Investigation of dysregulated lipid metabolism in diabetic mice via targeted metabolomics of bile acids in enterohepatic circulation.

RATIONALE: The mechanism of lipid metabolism disorder in type 2 diabetes (T2DM) remains unclear. This study aimed to reveal the mechanism underlying dysregulated lipid metabolism in T2DM through bile acid metabolism. METHODS: A db/db mouse model was employed to investigate the alteration of bile acid profiles in T2DM. Ultrahigh-performance liquid chromatography with tandem mass spectrometry was used to quantify the detailed bile acid levels in each compartment of enterohepatic circulation. The pathological change of mouse liver was assessed by liver histology and serum biochemical assays. The expression level of bile acid-related transporters and synthases was measured with Western blot analysis. RESULTS: The results showed that T2DM can result in severe liver fat accumulation and liver damage. In addition, compared to the control group, in T2DM mice, bile acid synthesis is reduced, while the level of bile acids is increased at the storage sites and the reabsorption sites, but there are subtle gender differences. Further, the ratio of conjugated bile acids in total bile acid in the liver of T2DM mice increased significantly relative to the control group for both female and male mice. CONCLUSIONS: In T2DM, bile acid metabolism is disordered in both male and female mice, which could be the underlying mechanism of dysregulated lipid metabolism in T2DM.

Biomarker identification and pathway analysis of rheumatoid arthritis based on metabolomics in combination with ingenuity pathway analysis.

Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease worldwide, but understanding its pathogenesis is still limited. In this study, plasma untargeted metabolomics of a discovery cohort and targeted analysis of a verification cohort were performed by gas chromatograph mass spectrometry (GC/MS). Univariate and multivariate statistical analysis were utilized to reveal differential metabolites, followed by the construction of biomarker panel through random forest (RF) algorithm. The pathways involved in RA were enriched by differential metabolites using Ingenuity Pathway Analysis (IPA) suite. Untargeted metabolomics revealed eighteen significantly altered metabolites in RA. Among these metabolites, a three-metabolite marker panel consisting of L-cysteine, citric acid and L-glutamine was constructed, using random forest algorithm that could predict RA with high accuracy, sensitivity and specificity based on a multivariate exploratory receiver operator characteristic (ROC) curve analysis. The panel was further validated by support vector machine (SVM) and partial least squares discriminant analysis (PLS-DA) algorithms, and also verified with targeted metabolomics using a verification cohort. Additionally, the dysregulated taurine biosynthesis pathway in RA was revealed by an integrated analysis of metabolomics and transcriptomics. Our findings in this study not only provided a mechanism underlying RA pathogenesis, but also offered alternative therapeutic targets for RA.

Impact of sample containers on gas chromatography mass spectrometry based plasma untargeted and targeted metabolomics.

Plasticware and glassware used in the sample processing stage could result in different analysis performance for macromolecules, which led to the speculation that a similar effect could happen to small molecules. To confirm the speculation, pooled human plasma sample spiked with and without metabolite standards was prepared with three most commonly used container materials (glass, deactivated glass and polypropylene) supplied by different manufacturers after a two-step liquid-liquid extraction, followed by gas chromatography mass spectrometry (GC-MS) based untargeted and targeted metabolomics. The results showed that both GC-MS-based targeted and untargeted metabolomics could be influenced significantly by not only the container material but also the manufacturing procedures employed by different vendors. As a general guideline, it would be highly practical to use the same type of container from the same manufacturer during a whole project. Our study could be extremely valuable for the fellow researchers when dealing with the sample containers for GC-MS-based untargeted and targeted metabolomics.

Combined Metabolomics with Transcriptomics Reveals Important Serum Biomarkers Correlated with Lung Cancer Proliferation through a Calcium Signaling Pathway.

