Measurement Of Total Protein Concentration Research Articles

Ethylenediaminetetraacetic acid (EDTA) anticoagulant affects the refractometric measurement of total protein concentration in equine synovial fluid

SummaryThe objectives of this study were to (i) determine the effect of ethylenediaminetetraacetic acid (EDTA) concentration on the refractometric measurement of the total protein (TP) concentration of equine synovial fluid and (ii) assess the effect of time and temperature on the refractometric measurement of TP concentration. Synovial fluid was retrieved aseptically from horses presenting to Liphook Equine Hospital either (a) immediately after euthanasia or (b) from those requiring arthrocentesis as part of their clinical investigation. Samples were aliquoted into commercially available (i) plain tubes (0.5 mL; control) and (ii) EDTA tubes (at different volumes to obtain serial dilutions of EDTA:synovial fluid). TP concentration was measured by two independent observers using a calibrated hand‐held refractometer. Samples from a separate group of horses were stored for 24 h at 4°C and 20°C before analysis to assess the effects of time and temperature. Results were compared using repeated measures ANOVA. TP concentration was significantly higher at all EDTA concentrations compared to control samples, with the greatest effect seen with higher concentrations (P<0.0001 for samples with ≥3.17 mg EDTA/mL). Storage time or temperature did not significantly affect TP concentration. Refractometric measurement of small volumes of synovial fluid in commercially available EDTA tubes leads to artificially elevated TP concentrations. Delays in sample analysis and alterations in storage conditions did not significantly affect TP concentration within the parameters studied. The magnitude of overestimation of synovial total protein concentration could result in misclassification of suspect synovial sepsis cases where EDTA tubes are used.

Review of methods for determination of total protein and peptide concentration in biological samples.

Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit-for-use, quality-by-design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein-reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 μg mL(-1) and 400 μg mL(-1) for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 μg mL(-1) (R(2) = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R(2) > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes.

Stability of commercial glucanase and β-glucosidase preparations under hydrolysis conditions.

The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R2 = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R2 = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R2 = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling.

Total protein measurement in canine cerebrospinal fluid: agreement between a turbidimetric assay and 2 dye‐binding methods and determination of reference intervals using an indirect a posteriori method

In veterinary clinical laboratories, qualitative tests for total protein measurement in canine cerebrospinal fluid (CSF) have been replaced by quantitative methods, which can be divided into dye-binding assays and turbidimetric methods. There is a lack of validation data and reference intervals (RIs) for these assays. The aim of the present study was to assess agreement between the turbidimetric benzethonium chloride method and 2 dye-binding methods (Pyrogallol Red-Molybdate method [PRM], Coomassie Brilliant Blue [CBB] technique) for measurement of total protein concentration in canine CSF. Furthermore, RIs were determined for all 3 methods using an indirect a posteriori method. For assay comparison, a total of 118 canine CSF specimens were analyzed. For RIs calculation, clinical records of 401 canine patients with normal CSF analysis were studied and classified according to their final diagnosis in pathologic and nonpathologic values. The turbidimetric assay showed excellent agreement with the PRM assay (mean bias 0.003 g/L [-0.26-0.27]). The CBB method generally showed higher total protein values than the turbidimetric assay and the PRM assay (mean bias -0.14 g/L for turbidimetric and PRM assay). From 90 of 401 canine patients, nonparametric reference intervals (2.5%, 97.5% quantile) were calculated (turbidimetric assay and PRM method: 0.08-0.35 g/L (90% CI: 0.07-0.08/0.33-0.39); CBB method: 0.17-0.55 g/L (90% CI: 0.16-0.18/0.52-0.61). Total protein concentration in canine CSF specimens remained stable for up to 6 months of storage at -80°C. Due to variations among methods, RIs for total protein concentration in canine CSF have to be calculated for each method. The a posteriori method of RIs calculation described here should encourage other veterinary laboratories to establish RIs that are laboratory-specific.

