Subjects
Sixteen male sprinters from a university sport club (aged 21.6 ± 1.5 years; body weight 76.9 ± 6.4 kg; height 180.5 ± 6.2 cm; BMI 23.5 ± 1.1 kg/m2; % body fat 11.8 ± 2.5 %; training experience 4.4 ± 1.4 years), all students of physical education, volunteered for the study. The exclusion criteria were the use of tobacco products, alcohol consumption, recent surgery or illness, and use of any medicine or dietary supplements in 4 weeks prior to the study. The protocol of the study was approved by the local Bioethical Committee at the Academy of Physical Education in Warsaw, and the research was conducted in accordance with the principles stated in the Declaration of Helsinki.
Experimental procedures
The double-blind, randomized, placebo (PL)-controlled crossover study was conducted during preparatory phase of yearly training cycle (after transition period). The two 4-week treatment periods (during which half of the subjects received GTE and the other half PL, and vice versa) were separated by a 4-week washout period. The duration of the washout period was selected based on the results of one previous study [26], in which 6-week supplementation with 800 mg catechins daily was used. In that study, plasma catechin concentration returned to its baseline level after at least 2 weeks of washout period.
Both GTE and PL were administered in the form of dark gelatin capsules (Olimp Labs, Dębica, Poland), identical in appearance (i.e., size, shape, and color); the same dosage regimen was used (two capsules twice a day). One GTP capsule contained 250 mg of standardized GTE (245 mg polyphenols, including 200 mg catechins, among them 137 mg epigallocatechin-3-galate) and additional substances (maltodextrin, microcrystalline cellulose, and magnesium stearate). Therefore, each participant was administered 980 mg polyphenols daily. PL capsules contained microcrystalline cellulose, magnesium stearate, and maltodextrin instead of GTP. The compliance was measured by capsule counting. The participants who returned no more than 15 % of their capsule dose were classified as “compliant”.
At the end of each of the two 4-week treatment periods, the sprinters performed a repeated cycle sprint test (RST) on a cycle ergometer (Ergomedic 839E, Monark, Sweden). The test consisted of four consecutive 15-s bouts (4 × 15 s), each of them with base set according to the Wingate procedure and separated by 1-min rest intervals. The subjects were asked to cycle for 15 s, as fast as possible, against a constant load (75 g/kg body weight). The test was preceded by a 5-min warm-up with submaximal load, until the heart rate reached 130–150 beats/min (Polar, Finland). The following exercise parameters were registered (Multi CykloErgometr MCE version 5.1) during each of four 15-s bouts: peak power (W/kg), mean power (W/kg), total work output (J/kg), and fatigue index (%). The tests were performed in the morning following 12-h overnight fast, at air temperature between 19 and 21 °C and with 40–60 % relative humidity. The subjects were instructed to not perform hard physical training for 48 h and avoid drinking tea and caffeinated beverages within 24 h prior to each of the RSTs. The subjects were asked to refrain from any dietary supplements for the duration of the study.
Dietary intake
The participants were asked to not modify their diet for the duration of the study, except for refraining from consuming any products containing green tea and limiting the intake of caffeine-containing drinks to one cup per day. Moreover, they were asked to maintain a similar diet for both treatment periods. During both the first and the second treatment periods (during 7 days preceding each RST), the participants filled out a 3-day dietary record (covering 2 week days and 1 day of the week end). The amount of ingested foods was estimated with an aid of dedicated picture book [27]. The daily dietary intake of vitamins C and E, beta-carotene, and selenium was calculated using Dietus software (based on national food tables) [28].
Blood sampling
Capillary blood samples were taken from a finger pulp, before the test and 3 min after completing the test. Venous blood samples were drawn into heparinized test tubes from the ulnar vein before completing the test (at rest), 5 min after completing the test, and following the 24-h recovery period. The venous blood was centrifuged (for 10 min at 3,000×g at a temperature of 4 °C) to separate erythrocytes from plasma. Subsequently, erythrocytes were washed three times with a cold isotonic saline solution. Both erythrocytes and plasma were frozen and stored at −80 °C until analysis.
