Refractometry as an alternative to the biuret method for measuring total serum proteins in Podocnemis expansa (Podocnemididae) and Phrynops geoffroanus (Chelidae) / Refratometria como alternativa ao método do biureto para mensuração de proteínas séricas totais em Podocnemis expansa e Phrynops geoffroanus (Podocnemididae, Chelidae)

Total serum protein is a significant indicator of health condition in animals. The aim of this study was to analyze the precision of the portable refractometer in determining the concentration of total serum proteins in Podocnemis expansa and Phrynops geoffroanus. A total of 26 animals were used. The blood samples were collected from the supraoccipital sinus and stored in tubes without anticoagulant. Total serum protein was determined using both the biuret reaction and refractometry. The total serum protein mean concentration (g dL-1) with biuret method and refractometry for P. expansa were 3.16 and 3.2; and for P. geoffroanus were 3.56 and 2.72, respectively. These results indicate that total serum protein values can be determined with precision in P. expansa and P. geoffroanus using a portable refractometer.

A proteína sérica total é um indicador significativo do estado de saúde em animais. O objetivo desse estudo foi analisar a precisão do refratômetro portátil para determinar a concentração de proteínas séricas totais em Podocnemis expansa e Phrynops geoffroanus. Foram utilizados um total de 26 animais. As amostras de sangue foram coletadas por punção do seio supraoccipital e armazenadas em tubos sem anticoagulante. A concentração de proteína sérica total foi determinada utilizando tanto a reação de biureto como um refratômetro portátil. A média da proteína sérica total (g dL-1) pela reação de biureto e pela refratometria para P. expansa foram de 3,16 e 3,2; e para P. geoffroanus foram de 3,56 e 2,72, respectivamente. Estes resultados indicam que os valores de proteínas séricas totais podem ser determinados com precisão em P. expansa e P. geoffroanus usando o refratômetro portátil.

The colorimetric detection and quantitation of total protein.

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

Albumin-based or albumin-linked calibrators cause a positive bias in serum proteins assayed by the biuret method.

BACKGROUND: Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%-5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. METHODS: We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. RESULTS: Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. CONCLUSIONS: Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%-5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91-101.

Effects of minimally invasive puncture and drainage of intracranial hematoma on the blood-brain barrier in patients with cerebral hemorrhage.

The effects of minimally invasive surgery on the blood-brain barrier (BBB) of 30 patients with cerebral hemorrhage were investigated. Difference of the BBB index and serum MBP concentration were assessed in 15 cases of conservative treatment group and 15 cases of minimally invasive surgery group. The BBB index in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and the BBB index in the two treatment groups was significantly higher than in control group (P<0.01). Serum MBP concentration in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and that in the two treatment groups was significantly higher than in control group (P<0.01). It was suggested the permeability of BBB in patients with cerebral hemorrhage was increased, and BBB index and serum MBP concentration in patients with cerebral hemorrhage were increased. Minimally invasive surgery can reduce the lesion of cytotoxicity to BBB and cerebral edema.

The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission.

Regulations recommend the routine application of biochemical tests, such as the Ninhydrin or Biuret tests, to confirm the efficacy of hospital sterile service department (SSD) washer-disinfector cycles in removing proteinaceous material, particularly with respect to prions. The effectiveness of these methods relies on both the effective sampling of the instruments and the sensitivity of the tests employed. Two commercially available contamination assessment tests were evaluated for their sensitivity to ME7 brain homogenate on surgical-grade stainless steel surfaces. Controls were visualized by the application of episcopic differential interference contrast/Epi-fluorecence microscopy (EDIC/EF) combined with the sensitive fluorescent reagent, SYPRO Ruby, which has been shown previously to rapidly visualize and assess low levels of contamination on medical devices. The Ninhydrin test displayed a minimum level of detection observed by 75% of volunteers (MLD(75)) of 9.25 microg [95% confidence interval (95% CI) 8.6-10.0 microg]. The Biuret test provided better sensitivity, with a MLD(75) of 6.7 microg (95% CI 5.4-8.2 microg). However, much lower concentrations of proteinaceous soiling (pg) were visualized using the EDIC/EF microscopy method. From these findings, it is clear that these approved colorimetric tests of cleaning are relatively insensitive. This investigation demonstrates how large amounts (up to 6.5 microg) of proteinaceous brain contamination could remain undetected and the instruments deemed clean using such methods. The application of more sensitive cleanliness evaluation methods should be applied to reduce the risk of iatrogenic transmission of prion disease in 'high-risk' instruments such as neurosurgical devices.

