Introduction

Phenomena-like French paradox (Renaud and Ruf 1994), obesity paradox (Dehlendoff et al. 2014) and dropping dementia rates (Prince et al. 2012) still coexist with projections of 115 million people with dementia in 2015 (Prince et al. 2013) and the related enormous socio-economic burden of Alzheimer’s disease (AD) (Larson et al. 2013). AD epidemiology, the absence of a cure against AD and the still difficult early diagnosis constitute three major challenges which make AD a unique social and medical issue of these decades. The recent development of AD research towards prevention strategies and the pathophysiological role of the vascular component have opened new investigation possibilities, in which oxidative stress-related mechanisms appear to confirm their pivotal position (recently reviewed in Polidori and Pientka 2012). The evidence of a link between vascular disease and AD was reported originally in a comprehensive review published by de la Torre (2002). The link was at that time under investigation since over ten years and supported by evidence that vascular risk factors and indicators of vascular disease are strongly associated with AD among over 7,000 older persons with and without dementia participating in the Rotterdam Study (Breteler et al. 1998, 2000). Several epidemiological studies in the last decade confirmed the relevance of the relationship between vascular disease and AD. However, reasons and mechanisms of such a relationship remain under debate. Meanwhile, there is an established epidemiological link between hypercholesterolemia and hypertension at midlife and increased risk for AD (reviewed in Polidori et al. 2012). Epidemiological risk assessment and clinical management of the axis vascular disease risk-dementia is extremely complex and cannot go beyond the attentive evaluation of subclinical and early clinically overt atherosclerosis. Atherosclerosis is known to cause ischemia- and inflammation-related oxidative stress as well as subsequent free radical production-related reperfusion and to be linked to dementia and AD (Luzzi et al. 2010). The conclusive statement for healthcare professionals from the American Heart Association/American Stroke Association cites that “Vascular contributions to cognitive impairment and dementia are important. There is a need for prospective, quantitative, clinical–pathological-neuroimaging studies to improve knowledge of the pathological basis of neuroimaging change and the complex interplay between vascular and Alzheimer disease. Studies of intensive reduction of vascular risk factors in high-risk groups are another important avenue of research”. Despite this need, however, there is still a substantial lack of assessment and management of vascular risk factors in the general population and in AD.

The involvement of oxidative stress at the neurovascular level includes several potential mechanisms. Neuronal mitochondria, known to be especially vulnerable to vascular oxidative damage in AD, undergo oxidation-mediated damage such as DNA oxidative changes with subsequent alterations of form and function, and endothelial cells might be the target for the toxic action of heavily aggregated proteins, glia-derived cytokines and stimuli-inducing oxidative and metabolic stress in AD brains (Salmina et al. 2010). Sustained hypoperfusion promoting oxidative stress of brain tissues might also stimulate secondary damage via the overexpression of inducible and neuronal-specific nitric oxide synthase and endothelin-1, and the continuous accumulation of oxidative stress products such as peroxynitrite accumulation may in turn accelerate the damage and compromise the blood–brain barrier (reviewed in Mariani et al. 2005). In the presence of vascular risk factors, a threshold for cerebral hypoperfusion might be critically attained (de la Torre 2000) in a way that resulting oxidative stress might damage the neurovascular unit and endothelium of the brain vessels, the latter producing in turn ROS able to favour Aβ formation and nourishing a vicious circle in which also plaque-induced ROS formation damages the neurovascular unit and the endothelium. In general, free radical production does play a critical role in vascular diseases and in AD and surely even more so in the combination of the two, especially according to ongoing research thoroughly reviewed by our group (Polidori et al. 2012).

Abnormal free radical-related lipid peroxidation is accompanied by a poor status of some antioxidant molecules in patients with atherosclerosis and cardiovascular diseases and F2-isoprostanes, robust biomarkers of lipid peroxidation in humans, are inversely correlated with increased intima–media thickness (IMT) of the carotid arteries (Polidori et al. 2007). An increase of the IMT is the earliest stage of atherosclerosis and is an independent risk factor for cardiovascular events (Tribble 1999; Lorenz et al. 2007; Lester et al. 2009). Several studies have investigated the possibility that carotid steno-occlusive disease may predispose to reduced cognitive function and multiple measures of carotid atherosclerosis have been shown to be associated with prospective risk of dementia (Wendell et al. 2012). In addition, lipid peroxidation, micronutrient status and endothelial function appear to be tightly connected mechanisms of neuronal damage in cognitive impairment with and without dementia (Dias et al. 2014).

