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PubMed2005

A simple precipitation method for measuring sialic acid in apolipoprotein B-containing lipoproteins in plasma.

BACKGROUND: A convenient method for the measurement of sialic acid in plasma apoB-containing lipoproteins is described. METHODS: Dextran sulphate-Mg(2+) precipitation and enzymatic sialic acid assay were combined and applied to analysis of plasma from 96 healthy controls and 136 hyperlipidaemic subjects of types IIa (n=46), IIb (n=43), and IV (n=47). RESULTS: The sialic acid concentrations (mean+/-SD) in plasma apoB-containing lipoproteins were 19.4+/-5.9, 24.3+/-4.7 (P<0.0001 versus normal), 23.0+/-4.7 (P<0.0001), 27.9+/-5.2 (P<0.0001), and 22.3+/-3.4 mg/L (P<0.002), for normal, all types of hyperlipidaemia, types IIa, IIb, and IV, respectively. The contents of sialic acid in apoB were 2.03+/-0.41%, 2.09+/-0.35% (no significance versus normal), 1.86+/-0.27% (P<0.0001), 1.97+/-0.26% (P<0.02), and 2.28+/-0.41% (P<0.002), for normal, all types of hyperlipidaemia, types IIa, IIb, and IV, respectively. CONCLUSION: The content of sialic acid in apoB decreased significantly in type IIa but increased in type IV hyperlipidaemia, which may reflect the presence of sialic acid in very low-density lipoprotein apolipoproteins other than apoB. This simple precipitation method will be useful to evaluate the sialic acid content in low-density lipoprotein in hyperlipidaemic subjects, especially of type IIa.

Adult↗
PubMed2005

Removing proteins from an aerated yeast fermentation by pulsing carbon dioxide: replacing salting-out as a method of protein precipitation.

Salting-out is a common technique used for precipitating proteins and other materials from fermentation and tissue culture processes. It leaves a salt residue in the system. Foam fractionation can also be used to remove proteins by protein precipitation from a dilute solution. In doing so, there is usually a trade-off between enrichment and recovery. An increase in the airflow rate will increase the recovery, but only at the expense of the enrichment. A new method for increasing the recovery in foam fractionations and in yeast fermentations is to add a burst of CO2 to the process and then restore the air. This CO2 acts like a temporary salt, but it does not leave behind a residue. The recovery increases as a result of the joint use of these gases, perhaps by more than 10-fold, without sacrificing the enrichment. Chicken egg albumin in a foam fractionation column can serve as a simple, experimental model for the proposed recovery process in lieu of the fermentation process.

Air↗
PubMed2005

RNA extraction from plant tissues: the use of calcium to precipitate contaminating pectic sugars.

Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca2+, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.

Calcium↗
PubMed1979

Cholesterol in high-density lipoproteins: a comparison between dextran sulfate-magnesium chloride precipitation and preparative ultracentrifugation.

The cholesterol in high density lipoproteins (HDL) has been determined in 140 serum samples after dextran sulfate-magnesium chloride precipitation of the apolipoprotein B-containing lipoproteins. The values correlated well with simultaneously measured values from preparative ultracentrifugation (slope 0.98, r = 0.93). Furthermore, there was a significant correlation in 60 specimen between the dextran sulfate-MgCl2 and heparin-MnCl2-precipitation method.

Chemical Precipitation↗
PubMed1998

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0.

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine beta-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using alpha-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of beta-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 microM; kinetic parameters for the substrate hydrolysis were: Km = 32 microM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

Amino Acids↗
PubMed1986

The effect of immediate polyethylene glycol precipitation on free insulin measurements in diabetic patients with insulin antibodies.

Free insulin (FI) measurements obtained by polyethylene glycol (PEG) precipitation within 3 min of drawing the blood sample (FI3) from four insulin-treated diabetic subjects with a wide range of insulin antibodies were compared with published methods of FI estimation. Comparison of FI values obtained by PEG precipitation in assays of replicate samples of the same specimens (N = 9) stored at 4 degrees C for 24 h (EFI) and FI3 were 4.76 +/- 1.5 microU/ml (mean +/- SEM) and 17.13 +/- 4.7 microU/ml, respectively (P less than 0.005). Comparison of FI values obtained in six groups of replicate samples (N = 12, 18 per group, a total of 91 specimens) from these four patients assayed immediately after thawing to 18 degrees C, and incubated at 37 degrees C for 30, 60, and 120 min, and FI3 showed a significant difference in at least one of these four comparisons (mean +/- SEM) in each of these six sample groups. In 18 of 24 comparisons there was a loss of FI when stored samples were used with or without incubation (12 of these were significant at the P less than 0.05-0.001 level), but in 6 of the 24 comparisons there was an increase in the FI against FI3 (3 of these 6 significant at the P less than 0.05-0.01 level). There was a trend toward a greater loss of FI in stored samples with higher FI3 content. Loss of FI during incubation occurred in all groups irrespective of the FI3 content.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Precipitation↗
PubMedDate not supplied

