, Investigating The Interaction and Exclusive Effect Of 8 Week β-Hydroxy- β-Methyl Butyrate

(HMB) Supplementation And Complex Training On Body Composition, performance, IGF-1 –

GH AXIS And TNF𝛼 serum levels Of Elite Basketball Players Aged 19-26 Years Old

𝑇𝑜𝑓𝑖𝑔ℎ𝑖 𝐴𝑠𝑔ℎ𝑎𝑟 1 .,𝑇𝑜𝑙𝑜𝑢𝑒𝑖 𝐴𝑧𝑎𝑟 𝐽𝑎𝑣𝑎𝑑 2 𝑆𝑎𝑙𝑎𝑡𝑖𝑛𝑇𝑎𝑚𝑎𝑟 𝑇𝑜𝑟𝑎𝑗 3 . , 𝐻𝑎𝑚𝑒𝑑𝐶ℎ𝑎𝑚𝑎𝑛 𝑁𝑎𝑑𝑒𝑟 4 .

1Associate Professor of exercise physiology, Urmia University, Uremia, Iran, 2 Assistant professor of exercise physiology,
Uremia University, Urmia, Iran, 3 State Universities of Urmia, MA degree in physiology of sport, 4 State Universities of Urmia,
MA degree in physiology of sport.

Abstract

BACKGROUND: Basketball is an activity that contributed from alternative energy systems is highly
correlated with the explosive power of lower body and muscular strength of athletes and production of
high levels of power in the shortest period of time (power), is introduced as necessary to achieve optimal
performance in the sport (6). To achieve the highest performance in this field of sport, taking advantage of
complex training and ergogenic supplement beta-hydroxy-beta-methyl-butyric acid(HMB) for its anabolic
and anti-catabolic traits, seems to make it available; therefore, the aim of this study was to investigate The
Interaction and Exclusive Effect Of 8 Week β-Hydroxy- β-Methyl Butyrate (HMB) Supplementation And
Complex Training On Body Composition, Strength And Hypertrophic/Atrophic Factors Of Elite
Basketball Players Aged 19-26 Years Old.

METHODS: In this semi-experimental, randomized, double-blind, placebo-controlled study with pre-test

and post-test design, 40 male basketball players of Division 1 Basketball League aged 26-19 years were
selected and randomly divided into four groups of 10 people, including: (1) Training-supplements(HMB),
(2)Training-placebo group,(3)supplement(HMB) and (4) control group. Complex training protocols was
performed in 8 weeks through 3 times a week (on odd days), on a regular basis by 60% to 80% of 1RM by
all four groups, while at the same time four group attended their regular basketball trainings on 3 days of
week (on even days). Data’s were expressed as mean (±SD) and analysis of variance (ANOVA),
Bonferroni post hoc tests to evaluate differences between the four groups (for difference between groups)
and dependent sample T-test for comparing the result of intra-group in different types in various times at
the significant level of p<0.05was performed. All statistical assessments were assessed by SPSS software
(21v).

RESULTS: The findings of this study showed that supplementation with HMB, complex training and
having both of these two factors through a chronic protocol, resulting in anabolic stimulus for a significant
increased levels of IGF-1 and GH ratio, (p <0.05), lean body mass (p <0.05) Based on BIA estimates and
the percentage of upper and lower body strength (p <0.05), so that the process of the increase in
supplementary-Training group was observed more than other groups, all these values was higher in the
training group in comparison to supplement group as well. In turn, cortisol serum levels were decreased in
same hierarchy of groups.

CONCLUSIONS: Beta-hydroxy-beta-methyl butyrate supplementation and complex training separately

contributed to preservation of muscle mass and strength increase and also lead to deliver anabolic and
anti-catabolic effects but the study indicates that the interaction of the these two factors might lead to a
synergistic effect on athletes especially when its applied in some specific periods of athlete's season where
there are high-intensity training periods. The results of this study suggests that complex training along
with HMB consumption could be beneficial for attenuating tissue catabolism and initiate muscle
anabolism particularly in athletes.

KEYWORDS: Complex Training, HMB supplementation, IGF-1 – GH axis, HPA axis.

