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Quantitative determination of resorcinol in presence of phenol.

A simple and accurate method for the quantitative determination of resorcinol in the presence of phenol is reported. The method is based on the formation of indophenol by reacting resorcinol with 2, 6-dibromoquinone-4-chlorimide. The concentration of indophenol can be measured spectrophotometrically. This method is recommended for the determination of resorcinol in resorcinol-phenol-boric acid solution and carbol-fuchsin solution. The decomposition products of resorcinol and phenol, e.g., colored quinones, do not interfere with the assay procedure.

Colorimetry↗

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction.

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.

Animals↗

Direct simultaneous determination of eight sweeteners in foods by capillary isotachophoresis.

A method for isotachophoretic determination of sweeteners of different character in candies and chewing gums was developed. A capillary of 0.8 mm ID and 90 mm effective length made of fluorinated ethylene-propylene copolymer is filled with an electrolyte system consisting of 10 mM HCl + 14 mM Tris, pH 7.7 (leading electrolyte) and 5 mM L-histidine + 5 mM Tris, pH 8.3 (terminating electrolyte). The analysis is performed at a driving current of 200 microA and for detection current is decreased to 100 microA. Boric acid is added to the aqueous sample solution to form borate complexes with substances of polyhydroxyl nature and make them migrate isotachophoretically. Using conductivity detection, the calibration curves in the tested concentration range up to 2.5 mM were linear for all components of interest: acesulfame K, saccharine, aspartame, cyclamate, sorbitol, mannitol, lactitol, and xylitol. The concentration detection limits ranged between 0.024 and 0.081 mM. Good precision of the ITP method is evidenced by favorable RSD values ranging from 0.8 to 2.8% obtained at the analyte concentration of 1.0 mM (n = 6). The analysis time was about 20 min. Simplicity, accuracy, and low cost of analyses make ITP an alternative procedure to methods used so far for the determination of ionizable sweeteners.

Calibration↗

Analysis of the heat-induced denaturation of proteins using temperature gradient gel electrophoresis.

A new technique, temperature gradient gel electrophoresis (TGGE), was applied to the study of heat-induced protein denaturation. The gels used contained 30 mM Borax + 75 mM boric acid, pH 8.4, and various concentrations of urea. When this technique was applied to bovine serum albumin, the protein showed discontinuous bands upon melting, indicating that the thermal transition is irreversible. The apparent melting temperature, calculated based on the relative intensity of two bands in the transition region, was 58 degrees C in 2 M urea. When the thermal denaturation of bovine serum albumin was analyzed spectroscopically, the transition was again irreversible, with an apparent transition temperature of 57 degrees C, consistent with the TGGE results. Recombinant stem cell factor, recombinant granulocyte macrophage-colony stimulating factor, and catalase were also analyzed by TGGE, indicating that the technique can be used to analyze denaturation of monomeric and multimeric proteins.

Animals↗

Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol.

A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of SOD activity is performed at pH 8.8, 37 degrees C, in 50 mM air-saturated 2-amino-2-methyl-1,3- propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT-01050 is stabilized in stock solution acidified at pH 1.5. The SOD activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One SOD activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied SODs. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of SOD activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known SODs such as Cu/Zn-, Mn-, or Fe-SOD.

Animals↗

Quantitation of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by chemical dephosphorylation and benzoylation.

A method for the determination of the relative amounts of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by benzoolysis is described. The procedure consists of simply heating the phospholipid with benzoic anhydride in the presence of boric acid for 5 h at 100 degrees C, followed by reaction with 4-dimethylaminopyridine for 2 h at room temperature and HPLC analysis of the lipidic products formed. With model compounds of the three subclasses it is shown that diacyl- and alkylacylglycerophosphocholines are completely dephosphorylated by this procedure, yielding quantitatively the corresponding diacyl- and alkylacylglycerobenzoates. The same procedure applied to a model alk-1-enylacylglycerophosphocholine gives only 53.5% of dephosphorylation, while the dephosphorylated products in turn are quantitatively converted into the corresponding acylglycerodibenzoates. The latter figure is shown to be fairly reproducible. The reduced dephosphorylation rate of plasmalogens appears to be due to complete disruption of the vinyl ether bond. The liberated fatty aldehyde gives rise to an addition product with benzoic anhydride, which was identified by gas chromatography-mass spectrometry. It is demonstrated that acyl migration occurring during the benzoolysis does not interfere with the HPLC separation of the glycerobenzoates and dibenzoates derived from the three distinct subclasses. Results of subclass determinations by benzoolysis of several natural diradylglycerophosphocholines are in good accordance with literature values. The agreement between the plasmalogen contents, determined by benzoolysis and by phosphorus determination following exposure to HCl and separation by thin-layer chromatography, is satisfactory. The reliability of the benzoolysis method is generally discussed.