Glycerol prevents insulin precipitation and interruption of flow in an implantable insulin infusion pump.

Insulin precipitation is a major obstacle to the use of implantable insulin infusion pumps. In one such pump (Infusaid, Infusaid Corporation, Norwood, Massachusetts), unprotected insulin precipitated and occluded nine pumps implanted in normal dogs within 43 days. In contrast, two similar pumps containing insulin mixed with 80% glycerol functioned normally for more than 250 days. In human studies, a similar mixture allowed insulin to be delivered to nine diabetic subjects for more than 6 mo in each case; total fluid flow rates from the pump were essentially unchanged after 460 patients-weeks of insulin infusion. A possible drawback of the mixture is a time- and temperature-dependent propensity to cause the formation of soluble, higher-molecular-weight insulin polymers, which apparently have lower biologic activity. Formation of such polymers and maintenance of biologic activity were largely prevented by the addition of phosphate buffer at neutral pH.

Animals↗
PubMed1981

Comparison of different analytical and precipitation methods for direct estimation of serum high-density lipoprotein cholesterol.

In this study two commercial enzymatic methods for the determination of serum cholesterol have been adapted for a continuous-flow analyser and a discrete multichannel analyser. The selective precipitation methods for lipoproteins using either dextran sulphate/MgCl2, heparin/MnCl2, phosphotungstate/MgCl2 or polyethylene glycol (mol. wt 6000) were compared with each other and with ultracentrifugal analysis as well as with agarose gel electrophoresis for lipoproteins. The results indicated that the dextran sulphate/MgCl2 precipitation procedure was the method of choice for the determination of HDL-cholesterol from clear and hyperlipemic sera. HDL-cholesterol values for 391 apparently health women (X +/- SD) were 1.58 +/- 0.34 mmol/l and for 432 men 1.27 +/- 0.29 mmol/L. The corresponding HDL-cholesterol/cholesterol ratios for women were 0.292 +/- 0.068 and for men 0.229 +/- 0.069. The HDL-cholesterol in serum was slightly age dependent in women, while the HDL-cholesterol/cholesterol ratio showed highly significant negative correlation with the age in both women and men.

Adolescent↗
PubMed1990

Diurnal rhythm in serum activity of wheat-germ lectin-precipitable alkaline phosphatase: temporal relationships with the diurnal rhythm of serum osteocalcin.

Serum levels of osteocalcin (S-OC) and lectin-precipitable alkaline phosphatase activity (S-LAP) are sensitive markers of osteoblastic activity. Diurnal variation has been found for S-OC but has not been reported for S-LAP. We measured S-LAP and serum total alkaline phosphatase (S-TAP) in samples drawn every 60 min during a 24-h study period in nine normal subjects and correlated the findings with the diurnal variation in S-OC. A significant (p less than 0.05) diurnal variation in S-LAP characterized by peaks at 1430 hours and 2330 hours and nadir at 0630 hours was found. Peak levels were 30% higher than nadir level (p less than 0.05). S-TAP also varied significantly (p less than 0.05) with nadir at 0630 hours, showing a difference of 23% between peak and nadir levels (p less than 0.05). Significant cross-correlation was found between S-OC and S-LAP and S-TAP when these lagged 4 h after S-OC: r = 0.51 (p less than 0.02) and r = 0.65 (p less than 0.003), respectively. In other words, changes in S-LAP and S-TAP preceded changes in S-OC by 4 h. There were no significant cross-correlations between the non-lectin-precipitable fraction of AP and S-OC. In conclusion, S-LAP varies in a diurnal rhythm closely related with the diurnal rhythm of S-OC. The almost similar patterns in the diurnal serum levels of these two osteoblastic products strongly suggest that osteoblastic activity fluctuates rhythmically during the day in humans.

