TITLE: Supplementation with Silk Amino Acids
improves physiological parameters defining stamina in elite
fin-swimmers

  ABSTRACT.BACKGROUND:
Previous animal study has shown that supplementation with silk amino
acid hydrolysate (SAA) increases stamina in mice. The presented study was the
first formal evaluation of the influence of SAA supplementation on parameters
defining physiological fitness level in humans.

ABSTRACT.METHODS:
It was a randomized controlled trial with a parallel-group design on
elite male fin-swimmers. The experimental group was supplemented with 500 mg of
SAA per kg of body mass, dissolved in 250 ml of a Carborade Drink®; the control
group with Carborade Drink® alone; 3 times a day, 30 minutes prior to the training
session.

ABSTRACT.RESULTS:
Changes discerned in the experimental group were more pronounced
than those observed in the control group. For example, the change in the serum
lactic acid concentration observed in the experimental group was sevenfold less
than in the control group [21.8 vs. -3.7 L% for the control and experimental
groups, respectively]. An analysis of a lactate profile as a function of a maximal
swimming velocity exposed a statistically significant positive shift in the
swimming velocity of 0.05 m/s, at the lactate concentration of 4 mmol/L in the
experimental group. There was also a positive, although statistically
insignificant, increase of 2.6 L% in serum testosterone levels in the experimental
group.

ABSTRACT.CONCLUSIONS:
This study showed that a 12-day SAA supplementation combined with an
extensive and rigorous training schedule was sufficient to increase an aerobic
stamina. However, this phenomenon was associated with an augmented level of
muscular damage (an increased level of creatine phosphokinase in the experimental
group).

BODY.INTRODUCTION:
A shorter running time, a longer jump, or a longer distance run during
a specific time, are results of not only a rigorous training regime but also of an
increase of understanding of human physiology combined with modern nutritional
techniques.  Recently, a hydrolysate of silk amino acids (SAA) from silk of
silkworms (Bombyx mori) comprising of peptides of
a length of 18 to 19 amino acids, has become very popular among people performing
leisure and competition sports.  According to a variety of findings employing animal models, SAA exerts
a vista of physiological actions including, among others, anti-diabetic
[1], anti-oxidant [2], and anti-tumor [3] properties. One study had even shown that SAA may also influence
levels of blood triglycerides [4].  Although some study indicated that branched-chain amino acids (BCAA)
supplementation (300 mg/kg of body mas/day) may enhance exercise capacity
[5] recent studies on an animal model,
performed by South Korean scientists, showed that high doses of SAA, i.e., 800 mg/kg
of body-mass [6] or 500 mg/kg
[7] also enhance stamina. The latter
also shows a clear augmentation of testosterone production.  Although, as stated above, SAA and BCAA evoke similar physiological
response, i.e., enhancement of exercise capacity, the obvious differences in their
amino acid compositions – BCAA comprises of Leu, Ile, and Val whereas SAA: Ala, Gly,
Ser, Val, and Thr - enforces, most probably, different modes of their metabolic
action. It is known that BCAAs are mainly oxidized in skeletal muscle and that
exercises promote oxidation of BCAA [8].
The mode of action of SAA is currently not established, although there are some
indications that its oxidation will occur mainly in liver and the main amino acid,
playing the paramount role in its physiological function, is L-alanine -through its
conversion to beta-alanine [9].  Since SAA is already on shelves of many sport nutrition shops, we
undertook the task of verifying its stamina-enhancing viability on a human model.
The presented study was a randomized controlled trial with a parallel-group design
on male competitive fin-swimmers.

BODY.METHODS.ETHICS:
All procedures used in this study were in accordance with the
ethical standards of the regional medical chamber. A written consent was obtained
in writing from participants and/or guardians of participants under the age of
18 years.

BODY.METHODS.STUDY SUBJECTS:
We conducted a randomized controlled trial with a parallel-group
design of competitive male fin-swimmers. All participants were recruited from the
national fin-swimmer team.  Both samples' size was eight (i.e., the control and the
experimental sample), and the age means and standard deviations were 17.6 ± 3.78
and 20.71 ± 4.82, respectively.

BODY.METHODS.TRAINING PROGRAM:
The training camp, during which the experiment was performed, was a
microcycle lasting 12 days and comprising three training units daily: (1)
7:00–9:30 am swimming, (2) 11:00–1:00 pm walking and/or running, every other day
stretching, and (3) 3:00–5:00 pm swimming.  Within this microcycle, each of the competitors performed 30
training units constituting of total 65training hours. Each of the subjects swam,
on average, 7 km per day.

