Deconjugation enzymes - pitfalls, contamination risks, and practical insights from human biomonitoring of bisphenols, phthalates, and oxidative stress markers

Abstract

Human biomonitoring assesses exposure to xenobiotics via human samples such as urine. Xenobiotics can undergo Phase I and II metabolism, with Phase II forming conjugates such as glucuronides and sulfates. Deconjugation enzymes hydrolyse conjugates to the free biomarker form for analytical quantification. Deconjugation enzymes are commercially available and include digestive enzyme mixtures from molluscs and from recombinant microorganisms. This paper provides contextual information on the metabolism of xenobiotics and the role of deconjugation enzymes, outlining good practices and pitfalls to avoid. Contamination of enzyme extracts is an important consideration, as the tested extracts contained background levels of bisphenol A and several oxidative stress biomarkers. Concentrations ranged from tens of ng/mL for bisphenol A (73 ng/mL) and 8-hydroxy-2′-deoxyguanosine (26 ng/mL) to thousands of ng/mL for N-carboxymethyllysine (2850 ng/mL) and N-carboxyethyllysine (1080 ng/mL). This work also demonstrates potential incompatibilities of deconjugation enzymes with workflows for certain compound classes. Several Helix pomatia extracts (e.g. HP-2) are shown to be incompatible with ester-containing analytes. Phthalates showed conversion from diesters to monoesters, with butyl phthalate the highest at >80 %. These monoesters are also Phase I metabolism products. If exposure assessment relied only on these monoesters as biomarkers, the use of Helix pomatia extract would result in overestimations, as at least part of these compounds would originate from enzymatic breakdown rather than true systemic human metabolism. When employed well, deconjugation enzymes are particularly valuable because they simplify analytical workflows and enhance data reliability and consistency between studies. This work provides guidance to support these objectives.