scholarly journals 3012 Functional consequences of pravastatin isomerization on OATP1B1-mediated transport

2019 ◽  
Vol 3 (s1) ◽  
pp. 107-107
Author(s):  
Jonathan Wagner ◽  
Melissa Ruggiero ◽  
J. Steven Leeder ◽  
Bruno Hagenbuch
Keyword(s):  
Sodium Salt ◽  
Total Protein ◽  
Protein Concentration ◽  
Linear Regression Analysis ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Total Protein Concentration ◽  
Pravastatin Sodium ◽  
Functional Consequences

OBJECTIVES/SPECIFIC AIMS: In the present study, we examined the functional consequences of 3α-PVA on OATP1B1-mediated PVA transport. To elucidate this, we determined the effect of SLCO1B1 genotype on PVA transport and the role of 3αPVA as a competitive inhibitor of OATP1B1, which could serve as another covariate that disrupts the systemic and hepatic exposure of pravastatin in children and adults. METHODS/STUDY POPULATION: Site directed mutagenesis was performed to generate SLCO1B1 genotypes of interest (*1a, *1b, *5, *15). Human embryonic kidney (HEK293) cells were grown and plated at 200 000 cells per well in 24-well plates. Twenty-four hours later the cells were transfected with the aforementioned plasmids. Forty-eight hours later cell-based transport was performed with radiolabelled [3H]-pravastatin sodium salt. Non-radioactive pravastatin sodium salt and 3’α-iso-pravastatin sodium salt was used for PVA transport and 3αPVA studies, respectively. Cells were washed 3 times with warm uptake buffer, incubated for 1 minute with uptake solutions containing PVA and 3αPVA at varying concentrations. Transport was terminated by four 1-ml washes with ice-cold uptake buffer. Cells were lysed with 300 µl 1% Triton X-100 in PBS at room temperature for 30 minutes prior to analysis. Radioactivity was measured in a MicroBeta2 liquid scintillation counter. The remaining cell lysates were transferred to 96-well plates to determine total protein concentration using the bicinchonic acid protein assay. All transport measurements were corrected by the total protein concentration. All experiments were performed 3 to 4 times independently with 2-3 determinations. Data were analyzed for significant differences amongst genotype groups using ANOVA followed by Tukey’s multiple comparisons test. IC50 and kinetic parameters were calculated using non-linear regression analysis. RESULTS/ANTICIPATED RESULTS: Pravastatin transport in SLCO1B1 variants (*5, *15) was significantly decreased compared to the reference genotype *1a and *1b (Km [µM]: *1a 18.2 ± 0.9; *1b 17.9 ± 3.3; *5 34.2 ± 9.7; *15 34.1 ± 6.1; p≤0.05; Vmax [pmol/mg/min]: *1a 104.9 ± 13.1; *1b 93.7 ± 16.7; *5 44.8 ± 15.9; *15 62.3 ± 22.5; p≤0.05). *1a and *1b were not significantly different with respect to pravastatin transport. Intrinsic clearance was diminished nearly 4 to 5-fold in SLCO1B1 variants compared to reference genotypes (Vmax/Km [µl/min/mg]: *1a 5.8 ± 0.8; *1b 5.7 ± 1.9; *5 1.3 ± 0.2; *15 1.8 ± 0.3; p≤0.01). Pravastatin transport was inhibited by 3αPVA for all genotypes. However, there was more pronounced inhibition in the SLCO1B1 variant genotypes compared to reference genotypes (IC50 [µM]: *1a 15.9 ± 1.9; *1b 18.6 ± 5.7; *5 3.9 ± 2.0; *15 4.4 ± 0.8; p≤0.01; Ki: *1a 15.0 ± 1.8; *1b 17.5 ± 5.4; *5 3.8 ± 2.9; *15 4.3 ± 0.8; p≤0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: In vitro PVA transport is altered according to SLCO1B1 genotype, consistent with previous in vitro and human experience. Our data suggest that the significantly different maximal transport velocity (Vmax) in variant versus non-variant genotypes is consistent with decreased membrane expression of OATP1B1 with the variant c.521T>C allele. However, in contrast to data involving typical model substrates (e.g. estrone-3-sulfate), the PVA binding affinity (Km) was significantly different between variant and non-variant genotypes, consistent with altered binding of the substrate to OATP1B1. Collectively, we conclude that decreased OATP1B1 expression and function in variant genotypes influence altered transport for PVA. Finally, the functional consequences of 3αPVA formation on PVA transport was confirmed in our study. Mechanistically, we confirmed our observation in humans that 3αPVA inhibits OATP1B1 transport. However, this effect is more pronounced in variant genotypes as shown by lower IC50 values compared to the reference genotypes. This highlights another source of variation that must be taken into consideration when trying to optimize the pravastatin dose-exposure relationship in humans.

