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Article ; Online: Analyte recovery in LC-MS/MS bioanalysis: An old issue revisited.

Kumar, Devendra / Gautam, Nagsen / Alnouti, Yazen

2022 Volume 1198, Page(s) 339512

Abstract: There are several challenges associated with LC-MS/MS bioanalytical method development and validation. Low and variable recovery of some analytes, especially the more hydrophobic ones, is often challenging. Analytes can be lost to various extents ... ...

Abstract	There are several challenges associated with LC-MS/MS bioanalytical method development and validation. Low and variable recovery of some analytes, especially the more hydrophobic ones, is often challenging. Analytes can be lost to various extents throughout the process of sample collection, storage, before, during, and/or after sample preparation and analysis. The calculation of overall extraction recovery can detect problems of low recovery during sample preparation but does not identify the source(s) of analyte losses. Low overall analyte recovery is the net result of losses that can happen for multiple reasons at all steps of sample preparation and analysis. Therefore, identifying the source(s) of analyte loss during sample preparation can help guide the optimization the bioanalysis conditions to minimize these losses. In this article we propose a practical protocol to systematically identify and quantify the sources of low analyte recovery. This allows the proper choice of strategies to optimize the relevant bioanalytical conditions to minimize analyte losses and improve overall recovery.
MeSH term(s)	Chromatography, Liquid/methods ; Specimen Handling ; Tandem Mass Spectrometry/methods
Language	English
Publishing date	2022-01-28
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1483436-4
ISSN	1873-4324 ; 0003-2670
ISSN (online)	1873-4324
ISSN	0003-2670
DOI	10.1016/j.aca.2022.339512
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Article ; Online: Crosstalk of hepatocyte nuclear factor 4a and glucocorticoid receptor in the regulation of lipid metabolism in mice fed a high-fat-high-sugar diet.

Lu, Hong / Lei, Xiaohong / Winkler, Rebecca / John, Savio / Kumar, Devendra / Li, Wenkuan / Alnouti, Yazen

Lipids in health and disease

2022 Volume 21, Issue 1, Page(s) 46

Abstract: Background: Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in ... ...

Abstract	Background: Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia. Methods: Adult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays. Results: Compared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes. Conclusions: induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.
MeSH term(s)	Animals ; Chromatography, Liquid ; Dietary Fats/metabolism ; Dietary Sugars/metabolism ; Fatty Liver/genetics ; Fatty Liver/metabolism ; Hepatocyte Nuclear Factor 4/genetics ; Hepatocyte Nuclear Factor 4/metabolism ; Hepatocyte Nuclear Factors/metabolism ; Lipid Metabolism/genetics ; Lipid Metabolism/physiology ; Lipids ; Liver/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; RNA, Messenger/metabolism ; Receptors, Glucocorticoid/genetics ; Receptors, Glucocorticoid/metabolism ; Sterol Regulatory Element Binding Protein 1/metabolism ; Tandem Mass Spectrometry
Chemical Substances	Dietary Fats ; Dietary Sugars ; Hepatocyte Nuclear Factor 4 ; Hepatocyte Nuclear Factors ; Lipids ; RNA, Messenger ; Receptors, Glucocorticoid ; Sterol Regulatory Element Binding Protein 1
Language	English
Publishing date	2022-05-25
Publishing country	England
Document type	Journal Article
ZDB-ID	2091381-3
ISSN	1476-511X ; 1476-511X
ISSN (online)	1476-511X
ISSN	1476-511X
DOI	10.1186/s12944-022-01654-6
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Article ; Online: Bile Acid sulfation: a pathway of bile acid elimination and detoxification.

Alnouti, Yazen

Toxicological sciences : an official journal of the Society of Toxicology

2009 Volume 108, Issue 2, Page(s) 225–246

Abstract: Sulfotransferase-2A1 catalyzes the formation of bile acid-sulfates (BA-sulfates). Sulfation of BAs increases their solubility, decreases their intestinal absorption, and enhances their fecal and urinary excretion. BA-sulfates are also less toxic than ... ...

