What is the ideal preconditioning time to reduce laparoscopic-induced oxidative stress?

Abstract

Introduction:

Ischaemic preconditioning is the most effective method for the prevention of ischaemic-reperfusion injury; however, no study has examined the question of the ideal time for ischaemic preconditioning.

Patients and Methods:

The patients were divided into five groups, each group including of 20 patients. The precondition was applied as 1, 5, 10 and 15 min in Groups I, II, III and IV and Group V was the control group. Repeated blood samples were taken to measure the total antioxidant status (TAS), total oxidant status and oxidative stress index (OSI) values, just before insufflation, at the end of the operation and at 6 and 24 h of the post-operative period.

Results:

A significant difference was observed between the TAS values at the end of the operation and at the sixth post-operative time of the four groups (P = 0.001, 0.000, 0.001, 0.019 and 0.033, respectively). Furthermore, a significant difference was observed between TAS values at the post-operative 24^th h of Group III and Group V, and also a significant difference was observed between the OSI values at the post-operative 6^th h of Groups III and V.

Conclusion:

The low OSI and TAS values may interpret as a low degree of oxidative damage. The OSI values at the post-operative 6 h of Groups I and II were lower than those of other groups. We accept this result as low oxidative damage.

INTRODUCTION

Today, laparoscopy is used successfully in many surgical procedures, even in complex operations, but pneumoperitoneum has some negative side effects.[1] Splenic ischaemia results from a decrease in the intramucosal pH of the intestinal tract due to an increase in intraabdominal pressure (IAP), which is the important cause of oxidative damage during laparoscopic.[2] In the post-operative period, reactive oxygen species (ROS) and inflammatory cytokines (TNF-α, IL-6) levels will increase which causes organ damage.[1] The magnitude of the surgical injury depends on the size and duration of the intra-abdominal pressure.[1] Pneumoperitoneum (Pp) and desufflation may consider typical ischaemic-reperfusion (I/R) injury.[3] Gasless surgery, excess fluid and various pharmacological means have been used methods to prevent Pp-related I/R injury.[3-6] Recently, ischaemic preconditioning (I/P) is used as an alternative method to reduce the I/R injury, which is based on the principle of short-term ischaemia and reperfusion before long-term ischaemia.[2-4] In various studies, I/P was shown to be used to prevent the adverse effects of I/R injury in cardiac, liver, kidney, bone, small intestine, lung and reconstructive surgery and is more effective than low-pressure pneumoperitoneum.[3,5-8] Thus, these cycles can be followed by increased resistance to aggravated cellular reoxygenation injury.[6] Although the efficacy of precondition for reducing oxidative damage has been demonstrated in many experimental and clinical studies performed at different pressure levels or modalities, there is no consensus on what should be an ideal time for I/P. This is very important for clinical practice because I/P itself prolongs anaesthesia and operating time.[9,10] On this subject, our study is the first report dealing with this question in humans and will bring about significant improvements for ideal IP time. We used total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) in laparoscopic cholecystectomy (LC) at constant pressure and different preconditioning periods for evaluating the ideal time of I/P in a prospective randomisation study.

PATIENTS AND METHODS

This study was conducted in the Derince Training and Research Hospital, between January 2016 and December 2016. The protocol of the study was approved by the Ethics Committee of Kocaeli University, Faculty of Medicine and conducted according to the Declaration of Helsinki, Good Clinical Practice Guidelines. The patients were informed about the nature of the study and informed consent was obtained. The patients, who have no systemic inflammatory disease, diabetes mellitus, autoimmune diseases, non-pregnant, with the American Society of Anaesthesiologists Score of II and patients otherwise incapable of providing informed consent were excluded from the study.

One hundred patients (30 men and 70 women) who underwent LC for symptomatic cholelithiasis were enrolled in the study. All patients underwent four-port LC. Patients were randomized to five groups each consisting of 20 patients; Group 1 (1-min I/P) was subjected to 3 min of 14 mmHg of pressure, followed immediately by 1 min of deflation, Group 2 (5-min I/P) was subjected to 3 min of 14 mmHg of pressure, followed immediately by 5 min of deflation; Group 3 (10-min I/P) was subjected to 3 min of 14 mmHg of pressure, followed immediately by 10 min of deflation; Group 4 (15-min I/P) was subjected to 3 min of 14 mmHg of pressure, followed immediately by 15 min of deflation and Group 5 (control) was subjected to operation period without IP. Repeated blood samples were taken before insufflation (A: pre-operative sample), just after the end of the operation (B: post-operative 0-h sample), at 6 and 24 h of the post-operative period (C: post-operative 6-h sample and D: 24-h sample) of each patient. Serum samples were centrifuged at 4000 rpm for 10 min and then transferred to Eppendorf tubes and stored at −80°C.

