Separation of double-label liquid scintillation samples by half-life

A method is presented for separation of double- or triple-labeled liquid scintillation samples based on the partial decay of one of the nuclides. Validity of the procedure has been demonstrated with the following combinations of nuclides commonly encountered in laboratory situations; ¹³¹I-¹⁴C, ³²P-¹⁴C, and ²⁰³Hg-¹⁴C, Separation of all nuclide pairs agrees favorably with predieted values except when the activity ratios exceed 10³. The determinations are independent of quenching and nuclide spectra and greatly augment the number of β-β and β-γ combinations resolved by liquid scintillation spectrometry. The utility of this method for separation of triple-labeled samples is discussed.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

The peroxidase-catalyzed oxidation of NADH as an indicator reaction for repetitive determinations by sample injection in closed flow-through systems: the determination of LDH in blood serum.

The air oxidation of reduced nicotinamide adenine dinucleotide (NADH), catalyzed by peroxidase, provides a useful “indicator reaction” for the determination of both NADH and serum lactate dehydrogenase (LDH). Application of this indicator reaction for repetitive determinations by sample injection into a continuously circulated reagent mixture, and by monitoring of oxygen depletion with an amperometric sensor, is described. At determination rates of 260 determinations/h, NADH and LDH have been determined in the range of 3.8 × 10^?4 to 7.6 × 10^?3m and 70 to 700 U/ml, respectively, and with relative errors and standard deviations (population) of about 2%. LDH has been determined in human blood serum and the injection procedure compared with a modified Wroblewski-Ladue procedure; the Pearson correlation coefficient was found to be 0.99₉ (40 samples). Recovery studies are also reported for LDH in serum calibration references and controls.     

Heterotrophic Potentials and Hydrocarbon Biodegradation Potentials of Sediment Microorganisms Within the Athabasca Oil Sands Deposit

Techniques for the enumeration and the determination of the potential activity of disturbed sediment mixed populations at control sites and sites within the Athabasca oil sands formation were applied to August and December samples. These techniques included the determination of general heterotrophic potential for the assimilation and respiration of glutamate, which indicated no oil sand-related changes in the sediments but which indicated a significant seasonal change. Enumeration by epifluorescence direct counts, oil sand hydrocarbon plate counts, and most-probable-number determinations of [¹⁴C]hexadecane and [¹⁴C]-naphthalene degraders indicated that only the plate count was sensitive to increased numbers of oil sand-related hydrocarbon-oxidizing microorganisms within the oil sands deposit. Unlike the most probable number determinations of [¹⁴C]hexadecane and [¹⁴C]naphthalene degraders, however, the biodegradation potential results of these substrates indicated a significant increase in activity at oil sands sites. These biodegradation potentials also showed a marked seasonal fluctuation. Although the biodegradation potentials and the endogenous hydrocarbon plate counts indicated an oil sand-adapted mixed sediment population, the results of these techniques did not correlate well with the concentrations of bituminous hydrocarbons in the sediments. The results suggest that a general capability for hydrocarbon oxidation exists in the Athabasca River system and that this capability is enhanced within the natural bounds of the Athabasca oil sands.     

Cobalt determination in serum and urine by electrothermal atomic absorption spectrometry

Cobalt determinations in biological fluids are of great interest in biological or toxicological research programs. Cobalturia is often chosen as an indicator for a biological monitoring program in occupational exposure to cobalt dusts. The method described here derives from the IUPAC reference method for nickel determination. It enables cobaltemia and cobalturia to be measured in small samples (1 mL). The mean usual values for cobalt in biological fluids are very low (2.7 nmol L⁻¹ for serum and 6.7 nmol L⁻¹ for urine), and therefore, thus require an analytical procedure with preconcentration and extraction. The sample is mineralized by wet acid digestion. After digestion, inorganic cobalt is extracted in form of ammonium pyrrolidine dithiocarbamate complex into isobutyl methyl ketone and measured in the organic layer by electrothermal atomic absorption spectrometry. The analytical parameters are described in detail. The extraction output is about 99%. The detection limits are 1.93 and 1.89 nmol L⁻¹ for serum and urine, respectively. Sensitivity (expressed as the concentration that gives a 0.044 absorbance) is 3.4 nmol L⁻¹ for serum and 3.3 nmol L⁻¹ for urine. Within-run precision ranged between 3.9 and 2.5% (coefficients of variation) for serum and 4.2 and 1.1% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. Between-run precision ranged between 4.3 and 3.3% (coefficients of variation) for serum and 4.2 and 2.3% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. At very low concentration, 5.7 nmol L⁻¹ for serum and 2.5 nmol L⁻¹ for urine, the between-run precision is, respectively, 19.5 and 28%. Linearity is effective between 0 and 272 nmol L⁻¹. Interferences and matrix effects are negligible for urine, serum, or plasma samples without hemoglobin. The method is easily applicable for routine determinations.     

