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PubMed1986

Analysis of plasma lipoproteins by ultracentrifugation in a new fixed angle rotor: evaluation of a phosphotungstic acid/MgCl2 precipitation and a quantitative lipoprotein electrophoresis assay.

A routine procedure for the ultracentrifugal analysis of human plasma lipoproteins by means of a fixed angle rotor was developed. It was applied as reference method for the evaluation of two widely used procedures of lipoprotein analysis, i.e. measurement of HDL-cholesterol after precipitation of the apolipoprotein B containing lipoproteins with a new phosphotungstic acid/MgCl2 reagent, and a quantitative lipoprotein electrophoresis system. A new statistical approach to the multivariate comparison of analytical methods, the linear structural relationship model, has been applied. The HDL-cholesterol levels measured after phosphotungstic acid/MgCl2 precipitation agreed well to those determined by ultracentrifugation, whereas electrophoretically quantified alpha-lipoprotein-cholesterol exceeded the ultracentrifugal HDL-cholesterol. The Friedewald formula obviously underestimated LDL-cholesterol due to an overestimation of VLDL-cholesterol, whereas the electrophoretically quantified pre-beta- and beta-lipoprotein-cholesterol levels fairly coincided with the respective ultracentrifugal measurements. The inter-assay reproducibility of the HDL-cholesterol determination after phosphotungstic acid/MgCl2 precipitation and subsequent LDL-cholesterol quantification according to Friedewald was statistically equivalent to that of ultracentrifugation, whereas the quantitative lipoprotein electrophoresis proved less precise.

Animals↗
PubMed1994

Precipitation procedures used to isolate high density lipoprotein with particular reference to effects on apo A-I-only particles and lipoprotein(a).

Analysis of high density lipoprotein (HDL) isolated from serum without major hypertriglyceridaemia and by five different precipitation methods showed that there were no significant differences in total cholesterol and triglyceride concentrations in the HDL supernatants prepared by the different methods but that free cholesterol, phospholipid, apolipoprotein (apo) A-I and HDL particles containing apo A-I but not apo A-II (LpAI) concentrations were significantly lower for heparin-manganese chloride method 2 (final manganese chloride concentration 92 mmol/l) compared with the other methods. Modest differences in HDL cholesterol, apo A-I and LpAI were observed between heparin-manganese chloride method 1 (final magnesium concentration 46 mmol/l) and the dextran sulphate, phosphotungstate and polyethylene glycol 6000 methods. Lipoprotein(a) (Lp(a)) and apo B were undetectable in the HDL supernatants, indicating that apo B-containing lipoproteins including Lp(a) were essentially completely removed by all the precipitation procedures.

Apolipoprotein A-I↗
PubMed1994

A new precipitation method with magnetic separation for high-density-lipoprotein cholesterol assay.

We describe a new precipitation method for high-density-lipoprotein cholesterol quantitation. The new method uses magnetic force instead of centrifugal force to separate high-density-lipoprotein from other lipoproteins that are fractioned with a precipitating reagent. The reagents used for the new method are the same as those for the conventional method except that magnetizable particles are included in the former. The magnetizable particles are used without any modifications. The correlation between the new and the centrifuge methods with dextran sulfate-magnesium chloride, sodium phosphotungstate-magnesium chloride and polyethylene glycol 6,000 were satisfactory (r = 0.990, 0.997 and 0.997, respectively). The new method, which can be combined with any precipitating reagents used in conventional methods, is very simple to perform and does not need any special equipment.

Chemical Precipitation↗
PubMed1984

Functional and structural analysis of acetylcholine receptor-rich membranes after negative staining.

Phosphotungstate (pH 7.4) used for negative staining of membranes from Torpedo electric tissue rich in acetylcholine receptor does not affect binding properties and cation permeability of the receptor and its ion channel. Uranyl salts, frequently used for negative staining, precipitate the receptor-rich membranes making measurements of ligand binding and ion-permeability regulation impossible. The gross ultrastructure in the two stains is not significantly different, but for future high-resolution electron microscopy aiming at visualizing structural details of functional receptor molecules it is necessary to resort to a stain preserving native and active receptor. Uranyl salts are not applicable for this purpose. The electron micrographs obtained with phosphotungstate reveal two distinct structures in the receptor-rich membrane: a closed ring ('doughnut') and an open ring ('horseshoe'), with a ratio of abundance of about 3:2.

Acetylcholine↗
PubMed1989

Shwartzman reaction in the brain induced by Haemophilus somnus and Escherichia coli lipopolysaccharide in rabbits.

Intracerebral inoculation of viable Haemophilus somnus resulted in suppurative or fibrino-suppurative meningitis of the brain and spinal cord in rabbits. Multiple fibrin thrombosis complicated with meningitis in the central nervous system was produced by intracerebral inoculation of H. somnus followed by intravenous inoculation with Escherichia coli lipopolysaccharide. The latter reaction may be attributable to a form of Shwartzman reaction.

Animals↗
PubMed1994

The use of a scanning proton microprobe to observe anti-HIV drugs within cells.

A series of inorganic polyanions (viz. heteropolytungstates) has been shown to have antiviral activity but there was no evidence to indicate that the drugs reached their site of antiviral (HIV) activity intact. We have shown that with a scanning proton microprobe it is possible to analyse the metal content of individual cells (PBLs) treated with such a polyoxometalate drug and to determine the atomic ratio of the metals within the cell. This was found to be near that in the drug. The distribution of the metals (tungsten and cobalt) within the cell was measured and it was shown that both metals were located in the same region within the cell. These findings would suggest that the drug had entered the cells intact.

Antiviral Agents↗