Determination of sorbitol dehydrogenase in microsamples of human serum

Abstract

The enzyme sorbitol dehydrogenase (SDH) is an emerging biomarker of drug-induced liver injury (DILI). This paper introduces determination of SDH in microliter samples of human serum at commercial glucose test strips. The determination relies on the oxidation of NADH cofactor, which is used by SDH reacting with its substrates. The strips could detect NADH down to 5.0 μM (5 pmol), which was two orders of magnitude better than the prior relevant limit of detection. The concentration of cofactors (NADH, NAD+) and substrates (fructose, sorbitol) for SDH determination at a strip was optimized via internally-calibrated amperometric assays at a chitosan/nitrogen-doped carbon nanotube electrode. Such an electrode provided reliable assay data for over 3 months with no need for its reactivation. The assays yielded kinetic parameters Km and kcat and demonstrated higher apparent affinity of SDH for NADH and fructose than NAD+ and sorbitol. The glucose strips detected SDH down to 98 pM (98 amol) in buffers and 200 pM (200 amol) in human serum after 20-min incubation with an optimized (c ≥ 10Km) mixture of cofactor + substrate. The charge ΔQ flowing through a strip was linear (R2, 0.994) up to 6.0 nM SDH, which covered enzyme's clinical range. The ΔQ was selective for SDH, independent of sample matrix, and free of interferences from indigenous glucose. The use of glucose strip as an electrolytic microcell to detect picomoles of NADH and attomoles of SDH is a step toward a point-of-care monitoring of DILI.