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PubMed1975

[Determination of urinary total proteins and some protein fractions].

In unconcentrated specimens measurements have been made of total proteins, by DOETSCH gel-filtration method, and of albumin, transferrin and of alpha-2-macroglobulin, by LAURELL electroimmunodiffusion technique. The results confirm that the gel-filtration method provides specific, reliable measurement of proteins in amount as little as 50 mg/1. The proteinuria of 21 samples of urine has been determined by the DOETSCH method and by the biuret method after protein precipitation by perchloric, trichloracetic and phosphotungstic acid. Only the use of phosphotungstic acid as precipitant provides a good correlation of results between the biuret method and the direct gel-filtration-biuret method. LAURELL electroimmunodiffusion technique was improved to allow the determination of 5 mg/1 of albumin, and transferrin, and of 1 mg/1 of alpha-2-macroglobulin. The AA. emphasize the advantages of using sensitive methods which do not require the preliminary concentration of the sample, for the analysis of urinary proteins.

Albuminuria↗
PubMed1995

Diagnostic analysis of total protein, albumin, white cell count and differential in ascitic fluid.

Accurate and precise procedures are described for the measurement of total protein, albumin, white cell count and differential in ascitic fluid. The total protein method (biuret) on the serum chemistry analyser, Bayer-Technicon Chem-1, was calibrated against the biuret reference method in the measuring range from 1-100 g/l, covering serum as well as ascitic fluid values. The albumin method (bromcresol green) on the Chem-1 was calibrated for the measurement range from 1-50 g/l against the new human plasma protein international reference preparations and compared to the nephelometric method on the Beckman Array, which was also calibrated against these reference preparations. A good correlation was obtained for total protein between the Chem-1 (y) and the biuret reference method (x) in 58 ascitic fluids (y = 1.02x -0.3; r = 1.00). A good correlation between the Chem-1 (y) and the Array (x) was also obtained for albumin (r = 0.99). The bromcresol green method, however, which is not fully specific, resulted in significantly higher results (y = 1.32x - 1.3). The immunochemical procedure is considered the more accurate and therefore the bromcresol green method is not suitable for the determination of albumin in ascitic fluids. The white cell count in ascitic fluid can be measured reliably by most haematology analysers if the sample is fresh and homogeneous, but the differential is flagged as a result of differing morphology. Handmade smears from ascitic fluid, however, show uneven distribution of cells and disturbed morphology.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1992

Determination of total protein in highly purified factor IX concentrates.

Protein determination by the methods of Kjeldahl, Biuret, Bradford and UV absorbance at 280 nm have been studied in regard to accuracy, precision and simplicity. A reference preparation of a highly purified factor IX concentrate, Nanotiv, reconstituted to 1/5 of ordinary volume was used in the study in order to make a comparison between the different procedures. The Kjeldahl method resulted in a protein concentration of 3.7 mg/ml, whereas the Biuret, Bradford (BSA) and UV absorbance at 280 nm resulted in protein concentrations of 3.6, 2.5 and 2.8 mg/ml, respectively. The corresponding values for specific activity were 136, 140, 200 and 179 IU/mg, respectively. These results demonstrate a great variation in the response obtained by different methods for determination of total protein.

Biuret Reaction↗
PubMed1991

External quality assessment of total urinary protein estimation in the United Kingdom.

Three surveys of total urinary protein quantitation have been carried out in 350 UK laboratories. The seven specimens comprised buffered saline or normal human urine with added human serum albumin or human serum, or urine from individuals with nephrotic syndrome. Principal method groups were: turbidimetry (57%), dye binding (25%) and biuret (15%). For all surveys, overall between-laboratory agreement was poor (CV 22.8% to 57.1%), with ranges of results from 24-fold (0.83-20 g/L) to 366-fold (0.05-18.3 g/L); there was no improvement with time. The most popular method (sulphosalicylic acid turbidimetry) consistently performed the worst, and performance of the direct biuret procedure was also unacceptable; both methods should be discontinued. There were no significant differences in performance between the other major method groups, and none can be specifically recommended. Within the individual calibrant groups, least variation was observed with human serum. A common calibrant for all participants yielded significantly better between-laboratory agreement for all methods except sulphosalicylic acid turbidimetry.