Abstract	Sulfotransferase-2A1 catalyzes the formation of bile acid-sulfates (BA-sulfates). Sulfation of BAs increases their solubility, decreases their intestinal absorption, and enhances their fecal and urinary excretion. BA-sulfates are also less toxic than their unsulfated counterparts. Therefore, sulfation is an important detoxification pathway of BAs. Major species differences in BA sulfation exist. In humans, only a small proportion of BAs in bile and serum are sulfated, whereas more than 70% of BAs in urine are sulfated, indicating their efficient elimination in urine. The formation of BA-sulfates increases during cholestatic diseases. Therefore, sulfation may play an important role in maintaining BA homeostasis under pathologic conditions. Farnesoid X receptor, pregnane X receptor, constitutive androstane receptor, and vitamin D receptor are potential nuclear receptors that may be involved in the regulation of BA sulfation. This review highlights current knowledge about the enzymes and transporters involved in the formation and elimination of BA-sulfates, the effect of sulfation on the pharmacologic and toxicologic properties of BAs, the role of BA sulfation in cholestatic diseases, and the regulation of BA sulfation.
MeSH term(s)	Animals ; Bile Acids and Salts/biosynthesis ; Bile Acids and Salts/metabolism ; Bile Acids and Salts/toxicity ; Enterohepatic Circulation ; Female ; Humans ; Male ; Sex Characteristics ; Species Specificity ; Sulfates/metabolism ; Sulfotransferases/biosynthesis ; Sulfotransferases/metabolism
Chemical Substances	Bile Acids and Salts ; Sulfates ; Sulfotransferases (EC 2.8.2.-) ; alcohol sulfotransferase (EC 2.8.2.2)
Language	English
Publishing date	2009-04
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	1420885-4
ISSN	1096-0929 ; 1096-6080
ISSN (online)	1096-0929
ISSN	1096-6080
DOI	10.1093/toxsci/kfn268
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Article: Urinary BA Indices as Prognostic Biomarkers for Complications Associated with Liver Diseases.

Li, Wenkuan / Alamoudi, Jawaher Abdullah / Gautam, Nagsen / Kumar, Devendra / Olivera, Macro / Gwon, Yeongjin / Mukgerjee, Sandeep / Alnouti, Yazen

International journal of hepatology

2022 Volume 2022, Page(s) 5473752

Abstract: Hepatobiliary diseases and their complications cause the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues, which may exacerbate the underlying condition and lead to unfavorable prognosis. To develop and validate prognostic ... ...

Abstract	Hepatobiliary diseases and their complications cause the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues, which may exacerbate the underlying condition and lead to unfavorable prognosis. To develop and validate prognostic biomarkers for the prediction of complications of cholestatic liver disease based on urinary BA indices, liquid chromatography-tandem mass spectrometry was used to analyze urine samples from 257 patients with cholestatic liver diseases during a 7-year follow-up period. The urinary BA profile and non-BA parameters were monitored, and logistic regression models were used to predict the prognosis of hepatobiliary disease-related complications. Urinary BA indices were applied to quantify the composition, metabolism, hydrophilicity, and toxicity of the BA profile. We have developed and validated the bile-acid liver disease complication (BALDC) model based on BA indices using logistic regression model, to predict the prognosis of cholestatic liver disease complications including ascites. The mixed BA and non-BA model was the most accurate and provided higher area under the receiver operating characteristic (ROC) and smaller akaike information criterion (AIC) values compared to both non-BA and MELD (models for end stage liver disease) models. Therefore, the mixed BA and non-BA model could be used to predict the development of ascites in patients diagnosed with liver disease at early stages of intervention. This will help physicians to make a better decision when treating hepatobiliary disease-related ascites.
Language	English
Publishing date	2022-03-30
Publishing country	United States
Document type	Journal Article
ZDB-ID	2621468-4
ISSN	2090-3456 ; 2090-3448
ISSN (online)	2090-3456
ISSN	2090-3448
DOI	10.1155/2022/5473752
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Article ; Online: Direct and indirect quantification of phosphate metabolites of nucleoside analogs in biological samples.

Gautam, Nagsen / Alamoudi, Jawaher Abdullah / Kumar, Sushil / Alnouti, Yazen

Journal of pharmaceutical and biomedical analysis

2019 Volume 178, Page(s) 112902

Abstract: Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is ... ...

Abstract	Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is challenging due to their instability, high hydrophilicity, and their low concentrations in blood and tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard technique for the quantification of NRTIs and their phosphorylated NMs. In this review, an overview of the publications describing the quantitative analysis of intracellular and total tissue concentration of NMs is presented. The focus of this review is the comparison of the different approaches and challenges associated with sample collection, tissue homogenization, cell lysis, cell counting, analyte extraction, sample storage conditions, and LC-MS analysis. Quantification methods of NMs via LC-MS can be categorized into direct and indirect methods. In the direct LC-MS methods, chromatographic retention of the NMs is accomplished by ion-exchange (IEX), ion-pairing (IP), hydrophilic interaction (HILIC), porous graphitic carbon (PGC) chromatography, or capillary electrophoresis (CE). In indirect methods, parent nucleosides are 1st generated from the dephosphorylation of NMs during sample preparation and are then quantified by reverse phase LC-MS as surrogates for their corresponding NMs. Both approaches have advantages and disadvantages associated with them, which are discussed in this review.
MeSH term(s)	Animals ; Chromatography, Liquid/methods ; Humans ; Nucleosides/analysis ; Nucleosides/metabolism ; Nucleotides/analysis ; Nucleotides/metabolism ; Phosphates/metabolism ; Tandem Mass Spectrometry/methods
Chemical Substances	Nucleosides ; Nucleotides ; Phosphates
Language	English
Publishing date	2019-10-03
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	604917-5
ISSN	1873-264X ; 0731-7085
ISSN (online)	1873-264X
ISSN	0731-7085
DOI	10.1016/j.jpba.2019.112902
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Article: Bile acid indices as biomarkers for liver diseases II: The bile acid score survival prognostic model.

