Astragaloside IV Ameliorates Isoprenaline-Induced Cardiac Fibrosis in Mice via Modulating Gut Microbiota and Fecal Metabolites.

Aim: Gut microbiota is of crucial importance to cardiac health. Astragaloside IV (AS-IV) is a main active ingredient of Huangqi, a traditional edible and medicinal herb that has been shown to have beneficial effects on cardiac fibrosis (CF). However, it is still uncertain whether the consumption of AS-IV alleviates cardiac fibrosis through the gut microbiota and its metabolites. Therefore, we assessed whether the anti-fibrosis effect of AS-IV is associated with changes in intestinal microbiota and fecal metabolites and if so, whether some specific gut microbes are conducive to the benefits of AS-IV. Methods: Male C57BL-6J mice were subcutaneously injected with isoprenaline (ISO) to induce cardiac fibrosis. AS-IV was administered to mice by gavage for 14 days. The effects of AS-IV on cardiac function, myocardial enzyme, cardiac weight index (CWI), and histopathology of ISO-induced CF mice were investigated. Moreover, 16S rRNA sequencing was used to establish gut-microbiota profiles. Fecal-metabolites profiles were established using the liquid chromatograph-mass spectrometry (LC-MS). Results: AS-IV treatment prevented cardiac dysfunction, ameliorated myocardial damage, histopathological changes, and cardiac fibrosis induced by ISO. AS-IV consumption increased the richness of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella. AS-IV also modulated gut metabolites in their feces. Among 141 altered gut metabolites, amino acid production was sharply changed. Furthermore, noticeable correlations were found between several specific gut microbes and altered fecal metabolites. Conclusions: An increase of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella abundance, and modulation of amino acid metabolism, may contribute to the anti-fibrosis and cardiac protective effects of Astragaloside IV.

Sensitive enantioseparation and determination of isoprenaline in human plasma and pharmaceutical formulations.

A simple, sensitive and fast RPHPLC method was developed and validated for the enantioselective determination of (RS)-isoprenaline (Ipn) in human plasma. The enantiomers were converted to diastereomeric derivatives using s-triazine (cyanuric chloride) based chiral derivatizing reagents. l-isoleucine and l-methionine were introduced as chiral auxiliary in s-triazine and two new monochloro-s-triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomeric derivatives of (RS)-Ipn spiked in human plasma. (RS)-Ipn was isolated (purified and characterized) from a commercial pharmaceutical formulation and was used as the standard racemic sample. Structures of the two diastereomeric derivatives were optimized for lowest energy using the Gaussian 09 Rev A. 02 program and hybrid density functional B3LYP with 6-31G* basis set which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomeric derivatives. The results were correlated with the mechanism of separation and elution order. Limit of detection values were found to be 24.6 and 26.8 ng mL-1 for the first and second eluting diastereomeric derivatives, respectively.

Rapid and mild fabrication of protein membrane coated capillary based on supramolecular assemble for chiral separation in capillary electrochromatography.

Exploration of the simple and stable coating methods is of great significance in capillary electrochromatography (CEC). In this work, a lysozyme assemble supramolecular membrane coated capillary column was developed for CEC chiral separation. Taking advantage of phase transformation of lysozyme, the coating process was achieved within 1.0 h thus to a large extent reduced the capillary preparation time and simplified the coating procedure. The successful fabrication of the supramolecular membrane coated capillary was verified by scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), fluorescence imaging, and electroosmotic flow (EOF). The separation capacity of the coated capillary was evaluated by analysis of different chiral analytes, including chiral amine drug and neurotransmitters, and good enantioseparation efficiency was achieved for the three pairs of enantiomers. For three consecutive runs, the relative standard deviations (RSD) for the migration time of the analytes for intra-day (n = 3), inter-day (n = 3) and column-to-column (n = 3) were in the range of 0.7-1.5%, 2.7-3.6%, and 4.5-5.8%, respectively. Additionally, the supramolecular membrane coated capillary column could run consecutively 100 times without observable change in the separation efficiency, proving the feasibility of the coating method based on the adhesion of the protein-based supramolecular membrane.

