Caffeine is the most widely used pharmacologically active compound, and it has been estimated that 80-90% of users report habitual consumption, with a daily average intake of approximately 200-250 mg (Barone and Roberts, 1996; Juliano and Griffiths, 2004). Caffeine has been shown to enhance cognitive function and feelings of mental alertness, mood, and arousal (Goldstein et al., 2010; Nehlig, 2010). In addition, caffeine ingestion has been demonstrated to maintain or enhance vigilance and choice reaction time (Lieberman et al., 2002; Mclellan et al., 2005), and is commonly used to alleviate the effects of sleep deprivation and fatigue (Beaumont et al., 2001; De Valck and Cluydts, 2001; Penetar et al., 1993).
Previous investigations have determined that caffeine is completely absorbed by the gastrointestinal tract within 1 hour, with peak plasma concentrations occurring between 15 and 120 minutes following ingestion (Magkos and Kavouras, 2005). Plasma caffeine concentrations have also been shown to rise in a dose-dependent manner and exhibit first order, linear kinetics resulting in a half-life of approximately 5 hours (Bonati et al., 1982; Kamimori et al., 2002). However, the half-life of caffeine has also been reported to range between 2.5 and 10 hours by other investigators (Magkos and Kavouras, 2005). Additionally, the nature of formulation can also directly influence the rate and extent of absorption following oral administration as caffeine has shown to have a greater rate of absorption from a capsule than from dietary sources such as coffee (Fredholm et al., 1999), cola, or chocolate (Mumford et al., 1996).
The efficacy of time-release caffeine capsules appears to be no different than regular caffeine capsules. Investigations have demonstrated that time-release caffeine can enhance alertness and reaction performance for up to 13 hours following ingestion (Lagarde et al., 2000), and improve vigilance and cognitive function during sleep deprivation as compared to a placebo (Beaumont et al., 2001; 2004; 2005; De Valck and Cluydts, 2001; De Valck et al., 2003; Doireau et al., 1997; Lagarde et al., 2000; Patat et al., 2000; Sicard et al., 1996). However, these previous studies have not compared the efficacy of time-release caffeine directly to regular caffeine capsule ingestion, nor have they examined performance changes relative to differences in the pharmacokinetics of caffeine uptake into the plasma. We hypothesized that a time-release caffeine containing supplement would alter the pharmacokinetics of caffeine, creating a sustained plateau of caffeine concentration in the plasma following consumption. In addition, the sustained effect of time-release caffeine may prolong the lipolytic, metabolic, and performance effects generally associated with caffeine ingestion.
Thus the primary objective of this study was to compare the pharmacokinetics of a multi-ingredient time-release caffeine containing supplement (TR-CAF) versus an equivalent dose of a regular caffeine capsule and a placebo. In addition, glycerol concentration, metabolic rate, reactive and cognitive performance, and subjective measures of mood, energy, focus, and alertness were assessed during an 8-hour period following ingestion.
Ten males (25.9 [+ or -] 3.2 y; 1.81 [+ or -] 0.08 m; 92.9 [+ or -] 9.9 kg; 13.3 [+ or -] 3.6% body fat) volunteered to participate in this acute randomized, double-blind, placebo-controlled study. Following an explanation of all procedures, risks, and benefits, each participant gave his informed consent prior to participation in this study. The Institutional Review Board of the University of Central Florida approved the research protocol. For inclusion in the study, participants had to be regular caffeine consumers (>200 mg per day) to increase homogeneity of the sample. Participants were excluded if they had any history of cardiovascular disease, metabolic, renal, hepatic, or musculoskeletal disorders or were taking any other medication as determined by a questionnaire.
Participants reported to the Human Performance Laboratory (HPL) for one familiarization session prior to experimental trials. During the familiarization session, participants were informed of all procedures and familiarized with all performance measures to reduce the possibility of a learning effect. Participants performed three trials with seven days between each trial. During each trial, participants consumed either a multi-ingredient supplement containing time-release caffeine (TR-CAF); a regular caffeine supplement (CAF); or a placebo (PL).
Participants reported to the HPL at 0800 hour following an 8-hour fast and were instructed not to exercise 24 hours prior to each trial. Assessments took place at baseline (prior to supplement ingestion) and at each hour following ingestion of the supplement for a total of 8 hours. Assessments consisted of blood measures, metabolic measures, cardiovascular measures, subjective measures, and performance measures. Between assessments, participants sat comfortably in a quiet room with out distraction wearing noise cancelling headphones (Bose, QuietComfort[R] 15, Framingham, MA). Participants were provided a standardized breakfast (310 kcal; 45 g carbohydrate, 17 g protein, 6 g fat) and lunch (290 kcal; 38 g carbohydrate, 19 g protein, 7 g fat) and were permitted to drink water ad libitum. The study protocol is depicted in Figure 1.
