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PubMed2001

Electroanalytical determination of tungsten and molybdenum in proteins.

Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.

Chemical Precipitation↗
PubMed1994

A stopped spectrophotometric assay for the dopa oxidase activity of tyrosinase.

A stopped spectrophotometric assay for the dopa oxidase activity of tyrosinase has been developed to enable large numbers of samples to be screened very rapidly. The assay measures the pink pigment formed by the reaction of Besthorn's hydrazone (3-methyl-2-benzothiazoninone hydrazone, or MBTH) with dopaquinone, the product of oxidation of L-dopa by tyrosinase. Addition of perchloric acid stops the reaction and precipitates protein, enabling turbid as well as non-turbid samples to be assayed. Stability of the pink product is enhanced in acid solution and the pigment has a sharp absorbance maximum at 505 nm such that it is easily measured spectrophotometrically. Using the stopped assay, tyrosinase is detectable only in mammalian cell lines expected to express the enzyme, and the specificity of the assay has also been confirmed using tyrosinase inhibitors. The stopped MBTH assay is approx. 15-times more sensitive than the widely used dopachrome assay and can reliably detect the formation of as little as 350 pmol of product.

Benzothiazoles↗
PubMed1996

[Proton magnetic resonance spectroscopy of excised pig liver].

After evaluating ex vivo pig liver by 1H magnetic resonance spectroscopy (1H-MRS) using a 1.5 Tesla super-conducting unit, the assignment of peaks was reevaluated in vitro using a 9.4 Tesla superconducting unit. The portal vein was cannulated and perfused by cooled Euro-Collins solution, and pig liver was removed and preserved in the solution. Five to 8 g of the liver was excised before and after preservation, then extracted by perchloric acid (PCA). In 1H-MRS of PCA extracted pig liver, large peaks of fatty acid disappeared, while peaks of the choline group from 3.2-3.3 ppm were clearly observed. Using high performance liquid chromatography (HPLC) and 31P (phosphorus)-MRS, the 3.23 ppm peak was determined as glycerophosphorylcholine (GPC), which diminished after preservation. The chronological change of GPC in PCA extracted pig liver was able to be observed by 1H-MRS. GPC peak may play a role as an indicator in evaluating preserved liver by 1H-MRS.

Animals↗
PubMed1967

A simple procedure for the estimation of very small amounts of nitrogen in lipids.

1. About 0.1mug.atom of combined nitrogen, in lipids and a few other compounds, can be determined quantitatively by the gentle digestion of dry samples in 10ml. test tubes with perchloric acid for 30min., followed by the estimation of the resulting ammonia as the stable blue colour (lambda(max.) 635mmu) produced by the addition of phenol and nitroprusside, an alkaline phosphate buffer and alkaline hypochlorite. 2. Deionized distilled water is required, but the other reagents need no special purification if chosen and handled with care. 3. The results are linear with from 0.015 to 0.15mug.atom of nitrogen, or up to 1mug.atom if the final solutions are diluted with water after full colour development.

Ammonia↗
PubMed1986

Separation of acylcarnitines from biological samples using high-performance liquid chromatography.

A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.

Animals↗
PubMedDate not supplied

Analysis of the purity of cetrimide by titrations.

The purity of cetrimide, trimethyl tetradecyl ammonium bromide (TTAB), which is an important preservative of many cosmetic and pharmaceutical products, was determined by three independent methods of titration. Traditionally, cetrimide was analysed by an assay method of the European Pharmacopoeia, which showed consistently a low purity of cetrimide with associated large standard deviations, however. A systematic 3% bias of the European Pharmacopoeia assay method was identified by comparing the result with results of two alternative methods of titration that exhibited high precision and high accuracy. Titration by perchloric acid showed a 99.69% +/- 0.05% purity of cetrimide, and titration by silver nitrate showed a 99.85% +/- 0.05% purity, while the traditional assay method predicted a purity of only 97.1% +/- 0.4%. It was found that the discrepancy could be identified as differences in selectivity during the extraction step of the European Pharmacopoeia assay method. The distribution coefficients between chloroform and water of cetrimide and the corresponding iodide species (TTAI) were thus determined as 2150 +/- 50 M(-1) and 68000 +/- 4000 M(-1), respectively.

Cetrimonium↗
PubMed1970

The methylation of transfer ribonucleic acid during regeneration of the liver.

