Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).     

Proteoglycan expression in the rat temporomandibular joint in response to unilateral bite raise

We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3alpha-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 microL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 microL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 microL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 microL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5-8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.     

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.     

Efficacy of gloves in reducing blood volumes transferred during simulated needlestick injury

This study was designed to evaluate factors that affect blood volumes transferred to skin during simulated needlestick injuries in an in vitro paper prefilter model and an ex vivo porcine tissue model. The effect of needle type and size, penetration depth, and glove use on the volume of radiolabeled blood transferred was determined in each model. Blood volumes ranged from 0.47 +/- 0.26 microL (30-gauge needle, 0.5-cm depth, in vitro model) to 5.88 +/- 1.45 microL (18-gauge needle, 2.0-cm depth, in vitro model). Needle size and penetration depth were significantly associated with transfer volume. Glove material reduced the transferred blood volume by 46%-86% in both models. Transfer volumes were within the same order of magnitude for all conditions. Hence, virus titer in the source blood may be a better predictor of needlestick infectivity than is exposure volume. Regardless, gloves may exert some protective effect and should be worn whenever needles are handled.     

An HPLC-GC/MS reference method for serum total cholesterol with control for ester hydrolysis

Currently used isotope-dilution mass spectrometry methods for serum total cholesterol are performed without control in each sample for completeness of hydrolysis of cholesterol esters. In order to monitor this step in the analysis, we developed a method based on both high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). 13C3-cholesterol and cholesteryl [1-14C] oleate were added to serum that was saponified and extracted into hexane. The extract was subjected to HPLC with collection of the fractions corresponding to cholesterol and cholesteryl oleate. The radio-activity of the latter was counted in order to estimate the nonhydrolysed fraction that on average amounted to less than 0.1% for commonly used ester hydrolysis procedures. The cholesterol fraction was subjected to GC/MS for quantitation using the traditional isotope-dilution principle. Evaluation of accuracy by assaying sera from the National Institute of Standards and Technology showed a deviation from the target value of < 1% with a coefficient of variation of < or = 1.0%. In conclusion, the present reference method for serum total cholesterol assures results based on complete hydrolysis of the cholesterol ester fraction.     

Comparison of two community alternatives to incarceration for chronic juvenile offenders

The relative effectiveness of group care (GC) and multidimensional treatment foster care (MTFC) was compared in terms of their impact on criminal offending, incarceration rates, and program completion outcomes for 79 male adolescents who had histories of chronic and serious juvenile delinquency. Results show that boys who participated in MTFC had significantly fewer criminal referrals and returned to live with relatives more often. Multiple regression analyses showed that assignment to a treatment condition (i.e., GC or MTFC) predicted official and self-reported criminality in follow-up beyond other well-known predictors of chronic juvenile offending (i.e., age at 1st offense, number of previous offenses, age at referral).     

Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients

A simple and inexpensive methacrylate rehydration chamber was built to accommodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 microL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.     

Purification and characterization of endothelin-1 degradation activity from porcine kidney

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway.     

Novel preconcentration technique for on-line coupling to high-speed narrow-bore capillary gas chromatography: sample enrichment by equilibrium (ab)sorption. II. Coupling to a portable micro gas chromatograph

The technique of equilibrium (ab)sorption has been proven to be a powerful method for preconcentration of gaseous samples for high-speed narrow-bore capillary gas chromatography (GC) in general and field-portable GC instruments, often referred as micro GCs, in particular. Using a simple experimental set-up equipped with an open-tubular enrichment column it is possible to produce a homogeneously enriched sample plug, allowing reproducible injections of an enriched sample into the micro GC. Using a non-polar trapping column enrichment factors found for n-alkanes in the range of C7 to C10 ranged from 15 to 150 and agree well with calculated values. Using a highly retentive Thermocap column, the enrichment factor observed for heptane was above 500. As the use of this new preconcentration method requires only minimum modification of the micro GC, the chromatographic performance of the instrument was not compromised by direct coupling to the preconcentration device. Examples of on-line enrichment with portable micro GC analysis of VOCs from air are shown. These examples clearly demonstrate the potentials of the new method in field analysis.     

