D-beta-Hydroxybutyrate reaction kinetics studied in nanoliter volumes using a capillary polarimeter

A laser-based capillary polarimetric detector (CPD) has been configured to allow for the analysis of optically active molecules at physiological tempe ratures and within a 40 nL sample volume. In the CPD, interference fringes contained within a 360 degrees fan of scattered light result from the inter action between a polarized laser beam and a 240 mum i.d. capillary tube. He re it is shown that these fringes change in a reproducible manner with solu te optical activity, facilitating a 3 sigma detection limit of 1.7 x 10(-3) M D-beta -hydroxybutyrate corresponding to 68 x 10(-12) mole (7 x 10(-9) g ram) of solute. Further, with the use of this improved and novel polarimete r, the noninvasive study of enzyme reaction kinetics for D-beta -hydroxybut yrate (50 mg/dL) and hydroxybutyrate dehydrogenase (100 muL of 50 units/L) has been evaluated for the first time in the absence of NAD. The rate const ant for this reaction was found to follow first-order kinetics and determin ed to be 2.2 x 10(-2) s(-1). The prospect of directly studying the influenc e of enzyme cofactors on reaction kinetics is at hand, and the observations presented suggest that the application of monitoring insulin therapy for p atients experiencing ketoacidosis is a tractable problem for the CPD.