The relation of sex,age, smoking status,birth rank and parental ages to pseudocholinesterase activity and phenotypes in a sample of Australian Caucasian adults

Summary The frequencies of the pseudocholinesterase alleles E₁ ^u, E₁ ^a and E₁ ^f have been determined in a random sample of Australian residents. The frequency of E₁ ^a is the highest yet reported in a large caucasian sample. The considerable variation in E₁ ^f frequencies in previously reported samples is discussed in terms of possible sources of error in fluoride number determinations.Enzyme activity was found to increase with age in adulthood and was higher in males than in females. It was also positively correlated with dibucaine number in type U subjects. These observations are in conflict with those reported in previous investigations.This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Tobacco Research Foundation.     

Measurement of DNA in cultured human cells

A simple microtechnique has been developed for the accurate determination of DNA in less than 1 × 10⁶ cultured human fibroblasts or lymphoblasts. This technique involves rapid separation of DNA from the cell extract supernatant and from the extraction buffer in order to avoid interference with the colorimetric diphenylamine assay. The method described does not involve complicated manipulation of samples and does not require special instrumentation. In addition, multiple samples can be conveniently prepared to be assayed at a later time and various other biochemical determinations such as enzyme assays can be done on soluble extracts of the same cell samples.     

A novel system of galangin–potassium permanganate–polyphosphoric acid for the determination of tryptophan and its chemiluminescence mechanism

A novel galangin–potassium permanganate (KMnO₄)–polyphosphoric acid (PPA) system was found to have an outstanding response to tryptophan (Trp). Trp determination using this KMnO₄–PPA system was enhanced significantly in the presence of galangin. A highly sensitive flow‐injection chemiluminescence (CL) method to determine Trp was developed based on the CL reaction of galangin–KMnO₄–Trp in PPA media. The presence of galangin, a member of the flavonol class of flavonoid complexes, greatly increased the luminous intensity of Trp in KMnO₄–PPA systems. Under optimized conditions, Trp was determined in the 0.05–10 µg/mL range, with a detection limit (3σ) of 5.0 × 10^?3 µg/mL. The relative standard deviation (RSD) was 1.0% for 11 replicate determinations of 1.0 µg/mL Trp. Two synthetic samples were determined selectively with recoveries of 98.4–100.1% in the presence of other amino acids. The possible mechanism is summarized as follows: excited states of Mn(II)^* and Mn(III ^* types are the main means of generating chemical luminescent species, and Trp concentration and luminescence intensity have a linear relationship, which enables quantitative analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

The determination of glutamine with flow‐injection chemiluminescence detection and mechanism study

The main purpose of this study was to develop an inexpensive, simple, rapid and sensitive chemiluminescence (CL) method for the determination of glutamine (Gln) using a flow‐injection (FI) system. Gln was found to strongly inhibit the CL signal of the luminol–H₂O₂–CuSO₄ system in Na₂B₄O₇ solution. A new FI‐CL method was developed for the determination of Gln. Parameters affecting the reproducibility and CL detection were optimized systematically. Under the optimized conditions, the corresponding linear regression equation was established over the range of 5.0 × 10^?7 to 2.5 × 10^?6 mol/L with the detection limit of 1.8 × 10^?8 mol/L. The relative standard deviation was found to be 1.8% for 11 replicate determinations of 1.5 × 10^?6 mol/L Gln. The proposed method has been satisfactorily applied for the determination of Gln in real samples (Marzulene‐s granules) with recoveries in the range of 98.7–108.6%. The minimum sampling rate was about 100 samples/h. The possible mechanism of this inhibitory CL was studied by fluorescence spectrophotometer and UV–vis spectrophotometer. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations.

