Exploring albumin-sulfonephthalein dyes interactions by a chemometric-assisted and multi-technique equilibria analysis in solution.

Albumin is undoubtedly the most studied protein thanks to its widespread diffusion and biochemistry; despite its binding ability towards different dyes, provoking dye's colour change, has been exploited for decades for quantification purposes, the joint effect of working pH, ionic strength, and dye's pKa still remains only sporadically discussed. In the present study, the interaction of Bovine Serum Albumin (BSA) with five dyes belonging to the sulfonephthalein group, Bromophenol Blue (BPB, pKa = 3.75), Bromocresol Green (BCG, pKa = 4.42), Chlorophenol Red (CPR, pKa = 5.74), Bromocresol Purple (BCP, pKa = 6.05) and Bromothymol Blue (BTB, pKa = 6.72), is investigated at four working pH values (3.5, 6.0, 7.5 and 9.0) and two ionic strength conditions by UV-Vis spectroscopy. Principal Component Analysis is then applied to rationalize dye behavior upon BSA addition at each pH value and to summarize the protein effect on dyes' spectral features, identifying three general behaviors. The most relevant systems are then submitted to further characterization involving a solution equilibria study aimed at determining conditional binding constants for the selected DSA-dye adducts and fluorescence, CD, and 1H NMR spectroscopy to evaluate the binding effect on the species involved.

Performance evaluation of different albumin assays for the detection of analbuminemia.

Analbuminemia is characterized by the near absence of albumin in the plasma. Different methods are available for measuring albumin levels, but they do not necessarily agree with one another. It is a concern that analbuminemic samples could be falsely characterized due to the incorrect estimation of albumin. The objective of the work was to evaluate the performance of different assays in detecting analbuminemia. Albumin knockout (Alb-/-) mouse plasma was used to test the suitability of different albumin assays for their ability to properly characterize extreme albumin deficiency. Bromocresol green (BCG), bromocresol purple (BCP), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gel electrophoresis were tested. The LC-MS/MS assay exhibited broad coverage of the amino acid sequence of albumin and indicated 8,400-fold lower (P<0.0001) albumin expression in Alb-/- than wildtype (WT), demonstrating its suitability for identifying extreme albumin deficiency. ELISA estimated albumin at 1.5±0.1 g/dL in WT and was below the detection limit in all Alb-/- samples. Gel electrophoresis yielded consistent results with LC-MS/MS and ELISA. The BCG assay overestimated albumin with apparently appreciable albumin concentrations in Alb-/- mice, yet the assay still indicated a significant difference between genotypes (Alb-/-, 1.2±0.05 g/dL, WT, 3.7±0.1 g/dL, P<0.0001). BCP drastically overestimated albumin and could not successfully identify the known analbuminemic phenotype of Alb-/- mice. By using Alb-/- plasma as a reference material and LC-MS/MS as a reference method, ELISA and gel electrophoresis appear appropriate for identifying analbuminemia, while BCG and BCP are not suitable. It is concluded that dye-binding assays should be avoided when extreme hypoalbuminemia or analbuminemia is suspected.

Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

An Adapted Protocol for Quantitative Rhizosphere Acidification Assay.

Acidification of the rhizosphere is a key process in the homeostasis of multiple essential nutrients, including iron. Under iron deficiency, the release of protons from the roots helps solubilize and increase the accessibility of iron in the soil. Rhizosphere acidification has been widely examined in many iron homeostasis studies, generally using a qualitative method based on the color change of bromocresol purple, a pH indicator dye, near the roots. In this chapter, we introduce an adapted version of a rhizosphere acidification assay protocol that allows for the quantitative assessment of small pH changes in the rhizosphere. This colorimetric method also utilizes bromocresol purple, but the ratio of its absorbance at 434 nm and 588 nm is considered to quantify protons released into the assay solution. Furthermore, the assay is compatible with small sample volumes, such as those with young Arabidopsis seedlings.

Evaluation of the two-point calibration bromocresol green method, showing reduced deviation from the bromocresol purple method in sera from patients with hypoalbuminemia.