Lung cancer (LC) is one of the most malignant cancers in the world, but currently, it lacks effective noninvasive biomarkers to assist its early diagnosis. Our study aims to discover potential serum diagnostic biomarkers for LC. In our study, untargeted serum metabolomics of a discovery cohort and targeted analysis of a test cohort were performed based on gas chromatography-mass spectrometry. Both univariate and multivariate statistical analyses were employed to screen for differential metabolites between LC and healthy control (HC), followed by the selection of candidate biomarkers through multiple algorithms. The results showed that 15 metabolites were significantly dysregulated between LC and HC, and a panel, comprising cholesterol, oleic acid, myo-inositol, 2-hydroxybutyric acid, and 4-hydroxybutyric acid, was demonstrated to have excellent differentiating capability for LC based on multiple classification modelings. In addition, the molecular interaction analysis combined with transcriptomics revealed a close correlation between the candidate biomarkers and LC proliferation via a Ca2+ signaling pathway. Our study discovered that cholesterol, oleic acid, myo-inositol, 2-hydroxybutyric acid, and 4-hydroxybutyric acid in combination could be a promising diagnostic biomarker for LC, and most importantly, our results will shed some light on the pathophysiological mechanism underlying LC to understand it deeply. The data that support the findings of this study are openly available in MetaboLights at https://www.ebi.ac.uk/metabolights/, reference number MTBLS1517.

Systematic evaluation of sample preparation strategy for GC-MS-based plasma metabolomics and its application in osteoarthritis.

Sample preparation plays a crucial part in plasma metabolomics. In order to obtain an optimal sample extraction method for gas chromatography mass spectrometry (GC-MS)-based plasma metabolomics, five different extraction strategies including protein precipitation, liquid-liquid extraction and solid-phase extraction were evaluated systematically for both plasma untargeted- and targeted-metabolomics. The comprehensive evaluation revealed that the all-in-one sample preparation method, MeOH-MTBE-H2O (1:5:1.5, v/v/v), was the optimal extraction method for both untargeted- and targeted-metabolomics. Next, the optimal sample preparation protocol was applied in plasma metabolomics of osteoarthritis (OA). A panel containing cholesterol, lactic acid, stearic acid, alpha-tocopherol and oxalic acid was selected as candidate biomarker to distinguish OA patients from healthy controls (HC) based on the support vector machine (SVM) classification model. The discriminating capability of the candidate biomarker panel was further validated successfully with logistic regression and principal components analysis (PCA) analysis. Therefore, the panel could potentially act as diagnostic biomarker for osteoarthritis.

Evaluating the interplay among stationary phases/ion-pairing reagents/sequences for liquid chromatography mass spectrometry analysis of oligonucleotides.

The correlation among stationary phases, ion-pairing reagents (IPR) and sequences for ion-pair reversed-phase liquid chromatography mass spectrometry (IP-RP LC-MS) analysis of oligonucleotide (ODN) remains unclear. The present study aimed to evaluate such correlation using particle-packed C18 columns in order to search for the optimal combination among them. Five C18 columns packed with core-shell silica, polymer, porous silica and hybrid particles, respectively, were evaluated for the analysis of synthetic and chemically modified ODNs with six different IPRs. Our results showed that silica-based porous particles, compared to other particles, retained ODN the strongest no matter which IPR was used. Meanwhile, among the six IPRs hexylamine (HA) produced the longest retention for all ODNs, regardless of the types of C18 particles. For the separation of ODNs, C18 columns performed similarly under identical LC conditions. However, the separation ability of C18 columns is highly dependent on the type of IPR and ODN sequences. Moreover, the type of particles has little impact on the signals of ODNs for the majority of synthetic sequences, but such impact could be dramatic for chemically modified sequences. On the other hand, both the type of IPR and ODN sequence have a significant effect on MS signals for synthetic and chemically modified ODNs.

Chronic Alcohol Consumption Increased Bile Acid Levels in Enterohepatic Circulation and Reduced Efficacy of Irinotecan.

