Acid In Rat Serum Research Articles

Mechanism of intestinal flora affecting SLC2A9 transport function to promote the formation of hyperuricemia

ObjectiveTo investigate the structural characteristics of the intestinal flora in obese-hyperuricemic (HUA-W) patients and the mechanisms by which they promote the formation of hyperuricemia. Methods120 human fecal samples (60 cases in HC, 30 cases in HUA-N, and 30 cases in HUA-W) and 40 cases in the colonic tissues (20 cases in HC, 10 cases in HUA-N, and 10 cases in HUA-W) were collected. The intestinal flora of faeces was detected by 16s rRNA method; and the expression of SLC2A9 on human colon tissues was detected by RT-qPCR method and immunofluorescence method. 40 obese-hyperuricemia rat models were established (10 rats in Model, 10 rats in HC-FT, 10 rats in HUA-N-FT, and 10 rats in HUA-W-FT), and 10 rats were set up in Control; and the level of uric acid in rat serum, the levels of xanthine oxidase (XOD) activity and uric acid in intestinal fluid were examined. SLC2A9+ Caco-2 cells were produced to simulate the Transwell uric acid transport model, and the Caco-2 cells and SLC2A9+ Caco-2 cells were grown in five different culture media (Blank, Germ-free, HC-germ, HUA-N-germ and HUA-W-germ), and the uric acid levels in the upper and lower layers of the chambers were detected. ResultsThe HUA-W intestinal flora showed significant specificity, with a decrease in Bacteroidota and Bacteroidia and an increase in Escherichia and Ruminococcus. There were no significant differences in the fluorescence intensity of the SLC2A9 protein and the SLC2A9 mRNA levels in the colon tissues of the HUA-N and HUA-W (P = 0.447, P = 0.152, P = 0.4799 and P = 0.965, respectively). In rat animal experiments, uric acid levels were significantly higher (P < 0.05) and XOD activity was significantly higher (P < 0.05) in intestinal fluid of HUA-W-FT. In Transwell experiments with SLC2A9+ Caco-2 cells, uric acid levels were increased in the upper compartment and decreased in the lower compartment of HUA-W-germ. ConclusionHUA-W intestinal flora may increase XOD activity in the intestinal tract and improve the ability of uric acid transporter protein SLC2A9 to reabsorb uric acid, providing a new theoretical basis for the pathogenesis of hyperuricemia.

Simultaneous determination of cis- and trans-palmitoleic acid in rat serum by UPLC–MS/MS

Palmitoleic acid, a monounsaturated fatty acid which could affect glucose and lipid metabolism and reduce insulin resistance has two isomers, i.e. cis-palmitoleic acid (cPOA) and trans-palmitoleic acid (tPOA). However, the pharmacokinetic, metabolic transformation and structure–activity relationship of the two isomers have not been reported. A precise and accurate ultra performance liquid chromatography–tandem mass spectroscopy (UPLC–MS/MS) method was developed to determine cPOA and tPOA simultaneously. Both the cPOA and tPOA were administered i.g. (intragastric gavage) to rats at 75 mg/kg. Serum samples were collected and analyzed for the two isomers by UPLC–MS/MS on a reverse-phase BDS C18 column equilibrated and eluted with water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The calibration curves for cPOA and tPOA were linear over the range 0.1–12 μg/mL. Analytes were monitored by selected-reaction monitoring in negative electrospray ionization mode. The Tmax of cPOA was 0.94 ± 0.44 h and the Cmax 8.17 ± 1.97 μg/L, and the Tmax and Cmax of tPOA were 1.50 ± 0.98 h and 14.77 ± 11.91 μg/L, respectively. AUC0–24 h of cPOA and tPOA were 59.45 ± 29.83 and 113.88 ± 72.25 mg/L·h. The method was applied in pharmacokinetic study of cPOA and tPOA in rat serum successfully. Besides, the concentrations of cPOA and tPOA in rat serums were observed fluctuating with a consistent trend, which may be due to reciprocal bio-convert in the body.

