biological functions.[1] They can act in redox signalling and antibacterial defence. In oxidative stress, production of free radicals exceeds the capacity of the mechanisms required to detoxify them, thus they can react and damage DNA, lipids, and proteins.  Surgical trauma elicits complex physiological stress response that involves the activation of inflammatory, endocrine, metabolic and immunologic mediators.[2] Furthermore, it produces oxidative stress. Despite the controversies in the literature, whether the laparoscopic surgery reduces oxidative stress[345] or not,[6] most of them indicated a reduction in oxidative stress with minimal invasive abdominal surgery. In laparoscopic surgery, oxidative stress generation has been attributed to inflation-deflation related splanchnic ischemia-reperfusion,[7] hemodynamic changes,[8] and CO2 pneumoperitoneum.[9] On the other hand, exposure of the peritoneal cavity to room air with higher oxygen concentration and manipulation of the bowel are thought to be responsible for oxidative stress in open surgery.[1011]  In acute appendicitis, it is not clear that whether oxidative stress is caused by an inflammatory response or the increase of free radicals are sufficient for the activation of the enzymatic defence system.[12] The association between acute appendicitis and oxidative stress has been showed in experimental[12] and clinical studies.[13141516] However, comparison of oxidative stress in laparoscopic or open appendectomy (OA) has not been established.  To date, there has been no report in the literature that compared oxidative stress markers between laparoscopic and open surgery for uncomplicated acute appendicitis. The aim of this double-blinded randomized trial was to compare oxidative stress in patients who underwent laparoscopic and OA for uncomplicated acute appendicitis by analysing the level of serum markers of oxidative stress.

BODY.PATIENTS AND METHODS:
The study was approved by the local ethics committee. The patients who were diagnosed as acute appendicitis between October 2012 and January 2013 were included. Adult patients older than 14 years were invited to participate in this trial if they had the clinical diagnosis of acute appendicitis. Enrolled patients were randomised to OA (n = 50) and laparoscopic appendectomy (LA) (n = 50) groups. Before randomisation, patients were informed concerning the risks and benefits of each procedure and signed a consent form for participation in the study. The patients, who have American Society of Anaesthesiologists (ASA) score of III or higher, presence of systemic inflammatory disease, diabetes mellitus, autoimmune diseases, pregnancy, and patients otherwise incapable of providing informed consent were excluded. Furthermore, patients with complicated appendicitis and patients who underwent conversion to the open procedure were excluded from the study too. No additional port placement was required in any of the patients in LA group.  Randomisation was done by a research assistant who was not involved in the enrolment. Just before the surgery patients were assigned to the LA or the OA group by means of sealed envelopes containing random numbers. The operator was informed about the procedure type by opening the envelope assigned to the patient in the theatre.  All the operations were performed by stuff surgeons of our institution, who were experienced in laparoscopic and open surgical techniques. All the patients were administered antibiotics preoperatively (cefoxitin 1 g intravenous [IV]). All patients were premedicated with IV midazolam 0.07 mg/kg 30 min prior to operation. Anaesthetic induction was performed with thiopental sodium (4-7 mg/kg), fentanyl (1.5 mcg/kg), and rocuronium (0.6 mg/kg). After endotracheal intubation, anaesthesia maintenance was provided with sevoflurane (1-2%) and an air/O2 mixture (30% O2).  All the OA procedures were performed through a McBurney's muscle-splitting incision. The base of the appendix was left uninvaginated (ligated 2 times with 2/0 vicryl). For the LA patients, 10mm umbilical trocar, and additional two trocars (10mm and 5mm) were placed in the lower abdomen. The appendicular artery was dissected and divided by a ligature device (Covidien LigaSure Advance Monopolar Tip Laparoscopic Sealer/divider LF5544, Valleylab, Boulder, CO., USA). The appendix was secured at the base by ligating two times intracorporeally with 2/0 vicryl, and divided above the two knots. The specimen was removed through the 10mm suprapubic trocar in a specimen bag. The base of the appendix was not invaginated. The resected appendix was routinely sent for histopathological examination. The operative time was calculated from the beginning of the incision to full closure of the wound.  Demographic characteristics, ASA score, operative time, length of hospital stay (LOS), intraoperative and postoperative complications were recorded. Blood samples for oxidative stress markers (total oxidant status [TOS]; and total antioxidant status [TAS]), C-reactive protein (CRP) and white blood cells (WBC's) were collected by two authors, just before the surgery and 24 h after surgery. After coagulation (30 min) at room temperature, the serum was separated and stored at −80°C until assay. Serum CRP level was determined by the nephelometric method (BeckmanArray 360 Protein System, Minnesota, Brea, CA, USA), and leukocytes were determined with an automatic haematology analyser (Beckman Coulter, Inc., Fullerton, CA, USA).

