Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
RESEARCH ARTICLE
Open Access
The effects of a multi-ingredient dietary supplement on body composition, adipokines, blood lipids, and metabolic health in overweight and obese men and women: a randomized controlled trial Michael J Ormsbee1,2,3*, Shweta R Rawal1, Daniel A Baur1, Amber W Kinsey1, Marcus L Elam1, Maria T Spicer1, Nicholas T Fischer1, Takudzwa A Madzima1 and D David Thomas1
Abstract Background: The present study investigated the effects of a multi-ingredient dietary supplement (MIDS) containing caffeine, conjugated linoleic acid (CLA), green tea, and branched-chain amino acids (BCAA) taken for 8 weeks on body composition, blood lipid profile, glucose, insulin, adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP) in overweight and obese men and women. Methods: Twenty-two participants completed the study (PL, n = 11; 7 women, 4 men; age, 34 ± 3.5 years; height, 169.2 ± 3.3 cm; body mass, 96.9 ± 6.8 kg; BMI, 34.1 ± 1.8 kg/m2; MIDS, n = 11; 9 women, 2 men; age, 36 ± 3.4 years; height, 173.2 ± 2.9 cm; body mass, 91.9 ± 5.6 kg; BMI, 30.0 ± 1.5 kg/m2). Participants were randomly assigned and stratified by body fat percentage to two groups: 1) a soybean oil placebo (PL) or 2) MIDS. Each group consumed two pills with breakfast and two pills with lunch. Body composition and android fat, waist and hip circumferences, blood pressure and heart rate were measured at baseline and after 8 weeks of supplementation. Results: There were no significant changes for any of the variables of body composition. Feelings of hunger were significantly higher in MIDS versus PL with no changes observed in satiety or desire to eat. Heart rate and blood pressure were unaltered in MIDS after 8 weeks of supplementation. Furthermore, lipid profile, food intake, mood state variables, fasting blood glucose, and endocrine markers did not significantly change regardless of group. Conclusion: MIDS intake does not appear to alter body composition or markers of cardiovascular health versus PL. Moreover, MIDS may actually increase feelings of hunger versus PL. Keywords: Green tea, Caffeine, Conjugated linoleic acid, Branched chain amino acids, Body composition, Obesity
Background Obesity is a growing trend in the United States with current estimates suggesting prevalence as high as 35% among adults [1]. These high rates potentiate a severe health crisis as obesity increases the likelihood of developing chronic diseases which include hypertension, insulin * Correspondence: [email protected] 1 Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA 2 University of KwaZulu-Natal, Durban, South Africa Full list of author information is available at the end of the article
resistance, type 2 diabetes mellitus (T2DM), arteriosclerosis, coronary heart disease, and metabolic syndrome [2]. Therefore, finding safe and effective methods for reducing body weight in obese individuals is essential. Reducing body weight requires manipulation of the energy balance equation to produce energy deficits. This can be accomplished through diet and exercise, pharmacological interventions, or surgical means. However, each of these methods comes with disadvantages. For instance, many diet and exercise lifestyle interventions suffer from a lack of long-term (≥1 yr) adherence [3]. Furthermore,
© 2014 Ormsbee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
pharmacological and/or surgical means to reduce body weight are typically expensive and are sometimes accompanied by potentially unpleasant and/or dangerous side effects [4,5]. As such, consideration of alternative weight loss methods is warranted. The consumption of natural ingredients and/or dietary supplements may provide a safe and effective means to induce weight loss and improve overall health. Indeed, recent evidence suggests that consumption of certain multi-ingredient dietary supplements may improve body composition and mood state [6,7]. The supplements utilized in these studies contained unique proprietary blends containing such ingredients as caffeine and green tea extract. These ingredients have been studied extensively and are now recognized as potential modulators of body weight, composition, adipokines, and metabolic health [8,9]. Beneficial effects of consuming green tea or caffeine, respectively, are likely a result of inhibited degradation of catecholamines (epinephrine and norepinephrine) or cyclic amino monophospates (cAMP) thereby enhancing thermogenesis and promoting lipolysis [10,11]. Of interest, the lipolytic effects reported with green tea and caffeine consumption may be the result of a synergistic effect as noted in a recent meta-analysis [12]. Supplementation with conjugated-linoleic acid (CLA) and branched-chain amino acids (BCAA) may also provide weight loss benefits [13,14]. These CLA-derived effects may be a result of enhanced β-oxidation via stimulation of enzymes responsible for transport of lipids into the mitochondria (i.e. carnitine palmitoyl transferase [CPT1]) [15], or through inhibition of adipocyte differentiation [16]. BCAA may improve body composition by enhancing protein synthesis, which maintains or increases lean mass [17]. While the efficacy of these ingredients has been extensively studied individually, there is a lack of research on the impact of combining these ingredients on body weight, composition and other markers of health. Furthermore, combining CLA and BCAA with green tea extracts and caffeine has also yet to be studied. Therefore, the purpose of the present study was to examine the effects of a multi-ingredient dietary supplement (MIDS) containing a proprietary blend of green tea extracts, caffeine, CLA, and BCAA on body composition, blood lipid profile, glucose, insulin, adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP) concentrations in overweight and obese men and women.
