Subjects were asked to step up (concentric muscle action) onto a 40 cm box then step down (eccentric muscular contraction) and the soreness in doing so was rated. The three scales (for the three mornings) were all contained on one sheet see more of paper, but marked soreness values from preceding mornings were covered on the second and third mornings to avoid comparison by the subject. Biochemical analyses Creatine kinase. Analysis of the muscle damage marker creatine kinase (CK), in serum collected before and 12, 36 and 60 hours
post damage, was carried out at a commercial blood testing laboratory (MedLab Central, Palmerston North, New Zealand). An enzymatic ‘reverse reaction’ method was employed, which photometrically measures the rate of NADPH formation as a final product of the last of three reactions, to quantify CK activity. Results are expressed as % change from pre-damage levels. Plasma protein carbonyls. Plasma protein carbonyls were measured using the method previous described by Levine et al.[24]. Briefly, 50 μL of plasma was added to an equal GS-9973 concentration volume of 2,4-dinitro-phenylhydrazine (DNPH, Sigma-Aldrich, Auckland, New Zealand) in 2 M HCl (control = DNPH/HCl in the absence
of plasma) and incubated in the dark for 1 hour. Protein was precipitated with 50% trichloroacetate (TCA, Sigma-Aldrich, Auckland, New Zealand) and the pellet washed GF120918 datasheet three times with ethanol:ethylacetate (1:1). The pellet was then re-suspended in 1 mL 6 M guanidine hydrochloride (Merck NZ Ltd., Palmerston North, New Zealand) at 37°C for approximately 15 min, followed by the absorbance being measured at 360 nm in a UV-visible 1601 spectrophotometer (Shimadza Corporation, Kyoto, Japan). Protein carbonyl levels were then calculated from the absorbance difference
between test and control many using the molar absorption coefficient (ϵ): 22,000 M-1 cm-1. Plasma protein levels were measured using the Bradford method [25] using commercial Bradford reagent (BioRad Laboratories). Results are calculated as nmol of protein carbonyls/mg total protein and expressed as % change from pre-damage levels. Plasma radical oxygen species (ROS)-generating potential. Hydrolysed carboxy-dihydro-2′,7′-dichlorohydrofluorescein diacetate (carboxy-H2DCFDA, Merck, Ltd., Palmerston North, New Zealand) was used to assess the ROS-generating capacity of plasma, using a method previously described by Hurst et al.[26]. Briefly, dihydro-2′,7′-dichlorohydrofluorescein (DCF), which is fluorescent when oxidised was added to diluted (1:4) plasma collected pre and post damage at 12, 36 and 60 hours in phosphate buffered saline [PBS], pH 7.4, Invitrogen NZ Ltd., Auckland, New Zealand), or PBS control, then 0.