Impact of Supplementing a Backgrounding Diet with Nonprotein Nitrogen on In Vitro Methane Production, Nutrient Digestibility, and Steer Performance.

Two experiments were conducted to evaluate the effect of nonprotein nitrogen (NPN) supplementation on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, incubations were conducted on three separate days (replicates). Treatments were control (CTL, without NPN), urea (U), urea-biuret (UB), and urea-biuret-nitrate (UBN) mixtures. Except for control, treatments were isonitrogenous using 1% U inclusion as a reference. Ruminal fluid was collected from two Angus-crossbred steers fed a backgrounding diet plus 100 g of a UBN mixture for at least 35 d. The concentration of volatile fatty acids (VFA) and ammonia nitrogen (NH3-N), in vitro organic matter digestibility (IVOMD), and total gas and methane (CH4) production were determined at 24 h of incubation. In experiment 2, 72 Angus-crossbred yearling steers (303 ±â€…29 kg of body weight [BW]) were stratified by BW and randomly allocated in nine pens (eight animals/pen and three pens/treatment). Steers consumed a backgrounding diet formulated to match the diet used in the in vitro fermentation experiment. Treatments were U, UB, and UBN and were isonitrogenous using 1% U inclusion as a reference. Steers were adapted to the NPN supplementation for 17 d. Then, digestibility evaluation was performed after 13 d of full NPN supplementation for 4 d using 36 steers (12 steers/treatment). After that, steer performance was evaluated for 56 d (24 steers/treatment). In experiment 1, NPN supplementation increased the concentration of NH3-N and VFA (P < 0.01) without affecting the IVOMD (P = 0.48), total gas (P = 0.51), and CH4 production (P = 0.57). Additionally, in vitro fermentation parameters did not differ (P > 0.05) among NPN sources. In experiment 2, NPN supplementation did not change dry matter and nutrient intake (P > 0.05). However, UB and UBN showed lower (P < 0.05) nutrient digestibility than U, except for starch (P = 0.20). Dry matter intake (P = 0.28), average daily gain (P = 0.88), and gain:feed (P = 0.63) did not differ among steers receiving NPN mixtures. In conclusion, tested NPN mixtures have the potential to be included in the backgrounding diets without any apparent negative effects on animal performance and warrant further studies to evaluate other variables to fully assess the response of feeding these novel NPN mixtures.

Nonprotein nitrogen (NPN) supplements can be used as a nitrogen source for ruminants fed low-protein diets. The most common NPN source is urea, included typically at a range between 0.5% and 1% of the diet dry matter in growing beef cattle. Although other NPN sources and mixtures are available, there is scarce information regarding their use in ruminant production. Two experiments were conducted to evaluate the effect of NPN sources on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, three different incubations were performed for 24 h. Treatments were control (without NPN), urea (U), urea­biuret (UB), and urea­biuret­nitrate (UBN) mixtures. In experiment 2, 72 crossbred yearling steers were randomly assigned to one of the following treatments: U, UB, and UBN mixtures. Diets were formulated to contain the same nitrogen concentration in both experiments. In experiment 1, supplementation of NPN increased the in vitro fermentation, but there were no differences among NPN sources. In experiment 2, steers performed similarly among NPN sources. These findings suggest that NPN mixtures have the potential to be included in the backgrounding diets without detrimental effects. Further studies should evaluate other variables (e.g., fermentation dynamic and microbial protein supply) when using these novel mixtures.

Comparison of the effect of Sodium Chloride concentration on protein determination: Bradford and Biuret methods.

Protein quantification methods using spectrophotometry are widely used in laboratory routines for different purposes. Samples generally contain non-protein components that can interfere with the quality of the analysis. A simple and quick test with different concentrations of sodium chloride demonstrated that the Bradford method is significantly affected by the presence of salt, while Biuret remains stable. Therefore, the choice of method is an important factor in reducing errors and ensuring more reliable results.

Preparation of novel fluorescent probe based on carbon dots for sensing and imaging Fe(III) and pyrophosphate in cells and zebrafish.

