Menadione-catalyzed luminol luminescent assay as a novel evaluation method of ethanol tolerance in yeast cells

In this study, ethanol inhibited the growth and glucose-induced proton release of yeast cells in a dose-dependent manner. On the other hand, ethanol tolerance of menadione-catalyzed luminol luminescence by yeast cells increased with increasing ethanol concentrations in the growth medium. The intracellular reduced-form nicotinamide adenine dinucleotide (NADH) concentration also increased with increasing ethanol concentrations in the medium and was enough to maintain constant menadione-catalyzed luminol luminescence. These facts suggest that the menadione-catalyzed luminol luminescent assay depending on a NADH:quinone reductase and NADH generation system is useful as a new evaluation assay for assessing the vitality of ethanol-stressed yeast cells, whereas the glucose-induced proton release assay is expected to be useful for the evaluation of cell growth under ethanol stress.     

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD⁺ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD⁺ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 × 10^?14 mol NADH or NAD⁺ per assay.     

Homogeneous amperometric immunoassay for theophylline in whole blood

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 μl whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of + 150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: Y = 0·90x − 1·01, (r = 0·98, N = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.     

Microanalysis of the metabolic intermediates of lactose synthesis in human milk and plasma using bioluminescent methods

Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small sample volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters sample. For the assays of the metabolites, recoveries ranged from 91 to 107%. For sample sizes of 2 to 5 microliters of protein free sample, the detection limits for milk were G1P, 0.09 microM; G6P, 0.05 microM; UDPhexose, 0.07 microM; UDP-Glc, 0.03 microM; glucose, 9 microM; and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.     

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution.

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.     

Spectrophotometric determination of oxidized and reduced pyridine nucleotides in erythrocytes using a single extraction procedure

Several methods are available for the extraction and quantitation of oxidized and reduced pyridine nucleotides in erythrocytes. Enzymatic methods, however, are complicated by the presence of hemoglobin, which causes oxidation of NADH and NADPH during extraction. Although hemoglobin-mediated oxidation can be prevented by the addition of reducing agents, these interfere with spectrophotometric cycling assays for these nucleotides. Therefore, we have developed a method for determining oxidized and reduced NAD and NADP in human erythrocytes using a single extract. Our extraction method eliminates the need for reducing agents and thus allows the use of a spectrophotometric cycling assay. Using this method, we obtained full recovery of all added nucleotides with both normal and reticulocyte-enriched red blood cells. Our method is suitable for the determination of NAD+, NADH, NADP+, and NADPH in normal human erythrocytes and in red cells from patients with hemolytic anemia with a higher proportion of reticulocytes.     

Enzymatic determination of the branched-chain alpha-keto acids

A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.     

An enzymatic determination of hydrogen peroxide by using spectrophotometry.

A new spectrophotometric method for determining low hydrogen peroxide concentrations by using horseradish peroxidase in the presence of NADH at pH 7.5 has been described. Both total NADH consumption and initial reaction rate may be used for the determination. Using the NADH consumption, a linear response with respect to hydrogen peroxide was observed in the concentration range 7 x 10(-8)-2.5 x 10(-6) M. Due to the presence of superoxide dismutase, hydrogen peroxide is partly regenerated and an amplification of the signal results, which explains the sensitivity.     

Development of a liquid chromatography-tandem mass spectrometry assay of six antimicrobials in plasma for pharmacokinetic studies in premature infants

This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50-300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85 to 110% and 92 to 110%, respectively, and within- and between-day precision of 89-111% and 91-109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants.     