Determination of Total Protein Concentration in Solution Using Gold Electrode Modified with Silver Nanoparticles

2014 ◽  
Vol 27 (1) ◽  
pp. 253-257 ◽  
Author(s):  
Patrick Marcel Seumo Tchekwagep ◽  
Charles Péguy Nanseu-Njiki ◽  
Emmanuel Ngameni ◽  
Ravi Danielsson ◽  
Thomas Arnebrant ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Total Protein ◽  
Gold Electrode ◽  
Protein Concentration ◽  
Total Protein Concentration

Albumin fraction and measurement of total protein concentration

1993 ◽  
Vol 264 (5) ◽  
pp. H1723-H1726 ◽  
Author(s):  
B. T. Peterson ◽  
R. W. Tate
Keyword(s):  
Total Protein ◽  
Protein Concentration ◽  
Gamma Globulin ◽  
Colorimetric Assay ◽  
Full Range ◽  
Computational Method ◽  
Total Protein Concentration ◽  
Albumin Fraction ◽  
Standard Curve ◽  
Protein Assays

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.

scholarly journals Role of fibronectin under conditions of doxorubicin action

2015 ◽  
Vol 6 (1) ◽  
pp. 17-22
Author(s):  
A. I. Shevtsova ◽  
G. A. Ushakova
Keyword(s):  
Blood Plasma ◽  
Total Protein ◽  
Protein Concentration ◽  
Male Rats ◽  
Antitumor Action ◽  
Total Protein Concentration ◽  
Monospecific Antibodies ◽  
Anthracycline Chemotherapy ◽  
In Blood Plasma

There is no standard as to treatment of anthracycline chemotherapy complications. The reduction of cytotoxic drugs toxicity without weakening of their antitumor action remains relevant. The extracellular matrix which key component is fibronectin is present in all tissues and it continuously undergoes controlled remodeling. So, the purpose of our work was to study the level of fibronectin in the experimental model of doxorubicin-induced cardiomyopathy and effects of this cytostatic and its co-administration with antioxidants of different nature.The level of fibronectin was measured by ELISA using monospecific antibodies against fibronectin (Sigma, USA), secondary anti-IgG labeled with horseradish peroxidase (Sigma, USA) and fibronectin standard (Sigma, USA). The study was conducted on Wistar male rats with weight of 210 ± 50 g which were divided into 4 groups by 8 animals in each group: 1 – control, rats receiving saline i/p; 2 – doxorubicin 1 mg/kg i/p once a week during 4 weeks; 3 – doxorubicin by the same scheme plus 1% 2-oxoglutarate in drinking water during 4 weeks;4 – doxorubicin by the same scheme and korvitin injection 30 min before doxorubicin application once a week during 4 weeks. Obtained data indicate the effect of doxorubicin to decrease in index mass heart in 38% of animals compared to control animals; decrease in total protein concentration by 8% (Р < 0,05) and increase of the level of fibronectin by 67% (P < 0,001) in blood plasma of rats and decrease in the level of fibronectin in the heart extract by 19% (Р < 0,05) under development of doxorubicin-induced cardiotoxicity. Increased fibronectin concentration in blood plasma had strong correlation with decreased total protein concentration in blood (r=0,80) and heart extract (r=0,59) in rats with doxorubicin-induced cardiomiophaty indicating the sensitive reaction of fibronectin to development of metabolic disorders under doxorubicin influence. 

Abstract WP263: Mutagenesis Studies Revealed Minimal Impact of Human A120T Variant of Protease-activated Receptor 4 on Receptor function or Pharmacological Response to a Potent and Selective Antagonist

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Mario F Callejo ◽  
Jeffrey Colin ◽  
Sophie Desmeules ◽  
Chi Sum ◽  
Tao Wang ◽  
...  
Keyword(s):  
African American ◽  
Cell Lines ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Directed Mutagenesis ◽  
Platelet Response ◽  
Functional Responses ◽  
Stroke Treatment

Introduction: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor and a novel target for ischemic stroke treatment. Recent reports suggested a subtle racial difference in platelet responses to submaximal concentrations of PAR4 agonist peptide (PAR4-AP). One of the PAR4 variants, A120T, is more common in African American than white (63% vs. 19%, Edelstein et al., Blood 2014). It was suggested that this variant contributes to the difference in response to PAR4-AP and impacts the in vitro response to YD-3, a PAR4 antagonist. In this study, site-directed mutagenesis was used to evaluate the effect of the A120T variant on PAR4 function and response to a newly discovered potent and selective PAR4 antagonist, UDM-001651. Unlike YD-3, UDM-001651 inhibited thrombin-induced platelet aggregation and prevented thrombosis in a monkey model and thus served as a relevant PAR4 pharmacology tool. Methods: Human PAR4 cDNAs expressing A120 and T120 variants were stably expressed in HEK293 cells. Cell surface expression of PAR4 was analyzed by FACS. Functional responses of cells were evaluated by monitoring calcium mobilization induced by BMS PAR4-AP, which was optimized based on AYPGKF. The potency of UDM-001651 was derived from an 11-point concentration response curve in the calcium assay using PAR4-AP at the EC80 concentration. Transient transfection studies were also performed to confirm the results. Results: Comparable levels of expression and functional responses were observed between A120 and T120 expressing cells. Results from side-by-side comparison between the two cell lines demonstrated no detectable difference in the mean EC50 values (0.42±0.054 versus 0.46±0.051 uM standard error, n=9) in calcium responses to PAR4-AP stimulation. Similarly, the potency of UDM-001651 in the same assay were similar between these two cell lines. Moreover, preliminary clinical results did not show differences in PAR4-mediated platelet response between African-American and white subjects. Conclusion: In contrast to the previous report, the results reported herein from site directed mutagenesis studies indicate that the A120T variant of PAR4 has no apparent impact on calcium signaling in response to agonist stimulation or response to a PAR4 antagonist.

