of 0.9 with 80% power to detect changes in intramuscular phosphocreatine (PCr) levels. Randomization Subjects were randomized, in a blinded 1:1 fashion, to receive identical-appearing capsules containing either pure creatine monohydrate or placebo (both provided by Jarrow Universal Herbs Inc., Union City, CA) for 14 weeks (Figure 1). A computer- generated randomization list was prepared by a statistical consultant and given directly to the research pharmacy at San Francisco General Hospital (SFGH), where the capsules were bottled and dispensed. All data were collected and analyzed without knowledge of treatment assignment. Three subjects (1 creatine, 2 placebo), including the one woman, withdrew after randomization but before baseline testing or initiation of treatment and are thus excluded from the analysis. Intervention Subjects began with a loading dose of 20 g/day (divided among four doses) or an equivalent number of placebo capsules for 5 days, followed by maintenance dosing of 4.8 g/day (in two 2.4 g doses) or placebo. Maintenance dosing was interrupted briefly for additional 5-day loading cycles at the beginning of weeks 7 and 13. This dosing regimen is similar to those employed in several recent studies (as reviewed by Nissen [13]). Subjects were given a non-caffeinated fruit juice beverage containing 20 g of simple carbohydrate to take with each dose of study medication [22]. Training Regimen. After two weeks, all subjects began a 12- week program of supervised PRT, while continuing on their assigned study medication. Training was performed using a Hoist 5000 Multi-Gym Fitness System (San Diego, CA). Subjects exercised for 1K hours 3 days weekly for 12 weeks. The training included ankle dorsiflexion, ankle plantar flexion, ankle plantar flexion with bent knee, leg press, leg curls, pec decks chest exercises, tricep pushdown, bicep curl and three types of abdominal crunches. Strength in each muscle group was assessed by the one repetition maximum (1RM) test [23] except in the rectus abdominus, in which the number of sit-ups performed in 30 seconds was recorded. Each training session included 4 sets of 8 repetitions at 80% of the 1RM for each exercise. The 1RM for each exercise was reassessed once every two weeks and training intensity adjusted to maintain 80% of the 1RM. All measurements were performed after two days of rest in absence of any symptoms of muscle soreness or fatigue. Objectives The primary aim was to compare the changes in muscle strength (1RM, sit-ups and MVC) from week 0 to week 14 in the two treatment groups. Secondary aims were to compare changes in muscle size, composition, energetics and fatigue, as well as body composition and biochemistry. Outcome Measurements Subjects were studied in the General Clinical Research Center (GCRC) at SFGH and in the Magnetic Resonance Unit at the San Francisco VA Medical Center. Height and weight were recorded with subjects wearing a hospital gown. Measurements of muscle strength, size, composition, energetics and fatigue, as well as body weight and composition and serum biochemistries, were made at baseline, after two weeks of treatment with creatine or placebo (before PRT began), and again after 12 weeks of PRT (study week 14). Safety was monitored throughout the study. Muscle Energetics and Fatigue. Non-invasive measures of intracellular phosphocreatine (PCr), inorganic phosphorous (Pi), Creatine and HIV+ Patients and pH were obtained from the dorsiflexor muscle of the right ankle by 31P magnetic resonance spectroscopy (MRS) using a 30-cm bore 1.9 T superconducting Oxford magnet, as described in detail elsewhere [24,25]. Subjects performed two contraction protocols on the same day, separated by 20 minutes of rest. The first protocol consisted of a 15-second maximum voluntary contraction (MVC), which was used to determine the muscle’s capacity for oxidative phosphorylation [24] reported as the half-time (T1/2) of PCr recovery following the contraction. The recovery analysis indicated that our MRS system can detect PCr changes of 0.5 mM. The second protocol was used to assess muscle function during high- intensity, fatiguing conditions. Muscle force and energetics were measured simultaneously during 36 consecutive MVCs over 6 minutes, one every 10 seconds. The fatigue profile was calculated as described by Karatzaferi et al [26]. Dorsiflexor Strength. Isometric MVC force was recorded during ankle dorsiflexion while the leg was positioned in the magnet for the MRS studies [24,25] The highest force from the 3 MVCs was used to quantify dorsiflexion strength [24] and scale performance during the aforementioned fatigue task. Muscle Size and Composition. Proton T1-weighted magnetic resonance imaging (MRI) was used to visualize the cross-sectional area (CSA) of the thigh and calf using a 1.5 T whole body Siemens Magnetom Vision System, as described previously [24]. Data were analyzed using a customized software program (Interactive Data Language Research Systems, Inc., Boulder, CO). The coefficient of variation of muscle CSA measurements was 0.6%. Body Composition. Whole body and regional fat and lean body mass (LBM) were measured using a Lunar model DPX dual energy x-ray absorptiometer (DEXA) (Madison, WI) [27,28]. Biochemical Measurements. Fasting blood samples were collected for determination of plasma creatine and lactate and serum creatinine, creatine kinase (CK), lipid, glucose, and insulin levels. Plasma creatine was determined by liquid chromatography – tandem mass spectrometry (LC/MS) using a Surveyor LC interfaced with a TSQ Quantum Ultra triple-stage quadrupole MS (Thermo-Finnigan, San Jose, CA) operated in the positive ion mode using atmospheric pressure chemical ionisation. Quantitation was achieved using selected reaction monitoring of the transitions m/z 114 to m/z 44 for creatine and m/z 117 to m/ z 47 for the internal standard. The lower limit of quantitation was 2 mg/mL (18 mmol/L). Intra-day precision ranged from 3.2% to 8.9%, and recovery from 100.3% to 103.4%. Plasma lactate was measured using a YSI STAT2300 analyzer (Yellow Springs, OH), and serum insulin by radioimmunoassay (Linco Research Inc., St. Charles, MO) in the GCRC Core Laboratory. The remaining assays were performed in the SFGH Clinical Laboratories. Insulin resistance was calculated by homeostasis model assessment (HOMA-IR [29]). Safety. Serum creatinine and CK levels were monitored biweekly. Study medication was suspended for one week if creatinine levels increased to .1.8 mg/dl or CK to .450 U/ liter. If, after one week, creatinine and/or CK levels returned to baseline levels, study medication was resumed at half of the initial dosing level and creatinine and/or CK levels were monitored weekly. If creatinine and/or CK levels again exceeded the aforementioned values, study drug was discontinued but subjects continued on study (including exercise). If subjects complained of soreness, the workload for the affected muscle group(s) was reduced until the soreness resolved. Statistical Analysis All safety analyses were performed on an intent-to-treat basis in all 40 subjects who underwent baseline testing. Efficacy analyses PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4605