Participants

15 male Taekwondo practitioners aged 16.9 ± 1.4 years with 3.3 ± 2.8 years of Taekwondo training experience, participating in national level competitions were recruited for this study from Sports Authority of India, Bangalore. All the athletes undertook 5-10 training sessions per week and exercised 1-2 hours per training session. The details regarding the health status, history of muscle injuries, hospitalization, medication and supplementary usage were obtained through the questionnaires. The study was approved by the institutional ethical committee and written informed consent was obtained from all the participants.

Training

Training programme comprised of two components namely Physical conditioning, Technical & Tactical training.

Physical conditioning

Physical conditioning was planned in the morning session (6.00 am to 7.30 am) and various exercises were given to meet the different conditioning abilities required for Taekwondo, such as strength, endurance, flexibility, speed, agility and so on.

This included-

Technical & tactical training

Technical & tactical training was scheduled in the evening from 4.00 pm to 6.00 pm. This training mainly focused on kicking skills, punching skills, distance, timing training, attacking, counterattacking and scoring.

The typical Taekwondo training session used in this investigation consisted of:

Specimen collection and assay

Blood samples were collected before and after competitive phase specific Taekwondo training having duration of 3 months. Samples were collected from the participants in seated position from antecubital vein into plain evacuated tubes in the morning in overnight fasting condition. All participants were refrained from training 24 hours prior to the test to avoid acute exercise-induced shifts in plasma volume. An aliquot of each sample was immediately mixed with EDTA solution to prevent clotting for haematology. The rest of the sample was left to coagulate at room temperature for 30 minutes and centrifuged at 1500 g for 10 minutes to separate the serum which was analysed for selected biochemical parameters such as triglycerides (mg.dl^-1), total cholesterol (mg.dl^-1), HDL-cholesterol (mg.dl^-1), LDL-cholesterol (mg.dl^-1), CPK (U/L), LDH (U/L), urea (mg.dl^-1), uric acid (mg.dl-1), serum iron (µg.dl^-1), total iron binding capacity-TIBC (µg.dl^-1), transferrin saturation (%).

Measurement of biochemical variables

Total Iron Binding Capacity (TIBC) & Unsaturated Iron Binding Capacity (UIBC)

TIBC was estimated calorimetrically by using Saturation – Precipitation method (spinreact). Serum transferrin is saturated with excess of Fe³⁺ and the unbound portion is precipitated with magnesium carbonate. The total amount of iron (TIBC) is then determined by using Ferrozine method. The difference between the TIBC and initial serum iron yields the unsaturated iron binding capacity (UIBC) and both the values were expressed as µg.dl^-1.

Transferrin Saturation (TS %)

Transferrin saturation is calculated from Serum iron and TIBC values by using the standard equation.

$\mathrm{T}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{ }\mathrm{S}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{f}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{ }\mathrm{\%}=\frac{\mathrm{S}\mathrm{e}\mathrm{r}\mathrm{u}\mathrm{m}\mathrm{ }\mathrm{I}\mathrm{r}\mathrm{o}\mathrm{n}}{\mathrm{T}\mathrm{I}\mathrm{B}\mathrm{C}}\times 100$

Triglycerides

Triglyceride Concentration was estimated calorimetrically by using GOD-POD method (Spinreact). Serum triglycerides incubated with lipoprotein lipase (LPL), liberate glycerol and free fatty acid. Glycerol is converted to glycerol-3 phosphate (G3P) and adenosine -5-diphosphate (ADP) by glycerol kinase and ATP. Glycerol -3- phosphate is then converted to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H₂O₂) by glycerol phosphate dehydrogenase. H₂O₂ reacts with 4-aminophenazone and p-chlorophenol in presence of peroxidase (POD) to give a red coloured dye. The optical density (OD) was measured at 505nm and the concentration is expressed as mg.dl^-1.

Total cholesterol (TC) and High density lipoprotein cholesterol (HDL-C)

Cholesterol esters liberate cholesterol and fatty acids; by the action of the cholesterol esterase (CHE). The cholesterol present in the sample is converted to 4-cholestenona and hydrogen peroxide (H₂O₂). H₂O₂ reacts with 4-aminophenazone and Phenol in presence of peroxidase (POD) to give a coloured product.

To estimate HDL-C, Chylomicrons, LDL and VLDL are precipitated from serum by adding phosphotungstate in the presence of divalent cations such as magnesium, the HDL-C remains unaffected in the supernatant is estimated using CHOD-POD method after centrifugation. The optical density (OD) was measured at 505nm and the concentration is expressed as mg.dl^-1.