Biochemical analysis

Total antioxidant status

TAS levels were measured using commercially available kits (Relassay, Turkey). The novel automated method is based on the bleaching of the characteristic colour of a more stable 2,2′-Azino-bis (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.[11]

Total oxidant status

TOS levels were measured using commercially available kits (Relassay, Turkey). In the new method, oxidants present in the sample oxidized the ferrous ion-o-dianisidine complex to the 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 colour 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 per litre (μmol H₂O₂ equivalent/L).[12]

Oxidative stress index

The ratio of TOS to TAS was accepted as the 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).[13-15]

Statistical analysis

All the data analysed using the Statistical Package for the Social Sciences (SPSS) software version 20.0 (IBM, Chicago, IL, USA). The normal distribution conformity was assessed using the Kolmogorov–Smirnov test. Numerical variables with normal distribution were given as mean ± standard deviation, numerical variables without normal distribution were given as median (25^th percentile–75^th percentile) and categorical variables were given as frequency (percentages). The differences between the groups were analysed by the one-way variance analysis for numerical variables with normal distribution and for numerical variables without normal distribution, the Kruskal–Wallis test was used. The Turkey, Dunnet and Dunn test were used for multiple comparisons. Since the differences between the repeated measurements are not provided by the assumption of normal distribution, so Friedman’s two-way analysis of variance test was used. P <0.05 was considered statistically significant.

RESULTS

Patients ranged from 18 to 82 years old, with a mean age of 51.87 ± 14.45 years. No significant difference in age was found between the five groups (P = 0.89). Seventy of the patients were female (55.6%) and 30 (23.8%) were male and a significant difference was observed between them (P = 0.049) [Table 1].

Table 1.

Gender comparison of groups

Group	Frequency, n (%)	Total	P
I
Female	9 (45)	20	0.049
Male	11 (55)
II
Female	16 (80)	20
Male	4 (20)
III
Female	13 (65)	20
Male	7 (35)
IV
Female	15 (75)	20
Male	5 (25)
V
Female	17 (85)	20
Male	3 (15)

A significant difference was observed between the TAS-C values of Groups I, II, III and IV (P = 0.001, 0.000, 0.001 and 0.019, respectively) and between the TAS-B values of Groups II and V, the TAS-D values of Groups III and V, the OSI-3 values of Groups III and V and the OSI-4 values of Groups I and III (P = 0.033, 0.040, 0.003 and 0.035, respectively) [Table 2].

Table 2.

Total oxidant status, total oxidant status and oxidative stress index values of the groups

	Group I	Group II	Group III	Group IV	Group V
Age	49.75±14.31	53.40±15.60	53.90±13.90	51.25±13.36	51.05±15.99
TAS
A	1.54±0.30	1.44±0.26	1.48±0.24	1.51±0.31	1.60±0.27
B	1.52 (1.42–1.72)	1.42 (1.32–1.69)e	1.53 (1.41–1.66)	1.42 (1.25–1.71)	1.65 (1.52–1.86)e
C	1.47±0.18a	1.40±0.23b	1.47±0.22c	1.55±0.36d	1.75±0.27a,b,c,d
D	1.58 (1.38–1.75)	1.47 (1.30–1.65)	1.49 (1.35–1.55)f	1.47 (1.33–1.69)	1.63 (1.53–1.75)f
TOS
A	10.91 (9.69–13.50)	9.51 (7.26–11.92)	11.26 (7.94–11.99)	10.64 (7.94–13.23)	9.47 (6.70–14.01)
B	10.48±3.26	9.76±4.98	11.24±5.11	10.57±5.68	13.05±9.39
C	5.64 (4.84–6.41)	5.75 (4.84–6.94)	6.33 (5.56–9.11)	5.64 (5.06–9.28)	5.69 (4.61–7.05)
D	6.06 (5.26–7.87)	5.87 (4.83–7.83)	8.83 (5.66–15.32)	6.32 (5.08–8.87)	6.78 (5.96–8.01)
OSI
1	7.63 (5.90–9.05)	6.75 (5.61–8.75)	7.38 (5.63–8.08)	7.18 (5.29–8.96)	5.96 (4.20–9.46)
2	7.17 (4.84–8.64)	5.91 (4.48–8.68)	6.54 (4.61–8.80)	5.98 (3.99–8.77)	6.21 (3.87–10.58)
3	3.73 (3.24–4.94)	3.83 (3.60–5.10)	4.19 (3.78–6.40)g	3.89 (3.15–5.81)	3.18 (2.71–4.02)g
4	3.77 (3.38–5.05)h	4.25 (3.69–5.20)	6.44 (3.81–10.16)h	4.20 (3.41–5.47)	4.29 (3.69–5.00)