Alamoudi, Jawaher Abdullah / Li, Wenkuan / Gautam, Nagsen / Olivera, Marco / Meza, Jane / Mukherjee, Sandeep / Alnouti, Yazen

World journal of hepatology

2021 Volume 13, Issue 5, Page(s) 543–556

Abstract: Background: Cholestatic liver diseases are characterized by an accumulation of toxic bile acids (BA) in the liver, blood and other tissues which lead to progressive liver injury and poor prognosis in patients.: Aim: To discover and validate ... ...

Abstract	Background: Cholestatic liver diseases are characterized by an accumulation of toxic bile acids (BA) in the liver, blood and other tissues which lead to progressive liver injury and poor prognosis in patients. Aim: To discover and validate prognostic biomarkers of cholestatic liver diseases based on the urinary BA profile. Methods: We analyzed urine samples by liquid chromatography-tandem mass spectrometry and investigated the use of the urinary BA profile to develop survival models that can predict the prognosis of hepatobiliary diseases. The urinary BA profile, a set of non-BA parameters, and the adverse events of liver transplant and/or death were monitored in 257 patients with cholestatic liver diseases for up to 7 years. The BA profile was characterized by calculating BA indices, which quantify the composition, metabolism, hydrophilicity, formation of secondary BA, and toxicity of the BA profile. We have developed and validated the bile-acid score (BAS) model (a survival model based on BA indices) to predict the prognosis of cholestatic liver diseases. Results: We have developed and validated a survival model based on BA (the BAS model) indices to predict the prognosis of cholestatic liver diseases. Our results demonstrate that the BAS model is more accurate and results in higher true-positive and true-negative prediction of death compared to both non-BAS and model for end-stage liver disease (MELD) models. Both 5- and 3-year survival probabilities markedly decreased as a function of BAS. Moreover, patients with high BAS had a 4-fold higher rate of death and lived for an average of 11 mo shorter than subjects with low BAS. The increased risk of death with high Conclusion: The BAS model is more accurate than MELD and non-BAS models in predicting the prognosis of cholestatic liver diseases.
Language	English
Publishing date	2021-05-27
Publishing country	United States
Document type	Journal Article
ZDB-ID	2573703-X
ISSN	1948-5182
ISSN	1948-5182
DOI	10.4254/wjh.v13.i5.543
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Article: Bile acid indices as biomarkers for liver diseases I: Diagnostic markers.

Alamoudi, Jawaher Abdullah / Li, Wenkuan / Gautam, Nagsen / Olivera, Marco / Meza, Jane / Mukherjee, Sandeep / Alnouti, Yazen

World journal of hepatology

2021 Volume 13, Issue 4, Page(s) 433–455

Abstract: Background: Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis.: Aim: To discover and validate diagnostic biomarkers of cholestatic liver ... ...

Abstract	Background: Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis. Aim: To discover and validate diagnostic biomarkers of cholestatic liver diseases based on the urinary BA profile. Methods: We analyzed urine samples by liquid chromatography-tandem mass spectrometry and compared the urinary BA profile between 300 patients with hepatobiliary diseases Results: Total and individual BA concentrations were higher in all patients. The percentage of secondary BA (lithocholic acid and deoxycholic acid) was significantly lower, while the percentage of primary BA (chenodeoxycholic acid, cholic acid, and hyocholic acid) was markedly higher in patients compared to controls. In addition, the percentage of taurine-amidation was higher in patients than controls. The increase in the non-12α-OH BA was more profound than 12α-OH BA (cholic acid and deoxycholic acid) causing a decrease in the 12α-OH/ non-12α-OH ratio in patients. This trend was stronger in patients with more advanced liver diseases as reflected by the model for end-stage liver disease score and the presence of hepatic decompensation. The percentage of sulfation was also higher in patients with more severe forms of liver diseases. Conclusion: BA indices have much lower inter- and intra-individual variability compared to absolute BA concentrations and changes of BA indices are associated with the risk of developing liver diseases.
Language	English
Publishing date	2021-01-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	2573703-X
ISSN	1948-5182
ISSN	1948-5182
DOI	10.4254/wjh.v13.i4.433
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Article ; Online: Direct Comparison of Chol-siRNA Polyplexes and Chol-DsiRNA Polyplexes Targeting STAT3 in a Syngeneic Murine Model of TNBC.

