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.

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
.

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.
.

Binding of bromocresol green and bromocresol purple to albumin in hemodialysis patients.

BACKGROUND: Colorimetric albumin assays based on binding to bromocresol purple (BCP) and bromocresol green (BCG) yield different results in chronic kidney disease. Altered dye binding of carbamylated albumin has been suggested as a cause. In the present study, a detailed analysis was carried out in which uremic toxins, acute phase proteins and Kt/V, a parameter describing hemodialysis efficiency, were compared with colorimetrically assayed (BCP and BCG) serum albumin. METHODS: Albumin was assayed using immunonephelometry on a BN II nephelometer and colorimetrically based on, respectively, BCP and BCG on a Modular P analyzer. Uremic toxins were assessed using high-performance liquid chromatography. Acute phase proteins (C-reactive protein and α1-acid glycoprotein) and plasma protein α2-macroglobulin were assayed nephelometrically. In parallel, Kt/V was calculated. RESULTS: Sixty-two serum specimens originating from hemodialysis patients were analyzed. Among the uremic toxins investigated, total para-cresyl sulfate (PCS) showed a significant positive correlation with the BCP/BCG ratio. The serum α1-acid glycoprotein concentration correlated negatively with the BCP/BCG ratio. The BCP/BCG ratio showed also a negative correlation with Kt/V. CONCLUSIONS: In renal insufficiency, the BCP/BCG ratio of serum albumin is affected by multiple factors: next to carbamylation, uremic toxins (total PCS) and α1-acid glycoprotein also play a role.

Determination of serum albumin, analytical challenges: a French multicenter study.

Among the biological markers of morbidity and mortality, albumin holds a key place in the range of criteria used by the High Authority for Health (HAS) for the assessment of malnutrition and the coding of information system medicalization program (PMSI). If the principle of quantification methods have not changed in recent years, the dispersion of external evaluations of the quality (EEQ) data shows that the standardization using the certified reference material (CRM) 470 is not optimal. The aim of this multicenter study involving 7 sites, conducted by a working group of the French Society of Clinical Biology (SFBC), was to assess whether the albuminemia values depend on the analytical system used. The albumin from plasma (n=30) and serum (n=8) pools was quantified by 5 different methods [bromocresol green (VBC) and bromocresol purple (PBC) colorimetry, immunoturbidimetry (IT), immunonephelometry (IN) and capillary electrophoresis (CE)] using 12 analyzers. Bland and Altman's test evaluated the difference between the results obtained by the different methods. For example, a difference as high as 13 g/L was observed for the same sample between the methods (p <0.001) in the concentration range of 30 to 35 g/L. The VBC overestimates albumin across the range of values tested compared to PBC (p <0.05). PBC method gives similar results to IN for values lower than 40 g/L. For IT methods, one of the technical/analyzer tandem underestimates the albumin values inducing a difference of performance between the immunoprecipitation methods (IT vs IN, p <0.05). Although, the albumin results are related to the technical/analyzer tandem used. This variability is usually not taken into account by the clinician. Thus, clinicians and biologists have to be aware and have to check, depending on the method used, the albumin thresholds identified as risk factors for complications related to malnutrition and PMSI coding.

The Roche Immunoturbidimetric Albumin Method on Cobas c 501 Gives Higher Values Than the Abbott and Roche BCP Methods When Analyzing Patient Plasma Samples.

BACKGROUND: Serum/plasma albumin is an important and widely used laboratory marker and it is important that we measure albumin correctly without bias. We had indications that the immunoturbidimetric method on Cobas c 501 and the bromocresol purple (BCP) method on Architect 16000 differed, so we decided to study these methods more closely. METHOD: A total of 1,951 patient requests with albumin measured with both the Architect BCP and Cobas immunoturbidimetric methods were extracted from the laboratory system. A comparison with fresh plasma samples was also performed that included immunoturbidimetric and BCP methods on Cobas c 501 and analysis of the international protein calibrator ERM-DA470k/IFCC. RESULTS: The median difference between the Abbott BCP and Roche immunoturbidimetric methods was 3.3 g/l and the Roche method overestimated ERM-DA470k/IFCC by 2.2 g/l. The Roche immunoturbidimetric method gave higher values than the Roche BCP method: y = 1.111x - 0.739, R² = 0.971. CONCLUSION: The Roche immunoturbidimetric albumin method gives clearly higher values than the Abbott and Roche BCP methods when analyzing fresh patient samples. The differences between the two methods were similar at normal and low albumin levels.

