Interference of ethanol in the determination of tryptophan by the method of spies and chambers.

Ethanol interference with the Spies and Chambers method (1–3) for the determination of tryptophan is shown in the results with cereal proteins. This negative interference of ethanol was confirmed by use of free tryptophan under the same conditions. The extent of ethanol interference varies with the sample. It is postulated that ethanol inhibits the tryptophan-p-dimethylaminobenzaldehyde (DAB) reaction by alkylation of the amino acid and possibly to a lesser extent, by acetal formation with DAB. A method for the removal of ethanol from samples before tryptophan determination is suggested.     

Measurements of polyamines and their acetylated derivatives in cell extracts and physiological fluids by use of an amino acid analyzer

A fast and sensitive method for the determination of free polyamines and their acetylated derivatives is presented. The separation is carried out on a Durrum DC-6A cation-exchange resin with an automated amino acid analyzer. The determination is based on a stepwise elution with a sodium chloride—sodium citrate buffer system. Detection is done by fluorescence of the o-phthaldialdehyde—polyamine conjugates. The sensitivity is in the picomole range. No prior purification step is needed. The method has been applied to cell extracts and urine samples.     

A catalytic method for determination of trace amounts of iron in biological material

A rapid, simple, and reproducible method for determination of iron in biological material is suggested using the oxidation of p-phenetidine hydrochloride with hydrogen peroxide in a reaction catalyzed by Fe(III) and activated by 1,10-phenanthroline. The high sensitivity of the reaction allows a single determination to be carried out with as much as 1–5 mg fresh tissue.     

Fluorometric Determination of Deoxyribonucleic Acid in Bacteria with Ethidium Bromide

A simple, sensitive, and rapid method is presented for the determination of deoxyribonucleic acid (DNA) in both gram-positive and gram-negative bacteria. It is based upon the fluorometric determination of DNA with ethidium bromide after alkaline digestion of the bacteria to hydrolyze the interfering ribonucleic acid. The assay takes less than 2 hr. Its sensitivity is at least 0.2 μg of DNA in a final solution of 4 ml and it uses commonly available filter or double monochromator fluorometers. Judicious choice of light source and filters allows an additional 10-fold increase in sensitivity with a filter fluorometer. Turbidity caused by bacteria or insoluble polysaccharides does not interfere with the fluorescence measurements. There was no significant difference between the results obtained with this method and those obtained with the indole and diphenylamine methods when these assays were applied to Escherichia coli and sucrose- or glucose-grown Streptococcus mutans. The method was also tested by determining the specific growth rate of E. coli. This new procedure should be especially useful for the determination of bacterial DNA in dilute suspensions and for the estimation of bacterial growth or DNA replication where more conventional methods are not applicable or sensitive enough.     

Molecular identification of species and ploidy of Carassius fishes in Lake Biwa,using mtDNA and microsatellite multiplex PCRs

We established an efficient and reliable method to identify the species and ploidy of Carassius fishes in Lake Biwa (C. cuvieri, C. buergeri, and the latter’s polyploid) using a combination of multiplex mitochondrial DNA haplotype-specific PCR and ploidy determination based on nuclear DNA microsatellite PCR. The high accuracy of ploidy determination was confirmed by flow cytometry. This method can also identify triploid clonal lines and provide information about their genetic diversity and structure. The method is applicable to ecological and evolutionary studies of Carassius fishes in Lake Biwa throughout their entire life history.     

Enzymatic determination of stevioside in Stevia rebaudiana

A simple enzymatic method is described for the determination of stevioside in Stevia rebaudiana. The method is based on the hydrolysis of stevioside with crude hesperidinase. The reaction is followed by monitoring the production of glucose with a glucose oxidase-peroxidase-2, 2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) system. The results for the stevioside content in S. rebaudiana leaves correlate with those obtained by other methods. The stevioside content in S. rebaudiana plants showed large variation.     

Determination of acetaldehyde in rat blood by the use of rat liver aldehyde dehydrogenase

A method has been developed for the determination of low concentrations of acetaldehyde in rat blood. The method involves extraction of blood in perchloric acid followed by a fluorimetric determination of acetaldehyde in neutralized extracts by the use of a low K_m aldehyde dehydrogenase isolated from rat liver mitochondria. Acetaldehyde concentrations down to 2 to 3 μm could be detected in blood samples of 0.1 ml containing high concentrations of ethanol (10–40 mm). Due to its simplicity, sensitivity, and the use of a low-cost fluorimeter, this enzymatic method should be a valuable complement to gas chromatographic methods for acetaldehyde determination.     