BODY.METHODS.SUPPLEMENTATION:
All the required supplements were bought directly from a
distributor (Fitness Authority®, http://www.fanutrition.pl/en) without an indication of the purpose of the purchase. According to
the producer, the supplement contained 85.95 g of SAA (alanine, glycine, serine,
valine, and threonine) per 100 g of dry mass. A required daily amount of SAA,
comprising total of 500 mg SAA per kg of body mass, was dissolved in 250 ml of a
Carborade Drink® containing 6.3 g of carbohydrates, 0.046 g of sodium, 2.4 mg of
niacin, 1.8 mg Vitamin E, 0.9 g pantothenic acid, 0.21 g Vitamin B6, and 7.5 μg of
Biotin per 750 ml. A respective nutrition, i.e., a mixture of SAA and Carborade
Drink®, and Carborade Drink® alone was served through a straw in lidded cups to
both the experimental and the control groups three times a day30 minutes before
each training session [10]. It has to
be stressed that all drinks were prepared by a person who did not have an access
to competitors.  During the study, we also monitored the total calories, proteins,
carbohydrates, and lipids consumed by the control and the experimental groups,
stratified into breakfast, lunch, and supper.

BODY.METHODS.DATA ACQUISITION:
A fasting blood draw was completed to measure the following
parameters: (1) creatine phosphokinase (CPK), (2) aspartate aminotransferase (AS),
(3) alanine transaminase (ALT), (4) lactate dehydrogenase (LDH), (5) serum
creatinine (SCr), (6) lactic acid (LH), (7) serum glucose (Gluc), (8) total blood
protein (TBP), (9) corticosterone (C), (10) testosterone (T), and (11) blood urea
nitrogen (BUN). All laboratory analyses were performed using the ARCHITECT ci8200
Integrated System, Abbott Diagnostic.

BODY.METHODS.LACTATE PROFILING PROTOCOL:
The incremental 7 × 200 m step test was used to provide objective
information on the aerobic fitness of a swimmer. All testing was conducted in a
50 m pool. Individualized target times, based on the personal best time for each
swimmer, were calculated before each test. The final swim was set to be for
maximum effort. The time for each 100 m split and the total 200 m was recorded
manually. After completion of each 200 m, heart rate was measured with a Polar
Sports Tester PE (Polar Electro Oy, Kempele, Finland) and a 25 mL capillary blood
sample was taken from the earlobe or finger tip and analyzed for lactate
concentration using the Accusport Blood Lactate Meter (Boehringer Mannheim,
Germany). Lactate tolerance was assessed graphically on a plot of swimming
velocity (swimming pace, m/s over the distance of 200 m) versus lactate
concentration.

BODY.METHODS.STATISTICAL ANALYSIS:
Changes induced by the training period in experimental and control
groups were examined using the Wilcoxon signed-rank test. Differences between the
groups were evaluated using the Wilcoxon rank sum test using the P-value of 0.05
as the statistical threshold.  Relative changes induced by the experiment were measured using the
"natural" relative difference, employing the natural logarithm, which was denoted
as log percent (L%) [11]: L% =100 *
ln (after/before).

BODY.RESULTS:
The basic statistics of the baseline and the experimental period are
shown in Table 1.Table 1
Differences in physiological parameters at baseline
and after 12-days training program in SAA supplemented group and a control
group

Variable

Cont (0)

Ex (0)

Cont (12)

Ex (12)

