*2.1. Study Population*

The study protocol was approved by the Ethical Review Committee of the Hospital of Careggi, Florence, Italy. Written informed consent was obtained from all eligible participants. A total of 71 subjects (54 patients with CD and 17 controls) were included in this observational study. Patients were recruited between January 2015 to January 2017 at the Digestive Surgery Unit of the Careggi Hospital, where all had severe relapse (CD activity index scores of >200) requiring surgery. The healthy volunteers were recruited among the personnel of the Careggi Hospital. Serum was obtained from blood samples, taken at surgery for CD patients or at enrolment for the control subjects, collected in Vacutainer ®collection tubes, coagulated at room temperature, and centrifuged at 1800× *g* for 10 min before the distribution of the supernatant in cryo-tubes, and stored at −20 ◦C until analysis.

Information on gender, age, disease duration, diagnostic delay, smoking habits, location, disease behavior, extra-intestinal manifestation, perianal disease, recurrence, number of operations, immunological comorbidity, familiarity IBD, and therapy were collected. Demographic and clinical characteristics of CD patients and healthy controls are reported in Table 1.

**Table 1.** Demographic and clinical characteristics of Crohn's patients and healthy volunteers enrolled in the study.


CDAI = Crohn disease activity index. Data are means ± SE or absolute and relative frequencies.

#### *2.2. Ethics Approval and Consent to Participate*

This study was approved by the Ethical Committee of Careggi-University Hospital (AOUC), Florence, Italy, on May 2, 2011, protocol no. 2011/0016888, rif. 95/10, authorization Gen Dir 17/572011 protocol no. 2011/0018055, and written informed consent was obtained from all study subjects.

#### *2.3. Ferric Reducing Activity of Plasma (FRAP)*

A FRAP reagen<sup>t</sup> solution was freshly prepared by mixing 300 mM acetate buffer, pH 3.6, TPTZ solution (10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl), and 20 mM FeCl3·6H2O in a volume ratio of 10:1:1. To perform the assay, 0.9 mL of FRAP reagent, 90 μL of distilled water, and 30 μL of serum were mixed and incubated at 37 ◦C for 30 min. The absorbance was measured at 595 nm. The antioxidant potential of samples was determined from a standard curve plotted using the FeSO4·7H2O [13].

#### *2.4. Advanced Oxidation Protein Product (AOPP)*

For AOPP determination, 20 μL of serum and 980 μL of PBS were mixed to 50 μL of KI 1.16 M and 100 μL of acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm. AOPP were quantified in μmol/mg of proteins using Chloramine-T (Sigma-Aldrich, Milan, Italy) as the standard for the calibration curve [14].
