Quantifying Serum Level of Glycochenodeoxycholic Acid Using High Performance Liquid Chromatography in Obstructive Jaundice Patients

Introduction. Accumulation of glycochenodeoxycholic acid (GCDC) in serum has a clinical significance as an inductor of pathological hepatocyte apoptosis, which impairs liver function. Inhibition of GCDC accumulation can be used as a marker in therapy. This study was aimed to quantify the serum level of GCDC in obstructive jaundice patients. Methodology. GCDC acid level in the serum was quantified using high performance liquid chromatography (HPLC) technique according to Muraca and Ghoos modified method. It was performed before and after decompression at day 7 and day 14. The sample was extracted with solid phase extraction (SPE) technique on SPE column. The results were analyzed using SPSS V 16.0 (P < 0.05) and quantified with standard curve on GCDC acid. Result. There were 21 cases with range of GCDC acid serum level before decompression was 90.9 (SD 205.5) μmol/L and day 7 after decompression decreased to 4.0 (SD 46.4) μmol/L and then increased to 11.3 (SD 21.9) μmol/L (P < 0.05). This method could separate GCDC acid on serum with good resolution, high precision and accuracy, and linear calibration curve on measured level range. Conclusion. HPLC can quantify GCDC acid serum on obstructive jaundice patients and can be used to support its pharmacokinetic study.


Introduction
e accumulation of glycochenodeoxycholic (GCDC) acid in obstructive jaundice patients is cytotoxic, can harm hepatocytes, and impairs liver function [1,2]. GCDC acid is a primary biliary acid directly divided from cholesterol in hepatocytes and conjugated with amino acid glycyne or taurin and becomes a conjugate of glycine or taurin [3,4]. GCDC acid, as a dose dependent inductor of apoptosis in hepatocyte and kinase protein C, can be used as a parameter of pathological apoptosis of hepatocyte and impaired liver function [5].
Several methods can be used to measure biliary acid level in the body �uid (serum or biliary �uid). Enzyme immunoassay method, which is mostly used to measure the total level of biliary acid, cannot be applied to measure primary, secondary, and tertiary biliary acid distinctively [6]. Liquid or gas chromatography is the chosen method for quantifying level of biliary acid [1]. Several animal studies have shown that high performance liquid chromatography (HPLC) can measure GCDC acid serum faster and precisely [6].
is study measures the GCDC acid serum in severe obstructive jaundice patients through biliary tract

Methodology
is study was performed on obstructive jaundice patients, mostly due to periampullary tumor, with serum bilirubin above 10 mg/dL, who were admitted in the period from December 2007 to January 2009. Patients with active hepatitis A, B, or C, liver malignancy, severe liver impairment with massive ascites, past experience of decompression procedure, and chronic liver failure were excluded. We used conventional open cholecystectomy as the bile decompression model to provide changes of GCDC acid level. Principle for quantifying conjugates of primary biliary acid (glycochenodeoxycholic, taurochenodeoxycholic, glycodeoxycholic, taurodeoxycholic acid), in the sample serum extracted by solid phase extraction (SPE) using Sepak C-18 column, with methanol solution aer being mixed with the base NaOH. e extract was concentrated using the N 2 gas evaporation method, then set by high performance liquid chromatography using analytical column C18 with eluated mixture of methanol-phosphate buffer solution pH 4.5 (75 : 25). e result of eluated separation is detected using ultra violet beam with the wave length of 205 nm. e chromatogram result was then recorded in the computer using the Empower soware. GCDC acid level was set using the calibration curve, as a regression line between the chromatographed areas versus standard level of GCDC acid. High performance liquid chromatography tool is made using Waters Alliance 2695 Separation Module system, Waters 2487 dual wave length, absorbance detector, with auto sampler, degasser and Empower soware. Separation of sample was done in an Atlantis C 18 5 m 3.9×150 mm Analytical column, protected by Sentry Guard Symmetry C 18 5 m 3.9 × 20 mm Guard column, using one set of vacuum �ltration from Millipore, SPE extractor from JT Baker, evaporization tool of N 2 gas with water bath 40 ∘ C.
Test sample: Spike sample is GCDC acid standard on model serum and serum sample from cholestasis patients.

Validation Of Setting Method
(1) Set the LOD and LOQ of GCDC acid standard solution.
(a) Produce the primary standard solution (std.P) and working standard solution (std.W) glycochenodeoxycholic acid and glycodeoxycholic acid on methanol.  methanol, add the model serum up to 2 mL (level 200 g/mL).   (3) Set the precision (coefficient of variation) and accuracy of the setting method.

