Abstract

Objective

To investigate changes in plasma oxidative stress markers associated with prolonged pneumoperitoneum during robot-assisted laparoscopic radical prostatectomy (RALP).

Methods

In all, patients of ASA Physical Status II–III who intended to undergo RALP were enrolled in the study. Arterial plasma levels of malondialdehyde (MDA) and intramucosal pH were measured 1 minute before and at 1 hour, 2 hours, 3 hours, and 4 hours after the introduction of pneumoperitoneum at an insufflation pressure of 15 mmHg; likewise, they were again measured every 10 minutes after deflation for 60 minutes, at 2 hours and 12 hours after deflation.

Results

The mean duration of pneumoperitoneum was around 4 hours. After induction of pneumoperitoneum, the MDA concentrations were significantly elevated at various time points as compared with the preinsufflation value. Significant increase of MDA value was noted also 30 minutes after deflation as compared with the pre-deflation levels. The intramucosal pH value decreased significantly after CO₂ insufflation compared with the preinsufflation values. It also increased significantly 2 hours after CO₂ deflation as compared with the pre-deflation values.

Conclusion

A prolonged pneumoperitoneum in RALP results in decreased splanchnic blood flow. Pneumoperitoneum itself produces oxidative stress, and ischemia-reperfusion model after deflation of pneumoperitoneum produces more oxidative stress.

Keywords

gastric mucosa; hydrogen-ion concentration; malondialdehyde; pneumoperitoneum, artificial; reperfusion injury;

1. Introduction

Minimally invasive surgery has seen tremendous growth. In 2000, Binder and Kramer¹ performed the first robot-assisted laparoscopic radical prostatectomy (RALP), which is a promising minimally invasive surgical approach with possible reduced blood loss, fewer blood product transfusions, shorter care in postanesthesia unit, and shorter hospital stay.2, 3 Although laparoscopic surgery is generally considered safe, increased intra-abdominal pressure associated with pneumoperitoneum during laparoscopic procedures greatly impairs the splanchnic perfusion as a result of compression. The detrimental effects of increased intra-abdominal pressure on the hepatic arterial, mesenteric, and intestinal mucosal blood flows have been described in several studies.4, 5 An ischemia-reperfusion human model has been observed during and soon after laparoscopic surgery.6, 7, 8, 9 After deflation of pneumoperitoneum, intra-abdominal pressure and splanchnic blood flow normalize, thereby representing the reperfusion phase. Clinical manifestations of ischemia-reperfusion injuries are diverse and may include myocardial hibernation/stunning, reperfusion arrhythmias, impaired cerebral function, breakdown of the gastrointestinal barrier, systemic inflammatory response syndrome, and multiorgan dysfunction syndrome.¹⁰ Previous studies have documented the impact of CO₂ pneumoperitoneum on oxidative stress during laparoscopic cholecystectomies and minor gynecological surgeries.11, 12 The duration of the pneumoperitoneum involved in these previous studies was roughly 1 hour. The severity of pneumoperitoneum-induced oxidative stress is suggested to be time dependent.¹³ However, data for long-term pneumoperitoneums are very limited.

Gastric intramucosal pH (pHi) reflects splanchnic perfusion and oxygenation.¹⁴ Its obtainment is minimally invasive, and its measurement is effective for assessing tissue oxygenation adequacy.¹⁴ Reactive oxygen species, which damage cellular components and initiate the lipid peroxidation process, are responsible for ischemia-reperfusion injuries.⁶ Harmful oxidative reactions may occur in organisms so that they are removed by means of enzymatic and nonenzymatic antioxidative mechanisms.¹¹ Malondialdehyde (MDA) is regarded to be the most reliable and producible markers of oxidative stress in the clinical setting.¹³ The main source of MDA is the peroxidation of polyunsaturated fatty acids with two or more methylene-interrupted double bonds.¹⁵ It is referred to as potentially mutagenic and atherogenic because of its interaction with DNA and proteins.¹⁵

The aim of the current study was to evaluate the alterations in oxidative stress and splanchnic perfusion during prolonged pneumoperitoneum in RALP. A prospective clinical study was designed, and plasma levels of MDA-lipid peroxidation marker and gastric pHi were measured.

