[Reversal effect and its mechanism of (- )-5-N-acetylardeemin on adriamycin resistance in multidrug-resistant cancer cells A549/Adr and MCF-7/Adr].

OBJECTIVE: To explore the reversal effect of (- )-5-N-acetylardeemin on adriamycin resistance in multidrug-resistant cancer cells including human breast cancer cells MCF-7/Adr and human non-small cell lung cancer cells A549/Adr in vitro. METHODS: The multidrug-resistant cancer cells MCF-7/Adr, A549/Adr and their respective parental cells were treated with different concentrations of (- )-5-N-acetylardeemin and adriamycin individually or in combination. Cell death was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. Intracellular accumulation of adriamycin was measured by the detection of fluorescence intensity of cell lysates using microplate reader. The expression of P-glycoprotein (P-gp) was evaluated by Western blot. RESULTS: (-)-5-N-acetylardeemin significantly reversed the adriamycin resistance in MCF-7/Adr and A549/ Adr in a dose-dependent manner, and the reversal folds were 10. 8 in MCF-7/Adr cells and 20.1 in A549/Adr cells with the treatment of 10 µmol/L (-)-5-N acetylardeemin. (- )-5-N-acetylardeemin also enhanced the sensitivity of parental MCF-7 and A549 cells to adriamycin. The fluorescence intensity in both MCF-7/Adr and A549/Adr cells, which reflected the intracellular accumulation of adriamycin, were significantly enhanced by ( -)5-N- acetylardeemin in a dose-dependent manner. The expressions of P-gp in MCF-7/Adr and A549/Adr cells were significantly inhibited by (- )-5-N-acetylardeemin. CONCLUSION: (- )5-N-acetylardeemin could reverse the multidrug resistance in cancer cells through inhibiting the expression of P-gp and enhancing the intracellular accumulation of cytotoxic drug.

[Relationship among ALDH2 gene polymorphism, alcohol metabolism and acetaldehyde level in peripheral blood].

OBJECTIVE: To explore alcohol pharmacokinetics as well as acetaldehyde level in peripheral blood in human subjects with different ALDH2 genotypes after drinking. METHODS: Venous blood samples of 14 unrelated volunteers were collected. Polymerase chain reaction-restriction fragment length polymorphism technology was adopted for DNA extraction and ALDH2 genotyping. The volunteers were asked to drink beer at certain doses. The concentration of alcohol and acetaldehyde were assayed by headspace gas chromatography method at different time. The pharmacokinetic parameters were calculated. RESULTS: According to the results of electrophoresis, 5 people carried ALDH2*1/*1 as wild group and 9 people carried ALDH2*1/*2 as mutation group. The good linear range of alcohol and acetaldehyde were 0-1 570.7 microg/mL and 0-5.1772 microg/mL, respectively. The AUC values of alcohol and acetaldehyde and the t1/2Z value of alcohol were higher in the mutation group than that in the wild group. But the CL/F value of alcohol was lower in the mutation group than that in the wild group (P<0.05). CONCLUSION: After the consumption of alcohol, alcohol and acetaldehyde metabolism in blood slow down in ALDH2*1/*2 mutation group influenced by the inhibition of enzyme activity, leading to the accumulation of acetaldehyde in peripheral blood, thus reinforcing their effects in the body.

[Determination of unknown impurities in cefotiam hexetil by HPLC-MS/MS].

OBJECTIVE: To detect unknown impurities in raw drug material of cefotiam hexetil. METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed for the determination of impurities in cefotiam hexetil. Agilent SB-C18 column (150 mm x 2.1 mm i. d. , 3.5 microm particles) was used for chromatographic separations of cofotiam hexetil dissolved in deionized water, with mobile phase consisting of (A) 0.1% formic acid and (B) acetonitrile and timed gradient program T (min)/B (%): 0/3, 5/3, 15/20, 20/40, 30/60, 40/80. The flow rate was set at 0. 3 mL/min with DAD detector wavelength fixed at 254 nm. Electrospray ionization source was applied and operated in positive ion MRM mode. The source voltage was kept at 4 kV and cone voltage was 100 V with the mass range m/z 50-1000. Nitrogen was used as nebulizing gas and the nebulizer pressure was 40 psi. The drying gas temperature was 350 degrees C and the drying gas flow was 10 L/min. Results Unknown impurities of cefotiam hexetil were identified. Substance 1 was delta3-isomer of cefotiam hexetil. The structures of 3 other substances were also determined. CONCLUSION: The method is sensitive, rapid and credible for the analysis of cefotiam hexetil and its related impurities, which can be applied in quality control of cefotiam hexetil.

