Development of an HPLC assay to study the effect of endogenous and exogenous substances on heat-induced aggregation of human serum albumin.

Abstract

The mechanism of action of non-steroidal anti-inflammatory drugs which are used in high doses in chronic inflammatory conditions is not clearly understood. Their known protein-stabilizing properties could play a significant role. The inhibition of cyclooxygenase may not be essential for their anti-rheumatic activity, since compounds with strong anti-denaturant properties and devoid of anti-inflammatory activity were shown to be effective in an experimental model of rheumatoid arthritis. Hence, to develop new anti-rheumatic drugs it is essential that a simple in vitro method to evaluate the anti-denaturant activity of endogenous and exogenous compounds is available. We developed a new assay, using gel permeation high performance liquid chromatography, to study the effect of endogenous and exogenous compounds on heat-induced aggregation of human serum albumin in conditions in which protein precipitation does not occur. Non-steroidal anti-inflammatory drugs like diclofenac, ibuprofen and naproxen inhibited the aggregation of albumin at low concentrations (EC50 10(-4)-10(-5) mol/l) comparable to those active in a classical turbidimetric method, whereas the effect of weak stabilizers, like sodium cloride and formic, fumaric, maleic, malonic, and succinic acid (EC50 10(-1)-10(-2) mol/l in the Mizushima test) was not detectable. Furthermore, the HPLC assay allowed the examination of a number of coloured substances, including bilirubin, which appeared to be a strong stabilizer of its physiological carrier, albumin. These data could be clinically relevant, since the drugs examined are used at very high doses in rheumatoid arthritis and related conditions, with plasma levels that could cause significant stabilization of serum albumin and perhaps other proteins.