Fasting/Refeeding Cycles Prevent Myocardial Dysfunction and Morphology Damage in the Spontaneously Hypertensive Rats / Ciclos de Jejum/Realimentação Previnem Disfunção Miocárdica e Danos Morfológicos em Ratos Espontaneamente Hipertensos

Abstract Background: Caloric restriction is known to impair the cardiac function and morphology in hypertrophied hearts of spontaneously hypertensive rats (SHR); however, the influence of fasting/refeeding (RF) is unknown. Objective: To investigate the fasting/refeeding approach on myocardial remodeling and function. In addition, the current study was designed to bring information regarding the mechanisms underlying the participation of Ca2+ handling and b-adrenergic system. Methods: Sixty-day-old male SHR rats were submitted to food ad libitum (C), 50% food restriction (R50) or RF cycles for 90 days. Cardiac remodeling was assessed by ultrastructure analysis and isolated papillary muscle function. The level of significance considered was 5% (a = 0.05). Results: The RF rats presented lower cardiac atrophy than R50 in relation to C rats. The C rats increased weight gain, R50 maintained their initial body weight and RF rats increased and decreased weight during RF. The RF did not cause functional impairment because the isotonic and isometric parameters showed similar behavior to those of C. The isotonic and isometric cardiac parameters were significantly elevated in RF rats compared to R50 rats. In addition, the R50 rats had cardiac damage in relation to C for isotonic and isometric variables. While the R50 rats showed focal changes in many muscle fibers, the RF rats displayed mild alterations, such as loss or disorganization of myofibrils. Conclusion: Fasting/refeeding promotes cardiac beneficial effects and attenuates myocardial injury caused by caloric restriction in SHR rats, contributing to reduce the cardiovascular risk profile and morphological injuries. Furthermore, RF promotes mild improvement in Ca2+ handling and b-adrenergic system.

Resumo Fundamento: A restrição calórica compromete a função e a morfologia cardíacas em corações hipertrofiados de ratos espontaneamente hipertensos (SHR). No entanto, a influência de ciclo de jejum/Realimentação é desconhecida. Objetivo: Investigar o efeito de ciclos de jejum/realimentação sobre a remodelação e função miocárdica. Além disso, o presente estudo foi desenhado para avaliar os mecanismos subjacentes à participação do trânsito de cálcio (Ca+2) e sistema beta-adrenérgico. Métodos: Neste estudo, SHR machos de 60 dias de idade foram submetidos a alimento ad libitum (grupo C), 50% de restrição alimentar (grupo R50) ou ciclos de RF (grupo RF) por 90 dias. A remodelação cardíaca foi avaliada por meio da análise ultraestrutural e função do músculo papilar isolado. Adotou-se o nível de significância de 5% (a = 0,05). Resultados: Os ratos do grupo RF apresentaram menor atrofia cardíaca do que os do grupo R50 em relação aos do grupo C. Os ratos do grupo C aumentaram peso corporal, os ratos do grupo R50 mantiveram seu peso corporal inicial e os ratos do grupo RF aumentaram e reduziram seu peso durante o ciclo RF. O ciclo RF não causou comprometimento funcional, pois os parâmetros isotônicos e isométricos apresentaram comportamento similar aos dos ratos do grupo C. Os parâmetros cardíacos isotônicos e isométricos mostraram-se significativamente elevados nos ratos do grupo RF em comparação aos dos ratos do grupo R50. Além disso, os ratos do grupo R50 apresentaram dano cardíaco em comparação aos ratos do grupo C quanto às variáveis isotônicas e isométricas. Os ratos do grupo R50 apresentaram alterações focais em muitas fibras musculares, enquanto os ratos do grupo RF apresentaram leves alterações, como perda ou desorganização de miofibrilas. Conclusão: Ciclos de Jejum/Realimentação promovem efeitos benéficos cardíacos e atenuam o dano miocárdico causado por restrição calórica em SHR, contribuindo para reduzir o risco cardiovascular e os danos morfológicos. Além disso, o ciclo de jejum/realimentação promove leve melhora do trânsito do Ca2+ e do sistema beta-adrenérgico.