Evidence from population studies has shown that both dietary (Klipstein Grobusch et al. 2001), plasma, or serum level of vitamin E (e.g. α-tocopherol) (Riemersma et al. 1996), vitamin C (ascorbic acid) (Tribble 1999; Riemersma 2001) and of the antioxidant enzyme superoxide dismutase (SOD) (Isogawa et al. 2009) are inversely associated with early atherosclerosis and cardiovascular mortality rate. Although RCTs of antioxidant compounds against vascular and neurodegenerative diseases led to largely conflicting results, the role of oxidative stress-lowering strategies cannot be considered disproven yet, due to severe methodological and design study limitations (Tribble 1999; Mecocci and Polidori 2012). Therefore, studies are particularly needed to identify the relationship between atherogenesis and degree of antioxidant defence prior to the clinical manifestation of overt atherosclerosis and neurodegeneration. B-mode ultrasound is, among several non-invasive imaging techniques (CT, echocardiography, MRI, carotid artery imaging), a well-established method to evaluate atherosclerosis of peripheral arteries. By means of ultrasonographic assessment, the thickness of the intima–media complex of easily accessible arteries can be calculated and has been also advocated as surrogate marker for less accessible vessels.

For its independent relationship with cardiovascular disease beyond standard risk factors and as non-invasive, nonionizing radiation test, carotid IMT (C-IMT) is recommended for risk assessment in asymptomatic adults with a class IIa level of evidence (2010 ACCF/AHA Guideline for assessment of cardiovascular risk in asymptomatic adults). We conducted the present study with the aim of exploring the relationship between plasma levels of the major antioxidant molecules of the organism and the carotid IMT in a consecutive sample of old age subjects with no or very mild cognitive impairment.

Subjects and methods

Subjects

One hundred ninety-two subjects (127 F, 65 M) aged 65 years and older (78.5 ± 4.3 years) admitted for routine check-up to the Outpatient Clinic of the Department of Gerontology and Geriatrics at the University Hospital of Perugia, Italy, were consecutively included in the study. The study conforms to the principles outlined in the Declaration of Helsinki as approved by the local Ethics Committee and all study participants gave their signed consent to participate. All participants underwent comprehensive geriatric assessment, including performance of the Mini-Mental State Examination (MMSE) and collection of information on comorbidities, drug therapy, smoking habit and physical activity level. Subjects with a body mass index (BMI) <18.5 or >30 kg/m2, current infections, existing diagnosis of chronic infection, carotid atherosclerotic plaque or major organ failure, as well as those taking antioxidant or iron supplements and consuming <100 or >400 g fruits and vegetables per day were excluded from the study. The rationale for the applied selection criteria was to minimize confounding factors.

IMT assessment

All participants underwent high-resolution B-mode ultrasonography (ESAOTE MAGAS GP, Genova Italy) for the measurement of IMT of the carotid artery using a 7.5 MHz array transducer. They were placed in a supine position and longitudinal, anterior, anterior-lateral and posterior-lateral projections were used to image the left and right carotid arteries. Briefly, IMT was determined as the distance between the blood to intima and media to adventitia interfaces along a 1-cm length at one centimetre of distance from the carotid bifurcation (Sidhu and Allan 1997). The average value of three measurements of the IMT at the common carotid artery (CCA) was calculated for each subject. One physician, blinded to the biochemical measurements, carried out all ultrasound examinations.

Biochemical analyses

All subjects under fasting conditions underwent blood withdrawal in a 10-ml heparinised tube. Blood was immediately centrifuged, and plasma was stored frozen (with 5 % metaphosphoric acid to stabilize vitamin C levels in the relative analysis aliquots) at −80 °C until analysis. Plasma samples were coded and analysis was carried out in a blind fashion. Plasma levels of vitamin C and uric acid were measured by high-performance liquid chromatography (HPLC) with electrochemical detection (Kutnik et al. 1987). Plasma levels of vitamins A and E were measured after extraction with methanol and exane by HPLC with UV detection (Nierenberg and Nann 1992). Activities of the plasma antioxidant enzymes superoxide dismutase (SOD) (L’Abbé and Fisher 1990) and glutathione peroxidase (GPx) (Flohé and Gunzler 1984) were measured spectrophotometrically.