In Vivo Implementation
(1) e extraction sample test 1.2 (serum) is suited with the extraction procedure used in the setting of the validation method (point A no. 2).
(2) e sample test level with HPLC according to the procedure was set using the validation setting (point A no. 3).
(3) e calibration curve was set in each sample test setting.

Result and Discussion
is study, which was performed on patients with characteristics mentioned in Table 2, succeeded in quantifying the level of GCDC and glycodeoxycholic (GDC) acid in the serum of severe obstructive jaundice patients using the modi�ed methods from Muraca and Ghoos. e chromatography method that is mostly used is high performance liquid chromatography (HPLC) with ultra violet (UV) detector or spectromass. Because the GCDC acid is a very polar molecule, the extraction or isolation of this molecule needs to use solidliquid extraction technique. e modi�cation was done on the SPE column to acquire the desired sensitivity. e modi�cation was also done on the chromatography system, which is an alternative to the degree of acidity on the mobile phase to 4.5 and using a more sensitive analytical homogenous column (C1�). With these modi�cations, the GCDC acid and glycodeoxycholic acid separation resulted in a better resolution and a sharp chromatogram peak. is modi�cation method can be used to quantify the GCDC acid and glycodeoxycholic acid serum (Figure 1). e glycodeoxycholic acid is a secondary bile acid used as an internal standard because it can be detected and gives a better chromatogram peak. With the chromatography system, the pessimistic hypothesis that glycodeoxycholic acid on the sample can decrease the accuracy of level estimation can be solved by decreasing the extracted estimated sample without using the glycodeoxycholic acid as the internal standard.
is study quanti�es the serial levels of GCDC acid serum in severe obstructive jaundice patients, which proves that there is an accumulation of glycochenodeoxycholate in the circulation of obstructive jaundice patients.
Several studies reported that the accumulation of bile acid can harm the liver via apoptosis and necrosis and can cause kidney failure, diarrhea, sepsis, arrhythmia, epistaxis, andasthma-like disease [7][8][9]. Although bile acid can cause several diseases, its serum level is still unfrequently included in clinical practices or in hospitals as a liver function test or hepatocelular damage test [10].
Accumulation of GCDC acid existed on each case of severe obstructive jaundice and was followed by pathological apoptosis of hepatocytes. is �nding suits the animal study result [11]. e aim of this study was to measure the difference between level of glycochenodeoxycholate in the pre-and postdecompression process of the bile duct. e decompression process signi�cantly decreased level of glycochenodeoxycholate as an apoptosis inductor of hepatocyte from 90.9 (SD 205.5) mol/L to 4.0 (SD 46.4) mol/L and then increased to 11.3 (SD 21.9) mol/L ( ( Figure 3). Decreased accumulation of glycochenodeoxycholate in the postdecompression stage is due to the regurgitation stop and reuptake process of glycochenodeoxycholate by hepatocytes and then it is excreted to canaliculi. Increasing of hepatocyte apoptosis and accumulation of glycochenodeoxycholate in obstructive jaundice patients showed the role of glycochenodeoxycholate as an inductor of hepatocyte apoptosis via extrinsic apoptosis pathway, and this �nding suits with the animal study result [5,12]. However, in postdecompression stage there was a signi�cant decrease in hepatocyte apoptosis and glycochenodeoxycholate level, so it can be concluded that a decrease on glycochenodeoxycholate level through bile duct decompression mechanism is an inhibitor of pathologic hepatocyte apoptosis.
e differences from the previous study are the glycochenodeoxycholate level and hepatocyte apoptosis, which are not dose dependent in this study [2,5]. is was due to difference in model and study design. Induction of apoptosis by glycochenodeoxycholate in the previous study was usually found in the hepatocyte culture, and on the other hand, this study showed that glycochenodeoxycholate was found on the liver tissue. e glycochenodeoxycholate level in this study is 30 times higher than normal level (according to Douglas, the glycochenodeoxycholate level in nonjaundice patient is between 0.2 and 3.2 mol/L) [13]. Aer decompression, the level of glycochenodeoxycholate on 12 cases was normal, mean while on the other 9 cases, the level decreased to 4 times lower than normal range.
A few recent studies were looking for glycochenodeoxycholate antagonist such as fructose, calpain, PMA, and brefeld in A as apoptosis inhibitors [2,5,11]. In this study, the antagonist form of glycochenodeoxycholate as inhibitor of pathologic hepatocyte apoptosis was reached through the decompression stage of bile duct.

Conclusion
From this study we can summarize that the modi�ed analysis method can be used to quantify level of GCDC acid on serum samples and furthermore the method can be used to support the GCDC acid pharmacokinetic study.