Adult↗
PubMed1975

Sodium urate accelerates precipitation of calcium oxalate in vitro.

Precipitation of calcium oxalate crystals from a metastable solution can be detected within 10 min if crystalline sodium urate is added at a solid to liquid ratio of 0.1 mM or more. Without urate, precipitation begins after 50 min. Uric acid is not effective. Pyrophosphate inhibits the effects of sodium urate.

Calcium↗
PubMed2003

Precipitation hardening in a dental low-gold alloy.

Age-hardening characteristics in a dental low-gold alloy composed of 40.0 wt% Au-35.0 wt% Ag-7.9 wt% Pd-7.0 wt% Cu-5.0 wt% In-3.5 wt% Zn-1.5 wt% Sn, were investigated by means of the hardness test, XRD study, SEM observations and EPMA. The following results were obtained. The age-hardening was characterized by a precipitation of Cu-rich alpha2 phase in the a phase. The softening that occurred following prolonged ageing was due to the heterogeneous formation of the fine nodular precipitates composed of the Ag-rich alpha1 phase and the Cu-rich alpha2 phase at the grain boundaries of the a phase.

Chemical Phenomena↗
PubMed1999

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.

Bifidobacterium↗
PubMed1994

Determination of apolipoprotein E in high density lipoprotein fraction by immunofixation method and turbidimetric immunoassay after precipitation.

Apolipoprotein E (apoE) in high density lipoprotein (HDL) fraction (HDL-fr) was determined by the immunofixation method and turbidimetric immunoassay (TIA) after precipitation with phosphotungstic acid/MgCl2 in normolipidemic control subjects and patients with type IV hyperlipemia and hyper HDL-cholesterolemia. Immunofixation assay revealed two major bands of apoE in whole serum: one in the alpha-area and the other in the pre beta-area. ApoE in alpha-area (alpha-apoE) was identical to alpha-apoE in HDL-fr separated by ultracentrifugation but not to alpha-apoE in HDL-fr separated by precipitation (pHDL-fr). alpha-ApoE in pHDL-fr lacked the slower area of the band. Agarose column chromatography and gradient gel electrophoresis indicated that alpha-apoE belongs to early fractionated HDL, and that the precipitatable alpha-ApoE belongs to the higher molecular size HDL alpha-ApoE (%) estimated by immunofixation showed a strong positive correlation with apoE (%) in pHDL-fr. ApoE (%) in pHDL-fr was higher in case of hyper HDL-cholesterolemia and lower in type IV hyperlipemia than in controls, and was inversely correlated with serum triglycerides (TG) and positively with HDL-cholesterol (especially HDL2-cholesterol) in these subjects. It is suggested that the variation of apoE in pHDL-fr depends on the level of HDL2. Also, it may be suggested that apoE in pHDL-fr and precipitatable alpha-apoE belong to low and high molecular HDL2, respectively, and that apoE content in these particles is correlated with HDL2.

Adult↗
PubMed1999

Survey of total error of precipitation and homogeneous HDL-cholesterol methods and simultaneous evaluation of lyophilized saccharose-containing candidate reference materials for HDL-cholesterol.

BACKGROUND: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs). METHODS: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated. RESULTS: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum-maximum) TE was 11.5% (2.7-25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of </=13% issued by the National Cholesterol Education Program (NCEP). In the homogeneous HDL-cholesterol method group, which included five laboratories, each performing two different methods, the mean (minimum-maximum) TE was 9.5% (6.0-17.3%) for the Boehringer assay and 15.7% (3.3-30.7%) for the Genzyme assay. For the Boehringer homogeneous assay, one of five laboratories did not meet the TE criterion, whereas for the Genzyme homogeneous assay, three of five laboratories exceeded the 13% criterion. The biases on the HDL-cholesterol values found by various precipitation methods were highly variable in all CRMs, irrespective of the quality, whereas the biases found by the homogeneous method from Boehringer were far less than +/-5% for the highest-quality CRMs (CRMs 4-6). CONCLUSIONS: The NCEP goal was met by 24 of 35 laboratories assessed by use of native human sera. Selectively pooled, lyophilized CRMs that are cryoprotected with 200 g/L saccharose have ample potential for use in the standardization of homogeneous HDL-cholesterol methods.