The bromocresol green assay, but not the modified bromocresol purple assay, overestimates the serum albumin concentration in nephrotic syndrome through reaction with α2-macroglobulin.

BACKGROUND: The bromocresol green (BCG) assay is commonly used for measuring albumin (ALB), but is affected by α1- and α2-globulins, which are elevated in systemic inflammation. The modified bromocresol purple (mBCP) assay is another dye-binding method developed to overcome non-specific reactions. Concentrations of α2-macroglobulin, a major α2-globulin component, are increased in nephrotic syndrome (NS), but not in inflammation. There is little direct evidence that α2-macroglobulin affects BCG or mBCP assays. METHODS: We measured serum albumin concentrations in 33 patients with NS and 13 reference healthy controls using BCG (ALBBCG) and mBCP (ALBmBCP) assays, and nephelometry (nALB) as a reference method. We also determined five specific proteins belonging to the α1- and α2-globulins by nephelometry. After adding purified α2-macroglobulin to albumin solutions, protein reactivity in these three assays was compared. RESULTS: Nephrotic syndrome patients were categorized to tertiles according to nALB concentration. In all tertiles, ALBBCG was significantly higher than nALB, especially in the severe hypoalbuminemia group, in which α2-macroglobulin was 43-49% higher. By contrast, ALBmBCP and nALB were almost identical in all three groups. The difference between ALBBCG and nALB was positively correlated with the α2-macroglobulin concentration. In vitro, when α2-macroglobulin was added to solutions containing identical albumin concentrations, α2-macroglobulin dose-dependently increased ALBBCG, but not ALBmBCP. CONCLUSIONS: In NS, α2-macroglobulin is a major factor for positive bias of ALBBCG, especially in patients with severe hypoalbuminemia. The mBCP assay is useful for measuring albumin concentrations in NS.

The impact of change in albumin assay on reference intervals, prevalence of 'hypoalbuminaemia' and albumin prescriptions.

BACKGROUND: We studied the impact on reference intervals, classification of patients with hypoalbuminaemia and albumin infusion prescriptions on changing from a bromocresol green (BCG) to a bromocresol purple (BCP) serum albumin assay. METHODS: Passing-Bablok regression analysis and Bland-Altman plot were used to compare Abbott BCP and Roche BCG methods. Linear regression analysis was used to compare in-house and an external laboratory Abbott BCP serum albumin results. Reference intervals for Abbott BCP serum albumin were derived in two different laboratories using pathology data from adult patients in primary care. Prescriptions for 20% albumin infusions were compared one year before and one year after changing the albumin method. RESULTS: Abbott BCP assay had a negative bias of approximately 6 g/L compared with Roche BCG method.There was good agreement (y = 1.04 x - 1.03; R(2 )= 0.9933) between in-house and external laboratory Abbott BCP results. Reference intervals for the serum albumin Abbott BCP assay were 31-45 g/L, different to those recommended by Pathology Harmony and the manufacturers (35-50 g/L). Following the change in method there was a large increase in the number of patients classified as hypoalbuminaemic using Pathology Harmony references intervals (32%) but not when retrospectively compared to locally derived reference intervals (16%) compared with the previous year (12%). The method change was associated with a 44.6% increase in albumin prescriptions. This equated to an annual increase in expenditure of £35,234. CONCLUSIONS: We suggest that serum albumin reference intervals be method specific to prevent misclassification of albumin status in patients. Change in albumin methodology may have significant impact on hospital resources.

A sensitive electrochemical DNA biosensor for antineoplastic drug 5-fluorouracil based on glassy carbon electrode modified with poly(bromocresol purple).

This paper describes an electrochemical sensor for the first time based on poly(bromocresol purple) (P(BCP)) developed to observe the interaction between 5-fluorouracil (5-FU) and fish sperm double strand DNA (dsDNA). The P(BCP) film was electrosynthesized by cyclic voltammetry method on the glassy carbon electrode (GCE). The dsDNA was electrochemically immobilized on the surface of P(BCP) modified GCE and the DNA biosensor was prepared. The interaction mechanism of dsDNA with 5-FU was investigated by differential pulse voltammetry using this biosensor. A decrease in the guanine oxidation peak current of the biosensor was observed after the interaction of dsDNA and 5-FU in 0.5 mol L(-1) acetate buffer (pH 4.8) containing 0.02 mol L(-1) NaCl. The accumulation time and dsDNA concentration were optimized to obtain the best peak current response. Under optimum conditions, the linear response on the guanine signal decreasing curve was observed in the 5-FU concentration range of 1.0-50 mg L(-1). The interaction mechanism between dsDNA and 5-FU was further investigated by UV-vis spectroscopy and viscometer. The results reveal that intercalation is the primary mode of interaction between 5-FU and dsDNA.

