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Effects of enzymatically modified isoquercitrin in supplementary protein powder on athlete body composition: a randomized, placebo-controlled, double-blind trial

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  • Effects of enzymatically modified isoquercitrin in supplementary protein powder on athlete body composition: a randomized, placebo-controlled, double-blind trial

    Study design and participants

    This randomized, parallel arm, placebo-controlled, double-blind study was conducted for 4 months in 2014. Primary outcomes were changes in lean mass and lower limb muscle mass, and secondary outcomes were changes in the antioxidant status. The study’s protocol was approved by the Ethics Committee of Tsukuba University (2014.7.30, no. 26–37), and the study was performed in accordance with the guidelines of the Declaration of Helsinki Declaration and the 2010 Consolidated Standards of Reporting Trials statement [23]. The trial was retrospectively registered in the University Hospital Medical Information Network Clinical Trial Registry (Japan, registration no. UMIN000036036) in 2019. Forty male Japanese students (from the Tsukuba University) who played American football (BMI ≥18.5 and 
    Exclusion criteria

    The following participants were excluded:
    • 1)

      Participants with food allergies.
    • 2)

      Participants who consume other supplementary protein powders, drugs, or supplements during the study.
    • 3)

      Participants who change their lifestyle, including dietary and exercise habits, during the study.
    • 4)

      Participants who eat unbalanced diet (consuming much polyphenol-rich food, including citrus fruit, buckwheat, and fermented soybeans), or consume excessive alcohol.
    • 5)

      Participants who refrained from practice for long periods (e.g., because of injury).
    One of the recruited participants was excluded because he failed to provide a blood sample.
    Characteristics of participants and training program

    Most participants had previously consumed supplementary protein powder (typically whey protein, 3–4 times a week after resistance training). The study’s flow chart and assessment schedule are shown in Figs. 1 and 2, respectively.

    Fig. 1Study flow chart. Forty young male Japanese university students who played American football were recruited, although one participant was excluded because he did not provide blood sample. Thus, 39 participants were randomized to receive either whey protein (W) or EMIQ in whey protein (EW)

    Fig. 2Study protocol. Participants consumed 20 g of their supplementary protein powder after exercise (6 days a week). Body composition was measured using dual-energy X-ray absorptiometry (DXA) at 0 and 4 months. Weight measurements and nutritional evaluations were performed at 0, 2, and 4 months. Medical check-ups were performed with blood sampling at 0 and 4 months. Oxidative stress was measured at 0 and 4 months. B: body composition measurement, W: weight measurement, N: nutritional evaluation, M: medical check-up, O: oxidative stress measurement

    The football team’s trainer provided input regarding the design of the training program. Resistance training to maintain or increase skeletal muscle mass and power was performed 3 times a week during the first month (from 0 month to 1 month). Participants also underwent skill training to increase their individual performance and teamwork. From 1 month to 2 month, the participants underwent moderately intense training (to develop physical strength and complete a training camp). From 2 month to 4 month, practice sessions/games were conducted 5–6 times a week (the competitive season).
    Supplementation protocols

    Participants consumed 20 g per day of whey supplementary protein powder (the W group) or 20 g of whey supplementary protein powder with 42 mg of EMIQ (the EW group). As calculated, participants consumed 0.26 g/kg whey protein powder, and which indicates 0.18 g/kg protein. Consumption amount of protein was determined on the basis of previous participants’ habits and previous reports [24, 25]. All supplements were consumed 6 times a week (immediately after practice). Nutritional components of the supplements are shown in Tables 1 and 2. Moisture and mineral contents were 0.8 g and 0.5–0.96 g, respectively.

