Analysis of the physicochemical interactions between Clostridium difficiletoxins and cholestyramine using liquid chromatography with post-column derivatization

A potential therapy for antibiotic-associated pseudomembranous colitis is t o bind Clostridium difficile toxins A and B using cholestyramine, a hydroph obic anion exchange medium. Frontal analysis in isotonic phosphate buffer w as studied using post-column derivatization with o-phthalaldehyde, which ga ve a highly sensitive (greater than or equal to 30 ng) flow-through analysi s. Following load (1.5-3.0 mug toxin/3.6 mg), toxin A was bound at a slight ly higher capacity than B, due to slower kinetics. A salt gradient eluted r oughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, he nce toxin A showed weaker electrostatic affinity. The remainder of toxin A (65%) and some of toxin B (10% out of 50%) were eluted using a subsequent g radient to 60% acetonitrile in normal saline, which measured predominantly hydrophobic binding. Low and high affinity populations of both toxins were observed. Glycocholic acid or amino acids were competitive binders, althoug h these components had little effect on the toxin A population bound primar ily through ionic interactions. Competitive protein constituents in hamster cecal contents were also profiled. These results help to explain the varia ble clinical response in using cholestyramine to treat colitis. Using quate rnary amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a tr end for increased binding at higher pH was observed, especially for toxin A . Binding to strong cation exchange resins (sulfonate-PHM) was not observed . A range of reversed phase media retained both toxins, although recovery w as very poor relative to protein standards. Size exclusion chromatography w ith light scattering detection showed that toxin B exists in different aggr egation states, while toxin A remains monomeric. (C) 2001 Elsevier Science B.V. All rights reserved.