Assay of cerebrospinal fluid protein: a rate biuret method evaluated.

1983 ◽  
Vol 29 (1) ◽  
pp. 126-129 ◽  
Author(s):  
P R Finley ◽  
R J Williams
Keyword(s):  
Cerebrospinal Fluid ◽  
Total Protein ◽  
Protein Concentration ◽  
Protein A ◽  
Colorimetric Method ◽  
Total Protein Concentration ◽  
Reaction Cell ◽  
Biuret Method ◽  
Automated Instrument ◽  
Cerebrospinal Fluid Protein

Abstract We evaluated a rate colorimetric method (Beckman) for measuring total protein in cerebrospinal fluid. The automated instrument we used was Beckman's ASTRA TM. A 100-microL sample of spinal fluid is introduced into the biuret reagent in the reaction cell and the increase in absorbance at 545 nm is monitored for 20.5 s. Solid-state circuits determine the rate of alkaline biuret-protein chelate formation, which is directly proportional to the total protein concentration in the sample. The linear range of measurement is 120 to 7500 mg/L. Day-to-day precision (CV) over the range of 150 to 1200 mg/L ranged from 15.2 to 2.3%. The method was unaffected by radical alteration of the albumin/globulin ratio, but there is a positive interference in the presence of hemoglobin, a suppression in the presence of bilirubin, and no effect by xanthochromia. The method is precise, accurate, rapid, and convenient. The method was compared with the trichloroacetic acid method as performed on the Du Pont aca III, giving a correlation coefficient (r2) of 0.9693. The method is precise, accurate, rapid, and convenient.

PAROTID SECRETION OF PROTEIN IN MAN

1958 ◽  
Vol 36 (10) ◽  
pp. 1001-1008 ◽  
Author(s):  
Marion H. Ferguson ◽  
H. P. Krahn ◽  
J. A. Hildes
Keyword(s):  
Total Protein ◽  
Protein Concentration ◽  
Serum Proteins ◽  
Amylase Activity ◽  
Early Morning ◽  
Total Protein Concentration ◽  
Zone Electrophoresis ◽  
Residual Radioactivity ◽  
Unstimulated Saliva ◽  
Protein Components

In unstimulated saliva, total protein concentration averaged 186 mg per 100 ml and amylase activity 146 units per 100 ml. The protein concentration was lower in the early morning than at midday. After dilute acetic acid stimulation, both total protein concentration and amylase activity were increased but the concentrations were not affected by rates of secretion above 0.1 ml per minute. Unlike protein, the potassium concentration fell with stimulation.Using zone electrophoresis on filter paper, as many as nine protein components were found, none of which corresponded to the serum proteins. The amylase activity was restricted to a component of low mobility which moved to the anode. There were two or three bands containing glycoproteins; all moved towards the cathode. There were qualitative and quantitative differences between stimulated and unstimulated secretions. Saliva collected 2 or 24 hours after a tracer dose of I131 showed less than 1% residual radioactivity after dialysis or treatment with an anion exchange resin, indicating that little if any of the salivary iodide is organically bound.

Effect of protein concentration on the determination of digoxin in serum by fluorescence polarization immunoassay.

1984 ◽  
Vol 30 (11) ◽  
pp. 1826-1829 ◽  
Author(s):  
W H Porter ◽  
V M Haver ◽  
B A Bush
Keyword(s):  
Serum Protein ◽  
Total Protein ◽  
Fluorescence Polarization ◽  
Protein Concentration ◽  
Total Serum Protein ◽  
Total Serum ◽  
Fluorescence Polarization Immunoassay ◽  
Total Protein Concentration ◽  
Protein Pellet

Abstract Determination of digoxin by fluorescence polarization immunoassay (FPIA) with the Abbott "TDx" is significantly influenced by the concentration of total serum protein. Each 10 g/L increase in serum protein results in an 8% decrease in measured digoxin. Studies with [3H]digoxin confirmed that digoxin binds to the protein pellet during the trichloroacetic acid precipitation step before the immunoassay. Serum protein, or equal concentrations of albumin or gamma-globulin, exert an equivalent effect on the apparent digoxin value. Because the total protein concentration of the assay calibrators is low (50 g/L) compared with its reference interval in serum (60-80 g/L), results by FPIA may be expected to be low by an average of 16% (range, 8-24%). Digoxin results by FPIA will be most nearly accurate when the calibrators include a total protein concentration of about 70 g/L. Patients' specimens with abnormally high or low protein content will give falsely high or low results for digoxin.

Sign in / Sign up

Export Citation Format

Share Document