Ferric ions (Fe3+) and pyrophosphate anions (PPi) are involved in many physiological processes and play important roles in biological systems. The abnormal level of Fe3+ and PPi will cause serious damage to the environment and life. At present, the application of such probes in life, especially in vivo, is still very scarce. So, the development of a fluorescent probe to simultaneously detect Fe3+ and PPi has great significance to the health of the environment and organisms. Herein, nitrogen-doped carbon quantum dots (N-CDs) were synthesized via solvothermal treatment, using biuret and citric acid as precursors. The synthesized N-CDs showed highly selective and sensitive detection of Fe3+ through a photoluminescence quenching effect. The fluorescence of N-CDs quenched by Fe3+ could be restored with PPi, rendering the N-CDs/Fe3+ sensor promising for PPi detection ('OFF-ON'). The linear ranges of detection for Fe3+ and PPi were 3-30 and 2-12 µM, and the limits of detection were 2.71 and 1.12 µM, respectively. The practical applications of N-CDs were tested using tap water samples. Furthermore, N-CDs can be used for the detection and imaging of Fe3+ and PPi in HeLa cells and zebrafish owing to their excellent optical properties.

Crystal Growth of Urea and Its Modulation by Additives as Analyzed by All-Atom MD Simulation and Solution Theory.

The crystal growth of urea was analyzed with all-atom molecular dynamics (MD) simulation for the (001) and (110) faces in contact with aqueous solutions. The local environment of a crystallizing molecule was treated in terms of the numbers of crystalline neighbors and the orientation relative to the crystal surface, and the molecular-level inhomogeneity of a growing surface was addressed by decomposing the overall rate of growth into a sum of the contributions conditioned by the local structure and orientation mode. The contrast of the growth mechanism between the (001) and (110) faces was then evidenced by the local contributions, and the roles of the outer layers of the crystal toward the liquid region were pointed out for (001). The effect of the additive species in the liquid on the crystal growth of urea was investigated with biuret, N,N-dimethylformamide (DMF), and acetone. The growth was observed to be suppressed more strongly in the order of biuret > DMF > acetone, and it was found that the ordering of suppression by the additive is common irrespective of the local environment of a crystallizing urea. This finding implies that the additive's effect on the crystal growth can be predicted by treating the flat surface, which is a convenient system for detailed analyses at atomic resolution. The correspondence to the free energy of adsorption of the additive was then examined for the additive-induced modulation of the growth rate. It was seen that the adsorption free energy correlates to the extent of modulation of the growth rate, and the interaction components that govern the adsorption propensity were identified.

Expeditious assembly of biuret-guanidine derivatives via the catalyst-free transformation.

We disclose a mild and practical catalyst-free transformation for the expeditious construction of biuret-guanidine derivatives using aromatic isocyanates. This synthetic transformation is featured with mild reaction conditions and high efficiency.

A procedure for removal of cyanuric acid in swimming pools using a cell-free thermostable cyanuric acid hydrolase.

Cyanuric acid (CYA) is used commercially for maintaining active chlorine to inactivate microbial and viral pathogens in swimming pools and hot tubs. Repeated CYA addition can cause a lack of available chlorine and adequate disinfection. Acceptable CYA levels can potentially be restored via cyanuric acid hydrolases (CAH), enzymes that hydrolyze CYA to biuret under mild conditions. Here we describe a previously unknown CAH enzyme from Pseudolabrys sp. Root1462 (CAH-PR), mined from public databases by bioinformatic analysis of potential CAH genes, which we show to be suitable in a cell-free form for industrial applications based upon favorable enzymatic and physical properties, combined with high-yield expression in aerobic cell culture. The kinetic parameters and modeled structure were similar to known CAH enzymes, but the new enzyme displayed a surprising thermal and storage stability. The new CAH enzyme was applied, following addition of inexpensive sodium sulfite, to hydrolyze CYA to biuret. At the desired endpoint, hypochlorite addition inactivated remaining enzyme and oxidized biuret to primarily dinitrogen and carbon dioxide gases. The mechanism of biuret oxidation with hypochlorite under conditions relevant to recreational pools is described.