Using conceptual models to teach BASIC computer programming.

Novices learned BASIC computer programming from a standard manual that emphasized the language's syntactic features or from a standard manual that included additional material emphasizing the underlying semantics (e.g., conceptual models). Although both kinds of training produced equivalent learning of the syntactic features of BASIC (i.e., syntactic knowledge), the semantically trained students developed fewer misconceptions (i.e., conceptual knowledge) and performed better on transfer tests of programming problem solving (i.e., strategic knowledge). In addition, problem-solving performance was strongly related to measures of conceptual knowledge. The results suggest that learning BASIC programming involves the growth of syntactic, conceptual, and strategic knowledge. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Self- and supervisory perspectives on age and work attitudes and performance.

Person- and context-oriented definitions of age were used to predict 3 sets of work outcomes: work attitudes, performance ratings, and reports of developmental practices. The 5 age measures included employee chronological age, employee subjective age (i.e., self-perceptions of age), and social age (i.e., others' perceptions of age), as well as self- and supervisors' perceptions of the employee's relative age (i.e., compared with the employee's work group). The study assessed (1) the relationships among the age measures, (2) the additive relationships among the age measures that predicted work outcomes, and (3) the interactive relationships among the age measures that predicted work outcomes. Each prediction received some support except for number (2). Furthermore, many of the age–work-outcome relationships were replicated in the managerial sample. Implications for the use of alternative age measures are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Influence of Zn Addition on Microstructures and Martensitic Transformation in CuZr-based Alloys

Compositional dependences on microstructures and martensitic transformation behaviors in(Cu_(0.5)Zr_(0.5))_(100-x)Zn_x(x=1.5,2.5,4.5,7.0,10.0,and 14.0at.%)alloys were investigated.It was found that CuZr martensites were present in the present alloys.With increasing Zn content,the volume fractions of CuZr martensitic crystals and B2 CuZr phase gradually decrease and increase,respectively.With the addition of high Zn contents(i.e.,7.0,10.0,and 14.0at.%),the matrix proves to be eutectic.Thermal analysis results show that the initial martensitic transformation temperature(M_s)decreases from(412±5)K to(329±5)K as the Zn content increases from 1.5at.% to14.0at.%.The values of Msof Cu-Zr-Zn shape memory alloys are inversely proportional to the number and concentrations of valence electrons(i.e.,e_v/a and c_v),respectively,implying that the martensitic transformation in CuZrZn alloys could be of electronic nature.     

进口含铁物料固体废物属性鉴别的探讨

含铁物料来源广泛、成分复杂,其中不乏我国限制或禁止进口的固体废物。不法商贩以废充好、掺杂废物等方式进口含铁物料的现象时有发生。这些物料仅从铁含量和外观上很难判别其属性。实验通过分析几种不同来源含铁物料样品的特征和来源,得出进口含铁物料固体废物属性鉴别的方法。其方法主要包括以下5个步骤:(1)考察样品的外观特征,包括样品颜色、状态、手感、气味、能否被磁铁吸附以及显微镜下观察等;(2)分析样品的理化组成,包括X射线荧光光谱(XRF)分析和X射线衍射(XRD)分析以及其他必要的测试分析,分别获得样品的元素组成、元素含量信息和样品的物相组成、物相含量信息等;(3)查证文献资料等,具体是根据以上获得的样品信息,查阅相关的文献资料、调研、咨询专家等,对样品可能产生的来源、工艺进行分析查证;(4)确立样品的来源,即通过以上的考察、分析、查证等,推断并确立样品的来源;(5)判别样品的固体废物属性,根据《固体废物属性鉴别导则》(试行)给出样品是否属于固体废物的判定。如果样品是固体废物,可参照《进口废物管理目录》给出限制类别的判定。根据上述鉴定步骤,对送检的含铁尘泥、浸出渣、磨屑泥和尾矿进行了鉴别,判定这些样品均属于我国目前禁止进口的固体废物。     

Limited-sample NMR using solenoidal microcoils, perfluorocarbon plugs, and capillary spinning

