Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

Selenium speciation profiles in selenite-enriched soybean (Glycine Max) by HPLC-ICPMS and ESI-ITMS

Soybean (Glycine Max) plants were grown in soil supplemented with sodium selenite. A comprehensive selenium profile, including total selenium concentration, distribution of high molecular weight selenium and characterization of low molecular weight selenium compounds, is reported for each plant compartment: bean, pod, leaf and root of the Se-enriched soybean plants. Two chromatographic techniques, coupled with inductively coupled plasma mass spectrometry (ICPMS) for specific selenium detection, were employed in this work to analyze extract solutions from the plant compartments. Size-exclusion chromatography revealed that the bean compartment, well-known for its strong ability to make proteins, produced high amounts (82% of total Se) of high molecular weight selenospecies, which may offer additional nutritional value and suggest high potential for studying proteins containing selenium in plants. The pod, leaf and root compartments primarily accumulate low molecular weight selenium species. For each compartment, low molecular weight selenium species (lower than 5 kDa) were characterized by ion-pairing reversed phase HPLC-ICPMS and confirmed by electrospray ionization ion trap mass spectrometry (ESI-ITMS). Selenomethionine and selenocystine are the predominant low molecular weight selenium compounds found in the bean, while inorganic selenium was the major species detected in other plant compartments.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

汞与硒在不同汞暴露水平地区猪肝脏和肾脏蛋白质中的分布

通过对贵州万山汞污染地区及北京地区猪肝脏和肾脏组织上清液进行凝胶过滤色谱分离(SephadexG 10 0 ) ,随后用原子荧光法测定它们蛋白质组分中汞和硒的含量 ,研究在汞暴露水平不同状态下微量元素汞和硒在动物体蛋白质分子水平上的分布 .发现这两个地区猪肝脏和肾脏组织上清液蛋白质组分中汞和硒的分布模式有明显差异 .贵州万山汞污染地区猪肝脏上清液中汞浓度比北京地区高 ,硒浓度也相应高 ,且前者与高分子量和低分子量蛋白结合的硒均明显高于后者 ;而北京地区猪肝脏上清液中的硒主要以与高分子量蛋白结合的形式存在 .贵州汞污染地区猪肝脏上清液中汞主要与高分子量蛋白结合 ,而北京地区猪肝脏上清液中汞则分布较为均匀 .贵州万山地区猪肾脏上清液中 ,含硒峰在高分子量蛋白区和低分子量区都有分布 ;而北京地区猪肾脏上清液中 ,硒则主要集中分布于高分子量蛋白范围 .这两个地区猪肾脏上清液中都有分子量约为 11kD的金属硫蛋白 (MT)存在 ,北京地区猪肾脏上清液中汞主要以与金属硫蛋白结合的形式出现 ,而贵州万山地区猪肾脏上清液中的汞除与金属硫蛋白结合外 ,尚有相当大部分是以与高分子量蛋白结合的形式存在 .研究结果表明 ,由于这两个地区汞暴露水平的差异 ,不仅使这两地区猪肝、肾上清液中的汞与硒含量     

Selenium Species and Their Distribution in Freshwater Fish from Argentina

The distribution and speciation of selenium (Se) in freshwater fish (muscle and liver tissue) from lakes in Argentina was investigated. Three introduced species, brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis), and one native species, creole perch (Percichthys trucha), were investigated. Values for total selenium in muscle ranged from 0.66 to 1.61 μg/g, while in the liver, concentrations were much higher, from 4.46 to 73.71 μg/g on a dry matter basis. Separation of soluble Se species (SeCys₂, selenomethionine (SeMet), SeMeSeCys, selenite and selenate) was achieved by ion exchange chromatography and detection was performed by inductively coupled plasma–mass spectrometry. The results showed that in fish muscle, from 47 to 55 % of selenium was soluble and the only Se species identified was SeMet, which represented around 80 % of soluble Se, while in the liver, the amount of soluble Se ranged from 61 to 76 % and the percentage of species identified (SeMet and SeCys₂) was much lower and ranged from 8 to 17 % of soluble Se.     

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.     