AIMS: To investigate the effect of ethanol intake on the whole enterohepatic circulation (EHC) of bile acids (BAs) and, more importantly, on pharmacokinetics of irinotecan. METHODS: The present study utilized a mouse model administered by gavage with 0 (control), 240 mg/100 g (30%, v/v) and 390 mg/100 g (50%, v/v) ethanol for 6 weeks, followed by BA profiles in the whole EHC (including liver, gallbladder, intestine and plasma) and colon using ultra-high performance liquid chromatography with tandem mass spectrometry analysis. Pharmacokinetic parameters of irinotecan were measured after administration of irinotecan (i.v. 5 mg/kg) on alcohol-treated mice. RESULTS: The results showed that compared with the control group, concentrations of most free-BAs, total amount of the three main forms of BAs (free-BA, taurine-BA and glycine-BA) and total BAs (TBAs) in 50% ethanol intake group were significantly increased, which are mostly attributed to the augmentation of free-BAs and taurine-BAs. Additionally, the TBAs in liver and gallbladder and the BA pool were markedly increased in the 30% ethanol intake group. Importantly, ethanol intake upregulated the expression of BA-related enzymes (Cyp7a1, Cyp27a1, Cyp8b1 and Baat) and transporters (Bsep, Mrp2, P-gp and Asbt) and downregulated the expression of transporter Ntcp and nuclear receptor Fxr in the liver and ileum, respectively. Additionally, 50% ethanol intake caused fairly distinct liver injury. Furthermore, the AUC0-24 h of irinotecan and SN38 were significantly reduced but their clearance was significantly increased in the disrupted EHC of BA by 50% ethanol intake. CONCLUSIONS: The present study demonstrated that ethanol intake altered the expression of BA-related synthetases and transporters. The BA levels, especially the toxic BAs (chenodeoxycholic acid, deoxycholic acid and lithocholic acid), in the whole EHC were significantly increased by ethanol intake, which may provide a potential explanation to illuminate the pathogenesis of alcoholic liver injury. Most importantly, chronic ethanol consumption had a significant impact on the pharmacokinetics (AUC0-24 h and clearance) of irinotecan and SN38; hence colon cancer patients with chronic alcohol consumption treated with irinotecan deserve our close attention.

Proteinase K Combining Two-Step Liquid-Liquid Extraction for Plasma Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics To Discover the Potential Mechanism of Colorectal Adenoma.

LC-MS-based untargeted metabolomics have been proven to be an extremely promising technique to discover biomarkers and explore the mechanisms underlying diseases, which, however, relies heavily on sample pretreatment for metabolite extraction. In the present study, a systematic and pragmatic evaluation of eight protocols employing conventional metabolites extraction strategies, protein precipitation (PPT), and liquid-liquid extraction (LLE), with and without proteinase K (PK) incubation, was performed simultaneously, using human plasma and a mixture of 39 endogenous metabolite standards. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) PPT with 2-propanol, (4) two-step LLE of CH2Cl2-MeOH, followed by MeOH-H2O, (5) PK incubation combining two-step LLE of CH2Cl2-MeOH followed by MeOH-H2O, (6) two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (7) PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (8) PK incubation combining MeOH-EtOH PPT. The results suggested that two-step LLE produced broader metabolome coverage than protein precipitation, and the addition of proteinase K enhanced the extraction performance further. Taken together, PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, was determined to be the most suitable extraction method, because of its broad metabolome coverage, high reproducibility, and satisfactory recovery. Next, the developed optimal sample preparation method was applied successfully to profile the plasma metabolome of colorectal adenoma and uncover its potential mechanism for significant differential changes in linoleic acid and phospholipid metabolism.

Triethylamine improves MS signals stability of diluted oligonucleotides caused by sample containers.

The effect of sample containers made of different materials on the MS-based analysis of oligonucleotides remains unknown. Here, we evaluated five types of sample containers on the MS signal stability of oligonucleotide, and they were normal glass insert, silanized glass insert with three different silanization techniques, and polypropylene sample vial. Also, we attempted to tackle signal stability issue by varying modifiers in dissolution solvent. Our results showed that sample containers made of different materials can significantly influence the MS signal stability of oligonucleotide at low concentration. Triethylamine (TEA) evidently improved both the signal stability and intensity of oligonucleotide.

The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides.