Transfer ribonucleic acid(1) is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-(14)C and (3)H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.

Acyltransferases↗
PubMed1991

A rapid one-step extraction procedure for the isolation of ubiquitin from human erythrocytes for antibody production.

A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Development and validation of an HPLC assay for fentanyl and related substances in fentanyl citrate injection, USP.

The stability indicating properties of the USP method for the assay of fentanyl in fentanyl citrate injection were evaluated [1] by analyzing fentanyl drug substance and product after acid, hydrogen peroxide, heat, and light treatment. N-phenyl-N-(4-piperidinyl)propionamide (PPA), which is a known degradation product/process impurity of fentanyl, was not adequately resolved from the fentanyl peak, and mobile phase adjustments did not improve the resolution (Fig. 1). Therefore, the USP method did not meet the requirements for a stability-indicating assay. In addition, the wavelength in the USP method was too high (230 nm) to provide adequate levels for the quantitation of the related substances of fentanyl and, in addition, the acetate ions in the mobile phase could interfere with a lower wavelength detection. An isocratic, reversed phase, stability indicating, high performance liquid chromatographic (HPLC) method for the assay of fentanyl and related substances in fentanyl citrate injection, USP has been developed and validated. The chromatographic conditions employed an Inertsil C8, 5 column (25 cm x 4.6 mm), a mobile phase of aqueous perchloric acid [0.23%, w/v]-acetonitrile [65:35, v/v], and ultraviolet (UV) detection at 206 nm. Under the chromatographic conditions of the method, PPA and seven other known process impurities were separated from the active. Degradation studies showed that the active eluted as a spectrally pure peak resolved from its degradation products.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

A highly selective spectrophotometric determination of niobium (V) using 3-hydroxy-2-[1'-phenyl-3'-(p-chlorophenyl)-4'-pyrazolyl]-4-oxo-4H-1-benzopyran as a complexing agent.

3-Hydroxy-2-[1'-phenyl-3'-(p-chlorophenyl)-4'-pyrazolyl]-4-oxo-4H-1benzopyran (HPCPB) is used as an analytical reagent for the spectrophotometric determination of niobium in trace amounts with which it forms a yellow coloured complex (4:1) in perchloric acid medium. The complex is extractable into chloroform and shows absorption maximum at 407-418 nm with a molar absorptivity of 2.79 x 10(4) L mol(-1) cm(-1) and Sandell's sensitivity equal to 0.0033 microg Nb(V) cm(-2), respectively. Beer's law holds good in the range 0-1.2 microg Nb ml(-1), with a standard deviation of +/- 0.0015 absorbance units. The method is free from the interference of a large number of elements and handles satisfactorily the analysis of various samples of varying complexity.

Absorption↗
PubMedDate not supplied

Effect of 5-aminolevulinic acid dose and estrogen on protoporphyrin IX concentrations in the rat uterus.

OBJECTIVES: To determine the in vivo dose-response relation between administered 5-aminolevulinic acid (ALA) and the concentration of protoporphyrin IX (PpIX) produced in rat uterine tissue, to determine the effect of estrogen on ALA-induced PpIX production in the rat endometrium and myometrium, and to determine the selectivity of ALA-induced PpIX production in uterine tissue. METHODS: Ovary-intact female rats (n = 53) received a subcutaneous estradiol-17 beta (E2) implant. Three days later, ALA dissolved in saline (0, 1, 2.5, 10, 25, or 50 mg/100 microL) was injected into one uterine horn. Three hours after ALA administration, the uterus was removed and the endometrium was scraped from the myometrium. In a second study, rats (n = 35) were ovariectomized and 8 days later given either an E2 or sham implant. After 3 days of hormonal or sham priming, ALA (10 or 25 mg) was injected into the uterine horn 3 hours before hysterectomy. In both studies, PpIX was extracted in a methanol/ perchloric acid (1:1) solution and quantified spectrofluorometrically. RESULTS: Five-aminolevulinic acid increased PpIX concentrations in the endometrium and myometrium in a dose-dependent fashion. Twenty-five milligrams of ALA produced maximum PpIX concentrations in both the endometrium and myometrium. In the second study, sham-implanted ovariectomized rats had endometrial PpIX concentrations approximately two times higher than those in the estrogen-primed rats after doses of either 10 or 25 mg ALA. In the third study, the endometrium had two to three times higher PpIX concentrations than the myometrium at 1, 10, 25, and 50 mg of ALA. CONCLUSIONS: An in vivo dose-response relation was demonstrated between ALA and uterine production of PpIX, with maximum PpIX concentrations occurring after 25 mg of intrauterine ALA. Because estrogen was not required to convert ALA to PpIX, complete endometrial ablation may best be achieved with an unstimulated endometrium.

