Study design and subjects
The present study was conducted at Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University and the Bio-gym Islamabad. Before onset of study, the purpose of study was verbally explained to all participants and they gave informed written consent showing their wiliness to participate in the study. The study was approved by ethical committee (BAS#256) for research on human subject, Quaid-i-Azam University, Islamabad. Male volunteer athletes who were not involved in any professional sports were selected for this study. A total of 150 adult males were included in this study. These included 93 athletes and 57 non-athletes (age: 25 ± 8 years; Range: 20–35 years). All the volunteers were informed about all the procedures and outcomes of the study.
Prior to this study self-reported questionnaire was done form all the volunteers who participated in study. Personal, social, demographical and reproductive health questions were asked in the questionnaires.
The subjects were categorized into following four groups. Control athletes and non-athletes, and athletes who, in addition to exercise, voluntarily took supplements were categorized into 2 groups on the basis of type of supplements taken.
Experimental design
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Control group I: Non athletes (n = 57)
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Control group II: Non-supplemental athlete group (n = 40)
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Supplemental athlete group (SAG) I*: (n = 28)
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Supplemental athlete group (SAG) II ¥: (n = 25)
*(SAG I) these athletes were taking supplement comprising human growth hormone = 4 IU daily, dihydrotestosterone, 1000 mg/week, Creatine = 40,000 mg daily; Glutamine = 40,000 mg daily; Branched chain amino acids = 200,000 mg protein/day.
¥ (SAG II) were taking supplements including dihydrotestosterone = 1000 mg/week; cytomel 13liothyronine = 0.1 mg/day; Clenbuterol = 0.02–0.1 mg/day; Caffeine, ephedrine, aspirin = 150,000 mg/day; L-arginine = 1500–3000 mg twice/day; L-carnitine = 500–1500 mg twice/day; Vanadyl sulfate = 50 mg twice/day; medium chain triglycerides = 50 mg twice/day.
Age and body mass index (BMI)
Age was self-reported. Weight and height were measured using the standard methods (Alam et al., 2013). BMI was calculated using weight and height and reference ranges were taken from World Health Organization defining underweight as BMI < 18.50; normal weight = 18.50–24.99; overweight≥25.00; and obese ≥30.00.
Blood collection and serum separation
A blood sample (10 ml) was anesthetically obtained from all the participants by a trained and certified technician. All the samples were centrifuged (kokusan, Japan), for 15 min at 3000 rpm to separate plasma which was transferred to eppendorf tubes and stored at − 70 °C respectively until analyzed.
Complete blood profile
Five milliliters of blood from syringe was transferred into lavender vacutainer, containing Ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Blood count was performed on an automated hematology analyzer (Medonic M16S, Sweden) at Life Care Diagnostic laboratory, Rawalpindi. Each count presented the data of white blood cell count (WBC), red blood count (RBC) platelets count (Plt), mean platelet volume (MPV), mean corpuscular volume (MCV), measurement of hemoglobin (Hgb), and measurement of hematocrit (Hct), red blood cell distribution width percentage (RDW %), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).
Plasma analysis
Plasma samples were analyzed for the determination of antioxidant enzymes activity of Catalase CAT) [20] Peroxidase (POD) [21] Glutathione Reductase (GR) [22], Reduced Glutathione (GSH) [23], lipid peroxidation (TBARS) [24] on a UV spectrophotometer (Agilent 8453) while total protein estimation was done by AMP Diagnostics reagent kit on chemistry analyzer and hormonal concentration was detected by Enzyme Immuno Assay test kit (Amgenix Inc., USA).
Determination of electrolytes/metal in blood
Electrolytes (Na+ and K+) and Zinc in blood was determined by following the method of [25] and [26] with some modification. It involves the following steps.
Acid digestion of serum samples
Acid digestion was done according to the following steps.