Fasting/Refeeding Cycles Prevent Myocardial Dysfunction and Morphology Damage in the Spontaneously Hypertensive Rats.

BACKGROUND: Caloric restriction is known to impair the cardiac function and morphology in hypertrophied hearts of spontaneously hypertensive rats (SHR); however, the influence of fasting/refeeding (RF) is unknown. OBJECTIVE: To investigate the fasting/refeeding approach on myocardial remodeling and function. In addition, the current study was designed to bring information regarding the mechanisms underlying the participation of Ca2+ handling and b-adrenergic system. METHODS: Sixty-day-old male SHR rats were submitted to food ad libitum (C), 50% food restriction (R50) or RF cycles for 90 days. Cardiac remodeling was assessed by ultrastructure analysis and isolated papillary muscle function. The level of significance considered was 5% (a = 0.05). RESULTS: The RF rats presented lower cardiac atrophy than R50 in relation to C rats. The C rats increased weight gain, R50 maintained their initial body weight and RF rats increased and decreased weight during RF. The RF did not cause functional impairment because the isotonic and isometric parameters showed similar behavior to those of C. The isotonic and isometric cardiac parameters were significantly elevated in RF rats compared to R50 rats. In addition, the R50 rats had cardiac damage in relation to C for isotonic and isometric variables. While the R50 rats showed focal changes in many muscle fibers, the RF rats displayed mild alterations, such as loss or disorganization of myofibrils. CONCLUSION: Fasting/refeeding promotes cardiac beneficial effects and attenuates myocardial injury caused by caloric restriction in SHR rats, contributing to reduce the cardiovascular risk profile and morphological injuries. Furthermore, RF promotes mild improvement in Ca2+ handling and b-adrenergic system.

Simultaneous determination of isoproterenol, acetaminophen, folic acid, propranolol and caffeine using a sensor platform based on carbon black, graphene oxide, copper nanoparticles and PEDOT:PSS.

We explored the use of carbon black (CB), graphene oxide (GO), copper nanoparticles (CuNPs) and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) as electrode materials for the simultaneous determination of isoproterenol, acetaminophen, folic acid, propranolol and caffeine. The designed nanostructured surface was widely characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), contact angle measurements and electrochemistry. From electrochemical characterization assays carried out towards the potassium ferricyanide redox probe, fast electron transfer kinetics and a considerably higher electroactive surface area were observed for the modified electrodic surface based on CB, GO, CuNPs and PEDOT:PSS film. Using square-wave voltammetry (SWV), well defined and resolved anodic peaks were detected for isoproterenol, acetaminophen, folic acid, propranolol and caffeine, with peak-to-peak potential separation not less than 170 mV. Then, the SWV technique was explored for the simultaneous determination of quinary mixtures of these analytes, resulting in analytical curves with linear ranges and limits of detection at micromolar concentration levels. The practical viability of the proposed voltammetric sensor was illustrated in the analysis of human body fluid samples. The proposed sensor showed good repeatability and a successful application using urine and serum matrices, with recoveries close to 100%.

Hydrogen bonding recognition and colorimetric detection of isoprenaline using 2-amino-5-mercapto-1,3,4-thiadiazol functionalized gold nanoparticles.

In this paper, we describe a rapid, low-cost and highly sensitive colorimetric method for the detection of isoprenaline, based on 2-amino-5-mercapto-1,3,4-thiadiazol (AMTD) functionalized gold nanoparticles (AMTD-AuNPs) as a sensing element. Hydrogen bonding interaction between isoprenaline and AMTD resulted in the aggregation of AuNPs and a consequent color change of AuNPs from red to blue. The concentration of isoprenaline could be detected with the naked eye or a UV-visible spectrometer. Results showed that the absorbance ratio (A650/A524) was linear with isoprenaline concentrations in the range of 0.2 to 2.6µM (R=0.997). The detection limit of this method was 0.08µM. The proposed method is simple, without using complicated instruments and adding salts for enhancing sensitivity. This probe could be successfully applied to the determination of isoprenaline in human serum samples and urine samples after deproteinization.

