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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↗

Comparative spermicidal performance of iodinated and non-iodinated contraceptive formulations of nonoxynol-9 co-precipitated with polyvinylpyrrolidone.

The objective of this study was to evaluate the spermicidal qualities of various combinations of nonoxynol-9 (N-9; whole molecule = oligomers 1-18) and its isolated fractions (oligomers 8-10, 4-6 and 1-3), co-precipitated with non-iodinated and/or iodinated (Io) polyvinylpyrrolidone (PVP) as possible vaginal contraceptives. Spermicidal qualities of known equimolar concentrations of various combinations of PVP/N-9 and PVP-Io/N-9 were tested via a modified Sander-Cramer test (SCT) using human spermatozoa. Spermicidal agents and semen samples were mixed 1:1 (v/v) and evaluated for sperm viability. Spermicidal activity was reported as the minimal concentration (microgram/mL) of spermicide capable of killing all spermatozoa within 20 sec after exposure to the spermicide. The spermicidal activity of PVP/N-9 and PVP-Io/N-9 preparations containing N-9 oligomers 1-18 and/or 8-10 was similar, and these preparations were more efficient in killing the spermatozoa than the ones containing N-9 oligomers 4-6 and 1-3. Polyvinyl-pyrrolidone proved to be an effective vehicle for PVP/N-9 and PVP-Io/N-9 preparations, especially those containing N-9 oligomers 4-6 and 1-3. Incorporation of Io into the spermicidal preparations brought about additional efficacy. The current findings could be of clinical significance in future studies when preparing and delivering those selected co-precipitates vaginally.

Cell Survival↗

Co-precipitation of intestinal p36 with a 73-K protein and a high molecular weight factor.

p36 is a major substrate of tyrosine kinases that co-localizes with spectrin in nonerythroid cells. Recent studies by Gerke & Weber [14] have shown that p36 can be isolated from intestine by selective extraction with the Ca2+-chelating agent EGTA. We now show that p36 can be re-precipitated by adding free Ca2+ to 1 mM with the co-precipitation of a high molecular weight (MW) factor and a polypeptide of 73K. The 73K protein was purified to apparent homogeneity, rabbit antibodies were raised to it and used in Western blots and immunofluorescence microscopy. The 73K protein is found in a wide range of tissues and is particularly concentrated in fibroblasts, where its distribution partially overlaps that of non-erythroid spectrin.

Animals↗

Glycine and other amino compounds prevent chromatin precipitation at physiological ionic strength.

Glycine, proline and taurine, when present in the range 0.1-0.60 M, inhibit chromatin precipitation by sodium chloride. Histone gel electrophoresis revealed that the linker histones H1 and H5 were largely depleted from the supernatant chromatin fraction at 0.2 M NaCl, while this depletion was absent in the presence of glycine. These observations are discussed in relation with the various factors which may be involved in the precipitation process.

Adrenal Gland Neoplasms↗

Concentrative biliary secretion of ceftriaxone. Inhibition of lipid secretion and precipitation of calcium ceftriaxone in bile.

The hepatic transport of ceftriaxone, a third-generation cephalosporin, was characterized in the rat and hamster; its effect on bile flow and bile acid-induced biliary lipid secretion was also measured. In anesthetized rats with biliary fistulae, the Tmax was about 5 mumol.min-1.kg-1, and in the hamster the Tmax was about 1 mumol.min-1.kg-1. The compound was not biotransformed. At high secretion rates, the concentration of cephalosporin in bile increased to 27 mmol/L, a concentration far exceeding the solubility product of its calcium salt [2 x 10(-6) (mol/L)2], which precipitated from bile. In the rat, ceftriaxone induced choleresis (22 microL/mumol ceftriaxone, the expected value for a dianionic compound). In the isolated perfused rat liver, ceftriaxone had a fractional hepatic extraction rate averaging 3%; the compound was concentratively secreted into bile, the bile-perfusate ratio ranging from 35-250. Ceftriaxone inhibited phospholipid and cholesterol secretion induced by endogenous or exogenous bile acids; the rate of inhibition was linearly proportional to the canalicular secretion rate of ceftriaxone. Hepatic transport of ceftriaxone had no influence on hepatic secretion of ursodeoxycholyltaurine. In contrast, the net hepatic transport of ursodeoxycholic acid, ursodeoxycholyltaurine, or cholyltaurine inhibited ceftriaxone transport in a dose-dependent manner. It is concluded that ceftriaxone and bile acids share a common mechanism for hepatic transport in the rat and also interact in the processes involved in biliary lipid secretion. Biliary secretion of unbiotransformed ceftriaxone occurs at high concentrations; secondary Ca2+ entry results in the formation of supersaturated canalicular bile and subsequent precipitation as a calcium salt in the biliary tract. These data explain the formation of biliary sludge that occurs in patients undergoing high-dose ceftriaxone therapy.

Animals↗

Integration of aqueous two-phase extraction and affinity precipitation for the purification of lactate dehydrogenase.

