Improved HPLC method for total plasma homocysteine detection and quantification

Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R=0.999), range (<2-100 microM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B(6), on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.     

A rapid method for simultaneous determination of zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol in traditional Chinese medicines by ultra-high-performance liquid chromatography-tandem mass spectrometry

A rapid and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of zearalenone (ZEN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) in traditional Chinese medicines (TCMs) was developed. The development of the method and investigations of the matrix influence were described in particular. After evaluation of the matrix effects of different TCMs, i.e., rhizomes and roots, seeds, flowers, grasses and leaves, by the post-extraction spiked method, a reliable sample clean-up method based on home-made clean-up cartridges, a suitable internal standard and the matrix calibration were combined using to minimize the matrix effects to ensure the accuracy of the method. The established method was further validated by determining the linearity (R(2)≥0.9990), sensitivity (limit of quantitation 0.11-0.99 ng mL(-1)), average recovery (86.6-113.5%) and precision (relative standard deviation ≤13.5%). It was shown to be a suitable method for simultaneous determination of ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL in different TCMs. Finally, the established method was successfully applied to the determination of the six mycotoxins in various TCMs and the results were presented to provide relevant insights to researchers in TCM analysis.     

Calculating glucose fluxes during meal tolerance test: a new computational approach

A new calculation method is proposed to quantify the endogenous glucose production (EGP), the glucose appearance rate due to meal ingestion (R(a meal)), and the glucose disposal (R(d)) during a three-tracer study design. The method utilizes the maximum likelihood theory combined with a regularization method to achieve a theoretically coherent computational framework. The method uses the two-compartment formulation of the glucose kinetics. Instead of assuming smoothness of unlabeled and labeled glucose concentrations, the method assumes that the EGP, the R(a meal), and the fractional glucose clearance are smooth, increasing plausibility of their individual estimates. The method avoids transformation of the measurement errors, which may skew the estimates of the EGP, R(a meal), and R(d) with the traditional approach. Finally, the sequential nature of the calculations is replaced by calculating the EGP, R(a meal), and R(d) in "one go" to avoid the propagation of the errors from the EGP and R(a meal) into R(d). An example study is shown demonstrating the utility of the approach. A better performance of the new method is demonstrated in a simulation study.     

Determination of component volumes of lipid bilayers from simulations.

An efficient method for extracting volumetric data from simulations is developed. The method is illustrated using a recent atomic-level molecular dynamics simulation of L alpha phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer. Results from this simulation are obtained for the volumes of water (VW), lipid (V1), chain methylenes (V2), chain terminal methyls (V3), and lipid headgroups (VH), including separate volumes for carboxyl (Vcoo), glyceryl (Vgl), phosphoryl (VPO4), and choline (Vchol) groups. The method assumes only that each group has the same average volume regardless of its location in the bilayer, and this assumption is then tested with the current simulation. The volumes obtained agree well with the values VW and VL that have been obtained directly from experiment, as well as with the volumes VH, V2, and V3 that require certain assumptions in addition to the experimental data. This method should help to support and refine some assumptions that are necessary when interpreting experimental data.     

A simplified method of quantitating protein using the biuret and phenol reagents.

A new modification of the Lowry method of quantitating protein is introduced, whereby the protein sample is mixed first with a diluted biuret reagent and later with 2 n phenol reagent (undiluted) for color development. The method is superior to the original in (i) extremely stable color development (0.3% change from 20 min to 2 hr), (ii) good reproducibility (±2% for 50–600 μg/ml of protein), (iii) elimination of the need to mix reagents for each assay, (iv) good storability (the diluted biuret reagent is storable for months), (v) simplicity (both reagents are available commercially), and (vi) the biuret method can be immediately converted to the Lowry method if the former does not yield a sufficient absorbance. It was found that the relationship between absorbance and protein concentration is expressed by a straight line with a slope of 1 in the Hill plot.     

Reliability of surface electromyography timing parameters in gait in cervical spondylotic myelopathy

The aims of this study were to validate a computerised method to detect muscle activity from surface electromyography (SEMG) signals in gait in patients with cervical spondylotic myelopathy (CSM), and to evaluate the test–retest reliability of the activation times designated by this method. SEMG signals were recorded from rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG), during gait in 12 participants with CSM on two separate test days. Four computerised activity detection methods, based on the Teager–Kaiser Energy Operator (TKEO), were applied to a subset of signals and compared to visual interpretation of muscle activation. The most accurate method was then applied to all signals for evaluation of test–retest reliability. A detection method based on a combined slope and amplitude threshold showed the highest agreement (87.5%) with visual interpretation. With respect to reliability, the standard error of measurement (SEM) of the timing of RF, TA and MG between test days was 5.5% stride duration or less, while the SEM of BF was 9.4%. The timing parameters of RF, TA and MG designated by this method were considered sufficiently reliable for use in clinical practice, however the reliability of BF was questionable.     

