Enzymatic-HPLC Method to Analyze D-3-Hydroxybutyric Acid in Rat Serum

An enzymatic-HPLC method to analyze the serum concentration of D-3-hydroxybutyric acid was developed. A deproteinized sample of rat serum was treated with 3-hydroxybutyrate dehydrogenase in the presence of NAD, and was analyzed by reversed-phase HPLC to separate and quantify NADH formed by the enzyme reaction, monitoring OD at 340 nm. Standard samples containing varying amounts of D-3-hydroxybutyric acid (0-10 nmol in 50 μl) were treated with 3-hydroxybutyrate dehydrogenase and analyzed by HPLC (the injected amount was 0-2.7 nmol of D-3-hydroxybutuyric acid), resulting in the peak area increasing proportionally with the injected amount. The method proved sensitive enough for as little as 0.2-2 nmol D-3-hydroxybutyric acid in 50 μl to be accurately analyzed. Only 10-20 μl of the rat serum protein-free extract is therefore required to obtain a reliable value. The values obtained with this method are identical to those observed by the conventional enzyme-spectrophotometric method. This method can be easily conducted in many laboratories because it is highly sensitive and only requires HPLC apparatus equipped with a UV meter.