The nature of the acid-volatile selenium in the liver of the male rat

1. The properties of rat liver acid-volatile selenium have been compared with those of H(2)Se and (CH(3))(2)Se. 2. In model experiments oxidation-sensitive H(2) (75)Se was trapped quantitatively under anaerobic conditions in 0.1m-AgNO(3), and (CH(3))(2) (75)Se was trapped quantitatively in 8m-HNO(3). The acid-labile selenium of a liver homogenate, and of a microsomal fraction, was found to behave quite unlike (CH(3))(2) (75)Se and in a manner indistinguishable from H(2) (75)Se. 3. It was concluded that the acid-volatile material is certainly not (CH(3))(2)Se and that it is probably H(2)Se. 4. The significance of these findings is discussed in relation to current knowledge about the metabolism and detoxication of selenium, and a scheme is proposed which incorporates this knowledge with recent observations on the interactions between trace amounts of selenium and tocopherol, and the production of acute selenium deficiency by Ag(+) in vitamin E-deficient rats.     

半散放猎豹毛发微量元素含量比较

许多微量元素与动物生理生化机能有着密切的联系,对机体的生长发育和维持健康起着重要作用.近年来,微量元素研究日渐得到重视,如Cu、 Zn、 Mn、 Se、 Pb等微量元素参与酶、激素和维生素代谢,影响动物生殖功能(Suchocki et al., 2003).     

原子荧光和电感耦合等离子体质谱法在硒形态分析中的应用

硒是人体必需的微量元素之一,具有抗肿瘤、防衰老、防辐射和增强机体免疫力等多种功能。近年来,随着人们生活水平的不断提高,各种各样的富硒产品及富硒保健品走进了我们的生活,硒的形态测定越来越受到人们广泛的关注。本文对目前主要检测硒形态的两种方法—原子荧光法(AFS)和电感耦合等离子质体谱法(ICP-MS)在食品、水果蔬菜、富硒保健品、生物样品等方面的应用进行了综述。     

Element concentrations in the archiacanthocephalan Macracanthorhynchus hirudinaceus compared with those in the porcine definitive host from a slaughterhouse in La Paz, Bolivia

Concentrations of lead and cadmium, determined by electrothermal atomic absorption spectrometry, and concentrations of the elements barium, cadmium, copper, iron, magnesium, manganese, nickel, lead, selenium and strontium, determined by inductively coupled plasma mass spectrometry, in the acanthocephalan Macracanthorhynchus hirudinaceus and its porcine final host, sampled at a slaughterhouse in La Paz, Bolivia, were compared. Inductively coupled plasma mass spectrometry analysis revealed that most of the elements were found at higher concentrations in the acanthocephalan than in different tissues of its host. The bioconcentration of elements in M. hirudinaceus compared with the host intestine, listed in order of decreasing values, was as follows: Cd > Pb > Ni > Sr = Cu > Mg > Se > Fe = Mn = Ba. Analysis by electrothermal atomic absorption spectrometry showed that M. hirudinaceus contained 85, 85, 56 and 24 times higher lead levels compared with hosts muscle, liver, kidney and intestine, respectively. The mean cadmium concentration of the parasite was 32 times higher than that of the liver and five times higher compared with porcine kidney. The metal distribution within the body of M. hirudinaceus was as follows: cement gland > testes > lemnisci > eggs = tegument for lead and lemnisci > testes > cement gland > tegument > eggs for cadmium. Therefore, the hypothesis that parasites excrete toxic metals with the shells of their eggs seems not to be valid for M. hirudinaceus. It is concluded, that not only eoacanthocephalans and palaeacanthocephalans parasitising fish, but also archiacanthocephalans from mammalian hosts, are able to bioaccumulate metals.     