Simultaneous determination of d- and l-propranolol in human plasma by high-performance liquid chromatography

A method for the determination of d- and l-propranolol in human plasma is described. The method involves extraction of propranolol from plasma, and the formation of diastereomeric derivatives with the chiral reagent N-trifluoroacetyl-1-prolylchloride. Separation and quantitation of the diastereomeric propranolol derivatives are carried out by a reversed-phase high-performance liquid-chromatographic system with fluorimetric detection. The reproducibility in the determination of d- and l-propranolol in human plasma was 4.5% (relative standard deviation) at drug levels of 10 ng/ml.In two subjects who received a single 40-mg tablet of racemic propranolol the plasma levels of the d-isomer were lower than of the l-propranolol. The half-lives of d- and l-propranolol were similar.     

Enzymatic determination of serum-free fatty acids: a colorimetric method

A simple and sensitive enzymatic method is described for the determination of serumfree fatty acids. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The reaction is followed as the production of hydrogen peroxide by using acyl-CoA oxidase/peroxidase/4-aminoantipyrine/phenol system. Results on human sera correlate well with those obtained by our previously described enzymatic method (Shimizu et al. (1979) Anal. Biochem.98, 341–345) and the chemical colorimetric method.     

Determination of tetrahydrothiophene formation as a probe of in vitro busulfan metabolism by human glutathione S-transferase A1-1: use of a highly sensitive gas chromatographic–mass spectrometric method

A method for the sensitive determination of tetrahydrothiophene (THT) in cytosolic incubation mixtures was developed. Busulfan conjugation with glutathione was predominantly catalysed by glutathione S-transferase A1-1 (GST A1-1) and THT was released from the primary metabolite by alkalization. After liquid–liquid extraction using n-pentane separation and quantification of the product was performed by gas chromatography with a mass-selective detector. The method showed good sensitivity, accuracy and reproducibility with a detection limit of 2 ng ml⁻¹ and a limit of quantification of 5 ng ml⁻¹. The suitability of the method is shown for enzyme kinetic studies in human liver cytosol as well as for determination of GST A1-1 activity.     

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts

The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24 h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.     

Biological and biomedical applications of isoelectric focusing : edited by N. Catsimpoolas and J. Drysdale, Plenum Press, New York, XV + 351 pp., Price US$ 39.00, ISBN 0-306-34603-6

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5–9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

Colorimetric assay of liver and Escherichia coli asparatate transcarbamylases in the presence of 2-mercaptoethanol

A modification of the method of Prescott and Jones (1) for the colorimetric determination of carbamyl aspartate has been developed to permit the assay of aspartate transcarbamylases in the presence of 2-mercaptoethanol. Interference by this compound is eliminated by means of N-ethylmaleimide. The usefulness of the modified method is illustrated by examination of the contrasting properties of the Escherichia coli and rat liver enzymes.     

Simultaneous Determination of Glass Transition Temperatures of Several Polymers

Aims

A simple and easy optical method is proposed for the determination of glass transition temperature (T_g) of polymers.

Methods & Results

T_g was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in T_g determination. So the determined T_g is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the T_g of several polymers in a single experiment, so it can greatly save experimental time and heating energy.

Conclusion

The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.     

Enantioselective high-performance liquid chromatographic method for the determination of methadone and its main metabolite in urine using an AGP and a C8 column coupled serially

A simple and sensitive method for the enantioselective high-performance liquid chromatographic determination of methadone and its main metabolite, EDDP, in human urine is described. (−)-(R)-Methadone, (+)-(S)-methadone, (+)-(R)-EDDP, (−)-(S)-EDDP and imipramine as an internal standard are detected by ultraviolet detection at 200 nm. The enantiomers of methadone and EDDP were extracted from human urine by a simple liquid–liquid extraction procedure. The extracted sample was reconstructed in mobile phase and the enantiomers of methadone and EDDP were quantitatively separated by HPLC on a short analytical LiChrospher RP8 column coupled in series with a chiral AGP column. Determination of all four enantiomers was possible in the range of 0.03 to 2.5 μM. The recoveries of methadone enantiomers and EDDP enantiomers added to human urine were about 90% and 80%, respectively. The method was applicable for determination of methadone enantiomers and the enantiomers of its main metabolite in urine samples from methadone maintenance patients and patients suffering from severe chronic pain.     

Determination of insulin in single pancreatic cells by capillary electrophoresis and laser-induced native fluorescence

Development of a method for the determination of insulin based on capillary electrophoresis with laser-induced native fluorescence detection is described. Under optimal conditions, insulin as low as 73 amol can be detected with a good signal-to-noise ratio (S/N=10 peak-to-peak). Application of this method for the determination of insulin content in single cells from the insulin-secreting cell lines RINm5F and βTC3 is demonstrated. Non-bonded poly(ethylene oxide)-coated and bare capillaries are evaluated for this purpose, with the latter found to be more suitable for single-cell analysis.     

A rapid microtechnique for the preparation of biological material for the simultaneous analysis of calcium,magnesium and protein

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r²) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.