Enzymatic antioxidants status in patients with recurrent aphthous stomatitis.

BACKGROUND: Oxidative stress (OS) has been thought to play a main role in the etiopathogenesis of recurrent aphthous stomatitis (RAS), which is one of the most common oral mucosal diseases characterized by recurrent and painful oral ulcers. The aim of this investigation was to evaluate the enzymatic antioxidants status in patients with RAS in the active stage and remission stage. METHODS: Ninety-seven patients with idiopathic minor RAS and 102 race-, age- and gender-matched healthy individuals were recruited. All these subjects were allocated to three groups: RAS patients with active lesion (group A); the same patients in group A in the remission stage of RAS (group B); and healthy individuals without RAS (group C). Following an overnight fast, blood samples were obtained. The serum levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) were measured by the spectrophotometric method. Independent-samples t-test and paired t-test were performed for statistical evaluation. RESULTS: The serum levels of SOD, GSHPx, and CAT (83.9 ± 17.1 U/ml, 6687.2 ± 2629.2 U/ml, 1789.7 ± 593.8 U/l) were found to be significantly lower in group A as compared to those of group B (99.8 ± 11.1 U/ml, 9364.1 ± 1607.9 U/ml, 2789.1 ± 1113.4 U/l; P < 0.05) or group C (97.3 ± 12.1 U/ml, 9246.2 ± 2376.1 U/ml, 2819.0 ± 914.8 U/l; P < 0.05). No significant differences were found between group B and group C with respect to any one of these enzymatic antioxidants (P > 0.05). CONCLUSIONS: Our results indicate that enzymatic antioxidant defense system is impaired in RAS patients with active lesion and seems to play a crucial role in its pathogenesis.

Effect of plasma nitriding and titanium nitride coating on the corrosion resistance of titanium.

STATEMENT OF PROBLEM: The passive film on the surface of titanium can be destroyed by immersion in a fluoridated acidic medium. Coating with titanium nitride (TiN) may improve the corrosion resistance of titanium. PURPOSE: The purpose of this in vitro study was to investigate the effect of duplex treatment with plasma nitriding and TiN coating on the corrosion resistance of cast titanium. MATERIAL AND METHODS: Cast titanium was treated with plasma nitriding and TiN coating. The corrosion resistance of the duplex-treated titanium in fluoride-containing artificial saliva was then investigated through electrochemical and immersion tests. The corroded surface was characterized by scanning electron microscopy (SEM) with energy-dispersive spectroscopy surface scan analysis. The data were analyzed using ANOVA (α=.05) RESULTS: Duplex treatment generated a dense and uniform TiN film with a thickness of 4.5 µm. Compared with untreated titanium, the duplex-treated titanium displayed higher corrosion potential (Ecorr) values (P<.001) and lower corrosion current density (Icorr) values (P<.001). SEM results showed that the surface of untreated titanium was more heavily corroded than that of duplex-treated titanium. Surface scan analysis of duplex-treated titanium that had been immersed in artificial saliva containing 2 g/L fluoride revealed fluorine on the titanium surface, whereas fluorine was not observed on the surface of untreated titanium after immersion in fluoride-containing artificial saliva. The concentration of titanium ions released from the treated titanium was less than the amount released from untreated titanium (P<.001). CONCLUSIONS: Duplex treatment by plasma nitriding and TiN coating significantly improved the corrosion resistance of cast titanium in a fluoride-containing environment.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

Recent advances in coupling capillary electrophoresis-based separation techniques to ESI and MALDI-MS.

Coupling CE-based separation techniques to MS creates a powerful platform for analysis of a wide range of biomolecules from complex samples because it combines the high separation efficiency of CE and the sensitivity and selectivity of MS detection. ESI and MALDI, as the most common soft ionization techniques employed for CE and MS coupling, offer distinct advantages for biomolecular characterization. This review is focused primarily on technological advances in combining CE and chip-based CE with ESI and MALDI-MS detection in the past five years. Selected applications in the analyses of metabolites, peptides, and proteins with recently developed CE-MS platforms are also highlighted.

Liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometric imaging with sprayed matrix for improved sensitivity, reproducibility and quantitation.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has been employed as a detection method for both capillary electrophoresis (CE)-MALDI and liquid chromatography (LC)-MALDI analyses. Based on our previous studies, here we report a new interface to couple LC with MSI by employing an automated matrix sprayer. The LC trace is directly collected on a ground stainless steel MALDI plate and dried. The matrix is sprayed on the MALDI plate using a programmable matrix sprayer. With the highly uniform matrix layers produced from the sprayer, the MS image signal quality is significantly improved with enhanced signal-to-noise ratios for analyte peaks. With the programmable matrix application and imaging MS data acquisition, the new LC-MSI platform exhibits highly stable and reproducible performance. A total of 87 bovine serum albumin (BSA) tryptic peptides and 295 putative neuropeptides from blue crab pericardial organs have been observed with LC-MSI analysis, exhibiting better performance in terms of peptide coverage than regular LC-MALDI with discrete spot collection and our previously reported LC-MSI interface with the matrix being delivered by a capillary. In addition to relative quantitation with isotopic labeling as we have previously demonstrated, we performed the first absolute quantitation using the new LC-MSI platform and obtained accurate quantitation results for neuropeptides, indicating great potential for quantitative analysis of complex samples.

