For the remaining two biopsies, attempts were made to extract tissue from approximately the same location as the initial biopsy by using the pre-biopsy scar, depth markings on the needle, and successive incisions that were made approximately 2 cm proximal to the former site. The initial leg was chosen by the flip of a coin and the contralateral leg was used during the cross-over.
After removal of adipose tissue, the muscle specimens were immediately frozen in liquid nitrogen and then stored at–80°C for later analysis. Three muscle samples were obtained (Days 0, 3, & 5) with the selleck compound same number repeated during crossover on the contralateral leg for a total of six muscle biopsies. Muscle tissue samples were prepared for spectrophotometric analysis for Cr using methods developed by Harris and colleagues [22, 24, 25]. Briefly, approximately 50–70 mg of muscle tissue was cut and transferred into a microfuge tube, followed by a dehydration process
in a vacuum centrifuge (SN-38 cell line Savant ISS110 SpeedVac Concentrator, Thermo Scientific, Milford, MA) and centrifuged for 18–24 hours. Connective tissue was removed from the dried samples which were then grinded into a powder in a porcelain plate and placed into pre-weighed microfuge tubes. selleck chemical Muscle metabolites were extracted in a 0.5 M perchloric acid/ 1 mM EDTA solution on ice for 15 minutes, while periodically vortexing. Samples were then centrifuged at 7,000 rpm for 5 minutes. The supernatant was transferred into a pre-weighed microfuge tube and neutralized with 2.1 M KHCO3/0.3 M MOPS solution. The samples were then centrifuged again at 7,000 rpm for 5 minutes Etomidate and the supernatant was removed and placed into microfuge tubes and frozen at–80°C. Muscle extracts and urine samples were assayed for Cr in the presence of 50 mM imidazole buffer, pH 7.4; 5 mM magnesium chloride; 20 mM
potassium chloride; 25 μM phosphoenolpyruvate; 200 μM ATP; 45 μM NADH; 1250 U/mL lactate dehydrogenase; 2000 U/mL pyruvate kinase. The assay was carried out in a standard fluorescence microplate reader using 10 μL of sample to 1 mL of reagent. The reactant solution was vortexed and read using a fluorometer (Shimadzu RFMini 150, Japan) with an excitation wavelength of 340 nm and an emission wavelength of 460 nm for baseline absorbance values. Five μL of CK (25 μ/mg) was added to 1 mL of the above buffer and stabilized using 1 mL of reagent. After 10 minutes the plate was read again for post-reaction absorbance values. Test to test reliability of duplicate muscle Cr assays was 0.01 ± 0.10 (r = 0.81) with a coefficient of variation of 2.62. Test to test reliability of duplicate of urine Cr assays was 0.01 ± 0.04 (r = 0.99) with a coefficient of variation of 1.13.