Cobalt determination in serum and urine by electrothermal atomic absorption spectrometry

Cobalt determinations in biological fluids are of great interest in biological or toxicological research programs. Cobalturia is often chosen as an indicator for a biological monitoring program in occupational exposure to cobalt dusts. The method described here derives from the IUPAC reference method for nickel determination. It enables cobaltemia and cobalturia to be measured in small samples (1 mL). The mean usual values for cobalt in biological fluids are very low (2.7 nmol L⁻¹ for serum and 6.7 nmol L⁻¹ for urine), and therefore, thus require an analytical procedure with preconcentration and extraction. The sample is mineralized by wet acid digestion. After digestion, inorganic cobalt is extracted in form of ammonium pyrrolidine dithiocarbamate complex into isobutyl methyl ketone and measured in the organic layer by electrothermal atomic absorption spectrometry. The analytical parameters are described in detail. The extraction output is about 99%. The detection limits are 1.93 and 1.89 nmol L⁻¹ for serum and urine, respectively. Sensitivity (expressed as the concentration that gives a 0.044 absorbance) is 3.4 nmol L⁻¹ for serum and 3.3 nmol L⁻¹ for urine. Within-run precision ranged between 3.9 and 2.5% (coefficients of variation) for serum and 4.2 and 1.1% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. Between-run precision ranged between 4.3 and 3.3% (coefficients of variation) for serum and 4.2 and 2.3% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. At very low concentration, 5.7 nmol L⁻¹ for serum and 2.5 nmol L⁻¹ for urine, the between-run precision is, respectively, 19.5 and 28%. Linearity is effective between 0 and 272 nmol L⁻¹. Interferences and matrix effects are negligible for urine, serum, or plasma samples without hemoglobin. The method is easily applicable for routine determinations.     

大阪鲫鱼（Carassius Auratus cuvieri Temminck et．Schlegel）雌性特异血清蛋白生化特性及免疫细胞化学定位的研究

雌性特异血清蛋白存在于成熟雌性大阪鲫鱼血清中,雄鱼则无。注射雌二醇可诱导雄鱼及成熟雌鱼合成这种蛋白质并引起鱼肝细胞粗面内质网增加,糖元颗粒减少。从诱导的大阪鲫鱼血清中分离出电泳纯的雌性特异血清蛋白的分子量为４６６０００±４０００（ｎ＝１０）,电泳谱带有３条,用纯的雌性特异血海蛋白制备的兔抗鱼抗血清不与雄鱼血清发生免疫反应,而与正常成熟雌鱼及诱导鱼的血清形成１条免疫沉淀线。免疫细胞化学定位诱导及成熟雌鱼的肝细胞核外细胞质有阳性颗粒,在卵母细胞外围有成团的阳性颗粒分布。     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

肺癌患者与肺结核患者血清蛋白的电泳分析

采用SDS聚丙烯酰胺凝胶电泳银染色的方法,对58例肺癌患者和32例肺结核患者的血清蛋白进行比较分析,发现有五种特异蛋白,分别存在于A区、C区、F区和G区。肺癌组和肺结核组比较,存在极显著性差异(P<0.01),提示有早期诊断肺癌的价值,有可能成为临床诊断肺癌的一个重要参考指标。     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.