Determination of main and trace element contents in human brain by NAA and ICP-AES methods

A study was undertaken to determine the average values for elements in normal human brain (11 individuals, age group 65–75). Twelve brain parts were selected from both hemispheres. Determinations were carried out by NAA and ICP-AES. The main elements (Na, K, Mg, Ca, Fe, P, S) and trace elements (Al, B, Co, Cr, Cu, Mn, Ni, Pb, Zn) were investigated. Quality control was ensured by using NBS Bovine Liver SRM. The results obtained with independent methods were compared, and the data show a good correlation. On the basis of these investigations, the regional distribution of elements can be given.     

Accurate determination of cobalt traces in several biological reference materials

A newly devised, very accurate (“definitive”) method for the determination of trace amounts of cobalt in biological materials was validated by the analysis of several certified reference materials. The method is based on a combination of neutron activation and selective and quantitative postirradiation isolation of radiocobalt from practically all other radionuclides by ion-exchange and extraction chromatography followed by γ-ray spectrometric measurement. The significance of criteria that should be fulfilled in order to accept a given result as obtained by the “definitive method” is emphasized. In view of the demonstrated very good accuracy of the method, it is suggested that our values for cobalt content in those reference materials in which it was originally not certified (SRM 1570 spinach, SRM 1571 orchard leaves, SRM 1577 bovine liver, and Czechoslovak bovine liver 12-02-01) might be used as provisional certified values.     

Multielemental analysis of human fetal tissues using inductively coupled plasma-mass spectrometry

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to study the distribution of 26 major and trace elements in six tissues from 21 human fetuses aged 16–22 wk. Brain, lung, spleen, kidney, heart, and liver were analyzed following a microwave oven digestion step carried out according to clean techniques designed for ultratrace metal analyses. Precision and accuracy controls were conducted using standard reference material #1577b Bovine Liver. Significant differences among tissues were found for most of the elements. Essential trace elements seem to be increasingly retained as fetal tissues mature and become physiologically functional. The ranges of concentrations measured in fetal tissues at this stage of development are generally lower and much narrower than in adult tissues. The age of the fetus, which is not given in most studies, as well as the different techniques and levels of quality assurance could be responsible for the discrepancies in the trace metal concentrations reported here and in the literature. Intratissue homogeneity was also assessed in five human fetal brains. Frontal, occipital, parietal and temporal lobes, striatum, hippocampus, and thalamus were isolated and analyzed separately. No significant differences were found in the distribution of any of the elements at this stage of development. Because of the relatively narrow ranges of concentrations found for most elements, we believe that the results presented in this study represent the inorganic fingerprint of the main tissues of normal fetuses at midpregnancy for the Greater Montreal area.     

Photon activation analysis of several kinds of marine invertebrates for trace elements

The concentrations of trace elements in several mollusk, arthropod, echinoderm, and tunicata species were determined by photon activation analysis. The samples were freeze-dried, pulverized by a mill, and fractionated through a 200-mesh sieve. Three standard materials were used for comparative standards: NIST SRM-1566a Oyster Tissue, NIST SRM-1577b Bovine Liver, and NRCC TORT-1 Lobster. The samples and comparator standards were sealed in silica tubes and irradiated for 3 h by 30-MeV bremsstrahlung from a linear accelerator at Tohoku University. By measuring gamma-ray spectra, the concentrations of 16 elements (As, Br, Ca, Cr, Cu, Fe, I, Mg, Mn, Mo, Na, Ni, Pb, Rb, Sr, and Zn) were determined.     

Differentiation of Nijmegen breakage syndrome from Fanconi anemia

Nijmegen breakage syndrome (NBS) is a rare auto-somal recessive condition with chromosomal instability. Clinical and biological overlap between Fanconi anemia and ataxia telangiectasia has been reported. We report two cases of NBS born to consanguineous parents. Case one had NBS and Falconi anemia clinical features but relatively little chromosome breakage. The second case had mild NBS features, while cytogenetic evaluation with mitomycin C induction showed chromosome damage. Chromosomal analysis of bone marrow cells revealed tetraploidy, which indicates progression towards leukemia. On the basis of clinical and cytogenetic evaluation, these two cases were confirmed as NBS. However, detailed molecular studies are essential for accurate diagnosis and management of this disease.     

The application of preirradiation combustion and neutron activation analysis technique for the determination of iodine in food and environmental reference materials

The pre-irradiation combustion (PC) of samples to liberate iodine, followed by trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has been used at the National Institute of Standards and Technology for the determination of iodine in biological materials. The applicability of this technique to numerous environmental and dietary matrices is illustrated by analysis of a range of certified reference materials (CRMs) and a powdered grass material that was prepared as an in-house reference material (RM). Because of the combustion step involved, samples with low or no fat content (e.g., cereal products, selected botanical specimens, and nonfat milk powder) and inorganic materials (e.g., coal fly ash and dried sediments) are more suited for analysis by this method. In general, the results for several types of samples obtained by this method agreed with those obtained by a second radiochemical (R) NAA, as well as by a third method using inductively coupled plasma mass spectrometry (ICP-MS). PC-NAA is a useful technique for determining iodine in biological and environmental samples, especially for verification of iodine results obtained from other methods.     