Aminolevulinic Acid↗
PubMed1997

Direct synthesis of aflatoxin B1-N7 guanine adduct: a reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies.

Aflatoxin B1-N7-guanine and aflatoxin B1-human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B1-8,9-epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B1-N7-guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B1-N7-guanine adduct using free guanine and m-chloroperbenzoic acid as the chemical oxidant for the production of AFB1-8,9-epoxide. At a molar ratio of 1:1 of AFB1-8,9-epoxide and guanine the recovery of the AFB1-N7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB1-N7-guanine adduct was found to be low (30-40%). HPLC analysis of the AFB1-N7 guanine adduct showed a retention time identical with the retention time of the AFB1-N7-guanine adduct synthesized using calf thymus DNA. TLC-fluorodensitometric analysis indicated that the Rf of the AFB1-N7-guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB1-N7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB1-N7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N7-AFB1 also cross-reacted with calf thymus DNA-AFB1 adduct, indicating specificity to the guanine-N7-AFB1 moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.

Aflatoxin B1↗
PubMedDate not supplied

Determination of selenium in infant formula and enteral formula by dry ash graphite furnace atomic absorption spectrometry with deuterium background correction.

A method was developed to determine selenium in infant formula using a graphite furnace equipped with deuterium background correction after dry ashing. The method circumvents the use of perchloric acid, 2,3-diaminonapthalene (DAN) and hydride generation without the use of Zeeman background correction. Twelve commercial infant and enteral formulas and corresponding spiked products (30-500 ng) were analyzed in triplicate for Se to evaluate this method. All test portions were digested on a hot plate after addition of magnesium nitrate-nitric acid. Following heating, digests were evaporated to dryness and placed in a 500 degrees C muffle furnace for 30 min to complete ashing. All Se was converted to Se+4 by dissolving the ash in HCl (5 + 1) and holding the solution for 20 min in a 60 degrees C water bath. Se+4 was subsequently reduced to Se(zero) with ascorbic acid and collected on a membrane filter. The membrane filters were digested in a small volume of nitric acid in a microwave oven. Following digestion, contents of the vessels were diluted and analyzed for Se by graphite furnace atomic absorption spectrometry. Selenium standards in starch or in unfortified formula containing trace levels of Se were carried through the entire process. The recovery range for Se was 85-127%, and analyzed reference materials fell within their certified range for Se. This method is as sensitive (detection limit 0.44 ng Se/g) as methods reported in the literature and may be applicable to other foods.

2-Naphthylamine↗
PubMed2002

[Use of acid enamel biopsy in examinations of enamel mineral content of permanent human teeth with reference to age and environmental conditions].

Human activity during the last decades has led to changes in the circulation of fluorine and other elements in the natural environment. Due to their narrow safety margin, environmental levels of fluorine compounds require careful monitoring. Studies on contamination with fluorine of the natural environment in Szczecin and its surroundings have a long history. Until recently, maximal allowable concentrations of this element in air, water and soil have substantially been exceeded. Markers reflecting adverse effects of fluorine on living organisms and man, among them magnesium deficit, continued to raise concern. The aim of this work was to apply a modified method for enamel sampling, the acid enamel biopsy, to determine the content of fluorine and magnesium in superficial layers of enamel of permanent teeth in 14-year-old children and adults residing in Szczecin or Białystok--two cities differing as to level of contamination of the environment with fluorine compounds. Correlations between age and content of fluorine and magnesium in superficial layers of permanent teeth were studied as well. Altogether, 40 premolars extracted for orthodontic reasons in children and for periodontologic or prosthetic reasons in adults were sampled and the mineral content in three superficial layers of enamel on the buccal surface was determined. Fluorine concentrations were measured with gas chromatography, while calcium and magnesium concentrations were determined with atomic absorption spectrometry. The mineral content was studied before and after removal of calcium fluorine deposits from the surface and pores of the premolars. In addition, the thickness and mass of each layer was calculated (Tables 1 and 2). To sample the layers, perchloric acid was applied three times to the same site and the depth of dissolution after each application was measured (Table 3). Total fluorine, structural fluorine and calcium fluorine content was measured in each layer (Table 4). Magnesium content was determined before and after removal of calcium fluorine deposits (Table 5). The highest content of fluorine and magnesium was found in the outermost layer of enamel (Figs. 1-4). The content decreased with depth demonstrating a logarithmic relationship. The following conclusions were drawn: 1. Increased content of fluorine in the natural environment is associated with accumulation of this element in superficial layers of the enamel of permanent teeth in the form of structural fluorine. This process takes place in children and adults alike. 2. The content of fluorine in enamel decreases with depth, irrespective of age and contamination level of the environment. 3. Increased content of fluorine in enamel of adults, as compared with children, appears to be caused by deposits of calcium fluorine. This process takes places independently of environmental contamination with fluorine. 4. A deficit of magnesium in the enamel was found in children residing in fluorine-contaminated areas. In adults, magnesium content was not related to the place of residence, suggesting a lack of effect of fluorine contamination of the content of magnesium in the enamel. 5. The content of magnesium in superficial layers of the enamel decreases with depth following a logarithmic curve. This finding was independent of the level of contamination with fluorine or age.

