Characterization of free radicals formed from COX-catalyzed DGLA peroxidation.

Like arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA) is a 20-carbon ω-6 polyunsaturated fatty acid and a substrate of cyclooxygenase (COX). Through free radical reactions, COX metabolizes DGLA and AA to form well-known bioactive metabolites, namely, the 1 and 2 series of prostaglandins (PGs1 and PGs2), respectively. Unlike PGs2, which are viewed as proinflammatory, PGs1 possess anti-inflammatory and anticancer activities. However, the mechanisms linking the PGs to their bioactivities are still unclear, and radicals generated in COX-DGLA have not been detected. To better understand PG biology and determine whether different reactions occur in COX-DGLA and COX-AA, we have used LC/ESR/MS with a spin trap, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), to characterize the carbon-centered radicals formed from COX-DGLA in vitro, including cellular peroxidation. A total of five types of DGLA-derived radicals were characterized as POBN adducts: m/z 266, m/z 296, and m/z 550 (same as or similar to COX-AA) and m/z 324 and m/z 354 (exclusively from COX-DGLA). Our results suggest that C-15 oxygenation to form PGGs occurs in both COX-DGLA and COX-AA; however, C-8 oxygenation occurs exclusively in COX-DGLA. This new finding will be further investigated for its association with various bioactivities of PGs, with potential implications for inflammatory diseases.

Characterization of novel radicals from COX-catalyzed arachidonic acid peroxidation.

The peroxidation of arachidonic acid (AA) catalyzed by cyclooxygenase (COX) is a well-known free radical-mediated process that forms many bioactive products. Because of a lack of appropriate methodologies, however, no comprehensive structural evidence has been found previously for the formation of COX-mediated and AA-derived free radicals. Here we have used a combination of LC/ESR and LC/MS with a spin trap, alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN), to characterize the carbon-centered radicals formed from COX-catalyzed AA peroxidation in vitro, including cellular peroxidation in human prostate cancer cells (PC-3). Three types of radicals with numerous isomers were trapped by POBN as ESR-active peaks and MS-active ions of m/z 296, 448, and 548, all stemming from PGF(2)-type alkoxyl radicals. One of these was a novel radical centered on the carbon-carbon double bond nearest the PGF ring, caused by an unusual beta-scission of PGF(2)-type alkoxyl radicals. The complementary nonradical product was 1-hexanol, another novel beta-scission product, instead of the more common aldehyde. The characterization of these novel products formed from in vitro peroxidation provides a new mechanistic insight into COX-catalyzed AA peroxidation in cancer biology.

Targeted delivery of methotrexate to skeletal muscular tissue by thermosensitive magnetoliposomes.

Thermosensitive magnetoliposomes (TMs) encapsulated with methotrexate (MTX) were prepared with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol by reverse-phase evaporation. Encapsulation efficiency of MTX and hydrophilic magnetite Fe(2)O(3)-glu, liposome particle size, zeta-potential, and in vitro and in vivo drug release were studied. More than 80% of loaded MTX was released from TMs within 30min when the environmental temperature increased from 37 degrees C to 41 degrees C, while 60% of the drug was remained inside TMs for up to 24h at 37 degrees C. Furthermore, the pharmacokinetics and tissue distribution study showed that TMs significantly increased the accumulation of MTX in the skeletal muscular tissue when exposed to an external constant magnetic field and heated to 41 degrees C compared to the absence of the magnetic field and heating. Therefore, the results in this study suggested that TMs prepared by reverse-phase evaporation can archive a good magnetic targeting effect and fast drug release in response to hyperthermia, which implies their great potential of application in cancer therapy.

Spectroscopic studies on the interaction of pazufloxacin with calf thymus DNA.

Fluorescence spectra, absorption spectra, DNA viscosity titrations, competition experiment, and iodide quenching experiment were used to study the interaction of DNA with pazufloxacin. DNA quenches the fluorescence of pazufloxacin significantly. No red shift and isobestic points are observed in UV titration experiment. DNA viscosity and iodide quenching results suggest that pazufloxacin does not intercalate into DNA. SsDNA has a stronger quenching effect on pazufloxacin than dsDNA has. Pazufloxacin interacts with DNA in a different mode from ethidium bromide, which is a typical intercalator of DNA. All these results indicate that pazufloxacin interacts with calf thymus DNA in the mode of groove binding. The quenching constant and thermodynamic constants have also been determined.