 1. Introduction
In the recent decade, the utilization of strength training interventions integrated with
basketball training has been receiving notable attention among coaches. Incorporating
strength training activities may improve more variables than basketball training alone.
Because of the growing popularity of youth strength and conditioning programs, along
with the potential health benefits associated with this methods (Faigenbaum & Westcott,
2009), it is important to find the most efficient method for enhancing fitness performance
in young athletes. One of these interventions is the combination of strength and
plyometric exercises in a single session named as complex training. More specifically,
complex training alternates biomechanically similar high load weight training exercises
with plyometric exercises, set for set, in the same workout. Hamza (2014) adds that the
concept of post activation potentiating considered modern training concepts in the field
of sports. where is intended to develop muscle strength using a preliminary stage, a
nervous activation and physiology of the muscles working, by raising the largest amount
of muscle fibers before starting to use strength training, composite training one of the
main forms of this phenomenon is the training (post activation potentiating).An example
of complex training would include performing a set of squats followed by a set of jump
squats. Anecdotal sources have described the application of complex training (Chu,
1998; Ebben, Blackard, 1998; Fees, 1997; Fleck, & Kontor.1986; Reddin, 1999;
Roque, 1999). The use of both plyometric and strength trainings in the same workout is
also an adequate strategy of training process organization, which has highly positive
effects on practice and training time management. Plyometric and strength training are
referred to as improving the most powerful motor performance skills in young basketball
players (Fulton, 1992; Matavulj, Kukolj, Ugark-ović, Tihanyi & Jarić., 2001; Radcliffe &
Farentinos, 1999) and they are a significant component of most conditioning program
designs. A number of studies demonstrate the effectiveness of plyometric compared to
non-exercising control groups. J.B. Blakey et al., 1987, O.Diallo et al., 2001, other
studies demonstrate an enhancement of motor performance associated with plyometric
training combined with Weight training or the superiority of plyometric, compared to
other methods of training K. Adams et al., 1991, E.J. McLaughlin, 2001, J.F. Vossen et
al., 2000. Andrejić (2012) found out that such strategy demonstrated enhancement in
motor performance among youth basketball players. Similarly, Santos and Janeiro
(2008) showed significant gains in the squat jump, countermovement jump, Abalakov
Test, and medicine ball throw using a similar program. Faigenbaum et al. (2007)
discovered significant improvements in strength training with plyometric in long jump,
medicine ball toss, and pro-agility shuttle than strength training alone. Lastly, Fatouros
et al. (2000) determined that combination of strength training with plyometric
participants produced greater vertical jump and leg strength when compared with
strength and plyometric training groups. Plyometric drills are often combined with
strength training, with the presumption that motor performance may be enhanced to a
significantly greater extent than each program performed alone (Adams, O'Shea,
O'Shea & Climstein, 1992; Faigenbaum et al., 2007; Fatouros et al., 2000; Myer, Ford,
Palumbo & Hewitt, 2005; Santos & Janeira, 2008). There seems to be paucity in
literature when using the previous intervention in basketball PE. On the other hand, use
of ergogenic nutritional supplements is becoming inseparable from competitive sports.
For just over a decade, β-hydroxy-β-methyl butyrate (HMB) has been studied for its
potential as an anabolic and anti-catabolic dietary supplement assisting gains in
strength, muscle size in humans when used in conjunction with a training program. With
some evidential support, HMB has become one of the more popular nutritional
supplements taken by strength and body building athletes looking to improve strength
and muscle size (12,16). Several mechanisms of action of HMB have been proposed,
largely on the basis of extrapolation from early research on animals and deduction from
indirect measures in humans (17, 27). Supplementation with HMB may attenuate
training-induced proteolysis in the muscle via down-regulation of proteolytic pathways
(15,17,27,30) and may also stimulate protein synthesis similarly to leucine (30). Despite
largely indirect evidence to support a mechanism, several trials of the proposed
ergogenic effects of HMB on strength, body composition, and markers of muscle
hypertrophy/atrophy have been conducted in humans in conjunction with training
program. Researchers have found both gains (11,17,21,33) and no change in strength
(13,14,19,28,35) with HMB supplementation in both acute and chronic methods. These
contradict results may be related to study-specific characteristics, such as training
experience of the subjects, the HMB daily dose, the intervention duration, or the
intensity, form, and volume of the accompanying exercise intervention. Additionally,
differential and inconsistent gains in upper vs. lower-body lift performances have been
reported, with the majority of improvements being in lower-body strength (6,17,33,35).
This apparent selectivity indicates regional differences in the scope for adaptation that
may have a physiological basis or that may be linked to previous training frequency or
the relative intensity of training on various muscle groups.

