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PubMedDate not supplied

Adaptation of the phosphotungstate method for the determination of vitamin C contents in animal and human tissues.

The usefulness of phosphotungstate reagent for vitamin C determination in tissue homogenates has been confirmed. An optimal homogenization medium was selected: 1.8 M solution of HPO3 in 1.3 M CH3COOH. With this medium the analytical curve (at 700 nm) demonstrated the right linearity, correlation and recovery coefficients were appropriately high (0.999 and 99.8%) and the values of intraserial and interserial variation coefficient were low (< 5% and < 10%, respectively). It makes this method sensitive, easily repeatable, and useful for vitamin C determination in animal and human tissues, including neoplastic ones.

Animals↗
PubMed1993

Multicenter evaluation of Reflotron direct dry-chemistry assay of high-density lipoprotein cholesterol in venous and fingerstick specimens.

The Reflotron HDL Cholesterol test (Boehringer Mannheim GmbH) directly separates and analyzes high-density lipoprotein (HDL) cholesterol in plasma collected with EDTA in an integrated dry-reagent system suitable for alternative site testing of lipoproteins. We describe a multicenter evaluation of this test by two US and six European laboratories experienced in lipid analysis. Each laboratory compared the Reflotron with the same conventional wet-chemistry method, Boehringer phosphotungstate-Mg2+ precipitation with enzymatic cholesterol assay. Imprecision was within accepted guidelines, with CVs of < or = 8% for fresh and frozen plasmas (median CV 1.7-3.9%) and for lyophilized sera (median CV 3.8-4.7%), similar to those of the conventional method. Results of linear-regression analysis were as follows: Reflotron HDL Cholesterol = 1.03 conventional - 3.9 mg/L, r = 0.987. The Reflotron results were somewhat low in the two US laboratories, demonstrating the need for general standardization of methods for measuring HDL cholesterol. Results from capillary fingerstick plasma agreed well with those from venous-derived plasma; capillary = 1.04 venous + 4.5 mg/L, r = 0.967. The system is relatively insensitive to interference from hemoglobin (< or = 0.75 g/L), ascorbic acid (< or = 0.3 g/L), bilirubin (< or = 50 mg/L), cholesterol (< or = 3.5 g/L), and triglycerides (< or = 4 g/L). The relative ease of operation and the rapid availability of results (within 90 s for plasma collected in EDTA) make the method appropriate for use by well-trained, but not necessarily technical, operators in the physician's office or other alternative sites.

Aminopyrine↗
PubMed2006

Histological assessment of the effect of laser irradiation on skin wound healing in rats.

OBJECTIVE: The purpose of this study was to evaluate, from the histological point of view, the effect of diode laser irradiation on skin wound healing in Sprague-Dawley rats. BACKGROUND DATA: Various biological effects have been described in different studies after low-level laser therapy (LLLT). METHODS: Two parallel full-thickness skin incisions were performed on the back of each rat (n = 49) and immediately sutured. After surgery, one wound of each rat was exposed to laser irradiation (continuous mode, 670 nm, daily dose 30 J/cm2), whereas the parallel wound was not irradiated and served as control. Both wounds were removed 24, 48, 72, 96, 120, 144, and 168 h after surgery and routinely fixed and embedded in paraffin sections, stained with hematoxylin and eosin, van Gieson, periodic acid Schiff + periodic acid Schiff diastase, Mallory's phosphotungstic hematoxylin, and azur and eosin, and histopathologically evaluated. RESULTS: As compared to nonirradiated control wounds, laser stimulation shortened the inflammatory phase as well as accelerated the proliferative and maturation phase, and positively stimulated the regeneration of injured epidermis and the reparation of injured striated muscle. CONCLUSION: LLLT at 670 nm positively influences all phases of rat skin wound healing.

Animals↗
PubMed1989

A rapid method for staining large chylomicrons.

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.

Animals↗
PubMed1997

Three generations of high-density lipoprotein cholesterol assays compared with ultracentrifugation/dextran sulfate-Mg2+ method.