Refractometric total protein concentrations in icteric serum from dogs

To determine whether high serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among refractometer measurements. Evaluation study. Sera from 2 healthy Greyhounds. Bilirubin was dissolved in 0.1M NaOH, and the resulting solution was mixed with sera from 2 dogs from which food had been withheld to achieve various bilirubin concentrations up to 40 mg/dL. Refractometric total protein concentrations were estimated with 3 clinical refractometers. A biochemical analyzer was used to measure biuret assay-based total protein and bilirubin concentrations with spectrophotometric assays. No interference with refractometric measurement of total protein concentrations was detected with bilirubin concentrations up to 41.5 mg/dL. Biases in refractometric total protein concentrations were detected and were related to the conversion of refractive index values to total protein concentrations. Hyperbilirubinemia did not interfere with the refractometric estimation of serum total protein concentration. The agreement among total protein concentrations estimated by 3 refractometers was dependent on the method of conversion of refractive index to total protein concentration and was independent of hyperbilirubinemia.

The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid

The aim of this study was to evaluate the influence of two commonly used anticoagulants (K 3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K 3EDTA, a solution of K 3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K 3EDTA. The solution of K 3EDTA in distilled water only caused TP overestimation at high K 3EDTA concentrations (>5 μmol/ml). By contrast, lithium heparin did not influence the refractometric values of TP. Neither anticoagulant modified TP values when measured by the biuret method. In conclusion, the use of K 3EDTA as anticoagulant may result in a significant overestimation of TP values of peritoneal fluid samples measured by refractometry.

A SU-8 fluidic microsystem for biological fluids analysis

This paper describes a fluidic microsystem fabricated in SU-8 epoxy photoresist suitable for on-chip liquid handling and mixing. The application is the concentration measurement of biomolecules in biological fluids, such as urine, blood, serum, plasma or cerebrospinal fluid. In general, fluidic microsystems on glass are fabricated using HF acid etching techniques, which requires careful handling, or using micromilling, which gives high-roughness surface. The SU-8 fabrication process is low-cost, biocompatible and UV lithography semiconductor compatible. It requires neither complex techniques nor expensive masks and is suitable for batch production. Moreover, SU-8-based processing enables the fabrication of deep microchannels with very low sidewall roughness, which is suitable, when it is used an optical absorption measurement method. The high light of this paper is the implementation of a fluidic microsystem for biological fluids analysis with SU-8 photoresist. The fluidic microsystem performance is successfully demonstrated in the spectrophotometric measurement of total protein concentration in urine, by optical absorption.

Assessment of the reliability of plasma electrophoresis in birds

To determine the reliability of plasma electrophoresis (EPH) in psittacine birds. 93 psittacine birds. Jugular venipuncture was performed on 93 awake psittacine birds. The plasma was centrifuged, separated, aliquoted into duplicate samples, frozen, and sent to 2 commercial laboratories that routinely perform avian EPH. Samples from 51 birds were sent to laboratory A, and samples from 42 birds were sent to laboratory B. The reliability of EPH results within each laboratory was assessed, but not between laboratories. To determine the reliability (agreement between duplicate samples) of total protein, albumin, prealbumin, alpha1-, alpha2-, beta-, and gamma-globulin concentrations, the intraclass correlation coefficient (r(i)) was calculated. Both laboratories had excellent agreement between samples for measurement of total protein concentration and only good agreement for albumin concentration. Except for the prealbumin concentration measured at laboratory B, both laboratories had poor agreement for all other values of the EPH. These data indicate that plasma EPH for measuring prealbumin, alpha1-, alpha2-, beta-, and gamma-globulin concentrations may not be a reliable tool for assessing avian health. Small amounts of these proteins in birds plus human variation in reading the EPH curves may lead to variable results. Avian veterinarians should cautiously interpret results from plasma EPH assays for these protein fractions.

Albumin fraction and measurement of total protein concentration.

