Ketone Body Tolerance Test

Materials

• Ketometer - Precision xtra ketone body / glucose monitor (Abbot)
• Ketone Body Test Strips
• Scale
• Bowl for weighing mice
• echoMRI if the mice differ in body fat levels (see below)
• Syringes
• 1.5 mg/mL bOHB in PBS (make as 15g bOHB in 10mL of PBS, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of bOHB, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.
• This may need to be adjusted depending on the ketone sensitivity of the mice, and this is based on PGC-1a on chow.
• In general you want the bOHB to increase blood bOHB levels by about 10 to 12-fold (bringing bOHB levels form around .5mmol/L up to the 5 to 6 mmol/L range if fasted or 1.5 to 2.5 mmol/L if fed in the most responsive of your two groups. If your response is lower or higher, you will probably have to change your dose and retry.
• Timer

Protocol

• If testing fasted ketone tolerance: Remove food from mice for about 6h by putting them in a fresh cage. Add “do not feed” acetate to cages, and ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on. Typically starve the mice at 8AM and aim to start injections at 2PM
• Prepare a 1 g/10mL solution of glucose in the unlikely case that some animals become hyperketonemic.
• Weigh/MRI mice ahead of time, mark tails if necessary with different colors for rapid identification and take fasting ketone measurement via a tail clip.
• Prepare bOHB syringes with 1.5 g/KG mouse weight (ie for a mouse with 15g lean body mass, use 15uL).
• Pre-open enough ketone test strips for one round of measurements.
• At approximately 40 sec to 1 min intervals, inject appropriate amount of bOHB into intraperitoneal cavity of the mouse.
  • Immobilize mouse and restrain tail with one hand.
  • Aim needle for peritoneal space, between the midline and the hip bone.
  • Insert syringe (do not inject) into cavity.
  • Eject syringe.
• At 15 minute intervals (normally 15, 30, 45, 60, 75, & 90 min), take blood bOHB measurements from tail vein. If needed re-snip the tail tip to expose the vein. When measuring BOHB just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse which can be more stressful.
• Analyze data by both % change from fasting ketones and absolute values. Our preferred outcome is to report fasting ketone levels and report percent change as a figure.
• The preferred statistical model is a mixed linear model using the time points as ordinate values and testing for a main effect or an interaction of the treatment/genotype. Use the R lme4 package for this.