We report on the analytical performance of three generations of HDL-cholesterol assays: phosphotungstic acid/Mg2+, Spinpro, and a homogeneous method, N-geneous. The run-to-run imprecision (CV) of all assays was < or = 4.9%, and all results correlated highly with those of a modified reference procedure (r > or = 0.96). At triglycerides concentrations < 4000 mg/L, these field methods showed an acceptable systematic error (y = 1.12x - 47, 1.05x - 23, and 0.96x + 8 for the phosphotungstate, Spinpro, and N-geneous assays, respectively), and the total error of the field methods met the current National Cholesterol Education Program (NCEP) performance goal of < or = 22%. Regression analyses of results for samples with triglycerides > 4000 mg/L produced the following results for the above respective assays: y = 1.08x - 4.2, 1.02x + 3.6, and 0.85x + 108. In this hypertriglyceridemic group, only the N-geneous assay (at an HDL-cholesterol content of 240 mg/L) had a total error (35%) that exceeded the NCEP limit. Bilirubin and ascorbate produced a negative interference with the phosphotungstate and Spinpro assays but had little effect on the N-geneous assay.

Ascorbic Acid↗
PubMed1982

[Carbohydrate content of lipoproteins after sequential precipitation with phosphotungstate magnesium chloride].

A method is described for rapid determination of protein-bound carbohydrates in human lipoproteins. The method is based on a successive precipitation of lipoproteins with sodium phosphotungstate and magnesium chloride. After purification of the separated lipoproteins by a twofold reprecipitation, a colorimetric determination of hexoses, hexosamines and N-acetylneuraminic acid (sialic acid) is performed. The coefficients of variation in the series are between 3.2% and 7.3%. The results for lipoprotein-bound carbohydrates after precipitation are compared with the corresponding values after separation by ultracentrifugation. The concentrations of hexoses, hexosamines and N-acetylneuraminic acid in the 3 lipoprotein classes VLDL, LDL and HDL of control persons are reported.

Carbohydrates↗
PubMed1998

New immunoseparation-based homogeneous assay for HDL-cholesterol compared with three homogeneous and two heterogeneous methods for HDL-cholesterol.

We evaluated four new commercial methods for HDL-cholesterol determination. The three completely homogeneous assays were an immunoseparation-based (IS) method from Wako, a polyethylene glycol-modified enzyme (PEG) method from Boehringer Mannheim, and a synthetic polymer-based (SP) method from Genzyme. The fourth method was a new heterogeneous method in which lipoproteins are removed using dextran sulfate-coated magnetic beads and Mg2+ (MB, Reference Diagnostics). We compared these methods with the conventional phosphotungstic acid/MgCl2 precipitation (PTA) procedure. The homogeneous assays had good intraassay imprecision with total CVs <2.3%, whereas the CVs of the MB assay were <5.9%. Adding HDL to serum to achieve HDL-cholesterol (HDL-C) concentrations up to 1000 mg/L revealed nearly complete recoveries in the IS, PEG, and MB assays, whereas the SP assay showed a lower recovery (approximately 70%). The SP HDL-C apparently increased at increasing LDL-cholesterol and VLDL-triglyceride concentrations, whereas the IS, PEG, and MB methods were not influenced by LDL-cholesterol up to 6000 mg/L (MB, 5000 mg/L) and VLDL-triglycerides up to 9000 mg/L. Free fatty acids above approximately 2 mmol/L produced falsely high HDL-C in the IS and SP assays, the error amounting to as much as 50% in some samples. An intermethod comparison in 291 fresh serum samples yielded correlation coefficients of at least r = 0.95 for all assays, when compared with the PTA procedure. The slopes and intercepts of the regression lines were 1.05 and 57 (IS), 1.12 and 9.9 (PEG), 1.00 and 39 (SP), and 1.0 and 38 mg/L (MB), respectively. The new assays are precise and simplify the determination of HDL-C, but in part they lack specificity or are susceptible to interferences, resulting in discrepancies when compared with the established PTA procedure.

Antibodies↗
PubMed1970

A fine structural analysis of intercellular junctions in the mouse liver.

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90-100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.

Acetates↗
PubMed1982

Evaluation of the Technicon bound uricase method for the determination of uric acid in urine.

