BHB Assay

Materials

• Wako Autokit Total Ketone Bodies: Cat#'s 415-73301, 411-73401 and 412-73791. Once R1 and R2 are resuspended, use within 3 weeks. Protocol modified from Wako Kit Protocol
• Serum, prepared according to Measuring Fasting Insulin
• Microtiter plate
• Plate reader, warmed to 37C

Procedure

1. Prepare R1 and R2 if not already resuspended
2. Pipet 4 uL of sample into each well (For ketogenic diet samples pipet 1 uL).
3. Pipet 0, 1, 2, 3, 4, 5, 6, and 7 uL of 40 uM standard into wells. If evaluating ketogenic diet samples, also pipet 1, 2, 3, 4, 5, 6, 7 and 8 uL of 300 uM standard
4. Using multichannel pipet add 270 uL of R1 to each well
5. Incubate at 37C for 5 minutes
6. Add 90 uL of R2 to each well and mix
7. Put in plate reader at 37C for 2 minutes
8. Measure A405 at 30s intervals. You may need to increase interval time in order to measure all your wells. Measure for at least 3 minutes total. Modify BHB Assay.prt on the plate reader if necessary.
9. Calculate Max V according to the software.
10. Calculate Ketone Levels by plotting Rate (Max V) versus BHB amount and adjusting by volume used.