|Year : 2015 | Volume
| Issue : 3 | Page : 133-138
The incidence of portal hypertension in children with choledochal cyst and the correlation of nitric oxide levels in the peripheral blood with portal pressure and liver histology
Karunesh Chand1, Veereshwar Bhatnagar1, Sandeep Agarwala1, Maddur Srinivas1, Nibhriti Das2, Manoj Kumar Singh3, Raju Sharma4
1 Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India
2 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
3 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
4 Department of Radiology, All India Institute of Medical Sciences, New Delhi, India
|Date of Web Publication||18-Jun-2015|
Prof. Veereshwar Bhatnagar
Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Aims: Symptomatic portal hypertension (PHT) as a complication of the choledochal cyst (CDC) is well-known, but the actual incidence of PHT in CDC has not been studied. This study was undertaken to evaluate the incidence of PHT in patients of CDC and correlate portal pressure (PP) with liver histology and blood nitric oxide (NO) levels. Materials and Methods: In this cross-sectional study, PP was measured after surgical access but before any mobilization of the cyst by directly cannulating a tributary of portal vein (preoperative PP) and at completion of surgery before closure (postoperative PP). Blood sample for NO and liver function tests (LFTs) was taken before surgery and during subsequent follow-up at 1-month, 3 months, and 6 months. Liver histology was assessed under parenchymal, bile duct, and portal parameters. Results: Measurement of PP and blood levels of NO was done in 20 patients. Mean preoperative PP was 16.45 ± 7.85 mmHg, and the median pressure was 14 mmHg (range 9-43). Mean of the postoperative PP was 14 ± 6.87 mmHg, and median pressure was 11.5 mmHg (range 7-37). The mean level of NO in the preoperative period was 11.85 ± 4.33 μmol/l, and median was 11.605 (range 5.24-22.77) μmol/l. NO levels at the first follow-up (1-month postoperative) were 5.96 ± 4.56 μmol/l and median value of 4.9 (range 1.74-23.56) μmol/l. Likewise, the mean and median values of NO at 3 months were 5.59 ± 7.15 μmol/l and median value of 3.71 (range 1.49-34.74) μmol/l. The mean and median levels of NO at 6 months postoperative were 5.08 ± 2.22 μmol/l and median of 4.59 (range 2.32-12.46) μmol/l. The fall in PP immediately after surgery was consistent and statistically significant (P = 0.001). There was statistically significant fall in the NO levels in the postoperative period as compared to the preoperative levels (P = 0.002). Bile duct proliferation was significantly correlated with PP (P = 0.05). Blood levels of NO closely followed the PP in the preoperative period and fell to baseline in subsequent follow-up. There was no statistically significant correlation between age at presentation, LFT and postoperative complications with either PP or NO levels. Conclusions: In this study, all patients with CDC had some degree of PHT. Measurement of PP and liver histology should be part of standard management protocol to take timely preventive measures so as to avoid life-threatening manifestations of PHT.
Keywords: Blood nitric oxide, choledochal cyst, liver histology, portal hypertension
|How to cite this article:|
Chand K, Bhatnagar V, Agarwala S, Srinivas M, Das N, Singh MK, Sharma R. The incidence of portal hypertension in children with choledochal cyst and the correlation of nitric oxide levels in the peripheral blood with portal pressure and liver histology. J Indian Assoc Pediatr Surg 2015;20:133-8
|How to cite this URL:|
Chand K, Bhatnagar V, Agarwala S, Srinivas M, Das N, Singh MK, Sharma R. The incidence of portal hypertension in children with choledochal cyst and the correlation of nitric oxide levels in the peripheral blood with portal pressure and liver histology. J Indian Assoc Pediatr Surg [serial online] 2015 [cited 2020 Jan 27];20:133-8. Available from: http://www.jiaps.com/text.asp?2015/20/3/133/159024
| Introduction|| |
Choledochal cyst (CDC), a congenital malformation of the biliary tract is predominantly a disease of childhood and can have a varied presentation. However, children with CDC have been reported to present with upper gastrointestinal bleed and splenomegaly. , Blood levels of nitric oxide (NO) is a known marker of portal hypertension (PHT), and its use in clinical practice has been suggested as a noninvasive method to monitor the severity of PHT.  Liver histology, which initially was not given much importance in CDC, has recently been extensively studied in cases of CDC and a wide spectrum of histological changes has been observed in the liver. ,,
This study has been designed to (i) evaluate the incidence of PHT in children with CDC, (ii) establish that blood levels of NO can be used as a marker to follow the postoperative changes in portal pressure (PP) noninvasively, and (iii) correlate PP and blood NO levels with histological changes in the liver.