Effects of Renshenjian Decoction on blood and urine metabonomics in type 2 diabetes rats based on ~1H-NMR

Proton nuclear magnetic resonance(~1H-NMR) is used to investigate the effect of Renshenjian Decoction on serum and urine metabolism of type 2 diabetic rats with insulin resistance induced by high-sugar and high-fat diet combined with low-dose streptozotocin(STZ). After the successful establishment of the insulin resistance model of type 2 diabetes, administration for 35 days, the serum and urine of rats were taken. Once the ~1H-NMR data have been collected and processed, PCA and OPLS-DA were used to analyze them. The results show that: compared with the blank group, the contents of methionine, taurine, α-glucose and β-glucose in the serum of the model group increased significantly(P&lt;0.001), while the contents of 3-hydroxybutyric acid, lactic acid and unsaturated fatty acids decreased significantly(P&lt;0.01). In the model group, the contents of trimethylamine oxide, glycine, α-glucose, β-glucose, taurine and phosphocholine in urine increased significantly(P&lt;0.05), while the contents of creatine, lactic acid, acetic acid and citric acid decreased significantly(P&lt;0.05). Compared with the model group, the contents of 3-hydroxybutyric acid and unsaturated fatty acids in serum of rats in the treatment group increased significantly(P&lt;0.05), while the contents of taurine, α-glucose and β-glucose decreased significantly(P&lt;0.01). In the treatment group, the contents of lactic acid, taurine and creatine in urine increased significantly(P&lt;0.05), while the contents of trimethylamine oxide, glycine, α-glucose, β-glucose and phosphocholine decreased significantly(P&lt;0.01). The results show that Renshenjian Decoction can regulate metabolic disorder and promote the metabolic phenotype to return to the normal range. It displayed therapeutic effect on type 2 diabetic rats with insulin resistance and provided a certain scientific basis for the biological basic research of Renshenjian Decoction by improving insulin resistance in diabetes mellitus.

An improved UPLC method for determining uric acid in rat serum and comparison study with commercial colorimetric kits

Uric acid (UA) is the final product of purine metabolism in humans. Elevated serum UA levels lead to the development of hyperuricemia, gout, kidney diseases, and metabolic syndrome. Accurate determination of UA plays a critical role in clinical diagnosis and laboratory investigation. An ultra-performance liquid chromatography (UPLC) with ultraviolet detection method has been developed and validated for UA analysis. Separation was achieved by a Waters ethylene bridged hybrid (BEH) Amide column (50 mm × 2.1 mm i.d., 1.7 μm) with acetonitrile and 0.1% acetic acid in deionized water in the proportion of 90 to 10 (v/v) as the mobile phase. The limit of detection and limit of quantification were 0.09 and 0.18 μmol/L, respectively. The method was validated by evaluating recovery (98.37–104.20%), accuracy (0.47–0.90%), and precision (1.24–1.81% for intra-batch and 1.76–3.98% for inter-batch). This method was then applied to UA determination in rat serum of hyperuricemia model. The results from UPLC, high-performance liquid chromatography (HPLC), and uric acid kits (phosphor-tungstic acid-based kit and uricase-based kit) were compared. The UPLC results were in very good agreement with HPLC. The developed method could be employed as a useful tool for the determination of UA in biofluids.

The possible mechanism of hydroxytyrosol on reducing uric acid levels

In this study, the inhibitory effect of hydroxytyrosol against xanthine oxidase (XOD) by in vivo animal model and in vitro inhibition assay was analysed. The results showed that hydroxytyrosol could reduce uric acid in rat serum, inhibit XOD activity in rat liver, and adjust the mRNA transcription of the renal uric acid transporters URAT1, GLUT9, OAT1, UAT and ABCG2 towards normal in the transcription level. Hydroxytyrosol was observed as an inhibitor of XOD activity, which’s IC50 value of restraining XOD activity was 8.75 mM. Circular dichroism (CD) spectroscopy indicated that the secondary structures of XOD were changed after incubation with hydroxytyrosol. The docking simulation showed that hydroxytyrosol could enter into the active site of XOD and form hydrogen bonds with amino acid residues (such as ARG880, GLU1261 and THR1010). The results suggested that hydroxytyrosol could be a potential XOD inhibitor for hyperuricaemia treatment.

