Site-specific analysis of N-glycans on haptoglobin in sera of patients with pancreatic cancer: a novel approach for the development of tumor markers

It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N-glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N-glycans. In the present study, site-specific N-glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di-, tri- and tetra-branched glycans of haptoglobin increased in the sera of PC patients. Tri-antennary N-glycans containing a Lewis X-type fucose markedly increased at the Asn211 site of haptoglobin N-glycans. While fucosylated N-glycans derived from serum haptoglobin of patients with CP slightly increased, di-fucosylated tetra-antennary N-glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site-specific analyses of N-glycans may be useful as novel tumor markers for PC.     

Toxicology effects of Morning Glory Seed in rat: A metabonomic method for profiling of urine metabolic changes

Ethnopharmacological relevance

Morning Glory Seed (MGS) is a valuable traditional Chinese medicine which is widely used for the treatment of edema, ascites, hydroncus, simple obesity, lung fever and ardent fever. In recent years, long-term exposure to Morning Glory Seed (MGS) has shown to pose progressive renal damage in clinical practice. We hypothesize that changes in metabolic profile could have occurred before symptoms were observed, which may allow early treatment.

Aim of the study

To investigate the metabolic changes caused by Morning Glory Seed-induced renal damage.

Method

Metabonomics method was used for chronic toxicology study of MGS in Wistar rats. With a therapeutic dose, the model rats were orally administered the extract of MGS for 10 weeks continuously. The urine samples of model and control rats were collected in various time-points and the endogenous metabolites were analyzed by ultraperformance liquid chromatography coupled with mass spectrometry. The identification of all the potential biomarkers was performed using reference standard by comparing their mass spectra, MS/MS fragmentation and retention time. Furthermore, histopathology and clinical chemistry studies were carried out to ensure the success of MGS-induced nephrotoxicity model.

Results

The difference in metabolic profiles between the control and the dosed rats was well observed by the principal component analysis (PCA) of the MS spectra. Significant changes of 12 metabolite biomarkers were detected in the rat urine samples. Metabonomics method could discriminate the model rats from the control rats in 2nd, 6th and10th week respectively before serious organic damage of kidney was found in 10th week by histopathology method.

Conclusion

We believe that metabolic profiling may act as a preclinical protocol for MGS exposure before symptoms are observed.     

Benzylidenemalononitrile compounds as activators of cell resistance to oxidative stress and modulators of multiple signaling pathways. A structure-activity relationship study

Benzylidenemalononitrile (BMN) tyrphostins are well known as potent tyrosine kinase inhibitors. Moreover, in recent years it has been recognized that members of the tyrphostin family possess additional biological activities independent of their ability to inhibit protein tyrosine kinases. In this study, we examined the relationship between the structure of 49 BMNs and related compounds, and their capacity to induce heme oxygenase 1 (HO-1) gene expression in U937 human monocytic cells, to activate upstream signaling pathways and to protect cells against menadione-induced oxidative stress. It was found that the electron-withdrawing (NO₂, CN, halogen) groups in BMN molecules and double meta-MeO substituents increased the HO-1 gene induction, while the electron-donating groups in ortho/para position (OH, MeO and N-morpholino) significantly decreased it. The magnitude of activation of c-Jun, Nrf2, p38 MAPK, and p70S6K correlated with specific substitution patterns in the BMN structure. BMN-dependent maximal up-regulation of HO-1 required parallel increase in Nrf2 and phospho-c-Jun cellular levels. Liquid chromatography mass spectrometry (LC–MS) analysis revealed that BMNs can generate conjugates with one or two glutathione equivalent(s). This study supports the hypothesis that BMNs induce the expression of protective genes by alkylating sensitive cysteine residues of regulatory factors.     

Melatonin levels, determined by LC-ESI-MS/MS, fluctuate during the day/night cycle in Vitis vinifera cv Malbec: evidence of its antioxidant role in fruits

The identification of melatonin in plants has inspired new investigations to understand its biological function and which endogenous and external factors control its levels in these organisms. Owing to the therapeutical and nutraceutical properties of melatonin, it should be important to develop reliable analytical methods for its quantification in vegetal matrices containing this indoleamine, such as grape and wine. The main objectives of the present study were to test whether melatonin levels fluctuate during the day in berry skins of Vitis vinifera L. cv Malbec, thereby possibly relating its abundance to its putative antioxidant function, to determine whether daylight reaching clusters negatively controls melatonin levels, and to evaluate whether total polyphenols and anthocyanins also change through a 24-hr period. Grapes were harvested throughout the day/night to determine the moment when high levels of these components are present in grapes. The presence of melatonin in grapes was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. It is shown for the first time that melatonin levels fluctuate during the day/night cycle in plants grown under field conditions in a fruit organ of the species Vitis vinifera. We also determined that the diurnal decay of melatonin in berry skins is induced by sunlight, because covered bunches retained higher melatonin levels than exposed ones, thus explaining at least part of the basis of its daily fluctuation. Evidence of melatonin's antioxidant role in grapes is also suggested by monitoring malondialdehyde levels during the day.     