[Recombinant soluble CD40 ligand enhances Wogonin-induced antitumor activity].

OBJECTIVE: To investigate the effect of recombinant soluble CD40 ligand (rsCD40L) on Wogonin mediated antitumor activity in cancer cells and the underlying molecular mechanisms. METHODS: Cell death was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. For morphological study of cell death, cells were stained with 50 microg/mL of acridine orange and 50 microg/mL of ethidium bromide and observed and photographed under a fluorescence microscope. Activation of apoptosis pathway was evaluated by Western blot. The effects of pan-caspase inhibitor Z-VAD-FMK and tumor necrosis factor alpha (TNF-alpha) neutralizing antibody on cell death induced by rsCD40L and Wogonin co-treatment were also investigated. RESULTS: rsCD40L significantly enhanced Wogonin-induced cell death of ovarian cancer cells SKOV3. A dose-dependent synergism was found with a fixed rsCD40L dose (1 microg/mL) and increased concentrations of Wogonin (5 micromol/L-15 micromol/L). rsCD40L and Wogonin co-treated cells showed typical apoptotic morphologies and enhanced activation of caspases pathway. As expected, the pan-caspase inhibitor Z-VAD-FMK inhibited synergistic cell death of rsCD40L and Wogonin co-treated SKOV3 cells. Interestingly, the TNF-alpha neutralizing antibody that blocks TNF-alpha binding to its receptor also significantly suppressed the cell death enhancing effect, indicating that autocrine TNF-alpha played a role of sensitization. CONCLUSION: rscCD40L sensitizes cancer cells to wogonin-mediated apoptosis, which may involve autocrine of TNF-alpha, and the combination of rsCD40L and Wogonin may have a potential for cancer therapy.

[Determination of cinobufagin and resibufogenin in liver tissue by HPLC-MS/MS].

OBJECTIVE: To develop a sensitive and accurate assay for detecting cinobufagin and resibufogenin in liver tissue using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). METHODS: The homogenization of liver tissue with internal standard dexamethasone was extracted with dichloromethane. The extracts with methanol were purified through ProElut C18 solid phase extraction and tested in positive electrospray ionization with multiple reaction monitoring of HPLC-MS/MS. RESULTS: The good linear relationship of cinobufagin and resibufogenin in liver tissue were 1-204 ng/g and 1-206 ng/g, respectively. The minimal detection threshold (S/N > or = 3) of this method was 0.3 ng/g for both cinobufagin and resibufogenin. The matrix effect was 96.5%-126.7%. The extraction recovery coefficient was 70.0%-82.3%. The precision of intra-day and inter-day was less than 10%. CONCLUSION: This method is sensitive and reliable, and can be used in forensic toxicological analysis.

[LC-MS/MS analysis of determination of strychnine and brucine in formaldehyde fixed tissue].

