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At least 19 recordsLinked to original sources
PubMed1984

Direct analysis for urinary protein with biuret reagent, with use of urine ultrafiltrate blanking: comparison with a manual biuret method involving trichloroacetic acid precipitation.

We describe a method for measuring urinary protein with a centrifugal analyzer. Biuret reagent is used, and blanking with an ultrafiltrate of urine eliminates interferences from the nonprotein, biuret-positive chromogens in urine. We compare results by this new method with those by a manual method in which trichloroacetic acid precipitation and biuret reagent are used. The new method shows good precision and excellent correlation (r = 0.997) with the manual method. The ease and convenience of this assay should make this a useful method for the routine clinical laboratory.

Anti-Bacterial Agents↗
PubMed2005

Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements.

BACKGROUND: Biuret assays for total protein measurement are considered to react with all peptides longer than 2 residues. Some studies using biuret assays of urine suggest that small peptides generally are more abundant than proteins in urine, but it is not clear whether this is a problem of assay specificity. METHODS: We analyzed the specificity and kinetics of a biuret reaction for solutions of amino acids, organic compounds, peptides, proteins, and ultrafiltered urine specimens and compared the results with standard clinical assays for protein measurement. RESULTS: The biuret assay cross-reacted with several amino acids, dipeptides, and other organic compounds able to form 5- or 6-member ring chelation complexes with copper. Reactions with amino acids and dipeptides had higher absorbance maxima (blue color) than with larger peptides and proteins (purple). Compounds forming potential 4-, 7-, 8-, or 9-member ring complexes with copper had low reactivity. Amino acid amides, dipeptides, and longer peptides had substantial reactivity, except those containing proline. Proteins and polypeptides had similar biuret reactivities per peptide bond, but reaction kinetics were slower for proteins than peptides. Urine specimens ultrafiltered through 3-kDa-cutoff membranes had substantial biuret reactivity, but absorbance maxima were consistent with cross-reactive amino acids rather than peptides. CONCLUSIONS: Many compounds, including amino acids, amino acid derivatives, and dipeptides, cross-react in biuret assays. Our studies improve understanding of the specificity of endpoint and kinetic biuret assays widely used in clinical laboratories. Amino acids, urea, and creatinine contribute to overestimation of urinary peptide content by biuret assays.

Amino Acids↗
PubMed2006

Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth.

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.

Allophanate Hydrolase↗
PubMed1991

[The determination of protein content of milk, cheese, and meat with the use of the biuret reaction].

A review is given of the different composition of biuret test solutions and experiments with the estimation of protein content of milk, cheese and meat by means of the biuret method are reported. The pure protein and the casein content of skimmed milk and the protein content of whole milk could be determined with biuret test solutions containing potassium hydroxide and a detergent. The use of the biuret method is advantageous in the case of cheese and meat, which are dissolved in potash lye or alkaline detergent solutions. Disturbances of the biuret method caused by lactose, fat or turbidity were eliminated after addition of hydrogen peroxide, by means of extraction or of additional measurements with a copper-free, zinc-containing biuret reagent. Deviations of the color intensity of biuret complexes were compensated by inclusion of standards in the measurement series.

Animals↗
PubMed2004

Daily and alternate-day supplementation of urea or biuret to ruminants consuming low-quality forage: II. Effects on site of digestion and microbial efficiency in steers.

Five steers (491 +/- 21 kg BW) were used in an incomplete 5 x 4 Latin square with four 24-d periods to determine the influence of supplemental non-protein N (NPN) source and supplementation frequency (SF) on nutrient intake and site of digestion in steers consuming low-quality grass straw (4% CP). Treatments (TRT) included an unsupplemented control and a urea- or biuret-containing supplement placed directly into the rumen daily (D) or every other day (2D) at 0700. The NPN treatments were formulated to provide 90% of the estimated degradable intake protein requirement. Daily TRT were supplemented CP at 0.04% of BW/d, whereas the 2D TRT were supplemented at 0.08% of BW every other day. Therefore, all supplemented TRT received the same quantity of supplemental CP over a 2-d period. Forage OM intake was not affected (P > 0.05) by NPN supplementation, NPN source, or SF; however, total OM and N intake were increased (P < 0.01) with CP supplementation. Duodenal flow of N was greater (P = 0.04) with CP supplementation compared with the control. In addition, duodenal bacterial N flow was increased with CP supplementation (P = 0.04) and for biuret compared with urea (P < 0.01). Bacterial efficiency (g bacterial N/kg OM truly digested in the rumen) was greater (P = 0.05) for biuret than for urea. Apparent total-tract N digestibility was increased with NPN supplementation (P < 0.01) but not affected by NPN source or SF. These results suggest that urea or biuret can be used effectively as a supplemental N source by steers consuming low-quality forage.