2. Methods

2.1. Patients

The study protocol was approved by the ethics committee of Chang Gung Memorial Hospital (IRB 98-2998B, Taoyuan, Taiwan). All patients provided written informed consent. Twelve adult patients whose physical statuses were graded as ASA Status II or III were recruited. The RALP was carried out by a single surgeon. Exclusion criteria included a history of sepsis or shock, cardiopulmonary disease clinically, tobacco smoking, or recent antioxidant or vasoconstrictor use.

2.2. Study protocol

General anesthesia was induced intravenously with fentanyl (3 μg/kg), midazolam (0.2–0.3 mg/kg), and rocuronium (0.5–0.6 mg/kg). A 20-gauge arterial catheter was inserted into the left brachial artery after the induction of anesthesia for blood sampling and arterial blood pressure monitoring. After tracheal intubation, anesthesia was maintained with isoflurane in oxygen at 1–1.5 end-tidal concentration, and the arterial blood pressure was kept within 20% of its preinduction value. The lungs were mechanically ventilated with pressure-controlled ventilation to maintain the end-tidal CO₂ at 30–40 mmHg. Bolus dose of rocuronium (0.2 mg/kg) was given every 30 minutes during surgery. Intraoperative electrocardiogram, pulse oximetry, central venous pressure, and arterial blood pressure were constantly monitored. After skin closure, patients were extubated in the operating room.

Pneumoperitoneum was created by intraperitoneal insufflation of CO₂ with the patient in the supine position. Patients were then placed in the steep Trendelenburg position (30° from horizontal). Intraperitoneal pressure was maintained at 15 mmHg during the induced pneumoperitoneum.

2.3. Measurement

Blood samples containing sodium heparin as anticoagulant were obtained for MDA measurements. Baseline value was obtained 1 minute before inducing the pneumoperitoneum. After pneumoperitoneum establishment, blood samples were collected 1 hour after and at 1-hour intervals during the procedure. At the end of the surgery, blood samples were obtained 10 minutes after the deflation of CO₂ and at 10-minute intervals for 1 hour, and then at 2 hours and 12 hours after the deflation of CO₂. Blood samples for MDA analysis were centrifuged (3500 rpm, 10 minutes) and the supernatants were immediately stored at −80°C until the analysis was performed within 1 week.

The MDA content was measured by a thiobarbituric acid assay to estimate the lipid peroxidation products. MDA was treated with thiobarbituric acid to form a colored complex. We measured the levels of thiobarbituric acid reactive substances by adopting the method reported by Yoshioka et al¹⁶ Briefly, 0.5 mL plasma, 2.5 mL thiobarbituric acid (200 g/L), and 1 mL TBA (6.7 g/L) were mixed together and boiled for 30 minutes. Two milliliters of butanol was added to the tubes, and the colored supernatant was extracted after centrifugation at 3000 rpm. The absorbance of the supernatant was spectrophotometrically measured at 535 nm. The MDA concentration was expressed in micromoles per mg protein (μmol/mg protein). The plasma level of MDA in each sample was analyzed by enzyme-linked immunoabsorbent assay and reported as femtograms per milligram of protein (fg/mg protein).

After the induction of anesthesia, a double-lumen tonometric nasogastric tube (Tonometrics Catheter; Datex-Ohmeda, Helsinki, Finland) was inserted to the stomach. The correct position of the tube was confirmed by positive aspiration of gastric juice. To calculate pHi, arterial blood and gastric juice samples were collected 1 minute before the insufflation of CO₂, 1 hour after the insufflation of CO₂, at 1-hour intervals thereafter during the procedure, 10 minutes after deflation, at 10-minute intervals until 1 hour after deflation, and 2 hours after deflation of CO₂.

