Date Published: July 25, 2016
Publisher: Public Library of Science
Author(s): Regina Michelis, Shifra Sela, Teuta Zeitun, Ronit Geron, Batya Kristal, Jaap A. Joles.
In clinical states associated with systemic oxidative stress (OS) and inflammation such as chronic kidney disease (CKD), oxidative modifications of serum albumin impair its quantification, resulting in apparent hypoalbuminemia. As the maintenance of oncotic pressure/colloid osmotic pressure (COP) is a major function of albumin, this study examined the impact of albumin oxidation on COP, both in-vivo and in-vitro.
Patients with proteinuria and patients on chronic hemodialysis (HD) with systemic inflammation and OS were enrolled. Blood samples were collected from 134 subjects: 32 healthy controls (HC), proteinuric patients with high (n = 17) and low (n = 31) systemic inflammation and from 54 patients on chronic hemodialysis (HD) with the highest levels of OS and inflammation.
In-vitro oxidized albumin showed significantly higher COP values than non-oxidized albumin at identical albumin levels. In vivo, in hypoalbuminemic HD patients with the highest OS and inflammation, COP values were also higher than expected for the low albumin levels. The contribution to COP by other prevalent plasma proteins, such as fibrinogen and immunoglobulins was negligible.
Albumin is the most abundant protein in human plasma with remarkably diverse functions including antioxidant activity, buffering properties, binding and transport capacities for numerous substances (free fatty acids, various ions, NO, bilirubin, peptides, uremic toxins and drugs). Physiologically, maintenance of oncotic pressure/colloid osmotic pressure (COP) is considered its major function as it controls the distribution of extracellular fluid between the vascular and extra-vascular compartments [1,2]. Albumin is predominantly an interstitial protein with only 40% of its total amount in the intravascular fluid [1–3]. Although albumin accounts for 50–60% of the plasma protein mass, it provides 75–80% of COP, due to its relatively low molecular mass (~67KDa, van’t Hoff law) . Its negative charge also plays a role in COP maintenance, by attracting cations such as sodium (Na+) and causing water molecules to shift across the semi-permeable capillary membrane into the intravascular space (Gibbs-Donnan effect) .
All chemicals and antibodies were obtained from SIGMA (St. Louis, MO, USA), unless specified otherwise.
For albumin purification from sera, Cibacron Blue 3GA Agarose (CB3GA) was inserted into a SigmaPrep spin column and washed four times by additions of 10mM Tris pH 8.0 and short centrifugations (8,000g, 10s). Serum (150μl) was added to the washed CB3GA, incubated at r.t. for 10 min and centrifuged (12,000g, 1 min). This step was repeated. The twice depleted serum was discarded and elution buffer (150μl, 10mM Tris pH 8.0 and 1.5M NaCl) was added to the column. After 10 min incubation the albumin samples were eluted by centrifugation (12,000g, 1 min) into a new collection tube. To deplete immunoglobulin contaminations, 100μl of the eluted albumin sample were incubated (with shaking) for 3hrs at 4°C with 20μl (washed and diluted into 900μl H2O) of protein A/G Ultralink Resin (Thermo Scientific, USA). The resin bound immunoglobulins were discarded by centrifugation. The supernatant was concentrated by SPEEDVAC centrifugation (approx. 3h).
Albumin levels were similar in proteinuric and HD groups but COP values in HD were significantly higher (Table 1). Albumin correlated with the index values of all subjects (Fig 1A insert). Albumin also correlated with COP when analyzed in all subjects, and in each group separately (Fig 1A and 1B). However, the regression lines in the HD group and in the proteinuria high inflammation group were clearly elevated compared to the regression lines in the proteinuria low inflammation and HC groups (Fig 1B). Partial depletion of albumin from HC sera, resulted in HC sera with decreased albumin levels, comprising a group of “hypoalbuminemic HC”, which enabled the comparison of COP values with those of hypoalbuminemic patients (<3.8g/dl). The COP values in these "hypoalbuminemic HC" sera were as low as in the proteinuria groups (Fig 1B). When compared to these albumin-depleted HC, serum COP was 5.1 and 2.9 mmHg higher in the HD and proteinuria patients with inflammation respectively, comprising an "oncotic gap". In order to clarify the significance of these high COP values, and to examine the role of albumin modifications in this observation, two further types of experiments were performed: (a) COP measurements of in-vitro oxidized albumin; and (b) measurements of COP in albumin purified from patients' sera. This study highlights the possibly misleading decrease in albumin concentrations, when measured by BCG, which complicates the interpretation of hypoalbuminemia in epidemiological and clinical studies. In patients with varying degrees of inflammation and oxidative stress such as proteinurics and patients on chronic HD therapy, the albumin detection efficacy of the BCG assay is greatly decreased. Consequently, the measured COP values are higher than expected for these "hypoalbuminemic" patients. The assumption that serum albumin levels are higher than measured was supported by COP measurements, used to indirectly assess albumin concentrations, in two types of experiments: in purified albumin from HD and HC subjects and in experiments where albumin was oxidized in-vitro. Source: http://doi.org/10.1371/journal.pone.0159839