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Assessing the Efficacy of Eufortyn - A Terclatrated CoQ10 and Creatine Combination Therapy on the Aging Rat Model

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Title:
Assessing the Efficacy of Eufortyn - A Terclatrated CoQ10 and Creatine Combination Therapy on the Aging Rat Model
Series Title:
Journal of Undergraduate Research
Creator:
Vorobyeva, Darya
Leeuwenburgh, Christiaan ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
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Language:
English

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serial ( sobekcm )

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Abstract:
With the progression of age, the organism enters a highly oxidized state resulting from impaired mitochondrial function. As the susceptibility to reactive oxygen species heightens, a decline of both size and number of skeletal muscle fibers manifests in chronic fatigue syndrome known as sarcopenia in the elderly. Consumption of antioxidant as part of a diet may help regulate oxidative damage by reducing the rampant activity of reactive oxygen species in the cell, yet the poor bioavailability of most commercial supplements limit the capacity of the antioxidant to quench free-radicals. Eufortyn is a terclatrated formulation of Coenzyme Q10, creatine, and ginseng extract that retains the integrity of the antioxidant moiety, while allowing maximal absorption into the intestinal mucosa. A Eufortyn pellet was fed orally to 19 and 27-month old Fischer 344 x BNF1 rats for six weeks. Grip strength improved (12%) in 19-mos, but not in 27-mos old rats. Water maze analysis exhibited sharpened cognitive performance in Eufortyn-treated old-age rats compared to their age-match peers. Significant advancement in mitochondrial calcium retention capacity (66% in 19-mos and 19% in 27-mos), diminished non-heme iron levels (54% in 27-mos), and suppressed nucleic acid oxidation (31% in 19-mos and 24% in 27-mos) were observed in skeletal muscle in Eufortyn-treated cohorts. These findings indicate that treatment is more effective in the middle-aged animals in comparison to the 27-month old rats suggesting that intervention with Eufortyn needs to be initiated at an earlier age than 27-month old to see optimal effects.

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Assessing the Efficacy of Eufortyn - A Terclatrated CoQ10 and

Creatine Combination Therapy on the Aging Rat Model


Darya Vorobyeva*


College of Medicine, University of Florida


With the progression of age, the organism enters a highly oxidized state resulting from impaired mitochondrial function. As the
susceptibility to reactive oxygen species heightens, a decline of both size and number of skeletal muscle fibers manifests in chronic
fatigue syndrome known as sarcopenia in the elderly. Consumption of antioxidant as part of a diet may help regulate oxidative damage
by reducing the rampant activity of reactive oxygen species in the cell, yet the poor bioavailability of most commercial supplements
limit the capacity of the antioxidant to quench free-radicals. Eufortyn is a terclatrated formulation of Coenzyme Q10, creatine, and
ginseng extract that retains the integrity of the antioxidant moiety, while allowing maximal absorption into the intestinal mucosa. A
Eufortyn pellet was fed orally to 19 and 27-month old Fischer 344 x BNF1 rats for six weeks. Grip strength improved (12%) in 19-
mos, but not in 27-mos old rats. Water maze analysis exhibited sharpened cognitive performance in Eufortyn-treated old-age rats
compared to their age-match peers. Significant advancement in mitochondrial calcium retention capacity (66% in 19-mos and 19% in
27-mos), diminished non-heme iron levels (54% in 27-mos), and suppressed nucleic acid oxidation (31% in 19-mos and 24% in 27-
mos) were observed in skeletal muscle in Eufortyn-treated cohorts. These findings indicate that treatment is more effective in the
middle-aged animals in comparison to the 27-month old rats suggesting that intervention with Eufortyn needs to be initiated at an
earlier age than 27-month old to see optimal effects.


Introduction

Aging is a natural, degenerative process occurring
throughout the life cycle of all organisms. In the elderly,
chronic fatigue syndrome is an expected, yet degenerative
symptom of aging evidenced by the decline of muscle
fibers, diminished cognitive function resulting in the
departure from a self-sufficient lifestyle '. The impairment
of mitochondrial bioenergetics is theorized to be the central
mechanism behind tissue dysfunction, fatigue and aging 2,
. In normal aging, cellular matter undergoes a highly
regulated form of cell death characterized by
morphological, biochemical, and molecular events referred
to as apoptosis 4-7. When apoptosis afflicts skeletal muscle,
the decline in both the size and number of both kinds of
muscle fibers, particularly the Type II (fast-twitch fiber)
contributes to the condition known as sarcopenia.
Sarcopenia is a debilitating condition commonly associated
with diminished muscle mass, strength and increased
disability and dependence 8. Apoptosis, in healthy cells,
allows for turnover and removal of defective cellular
matter as a way to promote tissue homeostasis.
Regrettably, once apoptosis strikes post-mitotic cells
(skeletal muscle fibers), regeneration is not possible. Thus,

*with Jinze Xu, Ph.D, Christiaan Leeuwenburgh, Ph.D.


progression of apoptosis is linked to the increasing frailty
experienced in aging. Being that apoptosis is strictly
regulated by controlled signaling platlh a ~ ', it is apparent
that any oxidative damage afflicting internal cellular
homeostasis will inevitably lead the cell to programmed
death. The central theory of aging states that mitochondrial
free-radical formation destabilizes the internal cellular
balance because endogenously occurring antioxidants fail
to manage the rampant pro-oxidant activity 9