Prior to the start of acid digestion all the apparatus was soaked with 10% Nitric acid for 24 h and then washed with 69% nitric acid. Before the process of digestion pre-digestion was done in which each sample of 0.5 ml of blood was taken and 5 ml of 69% HNO3 was added in it until it became clear.
Digestion
Samples were digested manually by boiling the whole blood in HNO3 on a hot plate at 400 °C until half of the liquid evaporated.
Filtration
After digestion these samples were filtered through filter paper and the final volume of filtrate was made to 15 ml by adding distilled water. Samples at this stage were ready for atomic absorption spectrometry.
Atomic absorption spectrometry
Prior to the start of atomic absorption spectrometry the following standard solutions were prepared.
Standard solution of each salt of 1000 ppm concentration was prepared by following formula.
$$ \frac{\mathrm{Molecular}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{salt}\ \mathrm{x}\ 0.1=\mathrm{x}}{\mathrm{Molecular}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{sample}} $$
x grams of salt were dissolved in 100 ml of distilled water to make 1000 ppm solution.
In order to calibrate the instrument, diluted solutions having concentration from 1 ppm to 50 ppm were used. The different calculations were made using the formula.
$$ {\mathrm{C}}_1{\mathrm{V}}_{1=}{\mathrm{C}}_2{\mathrm{V}}_2 $$
Where C1 represent concentration and V1 volume of stock solution of standard andC2 and V2 are the required concentration and volume respectively.
Determination of metal concentration
For detection of different metals by Fast Atomic Absorption Spectrometry (SAAS), different lamps specific for that particular metal were used. A series of standard elements under analysis were run. Each digested sample was subjected to Acetylene flame of atomic absorbance spectrometer (Varian AA240FS, USA) and concentration of microelements was noted. These concentrations were obtained in ppm and converted into mg/l by using the following formula
$$ \mathrm{Metal}\ \mathrm{concentration}\ \left(\mathrm{mg}/\mathrm{l}\right)=\frac{\mathrm{final}\ \mathrm{volume}\ \mathrm{of}\ \mathrm{dilution}\ \mathrm{x}\ \mathrm{metal}\ \mathrm{concentration}\ \left(\mathrm{ppm}\right)}{\mathrm{Volume}\ \mathrm{of}\ \mathrm{sample}} $$
Determination of plasma Luteinizing hormone
The kit contained antibody-coated microtitter plate coated with 96 wells, LH standard set, contains six vials (ready to use) 0, 5, 20 50, 100 and 200 mlU/ml, Enzyme conjugate reagent 12 ml, Trimethylbenzidine (TMB) substrate one bottle (ready to use) 12 ml, stop solution one bottle (ready to use) 12 ml, 50X wash buffer concentrate one bottle 15 ml.
Principle
This test is based on a solid phase ELISA. The assay system utilizes one anti-LH antibody for solid phase (microtiter wells) immobilization and another mouse monoclonal anti-LH antibody in the antibody-enzyme (horsereddish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the antibodies resulting in the LH molecules being sandwiched between the solid phase and enzyme-linked antibodies. Unbound LH and LH enzymes conjugate is washed off by washing buffer. Upon addition of substrate, the intensity of the color is inversely proportional to the concentration of LH in the sample; a standard curve is prepared relating color intensity to the concentration of the LH.
Procedure
The reagents provided with kit were gently mixed prior to the start of the experiment.
Desired number of wells were secured and 50ul of standard, control and specimen were dispensed into wells. 100ul of enzyme cognate was added and the samples were mixed and incubated at 37 °C for 60 mins. Washing was done of the plate 5 times with Elisa plate washer. TMB substrate 100ul was added in wells and again incubated for 20 min at 37 °C. later on stop solution 100ul was added in order to stop the solution and the absorbance was read at 450 nm on Elisa Plate reader.
Statistical analysis
All data is presented as the mean ± standard error of mean. Data of in vivo assays recorded was analyzed by one way ANOVA carried by computer software SPSS version 16.0. p < 0.05 was considered significantly significant.