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Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism.

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.

Cell Membrane↗

Acid-precipitated M protein compared with a column-eluted M protein preparation from type 12, group A Streptococci.

An M protein preparation of group A streptococci, precipitated with 0.03 m sodium acetate buffer (pH 4.0) was compared with a column-eluted M protein preparation. Absorption spectra and methyl pentose content were similar in both preparations. Acrylamide gel electrophoresis patterns were different. Gel diffusion demonstrated two lines of fusion in the preparations. More antigens could be demonstrated in both preparations by using immunoelectrophoresis. Neither the pH 4 precipitate nor the column-eluted preparation appeared to be a pure M protein preparation.

Antigens↗

Concentration of Rift Valley fever and Chikungunya viruses by precipitation.

Simple and efficient methods for concentrating Rift Valley fever (RVF) virus and chikungunya (CHIK) virus are described. Ammonium sulfate, potassium sulfate, or alcohol was used as a precipitating agent and the precipitate was resuspended to volumes suitable for further processing and purification. The methods permitted concentration of live RVF virus and CHIK virus about 100-fold with negligible losses of virus. RVF virus retained a high level of infectivity with potassium aluminum sulfate and alcohol, but CHIK virus retained a higher infectivity level with ammonium sulfate than with potassium aluminum sulfate. The data indicate that serum plays an important role in the concentration of both viruses, at least when the sulfate methods are used.

Animals↗

Nonspecific precipitation of serum proteins by sodium lauryl sulfate in agar diffusion and immunoelectrophoresis.

The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.

Agar↗

Clinical and immunochemical study of the serum IgG fraction not precipitated in a zinc-sodium salicylate reagent.

A reagent made of zinc sulphate (0-08 M) in a 0-4 M sodium salicylate solution at pH 7-3 precipitated most of the IgG when a small volume of human serum was added. Sera with normal IgG levels or polyclonal hyperglobulinaemia showed a close correlation between total IgG and zinc-precipitated IgG (r = + 0-95). In clinical material, not including IgG myeloma, zinc-soluble IgG varied between 0 and 6 mg/ml and was independent of the IgG serum concentration. In 31 normal subjects the average IgG concentration, as determined by the Technicon immunonephelometric method, was 10-2 +/- 1-7 mg/ml for total IgG and 2-2 +/- 1-0 mg/ml for the soluble fraction. Among 173 sera, including 24 from cord blood, 16 from pregnant women, and 133 from patients with miscellaneous diseases, no pathological conditions except three cases of IgG myeloma were found with a zinc-soluble IgG definitely above the normal values; zinc-soluble IgG levels were often low in patients with hyperglobulinaemia, and the difference was highly significant in liver disease. kappa and gamma light chains as well as the four IgG-Hp chain subclasses were found in both zinc-soluble fractions of normal IgG. A study of myeloma monoclonal IgG showed that globulins of classes 1, 3, and 4 could be either soluble or insoluble in the zinc reagent. One, G2, was mainly insoluble. Hexose and antistreptolysin contents per milligram normal IgG were not significantly different in either fraction. It is suggested that zinc-soluble IgG consists of the recently synthesized molecules, the zinc-solubility of which has not yet been decreased by protein association, lipid interaction, antigen binding, or enzymatic denaturation. Within this hypothesis, a low level of soluble IgG would mean either an increased precatabolic protein or a decreased synthesis.

Chemical Precipitation↗

Zinc acetate as a precipitant of unstable haemoglobins.

Whereas the addition of four moles of zinc acetate to one of normal human haemoglobin at pH 7.4 results in a clear solution, addition to unstable haemoglobin results in precipitate formation. Within certain limits no false-positive results are obtained with methaemoglobin, and with fetal haemoglobin. With excess zinc, normal haemoglobin also precipitates, but this process is reversible and can be used for a rapid concentration of dilute solutions of normal haemoglobin.

Acetates↗

Application of the concanavalin A precipitation technique in an evaluation of fenofibrate action on cholesterolemia in IIa and IIb hyperlipemic subjects.

