Participants

Thirty apparently healthy males with a mean age of 20.43 ± 1.71 years, height of 176.67 ± 8.02 cm, and total body mass of 80.35 ± 18.52 kg served as participants in the study. The participants were not resistance-trained [not following a consistent resistance training program (i.e. thrice weekly) for at least one year prior to the study], but were recreationally-active. All participants were cleared for participation by passing a mandatory medical screening. Participants with contraindications to exercise as outlined by the American College of Sports Medicine and/or who had consumed any nutritional supplements (excluding multi-vitamins) such creatine monohydrate or various androstenedione derivatives or pharmacologic agents such as anabolic steroids three months prior to the study were not allowed to participate. All eligible subjects signed a university-approved informed consent document. Additionally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code.

Testing sessions

The study included baseline testing at day 0, followed by testing sessions at days 6, 27, and 48 in which blood and muscle samples were obtained and where body composition and muscle performance tests were performed.

Strength assessment

The leg press and bench press maximal strength tests (Nebula, Versailles, OH) were performed by the participants to measure any changes in muscular strength during the course of the study. Four one repetition maximum (1-RM) strength tests were performed during the study at days 0, 6, 27, and 48. Initially, an estimated 50% (1-RM) measured from the previous testing 1-RM test, was utilized to complete 5 to 10 repetitions. After a two minute rest period, a load of 70% of estimated (1-RM) was utilized to perform 3 to 5 repetitions. Weight was gradually increased until a 1-RM was reached with each following lift, with a two-minute rest period in between each successful lift. Test-retest reliability of performing these strength assessments on subjects within our laboratory has demonstrated low mean coefficients of variation and high reliability for the bench press (1.9%, intraclass r = 0.94) and leg press (0.7%, intraclass r = 0.91), respectively.

Anaerobic power test

Anaerobic power was determined during each of the four testing sessions at days 0, 6, 27, and 48, and expressed relative to body mass. The determinations were made by performing a 30-second Wingate test on a computerized Lode cycle ergometer (Groningen, Netherlands). A warm-up of 30 rpm for 120 seconds was followed by maximal sprint for 30 seconds against a workload of 0.075 kg/kg of body weight. Correlation coefficients of test-retest reliability of performing these assessments of absolute peak power and mean power on participants within our laboratory has been found to be r = 0.692 and r = 0.950, respectively.

Body composition assessment

Total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Percent body fat, fat mass, and fat-free mass were determined using DEXA (Hologic Discovery Series W, Waltham, MA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALIBER Model DPA/QDR-1 anthropometric spine phantom) and a density step calibration phantom prior to each testing session. The DEXA scans were segmented into regions (right & left arm, right & left leg, and trunk). Each of these segments was analyzed for fat mass, lean mass, and bone mass. A sub-region was utilized to determine right thigh mass. The isolated region extended medially to the pubic symphysis down to the head of the femur. Total body water and compartment-specific fluid volumes were determined by bioelectric impedance analysis (Xitron Technologies Inc., San Diego, CA) using a low energy, high frequency current (500 micro-amps at a frequency of 50 kHz). Based on previous studies in our laboratory, the accuracy of the DEXA for body composition assessment is ± 2% as assessed by direct comparison with hydrodensitometry and scale weight.

Supplementation protocol

Participants were randomly assigned to one of three groups in a double blind manner in which they orally ingested capsules and powder which contained either dextrose placebo [PLC (AST Sport Science, Colorado Springs, CO)], creatine monohydrate [CRT (Integrity Nutraceuticals, Sarasota, FL)], or creatine ethyl ester [CEE (Labrada Nutritionals, Houston, TX)]. For CRT, each capsule contained 250 mg of creatine monohydrate; however, for CEE each capsule contained 700 mg of creatine ethyl ester. Quality control testing of the creatine ethyl ester supplement using NMR from an independent laboratory from the University of Nebraska determined the product to contain 100% creatine ethyl ester HCL, with no detectable creatine HCL or creatinine HCL. The creatine supplement was shown to contain 99.8% creatine monohydrate and 0.2% creatinine.