The pHi was estimated indirectly by the PCO₂ measured in the stomach lumen and the bicarbonate concentration measured in the arterial blood with the Henderson-Hasselbalch’s equation.¹⁷pHi=6.1+log[HCO3−]PCO2×0.031

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2.4. Statistical analysis

All data are presented as mean ± standard deviation. Calculation and data analysis were performed using SPSS version 17.0 (SPSS Inc., Troy, NY, USA). The levels of MDA and pHi were analyzed and compared using the Wilcoxon signed-rank test. Differences were considered to be statistically significant if p < 0.05.

3. Results

Twelve patients with prostate cancer, who underwent RALP performed by a sole surgeon, were enrolled for the study. Demographic and clinical data are presented in Table 1. Data of blood pressure and heart rate during and after the surgery are presented in Table 2.

Table 1. Patient demographic characteristics.

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Table 2. Data of mean arterial pressure and heart rate during and after the surgery. Mean arterial pressure was kept within 20% of its preinduction value.

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3.1. Malondialdehyde

After CO₂ insufflation, the plasma concentration of MDA, as a measure of free radical production, increased significantly as compared with the preinsuffltion value (Fig. 1). The mean peak value appeared 2 hours after CO₂ insufflation (95.3 ± 74.9 μmol/mg), which was significantly higher than the mean baseline value (47.7 ± 14.2 μmol/mg). After the deflation of CO₂, MDA values demonstrated a slight but insignificant increase. The significant increase appeared 30 minutes after deflation (106.7 ± 75.0 μmol/mg) as compared with the pre-deflation levels (75.3 ± 42.9 μmol/mg). The MDA values nearly returned to baseline levels some 12 hours after CO₂ deflation (Fig. 1).

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Fig. 1. Plasma MDA level in patients undergoing robot-assisted laparoscopic radical prostatectomy. Data are expressed as the mean ± standard deviation. Difference from preinsufflation value: *p < 0.05, **p < 0.01. Difference from pre-deflation value (+4 hours after pneumoperitoneum): #p < 0.05. MDA = malondialdehyde.

3.2. Intramucosal pH

The pHi values significantly decreased after CO₂ insufflation as compared with the preinsufflation values. After deflation, the pHi values were still significantly lower than the preinsufflation values. Two hours after CO₂ deflation, the pHi value increased and significantly differed as compared with the pre-deflation value (Fig. 2).

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Fig. 2. pHi value in patients undergoing robot-assisted laparoscopic radical prostatectomy. Data are expressed as the mean ± standard deviation. Difference from preinsufflation value: *p < 0.05. Difference from pre-deflation value (+4 hours after pneumoperitoneum): **p < 0.05. pHi = intramucosal pH.

4. Discussion

In the present study, significant increase in MDA concentration and decrease in pHi value were observed after the induction of pneumoperitoneum. After deflation, an unexpected increase in MDA value was noted. A prolonged pneumoperitoneum of 4 hours could lead to decreased splanchnic blood flow and increased oxidative stress, not only during the pneumoperutineum but also after the deflation.

Numerous outcome measures for oxidative stress can be used clinically, including endogenous antioxidant levels, peroxidation markers, derived gastric pHi, cytokine levels, and histological findings.¹³ Measurements of lipid peroxidation markers, especially MDA, are generally regarded to be most reliable and reproducible in the clinical setting.13, 15 The present data revealed that the maintenance of intra-abdominal CO₂ pressure at 15 mmHg for 4 hours could induce a significant increase in oxidative stress markers. There were two trends in regard to MDA changes throughout the surgery. One occurred during the pneumoperitoneum, and the other appeared after CO₂ deflation. During the induced CO₂ pneumoperitoneum, the plasma MDA level was significantly higher than its preinsufflation level. It peaked during the second hour and revealed a slight but insignificant decrease in the third and fourth hours. Previous studies suggested that the levels of oxidative stress markers associated with pneumoperitoneums could rise in a time-dependent manner.13, 18 The CO₂ insufflation pressure in our study was constant throughout the surgery, but the MDA level did not continuously increase during the third and fourth hours. The MDA level appeared to achieve homeostasis after being subject to pneumoperitoneum for 2 hours. No other study has evaluated oxidative stress markers during such a long pneumoperitoneum duration. To the best of our knowledge, there are no studies in this regard in prolonged pneumoperitoneum (i.e. 3 hours or 4 hours). In our speculation, there might be some substances that were produced to resist the lipid peroxidation process as time went by.