Biuret Reaction↗
PubMed1976

Amounts and sources of protein for dairy calves.

Female Holstein calves (178) were fed rations varying from 10 to 17% crude protein (air dry) from 30 to 102 days of age. Ten percent was inadequate resulting in less body weight, gain, withers height, and heart girth than rations containing 12 to 17% crude protein. In trial 1 addition of urea, biuret, or ammonia-molasses to a 12% diet to make a 16% crude protein diet allowed gains and weights indistinguishable from those on a 12 or 16% crude protein soybean meal ration. In another trial, addition of urea, biuret, or ammonium propionate to a 10% ration to make a 14% ration allowed insignificantly greater gains and body weight. Gains and body weight were greater for the 14% soybean meal ration than the three non-protein-nitrogen rations for calves to 71 days of age but equal for 71 to 102 days. Gain, body weight, dry matter, and protein intakes at most dates and intervals were greater for calves fed the 14% soybean meal ration than the 10% ration. When 10, 12, 14, and 16% soybean meal rations were compared simultaneously, only the 10% crude protein ration gave inferior performance. Gains were reduced when average daily crude protein intake for the 72 day trail was 258 g or less. Calves fed lupine or dried poultry waste did not gain or consume as much as those fed comparable soybean meal rations. Calves fed a 17% crude protein ration from several protein sources plus alfalfa hay gained no much than those fed the 12 to 16% crude protein rations. Regression analysis relating dry matter and protein intake to body weight or gains indicated both dry matter intake and protein percentage were about equally important from day 41 to 72 and that after this age dry matter intake was of much greater importance than protein percentage. Protein intake expressed as g/day was not of importance in this regression. Weaning at day 42 allowed for greater body weight and gains than weaning at day 32. The difference in body weight was 2.5 kg at day 42 and 7 kg at day 102.

Animal Feed↗
PubMed1986

The Coomassie Brilliant Blue method underestimates drug-induced tubular proteinuria.

The sensitivity of the Coomassie Brilliant Blue (CBB) procedure differs for different proteins. To investigate the impact of this variability on clinical measurements of proteinuria, we collected urine specimens from patients with glomerular disease or drug-induced tubular nephrotoxicity and compared urinary protein concentrations as determined by the CBB method with those obtained by the biuret method. The CBB method underestimated the protein concentration predicted by the biuret method by as much as eightfold in specimens with low proportions of albumin. Determinations of protein by the CBB method in serially diluted urine specimens from patients with tubular damage also deviated from linearity by as much as twofold for protein concentrations less than 1 g/L. We conclude that CBB binding is relatively insensitive for measuring proteins specifically associated with tubular damage and hence may be inappropriate for clinical use.

Biuret Reaction↗
PubMed1981

A candidate reference method for determination of total protein in serum. I. Development and validation.

We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.

Bilirubin↗
PubMed1982

Six methods for urinary protein compared.

Six assays for protein in urine were compared for linearity, ease of standardization, precision, comparability of assay values, technical ease of assay, and current cost. The assays investigated were three dye-binding techniques, a recent turbidimetric technique, the trichloroacetic acid--biuret reaction, and a tannic acid protein precipitation reaction with ferric chloride. All assays suffered from standardization problems, although the biuret method showed the best analytical recovery of albumin and gamma-globulin. The tannic acid/ferric chloride method is dependent on sample pH. The turbidimetric assay exhibited the greatest imprecision; i.e., CVs were 19.5% at a protein concentration of 0.13 g/L and 6.0% at a protein concentration of 1.3 g/L. On the basis of all the factors assessed, we conclude that the Pesce/Strande Ponceau-S and the Bio-Rad Coomassie Brilliant Blue dye-binding techniques offer certain advantages over the other assays studied.

Azo Compounds↗
PubMed1994

Measuring urinary protein with the new BioRad reagent kit: evaluation and comparison with five other methods.

