High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization

Ursodeoxycholic acid (3, 7β-dihydroxy-5β-cholanoic acid, UDCA) is a therapeutically applicable bile acid widely used for the dissolution of cholesterol-rich gallstones and in the treatment of chronic liver diseases associated with cholestasis. UDCA is more hydrophilic and less toxic than another therapeutically valuable bile acid, chenodeoxycholic acid (CDCA), the 7-epimer of UDCA. Procedures for sample preparation and HPLC determination of UDCA in blood serum were developed and validated. A higher homologue of UDCA containing an additional methylene group in the side chain was synthetized and used as an internal standard (IS). Serum samples with IS were diluted with a buffer (pH=7). The bile acids and IS were captured using solid phase extraction (C18 cartridges). The carboxylic group of the analytes was derivatized using 2-bromo-2′-acetonaphthone (a detection-oriented derivatization), and reaction mixtures were analyzed (HPLC with UV 245 nm detection; a 125–4 mm column containing Lichrospher 100 C18, 5 μm; mobile phase: acetonitrile–water, 6:4 (v/v)). Following validation, this method was used for pharmacokinetic studies of UDCA in humans.     

薄层扫描法测定熊胆引流物中胆汁酸含量

熊胆向以贵重药材闻名,被称之为稀有药品,为开发熊胆资源,解决熊胆奇缺问题,我校解剖教研室已成功地完成了人工引流熊胆汁技术,可随时进行人工引流获取熊胆汁。为了确定胆汁的质量指标,了解其主要成分,我们进行了引流胆汁与天然熊胆的分析。文献报道,熊胆中主要含熊去氧胆酸(ursodesoxycholic acid,UDCA)、鹅去氧胆酸(cheno desoxycholic acid,CDCA)、胆酸(cholic acid,CA)、去氧胆酸(deoxycholic,acid DCA)等。     

Effect of bile acids on absorption of nitrendipine in healthy subjects

AIMS: To examine whether bile acids such as ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) can influence the absorption of nitrendipine, a highly lipophilic calcium channel blocker. METHODS: Six healthy subjects received nitrendipine (10 mg) with and without UDCA (50 mg) and CDCA (200 and 600 mg) with an interval of 1 approximately 2 weeks between study phases. RESULTS: Bile acids decreased the Cmax (ng ml(-1)) [control 10.9 +/- 5.8 (mean+/- s.d.), UDCA 5.0 +/- 4.7 (95% confidence interval for difference; 3.9, 7.8, P = 0.0006), CDCA (600 mg) 5.0 +/- 3.9 (2.6, 9.2, P = 0.0059)] and AUC (ng ml(-1) h) [(control; 60 +/- 36, UDCA 15 +/- 13 (20, 73, P = 0.0064), CDCA (600 mg) 19 +/- 19 (21, 63, P = 0.0038)] of nitrendipine, while elimination half-life remained unchanged. CONCLUSIONS: These results suggest that the amount of nitrendipine absorbed was decreased when the drug was administered with UDCA and CDCA.     

Interactions of combined bile acids on hepatocyte viability: cytoprotection or synergism

Cholestasis results from hepatocyte dysfunction due to the accumulation of bile acids in the cell, many of which are known to be cytotoxic. Recent evidence implicates competitive antagonism of key cytotoxic responses as the mechanism by which certain therapeutic bile acids might afford cytoprotection against cholestasis. In this work, we compare the relative cytotoxicity of bile acids in terms of dose- and time-dependence. To better elucidate the controversy related to the therapeutic use of ursodeoxycholate (UDCA) in cholestatic patients, we also evaluated the effects of bile acid combinations. Viability of Wistar rat hepatocytes in primary culture was measured by LDH leakage after 12 and 24 h exposure of cells to the various bile acids. All unconjugated bile acids caused a dose-dependent decrease in cell viability. The tauro- and glyco-conjugates of chenodeoxycholate (CDCA) and UDCA were all less toxic than the corresponding unconjugated form. Although relatively non-toxic, UDCA caused synergistic cell killing by lithocholate (LCA), CDCA, glyco-CDCA (GCDC) and tauro-CDCA (TCDC). Glycoursodeoxycholate decreased the toxicity of GCDC, but potentiated the toxicity of unconjugated CDCA and LCA. The tauro-conjugate of UDCA had no significant effect. These data suggest that at cholestatic concentrations, bile acid-induced cell death correlates with the degree of lipophilicity of individual bile acids. However, these results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with UDCA is not due to a direct effect of UDCA on hepatocyte viability. Therefore, any therapeutic effect of UDCA must be secondary to some other process, such as altered membrane transport or nonparenchymal cell function.     