High sensitive and selective HPTLC method assisted by digital image processing for simultaneous determination of catecholamines and related drugs.

A highly sensitive and selective thin layer chromatographic (TLC) method was developed for simultaneous determination of catecholamines and their related drugs using a new detection method and digital image processing of chromatographic plates. For the quantitative evaluation of the investigated compounds, the chromatographic separation was followed by spraying the plate with 0.02% solution of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in ethanol. The BioDit Thin Layer Chromatography (TLC) Scanner device and advanced specific software (ImageDecipher-TLC, Sorbfil TLC Videodensitometer and JustTLC) were used for the detection and quantification of chromatographic spots. For an accurate determination, the RGB colored images of the bright-white spots detected against a purple background were inverted and processed after their conversion into green scale. The results showed a strongly linear correlation between area (R(2)>0.99) and volume (R(2)>0.99) of spots and concentration of investigated compounds in all cases. The limit of detection (LOD) and the limit of quantification (LOQ) were below 49.3 ng/spot and 69.6 ng/spot respectively in all cases. The evaluation of the method was performed using different pharmaceutical samples spiked with the investigated amines and validated with respect to accuracy and precision.

Fast capillary electrophoresis-time-of-flight mass spectrometry using capillaries with inner diameters ranging from 75 to 5 µm.

Fast electrophoretic separations in fused silica capillaries (CE) coupled to time-of-flight mass spectrometry (TOF-MS) are presented. CE separations of the model analytes (epinephrine, norepinephrine, dopamine, histidine, and isoproterenol) under conditions of high electric field strengths of up to 1.25 kV cm(-1) are completed in 20 s. Coupling of CE with TOF-MS is accomplished using a coaxial sheath liquid electrospray ionization interface. The influence of parameters inherent to the interface and their effects, including suction pressure and dilution, are discussed. In addition to standard capillaries of 75 and 50 µm inner diameter (ID), separations in capillaries with IDs of 25, 15, and 5 µm have been successfully applied to this setup. The analytical performance is compared over this range of capillary dimensions, and both advantages and disadvantages are discussed.

Voltammetric determination of isoproterenol using multiwall carbon nanotubes-ionic liquid paste electrode.

A sensitive and selective electrochemical method for the determination of isoproterenol (ISPT) was developed using multiwall carbon nanotubes and a room temperature ionic liquid (i.e. 1-butyl-3-methylimidazolium hexafluoro phosphate, ([C4mim]-[PF6])). This multiwall carbon nanotubes ionic liquid electrode (MWCNTILEE) is a very good alternative to previously described electrodes because the electrocatalytic effect is achieved without any electrode modification. The oxidation peak potentials in cyclic voltammogram of ISPT on MWCNTILEE was occurred around 470 mV vs Ag/AgCl (at pH 6.0) while this peak potential at carbon paste electrode was appeared around 605 mV at the same scan rate of 100 mV s⁻¹. The electrochemical parameters such as diffusion coefficient and charge transfer resistance were determined using cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimized conditions, the peak current was linear to ISPT concentration over the concentration range of 1.0 to 520 µmol L⁻¹ using differential pulse voltammetry. The detection limit was 0.85 µmol L⁻¹. The proposed method was successfully applied to the determination of ISPT in both ampoules and urine samples.

Determination of isoproterenol and uric acid by voltammetric method using carbon nanotubes paste electrode and p-chloranil.

