Analysis of Bile in Various Hepatobiliary Disease States: A Pilot Study

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12/09/14 11:36Analysis of bile in various hepatobiliary disease states: A pilot study

Pgina 1 de 9http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155632/?report=printable

J Indian Assoc Pediatr Surg. 2014 Jul-Sep; 19(3): 151155.

doi: 10.4103/0971-9261.136470

PMCID: PMC4155632

Analysis of bile in various hepatobiliary disease states: A pilot study

Ajay Verma, Veereshwar Bhatnagar, Shyam Prakash, andAbhay Kumar Srivastava

Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India

Department of Gastroentrology and Human Nutrition, All India Institute of Medical Sciences, New Delhi, India

Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India

Address for correspondence:Dr. Veereshwar Bhatnagar, Department of Pediatric Surgery, All India Institute of Medical

Sciences, New Delhi-110 029, India. E-mail: [email protected]

Copyright: Journal of Indian Association of Pediatric Surgeons

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike

3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

properly cited.

Abstract

Aim:

Our study aims to find various enzymatic and biochemical components of bile and their

clinical or prognostic correlation with regard to progression and severity of hepatobiliarydiseases.

Materials and Methods:

It was a cross-sectional study where all the patients suffering from choledochal cyst (CDC),

extrahepatic portal venous obstruction (EHPVO), and infantile obstructive cholangiopathy

undergoing diagnostic preoperative cholangiogram; and patients with history of total

parenteral nutrition (TPN) undergoing surgery for some other condition were included in the

study. Intraoperatively, bile was collected from the gallbladder and sent for estimation of

amylase, lipase, sodium, potassium, calcium, chloride, bicarbonate, total bilirubin, pH,cholesterol, triglycerides, and total bile acid.

Results:

A total of 80 patients were included in the study (20 in each of the four disease-based

groups). Amylase, lipase, and pH were significantly different among the patients of CDC

when compared with the presence or absence of dilated intrahepatic biliary radicals.

Similarly, amylase, lipase, and pH were also significantly different among the patients of

EHPVO when compared with presence or absence of biliopathy. Levels of cholesterol and

bile acid were significantly higher in patients who were evaluated after 1 year following TPN

than those who were evaluated before 1 year. The patients of infantile cholangiopathy, who

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Verma%20A%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Bhatnagar%20V%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Prakash%20S%5Bauth%5Dhttp://dx.doi.org/10.4103%2F0971-9261.136470http://www.ncbi.nlm.nih.gov/pmc/about/copyright.htmlmailto:dev@nullhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Srivastava%20AK%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Prakash%20S%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Bhatnagar%20V%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Verma%20A%5Bauth%5Dhttp://dx.doi.org/10.4103%2F0971-9261.136470


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had history of fever, had significantly higher level of calcium.

Conclusion:

The components of bile show close correlation with various clinical and prognostic markers,

there is a very close correlation between these parameters and the clinical severity, disease

progression, and final outcome.

KEY WORDS: Bile, choledochal cyst, extrahepatic portal venous obstruction, neonatal

cholangitis, total parenteral nutrition

INTRODUCTION

Bile has always been at the center of curiosity for the healers since ages. Even in the pre-

Hippocratic era, bile was considered as a pathological culprit for most of the diseases.

Yellow and black bile were two of the four vital fluids or humor of ancient and medieval

Greco-Roman medicine. Later in the 19 century, it was realized that bile is a normal

secretion of liver and is vital for normal function of the digestive system. This led to

investigations for understanding the composition of bile and hence the bile salt, bile pigment,

cholesterol, and various other products were discovered.

Various constituents of bile have been implicated in pathological states of the hepatobiliary

system, for example, the role of amylase in choledochal cyst (CDC), cholesterol in gallstone

disease, etc.[1] These discoveries have helped in understanding the natural history and

pathogenesis of these diseases. Constituents of bile and their changes in various disease

states especially in hepatobiliary conditions are not well studied. There is hardly any

literature available regarding role of bile in various hepatobiliary conditions, more so in thepediatric age-group.

