Chemicals and reagents
Methanol, acetonitrile, and formic acid were HPLC grade and purchased from Fisher Scientific (Loughborough, UK). Bile acid standards: cholic acids (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycoursodeoxycholic acid (GUDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), and taurolithocholic acid (TLCA), were also purchased from Sigma Chemical Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). HPLC grade water was obtained from Millipore pure water purification system (Diamond TΙΙ, USA).
Patients
The study was conducted in the period from October 2017 to August 2019. The study enrolled 2 groups of patients: chronic viral hepatitis (group 2) included 146 patients and their mean ages were 46.0 (39–51) years old, and NAFLD (group 3) included 39 patients and their mean ages were 47 (41.5–52) years old, beside normal healthy control subjects included 51 individuals (group 1) and their mean ages were 47.0 (38–52.5) years old. Patients in the chronic viral hepatitis group had chronic HBV or chronic HCV infection. Their diagnosis was based on positive hepatitis B surface antigen (HBsAg) and detectable HBV DNA and positive HCV antibody and detectable HCV RNA for more than 6 months respectively. In addition to established clinical, laboratory, and imaging findings of liver cirrhosis with no evidence of any hepatic focal lesion at the time of enrolment, NAFLD diagnosis was based on imaging analysis such as abdominal ultrasound and liver biopsy. All liver biopsy specimens at least 25 mm in length were obtained by percutaneous route. Liver sections were stained routinely with hematoxylin and eosin, Masson trichrome, silver reticulin, and occasionally with diastase-resistant periodic acid-Schiff and Perls’ Prussian blue. Liver biopsies had been read by a single pathologist who estimated semi-quantitatively the histopathological changes according to Brunt classification [8]. Fifty one normal healthy subjects were included matching the age and the gender of the other groups, with no clinical, laboratory, or radiological evidence of any type of liver diseases.
Ethical considerations
The study was conducted according to ethical standards for human experimentation (Helsinki Declaration). The ethics committee of the National Liver Institute approved the protocol, and written consents were filled and signed by all participants.
Sample collection
A sample (5 ml) of fasting venous blood was obtained in the early morning from each patient and control subject and divided into two tubes. Two millimeters was collected into an EDTA-containing tube for CBC assessment. Three millimeters was collected in plain vacutainer tube, after coagulation, and centrifugation of the sera was separated into aliquots for measurement of liver function tests and stored at − 80 °C until UPLC-analysis.
Laboratory investigations
Serum biochemical assay was performed with an automatic biochemistry analyzer (Daytona plus, Randox laboratories limited, UK) for analysis of blood chemistry as liver function tests including albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin total, alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (GGT). Alfafetoprotein (AFP) was performed by using (ARCHITECTi1000SR immunoassay analyzer, Abbott, Abbott Park, IL, USA). Hematological parameters and blood films were performed and measured using automatic analyzer (Sysmex KX-21, SysmexInc., Japan). The biochemical determinations were performed on the same day as blood was taken.
Serum sample preparation and bile acid detection
Sample preparation
The sample preparation method was based on a published method of [7] with modification. First, we added 100 μL of serum sample to 400 μL of ice cold methanol to precipitate proteins, vortex the mixture, then centrifugation of the mixture at 13,500 rpm for 15 min occurred; then, we separated the supernatant in a clean eppendorf bottle and centrifuged again at 13,500 rpm for 15 min; finally, 50 μL of the supernatant with 100 μL water/formic acid (1000: 1, v/v) solution was injected into the LC/MS/MS system. All chromatographic separations were performed with an ACQUITY HSS C18 column (1.7 μm, 100 mm × 2.1 mm internal dimensions) (Waters, Milford, MA). The injected sample volume was 10 μL, and the column temperature was maintained at 50 °C. Individual bile acids were eluted with a gradient at a flow rate of 0.5 ml/min.
Mobile phase A was (1/1000) formic acid/water, and mobile phase B was acetonitrile. The samples were eluted with 80% mobile phase A and 20% mobile phase B for an initial 2 min after injection, then with a linear gradient of mobile phase B of 20 to 30% over 5 min, followed by mobile phase B at 80% over 8 min, which was held for 0.50 min. Before the injection of the next sample, the column was equilibrated with 80% mobile phase A for 2 min. The mass spectrometer had an electrospray source operated in the negative ion mode using the multiple reaction monitoring (MRM) mode. UPLC-MS raw data obtained with MRM mode were analyzed using MassLynx applications manager version 4.1 (Waters Corp., Milford, MA) to obtain the calibration equations and the quantitative concentration of each bile acid in the samples. The method was validated ranging from 0.0010 to 20umol/L.
Statistical analysis
Data were analyzed using the IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Qualitative data were described using number and percent. The Kolmogorov-Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, standard deviation, and median. Significance of the obtained results was judged at the 5% level. The used tests were Chi-square test, for categorical variables, to compare between different groups; Monte Carlo correction for chi-square when more than 20% of the cells have expected count less than 5; F test (ANOVA), for normally distributed quantitative variables, to compare between more than two groups; post hoc test (Tukey) for pairwise comparisons; Kruskal-Wallis test for abnormally distributed quantitative variables to compare between more than two studied groups; and post hoc (Dunn’s multiple comparison test) for pairwise comparisons.