Methods Participants
Thirty-four inactive (2 times/week of planned physical activity for > 60 minutes), if they had uncontrolled hypertension (BP >140/ 90 mmHg), if they had high low-density lipoprotein cholesterol (LDL >160 mg/dL) or if they took cholesterol medication. In addition, those diagnosed with cardiovascular disease, stroke, diabetes, thyroid or kidney dysfunction, and smokers (>5 cigarettes per week) were excluded. Those who consumed any dietary supplements intended to alter body composition and body weight were excluded. In addition, those who had any allergies to soy, wheat, and grain products that would cause a health problem were excluded. This study was approved by Florida State University Institutional Review Board. Study design
This was a randomized, double-blinded, placebo-controlled study with two treatment groups. The participants were stratified based on body fat percentage and assigned to a placebo group (PL; soybean oil) or an isocaloric multiingredient dietary supplement group (MIDS; Suarez Corporation Industries, Canton, Ohio; 10 calories; 1 g fat, 99 mg caffeine, 1510 mg of a proprietary blend of: green tea, CLA, and BCAA per 2-pill serving [2 servings = 4 pills/day]). The green tea was standardized for 45% epigallocatechin gallate and 90% polyphenols. Both groups ingested 2 identical pills with both breakfast and lunch 7 days per week for 8 weeks. All laboratory procedures were conducted following an 8 – 10 h overnight fast and a 24 h abstinence from caffeine, alcohol intake and any intense physical activity. Additionally, participants abstained from consuming their assigned treatment on testing days. Every visit was completed between 7 am and 10 am. Anthropometrics
Height and body mass were measured using a wall-mounted stadiometer and a digital scale (SECA, Birmingham, UK) every 2 weeks under identical conditions (shorts, t-shirt) for each subject. Waist and hip circumferences were obtained at baseline and after 8 weeks using a standard tape measure (with strain gauge) at the maximum area around the waist line and around the largest component of the gluteus maximus. All circumference measurements were conducted by the same researcher. Total and regional body composition was determined by dual energy X-ray absorptiometry (iDXA; GE Lunar, Madison, WI) with participants in the supine position. Total body adiposity was expressed as percent body fat (% BF). Abdominal adiposity was determined by creating regions of interest (ROI) for the abdomen (region 1)
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
using the ROI option within the manual analysis menu of the iDXA software and included the area just below the last rib to just above the iliac crest, as described previously [18]. Abdominal adiposity was expressed as percent android fat (% android fat). A certified radiologist technician performed all iDXA analyses. Waist circumferences were measured at the maximum circumference around the umbilical point. Although this is not the standard measurement method for waist circumference, the abdominal obesity of many of the participants prevented the use of standard waist measurement techniques (i.e. 1–2 cm below the last rib typically corresponding with the narrowest part of the waist). Measurements were taken by the same researcher to minimize errors. Heart rate and blood pressure
Resting heart rate and blood pressure were measured at 0, 2, 4, 6, and 8 weeks. The participants were instructed to rest in a seated position, with both feet flat on the floor for 5 minutes before measurements were taken. Resting blood pressures were obtained by the same investigator on the same arm using an appropriately sized cuff and a sphygmomanometer (American Diagnostic Corp., Hauppauge, NY). Resting heart rate was obtained from a radial pulse. Two readings were taken for blood pressures and resting heart rate with 1 min intervals between each reading. The mean of the two readings were used for statistical analysis. Questionnaires: dietary intake, physical activity, hunger, and mood-state
