the free aldehyde of the open-chain GlcA and protein,
followed by an Amadori rearrangement to the more stable
adduct. The data suggest that although NAG can directly
and rapidly form an adduct that is unstable and hydrolyzed
almost as quickly as it is formed, intramolecular transa-
cylation may well be a prerequisite for formation of a stable
adduct. Presumably, the rapid complex formation of â-1-
O-acyl glucuronide with HSA may assist in the HSA-
mediated hydrolysis of NAG. We speculate that hydrolysis
of acyl glucuronides may proceed through an acylated
intermediate (acyl-HSA intermediate) such as an ester with
a serin residue, thioester with a cysteine residue and/or
acylimidazole with a histidine residue in HSA.33 In the
second step, the acyl-HSA intermediate is deacylated by
hydrolysis. Thus, this transient adduct may be less
important than the persistent one that arises from the 2-O-
acyl glucuronide.
15. Wilson, I. D.; Nicholson, J . K. Solid-phase extraction chro-
matography and NMR spectroscopy (SPEC NMR) for the
rapid identification of drug metabolites in urine. J . Pharm.
Biomed. Anal. 1988, 6, 151-165.
1
6. Sidelmann, U. G.; Gavaghan, C.; Carless, H. A. J .; Spraul,
M.; Hofmann, M.; Lindon, J . C.; Wilson, I. D.; Nicholson, J .
K. 750-MHz Directly coupled HPLC NMR: Application to
the sequential characterization of the positional isomers and
anomers of 2-, 3-, and 4-fluorobenzoic acid glucuronides in
equilibrium mixtures. Anal. Chem. 1995, 67, 7, 4441-4445.
1
7. Bradow, G.; Kan, L.-S.; Fenselau, C. Studies of intramolecu-
lar rearrangement of acyl-linked glucuronides using salicylic
acid, flufenamic acid, and (S)- and (R)-benoxaprofen and
confirmation of isomerization in acyl-linked ∆9-11-carbox-
ytetrahydrocannabinol glucuronide. Chem. Res. Toxicol.
1
989, 2, 316-324.
1
1
8. McGurk, K. A.; Remmel, R. P.; Hosagrahara, V. P.; Tosh,
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9. Smith, P. C.; Benet, L. Z. Characterization of the isomeric
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In conclusion, the present results indicate that covalent
adduct formation proceeds via the proposed two kinds of
mechanisms, transacylation and imine formation, accord-
ing to the degree of formation of isomeric forms of the acyl
glucuronide of NA.
20. Sidelmann, U.; Hansen, S. H.; Gavaghan, C.; Nicholls, A. W.;
Carless, H. A.; Lindon, J . C.; Wilson, I. D.; Nicholson, J . K.
Development of a simple liquid chromatographic method for
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Acknowledgments
We thank Dr Leslie Z. Benet for useful comments on this work
and Ms Toshiye Minematsu for the NMR analysis.
J S9802704
Journal of Pharmaceutical Sciences / 57
Vol. 88, No. 1, January 1999