A potential
therapy for
antibiotic-associated
pseudomembranous colitis is to bind Clostridium difficile toxins A and B using
cholestyramine, a hydrophobic
anion exchange medium. Frontal analysis in isotonic
phosphate buffer was studied using post-column derivatization with
o-phthalaldehyde, which gave a highly sensitive (> or =30 ng) flow-through analysis. Following load (1.5-3.0 microg toxin/3.6 mg), toxin A was bound at a slightly higher capacity than B, due to slower kinetics. A
salt gradient eluted roughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, hence 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 gradient 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, although these components had little effect on the toxin A population bound primarily through ionic interactions. Competitive
protein constituents in hamster cecal contents were also profiled. These results help to explain the variable clinical response in using
cholestyramine to treat
colitis. Using quaternary
amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a trend 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 was very poor relative to
protein standards. Size exclusion chromatography with light scattering detection showed that toxin B exists in different aggregation states, while toxin A remains monomeric.