^aP=0.001, ^bP=0.000, ^cP=0.001, ^dP=0.019, ^eP=0.033, ^fP=0.040, ^gP=0.003, ^hP=0.035. A: Pre-operative sample, B: Post-operative 0-h sample, C: Post-operative 6-h sample, D: Post-operative 24-h sample. 1: Pre-operative OSI sample, 2: Post-operative 0-h OSI sample, 3: Post-operative 6-h OSI sample, 4: Post-operative 24-h OSI sample. 1, 2, 3, 4 were given as median (25^th percentile–75^th percentile). TAS-A and C were given as mean±SD, TAS-B and D were given as median (25^th percentile–75^th percentile), TOS-A, C and D were given as median (25^th percentile–75^th percentile), TOS-B was given as mean±SD. Age was given as mean±SD. TOS: Total oxidant status, TAS: Total antioxidant status, OSI: Oxidative stress index, SD: Standard deviation

The repetition values of each group were compared and there were no significant differences between the TAS values of Groups I and IV (P = 0.37 and 0.12), however, Groups II and III showed significant differences (P = 0.032 and 0.00). There were significant differences between the TOS values of Groups I, II and IV (P = 0.00, 0.001 and 0.034), but no significant differences were observed in Group III. The OSI values of each group were compared and significant differences were found in Groups I, II and IV (P = 0.00, 0.00 and 0.007), however, no significant difference was found in Group III (P = 0.090) [Table 3].

Table 3.

Comparison of repetitive measurements within each group

	Groups
	I				II				III				IV
	A	B	C	D	A	B	C	D	A	B	C	D	A	B	C	D
TAS	1.48 (1.31–1.72)	1.52 (1.42–1.72)	1.46 (1.33–1.61)	1.58 (1.37–1.75)	1.43 (1.28–1.58)a	1.42 (1.32–1.69)	1.36 (1.26–1.59)	1.50 (1.30–1.64)	1.52 (1.26–1.64)b	1.53 (1.41–1.66)	1.47 (1.29–1.60)	1.49 (1.34–1.55)	1.45 (1.29–1.70)	1.42 (1.25–1.71)	1.47 (1.32–1.68)	1.47 (1.33–1.68)
TOS	10.91 (9.69–13.50)c	10.27 (7.64–12.46)	5.64 (4.84–6.41)	6.06 (5.26–7.87)	9.51 (7.26–11.92)d	9.35 (5.89–13.49)	5.75 (4.84–6.94)	5.87 (4.83–7.83)	11.26 (7.94–11.99)	10.49 (6.93–16.43)	6.33 (5.56–9.11)	8.83 (5.66–15.32)	10.64 (7.94–13.23)e	8.95 (6.24–14.71)	5.64 (5.06–9.28)	6.32 (5.08–8.87)
OSI	7.63 (5.90–9.05)f	7.17 (4.84–8.64)	3.73 (3.24–4.94)	3.77 (3.38–5.05)	6.75 (5.61–8.75)g	5.91 (4.48–8.68)	3.83 (3.60–5.10)	4.25 (3.69–5.20)	7.38 (5.63–8.08)	6.54 (4.61–8.80)	4.19 (3.78–6.40)	6.44 (3.81–10.16)	7.18 (5.29–8.96)h	5.98 (3.99–8.77)	3.89 (3.15–5.81)	4.20 (3.41–5.47)

^aP=0.032, ^bP=0.00, ^cP=0.00, ^dP=0.001, ^eP=0.034, ^fP=0.00, ^gP=0.00, ^hP=0.007. A: Pre-operative sample, B: Post-operative 0-h sample, C: Post-operative 6-h sample and D: 24-h sample. TOS: Total oxidant status, TAS: Total antioxidant status, OSI: Oxidative stress index