Metabonomics analysis of serum from rats given long-term and low-level cadmium by ultra-performance liquid chromatography–mass spectrometry

1. This study evaluated the toxicity of chronic exposure to low-level cadmium (Cd) in rats using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS). Forty male Sprague–Dawley rats were randomly assigned to four groups, namely, the control group, low-dose group (0.13 mg/kg·bw), middle-dose group (0.8 mg/kg·bw) and high-dose group (4.89 mg/kg·bw). The rats continuously received CdCl2 via drinking water for 24 weeks. Serum samples were collected for metabonomics analysis. The data generated from the UPLC–MS was analysed using principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). PLS-DA model with satisfactory explanatory and predictive ability is capable of discriminating the treatment groups from the control group.2. Finally, the 10 metabolites were identified and showed significant changes in some treatment groups compared with that in the control group (p < 0.0167 or p < 0.003). Exposure to Cd resulted in increased intensities of lysophosphatidic acid (P-16:0e/0:0), glycocholic acid, bicyclo-prostaglandin E2, lithocholyltaurine, sulfolithocholylglycine, lysophosphatidylethanolamine (20:5/0:0) and lysophosphatidylcholine (20:0), as well as decreased intensities of 3-indolepropionic acid, phosphatidylcholine (18:4/18:0) and 15S-hydroxyeicosatrienoic acid in rat serum.3. Results suggest that exposure to Cd can cause disturbances in the lipid metabolism, amino acid metabolism, nervous system, antioxidant defence system, liver and kidney function.

Inhibitory effect of verbascoside on xanthine oxidase activity

In this study, we analyzed the inhibitory effect of verbascoside against xanthine oxidase (XOD) in vitro by using animal model and in vivo by direct inhibition assay. Results showed that verbascoside could reduce uric acid in rat serum and inhibit XOD activity in rat liver. The IC50 value of restraining XOD activity was 81.11mgmL−1. Fluorescence chromatographic analysis and circular dichroism spectroscopy indicated that the secondary structures of XOD were changed after incubation with verbascoside. The docking simulation showed that verbascoside could enter into the active site of XOD and form hydrogen bonding with amino acid residues (such as Lys-1045, Arg-880, Arg-912, Glu-1261 and Gln-1194). The results suggested that verbascoside, which is a naturally occurring water-soluble antioxidant, could be a potential low-toxicity XOD inhibitor for hyperuricemia treatment.

Effect of Fuzheng Huayu capsule on serum metabolomics in rats with liver fibrosis induced by dimethylnitrosamine

To investigate the effect of Fuzheng Huayu capsule(FHC) on serum metabolomics in rats with liver fibrosis induced by dimethylnitrosamine(DMN). The metabolic profiles of rat serum of normal group, model group, and FHC group were established by liquid chromatography-mass spectrometry technology. Furthermore, the levels of endogenous metabolites such as amino acids and bile acids were measured in each group. The results showed that there were significant differences in the serum metabolic fingerprints between the FHC group and the model group. Moreover, 5 potential lysophosphatidylcholines biomarkers were identified by using principal component analysis(PCA) and partial least squares discriminant analysis (PLS-DA). Quantitative analysis of amino acids and bile acids in serum of rats showed that 14 kinds of amino acids and 5 kinds of bile acids returned to normal levels after four weeks of FHC treatment. In conclusion, the anti-hepatic fibrosis mechanisms of FHC may be related to the metabolic process of lysophosphatidylcholines, amino acids and bile acids.

An evaluation of acute hydrogen sulfide poisoning in rats through serum metabolomics based on gas chromatography-mass spectrometry.

Hydrogen sulfide (H2S) is the second leading cause of toxin-related deaths in the operational site. Its main target organs of toxic effects are the central nervous system and respiratory system. In this study, we developed a serum metabonomic method, based on gas chromatography-mass spectrometry (GC/MS), to evaluate the effect of acute poisoning by hydrogen sulfide on rats. Pattern recognition analysis, including both principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA), revealed that acute hydrogen sulfide poisoning induced metabolic perturbations. Compared to the control group, the level of urea, glucose, glyceryl stearate in rat serum of the poisoning group increased after two hours, and the level of glucose, docosahexaenoic acid, glyceryl stearate and arachidonic acid in rat serum of the poisoning group increased after 48 h, while the L-valine, galactose, L-tyrosine levels decreased. Our results indicate that metabonomic methods based on GC/MS may be useful to elucidate acute hydrogen sulfide poisoning through the exploration of biomarkers.

Enzymatic-HPLC Method to AnalyzeD-3-Hydroxybutyric Acid in Rat Serum

An enzymatic-HPLC method to analyze the serum concentration of D-3-hydroxybutyric acid was developed. A deproteinized sample of rat serum was treated with 3-hydroxybutyrate dehydrogenase in the presence of NAD, and was analyzed by reversed-phase HPLC to separate and quantify NADH formed by the enzyme reaction, monitoring OD at 340 nm. Standard samples containing varying amounts of D-3-hydroxybutyric acid (0-10 nmol in 50 microl) were treated with 3-hydroxybutyrate dehydrogenase and analyzed by HPLC (the injected amount was 0-2.7 nmol of D-3-hydroxybutuyric acid), resulting in the peak area increasing proportionally with the injected amount. The method proved sensitive enough for as little as 0.2-2 nmol D-3-hydroxybutyric acid in 50 microl to be accurately analyzed. Only 10-20 microl of the rat serum protein-free extract is therefore required to obtain a reliable value. The values obtained with this method are identical to those observed by the conventional enzyme-spectrophotometric method. This method can be easily conducted in many laboratories because it is highly sensitive and only requires HPLC apparatus equipped with a UV meter.