Selenium accumulation, distribution, and speciation in spineless prickly pear cactus: a drought- and salt-tolerant, selenium-enriched nutraceutical fruit crop for biofortified foods

The organ-specific accumulation, spatial distribution, and chemical speciation of selenium (Se) were previously unknown for any species of cactus. We investigated Se in Opuntia ficus-indica using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (μXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). μXRF showed Se concentrated inside small conic, vestigial leaves (cladode tips), the cladode vasculature, and the seed embryos. Se K-edge XANES demonstrated that approximately 96% of total Se in cladode, fruit juice, fruit pulp, and seed is carbon-Se-carbon (C-Se-C). Micro and bulk XANES analysis showed that cladode tips contained both selenate and C-Se-C forms. Inductively coupled plasma mass spectrometry quantification of Se in high-performance liquid chromatography fractions followed by LC-MS structural identification showed selenocystathionine-to-selenomethionine (SeMet) ratios of 75:25, 71:29, and 32:68, respectively in cladode, fruit, and seed. Enzymatic digestions and subsequent analysis confirmed that Se was mainly present in a "free" nonproteinaceous form inside cladode and fruit, while in the seed, Se was incorporated into proteins associated with lipids. μXRF chemical mapping illuminated the specific location of Se reduction and assimilation from selenate accumulated in the cladode tips into the two LC-MS-identified C-Se-C forms before they were transported into the cladode mesophyll. We conclude that Opuntia is a secondary Se-accumulating plant whose fruit and cladode contain mostly free selenocystathionine and SeMet, while seeds contain mainly SeMet in protein. When eaten, the organic Se forms in Opuntia fruit, cladode, and seed may improve health, increase Se mineral nutrition, and help prevent multiple human cancers.     

Quantification of biopharmaceuticals and biomarkers in complex biological matrices: a comparison of liquid chromatography coupled to tandem mass spectrometry and ligand binding assays

The quantification of proteins (biopharmaceuticals or biomarkers) in complex biological samples such as blood plasma requires exquisite sensitivity and selectivity, as all biological matrices contain myriads of proteins that are all made of the same 20 proteinogenic amino acids, notwithstanding post-translational modifications. This review describes and compares the two main approaches, namely, ligand binding assays (LBAs) and liquid chromatography coupled to tandem mass spectrometry in the selected reaction monitoring (SRM) mode. While LBAs remain the most widely used approach, SRM assays are gaining interest due to their generally better analytical performance (precision and accuracy) and their capacity for multiplex analyses. This article focuses on the possible reasons for the discrepancies between results obtained by LBAs and SRM assays.     

Human urinary excretion and metabolism of (82)Se-enriched selenite and selenate determined by LC-ICP-MS

Urinary excretion of selenium after ingestion of isotope labeled selenite and selenate was studied in seven healthy volunteers, 4 men and 3 women (age 28-50 years). An aqueous solution containing 330 μL (82)Se-selenate (corresponding to 74.3 μg (82)Se) was given orally and urine samples were subsequently collected during the following 24 hours. The scheme was repeated four weeks later with a 280 μL (82)Se-selenite solution (corresponding to 74.4 μg (82)Se). The amount of total Se in the urine samples was determined by inductively coupled mass spectrometry. The mean total urinary excretion of (82)Se following (82)Se-selenate administration was 33.7% (range 15.6-42.5%) while the mean total excretion of (82)Se after (82)Se-selenite administration was 3.2% (range 2.8-3.9%) of the ingested amount. LC-ICPMS analysis of the urine samples showed that the majority of the selenium excreted after selenate ingestion was unchanged selenate for 6 of the individuals while one individual had metabolized a fraction (approx. 20%) of the selenate to selenosugar. Ingestion of 10 times larger doses of selenite in two individuals resulted in 13-23% excretion primarily excreted as selenosugar. These results show that the human metabolic pathways of selenite and selenate are different and indicate that not all selenate, although well absorbed, may be available for the beneficial health effects.     

