Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Fast HPLC Estimation of γ-Aminobutyric Acid in Microdialysis Perfusates: Effect of Nipecotic and 3-Mercaptopropionic Acids

A method for rapid, automated (less than 5 min), and sensitive (detection limit 50 fmol/10 microliter) determination of gamma-aminobutyric acid (GABA) is described. The method is based on precolumn derivatization with o-phthaldialdehyde/t-butylthiol reagent and separation by reverse-phase HPLC with electrochemical detection under isocratic conditions. A 100 X 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3-mercaptopropionic acid is injected at a dose of 100 mg/kg i.p. There was a 15-fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.     

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.     

Alcohol dehydrogenase-coupled spectrophotometric assay of plasmalogenase

Plasmalogenase has been assayed by conversion of the fatty aldehydes, released by hydrolysis of the vinyl ether bond of plasmalogens, to long-chain alcohols by horse liver alcohol dehydrogenase. The reaction was followed spectrophotometrically by measuring the oxidation of NADH. The assay is sufficiently sensitive to enable plasmalogenase activity to be determined in isolated oligodendroglia and derived membranes and in brain microsomal membranes using 50-250 micrograms protein.     

Improved detection of iris severe mosaic virus in secondarily-infected iris bulbs

The influence of wounding and high-temperature treatment on the detection of iris severe mosaic virus (ISMV) in secondarily ISMV-infected iris bulbs was studied. Wounding of the bulbs just after lifting, followed by storage for 3 wk at 17°C or 20°C, increased the detectability of ISMV to 100% reliability. High-temperature treatment and consecutive storage at 17°C induced a similar improvement of detection. It is concluded that a certain degree of stress, such as wounding or high-temperature treatment, ultimately leads to an increase in viral antigens and thus to improvement of detection. It is hypothesised that the virus titre increases by the altered metabolism during the repair reactions as a response to stress applied to the bulbs.     

基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。