Separation/Determination of Flavonoids and Ascorbic Acid in Rat Serum and Excrement by Capillary Electrophoresis with Electrochemical Detection

A capillary electrophoresis with electrochemical detection was developed for the simultaneous determination of three flavonoids (naringenin, rutin, quercetin) and ascorbic acid. It was found that naringenin, rutin, quercetin and ascorbic acid were well separated within 5 min in borate buffer solution (pH 8.6, 24 mM). The detection limit was 1.0 microM for naringenin, 8.0 microM for rutin, 2.0 microM for ascorbic acid and 0.5 microM for quercetin. The protocol was successfully applied for the determination of the analytes in rat serum and excrement. Recovery results ranged from 90.9 to 108.6%.

Effect of Laggera alata on hepatocyte damage induced by carbon tetrachloride in vitro and in vivo

Ethnopharmacological relevance Laggera alata, as a traditional Chinese herbal medicine, has been widely used to ameliorate some ailments associated with inflammation including hepatitis in folk. Aim of the study Based on anti-inflammatory activity of total phenolics from Laggera alata (TPLA), to further validate the remarkable curative effect Laggera alata in hepatitis, hepatoprotective effect of TPLA was examined. Materials and methods TPLA was prepared and its principle components were quantificationally analyzed. The hepatoprotective effects of TPLA were studied using a CCl 4-induced injury model in primary cultured neonatal rat hepatocytes, and a CCl 4-induced acute and chronic damage model in vivo. Results TPLA significantly reduced cellular leakage of hepatocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and improved cell viability in vitro. TPLA markedly decreased the serum AST and ALT levels of the mice, the levels of AST, ALT, total protein, albumin, and sialic acid in rat serum, and the hydroxyproline level in rat liver. Meanwhile, severe hepatic lesions induced by CCl 4 in mice/rats were remarkably improved by the administration of TPLA. Conclusions This investigation verifies the hepatoprotective effect of TPLA in vitro/ in vivo and clarifies its active components dicaffeoylquinic acids responsible for hepatoprotective potential.

Simultaneous determination of geniposide, baicalin, cholic acid and hyodeoxycholic acid in rat serum for the pharmacokinetic investigations by high performance liquid chromatography–tandem mass spectrometry

A simple, rapid, and specific analytical method for simultaneous determination of geniposide, baicalin, cholic acid and hyodeoxycholic acid in 50 microL samples of rat serum was developed by high performance liquid chromatography-tandem mass spectrometry. The quantification of the target compounds was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The correlation coefficients of the calibration curves were better than 0.997. The intra- and inter-day accuracy, precision, and linear range had been investigated in detail. This method was subsequently applied to pharmacokinetic studies of geniposide, baicalin, cholic acid and hyodeoxycholic acid in rats successfully.

Detection of domoic acid in rat serum and brain by direct competitive enzyme-linked immunosorbent assay (cELISA)

In 1987 a large-scale incident of human poisoning in Canada was traced to commercial mussels contaminated with domoic acid (DOM). Since then, routine screening of shellfish domoic acid content has been carried out using a variety of assays, with liquid chromatography using ultraviolet absorbance detection (LC-UV) or mass spectrometric detection (LC-MS) being the currently accepted standard methodologies. Recently, a highly specific competitive enzyme-linked immunosorbent assay (cELISA) has been developed for the detection and analysis of DOM in commercial shellfish, but its accuracy relative to LC methods has not been independently verified in mammalian tissues. In this study we demonstrate that measurement of rat serum DOM concentration by cELISA gives a good correlation (r2 = 0.993) across a broad range of concentrations when compared to LC-MS analysis, with only a small (15%) overestimation of sample DOM content. In addition, we have developed an extraction method for analysis of DOM in rat brain by cELISA which yields complete recovery across a range of sample dilutions.