BACKGROUND: The bromocresol green (BCG) and bromocresol purple (BCP) methods are widely used for albumin measurements in routine testing, but the BCG method is known to react with globulin fractions and to have low specificity for albumin. We evaluated a calibration method using different concentrations of human serum albumin standards (two-point calibration BCG method) with the aim of reducing the effect of globulin fractions on the BCG method in patients with hypoalbuminemia. METHOD: In the two-point calibration BCG method, two concentrations of standard solutions and their calibration values are set based on the difference in albumin concentrations measured by the BCG method (BCG-HSA method) and the modified BCP (modified BCP-HSA method) calibrated with human serum albumin standard solution (HSA). Albumin concentrations were measured in 136 patient serum samples (healthy group: 52, hypoalbuminemic group: 84) by the two-point calibrated BCG method and compared with those obtained using the modified BCP-HSA method. RESULTS: The mean albumin concentrations obtained using the two-point calibrated BCG and modified BCP-HSA methods were 39.18 ± 3.42 g/L and 39.37 ± 3.14 g/L (healthy group) and 26.20 ± 6.23 g/L and 26.23 ± 5.67 g/L (hypoalbuminemia group), respectively. The results of the two-point calibration BCG method were in a close agreement over the entire concentration range tested compared to the modified BCP-HSA method. CONCLUSIONS: Based on these results, this calibration method reduces the influence of the globulin fraction on the BCG method. In the hypoalbuminemic group, the calibration method was shown to provide results consistent with the BCP method, which is highly specific for albumin.

Impact of Albumin Assays in the Diagnosis of Malnutrition in Hemodialysis Patients: A Cohort Study.

OBJECTIVE: In hemodialysis (HD) patients, malnutrition should be diagnosed by several assessment tools including a plasma albumin concentration of less than 3.8 g/dL or 3.5 g/dL using bromocresol green or immunonephelometry (IN), respectively. However, albumin measurement is not yet standardized and two alternative methods are also commonly used in laboratories: bromocresol purple (BCP) and immunoturbidimetry (IT). This study aimed to revisit the hypoalbuminemia thresholds for BCP and IT, in HD patients. METHODS: Plasma albumin was measured by the four analytical methods during the monthly HD nutritional assessment of 103 prospectively included patients. RESULTS: Significant differences in albumin levels were observed in HD patients depending on the method used. Using BCP or IT with the cut-off at 3.5 g/dL (determined for the general population) we obtained 33% and 9.7% of false hypoalbuminemia in comparison to IN (mean bias of -0.4 g/dL and -0.065 g/dL, respectively). The best hypoalbuminemia threshold for BCP was 3.05 g/dL and 3.4 g/dL for IT. Twenty percent of HD patients were classified as malnourished when albumin was determined by IN. Similar rates were obtained using the new hypoalbuminemia cut-offs for BCP (18.5%) and IT (19.5%). CONCLUSION: To avoid nutritional misclassification of HD patients, we should adjust hypoalbuminemia thresholds when BCP or IT methods are used in laboratories.

Development of a calcium hydroxide-dye kimchi ripening indicator and its application in kimchi packaging.

Intelligent packaging that provides real-time information on food quality is useful for consumers. We developed a kimchi ripening indicator that can determine the ripeness of kimchi inside packaging and evaluated its applicability and limitations. The indicator was made using calcium hydroxide, which captures CO2, and four pH-sensitive dyes (cresol red, bromothymol blue, bromocresol purple, and methyl red). Fourier-transform infrared spectra of the prepared powders showed shapes similar to that of calcium hydroxide, and the dyes were evenly distributed on the calcium hydroxide surfaces. When the developed indicators were evaluated for kimchi packaging application, the indicator made from synthesized calcium hydroxide and bromothymol blue was the most reliable and clearly reflected useful kimchi ripening information. The indicator developed in this study is judged to be practically usable at temperatures of 4-15 °C. However, its usefulness is limited in that the seller cannot change the packaging capacity or kimchi capacity.

Towards intelligent packaging: BCP-EVOH@ optode for milk freshness measurement.

Intelligent packaging represents an emerging trend in the food industry, especially for highly perishable foods like milk and dairy products. Despite the apparent simplicity, miniaturized BCP-EVOH@ sensor, made of bromocresol purple (BCP) covalently bound to ethylene-vinyl alcohol (EVOH) copolymer, meets the goal of milk freshness monitoring during chilled storage, allowing both naked-eye evaluation and chemometric-assisted spoilage modelling and measurements.

Bromocresol purple in the processes of cation-anionic interactions in aqueous solutions: UV-Vis spectroscopy and computer simulation of dissimilar association.