An electrochemical method is described for the voltammetric determination of isoproterenol in the presence of uric acid using p-chloranil-carbon nanotubes paste electrode. Cyclic voltammetric, chronoamperometric, and electrochemical impedance spectroscopic methods have used to investigate the suitability of p-chloranil as a mediator for the electrocatalytic oxidation of isoproterenol at pH=10.5. Using differential pulse voltammetry, isoproterenol (ISPT) and uric acid (UA) in a mixture can be measured simultaneously and individually with a potential difference of 360 mV. The electrocatalytic currents increase linearly with ISPT and UA concentrations over the concentration ranges of 0.015-100 µmol L(-1) ISPT and 3.0-310 µmol L(-1) UA. The detection limits for ISPT and UA were equal to 0.009 and 2.3 µmol L(-1), respectively. The diffusion coefficient and the kinetic parameters such as electron transfer coefficient and heterogeneous rate constant were determined for ISPT, using the electrochemical approaches. The proposed method was successfully applied to the determination of isoproterenol in both ampoule and urine samples.

[Determination of three adrenergic drugs using capillary electrophoresis with amperometric detection].

A method for the determination of three adrenergic drugs, including phenylephrine hydrochloride (PHE), metaraminol bitartrate (MR) and isoprenaline hydrochloride (IP), was developed using capillary electrophoresis with amperometric detection. The detection potential of working electrode was 0.950 V versus the reference electrode of Ag/AgCl. At the applied voltage of 18 kV, the three analytes were completely separated within 18 min in 50 mmol/L borate buffer (pH 10.00) with the injection time of 10 s. Good linear relationships were obtained for all the three analytes in the range of 2-100 micromol/L. The detection limits for PHE, MR and IP were 0.8, 0.8 and 1.0 micromol/L, respectively. The proposed method was applied to the analysis of some injection drugs, and the results were satisfactory.

[Expression and implication of angiotensin II type 1 receptor in myocardial fibrosis of rats].

OBJECTIVE: To investigate the expression and pathobiological implications of angiotensin II type 1 receptor (AT1R) in development of myocardial fibrosis of rats. METHODS: Rat myocardial necrosis model was established using isoproterenol injection (15 mg/kg). Rat serum aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzymes (CK-MB) were detected by MD-100 automatic biochemical analyzer. Masson staining was used to evaluate the morphological changes. The expression of AT1R protein was determined by immunohistochemistry and its mRNA expression was analyzed by RT-PCR. The expression of collage type I and III was determined by immunohistochemistry. RESULTS: Serum LDH, CK and CK-MB reached their peaks at 4 h (chi2 = 16.90, P < 0.05), and AST achieved its peak in 6 h (chi2 = 16.90, P < 0.05). AT1R mRNA expression was increased 2 - 12 h after isoproterenol injection, but no statistical significance (P > 0.05) was observed comparing with the control. However, a significant AT1R mRNA increase was present at 24 h and decreased gradually after 48 h, and back to the control level after 3 weeks. Protein expression of AT1R increased proportionally with the severity of the fibrosis. CONCLUSIONS: AT1R mRNA and protein expressions increase significantly during myocardial ischemia, and is closely correlated with the fibrosis. These findings indicate that AT1R may play an important role in the pathogenesis of myocardial fibrosis.

Sensitive determination of norepinephrine, synephrine, and isoproterenol by capillary electrophoresis with indirect electrochemiluminescence detection.

A new and sensitive method for the determination of norepinephrine (NE), synephrine, and isoproterenol was developed by CE separation and indirect electrochemiluminescence detection (ECL) based on their quenching effects on the tris(2,2'-bipyridyl)-ruthenium(II)/tripropylamine (TPA) system. The conditions for CE separation and ECL detection were investigated in detail. Under the optimum conditions, the three analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 2.6 x 10(-8) mol/L for NE, 6.6 x 10(-9) mol/L for synephrine and 8.4 x 10(-8) mol/L for isoproterenol, respectively. The precisions of intraday and interday are less than 4.4 and 6.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 2.6 x 10(-7) mol/L for NE, 8.8 x 10(-8 ) mol/L for synephrine, and 8.8 x 10(-7) mol/L for isoproterenol, respectively. The applicability of the proposed method was illustrated in the determination of 20 human urine samples from diabetic patients and healthy persons. The results obtained indicated that the level of NE in patients (mean value 0.41 micromol/L) was higher than that in healthy persons (mean value 0.24 micromol/L).

Determination of salbutamol enantiomers in human urine using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin in nonaqueous capillary electrophoresis.