A practical guide to the nonsurgical treatment of gallstones.

Until recently, cholecystectomy was the only treatment available for symptomatic gallstone disease. During the past 20 years, better understanding of the pathogenesis of cholesterol gallstone disease has led to alternative nonsurgical methods for treating gallstones in selected groups of patients. Use of 2 naturally occurring bile acids, chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), was reported in 1972 and 1975, respectively, for successful dissolution of cholesterol gallstones in humans. Both these bile acids act by reducing cholesterol secretion in bile, thus enabling it to solubilise more cholesterol from the stone surface. Micellar solubilisation is involved, together with liquid crystal formation in the case of UDCA. Having been extensively studied in clinical trials to assess efficacy and safety, both these compounds are now available for general use. The efficacy of CDCA can be enhanced by single bedtime dose administration and by taking a low cholesterol diet. Bedtime administration also enhances the effect of a suboptimal dose of UDCA. CDCA induces dose-related diarrhoea and hypertransaminaemia, and UDCA can induce calcification of gallstones, thus rendering them resistant to medical dissolution. A combination of the 2 bile acids at half the recommended dose for each has become an accepted practice for reducing adverse effects, and this may also enhance efficacy. One of the main problems of bile acid therapy is that dissolution of gallstones is a very slow process. Use of extracorporeal shockwave lithotripsy (ESWL) to break the stones into smaller fragments, with concurrent use of bile acids, has been shown to speed dissolution rate and to achieve complete gallstone dissolution in 78% of selected cases within 12 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibacterial and anti-atrophic effects of a highly soluble, acid stable UDCA formula in Helicobacter pylori-induced gastritis

Helicobacter pylori is one of the main causes of atrophic gastritis and gastric carcinogenesis. Gastritis can also occur in the absence of H. pylori as a result of bile reflux suggesting the eradication of H. pylori by bile acids. However, the bile salts are unable to eradicate H. pylori due to their low solubility and instability at acidic pH. This study examined the effect of a highly soluble and acid stable ursodeoxycholic acid (UDCA) formula on H. pylori-induced atrophic gastritis. The H. pylori infection decreased the body weight, mitochondrial membrane potential and ATP level in vivo. Surprisingly, H. pylori-induced expression of malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, at both the protein and mRNA levels. However, the UDCA formula repressed MDH expression and increased the membrane potential thereby increasing the ATP level and body weight in vivo. Moreover, UDCA scavenged the reactive oxygen species (ROS), increased the membrane potential, and inhibited apoptosis in AGS cells exposed to H₂O₂ in vitro through the mitochondria-mediated pathway. Taken together, UDCA decreases the MDH and ROS levels, which can prevent apoptosis in H. pylori-induced gastritis.     

Role of mitochondrial dysfunction in combined bile acid-induced cytotoxicity: the switch between apoptosis and necrosis.