| Materials and Methods|| |
The study was conducted in the Department of Pediatric Surgery, All India Institute of Medical Sciences over a period of 2 years (January 2012-November 2013). It was a cross-sectional observational study. All patients operated upon for CDC were included, unless the patient and parents refused to participation in the study. Those patients who died or were lost to follow-up were also excluded. Ethical clearance for the study was obtained from the Institutional Ethics Committee. All enrolled patients were evaluated with detailed history and clinical examination, blood was drawn for biochemical liver function tests (LFTs) and NO and imaging studies including ultrasonography, radionuclide scan, and magnetic resonance cholangio-pancreaticography were performed at presentation. All patients were treated with excision of the cyst and Roux en Y hepaticojejunostomy under general anesthesia by an upper abdominal laparotomy.
Estimation of blood levels of nitric oxide
A 2 ml blood sample was drawn from any peripheral vein and put in an ethylenediaminetetraacetic acid vial and immediately transported to the laboratory in an ice pack. Upon arrival in the laboratory, plasma was separated by centrifugation (+4°C, 3000 rpm, 10 min) and stored in 0.5 ml aliquots placed in cryo vials at −70°C until analyzed.
Blood concentration of NO was measured as its stable metabolite nitrate and nitrite. Nitrate was first reduced to nitrite by nitrate reductase, and then the blood level of nitrite was evaluated spectrophotometrically by Greiss reaction. The Greiss reagent, a 1:1 mixture of 0.2% N-(1 naphthyl)-ethylene-diamine and sulfanilamide in 5% phosphoric acid, was used in this procedure. 
Levels of NO in the normal population have been reported previously as 5.76 + 2.62 μmol/l. 
Measurement of portal pressure
Portal pressure was measured immediately after surgical access and before any mobilization of the liver and biliary tree. Datex Ohmeda S/5™ monitor (range of measurement = −40-320 mmHg, accuracy = ±5% or ±2 mmHg, sensitivity 5 μV/V/mmHg) attached to the Truwave disposable pressure transducer (Edward Life Sciences) was used for the pressure measurement. A continuous 3 ml/h flush device and stop cock was mounted at the level of the abdomen in the mid-axillary line to avoid the error due to height (10 mmHg for every 13.6 cm difference).  A 22-G cannula, attached to saline-filled pressure tubing was inserted directly into one of the tributaries of the gastroepiploic vein and PP was measured using the set up after proper zeroing for 3 times. The average of 3 measurements was taken as the preoperative PP. PP >8 mmHg was deemed as PHT. Similarly, the whole procedure was repeated at the end of the procedure just before closure of the abdomen, and that was recorded as the postoperative PP.
A wedge biopsy of the liver was taken before abdominal closure. Paraffin-embedded 4 μm thick sections were stained with hematoxylin and eosin and analyzed by a single senior pathologist (MKS) who was blinded for the tests. The histopathological changes were graded using the criteria as detailed in [Table 1] and [Table 2]. ,,
Postoperative and follow-up studies
In the postoperative period, the patients were followed-up at 1 month, 3 months, and 6 months interval. At each follow-up, blood was drawn for LFTs and estimation of NO levels.