Marine drugs--macrolactins.

The increasing demands for new lead compounds in pharmaceutical and agrochemical industries have driven scientists to search for new bioactive natural products. Marine microorganisms are rich sources of novel, bioactive secondary metabolites, and have attracted much attention of chemists, pharmacologists, and molecular biologists. This mini-review mainly focuses on macrolactins, a group of 24-membered lactone marine natural products, aiming at giving an overview on their sources, structures, biological activities, as well as their potential medical applications.

LC/ESR/MS study of spin trapped carbon-centred radicals formed from in vitro lipoxygenase-catalysed peroxidation of gamma-linolenic acid.

Gamma-linolenic acid (GLA) has been reported as a potential anti-cancer and anti-inflammatory agent and has received substantial attention in cancer care research. One of the many proposed mechanisms for GLA biological activity is free radical-mediated lipid peroxidation. However, no direct evidence has been obtained for the formation of GLA-derived radicals. In this study, a combination of LC/ESR and LC/MS was used with alpha-[4-pyridyl-1-oxide]-N-tert-butyl nitrone (POBN) to profile the carbon-centred radicals that are generated in lipoxygenase-catalysed GLA peroxidation. A total of four classes of GLA-derived radicals were characterized including GLA-alkyl, epoxyallylic, dihydroxyallylic radicals and a variety of carbon-centred radicals stemming from the beta-scissions of GLA-alkoxyl radicals. By means of an internal standard in LC/MS, one also quantified each radical adduct in all its redox forms, including an ESR-active form and two ESR-silent forms. The results provided a good starting point for ongoing research in defining the possible biological effects of radicals generated from GLA peroxidation.

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma.

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.

[Simultaneous analysis of six effective components in the anti-Alzheimer's disease effective component group of Xiao-Xu-Ming Decoction].

A method based on high performance liquid chromatography (HPLC) was developed for the quantitative determination of six components in an anti-Alzheimer medicine, Xiao-Xu-Ming Decoction, which is an effective prescription in treating stroke and the sequela of stroke by herbalist doctors for thousands years. The effective component group (ECG) was made according to the results of high-throughput screening, and the curative effect of ECG was validated on aging rats. In this method an ODS column was used. The mobile phase consisted of water-formic acid-ethylenediamine (A; 100: 0.1: 0.1, v/v) and methanol-formic acid (B; 100 : 0.05, v/v), eluted with gradient (0 - 5 min, 20% B; 5 - 100 min, 20% B - 40% B; 100 - 140 min, 40% B - 70% B). The flow rate was 1 mL/min. The detection wavelength was set at 240 nm. Under the above separation conditions, six components belonged to two different categories, indicans and alkaloids, were determined simultaneously. The relationships between the concentrations and the peak areas of these six components were all linear. The recoveries of the six components were 99.1% for paeoniflorin, 99.6% for prim-O-glucosylcimifugin, 98.4% for baicalin, 99.9% for 4'-O-beta-D-glucosyl-5-O-methylvisamminol, 99.6% for fangchinoline, and 102.0% for tetrandrine. The relative standard deviations (RSD) were 1.3%, 1.4%, 0.4%, 0.8%, 0.2%, and 1.4%, respectively. This method is simple and reproducible and it can be used for the quality control of the effective component group of Xiao-Xu-Ming Decoction.

Identification of polymethoxylated flavones from green tangerine peel (Pericarpium Citri Reticulatae Viride) by chromatographic and spectroscopic techniques.

