Background: Oxidative stress (OxS) is caused by the derangement of the balance between production of
oxidants, mainly represented by reactive oxygen species (ROS), and the antioxidant’s ability to detoxify
them. The consequent biomolecular oxidative damage tends to accumulate in aged biological systems and it
is widely believed to act as main trigger of aging- and metabolism- related diseases such as late onset
Alzheimer’s disease (LOAD), postmenopausal osteoporosis (PO) and metabolic syndrome (MetS). The
involvement of OxS in these pathologies has been nicely demonstrated in vitro and animal models. In
contrast, the data from human studies are scarce and highly controversial. Similar controversies feature the
body of population-based studies on the effects of antioxidant supplements or dietary interventions on OxS
and related diseases.
Objectives: To address these issues we have conducted three research projects with the following aims:
Project 1: to evaluate a possible cross-sectional and/or longitudinal association between systemic markers of
oxidative stress and the most common dementia-related diseases (Alzheimer’s disease and vascular
dementia). Projects 2: to evaluate a possible involvement of OxS in the development of PO. Project 3: to
evaluate in vitro the effects of flavonoids, in particular (-)-epicatechin (EC) and its metabolites (ECM), on
the regulation of expression and activity of NADPH oxidase in hepatic cells (HepG2) treated with palmitate
(Pal).
Material and methods: Project 1: The patients enrolled (n=476 subjects) were divided, in accordance to the
diagnosis of dementia, in: 105 late onset Alzheimer disease (LOAD), 54 vascular dementia (VaD), 199 mild
cognitive impairment (MCI), which is consider as an intermediate state between normal aging and dementia,
and 118 healthy controls. Among the MCI patients, a subgroup of 111 patients were follow-up for an
average of 2 years (2.0 ± 0.6 years). The serum markers of OxS assessed in each subjects were:
hydroperoxides (HY), advance oxidation protein products (AOPP), total antioxidant power (TAP), residual
antioxidant power (RAP), uric acid (UA), and thiols (TH)
Project 2: 290 women (age spanning from 21 to 65 years) were enrolled and divided in different groups
according to menopausal status (reproductive age, peri- and post-menopause). In these women, serum
markers of OxS (HY, AOPP, 8-isoprostane-F2-iso, 8-hydroxy-2'-deoxyguanosine-8-OH-dg, TAP, RAP, UA
and TH) and serum bone markers (Bone-specific Alkaline Phosphatase-BAP and C-terminal telopeptide of
type I collage-CTX-1) were assessed. Furthermore the areal bone density at lumbar spine, hip and total body
by was assessed by dual energy X-ray absorptiometry (DXA) scanner.
Project 3: HepG2 cells were incubated for 24 h with Pal (0.25 mM), in the absence/presence of EC (0.25-1
μM) and ECM (1 μM). By Real-Time PCR (RT-PCR) and western blot, mRNA and protein expression
levels of NADPH oxidase subunits (NOX3, p22phox, p47phox) were assessed. Moreover, protein expression
levels of insulin resistance pathway (IKK and JNK) were assessed by western blot as well. NADPH oxidase
activity and cell oxidants were measured respectively with chemiluminescence assay and oxidant-sensitive
probe 5-(and-6)-carboxy-2,7 dichlorodihydrofluorescein diacetate (DCF). Lipid accumulation was assessed
by Red Oil O staining.
Results: Project 1: Analysis of covariance showed that an oxidative derangement (high HY and/ low
antioxidants) were present in MCI, LOAD and, though less pronounced in VaD. Multivariate logistic
regression analysis showed that, compared with controls, high levels (over median value) of serum
hydroperoxides were independently associated with an increase in the likelihood of having MCI (Odd Ratio:
2.59, 95% Confidence Interval: 1.08-6.21) or LOAD (OR: 4.09, 95%CI: 1.36-11.81). Moreover, low levels
of residual antioxidant power were associated with increased risk of having MCI (OR: 3.97, 95% CI: 1.62-
9.72), but not LOAD (OR: 2.31, 95%CI: 0.83-6.63). No differences in either of these two OxS markers were
found by comparing MCI patients who converted (n = 29) or not converted (n = 82) to LOAD.
Project 2: Pearson’s correlation showed that increased serum levels of a lipid peroxidation marker, HY, were
negatively and independently associated with decreased bone mineral density (BMD) in total body (r=-0.192,
p<0.05), lumbar spine (r=-0.282, p<0.01) and total hip (r=-0.282, p<0.05), as well as with increased bone
resorption rate (r=0.233, p<0.05), as assessed by the serum concentration of CTX-1. Importantly, multiple
regression analysis revealed that HY is a determinant and independent factor for the association between
lumbar spine BMD and CTX-1 levels.
Project 3: Data obtained in vitro showed that Pal increases the production of oxidants in HepG2 through an
up-regulation of protein levels and mRNA expression of NOX3. Pal was able to “mimic” an insulin
resistance condition through an up-regulation of JNK and IKK expression. Moreover, our results
demonstrated that EC and ECM decrease the activity of NADPH oxidase and the ROS production;
furthermore, both EC and ECM decreased the expression of JNK and IKK, improving the insulin resistance
condition. The Oil Red O staining showed that EC and ECM cannot significantly prevent Pal-induced lipid
accumulation.
Conclusion: Systemic OxS might be a significant feature of LOAD, PO and MetS. Moreover, our results
suggest that rich-flavonoids food consumption may be an important way of prevention of age- and
metabolism-related diseases.
Abbreviations: OxS, oxidative stress; MCI, mild cognitive impairment; LOAD, late onset Alzheimer’s
disease; VaD, vascular dementia; HY, hydroperoxides; AOPP, advanced oxidation protein products; TAP,
total antioxidant power; RAP, residual antioxidant power; UA, uric acid; TH, thiol; BMD, bone mineral
density; OR, Odds Ratio; EC, (-)-epicatechin; ECM, (-)-epicatechin metabolites; Pal, palmitate