BODY.PATIENTS AND METHODS.DETERMINATION OF SERUM TOTAL OXIDANT STATUS LEVELS:
Total oxidant status levels were measured using commercially available kits (Rel Assay, Turkey). In the new method, oxidants present in the sample oxidized the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction was enhanced by glycerol molecules abundantly present in the reaction medium. The ferric ion produced a coloured complex with xylenol orange in an acidic medium. The color intensity, which could be measured spectrophotometrically, was related to the total amount of oxidant molecules present in the sample. The assay was calibrated with hydrogen peroxide, and the results were expressed in terms of micromolar hydrogen peroxide equivalent perlitre (μmol H2O2 equivalent/L).[17]

BODY.PATIENTS AND METHODS.DETERMINATION OF SERUM TOTAL ANTIOXIDANT STATUS LEVELS:
Total antioxidant status levels were measured using commercially available kits (Rel assay, Turkey). The novel automated method is based on the bleaching of characteristic color of a more stable 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation by antioxidants. The assay has excellent precision values, which are lower than 3%. The results were expressed as mmol Trolox equivalent/L.[18]

BODY.PATIENTS AND METHODS.CALCULATION OF OXIDATIVE STRESS INDEX:
The ratio of TOS to TAS was accepted as the oxidative stress index (OSI). For calculation, the resulting unit of TAS was converted to μmol/L and the OSI value was calculated according to the following formula: OSI (arbitrary unit) = TOS (μmol H2O2 equivalent/L)/TAS (μmol Trolox equivalent/L).[19]

BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS:
Statistical analysis of the data was performed using the Statistical Package for the Social Sciences (SPSS) version 19.0 (IBM, Chicago, IL, USA). The normality of quantitative variables was tested using the Kolmogorov–Smirnov test. Since the levels of all parameters studied herein were normally distributed, they were expressed as mean ± standard deviation. Between the groups, differences of the parameters were assessed using Student's t-test. The differences in patients' characteristics between the two groups of patients were assessed using Chi-square test (gender), Student's t-test (age), and the Mann–Whitney U-test (operative time, and hospital stay). All tests were two-tailed, and statistical significance was considered for P < 0.05. A power analysis using the Gpower computer program indicated that a total sample of 50 patients in both arms are adequate with 92% power using a t-test between means with alpha at 0.05.

BODY.RESULTS:
There were no statistically significant differences in the distribution of gender (P = 0.678), age (P = 0.347), and ASA grading (P = 0.456) between the two groups of patients. Operative time was significantly lower in patients who underwent OA than those who had LA (P = 0.001) LOS was similar between the groups (P = 0.209) Demographic and perioperative characteristics of the groups were presented in Table 1.   Table 1 Patient's characteristics with respect to the groups (LA or OA)   There were no differences in preoperative values of WBC and CRP between LA and OA groups (P = 0.523 and 0.424, respectively), however, in the postoperative 24th h, plasma level of CRP was significantly reduced in LA group (P = 0.031). Preoperative and postoperative values of WBC and CRP were presented in Table 2.  Table 2 Preoperative and postoperative values of WBC, CRP, TOS, TAS and OSI   There were no differences in preoperative levels of TOS, TAS, and OSI between LA and OA groups. However, in the postoperative 24th h, the plasma levels of TOS and OSI were found to be significantly higher in OA group when compared to LA group (P = 0.017 and 0.002, respectively) whereas no difference was detected in TAS level in the postoperative 24th h (P = 0.172). Preoperative and postoperative values of TOS, TAS and OSI, were presented in Table 2.  No intraoperative or postoperative complication were seen in any of the patients.