Two-day diet logs were completed at weeks 0, 4, and 8 to quantify calorie and macronutrient intake. The participants were asked to maintain their food intake throughout the duration of the study. The Food Processor software (version 10.9 ESHA Research Salem, OR, USA) was used to analyze the food logs. Participants were asked to not change their physical activity during intervention period. Physical activity logs completed by each participant were collected at weeks 0, 4 and 8. Adverse events forms were collected and recorded at weeks 2, 4, 6 and 8 by research staff. Mood state was accessed via the completion of a 65question mood state questionnaire which assessed mood state at weeks 0, 4 and 8. This was a simple likert scale (0, very – 4, not at all) questionnaire. Hunger was assessed at the same time points using a simple visual analog scale (VAS) (1 to 100 mm). Participants were instructed to place a mark on the 100 mm line to indicate their levels of hunger, satiety, and desire to eat. A mark at 0 indicated a complete lack of hunger, satiety, or desire to eat and a mark at 100 indicated extreme hunger, satiety, or desire to eat for each VAS, respectively. For each of the three measures (hunger, satiety, and desire to eat), the degree in which each sensation was felt was quantified by measuring
Page 3 of 10
how far the mark was from the 0 mm mark. For this measurement, a standard millimeter ruler was used and all scores were computed by the same investigator. VAS scales were completed at baseline (week 0) and postintervention (week 8) in a fasted state. Hormones, blood lipids and blood glucose
A venous blood sample (20 ml) was obtained from the antecubital vein at baseline and after 8 weeks of supplementation (post). Whole blood was used to measure total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TRG), and glucose concentrations using the Cholestech LDX blood analysis system (Hayward, CA). Inter-assay coefficients of variation were 2.1%, 4.0%, 4.1%, 4.7%, and 2.3% for TC, HDL, LDL, TRG, and glucose, respectively. Remaining samples were centrifuged (IEC CL3R Multispeed Centrifuge, ThermoElectron Corporation, Needham Heights, MA) for 15 minutes at 3500 rpm at 4°C. Serum aliquots of 300 μL were transferred into microtubes and stored at −80°C for later analysis of insulin, leptin, adiponectin, and hs-CRP. All assays were performed in duplicate in a single assay using commercially available ELISA kits according to the manufacturer’s instructions (leptin and adiponectin: R&D Systems Inc., Minneapolis, MN, USA; hs-CRP and insulin: IBL International, Inc., Hamburg, Germany). The mean of duplicate samples were used for statistical analysis. Interassay coefficients of variation were 1.3%, 6.2%, and 5.2% for leptin, adiponectin, and hs-CRP, respectively. Inter-assay data were missing for insulin due to technical errors. Intraassay coefficients of variation were 5.6%, 13.8%, 3.8%, and 8.1% for insulin, leptin, adiponectin, and hs-CRP respectively. Insulin resistance was assessed using the homeostatic model of assessment, as described previously [19]. Compliance
Compliance was checked by asking participants to bring supplement containers every 4 weeks. Weekly calls or emails were sent to remind participants to take the supplement and maintain their food and physical activity logs for the duration of the study. Statistical analysis
An a priori power analysis was performed which revealed a need for a minimum of 6 participants per group to achieve a power of 0.80, α = 0.05, standard deviation = 1.1, difference = 6 (41). One way analysis of variance (ANOVA) was performed to examine possible group differences at baseline. Data were analyzed using a 2X5 repeated measures ANOVA ([PL × MIDS]) × ([week 0 × week 2 × week 4 × week 6 × week 8]). Data were analyzed using JMP PRO 9 software (Cary, NC). If significant main effects were identified by ANOVA, a Tukey post hoc comparison test was performed to locate differences.