CDC is a congenital dilatation of the common bile duct (CBD); it may involve intrahepatic

and extrahepatic ducts as well. Its pathogenesis is still obscure; since it is commonly

diagnosed in infants and children, a congenital origin has been proposed.[2,3] The

cholesterol contained in bile will occasionally accrete into lumps in the gallbladder, forming

gallstones. Nonhemolytic cholelithiasis is seen in neonates and infants and is due to use of

total parenteral nutrition (TPN).[4]

Composition of bile

Bile is made up of bile salts (0.7%), bile pigments (0.2%), and other substances mixed in

alkaline electrolyte solution that resembles pancreatic secretion. Approximately 500 ml is

secreted in 1 day; some of these substances are reabsorbed in the intestine and then excreted

again by the liver also known as enterohepatic circulation. The glucuronides of bile

pigments, bilirubin, and biliverdin are responsible for golden yellow color of bile, which are

breakdown products of heme part of hemoglobin. The bile salts are sodium and potassium

salts of bile acids and those secreted in bile are conjugated to glycine or taurine, a derivative

of cysteine. The bile acids are breakdown product of cholesterol. The two primary bile acidsformed in liver are cholic acid and chenodeoxycholic acid; in colon the intestinal bacteria

th
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convert cholic acid to deoxycholic acid and chenodeoxycholic acid into lithocholic acid,

these are called secondary bile.

With this limited information regarding composition of bile and its pathological effects in

various hepatobiliary disease states, it is noteworthy to investigate the role of various

constituents of bile in pediatric hepatobiliary diseases. With this aim we conducted this study

to find various enzymatic and biochemical components of bile, and their scientificcorrelation with various hepatobiliary disease states.

MATERIALS AND METHODS

This study was conducted at our institute after receiving ethical clearance from the institutes'

ethics committee. This was a cross-sectional study where all the patients suffering from CDC

(Group 1), extrahepatic portal venous obstruction (EHPVO) (Group 2), infantile obstructive

cholangiopathy undergoing diagnostic preoperative cholangiogram (Group 3), and patients

with history of TPN (Group 4) undergoing surgery for some other condition were included in

the study.

For the purpose of analysis, we subdivided the patients of Group 1 into two subgroups as per

presence or absence of intrahepatic biliary radical dilatation (IHBRD). The patients of Group

2 were subdivided as per presence or absence of biliopathy. Biliopathy is defined as dilated

intrahepatic biliary radicals in any of the radiological investigations such as ultrasonography,

computerized tomography portogram or magnetic resonance cholangiopancreatography

(MRCP).

The parents were informed and consent for inclusion in the study was taken. The

demographic and preoperative data was noted. The peroperative cholangiogram was done toconfirm the diagnosis in patients of CDC and in patients who were suspected to have

extrahepatic biliary atresia (EHBA). During surgery 5 ml of bile was aspirated from cyst in

patients of CDC and gallbladder of other patients with help of 24 G hypodermic needle. Two

milliliter of bile was sent for analysis immediately and the remaining sample was stored at

!80 C for the analysis later.

The first sample was sent to laboratory immediately and analyzed for amylase, lipase,

sodium, potassium, calcium (total/ionized), chloride, bicarbonate, total bilirubin, and pH.

Amylase, total bilirubin, and lipase estimations were done based on the principle of 2-chloro-4-nitrophenyl maltotrioside (CNP-G) as substrate, dichloroaniline (DCA), and kinetic

colorimetry, respectively on Hitachi 902 analyzer (Roche, Switzerland). Bicarbonate

estimation was done on Roche cobas 221 blood gas analyzer (Roche, Switzerland), while

pH, sodium, potassium, chloride and calcium estimations were done on Carelyte electrolyte

analyzer (Carewell Biotech Pvt Ltd) using ion-selective electrode method.

The second part of the sample was analyzed for cholesterol, triglycerides, and total bile acid.

Cholesterol-bile acid ratio was calculated and noted. The bile was diluted one to five times

with methanol and the protein precipitate was removed by centrifugation (deproteinated).

The methanol was then evaporated and residue was taken for analysis. The cholesterol and

triglycerides were analyzed on glycerol-3-phosphate oxidase, phenol aminophenazone


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(GPO-PAP) principle by photometric analysis on Beckman CX4 pro autoanalyzer (Beckman

Coulter, USA). Bile acid was analyzed by 3 "-hydroxysteroid dehydrogenase enzymatic

assay.[5]

The data analysis was done using STATA software version 9.0 (StataCorp LP, Texas, USA).

Data was represented as median and the difference in medians across various categories of

observation, for example, demographic profile and clinical parameters were compared usingWilcoxon-Rank sum/Kruskal-Wallis test. P-value less than 0.05 was taken as significant.

RESULTS

Total patients included were 80, that is, 20 in each of four groups. The demographic profile

and bile analysis of each group is mentioned in Table 1. In Group 1, all our patients were

type 1 CDC, lump was present in eight (16%), pain abdomen was present in 29 (58%),

history of jaundice was present in 11 (22%), history of fever was present in 12 (24%), ascites

was present in one (2%), splenomegaly was not detected in any patient, and IHBRD was

present in 11 (22%). We subdivided this group in two as per presence or absence of IHBRDand analyzed their constituents [Table 2]. We found that levels of amylase, lipase, pH,

calcium, and ionized calcium were significantly higher in patients with IHBRD.

