Muscle biopsies

During the study, we collected and analyzed muscle biopsy samples to assay for creatine concentration. All samples were collected while the participant was in the supine position, where the region around the biopsy site was shaved and sterilized with 3 povidone-iodine swab sticks (Professional Disposables International, Inc., Orangeburg, NY). Lidocaine HCl (1 %) was injected underneath the skin, followed by an injection through the fascia and to the epidermis using a 10 mL syringe to anesthetize the biopsy region. After approximately 5 – 10 min a small incision of about 0.5 cm was made at the biopsy site using a sterile scalpel (Aspen Surgical, Caldedonia, MI). Pressure was applied with sterile gauze after the incision was made. A 5 mm biopsy needle was inserted into the incision and into the ‘belly’ of the vastus lateralis muscle. Once the biopsy needle was pushed through the fascia and settled into the correct location suction was applied using 60 mL syringe, a small muscle sample was collected with the biopsy needle. The biopsy needle was in the muscle for approximately 5 – 20 sec until the procedure was completed.

After the biopsy was obtained, the sample was removed from the needle by forcing air through the syringe connected to the biopsy needle. The muscle was quickly blotted on a sterile cover sponge to remove excess blood and then snap frozen into liquid nitrogen. The sample was then stored at −80 °C for later analysis. Immediately following the biopsy procedure, pressure was applied for at least 10 min to the incision site to halt bleeding. After bleeding had stopped, steristrips (3 M Health Care, St. Paul, MN) were applied to ensure closure of the incision. A tegaderm film (3 M Health Care, St. Paul, MN) was placed over the steristrips, followed by gauze and a self-adherent pressure bandage. Participants were provided with a biopsy care kit including multiple steristrips, tegaderm films, and the contact information of the study coordinator.