*2.1. Participants*

Participants were community-dwelling men and women aged 70 years or older. Healthy young adults between the ages of 18 and 35 years were recruited as controls. Participant recruitment was coordinated by the Recruitment Core of the University of Florida Claude D. Pepper Older Americans Independence Center, as detailed elsewhere [23–25].

A set of eligibility criteria was chosen to minimise the possible confounding e ffect of co-morbid conditions, medications, or lifestyle habits on the relationship among physical performance, iron metabolism, and indexes of muscle mitochondrial damage [26]. Briefly, candidates were not included if presenting with any of the following characteristics: smoking in prior 12 months; engagemen<sup>t</sup> in regular physical exercise; history of drug or alcohol abuse; active treatment for cancer or cancer in the past three years; heart failure New York Heart Association class III–IV; stroke with upper and/or lower extremity involvement; Parkinson's disease or other neurological disorders likely to interfere

with physical function; major psychiatric illnesses; peripheral vascular disease Lériche–Fontaine stage 3–4; history of life-threatening cardiac arrhythmias; cognitive impairment (i.e., Mini Mental State Examination score ≤ 21); renal disease requiring dialysis; lung disease requiring steroids; chronic viral diseases (e.g., hepatitis B and C, HIV); lower extremity amputation; severe knee or hip osteoarthritis limiting mobility; diabetes with visual, vascular or neuropathic complications; inflammatory diseases (e.g., rheumatoid arthritis, vasculitis, autoimmune disorders, inflammatory bowel disease); taking growth hormone, oestrogen replacement, testosterone, anticoagulants, steroids, or non–steroidal anti–inflammatory drugs on a regular basis; severe obesity [i.e., body mass index (BMI) ≥35]; underweight (i.e., BMI ≤18.5); active weight loss >5 kg in prior three months; life–threatening illnesses with an estimated life expectancy <1 year. Candidates on statin treatment were asked to refrain from drug administration one month prior to blood drawn upon their general practitioner's approval.

Old enrolees were categorised as high–functioning (HF) and low–functioning (LF) based on their Short Physical Performance Battery (SPPB) summary score [27]. Specifically, participants with a SPPB score ≥11 were classified as HF, while those who scored ≤7 were categorised as LF. These cut-o ffs were selected based on their ability to predict several relevant health outcomes in older adults (e.g., functional limitations, institutionalisation, mortality) [27–31]. Individuals scoring 8–10 on the SPPB were excluded to allow greater discrimination in physical function and possibly biochemical parameters between groups.

Prior to enrolment in the study, all participants provided written informed consent. The study protocol was approved by the University of Florida's Institutional Review Board (IRB201300790).

#### *2.2. Blood Collection and Processing*

Blood samples were obtained in the morning by venipuncture of the median cubital vein after overnight fasting, using commercial ethylenediaminetetraacetic acid (EDTA) collection tubes (BD Medical, Franklin Lakes, NJ, USA). Samples were immediately centrifuged at 1000× *g* for 10 min at 4 ◦C, aliquots were prepared, and stored at −80 ◦C until analysis.

#### *2.3. Collection of Muscle Biopsies*

Muscle samples were obtained from the vastus lateralis of the dominant lower extremity by percutaneous needle biopsy, under local anaesthesia, as previously described [25]. Muscle specimens were cleaned of any visible blood and fat, snap-frozen in liquid nitrogen, and subsequently stored at −80 ◦C until analysis.

#### *2.4. Measurement of Circulating Iron Transporters and Inflammatory Biomarkers*

Plasma levels of the iron transporter ferritin and the iron regulator hepcidin as well as those of C-reactive protein (CRP) and interleukin (IL) 6 were measured using enzyme-linked immunosorbent assays (ferritin: Human ELISA Kit, Thermo Scientific (Waltham, MA, USA); hepcidin: Intrinsic Hepcidin IDx ™ ELISA Kit, Intrinsic LifeSciences (La Jolla, CA, USA); CRP: Human C-Reactive Protein/CRP Quantikine ELISA Kit, R&D Systems (Minneapolis, MN, USA); IL6: Human IL-6 Quantikine HS ELISA Kit, R&D Systems). Plate processing and data collection were carried out according to the manufacturer's instructions. Absorbance was read on a Synergy HT Multi-Detection microplate reader (BioTek, Winooski, VT, USA). Concentrations of ferritin, hepcidin, and CRP are shown as ng/mL, whilst IL6 levels are reported in pg/mL.

#### *2.5. Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) Determination of Total Iron in Muscle Biopsies*

Total iron content in muscle samples was determined by ICP-MS as described previously with modifications [32]. Briefly, 15–30 mg of vastus lateralis muscle samples were digested in 1 mL concentrated nitric acid (HNO3 Optima-grade) in capped Teflon (Savillex Corporation, Eden Prairie, MN, USA) vials for 24 h. Afterwards, 1 mL of 30% hydrogen peroxide (H2O2 Optima-grade) was added to each vial and placed opened on a hot plate (100 ◦C) to let the mixture evaporate. Subsequently, 1 mL of HNO3 and 1 mL of H2O2 were added to the dry residue and incubated on the hot plate (100 ◦C) overnight to digest any remaining organic material. After this second digestion, samples were evaporated to dryness, followed by addition of 0.8 N HNO3 spiked with 8 parts per billion (ppb) rhenium (Re) and rhodium (Rh). Vials were then incubated at 100 ◦C overnight to ensure complete dissolution. A fraction of the sample solution was removed and further diluted with 0.8 N HNO3 spiked with 8 ppb Re and Rh to obtain a final dilution of approximately 300×. The exact final dilution for elemental analyses was achieved according to the weight of each sample. Trace element analysis was conducted on a Thermo Finnigan Element2™ high–resolution ICP-MS (Thermo Fisher Scientific, San Jose, CA, USA) in medium resolution using Re and Rh as internal standards. In order to avoid analytical biases, all samples were run in the same day and in the same sequence. Results were quantified by external calibration using a combination of gravimetrically prepared ICP-MS standards obtained from QCD Analysts (www.qcdanalysts.com). Iron concentrations are reported in parts per million (ppm), with an analytical error < ±5%.