Serum levels of reduced glutathione, oxidized glutathione, and glutathione reductase activity in minor recurrent aphthous stomatitis patients.

Background/purpose: Recurrent aphthous stomatitis (RAS) is one of the most prevalent oral mucosa diseases with unknown etiology. Reduced glutathione (GSH) is a major intracellular non-protein physiological antioxidant, and it has been demonstrated that GSH deficiency may be related to cardiovascular, immune, and diabetes. The purpose of this investigation was to evaluate the potential roles of GSH, oxidized glutathione (GSSG), and glutathione reductase (GR) in the etiopathogenesis of minor recurrent aphthous stomatitis (MiRAS). Materials and methods: The study comprised 87 patients with idiopathic MiRAS and 90 race-, age-, and gender-matched healthy individuals. The spectrophotometric method was used to determine serum GSH and GSSG concentrations as well as GR activity. The GSSG/GSH ratios were subsequently computed. For statistical evaluation, the independent sample t test, Pearson's chi-square test, Mann-Whitney U test, Kruskal-Wallis H test, and Binary logistic regression analysis were used. Results: The serum GSSG level, GR activity and GSSG/GSH ratio were statistically higher in MiRAS patients, whereas the concentration of serum GSH was significantly decreased. With the exception of GR, serum GSSG, GSH, and GSSG/GSH were all significantly associated with MiRAS. Serum GSSG can be regarded as a risk factor, whereas serum GSH and GSSG/GSH maybe considered as protective factors against the occurrence of MiRAS. Conclusion: GSSG may be a potential danger factor to MiRAS and GSH may be a protective factor, while GR may not play an important role in the aetiopathogenesis of MiRAS.

Pressure-assisted capillary electrophoresis coupling with matrix-assisted laser desorption/ionization-mass spectrometric imaging for quantitative analysis of complex peptide mixtures.

Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

Poly(glycidyl methacrylate-divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis.

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing-matrix-assisted laser desorption/ionization mass spectrometry (CIEF-MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB)-based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off-line coupling of IPG-CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG-CIEF. Enhanced neuropeptide detection is also observed after coupling IPG-CIEF with MALDI MS.

Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging.

Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques.

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.

Advancing matrix-assisted laser desorption/ionization-mass spectrometric imaging for capillary electrophoresis analysis of peptides.

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.

Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins.

Recombinant adeno-associated viruses (AAVs) have emerged as the leading gene delivery platform owing to their nonpathogenic nature and long-term gene expression capability. The AAV capsid, in addition to protecting the viral genome, plays an important role in viral infectivity and gene transduction, indicating the value of the constituent viral proteins (VPs) being well-characterized as part of gene therapy development. However, the limited sample availability and sequence homology shared by the VPs pose challenges to adapt existing analytical methods developed for conventional biologics. In this study, we report the development of reversed-phase liquid chromatography/mass spectrometry-based methods for characterization of AAV capsid proteins at intact protein and peptide level with reduced sample consumptions. The developed methods allowed the measurement of VP expression with fluorescence detection and intact mass/post-translational modifications (PTMs) analysis through a benchtop time-of-flight mass spectrometer. The general applicability and validity of the methods for gene therapy product development were demonstrated by applying the optimized methods to multiple common AAV serotypes. A 1-h enzymatic digestion method was also developed using 1.25 µg of AAV VPs, providing >98% protein sequence coverage and reproducible relative quantification of various PTMs of the VPs. The efficient and sensitive analyses of AAV capsid proteins enabled by the reported methods provide further understanding and offer guidance in the development and manufacturing of AAV-related therapeutics.

Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.

Galectin-3 and acute heart failure: genetic polymorphisms, plasma level, myocardial fibrosis and 1-year outcomes.

Aim: This study sought to investigate the relationship between galectin-3 (Gal-3), myocardial fibrosis (MF) and outcomes in acute heart failure. Materials & methods: The single-nucleotide polymorphisms (SNPs) of LGALS3 at rs4644 and rs4652, plasma Gal-3 level, MF and major adverse events (MAEs) were obtained. Results: There was no significant difference in MAEs when categorizing patients by the LGALS3 SNPs at rs4644 and rs4652. The circulating Gal-3 was related to the degree of MF (p < 0.001). Plasma Gal-3 level and MF can predict an increased risk of MAEs (p < 0.001, p = 0.023, respectively). Conclusion: Not the SNPs of LGALS3 but Gal-3 and MF can predict MAEs in acute heart failure at 1 year of follow-up.

