Abstract
Bovine serum albumin (BSA) is a commonly used model protein in the development of pharmaceutical formulations. In order to assay its release from various dosage forms, either the bicinchoninic acid (BCA) assay or a more specific size-exclusion high performance liquid chromatography (SE-HPLC) method are commonly employed. However, these can give erroneous results in the presence of some commonly used pharmaceutical excipients. We therefore investigated the ability of these methods to accurately determine BSA concentrations in pharmaceutical formulations that also contained various polymers and compared them with a new reverse-phase (RP)-HPLC technique.
We found that the RP-HPLC technique was the most suitable method. It gave a linear response in the range of 0.5–100 μg/ml with a correlation co-efficient of 0.9999, a limit of detection of 0.11 μg/ml and quantification of 0.33 μg/ml. The performed ‘t’-test for the estimated and theoretical concentrations indicated no significant difference between them providing the accuracy. Low % relative standard deviation values (0.8–1.39%) indicate the precision of the method. Furthermore, the method was used to quantify in vitro BSA release from polymeric freeze-dried formulations.
Manish Umrethiaa, Vicky L. Kett, a, , Gavin P. Andrewsa, R. Karl Malcolma and A. David Woolfsona
a School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK
Selection of an analytical method for evaluating bovine serum albumin concentrations in pharmaceutical polymeric formulations
The redox state of human serum albumin in eye diseases with and without complications
ABSTRACT
Purpose: To investigate the redox state of human serum albumin concerning cysteine-34 as a possible systemic redox marker in patients with different eye diseases with and without complications and with consideration of possible effects of age.
Methods: Cataract (CAT), glaucoma, age-related macular degeneration (AMD), diabetes mellitus (DM), diabetic retinopathy and hypertension were the pathologies investigated. Albumin redox state concerning cysteine-34 was measured by high-performance liquid chromatography with fluorescence detection. The separation gives three fractions: the fully reduced form containing a thiol group, the disulphide form and a higher oxidized form. Statistical analysis was done by Student's t-test, analysis of variance and stepwise regression analysis.
Results: Albumin as a systemic marker for oxidative stress was shifted to a more oxidized state by DM. An even stronger shift to the oxidized form was observed in patients with proliferative diabetic retinopathy. Notably, these effects were independent from age. In contrast, CAT and AMD had no influence on serum albumin redox state.
Conclusion: Serum albumin is not shifted to more oxidized forms by localized oxidative stress, but it is in systemic diseases like DM.
Karl Oettl 1 , Gilbert Reibnegger 1 and Otto Schmut 2
1 Institute of Physiological Chemistry, Center for Physiological Medicine, Medical University of Graz, Graz, Austria
2 University Eye Hospital, Medical University of Graz, Graz, Austria
Purpose: To investigate the redox state of human serum albumin concerning cysteine-34 as a possible systemic redox marker in patients with different eye diseases with and without complications and with consideration of possible effects of age.
Methods: Cataract (CAT), glaucoma, age-related macular degeneration (AMD), diabetes mellitus (DM), diabetic retinopathy and hypertension were the pathologies investigated. Albumin redox state concerning cysteine-34 was measured by high-performance liquid chromatography with fluorescence detection. The separation gives three fractions: the fully reduced form containing a thiol group, the disulphide form and a higher oxidized form. Statistical analysis was done by Student's t-test, analysis of variance and stepwise regression analysis.
Results: Albumin as a systemic marker for oxidative stress was shifted to a more oxidized state by DM. An even stronger shift to the oxidized form was observed in patients with proliferative diabetic retinopathy. Notably, these effects were independent from age. In contrast, CAT and AMD had no influence on serum albumin redox state.
Conclusion: Serum albumin is not shifted to more oxidized forms by localized oxidative stress, but it is in systemic diseases like DM.
Karl Oettl 1 , Gilbert Reibnegger 1 and Otto Schmut 2
1 Institute of Physiological Chemistry, Center for Physiological Medicine, Medical University of Graz, Graz, Austria
2 University Eye Hospital, Medical University of Graz, Graz, Austria
Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin
Journal of Inorganic Biochemistry
Volume 103, Issue 7, July 2009, Pages 1005-1013
Abstract
Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS).
Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 104 M−1, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.
Magdalena Sokołowskaa, Małgorzata Wszelaka-Rylikb, Jarosław Poznańskib, c and Wojciech Balc, d, ,
aDepartment of Hygiene, Wroclaw Medical University, Mikulicza-Radeckiego 7, 50-368 Wroclaw, Poland
bInstitute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
cInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
dCentral Institute for Labour Protection – National Research Institute, Czerniakowska 16, 00-701 Warsaw, Poland
Volume 103, Issue 7, July 2009, Pages 1005-1013
Abstract
Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS).
Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 104 M−1, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.
Magdalena Sokołowskaa, Małgorzata Wszelaka-Rylikb, Jarosław Poznańskib, c and Wojciech Balc, d, ,
aDepartment of Hygiene, Wroclaw Medical University, Mikulicza-Radeckiego 7, 50-368 Wroclaw, Poland
bInstitute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
cInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
dCentral Institute for Labour Protection – National Research Institute, Czerniakowska 16, 00-701 Warsaw, Poland
Oxidative Modification of Albumin in Predialysis, Hemodialysis, and Peritoneal Dialysis Patients
Mitrogianni Z, Barbouti A, Galaris D, Siamopoulos KC.
Department of Nephrology, Medical School, University of Ioannina, Ioannina, Greece.
Background/Aims: Oxidative damage has been reported to be involved in the pathophysiology of chronic kidney disease (CKD) as well as in the pathogenesis of cardiovascular complications of CKD patients. The aim of the present investigation was to evaluate the levels of plasma carbonyl formation, a sensitive marker of enhanced oxidative stress in predialysis, hemodialysis (HD) and peritoneal dialysis (PD) patients. Methods: Plasma samples from 20 apparently healthy control individuals and 127 CKD (stages 2, 3, 4, HD and PD) patients were evaluated by Western blot analysis for the estimation of the levels of protein carbonyl formation.
Results: Albumin represented the main plasma carbonylated protein. Increasing carbonylation of albumin was detected along with the severity of CKD, reaching significance at stages 3 and 4 (p < 0.01, compared to healthy controls). The carbonylation of albumin was even higher in the plasma of HD patients (p < 0.001), while in PD patients it was not statistically significant compared to controls (p = 0.224).
Conclusions: The data presented in this work indicate that oxidative stress in CKD patients gradually increased during the development of the disease. This stress is probably intensified during HD, but not in PD subjects.
Department of Nephrology, Medical School, University of Ioannina, Ioannina, Greece.
Background/Aims: Oxidative damage has been reported to be involved in the pathophysiology of chronic kidney disease (CKD) as well as in the pathogenesis of cardiovascular complications of CKD patients. The aim of the present investigation was to evaluate the levels of plasma carbonyl formation, a sensitive marker of enhanced oxidative stress in predialysis, hemodialysis (HD) and peritoneal dialysis (PD) patients. Methods: Plasma samples from 20 apparently healthy control individuals and 127 CKD (stages 2, 3, 4, HD and PD) patients were evaluated by Western blot analysis for the estimation of the levels of protein carbonyl formation.
Results: Albumin represented the main plasma carbonylated protein. Increasing carbonylation of albumin was detected along with the severity of CKD, reaching significance at stages 3 and 4 (p < 0.01, compared to healthy controls). The carbonylation of albumin was even higher in the plasma of HD patients (p < 0.001), while in PD patients it was not statistically significant compared to controls (p = 0.224).
Conclusions: The data presented in this work indicate that oxidative stress in CKD patients gradually increased during the development of the disease. This stress is probably intensified during HD, but not in PD subjects.
Serum Albumin may not correlate with weight status
Anorexia nervosa is a difficult disease to treat effectively. Inpatient treatment in facilities with specialized expertise heightens the chance for success. Patients with the most severe degrees of anorexia nervosa are especially in need of hospitalization. Authorization from insurers can be a barrier to admitting these patients to reputable treatment facilities.
Therefore, familiarity with accurate markers of disease severity is important to understand in order to effectively advocate for these patients. Albumin , a commonly used marker for nutritional status is surprisingly normal even in patients with severe anorexia nervosa. Understanding that albumin levels do not correlate with the severity of anorexia nervosa is an important lesson to understand in the process of facilitating the most effective care settings for patients with severe anorexia nervosa
University of Colorado Health Sciences Center and Internal Medicine, Denver Health, Denver, Colorado, USA
Therefore, familiarity with accurate markers of disease severity is important to understand in order to effectively advocate for these patients. Albumin , a commonly used marker for nutritional status is surprisingly normal even in patients with severe anorexia nervosa. Understanding that albumin levels do not correlate with the severity of anorexia nervosa is an important lesson to understand in the process of facilitating the most effective care settings for patients with severe anorexia nervosa
University of Colorado Health Sciences Center and Internal Medicine, Denver Health, Denver, Colorado, USA
Three new Cardiovascular Risk Markers -Do They Improve Risk Prediction and Influence Treatment
Urine Albumin/Creatinine Ratio, High Sensitivity C-Reactive Protein and N-Terminal Pro Brain Natriuretic Peptide -Three new Cardiovascular Risk Markers -Do They Improve Risk Prediction and Influence Treatment
In order to prioritize limited health resources in a time of increasing demands optimal cardiovascular risk stratification is essential. We tested the additive prognostic value of 3 relatively new, but established cardiovascular risk markers: N-terminal pro brain natriuretic peptide (Nt-proBNP), related to hemodynamic cardiovascular risk factors, high sensitivity C-reactive protein (hsCRP), related to metabolic cardiovascular risk factors and urine albumin/creatinine ratio (UACR), related to hemodynamic as well as metabolic risk factors. In healthy subjects with a 10-year risk of cardiovascular death lower than 5% based on HeartScore and therefore not eligible for primary prevention, the actual 10-year risk of cardiovascular death exceeded 5% in a small subgroup of subjects with UACR higher than the 95-percentile of approximately 1.6 mg/mmol. Combined use of high UACR or high hsCRP identified a larger subgroup of 16% with high cardiovascular risk in which primary prevention may be advised despite low-moderate cardiovascular risk based on HeartScore. Furthermore, combined use of high UACR or high Nt-proBNP in subjects with known cardiovascular disease or diabetes identified a large subgroup of 48% with extremely high cardiovascular risk who should be referred for specialist care to optimize treatment.