RATIONALE: Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion-pairing reversed-phase liquid chromatography/mass spectrometry (IP-RP-LC/MS) analysis of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS analysis of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. METHODS: The study was performed using an Agilent 1290 ultra-high-performance liquid chromatography (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 µm). Buffer systems containing ion-pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N-dimethylcyclohexylamine, and octylamine) and fluoroalcohols (HFIP and hexafluoro-2-methyl-2-propanol (HFTP)) were compared chromatographically and mass spectrometrically. RESULTS: Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the separation of oligonucleotides sized from 10mer to 40mer for most of ion-pairing reagents, with the exception of triethylamine and N,N-dimethylcyclohexylamine, where HFIP performed better than HFTP. CONCLUSIONS: The choice of fluoroalcohols in IP-RP-LC/MS analysis of oligonucleotides depends on the type of ion-pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA-HFIP or HA-HFTP for small oligonucleotides, but TPA-HFTP for large oligonucleotides for IP-RP-LC/MS analysis of synthetic oligonucleotides.

Analysis of oligonucleotides by ion-pairing hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC-LC/ESI-MS) has been proved to be useful for the quality control of oligonucleotides. However, the lack of separation for some oligonucleotides using HILIC-LC/MS has proved problematic. This study aimed to improve the resolving ability of HILIC-LC/MS. METHODS: The study was performed on a Waters UPLC® system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer using a Zorbax® RRHD HILIC column (2.1 mm × 100 mm, 1.8 µm). Buffer systems contained triethylammonium acetate (TEAA) and acetonitrile. The effects of the concentration of TEAA and the type of organic modifiers on the separation of oligonucleotides were investigated. RESULTS: The results showed that the optimum concentration of TEAA is 10 mM and acetonitrile is a better organic solvent than methanol. The addition of TEAA in the HILIC mobile phase improved the separation of N from N + A significantly compared to the HILIC method buffered with ammonium acetate. The IP-HILIC chromatography has demonstrated that the separation of oligonucleotides is sequence dependent. In addition, the IP-HILIC method produces a much simpler mass spectrum of an oligonucleotide with very efficient desalting. CONCLUSIONS: The HILIC-LC/MS method with the addition of TEAA at a MS-compatible concentration has improved the separation of oligonucleotides. The IP-HILIC-LC/MS method also produces very simple mass spectra with high desalting efficiency.

Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation.

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N(6)-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66-292) to 2.0 Å resolution. ALKBH566₋292 has a double-stranded ß-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (ßIV-V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX-nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.

Comparing ion-pairing reagents and counter anions for ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of synthetic oligonucleotides.

RATIONALE: Ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-LC/ESI-MS) has been widely used for the quality control of oligonucleotides. However, researchers are still looking to improve mobile phase systems for IP-RP-LC/ESI-MS analysis of oligonucleotides. This study compared the performance of six ion-pairing reagents with three different counter anions for IP-RP-LC/ESI-MS analysis of oligonucleotides. METHODS: The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT Premier XE mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing acetate, bicarbonate, and hexafluoroisopropanolate salts of six ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine), respectively, were optimized for IP-RP-LC/ESI-MS analysis of oligonucleotides, and then the optimized conditions were applied for the separation of oligonucleotides. RESULTS: Counter anions definitely play a role in IP-RP-LC/ESI-MS analysis of oligonucleotides. Buffer containing 30 mM diisopropylethylamine and 200 mM hexafluoroisopropanol provided the highest separation of unmodified heterogeneous oligonucleotides, but tripropylammonium hexafluoroisopropanolate achieved the most enhanced separation of sequence isomers. However, triethylammonium acetate and bicarbonate had equally the highest separation for positional isomers. CONCLUSIONS: IP-RP-LC/ESI-MS separation of oligonucleotides is mainly sequence dependent, but it is also dependent on both the type of ion-pairing reagent and counter anion present in the mobile phase.

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides.