Total plasma cholesterol and lipoprotein fractions were studied before and after 4 months fenofibrate (Lipanthyl) treatment of 34 hypercholesterolemic adults (16 IIa and 18 IIb), using a concanavalin A precipitation technique. The total plasma cholesterol levels decreased significantly for both IIa and IIb (24 and 18%, respectively, p less than 0.001). Fenofibrate reduced the apoprotein B bound cholesterol in IIa and IIb (29 and 27%, respectively, p less than 0.001). Apoprotein A bound cholesterol increased significantly in IIb (29%, p less than 0.001), but there was no significant increase in IIa (11%). Triglycerides were significantly reduced in both groups. In the light of concanavalin A specificity for apoprotein B and the utility of this technique, it is suggested that the concanavalin A precipitation technique may be applied to hyperlipidemic subjects undergoing treatment with lipid-lowering agents.

Adult↗

First experiences with the heparin-induced extracorporeal low-density lipoprotein precipitation in the treatment of critical limb ischaemia: a new therapeutical approach?

The heparin-induced extracorporeal low-density lipoprotein (LDL) precipitation (HELP) system is based on the fact that besides lipoproteins even fibrinogen, which seems to be a very important factor in pathogenesis of peripheral arterial disease, is precipitated by high-dose heparin at acid pH values. The elimination of excess fibrinogen and lipoproteins, in particular LDL, leads to a markedly improved microcirculation. The restoration of the latter obviously enabled us to perform limb-saving surgical procedures instead of mutilating amputations. 12 patients suffering from peripheral arterial disease were submitted to 18 HELP treatments in each case instead of having their critically ischaemic legs removed. Surgery could be limited to necrosectomy only and the wounds were either primarily sutured or covered with skin grafts. We were able to avoid 13 amputations in these 12 patients who could finally walk out of the hospital. HELP treatment was free of side effects in all of them.

Adult↗

Partial defluoridation of drinking water using fluorapatite precipitation.

Ion adsorption and ion exchange are two methods commonly used in small home units to treat drinking water to bring the fluoride concentration to within acceptable limits. However, the necessary flowthrough system is often difficult to arrange where there is no piped supply and gradual exhaustion of the active agent is not easily detected. In an attempt to overcome these problems a defluoridation method based on the precipitation of a sparingly soluble fluoride salt, fluorapatite, has been studied. Samples of simulated high-fluoride drinking waters, approximately 10 ppm F, were saturated with brushite, resulting in a state of supersaturation with respect to fluorapatite. Subsequent seeding with hydroxyapatite caused a lowering of the calcium, phosphate, and fluoride concentrations in solution, indicative of fluorapatite precipitation. Repeating the process had an additive effect. Bone char was a less effective seed than hydroxyapatite with water containing fluoride only, but was a more effective seed with simulated Kenyan borehole water containing additional salts. Sixty-minute brushite saturation and apatite seeding steps were generally more effective than 10-min steps. The results suggest that apatite coprecipitation may be a convenient low-technology way to defluoridate drinking water, although prior testing might be useful to ensure adequate removal of fluoride.

Apatites↗

Gamma-crystallin-dodecyl sulfate complexes show discrete precipitation in non-ionic detergents.

Gamma-crystallin-sodium dodecyl sulfate (SDS) complexes are precipitated by Triton X-100 and other non-ionic detergents. This is accomplished as discrete lines by double diffusion, radial diffusion and other immunotechniques. Many other protein-SDS complexes including those of alpha- and beta-crystallins remain soluble. Immunoglobulin G tends to precipitate under similar conditions, a reason why the use of detergents in immunoprecipitation reactions has to be carefully controlled. The phenomenon can be used for the estimation of SDS or non-ionic detergents in the presence of gamma-crystallin.

Animals↗

Studies on the purification of antihemophilic factor (factor 8. I. Precipitation of antihemophilic factor by concanavalin A.

Concanavalin A precipitates antihemophilic factor from normal plasma. Combining this precipitation with other techniques, we were able to separate fractions rich in antihemophilic activity from human plasma with rapidity. The molecular weight of antihemophilic factor was estimated to be greater than 2 million. Presumably, antihemophilic factor is a large glycoprotein.

Animals↗

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

Amino Acids↗

Glycochenodeoxycholic acid inhibits calcium phosphate precipitation in vitro by preventing the transformation of amorphous calcium phosphate to calcium hydroxyapatite.

