Degradation of oxidative stress-induced denatured albumin in rat liver endothelial cells.

We previously identified conformationally denatured albumin (D2 and D3 albumin) in rats with endotoxicosis (Bito R, Shikano T, and Kawabata H. Biochim Biophys Acta 1646: 100-111, 2003). In the present study, we attempted first to confirm whether the denatured albumins generally increase in conditions of oxidative stress and second to characterize the degradative process of the denatured albumin using primary cultured rat liver endothelial cells. We used five models of oxidative stress, including endotoxicosis, ischemic heart disease, diabetes, acute inflammation, and aging, and found that serum concentrations of D3 albumin correlate with the serum levels of thiobarbituric acid-reactive substance (R = 0.87), whereas the concentrations of D2 albumin are 0.52. Ligand blot analysis showed that the D3 albumin binds to gp18 and gp30, which are known endothelial scavenger receptors for chemically denatured albumin. Primary cultured rat liver endothelial cells degraded the FITC-D3 albumin, and the degradation rate decreased to approximately 60% of control levels in response to anti-gp18 and anti-gp30 antibodies, respectively. An equimolar mixture of these antibodies produced an additive inhibitory effect on both uptake and degradation, resulting in levels approximately 20% those of the control. Furthermore, filipin and digitonin, inhibitors of the caveolae-related endocytic pathway, reduced the FITC-D3 albumin uptake and degradation to <20%. Laser-scanning confocal microscopic observation supported these data regarding the uptake and degradation of D3 albumin. These results indicate that conformationally denatured D3 albumin occurs generally under oxidative stress and is degraded primarily via gp18- and gp30-mediated and caveolae-related endocytosis in liver endothelial cells.

Isolation and characterization of denatured serum albumin from rats with endotoxicosis.

Due to its rapid breakdown in the body, denatured serum albumin has not been identified in biological samples. In this study we attempted to determine whether denatured albumin could be identified in rats with endotoxicosis. Male Wistar rats were injected with lipopolysaccharide (LPS; 5 mg/kg body weight). Plasma albumin concentration decreased to one-third the normal level at 2 days after the injection. By using the purified IgG against the specific epitope of chemically denatured albumin, two immunoreactive plasma proteins (bands D2 and D3) were identified by native PAGE followed by Western blot analysis. The plasma concentration of these two proteins increased significantly at 1 and 1.5 days after LPS injection. Peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/TOF-MS) identified these two proteins as serum albumin. In order to characterize their conformational nature, ion-exchange chromatography was used to isolate D2 and D3 albumins from rats injected with LPS. Far- and near-UV circular dichroism (CD), tryptophan and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence, and proteolytic susceptibility showed conformational alterations in the D2 and D3 albumins as compared with native albumin. These data indicate the presence of denatured albumin in circulating rat plasma, and this fact may contribute to a further understanding of the molecular mechanisms of albumin breakdown in physiological and pathophysiological conditions.