Ye, Zhen / Abdelmoaty, Mai Mohamed / Curran, Stephen M / Dyavar, Shetty Ravi / Kumar, Devendra / Alnouti, Yazen / Coulter, Don W / Podany, Anthony T / Singh, Rakesh K / Vetro, Joseph A

Non-coding RNA

2022 Volume 8, Issue 1

Abstract: RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified ... ...

Abstract	RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED
Language	English
Publishing date	2022-01-13
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2813993-8
ISSN	2311-553X ; 2311-553X
ISSN (online)	2311-553X
ISSN	2311-553X
DOI	10.3390/ncrna8010008
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Article ; Online: Discovery, synthesis and biological characterization of a series of

Sharma, Swagat / Lesiak, Lauren / Aretz, Christopher D / Du, Yu / Kumar, Sushil / Gautam, Nagsen / Alnouti, Yazen / Dhuria, Nikilesh V / Chhonker, Yashpal S / Weaver, C David / Hopkins, Corey R

RSC medicinal chemistry

2021 Volume 12, Issue 8, Page(s) 1366–1373

Abstract: The present study describes the discovery and characterization of a series ... ...

Abstract	The present study describes the discovery and characterization of a series of
Language	English
Publishing date	2021-06-21
Publishing country	England
Document type	Journal Article
ISSN	2632-8682
ISSN (online)	2632-8682
DOI	10.1039/d1md00129a
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Article ; Online: A combination of Omega-3 PUFAs and COX inhibitors: A novel strategy to manage obesity-linked dyslipidemia and adipose tissue inflammation.

Saraswathi, Viswanathan / Heineman, Robert / Alnouti, Yazen / Shivaswamy, Vijay / Desouza, Cyrus V

Journal of diabetes and its complications

2019 Volume 34, Issue 2, Page(s) 107494

Abstract: We previously reported that fish oil in combination with cyclooxygenase (COX) inhibitors exerts enhanced hypolipidemic and anti-inflammatory effects in mice. Here, we sought to determine the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in ... ...

Abstract	We previously reported that fish oil in combination with cyclooxygenase (COX) inhibitors exerts enhanced hypolipidemic and anti-inflammatory effects in mice. Here, we sought to determine the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in combination with naproxen (NX), a COX inhibitor, on dyslipidemia and gene expression in adipose tissue (AT) in humans. Obese dyslipidemic patients were randomly assigned to one of these interventions for 12 wk: 1) Standard nutrition counseling (control), 2) ω-3 PUFAs (2 g twice daily), 3) NX (220 mg twice daily), and 4) ω-3 PUFAs (2 g twice daily) + NX (220 mg twice daily). The serum triglycerides showed a trend towards a reduction and a significant reduction (P<0.05) in ω-3 and ω3 + NX-treated subjects, respectively, compared to control. The mRNA expression of vascular cell adhesion molecule-1 (Vcam1), an inflammatory marker, increased significantly in AT of ω-3 PUFA-treated subjects but not in ω-3 PUFAs+NX-treated group. The plasma level of glycine-conjugated hyodeoxycholic acid, a secondary bile acid with hypolipidemic property, increased significantly in ω-3 PUFAs + NX-treated group. Our data suggest that combining NX with ω-3 PUFAs increases their effectiveness in reducing serum TG and favorably altering AT gene expression and plasma bile acid profile.
MeSH term(s)	Adipose Tissue/pathology ; Adult ; Biopsy ; Cyclooxygenase Inhibitors/therapeutic use ; Dyslipidemias/blood ; Dyslipidemias/drug therapy ; Dyslipidemias/etiology ; Fatty Acids, Omega-3/therapeutic use ; Female ; Fish Oils/therapeutic use ; Humans ; Inflammation/blood ; Inflammation/drug therapy ; Inflammation/physiopathology ; Male ; Middle Aged ; Naproxen/therapeutic use ; Obesity/blood ; Obesity/complications ; Overweight/blood ; Overweight/complications ; Pilot Projects ; Prospective Studies ; Triglycerides/blood
Chemical Substances	Cyclooxygenase Inhibitors ; Fatty Acids, Omega-3 ; Fish Oils ; Triglycerides ; Naproxen (57Y76R9ATQ)
Language	English
Publishing date	2019-11-22
Publishing country	United States
Document type	Journal Article ; Randomized Controlled Trial ; Research Support, Non-U.S. Gov't
ZDB-ID	1105840-7
ISSN	1873-460X ; 1056-8727
ISSN (online)	1873-460X
ISSN	1056-8727
DOI	10.1016/j.jdiacomp.2019.107494
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