Delivery of orally supplemented alpha-tocotrienol to vital organs of rats and tocopherol-transport protein deficient mice

Abstract

The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508). Tocopherol-transport protein (TTP) represents the primary mechanism for maintaining normal alpha-TCP concentrations in plasma and extrahepatic tissues. TTP primarily transports alpha-TCP and has low affinity for alpha-TCT. There are no studies that have investigated tissue delivery of alpha-TCT when orally gavaged on a long-term basis. A long-term study was conducted to examine the effects of alpha-TCT or alpha-TCP supplementation, either alone or in combination, on tissue levels. Rats were maintained on a vitamin E-deficient diet and gavaged with alpha-TCT or alpha-TCP alone or in combination. Five generations of rats were studied over 60 weeks. TTP-deficient mice were supplemented with TCT and bred to examine tissue delivery of oral alpha-TCT. Orally supplemented alpha-TCT was effectively delivered to most tissues over time. When co-supplemented, alpha-TCP outcompeted alpha-TCT for transport systems delivering vitamin E to tissues. To evaluate the significance of TTP in alpha-TCT delivery to tissues, tissue levels of alpha-TCT in supplemented TTP-deficient mice were studied. alpha-TCT was transported to several vital organs in TTP-deficient mice. alpha-TCT restored fertility in TTP-deficient mice. In sum, orally supplemented alpha-TCT was successfully delivered to several vital organs. The transport efficiency of alpha-TCT to tissues may be maximized by eliminating the co-presence of alpha-TCP in the oral supplement. Examination of whether alpha-TCT may benefit humans suffering from neurological disorders because of congenital TTP deficiency is warranted.

Figure 1

Schematic representation of the study design aimed at examining the long-term effects of oral vitamin E supplementation in rats. Female rats were maintained on vitamin E deficient diet (TD 88163, Harlan) and divided into the following four groups supplemented (5d/week) with: (i) α-TCT (5mg/kg body weight) (ii) α-TCP (5mg/kg body weight), (iii) α-TCT+α-TCP (2.5 + 2.5 mg/kg body weight), (iv) placebo vitamin E-stripped corn oil (volume matched). These rats were identified as first generation (G) breeders, i.e G = 1. Offspring from all groups nursed from their mother until four weeks of age. On the fifth week of age, the offspring were weaned and supplemented with their respective isoform of vitamin E for one week. This was followed by tissue harvest from vitamin E supplemented rats in G =2, 3 and 5. The placebo group females, fed with E-deficient corn oil alone, lost fertility and did not produce G = 3. The mean duration taken to generate each generation is indicated in weeks against each G row. Sample size: For G = 2, α-TCP n = 3M and 4F, α-TCT+α-TCP n = 4M and 3F, and α-TCT n = 3M and 3F. For G = 3, α-TCP n = 4M and 3F, α-TCT + α-TCP n = 4M and 0F (no females were born), α-TCT n = 3M and 3F. For G = 5, α-TCP n = 4M and 6F, α-TCT+α-TCP n = 4M and 4F, α-TCT n = 4M and 4F. M, male; F, female.

Figure 2

α-Tocotrienol and α-tocopherol levels in the skin of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-e. a, higher than in corresponding gender-matched group in the same generation; b, lower than in corresponding gender-matched group in the same generation; c, higher than in corresponding gender-matched and supplementation-matched group in G2; d, higher than in corresponding gender-matched and supplementation-matched group in G3; e, higher in females compared to corresponding males in the same generation and supplementation group. G, generation.

Figure 3

α-Tocotrienol and α-tocopherol levels in abdominal adipose and gonads of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Data from G5 are shown. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-c. a, higher than in corresponding gender-matched in the same supplementation group; b, higher in females compared to corresponding males in the same generation and supplementation group; c, lower compared to α-TCP levels in the corresponding gender-matched co-supplemented group.

Figure 4

α-Tocotrienol and α-tocopherol levels in the heart of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-e. a, higher than in corresponding gender-matched α-TCP supplemented group in the same generation; b, lower than α-TCP levels in the corresponding gender-matched α-TCP supplemented group in the same generation; c, lower than corresponding gender-matched α-TCP levels in the same tissue in co-supplemented rats; d, higher than corresponding supplementation-matched females in G2. e, higher in females compared to corresponding males in the same generation and supplementation group. G, generation.

Figure 5

α-Tocotrienol and α-tocopherol levels in the lungs of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-e. a, higher than in corresponding gender-matched α-TCP supplemented group in the same generation; b, lower than α-TCP levels in the corresponding gender-matched α-TCP supplemented group in the same generation; c, lower than in corresponding gender-matched α-TCP levels in the same tissue of co-supplemented rats; d, higher than in corresponding gender-matched co-supplemented rats in G2; e, higher in females compared to corresponding males in the same generation and supplementation group. G, generation.

Figure 6

α-Tocotrienol and α-tocopherol levels in the vastus lateralis skeletal muscle of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-d. a, higher than in corresponding gender-matched α-TCP supplemented group in the same generation; b, lower than in corresponding gender-matched α-TCP levels in the same tissue of co-supplemented rats; c, higher than in corresponding gender-matched α-TCT-supplemented rats in G2; d, higher in females compared to corresponding males in the same generation and supplementation group. G, generation.

Figure 7

α-Tocotrienol and α-tocopherol levels in the brain and spinal cord of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-d. a, higher than in corresponding gender-matched α-TCP supplemented group in the same generation; b, lower than α-TCP levels in the corresponding gender-matched α-TCP supplemented group in the same generation; c, lower than in corresponding gender-matched α-TCP levels in the same tissue of co-supplemented rats; d, higher in females compared to corresponding males in the same generation and supplementation group. G, generation.

Figure 8

α-Tocotrienol and α-tocopherol levels in the blood of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Blood was collected 12h after last supplementation. Data represent mean ± SD. p < 0.05 is designated by letters a-d. a, higher than in corresponding gender-matched α-TCP supplemented group; b, lower than in corresponding gender-matched α-TCP levels in the TCP-supplemented group in the same generation; c, lower than in corresponding gender-matched α-TCP levels in the same tissue in co-supplemented rats; d, higher in females compared to corresponding males in the same generation and supplementation group.

Figure 9

α-Tocotrienol and α-tocopherol levels in the liver of rats. Animals were maintained on vitamin E deficient diet and supplemented with either α-TCT, α-TCP or co-supplemented with α-TCT+α-TCP as indicated in the figure. Open and closed bars represent α-TCP data from male and female rats, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female rats, respectively. Data represent mean ± SD. p < 0.05 is designated by letters a-d. a, higher than in corresponding gender-matched α-TCP supplemented group; b, lower than α-TCP levels in the corresponding gender-matched α-TCP supplemented group; c, lower than corresponding gender-matched α-TCP levels in the same tissue of co-supplemented rats; d, higher in females compared to corresponding males in the same generation and supplementation group.

Figure 10

α-Tocotrienol and α-tocopherol levels in tocotrienol supplemented TTP deficient mice. Top left: An adult (7 week old) TTP deficient (-/-) mouse born from homozygous parents maintained on tocotrienol-supplementation compared to a wild-type (+/+) mouse of the same background. Mice were maintained on long-term tocotrienol supplementation as described in Materials & Methods. Open and closed bars represent α-TCP data from male and female mice, respectively. Hatched and cross-hatched bars represent α-TCT data from male and female mice, respectively. Data represent mean ± SD. ^*, p < 0.05, represents significantly higher (lower, in the case of liver) than α-TCP level in the corresponding gender-matched tissue.