After baseline testing procedures and fat-free mass determination by DEXA, supplements placebo were ingested relative to fat-free mass based on previous guidelines [17] for 48 days (loading from days 1–5 and maintenance from days 6–48.). Specifically, supplements were ingested at a relative daily dose of 0.30 g/kg fat-free body mass (approximately 20 g/day) during the loading phase, and at a relative daily dose of 0.075 g/kg fat free mass (approximately 5 g/day) during the maintenance phase. After the initial baseline assessment of body composition at day 0, supplement dosages were subsequently adjusted based on body composition assessments performed at days 6 and 27.

In order to standardize supplement intake throughout the study, participants were instructed to ingest the supplements in two equal intervals, one in the morning and one in the evening, throughout the day during the loading phase [13], and at one constant interval, in the morning, during the maintenance phase. Compliance to the supplementation protocol was monitored by supplement logs and verbal confirmation. After completing the compliance procedures the subjects were given the required supplement dosage for the following supplementation period.

Resistance training protocol

Participants engaged in a 4-day per week resistance-training program split into two upper and two lower extremity workouts per week for a total of seven weeks. The upper body resistance-training program consisted of nine exercises (bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flies, biceps curl, triceps press down, and abdominal curls) twice per week and a seven exercise lower extremity program (leg press or squat, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches) performed twice per week. We have previously shown this program to be effective at promoting significant gains in muscle strength and mass [18]. Participants performed 3 sets of 8–10 repetitions with 70–80% 1-RM. Rest periods between exercises lasted no longer than three minutes and rest between sets lasted no longer than two minutes. Training sessions were not supervised, but were documented in training logs, and signed off to verify compliance and to monitor progress.

Muscle biopsies and venous blood sampling

Based on our previously-established guidelines [18], at each of the four testing sessions at days 0, 6, 27, and 48 percutaneous muscle biopsies (50–70 mg) were obtained using a Bergstrom (5 mm) needle. Muscle samples were obtained from the middle portion of the vastus lateralis muscle of the dominant leg at the midpoint between the patella and the greater trochanter of the femur, at a depth between one and two cm. For the remaining three biopsies, attempts were made to extract tissue from approximately the same location as the initial biopsy by using the pre-biopsy scar, depth markings on the needle, and a successive incision that was made approximately 0.5 cm to the former from medial to lateral. After removal, the muscle specimens were immediately frozen in liquid nitrogen and then stored at -80°C for later analysis.

At each of the four testing sessions, venous blood samples were obtained from the antecubital vein using a standard Vacutainer apparatus. Once collected, the samples were centrifuged for 15 minutes. The serum was removed and frozen at -80°C for later analysis. An 8-hour fast prior to blood donation was required for the participants before each of the four testing sessions.

Muscle and serum creatine analysis

Muscle tissue samples were analyzed spectrophotometrically for total creatine by the diacetyl/α-napthtol reaction [19]. Using similar methods, serum samples were measured in duplicate for creatine concentration. Serum samples were immediately ready for creatine analysis, whereas muscle tissue had to first be prepared. For serum creatine analysis, duplicates for all samples yielded a coefficient of variation of 5.4%.

Approximately 10–15 mg of muscle tissue was cut and placed in a microfuge tube, and then placed in a vacuum centrifuge (Savant ISS110 SpeedVac™ Concentrator, Thermo Scientific, Milford, MA) to be spun for 18–24 hours. After sufficient muscle drying, the samples were then placed in an ultra-low freezer at -80°C. Dried muscle was powdered by grinding on a porcelain plate with a pestle. Connective tissue was removed and discarded, whereas powdered muscle was placed into pre-weighed microfuge tubes. Powdered muscle was extracted in a 0.5 M perchloric acid/1 mM EDTA solution on ice for 15-minutes, while periodically vortexing. Samples were then spun at 15,000 rpm at 4°C for 5-minutes. The supernatant was transferred into a microfuge tube and neutralized with 2.1 M KHCO₃/0.3 M MOPS solution and then centrifuged again at 15,000 rpm for 5-minutes. In order to determine muscle total creatine concentration, supernatant from the above reaction was combined with ddH₂O and 0.4 N HCl and heated at 65°C for 10-minutes to hydrolyze phosphate groups. The solution was then neutralized with of 2.0 N NaOH and the samples were allowed to incubate at room temperature allowing for color formation, which was detected by a spectrophotometer at 520 nm. Then the samples were run in duplicate against a standard curve of known creatine concentrations. The mean correlation coefficient of variation between duplicates was 1.53%. The standard curve correlation coefficient between plates for total muscle creatine was 0.998.

