MESSA PUNTO E VALIDAZIONE DI UN METODO PER LA DETERMNAZIONE DELL¿ACIDO SALICILICO E ACIDO FERULICO NELL¿UOMO, E STUDIO DELL'INFLUENZA DEL PATTERN DI MEDITERRANEITÀ DELLA DIETA SULLE CONCENTRAZIONI EMATICHE DI DUESTE DUE MOLECOLE.

A chronic systemic inflammation is reported to be involved in atherosclerosis, coronary heart disease, diabetes and the metabolic syndrome. Evidences suggest that elevated levels of circulating pro-inflammatory markers (interleukin 6, C-reactive protein and tumor necrosis factor alpha) are risk factors for many chronic diseases (Battezzati A, 2005). Food choices play an important role because of macro and micronutrients may interact with human body producing specific metabolic and inflammatory responses relevant to the prevention of chronic-degenerative diseases (Dauchet L, 2009, Pelucchi C, 2009). Particularly, fruit and vegetable provide measurable amounts of bioactive compounds, such as Salicylic acid (SA) that is an ubiquitous hormone in plants. Circulating SA in human is related to fruit and vegetable consumption [Spadafranca et al 2006]. However, only few studies report the effect of acute meal administration on the blood concentrations of salicylic acid. The aim of this study therefore was to investigate the short-term effect of a single meal of fruit on blood concentrations of salicylic acid and the effects of fruit intake on metabolic and inflammatory parameters are unknown. Based on these considerations, a simple, highly selective, and sensitive method using stable isotope dilution and gas chromatography–mass spectrometry has been developed to quantify Salicylic acid and Ferulic acid at concentrations naturally occurring in human serum. This method involves: • deproteinization with ACN; • enzymatic hydrolysis with beta-glucuronidase. • Liquid-liquid extraction with ethylacetate and diethyl ether • derivatization with BSFTA+TMS1% • GC-MS- electron impact-selective ion monitoring analysis. We therefore administrated to 26 healthy subjects a peach shake (PSM), of known SA content and centesimal composition, and an isoglucidic solution (MSM). The evening before the tests Group1 (n=16) abstained from FV consumption; Group2 (n=10) kept a free diet. During the tests circulating SA, glucose, insulin, FFA, IL-6 and CRP were measured. Basal SA was lower (p<0,001) in Group1 (0.09±0.02 μmol/l) than in Group2 (0,30±0,03 μmol/l). After PSM, SA peaked at 90 min in Group1 (0,18±0,01 μmol/l, p<0.01) and Group2 (0,38±0,02 μmol/l, p<0.05) and remained above baseline (p<0.05) up to 3 hours. Glycemia increased more slowly with PSM than MSM (+9,69±0,70 vs +28,21±3,15 mg/dl at 15 min, p<0.01) with a lower average glucose excursion (p<0.05). Insulin peaked at 45 min with PSM and MSM (41,49±5,94 vs 38,40±5,56 µIU/ml) and decrease less rapidly with PSM. FFA were percentually more suppressed (p<0,01) after PSM. IL-6 increased less (p<0.05) with PSM than MSM. CRP was similar between the meals. In conclusion, peach consumption increases circulating SA for more than three hours. Compared to a watery carbohydrates mix, fruit results in a slower plasma glucose increment, and a lower glucose excursion a smaller increment of inflammatory cytokines. Our results confirm that SA is normally present in the blood of people that does not take salicylates drugs. Fruit and vegetables are the main dietary sources of SA and subjects on a free diet, showed concentrations of SA three times higher than individuals who abstained from fruit and vegetables consumption. However, we noticed that SA is still present in blood in individuals who abstained from eating fruits and vegetables in the previous twelve hours. This phenomenon could be due to the long half-life of SA (Hare LG, 2003) either to endogenous metabolic pathways as recently suggested by Paterson (Paterson JR, 2008). Moreover it is interesting to notice that the concentrations of SA achieved in our study are similar to those found by Xu et al (Xu, 1999) to be effective in inhibiting COX-2 gene transcription in HUVE cells. Even though ASA is clinically used at doses that are two orders of magnitude greater than the doses naturally delivered in our study, we demonstrated that after a fruit meal, basal circulating SA may be reach concentrations comparable with those found by Blacklock et al, 2001 in vegetarians and may overlap those of subjects chronically taking aspirin 75 mg/die.