**3. Results**

#### *3.1. Characteristics of Study Participants*

A total of 34 volunteers were enrolled, 11 young (six men and five women; mean age: 24.7 ± 4.4 years) and 23 older persons (14 men and nine women; mean age: 77.5 ± 8.0 years). Participant characteristics according to age groups and physical performance categories are shown in Table 1. No di fferences were observed among groups for gender distribution, BMI, or number of disease conditions and medications. The two subgroups of older adults did not di ffer for age. As per the study design, HF participants showed higher SPPB scores than LF older adults (*p* = 0.0002).


**Table 1.** Participant characteristics according to age groups and physical performance categories.

Abbreviations: BMI, body mass index; HF, high functioning; LF, low functioning; SD, standard deviation; SPPB, Short Physical Performance Battery. \* *p* < 0.05 vs. young group. § *p* < 0.05 vs. HF. ¥ includes hypertension, coronary artery disease, prior stroke, peripheral vascular disease, diabetes, chronic obstructive pulmonary disease, and osteoarthritis. # includes prescription and over-the-counter drugs.

#### *3.2. Quantification of Total Iron and Selected Metal Transporters in Vastus Lateralis Muscle Biopsies*

To evaluate whether iron levels in muscle were associated with age and physical performance, total iron content was quantified by ICP-MS. Iron levels were significantly greater in muscles of old enrolees compared with the young group (*p* < 0.05), with no di fferences between SPPB categories (Figure 1).

**Figure 1.** Total content of iron in the vastus lateralis muscle of young and old participants. Bars represent mean values (± standard deviation) in the three experimental groups. Values are expressed in ppm. \* *p* < 0.05 vs. young group (*n* = 11). HF: high-functioning (*n* = 16); LF: low-functioning (*n* = 7).

Protein levels of selected iron transporters (TFR1, ZIP8, and ZIP14) were assayed by Western immunoblotting. The expression of TFR1, the primary cellular iron importer, was significantly lower in old LF participants compared with the young group (*p* < 0.05; Figure 2A). Also, lower protein levels of ZIP8 were detected in old enrolees compared with their younger counterparts (Figure 2B). A pattern similar to TFR1 was found for ZIP14 (*p* < 0.05; Figure 2C).

**Figure 2.** Protein expression of (**A**) transferrin receptor 1 (TFR1), (**B**) Zrt-Irt-like protein (ZIP) 8, and (**C**) ZIP14 in the vastus lateralis muscle of young and old participants. Bars represent mean values (±standard deviation) in the three experimental groups. Values are expressed in arbitrary units (a.u.). Representative blots are shown. \* *p* < 0.05 vs. young group (*n* = 11). HF: high-functioning (*n* = 16); LF: low-functioning (*n* = 7).

#### *3.3. Circulating Levels of Ferritin, Hepcidin, and Selected Inflammatory Biomarkers*

Perturbations in iron status have been associated with chronic low-grade inflammation during ageing [41]. In turn, inflammation is acknowledged as a major mechanism contributing to functional impairment [42]. We, therefore, verified whether circulating levels of ferritin, hepcidin, and selected inflammatory biomarkers were associated with age and functional status.

An age-dependent increase was observed for plasma ferritin concentrations (*p* = 0.0291; Figure 3A), with no differences between SPPB categories. Circulating levels of the defensin-like hormone hepcidin were also increased with age (*p* = 0.0232; Figure 3B). The post-hoc test revealed significantly higher hepcidin concentrations in LF older adults compared with young enrolees.

**Figure 3.** Plasma levels of (**A**) ferritin and (**B**) hepcidin in young and old participants. Bars represent mean values (± standard deviation) in the three experimental groups. \* *p* < 0.05 vs. young group (*n* = 11). HF: high-functioning (*n* = 16); LF: low-functioning (*n* = 7).

A similar pattern was described for plasma IL6 (*p* = 0.0174; Figure 4A) and CRP (*p* = 0.0488; Figure 4B).

We performed a correlation analysis to test the hypothesis of an association between inflammation and iron status in LF older adults. As reported in Table 2, ZIP14 was the only iron transporter showing a significant negative correlation with IL6.

**Figure 4.** Plasma levels of (**A**) interleukin 6 (IL6) and (**B**) C-reactive protein (CRP) in young and old participants. Bars represent mean values (±standard deviation) in the three experimental groups. \* *p* < 0.05 vs. young group (*n* = 11). HF: high-functioning (*n* = 16); LF: low-functioning (*n* = 7).

**Table 2.** Relationship between plasma concentrations of IL6 and protein expression of iron transporters in muscle in old low-functioning participants.


Abbreviations: TFR1, transferrin receptor 1, ZIP, Zrt-Irt-like protein.

#### *3.4. Determination of mtDNA Content and Damage*

mtDNA homeostasis in muscle becomes impaired during ageing and in the setting of atrophying conditions [43]. However, whether the abundance and integrity of mtDNA in muscle are associated with physical function in old age is still debated [44]. In the attempt to shed light on this relevant research question, we determined the relative content of mtDNA and mtDNA damage load in muscle samples of young and old enrollees. As depicted in Figure 5, older participants showed lower mtDNA content (*p* = 0.0012, Figure 5A) and greater mtDNA damage (*p* = 0.0001, Figure 5B) compared with young controls, with no differences between HF and LF individuals.

**Figure 5.** (**A**) mtDNA content and (**B**) mtDNA damage in the vastus lateralis muscle of young and old participants. Bars represent mean values (± standard deviation) in the three experimental groups. \* *p* < 0.05 vs. young group (*n* = 11). HF: high-functioning (*n* = 16); LF: low-functioning (*n* = 7).

#### *3.5. Protein Levels of Selected Markers of Oxidative*/*Nitrosative Damage*

Protein expression of the repair enzyme OGG1 and levels of nitrosative stress-associated 3-NT were determined in muscle samples to obtain indications on the extent of oxidative-related molecular damage [45].

OGG1 protein expression declined with ageing (*p* = 0.0435), with no differences among individual groups (Figure 6A). An age-related increase in 3-NT levels was observed (*p* = 0.0005, Figure 6B), without differences between the two old groups.

**Figure 6.** Protein expression of (**A**) 8-oxoguanine DNA glycosylase (OGG1) and (**B**) levels of 3-nitrotyrosine (3-NT) in the vastus lateralis muscle of young and old participants. Bars represent mean values (±standard deviation) in the three experimental groups. Representative blots of OGG1 and 3-NT are shown in panel (**C**) a.u. arbitrary units. \* *p* < 0.05 vs. young group (*n* = 11). HF: high functioning (*n* = 16); LF: low functioning (*n* = 7).