    Table 1 Nutrition facts per 20 g supplementation

    Table 2 Amino acid composition
    EMIQ in the supplements was analyzed using a previously described high-performance liquid chromatography method [26] that revealed that the whey protein powder contained 0.0 mg of EMIQ and whey protein powder with EMIQ contained 42 mg of EMIQ. All supplements were prepared by Morinaga & Co. Ltd. (Tokyo, Japan).
    Body composition measurements

    Body weights were measured using a body composition meter (MC-190; TANITA, Japan) at baseline and 4 months. Body composition parameters (bone mineral content, fat mass, and muscle mass) were measured using dual-energy X-ray absorptiometry (DXA; QDR-4500A; Hologic, Japan) at baseline and 4 months. DXA measurements were conducted following overnight fast and 24-h absence of strenuous exercise. Participants wore typical athletic clothing and removed all metal jewelry. Participants were laid on their back on the DXA table with their arms at their sides and feet together. The same investigator conducted all analyses, and the second measurement was performed as a comparable mode. The investigator checked the setting of analysis region. Lower limb were separated from the trunk by a horizontal line just below the lower pelvic. Analysis provided data on lean body mass, fat mass, and bone mineral content for the total body and lower limb. Lower limb fat-free mass was calculated from lower limb lean mass plus lower limb bone mineral content. Lower limb lean mass was expressed as lower limb muscle mass because lower limbs consist mostly of skeletal muscle, bone, and fat [27].
    Nutritional evaluations

    To assess food intake, participants completed food frequency questionnaires (FFQg version 3.5; Kenpaku-sha, Tokyo, Japan) at baseline, 2 months, and 4 months (on the same day as their body weight measurements). Supplementation with protein powder for this study was not included in these nutritional evaluations.
    Blood sampling

    Blood sampling was performed at baseline and 4 months. The venous blood samples were collected in vacutainers in the morning after overnight fasting and 24-h absence of strenuous exercise; 2 mL was drawn for general testing and 9 mL for liver and renal function testing. All blood samples were analyzed at the Tsukuba i-Laboratory. The blood test parameters were red blood cell (RBC), and white blood cell (WBC) counts, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total bilirubin (T-BIL), creatinine (Crea), uric acid (UA), urea nitrogen (UN), aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), platelet (PLT), and γ-glutamyl transpeptidase (γ-GTP). RBC and WBC were measured using the electric resistance method. Hb was measured using the sodium lauryl sulfate-Hb method. Ht was measured using the RBC pulse peak method. MCV was calculated as follows: Ht(%)/RBC (106/ mm3)  10. MCHC was calculated as follows: Hb(g/dL)/Ht (%)  100. T-BIL, Crea, and UA were measured using the enzymatic method. UN was measured the urease-UV method. AST, ALT, LDH, PLT, and γ-GTP were measured the Japan Society of Clinical Chemistry standardization method.
    Oxidative stress analysis

    Blood samples were collected from participants’ fingertips in the morning after overnight fasting and 24-h absence of strenuous exercise at 0 and 4 months. The d-ROMs were measured using a free radical system (FRAS4; Health & Diagnostics Ltd., Italy) and measurement kits (DIACRON, Italy). The d-ROMs results were expressed in arbitrary units, with one unit corresponding to 0.08 mg/dL of hydrogen peroxide. BAP was also measured using the FRAS4 system and DIACRON measurement kits. The BAP results were expressed in mmol/L of reduced ferric ions.
    Statistical analysis

    Data were expressed as mean  standard deviation, and changed data were expressed as mean change 95% CI. Data were analyzed using a general linear model (GLM) with repeated measures two-way analysis of variances (ANOVA), with two levels by time (pre- and post-test or pre-, 2 months, and post-test) and groups (W and EW) as the Levene’s test revealed homoscedasticity and the Kolmogorov-Smirnov test revealed normality. In some cases, simple main effect test was performed following repeated measures two-way ANOVA. Changed data were analyzed using a GLM with one-way ANOVA as the Levene’s test revealed homoscedasticity and the Kolmogorov-Smirnov test revealed normality. In addition, effect size (ES) was calculated with Cohen’s d as a standardized measurement based on SD differences. Values closer to 1 is indicated substantive significance. Statistical analyses were performed using the SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA), and differences were considered statistically significant at p
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