In vivo gamma-tocopherol supplementation decreases systemic oxidative stress and cytokine responses of human monocytes in normal and asthmatic subjects

Abstract

We have recently reported that gamma-tocopherol (gammaT) reduces allergen- and zymosan-induced inflammation using rodent models. As an initial step in extending these observations to humans, we conducted an open-label, Phase I dosing study of two doses (one or two capsules daily for 1 week) of a gamma-tocopherol-rich preparation containing 623 mg of gamma-tocopherol, 61.1 mg of d-alpha-tocopherol, 11.1 mg of d-beta-tocopherol (11.1 mg), and 231 mg of d-sigma-tocopherol per capsule. Endpoints for this study include serum levels of 5-nitro-gamma-tocopherol, as a marker of oxidative stress, and changes in serum gamma-, alpha-, and delta-tocopherol and gamma-2'-carboxyethyl-6-hydroxychroman (CEHC) 6 and 24 h after the first dose and after 1 week of treatment. To assess the biological activity of this treatment, we obtained peripheral blood mononuclear cells at baseline and after 1 week of treatment with two capsules of a gamma-tocopherol-rich preparation/day and examined the inflammatory cytokine response of these cells in culture to ex vivo endotoxin/LPS (0.01 ng/ml) challenge. We also monitored a number of safety endpoints to examine how well this preparation is tolerated in eight normal volunteers (four allergic and four nonallergic) and eight allergic asthmatics. We further obtained human monocytes from a subset of these volunteers and treated them ex vivo with gammaT, alphaT, gamma-CEHC, and alpha-CEHC and assessed their actions on LPS-induced degradation of IkappaBalpha and JNK signaling and ROS generation. As detailed herein, this open-label study demonstrates that gamma-tocopherol-enriched supplementation decreased systemic oxidative stress, increased serum levels of gamma-tocopherol, and inhibited monocyte responses to LPS without any adverse health effects. Further, in vitro treatment of human monocytes with gamma-CEHC and alpha-CEHC inhibits ROS generation and LPS-induced degradation of IkappaB and JNK activation.

Figures

Figure 1

Phase I study protocol for examination of the effect of gamma tocopherol enriched capsules on blood levels of alpha, delta and gamma tocopherol, gamma CEHC, safety endpoints an effect on ex vivo response of peripheral blood monocytes to LPS in 8 allergic asthmatic and 8 non-asthmatic volunteers. There was 623mg of γ tocopherol, 61.1mg of d-α-tocopherol, 11.1 mg of d-β-tocopherol (11.1mg), and 231 mg of d-σ-tocopherol per geltab.

Figure 2

Levels of gamma CEHC recovered from serum of 16 volunteers prior to initial dose of the tocopherol preparation, 6 and 24 hours after the initial dose of the 1 geltab preparation, 6 hours after the eighth daily dose of 1 geltab. There was a 1 week period between the 1 and 2 geltab dosing periods and then blood sampling occurred immediately prior to initial administration of the 2 geltab dose, 6 and 24 hours after that dose, 6 hours after the eighth daily dose of 2 geltabs and 1 week after dosing ended.

Figure 3

Levels of 5-nitro-gamma tocopherol recovered from serum of 16 volunteers prior to initial dose of the tocopherol preparation, 6 and 24 hours after the initial dose of the 1 geltab preparation, 6 hours after the eighth daily dose of 1 geltab. There was a 1 week period between the 1 and 2 geltab dosing periods and then blood sampling occurred immediately prior to initial administration of the 2 geltab dose, 6 and 24 hours after that dose, 6 hours after the eighth daily dose of 2 geltabs and 1 week after dosing ended.

Figure 4

IL-1β (Panel A), IL-6 (Panel B) and TNFα (Panel C) secretion of PBMCs recovered at baseline and after gamma-tocpherol enriched supplementation following control (open bars) and LPS (closed bars) treatment of PBMCs. *=p

Figure 5

MCP-1 (Panel A) and MIP-a (Panel B) secretion of PBMCs recovered at baseline and after gamma-tocpherol enriched supplementation following control (open bars) and LPS (closed bars) treatment of PBMCs. *=p

Figure 6

IL-1RA (Panel A) and IL-10 (Panel B) secretion of PBMCs recovered at baseline and after gamma-tocpherol enriched supplementation following control (open bars) and LPS (closed bars) treatment of PBMCs. There was no significant change in LPS response at baseline vs, after supplementation.

Figure 7

Effect of tocopherol species on PMA-Induced ROS generation, A. Effect of α and γ tocopherol (α-T and γ-T) on PMA-induced ROS production in PBMCs. α-T could significantly inhibit PMA-induced ROS production. B, Effect of α and γ CEHC (α-CEHC and γ-CEHC) on PMA-induced ROS production in PBMCs. α-CEHC and γ-CEHC significantly inhibited PMA-induced ROS production.

Figure 8

Effect of tocopherols on IκBα degradation. A, Effect of α and γ tocopherol (α-T and γ-T) on LPS-induced IkBα degradation (NFkB activation) in PBMCs. γ-T could moderately inhibit LPS-induced IkBα degradation. B, Effect of α and γ CEHC (α-CEHC and γ-CEHC) on LPS-induced IkBα degradation in PBMCs. α-CEHC and γ-CEHC could differentially inhibit LPS-induced NFkB activation.

Figure 9

Effect of tocopherols on JNK phosphorylation. A, Effect of α and γ tocopherol (α-T and γ-T) on LPS-induced JNK phosphorylation in PBMCs. γ-T could moderately inhibit LPS-induced JNK phosphorylation. B, Effect of α and γ CEHC (α-CEHC and γ-CEHC) on LPS-induced JNK phosphorylation in PBMCs. α-CEHC and γ-CEHC could differentially inhibit LPS-induced JNK phosphorylation.