Very low-density lipoprotein cholesterol (VLDL-C)

Very low-density lipoprotein cholesterol (VLDL-C) was indirectly assessed using standard equation

$\mathrm{V}\mathrm{L}\mathrm{D}\mathrm{L}-\mathrm{C}\left(\mathrm{m}\mathrm{g}.{\mathrm{d}\mathrm{l}}^{-1}\right)=\frac{\mathrm{T}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{l}\mathrm{y}\mathrm{c}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{s}}{5}$

Low-density lipoprotein cholesterol (LDL-C)

Low-density lipoprotein cholesterol (LDL-C) was indirectly assessed using standard equation

$\mathrm{L}\mathrm{D}\mathrm{L}-\mathrm{C}\left(\mathrm{m}\mathrm{g}.\mathrm{d}\mathrm{l}-1\right)=\mathrm{T}\mathrm{C}-(\mathrm{H}\mathrm{D}\mathrm{L}-\mathrm{C}+\mathrm{T}\mathrm{G}/5)$

Creatine Kinase (CK)

Estimation of CK was done by kinetic method (Spinreact). CK catalyses the reversible transfer of phosphate group from phophocreatine to ADP and forms ATP. ATP and Glucose reacts in the presence of Hexokinase (HK) and forms glucose -6-phosphate (G6P) and ADP.G6P and NADP⁺ reacts in the presence of glucose -6- phosphate dehydrogenase and produces NADPH⁺ along with 6-Phosphogluconate. The rate of NADPH formation is measured photometrically at 340nm and is proportional to the catalytic concentration of CK present in the sample and is expressed as U/L.

Lactate Dehydrogenase (LDH)

LDH was estimated by using kinetic method (Spinreact). Lactate dehydrogenase; present in the sample catalyses the reduction of Pyruvate by NADH. The rate of decrease in concentration of NADH, measured photometrically at 340nm is proportional to the catalytic concentration of LDH present in the sample and is expressed in U/L.

Complete Blood Picture (CBP)

Haematological parameters namely hemoglobin (Hb), red blood cells (RBC), white blood cells (WBC), jematocrit (Hct), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), absolute neutrophil count, absolute lymphocyte count, mixed cell count, neutrophil % and lymphocyte %, red cell distribution width (RDW-CV) were assessed in Sysmex –PocH100i^TM automated haematology analyzer (Japan).

Statistics

IBM SPSS statistics (version 23.0) was used for analysis. Data were expressed as Mean ± standard deviation. Shapiro-Wilk test was used to check Normal distribution. The paired-t test was used to determine differences between two independent means, statistical significance was set at P = 0.05. Hedge’s g (unbiased) effect size also calculated between pre and post tests to measure the magnitude of the training effect.

Lipid Profile

A significant increase (P< 0.05) of total cholesterol & HDL-cholesterol levels and a significant decline (P< 0.05) of TC/HDL-C Ratio (from 3.4 ± 0.57 to 2.9 ± 0.44) were observed after the 12 weeks of training besides this an insignificant decrease of triglycerides and VLDL-cholesterol was also observed.

Serum Enzymes

Serum levels of skeletal muscle enzymes are markers of the functional status of muscle tissue and fluctuate widely in both pathological and physiological conditions (Brancaccio et al., 2007). Serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) give an indication of the degree of metabolic adaptation to physical training of skeletal muscles. The enzyme creatine kinase uses phosphocreatine (PCr) as its substrate and transfers a phosphate group to ADP, thus making the products ATP and creatine (Cr). This reaction is highly active within the first 10 seconds of maximal exercise; provide a rapid source of ATP production to fuel muscle contraction (Parolin et al., 1999). LDH is an oxidoreductase enzyme that catalyses the inter conversion of pyruvate and lactate. Both enzymes are involved in muscle metabolism, and their serum concentration is normally very low. As a result of physiological wear and tear of the cells, their levels increase considerably after intensive exercise and in muscle pathology (Garry JP et al 2000). CPK and LDH enzymes are widely used in sports medicine to monitor acute or chronic muscle tissue damages and to understand the biochemical adaptation to physical load. The increase in CPK and LDH activities in plasma after exercise is connected with changes of cell membrane integrity and mechanical damage of muscle fibres. Many studies have shown a significant increase in serum LDH levels after exhaustive exercise (Anugweje et al., 2014, Brancaccio et al., 2007). In our study, no significant change is observed in basal CPK levels after the training, but there is a significant decrease in basal LDH level. This might be due to muscular membrane adaptations after the training. Phospholipids are the major structural lipids of membranes and the functional properties of the membranes such as fluidity, permeability and anchoring of membrane proteins depend considerably on membrane phospholipid fatty acid composition (Stubbs CD et al., 1984, Hagve TA., 1988). Helge JW et al.(1985) have shown a significant effect of regular exercise training on the composition of the membrane phospholipid fatty acids. Another cause for different CPK and LDH responses in blood after training might be the disparity of enzymatic protein particles size. CK is a smaller protein (dimer with mass of 84,000 Da) compared to the LDH (tetramer with mass of 150,000 Da), consequently CPK can penetrate easily through muscle cell membrane disrupted by exercise (S. Poprzęcki et al, 2004).

Iron Profile

Serum Iron levels were significantly increased (P< 0.05) from pre training to post training, without any intervention of iron supplements. This change might be due to the adoption of good dietary habits or due to the training induced changes in hepcidin levels through altering basal interleukin -6 (IL-6) levels. A study by Kaya 2016 had shown that the serum IL-6 levels have been significantly reduced after 4 weeks of Taekwondo training in 10 elite male Taekwondo players and different research groups discovered that IL-6 is involved in regulation of hepcidin levels in response to inflammatory stimuli, through the Jak/STAT-3 signalling pathway (Wrighting DM et al 2006; Verga Flazacappa MV et al 2007). Iron is an essential trace element in the human body. It is very important for the synthesis of hemoglobin, myoglobin and plays an important role in the electron transport chain (ETC) and production of energy in mitochondria. Iron content in the body is largely controlled through absorption rather than excretion (Mccance, R. et al 1937). Hepcidin is the principle iron regulatory hormone, responsible for the maintenance of iron homeostasis (Ganz, T. 2011). Hepcidin levels that are inappropriately low or high result in iron overload or iron deficiency, respectively. Hepcidin by acting on ferroportin, the iron exporter located on the basolateral membrane and on macrophages controls the main inflows of iron into plasma. When hepcidin concentration is high ferroportin is internalized and iron is trapped in enterocytes, macrophages and lymphocytes. Hepcidin synthesis was markedly induced by infection and inflammation. IL-6 the major cytokine produced in response to a bout of exercise, itself is sufficient to induce hepcidin expression during inflammation. Studies by Peeling et al (2009) have shown that the production of IL-6 in contracting skeletal muscles results in the exercise induced hepcidin increase. In addition animals treated with cyclosporine-A, a calcineurin inhibitor to blunt plasma IL-6 during exercise showed lower hepcidin levels as compared to the exercised group without cyclosporine-A treatment (Banzet S et al 2012). So improvement in iron profile observed in our study might be due to the training induced reduction of basal IL-6 and plasma hepcidin levels.

Haematological parameters

In the present study significant changes (p< 0.001) were observed in hematocrit (Hct) and RBC indices namely mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) after 12 weeks exercise training and no significant change is observed in mean corpuscular hemoglobin (MCH). Hct and MCV values were significantly increased after the training but the MCHC is significantly reduced. MCV (fL) is the average volume of a red blood cell and is calculated by dividing the haematocrit (%) by the red blood count (10¹² cells/L). MCHC (g.dl^-1) is the average concentration of hemoglobin per unit volume of red blood cells, calculated through dividing the hemoglobin (g.dl^-1) by the haematocrit (%) values. Significant increase in Hct and insignificant rise in RBC were observed in this study after the training however no significant changes were observed in Hemoglobin levels. This can be due to the exercise induced erythropoiesis or due to the release of young RBC from the bone marrow under the influence of stress hormones such as Catecholamines and cortisol (Ghigo E et al, 2011). It is well documented in the literature that exercise training can stimulate erythropoiesis and increases red cell mass as well as plasma volume, resulting in increased blood volume (Convertino VA., 1991; Min Hu., Wentao Lin., 2012). Consistent hemoglobin concentration with increased haematocrit resulted in the decreased mean corpuscular hemoglobin concentration. With exercise training, significant increase in serum iron, Hct and MCV were noted but no significant improvement is observed in basal hemoglobin levels.

No significant changes were observed in total white blood cell (WBC), WBC cell types and platelet after the training. But NLR was decreased insignificantly from 1.41 ± 0.87 to 1.36 ± 0.63. It has been proposed that the NLR can be used to monitor the exercise recovery status of an individual following a bout of exercise (Bessa et al., 2016; Gleeson, 2002). Researchers have suggested that the NLR can provide a good measure of exercise stress and subsequent recovery (Nieman, 1998). The NLR usually returns to normal within 6-9 hours after exercise, but where the exercise has been particularly prolonged and stressful, the NLR may still be elevated at 24 hours post-exercise.