DETERMINATION OF TOTAL CARBON DIOXIDE IN HORSES TCO

DETERMINATION OF TOTAL CARBON DIOXIDE IN HORSES TCO 2 TESTING George Maylin Phil Lorimer

MILKSHAKES • Horses develop acidosis during racing • Acidosis impairs performance • Originally blamed on lactic acid build-up • Now known to be accumulation of Hydrogen Ion • (H+) in tissues. Alters cell metabolism. • Acidosis prevented and treated with sodium bicarbonate or baking soda…. Milkshakes

CARBON DIOXIDE BUFFERING SYSTEM • CO 2 + H 2 O H 2 CO 3 • PH 7. 4 HCO 3 - + H+

Chemical Reaction to Determine TCO 2 Specific Ion Electrode CO 2 + H 2 CO 3 + HCO 3 - + CO 3= + RNHCOO- + H+ → CO 2 + H 2 O The p. H electrode coated with a silicone rubber membrane is used to measure CO 2 by a differential change in p. H. When the sample is mixed with acid, all forms of carbon dioxide are converted to their gaseous form. The gas diffuses through the membrane and lowers the p. H of a bicarbonate solution. The change in p. H is proportional to the carbon dioxide in the sample which is recorded as a result.

Threshold Values for TCO 2 • 39 mmoles TCO 2/ml plasma Furosemide • 37 mmoles TCO 2 /ml plasma No Furosemide • 37 mmoles TCO 2/ml plasma For both (Proposed) • Test sample at least 4 hrs post furosemide • Test sample within 3 hrs can be problematic

TCO 2 mmoles/L NY Harness Track Pre Race Samples No Furosemide Post Race Samples Furosemide Pre Race Samples Furosemide <3 hrs Post Race Samples Furosemide > 5 hrs n= 29 n= 20 n=79 n=35 Range : 29. 3 -34. 4 Range: 26. 4 -33. 1 Range: 30 -38. 5 Range: 27. 6 -33. 8 Ave: 32. 0 Ave: 29. 5 Ave: 33. 4 Ave: 29. 8 Doses of furosemide not recorded. Usual dose 5 ml. Pre race with furosemide: The higher levels associated with 2 hr time interval. Impact of furosemide can be significant.

Longitudinal Drug Testing • Testing horses over a period of time. • Now done in IOC and human athletes. • Has potential for incriminating as well as exonerating positive findings. • “My horse is an outlier”.

Summary • TCO 2 testing to detect alkalinizing agents. • Must distinguish natural levels of TCO 2 from feed supplements that may have alkalinizing agents in them. • Detect illegal milkshakes. • Furosemide can increase the TCO 2 levels depending on dose and time interval between injection and blood sampling. • Sampling time within 3 hours of injection is problematic.

QUARANTINE PROTOCOL NY § § § Request by trainer for quarantine Quarantine will last for 3 days Trainer has 3 days to decide Quarantine in a secure barn, 24 hr watch for 72 hrs Access to horse by a pre-determined groom and an additional designated person. Only in the presence of an investigator Horse fed hay, oats and water. No electrolytes or supplements Horse feed supplied by the State Work schedule negotiated to facilitate feeding times and sampling Trainer has to pay for security expenses Tested for TCO 2 on arrival and then daily Split sample testing can be done