Chromatography, High Pressure Liquid↗

Pharmacokinetic studies of oral L-threo-3,4-dihydroxyphenylserine in normal subjects and patients with familial amyloid polyneuropathy.

The pharmacokinetics of oral L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) was studied in 7 normal subjects and 7 patients with familial amyloid polyneuropathy. Each person swallowed a single 300 mg dose in the fasting state, and L-threo-DOPS in plasma and urine was determined by high performance liquid chromatography with an electrochemical detector after separation on a boric acid gel column. L-threo-DOPS was slowly absorbed by normal subjects; the maximum plasma concentration occurred 3 h after administration and 20% of the oral dose was recovered unchanged in the urine within 12 h. It induced a substantial elevation of plasma norepinephrine levels, the peak being attained at 5 h, but without any change in blood pressure. In the patients, the absorption and metabolism of L-threo-DOPS were delayed, and a prolonged pressor response was observed, with a peak after 8 h. It was concluded that the effects on plasma norepinephrine and blood pressure of oral L-threo-DOPS were essentially equal to those of twice as large a dose of DL-threo-DOPS.

Adult↗

Diethylpyrocarbonate reactivity of Klebsiella aerogenes urease: effect of pH and active site ligands on the rate of inactivation.

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.

Binding Sites↗

Quantitative aspects of two methods to dissolve collagen-free muscle proteins from acetone dry powders of Guelders ring sausage.

The purpose of this study was to find experimental conditions for the complete solubility of collagen-free muscle proteins (CFMP) using acetone powder of Guelders ring sausage. Preliminary experiments were carried out to choose the best procedure for preparing the acetone dry powder. Two different methods of acetone extraction of minced sausage were compared. The acetone dry mass (ADM) method using continuous extraction in a Soxhlet [2] apparatus gave better results than the acetone powder (ACP) method, which used a blender [1]. The ADM method was used for further investigations. ADM was extracted with two types of sodium dodecyl sulphate (SDS), containing solvents A and B. Solvent A contains a Tris-boric acid buffer (pH 8.2) with 1.5% (m/v) SDS and 0.05% (m/v) dithioerythreitol [3]. Solvent B is a borate-chloric acid buffer (pH 9.0) with 2.0% (m/v) SDS and 1.0% (m/v) mercapto-ethanol [2]. Both solvents showed a linear relationship between the quantities of CFMP in ADM and the dissolved CFMP. The linear relationships were found between quantities of 10.0 and 30.0 mg (solution A) and of 5.0 and 30.0 mg ADM (solution B) per ml solvent. The solubility of CFMP was better in solvent B than in solution A. Completely dissolved CFMP from ADM was only obtained in the case of 5.0 mg ADM in 1.0 ml solution B. These conditions will be used in liquid chromatography experiments, the results of which will be reported later.

Acetone↗

Interactive effects of boron, selenium, and dietary protein on survival, growth, and physiology in mallard ducklings.

High concentrations of boron (B) and selenium (Se) have been found in aquatic food chains associated with irrigation drainwater. Total biomass of invertebrates, a major source of protein for wild ducklings, is sometimes diminished in agricultural drainwater ponds contaminated with Se and B. Day-old mallard (Anas platyrhynchos) ducklings received an untreated diet (controls) containing 22% protein or diets containing 15 ppm (microgram/g) Se (as selenomethionine), 60 ppm Se, 1,000 ppm B (as boric acid), 15 ppm Se with 1,000 ppm B, or 60 ppm Se with 1,000 ppm B. In a concurrent experiment, the above sequence was repeated with a protein-restricted (7%) but isocaloric diet. After four weeks, blood and tissue samples were collected for biochemical and histological examination. With 22% protein and 60 ppm Se in the diet, duckling survival and growth was reduced and histopathological lesions of the liver occurred. Boron alone caused some reduction in growth. Several interactive effects occurred between B and Se, including further reduction in growth, and increases in plasma glutathione reductase activity, hematocrit, hemoglobin and plasma protein concentrations. With 7% protein, the growth of controls was less than that with 22% protein, 60 ppm Se caused 100% mortality, and growth effects of 15 ppm Se and 1,000 ppm B alone were more pronounced than with 22% protein. Selenium accumulation increased in the liver with 7% protein. Interactive effects were greater for Se and B with 7% protein than with 22% protein and included significant mortality and enhanced accumulation of Se in the liver. These findings suggest the potential for more severe toxicological effects of Se and B independently and interactively on duckling survival and development when dietary protein is diminished.

Animals↗

The bactericidal activity of aqueous disinfectants applied on living tissues.

