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PubMed1996

Homogeneous assay for direct determination of high-density lipoprotein cholesterol evaluated.

We evaluated a new homogeneous assay for quantifying high-density lipoprotein cholesterol (HDL-C). The assay included four reagents: polyethylene glycol for "wrapping" chylomicrons, very-low-density lipoproteins (VLDL), and low-density lipoproteins (LDL); antibodies specific for apolipoprotein (apo) B and apo C-III to produce aggregates of chylomicrons, VLDL, and LDL; enzymes for the enzymatic cholesterol determination of the noncomplexed lipoproteins with 4-aminoantipyrine as the color reagent; and guanidine salt to stop the enzymatic reaction and to solubilize the complexes of apo B-containing lipoproteins, which would otherwise interfere with the reading of absorbance. The total CVs of the new method ranged between 2.4% and 8.4%. The HDL-C values (y) were in good agreement with those by a comparison phosphotungstic acid/MgCl2 method (x): y= 0.987x + 17.2 mg/L (68th percentile of the residuals on the regression line= 21.49, r= 0.970). At triglyceride concentrations of 20 g/L (Intralipid) the homogeneous HDL-C concentrations increased by 2%. Hemoglobin markedly increased the results, whereas bilirubin reduced them. The homogeneous HDL-C assay was easy to handle and allows full automation. This test should considerably facilitate the screening of individuals at an increased risk of cardiovascular disease.

Apolipoprotein A-I↗
PubMed1997

Precipitation methods for high-density lipoprotein cholesterol measurement compared, and final evaluation under routine operating conditions of a method with a low sample-to-reagent ratio.

We evaluated six precipitation methods for high-density lipoprotein cholesterol (HDL-chol) determination: the heparin/Mn2+ precipitation reagent method (Hep), two variants of the phosphotungstic acid/Mg2+ method (Tung-L and Tung-B), the dextran sulfate 50,000/Mg2+ method (Dex), the PEG 6000 method (PEG), and the PEG 6000/dextran sulfate 15,000 (PEG/Dex) method. The Tung-B and PEG/Dex precipitation methods have a low sample/precipitation reagent volume ratio (< 0.4). The Tung-B, Dex, PEG, and PEG/Dex methods gave similar values, averaging within 0.1 mmol/L of each other, showing that the precipitation selectivity of these methods is comparable. The precipitation efficiency of Tung-B and Peg/Dex, however, was superior. Ultrafiltration of the supernatants was needed only at triglyceride concentrations > 16.4 mmol/L (undiluted sample) or > 28.0 mmol/L (sample diluted twofold); however, ultrafiltration without dilution was the most accurate method. Results of Tung-B under routine conditions (33 technicians) agreed well with those of the PEG method for 406 normo- and hyperlipidemic plasma samples. By comparison with the HDL-chol method from the Centers for Disease Control and Prevention, the Tung-B method showed a total error of 10.6% which fulfills the criteria of the National Cholesterol Education Program for HDL-chol analysis. In conclusion, with motivated personnel, Tung-B is a reliable, cost-effective method for routine HDL-chol analysis.

Chemical Precipitation↗
PubMed1988

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage.

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.

Acid Phosphatase↗
PubMed1968

Ultrastructure of the surfaces of cells infected with avian leukosis-sarcoma viruses.

When stained with ruthenium red (RR), chick embryo cells infected with various strains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 and RAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) at their surfaces as compared with uninfected cells. This increase was most prominent in cells infected with the Fujinami strain of RSV. The layer was resistant to digestion with neuraminidase or trypsin but was readily removed by exposure to hyaluronidase. The thickness of this AMPS layer was not correlated with the varying degree of loss of contact inhibition exhibited by cells infected with the different strains of virus. The staining of the cell envelope with a solution of phosphotungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins. The outer surface of the virions showed the same staining as the cell surface with RR and PTA-CR, and the budding virus particle was seen to incorporate the RR layer of the cell into its structure. The RR layers of cells and virions appeared to fuse, as did those between virus particles, suggesting that these layers play a role in the aggregation of virus particles and in their adherence to the surface of the cell.

Alpharetrovirus↗
PubMed1975

Effect of paracetamol on uric acid determination.