We have evaluated the Technicon bound uricase method for the determination of uric acid in urine with the AutoAnalyzer II. The general analytical characteristics of the method, and the effect of urine on the immobilized uricase nylon tube reactor were investigated. The method was found to be a linear up to 7.0 mmol/l with aqueous standards and up to 5.0 mmol/l with urine samples. Between-days imprecision had a coefficient of variation (CV) of 1.6% for values of about 1.2 mmol/l, and 0.8% for values above 3.5 mmol/l. Within-run imprecision gave a CV of 0.7% for values of 1.07 and 2.95 mmol/l and a CV of 0.4% for a value of 4.77 mmol/l. The mean analytic recovery was 99.1% (range 93.8-103.0%). The sample interaction was 0.9%. The correlation with the phosphotungstate method and the manual Dutch standard method was good, but the enzymatic values were 20% lower than the phosphotungstate values. Storing the immobilized uricase nylon tube reactor at 4 degrees C, when not in use, prolonged the lifetime by nearly 50%. Urine samples were not different from aqueous uric acid standards in their effect on the stability of the uricase nylon tube reactor.

Enzymes, Immobilized↗
PubMed1980

[Density-gradient centrifugation in lithium metatungstate and tris-(hydroxymethyl)aminomethane phosphotungstate].

New substances--lithium metatungstate (MTL) and tris-(hydroxymethyl)aminomethane phosphotungstate (PTT)--have been presented for density-gradient preparation. The buoyant densities of protein, RNA, DNA and some nucleoproteins were determined in solutions of these salts. Nucleic acids have been smaller buoyant density (1.1 g/cm3) than the proteins in contrast to CsCl-gradients. The protein in PTT solution have buoyant density 1.5 g/cm3 and in MTL solution 2.0-2,3 g/cm3. It was shown that MTL gradients allow to reach better resolution in nucleoprotein analysis than CsCl gradients.

Centrifugation, Density Gradient↗
PubMed1979

Hyperxanthinemia interferes with serum uric acid determinations by the uricase method.

Serum xanthine concentrations as high as 148 mg/L were noted after treatment of a patient with Burkitt's lymphoma who was receiving allopurinol. These markedly above-normal values for xanthine led to spuriously low values for serum uric acid as measured by the uricase method. Rapid tumor lysis in patients who are receiving allopurinol may lead to marked hyperxanthinemia, which in turn may obscure hyperuricemia in such patients when the uricase method is used for uric acid analysis. In such situations, uric acid concentrations should be measured by the phosphotungstate colorimetric assay.

Adult↗
PubMedDate not supplied

Adaptation of the phosphotungstate method to determine reduced and oxidized vitamin C in blood plasma.

The phosphotungstate reagent (PTR) was used for quantitative spectrophotometric determination of physiological forms of vitamin C in blood plasma. An immediate action of PTR on the first half of the tested samples allowed to determine reduced vitamin C concentrations (I) at 700 nm. 10 mM dithiothreitol added to the second half of the samples reduced oxidized vitamin C in it--hence the total amount of this vitamin was reduced with a concentration (II) determined as above (remains of dithiothreitol were removed with N-ethylmaleimide). The difference of results (II) and (I) gave the concentration of oxidized vitamin C. The method is characterised by fault-less analytical parameters: correlation coefficients of analytical curves > 0.99, recovery factor 100.5%, variation coefficients intra- and inter-serial < 3% and < 5%, respectively, detection limit 0.05 microM. The simplicity of the method enables an easy control of the ratio of oxidized and reduced vitamin C concentrations in blood plasma--the biomarker of the level of oxidative damage to cells.

Animals↗
PubMed1978

Analytical interferences of drugs in clinical chemistry.

The effects of several drugs were tested on analytical methods used in the clinical laboratory to measure the concentration of certain chemical constituents and activity of enzymes in body fluids. Drugs were studied initially at reported toxic concentrations, but if they had an effect at this level effects of lower concentrations were also investigated. The drugs were first studied on the methods of the Technicon SMACTM analyzer. Subsequently, methods in use with the Du Pont ACA and Technicon AutoAnalyzerTM I or II were evaluated. The most commonly affected method was the phosphotungstate reduction procedure for measuring uric acid. However, methods for measuring total protein and albumin were also affected frequently, as were determinations of alkaline phosphatase and lactate dehydrogenase activity.

Chemistry, Clinical↗
PubMed1981

Analyses of cystine in human hair: its level in women's hair of former times.