Liquid chromatography method for plant and mammalian lignans in human urine

Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC-MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.     

Degradation of bacterial cyclopropane acids with boron trihalide reagents

The cellular fatty acid compositions of Pseudomonas diminuta UC 501 and Streptococcus mutans OMZ-61 were compared in samples processed by a saponification-methylation procedure (method S) and in samples processed by a transesterification procedure (method T). All samples were heated in an inert atmosphere of nitrogen. The major acids found in samples of P. diminuta treated by method S included 16:0 (palmitic), 18:1 (octadecenoic) and 19 cyc (19 carbon cyclopropane acid). Those found in samples of S. mutans treated by the same method included 16:0, 18:1. 18:0 (stearic), 20:1 (eicosenoic), 20:0 (eicosanoic), 19 cyc and 21 cyc. When method T was used to process samples of both cultures, the cyc acids were degraded and artifacts were produced. Transesterification with boron trihalide reagents is therefore not recommended for routine analysis of bacterial fatty acids.     

Determination of cocaine, its metabolites, pyrolysis products, and ethanol adducts in postmortem fluids and tissues using Zymark automated solid-phase extraction and gas chromatography-mass spectrometry

Demonstrating the presence or absence of cocaine (COC) and COC-related molecules in postmortem fluids and/or tissues can have serious legal consequences and may help determine the cause of impairment and/or death. We have developed a simple method for the simultaneous determination of COC and the COC metabolites benzoylecgonine (BE), norbenzoylecgonine (NBE), ecgonine methyl ester (EME), ecgonine (E), and norcocaine (NCOC), as well as anhydroecgonine methyl ester (AEME) (a unique byproduct of COC smoking), cocaethylene (a molecule formed by the concurrent use of COC and ethanol) and their related metabolites, anhydroecgonine (AE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE). This method incorporates a Zymark RapidTrace automated solid-phase extraction (SPE) system, gas chromatography/mass spectrometry (GC/MS) and 2,2,3,3,3-pentafluoro-1-propanol (PFP)/pentafluoropropionic anhydride (PFPA) derivatives. The lower limits of detection ranged from 0.78 to 12.5 ng/mL and the linear dynamic range for most analytes was 0.78-3200 ng/mL. The extraction efficiencies were from 26 to 84% with the exception of anhydroecgonine and ecgonine, which were from 1 to 4%. We applied this method to five aviation fatalities. This method has proven to be simple, robust and accurate for the simultaneous determination of COC and 11 COC metabolites in postmortem fluids and tissues.     

Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein

The activation of peroxisome-proliferator-activated receptor-γ (PPARγ), which plays a central role in adipocyte differentiation, depends on ligand-dependent co-activator recruitment. In this study, we developed a novel method of PPARγ ligand screening by measuring the increase in fluorescent polarization accompanied by the interaction of a fluorescent co-activator and PPARγ. Sterol receptor co-activator-1 (SRC-1), a major PPARγ co-activator, was probed by fluorescent TAMRA by the Amber codon fluorescence probe method. Polarization was increased by adding PPARγ ligands to a solution containing labeled SRC-1 (designated TAMRA-SRC-S) and PPARγ. The disassociation constants (Kd) of the PPARγ synthesized ligands, pioglitazone (221 nM), troglitazone (83.0 nM), and 15-deoxy-Δ12,14-prostaglandin J(2) (15d-ΔPGJ(2)) (156 nM), were determined by this method. Farnesol (2.89 μM) and bixin (21.1 μM), which we have reported to be PPARγ ligands, increased the fluorescent polarization. Their Kd values were in agreement with the ED(50) values obtained in the luciferase assay. The results indicate that the method is valuable for screening natural PPARγ ligands.     

Local sequence information-based support vector machine to classify voltage-gated potassium channels

In our previous work,we developed a computational tool,PreK-ClassK-ClassKv,to predictand classify potassium (K~ ) channels.For K channel prediction (PreK) and classification at family level(ClassK),this method performs well.However,it does not perform so well in classifying voltage-gatedpotassium (Kv) channels (ClassKv).In this paper,a new method based on the local sequence information ofKv channels is introduced to classify Kv channels.Six transmembrane domains of a Kv channel protein areused to define a protein,and the dipeptide composition technique is used to transform an amino acid sequenceto a numerical sequence.A Kv channel protein is represented by a vector with 2000 elements,and a supportvector machine algorithm is applied to classify Kv channels.This method shows good performance withaverages of total accuracy (Acc),sensitivity (SE),specificity (SP),reliability (R) and Matthews correlationcoefficient (MCC) of 98.0%,89.9%,100%,0.95 and 0.94 respectively.The results indicate that the localsequence information-based method is better than the global sequence information-based method to classifyKv channels.     