Comparison of plasma total solids concentration as measured by refractometry and plasma total protein concentration as measured by biuret assay in pet rabbits and ferrets.

OBJECTIVE: To determine the agreement between plasma total solids (TS) concentration as measured by refractometry and plasma total protein (TP) concentration as measured by biuret assay in pet rabbits and ferrets. SAMPLE: 253 and 146 blood samples from 146 and 121 ferrets and rabbits, respectively, with results of CBC and plasma biochemical analyses. PROCEDURES: Data were collected from medical records regarding plasma TS and TP concentrations, PCV, plasma biochemical values, plasma appearance, and patient signalment. Agreement was determined between refractometer and biuret assay (reference method) values for plasma TS and TP concentration. Other variables were examined for an impact on this agreement. RESULTS: Mean ± SD plasma TP and TS concentrations were 6.4 ± 0.8 mg/dL and 6.6 ± 0.8 mg/dL, respectively, for rabbits and 6.3 ± 1.2 mg/dL and 6.4 ± 1.1 mg/dL for ferrets. On average, refractometer values overestimated plasma TP concentrations as measured by biuret assay. Plasma cholesterol, glucose, and BUN concentrations and hemolysis and lipemia had significant effects on this bias for ferrets; only BUN concentration had an effect on bias for rabbits given the available data. Other variables had no influence on bias. The limits of agreement were wider than the total allowable analytic error, and > 5% of the data points were outside acceptance limits, indicating that the 2 methods were not in clinical agreement. CONCLUSIONS AND CLINICAL RELEVANCE: Refractometer measurements of plasma TS concentration failed to provide a good estimation of biuret assay measurements of plasma TP concentration in rabbits and ferrets, suggesting that these 2 analytic methods and the results they yield cannot be used interchangeably in these species.

Comparison of tests for failure of passive transfer in neonatal calf serum using total protein refractometry and the biuret method.

Failure of passive transfer (FPT) is defined as failure to absorb colostral antibodies sufficient to achieve a serum Immunoglobulin G (IgG) concentration >10 g/L. Immunoglobulin G can be measured directly in calf serum using radial immunodiffusion (RID), or indirectly estimated by measuring total protein (TP). Indirect TP measures are usually favoured because of their relatively lower costs. The aim of this work was to compare TP measurements using refractometry and biuret methods against the reference RID test in neonatal dairy calves, and to assess agreement between these indirect measures. Neither the biuret nor the refractometer method provided a high sensitivity for detection of FPT, as defined by RID. There was no systematic difference between the methods in their estimation of TP, although the biuret method was more accurate than the refractometer method when tested against the reference RID test (accuracy = 83.1 % v 69.3 %) and the refractometer was more likely to overestimate the number of calves with FPT. Specificity for the biuret test was 93.9 % compared with the refractometer specificity of 74.4 %. Mean TP as estimated by the biuret method was higher than the mean TP estimated by the refractometer (6.25 g/dL versus 5.52 g/dL), and the Pearson correlation coefficient for the two assays was only moderate, at 0.58. This suggests that the biuret method is preferable to the refractometer for detecting FPT in calves, despite the superior convenience of the refractometer.

A biuret-derived, MS-cleavable cross-linking reagent for protein structural analysis: A proof-of-principle study.

Chemical cross-linking combined with mass spectrometry (XL-MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross-linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross-linkers containing an MS-labile urea group, we now present the biuret-based, CID-MS/MS-cleavable cross-linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross-linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.

Cyanuric Acid Biodegradation via Biuret: Physiology, Taxonomy, and Geospatial Distribution.