Adsorption and transport of charged vs. neutral hydrophobic molecules at the membrane of murine erythroleukemia (MEL) cells.

The adsorption and transport of hydrophobic molecules at the membrane surface of pre- and post-DMSO induced differentiated murine erythroleukemia (MEL) cells were examined by time- and wavelength-resolved second harmonic light scattering. Two medium (<600 Da) hydrophobic molecules, cationic malachite green (MG) and neutral bromocresol purple (BCP), were investigated. While it was observed that the MG cation adsorbs onto the surface of the MEL cell, neutral BCP does not. It is suggested that an electrostatic interaction between the opposite charges of the cation and the MEL cell surface is the primary driving force for adsorption. Comparisons of adsorption density and free energy, measured at different pH and cell morphology, indicate that the interaction is predominantly through sialic acid carboxyl groups. MG cation adsorption densities have been determined as (0.6±0.3)×10(6) µm(-2) on the surface of undifferentiated MEL cells, and (1.8±0.5)×10(7) µm(-2) on differentiated MEL cells, while the deduced adsorption free energies are effectively identical (ca. -10.9±0.1 and -10.8±0.1 kcal mol(-1), respectively). The measured MG densities indicate that the total number of surface carboxyl groups is largely conserved following differentiation, and therefore the density of carboxylic groups is much larger on the differentiated cell surface than the undifferentiated one. Finally, in contrast to synthetic liposomes and bacterial membranes, surface adsorbed MG cations are unable to traverse the MEL cell membrane.

One-pot and one-step synthesis of bioactive urease/ZnFe2O4 nanocomposites and their application in detection of urea.

This communication describes a novel environmentally friendly method to prepare bioactive urease/ZnFe2O4 nanocomposites through a one-pot and one-step process. The synthetic procedure is triggered through a biological mineralization process of decomposition of urea catalyzed by urease. During the growth of ZnFe2O4, urease molecules are immobilized by original ZnFe2O4 nanoparticles. As a consequence, the bioactive urease/ZnFe2O4 nanoparticle composites are assembled. This simple route is expected to endow the bioactive nanocomposites with new properties for various interesting fields.

Carbamylation of albumin is a cause for discrepancies between albumin assays.

BACKGROUND: Several investigators have reported discrepancies between the bromocresol-purple (BCP), bromocresol-green (BCG) and immunonephelometric (INP) assays in dialysis patients. This study compared the abovementioned assays and investigated whether hemodialysis itself or carbamylation of albumin is the cause for this discrepancy. METHODS: Samples obtained from hemodialysis patients were analyzed by BCP, BCG and INP. Furthermore, albumin was carbamylated in vitro using isocyanate. Isocyanate converts lysine to homocitrulline. RESULTS: No differences were observed between samples of the pre- and post-hemodialysis groups for BCP. In the control group, BCG averaged 6 g/L higher than INP. BCP did not statistically deviate from INP. In the dialysis group BCG averaged 5 g/L higher when compared to INP, whereas BCP averaged 2 g/L lower. BCP was affected by carbamylation of albumin. BCG and INP measurements were affected to a much lesser extent. Homocitrulline content of hydrolysates was increased in both the carbamylated albumin as well as in the dialysis population. CONCLUSION: In a hemodialysis population albumin concentrations are not consistently estimated by both BCG and BCP methods. Relative to INP measurements BCG overestimates the albumin concentration (4-10 g/L), whereas BCP leads to an underestimation (0-4 g/L). Carbamylation of albumin is the main attributor to the discrepancy found with BCP.

Albumin concentration determined by the modified bromocresol purple method is superior to that by the bromocresol green method for assessing nutritional status in malnourished patients with inflammation.