Interference of dextran in biuret-type assays of serum proteins.

Dextran interference in biuret-type assays of total serum proteins was investigated in a Belgian National External Quality Assurance Survey with 256 participants. In vitro supplementation of therapeutic (10% Gentran 70) dextran concentrations showed a broadly varying (from 0 to 20%) negative interference. The analytical interference was found to depend on both the sodium hydroxide and tartrate concentrations in the reagent formulation. The dry chemistry biuret method was not affected by the dextran interference. In a number of cases, the effects observed may be of clinical importance. Both clinicians and laboratory staff should be aware of the persistence of this analytical problem.

The colorimetric detection and quantitation of total protein.

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

[Materials for carrying out external quality control in the determination of total protein]. / Material dlia osushchestvleniia vneshnego kontrolia kachestva opredeleniia obshchego belka.

Quaternary bis-ammonium compound No. 82 (QAC-82) is a highly effective antibacterial biochemical stabilizer of total protein. In concentrations of 3.4-13.6 mmol/liter, it preserves the content of protein in the blood serum for 40 days at 18-20 degrees C. For preparing reference material, 5 ml human or animal serum is added to 95 ml 5.25 mM aqueous solution of QAC-82, mixed, stored at room temperature, and used for assessing the quality of total protein measurements by the biuret method.

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).

Estudo da aplicabilidade do Método de Bradford para a dosagem de proteínas na urina / The applicable of the Bradford method for measuring urine protein

Neste trabalho é apresentado um método direto para dosagem de proteínas na urina, o Método de Bradford. O método caracteriza-se por ser colorimétrico, direto, rápido e sem necessidade de um tempo crítico para o ensaio, o que indica a sua aplicabilidade nas dosagens efetuadas em Laboratórios de Análises Clínicas. Este estudo teve como finalidade a aplicaçäo desta metodologia à dosagem de proteínas na urina e a demonstraçäo de sua utilizaçäo para detectar a microproteinúria, sinal precoce de patologia renal no paciente com Diabete mellitus. Foram dosadas 114 amostras de urina de 24 horas, incluindo as de indivíduos normais, pacientes com patologias renais e diabéticos. As amostras foram determinadas em comparaçäo com o método do Biureto. Os resultados indicam que o método de Bradford, com suas respectivas modificaçöes, pode ser aplicado à dosagem de proteínas na urina e por ser direto, facilita o trabalho de rotina laboratorial

[The use of different albumin preparations as calibrators in determining the total protein in blood serum by the biuret method]. / Ispol'zovanie raznykh preparatov al'bumina v kachestve kalibratorov pri opredelenii obshchego belka v syvorotke krovi biuretovym metodom.

The authors have investigated the possibility of using various albumin preparations as calibrators in measurements of human blood serum total protein by the biuret method. Analysis of Precinorm U and Precipath U reference sera has demonstrated that use of various albumin preparations as calibrators may result in significant deviations (as much as 27%) of the resultant values from the due ones.

Effects of low and high relative molecular protein mass on four methods for total protein determination in urine.

Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.

Prevention of interference by dextran with biuret-type assay of serum proteins.

In assay of serum proteins by use of the biuret reaction, dextran can cause turbidity by formation of an insoluble complex of dextran with copper and tartrate (or EDTA) in strongly alkaline solution. Whether or not the turbidity occurs depends on the tartrate concentration: turbidity is maximal at about 10 g/L, absent at 20 g/L or more, and only slight and delayed at 4 g/L. Two biuret reagents, containing respectively 5.6 and 22.5 g of tartrate per liter, obviate the interference, but the former is suitable only when a short (5 min) incubation is used. Both reagents show linear calibration curves and yield virtually identical results.

[Quantitative protein estimation following standardized concentration of diluted physiological fluids (author's transl)]. / Quantitative Proteinbestimmung nach standardisierter Konzentrierung verdünnter physiologicher Flüssigkeiten

After the tenfold concentration of 1:100 diluted standard solutions in the Minicon B-15 concentrator, the quantitative determination of total protein and of ten single proteins showed a decreased yield. These losses are not standardizable. The reason is analyzed and discussed. Therefore, the quantitative estimation of single proteins by means of immunochemical methods as well as the densitometry of electrophoreses of dilute protein solutions following concentration should be interpreted with caution, because changes in the absolute and relative concentration of proteins are to be expected. These methods are not suitable for exact measurements. An unstandardizable correction coefficient ought to be considered.