Cyanuric acid is an industrial chemical produced during the biodegradation of s-triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a single model system, Pseudomonas sp. strain ADP, in which cyanuric acid hydrolase (AtzD) opens the s-triazine ring and AtzEG deaminates the ring-opened product. A significant question remains as to whether the metabolic pathway found in Pseudomonas sp. ADP is the exception or the rule in bacterial genomes globally. Here, we show that most bacteria utilize a different pathway, metabolizing cyanuric acid via biuret. The new pathway was determined by reconstituting the pathway in vitro with purified enzymes and by mining more than 250,000 genomes and metagenomes. We isolated soil bacteria that grow on cyanuric acid as a sole nitrogen source and showed that the genome from a Herbaspirillum strain had a canonical cyanuric acid hydrolase gene but different flanking genes. The flanking gene trtB encoded an enzyme that we show catalyzed the decarboxylation of the cyanuric acid hydrolase product, carboxybiuret. The reaction generated biuret, a pathway intermediate further transformed by biuret hydrolase (BiuH). The prevalence of the newly defined pathway was determined by cooccurrence analysis of cyanuric acid hydrolase genes and flanking genes. Here, we show the biuret pathway was more than 1 order of magnitude more prevalent than the original Pseudomonas sp. ADP pathway. Mining a database of over 40,000 bacterial isolates with precise geospatial metadata showed that bacteria with concurrent cyanuric acid and biuret hydrolase genes were distributed throughout the United States.IMPORTANCE Cyanuric acid is produced naturally as a contaminant in urea fertilizer, and it is used as a chlorine stabilizer in swimming pools. Cyanuric acid-degrading bacteria are used commercially in removing cyanuric acid from pool water when it exceeds desired levels. The total volume of cyanuric acid produced annually exceeds 200 million kilograms, most of which enters the natural environment. In this context, it is important to have a global understanding of cyanuric acid biodegradation by microbial communities in natural and engineered systems. Current knowledge of cyanuric acid metabolism largely derives from studies on the enzymes from a single model organism, Pseudomonas sp. ADP. In this study, we obtained and studied new microbes and discovered a previously unknown cyanuric acid degradation pathway. The new pathway identified here was found to be much more prevalent than the pathway previously established for Pseudomonas sp. ADP. In addition, the types of environment, taxonomic prevalences, and geospatial distributions of the different cyanuric acid degradation pathways are described here.

Quantitative measurement of urinary proteins in 2018: advantages, disadvantages, limits.

Today, there is no reference method for the measurement of urinary proteins. The difficulties are that urine is a very complex biological fluid, and that there are a high intra-and inter-individual variability in the protein excretion rate. Progress has been made during the last thirty years, but high analytical variability persists among the colorimetric or turbidimetric methods used for urinary proteins measurement.

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min-1 mg-1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems.

Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841.

Biuret deamination is an essential step in cyanuric acid mineralization. In the well-studied atrazine degrading bacterium Pseudomonas sp. strain ADP, the amidase AtzE catalyzes this step. However, Rhizobium leguminosarum bv. viciae 3841 uses an unrelated cysteine hydrolase, BiuH, instead. Herein, structures of BiuH, BiuH with bound inhibitor and variants of BiuH are reported. The substrate is bound in the active site by a hydrogen bonding network that imparts high substrate specificity. The structure of the inactive Cys175Ser BiuH variant with substrate bound in the active site revealed that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of BiuH variants. Finally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface: a persistent tunnel gated by residues Val218 and Gln215 forming a potential substrate channel and two smaller channels formed by Val28 and a mobile loop (including residues Phe41, Tyr47 and Met51) that may serve as channels for co-product (ammonia) or co-substrate (water).

Comparison of biuret and refractometry methods for the serum total proteins measurement in ruminants.

BACKGROUND: Determination of serum total protein concentration is commonly performed by the biuret method. Refractometric measurement is a faster and less expensive alternative but its accuracy has not been determined in ruminants. OBJECTIVE: The purpose of the study was to compare the serum total protein concentrations in cattle, sheep, and goats measured by the biuret method with those obtained by refractometry. METHODS: Serum total protein concentration was determined in 120 cattle, 67 sheep, and 58 goat blood samples refractometrically and with the biuret method. The data were analyzed with a paired samples t-test, and Passing and Bablok regression equations and Bland and Altman plots were generated. RESULTS: There was a strong linear relationship between the total protein values determined with the refractometer and the biuret method in cattle, sheep, and goats. The statistical accuracy, which represents a bias correction factor that measures the deviation of the best-fit line from the 45° line through the origin, was 90.63% for cattle, 93.05% for sheep, and 91.76% for goats. The mean protein values determined with the refractometer were significantly lower than those measured with the biuret method in cattle and goats (P < .05) but not in sheep (P > .05). CONCLUSIONS: The evaluated refractometer was sufficiently accurate for the determination of serum total proteins in cattle, sheep, and goats, although it cannot be used interchangeably with the biuret method. The RIs should be corrected for negative bias based on the created equations.