BACKGROUND: The controlling nutritional status (CONUT) score (CS), a simple score for assessing nutritional status, is calculated using laboratory data, including serum albumin concentration. Although dye-binding assays such as the bromocresol green (BCG) and modified bromocresol purple (mBCP) methods are widely used for albumin measurement, acute-phase proteins interfere with the BCG method. OBJECTIVE: We aimed to determine whether the choice of albumin assay affects assessment of nutritional status using CONUT scores (CSs). DESIGN: We measured serum albumin concentrations by the BCG (ALBBCG) and mBCP (ALBmBCP) methods in 44 malnourished inpatients, 27 of whom underwent nutritional intervention, and compared them to 30 age-matched healthy volunteers. In treated patients, CSs were calculated by ALBBCG (CS-BCG) and ALBmBCP (CS-mBCP). RESULTS: C-reactive protein (CRP) concentrations were positively correlated with the difference between ALBBCG and ALBmBCP in malnourished inpatients (r = 0.59, p < 0.001). CS-BCG was always lower than CS-mBCP (lower CS indicates superior nutritional status) in treated patients with persistently high CRP levels. However, in patients whose CRP decreased gradually, this difference diminished over the clinical course. CS-BCG and CS-mBCP were similar throughout their courses in patients with normal CRP concentrations. Adding haptoglobin to the human albumin solutions increased ALBBCG in a dose-dependent manner. CONCLUSIONS: The choice of albumin assay affected the assessment of nutritional status using CSs in patients with inflammation. We recommend that the modified BCP assay be used to assess nutritional status, particularly in patients with inflammation.

Laboratory performance in albumin and total protein measurement using a commutable specimen: results of a College of American Pathologists study.

CONTEXT: Discrepant results for serum constituents were observed among peer groups in the College of American Pathologists Comprehensive Chemistry Survey. OBJECTIVES: To assess the performance of serum albumin and total protein measurement procedures and to evaluate the commutability of the conventional survey specimens. DESIGN: A fresh frozen, off-the-clot serum sample was included along with 4 conventional survey specimens. The fresh frozen, off-the-clot serum sample was prepared in a manner expected to confer commutability with native clinical samples. RESULTS: For the fresh frozen, off-the-clot serum sample, the mean values for 17 peer-groups were -0.07 to 0.32 g/dL from the bromocresol green albumin designated comparison method, whereas 4 VITROS (Ortho Clinical Diagnostics, Rochester, New York) peer groups differed by -0.29 to -0.37 g/dL (15 of 21 differences [71%] had P < .001). For bromocresol purple albumin methods, the mean differences from the designated comparison method from 8 peer groups were 0.25 to 0.47 g/dL (all had P < .001). For total protein methods, 23 peer group mean values were -0.07 to 0.15 g/dL from the reference measurement procedure (12 of 24 [50%] had P < .001). The Beckman (Fullerton, California) Synchron LX20 had a bias of -0.30 g/dL (P <.001). The commutability of the conventional specimens was acceptable for 23 of 24 bromocresol green method-material combinations (96%) and 13 of 16 bromocresol purple albumin method-material combinations (81%). All (100%) of the 36 method-material combinations had acceptable commutability for total protein. CONCLUSIONS: One (2.2%) of the instrument systems (Synchron) using bromocresol green and none (0%) of the instrument systems using bromocresol purple had satisfactory total-error performance for albumin measurement. Differences in results between bromocresol green and bromocresol purple methods precluded using common reference intervals for interpreting results for serum albumin. Eight of 9 instrument systems (86.5%) had satisfactory total-error performance for total protein measurement.

Use of two sulfonthalein dyes in the extraction-free spectrophotometric assay of tramadol in dosage forms and in spiked human urine based on ion-pair reaction.

Tramadol is a centrally acting analgesic used in the prevention and treatment of moderate to severe pain. Two sensitive, selective, and rapid spectrophotometric methods are described for the determination of tramadol in its dosage forms and in spiked human urine. The methods are based on formation of yellow ion-pairs between tramadol and two sulfonthalein dyes; bromocresol purple (BCP) and bromocresol green (BCG) in dichloromethane medium followed by absorbance measurement at 400 and 410 nm, respectively. Under the optimum conditions, tramadol could be assayed in the concentration ranges, 1-15 and 1-16 µg ml(-1) with correlation coefficient greater than 0.999 in both cases. The molar absorptivity values are calculated to be 1.84 × 10(4) and 1.97 × 10(4) l mol(-1) cm(-1) for BCP and BCG methods, respectively; and the corresponding Sandell sensitivity values are 0.0143 and 0.0134 µg cm(-2). The limits of detection (LOD) and quantification (LOQ) have also been reported. The stoichiometry of the reaction was found to be 1:1 in both cases and the conditional stability constant (K(f)) values of the ion pairs have been calculated. The within-day and between-day RSD were 0.9-1.96% and 1.56-3.21%, respectively. The methods were successfully applied to the determination of tramadol in tablets and injections and also in spiked human urine with good recoveries. The procedures are simple, accurate, and suitable for quality control application.