Subjects
Fifty two endurance trained subjects, most of them cyclists and triathletes, participated in this trial. Inclusion criteria: healthy males or females, 25 – 50 years, aerobically trained (VO2max from 45 mL/kg . min-1 to 65 mL/kg . min-1), non-smokers, no dietary or nutritional supplement use within four weeks prior to the first blood drawing/stool collection. Exclusion criteria: smokers, subjects outside the predefined VO2max range, subjects who underwent major changes in training regimens during the study, chronic or excessive alcohol consumption, recent surgery or illness, body fat > 20 %. Body fat content and distribution was estimated by a computerized optical device Lipometer (Möller Messtechnik, Graz, Austria), as described by Möller, et al [17]. Besides inclusion and exclusion criteria, a standard blood chemistry panel was determined and all subjects completed a medical history.
Ethical aspects, recruitment and randomization
All subjects provided written informed consent prior to participating in this investigation. This study was conducted according to the guidelines of the Declaration of Helsinki for Research on Human Subjects 1989 and was approved by the Ethical Review Committee of the Medical University of Graz, Austria. The trial was registered under www.clinicaltrials.gov, identifier: NCT01831492.
The study focused trained men and women and was announced in the largest sport magazine of Austria. After a telephone screening conducted by the research team, 61 subjects volunteered for eligibility testing. From those, 56 men and women were selected and entered the study program.
Before randomization VO2max tests were conducted and VO2max data were ranked from highest to lowest. Subjects were randomized into blocks of four and sequentially numbered. To guarantee a balanced VO2max distribution between groups (zeolite versus placebo) we conducted stratification via VO2max rank statistics. Randomization code was held by a third party (Union of Sport and Exercise Scientists Austria) and handed over for statistical analyses after collection of all data.
Study design and time schedule
This was a randomized, placebo controlled, double-blinded study. All eligibility testing - such as blood panel, eligibility for exercise, clinic check-up, medical history questionnaire, one-on-one interview - was finalized prior to the 1st VO2max testing. On the day of the 1st VO2max testing blood and stool samples were collected before the subjects conducted the cycle step test ergometries (= first visit and 1st VO2max testing) to exhaustion. After completion of these 56 ergometer step tests, the investigators dispensed the randomized capsule supply according to the subject’s VO2max-ranking. After 12 weeks taking the capsules as instructed, subjects returned to the study side for the second blood and stool collection and another VO2max testing, and returned their remaining capsules to adjust compliance.
Dietary assessment
Subjects were instructed to maintain their habitual diet, lifestyle and training regimen during the 12 weeks study and to duplicate their diet before each blood/stool collection. Before the first visit at the study site, subjects completed a 7-day food record for nutrient intake assessment. They subsequently received copies of their 7-day diet records and were instructed to replicate the diet prior to the second visit. Diet records were analyzed for total calories, protein, carbohydrate, fat, cholesterol, fiber, water, alcohol, several vitamins, minerals, and fatty acids using “opti diet” software 5.0 (GOEmbH, Linden, Germany).
Treatment
The subjects randomized to zeolite-clinoptilolite (n = 28) received boxes with capsules containing (per capsule): Zeolite 307,50 mg, Dolomite 75,17 mg (thereof Magnesium 15,79 mg; Calcium 34,9 mg), Maca 27,33 mg, Cellulose 90 mg (PANACEO SPORT®, Panaceo International Active Mineral Production GmbH, Villach, Austria). The placebo consisted of identical appearing capsules with 500 mg Cellulose per capsule. All subjects were instructed to take 6 capsules per day, 3 capsules in the morning with breakfast and the other 3 capsules with the last meal of the day throughout 12 weeks. To ensure good compliance, subjects were called and e-mailed biweekly to remind and motivate them to adhere to the suggested instructions.
VO2max and performance tests
To assess the supplement’s impact on VO2max and maximum performance in watt (W), and to avoid biased responses caused by differences in aerobic fitness levels, we conducted step wise incremental ergometries until exhaustion to evaluate VO2max and maximum performance of each subject, at the beginning and the end of the study. Exhaustion was reached/defined when cadence could not be maintained with maximum exertion on the last (heaviest) step. They were instructed not to perform physical training 3 days prior to the exercise test. All subjects performed an incremental cycle ergometer exercise test (EC 3000, Custo med GmbH, Ottobrunn, Germany) at 80 rpm. After a three minute rest phase sitting inactive on the ergometer, work rate started at 60 W (women 40 W) for three minutes and was increased 15 W every minute until voluntary exhaustion. A standard electrocardiogram was recorded during the entire test, which was supervised by a physician. Respiratory gas exchange variables were measured throughout the incremental exercise tests using a breath-by-breath mode (Metalyzer 3B, Cortex Biophysik GmbH, Leipzig, Germany). During these tests, subjects breathed through a facemask. Oxygen uptake (VO2), carbon dioxide output (VCO2), minute ventilation (VE), breathing rate (BR) and tidal volume (VT) were continuously obtained. Blood pressure as well as heart rate (HR) were monitored throughout the tests using a commercially available heart rate monitor (Polar Vantage NV, Polar Electro Finland).
Feces and blood collection
For the measurements of zonulin and alpha1-antitrypsin from feces the subjects collected samples at baseline and after 12 weeks. They used standardized stool tubules and brought the samples with a cooling bag, within 24 h after collection, to the laboratory. All samples were analyzed within 48 h after dispensing. Throughout the 12 weeks treatment the subjects recorded a stool protocol to monitor stool appearance with help of the Bristol stool scale/chart [18].
Blood collections were conducted in supine position from a medial cubital vein at baseline and after 12 weeks of treatment. Venous blood was collected to determine carbonyl proteins (CP), 8-iso-prostaglandin F2α (8-iso-PGF2α), plasma glutathione peroxidase (GPx3), uric acid, vitamin C, DNA strand-breaks, cytokines like tumor necrose factor alpha (TNF-α), interleukines 6, 8, 10, 22 (IL-6, IL-8, IL-10, IL-22). For determination of a blood clinical chemistry panel we measured Mg, Ca, Fe, K, Na, AI, P, Cl, ferritin, transferrin, liver and kidney parameters (ALT, AST, GGT, creatinine), testosterone, sex hormone binding globulin (SHBG), free androgen index (FAI), and a hemogram.
Stool analyses
Zonulin and α1-antitrypsin were analyzed with commercially available ELISA kits (Immundiagnostik AG, Bensheim, Germany).
Analyses of blood parameters
The quantity of carbonyls in protein samples (carbonyl proteins, CP) was analyzed with a commercially available ELISA kit from Immundiagnostik AG, Bensheim, Germany.
An isotope diluted liquid chromatography tandem mass spectrometry (LC-MS) approach was used for the detection of 8-iso-PGF2α.
For analyses of plasma GPx3, the Human Glutathione Peroxidase ELISA Kit assay was used (Cusabio, antibodies-online GmbH, Aachen, Germany).
For DNA strand-break analyses in mononuclear cells the Comet assay was used.
For analyses of cytokines IL-6, IL-8, IL-10 and IL-22, an ELISA kit was used (Cusabio; antibodies-online GmbH; Aachen, Germany).
For quantitative determination of TNF-alpha an enzyme-linked-immunosorbent Assay (TNF-alpha ELISA; Immundiagnostik AG, Bensheim, Germany) was used.
For analyses of vitamin C, uric acid, minerals, functional liver and kidney markers, as well as androgens, fully automated routine clinical chemistry analyzers were used (vitamin C: LaChrom L7200, Merck KG, Darmstadt, Germany; all the others: CI 8200, Abbott Diagnostics, Green Oaks, IL).
The hemogram was measured with the ABX Micros 60 (AxonLab, Baden, Switzerland) from EDTA-blood.
Statistical analyses and sample size calculation
Per protocol analyses were performed using SPSS for Windows software, version 19.0. Data are presented as mean ± SD. Statistical significance was set at P < 0.05. The Shapiro-Wilk test was used to determine normal distribution. Baseline characteristics, nutrient and clinical chemistry data, were compared by unpaired Student’s t-test. Data obtained for zonulin and α1-antitrypsin from feces, as well as CP, 8-iso-PGF2α, GPx3, uric acid, vitamin C, DNA strand-breaks, IL-6, IL-8, IL-10, IL-22 from blood and performance data were analyzed using a univariate, two-factorial, repeated measures analysis of variance (ANOVA). Factors: treatment (zeolite supplementation versus placebo), time (baseline versus 12 weeks of treatment). Significant interactions and main effects were post-hoc analyzed by using Bonferroni correction. Performance data were analyzed separately for men and women.
Sample size calculation was based on zonulin, redox markers and cytokines. We estimated 12 (zonulin) - 25 (TNF-α) subjects per group (1 : 1 distribution) - depending on the calculated parameter, standard deviation and effect size - to reach a probability of error (alpha/2) of 5 % (α = 0.05) and 80 % power. TNF-α sample size calculation resulted in the highest number of all parameters: 50 subjects. Hence, and including a drop-out rate of 10 %, 56 subjects were recruited.