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the resolution and the determination of salbutamol enantiomers in urine samples using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD). After optimization of the electrophoretic parameters, namely the background electrolyte (BGE) composition and the HDAS-beta-CD concentration, salbutamol enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 15 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. Isoprenaline was selected as internal standard. Solid-phase extraction (SPE) was used for sample cleanup prior to the CE separation. Different sorbents involving polar, nonpolar interactions or dual retention mechanisms were evaluated and extraction cartridges containing both nonpolar and strong cation-exchange functionalities were finally selected. Salbutamol enantiomers recoveries from urine samples were determined. The method was then successfully validated using a new approach based on accuracy profiles over a concentration range from 375 to 7500 ng/mL for each enantiomer.

Chiral analysis of neurotransmitters using cyclodextrin-modified capillary electrophoresis equipped with microfabricated interdigitated electrodes.

We present cyclodextrin-modified capillary electrophoresis equipped with a microfabricated chip consisting of an array of eight interdigitated microband platinum electrodes (IDs) for simultaneous analysis of three chiral models: epinephrine, norepinephrine and isoproterenol. The IDE chip, positioned very close to the capillary outlet, served as an amplification/detection system. Emerging neurotransmitters at the IDE surface were oxidized at +1.1 V by seven electrodes of the array and then detected by the remaining electrode, poised at +0.0 V. There was an amplification effect on the detecting electrode owing to the recycle between the reduced and oxidized forms of the optical isomers at the electrode surface. The detecting "amplification" current response was governed by the applied potential, the detecting electrode position, the number of adjacent electrodes used for recycling and the distance between the oxidative and reductive electrodes. The six chiral forms of the three neurotransmitters were resolved using 25 mM heptakis(2,6,di-o-methyl)-beta-cyclodextrin with a detection limit of approximately 5 microM. The scheme detected a reduced compound at a reducing potential instead of conventional oxidation detection to alleviate electrode fouling and electroactive interferences. The concurrent oxidation/reduction detection of compounds also facilitated and ascertained peak identification as interfering compounds were unlikely to have the same oxidative/reductive characteristics and mobilities as the analytes of interrogation.

Deviceless decoupled electrochemical detection of catecholamines in capillary electrophoresis using gold microband array electrodes.

Samples containing microM concentrations of dopamine, (+/-)-isoproterenol, para-aminophenol and chlorogenic acid have been separated by capillary electrophoresis (CE) and detected using end-column amperometric detection based on a novel decoupling method. The present decoupling approach involves the use of an electrochemical detector chip containing an array of microband electrodes where the working and reference electrodes are positioned only 10 microm from each other. The short distance between the working and reference electrodes ensures that both electrodes are very similarly affected by the presence of the CE electric field. With this method, no shift in the detection potential was seen when the CE high voltage was applied. This eliminated the need for a reoptimization of the detection potential to compensate for the influence of the separation voltage on the detection. It is also demonstrated that catecholamines can be detected using gold microband electrodes by careful adjustment of the detection potential to avoid the formation of gold oxide. Such careful adjustments of the detection potential are straightforward using the present decoupling method.

Electrospray tandem mass spectrometry of aminochromes.

The catecholamines adrenaline, noradrenaline, dopamine, dopa and isoprenaline were oxidized into their respective aminochromes: adrenochrome, noradrenochrome, dopaminochrome, dopachrome and isoprenochrome. Tandem mass spectrometry (MS/MS) fragmentation patterns were examined for the five aminochromes in order to establish a general structural assignment of these oxidation products by electrospray mass spectrometry. Although protonated aminochromes undergo similar fragmentation patterns with a characteristic consecutive loss of two carbonyl groups, the presence of different substituents in the parent compounds led to significant changes in the CID spectra. This feature is more evident for isoprenochrome and dopachrome, especially for the latter where the MS/MS spectrum is dominated by the loss of formic acid. A general pattern of fragmentation for aminochromes is proposed, which should provide a suitable basis to aid their characterization in studies in vivo or in vitro.