Animal Feed↗
PubMed2004

Daily and alternate-day supplementation of urea or biuret to ruminants consuming low-quality forage: III. Effects on ruminal fermentation characteristics in steers.

Five ruminally and duodenally cannulated steers (491 +/- 21 kg BW) were used in an incomplete 5 x 4 Latin square with four 24-d periods to determine the influence of supplemental nonprotein N (NPN) source and supplementation frequency (SF) on the dynamics of ruminal fermentation in steers consuming low-quality grass straw (4% CP). Treatments (TRT) included an unsupplemented control (CON) and a urea or biuret supplement that were placed directly into the rumen at 0700 daily (D) or every other day (2D). The NPN treatments were formulated to provide 90% of the estimated degradable intake protein requirement; therefore, the urea and biuret treatments received the same amount of supplemental N over a 2-d period. Daily TRT were supplemented with CP at 0.04% of BW/d, whereas the 2D TRT were supplemented at 0.08% of BW every other day. Forage was provided at 120% of the previous 5-d average intake in two equal portions at 0715 and 1900. Ruminal fluid was collected 0, 3, 6, 9, 12, and 24 h after supplementation on a day of and a day before supplementation for all TRT. Ruminal NH3-N increased (P < 0.04) with CP supplementation on the day all supplements were provided and on the day on which only daily supplements were provided compared with the CON. However, an NPN source x SF interaction (P = 0.03) on the day all supplements were provided indicated that NH3-N increased at a greater rate for urea as SF decreased compared with biuret. Ruminal NH3-N on the day only daily supplements were provided was greater (P = 0.02) for D compared with 2D. On the day all supplements were provided, D increased (P = 0.05) ruminal indigestible acid detergent fiber passage rate and ruminal fluid volume compared with 2D. These results suggest that urea or biuret can be used effectively as a supplemental N source by steers consuming low-quality forage without adversely affecting ruminal fermentation, even when provided every other day.

Animal Feed↗
PubMed2001

Application of an improved biuret method to the determination of total protein in urine and cerebrospinal fluid without concentration step by use of Hitachi 7170 auto-analyzer.

A biuret automated colorimetric assay for total protein in urine and cerebrospinal fluids was established. The procedures were as follows. Acidify all urine sample before analysis. Add precipitant Na(2)WO(4) to urine samples. After 10 min, centrifuge, decant the supernatant fluid, drain the inverted tubes on absorbent tissue, dissolve the precipitation with 0.1 mol/L NaOH, and finally adapt the reconstituted urine to the Hitachi 7170 analyzer. A cell-free cerebrospinal fluid sample produced by centrifugation can be inserted in an auto-analyzer for protein measurement directly. The program: mix 35 microl sample (CSF or reconstituted urine) and standard with 0.2 mol/L NaOH; incurable at 37 degrees C for 5 min, and real A1. Add concentrated biuret reagent, and 10 min later measure absorbance A2 at 546 nm vs. reagent blank. Secondary wavelength was 700 nm. The test results were calculated against a one-point standard. This biuret colorimetric method was relatively simple, fast, and accurate for the determination of protein in urine and cerebrospinal fluid, with a wide linearity extending from 0.125 g/L up to 6 g/L, had a good correlation with Benzethonium chloride turbidimetry technique, and was a practical routine method.

Albumins↗
PubMed2004

Daily and alternate day supplementation of urea or biuret to ruminants consuming low-quality forage: I. Effects on cow performance and the efficiency of nitrogen use in wethers.