Polymethoxylated flavones (PMFs) were extracted from Pericarpium Citri Reticulatae Viride using a procedure that obtained a consistent mixture of PMFs both in identity and proportion. The mixture consisted of isosinensetin (0.2%) (1), sinensetin (1.7%) (4), tetramethyl-o-isoscutellarein (0.3%) (5), nobiletin (40.5%) (6), tetramethyl-o-scutellarein (1.2%) (7), tangeretin (45.6%) (10), 5-demethylnobiletin (8.7%) (12), 5-demethyl tangeretin (0.8%) (14) and other flavonoids including heptamethoxyflavone (1.0%) (9), among which, compounds 1, 4, 5, 7 and 9 were identified based on their UV spectra, MS data and elution order described in the literature while compounds 6, 10, 12 and 14 were isolated and identified by UV, IR, MS, (1)H NMR, (13)C NMR and 2D NMR spectral studies. In addition, compound 14 was isolated and identified for the first time from Citrus.

Association of IL1B polymorphisms with gastric cancer in a Chinese population.

OBJECTIVES: To investigate the association between IL1B polymorphisms and risk of gastric cancer in a Chinese population, seven SNPs in the IL1B gene were selected for this study. METHODS: A multiplex genotyping method, which is based on adapter ligation and allele-specific amplification, was established to type seven SNPs on the IL1B gene simultaneously. One hundred and forty-one non-cancer outpatients and 97 patients with gastric cancer were genotyped, and the relation between IL1B polymorphisms and gastric cancer was statistically analyzed. RESULTS: The seven SNPs were successfully typed and the results were consistent with those obtained by both Sanger's sequencing and PCR-RFLP. Handling with statistical analysis, we observed significantly different genotype frequencies of 0794C>T (chi(2)=6.11, P=0.05), 1274C>T (chi(2)=6.86, P=0.03) and 2143T>C (chi(2)=6.86, P=0.03) between patients and controls. CONCLUSION: ALM-ASA is a potential method for multiplex SNP typing with a low consumption of genomic DNA. SNPs 0794C>T, 1274C>T, and 2143T>C are associated with gastric cancer.

Determination of vertilmicin sulfate and its related substances by HPLC-ELSD and HPLC-MS2.

A new and simple high-performance liquid chromatography (HPLC)-evaporative light scattering detection (ELSD) method for the determination of vertilmicin sulfate and its related substances is developed. The column is an Agilent SB-C(18) (250 x 4.6 mm, 5 microm). The mobile phase is 0.05 mol/L trifluoroacetic acid-methanol (85:15). Good separation of vertilmicin from the main related substances is achieved. The standard curve is rectilinear in the range of 270-1350 microg/mL (r = 0.9998). The average recovery is 99.8%. The limit of detection is 10 microg/mL. The HPLC-mass spectrometry-mass spectrometry (MS(2)) method is used to characterize the structures of vertilmicin sulfate and its related substances. In positive mode, vertilmicin sulfate and its related substances are elucidated by use of electrospray ion trap MS in the multi-stage MS full scan mode. The possible structure of an unknown impurity in vertilmicin is deduced based on the HPLC-MS(2) data.

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification.

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.

Determination of spectinomycin hydrochloride and its related substances by HPLC-ELSD and HPLC-MSn.

A new and simple high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C(18) (250 mm x 4.6 mm, 5 microm). The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 degrees C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07-3.8 mg/ml (r = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 microg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC-MS(n) method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC-MS(n) data.

[Adapter-ligation mediated allele-specific amplification (ALM-ASA) for multiplex SNP genotyping].

To establish adapter-ligation mediated allele-specific amplification ("ALM-ASA" for short) for multiplex SNP genotyping, five SNPs, 100C>T, 1661G>C, 1758G>T, 2470T>C and 2850C>T in CYP2D6 gene were used as an example for evaluating the method. Firstly, a preamplification was carried out for producing a long target containing all SNPs of interest. Secondly, the preamplified DNA fragments were digested with a restriction endonuclease to form sticky ends. Thirdly, an adapter was ligated to either end of the digested fragment. One end of the adapter was designed as a sequence sticky to the ends of the enzymatically digested fragments, and the other end had a common sequence. Fourthly, an allele-specific amplification was performed by allele-specific primers and a universal primer in one tube by using the adapter-ligated fragments as templates. Finally, the allele-specific amplification products were separated by agarose gel electrophoresis. Because each tube corresponds to one kind of allele-specific primers, the genotype of an SNP can be easily discriminated by the length of the amplified products in each tube. The products of 5-plex allele-specific amplification can be separated by agarose gel electrophoresis. Five SNPs in the CYP2D6 gene were successfully typed for 20 healthy Mainland Chinese and the results were in agreement with those by RFLP. By ALM-ASA, n+1 primers (n SNP allele-specific primers and a universal primer) can be used for an n-plex PCR amplification; the specificity of PCR is thus enhanced significantly. It is concluded that ALM-ASA can be used for typing multiple SNPs simultaneously.