流式细胞术检测组蛋白乙酰化水平方法的建立与应用

组蛋白去乙酰化酶抑制剂是近年来出现的一类新的抗肿瘤药物,在艾滋病等其他疾病中同样也受到关注。但是在基础和临床研究中,目前还缺乏统一可靠的组蛋白乙酰化水平的检测手段。本文利用全血和外周血单个核细胞,通过一系列的对比实验,比较了不同样品处理温度(冰上和室温)、破膜方法(细胞内因子染色破膜和核内因子染色破膜)、抗体剂量(抗体滴定)和抗体孵育时间(时间梯度)等实验条件对流式细胞术检测的影响,最终建立了一套基于流式细胞术的组蛋白乙酰化水平检测手段。同时,将优化后的流式细胞检测技术应用于西达本胺(目前国内唯一上市的组蛋白去乙酰化酶抑制剂)的体外实验和临床试验,结果均证明本文建立的组蛋白乙酰化流式细胞检测方法可以作为基础和临床研究中一个可靠、快速、便捷的检测手段。     

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography-mass spectrometry

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI-) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.     

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.     

Detection and quantitation of human enteric viruses in waste waters: increased sensitivity using a human immune serum globulin--immunoperoxidase assay on MA-104 cells

This study demonstrates that the most sensitive method for the detection and quantitation of cultivable human enteric viruses in water samples after repassage in the MA-104 cell line is the detection of infected cells by the human immune serum globulin--immunoperoxidase (HISG-IP) method recently described by the authors. This immunoperoxidase method is up to 50 times more sensitive than a liquid overlay assay by cytopathic effect in BGM cells. The viral content of waste waters was evaluated with this new methodology. By this method the average viral content of raw sewage (RS) was 900 mpniu/L (most probable number of infectious units per litre), 1056 mpniu/L in primary effluent (PE), and 106 mpniu/L in secondary effluent (SE). With a cytopathic effect assay on BGM cells, values of 85 (RS), 56 (PE), and 2 (SE) mpniu/L were observed, a striking underestimation of the viral content of secondary effluents.     

棉叶中棉酚的快速提取与测定方法研究

棉酚(G)及其相关物甲氧基半棉酚(DHG)、半棉酚酮(HGQ)、半棉酚(HG)、杀实夜蛾素(H1-4)等是棉花中重要的抗虫性萜烯类次生物质.利用高效液相色谱法(HPLC)对棉酚及其相关物进行了分离,测定了棉叶中的棉酚含量,讨论了不同的提取方法和测定条件对结果的影响,给出了一套简便、快速的分析测试方法,同时与紫外-可见分光光度法(苯胺法)的结果进行了对比,认为对于棉花的抗虫性的研究来说,HPLC是比较适宜的方法.     

300 株鲍曼不动杆菌琼脂稀释法与纸片扩散法药敏检测结果比较

目的:比较琼脂稀释法和纸片扩散法对鲍曼不动杆菌的药敏试验结果。方法:随机挑选的300株鲍曼不动杆菌,检测其标本及科室的分布情况,并采用琼脂稀释法和纸片扩散法检测鲍曼不动杆菌对环丙沙星(CIP)、庆大霉素(CN)、阿米卡星(AK)、头孢他啶(CAZ)、头孢吡肟(FEP)、左氧氟沙星(LEV)、氨苄西林-舒巴坦(SAM)、妥布霉素(TOB)、美洛培南(MEN)、米诺环素(MH)、头孢哌酮-舒巴坦(SCF)11种抗菌药物的敏感性,比较两种检测结果的差异。结果:300株鲍曼不动杆菌主要分布在痰液标本中,共214株,占71.3%,主要来源于ICU 101株(33.7%)及脑外科59株(19.7%)。药敏检测结果显示,两种检测方法所得的SCF和MH的敏感性差异具有统计学意义(P0.05),其他9种抗菌药物的药敏检测结果差异没有统计学意义(P0.05)。结论:琼脂稀释法和纸片扩散法对鲍曼不动杆菌药敏试验结果并不完全一致,临床用药时尤其要注意SCF和MH这两种药物药敏结果的可靠性。     

Changes in activity coefficient gamma(w) of water and the foaming capacity of protein during hydrolysis

The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity a(w), and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for a(w), and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, gamma(w) (=a(w)/x(w), where x(w) is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate x(w) in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2,4,6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum gamma(w) value and maximum foaming capacity.