Statistical analysis

Participants’ characteristics were described grouping them according to IMT quartiles. Variables with symmetric distribution were reported as means and standard deviations (SD). Variables with asymmetric distribution were summarized as medians and interquartile ranges. Statistical comparisons across groups were performed using the ANOVA analysis of variance with Bonferroni post hoc test or the Chi-square test, as appropriate. The Mantel–Haenszel Chi-square test was used for trends among IMT quartiles. The significance level used for 2-sided tests was p < 0.05. The associations between IMT quartiles and plasma levels or activity of antioxidants were evaluated using multivariate regression model based on generalized linear model procedure. The probability of a IMT >1.2 mm among participants stratified by quartiles of vitamin C and E plasma levels was tested using multivariate logistic analysis. All analyses were performed using the SAS statistical package, version 8.2 (SAS Institute, Cary, NC).

Results

Demographics and clinical characteristics of the participants for the entire sample and according to IMT quartile distribution are presented in Table 1. Overall, participants had IMT 1.2 ± 0.2 mm (mean ± SD), with the lowest and the highest estimates ranging from 0.70 to 1.50 mm, respectively. Participants were over 75 years old, mainly women, living almost fully independent in ADL and IADL and showing a borderline performance at MMSE. Of note, the IADL score tended to decline across IMT strata (p < 0.003), while the prevalence of smokers was very low in the entire sample and almost superimposed across IMT quartiles. There was no difference with respect to the number of comorbidities and current drug therapy across IMT quartiles. Hypertension and CVD were the most prevalent diseases, with three on four participants (76.6 %) taking low dose antiplatelets, while less than 10 and 5 % of them had diabetes or stroke, respectively. Statins (5.7 %) and oral hypoglycaemic drugs (4.7 %) tended to be more prescribed among participants belonging to the third and fourth IMT quartiles (p = 0.7 and p = 0.05, respectively). Plasma levels of vitamin C, uric acid and vitamin E significantly decreased among participants from the first to the fourth IMT quartile, with a linear slope only for vitamin C. Plasma levels of vitamin A, cholesterol and SOD activity were similar across IMT strata (Table 1). The associations between IMT quartile distribution and both vitamin C and E plasma levels are presented in Table 2. Compared to participants with the lowest IMT, those belonging to the higher quartiles showed declining levels of Vitamin C independent of age, gender, smoking habit, hypertension, CVD, stroke, diabetes, antiplatelets, statins, uric acid levels. Only participants with the highest IMT showed significant lower levels of vitamin E compared to those in the lowest IMT quartiles independent of confounders, including age, gender, smoking habit, hypertension, CVD, stroke, diabetes, antiplatelets, statins, uric acid and cholesterol levels. Finally, we tested the probability of having IMT >1.2 mm among participants stratified by quartile distribution of vitamins C and E plasma levels (Fig. 1, panel a and b). Compared to participants with the lowest vitamin C quartile, the probability to have IMT >1.2 mm significantly decreased among persons from the second to the fourth quartile independent of confounders (Fig. 1, panel a). The probability of having IMT >1.2 mm was not influenced by vitamin E plasma levels when confounders were taking into account (Fig. 1, panel b).

Table 1 Participants’ characteristics of the entire sample and according to carotid IMT quartile distribution
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Table 2 Association between carotid IMT quartile distribution and plasma levels and activity of antioxidants
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Fig. 1
figure 1

The odds ratio of carotid IMT >1.2 mm in older subjects in the middle and the highest quartile of vitamin C (panel a) and vitamin E (panel b) compared to those in the lowest quartile. Adjusted for age, gender, smoking habit, age, hypertension, cardiovascular disease, diabetes, stroke, statins, antiplatelets, oral antidiabetics, total cholesterol plus plasma levels of uric acid and vitamin E in panel a, while plasma levels of vitamin C in panel b, respectively

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Finally, although no statistically significant difference was observed in MMSE scores among vitamin C strata and vitamin C levels among MMSE score strata, there was a tendency of CC-IMT values to decline across MMSE quintiles (p = 0.09 adjusted for age and sex).

