Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry

The effect of the feeding on the C-13/C-12 isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C-3 and C-4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the C-13/C-12 isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C-4 to C-3 plants significant changes of the delta C-13 parts per thousand values were observed from the -19 to -23 parts per thousand level to the -24 to -32 parts per thousand level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C-3 to C-4 plants (after 222 days), the measured delta C-13 parts per thousand values have been stabilised at the original level. The results show that in case of the feeding with only C-3 plants the endogenous 3 values of -32 parts per thousand can be reached. In this case the contribution of exogenous material with a delta value of -32 parts per thousand could not be detected independently of the concentration. If the diet contains C-4 plants the difference or the ratio of the delta C-13 parts per thousand values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta C-13 parts per thousand values were measured in incurred human and cattle urine. (c) 2004 Elsevier B.V. All rights reserved.