The caffeine containing supplements (TR-CAF and CAF) and PL were ingested in tablet form, and two tablets were consumed during each trial. Tablets for all trials were similar in appearance and taste. The TR-CAF supplement contained 194 mg time-release caffeine, 5.2 mg vitamin B1, 25 mg vitamin B6, 200 [micro]g folate, 3 [micro]g vitamin B12, 150 mg magnesium, 971 mg L-tyrosine, 250 mg glucuronolactone, 75 mg theobromine, 75 mg rhodiola rosea extract, 25 mg Korean ginseng powder, and 10 mg octacosonal. To compare the supplement with an equivalent amount of regular caffeine, CAF contained 194 mg regular caffeine and rice powder, while the PL contained rice powder only. CAF served as a typical caffeine ingestion ordinarily used by habitual caffeine users.
During each experimental trial, all blood samples were obtained using a 20-gauge Teflon cannula placed in a superficial forearm vein using a three-way stopcock with a male luer lock adapter. The cannula was maintained patent using an isotonic saline solution (Becton Dickinson, Franklin Lakes, NJ). The first blood draw occurred at baseline (BL) prior to supplementation and breakfast. Following ingestion of the supplement and breakfast, blood draws occurred at every hour over the 8 hour period (9 total blood draws). Each participant's blood samples were obtained at the same time of day during each session.
All blood samples were collected into two Vacutainer[R] tubes, one containing SST[R] Gel and Clot Activator and the second containing sodium heparin. The sodium heparin tube was kept chilled prior to each blood draw. The blood in the first tube was allowed to clot at room temperature for 2 hours and subsequently centrifuged at 3,000 x g for 15 min along with the remaining whole blood from the second tube. The resulting plasma and serum was placed into separate 1.8-mL microcentrifuge tubes and frozen at -80[degrees]C for later analysis.
Plasma caffeine concentrations were quantified using high performance liquid chromatography (HPLC). Chromatographic conditions were based upon a modified version of Agilent Technologies application brief (Agilent Technologies, Santa Clara, CA). Chromatography was performed on an Agilent Infinity 1260 HPLC (Agilent Technologies, Santa Clara, CA) consisting of a degasser, binary pump, auto-sampler, column thermostat, and photodiode array detector. A Zorbax Eclipse Plus C18 (4.6 x 150mm, 5pm) column and Zorbax analytical guard column (4.6 x 12.5 mm, 5-pm) were used for separation. Data were collected using OpenLAB chromatography data system, ChemStation edition.
All reagents were of HPLC grade. Caffeine, betahydroxyethyl-theophylline, sodium phosphate monobasic and sodium phosphate dibasic were purchased from Sigma-Aldrich (St. Louis, MO) to create the stock solution. Acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA). HPLC grade water was prepared by reverseosmosis and purified using a Milli-Q Direct 8 water purification system (EMD Millipore, Billerica, MA).
A 40 [micro]g x m[L.sup.-1] stock solution of caffeine, theobromine and beta-hydroxyethyl-theophylline was prepared in water and sonicated. Twelve calibration standards were prepared from the stock solution in the range of 0.039-40 [micro]g x m[L.sup.-1] by serial dilution of 1 mL of the stock solution. Beta-hydroxyethyl-theophylline (internal standard; IS) working solution was prepared in water (10 [micro]g x m[L.sup.-1]).
An internal plasma sample was collected to serve as control and analyzed every 50 samples. Calibration standards, samples, and controls were prepared in the same fashion for linearity. Sixty microliters of the calibration standards or 50 [micro]L of sample or quality control sample was added to 1.5 mL microcentrifuge tubes. Ten microliters of IS was subsequently added to the samples and controls, followed by 140 [micro]L of chilled acetonitrile for deproteinization. Standards, samples, and controls were then vortexed vigorously for 30 seconds and placed in a refrigerator (4[degrees]C) for two hours followed by centrifugation at 14000g for 15 minutes in a microcentrifuge to allow the protein to form a pellet. The supernatant (150 [micro]L) was collected and subsequently transferred to a 0.45 pm polytetrafluoroethylene syringeless filter vial (GE Healthcare Mini-Uniprep[TM], Piscataway, NJ). A concentration of 300 [micro]L of sodium phosphate buffer (mobile phase) was then added to the vial. The solution was...