Development of a stable isotope approach for the inductively coupled plasma-mass spectrometry determination of oxidized metallothionein in biological materials

The use of isotope dilution analysis (IDA) with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope ((109)Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements ((114)Cd/(111)Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP-MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP-MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive (109)Cd. The final procedure was applied to the quantification of MT in Long-Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference (109)Cd methodology. A good agreement between the analytical values obtained by both methods was observed.     

Comparison of SR-excited X-ray fluorescence analysis with neutron activation analysis for hair and fiber

Human scalp hair and some kinds of vegetable and animal fibers were analyzed by means of the SR excited X-ray fluorescence method (SRXFA) and the neutron activation method (NAA). Human hair samples collected from five males and five females were washed by the IAEA method prior to analysis. In the SRXFA analysis, samples were excited by monochromated X-rays. Fluorescence X-rays were measured by an Si(Li) detector. The elements detected in all hair samples were S, Ca, Cu, Fe, Zn, Br, and Sr. The elements K, Ti, Cr, Mn, Ni, Se, Hg, and Pb were also detected in several samples. After SRXFA analysis these same samples were analyzed by the NAA method. Elements such as Cu, Zn, and Br were detected by both methods, and their relative concentrations show a good agreement of variation between individuals. However, Pb was only detected by SRXFA, and Na, Au, and Sb were only detected by NAA. Therefore, these two methods are complementary to each other for trace element analysis.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

NBS1基因SNPs与中国汉族人群原发性肝癌的遗传易感性

为分析DNA损伤修复相关基因NBS1单核苷酸多态性(SNPs)与原发性肝癌遗传易感性的关系,并对高分辨率单链构象多态性(SSCP)检测技术在SNPs分型中的适用性进行评估,本研究对来自中国汉族人群的327例原发性肝癌以及295例阴性对照中NBS1基因常见SNPs的稀有等位基因频率进行检测和分析．此外,对NBS1基因6个常见SNPs分别选择部分样本同时进行直接序列测定,以比较2种方法的检测效果．119例原发性肝癌以及95例肝硬化/慢性肝炎组织标本的SSCP分析结果表明,6个常见NBS1基因SNPs位点(102G>A, 320+208G/A, 553G>C, 1197T>C, 2016A>G和2071-30A>T)中,SNP 1197T>C的稀有等位基因频率为68.1%,显著高于肝硬化/慢性肝炎对照的57.9% (P = 0.0298)．对该SNP位点另外采用208份肝细胞癌和200份健康人群血液标本进一步分析, 肝细胞癌SNP 1197T>C的稀有等位基因频率为66.8%,显著高于健康人群对照的58.8% (P = 0.0170)．其他5个SNPs的稀有等位基因频率在原发性肝癌与肝硬化/慢性肝炎之间均无显著性差异．高分辨率SSCP分析法与直接序列测定法对所选样本的SNPs基因分型结果完全一致,而且直接测序法对PCR扩增产物质量的要求相对高分辨率SSCP分析更高．研究表明,中国汉族人群NBS1基因SNP 1197T>C可能与原发性肝癌的发生相关,高分辨率SSCP技术准确度与直接测序法相当,且操作更加简便易行,非常适用于大量样本多个已知SNPs的基因分型．     

Validation of model virus removal and inactivation capacity of an erythropoietin purification process

Human erythropoietin (hEpo) production requires mammalian cells able to make complex post-translational modifications to guaranty its biological activity. As mammalian cell can be reservoir of pathogenic viruses and several animal origin components are usually used in the cultivation of mammalian cells, hEpo contamination with viruses is something of great concern. As consequence, this study investigated the viral removal and inactivation capacity of a recombinant-hEpo (rec-hEpo) purification process. Canine parvovirus, Human poliovirus type-2, Bovine viral diarrhea virus and Human immunodeficiency virus type-1 were used for measuring process viral removal and inactivation capacities. In conclusion, this study corroborated that the assessed rec-hEpo purification process has enough capacity (5.0–19.4 Logs) for removing and inactivating these model viruses and sodium hydroxide demonstrated to be a robust sanitization solution for chromatography columns (5.0 (PV-2)-6.7 (CPV) Logs).     

Multielement determination in small samples of human milk by inductively coupled plasma atomic emission spectrometry

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for routine analysis of small samples of human milk. The concentrations of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), and zinc (Zn) were determined in 203 milk samples from postpartum women at different stages of lactation after stepwise digestion in HNO₃, HCIO₄, and H₂O₂ under heat. Validation of the procedure was achieved using certified reference material of bovine liver (NBS 1577) with mean recoveries of 103.5%. The concentrations of the above elements in milk matrix were comparable with previously reported values. The analytical results from breast milk will provide reference information for mineral studies of Brazilian mothers and breast-fed infants.     