Recent developments in analytical determination of furosemide

Furosemide (FUR), a drug that promotes urine excretion, is used in the pharmacotherapy of various diseases and is considered as a doping agent in sports. FUR is a powerful diuretic (water pill). This medicine is used to treat excessive fluid accumulation and swelling (edema) of the body caused by heart failure, cirrhosis, chronic kidney failure, and nephrotic syndrome. Owing to its extensive use as a powerful diuretic, FUR has long attracted the attention of many analysts. A variety of analytical methods have been proposed for the determination of FUR in biological fluids and pharmaceutical samples. The revision includes the most relevant analytical methodologies used in its determination from the nineties decade at present.     

Determination of mianserin in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS): application to a bioequivalence study in Chinese volunteers

This study aims to develop a standard protocol for the bioequivalence study of mianserin hydrochloride tablets--a tetracyclic antidepressant drug. For this purpose, a rapid, convenient and selective method using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI/MS) has been developed and validated to determine mianserin in human plasma. Mianserin and the internal standard (I.S.), cinnarizine were extracted from plasma by N-hexane:dimethylcarbinol (98:2, v/v) after alkalinized with sodium hydroxide. LC separation was performed on a Thermo Hypersil-Hypurity C18 (5 microm, 150 mm x 2.1 mm) with the mobile phase consisting of 10mM ammonium acetate (pH 3.4)-methanol-acetonitrile (35:50:15, v/v/v) at 0.22 ml/min. The retention time of mianserin and cinnarizine was 3.4 and 2.1 min, respectively. Quadrupole MS detection and quantitation was done by monitoring at m/z 265 [M+H]+ for mianserin and m/z 369 [M+H]+ for cinnarizine. The method was validated over the concentration ranges of 1.0-200.0 ng/ml for mianserin. The recovery was 81.3-84.1%, intra- and inter-day precision of the assay at three concentrations were 9.6-11.4% with accuracy of 97.5-101.2% and the lower limit of quantitation (LLOQ) detection was 1.0 ng/ml for mianserin. The stability of compounds was established in a battery of stability studies, i.e., short-term and long-term storage stability as well as freeze-thaw cycles. This method proved to be suitable for the bioequivalence study of mianserin hydrochloride tablets in healthy human male volunteers.     

Separation and detection of all phosphoinositide isomers by ESI-MS

Phosphoinositides (PIs) play fundamental roles as signalling molecules in numerous cellular processes. Direct analysis of PIs is typically accomplished by metabolic labelling with ³H-inositol or inorganic ³²P followed by deacylation, ion-exchange chromatography and flow scintillation detection. This analysis is laborious, time-consuming, and involves massive amounts of radioactivity. To overcome these limitations we established a robust, non-radioactive LC–ESI–MS assay for the separation and analysis of deacylated PIs that allows discrimination of all isomers without the need for radioactive labelling. We applied the method to various cell types to study the PI levels upon specific stimulation.     

栀子水提物的HPLC-ESI-MSn分析

目的:建立栀子水提物中栀子苷和绿原酸两种成分的分析方法,全面分析水提物的化学成分。方法:采用加热回流法获得栀子的水溶性总提物,外标法测定水提物中栀子苷和绿原酸的含量,通过离子阱多级质谱法(ESI/MSn)全面分析水提物的化学成分。结果:测得水提物中栀子苷和绿原酸的含量分别为2.52%和0.08%,试验共对13种化合物进行了结构分析,其中2种尚未见报道。结论:该方法专属性强,可作为栀子的准确定性分析方法。     

HPLC-ESI-MS测定冬虫夏草和蚕蛹虫草中腺苷和虫草素含量

目的 :建立测定冬虫夏草及蚕蛹虫草中腺苷和虫草素的方法。方法 :HPLC-ESI-MS法 ,用甲醇为提取溶剂 ,采用选择性离子检测 (SIM)和电喷雾离子化 (ESI) ;色谱条件 :ShimadzuVP-ODS色谱柱 ;流动相水 甲醇 甲酸(94∶5∶1) ,以 2 氯腺苷为内标。结果 :腺苷回归方程Y=0 .1346X +0.0129,r=0.9984 ,线性范围 0.5～124.5mg·L^-1;虫草素回归方程Y=0.2164X +0.0215 ,r=0.9991,线性范围 0.5～ 136 .5mg·L^-1;腺苷和虫草素加样回收率分别为 95 .8%及 98.1%。结论 :方法灵敏、快速和选择性好 ,可用于冬虫夏草及其代用品中腺苷和虫草素的分析及质量控制。     

液质联用鉴定重组人血管内皮抑制素

目的：用液质联用技术鉴定重组人血管内皮抑制素（rhEndostatin）。方法：利用ESI—Q—TOF—MS法测定rhEndostatin的精确相对分子质量，通过HPLC—ESI—Q—TOF—MS／MS法测定其胰蛋白酶酶切后肽段的部分氨基酸序列并结合数据库检索进行结构鉴定。结果：重组人血管内皮抑制素的实测相对分子质量为21114．5，与理论相对分子质量21113．8相比非常接近。HPLC—ESI—Q—TOF—MS／MS测定m／z802．0的肽段的部分氨基酸序列为Ala—Pro—Ser—Ala-Thr—Gly—Gin—Ala—Ser—Ser—Leu—Leu。将其m／z和氨基酸序列在MASCOT数据库检索，结果表明重组人血管内皮抑制素的结构正确。结论：液质联用是鉴定蛋白质的灵敏、快速、准确的新方法。