2.1. Chemical Characterization of EVOOs
The study was first focused on the chemical characterization of seven EVOOs derived from organic cultivation from Frantoio, Leccino, Moraiolo (Tuscany, Italy), and Intosso (Abruzzo, Italy) olive varieties. As summarized in Table 1
, EVOOs named Frantoio (FR), Leccino (LE), Moraiolo (MO), Moraiolo (MOS), and Intosso (IN) were monocultivar, whereas Leccino (33.3%), Moraiolo (33.3%) and Frantonio (33.3%) was a Tuscan blend (TB) and Leccino (95%), Intosso (5%) was a Tuscan and Abruzzo blend (TAB). In particular, TB was a blend of three cultivars in equal parts (v/v
): Frantoio, Leccino, and Moraiolo, in accordance to the Chianti (Tuscany) disciplinary; TAB was a blend of Leccino (95%, v/v
) and Intosso (5%, v/v
All olive trees were grown organically; those from which MOS was obtained were also under copper-free conditions. Olives were crushed in the olive oil mills of MORI-TEM (Florence, Italy) using innovative equipment to obtain good quality and low oxidation impact products (see Materials and Methods, Section 3.2
Acidity, peroxides, and polyphenols were determined for all EVOOs using the OxiTester Analysis System—a fast, simple, and reliable analyzer based on a photometric technology. The content of polyphenols was related to the antioxidant capacity of the sample. A study carried out in 2008 has validated the data of the antioxidant capacity obtained by the OxiTester analyzer of a wide range of EVOOs samples with those of the official method—the Rancimat method [24
As reported in Table 2
, the acidity, expressed as a percentage of oleic acid, ranged from 0.15 for LE, MOS, and IN to 0.25 for FR; the peroxides, reported in meqO2
/Kg, varied from 4.49 for IN to 8.80 for TB; the polyphenols, expressed as mg tyrosol/Kg, reached for all samples significant values varying from 342 for TB to 890 for TAB. Among monocultivar EVOOs, LE was characterized by the highest value (791 mg tyrosol/Kg) while IN by the lowest value (400 mg tyrosol/Kg).
After the preliminary analysis with the OxiTester system, all EVOOs were analyzed by HPLC-DAD-MS to identify and quantify the single MCPs found in each sample. As reported in Table 3
, the highest value of MCPs was found for TAB and LE, respectively with 1145.98 and 891.70 mg/L, followed by MOS (706.83 mg/L), MO (686.96 mg/L), FR (654.90 mg/L), TB (496.80 mg/L), and IN (417.96 mg/L). A similar trend was observed for the total phenolic compounds; noteworthy is the high content of lignans in all samples and, in particular, TAB and MO with 208.17 and 205.02 mg/L, respectively. FR, LE, MO, MOS, TB, and TAB satisfy the above reported EFSA’s health claim with the highest value of hydroxytyrosol and derivatives content for TAB (986.60 mg/L), LE (799.36 mg/L), and MOS (351.25 mg/L).
Based on the chemical characterization of all samples, the selected EVOOs for the in vivo study in CKD patients were MOS and TB, characterized by a different MCPs content, respectively 706.83 and 496.80 mg/L. These samples showed values of total phenolic compounds of 480.73 and 358.87 mg/L and a content of hydroxytyrosol and derivatives of 351.25 and 296.73 mg/L, respectively (Table 3
). MOS was selected despite being a monocultivar EVOO produced from organic cultivation under copper-free conditions, while TB is a blend of three different cultivars (Leccino, Moraiolo, and Frantoio). IN was not chosen for the lowest value of total phenolic compounds (310.75 mg/L) and hydroxytyrosol and derivatives content (216.08 mg/L), which does not respect the health claim [19
]. However, TAB was not chosen for the lack of literature about possible in vivo pro-oxidative effects of EVOOs characterized by a high polyphenol and hydroxytyrosol content.
For CKD patients, MOS and TB are included in their daily diet as the only plant lipid source.
2.2. Human Study
The main epidemiological characteristics of the two groups of patients are summarized in Table 4
. Patients assumed two EVOOs at different MCP content: 14 patients (age mean 70.8 ± 12.4 years) took MOS and 13 patients (age mean 65.9 ± 11.4 years) took TB.
Primary causes of CKD were chronic glomerulonephritis (7.4%), nephroangiosclerosis (37.2%), diabetic nephropathy (11%), chronic pyelonephritis (7.4%), and other causes (37%).
The results of the laboratory parameters are reported in Table 5
. We observed at T1, compared to T0, a significant increase of estimated glomerular filtration rate (e-GFR) in the group of patients that consumed MOS (35.4 ± 16.32 mL/min vs. 38.1 ± 16.69 mL/min; p
In the same group, we observed a significant decrease of uric acid (6.36 ± 1.9 mg/dL vs. 5.0 ± 1.2 mg/dL, p = 0.049) and an improvement of lipid profile with a significant decrease of triglycerides (165.75 ± 83.18 mg/dL vs. 145.08 ± 70.9 mg/dL, p = 0.016).
In both groups, we observed a significant increase of serum albumin (4.17 ± 0.26 g/dL vs. 4.31 ± 0.27 g/dL, p = 0.021; 4.12 ± 0.25 g/dL vs. 4.39 ± 0.38 g/dL; p = 0.032).
In both groups, we have not observed any statistically significant differences for the atherogenic and inflammatory indices.
At the end of the study, we observed a trend of reduction of the Free Oxygen Radical Test (FORT) and Free Oxygen Radical Defense (FORD), although not significant, in both groups, as reported in Figure 2
Statistical analysis shows no significant changes in eating habits, monitored through the Prevención con Dieta Mediterránea (PREDIMED) questionnaire, administered at two times of the study (T0 and T1), and in physical activity, monitored through the International Physical Activity Questionnaire (IPAQ), administered at the same time-points (Table 6
and Table 7
). Therefore, there is no statistically significant difference between the scoring of both questionnaires administered in both groups. This makes the results obtained from the study even more reliable as they are not influenced by modifiable factors such as dietary habits and degree of physical activity.
The preliminary data obtained from the present study seem to highlight the potential beneficial role of MOS, the EVOO characterized by a high value of MPCs in CKD patients. In fact, the intake of 40 mL per
day of MOS would seem to increase the e-GFR after 9 weeks. This data could be supported by the observed reduction trend of oxidative stress (OS); in fact, the latter plays a key role in the progression of the CKD because OS is related to CKD complications like arterial hypertension, low-grade chronic inflammation, and anemia [25
]. The OS reduction observed confirms previous studies [27
] such as the CMPs of EVOO play a key role in the mechanisms that favor the induction of antioxidant activity [31
The GFR improvement could also be related to the statistically significant reduction in uric acid levels that we observed in patients treated with 40 mL per
day of MOS, after 9 weeks of assumption. In this regard, many studies have shown that the use of urate-lowering therapy is related to the slowing of the progression of CKD [34
Previous in vivo studies demonstrated that EVOO can improve the lipid profile by the enhancement of high-density lipoprotein-cholesterol (HDL-C) and the reduction of LDL-C and triglyceride concentration [38
]. This reduction seems to be related to the concentration of EVOO MPCs, as reported by several authors [38
Our data confirm a significant reduction of triglyceride levels due to the consumption of 40 mL per day of EVOO (MOS) in CKD patients.