Sai Praneeth R. Bathena

Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC–MS/MS

Sulfation is a major metabolic pathway involved in the elimination and detoxification of bile acids (BAs). Several lines of evidence are available to support the role of sulfation as a defensive mechanism to attenuate the toxicity of accumulated BAs during hepatobiliary diseases. Individual BAs and their sulfate metabolites vary markedly in their physiological roles as well as their toxicities. Therefore, analytical techniques are required for the quantification of individual BAs and BA-sulfates in biological fluids and tissues. Here we report a simple, sensitive, and validated LC-MS/MS method for the simultaneous quantification of major BAs and BA-sulfates in mouse liver, plasma, bile, and urine. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for liver and plasma) was used to extract BAs and BA-sulfates. Base-line separation of all analytes (unsulfated- and sulfated BAs) was achieved in 25min with a limit of quantification of 1ng/ml. This LC-MS/MS method was applied to simultaneously quantify BAs and BA-sulfates in both male and female mouse tissues and fluids. Less than 3% of total BAs are present in the sulfate form in the mouse liver, plasma, and bile, which provides strong evidence that sulfation is a minor metabolic pathway of BA elimination and detoxification in mice. Furthermore, we report that the marked female-predominant expression of Sult2a1 is not reflected into a female-predominant pattern of BA-sulfation.

The profile of bile acids and their sulfate metabolites in human urine and serum.

The role of sulfation in ameliorating the hepatotoxicity of bile acids (BAs) in humans remains unknown due to the lack of proper analytical methods to quantify individual BAs and their sulfate metabolites in biological tissues and fluids. To this end, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to characterize the detailed BA profile in human urine and serum. The limit of quantification was 1ng/mL and baseline separation of all analytes was achieved within in a run time of 32min. The method was validated over the dynamic range of 1-1000ng/mL. The LC-MS/MS method was more accurate, precise, and selective than the commercially available kits for the quantification of sulfated and unsulfated BAs, and the indirect quantification of individual sulfated BAs after solvolysis. The LC-MS/MS method was applied to characterize the BA profile in urine and serum of healthy subjects. Thirty three percent of serum BAs were sulfated, whereas 89% of urinary BAs existed in the sulfate form, indicating the role of sulfation in enhancing the urinary excretion of BAs. The percentage of sulfation of individual BAs increased with the decrease in the number of hydroxyl groups indicating the role of sulfation in the detoxification of the more hydrophobic and toxic BA species. Eighty percent of urinary BAs and 55% of serum BAs were present in the glycine-amidated form, whereas 8% of urinary BAs and 13% of serum BAs existed in the taurine-amidated form.

Urinary bile acids as biomarkers for liver diseases I. stability of the baseline profile in healthy subjects

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.

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues.

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 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(®)BEH Shield RP 18 column. All compounds eluted within a 7 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.

Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry.

Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5-20 to 500-4000 ng/ml. This LC-MS/MS method was validated for biomarker discovery in models of human neurological disorders.

Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection

Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.

Urinary Bile Acids as Biomarkers for Liver Diseases II. Signature Profiles in Patients

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.

Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC–MS/MS

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 (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM 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 validated over 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 the pathogenesis of periodontal diseases.

Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids

BACKGROUND & AIMS Chronic liver disease is characterized by fibrosis that may progress to cirrhosis. Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to the ligand muramyl dipeptide (MDP). Here, we investigated the role of Nod2 in the development of liver fibrosis. METHODS We studied experimental cholestatic liver disease induced by bile duct ligation or toxic liver disease induced by carbon tetrachloride in wild type and Nod2(-/-) mice. RESULTS Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Most notably, the hepatic bile acid concentration was lower in Nod2(-/-) mice than wild type mice following bile duct ligation for 3 weeks. In contrast to wild type mice, Nod2(-/-) mice had increased urinary excretion of bile acids, including sulfated bile acids, and an upregulation of the bile acid efflux transporters MRP2 and MRP4 in tubular epithelial cells of the kidney. MRP2 and MRP4 were downregulated by IL-1β in a Nod2 dependent fashion. CONCLUSIONS Our findings indicate that Nod2 deficiency protects mice from cholestatic liver injury and fibrosis through enhancing renal excretion of bile acids that in turn contributes to decreased concentration of bile acids in the hepatocyte.

Pharmacokinetics, protein binding and metabolism of a quinoxaline urea analog as an NF-κB inhibitor in mice and rats by LC-MS/MS

13-197 is a novel NF-κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13-197 in mice and rats. A valid, sensitive and selective LC-MS/MS method was developed. This method was validated for the quantification of 13-197, in the range of 0.1 or 0.2-500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13-197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12-fold shorter Tmax than in rats. Tissue concentrations were 1.3-69.2-fold higher in mice than in rats at 72 h after intravenous administration. 13-197 is well distributed to the peripheral tissues and has relatively high tissue-plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13-197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13-197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion.