Time Since Dose and Dietary Vitamin A Intake Affect Tracer Mixing in the 13C-Retinol Isotope Dilution Test in Male Rats.

BACKGROUND: Retinol isotope dilution (RID) estimates total liver vitamin A reserves (TLRs), the gold-standard vitamin A (VA) biomarker. RID equation assumptions are based on limited data. OBJECTIVES: We measured the impact of tracer choice, mixing period, and VA intake on tracer mixing [ratio of tracer enrichment in serum to that in liver stores (S)] in VA-deficient, -adequate, and hypervitaminotic rats. METHODS: Study 1 was a 3 × 2 × 3 design (18 groups, n = 5/group). Male Sprague-Dawley rats (21 d old) received 50, 100, or 3500 nmol VA/d for 21 d, were administered 52 nmol 13C2- or 13C10-retinyl acetate orally, and killed 5, 10, or 15 d later. Unlabeled VA (50 nmol/d) was given on days 11-14. Study 2 used 100 nmol VA/d for 21 d with 3 groups (n = 6-7): 52 nmol 13C2- or 13C10-retinyl acetate and 100 nmol VA/d throughout 14-d mixing, or 13C2-retinyl acetate without VA. Repeated-measures, 1-factor, and 3-factor ANOVAs were used for analysis. RESULTS: Mean ± SD TLRs (µmol/g liver) reflected intake: 0.11 ± 0.04 (50 nmol VA/d), 0.16 ± 0.04 (100 nmol VA/d), and 5.07 ± 1.58 (3500 nmol VA/d) in Study 1 and 0.24 ± 0.08 (100 nmol VA/d) in Study 2. In Study 1, mean ± SD S was 1.65 ± 0.26 (5 d), 1.16 ± 0.09 (10 d), and 0.92 ± 0.08 (15 d). The interactions tracer*VA intake and time*VA intake were significant between days 10 and 15 (P < 0.05). In Study 2, mean ± SD S was 1.07 ± 0.02 without VA during mixing, and 0.81 ± 0.04 (13C2) and 0.79 ± 0.03 (13C10) with VA intake throughout. Estimated:measured TLRs varied by VA intake and time in Study 1 but not between groups in Study 2. CONCLUSIONS: The 13C-content effect on RID through S is inconsistent. S is highly variable at 5 d, contraindicating early-time point RID. VA intake effects on S vary with timing and quantity. Assuming S = 0.8 at 14 d with consistent VA intake in human studies is likely appropriate.