Studies on the reactivity of acyl glucuronides--III. Glucuronide-derived adducts of valproic acid and plasma protein and anti-adduct antibodies in humans.

The major metabolite of the anti-epileptic agent valproic acid (VPA) is its acyl glucuronide conjugate (VPA-G), which undergoes non-enzymic, pH-dependent rearrangement via acyl migration to a mixture of beta-glucuronidase-resistant forms (collectively VPA-G-R). We have compared the reactivity of VPA-G and VPA-G-R towards covalent VPA-protein adduct formation by incubation in buffer, human serum albumin (HSA) and fresh human plasma at pH 7.4 and 37 degrees. In all three media, the predominant reaction of VPA-G over 30 hr was rearrangement to VPA-G-R (ca. 24%). Hydrolysis was quite minor (ca. 2%) and covalent adduct formation negligible (when protein was present). On the other hand, both hydrolysis (ca. 27%) and adduct formation (ca. 7%) were extensive when VPA-G-R was incubated with HSA or plasma. These data do not support a transacylation mechanism for VPA-protein adduct formation, since this pathway should be much more highly favoured by VPA-G (an acyl-substituted acetal) than VPA-G-R (simple esters). VPA-protein adducts were found in the plasma of epileptic patients taking VPA chronically (mean 0.77 +/- SD 0.63 microgram VPA equivalents/mL, N = 17). An enzyme linked immunosorbent assay was developed, using HSA modified by incubation with VPA-G-R, to test the immunoreactivity of the patients' plasma. Of 57 patients tested, nine showed measurable levels of antibodies to these adducts, but the titres were very low, with no difference in response to modified and unmodified protein detectable at plasma dilutions of 1:16 or greater. These results suggest that the VPA-protein adducts have little immunogenicity, and are in agreement with clinical observations that drug hypersensitivity responses have not been associated with VPA therapy. Thus, although the in vitro data show that VPA-G is an example of a relatively unreactive acyl glucuronide, covalent VPA-plasma protein adducts and anti-adduct antibodies are nonetheless formed in vivo, at least in some patients on chronic therapy with the drug.