Discussion

The main results of this study are that (1) belonging to the higher IMT quartiles is associated with lower levels of vitamins C and E, and that (2) the probability of having IMT >1.2 mm significantly decreases among persons from the second to the fourth vitamin C but not E quartile independently of confounders. The risk of having a carotid IMT >1.2 mm—i.e. largely above the cut-off for pathologic values (European Society of Hypertension and the European Society of Cardiology 2007) has been independently associated with an increased risk for vascular disease (ACCF/AHA Guidelines 2011). Our results fully support the observations of large longitudinal cohort studies on vitamin C and cardiovascular risk. An investigation on participants of the NHANES II study (n = 8,453) concluded that individuals with vitamin C levels ≥45.4 µM had a significantly 21–25 % lower risk of CVD-related deaths and a significant 25–29 % lower risk of all-cause mortality compared to the participants with low levels of vitamin C <23 µM (Simon et al. 2001). In the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study (Boekholdt et al. 2006) a 33 % lower risk of developing coronary heart disease was shown for subjects with the highest plasma vitamin C levels compared to the lowest (mean 27.6 µM) over six years of follow-up. Long in advance to these results, the EPIC-Norfolk study had found that plasma vitamin C concentrations in 8,860 men and 10,636 women were inversely correlated to mortality from all causes and CVD (Khaw et al. 2001). In this study, each 20 µmol/L increase in plasma vitamin C was associated with a 20–30 % risk reduction for all-cause and CVD mortality.

Vitamin C is a remarkable water-soluble antioxidant concentrated predominately in citrus fruits, strawberries and vegetables (e.g. spinach and broccoli) and found in many supplement formulations (LPI Micronutrient Information Center). The adequate functioning of the human organism in general, and of the brain in particular, is highly dependent on vitamin C, but humans are completely dependent on dietary sources due to the evolutionary process leaving us a gene incapable of producing the enzyme gulonolactone oxidase needed to yield ascorbic acid from glucose (Nishikimi et al. 1994). Thus, humans require a constant stream of ascorbic acid from the diet and rely on robust “carrier” transport and “barrier” integrity mechanisms to meet the brain’s demand. Vitamin C is the most powerful water-soluble antioxidant of the organism, and key to prevent oxidative lipid damage in biological systems (Frei et al. 1989). It forms the first line of antioxidant defence under many types of oxidizing conditions. It can rapidly intercept free radicals in the aqueous phase before they attack lipids (Berger et al. 1997). As an antioxidant, vitamin C also provides protection against oxidative stress-induced cellular damage by neutralization of lipid hydroperoxyl (LHP) radicals and by protecting proteins from alkylation by electrophilic lipid peroxidation activity (for review, see Harrison et al. 2010).

Vitamin C plays a role in the function of endothelial nitric oxide synthase (eNOS) by recycling the eNOS cofactor, tetrahydrobiopterin, relevant to arterial elasticity and blood pressure regulation (Traber and Stevens 2011). In addition to the more well-known functions of vitamin C, such as synthesis and deposition of collagen in the basement membrane and antioxidant defence against oxygen and other radicals, roles include stimulating endothelial proliferation, inhibiting apoptosis, and sparing endothelial cell-derived nitric oxide to help modulate blood flow (May et al. 2013). While the position of vitamin C in vascular pathology is well established, this might have much larger implications in age-related diseases, as more recently vascular comorbidities including atherosclerosis and increased intima–media thickness are being acknowledged as important factors in age related dementias (de la Torre et al. 2000; Polidori et al. 2001, 2004; Knopman et al. 2001; Wendell et al. 2009; 2012). Some of the vitamin C-mediated protective “vascular” mechanisms negatively affecting dementia onset and progression may include: (1) reducing carotid intima–media-thickness; (2) reducing lipid peroxidation and (3) reducing endothelial dysfunction (for review, see Harrison et al. 2010).

When 563 elderly men were randomly allocated to one of four treatment groups in a clinical trial that included dietary intervention, omega-3 supplementation, both or neither, carotid IMT progression over a three-year term was reduced in those undergoing dietary intervention that included daily vitamin C intake (Ellingsen et al. 2009). Vitamin E (Hodis et al. 2002) or combined antioxidants (Zureik et al. 2004) in the dietary intervention groups were not successful. These data suggest that vitamin C has a role in modifying vascular risk factors and vascular disease, which could represent mechanisms by which atherosclerosis might reduce dementia risk in people carrying this vascular risk profile (Luzzi et al. 2010). As far as cognitive performance in our study is concerned, the absence of statistically significant differences in MMSE scores among groups do not allow, unfortunately, the exploration and detection of an influence of the vitamin C/C-IMT relationship on cognitive status. However, the trends to significance of decreasing CC-IMT values across MMSE quintiles might warrant further research in patients with different degrees of cognitive performance and C-IMT.

In conclusion, higher vitamin C plasma levels appear to be protective against elevated carotid IMT values in the older persons. In light of the increased risk for cardiovascular disease, cerebrovascular disease and cognitive impairment shown by patients with increased IMT, the achievement of a better antioxidant status in general and of vitamin C in particular should be always encouraged through natural nutrition in the elderly.