Two experiments were conducted to determine the influence of supplemental nonprotein N (NPN) provided daily (D) or every other day (2D) on ruminant performance and N efficiency. Treatments included an unsupplemented control (CON) and a urea (28.7% CP) or biuret (28.6% CP) supplement provided D or 2D at 0700. In Exp. 1, five wethers (39 +/- 1 kg BW) were used in an incomplete 5 x 4 Latin square with four 24-d periods to determine the influence of supplemental NPN source and supplementation frequency (SF) on the efficiency of N use in lambs consuming low-quality grass straw (4% CP). The amount of CP supplied by each supplement was approximately 0.10% of BW/d (averaged over a 2-d period). In Exp. 2, 80 Angus x Hereford cows (540 +/- 8 kg BW) in the last third of gestation were used to determine the effect of NPN source and SF on cow performance. The NPN treatments were formulated to provide 90% of the estimated degradable intake protein requirement. The supplemented treatments received the same amount of supplemental N over a 2-d period; therefore, the 2D treatments received double the quantity of supplemental N on their respective supplementation day than the D treatments. In Exp. 1, total DM, OM, and N intake; DM, OM, and N digestibility; N balance; and digested N retained were greater (P < 0.03) for supplemented than for CON wethers, with no difference (P > 0.05) between NPN sources or SF. Plasma urea-N (PUN) was increased with N supplementation compared with CON (P < 0.01), and urea treatments had greater PUN than biuret (P < 0.01). In addition, PUN was greater (P = 0.02) for D than for 2D treatments. In Exp. 2, pre- and postcalving (within 14 d and 24 h after calving, respectively) cow weight and body condition score change were more positive (P < 0.05) for supplemented groups than for CON. These results suggest that supplements containing urea or biuret as the primary source of supplemental N can be effectively used by lambs and cows consuming low-quality forage, even when provided every other day.

Animal Feed↗
PubMedDate not supplied

The measurement of total serum proteins by the Biuret method.

The biuret reaction for proteins provides a simple and precise method for measuring serum proteins; Beer's law is obeyed to at least 10 g per dl. Several stable biuret reagents are available. Hemoglobin is the only important cause of interference which cannot be minimized by use of a sample blank. The mechanism of the biuret reaction is described and attention is drawn to the heterogeneity of the serum proteins and to the use of a certified albumin standard.

Biuret Reaction↗
PubMed1978

[Refractometric measurement of total serum protein, comparison of refractometry and biuret test (author's transl)].

Study on the Refractometric Determination of Total Protein in Serum, Comparison of the Refractometric and Biuret Method. Total protein concentration in serum was determined by the aid of the Abbé-refractometer and the biuret method. Both methods showed a good precision and accuracy. The investigation was carried out in 241 sera with normal bilirubin (up to 1 mg/100 ml), cholesterol (up to 200 mg/100 ml) and urea (up to 23,0 mg/100 ml) concentration, in 43 sera with increased (10,6-26,6 mg/100 ml) bilirubin concentration, in 129 sera with increased (200-520 mg/100 ml) cholesterol concentration and in 43 sera with increased (23,0-155,3 mg/100 ml) urea concentration. The comparison of the refractometric values with the values obtained by the biuret method in the 241 sera with normal bilirubin, cholesterol and urea concentration (correlation coefficient = 0,971) showed a close correlation and in the 43 sera with increased bilirubin concentration (correlation coefficient = 0,958) an acceptable correlation. However no close correlations were observed in the 129 sera with increased cholesterol concentration and in the 43 sera with increased urea concentration. The correlation lines diverged proportional with the increase of cholesterol and urea concentration from the expected correlation lines.

Bilirubin↗
PubMed1993

Determination of proteins by a reverse biuret method combined with the copper-bathocuproine chelate reaction.