DISCUSSION

Today laparoscopic technique has become the gold standard in many surgical procedures and high percentage of abdominal surgeries are carried out by laparoscopy and soon we can expect more difficult and long operations to be performed by this method, even in elderly patients.[16] The laparoscopic procedure is more beneficial than conventional techniques because of less post-operative pain, shorter hospitalisation, better wound healing and less chance of post-operative herniation.[1] Decreased venous return and increased systemic vascular resistance are the undesirable haemodynamic changes in laparoscopic surgery, which were mainly associated with an increase in IAP caused by Pp.[17] It has been shown that abdominal visceral perfusion and portal venous flow are decreased in patients who underwent LC.[18] Decreased abdominal visceral perfusion may be the most important cause of oxidative stress during laparoscopic surgical procedures.[17] The oxidative effects of laparoscopic procedures occur in two phases Pp and deflation.[2] Pp produces ischaemia during insufflation, but reperfusion injury occurs during the desufflation phase.[2] Furthermore, reactive oxygen radicals, the most important mechanisms of organ dysfunction related to laparoscopy, are generated in the process of restoring blood flow in the deflation phase.[19] Although decreased splenic blood flow by I/R leads to silent clinical results and does not cause problems in healthy individuals, it has the potential to develop severe organ dysfunction in patients with limited organ function.[18] The most important mechanism of organ dysfunction following an I/R injury is oxidative stress, which is the result of ROS produced following the restoration of bloodstream.[20] So far, many methods have been attempted to prevent or reduce I/R damage; one of these is I/P.[21,22] It is an endogenous protective mechanism which prepares the tissue for subsequent laparoscopic injury.[23] It has been reported that Pp above the normal physiological portal circulation pressure (7–10 mmHg) causes ischemia in the intra-abdominal organs, and this ischemia is normalised by deflation.[24] However, there is no ideal or standard preconditioning procedure, various cycles or periods of preconditioning manoeuvres have been applied to reduce oxidative injury related to laparoscopy. It was shown that Pp-induced oxidative damage at 15 mmHg pressure reduced by 5 or 10 min I/P.[23-25] On the other hand, the optimal course of I/R time for IP, which varies by different organ systems, is unknown and needs to be elucidated. IP was succeeded in the 5/10, 10/10 and 15/10 min cycles for the liver in the experimental studies and mostly IP was applied as much as the time of Pp.[3,4,26] However, only a few studies are currently available in the literature on this topic in laparoscopic surgery. In a study by Yilmaz et al. showed that a 10-min insufflation followed by 10-min desufflation decreases oxidative stress.[25] In our study, we applied IP after 3 min of 14 mmHg IAP. This short period of Pp is because if we applied IP as much as the time of Pp, it would prolong the duration of the operation from 10 to 30 min which causes a long period of inhalation of anaesthetic gas. There is no single biomarker that can objectively measure oxidative stress and there is no biomarker associated with tissue oxidative stress in peripheral blood.[27] Various oxidative stress markers, such as nitric oxide, tissue/blood malondialdehyde (MDA), total antioxidant levels, lipid peroxidation (LPO), protein carbonyl content and protein sulfhydryl levels have been studied.[28] In addition to these methods, LPO was used to reflect oxidative stress and total glutathione (GSH) was used to reflect actions against oxidative stress.[28] In the early 1990s, Kusano C et al. developed a new test called total antioxidant capacity (TAC), which measures TAS. Thus, the antioxidant capacity of all antioxidants in a biological sample could be measured.[29] The OSI indicates the degree of oxidative stress and can be expressed as the ratio of total peroxide to total antioxidant potential.[15] Liver I/R injury can result from 60 min of 15 mmHg Pp, manifested by a significant increase in LPO and transaminase levels and a decrease in TAS.[3] However, in the early post-operative period, IP can prevent oxidative stress and preserve liver function if it is established like 15 min of ischaemia followed by 10 min of reperfusion followed by 60 min of Pp.[3] The decrease in TAS value at 24-h gas dissolution is due to the decrease in TAC, which indicates the amount of free radicals production.[3] Stipancic et al. reported that there was no statistically significant change in TAS value amongst patients operated with the laparoscopic or open technique, however, Glantzounis et al. showed that the value of TAS decreased at 24 h of LC.[10,16] While the elevation of LPO and liver enzymes and a decrease in the TAS value are indicative of hepatic I/R damage, a decrease in OSI and TAS values are considered as indicators of low oxidative damage.[3] In this respect, our study revealed a significant difference between the OSI values of the Groups I, II and IV (P = 0.00, 0.00 and 0.007, respectively), and the lowest OSI levels were observed in Group I and Group II and were interpreted as a low oxidative damage. Despite the variable application of pre-condition periods in experimental studies, little information was found in the literature on the application of pre-condition periods in the clinical practise. In a study, 5 min of 15 mmHg Pp and then precondition with 5-min of desufflation showed that I/P significantly corrected the increase in MDA and GSH levels declined.[19] Arioz et al. recommended 5 min of precondition to improve the MDA and GSH values.[9] In our study, we found no significant differences between 1, 5, 10 and 15 min of I/P, but the lowest OSI values were observed at 1 and 5 min of I/P, which can be considered as low oxidative stress. Previous studies on this subject have shown that pneumoperitoneum causes oxidative stress and has negative side effects, and I/P can be prevented with a 5 or 10 min precondition.[2,9] As a consequence of our results, 1 or 5 min of deflation reduced oxidative damage, but much more clinical studies are needed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.