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
Page 4 of 10
Results Participant demographics
A total of 160 individuals were pre-screened for participation in this study. Of the 68 individuals eligible for the study, 34 decided to participate. Out of these 34 participants, 5 participants withdrew from the study due to personal reasons. A total of 29 participants completed the study, however, data from 7 participants was excluded due to low compliance (
RESEARCH ARTICLE
Open Access
The effects of a multi-ingredient dietary supplement on body composition, adipokines, blood lipids, and metabolic health in overweight and obese men and women: a randomized controlled trial Michael J Ormsbee1,2,3*, Shweta R Rawal1, Daniel A Baur1, Amber W Kinsey1, Marcus L Elam1, Maria T Spicer1, Nicholas T Fischer1, Takudzwa A Madzima1 and D David Thomas1
Abstract Background: The present study investigated the effects of a multi-ingredient dietary supplement (MIDS) containing caffeine, conjugated linoleic acid (CLA), green tea, and branched-chain amino acids (BCAA) taken for 8 weeks on body composition, blood lipid profile, glucose, insulin, adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP) in overweight and obese men and women. Methods: Twenty-two participants completed the study (PL, n = 11; 7 women, 4 men; age, 34 ± 3.5 years; height, 169.2 ± 3.3 cm; body mass, 96.9 ± 6.8 kg; BMI, 34.1 ± 1.8 kg/m2; MIDS, n = 11; 9 women, 2 men; age, 36 ± 3.4 years; height, 173.2 ± 2.9 cm; body mass, 91.9 ± 5.6 kg; BMI, 30.0 ± 1.5 kg/m2). Participants were randomly assigned and stratified by body fat percentage to two groups: 1) a soybean oil placebo (PL) or 2) MIDS. Each group consumed two pills with breakfast and two pills with lunch. Body composition and android fat, waist and hip circumferences, blood pressure and heart rate were measured at baseline and after 8 weeks of supplementation. Results: There were no significant changes for any of the variables of body composition. Feelings of hunger were significantly higher in MIDS versus PL with no changes observed in satiety or desire to eat. Heart rate and blood pressure were unaltered in MIDS after 8 weeks of supplementation. Furthermore, lipid profile, food intake, mood state variables, fasting blood glucose, and endocrine markers did not significantly change regardless of group. Conclusion: MIDS intake does not appear to alter body composition or markers of cardiovascular health versus PL. Moreover, MIDS may actually increase feelings of hunger versus PL. Keywords: Green tea, Caffeine, Conjugated linoleic acid, Branched chain amino acids, Body composition, Obesity
Background Obesity is a growing trend in the United States with current estimates suggesting prevalence as high as 35% among adults [1]. These high rates potentiate a severe health crisis as obesity increases the likelihood of developing chronic diseases which include hypertension, insulin * Correspondence: [email protected] 1 Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA 2 University of KwaZulu-Natal, Durban, South Africa Full list of author information is available at the end of the article
resistance, type 2 diabetes mellitus (T2DM), arteriosclerosis, coronary heart disease, and metabolic syndrome [2]. Therefore, finding safe and effective methods for reducing body weight in obese individuals is essential. Reducing body weight requires manipulation of the energy balance equation to produce energy deficits. This can be accomplished through diet and exercise, pharmacological interventions, or surgical means. However, each of these methods comes with disadvantages. For instance, many diet and exercise lifestyle interventions suffer from a lack of long-term (≥1 yr) adherence [3]. Furthermore,
© 2014 Ormsbee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
pharmacological and/or surgical means to reduce body weight are typically expensive and are sometimes accompanied by potentially unpleasant and/or dangerous side effects [4,5]. As such, consideration of alternative weight loss methods is warranted. The consumption of natural ingredients and/or dietary supplements may provide a safe and effective means to induce weight loss and improve overall health. Indeed, recent evidence suggests that consumption of certain multi-ingredient dietary supplements may improve body composition and mood state [6,7]. The supplements utilized in these studies contained unique proprietary blends containing such ingredients as caffeine and green tea extract. These ingredients have been studied extensively and are now recognized as potential modulators of body weight, composition, adipokines, and metabolic health [8,9]. Beneficial effects of consuming green tea or caffeine, respectively, are likely a result of inhibited degradation of catecholamines (epinephrine and norepinephrine) or cyclic amino monophospates (cAMP) thereby enhancing thermogenesis and promoting lipolysis [10,11]. Of interest, the lipolytic effects reported with green tea and caffeine consumption may be the result of a synergistic effect as noted in a recent meta-analysis [12]. Supplementation with conjugated-linoleic acid (CLA) and branched-chain amino acids (BCAA) may also provide weight loss benefits [13,14]. These CLA-derived effects may be a result of enhanced β-oxidation via stimulation of enzymes responsible for transport of lipids into the mitochondria (i.e. carnitine palmitoyl transferase [CPT1]) [15], or through inhibition of adipocyte differentiation [16]. BCAA may improve body composition by enhancing protein synthesis, which maintains or increases lean mass [17]. While the efficacy of these ingredients has been extensively studied individually, there is a lack of research on the impact of combining these ingredients on body weight, composition and other markers of health. Furthermore, combining CLA and BCAA with green tea extracts and caffeine has also yet to be studied. Therefore, the purpose of the present study was to examine the effects of a multi-ingredient dietary supplement (MIDS) containing a proprietary blend of green tea extracts, caffeine, CLA, and BCAA on body composition, blood lipid profile, glucose, insulin, adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP) concentrations in overweight and obese men and women.