Homocysteine, a risk factor for atherosclerosis, biphasically changes the endothelial production of kynurenic acid

Increased serum level of homocysteine is an independent risk factor for vascular disease. The effect of dl-homocysteine on the endothelial production of kynurenic acid, an antagonist of α7-nicotinic and N-methyl- d-aspartate (NMDA) glutamate receptors, has been evaluated in vitro and in vivo. In rat aortic rings, dl-homocysteine at 40–100 μM enhanced, whereas at ≥ 400 μM decreased the synthesis of kynurenic acid. S-adenosylhomocysteine mimicked the biphasic action of dl-homocysteine. On the contrary, thiol-containing compounds, l-cysteine and l-methionine, were only inhibiting kynurenic acid production. l-kynurenine uptake blockers, l-phenylalanine and l-leucine, reversed the stimulatory effect of S-adenosylhomocysteine. l-glycine, co-agonist of NMDA receptor, and cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), an antagonist of NMDA receptor, have not influenced kynurenic acid formation. In vivo, dl-homocysteine (1.3 mmol, i.p.) increased the level of kynurenic acid in rat serum from 23.7 ± 7.1 to 60.7 ± 14.2 (15 min, P < 0.01) and 55.7 ± 13.6 (60 min, P < 0.01) pmol/ml, respectively; the endothelial content of kynurenic acid was also increased (51.6 ± 5.8 vs. 73.2 ± 9.4 fmol/μg of protein; 15 min; P < 0.01). dl-homocysteine seems to modulate the production of kynurenic acid both directly and indirectly, possibly following the conversion to S-adenosylhomocysteine. The obtained data suggest a potential contribution of altered formation of kynurenic acid to the endothelial changes induced by hyperhomocysteinemia.

Analysis of n‐acetyl and n‐glycolylneuraminic acid in rat serum and tissues with walker 256 carcinoma by high‐performance liquid chromatography

Analysis of n‐acetyl and n‐glycolylneuraminic acid in rat serum and tissues with walker 256 carcinoma by high‐performance liquid chromatography

Analysis of N-acetyl and N-glycolylneuraminic acid in rat serum and tissues with Walker 256 carcinoma by high-performance liquid chromatography.

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

Analysis of n‐acetyl and n‐glycolylneuraminic acid in rat serum and tissues with walker 256 carcinoma by high‐performance liquid chromatography

Analysis of n‐acetyl and n‐glycolylneuraminic acid in rat serum and tissues with walker 256 carcinoma by high‐performance liquid chromatography

Fluorometric Assay for the Determination of Anthranilic Acid in Biological Materials

In the brain, anthranilic acid may serve as a bioprecursor of the endogenous excitotoxin quinolinic acid. Using a novel isolation procedure followed by HPLC and fluorimetric detection, we have developed an assay which is sufficiently sensitive to determine anthranilic acid in small (≥3 mg) samples of rat brain tissue (sensitivity limit: 50 fmol). Anthranilic acid was identified by its retention time in three chromatographic systems. The assay was applied to the measurement of anthranilic acid in rat serum (131 ± 7 nM) and urine (9.9 ± 118 nmol/mg creatinine) and in several organs which contained between 0.5 and 2 pmol anthranilic acid/mg protein. Only small differences in anthranilic acid content were found among 10 regions of the rat brain. Neuronal depletion induced by an intrastriatal excitotoxin injection resulted in an increase in anthranilic acid levels, suggesting a nonneuronal localization of the metabolite in the brain. This assay should provide an improved means for the investigation of the neurobiology of anthranilic acid.

High-performance liquid chromatographic determination of nalidixic acid in rat serum, brain and cerebrospinal fluid

A high-performance liquid chromatographic method for the determination of nalidixic acid (NA) in rat serum, brain and cerebrospinal fluid (CSF) was developed. NA in rat serum and brain homogenate was extracted and injected onto a reversed-phase column. CSF was directly analysed without extraction procedure. The limits of detection were 0.05 μg ml −1 for serum, 0.07 μg g −1for brain and 0.02 μg ml −1 for CSF, respectively. Calibration curves were linear over the concentration ranges 0.1–50 μg ml −1 for serum, 0.12–9 μg g −1 for brain and 0.05–10 μg ml −1 for CSF, respectively. The reproducibility of NA assay in rat biological media ranged from 1 to 4% relative standard deviations (RSD). The recoveries of NA added to serum and brain were higher than 96% with an RSD of less than 4%. The present method was found to be applicable to pharmacokinetic study of NA in rat serum, brain and CSF.