Vitamin A status assessment in rats with 13C4-retinyl acetate and gas chromatography/combustion/isotope ratio mass spectrometry

Vitamin A assessment methods that indirectly determine liver reserves are still in development. The deuterated vitamin A assay has been successfully applied in several population groups, but large doses of vitamin A must be used and the gas chromatography/mass spectrometry analysis is not very sensitive. Therefore, 10,11,14,15-C-13(4)-retinyl acetate was synthesized using a modified Wittig-Horner procedure. Thereafter, female Sprague-Dawley rats (n = 47) were fed a vitamin A-deficient diet and divided into three groups: low (L), moderate (M) and high (H) vitamin A. Groups L, M and H were supplemented with 35, 70 and 350 nmol of unlabeled retinyl acetate/d for 17 d. On d 18, three rats from each group were killed to determine baseline C-13 levels. Serum was prepared, and livers were collected and stored at -70 degreesC until analyzed with HPLC and gas chromatography/combustion/isotope ratio mass spectrometry. The remaining rats were supplemented with 52 nmol of C-13(4)-retinyl acetate. Rats were killed on d 1, 2, 4 and 10. The calculated and measured values of total body reserves (TBR) of vitamin A were within 7% of each other overall, and the relationship was linear (r = 0.98, P < 0.0001). The calculated mean TBR were 0.49 +/- 0.03, 0.82 +/- 0.007 and 3.72 +/- 0.40 mol, and the measured mean TBR were 0.50 +/- 0.045, 0.69 +/- 0.10 and 3.6 +/- 0.29 mu mol for groups L, M and H, respectively. In contrast, serum retinol concentrations did not show a difference among the dietary groups: 1.32 +/- 0.14, 1.35 +/- 0.17 and 1.28 +/- 0.15 mu mol/L for groups L, M and H, respectively (P = 0.25). In conclusion, this method offers more sensitivity than traditional methods and may be applicable to human vitamin A status assessment when TBR estimations are desired.