Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Independent Bioethics Committee for Scientific Research at the Medical University of Gdansk (NKBBN/354-201/2017) and registered in the ClinicalTrials.gov Registry (NCT05120011). Before starting the experimental procedure, all participants were informed about the procedure, risks, and expected outcomes and they provided their written informed consent for participation.
Participants
Postmenopausal women, with no cardiovascular disease, liver or kidney disease, gastrointestinal disorder, including stomach ulcer or erosions, cancer, diabetes, musculoskeletal disease, and other severe chronic diseases were recruited. All included participants presented a physician’s certificate indicating a lack of contradictions to strength training. Thirty-six women without a history of osteoporosis, low-energy fractures, or antiresorptive treatment were included in the study (Fig. 1).
Experimental design and study procedure
Participants were randomly assigned (1:1 ratio) to one of the two groups using the Random Sequence Generator (RANDOM.ORG, Dublin, Ireland). The randomization sequence was stored by a researcher who had no contact with the participants. Over the 12 weeks, the participants were supplemented with either 1 g of LC-L-tartrate and 3 g of leucine per day (LC group) or 4 g of leucine per day as a placebo (PLA group), in a double-blind fashion. The supplements were encapsulated in identical gelatin capsules, and the participants were instructed to consume the supplements once a day with their main meal.
RT protocol
During the study period, all subjects participated in the RT program, which was held in a commercial gym, twice a week. Each participant attended the program on Mondays and Wednesdays, or Tuesdays and Thursdays. Professional coaches conducted each training session. Over the 12 weeks, 24 training sessions, each lasting for 45-60 min were performed for each group. The RT protocol was based on a previously described procedure [17]. Each training session started with a 10-min warm-up on a treadmill (walking). The participants then performed three sets of the following four exercises: leg press, leg extension, shoulder press or horizontal row, and chest press or lateral pulldown. The leg press and extension were performed at every training session, but the shoulder press and lateral pulldown were performed only on Monday or Tuesday, and the horizontal row and chest press only on Wednesday or Thursday. Each session ended with a 10-min cooldown on a cycle ergometer.
Before starting the training protocol, a one-repetition maximum (RM) test was performed. During the first 2 weeks, the workload was set at 65% of RM for each exercise, and the exercise was performed in three sets of 10-12 repetitions. After 2 weeks, the workload was increased to 80% of RM, and each exercise was performed in three sets of 6-8 repetitions.
Blood collection and analysis
During the week before initiation of the experimental protocol and 12 weeks after the initiation, fasting blood samples were taken from the antecubital vein into BD Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). After collection, the samples were centrifuged at 2000 g and 4°C for 10 min, and aliquots were stored at -80°C for later analyses. Plasma TMAO level was measured as described previously [18]. For total carnitine (TC), 5 μL of the sample (plasma, calibration points) was transferred into a 1.5 mL test tube. Then 200 μL of acetonitrile containing the internal standard was added for protein precipitation, and 100 μL of 1 M KOH in methanol was added to hydrolyze acylcarnitines. The solution was incubated at 50°C for 60 min, and 100 μL of 1 M HCl in methanol was added to neutralize the mixture [19]. Samples were centrifuged for 2 min at 14000 rpm and injected into the liquid chromatography-mass spectrometry system at the Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Serum dickkopf-1 (DKK1), osteopotegerin (OPG), osteopontin (OPN), sclerostin (SOST), and fibroblast growth factor-23 (FGF-23) levels were determined using the MILLIPLEX Human Bone Magnetic Bead Panel (catalog number [cat. #]: HBNMAG-51K; Merck KGaA, Darmstadt, Germany). Insulin-like growth factor-1 (IGF-1), IL-6, TNF-α, decorin, and osteonectin/secreted protein acidic and rich in cysteine (SPARC) levels were measured using commercially available enzyme immunoassay kits (total IGF-1, cat. # DG100; IL-6, cat. # HS600B; TNF-α, cat. # HSTA00E; decorin, cat. # DY143 and # DY008; SPARC, cat. #DY941-05; R&D Systems, Minneapolis, MN, USA), and C-reactive protein (CRP) level using cat. # EIA-3954 kit (DRG Instruments GmbH, Marburg, Germany).
Dual-energy X-ray absorptiometry (DXA)
Areal BMDs of the lumbar spine (L1–L4), hip, and whole body were measured on separate days, at baseline and after 12-week RT. Measurements were performed using a Hologic Discovery Wi DXA scanner and analyzed using APEX software version 13.4. All DXA scans were performed by the same trained technician according to the manufacturer’s instructions, and analyzed by the same investigator who was blinded to the intervention. T-scores and Z-scores were calculated using the manufacturer's reference ranges.
Diet
Three-day food records were self-reported for 2 weekdays and 1 weekend day at the beginning of the study. The diet was analyzed in terms of the amount of energy, protein, carbohydrates, and fat consumed.
Statistical analysis
Participants included in the statistical analyses completed a minimum of 80% of the training sessions. All calculations were performed using Statistica 13.1 software (Dell Inc., Tulsa, OK, USA). Analysis of variance (ANOVA) for repeated measurements was performed to examine the interaction between the treatment and time. In case ANOVA yielded a significant effect, Tukey’s HSD test was used for post hoc comparisons. Correlations between the changes in absolute values from before to after the RT intervention were calculated using Pearson and Spearman correlation tests for normally and nonnormally distributed data, respectively. T-student test was used for comparison of baseline characteristics of participants. A probability level of p < 0.05 was considered significant. All data are expressed as mean ± standard deviation, unless otherwise stated.
-