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

Validated LC-MS/MS methods for the determination of risperidone and the enantiomers of 9-hydroxyrisperidone in human plasma and urine

Two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods are described, one for the quantitative determination of risperidone and the enantiomers of its active metabolite 9-hydroxyrisperidone (paliperidone) in human plasma and the other for the determination of the enantiomers of 9-hydroxyrisperidone in human urine. The plasma method is based on solid-phase extraction of 200 microl of sample on a mixed-mode sorbent, followed by separation on a cellulose-based LC column with a 13.5-min mobile phase gradient of hexane, isopropanol and ethanol. After post-column addition of 10 mM ammonium acetate in ethanol/water, detection takes place by ion-spray tandem mass spectrometry in the positive ion mode. Method validation results show that the method is sufficiently selective towards the enantiomers of 7-hydroxyrisperidone and capable of quantifying the analytes with good precision and accuracy in the concentration range of 0.2-100 ng/ml. An accelerated (run time of 4.3 min) and equally valid method for the enantiomers of 9-hydroxyrisperidone alone in plasma is obtained by increasing the mobile phase flow-rate from 1.0 to 2.0 ml/min and slightly adapting the gradient conditions. The urine method is based on the same solid-phase extraction and chromatographic approach as the accelerated plasma method. Using 100 microl of sample, (+)- and (-)-9-hydroxyrisperidone can be quantified in the concentration range 1-2000 ng/ml. The accelerated method for plasma and the method for urine can be used only when paliperidone is administered instead of risperidone, as there is insufficient separation of the 9-hydroxy enantiomers from the 7-hydroxy enantiomers, the latter ones being present only after risperidone administration.     

Optimisation of an extraction procedure and chemical characterisation of Croatian propolis tinctures

Three spectrophotometric methods for the quantitative determination of different flavonoid groups and total phenolics in Croatian propolis samples were optimised and validated. The assay based on the formation of aluminium chloride complex (with galangin as a standard) was applied to the quantification of flavones and flavonols, while the 2,4-dinitrophenylhydrazine method (with pinocembrine as a reference) was used for the quantification of flavanones. Total phenolic content was measured by the Folin-Ciocalteau method using reference solution of caffeic acid:galangin:pinocembrine (1:1:1). Through analytical validation, the most suitable extraction conditions (with respect to time, temperature and concentration of extraction solvent) were determined, and final conditions for the extraction were established (80% ethanol, 1 h at the room temperature). The appropriate ratio between the mass of raw propolis and the extraction solvent volume was also established. By the application of the optimised method of extraction, 10 propolis tinctures were prepared and subjected to the analysis of general pharmacopoeial parameters, which are fundamental for the creation of quality specification (relative density, dry residue of extract, content of ethanol, methanol and 2-propanol). Additionally, the content of waxes as the main inactive constituents was determined in order to observe the level of their migration from crude propolis to the prepared tinctures.     

NADH-coupled spectrophotometric assay of tyrosine aminotransferase

A rapid spectrophotometric method for estimation of tyrosine aminotransferase activity (TAT) is described, based on a coupled reaction with NADH-dependent aromatic ketoacid reductase. 3-iodo-L-tyrosine, upon TAT action, is transformed into 3-iodo-4-hydroxyphenylpyruvate which quickly reacts with NADH in the presence of aromatic ketoacid reductase; oxidation rates at 340 nm are linear with protein concentration over the whole range of purification steps of TAT. This new method, for its sensitivity, easy performance and possibility of a continuous monitoring of TAT reaction, may be considered comparable to the more diffuse spectrophotometric standard method, and also as an alternative, advantageous procedure in some instances. The method for purification of the coupled aromatic ketoacid reductase is also described.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