Total urinary protein was measured by five methods: BioRad Total Protein Test (TPT), pyrogallol red, benzethonium chloride, sulfosalicylic acid, trichloroacetic acid, and the results compared to those obtained by a method combining preparative ultrafiltration and the biuret reaction. TPT was linear to 1.5 g protein/l, the detection limit 0.0135 g/l, and it was 3-5 times more sensitive than the other methods. Within-day precision (CV) was 4.3%, (0.60 g/l), the day-to-day precision was 4.5%. The protein contents of 35 selected urine samples assigned to one of five groups according to their electrophoretic pattern were assayed by the five methods. No method accurately measured physiological proteinuria, but the values for light chain (Bence Jones), glomerular, tubular and overload proteinurias measured by TPT did not differ significantly from the biuret value. The other methods differed significantly for at least three groups. Alpha 1 acid glycoprotein slightly inhibited TPT, but peptones, amino acids, antibiotics or normal urine constituents had little or no effect. The TPT method has been automated (Kone Progress); normal 24-h urinary protein excretion was 36 mg/day (range 12-114), the protein creatinine ratio was 34 mg/g (12-106 mg/g).

Autoanalysis↗
PubMed1980

The radiolysis of aqueous ammonium cyanide: compounds of interest to chemical evolution studies.

Oxygen-free aqueous solutions of NH4CN (0.1 M, pH9) were exposed to gamma rays from a 60Co source, the mixture of nonvolatile products was fractionated, and the fractions were analyzed. The procedures were chosen to make effective investigations of radiolytic products, and to minimize the contributions of chemical changes which are known to occur in aqueous solution in the absence of ionizing radiation. It has been found that the main constituents are: urea, 25.9%; an oligomer, very likely oligoimine (18.4%); and several fractions (about 50%) which release amino acids on hydrolysis. These fractions differ considerably, as shown by amino acid assay, enzymatic digestion, IR spectra, and biuret reaction. All these tests were found to be positive for two fractions; in two further fractions the enzymatic cleavage was absent, but other tests were positive. Negative enzymatic and biuret tests, and no bands characteristic of amide or peptide, were found for a fraction whose hydrolysate consisted of 55% glycine. Although most of the isolated materials were found to be composite, the results of the analyses were sufficient for getting a reliable over-all picture of the chemical action of the ionizing radiation. The role of free radicals in reactions leading to the formations of radiolytic products was considered.

Amino Acids↗
PubMed1986

Comparison of five techniques for the determination of protein content in mixed human saliva.

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.

Amino Acids↗
PubMed1983

Concentration and composition of plasma proteins in wild mammals.

Total protein concentrations from a variety of wild mammalian species were measured by the Lowry and Biuret techniques and compared with results obtained or derived by refractometry. Values obtained with the Lowry method were the highest and certain derived refractometry results, the lowest. It is suggested that the Biuret method provides the most acceptable values but that care should be exercised in using the correct standard. The percentage composition of plasma from 22 species of wild mammals is also presented and the observations discussed with relation to methodology and A/G ratio.

Animal Population Groups↗
PubMed2005

Urinary-peptide excretion by patients with and volunteers without diabetes.

Large quantities of peptides (1-4 g) are excreted in human urine each day. In this study we sought to analyze how peptide excretion varies with increasing albuminuria associated with diabetes, as well as to characterize the size distribution of albumin-derived peptides in urine from volunteers without diabetes and from patients with macroalbuminuria and diabetes. We detected albumin-derived peptides by injecting tritiated albumin intravenously into human volunteers and patients with diabetes. Urine was collected after 24 hours and fractionated on a size-exclusion column. This fractionation revealed peptides with molecular weights ranging from 300 to 500 Da in volunteers without diabetes. The albumin-derived peptides were of higher molecular weight in the urine of a patient with macroalbuminuria and diabetes. The molecular-weight distribution of the peptides derived from tritiated albumin peptides was paralleled by the distribution of all protein peptides (including albumin) as determined with the use of the Biuret protein assay or absorbance at 214 nm. We determined peptide-excretion rates by filtering urine from patients with diabetes through a 10,000 Da molecular-weight-cutoff membrane and then measuring the filtrate with the use of the Biuret assay. This analysis revealed that the peptide-excretion rate increased with increasing total protein excretion, regardless of whether the patient demonstrated normoalbuminuria or microalbuminuria. Among patients with macroalbuminuria, the peptide-excretion rate leveled off and even decreased in the face of an increasing albumin concentration or protein-excretion rate. This study confirms that albumin-derived and protein-derived peptides exist at high concentrations in urine. Although peptide-excretion rates are maintained at similar levels up to macroalbuminuric states, the relative proportion of peptide excretion is significantly reduced compared with total protein.