Olsen MH, Sehestedt T, Lyngbæk S, Hansen TW, Rasmussen S, Wachtell K, Torp-Pedersen C, Hildebrandt PR, Ibsen H.
Research Center for Prevention and Health, Gentofte University Hospital, Holbaek Hospital, Denmark
In order to prioritize limited health resources in a time of increasing demands optimal cardiovascular risk stratification is essential. We tested the additive prognostic value of 3 relatively new, but established cardiovascular risk markers: N-terminal pro brain natriuretic peptide (Nt-proBNP), related to hemodynamic cardiovascular risk factors, high sensitivity C-reactive protein (hsCRP), related to metabolic cardiovascular risk factors and urine albumin/creatinine ratio (UACR), related to hemodynamic as well as metabolic risk factors. In healthy subjects with a 10-year risk of cardiovascular death lower than 5% based on HeartScore and therefore not eligible for primary prevention, the actual 10-year risk of cardiovascular death exceeded 5% in a small subgroup of subjects with UACR higher than the 95-percentile of approximately 1.6 mg/mmol. Combined use of high UACR or high hsCRP identified a larger subgroup of 16% with high cardiovascular risk in which primary prevention may be advised despite low-moderate cardiovascular risk based on HeartScore. Furthermore, combined use of high UACR or high Nt-proBNP in subjects with known cardiovascular disease or diabetes identified a large subgroup of 48% with extremely high cardiovascular risk who should be referred for specialist care to optimize treatment.
Olsen MH, Sehestedt T, Lyngbæk S, Hansen TW, Rasmussen S, Wachtell K, Torp-Pedersen C, Hildebrandt PR, Ibsen H.
Research Center for Prevention and Health, Gentofte University Hospital, Holbaek Hospital, Denmark
Interaction of perfluorooctanoic acid with human serum albumin.
ABSTRACT: BACKGROUND: Recently, perfluorooctanoic acid (PFOA) has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA) is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands.
RESULTS: The interaction of PFOA with Human serum Albumin was investigated in the normal physiological condition by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC) and circular dichroism (CD). The non-covalent interaction is resulted from hydrogen bond, van der Waals force e and hydrophobic stack. PFOA binding to Human Serum Albumin accorded with two-step binding model with the saturation binding numbers of PFOA, only 1 in the hydrophobic intracavity of Human Serum Albumin and 12 on the exposed outer surface. The interaction of PFOA with Human Serum Albumin is spontaneous and results in change of HSA conformation. The possible binding sites were speculated.
CONCLUSION: The present work suggested a characterization method for the intermolecular weak interaction. It is potentially useful for elucidating the toxigenicity of perfluorochemicals when combined w: BMC Struct Biol. 2009 May 14;9(1):31.ith biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.
RESULTS: The interaction of PFOA with Human serum Albumin was investigated in the normal physiological condition by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC) and circular dichroism (CD). The non-covalent interaction is resulted from hydrogen bond, van der Waals force e and hydrophobic stack. PFOA binding to Human Serum Albumin accorded with two-step binding model with the saturation binding numbers of PFOA, only 1 in the hydrophobic intracavity of Human Serum Albumin and 12 on the exposed outer surface. The interaction of PFOA with Human Serum Albumin is spontaneous and results in change of HSA conformation. The possible binding sites were speculated.
CONCLUSION: The present work suggested a characterization method for the intermolecular weak interaction. It is potentially useful for elucidating the toxigenicity of perfluorochemicals when combined w: BMC Struct Biol. 2009 May 14;9(1):31.ith biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.
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