Showing papers on "Serum albumin published in 2005"

Interpretation of Protein Adsorption: Surface-Induced Conformational Changes

Abstract: Protein adhesion plays a major role in determining the biocompatibility of materials. The first stage of implant integration is the adhesion of protein followed by cell attachment. Surface modification of implants (surface chemistry and topography) to induce and control protein and cell adhesion is currently of great interest. This communication presents data on protein adsorption (bovine serum albumin and fibrinogen) onto model hydrophobic (CH3) and hydrophilic (OH) surfaces, investigated using a quartz crystal microbalance (QCM) and grazing angle infrared spectroscopy. Our data suggest that albumin undergoes adsorption via a single step whereas fibrinogen adsorption is a more complex, multistage process. Albumin has a stronger affinity toward the CH3 compared to OH terminated surface. In contrast, fibrinogen adheres more rapidly to both surfaces, having a slightly higher affinity toward the hydrophobic surface. Conformational assessment of the adsorbed proteins by grazing angle infrared spectroscopy (GA...

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.

Abstract: Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

Revisiting mortality predictability of serum albumin in the dialysis population: time dependency, longitudinal changes and population-attributable fraction

Abstract: Background. Hypoalbuminaemia is a marker of malnutrition–inflammation complex syndrome (MICS) and a strong predictor of cardiovascular (CV) death in maintenance haemodialysis (MHD) patients. However, serum albumin may change over time. Hence, its time-varying associations with outcome may be different. Methods. Associations between 3-month averaged serum albumin levels, measured in a single laboratory using bromocresol green, and CV mortality were studied longitudinally in a 2-year cohort of 58 058 MHD patients. Mortality predictability of fixed baseline and trimonthly-varying serum albumin concentrations were compared. Results. Hazard ratios (HRs) of CV death strictly increased across decrements of baseline serum albumin, whereas the HR for time-varying serum albumin decrements below 3.8 g/dl did not differ. A drop in serum albumin in the first 6 months was associated with increasing all-cause and CV death risks in the subsequent 18 months, while a rise in serum albumin was a predictor of better survival independent of baseline serum albumin. The multivariate adjusted population-attributable fraction of death due to baseline serum albumin 3.8 g/dl might reduce the number of MHD deaths in the USA by � 10 000 annually. Nutritional interventions examining benefits of increasing serum albumin in MHD patients are urgently needed.

Albumin depletion of human plasma also removes low abundance proteins including the cytokines.

Abstract: The use of proteomics for efficient, accurate, and complete analysis of clinical samples poses a variety of technical challenges. The presence of higher abundance proteins in the plasma, such as albumin, may mask the detection of lower abundance proteins such as the cytokines. Methods have been proposed to deplete the sample of these higher abundance proteins to facilitate detection of those with lower abundance. In this study, a commercially available albumin depletion kit was used to determine if removal of albumin would measurably reduce detection of lower abundance cytokine proteins in human plasma. The Montage Albumin Deplete Kit (Millipore) was used to deplete albumin from LPS-stimulated whole blood from 15 normal human donors. Albumin depletion was measured using the BCG reagent and SDS-PAGE, and cytokine recovery was determined by a microassay immunoassay that measures both pro- and anti-inflammatory cytokines. Average albumin depletion from the samples was 72%. However, several cytokines were also significantly reduced when the albumin was removed from the plasma. Additionally, there was a variable reduction in cytokine recovery from a known mixture of cytokines in a minimal amount of plasma that were loaded onto the columns. These data demonstrate that there may be a non-specific loss of cytokines following albumin depletion, which may confound subsequent proteomic analysis.

A modified protein precipitation procedure for efficient removal of albumin from serum

Abstract: Proteomic analysis of sera and the quest for identifying serum proteins as disease markers have often been hampered by the predominance of several highly abundant proteins including albumin and immunoglobulins. Prior albumin depletion so as to enrich for otherwise undetectable serum components is therefore a prerequisite in mining the serum proteome. In the course of evaluating several available methods and commercial kits, we have been able to refine the albumin depletion protocols and establish a modified albumin removal method using trichloroacetic acid (TCA)/acetone. Changes in major protein bands were monitored by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and used as the first screening strategy to evaluate and optimize for the precipitation experimental conditions. Our method showed better performance in efficiency, specificity, and costs in comparison with two commercially available albumin removal kits, and provides a simple pre-fractionation step for the proteomic analysis of serum biomarkers. Albumin isolated by the modified method is in the native state. Our method may offer a rapid method for purifying serum albumin in large scale.

Heterogeneity and oxidation status of commercial human albumin preparations in clinical use

Abstract: Objective:Human serum albumin is indicated for the treatment of shock, acute restoration of blood volume, and in hypoalbuminemia. Conflicting reports are found in the literature for the clinical safety and efficacy of human serum albumin administration to critically ill patients. We sought to analyz

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice.