In Group 2, jaundice was present in five (25%), history of fever was present in seven (35%),

ascites was present in three (15%), history of hematemesis or melena was present in seven

(35%), hepatomegaly was detected in seven (35%), splenomegaly was present in all patients,

and biliopathy was present in seven (35%) patients. We subdivided this group into two as per

presence or absence of biliopathy and found that levels of amylase, lipase, and pH were

significantly higher in patients with biliopathy [Table 3].In Group 3, progressive jaundice was the main presenting symptom, and was present in all

(100%) patients, history of fever was present in 10 (50%), ascites was detected in eight

(40%), hepatomegaly was present in all (100%) patients, and splenomegaly was present in

four (20%). This was a very heterogeneous group, only three patients fulfilled the inclusion

criteria, that is had neonatal hepatitis with patent proximal and distal ducts. We aspirated

content of gallbladder and analyzed them separately [Table 1]. Three out of 20 patients had

CDC with distal biliary atresia. Nine out of 20 had mucus-like fluid in the gallbladder,

remaining five patients had collapsed bladder, with no content in it.

In Group 4, median duration of TPN was 7 days (515 days) and median duration of time

between TPN institution and analysis of bile was 11.5 months (1 day85 months). We

subdivided this group in patients who were being analyzed before and after 1 year of TPN,

and we found that levels of cholesterol and bile acid were significantly higher in patients

who were analyzed after 1 year of TPN [Table 4].

DISCUSSION

Bile has often being implicated in pathogenesis of various hepatobiliary disease states, but

the various components of bile and their correlation with various conditions have never beenstudied. Level of amylase in bile has been correlated with etiopathogenesis of CDC.[1] A
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study published by Shimada K et al., reported increased levels of lysophosphatidylcholine

and amylase in patients with anomalous pancreatic biliary junction.[6] Up to 43% of

children, receiving long-term TPN develop gallstones.[7] The mechanism of gallstone

formation related to TPN remains unclear, but may be associated with changes in bile

composition caused by amino acid infusion or the lack of enteral feeding.[8] We conducted

this pilot study to evaluate the various components of bile and to document their correlation

with the hepatobiliary diseases in study.

In patients of CDC, the levels of amylase and lipase correlated with the information in

available literature when we compared the two subgroups of Group 1, the level of amylase

(P< 0.001) and lipase (P< 0.001) was significantly higher in patients who had IHBRD. The

pH was significantly lower in this group (P< 0.001). As one of the theories for pathogenesis

of CDC, these levels again support the cause and effect relationship between the pancreatic

enzymes and CDC. The low pH in this subgroup signifies the maximum mucosal damage

and altered mucosal secretions, thus altering the pH of its content. This fact was further

supported by the evidence that the subgroup with IHBRD had higher levels of calcium (P0.001) and ionized calcium (P< 0.001). The role of pancreatic enzymes and bile acid in

pathogenesis of biliary carcinoma has been reported by Sugiyama et al.[9] The presence of

biliary sludge and cholesterol stones in the dilated biliary radicals has also been supported by

the findings of this study, but the role of calcium in biliary stone formation and its role in

biliary carcinogenesis is a matter of further research.

In patients of EHPVO (Group 2), the patients who had jaundice (5/20) had significantly

higher levels of amylase (P= 0.0034), lipase (P= 0.0059), and pH (P= 0.0495). These

findings might be because of the dilated tortuous vessels in and around pancreas due to the

portal hypertension, these cavernous dilatations cause relative obstruction to the bile outflow

and possible reflux of the pancreatic secretions. The patients who had history of hematemesis

also had higher levels of amylase (P= 0.0003) and lipase (P= 0.0008), thus supporting the

aforementioned statement. The patients who had fever (7/20) had significantly higher levels

of calcium (P= 0.0474), ionic calcium (P= 0.0166), and triglyceride (P= 0.0286),

indicating underlying inflammatory pathology. The patients who had hepatomegaly (7/20)

had significantly higher levels of bile acid and cholesterol. The level of cholesterol and bile

acid in patients with hepatomegaly has been already reported by Nagi et al., and is possibly

due to large varices at porta causing obstruction and hepatocyte damage.[10] While

comparing the two subgroups of Group 2, we observed that patients with biliopathy had

significantly higher levels of amylase (P= 0.0003), lipase (P= 0.008), and pH (P= 0.0100).

The genesis of biliopathy in patients of EHPVO, if thought to be due to partial or complete

obstruction at porta due to the dilated varices.[11] Dilawari and Chawla had observed biliary

changes in all the 20 patients and labeled the radiological abnormalities as pseudosclerosing

cholangitis.[12] Williams et al., were the first to report cholangiographic changes caused by

choledochal varices.[13] Our study clearly shows that the pancreatic enzymes also cause

weakness of wall of bile duct and along with biliary obstruction (due to cavernous

malformation) increases the severity of portal biliopathy. In patients of Group 3, the patientwho had fever had significantly higher levels of calcium in the bile indicating inflammatory
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pathology in the ongoing process.