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.     

靶向NBS1基因的 microRNA真核表达载体的构建及其活性鉴定

目的:构建靶向NBS1基因的 microRNA真核表达载体,鉴定其转染宫颈癌细胞株Hela后的生物活性?方法:根据NBS1mRNA序列设计合成四对pre- microRNA片段,定向克隆到pcDNA 6.2- GW/ EmGFP-miR真核表达载体上,并将其转染至Hela细胞株中?采用菌落PCR和测序分析鉴定插入序列的完整性;采用实时定量PCR分析鉴定重组体对NBS1mRNA表达的干扰效果以确定其生物活性? 结果:构建的四组重组体插入片段的碱基序列完全正确?重组体能干扰Hela细胞NBS1基因的表达,四组重组体NBS1 mRNA表达量分别为: 0.24±0.17 (NBS1mi-1组)?0.12±0.12 (NBS1mi-2组)?0.41±0.97 (NBS1mi-3组)?0.48±0.93 (NBS1mi-4组),其中NBS1mi-2组表达最低? 结论: 构建的四组NBS1 microRNA重组体在Hela细胞株中都具有生物活性, 且NBS1mi-2组的干扰作用最强? 载体构建成功,为应用microRNA靶向NBS1的肿瘤基因治疗的研究奠定了基础。     

Influence of ashing techniques on the analysis of trace elements in biological samples

Dry ashing and wet ashing are two commonly used methods for the preparation of biological materials for trace element analysis by atomic absorption spectrophotometry. In this paper, National Bureau of Standards (NBS) bovine liver was dry ashed at 450°C for 24 h in silica glass (Vycor) or procelain crucibles; the resulting ash was dissolved in either concentrated nitric or hydrochloric acid. Dry ashing efficiency was evaluated by comparing iron, copper, zinc, and manganese concentrations of the samples with the values certified by NBS. Highest recoveries were obtained by dry ashing in silica glass (Vycor) crucibles. Dissolving the resultant ash in either hydrochloric or nitric acids did not significantly alter the results. A comparison between dry and wet ashing shows the latter method to be superior for the preparation of biological tissues for analysis of iron, copper, zinc, and manganese.     

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest.     

Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry

The analysis of trace elements in biological samples will extend our understanding of the impact that environmental exposure to these elements has on human health. Measuring arsenic content in nails has proven useful in studies evaluating the chronic body burden of arsenic. In this study, we developed methodology with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of total arsenic in nails. We assessed the utility of the washing procedures for removing surface contamination. Four types of preanalysis treatments (water bath, sonication, water bath plus sonication, and control) after sample decomposition by nitric acid were compared to evaluate the digestion efficiencies. In addition, we studied the stability of the solution over 1 wk and the effect of acidity on the arsenic signal. Arsenic content in the digested solution was analyzed by using Ar-N₂ plasma with Te as the internal standard. The results suggest that washing once with 1% Triton Χ-100 for 20 min for cleaning nail samples prior to ICP-MS analysis is satisfactory. Repeated measurement analysis of variance revealed that there was no significant difference among the various sample preparation techniques. Moreover, the measurements were reproducible within 1 wk, and acidity seemed to have no substantial influence on the arsenic signal. A limit of detection (on the basis of three times the standard deviation of the blank measurement) of 7 ng As/g toenail was achieved with this system, and arsenic recoveries from reference materials (human hair and nails) were in good agreement (95–106% recovery) with the certified/reference values of the standard reference materials. ICP-MS offers high accuracy and precision, as well as highthroughput capacity in the analysis of total arsenic in nail samples.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Determination of trace elements in human serum by inductively coupled plasma-mass spectrometry

The determination of trace and ultratrace elements in human serum by ICP-MS is described. The accuracy of the method is tested using a “second generation” human serum reference material. Elements determined include Fe, Co, Cu, Zn, Br, Rb, Sr, Mo, and Cs. The method is compared to nuclear analytical methods (NAA, PIXE). Perspectives for the future are also discussed.     

Trace-element contents of human fetal liver of 12–22 weeks gestation

There are some papers in the literature on the trace element contents of fetal livers of 20-wk gestation time and over. However, there is very little information on this subject for fetal livers of less than 20-wk gestation. We have initiated a program on the measurement of trace elements in fetal livers of 12–22-wk gestation, using thick-target X-ray fluorescence analysis. The liver samples were obtained from freshly aborted fetuses. After removing blood from the samples, they were chopped into small pieces and freeze dried. The resulting material was ground into fine powder and compressed into 3-mm thick pellets, with boric acid backing. A similar pellet was also made of NBS—Bovine Liver—which was used as the standard for calculating the absolute concentrations of different trace elements. The measurements were carried out using a commercial Wave-Length-Dispersive XRF-System. Different X-ray tubes were used for different sets of elements in order to maximize the detection sensitivity. The results are compared with those of fetal liver of longer gestation and adult livers.