Laboratory analysis
Capillary blood was assayed for the concentration of lactate, as well as for the parameters of acid–base equilibrium. The concentration of lactate was determined with a diagnostic cuvette kit (Dr. Lange, catalogue no. LKM 140, Germany) in a Miniphotometer Plus LP 20 (Hach Lange, Germany). An automated analyzer (OMNI-C analyzer, Roche Diagnostics, Austria) was used to measure the acid–base balance parameters in capillary blood: pH value, base excess, and anion gap, as well as hematocrit. According to the manufacturer, the intra-assay coefficients of variation for pH and hematocrit measurements were below 2.0 and 3.0 %, respectively.
Plasma concentrations of total polyphenols, UA, AL, and MDA were determined, along with TAC and the activity of CK. Erythrocytes were analyzed for the activity of SOD and GPx.
Analysis of total plasma polyphenols was performed using the Folin–Ciocatleau method, as described by Maskarinec et al. [29]. Gallic acid in ethanol served as the standard solution, and the results for total polyphenols were given as µg/ml gallic acid equivalents (GE). The average intra-assay coefficient of variation (calculated for ten duplicated samples) was 7.8 %.
The total antioxidant capacity of plasma (TAC) to scavenge ABTS radicals was measured by a chromogenic method with commercially available kit (Cat. No. NX 2332, Randox, Crumlin, UK). Antioxidant capacity of samples was expressed as millimoles per liter of Trolox equivalents (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The average intra-assay coefficient of variation (calculated for ten duplicated samples) was 4.9 %.
Plasma UA and AL concentrations were determined with commercially available kits (Cat. No. K6580-200 and A6502-100, respectively; Alpha Diagnostics, USA). The average intra-assay coefficients of variation (calculated for ten duplicated samples) for UA and AL were 6.1 and 4.3 %, respectively.
The SOD and GPx activities in erythrocytes were determined with commercially available kits (RANSOD Cat. No. SD 125 and RANSEL Cat. No. RS 505, respectively; Randox, Crumlin, UK). The antioxidant enzyme activities were measured at 37 °C and expressed in U/g Hb. Hemoglobin was assessed by a standard cyanmethemoglobin method, using a diagnostic kit (HG 1539; Randox, Crumlin, UK). The average intra-assay coefficient of variation (calculated for ten duplicated samples) for SOD, GPx, and Hb was 3.8, 6.9, and 2.9 %, respectively.
Plasma MDA levels were determined with a commercially available kit (LPO-586, OXIS Internatl., Portland, OR). Plasma CK activity was determined with the use of a diagnostic kit (Cat. No. C6512-100, Alpha Diagnostics, USA). The average intra-assay coefficient of variation (calculated for ten duplicated samples) for MDA and CK was 7.5 and 8.3 %, respectively. Due to the decreased plasma volume following exercise [30], plasma volume was corrected for post-exercise changes by considering the changes in hematocrit according to the Dill and Costill method [31].
Statistical analysis
Statistical analysis was performed with Statistica version 6.0 software package. Paired t test was used to compare the results of the dietary survey and RST, and resting total plasma polyphenols levels documented after two 4-week treatment periods. The data regarding biochemical parameters were analyzed using 2 (treatment: PL and GTE) × 3 (time points: at rest, 5 min, and 24 h after completion of the test) factorial (two-way) ANOVA. If a significant time and treatment interaction was detected, the data within each treatment (at rest, 5 min post-exercise, and after 24-h of recovery) were analyzed and compared between treatments (PL and GTE) using the Bonferroni corrected paired t test. The normal distribution of all dependent variables was confirmed with the Shapiro–Wilk test and visual inspection (quantile distribution plots). All values were reported as mean ± SEM. The level of statistical significance was set at p < 0.05.