In Group 4, the patients who were analyzed after 1 year of TPN had higher levels of

cholesterol (P= 0.0002) and bile acid (P= 0.0017). This subgroup analysis supported the

evidence that patients with history of TPN have high chances of forming cholesterol stones,

and chances to form biliary stones increases with duration following TPN. Komura et al.,

suggested that children with history of TPN have high chances of developing gallbladderstones. The incidence increases if ratio of protein to calorie is more; high fat content

decreases the chance of developing such stones.[7,14] King et al., reported high incidence of

cholelithiasis in patients who received TPN for more than 30 days.[4] They did not compare

the duration following TPN and the incidence of gallstone. Our data shows that there is a

clearly high chance of gallstone formation in these patients, which increases with increase in

time following TPN; however, the comparison with age-matched control was not done and

the final comment on this issue is beyond the scope of this study. The analysis of our data

suggests that even a short duration of TPN induces change in the liver secretions, altering its

composition which progresses in due course of time and leads to stone formation.

CONCLUSION

This was a pilot study to identify various components of bile and their comparative levels in

pediatric patients suffering from various hepatobiliary diseases. This study provides an

insight to various components of bile and their levels correlating with clinical parameters and

disease progression. This study stimulates the need for further investigation into these

parameters so that their proper role could be established.

FootnotesSource of Support:Nil

Conflict of Interest:None declared.

REFERENCES

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dilatations. J Pediatr Surg. 1995;30:4747. [PubMed: 7539079]

2. O'Neill JA., Jr Choledochal cyst. Curr Probl Surg. 1992;29:361410. [PubMed: 1582241]

3. Lipsett PA, Pitt HA, Colombani PM, Boitnott JK, Cameron JL. Choledochal cyst disease.

A changing pattern of presentation. Ann Surg. 1994;220:64452. [PMCID: PMC1234452]

[PubMed: 7979612]

4. King DR, Ginn-Pease ME, Lloyd TV, Hoffman J, Hohenbrink K. Parenteral nutrition with

associated cholelithiasis: Another iatrogenic disease of infant and children. J Pediatr Surg.

1987;22:5936. [PubMed: 3112362]

5. Turley SD, Dietschy JM. Re-evaluation of the 3-"hydroxysteroid dehydrogenase assay

for total bile acids in bile. J Lipid Res. 1978;19:9248. [PubMed: 712252]

6. Shimada K, Yanagisawa J, Nakayama F. Increased lysophosphatidylcholine and
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pancreatic enzyme content in bile of patients with anomalous pancreticobiliary ductal

junction. Hepatology. 1991;13:43844. [PubMed: 1999314]

7. Komura J, Yano H, Tanaka Y, Tsuru T. Increased incidence of cholilithiasis during total

parenteral nutrition in children--factors affecting stone formation. Kurume Med J.

1993;40:711. [PubMed: 8355476]

8. George W, Holcomb, Walter SA. Gallbladder diseases and hepatic infections. In:Grossfeld JL, O'Neill JA, Coran AG, Fonkalsrud E, editors. Paediatric Surgery. 6th ed.

Philadelphia: Mosby Elsevier; 2006. p. 1635.

9. Sugiyama Y, Kobori H, Hakamada K, Seito D, Sasaki M. Altered bile composition in gall

bladder and common bile duct of patients with anomalous pancreaticobiliary ductal junction.

World J Surg. 2000;24:1720. [PubMed: 10594197]

10. Nagi B, Kochhar R, Bhasin D, Singh K. Cholangiopathy in extrahepatic portal venous

obstruction. Radiological appearances. Acta Radiol. 2000;41:6125. [PubMed: 11092484]

11. Sarin SK, Bhatia V, Makwane U. Portal biliopathy in extrahepatic portal vein

obstruction. Indian J Gastroenterol. 1992;2:A82.

12. Dilawari JB, Chawla YK. Pseudosclerosing cholangitis in extrahepatic portal venous

obstruction. Gut. 1992;33:2726. [PMCID: PMC1373944] [PubMed: 1541425]

13. Williams SM, Burnett DA, Mazer MJ. Radiographic demonstration of common bile duct

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Figures and Tables

Table 1


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Demographic profile and levels of various constituents of bile

Table 2

Constituents of bile in subgroups of Group 1

Table 3


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Table 4


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Analysis of Bile in Various Hepatobiliary Disease States: A Pilot Study