The formation of associates between single- or double-charged anions of bromocresol purple (3,3'-dimethyl-5,5'-dibromophenolsulfonephthalein) and single-charged cations of cyanines (quinaldine blue, quinaldine red) has been investigated. The equilibrium constants of the association have been determined on the basis of UV-Vis spectroscopy data. The energy of the cation-anion interaction (the standard enthalpy of formation of ions and associates), as well as the probable structure of associates, were established using the PM7 semiempirical method. The presented results can be useful in the development of new spectroscopic methods for the detection of organic additives in water (e.g. acetone) due to the spectral properties of dissimilar associates.

[Comparison of three albumin assay methods: a need for standardization within a healthcare area]. / Comparaison de trois techniques de dosage de l'albumine : un argument pour la standardisation des pratiques au sein d'un territoire de santé.

Albumin is a main biomarker of denutrition severity. Since 2019, the French National Authority of Health (HAS) recommends an immunologic technique as a reference for measuring albuminemia. In a context of partial recovery by the laboratory of GHBS (using colorimetric method with bromcresol purple) of the biological activity of the dialysis center, until then carried out by the Ouest biology - Biolor laboratory (using colorimetric method with bromcresol green) and in order to standardize practices in the healthcare sector, we compared 3 albumin assay techniques: immunoturbidimetric (Diagam®), BCG (Siemens®), BCP (Siemens®) on Siemens Atellica PLCs. The albumin level of 183 patient samples was measured. We show an overestimation of the dosage by the BCG (+ 5.46 g/L compared to IT) and an interference related to the inflammatory state of the patient during the dosage with this technique, as well as an underestimation of the dosage by the BCP (- 0.91 g/L relative to the IT), increased in patients with renal injury. This biological difference is added to a discrepancy in clinical conclusions. A change in dosing technique from BCG to BCP may impact the biological follow-up of patients and the PMSI score (mean difference of - 6.67 g/L between BCG and BCP while the classification of the severity of denutrition uses thresholds set in steps of 5 g/L). This variability of results should be taken into account by the clinicians and constitutes an argument in favor of the standardization of laboratories around a reference technique.

Isotherm, Kinetic and Thermodynamic Studies on Adsorption of Bromocresol Purple, Acid Red 66 and Acid Blue 40 Using Activated Carbon.

This study was conducted with a batch reaction to equilibrium isotherm, kinetic and thermodynamic parameters on adsorption of bromocresol purple (BCP), acid red 66 (AR 66) and acid blue 40 (AB 40) from aqueous solution by using activated carbon with nanopores. Freundlich and Temkin isotherm models were used to evaluate the suitability of isotherm for adsorption equilibrium data. The adsorption equilibrium was best fitted by Temkin model. The Freundlich separation factor values indicated that adsorption on the nanoporous activated carbon could effectively treat three dyes. The kinetic analysis of the adsorption process confirmed that it was more consistent with the pseudo second order model. The intraparticle diffusion was rate limiting step. The adsorption process of three dyes were endothermic because they were positive enthalpy values. The free energy values of three dyes decreased with increasing temperature, so that the spontaneity becomes higher with temperature increase. The activation energy value of three dyes were confirmed the physical adsorption.

Dye-decolorization of a newly isolated strain Bacillus amyloliquefaciens W36.

Dye-decolorization is one of the most important steps in dye-polluted wastewater treatment. The dye-decolorization bacteria were isolated from active sludge collected from wastewater treating pond of a dyeing and printing plant using serial dilution method. Among the 44 bacteria isolates from the active sludge, the strain Bacillus amyloliquefaciens W36 was found to have strong ability in dye-decolorization. The effects of carbon source, nitrogen sources, C/N, metal ions, temperature, pH, and rotation speed for dye-decolorization were investigated. The optimum decolorization conditions were that the strain was grown in enriched mineral salt medium (EMSM) using maltose 1 g/L, (NH4)2SO4 1 g/L as carbon and nitrogen source respectively, supplemented with 100 mg/L different dyes (pH 6.0), at 30 °C, 200 rpm from 48 to 96 h. The bacteria could aerobically decolorize dyes, such as Coomassie brilliant blue (95.42%), Bromcresol purple (93.34%), Congo red (72.37%) and Sarranine (61.7%), within 96 h. The dyes decolorization products were analyzed by ultra-violet and visible (UV-vis) spectroscopy before and after decolorization, which indicated that the four dyes were significantly degraded by the strain. The results indicated that the bacteria Bacillus amyloliquefaciens W36 could be used in dye-polluted wastewater treatment.

Simultaneous Determination of Ferulic Acid and Vanillin in Vanilla Extracts Using Voltammetric Sensor Based on Electropolymerized Bromocresol Purple.

