Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

Synthesis and characterization of a molecularly imprinted polymer and its application as SPE enrichment sorbent for determination of trace methimazole in pig samples using HPLC-UV

A novel molecularly imprinted polymer that could be applied as enrichment sorbent was prepared using methimazole (MMZ) as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker. Though evaluated by static, kinetic and competitive adsorption tests, the polymer exhibited high adsorption capacity, fast kinetics and good selective ability. A method for determination of trace MMZ was developed using this polymer as enrichment sorbent coupled with high performance liquid chromatography focusing on complex biological matrices. Under the optimum experimental conditions, the MMZ standard is linear within the concentration range studied, that is, from 0.5 μg L⁻¹ to 150 μg L⁻¹ (r² = 0.9941). Lower limits of detection (LOD, at S/N = 3) and quantification (LOQ, at S/N = 10) in pig samples were 0.63 μg kg⁻¹ and 2.10 μg kg⁻¹ for kidney, 0.51 μg kg⁻¹ and 1.70 μg kg⁻¹ for liver, 0.56 μg kg⁻¹ and 1.86 μg kg⁻¹ for muscle, respectively. Recoveries and relative standard deviation (RSD, n = 9) values for precision in the developed method were from 71.14% to 88.41% and from 2.53% to 6.18%.     

Analyses for phosphatidylcholine hydroperoxides by LC/MS

A new liquid chromatography mass spectrometry (LC/MS) method has been developed for the qualitative and quantitative analyses of phosphatidylcholine hydroperoxides (PC-OOH) in human plasma using a synthetic hydroperoxide (1-stearoyl-2-erucoyl-PC monohydroperoxide, PC 18:0/22:1-OOH) as an internal standard. 1-Stearoyl-2-linoleoyl-PC monohydroperoxide (PC 18:0/18:2-OOH) was identified in plasma by LC/MS by comparison with an authentic standard. The calibration curves obtained for 1-palmitoyl-2-linoleoyl-PC monohydroperoxide, PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were linear throughout the calibration range (0.1–1.0 pmol). The limit of detection (LOD) (S/N = 3:1) was 0.01 pmol, and the limit of quantification (LOQ) (S/N = 6:1) was 0.1 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 89 and 32 nM, respectively, in a healthy volunteer.     

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20 μM). Importantly, when the 5 μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.     

Tracheal compliance and limit flow rate changes in a murine model of asthma

Trachea is the unique passage for air to flow in and out. Its tone is of importance for the respiration system. However, investigation on how tracheal tone changes due to asthma is limited. Aiming at studying how the mechanical property changes due to asthma as well as the compliance and flow limitation, the following methods are adopted. Static and passive pressure-volume tests of rats' trachea of the asthmatic and control groups are carried out and a new type of tube law is formulated to fit the experimental data, based on which changes of compliance and limit flow rate are investigated. In order to give explanation to such changes, histological examinations with tracheal soft tissues are made. The results show that compliance, limit flow rate and material constants included in the tube law largely depend on the longitudinal stretching ratio. Compared with the control group, the tracheal compliance of asthmatic animals decreases significantly, which results in an increased limit flow rate. Histological studies indicate that asthma can lead to hyperplasia/hypertrophy of smooth muscle cells, and increase elastin and collagen fibres in the muscular membrane. Though decreasing compliance increases sta- bility, during the onset of asthma, limit flow rate is much smaller due to the lower transmural pressure. Asthma leads to a stiffer trachea and the obtained results reveal some aspects relevant to asthma-induced tracheal remodelling.     

Growth response of pikeperch larvae in relation to body size and zooplankton abundance

The most critical period at onset of feeding in pikeperch Stizostedion lucioperca is short (<5 days at 20° C). The larvae are sensitive to prey density during the first week of exogenous feeding. First-feeding larvae of 6.5 mm total length ( L_T ) needed prey densities of >585 prey l⁻¹ to maintain mass (C_maint), whereas 5 days older larvae of 7 mm L _T C_maint= 55 prey l⁻¹ and for 11 mm L _T larvae C_maint<10 prey 1⁻¹. Changes in specific growth rates for larvae of 7 and 11 mm were similar to a type-II functional response curve reaching a specific growth rate of 26 and 30% day⁻¹, respectively, at 1000 prey l⁻¹, whereas the 6·5 mm larvae showed a linear growth response reaching a specific growth rate of only 9% day ⁻¹ at 1000 prey l⁻¹. The results suggest that prey density is a limiting factor, which might contribute to the high variation between year-class Strengths.     

衰老及相关基因群

综述20世纪与基因相关的衰老原理的探索及其进展,整体动物水平的衰老研究归纳了衰老了诸多表象但疏于对衰老本质的探讨。线粒体－自由基衰老学说阐述了线粒体DNA的损伤与衰老有很大的相关性,由Hayflic k分裂限制衍生的端粒衰老学说给衰老机制提供了重要信息,目前狭隘的基因程序化衰老学说已和损伤衰老概念有机的联系在了一起。总之,自由基衰老学说得到了氧化衰老学说和糖基化衰老学说的补充逐渐形成了生化副反应与基因衰老学说的大统一衰老机制板块理论。