The data analysis was done using STATA software version 11 (Stata Corp LP, Texas, USA). Data were presented as number (%) or mean ± standard deviation/median (range) as appropriate. The levels of NO in the patients of CDC were compared with those of controls as determined in a previous study  and also within themselves at various stages of management using paired t-test. The correlation between fall in the NO levels and the PPs following surgery was calculated using Spearman rank correlation coefficient. The (P < 0.05) were considered statistically significant.
| Results|| |
A total of 20 cases were included in the study. M:F ratio was 13:7. Mean age at presentation was 61.4 months, median age was 72 months, the age range was between 4 and 150 months. All patients had type I (fusiform) CDC. Clinical manifestations of PHT, e.g., gastrointestinal bleeding, ascites, splenomegaly etc., were not evident in any patient.
Portal pressure measurement
The mean preoperative PP was 16.45 ± 7.85 mmHg, and the median pressure was 14 mmHg (range 9-43). Mean of the PP recorded at the completion of surgery (postoperative) was 14 ± 6.87 mmHg and median pressure was 11.5 mmHg (range 7-37). In view of the small number of cases and large standard deviations of 7.85 and 6.87 in the pre and postoperative pressures, respectively, median values of PP were used for purposes of correlation. Since the PPs were >8 mmHg in the preoperative and postoperative periods, it was inferred that all patients had some degree of PHT prior to surgery, and this persisted at the end of surgery also. The fall in PP immediately after surgery was consistent and statistically significant (P = 0.001).
Levels of nitric oxide
The mean level of NO in the preoperative period was 11.85 ± 4.33 μmol/l, and median was 11.605 μmol/l (range 5.24-22.77) μmol/l. Mean and median levels of NO levels at the first follow-up (1-month postoperative) were 5.96 ± 4.56 μmol/l and 4.9 μmol/l (range 1.74-23.56), respectively. Likewise, the mean and median values of NO at 3 months were 5.59 ± 7.15 μmol/l and 3.71 μmol/l (range 1.49-34.74), respectively. The mean and median levels of serum NO at 6 months postoperative were 5.08 ± 2.22 μmol/l and 4.59 μmol/l (range 2.32-12.46), respectively. On correlating the NO levels in the preoperative and postoperative period, there was a statistically significant fall in the NO levels in the postoperative period at 1 st follow-up as compared to preoperative NO levels (P = 0.002). However, there was no significant fall in NO levels in the postoperative period during subsequent follow-up, that is, between 1 st and 2 nd follow-up and 2 nd and 3 rd follow-up (P = 0.52 and 0.13, respectively). Thus, the preoperative NO levels were elevated in all patients as compared to the normal population. The NO levels in the different follow-up periods postoperatively were similar to the levels in the normal population.
On correlating the PP with the histopathological changes in the liver, bile duct proliferation was the only factor which had a significant correlation with the PP (P = 0.04). The other liver histology parameters did not show significant correlation with the PP [Table 3]. Similarly, the NO levels also correlated with bile duct proliferation only (P = 0.05) [Table 4].
|Table 3: Correlation of various histology parameters with portal pressure|
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|Table 4: Correlation of various histology parameters with nitric oxide levels (< median vs. > median levels)|
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On correlating the preoperative PP and preoperative NO levels, it was evident that the NO levels followed the PP closely as depicted in the line diagram [Figure 1].
|Figure 1: Comparison of portal pressure (mmHg) with nitric oxide levels (mmol/l)|
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During postoperative follow-up, all patients remained asymptomatic.
| Discussion|| |
Portal hypertension, in cases of CDC, may develop by various mechanisms. The most important mechanism which has been proposed in children is the direct compression of the portal vein by the CDC by virtue of its location anterior to the portal vein and pressure on the vein by the sheer weight of cyst. Other mechanisms which have been described include biliary obstruction leading to secondary biliary cirrhosis and portal vein thrombosis resulting from recurrent cholangitis. ,,
The splanchnic and systemic circulations have been observed to be in a hyperdynamic state in all forms of PHT. This hyperdynamic circulation is initiated by arterial vasodilatation. NO, an endothelial-derived relaxing factor, is a key molecule in arterial vasodilatation in the splanchnic and systemic circulation. Enhanced NO production is one of the important features observed in the arteries of the systemic and particularly, the splanchnic circulation in PHT.  Goel et al. demonstrated increased NO levels in patients with PHT due to extrahepatic portal vein obstruction; these levels declined after successful shunt surgery and remained low till the shunt was patented. A shunt block was accompanied by rising NO levels.  Thus, NO levels have been shown to be a useful marker for PHT and its severity.
