NMR Derivatives for Quantification of ²H and ¹³C‐Enrichment of Human Glucuronide from Metabolic Tracers

Abstract

Quantification of ²H and ¹³C enrichment distributions in human urinary glucuronide following ingestion of ²H₂O and ¹³C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone and 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well‐resolved and narrow ²H and ¹³C NMR signals. The 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided resolution of the six glucuronide ¹³C signals and numerous ¹³C isotopomer populations through one‐ and two‐bond ¹³C‐¹³C‐coupling, while the 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided complete resolution of the ²H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal ²H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.

Keywords:

• Isotopomers
• Gluconeogenesis
• Intermediary metabolism
• Glycogen
• Liver

Acknowledgements

This work was supported by a grant from the Juvenile Diabetes Research Foundation International (Grant # 5‐2004‐306) and the Portuguese Foundation of Science and Technology (Grant # POCTI/QUI/55603/2004).