Dietary intake records and supplementation compliance

Throughout the course of the study, participants' dietary intake was not supervised; however, it was required that all participants keep detailed dietary records and not change their routine dietary habits throughout the course of the study. As such, participants were required to keep weekly physical activity records and four-day dietary records (three weekdays and one weekend) prior to each of the four testing sessions. The four-day dietary recalls were evaluated with the Food Processor dietary assessment software program (ESHA Research, Salem, OR) to determine the average daily macronutrient consumption of fat, carbohydrate, and protein. The participants were instructed to turn in their dietary records during each testing session. Each participant returned all of their dietary evaluations at the required time points for a 100% compliance rate. In an effort to ensure compliance to the supplementation protocol, participants were supplied with the appropriate amount of supplement to be ingested during the time between last three testing sessions. Upon reporting to the lab for each testing session at days 6, 27, and 48, participants returned the empty containers they had acquired between testing sessions

Reported side effects from supplements

At the last three testing sessions, participants reported by questionnaire whether they tolerated the supplement, supplementation protocol, as well as report any medical problems/symptoms they may have encountered throughout the study.

Statistical analysis

Data were analyzed using separate 3 (group) × time [4] univariate analysis of variance (ANOVA) with repeated measures on the time factor with SPSS for Windows Version 16.0 software (SPSS inc., Chicago, IL). Significant differences among groups were identified by a Tukey HSD post-hoc test. A probability level of ≤ 0.05 was adopted throughout.

Subject Demographics

Dietary analysis, supplement compliance, and side effects

Serum creatine

A significant difference among the three groups was observed indicating significantly higher serum creatine concentrations in the CRT group when compared to PLA (p = 0.007) and CEE (p = 0.005) (Figure 1). Also, significant differences for CRT occurred at days 6 (p = 0.028), 27 (p = 0.014), and 48 (p = 0.032). Muscle creatine

A significant difference among groups for total muscle creatine indicated that total muscle creatine content was significantly higher in the CRT (p = 0.026) and CEE (p = 0.041) groups when compared to the PLA group. Significant differences over the course of the four testing sessions were observed indicating that the CRT group underwent increases in total muscle creatine at day 6 (p = 0.041) and 27 (p= 0.036), whereas CEE only increased at day 27 (p = 0.043) (Figure 2).

Serum creatinine

A significant difference over the course of the four testing sessions (p = 0.001) and significant difference between groups (p = 0.001) was observed for serum creatinine. Serum creatinine was greater in the CEE group compared to the PLA (p = 0.001) and CRT (p = 0.001) groups. Further analysis revealed significant elevations in serum creatinine with the CEE group that occurred days 6 (p = 0.007), 27 (p = 0.005), and 48 (p = 0.005) (Figure 3).

Body composition

Body water

Muscle strength

Anaerobic Power

Serum and Muscle Creatine

Studies have shown the acute ingestion of 5 g and 20 g of creatine monohydrate to increase serum levels of creatine [5]. The recommended loading and maintenance dosages for creatine ethyl ester are 10 g and 5 g, respectively. As a result, in the present study participants ingested twice the recommended dose of creatine ethyl ester, yet the CRT group resulted in significantly higher levels of serum creatine than the CEE group (Figure 1). Total muscle creatine for the CRT group was significantly greater than the PLA group, but not the CEE group. However, in light of ingesting twice the recommended dose of creatine ethyl ester, total muscle creatine concentration for the CEE group was not significantly different from either the PLA or CRT groups (Figure 2). There was a significant increase in total muscle creatine levels for the CRT at day 6 and 27; however, for CEE an increase was observed to occur at day 27. This is in agreement with most other studies showing significant increases in muscle creatine [3, 20–22].