L% Cont

L% Ex
LA0.82 (0.76,0.9)1.1 (0.98,1.1)1.02 (0.92,1.15)1.06 (0.905,1.2)21.8−3.7G94 (92,101)90 (90,93)80 (74,81)85 (80,87)−16.1−5.7CPK195 (168,215)168 (113,368)290 (288,297)413 (331,568.5)39.789.9LDH310 (272,314)298 (266,310)389 (303,391)339 (305.5,398.5)22.712.9AST18 (17,18)21 (15,26)29 (21,29)27 (25,32.5)47.725.1TBP6.7 (6.5,6.9)6.8 (6.8,7)6.5 (6.4,6.8)6.6 (6.5,6.7)−3.0−3.0ALT17 (15,20)22 (15,29)25.5 (22.75,28.75)22 (20.5,27)40.60SCr1.08 (1.01,1.08)1.19 (1.16,1.27)0.93 (0.91,0.99)1.26 (1.165,1.32)−15.05.7BUN28 (26,29)32 (30,32)24 (24,27)27 (26,30)−15.4−17.0C39.7 (30.3,50.3)52.3 (40.8,53)38.9 (33.8,39.2)58.3 (52.2,64.55)−2.010.9T23.92 (21.01,28.07)20.57 (19.43,26.64)22.51 (21.49,22.8)21.12 (15.73,24.69)−6.12.6Ex = experimental group; Cont = control group; LA = Lactic Acid
(mmol/L); G = blood glucose (mg/dL); CPK = creatine kinase (U/L);
LDH = lactate dehydrogenase (U/L); AST = aspartate aminotransferase (U/L);
TBP = Total Blood Protein (g/dL); ALT = alanine transaminase (U/L);
SCr = Serum creatinine (mg/dL); BUN = blood urea nitrogen (mg/dL),
C = corticosterone (nmol/L); T = Testosterone (nmol/L). The values are
presented as a median and an inter-quartile range.  An analysis of changes induced by SAA supplementation in experimental
and control groups revealed the lack of statistically significant changes. However,
the majority of changes observed in the experimental group are more pronounced than
those observed in the control group.  A statistical comparison of the total calories, proteins,
carbohydrates, and lipids consumed during the breakfast, lunch, and supper, revealed
the lack of significant differences between the control and experimental
group.  A concentration of serum lactic acid in the control group elevated
(21.8 L%), and decreased (−3.7 L%) in experiment group.  Corticosterone concentration decreased in the control group (−2.0 %L)
while in the experimental group, it increased (10.9 L%). Analogous, although not so
pronounced, a decrease of −6.1 L% and an increase of 2.6 L% of testosterone levels
in the control and experimental groups was observed. It has to be noted that in the
previous study an increase of only 1.3 L% in mean testosterone levels after 44 days
of exercise program was observed [7].  An increase of 47.7 L% in the control group versus the lack of any
changes in the experimental group was observed for ALT. AST concentration increased
in both groups, and an increase in the control group was more pronounced than in the
experimental group.  There was also a substantial increase in serum creatine kinase levels
in both control and experimental groups.  An analysis of a lactate profile, Figure 1A-B, as a function of a maximal swimming velocity, exposed a
statistically significant positive shift of swimming velocity, at the reference of
the lactate concentration of 4 mmol/L, between the experimental group and the
control group.Figure 1
Blood Lactic Acid (mmol/L) concentration in the
control group (A) and the experimental group (B) of elite male
fin-swimmers athletes before and after 12 days training
microcycle.


BODY.CONCLUSIONS:
A search for novel supplements, other than anabolic steroids, lead to
founding of a well-prospering industry, providing amateur and professional sportsmen
with a vista of nutrition increasing muscular gain and stamina.  Among them, SAA has become very popular due to their supposed
positive influence on physical stamina. Unfortunately, the popularity of this
supplement is rendered rather by claims of a manufacturer and not lucid scientific
reports on human models. To address this shortcomings we performed this study, which
is the first ever attempt to analyze an impact of SAA supplementation on physical
stamina in humans.  Taking into account the current literature on translation of drugs
dose from animal to human [12], we
primarily contemplated the use of ~41 mg of SAA per kg of body mass/day, not
500 mg/kg/day as presented in this study. However, we did not consider SAA as drugs,
per se, but only as a nutritional supplement.
Additionally a limited duration of an experiment i.e., 12-day training regime, and
demands of team coaches inclined us to employ a very high dose of SAA. i.e.,
500 mg/kg body mass/day.  In this study, performed on a group of competitive male swimmers, we
confirmed the earlier finding, that indicated SAA induced increase in physical
stamina; vide an increase of a maximal swimming
velocity at the lactate threshold of 4 mmol/L [13,14] in the
experimental group.  Although the previous study showed a positive influence of SAA
supplementation on the elevation of testosterone levels on the
44th day program, in which mice were administered to a
weight-loaded (5% of body weight) forced swimming, this study, i.e., a 12-day SAA
supplementation program combined with an extensive and rigorous training program,
resulted only in an slight increase in serum testosterone levels.  There is a significant discrepancy regarding the levels of creatine
phosphokinase (CPK), a marker of muscle damage, between this report and the previous
one [7]. Opposite to the animal model
study, this analysis shows a perspicacious increase in CPK levels in the
experimental group as compared to the control group; a change of 36.7 L% in the
former versus 89.9 L% in the latter. This observation allows us to conjecture that
SAA supplementation induces a higher stamina, but it does not prevent a muscular
damage caused by an extensive training effort. However, an analysis of the current
literature indicated that supplementation with branched-chain amino acids and
taurine-the supplement diminishing or preventing muscular damage caused by extensive
training-may be the right direction for utilizing the whole potential of SAA
supplementation [15].  The weak point of this study is small sample size, which may be
responsible for the lack of more pronounced changes induced by SAA supplementation.
Nevertheless, a comparison of our study's data with those performed on an animal
model [7], where the study sample
comprised only 10 animals, supported the practical viability of the obtained
results.

Question: Compared to control group with Carborade Drink® alone what was the result of Intervention on Corticosterone concentration?
Evidence: An analysis of a lactate profile as a function of a maximal
swimming velocity exposed a statistically significant positive shift in the
swimming velocity of 0.05 m/s, at the lactate concentration of 4 mmol/L in the
experimental group. 
Answer: significantly increased