A rapid and simple radiometric assay for cholinesterase, suitable for multiple determinations, has been developed. (³H-acetyl) choline is enzymatically hydrolyzed in a small reaction volume in a scintillation vial. The released [³H]acetate is then extracted into a toluene-based scintillator added directly to the vial, without removing the reaction volume. The extracted [³H]acetate counts efficiently, but the unhydrolyzed [³H]acetylcholine remains unextracted in the small aqueous reaction volume, from which its weak β-particles of decay do not escape to excite the scintillator. The assay is highly reproducible, quite sensitive, and useful for applications in which multiple samples must be quickly assayed.     

Development of a dispersive liquid-liquid microextraction method for spectrophotometric determination of barbituric acid in pharmaceutical formulation and biological samples

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid–liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml⁻¹ with limit of detection of 2.0 ng ml⁻¹. Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml⁻¹ concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples.     

Effect of 2,4-dichlorophenoxyacetic acid on the structure and function of developing chloroplasts

Dark-grown radish seedlings (Raphanus sativus L.) were sprayed with 10^-3 mol·l^-1 2,4-dichlorophenoxyacetic acid and then were exposed to a 14:10 light: dark cycle. Cotyledon samples from these seedlings and unsprayed controls were taken for electron microscopy, chlorophyll determinations, and photosynthetic rate measurements at regular intervals for 72 h. A normal development of etioplasts to chloroplasts with formation of typical grana-fret work system was observed in the control cotyledons. The chloroplasts in the 2,4-D-treated cotyledons showed changes in the organization of the grana thylakoids; these thylakoids being more appressed to each other than in the controls. The chlorophyll content of treated plants was less than that of controls but the rate of chlorophyll biosynthesis was unaffected. The photosynthetic rate/mg chlorophyll was considerably higher for treated plants suggesting that 2,4-D treatment resulted in decreased size of the photosynthetic unit.     

Use of reverse isotope dilution analysis to determine blood plasmal(+)-14C-lactate specific radioactivity

A method for estimation of the specific radioactivity of blood plasmal(+)-¹⁴C-lactate is described. It is based on the reverse isotope dilution principle and involves a maximum error on any individual sample of 4%. The mean error for a number of determinations is reduced to 0.8%. Duplicate specific radioactivity values can conveniently be obtained on six blood samples by one worker in a normal 8 hr working day.     

Adsorption chromatography of cyclic nucleotides on silica gel and alumina thin-layer sheets

Several solvent combinations of graded polarity have been developed, capable of separating cAMP or cGMP, not only from related nucleotides but from bases and nucleosides on alumina and silica gel thin-layer sheets (tls). The solvent system chloroform (C), methanol (M), water (W) (40:20:3) gave effective separations of cAMP on silica gel TLS. Identical results were obtained when this adsorption chromatography method was compared with the ion-exchange chromatography method involving PEI for its effectiveness in the separation of [α-³²P]cAMP formed from [α-³²P]ATP by a preparation of bovine sperm cells. In addition, this solvent system effectively separates cAMP from inosine, hypoxanthine, xanthine, and uric acid which may be useful in determinations of cAMP arising from ³H- or ¹⁴C-prelabeled ATP. Effective separation of cGMP on silica gel tls was accomplished with C:M:W (30:30:5). On alumina tls, cAMP and cGMP were separated from related compounds with M:W combinations; the solvent M:W (50:50) gave the lowest blanks (0.02%) for cGMP and it may prove useful in cGMP determinations.     

Functional hemizygosity in the human genome: direct estimate from twelve erythrocyte enzyme loci

Summary Cord blood samples from 2020 unrelated newborns were screened for levels of enzyme activity for twelve enzymes. The level of enzymatic activity for 100 determinations were consistent with the existence of an enzyme-deficiency allele. The frequency of deficiency alleles in the Black population (0.0071) was four times higher (after removal of the G6PD^*A^- variant) than in the Caucasian sample (0.0016). These frequencies are approximately double the frequency of rare electrophoretic mobility variants at similar loci in the same population. Given the number of functionally important loci in the human genome, these enzyme deficiency variants could constitute a significant health burden.     

