Abstracts

Rationale: Ketone bodies have been studied for many years and it is generally accepted that D-BHB, the stereo-selective product of D-3-hydroxybutyrate dehydrogenase, is the clinically significant (i.e., physiological) stereoisomer (Williamson, 1970; Moore et al., 1976). In contrast, L-BHB, the dextrorotatory enantiomer has traditionally been viewed as the non-physiological isomer. While evidence for the biological relevance of L-BHB in mammals has existed for many years, it was not until recently that this ketone isomer was actually measured in rat serum (Tsai et al., 2003). However, the reported procedure is hampered by an inability to detect low concentrations such as those found in small volumes of mouse cerebrospinal fluid (CSF). Here, we present an improved assay that is capable of measuring L-BHB in as little as 10 μL of mouse CSF. This technical enhancement may contribute to mechanistic studies of the ketogenic diet which is used in the treatment of medically refractory epilepsy. Methods: Microdialysate was taken from juvenile C3H mice and the racemic mixture of D-3 hydroxybutyrate and L-3 hydroxybutyrate was extracted from CSF with a solid phase extraction procedure (SPE) on Oasis MCX cartridges. The analyses were carried out on two Diacel OD-RH chiral columns (150mm×4.6 mm, 5μm), using an isocratic mobile phase composed of 40% acetonitrile and 60% of a mixture of potassium phosphate buffer (50.0 mM, pH 2.0) and potassium hexafluorophosphate (100mM) , flowing at 0.5 mL/min. An excitation wavelength of 491 nm and an emission wavelength of 547 nm were used for the analysis. Results: A 5-point calibration curve was established in which mouse CSF was spiked with L-BHB. The calibration function was linear in the range of 7.8 ng/mL to 250 ng/ml with a correlation coefficient of 1. Extraction yield assays led to a mean absolute recovery of 98% and interassay variability was expressed as %Cv and was found to be less than 5%. This assay was successfully applied to mouse CSF samples. Conclusions: Our data indicate that L-BHB can be consistently measured in small volumes of mouse CSF using an improved high performance liquid chromatography assay. While the biological role(s) of the putative non-physiological L-BHB isomer remain(s) to be elucidated, our assay should prove useful in further investigations of ketone body metabolism in both normal and epileptic rodents.