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

A practical method for extending the biuret assay to protein determination of corn-based products.

A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.

The effect of urea on refractometric total protein measurement in dogs and cats with azotemia.

BACKGROUND: While protein is the predominant solute measured in plasma or serum by a refractometer, nonprotein substances also contribute to the angle of refraction. There is debate in the current literature regarding which nonprotein substances cause factitiously high refractometric total protein measurements, as compared to the biuret assay. OBJECTIVES: The purpose of the study was to determine if the blood of azotemic animals, specifically with increased blood urea concentration, will have significantly higher refractometric total protein concentrations compared to the total protein concentrations measured by biuret assay. METHODS: A prospective case series was conducted by collecting data from azotemic (n = 26) and nonazotemic (n = 34) dogs and cats. In addition, an in vitro study was performed where urea was added to an enhanced electrolyte solution at increasing concentrations, and total protein was assessed by both the refractometer and spectrophotometer. Statistical analysis was performed to determine the effect of urea. RESULTS: The refractometric total protein measurement showed a positive bias when compared to the biuret protein measurement in both groups, but the bias was higher in the azotemic group vs the nonazotemic group. The mean difference in total protein measurements of the nonazotemic group (0.59 g/dL) was significantly less (P < .01) than the mean difference of the azotemic group (0.95 g/dL). The in vitro experiment revealed a positive bias with a proportional error. CONCLUSIONS: This study demonstrated that increasing concentrations of urea significantly increased the total protein concentration measured by the refractometer as compared to the biuret assay, both in vivo and in vitro.

Characterization of bacterial diversity in an atrazine degrading enrichment culture and degradation of atrazine, cyanuric acid and biuret in industrial wastewater.

An enrichment culture was used to study atrazine degradation in mineral salt medium (MSM) (T1), MSM+soil extract (1:1, v/v) (T2) and soil extract (T3). Results suggested that enrichment culture required soil extract to degrade atrazine, as after second sequential transfer only partial atrazine degradation was observed in T1 treatment while atrazine was completely degraded in T2 and T3 treatments even after fourth transfer. Culture independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique confirmed selective enrichment of genus Bacillus along with Pseudomonas and Burkholderia. Degradation of atrazine/metabolites in the industrial wastewater was studied at different initial concentrations of the contaminants [wastewater-water (v/v) ratio: T1, 1:9; T2, 2:8; T3, 3:7; T4, 5:5 and T5, undiluted effluent]. The initial concentrations of atrazine, cyanuric acid and biuret ranged between 5.32 and 53.92 µg mL(-1), 265.6 and 1805.2 µg mL(-1) and 1.85 and 16.12 µg mL(-1), respectively. The enrichment culture was able to completely degrade atrazine, cyanuric acid and biuret up to T4 treatment, while no appreciable degradation of contaminants was observed in the undiluted effluent (T5). Inability of enrichment culture to degrade atrazine/metabolites might be due to high concentrations of cyanuric acid. Therefore, a separate study on cyanuric acid degradation suggested: (i) no appreciable cyanuric acid degradation with accumulation of an unidentified metabolite in the medium where cyanuric acid was supplemented as the sole source of carbon and nitrogen; (ii) partial cyanuric acid degradation with accumulation of unidentified metabolite in the medium containing additional nitrogen source; and (iii) complete cyanuric acid degradation in the medium supplemented with an additional carbon source. This unidentified metabolite observed during cyanuric acid degradation and also detected in the enrichment culture inoculated wastewater samples, however, was degraded up to T4 treatments and was persistent in the T5 treatment. Probably, accumulation of this metabolite inhibited atrazine/cyanuric acid degradation by the enrichment culture in undiluted wastewater.