The goal of this investigation was to determine whether chenodeoxycholic acid (CDCA)-induced apoptosis is prevented by ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDC) and to characterize the involvement of mitochondria in the process. Cultured human HepG2 cells were treated in a dose- and time-dependent protocol in order to establish a sufficiently low exposure to CDCA that causes apoptosis but not necrosis. Low-dose CDCA induced an S-phase block and G2 arrest of the cell cycle, as determined by flow cytometry. As a result, cell proliferation was inhibited. CDCA-induced apoptosis, as determined by fluorescence microscopy of Hoechst 33342-stained nuclei, was evident upon coincubation with TUDC. Additionally, after exposure to UDCA plus CDCA, the cell membrane was permeable to fluorescent dyes. Caspase-9-like activity, poly(ADP-ribose) polymerase (PARP) cleavage, and extensive DNA fragmentation were detected in CDCA-exposed cells and in cells coincubated with TUDC, but not UDCA. CDCA caused a decrease in mitochondrial membrane potential and depletion of ATP, both of which were potentiated by UDCA but not TUDC. The results suggest that UDCA potentiates CDCA cytotoxicity, probably at the level of induction of the mitochondrial permeability transition (MPT). Consequently, as suggested by the lack of the main hallmarks of the apoptotic pathway, in the presence of UDCA, CDCA-induced apoptosis is not properly executed but degenerates into necrosis.     

Effects of fatty acid esters of cheno- and ursodeoxycholic acids on gallstone formation in hamsters

Fatty acid esters (at the 7 position) of chenodeoxycholic (CDCA) and ursodeoxycholic (UDCA) acids have been tested for their effects on formation and dissolution of gallstones in hamsters. The free bile acids were fed at a level of 0.2% of the diet and esters were fed at equimolar levels. The earlier finding that CDCA does not affect gallstone formation in hamsters fed the Dam and Christensen diet were confirmed. The acetic, butyric and lauric acid esters of CDCA had a very slight inhibitory effect on lithogenesis but CDCA 7 oleate and linoleate completely inhibited gallstone formation. UDCA and its 7 oleate inhibited both formation and progression of gallstones. The observed effects are probably a function of the form of the bile acid and not of the esterifying acid. The observation that ethyl oleate has a slight litholytic effect suggests that the acid moiety of the ester may exert a slight influence.     

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated. Male Golden Syrian hamsters were divided into five groups: a control group, two groups that received 0.5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days. After ether anaesthesia the gallbladder was removed and bile was immediately aspirated. Bile acids were determined by high performance liquid chromatography (HPLC). Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups. The glyco/tauro ratio of 0.69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1. UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives. A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives.     

Permeation enhancement of octreotide by specific bile salts in rats and human subjects: in vitro, in vivo correlations.

1. The potential of bile salts to improve the enteral absorption of octreotide, an orally active somatostatin analogue, was investigated by a combination of in vitro, in situ and in vivo experiments. 2. Incorporation of octreotide into lipid monolayers (as measured by area increase of the monolayer at constant surface pressure using a Langmuir-Blodgett trough set-up) depended on the type of bile salt used for monolayer pre-treatment. Addition of 20 microM octreotide to the subphase containing 20 microM of the dihydroxylated bile salt ursodeoxycholate (UDCA) causes a 9% increase in area, whereas addition of octreotide to the subphase containing the 7 alpha-enantiomer of UDCA, chenodeoxycholate (CDCA), resulted in an area increase of the lipid monolayer of 20%. Area increase by octreotide alone was not significantly different from the increase of octreotide and UDCA in combination. 3. CDCA and UDCA in combination with octreotide increased the permeability of liposomal membranes for rubidium ions, whereas octreotide alone did not significantly change the permeability. This indicates membrane distortion as a possible cause for the enhanced absorption of octreotide by bile salts. 4. In polarized Caco-2 cell monolayers octreotide exhibited a permeation coefficient of 0.008 +/- 0.004 cm h-1. Addition of 0.2-1% of UDCA to the apical incubation medium had no significant effect upon the permeation coefficient. In contrast, 0.2-1% CDCA in the incubation medium resulted in a significant increase (P < 0.05) of the monolayer permeability of octreotide (0.015-0.037 cm h-1). 5. Octreotide was absorbed as the intact peptide from the gastrointestinal tract in rats with an absorption efficiency of 0.26%. Coadministration of bile salt resulted in a dose-dependent increase in absorption efficiency of the peptide up to 20.2%. The observed effect was more pronounced for CDCA than for UDCA. 6. The effect of CDCA and UDCA on octreotide absorption in vivo was assessed in a pharmacokinetic study with healthy volunteers. After oral administration of 4 mg octreotide in the presence of 100 mg bile salt, an average bioavailability of the peptide of 1.26% was achieved in the presence of CDCA, whereas in the presence of UDCA a bioavailability of only 0.13% was reached. This difference was statistically significant (P < 0.01). 7. In conclusion, the co-administration of CDCA is able to enhance the enteral absorption of octreotide. The in vitro and in situ experiments were predictive for the observed effect in human subjects.     