Choledochal cyst is considered a disease of bile ducts only and going by the external appearance of liver the histological features in the liver have long been ignored. Since then focused work in this field by various workers ,,, have led us to believe that CDC is not only a disease of bile ducts but also cause pathological changes in the liver.
The present study was based on certain premises. The first premise was that symptomatic PHT is a complication of CDC. The second premise was that blood NO level is a surrogate marker of PHT. And, the third premise was that histological changes in liver vary with the severity of PHT.
Portal pressure measurement
Median preoperative pressure was 14 mmHg (range 9-47). Median of the PP recorded at the completion of surgery (postoperative) was 11.5 mmHg (range 7-37). Since PHT is defined as PP more than 8 mmHg, in this study, all the patients had PHT preoperatively. Thus, it can be confidently stated that some degree of PHT is expected in all cases of CDC. PP measurement immediately following the excision of CDC consistently showed a fall in pressure to the tune of 6 mmHg. This fall in pressure was statistically significant (P = 0.001).
The immediate fall in PP postsurgery can be assumed as proof that direct compression of the portal vein by the cyst is the dominant mechanism of PHT. However, the PP does not normalize immediately postsurgery. If it does, then it may take a few months before doing so, as seen from the fall in levels of NO during subsequent follow-up at 1 month, 3 months, and 6 months. This highlights that other mechanisms, e.g., pathological changes in the liver, may also have a role to play in the causation of PHT in CDC. In a recent study, Sugandhi et al. have shown that the majority of histopathological changes in the liver either resolve completely or decrease in severity postoperatively. However, portal fibrosis did not show any significant reduction and central venous distension increased in severity.  Perhaps the time taken by the histological changes in the liver to revert to normal is the same time which the PP takes, to fall back to normal. Persistence of portal fibrosis may not allow the PHT to revert to normal, and these cases may require long-term follow-up.
In various published reports of symptomatic PHT with CDC, PHT actually resolved after excision of cyst and hepaticojejunostomy and the children were asymptomatic during subsequent follow-up. , In this cohort of patients, although all patients remained asymptomatic after successful surgery of the CDC cyst, the range of NO levels at 1 month, 3 months, and 6 months follow-up helped to identify the patients with persistently elevated NO levels thereby indicating persistently elevated PP. Thus, successful surgery of CDC does not rule out PHT in the subsequent follow-up, and these patients need to be under surveillance.
Since levels of NO closely follow the PP as depicted by the line diagram, it can be confidently stated that the fall of NO levels to normal indicates that PPs have also normalized in the postoperative period.
On correlating PP with histopathological changes in this study, only ductular proliferation correlated significantly with PP. Ductular proliferation is seen with variable severity in many conditions, including biliary obstruction, acute or chronic cholestasis, and cirrhosis of diverse etiology. A consistent finding in biliary obstruction is the proliferation of bile ductules, characteristically along the borders of portal tracts.  Portal biliopathy is well-known in cases of PHT. Does the PHT in CDC have the potential to cause secondary changes in the liver or worsen those already present needs to be studied in detail and can be a subject for further studies.
| Conclusions|| |
The genesis of PHT in patients of CDC is multifactorial. PHT due to compression of the portal vein by the cyst seems to be the main cause, and it resolves after surgery. However, pathological changes in the liver may also play a significant part and cause the PHT to persist. In order to identify these patients, it is important to monitor PP in the follow-up period and blood NO levels can be effectively used for this. Hence, it is recommended that measurement of PP during surgery and liver biopsy should form a part of the standard management of CDC. The patients in whom NO levels suggest the presence of elevated PPs needs to be followed up more closely for appropriate and timely investigations and interventions.
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[Table 1], [Table 2], [Table 3], [Table 4]