ß-Carotene, α-tocopherol and ascorbic acid: differential profile of antioxidant, inflammatory status and regulation of gene expression in human mononuclear cells of diabetic donors.

BACKGROUND: Diabetic patients are exposed to increased oxidative stress due to several mechanisms, mainly hyperglycaemia. Pathological processes, such as those in type 1 diabetes, include diminished activity of the antioxidant defense system(s) or excessive oxidative generation resulting in an oxidative/antioxidant imbalance and development of oxidative stress. METHODS: The purpose of this study was to evaluate the production of reactive oxygen species (ROS) (chemiluminescence) and reduction capacity (MTT dye reduction), the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, superoxide dismutase and catalase using quantitative reverse-transcriptase polymerase chain reaction, and the levels of cytokines [interleukin (IL)-6, tumour necrosis factor-α, IL-8, IL-10 and IL-4] by sandwich enzyme-linked immunosorbent assay in mononuclear cells from non-diabetic and diabetic donors treated with a vitamin complex (ascorbic acid, ß-carotene and α-tocopherol) in two different concentrations ([A] = ascorbic acid = 0.08 µM, α-tocopherol = 0.04 µM, ß-carotene = 0.0008 µM and [20A] = ascorbic acid = 1.6 µM, α-tocopherol = 0.82 µM, ß-carotene = 0.016 µM). RESULTS: Concentration [A] was antioxidant reducing ROS production, expression of NADPH oxidase subunits and pro-inflammatory cytokines while raising the expression of antioxidant enzymes and reducing pro-inflammatory cytokines in both groups. Concentration [20A] was pro-oxidant by raising ROS production, NADPH oxidase subunits and pro-inflammatory cytokines and reducing antioxidant enzymes and anti-inflammatory cytokines in the non-diabetic group but antioxidant in cells of type 1 diabetic patients by raising antioxidant enzymes and anti-inflammatory cytokines and reducing pro-inflammatory cytokines. CONCLUSION: The vitamin complex has a dual effect, pro-oxidant and antioxidant, being also dose dependent with different profiles of cells of non-diabetic and type 1 diabetic patients.

High doses of in vitro beta-carotene, alpha-tocopherol and ascorbic acid induce oxidative stress and secretion of IL-6 in peripheral blood mononuclear cells from healthy donors.

BACKGROUND: Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or repair the resulting damage. Our objective was to verify the existence of an in vitro dual effect of alpha-tocopherol, beta-carotene and ascorbic acid in peripheral blood mononuclear cells (PBMNC) of healthy donors and the inflammatory capacity by IL-6 production. METHODS: PBMNC were incubated with two concentrations of vitamin complex: [A] = Ascorbic Acid = 0.08µM, α- tocopherol = 0.04µM, ß-carotene = 0.0008 µM and [20A] = Ascorbic Acid = 1.6µM, α-tocopherol = 0.82µM, ß-carotene = 0.016µM. Oxidizing and reducing response were measured by chemiluminescence and MTT assays, respectively. IL-6 production was measured by sandwich ELISA. RESULTS: Ours results demonstrated that PBMNC (from 20-39-year-old donors) incubated with vitamins activated free radical production only in [20A] concentration. However, in the age groups of 40-59 and 60-80 years old, there was a significant reduction and activation of the oxidizing response with both concentrations, respectively. The inflammatory profile showed an elevation of IL-6 production in pro-oxidant and a decrease in antioxidant conditions. Correlation between ROS production and IL-6 releasing was observed. CONCLUSIONS: With this experiment we concluded that vitamins can exert an antioxidant effect and a pro-oxidant effect according to their concentration, and could be an inductor of an inflammatory process in vitro generating severe complications to the body in cellular levels.

Ascorbic acid, alpha-tocopherol, and beta-carotene reduce oxidative stress and proinflammatory cytokines in mononuclear cells of Alzheimer's disease patients.

OBJECTIVES: The in vitro effect of a vitamin complex in generating and reducing oxidative species in peripheral blood mononuclear cells (PBMNC) and plasma of patients with Alzheimer's disease (AD) and healthy subjects (HS) was evaluated. METHODS: Two concentrations of a vitamin complex ([A] and [20A]) with ascorbic acid, alpha-tocopherol, and beta-carotene were incubated with either mononuclear cells or plasma. The generation of oxidizing species was measured in a luminol-dependent chemiluminescence assay and the reducing response by the MTT dye reduction assay. The levels of cytokines (interleukin [IL]-1ß, IL-6, and IL-4) were measured by sandwich enzyme-linked immunosorbent assay. RESULTS: Our results demonstrated that the increase in the vitamin complex concentration reduced the reactive oxygen species (ROS) production and enhanced cellular reduction capacity in cells of AD patients in concentration [20A]. Plasma reduction capacity rose significantly for both groups (AD and HS). Concentration [A] did not alter the IL-1ß production, increased IL-4 production in both groups and lowered IL-6 production in AD cells. Concentration [20A] increased pro-inflammatory cytokines (IL-1ß and IL-6) and decreased IL-4 production by PBMNC of HS leading to a pro-inflammatory status. DISCUSSION: The antioxidant vitamin complex was effective in reducing oxidative stress in PBMNC of AD patients by lowering ROS production, improving cellular antioxidant capacities and modifying cytokine induced inflammation.

Role PKA and p38 MAPK on ROS production in neutrophil age-related: Lack of IL-10 effect in older subjects.

BACKGROUND: There is a large increase in the number of elderly people in modern societies. This demographic phenomenon has been paralleled by an epidemic of chronic diseases and inflammatory processes usually associated with advanced age. OBJECTIVE: We studied the role of protein kinase A (PKA), protein kinase B (Akt/PKB) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways in ROS produced by neutrophils induced by pro-interferon-gamma (IFN-gamma) or anti-inflammatory interleukin 10 (IL-10) cytokines age-related. METHODS: The ROS generation was studied in healthy subjects in age ranging from 20 to 80 years old divided in five age groups: (20-39), (40-49), (50-59), (60-69) and (70-80) years old. ROS production was quantified in a luminol-dependent chemiluminescence assay and the results were expressed as relative light units/min). RESULTS: ROS production in human neutrophil was activated by IFN-gamma in all the groups studied. This activation was down-regulated by IL-10. The inhibitory effect of IL-10 on 20-49 years old group was reversed by the pre-treatment with H89 (PKA inhibitor) but not with PD169316 (p38 MAPK inhibitor). This differential effect of IL-10 associated with age was not observed with the neutrophil pre-treatment with Akt/PKB or NADPH-oxidase inhibitor (DPI). Lack of IL-10 effect on ROS production was observed in older subjects (50-80 years old). The effect of IL-10 showed a significant inhibition of ROS production similar to those got with PD169316 alone as compared to that of p38 MAPK. CONCLUSION: The results suggest that inhibitory effect of the ROS production mediated by IL-10 depends on PKA for the younger and the lack effect on the elderly is p38 MAPK dependent.