Arsenic speciation in human urine reference materials using high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

Six arsenic compounds including arsenocholine, arsenobetaine, dimethylarsinic acid, methylarsonic acid, arsenous acid and arsenic acid were separated by high-performance liquid chromatography (HPLC) on a Hamilton PRP-X100 anion-exchange column using isocratic elution and detected by inductively coupled plasma mass spectrometry (ICP-MS). This analytical procedure was applied to the speciation of arsenic compounds in human urine. The influence of urine matrix on the separation of arsenic compounds was evaluated and the determination of arsenic compounds was not hampered by the ArCl interference which has often been encountered in ICP-MS. Three human urine reference materials, SRM 2670 normal level, SRM 2670 elevated level and Lyphocheck urine metal control 1, were analyzed with respect to arsenic compounds by HPLC-ICP-MS. The results were found to be in good agreement with the certified total arsenic concentration in the reference materials. Six arsenic compounds were detected. Arsenobetaine was found to be present in all of the investigated human urine reference materials.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

Determination of neonicotinoid insecticides residues in bovine tissues by pressurized solvent extraction and liquid chromatography-tandem mass spectrometry

A rapid, sensitive, and environmental-friendly method has been developed for the simultaneous determination of seven neonicotinoid insecticides residues in bovine muscle and liver. The sample preparation procedure was based on a high automated pressurized solvent extraction (PSE) combined with solid-phase extraction (SPE) clean-up. The target compounds were identified and quantitatively determined by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operated in multiple reaction monitoring mode. Average recoveries of the seven analytes from fortified samples ranged between 83.2% and 101.9%, with relative standard deviations (RSDs) lower than 10.8%. The limits of detection (LODs) and quantification (LOQs) for neonicotinoids were in the ranges of 0.8-1.5 μgkg?1 and 2.5-5.0 μgkg?1, respectively. This validated method was successively applied to the determination of neonicotinoid insecticides in real samples from markets.     

Investigation of selenium distribution in subcellular fractions of human liver by neutron activation analysis

Selenium is an important and essential trace element to living systems. In the article, two methods of instrumental neutron activation analysis and hydride generation-atomic fluorescence spectrometry were applied to determine Se in biological samples and the accuracy was evaluated by several reference materials. The subcellular distribution of selenium in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation combined with INAA. Selenium was mainly enriched in mitochondria, nuclei, and cytosol. Almost half of the total Se content existed in nuclei as a result of the large amount in liver and the high Se concentration. Generally, the highest Se concentration in the mitochondrial fractions of each liver sample suggested that Se had important functions in this liver component.     

Distribution and metabolism of selenite and selenomethionine in the Japanese quail

Compared to the many studies on the physiological and toxicological effects of selenium (Se) in mammals, avian Se metabolism is still an unexplored topic. Some birds are useful as poultry for human nutrition. Moreover, birds belong to higher trophic levels in the biosphere and thus may play an important role in Se circulation in the ecosystem in the same way as mammals do. In this study, we analyzed the distribution and metabolism of Se in an experimental bird, the Japanese quail, which was fed drinking water containing sodium selenite or selenomethionine (SeMet). The highest concentration of Se was detected in the pancreas, followed by down feathers, liver, and kidneys. SeMet was more efficiently incorporated into the quail than selenite. The specific and preferable distribution of Se to the high molecular weight fraction in the serum of the quail was observed only in the SeMet-ingestion group. As in mammals, selenosugar and trimethylselenonium were the major metabolites in quail excreta. Three unknown Se metabolites were detected by HPLC-ICP-MS. Although part of the metabolic pathway of Se in the Japanese quail fed selenite and SeMet was the same as that observed in mammals, the bird also showed certain avian-specific metabolic process for Se.     

Effects of dietary selenite,copper, and zinc on tissue trace mineral levels in chicks

Studies were conducted to determine whether nutritional selenium (Se) status affects the nutritional status of the chick with respect to other trace elements, particularly copper (Cu) and Zinc (Zn). Severe Se deficiency was produced in chicks by the use of diets that contained exceedingly low contents (less than 0.010 ppm) of Se, but contained adequate amounts of all other known essential nutrients. This diet was based upon corn and soybean meal produced in areas of China with endemic Se deficiency of geobotanical origin. A level of at least 0.10 ppm Se was found to be required to maintain normal Se status of chicks fed this diet, and Se deficiency resulted in decreased levels of Cu, Zn, and molybdenum in the pancreas (liver and plasma levels were not affected). High dietary supplementation of Zn nor Cu did not affect the short-term utilization of Se, as indicated by the 18-h responses of Se-dependent glutathione peroxidase in plasma and liver.