Integration of extraction in aqueous two-phase system and affinity precipitation was investigated as a technique for purification of lactate dehydrogenase (LDH) from porcine muscle extract. An enteric coating polymer, Eudragit S 100, which can be made reversibly soluble and insoluble by change in pH was used as the ligand carrier. The ligand used was Cibacron blue 3GA. The polymer is nearly totally partitioned to the top phase (> 98%) in PEG-dextran aqueous two-phase system. The enzyme, lactate dehydrogenase, was first spontaneously partitioned to the bottom phase in a 6% (w/w) PEG 8000-8% (w/w) dextran T250 phase system. New PEG phase and Eudragit-dye were then added to the bottom phase, which helped in extraction of LDH to the top phase. After a washing step with a fresh bottom phase, Eudragit-dye-target protein affinity complex was precipitated out from the top phase by lowering the pH to 5.1. The enzyme was recovered by treatment of the complex with 0.5 M NaCl with a yield of 54% and a specific activity of 245 units/mg. The purification of LDH by this procedure was better than that obtained by a single step of affinity partitioning.

Acrylic Resins↗

Modification of the Farr assay using ethanol-ammonium acetate precipitation and its application to the measurement of affinity of anti-HCG produced in several species.

A double isotope modified Farr assay was used to determine the total binding sites and affinity of antibodies to human chorionic gonadotrophin. Precipitation of the antigen--antibody complex at equilibrium with ammonium sulphate gave very high levels of nonspecific binding. Good discrimination over background was observed using a specific anti-immunoglobulin serum. However since we were interested in measuring the affinity of antibodies raised in several animal species it was more appropriate to use a single nonspecies precipitating reagent. We found that the use of a mixture of ethanol-ammonium acetate gave very low levels of non-specific binding in baboons, marmosets, rabbits and mice.

Animals↗

Radioimmune double PEG precipitation technique for detecting complexed IgE.

The presence of IgE in immune complex-enriched fractions prepared by 3% polyethylene glycol (PEG) precipitation was studied. The dissolved PEG-insoluble fraction was incubated with [125I]anti-IgE antibodies an then precipitated again with 3% PEG. Radioactivity in the washed pellets was taken as indicating IgE present in immune complex form. A calibration curve was constructed using artificially prepared IgE-anti-IgE complexes. The method developed may be useful to detect other components present in immune complex fractions.

Antibodies, Anti-Idiotypic↗

Quantification of human immunoglobulins by semiautomatic polyethylene glycol precipitation radioimmunoassays. Estimation of circulating immune complexes and immunoglobulin synthesized in vitro.

Simple, reliable semiautomatic radioimmunoassays have been developed for the measurement of human immunoglobulins (IgG, IgA, IgM, IgK and Ig lambda). The assays are based on the separation of free from antibody-bound radiolabelled fragments of immunoglobulin by precipitation with 13% polyethylene glycol. The precipitate is harvested and washed on glass fibre filters by means of a commercial cell harvester. Radiolabelled lambda chain from Bence Jones urine was used for lambda chain assay and Fab' gamma from pooled IgG for the kappa assay. Labelled Fab or Fc fragments of IgG, IgA and IgM were used for the class specific assays. Selected commercial antisera were used throughout. The sensitivity limits of the assays performed according to the standard procedure were about 20 ng Ig per ml. The assays have been used for the quantification of circulating immune complexes and both cellular and secreted immunoglobulin produced in cell cultures. The results demonstrate the importance of the simultaneous quantification by L chain and H chain specific assays.

Animals↗

A filtration double antibody radioimmunoassay that simplifies and semi-automates the isolation of immune precipitates.

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.

Animals↗

Jacalin, a jackfruit lectin, precipitates IgA1 but not IgA2 subclass on gel diffusion reaction.

Precipitation reaction in agarose gel between jacalin, a lectin from jackfruit seeds, and purified immunoglobulins of various classes and their components was studied. Single precipitation arcs were observed with monomeric and dimeric IgA1 subclass as well as secretory IgA1, but not with IgA2 of both allotypes, IgG, IgM, IgD, IgE, free secretory component or with the J chain. Thus, jacalin can be used for differentiating IgA1 from IgA2 subclass by a simple agarose gel diffusion.

Chemical Precipitation↗

Differential binding of dexamethasone to ammonium sulfate precipitates of human adipose tissue cytosols.

Saturation analysis of the binding of [3H]dexamethasone [( 3H]DEX) to ammonium sulfate precipitates (ASPs) confirmed the presence of a limited-capacity, high-affinity binder in human adipose tissue cytosols. Various non-radioactive steroids competed with [3H]DEX for binding to the ASPs in the following sequence: dexamethasone (DEX) approximately equal to triamcinolone acetonide (TA) greater than progesterone (P) much greater than estradiol (E2). The steroid specificity of the binder precipitated by AS was consistent with the specificities reported for glucocorticoid receptors in a number of systems. In order to investigate possible regional differences, glucocorticoid binding to ASPs derived from adipose tissues removed from two different sites in the same subject was quantitated. ASPs of human omental adipose tissue bound significantly more [3H]DEX than did similar preparations of subcutaneous adipose tissue from the abdominal wall (116 +/- 32 vs. 50 +/- 22 fmol/mg protein; mean +/- SD; p less than 0.02). The findings are consistent with reports from other laboratories suggesting that intra-abdominal fat is more responsive to glucocorticoids than is subcutaneous adipose tissue.

