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The role of bile acids (BAs) as biomarkers for liver injury has been proposed for decades. However, the large inter- and intra-individual variability of the BA profile has prevented its clinical application. To this end, we investigated the effect of covariates such as food, gender, age, BMI, and moderate alcohol consumption on the BA profile in healthy human subjects. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Both inter- and intra-individual variabilities of BA indices were low in serum and even lower in urine compared with those of absolute concentrations of BAs. Serum BA concentrations increased with consumption of food, whereas urinary BA concentrations were mildly affected by food. Gender differences in the urinary and serum BA profile were minimal. The serum and urinary BA profiles were also not affected by age. BMI showed minimal effect on the urine and serum BA profile. Moderate alcohol consumption did not have a significant effect on the BA profile in both urine and serum. When the effect of the type of alcohol was studied, the results indicate that moderate drinking of beer does not affect BA concentrations and has minimal effect on BA indices, whereas moderate wine consumption slightly increases BA concentrations without affecting the BA indices. In summary, urinary BA indices showed lower variability and higher stability than absolute BA concentrations in serum and showed minimal changes to covariate effects suggesting their utility as biomarkers in clinic.

Hepatobiliary diseases result in the accumulation of bile acids (BAs) in the liver, systemic blood, and other tissues leading to an unfavorable prognosis. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Comparison of the urinary BA profiles between healthy subjects and patients with hepatobiliary diseases demonstrated significantly higher absolute concentrations of individual and total BAs in patients. The percentage sulfation of some individual BAs were different between the two groups. The percentage amidation of overall and most individual BAs was higher in patients than controls. The percentage of primary BAs (CDCA and CA) was higher in patients, whereas the percentage of secondary BAs (DCA and LCA) was lower in patients. BA indices belonging to percentage amidation and percentage composition were better associated with the severity of the liver disease as determined by the model for end-stage liver disease (MELD) score and disease compensation status compared with the absolute concentrations of individual and total BAs. In addition, BA indices corresponding to percentage amidation and percentage composition of certain BAs demonstrated the highest area under the receiver operating characteristic (ROC) curve suggesting their utility as diagnostic biomarkers in clinic. Furthermore, significant increase in the risk of having liver diseases was associated with changes in BA indices.

Summary Rapid determination of in vitro metabolic stability and metabolite profiling of new chemical entities using microsomes or other liver preparations is one of the most important steps in drug discovery. In this paper, we report the use of liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry for the simultaneous analysis of metabolic stability, metabolite profiling, and the kinetics of metabolite formation of praziquantel and three structural analogs. Multiple reaction monitoring (MRM) scans were used to quantify the disappearance of parent compounds and the formation of metabolites. MRM-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) scans were used for the identification of the metabolites formed. Metabolic stability of these anthelmintics were studied in human liver microsomes (HLM) using MRM as a survey scan, which resulted in the identification of a higher number of metabolites compared to neutral loss (NL), precursor ion (PI), and enhanced mass spectrometry (EMS) scans. MRM-IDA-EPI scans resulted in the generation of similar calibration curves to MRM-only quantitative analysis. Therefore, the quantitative capabilities of the method was not affected by the additional qualitative information obtained during the same run. The formation of major metabolites was also simultaneously monitored, which could be used to understand the kinetics and mechanism of metabolite formation. Finally, our data demonstrate that the three analogs had higher metabolic stability than the anthelmintic prototype (praziquantel).

Human sulfotransferase 2A1 (SULT2A1) is a member of the hydroxysteroid sulfotransferase (SULT2) family that mediates sulfo-conjugation of a variety of endogenous molecules including dehydroepiandrosterone (DHEA) and bile acids. In this study, we have constructed a stable cell line expressing SULT2A1 by transfection into HEK293 cells. The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human bile acids by SULT2A1. Formation of DHEA sulfate demonstrated Michaelis-Menten kinetics with apparent K(m) and V(max) values of 3.8 muM and 130.8 pmol min(-1) mg(-1) protein, respectively. Sulfation kinetics of bile acids also demonstrated Michaelis-Menten kinetics with a marked variation in apparent K(m) and V(max) values between individual bile acids. Sulfation affinity was inversely proportional to the number of hydroxyl groups of bile acids. The monohydroxy- and most toxic bile acid (lithocholic acid) had the highest affinity, whereas the trihydroxy- and least toxic bile acid (cholic acid) had the lowest affinity to sulfation by SULT2A1. Intrinsic clearance (CL(int)) of ursodeoxycholic acid (UDCA) was approximately 1.5- and 9.0-fold higher than that of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), respectively, despite the fact that all three are dihydroxy bile acids.

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors.LPAs are known to be key mediators in inflammation,and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125mm × 2.0mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile Phase A) and methanol/water : 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validatedover the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in thepathogenesis of periodontal diseases.

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5 mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC® BEH Shield RP 18 column. All compounds eluted within a seven min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2–1000 ng/mL for RTV, IDV, and ATV, and 0.5–1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5–4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.