Abstract: Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN-γ from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 μg/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10–14 and 17–21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN-γ from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19–57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4+ and CD8+ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.

Serum creatinine is a poor marker of GFR in nephrotic syndrome.

Abstract: Background. In daily clinical practice creatinine clearance is used as marker of glomerular filtration rate (GFR). As a result of the tubular secretion process endogenous creatinine clearance (ECC) overestimates glomerular filtration rate, particularly in patients with impaired renal function. It has been suggested that the tubular handling of creatinine is altered in patients with a nephrotic syndrome. Methods. Inulin clearance (GFR) and creatinine clearance (ECC) have been simultaneously measured in a cohort of 42 patients with proteinuria and 45 healthy controls. The clearance of creatinine by tubular secretion (TScreat) can be estimated by ECC–GFR. TScreat was calculated in both groups. Regression analysis was performed to identify factors that independently influence tubular creatinine secretion. Results. The mean age (±SD) of the patients was 41±13 years, serum albumin 26±9 g/l, median (IQR) proteinuria 4.5 (3.6–8.2) g/10 mmol creatinine, serum creatinine 103 (84–143)mmol/l, ECC 85 (69–118) ml/ min/1.73 m 2 , and GFR 54 (36–83) ml/min/1.73 m 2 . Median TScreat amounted to 29 (21–36) ml/min/ 1.73 m 2 . In the healthy controls serum creatinine was 75 (70–81)mmol/l, ECC 118 (109–125) ml/min/ 1.73 m 2 , GFR 106 (102–115) ml/min/1.73 m 2 , and TScreat 11 (3.5–19) ml/min/1.73 m 2 . By regression analysis serum albumin was identified as an independent predictor of tubular creatinine secretion. We divided the patients in two subgroups based on serum albumin levels. TScreat was 24 (14–29) ml/min/1.73 m 2 in patients with serum albumin levels >25.8 g/l, and 36 (28–54) ml/min/1.73 m 2 in patients with serum

Albumin administration--what is the evidence of clinical benefit? A systematic review of randomized controlled trials.

Abstract: SummaryBackground and objective: The advantages of albumin over less costly alternative fluids continue to be debated. Meta-analyses focusing on survival have been inconclusive, and other clinically relevant end-points have not been systematically addressed. We sought to determine whether albumin confers significant clinical benefit in acute illness compared with other fluid regimens.Methods: Database searches (MEDLINE, EMBASE, Cochrane Library) and other methods were used to identify randomized controlled trials comparing albumin with crystalloid, artificial colloid, no albumin or lower-dose albumin. Major findings for all end-points were extracted and summarized. A quantitative meta-analysis was not attempted.Results: Seventy-nine randomized trials with a total of 4755 patients were included. No significant treatment effects were detectable in 20/79 (25%) trials. In cardiac surgery, albumin administration resulted in lower fluid requirements, higher colloid oncotic pressure, reduced pulmonary oedema with respiratory impairment and greater haemodilution compared with crystalloid and hydroxyethylstarch increased postoperative bleeding. In non-cardiac surgery, fluid requirements, and pulmonary and intestinal oedema were decreased by albumin compared with crystalloid. In hypoalbuminaemia, higher doses of albumin reduced morbidity. In ascites, albumin reduced haemodynamic derangements, morbidity and length of stay and improved survival after spontaneous bacterial peritonitis. In sepsis, albumin decreased pulmonary oedema and respiratory dysfunction compared with crystalloid, while hydroxyethylstarch induced abnormalities of haemostasis. Complications were lowered by albumin compared with crystalloid in burn patients. Albumin-containing therapeutic regimens improved outcomes after brain injury.Conclusions: Albumin can bestow benefit in diverse clinical settings. Further trials are warranted to delineate optimal fluid regimens, in particular indications.

Attenuation of endotoxin-induced acute lung injury by the Rho-associated kinase inhibitor, Y-27632.

Abstract: A small GTPase, Rho, plays key roles in cell adhesion, motility, and contraction after stimulation. Among Rho effectors isolated, the family of Rho-associated coiled-coil–forming protein kinases (ROCK) is implicated in Rho-mediated cell adhesion and smooth muscle contraction. The effect of a specific inhibitor of ROCK, Y-27632, was evaluated in a murine model of acute lung injury induced by intravenous injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]). Lung edema was evaluated by measuring extravascular leakage of radio-labeled serum albumin, and neutrophil emigration into the lung parenchyma by morphometric observation and measuring myeloperoxidase activity. Pretreatment with Y-27632 attenuated both lung edema and neutrophil emigration after LPS. We also measured albumin transfer through cultured endothelial cell monolayers on a porous filter. Tumor necrosis factor-α significantly increased albumin transfer, which was attenuated by pretreatment with Y-27632. Fluorescence microscopy revea...

Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3.