The effect of paracetamol on uric acid determination using a phosphotungstate reduction method has been investigated. The results show that apparently raised values of serum uric acid may be reported in patients receiving therapeutic levels of this drug. Levels of serum uric acid were determined over a five-hour period in three patients who had received therapeutic doses of paracetamol. The assays of serum uric acid were performed by a method based on phosphotungstate reduction and by one employing uricase. The results illustrate the problems of using the former method for clinical purposes.

Acetaminophen↗
PubMed1978

[Polysulfates, anionic detergents, sodium phosphotungstate and electrophoretic mobility of plasmatic proteins].

Anions with strong electro-negative charges, like sulphuric esters of polysaccharides (heparin, dextran sulphate M.W. 15 00), the sodium salt of polyanethol sulfonic acid (liquoid, Roche), anionic detergents (sodium dodecylsulphate, sodium oleate, sodium deoxycholate), and the sodium salt of phosphotungstic acid were added to human serum or citrated plasma. For each compound several final concentrations were adopted, the highest being 4%. By using microimmunoelectrophoresis and numerous specific antisera against human plasma proteins, it was demonstrated that at pH 8.60 anions increase electrophoretic mobility of the following antigens: lipoproteins alpha and beta; fibrinogen; beta 2-glycoprotein I; beta 2-glycoprotein II ; antithrombin III. All reagents utilized do not react with all these proteins; for instance, only detergents accelerate the migration rate of a lipoproteins. Besides, depending on the protein, this or that reagent may be the most active. Thus, in the polysulphate group, heparin has the highest affinity for antithrombin III, liquoid for fibrinogen and dextran sulphate for beta 2-glycoprotein I.

Blood↗
PubMed1976

Cytochemical characterization of the modified Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. II. Substitution of dyes for biebrich scarlet.

A number of acidic dyes, including various fluorochromes, were substituted for biebrich scarlet in the modified Guard (1959) procedure, a regressive staining method which appears to demonstrate basic chromosomal proteins. These substitutions were made to test the possibility that dyes other than biebrich scarlet might provide advantages in sensitivity and/or contrast, or that more control could be exerted over the "differentiation" step in which solutions of phosphotungstic and phosphomolybdic acids (polyacids) are used. Of the dyes tested in this investigation, six were found to be especially suitable: procion brilliant blue, procion yellow, geranine G, brilliant sulfoflavine, eosin Y, and eosin B. While procion brilliant blue could be used as an absorption dye only, the other dyes were used more profitably as fluorochromes. The various dyes displayed considerable variability in the ease with which they could be displaced from substrates with polyacid solutions during the differentiation step. Procion brilliant blue, procion yellow, and geranine G were displaced gradually and thus resembled biebrich scarlet. In contrast, eosin B, eosin Y, and brilliant sulfoflavine were displaced more easily from all but the most highly condensed chromatin in substrates. Brilliant sulfoflavine yeilded exceptionally bright and nearly selective fluorescence of consensed chromosomes in division, "X" chromosomes of grasshopper spermatocytes, and sperm heads. Weak, but selective fluorescence was observed when monazo sulfonated dyes, including ponta chrome violet SW, eriochrome black, diamond red, and ponta chrome blue black, were substituted in the modified Guard procedure. Similar results were obtained with solochrome cyanin R. As expected, these dyes seemed to function as weakly acidic dyes.

Animals↗
PubMedDate not supplied

Effect of succinylation on images of negatively stained arrays of mitochondrial outer membrane channels.

The voltage-dependent anion-selective channels of the outer membrane of Neurospora mitochondria occur in two-dimensional crystalline arrays. Electron microscopic images of negatively stained arrays have been compared for normal membranes and membranes pretreated with succinic anhydride, which changes the functional characteristics of the channel. Succinic anhydride does not alter the lattice parameters or the long-range order in the arrays. Also, it has no significant effect on correlation averages of channel arrays embedded in uranyl acetate. Thus, functional changes induced in the channel by succinic anhydride are probably not due to large-scale conformational changes. The distribution of the anionic stain phosphotungstate on the mitochondrial channel arrays is significantly altered by succinic anhydride pretreatment. There are loci on the channels of reduced phosphotungstate accumulation following succinylation. Since phosphotungstate selectively stains positively charged amino acids, it is proposed that these loci may represent clusters of functionally important, exposed basic amino acids.