Thirteen antiseptic aqueous solutions intended for the disinfection of living tissues were compared in regard to their microbicidal effectiveness towards. Staphylococcus aureus and Pseudomonas aeruginosa. Six antiseptics, which contain boric acid, eosine, hydrogen peroxide or an organic mercury compound as the active substance, did not fulfil the requirements of the preliminary in vitro test. The seven other preparations were examined in a practical test, in which bactericidal activity was assessed on artificially contaminated intact skin after exposures of 15 s and 60 s. The most active solution appeared to be 0.5% tosylchloramide sodium, followed by 0.05% chlorhexidine with 0.5% cetrimide. The other preparations, namely 0.05% chlorhexidine without cetrimide, 0.245% chloroxylenol, 0.04% clorofene, 10% povidone-iodine and 0.2% tosylchloramide sodium, were less active in this practical test.

Bacteria↗

Effects of some chemical factors on flagellation and swarming of Vibrio alginolyticus.

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0-9.0% (w/v) NaCl, cells with polar tufts of 2-4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3-9.8% (w/v) Na2SO4 had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1-0.3% boric acid or 0.05-0.1% aluminum hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01-0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05-0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.

Agar↗

Samuel Theobald M.D. Ophthalmology at the Johns Hopkins Hospital before the Wilmer Institute 1889-1925.

Although ophthalmology today at the Johns Hopkins Hospital is synonymous with the name of Wilmer, it should be remembered that the Wilmer Ophthalmological Institute was not founded until 1925. Thus, to appreciate fully the ophthalmic heritage of Johns Hopkins we must look back to the beginnings of the medical institutions. When the Johns Hopkins Hospital opened in 1889 and the medical school followed in 1893, Samuel Theobald, M.D., was appointed ophthalmic and aural surgeon, and later clinical professor of ophthalmology and otology. Dr. Theobald, a native Baltimorean, was a member of the prestigious Smith family, which distinguished itself in early American medicine. He was raised in the home of his grandfather, Dr. Nathan Ryno Smith who directed his education. Dr. Theobald studied ophthalmology and otology abroad before opening a practice in Baltimore in 1871. Before his appointment at Johns Hopkins he was one of the founders of the Baltimore Eye, Ear and Throat Charity Hospital in 1882. In addition to his teaching at the medical school and his work in the dispensary, he contributed many articles in journals, and presentations to society meetings. He is especially remembered for his development of 'Theobald lacrimal probes', the introduction of boric acid as a collyrium, and his text book Prevalent Diseases of the Eye. He was a member of the American Ophthalmological Society for 50 years and its 14th President. In 1925 at the age of 79 years, he retired and became emeritus.

Baltimore↗

Considerations in the determination of boron at low concentrations.

Experimental evidence now supports the nutritional essentiality of boron (B) in some biological systems, and accordingly, the need for reliable analytical B data is increasing. However, the accurate determination of B in biological materials is a formidable challenge at low concentrations (<1 mg B/kg). Recent studies still show significant analytical discrepancies in the analysis of animal tissues and fluids, despite the development of instrumental techniques such as TIMS, ICP-MS, ICP-ES, ICAP, SIMS, NA-MS, PGAA, NRA, and so forth, which have demonstrated detection limits approaching or exceeding (microg B/kg concentrations. Since boric acid is both volatile and ubiquitous in nature, the chemical and physical pathways for B contamination and its loss are manifold, especially during sample preparation. An added obstacle is the inadequacy of biological reference materials certified for B below mg B/kg. With an emphasis toward sample preparation and ICP-MS analysis, examples are provided in this article to help the analyst avoid common problems associated with the analysis of B from biological sources. Topics that are discussed include contamination from Teflon vessels during microwave digestion, losses owing to freeze-drying, B isotopic variations, standards preparation, reagent backgrounds, and instrumental interferences.

Animals↗

Boron exposure from consumer products.

For a natural substance that is mined and for which the world total production is in excess of 3 million tons, it would not be surprising to discover there is considerable exposure to boron (B) and its salts. Human exposure can arise from a variety of natural sources, such as soil and water, and artificially in soils via fertilizer and in waters via discharges through its use in household products and cosmetics, or via its use in pesticides, preservatives, and pharmaceuticals. Receiving waters can also obtain B through industrial usage. Indirect and probably insignificant consumer exposure can arise via the use of borates in glass products, flame retardants, and enamels, these applications comprising the major proportion of borate usage. The general intake of B via its presence in food and water is around 1-7 mg/d, but the range of intake values is large and depends on geographical region, dietary habits (with nuts, fresh and dried fruits, and wine providing particularly rich sources), as well as the method of analysis employed. Drinking waters typically contain <1 mg B/L, but the range is large, and some populations will be exposed to considerably more than 1 mg B/L. B via food and water represents the greatest source of exposure for the general consumer. Sodium borate and boric acid are widely used in consumer products, and perborate is used in detergents, but even though there is a high potential for exposure, the internal dose of B to consumers via its use in household and personal products is very low partly owing to insignificant amounts of dermal absorption across normal skin.