Serum Creatinine

For serum creatinine, the CEE group underwent significant increases compared to the PLA and CRT groups at days 6 and 48 (Figure 3). In the CEE group, creatinine levels increased 3-fold after the loading phase, and continued to be elevated above normal values throughout the study. This observation can likely be based on the premise that creatine ethyl ester has been shown to be degraded to creatinine in stomach acid (Tallon). Creatinine levels for the CRT group did elevate, but stayed within the normal range of 0.8–1.3 mg/dL, while the PLA group stayed near baseline levels. Serum creatinine is of importance because creatinine is the by-product of creatine degradation. Creatine is non-enzymatically converted into creatinine at approximately 1.7% daily for a typical 70 kg individual [23]. Creatine is also degraded by the gut into creatinine at an estimated rate of 0.1 g of a 5 g dose per hour. This indicates that the GI tract is not a major source of creatinine production; therefore, skeletal muscle is the primary site of creatinine production. [13, 24]. With increases in muscle saturation of creatine, creatinine levels will increase due to reduction in the skeletal muscle uptake [1]. In the CRT group, skeletal muscle total creatine content underwent a significant increase at day 6 and 27, whereas the CEE group only increased at day 27. In light of the results for serum creatine and total muscle creatine, based on the premise that serum creatinine levels for CEE were significantly increased at days 6 and 48 (Figures 2 &3) our results seem to indicate that creatine esterification does not provide a superior alternative to creatine monohydrate for muscle creatine uptake.

Supplementation was based on fat-free mass for all groups but was comparable to a 20 g loading phase and a 5 g maintenance phase typically seen with creatine supplementation. When creatine is esterified with an alcohol group, the structure yields approximately 17.4 g of creatine for a 20 g dose and 4.37 g for a 5 g dosage [14]. The recommended loading and maintenance dosages for creatine ethyl ester are 10 g and 5 g, respectively. The supplement loading phase in the present study consisted of two 10 g dosages based on the premise that for a 10 g dose, maximal absorption usually occurs within two hours [13]. Blood draws were not taken specifically after supplementation, yet serum creatinine levels were approximately tripled at day 6 (2.68 ± SD 1.53 mg/dL) compared to baseline (0.95 ± SD 0.18 mg/dL) for the CEE group.

Muscle Mass and Body Composition

Non-resistance trained participants were selected to perform a 47-day (4 days/week) training program and were expected to have changes in muscle mass and body composition, independent of supplementation. Compared to day 0, all groups showed significant increases in body weight at each of the three testing sessions (Table 3). While all groups increased in total body mass, there was no significant difference between the three groups. Various studies have shown an average of 1–2 kg of total body mass increases with 20 g/day of creatine supplementation for 5–7 days [4, 21, 23, 25]. Total body mass increases after the 5-day loading phase were 0.03 ± 0.60 kg, 1.39 ± 0.46 kg, and 0.80 ± 0.51 kg for PLA, CRT, and CEE, respectively. Kreider [8] indicated that short duration (5–7 days) of creatine supplementation at 20–25 g/day typically leads to increases of up to 1.6 kg in total body mass. The total body mass increase observed with the CRT group was within typical ranges previously seen [26, 27], even though there were no significant differences between the groups. For fat mass, fat-free mass, and thigh mass there were no significant differences between any of the three groups. However, collectively fat-free mass was shown to increase at days 6, 27, and 48 compared to day 0. Fat-free mass was also significantly increased at days 27 and 48 compared to day 6 (Table 3). Fat-free mass increases after the 5-day loading phase were 0.55 ± 0.46 kg, 1.41 ± 0.29 kg, and 0.68 ± 0.42 kg for PLA, CRT, and CEE, respectively. Previous studies have shown that longer duration (12 weeks) of creatine supplementation with resistance exercise [28] and shorter duration (5 days loading and 4 days of maintenance) creatine supplementation to increase fat-free mass [29]. As anticipated with an untrained population, increases in body mass and fat-free mass were expected due to a training effect. In line with fat-free mass increases, thigh muscle mass increases were also observed throughout the duration of the study. Thigh mass increases after the 5-day loading phase were 0.10 ± 0.04 kg, 0.24 ± 0.53 kg, and 0.48 ± 0.02 kg for PLA, CRT, and CEE, respectively. In contrast to total body mass and fat-free mass, the CRT group showed the largest increase in thigh muscle mass (Table 3). Fat mass was shown to significantly decrease at days 6, 27, and 48. Both PLA and CRT groups had reductions in fat mass throughout the study, whereas CEE underwent a slight increase (Table 3). Specifically, fat mass was shown to decrease 0.64 ± 0.08 kg and 1.47 ± 0.35 kg, respectively, whereas the CEE group increased 0.44 ± 0.68 kg. Although not statistically significant, it should be noted that the CRT group had a higher baseline fat mass than the PLA and CEE groups. Even though total body mass and fat-free mass were not statistically different, the CRT group may have had a greater potential for reductions in fat mass than the CEE group. As such, the reduction of fat mass observed with the PLA, CRT, and CEE groups was mostly likely due to the resistance training rather than supplementation.

