D-β-Hydroxybutyrate Reaction Kinetics Studied in Nanoliter Volumes Using a Capillary Polarimeter

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Abstract
A laser-based capillary polarimetric detector (CPD) has been configured to allow for the analysis of optically active molecules at physiological temperatures and within a 40 nL sample volume. In the CPD, interference fringes contained within a 360° fan of scattered light result from the interaction between a polarized laser beam and a 240 μm i.d. capillary tube. Here it is shown that these fringes change in a reproducible manner with solute optical activity, facilitating a 3σ detection limit of 1.7 × 10−3 M d-β-hydroxybutyrate corresponding to 68 × 10−12 mole (7 × 10−9 gram) of solute. Further, with the use of this improved and novel polarimeter, the noninvasive study of enzyme reaction kinetics for d-β-hydroxybutyrate (50 mg/dL) and hydroxybutyrate dehydrogenase (100 μL of 50 units/L) has been evaluated for the first time in the absence of NAD. The rate constant for this reaction was found to follow first-order kinetics and determined to be 2.2 × 10−2 s−1. The prospect of directly studying the influence of enzyme cofactors on reaction kinetics is at hand, and the observations presented suggest that the application of monitoring insulin therapy for patients experiencing ketoacidosis is a tractable problem for the CPD.