RATIONALE: A sensitive and selective liquid chromatography/mass spectrometry (LC/MS) method is essential for quality control of synthetic oligonucleotides. However, researchers are still searching for improvements to ion-pairing reagents for ion-pairing reversed-phase LC/MS. This study performed a comprehensive comparison of six ion-pairing reagents to determine their performance as mobile phase modifiers for oligonucleotide LC/MS. METHODS: The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine) and hexafluoro-2-propanol were compared by measuring the adduct ion formation, chromatographic separation, and MS signal intensity of four oligonucleotides (10mer to 40mer). The effect of dissolution solvents on MS signal intensity and adduct ion formation was also investigated. RESULTS: Results showed that the type of dissolution solvent can have a signficiant impact on adduct ion formation with oligonucleotides. Results also showed that the maximum separation for small, medium and large oligonucleotides occured when using tripropylamine, N,N-dimethylbutylamine, and dibutylamine, respectively. However, on average 15 mM hexylamine and 50 mM hexafluoro-2-propanol provided the best chromtatographic performance (resolution values: 14.1 ± 0.34, 11.0 ± 0.17, and 6.4 ± 0.11 for the pairs of oligonucleotides T10 & T15, T15 & T25, and T25 & T40, respectively (3 replicates)). CONCLUSIONS: The impact of dissolution solvent on the MS signal of oligonucleotides depends on the type of ion-pairing reagent. Buffer combining 15 mM hexylamine and 50 mM hexafluoro-2-propanol produced the highest overall performance for oligonucleotides (10mer to 40mer) with respect to chromatographic resolution and mass detection.

Asparagine and aspartate hydroxylation of the cytoskeletal ankyrin family is catalyzed by factor-inhibiting hypoxia-inducible factor.

Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the ß-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13-24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.

Combined metabolomics with transcriptomics reveals potential plasma biomarkers correlated with non-small-cell lung cancer proliferation through the Akt pathway.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is one of the main types of lung cancer. Due to lack of effective biomarkers for early detection of NSCLC, the therapeutic effect is not ideal. This study aims to reveal potential biomarkers for clinical diagnosis. METHODS: The plasma metabolic profiles of the patients were characterized by liquid chromatography-mass spectrometry (LC-MS). Differential metabolites were screened by p less than 0.05 and VIP greater than 1. Multivariate statistical analysis was used to search for potential biomarkers. Receiver operating characteristic (ROC) curve was used to evaluate the predictors of potential biomarkers. Pathway enrichment analysis was performed on metabolomics data by Ingenuity Pathway Analysis (IPA) and transcriptomics data from GEO were used for validation. RESULTS: A plasma metabolite biomarker panel including 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) and arachidonic acid was chose. The area under the ROC curve were 0.917, 0.900 and 0.867 for the panel in the different algorithm like Partial Least Squares Discrimination Analysis (PLS-DA), Support Vector Machine (SVM), Random Forest (RF). The candidate biomarkers were associated with the Akt pathway. Genes involved in the biological pathway had significant changes in the expression levels. CONCLUSION: 13(S)-HODE and arachidonic acid may be potential biomarkers of NSCLC. The Akt pathway was associated with this biomarker panel in NSCLC. Further studies are needed to clarify the mechanisms of disruption in this pathway.

Simultaneous determination of oxalate and citrate in urine and serum of calcium oxalate kidney stone rats by IP-RP LC-MS/MS.