Body Water

Total, intracellular, and extracellular body water are of particular interest for the CEE group. Claims by the manufactures of creatine ethyl ester have stated a difference in the retention of body water compared to other forms of creatine, specifically creatine monohydrate. Through the use of the esterfication process, creatine is alleged to become more permeable to the sarcolemma and bypass the creatine transporter, thereby allowing more creatine to enter the cell and minimize the amount of extracellular water retained during supplementation. A potential benefit of creatine supplementation is through the action of an anabolic signal for skeletal muscle hypertrophy, with increases in total and intracellular water [5, 13]. Roughly two-thirds of the increases in total body water seen during supplementation are intracellular, with no fluid shift occurring [30, 31]. Mean increases in total body water (Table 4) from day 0 to day 48 were 2.43 ± 1.19 L, 2.64 ± 0.31 L, and 1.95 ± 0.90 L for PLA, CRT, and CEE groups, respectively. For all groups, total body water was shown to significantly increase at days 27 and 48 compared to day 0. Mean increases in intracellular body water (Table 4) from day 0 to 48 were 2.52 ± 1.63 L, 2.52 ± 0.006 L and 1.01 ± 0.65 L for PLA, CRT, and CEE groups, respectively, and intracellular body water was significantly increased at days 27 and 48. For extracellular water, mean increases from day 0 to 48 were 0.42 ± 0.37 L 0.11 ± 0.18 L and 0.50 ± 0.21 L for PLA, CRT, and CEE groups, respectively, whereas extracellular body water was only significantly increased at day 27 (Table 4).

Collectively, changes in total, intracellular, and extracellular body water were not significantly different between the supplement and placebo groups. However, the mean increases for total and intracellular body water from day 0 to 48 were greatest for the CRT group. Extracellular water increases from baseline were actually largest for the CEE groups. Therefore, claims by the manufactures of creatine ethyl ester stating that extracellular water retention is minimized were shown to be unfounded by the present study. Previous research has shown creatine supplementation to increase total body water, yet no fluid shift occurs [30]. In resistance-trained participants, increases in total body water with creatine supplementation, but not a placebo, during resistance training have been observed [32]. In contrast, in the present study the participants were not resistance-trained, with increases in body water observed in the PLA group. Because resistance training is associated with increases in body water [33], the changes observed in the present study were mostly likely due to the resistance training program itself rather than the supplementation.

Muscle Strength and Power

Various studies have shown improvements in muscle strength and power through the use of creatine supplementation [1, 20, 28]. Bench press strength was shown to increase at days 27 and 48 compared to day 0 (Figure 1), whereas leg press strength showed an increase at day 6, 27, and 48 compared to day 0 (Table 5). However, in both instances there were no differences between the three groups. Mean and peak power showed a significant improvement over the course of the study (Table 6). However, the muscle power measures had no significant differences between the three groups. Other studies have shown no benefits for increases in muscle power with supplementation [34].

An increase in muscle performance typically correlates with an increase in creatine muscle uptake [20]. Even though there was no significant increase in total muscle creatine content with the supplement groups over the course of the study. The PLA group, which did not consume creatine, showed similar improvements in muscle strength and performance. Therefore, our data indicates the improvements that were observed were most likely from the strength training program, not due to the creatine supplements.