Original Contribution Homocysteine and inflammation as main determinants of oxidative stress in the elderly Emilie Ventura a,b , Richard Durant c , Audrey Jaussent d , Marie-Christine Picot d , Marion Morena b , Stéphanie Badiou a,b , Anne-Marie Dupuy a , Claude Jeandel c , Jean-Paul Cristol a,b, ⁎ a Biochemistry Department, Lapeyronie Hospital, CHU de Montpellier, 34295 Montpellier Cedex 5, France b EA 4188, Nutrition Humaine, Biodisponibilité et Athérogénèse, Institut Universitaire de Recherche Clinique, Université Montpellier I, Montpellier, France c Pôle de Gérontologie, Antonin Balmes Center, CHU de Montpellier, 34295 Montpellier Cedex 5, France d Biostatistics Department, Arnaud de Villeneuve Hospital, CHU de Montpellier, 34295 Montpellier Cedex 5, France abstract article info Article history: Received 20 August 2008 Revised 10 November 2008 Accepted 10 November 2008 Available online 19 November 2008 Keywords: Superoxide anion Hyperhomocysteinemia Inflammation Aging Free radicals Oxidative stress is commonly observed in the elderly and could be involved in age-related diseases. However, the determinants of superoxide anion overproduction are not clearly understood. Superoxide anion production was evaluated using a lucigenin-based chemiluminescence method in 478 elderly subjects (304 women,174 men; 79.5±7.1 years). Homocysteine (HCy) metabolism (homocysteinemia, vitamin B12, plasma, and erythrocyte folates), inflammation (CRP, fibrinogen, α-1 acid glycoprotein), lipid parameters (total cholesterol, triglycerides, HDL and LDL cholesterol), and nutritional parameters (albumin, transthyretin) were determined. The results show that HCy levels (p b 0.001) and superoxide anion production (p = 0.04) increase with aging, but CRP does not. Highest HCy (N 20 μM) (OR 1.83 (1.09–3.07), p =0.02) and CRP over 5 mg/L (adjusted OR 2.01 (1.15–3.51), p =0.01) are the main determinants in superoxide anion production in the elderly. These clinical data are confirmed in an in vitro study using THP-1 monocyte-like cells. Incubation with HCy thiolactone (HTL) (0–200 μM) and LPS (0–20 ng/ml) dramatically enhances NADPH oxidase expression and activation. Moreover, a synergic action was evidenced for low concentrations of HTL (20 μM) and LPS (5 ng). Taken together, the clinical data and in vitro experiments support the hypothesis that moderate homocysteinemia and low-grade inflammation synergically enhance NADPH oxidase activity in the elderly. © 2008 Published by Elsevier Inc. Increased blood levels of homocysteine (HCy) are commonly found in the elderly as the result of aging itself, age-related reduction in renal function, and vitamin dietary deficit (mainly folate and vitamin B12) [1,2]. Hyperhomocysteinemia is of growing clinical interest owing to its involvement in atherosclerosis [3,4] and chronic heart failure [5]. Beyond thrombosis, impairment of endothelium- dependent vasorelaxation [6], and proliferation of vascular smooth muscle cells [7], HCy can also promote the synthesis of proinflam- matory mediators [8–10]. In turn, chronic systemic low-grade inflammation, which could be defined in clinical studies as a mode- rate increase in CRP levels, has emerged as a major mechanism involved in atherosclerosis during aging [11–13]. Researchers have focused their attention on the relationship between increased plasma HCy and elevation of CRP in aging [14,15]. However, the molecular mechanism by which elevated plasma concentrations of HCy and inflammation are related to the pathogen- esis of atherothrombotic disease remains incompletely understood. Most hypotheses suggest that HCy contributes to enhanced vascular inflammation in part via oxidative stress [16–18]. It has been proposed that the thiol group of HCy readily undergoes autoxidation in plasma to generate reactive oxygen species (ROS), thereby leading to endothelial cell injury and/or dysfunction [19,20]. Indeed, homo- cysteine thiolactone (HTL) has been involved in LDL oxidation, a key step in foam cell formation [21]. Interestingly, oxidative stress per se has been implicated as a potential mediator of the aging process [22]. In parallel, increased activity of NADPH oxidase, the main source of superoxide anion production [23], is reported in atherosclerotic lesions [24]. NADPH oxidase could be activated by HCy [25,26] as well as by inflammatory mediators such as cytokines or CRP [27]. In addition, interactions between endothelial cells and monocytes promoted by HCy [28,29] or inflammation [30] enhance NADPH oxidase activity [31]. Thus, it could be hypothesized that HCy, inflammation, and superoxide anion overproduction by NADPH oxidase could be linked in elderly people and conspire as vascular risk factors. The present study was performed to analyze biological determi- nants of superoxide anion production in the elderly, with special attention to homocysteine metabolism and low-grade inflammation. In a second step, in vitro studies were performed to evaluate an Free Radical Biology & Medicine 46 (2009) 737–744 ⁎ Corresponding author. Fax: +33 4 67 33 83 93. E-mail address: jp-cristol@chu-montpellier.fr (J.-P. Cristol). 0891-5849/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.freeradbiomed.2008.11.002 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed