 |
|
|
|
|
By Anita S. Andrew, Andrew J Bryce, Allen Stenhouse and Jill Rogerson
November 2005
RIRDC Publication No 05/167 RIRDC Project No RSC-1A
Executive Summary
The object of this study
was to test the feasibility of using the stable isotopes of carbon to confirm
the detection of the use of alkalizing agents. The test employs carbon
isotope ratio mass spectrometry and measures the isotope ratio of 13C to
12C (the normalized measurement is defined as ä13C) to differentiate
between exogenous and endogenous CO2 in blood and possibly urine. The proposed
test was devised to be additional to, and independent of, the currently
used total CO2 concentration defined in the rules of racing and pacing.
In this project, the ä13C value for CO2 liberated from normal horse blood is approximately -24‰ VPDB and the CO2 liberated from sodium bicarbonate (NaHCO3) is -5.1‰ VPDB. Hence, it was expected that when a horse is administered NaHCO3 the ä13C value of CO2 liberated from the blood would become less negative than the CO2 in blood prior to the bicarbonate administration. A preliminary study confirmed this hypothesis in two horses with the blood CO2 ä13C values rising from –25.2 to –20.6‰ VPDB after the administration of 500 g of NaHCO3 to the horses.
Sample collection was optimized by measuring ä13C differences in blood collected in lithium heparin tubes, plain tubes and clot activator tubes. Different fill volumes were investigated with the optimum sampling procedure was found to be 5 to 7 mL of blood collected in a plain 10 mL Vacutainer tube, stored at 4oC and with measurement within 7 days of collection. Methods of measuring of CO2 from blood tested were by sampling the headspace above the blood and with and without the addition of acid to the blood. The optimum method of measurement uses injection of headspace CO2 from the collection tube into a modified elemental analyzer attached to an isotope ratio mass spectrometer (EAIRMS).
The precision was quite acceptable (1ó = 0.03).
Using the optimum sampling and measuring methods, the following research was conducted:
Pre-administration samples in two of the administration trials showed high ä13C values indicating a significant influence from the diet of the animals. Results from the third administration trial, where diet was carefully controlled, gave similar results to trial carried out prior to this investigation.
The dietary effect noticed in the two administration trials can be accounted for by using a measurement Ä (defined as ä13Cblood CO2 - ä13Cdried blood). However, the comparison of the measurement of total CO2 (TCO2) concentration and Ä of the samples collected on race day are not well correlated.
The Ä value suggested too many false positives.
Whilst the blood concentration of TCO2 returns to normal within 24 hours of a substantial dose of NaHCO3 to a horse, the ä13C takes longer to return to normal. Hence, diet can significantly influence the ä13C value. Ä values do go a good way towards accounting for dietary influences but more work is required before this is useful in a legal or quasi-legal situation.
