Iron Supplementation and Eccentric Exercise

An isokinetic dynamometer (Cybex, Ronkonkoma, NY) will be used for the estimation of changes in isometric knee extensor's peak torque at 90o knee flexion between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise. The average of the three best maximal voluntary contractions with the subjects' one lower extremity will be recorded. To ensure that the subjects provide their maximal effort, the measurements will be repeated if the difference between the lower and the higher torque value exceed 10%. There will be two minutes rest between isometric efforts.

An isokinetic dynamometer (Cybex, Ronkonkoma, NY) will be used for the estimation of changes in isokinetic knee extensor's peak torque at 60o/sec angular velocity between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise. The higher absolute value of five maximal voluntary contractions with the subjects' one lower extremity will be recorded.

An isokinetic dynamometer (Cybex, Ronkonkoma, NY) will be used for the estimation of changes in isokinetic knee extensor's peak torque at 60o/sec angular velocity between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise. The higher absolute value of five maximal voluntary contractions with the subjects' one lower extremity will be recorded.

The assessment of changes in pain-free ROM between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise, will be performed manually using the isokinetic dynamometer. The investigator will move the calf at a very low angular velocity from 0 knee extension to the position where the subject will feel any discomfort.

Each participant will assess changes in delayed onset of muscle soreness (DOMS) during walking and squat movement (90o knee flexion) and perceived soreness between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise. DOMS and perceived soreness will be rated on a scale ranging from 1 (normal) to 10 (very sore).

CK activity will be measured as a general indicator of muscle damage. Changes in CK activity between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be estimated in a Clinical Chemistry Analyzer Z1145 (Zafiropoulos Diagnostica, Athens, Greece) with commercially available kits (Zafiropoulos, Athens, Greece).

GSH will be measured as a general index of oxidative stress. Changes in GSH between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be assessed. For GSH, 20 μL of erythrocyte lysate will be treated with 5% TCA mixed with 660 μL of 67 mM sodium potassium phosphate (pH 8.0) and 330 ΜL of 1 mM 5,5-dithiobis-2 nitrobenzoate. The samples will be incubated in the dark at room temperature for 45 min, and the absorbance will be read at 412 nm.

GSSG will be measured as a general index of oxidative stress. Changes in GSSG between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. GSSG will be assayed by treating 50 μL of erythrocyte lysate with 5% TCA and neutralized up to pH 7.0-7.5. One microliter of 2-vinylpyridine will be added, and the samples will be incubated for 2 h. Sample will be treated with TCA and will be mixed with 600 μL of 143 mM sodium phosphate 100 ΜL of 3 mM NADPH, 100 ΜL of 10 mM 5,5-dithiobis-2-nitrobenzoate, and 194 μL of distilled water. After the addition of 1 μL of glutathione reductase, the change in absorbance at 412 nm will be read for 3 min.

TBARS will be measured as an index of lipid peroxidation. Changes in TBARS between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. For TBARS determination, 100 μL of plasma will be mixed with 500 ΜL of 35% TCA and 500 μL of Tris-HCl (200 mM, pH 7.4) and will be incubated for 10 min at room temperature. One milliliter of 2 M Na2SO4 and 55 mM thiobarbituric acid solution will be added, and the samples will be incubated at 95O C for 45 min. The samples will be cooled on ice for 5 min and then will be vortexed after adding 1 mL of 70% TCA. The samples will be centrifuged at 15,000g for 3 min, and the absorbance of the supernatant will be read at 530 nm.

Carbonyls will be measured as an index of protein oxidation. Changes in PC between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. Protein carbonyls will be determined adding 50 μL of 20% TCA to 50 μL of plasma. Samples will be incubated in the dark at room temperature for 1 hour. The supernatant will be discarded, and 1 mL of 10% TCA will be added. The supernatant will be discarded, and 1 mL of ethanol-ethyl acetate will be added and centrifuged. The supernatant will be discarded, and 1 mL of 5 M urea will be added, vortexed, and incubated at 37C for 15 min. The samples will be centrifuged at 15,000g for 3 min at 4C, and the absorbance will be read at 375 nm.

Catalase will be measured as one of the main antioxidant enzyme of erythrocytes. Changes in catalase between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. Catalase activity will be determined adding 4 μL of erythrocyte lysate, 2955 μL of 67 mM sodium potassium phosphate (pH 7.4), and the samples will be incubated at 37C for 10 min. Five microliters of 30% hydrogen peroxide was added to the samples, and the change in absorbance will immediately read at 240 nm for 1.5 min.

Changes in TAC between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. TAC will be determined adding 20 μL of plasma to 480 ΜL of 10 mM sodium potassium phosphate (pH 7.4) and 500 μL of 0.1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, and the samples will be incubated in the dark for 30 min at room temperature. The samples will be centrifuged for 3 min at 20,000g, and the absorbance will be read at 520 nm.

Uric acid will be measured as the main antioxidant component of blood plasma. Changes in Uric acid between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. It will be measured in a Clinical Chemistry Analyzer Z1145 (Zafiropoulos Diagnostica, Athens, Greece) with commercially available kits (Zafiropoulos, Athens, Greece).

Changes in Bilirubin between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. Bilirubin will be measured in a Clinical Chemistry Analyzer Z1145 (Zafiropoulos Diagnostica, Athens, Greece) with commercially available kits (Zafiropoulos, Athens, Greece).

Changes in Iron concentration between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. Iron concentration will be measured in a Clinical Chemistry Analyzer Z1145 (Zafiropoulos Diagnostica, Athens, Greece) with commercially available kits (Zafiropoulos, Athens, Greece).

Changes in TIBC between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. TIBC will be measured in a Clinical Chemistry Analyzer Z1145 (Zafiropoulos Diagnostica, Athens, Greece) with commercially available kits (Zafiropoulos, Athens, Greece).

Changes in TS between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. Transferrin saturation will be calculated from iron and TIBC values.

Changes in Ferritin concentration between baseline and after 3 weeks of supplementation (pre-eccentric exercise), and also between pre-eccentric exercise and 24 hours, 48 hours, 72 hours, 96 hours after the eccentric exercise will be investigated. For the determination of serum Ferritin an immunoenzymometric assay (EIA) kit based on sandwich ELISA will be used (Accubind, Monobind Inc., USA®) which contains all the necessary reagents. The absorbance in each well will be read at 450nm using a microplate reader (Biochrom Asys Expert 96, UK). For the calculation of the concentration of ferritin, a dose response curve was used according to the assay directions.