Adolescent↗
PubMed2002

A comparison of cell and tissue extraction techniques using high-resolution 1H-NMR spectroscopy.

Analysis of brain metabolites by a wide range of analytical techniques is typically achieved using biochemical extraction methodologies that require either two separate samples or two separate extraction steps to prepare both aqueous and organic metabolite fractions. However there are a number of brain pathologies in which both aqueous metabolite and lipid changes occur so that a simultaneous extraction of both fractions would be valuable. The methanol-chloroform (M/C) technique enables extraction of both aqueous metabolites and lipids simultaneously. It is already well established for lipid extraction of cells and tissue but its efficiency and reproducibility for extraction of aqueous metabolites is unknown. Therefore, we compared the aqueous metabolite yield and the reproducibility of the M/C method to the commonly used perchloric acid (PCA) method, using 1H-NMR spectroscopy of adult rat brain and purified rat astrocyte culture extracts. The results indicate that M/C is a superior technique for aqueous metabolite extraction from both brain tissue and cells when compared to the PCA method. The M/C extraction technique enables the simultaneous extraction of both lipids and aqueous metabolites from a single sample using small solvent-volumes, making it well suited for NMR investigations of both tissues and cells.

Animals↗
PubMed1978

A simplified solid-phase radioimmunoassay for carcinoembryonic antigen.

A simple and rapid solid-phase radioimmunoassay for the measurement of carcinoembryonic antigen (CEA) levels in sera or plasma is described. The procedure involves perchloric acid (PCA) extraction of the samples, followed by a rapid neutralization of excess acid with alkaline buffer solution. The PCA extracts are assayed uing antibody-coated plastic tubes and radio-labeled anti-CEA antibody as a marker. The assay may be completed in one day. The solid-phase direct-binding assay demonstrated a sensitivity of 0.5 ng CEA/ml plasmin and a marked tolerance to variations in pH and ionic strength of the system. A fair correlation between the CEA levels determined by the solid-phase radioimmunoassay and zirconium phosphate gel method was observed.

Carcinoembryonic Antigen↗
PubMed1989

Simple determination of acetylcholine and choline within 4 min by HPLC-ECD and immobilized enzyme column in mice brain areas.

A rapid and simple method using a combination of high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column herein is described for simultaneous assay of acetylcholine (ACh) and choline (Ch) in discrete brain areas of the mouse. Perchloric acid extracts of small brain tissues were injected directly onto the HPLC system with no pre-cleanup procedure. The ACh and Ch levels were determined within 4 min for each chromatographic run. Recoveries were 100.0 +/- 4.8% for ACh and 96.1 +/- 1.4% (mean +/- S.D., n = 10) for Ch. Detection limits for both compounds were 0.25 pmol (signal-to-noise ratio of three). The present method has been applied to the measurement of Ch and ACh in several discrete brain areas of the mouse.

Acetylcholine↗
PubMed1987

Metabolism of excess methionine in the liver of intact rat: an in vivo 2H NMR study.

L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo 2H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-[methyl-2H3]methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small 2H resonances assigned to N1-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.

Animals↗