The standard curve of a typical colorimetric assay for total protein is often nonlinear and dependent on the albumin fraction of the protein standard. We developed a simple mathematical transformation to make the standard curve linear and a computational method to correct for differences in albumin concentrations among the samples. This method uses data from total protein assays on two sets of standards (albumin and gamma globulin) and provides accurate measures of total protein over the full range of albumin fractions. Comparison of this two-standard method with the a method that uses only albumin as a standard shows that this method prevents physiologically significant overestimations in total protein concentration and calculated protein osmotic pressure differences in the lungs.

A sound-speed sensor for the measurement of total protein concentration in disposable, blood-perfused tubes

The aim of this investigation was to develop a sensor containing a disposable measuring cell capable of measuring fluid shifts in blood-perfused circulation systems. Usually, the determination of fluid shifts in such systems is achieved by tracking the concentration of particles or solutes in the perfusate. Since acoustic properties of blood are closely related to the total protein concentration (TPC) of the sample, a new approach for the calculation of fluid shifts is made by the continuous measurement of sound speed. The measuring principle is based upon the evaluation of the propagation time of short acoustic pulses that are repeatedly transduced to the sample. The retransmission is triggered by means of a new type of delay line. The method stands out for its accuracy (better than 0.1 m/s) and for the short acoustic propagation path (5–10 mm). In order to develop the disposable measuring cell, the acoustic properties of porcine blood were determined at different hematocrits and TPC at temperatures ranging from 20–40 °C. The dependence of sound speed on temperature and on TPC is described by a polynomial. The new sensor was tested in vitro simulating mass shifts by adding a mass (Msaline) of isotonic saline solution to the circulating blood mass (M). The acoustic device containing the disposable cell proved capable of sensing mass fractions [Msaline/(M+Msaline)] in the order of 0.5 g/100 g.

Determination of serum protein concentration in nanoliter blood samples using fluorescamine or o-phthalaldehyde

Fluorometric procedures were developed to permit measurement of total protein concentration in nanoliter serum samples, using either fluorescamine or o-phthalaldehyde. The sensitivities of assays using these two reagents were similar, but the o-phthalaldehyde method was found to be somewhat simpler and more reproducible. Accurate measurements could be obtained on serum samples of 4 to 5 nl with either reagent, by using a serum standard. Fluorescence differed considerably among individual serum proteins, albumin generally showing greater fluorescence than globulins. Small molecular weight species in serum did not contribute appreciably to total serum fluorescence with either reagent.

On estimating colloid osmotic pressure in pre- and postglomerular plasma in the rat

Currently in renal physiology much investigative effort is being directed toward an assessment of the role of plasma colloid osmotic pressure (COP) in influencing both the rate of formation of glomerular ultrafiltrate and the rate of reabsorption of this ultrafiltrate by the renal tubules [1–4]. One important consequence of this process of ultrafiltration is that plasma undergoes a progressive increase in its protein concentration (and therefore in COP) in the course of traversing the glomerular capillary network, rising from approximately 6 g/100 ml in the afferent arteriole to values as high as 9 to 11 g/l00 ml in the efferent arteriole [1–3, 5, 6]. Thereafter, as a result of the return of reabsorbate to the postglomerular microcirculation, the concentration of protein declines from these high levels to values approaching pre-glomerular concentrations, the exact level being determined primarily by the volume of ultrafiltrate which escapes return to the renal circulation and instead leaves the kidney either as urine or lymph. At present no method is available for the direct measurement of COP in the small samples of blood which are obtainable from vessels in the postglomerular microcirculation using conventional micropuncture techniques. Instead the current approach to the estimation of COP in pre- and postglomerular blood makes use of 1) the measurement of total protein concentration in systemic and efferent arteriolar blood plasmas [1–3, 5–7], or the measurement of systemic protein concentration and the calculation of postglomerular protein concentration from simultaneous determination of filtration fraction [8], and 2) the application of one of several empirical equations which relate total protein concentration to COP [9]. The equation most frequently employed is that derived by Landis and Pappenheimer [9] for human plasma containing approximately equal concentrations of albumin and globulin (see equation 2 below).