[Approaches for SNP genotyping].

With the completion of the Human Genome Project (HGP), typing single nucleotide polymorphisms (SNP) has become one of the main tasks in the post-genome era. Consequently, a robust, flexible and cost-effective technique for SNP typing is essential to analyze a large number of SNPs. The latest genotyping technologies and the relative detection platforms were introduced systematically. The principle of SNP typing was described in detail in respect of allele-specific hybridization, restriction enzyme digestion, primer extension and oligonucleotide ligation assay, as well as the genotyping platforms of plate readers, genechips, bead array and mass spectrometry. Moreover, the way to the high-throughput genotyping in the future was briefly discussed.

[Interaction between balofloxacin and DNA and the influence of Mg2+ on the interaction].

AIM: To study the binding mode of balofloxacin with DNA and evaluate the influence of Mg2+ on the binding between balofloxacin and DNA. METHODS: Fluorescent spectroscopy was used to study the interaction of balofloxacin with DNA and to calculate the thermodynamic constants. UV-Vis spectra, DNA viscosity titration, competition experiment and the effect of dsDNA and ssDNA on the fluorescense intensity were used to identify the binding mode. RESULTS: Balofloxacin interacted with CT-DNA with a quenching constant of (5.43 +/- 0.07) x 10(3) L x mol(-1). The interaction was exothermic with a Van't Hoff enthalply of - 8.03 kJ x mol(-1) x Mg2+ cation could enhance the quenching constant between balofloxacin and DNA. CONCLUSION: Balofloxacin interacted with CT-DNA in the mode of groove binding and Mg2+ could mediate the binding of balofloxacin to DNA.

Structural characterization of chlorobenzylidine.

AIM: To study the structure and crystal forms of chlorobenzylidine. METHODS: Karl Fischer titrimetry, FTIR, thermal analysis, single and powder X-ray diffraction were used for the studies of the structure of chlorobenzylidine and for the identification of two forms of chlorobenzylidine. RESULTS: Chlorobenzylidine and its diastereoisomer have been studied in this article. They can be distinguished by their different melting points. Two crystal forms of chlorobenzylidine (form A and form B) have also been detected and studied. Form A was studied by single-crystal X-ray diffraction, it crystallized in the triclinic system, space group P1(-), with two formula units per cell, is monohydrate. Karl Fischer titrimetry, FTIR, thermal analysis and powder X-ray diffraction were used for identification of the two forms. CONCLUSION: The studies of structure and crystal forms of chlorobenzylidine are very useful for the clinical research and the selection of recrystallization process.

The interaction of human serum albumin with a novel antidiabetic agent--SU-118.

SU-118 is a newly synthesized antidiabetic agent and shows the best hypoglycemic effect among a series of analogs. Its binding properties and binding sites located on human serum albumin (HSA) have been studied using UV absorption and fluorescence spectroscopy. The results of spectroscopic study and the thermodynamic parameters obtained suggest that SU-118 binds to the hydrophobic cavity of human serum albumin and the hydrophobic interaction is the predominant intermolecular force stabilizing the complex. Fluorescent probe displacement studies show that SU-118 can displace competitively both dansylamide and dansylsarcosine from HSA. It is suggested that SU-118 can bind to both site I and site II, but the primary interaction may take place at site I. A binding constant of 1.4 x 10(4) M(-1) and a binding site of 2.0 are obtained from absorbance titration data. The value of binding constant is of the same order of magnitude as that from fluorescence titration. This study provides a molecular basis for elucidating the mechanism of drug acting and predicting unfavorable drug interaction.