Description of analytical problems arising from elevated serum solids

There has always been a problem with the collection of data and interpretation of the results obtained from any biological fluid in which the solids content was increased to a great extent. Of these solids, the triglycerides of the lipids may cause a plasma (serum) to vary in appearance from opalescent to milky. This condition of the specimen and the concomitant turbidity upon its addition to reagents creates the well-documented optical aberrations of spectrophotometric measurements. In addition, the lipids, in conjunction with the proteins, can act as diluents when they are elevated, thereby decreasing what might be termed the residual true plasma volume. Thus the water content of an aliquot sampled for a particular analytical procedure is diminished, and by that means a situation is created in which a short sample is drawn. This dilution effect by the solids results in a lowering of the assay values obtained for the measured constituents of such a serum sample. An associated phenomenon of high concentrations of solids, especially proteins, is the increase in viscosity of a specimen, a condition that also causes an error of short sampling when certain peristaltic pumping devices are used. This review considers several aspects of problems encountered when dealing with a number of circumstances that are critical to the measurement of analytes in severely hyperlipemic and/or hyperproteinemic specimens. These include the problems of short sampling; the potential amelioration of the problem by corrective mathematics, extraction of the lipids, or ultracentrifugation of the true plasma from the lipids; the important need to include most analytes into our considerations; the difference in reference base values for the calculation of concentrations of lipids of serum versus other analytes; the concept of the use of ratios when the reference base values differ, numerator analyte from denominator analyte; and the problems of using serum blanks when necessary corrective action for the solids volume is neglected. Thus, in the final analysis, problems with underestimated volumes of samples used for many spectrophotometric determinations are considered here along with the other difficulties encountered when the need to measure analytes in serums with extremely high solids content presents.     

栽培青蒿中总黄酮提取工艺

利用超声波辅助技术,获得最大限度提取青蒿中总黄酮的新工艺。用正交设计理论,结合分光光度法,优化超声波辅助醇提法提取青蒿总黄酮工艺中的关键技术参数。最佳提取．工艺为：超声波频率59kHz,乙醇体积分数60％,提取时间40min,料液比1：40。超声波辅助提取法能够实现青篙中总黄酮的高效提取,产率达1．497％。     

Determination of total insulin (TIRI) in plasma of insulin-treated diabetics and newborn infants of insulin-treated diabetic mothers.

Plasma of insulin-treated diabetics and of newborn infants of insulin-treated diabetic mothers contains insulin antibodies which invalidates the radioimmunoassay of insulin. Therefore, the endogenous insulin antibody complex must be splitted at a pH lower than 5 and the total IRI (TIRI) is separated by ethanol extraction. It was investigated the recovery rate in dependence upon plasma volume used for extraction. By reduction of used plasma volume from 500 to 200 mul per extraction the recovery rate was increased from 65.1 +/- 8.4 to 88.3 +/- 4.2% (mean +/- SEM). The low plasma volume of 200 mul for TIRI extraction made it possible to determine TIRI during glucose loads of newborn infants. To eliminate different conditions of incubation for standard and unknown plasma samples the TIRI levels were computed by means of so-called "extracted" standard curve, obtained with extracted insulin from standard insulin dilution in insulin-free pooled human plasma. Using the described method a temporary regeneration of insulin secretion of a newly diagnosed juvenile diabetic after insulin treatment could be shown. In contrast to newborn infants of healthy mothers a biphasic/insulin release was found during the intravenous glucose loads in newborn infants of insulin-treated diabetic mothers.     

Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD⁺/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.     

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).     

Development of a microporous membrane liquid–liquid extractor for organophosphate esters in human blood plasma: identification of triphenyl phosphate and octyl diphenyl phosphate in donor plasma

An extractor has been developed for microporous membrane liquid–liquid extraction (MMLLE) of lipophilic xenobiotics at trace levels in biological fluids. This new construction allows the sample phase to be stirred, while the organic phase is pumped. The extractor was evaluated using human blood plasma with added organophosphate esters. The size exclusion properties of the membrane reduced lipid co-extraction by 94% compared to ordinary liquid–liquid extraction. In combination with a solid-phase extraction (SPE) step, the method was shown to remove plasma lipids efficiently and thus allow gas chromatographic separation of the compounds. The clean-up method described, including the SPE step, showed a high level of reproducibility, and recoveries of between 72 and 83% were obtained for five of the organophosphate esters after a 200-min extraction period. Using this technique, triphenyl phosphate and an isomer of octyl diphenyl phosphate were detected in human plasma obtained from blood donors. The concentration of triphenyl phosphate ranged between 0.13 and 0.15 μg/g plasma.