鹅去氧胆酸对胆汁淤积孕鼠血生化指标、肝脏病理及胎鼠预后的影响

目的以熊去氧胆酸(UDCA)为对比,研究法尼醇X受体(FXR)激动剂——鹅去氧胆酸(CDCA)对妊娠期肝内胆汁淤积(ICP)孕鼠血生化指标、肝脏病理及胎鼠预后的影响。方法建立ICP孕鼠模型,将孕17d的SD大鼠40只随机分成对照组、ICP组、UDCA组和CDCA组。比色法检测用药前后各组丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST),碱性磷酸酶(ALP)和总胆酸(TBA)水平,光镜下观察孕鼠肝脏病理学变化,记录胎鼠身长、尾长、体重及死胎数。结果ICP组ALT、AST、ALP、TBA明显高于对照组(P<0.008 3);与ICP组比较,UDCA组ALT、AST、ALP明显降低(P<0.008 3),但TBA无明显改善(P>0.008 3);CDCA组ALT、AST、ALP无明显改善(P>0.008 3),但TBA显著降低(P<0.008 3);2治疗组胎鼠的身长、尾长、体重及死亡率与ICP组比较均无统计学差异(P>0.008 3)。光镜下见ICP组肝细胞肿胀变性,肝血窦变窄,胆管扩张;UDCA组肝细胞脂肪变性明显,小叶结构正常;CDCA组小部分肝细胞脂肪变性,小叶结构正常。结论UDCA能改善ICP孕鼠肝功能指标,但不能有效降低血胆汁酸水平及胎鼠死亡率;CDCA能明显降低胆汁酸,促进胆汁酸代谢,但其安全性还有待确定。     

High-throughput multi-analyte screening for renal disease using capillary electrophoresis

End-state renal disease (ESRD) affects 300 000 people in the United States each year. A large percentage of these individuals (20%) die within the first year after diagnosis. Current methods of determining renal function rely on the measurement of a single marker using slow and frequently non-specific colorimetric methods. In this report, capillary zone electrophoresis was used to perform a multi-analyte assay for markers of renal function in urine. This method tested for creatinine (Cr), creatine (Cn), uric acid (UA), and p-aminohippuric acid (PAH) levels. The limits of detection (S/N=3) were found to be 5 μM for Cr, 0.75 μM for Cn, and 1.5 μM for UA and PAH. Linear ranges were determined to be 5–500 μM for Cr, 0.75–500 μM for Cn, and 1.5–250 μM for UA and PAH. These ranges included the expected concentrations of the markers in human urine after 50-fold dilution. This screening method proved to be a simple and fast way to perform a high throughput analysis for multiple renal function indicators.     

CDCA8通过调控增殖促进前列腺癌发生的作用机制研究

目的 探讨细胞分裂周期相关蛋白8（CDCA8）在前列腺癌（PCa）中的表达及作用机制。方法 通过生物信息学方法分析正常前列腺组织和PCa组织中CDCA8 mRNA水平差异。利用癌症基因组图谱（TCGA）数据库中RNA表达测序数据分析CDCA8表达相关的PCa患者无病生存期（DFS）。免疫组织化学方法检测根治性前列腺切除...     

Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring (S)-glutamic acid ((S)-Glu) receptors. We report here the separation of (+)- and (−)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and >99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of (S)-(+)-ATPA and (R)-(−)-ATPA. (S)- and (R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6+ KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, (S)-ATPA showed AMPA receptor agonist effects (EC₅₀=23 μM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (K_i=253 μM) and by (S)-ATPA (K_i=376 μM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (K_i=301 μM and 1115 μM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC₅₀ values of approximately 8 μM and at GluR1 (EC₅₀=22 μM), producing maximal steady state currents only 5.4–33% of those evoked by kainic acid. (R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with K_i values of 33–75 μM. (S)-ATPA produced potent agonist effects at GluR5 (EC₅₀=0.48 μM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, (S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by (S)-ATPA and kainic acid were not significantly different. (R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither (S)- nor (R)-ATPA showed significant effects at GluR6. (S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6+KA2 receptors, whereas (R)-ATPA was inactive. Thus, (S)- and (R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA (S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.     

Voltammetric determination of isoxsuprine and fenoterol in dosage forms and biological fluids through nitrosation

A simple and highly sensitive voltammetric method was developed for the determination of isoxsuprine HCl (I) and fenoterol HBr (II) in dosage forms and biological fluids. The method is based on treatment of the two compounds with nitrous acid followed by measuring the cathodic current produced by the resulting nitroso derivatives. The voltammetric behavior was studied adopting Direct Current (DC_t), Differential Pulse (DPP) and Alternating Current (AC_t) polarography. Both compounds produced well-defined, diffusion-controlled cathodic waves over the whole pH range in Britton–Robinson buffers (BRb). At pH 11 and pH 9, the values of diffusion-current constants (Id), were 9.4±0.3 and 7.7±0.4 for I and II, respectively. The current–concentration plots for I were rectilinear over the range of 0.6–12 μg/ml and 0.1–12 μg/ml in the DC_t and DPP modes, respectively. As for II, the range was 1–20 μg/ml and 0.1–20 μg/ml in the DC_t and DPP modes, respectively. The minimum detectability (S/N=2) were 0.02 μg/ml (≈6×10⁻⁸ M) and 0.01 μg/ml (≈2.6×10⁻⁸ M) for I and II, respectively, adopting the DPP mode. The proposed method was applied to the determination of both compounds in dosage forms and the results obtained were in good agreement with those obtained using reference methods. The proposed method was further applied to the determination of isoxsuprine in spiked human urine and plasma. The percentage recoveries adopting the DPP mode were 98.84±1.18 and 99.26±0.97, respectively.     

The Influence of 2-Hydroxypropyl-β-cyclodextrin on the Haemolysis Induced by Bile Acids

Cyclodextrins improve the water-solubility of drugs and can mask their haemolytic effect in parenteral use. Because the mechanism by which bile acids induce haemolysis is poorly understood, it has been investigated in the presence of 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD). The haemolytic effect of 1.8 mm solutions of cholic acid, chenodeoxycholic acid (CDCA), deoxycholic acid and ursodeoxycholic acid (UDCA) in isotonic buffer at pH 7.4 was investigated at 37°C in the presence of HP-β-CyD at concentrations from 0.18 to 32 mm . No haemolytic effect was evident for cholic acid and UDCA. The haemolytic effect of the other bile acids was reduced by addition of HP-β-CyD and was prevented at a molar ratio of 1:1 owing to complex formation. An HP-β-CyD:bile acid molar ratio greater than 5:1 had a different effect on the erythrocyte membrane, irrespective of the identity of the bile acid; the effect was in accordance with the complexion affinities. In the absence of HP-β-CyD, the haemolytic effect of CDCA and deoxycholic acid appeared related to their capacity to form a surface monolayer and to solubilize the components of the erythrocyte membrane. The haemolytic effect observed after complexation of the bile acids appeared to be solely the effect of HP-β-CyD, which was able to form a reversible inclusion complex with lipophilic components of the erythrocyte membranes at concentrations higher than 12 mm .