Albumins↗
PubMed2005

The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid.

The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The solution of K3EDTA in distilled water only caused TP overestimation at high K3EDTA concentrations (>5 micromol/ml). By contrast, lithium heparin did not influence the refractometric values of TP. Neither anticoagulant modified TP values when measured by the biuret method. In conclusion, the use of K3EDTA as anticoagulant may result in a significant overestimation of TP values of peritoneal fluid samples measured by refractometry.

Animals↗
PubMed1990

Electrochemical detection of dipeptides and dipeptide amides.

A postcolumn reagent is used to create electroactive species from non-electroactive peptides. The reagent, based on the classical biuret reagent, consists of Cu(II), tartrate, bicarbonate and base. The detection is by dual electrode electrochemical detection. N-Acetylated dipeptides are oxidized at pH 12 and low potential. Dipeptides and carboxy terminal dipeptide amides give useful signals at lower pH. The dipeptide amides react with Cu(II) as do the tripeptides to yield the electronic spectrum and electrochemistry of the biuret complex. The complexes formed from the dipeptides are reversibly oxidized at potentials greater than 0.85 V vs. Ag/AgCl, 3 M NaCl. Analytically useful signals are obtained for the dipeptide amides at sensitivities equivalent to the sensitivities for longer peptides, 3-5 nA/microM, while the sensitivities for the dipeptides are about an order of magnitude lower.

Amides↗
PubMed2003

An evaluation of protein assays for quantitative determination of drugs.

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.

Analgesics↗
PubMedDate not supplied

Development and application of a dichotomous vapor/aerosol sampler for HDI-derived total reactive isocyanate group.

A dichotomous vapor/aerosol sampler was developed for measurement of HDI (1,6-hexamethylene diisocyanate)-derived total reactive isocyanate group (TRIG). The sampler consisted of an impactor or cyclone inlet, followed by an annular diffusional denuder, and a glass-fiber filter backup. The denuder walls and backup filter were each coated with 20 mg tributylphosphate and 1 mg MAMA reagent (9-N-methylamino-methylanthracene). After collection, MAMA-derivatized isocyanates were desorbed from the sampler and determined by high-performance liquid chromatography with dual-wavelength ultraviolet absorbance and fluorescence detection. Test atmospheres of HDI vapor and of HDI/HDI-biuret aerosols were generated in the laboratory and sampled with the optimized dichotomous sampler. Vapor phase HDI was completely collected by the diffusional denuder. When a mixture of HDI-biuret and HDI (approximately 30 ppb) was nebulized and collected with the dichotomous sampler, approximately 78% of the HDI was in the vapor phase, whereas about 22% was associated with the aerosol fraction. The dichotomous sampler was then used to measure vapor and condensed phase TRIG in a paint spray booth during application of a polyurethane paint. Measured levels of TRIG during the spraying operation averaged 391 +/- 154 micrograms/m3. Concentrations of HDI monomer averaged only 14 +/- 6.5 micrograms/m3. HDI-biuret was the largest component of TRIG found in these samples and was completely in the condensed aerosol phase. In contrast, the majority of the HDI was in the vapor phase, but significant (15-26%) amounts were measured in the aerosol fraction of the paint overspray. Thus, significant partitioning of HDI between vapor and condensed phases was demonstrated in both the laboratory and field, even when its concentration was well below the vapor saturation point.

Aerosols↗
PubMed2001

Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP.

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPbeta plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.

Amino Acid Sequence↗