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.

Oxidizing and reducing responses of granulocytes from type 2 diabetic patients.

Oxidizing/reducing response by granulocytes and a potential correlation between reactive oxygen species generation and triglyceride, total cholesterol, low-density lipoprotein, high-density lipoprotein, body mass index, fasting plasma glucose, glycemic control (hemoglobin A(1c)), or duration of diabetes were examined in type 2 diabetic patients and in healthy subjects. An increase in both oxidizing and reducing responses was observed in cells from diabetic patients relative to normoglycemic individuals. The increase in oxidizing response was nearly 2-fold higher, whereas the antioxidant response increased by 50%. Although reactive oxygen species generation from healthy subjects was correlated with levels of low-density lipoprotein (positive correlation), high-density lipoprotein (negative correlation), and body mass index (positive correlation), no such associations were observed in diabetic subjects, suggesting either an intrinsic perturbation in the oxidant/antioxidant response or possibly due to the effect of the various medications being taken by these patients. These issues need further study.

Aging: functional metabolic balance among cAMP, cGMP and reactive oxygen intermediate generation by human granulocytes.

BACKGROUND: The nature of the aging process has been the subject of considerable speculation. It has been reported that in the aging process several components of the signal transduction pathways, including phosphoinositide, protein kinase C, protein kinase A and reactive oxygen intermediate (ROI) generation, are altered. OBJECTIVE: The aim of our study was to evaluate the functional metabolic balance among cAMP, cGMP and ROI generation by human neutrophils in relation to age. METHODS: The age-induced ROI generation was studied in healthy subjects ranging in age from 20 to 80 years old, divided into 6 age groups: 20-29, 30-39, 40-49, 50-59, 60-69 and 70-80 years old. The oxidizing cellular generation was quantified in a luminol-dependent (ROI production) chemiluminescence assay and the results expressed as relative light units per minute. RESULTS: Our results show a differential functional metabolic balance of cAMP and cGMP in relation to age from 50 years on. This phenomenon is reflected by the increase in ROI generation by neutrophil stimulation with cGMP at all ages and a simultaneous lack of effect of cAMP on cGMP from 50 years old. The same results were observed when neutrophil reacted with endogenous contents of cGMP (levamisole, an inhibitor of cGMP phosphodiesterase) or cAMP (aminophylline, an inhibitor of cAMP phosphodiesterase). Our results show that the lack of modulation of the endogenous or exogenous contents of cAMP or cGMP on ROI generation altered the age-related functional metabolic balance. CONCLUSIONS: This altered functional metabolic balance in cAMP, cGMP and ROI generation of neutrophils may certainly have consequences on host defenses, mainly on inflammatory processes, in healthy subjects from 50 years old. However, the exact consequences of this phenomenon on the aging process remain unknown.

Correlation between NADPH oxidase and protein kinase C in the ROS production by human granulocytes related to age.

BACKGROUND: Aging may be defined as gradual and progressive changes in an organism that increase the probability of death. Accumulating evidence now indicates that the sum of deleterious free radical reactions going on continuously throughout cells and tissues constitutes the aging process or is a major contributor to it. OBJECTIVE: The aim of this paper was to study the correlation between NADPH oxidase and protein kinase C (PKC) in the reactive oxygen species (ROS) production related to age. METHODS: The age-induced ROS generation was studied in healthy subjects ranging in age from 20 to 80 years, divided into six age groups: (1) 20-29, (2) 30-39, (3) 40-49, (4) 50-59, (5) 60-69, and (6) 70-80 years. The ROS were quantified using a chemiluminescence assay (luminol dependent) and the results expressed as RLU/s at maximum peak and total chemiluminescence (integral under the curve RLU/s). RESULTS: Our results demonstrate a significant increase of the ROS production from 40 years of age (age groups 3-6). In the age groups 1 and 2, we did not observe a significant difference (p > 0.05). These data suggest an increase of the ROS production from 40 to 49 years of age which may be induced by the PKC activity. The selective PKC inhibitor (calphostin C) abrogated the stimulatory effect of phorbol-12,13-dibutyrate on the ROS production. However, the NADPH oxidase inhibitor diphenylene iodonium did not inhibit the total ROS production by granulocytes in relation to age. CONCLUSIONS: These data suggest a correlation between age-related PKC activity, NADPH oxidase phosphorylation, and ROS production. The above correlations between unspecific and inflammatory responses related to age are discussed.