Abstract: Suppression of angiogenesis during diabetes is a recognized phenomenon but is less appreciated within the context of diabetic retinopathy. The current study has investigated regulation of retinal angiogenesis by diabetic serum and determined if advanced glycation end products (AGEs) could modulate this response, possibly via AGE-receptor interactions. A novel in vitro model of retinal angiogenesis was developed and the ability of diabetic sera to regulate this process was quantified. AGE-modified serum albumin was prepared according to a range of protocols, and these were also analyzed along with neutralization of the AGE receptors galectin-3 and RAGE. Retinal ischemia and neovascularization were also studied in a murine model of oxygen-induced proliferative retinopathy (OIR) in wild-type and galectin-3 knockout mice (gal3 / ) after perfusion of preformed AGEs. Serum from nondiabetic patients showed significantly more angiogenic potential than diabetic serum (P < 0.0001) and within the diabetic group, poor glycemic control resulted in more AGEs but less angiogenic potential than tight control (P < 0.01). AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (P < 0.001), and AGE receptor neutralization significantly reversed the AGE-mediated suppression of angiogenesis (P < 0.01). AGE-treated wild-type mice showed a significant increase in inner retinal ischemia and a reduction in neovascularization compared with non-AGE controls (P < 0.001). However, ablation of galectin-3 abolished the AGE-mediated increase in retinal ischemia and restored the neovascular response to that seen in controls. The data suggest a significant suppression of angiogenesis by the retinal microvasculature during diabetes and implicate AGEs and AGEreceptor interactions in its causation. Diabetes 53: 785‐794, 2005

Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins.

Abstract: New negatively charged water-soluble bacteriochlorophyll (Bchl) derivatives were developed in our laboratory for vascular-targeted photodynamic therapy (VTP). Here we focused on the synthesis, characterization and interaction of the new candidates with serum proteins and particularly on the effect of serum albumin on the photocytotoxicity of WST11, a representative compound of the new derivatives. Using several approaches, we found that aminolysis of the isocyclic ring with negatively charged residues markedly increases the hydrophilicity of the Bchl sensitizers, decreases their self-association constant and selectively increases their affinity to serum albumin, compared with other serum proteins. The photocytotoxicity of the new candidates in endothelial cell culture largely depends on the concentration of the serum albumin. Importantly, after incubation with physiological concentrations of serum albumin (500–600 μM), WST11 was found to be poorly photocytotoxic (>80% endothelial cell survival in cell cultures). However, in a recent publication (Mazor, O. et al. [2005] Photochem. Photobiol. 81, 342–351) we showed that VTP of M2R melanoma xenografts with a similar WST11 concentration resulted in ∼100% tumor flattening and >70% cure rate. We therefore propose that the two studies collectively suggest that the antitumor activity of WST11 and probably of other similar candidates does not depend on direct photointoxication of individual endothelial cells but on the vascular tissue response to the VTP insult.

Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

Abstract: The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems.

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two‐dimensional gel electrophoresis

Abstract: Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.

Serum albumin and muscle strength: a longitudinal study in older men and women

Abstract: OBJECTIVES: To examine whether low serum albumin is associated with low muscle strength and future decline in muscle strength in community-dwelling older men and women. DESIGN: Population-based cohort study. SETTING: The Longitudinal Aging Study Amsterdam. PARTICIPANTS: Six hundred seventy-six women and 644 men aged 65 to 88. MEASUREMENTS: Serum albumin was determined at baseline. Muscle strength was assessed using grip strength at baseline, after 3 (n 51,009), and 6 (n 5741) years. The outcomes were continuous baseline muscle strength, 3- and 6-year change in muscle strength, and a dichotomous indicator for substantial decline (a decrease if � 1 standard deviations for women 511kg, for men 512kg) in muscle strength. RESULTS: Mean serum albumin concentration � standard deviation was 45.0 � 3.3g/L for women and 45.2 � 3.2g/L for men. At baseline, adjusting for age, lifestyle factors, and chronic conditions, lower serum albumin was cross-sectionally associated with weaker muscle strength (Po.001) in women and men. After 3 years of follow-up, mean decline in muscle strength was � 5.6 � 10.9kg in women and � 9.6 � 11.9kg in men. After adjustment for potential confounders, lower serum albumin was associated with muscle strength decline over 3 years (Po.01) in women and men (b 50.57, standard error (SE) 5 0.18; b 50.37, SE 50.16, respectively). Lower serum albumin was also associated with substantial decline in muscle strength in women (per unit albumin (g/L) adjusted odds ratio (OR) 51.14, one-sided 95% confidence limit (CL) 51.07) and men (per unit albumin (g/L) adjusted OR 51.14, 95% CL 51.08). Similar but slightly weaker associations were found between serum albumin and 6-year change in muscle strength (Po.05). CONCLUSION: These results suggest that low serum albumin, even within the normal range, is independently associated with weaker muscle strength and future decline in muscle strength in older women and men. J Am Geriatr Soc 53:1331–1338, 2005.