OBJECTIVE: To establish a method for determination of strychnine and brucine in formaldehyde fixed tissue by LC-MS/MS analysis. METHODS: The samples were pretreated with solid phase extraction using SCX cartridges and separated on SB-C18 column with mobile phase 0.1% formic acid : 0.1% formic acid-acetonitrile (75:25). Electrospray ionization (ESI) source was utilized and operated in positive ion mode. Multiple reactions monitoring (MRM) mode was applied. External standard method was applied for quantitation. RESULTS: The chromatographic separation of strychnine and brucine in formaldehyde fixed nephritic and hepatic tissues resulted successfully. The standard curve was linear in the range of 0.002-2.0 microg/g for strychnine and brucine in formaldehyde fixed tissues, and the correlation coefficient was more than 0.996. The limits of detection (LOD) of strychnine and brucine in nephritic tissues were 0.06ng/g and 0.03 ng/g, respectively. The LOD of both chemicals were 0.3 ng/g in hepatic tissues. The extraction recovery rate was more than 74.5%. The precision of intra-day and inter-day were both less than 8.2%. CONCLUSION: Strychnine and brucine can be sensitive to be determined in formaldehyde fixed tissue by LC-MS/MS analysis. It can be applied in the forensic toxicological analysis.

[Effect of heat shock protein 90 inhibitor 17-dimethylamino-ethylaminogeldanamycin on TNfalpha-mediated apoptosis and TNF-induced NF-kappaB activation].

OBJECTIVE: To investigate the effect of heat shock protein 90 (HSP90) inhibitor 17-dimethylaminoethylaminogeldanamycin (17DMAG) on tumor necrosis factor alpha (TNFalpha) mediated apopotosis in cancer cells and the underlying molecular mechanisms. METHODS: Cell death treated with different concentration of 17DMAG and TNFalpha was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. For morphological study of cell death, cells were stained with 50 microg/mL of acridine orange and 50 microg/mL of ethidium bromide and observed and photographed under a fluorescence microscope. Activation of apoptosis and NF-kappaB pathway were evaluated on the change of caspase-8, caspase-3, poly (ADP-ribose) polymerase (PARP), receptor-interaction protein (RIP), IkappaB kinase beta (IKKbeta) and inhibitor of IkappaB (IkappaBalpha) by Western blot. RESULTS: 17DMAG sensitized cervical cancer cells HeLa and ovarian cancer cells SKOV3 to TNFalpha-induced cell death in a dose-dependent manner, which was accompanied with degradation of RIP and Ikappakappabeta, and consequent blockage of TNFalpha-induced NFkappaB activation. 17DMAG and TNFalpha cotreated cells showed typical apoptotic morphologies and enhancing of activation of caspases. CONCLUSION: 17DMAG sensitizes cancer cells to TNFalpha-mediated apoptosis through blockage of TNF-induced NF-kappaB activation, and disabling this survival signal with 17DMAG followed by TNF treatment could be an effective new therapeutic strategy for improving the anti-cancer value of TNFalpha.

[Analysis of multiple cephalosporins in blood and urine by HPLC].

OBJECTIVE: To establish a new high performance liquid chromatography (HPLC) method for determining the concentration of cefazolin, cefradine, cefoperazone and cefotaxime in blood and urine, as well as to investigate its applicability. METHODS: Protein in blood and urine was precipitated directly by acetonitrile with acetanilide was used as the internal standard using Agilent Zorbax SB-Aq column (250 mm x 4.6 mm, 5 microm). The mixed solvents of water (triethylamine 0.12%, acetic acid 0.12%) and acetonitrile were used as the mobile phase to separate cephalosporins using gradient elution method at 1 mL/min (flow rate) and 254 nm (detection wavelength). RESULTS: The working curve of four cephalosporins showed a good correlation (r = 0.9993), with the detection limit up to 0.01 microg/mL. The recovery rate was more than 81.2%. CONCLUSION: This method is fast, easy and accurate. It is suitable for biological analysis of the 4 cephalosporins of the blood and urine in practical cases.

[Determination of ketamine and norketamine in blood and urine by high performance liquid chromatography].

