Total Serum Bile Acid as a Potential Marker for the Diagnosis of Cholangiocarcinoma without Jaundice

Cholangiocarcinoma (CCA) is a cancer of biliary epithelial cells. The prevalence of CCA shows a wide geographic variability, with the highest rates in Asia, especially Southeast Asia (Blechacz and Gores, 2008). The incidence of intrahepatic CCA is highest in the Northeast Thailand, especially in Khon Kaen province (44.3/100,000 in men and 17.6/100,000 in women) (Wiangnon et al., 2012), because of the high prevalence of carcinogenic liver fluke, Opisthorchis viverrini, infection (Sripa et al., 2007; Sripa et al., 2011). CCA patients are asymptomatic in an early stage, but often become symptomatic later in relation to obstructive jaundice, for example, clay-colored stools, bilirubinuria, bilirubinemia, pruritus, dyspepsia, weight loss, and abdominal pain (Braconi and Patel, 2010; Zografos et al., 2011; Bhudhisawasdi et al., 2012). Thus, CCA patients can be divided into two groups, those with obstructive jaundice and without jaundice, which can be distinguished by high and low levels of total bilirubin level in serum. Diagnosis of CCA without jaundice is often delayed and only made


Introduction
Cholangiocarcinoma (CCA) is a cancer of biliary epithelial cells.The prevalence of CCA shows a wide geographic variability, with the highest rates in Asia, especially Southeast Asia (Blechacz and Gores, 2008).The incidence of intrahepatic CCA is highest in the Northeast Thailand, especially in Khon Kaen province (44.3/100,000 in men and 17.6/100,000 in women) (Wiangnon et al., 2012), because of the high prevalence of carcinogenic liver fluke, Opisthorchis viverrini, infection (Sripa et al., 2007;Sripa et al., 2011).
CCA patients are asymptomatic in an early stage, but often become symptomatic later in relation to obstructive jaundice, for example, clay-colored stools, bilirubinuria, bilirubinemia, pruritus, dyspepsia, weight loss, and abdominal pain (Braconi and Patel, 2010;Zografos et al., 2011;Bhudhisawasdi et al., 2012).Thus, CCA patients can be divided into two groups, those with obstructive jaundice and without jaundice, which can be distinguished by high and low levels of total bilirubin level in serum.Diagnosis of CCA without jaundice is often delayed and only made
Alterations in serum bile acid metabolism are considered as the reflection of liver function (Heaton, 1979).Since bile acid metabolism mostly occurs in hepatocytes, accumulation of bile acids will occur if there is impairment in hepatocyte uptake, synthesis or secretion (Webster et al., 2002).The elevation of total serum bile acids (TSBA) was reported in patients with hepatocellular carcinoma and CCA (Changbumrung et al. 1990).In both patients groups, 86 and 92% of the patients showed higher level of serum total bilirubin than the upper limit of the normal controls.
The aim of this study is to evaluate whether the total serum bile acid (TSBA) can be used for the diagnostic marker for CCA patients without jaundice.For this purpose, we divided CCA patients into two entities, those with total serum bilirubin ≤2 mg/dL (LTB) and those with total serum bilirubin >2 mg/dL (HTB).Healthy control was used as the reference.Diagnostic value of total bile acid for CCA was evaluated by ROC analysis.In addition, the correlation between TSBA and liver function parameters were analyzed.The results showed that the TSBA level of CCA patients can be used as the diagnostic marker for CCA patients without jaundice, especially when it was combined with the serum alkaline phosphatase (ALP) level.

Subjects
Serum samples from 92 patients (57 males and 35 females) with CCA and 46 patients (28 males and 18 females) with benign biliary disease (BBD) who have total serum bilirubin ≤2 mg/dL (low total bilirubin; LTB) were selected at the Liver Fluke and Cholangiocarcinoma Research Center, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand.Data of the patients included age, sex and biochemical analysis profile of serum such as cholesterol, albumin, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP).
In addition, CCA patients were divided into two categories; those who had total serum bilirubin ≤2 mg/dL (low total bilirubin; LTB) group (37 males and 23 females) and those who had total serum bilirubin >2 mg/dL (high total bilirubin; HTB) group (20 males and 12 females).Sera were collected also from 115 healthy controls (39 males and 76 females) who visited for health check-up at the office of Medical Technology and Physical Therapy Health Service, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand.Data for control samples also included age, sex and the profile of serum biochemical analysis including cholesterol, ALT, AST and ALP.This study was approved by the Ethics Committee for Human Research (HE561280) of Khon Kaen University.

Total serum bile acids (TSBA) measurement
Total serum bile acids (TSBA) was determined using the Total Bile Acids Assay kit (Diazyme Laboratories, Poway, CA) based on the enzyme cycling method.Serum sample of 3 µL was used for the analysis on the Beckman Synchron CX4 clinical chemistry analyzer.The results were presented in µmol/L.

Determination of diagnostic value
The cut-off values were calculated by the receiver operating characteristic (ROC) curve analysis.The percentages of the sensitivity, specificity and accuracy of total bile acids were estimated.

Statistical analysis
Results were presented as median ± quartile deviation and the range (minimum to maximum).Data were analyzed using Mann-Whitney U test and Spearman's rank correlation test.The significant level was set at p-value less than 0.05.

Correlation between the total serum bile acid level and total bilirubin or other liver function parameters
Total serum bile acid (TSBA) levels (median±quartile deviation) of CCA patients with HTB or LTB and that of the control group were 110.6±122.8,5.6±3.3, and 3.9±1.1 µmol/L, respectively.
TSBA levels of overall CCA patients were significantly correlated with total bilirubin (Figure 1A), ALT (Figure 1C), AST (Figure 1D) and ALP (Figure 1E) but not with cholesterol (Figure 1B) and albumin (Figure 1F).When the CCA patients were divided into HTB and LTB groups, TSBA was significantly correlated with total bilirubin (Figure 1A), cholesterol (Figure 1B), and AST (Figure 1D) in HTB group.In LTB group of CCA patients, TSBA levels did not correlate with any one of those tests (Figure 1A-1F, Table 1).

