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Changes of nitric oxide and endothelin, thromboxane A2 and prostaglandin in cirrhotic patients undergoing liver transplantation

AIM: To investigate the perioperative changes of nitric oxide (NO) and endothelin (ET), thromboxane A 2 (TXA 2 ) and prostaglandin (PGI 2 ) during liver transplantation in end-stage liver disease patients. METHODS: Twenty-seven patients with end-stage cirrhosis undergoing liver transplantation were enrolled in this prospective study. Blood samples were obtained from superior vena at five different surgical stages. Plasma concentrations of nitrate and nitrite were determined to reflect plasma NO levels. Plasma levels of ET-1, 6-keto-PGF1 alpha and thromboxane B 2 (TXB 2 ), the latter two being stable metabolites of PGI 2 and TXA 2 respectively, were measured. RESULTS: The NO level decreased significantly after vascular cross-clamping and increased significantly at 30 min after reperfusion. While the ET levels at 30 min after clamping and after reperfusion were significantly elevated. The ratio of NO/ET decreased significantly at 30 min after vascular cross-clamping and at the end of surgery. The PGI 2 level and the TXA 2 during liver transplantation were significantly higher


INTRODUCTION
Liver transplantation is becoming a more and more common treatment method of end-stage liver disease. The hemodynamic alterations occurring after graft reperfusion and the subsequent complications may result from the changes of some biotic activators [1] . Nitric oxide (NO) and endothelin (ET), thromboxane A2 (TXA2) and prostaglandin (PGI2) are two groups of important vasoactive substance. The clinical and animal experiment documented that the two groups of substance are involved in a variety of liver transplant-relevant processes, including ischemia-reperfusion injury, acute cellular rejection, and circulatory changes characteristic of advanced liver disease [2] . However, there is considerable controversy in the perioperative plasma concentrations of the two groups of substance and the precise mechanism of these changes is not fully known.
This study monitored blood levels of NO and ET, TXA2 and PGI2 at different time points perioperatively in cirrhotic patients undergoing liver transplantation.

Patients
During a 10-mo period, all patients with end-stage cirrhosis undergoing liver transplantation were enrolled in this prospective study. With the approval of the institutional ethics committee from Sun Yet-Sen University, informed consent was obtained from patient before each assessment. We excluded patients receiving liver transplantation more than one time and patients losing blood more than 3500 mL during operation.

METHODS:
Twenty-seven patients with end-stage cirrhosis undergoing liver transplantation were enrolled in this prospective study. Blood samples were obtained from superior vena at five different surgical stages. Plasma concentrations of nitrate and nitrite were determined to reflect plasma NO levels. Plasma levels of ET-1, 6-keto-PGF1 alpha and thromboxane B2 (TXB2), the latter two being stable metabolites of PGI2 and TXA2 respectively, were measured.

RESULTS:
The NO level decreased significantly after vascular cross-clamping and increased significantly at 30 min after reperfusion. While the ET levels at 30 min after clamping and after reperfusion were significantly elevated. The ratio of NO/ET decreased significantly at 30 min after vascular cross-clamping and at the end of surgery. The PGI2 level and the TXA2 during liver transplantation were significantly higher than the baseline level, but the ratio of TXA2/PGI2 decreased significantly at 30 min after clamping.
CONCLUSION: NO/ET and TXA2/PGI2 change during liver transplantation. Although the precise mechanism r e m a i n s u n k n o w n , t h e y m a y p l a y a r o l e i n t h e pathobiology of a variety of liver transplant-relevant processes.

Detection of NO
Two milliliters of superior vena cava blood were collected and placed in ice-cooled polypropylene tubes. These samples were immediately centrifuged at 3000 r/min for 10 min and stored at -40℃ for late assay. Test kits (Science and Technology Development Center of The General Hospital of PLA, Beijing, China) for NO analysis were used. The optical density value was read in a spectrophotometer set at 546 nm. The NO numeric value was calculated based on a standard and empty tube according to the equation. The calculated results were expressed as μmol/L.

Detection of ET
Two milliliters of superior vena cava blood were collected and placed in ice-cooled polypropylene tubes containing 40 μL aprotinin and 30 μL EDTA. These samples were immediately centrifuged at 3000 r/min for 10 min. Collected plasma was stored at -40℃ until batched assays were performed. Plasma ET levels were evaluated with ET detection kit (Science and Technology Development Center of The General Hospital of PLA, Beijing, China) according to the manufacturer's instructions.

Detection of TXA2 and PGI2
Three milliliters of superior vena cava blood were collected with a five-milliliter syringe containing 200 μL EDTA·2Na, mixed fully and injected into a tube. These tubes were immediately centrifuged at 3500 r/min for 15 min. Collected plasma was stored at -40℃ until use. Detection of the level of TXA2 and PGI2 was performed according to the manufacturer's instructions (Science and Technology Development Center of The General Hospital of PLA, Beijing, China).

Statistical analysis
Data are shown as mean ± SD. Data of the various time points were compared using ANOVA analysis (SPSS statistical package, version 11.0). Significant difference was accepted at P < 0.05.