A method of protein determination has been developed which combines the biuret reaction and the copper(I)-bathocuproine chelate reaction. Protein in the specimen forms a Cu(2+)-protein chelate complex (biuret reaction) during the first step. Excess Cu2+ is reduced to Cu+ by ascrobic acid, allowing the Cu+ to form a Cu(+)-bathocuproine chelate complex during the second step. The amount of Cu(+)-bathocuproine chelate complex formed is inversely proportional to the protein concentration. The sensitivity (epsilon = 1.4 x 10(6) 1.mol-1.cm-1 against human albumin) of this method was higher than that of the original Lowry (9.8 x 10(5)), pyrogallol red (1.0 x 10(6)) and commercially available Coomassie Brilliant Blue G.250 methods (6.7 x 10(5)). The color intensities of human gamma-globulin, human globulin (fractions IV-1 and IV-4), bovine albumin, egg albumin and horse gamma-globulin against human albumin (100%) ranged from 92 to 101%. The results obtained with the present method (y) correlated well with those determined by the biuret method (r = 0.998, y = 0.98 chi - 0.002, x = 1.31, y = 1.29 g/l) in 30 diluted sera. These results confirm that this assay is similar in sensitivity to the original Lowry method, is rapid and has similar reactivity to each of the various proteins in biological fluids.

Ascorbic Acid↗
PubMed2001

Comparison of refractometer and biuret methods for total protein measurement in body cavity fluids.

Most hand-held medical refractometers have internal scales that limit protein measurement to results >/=2.5 g/dL. Tables for conversion of refraction (r) to protein concentration for values as low as 0.1 g/dL were published in the 1960s, but their accuracy for use on body fluids has not been established. The purpose of this study was to assess the reliability of body cavity fluid protein determination by refractometry. We compared the protein concentration of 25 body cavity fluids as determined by 2 Goldberg type hand-held refractometers with results obtained by the biuret method. Published charts converting refraction (r) to protein concentration were used to determine protein concentration in samples with protein <2.5 g/dL. Higher protein values were read directly from the instruments. The range of comparison was limited to >/=0.6 g/dL, the lowest concentration of the biuret method's standard curve. Twenty-one peritoneal fluid, 2 pleural fluid and 2 pericardial fluid samples from 16 horses, 5 cattle, 3 dogs, 2 llamas and 1 cat were tested. The results obtained by the two refractometers were closely and linearly related to biuret results (P<.001), with slopes by linear regression analysis close to 1, and correlation coefficients >0.977. Based on this study, the range for quantification of body cavity fluid protein concentration by refractometry can be extended below 2.5 g/dL, allowing for quantitative assessment of most clinical samples.

Journal Article↗
PubMed1980

[Determination of proteins with the Coomassie brilliant blue G 250 method. IV. Use with cerebrospinal fluid proteins and comparative analysis with the biuret and Lowry methods].

The method of Bradford with Coomassie Brillant Blue G 250 for protein assay has been applyed to CSF in comparison with biuret and Lowry's methods. The results of Coomassie method closely agree with Lowry, and are similar to those of biuret. It is suggested to replace the previously used methods for CSF protein assay by the Bradford's method, because it keeps the sensitivity of Lowry and the simplicity of biuret. It adds too the advantage of requiring less time.

Cerebrospinal Fluid Proteins↗
PubMed1980

Automated multiple flow-injection analysis in clinical chemistry: determination of total protein with Biuret reagent.

We have examined the feasibility of the automated multiple flow-injection technique for application to clinical chemistry by adapting to this system the biuret method for the determination of total protein. Samples were discretely and rapidly introduced into a continuously flowing, nonsegmented reagent stream by means of an automatic sampler and high-pressure injection valve. Pumps operating at 1380-2070 kPa (200-300 psi) were utilized to introduce the biuret reagent and saline diluent into the system separately at flow rates of 72 and 47 microL/s, respectively. Use of 20-microL sample and a 3.0-s reaction-delay coil was adequately sensitive for analysis for total protein by this method. Samples were analyzed at a rate of 150/h with no detectable between-sample carryover. Within-run precision studies yielded relative standard deviations of 2.5% and less. Total protein values obtained by this method correlated well with those obtained by centrifugal analyzer and bubble-segmented continuous-flow biuret methods.

Autoanalysis↗
PubMed1984

Postcolumn detection of serum proteins with the biuret and Lowry reactions.

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.

Biuret Reaction↗