Adipose Tissue↗

Evidence that changes in fibrinogen quality during acute phase reactions are of major importance for the amount of heparin precipitable fraction (HPF).

The influence of qualitative and quantitative changes in plasma fibrinogen upon the amount of heparin precipitable fraction (HPF), obtained during the course of acute disease states, were examined. Whereas fibrinogen in plasma includes 3 species of different molecular weights (HMW, LMW, LMW'), fibrinogen in HPF, from normal as well as from "acute phase" plasma, consisted almost exclusively of HMW-fibrinogen (3% SDS-PAGE). Sub-unit chain electrophoresis (10% PAGE-SDS and two-dimensional electrophoresis) did not disclose any signs of abnormalities in the fibrinogen precipitated in HPF. Studies on patients revealed that the composition of plasma fibrinogen changed (increased HMW) during the course of acute myocardial infarction and following hip operations, and indicated that the amount of HPF was governed by the relative amount of HMW-fibrinogen as well total fibrinogen. This assumption was strengthened by quantitating the amount of HPF obtained after addition of highly purified HMW and LMW fibrinogen to normal and "acute phase" plasma. It is concluded that fibrinogen quality (per cent HMW or HMW/LMW ratio) is of major importance for the amount of HPF, thereby explaining some of the HPF-variations seen during the course of acute diseases.

Acute Disease↗

Precipitation methods for quantification of lipoproteins.

In the foregoing discussion we have included precipitation methods for HDL analysis that we consider to be among the most accurate and extensively characterized compared to analyses performed with the ultracentrifuge. The emphasis has been on the precipitation steps rather than on the analysis of cholesterol in the lipoprotein fractions. It is evident, however, that the accuracy of the cholesterol method itself will influence the overall reliability of the measurements even when the lipoprotein fraction has been separated adequately. Ideally, the cholesterol measurements would be made with reference methods. Since this is usually not practical with large numbers of samples, the accuracy and precision of the method employed should be established with respect to a reference method, such as that of Abell et al., before being used for routine cholesterol measurements. Finally, it should be mentioned that the conditions used in the methods discussed here were developed for human plasma or serum, and in the authors' experience may have to be modified for the adequate separation of plasma lipoproteins in other species.

Blood Specimen Collection↗

Precipitation of proteins with polyethylene glycol.

Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.

Chemical Precipitation↗

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin.

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.

Animals↗

Polymer-protein conjugates. II. Affinity precipitation separation of human immunogammaglobulin by a poly(N-isopropylacrylamide)-protein A conjugate.

The conjugate of protein A with poly(N-isopropylacrylamide) was synthesized and utilized in the separation of human immunogammaglobulin. In the separation process, poly(N-isopropylacrylamide)-protein A conjugate binds to the immunoglobulin with high specificity to form the poly(N-isopropylacrylamide)-protein A/immunoglobulin complex. The complex can be conveniently separated by precipitation upon heating above the lower critical solution temperature of the poly(N-isopropylacrylamide)-protein A/immunogammaglobulin complex. The separation capacity of poly(N-isopropylacrylamide)-protein A conjugate for human immunogammaglobulin was studied and it was demonstrated that approximately one out of every four protein A molecules binds to human immunogammaglobulin with a dissociation constant (Ks) of 3 x 10(-6) M. The affinity precipitation separation of human immunogammaglobulin is a rapid process which avoids the need for chromatographic columns. It can also be designed to run in a continuous mode.

Acrylic Resins↗

Effect of polyethylene glycol on the rate of immune complex formation by non-precipitating antibody.

A kinetic study formation of large size complexes of non-precipitating pig-Dnp anti-Dnp antibody and multivalent dinitrophenylated serum albumin was performed using light scattering and absorption spectroscopy of the Dnp-group. A very rapid phase of the process resulted in the formation of complexes having molecular weight of about 2 X 10(6). Further increase of the complex size was much slower. Addition of PEG affected positively the rate of complex growth even in concentrations below 1%. The spectroscopic kinetic curves also showed a rapid and a slow phase, sensitive to the presence of PEG. The character of the kinetic data does not support the simple view that polymers enhance precipitate-formation by the steric exclusion of complexes from the polymer domains. It can be assumed that the interaction of the polymer with the antigen-antibody system consists of a subtle temporary attachment of the polymer to the antibody molecule resulting in a change of the shape and/or flexibility of the antibody molecule, favouring its cross-linking capacity.

Absorption↗