This study demonstrates three improvements to mass-limited NMR using solenoidal microcoils as detectors: (1) sample confinement using liquid perfluorocarbon plugs to increase the observe factor, (2) design and incorporation of a capillary spinner to improve spectral line widths, and (3) facile sample changing via the use of a capillary insert. The probe is constructed to spin a fused silica capillary of 530 microns i.d., 700 microns o.d. inside a solenoidal coil wound around a 0.8 mm i.d., 1 mm o.d. glass capillary. The smaller capillary contains the sample, and capillaries with different samples can be exchanged easily. In high-resolution limited sample microcoil NMR studies published thus far, the length of the sample plug has been 7-10 times the length of the solenoid to avoid line broadening from volume magnetic susceptibility (chi v) mismatches at both ends of the sample. This arrangement is not efficient since it places most of the sample volume outside of the coil observe volume. It is shown here that the observe factor cannot exceed 23% if the sample plug is bracketed by air, without substantial line broadening occurring. However, if the sample is bracketed by two liquid perfluorocarbon plugs, the observe factor can be increased to 70% while maintaining high spectral resolution. This is equivalent to improving the limit of detection by a factor of 3, or reducing the total data acquisition time for a given signal-to-noise by a factor of 9. It is also shown that, for the 440-nL sample plug used in this study (bracketed by the perflurocarbon plugs), sample spinning can improve the spectral resolution from 1.5 (nonspinning) to 0.6 Hz (spinning). This corresponds to a further improvement in the limit of detection of 2.5, or just over a factor of 6 decrease in data acquisition time.     

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.     

Schoolwide social skills training: Providing preventive services to students at-risk.

Describes a schoolwide social skills assessment and training model including a 2-gate student identification procedure (i.e., in-class social skills training for all students and small group intensive training for at-risk target students). The model is directed at reducing future serious problems by addressing identified difficulties early (i.e., through secondary prevention procedures). Specific and model objectives are to (1) use a comprehensive, multigate procedure to identify students at risk; (2) implement schoolwide training procedures to teach a number of important prosocial skills identified by teachers; (3) treat a subgroup of target Ss considered at highest risk for developing pervasive social problems; and (4) implement procedures to encourage the transfer and generalization of all students' skills across untrained stimuli, settings, and situations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The mental representation of numerical fractions: Real or integer?

Numerical fractions are commonly used to express ratios and proportions (i.e., real numbers), but little is known about how they are mentally represented and processed by skilled adults. Four experiments employed comparison tasks to investigate the distance effect and the effect of the spatial numerical association of response codes (SNARC) for fractions. Results showed that fractions were processed componentially and that the real numerical value of the fraction was not accessed, indicating that processing the fraction's magnitude is not automatic. In contrast, responses were influenced by the numerical magnitude of the components and reflected the simple comparison between numerators, denominators, and reference, depending on the strategy adopted. Strategies were used even by highly skilled participants and were flexibly adapted to the specific experimental context. In line with results on the whole number bias in children, these findings suggest that the understanding of fractions is rooted in the ability to represent discrete numerosities (i.e., integers) rather than real numbers and that the well-known difficulties of children in mastering fractions are circumvented by skilled adults through a flexible use of strategies based on the integer components. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Optimization of Settling Tank Design to Remove Particles and Metals

Mass reduction rates of particles and metals were simulated for a two-compartment settling tank composed of a storage compartment and a continuous flow compartment. Particle-size distribution, rainfall, and flow data from 16 storm events measured at three highway sites were used. The volume ratio (i.e., ratio of surface areas for a given depth) between storage and continuous flow compartment was optimized for a given design storm size to maximize total mass reduction rates of particles and heavy metals. Measured settling velocity profiles of runoff samples were used in the simulation. Simulation results showed that in a given total design storm, larger storage compartment fractions (>0.95) enhanced the removal of smaller particles (2–104?μm) and particulate phase metals, and even a small fraction (<0.05) of continuous flow compartment effectively removed larger particles (104–1,000?μm). A volume fraction of 0.75 for the storage compartment is suggested to optimize annual reductions of particles and associated heavy metals.