BODY.DISCUSSION:
The term "oxidative stress" is broad, briefly refers to an imbalance between formation of free radicals and antioxidant defence systems. Underlying pathophysiology of the creation of free radicals and response of the organism is complex. Local and systemic factors can affect the tissue oxygenation, also, oxidant molecules could be taken from the outer environment.[20] In an inflammatory condition, such as acute appendicitis, mast cells and polymorphonuclear leukocytes are recruited to the site of inflammation, they subsequently causes to a "respiratory burst" due to an increased uptake of oxygen, and thus free radicals. Free radicals causes lipid peroxidation in cellular membranes, which resulting in increased microvascular permeability, interstitial edema, inflammatory cell infiltration and activation. Activated inflammatory cells produce soluble mediators, including metabolites of arachidonic acid, cytokines, and chemokines, and thus resulting in more free radical production, and this cascade may cause eventually cell death. To date, a variety of oxidant species have been measured to reflect the tissue oxidative stress. In this study, we aimed to evaluate the additive oxidant effect of different oxidant molecules and antioxidant balance; therefore we used TOS, TAS, and OSI. The association between acute appendicitis and oxidative stress have been showed in experimental[12] and clinical studies.[13141516] However, to the best of our knowledge, this is the first study to aimed to investigate the comparison of oxidative stress in laparoscopic or OA.  To exclude the effect of excessive inflammatory and infectious changes on oxidative status in complicated appendicitis, we included only uncomplicated cases. Preoperative values of WBC and CRP, which were predictors of inflammation severity,[2122] were found to be similar in open and LA groups. Also, preoperative TOS, TAS, and OSI values showed no difference between the groups. Thus, the preoperative inflammatory, infectious, and oxidative status of the groups were proven to be similar. To exclude the pharmacologic effect on oxidative stress, sevoflurane anaesthesia was used in all operations rather than propofol, which was suggested as an antioxidant.[23]  TOS and OSI reduced postoperatively in open and laparoscopic surgery groups, however; they were found to be significantly higher in OA group in postoperative 24th h. Suggesting that, open and LA reduced acute appendicitis related oxidative stress, but open surgery was generated more free radicals than laparoscopic surgery. The association of the severity of oxidative stress and progression of inflammation to the perforation in acute appendicitis was showed previously,[2425] thus, the reduction in oxidative stress was an expected finding when the inflamed appendix was resected. Although, the literature have contradictory results[6] about the effect of laparoscopic surgery in oxidative stress, a recent systematic review[4] have showed increased oxidative stress immediately following open surgery. The advantage of laparoscopic surgery on oxidative status was confirmed in different status of basal oxidative stress; Tsuchiya et al.[5] suggested an association between open abdominal surgery (sigmoid resection) and increased oxidative stress, also, Pappas-Gogos et al.[3] showed lower oxidative stress with laparoscopic colectomy for colorectal cancer. Theoretically, the resection of the inflamed appendicitis and the resection of free radical generating tumor may show similar oxidative status, when it compared with surgery related oxidative stress induction. In both conditions, the changes in oxidative stress could be explained as a consequence of the removal of the pathological organ. Assuming that the resection of the inflamed appendix reduces oxidative stress in open and laparoscopic surgery. The lower oxidative stress observed in LA compared to OA may be attributed to the following factors: Minor surgical trauma in laparoscopic surgery and exposure of the peritoneal cavity to room air in open surgery. In both groups, preoperative antioxidant status reduced minimally in postoperative 24th h while oxidative status reduced significantly. This findings may be interpreted as meaning that the preoperative antioxidant status of the patients are in maximum limits of the host defense system, and they reduced with the combined effect of detoxification and the decreased response to decreased volume of the antioxidants. Also, this theory was supported by the significant reduction on CRP values in LA group in postoperative 24th h.  Assessing oxidative stress and the response of the human body is challenging. There are no single biomarker can objectively measure oxidative stress,[26] also there are no biomarker in peripheral blood that correlates with tissue oxidative stress.[4] The studies, which were compared tissue levels of oxidative stress in open versus laparoscopic surgery have showed greater oxidative stress in the laparoscopic group.[1127] On the other hand, Dumlu et al.[24] suggested that serum oxidative stress markers can better predict oxidative imbalance in acute appendicitis compared with tissue markers. It has been shown that oxidative damage in tissue level can occur without any measurable changes in the peripheral blood.[28] Furthermore, blood and tissue biomarkers of oxidative stress may not follow the same pattern of change.[29] Beside the absence of the ideal method for the measurement of oxidative stress, unknown clinical significance of the alteration of oxidative stress is seems to be the main limitations of oxidative stress measuring studies. Even in the settings of uncomplicated surgery in relatively healthy individuals with no measurement of clinical recovery.  As a conclusion, this double-blind, randomized clinical trial provides evidence that LA for uncomplicated appendicitis is associated with significantly lower oxidative stress compared to OA. Some of the advantages of LA may be attributed to the significant reduction of oxidative stress in these patients. TOS reduction in postoperative 24th h is correlates with the reduction of CRP, which was suggested as a reliable biomarker of inflammation, in laparoscopic surgery.

Question: Compared to Open appendectomy what was the result of Laparoscopic appendectomy on C-reactive protein?
Evidence: TOS and OSI were found to be significantly higher in OA group when compared to LA group (P = 0.017 and 0.002) whereas no difference was detected in TAS level in the postoperative 24th h (P = 0.172).
Answer: significantly decreased