Serum levels of total antioxidant status, nitric oxide and nitric oxide synthase in minor recurrent aphthous stomatitis patients.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is one of the most common inflammatory ulcerative conditions of oral cavity with uncertain etiology. Several studies have reported that oxidative stress may be associated with RAS. The aim of this study was to compare the serum levels of total antioxidant status (TAS), nitric oxide (NO) and nitric oxide synthase (NOS) in minor RAS (MiRAS) patients with healthy individuals and determine the possible association of MiRAS with the 3 physiological parameters mentioned above. METHODS: Ninety patients with idiopathic MiRAS and 90 race-, age- and sex-matched healthy individuals were included in this study. All these subjects were allocated to 3 groups: MiRAS patients in the active stage (Group A); the same MiRAS patients in Group A in the inactive stage (Group B); healthy individuals without MiRAS (Group C). Serum levels of TAS, NO and NOS were determined by the spectrophotometric method. Independent sample t test and paired t test were performed for statistical evaluation. RESULTS: Serum TAS level of Group A was significantly decreased than that of Group C, whereas the serum level of NO was significantly higher in Group A as compared to Group C (P < .05). The serum levels of TAS and NO in Group B were no significant differences when compared with those in Group A or Group C. No significant differences in NOS activities were also found between the 3 groups (P > .05). CONCLUSIONS: MiRAS is associated with decreased TAS and increased NO levels, but NOS may not play an important role in the aetiopathogenesis.

Metabolic analysis of four phenolic acids in rat by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC-DAD-ESI-MS(n)) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid-liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C(18) reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.

Determination of danshensu in rat plasma and tissues by high-performance liquid chromatography.

A systematic study on pharmacokinetics and tissue distribution of danshensu (one of the major active components from Salvia miltiorrhizae) is conducted using a rapid and sensitive high-performance liquid chromatographic (HPLC) method. Before HPLC analysis, biological samples are pretreated with a liquid-liquid extraction. Separation of danshensu and internal standard is achieved on an Agilent Zorbax C18 column with a mobile phase made up of acetonitrile and 0.05% trifluoracetic acid at a flow rate of 0.8 mL/min. The calibration curves in plasma and tissues are linear in the given concentration ranges, with r2 no less than 0.99. The intra-day and inter-day relative standard deviations in the measurement of quality control samples are less than 15%, and the accuracies are in the range of 86-115%. The recoveries of danshensu in plasma and tissues are among 80% to 118%. Meanwhile, the multi-peaks in pharmacokinetic profiles are observed. The method is successfully applied to the pharmacokinetics and tissue distribution study of danshensu after a single oral administration of 50.0 mg/kg sodium danshensu to rats.

Chronic heart failure in patients with nonalcoholic fatty liver disease: prevalence, clinical features, and relevance.

Objective This study was performed to assess the prevalence of nonalcoholic fatty liver (NAFL) in patients with symptomatic congestive heart failure (CHF) and compare the clinical features with those of patients without NAFL. Methods In total, 102 patients with CHF were divided into NAFL and non-NAFL groups according to their hepatic ultrasonography findings. All patients underwent transthoracic echocardiography and cardiac magnetic resonance examination. Follow-up was performed for major cardiovascular events (MACE) and readmission due to heart failure at 1, 3, 6, and 12 months after the index hospitalization. Results NAFL was detected in 37 of 102 patients (36.27%). Compared with the non-NAFL group, patients with NAFL were younger, had a higher body mass index and left ventricular (LV) mass index, and had more severe fibrosis. MACE and readmission occurred in 15 patients in the NAFL group and 29 patients in the non-NAFL group, without a significant difference. Linear regression analysis revealed that after adjusting for confounders, NAFL was independently associated with the LV fibrosis size and the ratio of the LV fibrosis size to the LV mass index. Conclusions NAFL is present in more than one-third of patients with CHF and is associated with the severity of LV fibrosis.

Liquid chromatography-tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat.

A method using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) analysis was established for the identification of metabolites in rat after oral administration of protocatechuic aldehyde, a major bioactive phenolic acid in the roots of Salvia miltiorrhiza. Eleven metabolites in rat plasma and urine were firstly identified as protocatechuic aldehyde, protocatechuic acid and their methylated, glucuronized or glycine conjugates on the basis of their MS fragmentation behaviors, while nine of these metabolites (except protocatechuic aldehyde and protocatechuic acid) were detected in rat bile. In addition, the possible metabolic pathway was proposed for the first time. In the phase I metabolism, protocatechuic aldehyde could be oxidized to protocatechuic acid. The conjugates would be formed in rat intestine, liver and kidney and excreted from rat urine and bile. Enthrohepatic circulation played an important role in the metabolism of protocatechuic aldehyde. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of protocatechuic aldehyde and to better understand its in vivo metabolism.

Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography-mass spectrometry.

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC-MS(n) analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS(n) fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.