Diagnostic value of TSBA in CCA with LTB
Since TSBA levels did not correlate with any one of the liver function tests in the CCA with LTB group, we analysed further whether TSBA had a diagnostic value for  When the CCA-LTB group was divided into two subgroups according to TSBA cut-off value of >6.05 µmol/L, and various CCA-associated parameters were compared, only ALP was found to be significantly different between two subgroups (Table 2).Based on the significant difference of ALP in two subgroups, we calculated the cut-off value of ALP using ROC curve analysis.The results showed that when cut-off value of ALP was set at >82 U/L (Figure 2A), the sensitivity, specificity and accuracy of the diagnostic value of ALP were 90.0%, 88.7% and 89.1%, respectively.When TSBA and ALP were combined as the diagnostic markers, the specificity was increased to 97.4% as compared with the control value.
Then, we compared the diagnostic efficiency of the TSBA and ALP for CCA with LTB and BBD with LTB.Using ROC curve analysis the cut-off values of the TSBA and ALP were >6.65 µmol/L and >82.5 U/L, respectively.When the TSBA and ALP were combined as the diagnostic markers, the specificity increased to 72.9% compared with TSBA alone (67.4%) or ALP alone (43.5%) (Figure 2B).
Since TSBA level seems to have a diagnostic value for CCA with LTB, we compared the TSBA levels in CCA with LTB, benign biliary diseases (BBD) with LTB and control group.The results demonstrated that TSBA level in CCA with LTB was 5.6±3.3(median±quartile deviation), that in BBD with LTB was 5.2±3.1 and that in normal control was 3.9±1.1 µmol/L.TSBA in CCA with LTB and BBD with LTB was significantly higher compared with that in normal group (Figure 3).However, TSBA was not statistically different between CCA with LTB and BBD with LTB groups, but CCA demonstrated a trend to be higher.However, ALP in CCA with LTB and BBD with LTB were significantly higher when compared with that for the normal group (Figure 4).    the CCA with LTB group.For this purpose, we performed a ROC curve analysis for the TSBA levels in the sera of the CCA patients with an LTB group and a normal group.The results showed that the cut-off value of the TSBA for the diagnosis of CCA-LTB was >6.05 µmol/L (Figure 2A) with the sensitivity, specificity and accuracy of 46.7%,

Discussion
In CCA with HTB group, the level of TSBA was significantly correlated with the levels of total bilirubin, cholesterol, and AST.In contrast, no significant correlation was observed between TSBA and those liver function tests.This result suggested that the damage of liver cells might not occur in LTB group.The non-jaundiced group had a significantly higher survival rate than the jaundiced group (Sugiyama et al., 1997).The TSBA in CCA with LTB was not correlated with liver function, but more specific to cholangiocytes.As a result, TSBA might be useful as a diagnostic marker for CCA without jaundice.
Bile acid is the end product of cholesterol breakdown, which takes place in hepatocytes.Thus, accumulation of bile acid will occur if there is an impairment in hepatocyte uptake, synthetic or secretion function (Fausa and Gjone, 1976;Shaffer and Gordon, 1978;Tabibian, 1988).In our study, the levels of TSBA in CCA and BBD with LTB were significantly higher than that of the normal group.However, no statistically significant difference was found between CCA and BBD with LTB groups, but CCA has a trend to be higher.Changbumrung et al. (1990) reported that TSBA and conjugated bile acids were elevated in CCA patients.Furthermore, patients with hepatocellular carcinoma also showed significantly higher TSBA than control subjects (Changbumrung et al., 1990;Motawa El-Houseine et al., 2000).However, our results showed that TSBA level alone was not sufficient for discrimination of CCA and BBD.
The concentration of TSBA found in CCA with HTB group was significantly higher than LTB group.Similar to our results, Jusakul et al. (2012) analyzed total bile acids in gallbladder bile, and suggested that the increase in the TSBA levels might be caused by high total bile acids levels in bile and by impaired enterohepatic circulation system (Jusakul et al., 2012).
The serum bile acid concentrations is considered to be more sensitive tests for hepatobiliary diseases than bilirubin, AST and ALT (Fausa and Gjone, 1976).In this study, the liver enzymes such as ALT, AST and ALP in CCA with HTB group was much higher than those in the CCA with LTB group, suggesting that the high total bile acids in HTB group might be caused by liver cell damage (Changbumrung et al., 1990).Determination of TSBA at the cut-off >6.05 µmol/L gave high specificity but low sensitivity in diagnosis of CCA with LTB patients.When CCA with LTB patients were divided into two subgroups by total bile acids >6.05 µmol/L, ALP was found to be significantly different between the two subgroups.Consequently, we found that the combination of TSBA with ALP increased the specificity compared with the use of TSBA or ALP alone.The persistent elevation of serum ALP is usually the biochemical abnormality leading to the diagnosis of primary sclerosing cholangitis and generally it remains elevated during the course of the disease (Stanich et al., 2011;Al Mamari et al., 2013).
In conclusion, CCA with LTB was found to have significant elevation of TSBA than normal controls.The TSBA level in LTB patients did not correlate with any liver function tests.Nevertheless, the TSBA, in combination with ALP, might be a helpful marker for the detection of CCA suspected patients without jaundice.

Figure 1 .
Figure 1.Correlations between Total Serum Bile Acid (TSBA) and Total Bilirubin, Cholesterol, ALT, AST, ALP and Albumin of CCA Patients