Natural phenolic antioxidants are one of the widely studied compounds in life sciences due to their important role in oxidative stress prevention and repair. The structural similarity of these antioxidants and their simultaneous presence in the plant samples stipulate the development of methods for their quantification. The current work deals with the simultaneous determination of vanillin and its bioprecursor ferulic acid using a voltammetric sensor for the first time. A sensor based on the layer-by-layer deposition of the polyaminobenzene sulfonic acid functionalized single-walled carbon nanotubes (f-SWCNTs) and electropolymerized bromocresol purple has been developed for this purpose. The best response of co-existing target analytes was registered for the polymer obtained from the 25 µM dye by 10-fold potential cycling from 0.0 to 1.2 V with the scan rate of 100 mV s-1 in 0.1 M phosphate buffer (PB), pH 7.0. Scanning electron microscopy (SEM), cyclic voltammetry and electrochemical impedance spectroscopy (EIS) confirmed the effectivity of the sensor developed. The linear dynamic ranges of 0.10-5.0 µM and 5.0-25 µM for both analytes with the detection limits of 72 nM and 64 nM for ferulic acid and vanillin, respectively, were achieved in differential pulse mode. The sensor was applied for the analysis of vanilla extracts.

Application of bromocresol purple nanofilm and laser light to detect mutton freshness.

Optical waveguide gas sensor with bromocresol purple (BCP) nano-film (70-80 nm) composited with silicone deposited on surface of K+-ion exchanged glass slide was fabricated and applied to detect amine gases released during mutton spoilage to test mutton freshness with the help of laser light (632 m). Gas sensing measurements (output light intensity vs. time) proved its high selectivity and good sensitivity (0.01 ppm) toward amines among volatile organic compounds. The optimal fabricating condition (2600 rpm, 0.10% BCP, 5.17% silicone) was selected experimentally, displaying 1 s and 12 s response-recovery time toward gases released from mutton samples stored at 5 °C and 25 °C respectively. The sensing mechanism was explained by combination and competition of diffusion-reaction, considering deprotonating reaction of bromocresol purple molecules and molecular size of analyte gases in diffusion process. UV-vis spectroscopy was used to select the specific wavelength of laser light source in optical waveguide performance and to detect the total volatile basic nitrogen contents in mutton sample to confirm the practicability of as-prepared optical waveguide sensor in detecting mutton freshness. The fabricated sensor is able to detect 7.2 mg/100 g gases released from mutton decomposition, providing a simple, fast, and cheap method to detect meat freshness.

The bias between different albumin assays may affect clinical decision-making.

Differences between laboratory assays can have important clinical implications. For creatinine assays this became apparent soon after the introduction of the Modification of Diet in Renal Disease formula and resulted in international efforts towards standardization. Albumin in blood is measured by different assays, either chromogenic using Bromocresol green (BCG) or Bromocresol purple (BCP), or by an immunoassay. Since differences between these assays have received limited attention we evaluated bias and imprecision of BCG and BCP assays in comparison to the immunoassay using blood samples from patients with membranous nephropathy and nephrotic syndrome. For the BCG assay, the mean bias was high (6.2 g/l, with a standard deviation of 2.4 g/l) compared to a bias of 0.3 g/l (standard deviation 1.5 g/l) for the BCP assay. Importantly, we questioned clinical relevance by evaluating the accuracy of the decision toward the use of prophylactic anticoagulant therapy. Notably, nephrologists may reach inappropriate treatment decisions using the BGC assay in up to 59% of patients. Thus, our study should stimulate efforts towards standardization of the albumin assays.

High-efficiency extraction of bromocresol purple dye and heavy metals as chromium from industrial effluent by adsorption onto a modified surface of zeolite: Kinetics and equilibrium study.