OBJECTIVE: To develop a high-performance liquid chromatographic method for determination of ketamine and norketamine in blood and urine. METHODS: The compounds were extracted from blood or urine by liquid-liquid extraction using toluene after blood or urine was adjusted pH to 14. The extracts were analyzed by HPLC. RESULTS: Linear limits of ketamine and norketamine determination in blood ranged from 0.05 microg/mL to 10 microg/mL (R2 > 0.9993) and in urine ranged from 0.01 microg/mL to 200 microg/mL (R2 > 0.9995). Limits of detection (LODs) for ketamine and norketamine were 0.006 microg/mL and 0.003 microg/mL (S/N > or = 3), respectively. The mean extraction recovery was over 82.4% and its coefficients of variation were less than 10.0% for ketamine and norketamine. Concentration-time curves and urinary drug velocity curves of ketamine norketamine were obtained by determinations of them in blood and urine in rat using the developed method. CONCLUSION: The method is sensitive, simple, rapid and suitable for determination of ketamine and norketamine in blood and urine for toxicological and clinical pharmaceutical analysis.

[Analysis of paraquat in blood and urine by sodium borohydride/nickel chloride chemical reduction-gas chromatography/thermionic specific detector].

OBJECTIVE: To establish a new method for the analysis of paraquat in blood and urine by sodium borohydride/nickel chloride chemical reduction-gas chromatography/thermionic specific detector. METHODS: An initial procedure of precipitation was performed by adding hydrochloric solution with sodium chloride and a mixture of chloroform and ethanol. Then the analyte contained in supernatant was reduced by a reduction system of sodium borohydride and nickel chloride and extracted by acetic ether. Ethyl paraquat (EPQ) was used as internal standard. GC/TSD was used to identify and quantify the analyte. RESULTS: The limits of detection (S/N=3) in blood and urine were 0.002 and 0.004 microg/mL, respectively. The linear ranges were 0.050-30.0 microg/mL. Correlation coefficients in blood and urine were 0.999 and 0.998, respectively. The recoveries exceeded 80% both in blood and urine. CONCLUSION: This method is applicable for quantification of paraquat in biological fluids.

Urinary metabonomics of rats with ketamine-induced cystitis using GC-MS spectroscopy.

Ketamine abuse has dramatically increased in recently years. With the widely application of ketamine, its side effects, especially cystitis induced by long-term use, have attracted more and more attention from the public. In the present study, we aimed to explore the potential generative mechanism of ketamine-induced cystitis by determining the endogenous metabolites at different time points after ketamine treatment. Body weight, bladder/body coefficient, urinary frequency, urinary potassium, serum IL-6, and TNF-α were determined at different time points after ketamine treatment. H&E staining was used to observe the changes of histopathology. Metabonomics was performed to determine the changes of endogenous metabolites. After 12 weeks of treatment, obvious inflammatory reaction was noticed in the KET group; the body weight and urinary potassium of the KET group were significantly lower than the NS group (P < 0.05) and other factors, such as urinary frequency, bladder/body coefficient, serum TNF-α and IL-6 were higher than the NS group (P < 0.05). A total of 30, 28, and 32 significantly changed metabolites were identified at the 1st week, 4th week and 12th week, respectively. Metabolic pathway analysis showed that different metabolic pathways were affected during the treatment process. Linoleic acid metabolism, beta-alanine metabolism, glyoxylate and dicarboxylate metabolism were only affected following long-term administration of ketamine. Those metabolic pathways may have a close relationship with cystitis induced by ketamine.

[Detecting methamphetamine and amphetamine with high performance liquid chromatography].

OBJECTIVE: To establish a method for detecting methamphetamine (MA) and amphetamine (AMP) with high performance liquid chromatography (HPLC). METHODS: Both MA and AMP were isolated on a C18 column and methanol-phosphate buffer (0.015 mol/L NaH2PO4) at a flow rate of 1.0 mL/min. The 190-360 nm ultraviolet spectrum was examined, with 215 nm as the detection wavelength. RESULTS: The MA and AMP were well isolated and determined. The MA determined by the HPLC had good linearity with the real value at the range from 1.4 to 270 microg/mL (R2=1), with an average recovery rate of 102.5%. The detectable Limit was 0.73 microg/mL (S/N > or =3). The AMP determined by the HPLC had a good linearity with the real value at the range from 0.9 to 580 microg/mL (R2 = 0.9999), with an average recovery rate of 101.7%. The detectable limit was 0.52 microg/mL (S/N > or =3). Both intra-day and inter-day precisions expressed by relative standard deviations of the MA and AMP were less than 2.4%. CONCLUSION: This is a simple, rapid and accurate method for detecting methamphetamine and amphetamine.