After CO₂ deflation, MDA values increased steadily and reached a peak 30 minutes after deflation, which was significantly higher than that during CO₂ insufflation. Various studies have investigated the ischemia-reperfusion injury that occurs as a result of pneumoperitoneum.6, 7, 11, 12 Pneumoperitoneum for laparoscopic surgery at intra-abdominal pressure of 12–15 mmHg is much higher than the normal pressure of the portal system (7–10 mmHg). Previous experimental and human studies have shown that CO₂ pneumoperitoneum impairs splanchnic perfusion by compressing vessels.⁵ Portal venous flow is also reduced during laparoscopic procedures,¹⁹ and there have been several reports about intestinal ischemia after laparoscopic procedures.20, 21, 22, 23 These findings suggest that pneumoperitoneum for laparoscopic surgery, which consequentially raises the intra-abdominal pressure, may produce significant organ ischemia followed by reperfusion injury on deflation of the abdomen.24, 25 Consistent with these findings, our results suggest that an ischemia-reperfusion injury would be present at the end of a prolonged induced pneumoperitoneum. A previous study in women who underwent laparoscopic enucleation of uterine myoma demonstrated increased levels of MDA immediately after surgery, which normalized 24 hours after surgery.²⁶ In our study, plasma MDA levels normalized 12 hours after CO₂ deflation.

Fall of gastric pHi is an early indicator of inadequate splanchnic perfusion.13, 17 It is based on the measurement of increased tissue CO₂ production that accompanies anaerobic metabolism in the gut. The pHi is an estimate of the gastric mucosal CO₂ and bicarbonate concentration in arterial blood. The pHi value was significantly decreased after CO₂ insufflation in our study. Previous studies have reported a measurable and continuous reduction in gastric mucosal pH during laparoscopic cholecystectomy.27, 28 These results indicate that splanchnic ischemia occurs during the induced pneumoperitoneum. In our study, the decrease in pHi persisted up to 1 hour after CO₂ deflation. Koivusalo et al²⁸ reported a decrease in pHi after the creation of a pnemoperitoneum for laparoscopic cholecystectomy, and this decrease persisted for up to 3 hours after CO₂ deflation. Persistent decrease of pHi indicates that although the splanchnic blood flow increases after deflation, however, impaired splanchnic circulation persists even after the termination of the pneumoperitoneum.

Decreased splanchnic blood flow and increased oxidative stress were observed during the existing pneumoperitoneum and after its deflation. The clinical relevance of oxidative stress after pneumoperitoneum is related to hepatic injury,²⁹ lung injury,³⁰ and renal injury.³¹ Light microscopy has also revealed histopathological findings concerning ischemia-reperfusion injuries in the liver and ileum.32, 33 It is reported that hypoperfusion-reperfusion injury would initiate a wide and complex array of inflammatory responses that may aggravate local injury and induce impairment of remote organ function as well.³⁴ Given the increasing complexity and duration of laparoscopic procedures, organ ischemia-reperfusion injury and oxidative stress associated with prolonged pneumoperitoneum may become more pervasive.

In conclusion, a prolonged pneumoperitoneum of 4 hours results in decreased splanchnic blood flow and increased oxidative stress both during the pneumoperitoneum and after deflation. Although RALP is a promising and minimally invasive surgical approach with many advantages, the oxidative stress generated during a prolonged pneumoperitoneum should be noted. The clinical significance of prolonged pneumoperitoneum-associated oxidative stress and the impact of its reduction on defined clinical endpoints need further investigation.

Acknowledgment

This study was supported by Chang Gung Medical Grant (CMRPG390881).