An appropriate procedure for analysis of cystine in the hair has been established, and this procedure was applied to the hair of Japanese women. The recovery of authentic cystine added to a hair sample was 85-90% in 6N HCl hydrolysis, and 96% in acid hydrolysis after performic acid oxidation. The 18-hr acid hydrolysis, but not 4-hr one, was sufficient to digest the hair. In colorimetry by using phosphotungstate, Brown's reagent gave a stable color development. The cystine content by amino acid autoanalysis was significantly correlated with that by the phosphotungstate colorimetry by the modified method of Kassel et al. or of Shinohara. The cystine content in women's hair, which was collected from specimens of different individuals cut during the period from 1910s to 1980, indicated a wide variation ranging from 0.654 to 1.607 mmol half-cystine per g of hair after washing with 0.5% sodium laurylsulfate.

Adult↗
PubMed1990

The enhancing effects of anion channel blockers on sperm activation by bicarbonate.

We found that anion channel blockers such as phosphotungstate and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) enhanced HCO3(-)-induced activation on porcine epididymal sperm. In the presence of these compounds, HCO3- increased the motility, respiration rate and especially the cAMP content of the sperm to a greater extent than did HCO3- alone. The enhancing effects were not observed in the absence of HCO3-, but were evident when the concentration of HCO3- was low. These compounds did not significantly alter the intracellular pH and did inhibit the adenylate cyclase activity of the sperm plasma membrane. When these compounds were added to sperm homogenate with ATP, the cAMP formed was reduced compared to the control. In addition, these compounds inhibited both the SO4(2-) influx and efflux of the sperm. From these results, we conclude that the anion channel blockers tested principally inhibit the efflux of endogenous HCO3- derived from metabolic CO2, so that HCO3- accumulates intracellularly and stimulates the adenylate cyclase of the sperm.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

Construction and performance characterization of ion-selective electrodes for potentiometric determination of pseudoephedrine hydrochloride applying batch and flow injection analysis techniques.

New pseudoephedrine selective electrodes have been constructed of the conventional polymer membrane type by incorporation of pseudoephedrine-phosphotungstate (PE-PT) or pseudoephedrine-silicotungstate (PE-SiT) ion-associates in a poly vinyl chloride (PVC) membrane plasticized with dibutyl phthalate (DBP). The electrodes were fully characterized in terms of the membrane composition, temperature, and pH. The electrodes exhibited mean slopes of calibration graphs of 57.09 and 56.10 mV concentration decade(-1) of PECl at 25 degrees C for (PE-PT) and (PE-SiT) electrodes, respectively. The electrodes showed fast, stable, and near-Nernstian response over the concentration ranges 6.31 x 10(-6)-1.00 x 10(-2) and 5.00 x 10(-5)-1.00x10(-2) M in the case of PE-PT applying batch and flow injection (FI) analysis, respectively, and 1.00 x 10(-5)-1.00 x 10(-2) and 5.00 x 10(-5)-1.00x10(-2) M in the case of PE-SiT for batch and FI analysis system, respectively. Detection limit was 5.01x 10(-6) M for PE-PT electrode and 6.31x10(-6) M for PE-SiT electrode. The electrodes were successfully applied for the potentiometric determination of pseudoephedrine hydrochloride (PECl) in pharmaceutical preparations with mean recovery 101.13 +/- 0.85% and 100.77+0.79% in case of PE-PT applying batch and flow injection systems, respectively, and 100.75+0.85% and 100.79 +/- 0.77% in case of PE-SiT for batch and flow injection systems, respectively. The electrodes exhibited good selectivity for PECl with respect to a large number of inorganic cations, sugars and amino acids.

Calibration↗
PubMed1998

Three-dimensional electron microscopy of the photosystem II core complex.

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochrome b-559, and psbI gene product was reconstructed at 20-A resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had a p22121 symmetry. One PSII core complex was measured to be 80 x 80 A in the membrane plane and 88 A normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.

Crystallization↗
PubMed1984

Evaluation of the enzymatic assay of serum uric acid with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as chromogen.

A spectrophotometric method for the determination of serum uric acid based on the oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonate) by use of uricase and peroxidase has already been reported. The method is very precise (CV less than 4.7%). The standard curve is linear up to 4640 mumol/L. Comparison with other enzymatic methods gave good correlation. The method gave results 9% lower than the phosphotungstate method. The effects of bilirubin, haemoglobin, glucose, ascorbic acid, anticoagulants and purine compounds were studied. The reference values for this method are 140.8-407.8 mumol/L for female subjects and 145.6-514.7 mumol/L for male subjects.

Anticoagulants↗