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

C-H?π interactions in the [Co(N-(2-aminomethylpyridyl)ethylenediamine)(2- aminomethylpyridine)Cl] system: syntheses, 2D NMR, X-ray structures and energy minimizations

Ten asymmetric isomers exist for [Co(pema)(ampy)Cl]²⁺ (pema=N-(2-aminomethylpyridyl)ethylenediamine, ampy=2-aminomethylpyridine) which involve unsymmetrical triamine and diamine ligands. Four of these have been synthesized, two facial (f3, f2^′) and two mer (m3 and m4) isomers. Ab initio energy calculations for the [Co(pema)(ampy)Cl]²⁺ and [Co(pema)(ampy)OH]²⁺ systems show that the isomers containing a C-H?π interaction are the more stable forms. The f3 is the most stable isomer in the chloro system and the m3 form in the hydroxo system. The structures are deduced from the 2D NMR spectra and confirmed by the X-ray crystal structure determinations for the four chloro isomers.     

A semiautomated fluorometric determination of 5-hydroxyindoles in the nanogram range

Automated determinations of 5-hydroxytryptamine and its main metabolite, 5-hydroxyindoleacetic acid, have been described (Technicon autoanalyzer). The determinations are based on an extraction procedure from deproteinized tissue extracts or cerebrospinal fluid by means of butanolheptane mixtures. The indoles are transferred from the organic phase to a water phase and determined fluormetrically with the cysteine-o-phthaldialdehyde method. The method is highly sensitive: solutions containing 1 ng/ml can be reproducibly determined. Twenty samples per hour can be passed through the system. The determination of both 5-hydroxyindoles is performed with the same manifold.     

Experimental acute Babesia caballi infections: I. Red blood cell dynamics

Hematological determinations were made on blood samples from six ponies acutely infected with two dosage levels of Babesia caballi (Group 1: divided into two subgroups of three ponies each). Similar determinations were made on blood samples from three premunized ponies given challenge inoculations (Group 2), and three equidae given uninfected red blood cells (Group 3).A trend towards decreases in RBC counts, hemoglobin concentrations, and hematocrits within one to four days after inoculation (AI) was observed in all groups. However, it was marked only in Group 1. In addition, only in Group 1 was there observed a concerted anemia occurring between Days 7 and 16.Those surviving ponies in Group 1 which developed a higher parasitemia between Days 5 and 6 AI (first parasitemia peak) developed a more severe anemia between Days 7 and 16. Ponies which developed parasitemias higher than 40 × 10³ parasitized cells/mm³ at the first parasitemia peak subsequently died.Free bilirubin in Group 1 animals increased immediately after inoculation, and repeatedly exceeded normal ranges until after Day 20 AI when the RBC counts were rising. Similar changes in free bilirubin did not occur in either Groups 2 or 3. Conjugated bilirubin levels did not exceed normal ranges in any of the experimental animals.Active erythrophagocytosis was evident in histological preparations of lymph node, spleen, liver, and lung from ponies which died. Cytosiderin pigment was present in liver parenchyma, and hematin was scattered throughout lymph nodes and spleen.     

High-sensitive chemiluminescent assay for cholesterol

Chemiluminescent measurement of cholesterol can be performed in various biological tissues and fluids. The method described in this study has a sensitivity of 54 pmol. The tissue samples used for the determination of cholesterol can be reduced to as little as 1 mg and assay can be performed on diluted biological fluids, allowing sampling of plasma or serum as little as 5 μl. Cholesterol is solubilized in sodium cholate and aliquots are added to a reaction mixture containing cholesterol oxidase, luminol and peroxidase. Cholesterol oxidase, in the presence of cholesterol yields H₂O² which produces light in presence of luminol and peroxidase. Emitted light is quantified at a wavelength of 420 nm by means of a photomultiplier. Optimal conditions of the assay were determined and examples of cholesterol determinations, in blood plasma and nervous tissues, are presented.