Dark Chocolate Acutely Improves Walking Autonomy in Patients With Peripheral Artery Disease

NOX-2, the catalytic subunit of NADPH oxidase, has a key role in the formation of reactive oxidant species and is implicated in impairing flow-mediated dilation (FMD). Dark chocolate exerts artery dilatation via down-regulating NOX2-mediated oxidative stress. The aim of this study was to investigate whether dark chocolate improves walking autonomy in peripheral artery disease (PAD) patients via an oxidative stress-mediated mechanism. FMD, serum levels of isoprostanes, nitrite/nitrate (NOx) and sNOX2-dp, a marker of blood NOX2 activity, maximal walking distance (MWD) and maximal walking time (MWT) were studied in 20 PAD patients (14 males and 6 females, mean age: 69±9 years) randomly allocated to 40 g of dark chocolate (>85% cocoa) or 40 g of milk chocolate (≤35% cocoa) in a single blind, cross-over design. The above variables were assessed at baseline and 2 hours after chocolate ingestion. Dark chocolate intake significantly increased MWD (+11%; P<0.001), MWT (+15%; P<0.001), serum NOx (+57%; P<0.001) and decreased serum isoprostanes (-23%; P=0.01) and sNOX2-dp (-37%; P<0.001); no changes of the above variables were observed after milk chocolate intake. Serum epicatechin and its methylated metabolite significantly increased only after dark chocolate ingestion. Multiple linear regression analysis showed that Δ of MWD was independently associated with Δ of MWT (P<0.001) and Δ of NOx (P=0.018). In vitro study demonstrated that HUVEC incubated with a mixture of polyphenols significantly increased nitric oxide (P<0.001) and decreased E-selectin (P<0.001) and VCAM1 (P