Calcium hydroxyapatite can be a significant component of black pigment gallstones. Diverse molecules that bind calcium phosphate inhibit hydroxyapatite precipitation. Because glycine-conjugated bile acids, but not their taurine counterparts, bind calcium phosphate, we studied whether glycochenodeoxycholic acid inhibits calcium hydroxyapatite formation. Glycochenodeoxycholic acid (2 mM) totally inhibited transformation of amorphous calcium phosphate microprecipitates to macroscopic crystalline calcium hydroxyapatite. This inhibition was not mediated by decreased Ca2+ activity. Taurocholic acid (2-12 mM) did not affect hydroxyapatite formation, but antagonized glycochenodeoxycholic acid. Both amorphous and crystalline precipitates contained a surface fraction relatively rich in phosphate. The surface phosphate content was diminish by increasing glycochenodeoxycholic acid concentrations, and this relationship was interpreted as competition between bile acid and HPO4(-4) for binding sites on the calcium phosphate surface. A phosphate-rich crystal surface was associated with rapid transition from amorphous to crystalline states. These results indicate that glycochenodeoxycholic acid prevents transformation of amorphous calcium phosphate to crystalline hydroxyapatite by competitively inhibiting the accumulation of phosphate on the crystal embryo surface.

Calcium↗

Why the assay of serum cystine by protein precipitation and chromatography should be abandoned.

The higher bias of serum cystine estimations by a HPLC method compared with those by ion exchange techniques is shown to be largely due to differences in the sample preparation procedures of the two techniques. The ion exchange methods utilised sulphosalicylic acid serum protein precipitation and post-column ninhydrin derivatisation of cystine, whilst the high pressure liquid chromatography technique employed automated dialysis for removal of proteins and pre-column ortho-phthalaldehyde derivatisation of cystine after its conversion to cysteine and then to S-carboxymethylcysteine. Examination of these procedures showed that whilst the high pressure liquid chromatographic method accurately estimates total serum cystine and cysteine, many factors affect the precision and accuracy of serum cystine estimations using the ion exchange techniques. In particular, serum protein precipitation techniques that are currently employed for the preparation of samples for cystine analysis by ion exchange chromatography should be abandoned.

Blood Proteins↗

FDA safety alert: hazards of precipitation associated with parenteral nutrition.

In April 1994, the Food and Drug Administration issued a Safety Alert regarding the hazards of precipitation associated with parenteral nutrition in response to reports of two deaths and at least two cases of respiratory distress in patients receiving three-in-one admixtures. The admixtures were thought to contain a precipitate of calcium phosphate, and patient autopsies revealed diffuse microvascular pulmonary emboli. The American Society for Parenteral and Enteral Nutrition took an active role in informing its members of the alert and responding to their concerns. Release of the alert has caused nutrition practitioners to reevaluate their compounding practices, use of filters, prescribing practices for calcium and phosphate in parenteral nutrition solutions, and use of three-in-one admixtures.

Adverse Drug Reaction Reporting Systems↗

Protein precipitation as a possible important pitfall in the clinical chemistry analysis of blood samples containing monoclonal immunoglobulins: 2 case reports and a review of the literature.

Two case reports are presented, both illustrating an analytical interference caused by monoclonal immunoglobulins. Falsely low results were obtained in the routine analysis of glucose, CRP and HDL-cholesterol. When analysing samples containing paraproteins, various problems can be encountered in the clinical laboratory: next to the antibody effect, pseudohyponatraemia, hyperviscosity, cryoglobulinaemia and gel formation have to be taken into account. In our two cases the interference was caused by paraprotein precipitation, causing an increased turbidity and an apparent increase of light absorbance at every wavelength due to light scattering, including the wavelengths used in the clinical chemistry assays. We review the literature on this sometimes overlooked interference in photometric/turbidimetric assays. This reaction is based on the insolubility of these proteins in specific physico-chemical circumstances in which many variables are involved, among others: pH and ionic strength, presence of preservatives and surfactants in the assays, pI and other specific properties of the monoclonal immunoglobulins. The complexity of the problem makes predicting or preventing this probably infrequent interference usually impossible. This artifact can cause both false positive and false negative results in multiple parameters (e.g. bilirubin, creatinine, iron, urea, uric acid), the most frequently reported analyte being phosphate. The Sia water test (Sia euglobulin precipitation test) can provide a first clue to a paraprotein aggregation; confirmation can be obtained by observing the time/ absorbance curves of the analysis, performing the test manually or setting up a serial dilution of the sample. The problem can be solved by avoiding the presence of the proteins in the assay, performing the analysis using an alternative method or diluting out the interference. Both laboratorians and clinicians should be aware of interferences in the clinical laboratory since the clinical consequences could be important.

Aged↗