[Comparison of the contents of ATP, ADP, and AMP in the cardiac muscle of rat killed by different manners].

OBJECTIVE: The content changes of energy substances in the cardiac muscle of rat killed by different manners were investigated to elucidate evidence that can be used to determine the modes of death and postmortem interval. METHODS: One hundred and eighty rats were randomly allocated into 3 groups and killed by bleeding, suffocating, and neck breaking, respectively. The contents of ATP, ADP, and AMP in the cardiac muscle of rats killed by the different manners at different death intervals (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 h) were measured by HPLC. RESULTS: There were significant differences observed in the contents of ATP and AMP in the rats' cardiac muscle in different groups at most of the intervals (P < 0.05) and at all of the intervals within the same group (P < 0.01), but no differences were found in the ADP contents in any of the group at most of the intervals. CONCLUSION: The content changes of energy substances (ATP and AMP) in the cardiac muscle of dead rats may provide a basis for determination of the death manners and postmortem intervals.

[Detection of pentachlorophenol in biomaterial using HPLC].

OBJECTIVE: To develop a method of detecting pentachlorophenol in biomaterial. METHODS: The samples were extracted by ether, and the characters of the high performance liquid chromatography (HPLC) method were studied. RESULTS: A method for the detection of pentachlorophenol in fish tissue samples was set up with the conditions of chromatography as follows: the compounds are separated on Hypersil ODS column(4.6 mm i.d. x 150 mm, 5 microns) and eluted with methanol which contains 2 g/L NH4Ac (20:80) using 305 nm as the detecting wavelength, the recovery of method being 76%, the linear range 0.042 microgram/g-1.344 micrograms/g, and the correlation coefficient 0.9994, the limit of quantification 0.014 microgram/g. Finally, pentachlorophenol was successfully detected in the fish tissue samples. CONCLUSION: The method has been proven useful in the forensic cases involved in the poisoning of pentachlorophenol.

[A study on the effect of methanal during determination of carbon monoxide in blood].

OBJECTIVE: To confirm whether formaldehyde disturb detecting carbon monoxide in blood. To give an evidence that can be used for detecting carboxyhemoglobin more accurately in carbon monoxide posioning appraises. METHODS: Blood samples came from carbon monoxide poisoning and the health were collected. Regular methods for detecting carboxyhemoglobin were used. Observing and comparing the detection results between which were spiked with methanal and no spiked one were performed. RESULTS: Methanal will affect the result of following experiments such as heating, adding NaOH, absorbed by PdCl2 and spectrophotometry. CONCLUSION: The samples which contaminated by formaldehyde couldn't be used for detecting carboxyhemoglobin.

[The research of the heroin and its metabolites analysis in clinical samples].

Heroin can be metabolized easily in body and the mail metabolites are 6-MAM, morphine and so on. At present, there are urine, blood, hair and so on as specimens for detection, while the analytical technology conclude TLC, GC, HPLC, GC/MS, LC/MS, IA, CE etc. In this paper, these technologies used for heroin's metabolites were viewed in order to provide some reference to the study in relative field.

Absence of association between CD86 +1057G/A polymorphism and coronary artery disease.

CD86, one of the key costimulatory molecules, is not only involved in the initiation of T-cell immunity but also plays important roles in the development of cardiovascular diseases. The purpose of this study was to investigate the association between the CD86 polymorphism and the risk of coronary artery disease (CAD) in a Chinese population. We analyzed single-nucleotide polymorphism of CD86 +1057G/A (rs1129055) in 164 patients with CAD and 299 healthy controls by performing polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing assay. No significant association was observed in the genotype and allele frequencies of +1057G/A polymorphism between cases and controls, indicating that CD86 +1057G/A polymorphism may not be associated with CAD in the Chinese population.