Anions↗
PubMed2001

Conductimetric determination of reproterol HCl and pipazethate HCl and salbutamol sulphate in their pharmaceutical formulations.

A simple and sensitive conductimetric method for the determination of salbutamol sulphate and reproterol and pipazethate hydrochlorides is presented based on their ion associates with phosphotungstic and phosphomolybdic acids. The effect of solvent, molar ratio, reagent concentration and temperature were studied, and the solubility products of the formed ion associates were calculated. The method was applied to the determination of the drugs in their pure state or pharmaceutical preparations with mean recovery values of 99.82-100.54, 99.75-100.12 and 99.95-100.40%, and coefficient of variation 0.28-0.52, 0.16-0.36 and 0.19-0.33 for salbutamol sulphate, reproterol HCl and pipazathate HCl, respectively.

Albuterol↗
PubMed1991

Two distinct steps for spontaneous generation of subprotoplasts from a disintegrated bryopsis cell.

The unusual nature of protoplasm to generate subprotoplasts spontaneously from disintegrated Bryopsis cells was examined. Protoplasm extruded from algal cells aggregated rapidly in cell sap which was derived mainly from huge central vacuoles of the cells. Electron microscopic observations revealed extensive agglutination of algal cellular membranes in the protoplasmic masses, suggesting that this is of primary importance for the wound-healing ability of the alga. Seawater caused spheration of the resultant protoplasmic aggregates. Gelatinous sheaths were formed temporarily surrounding the spherical protoplasmic masses before reformation of cell membrane. Staining with phosphotungstic and chromic acids suggested that new cell membrane was formed by fusion of the disintegrated original cell membrane with cytoplasmic vesicles on the surfaces of the protoplasmic masses. Both pH and salts were found to be essentially important at the two steps of subprotoplast generation. The newly formed cell membranes were responsible for subsequent notable plasmolysis of the wounded cells in seawater. Thus, it is suggested that unicellular marine algae Bryopsis spp. naturally contain effective materials for agglutinating and fusing particular cellular membranes through the sequential aid of acidic cell sap and alkaline seawater after disintegration of the giant cells.

Journal Article↗
PubMed1981

Serum uric acid concentrations and arthralgia among patients treated with pyrazinamide-containing regimens in Hong Kong and Singapore.

The serum uric acid concentrations of patients being treated in Hong Kong and Singapore with a daily regimen consisting of streptomycin, isoniazid, rifampicin and pyrazinamide or a control regimen of daily streptomycin, p-amino-salicylic acid (PAS) and isoniazid were determined by the phosphotungstate method. Arthralgia and elevated serum uric acid levels were encountered only during the period that patients were being treated with pyrazinamide. Although daily treatment with pyrazinamide increased serum uric acid concentrations approximately 2.5-fold, the concentrations of the 6 patients who developed arthralgia were closely similar to those of matched controls without arthralgia. This fails to confirm a previous suggestion that patients with arthralgia might have higher values. Rifampicin appeared not to influence the hyperuricaemic effect of pyrazinamide.

Humans↗
PubMed1977

[Cytochemical studies of ultrathin frozen sections of blood platelets].

Results obtained by conventional techniques and freeze-ultramicrotomie have been compared. Ultrathin frozen sections of platelets, which are fixed by glutaraldehyde, freeze-protected by polyvinyl-pyrrolidone and encapsulated in gelatin show a well preserved fine structure after staining with buffered OsO4, phosphotungstic, phosphomolybdic or ammoniummolybdic acid. The preservation of polysaccharides or mucopolysaccharides depends on a suitable fixation, that of glycogen in certain cases depends on a double fixation (glutaraldehyde and OsO4). Cytoplasmic nucleoproteins like ribosomes are visible only after drying of sections and staining with uranyl-acetate. Lipids, both membrane lipoproteins as well as lipids in the matrices of cell organelles, are well preserved. For example the zinc-iodide osmiumtetroxide reaction yields positive results in frozen sections contrary to such obtained by conventional methods. Furthermore, the extraction of altered lipids after staining with dyes like acridine orange does not take place with this method. For this reasons the freeze-ultramicrotomy seems to be an useful alternative method.

Animals↗