Adverse Drug Reaction Reporting Systems↗

Adverse reproductive and developmental effects in Xenopus from insufficient boron.

Frog embryo teratogenesis assay--Xenopus (FETAX) was utilized as a model system to evaluate the effects on embryo-larval development at various low boron (B) exposure levels in the culture media. Concentrations tested ranged from < 1 to 5000 microg B/L. A statistically significant (P < 0.05) increase in malformations was observed at < or = 3 microg B/L, but not at the greater concentrations. Abnormal development of the gut, craniofacial region and eye, visceral edema, and kinking of the tail musculature (abnormal myotome development) and notochord were observed. In subsequent studies, adult frogs were maintained for 28 d on two diets: (1) low B (LB, 62 microg B/kg) or (2) boric acid supplemented (BA, 1851 microg B/kg); the frogs were subsequently mated, and their offspring were cultured in media containing various levels of B. Results of the 28-d depletion studies indicated that frogs maintained under LB conditions produced a greater proportion of (1) necrotic eggs and (2) fertilized embryos, which abnormally gastrulated at a greater rate and were substantially less viable than embryos from frogs fed the BA diet. Malformations similar to those seen in the initial study were observed in embryos from the B-depleted adults maintained in an LB environment; 28 d on the LB diet enhanced the incidence of malformations associated with the LB culture media. These abnormalities were not observed in embryos cultured in > or = 4 microg B/L from adults cultured on the BA diet. These studies showed that insufficient B reproducibly interfered with normal Xenopus laevis development during organogenesis, substantially impaired normal reproductive function in adult frogs, and thus represent the first studies demonstrating the nutritional essentiality of B in an amphibian species.

Animals↗

Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems.

To date, boron (B) essentiality has not been conclusively shown in mammals. This article summarizes the results of a series of in vitro and in vivo experiments designed to investigate the role of B in mammalian reproduction. In the first study, rat dams were fed either a low (0.04 microg B/g) or an adequate (2.00 microg B/g) B diet for 6 wk before breeding and through pregnancy; reproductive outcome was monitored on gestation day 20. Although low dietary B significantly lowered maternal blood, liver, and bone B concentrations, it had no marked effects on fetal growth or development. The goal of the second study was to assess the effects of B on the in vitro development of rat postimplantation embryos. Day 10 embryos collected from dams fed either the low or adequate B diets for at least 12 wk were cultured in serum collected from male rats exposed to one of the two dietary B treatments. Dams fed the low B diet had a significantly reduced number of implantation sites compared to dams fed the B-adequate diet. However, embryonic growth in vitro was not affected by B treatment. The aim of study 3 was to define the limits of boric acid (BA) toxicity on mouse preimplantation development in vitro. Two-cell mouse embryos were cultured in media containing graded levels of BA (from 6 to 10,000 microM). Impaired embryonic differentiation and proliferation were observed only when embryos were exposed to high levels of BA (>2000 microM), reflecting a very low level of toxicity of BA on early mouse embryonic development. Study 4 tested the effects of low (0.04 microg B/g) and adequate (2.00 microg B/g) dietary B on the in vitro development of mouse preimplantation embryos. Two-cell embryos obtained from the dams were cultured in vitro for 72 h. Maternal exposure to the low B diet for 10, 12, and 16 wk was associated with a reduction in blastocyst formation, a reduction in blastocyst cell number, and an increased number of degenerates. Collectively, these studies support the concept that B deficiency impairs early embryonic development in rodents.

Animals↗

Multi-element trace determinations in pure alkaline earth fluoride powders by high-resolution ICP-MS using wet-chemical sample preparation and laser ablation.

Four alternative analytical procedures for the determination of ten important trace impurities (Mg, Cr, Fe, Cu, Zn, Sr, Zr, Cd, Ba, and Pb) in pure alkaline earth fluoride powders were applied using high-resolution inductively coupled plasma mass spectrometry (ICP-MS). Two procedures are based on a wet-chemical microwave digestion with boric acid and quantification by the standard addition technique and isotope dilution mass spectrometry (IDMS), respectively. In addition, analyses are also performed by laser ablation as a direct solid sampling technique applying matrix-matched external calibration as well as isotope dilution of the powdered sample. For most elements good agreement between the different methods is found. Detection limits for laser ablation vary between 0.05 ng g(-1) for Zr and 20 ng g(-1) for Mg. They are about one to two orders of magnitude lower than those of the wet-chemical procedures, which is mainly due to the high dilution factor during the sample preparation step. Advantages and restrictions of the different analytical procedures are discussed with respect to their routine applicability. Due to its relatively high accuracy, low detection limits, and time-efficiency LA-ICP-IDMS is the preferred choice if no standard reference materials are available.

Journal Article↗