Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extra-hepatic vitamin E metabolism

Abstract

The widely conserved preferential accumulation of α-tocopherol (α-TOH) in tissues occurs, in part, from selective postabsorptive catabolism of non-α-TOH forms via the vitamin E-ω-oxidation pathway. We previously showed that global disruption of CYP4F14, the major but not the only mouse TOH-ω-hydroxylase, resulted in hyper-accumulation of γ-TOH in mice fed a soybean oil diet. In the current study, supplementation of Cyp4f14(-/-) mice with high levels of δ- and γ-TOH exacerbated tissue enrichment of these forms of vitamin E. However, at high dietary levels of TOH, mechanisms other than ω-hydroxylation dominate in resisting diet-induced accumulation of non-α-TOH. These include TOH metabolism via ω-1/ω-2 oxidation and fecal elimination of unmetabolized TOH. The ω-1 and ω-2 fecal metabolites of γ- and α-TOH were observed in human fecal material. Mice lacking all liver microsomal CYP activity due to disruption of cytochrome P450 reductase revealed the presence of extra-hepatic ω-, ω-1, and ω-2 TOH hydroxylase activities. TOH-ω-hydroxylase activity was exhibited by microsomes from mouse and human small intestine; murine activity was entirely due to CYP4F14. These findings shed new light on the role of TOH-ω-hydroxylase activity and other mechanisms in resisting diet-induced accumulation of tissue TOH and further characterize vitamin E metabolism in mice and humans.

Fig. 1.

Effect of Cyp4f14 disruption on vitamin E metabolism and tissue accumulation in mice supplemented with γ- and δ-TOH for 12 weeks. A: 24 h ω-hydroxy, ω-1/ω-2 hydroxy and total vitamin E metabolite excretion in wild-type (solid bar) and Cyp4f14^−/− mice (open bar) were quantified by GC-MS. Asterisks indicate significant differences from wild-type. B: 24 h fecal excretion of unmetabolized TOH in wild-type (solid bar) and Cyp4f14^−/− mice (open bar). C: Concentration of TOH in plasma and tissues of wild-type (solid bar) and Cyp4f14^−/− mice (open bar) fed the supplemented diet for 12 weeks. Asterisks indicate significant differences from wild-type mice (P < 0.05).

Fig. 2.

Effect of the L-Cpr disruption on vitamin E metabolism and tissue accumulation in mice supplemented with γ- and δ-TOH for 4 weeks A: 24 h ω-hydroxy, ω-1/ω-2 hydroxy and total vitamin E metabolite excretion in wild-type (solid bar) and L-Cpr^−/− mice (open bar) were quantified by GC-MS. Asterisks indicate significant differences from wild-type. B: 24 h fecal excretion of unmetabolized TOH in wild-type (solid bar) and L-Cpr^−/− mice (open bar). C: Concentration of TOH in plasma and tissues of wild-type (solid bar) and L-Cpr^−/− mice (open bar) fed the supplemented diet for 4 weeks. Asterisks indicate significant differences from L-Cpr^−/− mice (P < 0.05).

Fig. 3.

Evidence of TOH-ω-hydroxylase activity in microsomes prepared from mouse liver (A–C) and small intestinal mucosa (D–F). GC-MS chromatograms from incubations of liver microsomes from L-Cpr^+/+ mice with (A) 250 µM α-TOH, (B) 80 µM γ-TOH, and (C) 80 µM δ-TOH, illustrating formation of the corresponding 13′-OH metabolites. Microsomes from L-Cpr^−/− mouse small intestinal mucosa were incubated with (D) 250 µM α-TOH, (E) 80 µM γ-TOH, and (F) 80 µM δ-TOH, illustrating formation of the corresponding 13′-OH metabolites and demonstrating vitamin E-ω-hydroxylase activity in mouse intestine. Retention times and the ratio of the molecular ion to the fragment ion of intestinal samples were consistent with that of the liver.

Fig. 4.

TOH-ω-hydroxylase activity in pooled human liver microsomes (A–C) and pooled human intestinal microsomes (D–F). GC-MS chromatograms from incubations of human liver microsomes with (A) 250 µM α-TOH, (B) 80 µM γ-TOH, and (C) 80 µM δ-TOH, illustrating formation of the corresponding 13′-OH metabolites. Human intestinal mucosal microsomes were incubated with (D) 250 µM α-TOH, (E) 80 µM γ-TOH, and (F) 80 µM δ-TOH, illustrating formation of the corresponding 13′-OH metabolites and demonstrating vitamin-E-ω-hydroxylase activity in human intestine. Retention times and the ratio of the molecular ion to the fragment ion of intestinal samples were consistent with that of the liver.