Methods Participants
Thirty-four inactive (2 times/week of planned physical activity for > 60 minutes), if they had uncontrolled hypertension (BP >140/ 90 mmHg), if they had high low-density lipoprotein cholesterol (LDL >160 mg/dL) or if they took cholesterol medication. In addition, those diagnosed with cardiovascular disease, stroke, diabetes, thyroid or kidney dysfunction, and smokers (>5 cigarettes per week) were excluded. Those who consumed any dietary supplements intended to alter body composition and body weight were excluded. In addition, those who had any allergies to soy, wheat, and grain products that would cause a health problem were excluded. This study was approved by Florida State University Institutional Review Board. Study design
This was a randomized, double-blinded, placebo-controlled study with two treatment groups. The participants were stratified based on body fat percentage and assigned to a placebo group (PL; soybean oil) or an isocaloric multiingredient dietary supplement group (MIDS; Suarez Corporation Industries, Canton, Ohio; 10 calories; 1 g fat, 99 mg caffeine, 1510 mg of a proprietary blend of: green tea, CLA, and BCAA per 2-pill serving [2 servings = 4 pills/day]). The green tea was standardized for 45% epigallocatechin gallate and 90% polyphenols. Both groups ingested 2 identical pills with both breakfast and lunch 7 days per week for 8 weeks. All laboratory procedures were conducted following an 8 – 10 h overnight fast and a 24 h abstinence from caffeine, alcohol intake and any intense physical activity. Additionally, participants abstained from consuming their assigned treatment on testing days. Every visit was completed between 7 am and 10 am. Anthropometrics
Height and body mass were measured using a wall-mounted stadiometer and a digital scale (SECA, Birmingham, UK) every 2 weeks under identical conditions (shorts, t-shirt) for each subject. Waist and hip circumferences were obtained at baseline and after 8 weeks using a standard tape measure (with strain gauge) at the maximum area around the waist line and around the largest component of the gluteus maximus. All circumference measurements were conducted by the same researcher. Total and regional body composition was determined by dual energy X-ray absorptiometry (iDXA; GE Lunar, Madison, WI) with participants in the supine position. Total body adiposity was expressed as percent body fat (% BF). Abdominal adiposity was determined by creating regions of interest (ROI) for the abdomen (region 1)
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
using the ROI option within the manual analysis menu of the iDXA software and included the area just below the last rib to just above the iliac crest, as described previously [18]. Abdominal adiposity was expressed as percent android fat (% android fat). A certified radiologist technician performed all iDXA analyses. Waist circumferences were measured at the maximum circumference around the umbilical point. Although this is not the standard measurement method for waist circumference, the abdominal obesity of many of the participants prevented the use of standard waist measurement techniques (i.e. 1–2 cm below the last rib typically corresponding with the narrowest part of the waist). Measurements were taken by the same researcher to minimize errors. Heart rate and blood pressure
Resting heart rate and blood pressure were measured at 0, 2, 4, 6, and 8 weeks. The participants were instructed to rest in a seated position, with both feet flat on the floor for 5 minutes before measurements were taken. Resting blood pressures were obtained by the same investigator on the same arm using an appropriately sized cuff and a sphygmomanometer (American Diagnostic Corp., Hauppauge, NY). Resting heart rate was obtained from a radial pulse. Two readings were taken for blood pressures and resting heart rate with 1 min intervals between each reading. The mean of the two readings were used for statistical analysis. Questionnaires: dietary intake, physical activity, hunger, and mood-state