Tannery industrial effluent is rich in heavy metals and basic dyes as bromocresol purple (BCP), poses an economic problem and a serious danger to the environment. This research had evaluated the importance of the adsorption properties of a modified clinoptilolite (CL) (a type of zeolite) for the removal of BCP dye and some heavy metals as total chromium (tCr) in the ammoniac phase. The modified adsorbent was prepared by mixing solid waste (SW) and CL in a ratio 10:1. The CL, SW, and CL-SW materials were characterized and the adsorption behavior of the later to BCP and tCr was completely studied. The batch removal showed the optimal conditions for BCP adsorption: pH (6.5), time (t) (60 min), temperature (T) (303.15 K), sorbent dosage (m) (60.4 mg), and initial concentration (Co) (11.7 mg/L). Moreover, the optimum conditions for tCr removal were: pH (8.8), t (55 min), T (303.15 K), m (400 mg), and Co (16.0 mg/L). Cr desorption mechanism was an ion exchange reaction. The experimental data of tCr were best fitted by the Freundlich isotherm and the pseudo-second-order kinetic model. The maximum adsorption capacities of BCP and tCr onto the CL-SW were 175.5 mg/g and 37 mg/g, respectively. Thermodynamic studies revealed that the adsorptions were spontaneous and endothermic with an increase of entropy. The CL modified adsorbent seems to be a good and efficient for the removal of dyes as BCP and such heavy metals including Cr. Surprisingly, this treatment has largely improved the physicochemical properties of the industrial wastewater and proved a new concept "Polluter Cleans Polluters (PoClPos)".

Overestimation of Albumin Measured by Bromocresol Green vs Bromocresol Purple Method: Influence of Acute-Phase Globulins.

BACKGROUND: Usually serum albumin is measured with dye-binding assay as bromocresol green (BCG) and bromocresol purple (BCP) methods. The aim of this paper was to examine the differences in albumin measurements between the Advia2400 BCG method (AlbBCG), Dimension RxL BCP (AlbBCP) and capillary zone electrophoresis (CZE). METHODS: Albumin concentrations from 165 serum samples were analysed using AlbBCG, AlbBCP and CZE. CZE was employed to estimate different serum protein fractions. Influence of globulins on albumin concentration discrepancies between methods was estimated as well as the impact of the albumin method on aCa concentrations. Medcalc was employed for statistical analysis, setting a value of P < 0.05 as significant. RESULTS: Correlation of AlbBCG and AlbBCP was r = 0.948 (p < 0.0001), but mean difference was large. Bland-Altman plots showed greater bias at lower albumin concentrations. AlbBCG were positively biased versus CZE (3.54 g/L). There was good agreement between CZE and ALbBCP (< 1 g/L). The AlbBCG assay bias shows a good correlation with alpha-1-globulin concentrations (r = 0.758); moderate and weak correlations were observed with CRP (r = 0.729) and alpha-2-globulin (r = 0.585); we found no correlation with beta-globulin (r = 0.120) or gamma-globulin (r = -0.303). Mean aCa based on AlbBCG and AlbBCP methods were 2.34 ± 0.15 mmol/L and 2.46 ± 0.16 mmol/L (p < 0.01), with a mean BCG-BCP difference of -0.12. CONCLUSION: Albumin results from the BCP and BCG methods may result in unacceptable differences and clinical confusion, especially at lower albumin concentrations. Serum acute phase proteins contribute to overestimating the albumin concentration using AlbBCG.

Comparison of hypoalbuminemia-corrected serum calcium using BCP albumin assay to ionized calcium and impact on prescribing in hemodialysis patients
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BACKGROUND: Albumin-corrected calcium (cCa) is recommended over ionized calcium (iCa) in hemodialysis (HD) patients per the Kidney Disease: Improving Global Outcomes position statements due to cost and feasibility. Two common albumin assays, bromocresol green (BCG) and bromocresol purple (BCP), produce differing results in uremic patients. All previous studies compared iCa to cCa from a BCG assay. This study, using the BCP assay, aimed to compare cCa and total calcium, respectively, to iCa. We also sought to assess phosphate binders and dialysis prescribing patterns following abnormal calcium measurements. MATERIALS AND METHODS: Retrospective review of 122 stable chronic HD patients with iCa, serum calcium, and albumin measured together throughout 6 blood work periods for a total of 338 sets of comparison values. Payne and Jain calcium correction equations were used. Prescription changes within 2 weeks of abnormal iCa values were recorded. RESULTS: Mean iCa, cCa, and total calcium were 1.17 ± 0.08, 2.37 ± 0.16, and 2.28 ± 0.15 mmol/L, respectively. Total calcium and cCa compared to iCa had κ-coefficients of 0.19 and 0.08, respectively, for hypocalcemia, 0.19 and -0.02 for normocalcemia and 0.59 and 0.46 for hypercalcemia. 21 interventions were made in hypocalcemic patients using iCa as reference; however, if total or corrected calcium values were used, only 8 and 5 interventions, respectively, would result. CONCLUSION: When BCP assay is used, conventional correction equations should not be utilized in hemodialysis patients; uncorrected serum calcium has a better predictive value.
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