Negative interference of icteric serum on a bichromatic biuret total protein assay.

BACKGROUND: Bilirubin is stated to be a negative interferent in some biuret assays and thus could contribute to pseudohypoproteinemia in icteric samples. OBJECTIVE: The purpose of the study was to evaluate the magnitude of and reason for a falsely low total protein concentration in icteric serum when the protein concentration is measured with a bichromatic spectrophotometric biuret assay. METHODS: Commercially available bilirubin was dissolved in 0.1 M NaOH and mixed with sera from 2 dogs to achieve various bilirubin concentrations of up to 40 mg/dL (first set of samples) and 35 mg/dL (second set of samples, for confirmation of first set of results and to explore the interference). Biuret total protein and bilirubin concentrations were determined with a chemistry analyzer (Cobas 6000 with c501 module). Line graphs were drawn to illustrate the effects of increasing bilirubin concentrations on the total protein concentrations. Specific spectrophotometric absorbance readings were examined to identify the reason for the negative interference. RESULTS: High bilirubin concentrations created a negative interference in the Cobas biuret assay. The detectable interference occurred with a spiked bilirubin concentration of 10.7 mg/dL in one set of samples, 20.8 mg/dL in a second set. The interference was due to a greater secondary-absorbance reading at the second measuring point in the samples spiked with bilirubin, which possibly had converted to biliverdin. CONCLUSION: Marked hyperbilirubinemia is associated with a falsely low serum total protein concentration when measured with a bichromatic spectrophotometric biuret assay. This can result in pseudohypoproteinemia and pseudohypoglobulinemia in icteric serum.

Validation for the determination of biuret in water-soluble, urea-based commercial inorganic fertilizer materials, urea solutions, and sulfur-coated urea products by reversed-phase liquid chromatography: single-laboratory validation of an extension of AOAC official methods 2003.14.

A single-laboratory validation (SLV) study for the LC determination of biuret in dry and liquid urea-based commercial fertilizers was conducted. A total of 23 samples were used: 11 commercial dry urea products, two urea ammonium nitrate products, eight liquid urea-based commercial fertilizers, and four sulfur-coated urea samples from different sources. In addition, one biuret standard from Aldrich and one sample from the Magruder check sample program were used as validation samples. The proposed method is an extension of AOAC Official Method 2003.14 and is based on dissolution of the test portion in the LC mobile phase and determination by HPLC. The system is linear over a concentration range of 1.00-4.50 mg/L biuret, with a correlation coefficient > or = 0.9999. The biuret was well- separated from urea in the commercial urea samples, and from other constituents in the commercial liquid fertilizer with no observed interferences. Recoveries were determined by spiking four of the validation materials with a known amount of biuret standard and measuring the biuret level according to the method. The averaged recovery was 97%. Method precision was determined by quadruplicate analyses of four of the liquid and six of the commercially available dry urea validation materials using three and four replicate analyses. For the liquid fertilizer analyses, the RSD ranged from 7.04 to 13.31%. For the dry urea analyses, the RSDs ranged from 5.68 to 14.34%. Instrument precision was evaluated at the test initiation by using seven injections of five biuret standard solutions. SD varied from 0.27 to 1.02%, with RSDs averaging 1.14%. The LOD was determined to be 0.009% biuret in material, while the LOQ was determined to be 0.031% biuret in material. In addition to the intralaboratory study, interlaboratory studies were performed by two other outside laboratories using this method. Over a concentration range of 0.2 to 0.9% biuret, the average SD was 0.11%, the average RSD was 21.16%, and the average HorRat value was 4.73%. Furthermore, comparative studies for biuret using AOAC Official Methods 960.04 and 976.01 with the proposed LC method were performed. The three methods produced very close results; however, the two AOAC methods generate hazardous wastes and are more tedious. On the basis of accuracy and precision of the results for this SLV study, it is recommended that this method be collaboratively studied for the determination of biuret in dry and liquid urea-based commercial fertilizer materials.