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

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 < 30) were recruited to participate in the study. All participants provided written informed consent and were assigned to receive either EMIQ in whey protein (the EW group) or contrast whey protein (the W group), and the groups were stratified according to grade (junior vs. senior) and football position (back vs. lineman) using the “rand” function of Microsoft Excel (version 14.4.7). Time limits of whey protein powder with/without EMIQ are 18 months, and the protein was consumed within this limit. During the study period, the groups were identified as group A (the W group) and group B (the EW group) to ensure blinding.

The following participants were excluded:

One of the recruited participants was excluded because he failed to provide a blood sample.

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.

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).

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.

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 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].

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 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.

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.

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 < 0.05.

The FFQg questionnaire was used to evaluate nutrient intake; supplementary protein intake was not included in this questionnaire. There were no significant differences in nutrient intake differences during the study periods. There were no significant differences in nutrient intake during between the W and EW groups at 4 months, suggesting that dietary habits had no effect on the results (Table 2).

Results of blood tests at baseline and 4 months are shown in Table 3. One of the participants in the W group and three of those in the EW group could not provide blood samples at baseline because of not meeting the schedules. Only AST values were higher in the W group than in the EW group during the experiment. There was no significant changes due to EMIQ supplementation. RBC, and WBC counts, Hb, Ht, MCV, Crea, ALT, PLT, and γ-GTP increased from baseline to 4 months, possibly because of the strenuous training. However, the participants exhibited no significant deviations from the reference ranges for blood parameters during the study period. No adverse events were associated with the investigational product during the study period.

Basic information of the two groups is shown in Table 4, and body composition data at baseline and 4 months are shown in Table 5. There were no differences in the basic information between the two groups. Lower limb fat-free mass, lower limb muscle mass, and lower limb fat mass increased from baseline to 4 months, possibly because of the strenuous training and protein consumption. Increase in lower limb fat-free mass and lower limb muscle mass was markedly observed in the EW group (Group × Time interaction). Changes in the body composition from baseline to 4 months are shown in Fig. 3. Increases in lower limb fat-free and muscle masses were significantly greater in the EW group than in the W group (p = 0.030, and 0.020, respectively), with a large effect size (SE = 0.740, and 0.800, respectively). There were no significant differences in changes in lean body mass, fat mass and lower limb fat mass between the groups.

Antioxidant measurements are shown in Table 6. The EW and W groups did not exhibit any significant differences in BAP and d-ROMs at baseline. From baseline to 4 months, d-ROMs increased, indicating that the strenuous training increased plasma d-ROMs. Group interaction of BAP/d-ROMs showed 0.075; therefore, we analyzed simple main effect of groups. BAP/d-ROMs ratio at 4 months was significantly higher in the EW group than in the W group (p = 0.028, and ES = 0.750) Table 7.

The present study evaluated only young male athletes because participants included were American football players of the university. Thus, there is a risk of age-related bias as age can be a factor affecting muscle hypertrophy. Furthermore, it remains unclear whether these results can be observed in individuals who exercise less frequently because exercise habits are also a factor. Further studies on other participants based on these limitations may clarify the effectiveness of EMIQ.