Chemical Precipitation↗
PubMed2000

Supercritical fluid precipitation of recombinant human immunoglobulin from aqueous solutions.

Supercritical carbon dioxide was used as an antisolvent for producing recombinant human immunoglobulin G (rIgG) particulate powders. Liquid carbon dioxide (CO(2)) was premixed with ethanol to create a single-phase, modified supercritical fluid (SCF). The modified SCF was then vigorously mixed with a pharmaceutically acceptable, aqueous formulation of rIgG, and the mixture was immediately atomized into a pressurized vessel where rapid expansion of the modified SCF extracted the aqueous phase, resulting in precipitation of the protein powder. The process was reproducible, and resulting powder products were characterized by their aqueous solubilities, and the spectroscopic profile, molecular integrity, and antigen binding activity of the individual soluble fractions. Molecular integrity was assessed via size-exclusion high-performance liquid chromatography (SEC), whereas antigen binding activity was determined using an enzyme-linked immunosorbent assay (ELISA). Attempts to characterize particle size and morphology were confounded due to the extremely deliquescent nature of the powders, causing them to absorb moisture rapidly and become gummy. Operational conditions were optimized to a point which yielded powders that were completely soluble, and had ultraviolet (UV) spectroscopic and SEC profiles indistinguishable from those of the reference standard starting solution from which the powders were derived. Antigen binding activities of the powders, however, were </=50% of expected levels, revealing the need for improvement of this SCF processing approach for rIgGs. From this study, two important conclusions were drawn: (1) Biopharmaceutical product quality assessments must include definitive activity analyses, because profound activity losses, such as those suffered here by the rIgGs, may not be revealed when using standard physicochemical analyses alone; and, (2) A more comprehensive understanding and control of the range of SCF processing effects, particularly on aqueous-based macromolecular protein therapeutics, is necessary for SCF precipitation to gain full acceptance as a viable option for secondary manufacturing of biopharmaceutical powders.

Biotechnology↗
PubMed1999

Marine Bacillus spores as catalysts for oxidative precipitation and sorption of metals.

The oxidation of soluble manganese(II) to insoluble Mn(III,IV) oxide precipitates plays an important role in the environment. These Mn oxides are known to oxidize numerous organic and inorganic compounds, scavenge a variety of other metals on their highly charged surfaces, and serve as electron acceptors for anaerobic respiration. Although the oxidation of Mn(II) in most environments is believed to be bacterially-mediated, the underlying mechanisms of catalysis are not well understood. In recent years, however, the application of molecular biological approaches has provided new insights into these mechanisms. Genes involved in Mn oxidation were first identified in our model organism, the marine Bacillus sp. strain SG-1, and subsequently have been identified in two other phylogenetically distinct organisms, Leptothrix discophora and Pseudomonas putida. In all three cases, enzymes related to multicopper oxidases appear to be involved, suggesting that copper may play a universal role in Mn(II) oxidation. In addition to catalyzing an environmentally important process, organisms capable of Mn(II) oxidation are potential candidates for the removal, detoxification, and recovery of metals from the environment. The Mn(II)-oxidizing spores of the marine Bacillus sp. strain SG-1 show particular promise, due to their inherent physically tough nature and unique capacity to bind and oxidatively precipitate metals without having to sustain growth.

Bacillus↗
PubMed1979

Detection of immune complexes by a new assay, the polyethylene glycol precipitation-complement consumption test (PEG-CC).

A new assay for the detection of circulating immune complexes, the polyethylene glycol precipitation-complement consumption assay (PEG-CC), is described. The test is both simple and sensitive, and exhibits a high degree of specificity. Immune complexes are first isolated from serum by precipitation in 2.5% polyethylene glycol (PEG) and concentrated. They are then assayed functionally by measuring their ability to fix complement using a sensitive kinetic assay for total haemolytic complement. The test can detect aggregated IgG in serum at concentrations around 6.0 micrograms/ml (about 2.0 micrograms absolute. Using DNA-anti-DNA and ovalbumin-anti-ovalbumin immune complexes prepared in vitro, antigen concentrations less than 0.5 micrograms/ml can be detected. Interference by endogenous complement, polyanions and other factors in test sera has been virtually eliminted by the design of the assay. The increased specificity of the PEG-CC test for immune complexes, should prove useful in the diagnosis and monitoring of immune complex-mediated diseases.

Animals↗