Bone turnover markers in response to short-term high energy availability high carbohydrate or ketogenic, or low energy availability diets in elite athletes

This study has been submitted for publication. Ethics approval was obtained from the ethics committees of the Australian Institute of Sport (ref: 20181203) and the Australian Catholic University (ref: 2020-238HC). This study took place over 2 training camps, each comprised of two, 6 day phases. Prior to study commencement, baseline characteristics were measured (bone mineral density, body composition, VO2max). During phase 1 (Baseline), all athletes adhered to a high carbohydrate /high EA (> 40kcal.kg-1 FFM.d-1) control (CON) diet. For phase 2 (Adaptation), athletes were assigned to one of three diets: high carbohydrate/high energy availability (EA) (CON, n=10), low carbohydrate/high fat/high EA (LCHF, n=8), or low EA (< 15kcal.kg-1 FFM.d-1; LEA, n=10). Due to the inability to blind participants to the diet, allocations to dietary interventions were based on athlete preference whilst matching for individual characteristics (age, 20 km personal best time, training status). During both phases, a structured training plan was followed to ensure similar training volume and intensity among groups. On the final day of each phase, a 25 km racewalking protocol was performed, where venous blood samples were taken to measure bone turnover markers. Blood samples were taken at rest (fasted), pre-exercise, and immediately, 1 h, and 3 h post-exercise. Samples were collected into BD Vacutainer SST II tubes (East Rutherford, NJ, USA), which were left to clot at room temperature for 30 min prior to being centrifuged at 1500g at 4°C for 10 min. Serum was aliquoted into 1 ml Eppendorf tubes and frozen at -80°C for batch analysis. Concentrations of carboxy-terminal telopeptide (CTX), procollagen-1 N-terminal peptide (P1NP), carboxylated osteocalcin (gla-OC), and undercarboxylated osteocalcin (glu-OC) were measured from each sample.

Body composition and bone mineral density were measured with the Lunar iDXA machine (v16, GE Healthcare). CTX and P1NP concentrations were assessed by electrochemiluminescence immunoassay (Cobas e411, Roche Diagnostics, Basel, Switzerland). Carboxylated and undercarboxylated osteocalcin measurements were performed using enzyme immunoassay (EIA) kits (Takara Bio inc., Shiga, Japan) analyzed on a FLUROstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany). Diets were constructed and analysed with Foodworks Professional Edition 9 (Xyris Software). Area under the curve concentrations (pre- to 3h post-exercise) were calculated with PKSolver add-in in Microsoft Excel (v16.48). Statistical analysis was conducted with R Studio (v1.4.1106) using linear mixed models through the 'lme4' package.