Two-day diet logs were completed at weeks 0, 4, and 8 to quantify calorie and macronutrient intake. The participants were asked to maintain their food intake throughout the duration of the study. The Food Processor software (version 10.9 ESHA Research Salem, OR, USA) was used to analyze the food logs. Participants were asked to not change their physical activity during intervention period. Physical activity logs completed by each participant were collected at weeks 0, 4 and 8. Adverse events forms were collected and recorded at weeks 2, 4, 6 and 8 by research staff. Mood state was accessed via the completion of a 65question mood state questionnaire which assessed mood state at weeks 0, 4 and 8. This was a simple likert scale (0, very – 4, not at all) questionnaire. Hunger was assessed at the same time points using a simple visual analog scale (VAS) (1 to 100 mm). Participants were instructed to place a mark on the 100 mm line to indicate their levels of hunger, satiety, and desire to eat. A mark at 0 indicated a complete lack of hunger, satiety, or desire to eat and a mark at 100 indicated extreme hunger, satiety, or desire to eat for each VAS, respectively. For each of the three measures (hunger, satiety, and desire to eat), the degree in which each sensation was felt was quantified by measuring
Page 3 of 10
how far the mark was from the 0 mm mark. For this measurement, a standard millimeter ruler was used and all scores were computed by the same investigator. VAS scales were completed at baseline (week 0) and postintervention (week 8) in a fasted state. Hormones, blood lipids and blood glucose
A venous blood sample (20 ml) was obtained from the antecubital vein at baseline and after 8 weeks of supplementation (post). Whole blood was used to measure total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TRG), and glucose concentrations using the Cholestech LDX blood analysis system (Hayward, CA). Inter-assay coefficients of variation were 2.1%, 4.0%, 4.1%, 4.7%, and 2.3% for TC, HDL, LDL, TRG, and glucose, respectively. Remaining samples were centrifuged (IEC CL3R Multispeed Centrifuge, ThermoElectron Corporation, Needham Heights, MA) for 15 minutes at 3500 rpm at 4°C. Serum aliquots of 300 μL were transferred into microtubes and stored at −80°C for later analysis of insulin, leptin, adiponectin, and hs-CRP. All assays were performed in duplicate in a single assay using commercially available ELISA kits according to the manufacturer’s instructions (leptin and adiponectin: R&D Systems Inc., Minneapolis, MN, USA; hs-CRP and insulin: IBL International, Inc., Hamburg, Germany). The mean of duplicate samples were used for statistical analysis. Interassay coefficients of variation were 1.3%, 6.2%, and 5.2% for leptin, adiponectin, and hs-CRP, respectively. Inter-assay data were missing for insulin due to technical errors. Intraassay coefficients of variation were 5.6%, 13.8%, 3.8%, and 8.1% for insulin, leptin, adiponectin, and hs-CRP respectively. Insulin resistance was assessed using the homeostatic model of assessment, as described previously [19]. Compliance
Compliance was checked by asking participants to bring supplement containers every 4 weeks. Weekly calls or emails were sent to remind participants to take the supplement and maintain their food and physical activity logs for the duration of the study. Statistical analysis
An a priori power analysis was performed which revealed a need for a minimum of 6 participants per group to achieve a power of 0.80, α = 0.05, standard deviation = 1.1, difference = 6 (41). One way analysis of variance (ANOVA) was performed to examine possible group differences at baseline. Data were analyzed using a 2X5 repeated measures ANOVA ([PL × MIDS]) × ([week 0 × week 2 × week 4 × week 6 × week 8]). Data were analyzed using JMP PRO 9 software (Cary, NC). If significant main effects were identified by ANOVA, a Tukey post hoc comparison test was performed to locate differences.
Ormsbee et al. Journal of the International Society of Sports Nutrition 2014, 11:37 http://www.jissn.com/content/11/1/37
Page 4 of 10
Results Participant demographics
A total of 160 individuals were pre-screened for participation in this study. Of the 68 individuals eligible for the study, 34 decided to participate. Out of these 34 participants, 5 participants withdrew from the study due to personal reasons. A total of 29 participants completed the study, however, data from 7 participants was excluded due to low compliance (