Oxalate and citrate in 24 h urine and serum are considered to be associated with the incidence and recurrence risk of calcium oxalate kidney stones. The quantification of oxalate and citrate contributes to understand the pathological metabolism of kidney stones and guide the early diagnosis and recurrence monitoring. Although simultaneous quantification of oxalate and citrate in urine using liquid chromatography tandem mass spectrometry (LC-MS/MS) have been reported, the optimization of chromatographic column, mobile phase and mass spectrometry (MS) parameters has not been performed. In addition, these is a lack of suitable method for simultaneous detection of oxalate and citrate both in serum and urine. Therefore, we developed a method for the simultaneous determination of oxalate and citrate in urine and serum based on ion-pairing reversed-phase (IP-RP) LC-MS/MS. Herein, five ion-pair reagents, namely, triethanolamine, dimethylbutyl amine, diisopropenyl amine, N,N-dimethylcyclohexylamine and tripropylamine, and three ion-pairing reagent (IPR) buffers, namely, acetic acid, hexafluoro-2-isopropanol, and hexafluoro-2-methyl-2-propanol, were compared in regard to their chromatographic peak abundance and separation of oxalate and citrate. Moreover, MS parameters and the multiple reaction monitoring (MRM) conditions were also evaluated and optimized to obtain the maximum peak abundance. After that, the method was validated in the linear range of 0.25-1000 µM, and the correlation coefficient was ≥ 0.99. The precision and accuracy were < 14.70% and < 19.73%, respectively. The extraction recovery was 80.53-108.79%, and the matrix effect was < 8.79%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, and for 3 freeze-thaw cycles. Finally, this method was applied to the determination of oxalate and citrate in the serum and urine of rats with calcium oxalate kidney stones. The establishment of a stable and effective oxalate and citrate detection method is conducive to the diagnosis and monitoring of kidney stones.

Integrated plasma and liver gas chromatography mass spectrometry and liquid chromatography mass spectrometry metabolomics to reveal physiological functions of sodium taurocholate cotransporting polypeptide (NTCP) with an Ntcp knockout mouse model.

Sodium taurocholate cotransporting polypeptide (NTCP) is an important hepatocyte transporter, while its physiological functions require further investigation. In our study, an integrated plasma and liver GC-MS- and LC-MS-based metabolomics strategy with an optimized two-step liquid-liquid extraction was utilized to explore the physiological functions of NTCP via a knockout (KO) mouse model. The present study found that NTCP deficiency resulted in obvious metabolic change in the plasma and liver of mice. Totally, 102 and 87 differential metabolites were discovered in the liver and plasma, respectively. Pathway analysis revealed that the metabolism of tyrosine, glycine, taurine, fatty acid and glycerophospholipid as well as the biosynthesis of tryptophan, pantothenate and CoA were significantly dysregulated in the Ntcp KO mice, indicating that NTCP is closely involved in these metabolic pathways. Moreover, L-tryptophan, cadaverine and D-pantothenic acid could serve as the diagnostic biomarker for NTCP deficiency. Our study provided deep insights into the physiological functions of NTCP, and the findings would hold the great potential to be used for the discovery of new therapeutic and diagnostic strategies for NTCP deficiency clinically.

The Function of Multidrug Resistance-associated Protein 3 in the Transport of Bile Acids under Normal Physiological and Lithocholic Acid-induced Cholestasis Conditions.

BACKGROUND: The role of multidrug resistance-associated protein 3 (Mrp3) in the transport of bile acid (BA) in drug-induced cholestasis has not been well studied. OBJECTIVE: In this study, wild type and Mrp3 knockout (Mrp3-/-) mice under normal physiological and lithocholic acid (LCA)-induced cholestatic conditions were employed to investigate the role of Mrp3 in BA transport. METHODS: The levels of BA in serum, liver, gallbladder, intestine, kidney, feces and urine were quantified in both wild type and Mrp3-/- mice via ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS). Quantitative real-time PCR (RT-PCR) analysis was used to measure the expression of genes related to the transport and synthesis of BA. RESULTS: The results showed that the liver did not suffer more serious damage as a result of cholestasis when Mrp3 was depleted. The level of some individual bile acids changed apparently in the compartments of enterohepatic circulation (EHC) between the two control and model groups, respectively, but the level of serum total bile acid was only slightly reduced for Mrp3-/- groups. In addition, the level of BA-related efflux transporters and synthases increased significantly when Mrp3 was knocked out under normal physiological conditions, but a negligible alteration appeared under cholestatic conditions. CONCLUSION: Our results indicated that Mrp3 could be responsible for the transport of some specific bile acids, and part of the Mrp3 role could be compensated for by other transporters. Moreover, Mrp3 deficiency has a direct effect on the expression of BA-related synthases and efflux transporters under normal physiological conditions, but this effect could be less prominent under cholestatic conditions. This study could provide much valuable insight into the physiological function of Mrp3 in the transport of bile acids.