Through access to what may distinguish this study from previous studies, is the
absence of any research on accompanying complex training with HMB supplementation
and also the link between the these two factor with a new cytokine which is a recently
discovered growth factor named interleukin-15 (IL-15), that expressed in skeletal
muscle (Grabstein et al.1994). In human skeletal muscle cell cultures, IL-15 induces an
accumulation of myosin heavy chain (MHC) protein in differentiated myotubes,
suggesting IL-15 as an anabolic factor in muscle growth (Furmanczyk & Quinn,2003).
IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured
skeletal myotubes (Quinnet al.2002) suggesting that skeletal muscle act as a cytokine-
producing organ. It has been demonstrated that skeletal muscles produce and express
cytokines belonging to distinct different families. However, whereas expression of
several cytokines in skeletal muscle is very low and of unknown physiological
significance, it has recently been demonstrated that the expression of some cytokines is
markedly enhanced by muscle contractions. The regulatory role of muscle contraction in
regard to IL-15 is not clear. Previous human studies have reported that skeletal muscle
IL-15 mRNA levels were not changed immediately after a 3 h run (Nieman et al. 2003)
and that plasma IL-15 (measured up to 6 h into recovery) did not change in response to
2.5 h of treadmill running (Ostrowski et al. 1998). Skeletal muscle IL-15 mRNA levels,
measured immediately after a 2 h weight training bout, did not differ from baseline
(Niemanet al.2004), whereas plasma IL-15 protein was increased immediately after
acute resistance exercise in one study (Riechman et al. 2004).
Bearing all these contradict results in mind, and the relatively low number of studies
investigating the effectiveness of HMB and complex training together over a longer
period on Hypertrophic/Atrophic trained athletes, the primary purpose of this
investigation was to test the hypothesis of the efficacy of an ergogenic nutritional
supplement and also the effect of a chronic heavy complex training bout on hypertrophy
indexes following expression of anabolic (IL-15, Testosterone)/catabolic (𝑇𝑁𝐹𝛼 ) indexes in
serum levels of the subjects, In addition, we studied the interaction effect of both these
factors on IL-15 expression in serum of the subjects in four different groups. The output
of this review will then have practical implications for sport coaches, nutritionists and/or
physicians who want to know the current evidence about the efficacy of these two
factors in their specific sport discipline.

2. Materials and Methods

2.1. Subjects

Forty male basketball players from League One team of Iran Municipality club
volunteered to participate in this randomized, double-blind, controlled and parallel design
study. Following an orally and writing explanation of all procedures, risks and benefits,
each participant signed his informed consent prior to participation in the study.
Participants were not permitted to use any additional dietary supplementation and did not
consume any androgens or any other performance enhancing drugs. Screening for
performance enhancing drug use and additional supplementation was accomplished via a
health questionnaire completed during participant recruitment. Subjects were from the
same team and were randomly assigned to one of four groups. They underwent a medical
examination and a standard set of blood tests. Subject characteristics are listed in Table 1
as mean±s.d.

Table 1. Subject characteristics

𝑮𝒓𝒐𝒖𝒑 𝟏 𝑮𝒓𝒐𝒖𝒑 𝟐 𝑮𝒓𝒐𝒖𝒑 𝟑 𝑮𝒓𝒐𝒖𝒑 𝟒

𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒔𝒖𝒃𝒋𝒆𝒄𝒕𝒔 10 10 10 10
𝑨𝒈𝒆 (𝒚𝒆𝒂𝒓𝒔) 23/7 ± 4/5 23/7 ± 4/5 23/7 ± 4/5 23/7 ± 4/5
𝑯𝒆𝒊𝒈𝒉𝒕 (𝒄𝒎) 178/2 ± 8/9 178/2 ± 8/9 178/2 ± 8/9 178/2 ± 8/9
𝑩𝒐𝒅𝒚 𝒎𝒂𝒔𝒔 (𝒌𝒈) 78/5 ± 9/25 78/5 ± 9/25 78/5 ± 9/25 78/5 ± 9/25
𝑩𝑴𝑰 (𝒌𝒈/𝒎−𝟐 ) 24/2 ± 2/5 24/2 ± 2/5 24/2 ± 2/5 24/2 ± 2/5

2.2. Study protocol

Athletes were members of the League One team of Iran Municipality club with a record of
at least 10 years of training experience, occupying high positions in national competitions.
Athletes were subjected to the randomization procedure and assigned into 4 groups: HMB
group, complex training group (CTG), HMB-complex training group (HMB-CTG) and
control group (CG). All players performed their same weekly basketball trainings sessions
𝒅𝒂𝒚
for 3𝒘𝒆𝒆𝒌 on odd days during this 56-day study protocol. The experiments were conducted
𝒎𝒈
using a preparation of HMB produced by Karen Laboratories by dosage of 𝟑𝟖 𝒌𝒈 (of total
weight) per day in three doses as follows: upon waking, immediately after training, and
before sleep; lower doses were found significantly less effective, and higher doses were not
significantly more effective (Gallagher et al.2000). The producer also prepared a placebo
preparation containing maltodextrin.
The consumed HMB dose was equivalent to the most commonly recommended intake of
𝒎𝒈
𝟑𝟖 𝒌𝒈 (of total weight) HMB per day.
Subjects of CTG and HMB-CTG perform their complex training protocol on even days, all
sets of the weights exercise with a recovery of 60 seconds/set. This is followed by a three
minute rest before performing all sets of the matched plyometric exercise with a recovery
of 90 second/set. Load intensity was ranged between %60-%80, the complex training
program is described in Table 2, and on the other hand HMB group and HMB-CTG
consume their determined dosage of HMB supplementation daily on specific times.
The schematic of study design is available in figure 1.