RESULTS
A total of 27 patients (81% men, age 49 ± 11 year, BSA 1.72 ± 0.15) were enrolled in this study. Among these patients, 15 had hepatitis B virus associated cirrhosis, 12 had cirrhosis and liver cancer.
The changes of nitrate, nitrite and ET levels in plasma during liver transplantation are shown in Table 1. The mean baseline value of NO (reflected by nitrate and nitrite) was 27.45 ± 5.70 μmol/L. The NO level decreased significantly after vascular cross-clamping, from 35.19 ± 7.72 μmol/L to 25.64 ± 5.64 μmol/L (P < 0.05). At 30 min after reperfusion, a significant elevation of NO was found, from 25.64 ± 5.64 μmol/L to 35.30 ± 11.23 μmol/L (P < 0.05).
The baseline ET concentration was 61.21 ± 25.38 ng/L. Compared with the baseline level, the ET levels at 30 min after clamping and after reperfusion were significantly elevated (110.61 ± 24.65 ng/L, 114.00 ± 26.73 ng/L respectively).
The changes of TXB2 and 6-keto-PGF1a levels in plasma during liver transplantation are shown in Table 2. The prostaglandin level and the TXA2 level presented the same changes. The concentrations at every time point were significantly higher than the baseline level. Peak levels of prostaglandin were 932.71 ± 25.66 ng/L and 895.83 ± 300.71 ng/L, detected at 60 min after operation and 30 min after vascular cross-clamping respectively.
The mean baseline value of NO/ET was 0.54 ± 0.25. The ratio decreased significantly at 30 min after vascular cross-clamping and at the end of surgery. The mean Table 1 Changes of NO2/NO3 and ET levels in plasma during liver transplantation (mean ± SD, n = 27)    baseline value of TXA2/ PGI2 was 0.89 ± 0.37, the ratio decreased significantly at 30 min after clamping. Compared with the value at 30 min after clamping, the ratio increased significantly at 30 min after reperfusion, from 0.33 ± 0.14 to 0.49 ± 0.21.

DISCUSSION
In this study we measured systemic NO and ET, TXA2 and PGI2 at five different surgical stages: basal, hepatectomy, anhepatic, 30 min after graft reperfusion and the end of surgery. Overall results showed that the levels of these two groups of vasoactive substances changed at each stage during liver transplantation. Previous studies have demonstrated that NO and ET, TXA2 and PGI2 play an important role in the pathobiology of ischemiareperfusion injury and postreperfusion syndrome [3,4] . It suggests that disturbed balance between these vasodilators and vasoconstrictors may contribute to some liver transplantation-relevant syndromes. NO and ET are the most important local vasodilator and vasoconstrictor respectively. They seem to play a role in almost every organ and tissue. However, there is considerable confusion in understanding their roles. Some researches sug gest that the important factors in determining the beneficial versus harmful effects of NO are the amount, duration, and site of NO production [5] . Ovadia et al demonstrated that the fetal pulmonary vasoconstriction after acute constriction of the ductus arteriosus is mediated by NO/ET-1 interactions [6] . Shirakami et al reported that the plasma ET level was increased before transplantation compared with that of healthy children, but decreased during the anhepatic phase [7] . It increased again after reperfusion and remained at high level in the early postoperative period. These suggest that ET production in the cirrhotic liver is augmented and ET plays some role in circulatory regulation during the perioperative period of pediatric liver transplantation. In the present study, although both NO and ET increased after graft reperfusion, the ratio of them was decreased. It suggested that the imbalance of NO and ET level may participate in the pathophysiology of systemic and local circulation disorders.
Many data about the relationship between another g roup of vasoactive substances, PGI2/TXA2 and hemodynamics have been reported [8,9] . TXA2 is both a vasoconstrictor and a potent stimulus for platelet aggregation. Its effect is antagonized by prostacylin, which is released by vascular endothelial cells. Prostacylin exerts a variety of effects on the cardiovascular system, including a decrease in blood pressure associated with a decrease in systemic vascular resistance. In clinical liver transplantation, Khoury et al [10] demonstrated 60% of patients undergoing orthotopic liver transplantation accumulated prostacylin in the portal vein, which could be one of the causes of hypotension seen at reperfusion of the donor liver. As previously demonstrated, we found that the baseline levels of PGI2 and TXA2 in our patients were higher than normal values. It was most likely due to the decreased metabolism of them in patients with endstage liver disease. In addition, we found PGI2 and TXA2 had the same changes during liver transplantation. The levels of PGI2 and TXA2 after reperfusion were elevated compared with the baseline level. However, the ratio of TXA2 and PGI2 was significantly lower than that of prereperfusion. This indicated that the disorder of TXA2 and PGI2 might also be involved in the circulation disorders during orthotopic liver transplantation.
In summary, two groups of endogenous vasoactive substance, NO/ET and TXA2/PGI2, are changed during liver transplantation. Although the precise mechanism remains unknown, they may play a role in the pathobiology of a variety of liver transplant-relevant processes.