Week One Two Three Four

Intensity %60 - %65 1RM %65 - %70 1RM %70 - %75 1RM %75 - %80 1RM
 2.3 Testing Procedures

Subjects were assessed before and after an 8-week of protocol. All measurements were
taken during one day before and 48h after training at the same times of day. Tests
followed a general warm-up that consisted of running, calisthenics, and stretching.

Vertical Jump Test (VJ):

The subject stands side on to a wall and reaches up with the hand closest to the wall.
Keeping the feet flat on the ground, the point of the fingertips is marked or recorded. This
is called the standing reach height. The athlete then stands away from the wall, and leaps
vertically as high as possible using both arms and legs to assist in projecting the body
upwards. Attempt to touch the wall at the highest point of the jump. The difference in
distance between the standing reach height and the jump height is the score. The best of
three attempts is recorded.

Seated Medicine Ball Throw (SMBT):

The subject sits with their back to a wall, on a mat facing the area to which the ball is to be
thrown, and with the feet extended and slightly apart. The ball is held with the hands on
the side and slightly behind the center. The ball is brought to the chest, and then thrown
vigorously out as far as possible. The back should remain in contact with the wall at all
times. Three attempts are allowed. The distance from the wall to where the ball lands are
recorded. The measurement is recorded to the nearest 10 cm. The best result of three
throws is used.

Statistical analysis
All statistical analyses were calculated by the SPSS statistical package (version 22.0.1).
The results are reported as means and standard deviations (SD). Differences between two
groups were reported as mean difference ±95% confidence intervals (meandiff ± 95% CI).
Student’s t-test for independent samples was used to determine the differences in fitness
parameters between the two groups. The p<0.05 was considered as statistically significant.

Blood measurements
Sample collection was performed in the morning (be-tween 7:00 and 8:00 a.m.), after an
overnight fasting and a minimum of 30 min rest before sampling, also they were obtained
prior to pre-test and during 48h after post-test session. These blood samples were obtained
from an antecubital arm vein using a 20-gauge disposable needle equipped with a
Vacutainer® tube holder. Each participant's blood samples were obtained at the same
time of day during each session following an overnight fast. All blood samples were
collected into Vacutainer® tubes, one containing no anti-clotting agent and the second
containing gel and clot activator. The blood samples in the tubes were allowed to clot at
room temperature for 30 min and subsequently centrifuged at 3000 ×g for 15 min. The
resulting plasma and serum was placed into separate 1.8-mL micro centrifuge tubes and
isolated and frozen at−80 °C for later analysis.

Biochemical analysis
Serum concentrations of IL-15 were analyzed using a commercially available Human
Interleukin 15 (IL-15) ELISA Kit (Cat.No：CK-E91025), per manufacturer's instructions,
without modification. Listed sensitivities for IL-15 were 0.007–0.056 ng·ml−1. The intra-
assay coefficient of variation for IL-15 assay was less than 5%. Serum testosterone
concentration was determined using an electrochemiluminescence immunoassay kit
(Roche Diagnostics), with a measurement range of 0.025–15 ng/mL. The intra- and inter-
assay CVs were 4.7 and 8.4%, respectively. The serum concentration of tumour necrosis
factor (TNF)-α was determined using a commercially available ELISA kit (Canine TNF-α
ELISA Kit, MyBioSource). The sensitivity of this kit was 2.0 pg/mL, and the detection
range was 15.6–500.0 pg/mL. Both the intra-and inter-assay CVs were<15%. To eliminate
inter assay variance, all samples for a particular assay were thawed once and analyzed in
the same assay run by a single technician.
 Results

Table 3 presents the results of ANOVA for repeated measures models for the different
tests. It can be observed that baseline pre-test values, Biochemical markers, Strength and
𝑩𝑴𝑰 did differ significantly (p<0.05) after intervention, and also same modifications were
true comparing to the control group.

Table 3. Mean values (standard deviation) of the different variables in 4 groups at pre and post-test
times.
HMB Group CTG HMB-CTG CONTROL GROUP
Pre post Pre post Pre post Pre post

IL-15
Biochemical
Testosterone
markers
TNFα

Vertical Jump
Test
Strength
Seated
Medicine Ball
Throw

BMI

Concerning motor proficiency, there were obvious changes over the course of the
intervention program. Although all 3 intervention groups showed improvements in the
course of 8 weeks, the change in the HMB-CTG is more significant. After the training
program there was a significant progress in lean body mass, upper and lower body
(p<0.05). Anabolic indexes improved significantly with the training program, and
subsequently; catabolic index showed a reduction in serum levels of all 3 groups with
notable changes occurring in the HMB-CTG.

Discussion