Free-Radical Chemistry

Senescence is tied to the accrual of tissue damage by
free-radicals that results from a marked shift in pro-oxidant
activity 10 11. Normally, there is a balance between pro-
oxidant generation and anti-oxidant defense. However, in
aging, the ratio of pro-oxidants increases relative to the
endogenously occurring anti-oxidant activity 12. The free-
radical formation pathway begins in the mitochondria of
the skeletal muscle where superoxide anion (02-) and
hydrogen peroxide (H202) diffuse in and out of skeletal
muscle cells. When ferrous (Fe2+) iron, a pro-oxidant,
reacts with diatomic oxygen, the produced H202 is
converted to the highly unstable hydroxyl radical (.OH) via
Fenton chemistry (Fig. 1). Additionally, ferric (Fe3+) iron
can in turn react with hydroxyl radical and hydroxide ion to
further contribute to overall superoxide ion concentration
via Haber-Weiss reactions 3. The resulting oxidation is


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009





DARYA VOROBYEVA


Extracellular

Intracellular Fe2++ 0
H202
Fenton rac"on FeZ+ O-
,OH-+ *OH Fe3+

I- Calorie Restriction

RNA/DNA damage
Protein modification (Aiu eta . 2oT
Lipid peroxidation (Cook anYd vi19



Sarcopenia

Figure 1: Intracellular Free-Radical Generation Cycle. The
release of iron from heme-protein or ferritin protein via H202
and superoxide ion results in free iron capable of reacting with
diatomic oxygen to generate the highly-reactive hydroxyl
radical via Fenton chemistry. This free-radical, in turn, has the
capacity to alter the physiological structure of nucleic acids,
proteins, and lipids, thus compromising the integrity of the cell.
This ultimately leads to cellular apoptosis - a widely
recognized contributor of sarcopenia.

then at liberty to modify the delicate structure of nucleic
acids, lipids, and proteins 14 15. Cell death, the theorized
culprit behind degenerative disease, is facilitated by the
compromised integrity of the cell.

Coenzyme Q10

Naturally present within the inner mitochondrial
membranes is Coenzyme Q10 (CoQ10), a critical
biomolecule crucial to ATP synthesis. Functioning as a
ubiquinone and an important Complex II electron carrier in
the mitochondrial electron transport chain (ETC), CoQ10 is
a powerful antioxidant that scavenges free-radicals and
thus, maintains tissue health, potentiates cell growth, and
enhances vigor 16. Comparative studies on various
mammalian species have observed an inverse relationship
between the generation of super oxide anion radical and
sub mitochondrial CoQ10 content, implying the effective
antioxidant properties of CoQ10 16. Another study
investigated age-related changes in lipid peroxidation and
functionality of liver and skeletal-muscle mitochondria in
rats fed a diet rich in polyunsaturated fatty acids that was
either supplemented or not with CoQ10. Results for the


supplemented groups showed a decrease in peroxidizability
index, an increase in catalase activity in skeletal muscle,
and modulation of the age-related changes in the
mitochondrial ETC components. The shifts in these
biomarkers from CoQ10 supplementation suggest key
underlying mechanisms associated with the age-delaying
properties of CoQ10 17

Creatine Monohydrate

Creatine monohydrate (C) is a high energy compound
that anaerobically recycles ATP during intense muscular
exertion and is therefore concentrated in fast-twitch (Type
IIB fiber) muscle. Synthesized in the liver, pancreas, and
kidneys, creatine is transported in the bloodstream to
muscle cells where 95% of all creatine is found.
Supplementation can improve stores of phosphocreatines
and thus optimize muscular output during periods of high-
intensity exercise as well as lessen recovery time thereafter
18 Recent studies suggested that created aids patients
suffering from muscular dystrophy as well as attenuate
sarcopenia by rehabilitating disuse atrophy 19. Resistance
training for the elderly has proved beneficial, yet some
muscular loss is still problematic in the elderly; a lack of a
nutritional component may be the culprit. Creatine
supplementation has the potential to override muscle
atrophy during resistance training, although the mechanism
for its ergogenic effect is unclear 20. Additionally, creatine
descreases cytoplasm Ca2+ levels and increases
phosphocreatine stores intramuscularly which allows for
possible musculoskeletal effects, including cellular
hydration, increases in myogenic transciption factors, and
up-regulation of myosin heavy chains possibly involved in
muscle hypertrophy 19

Ginseng Extract

Ginseng extract is regarded widely as an effective
adaptogen - a natural herb product used to increase an
organism's resistance to stress, trauma, anxiety, and fatigue
21. Prolonged administration of standardized ginseng to rats
reduced oxidative stress in certain tissues by modifying
specific antioxidant enzyme activities that are required to
eliminate free radicals, thus mitigating tissue peroxidation
end-products 22, 23. Some earlier studies reported greater
oxygen uptake and transport in elderly subjects as well as
enhanced energy levels in athletes.

Recognizing the Need for Bioavailable Combination
Therapy

With the progression of age, the susceptibility to
reactive oxygen species expedites the deterioration of


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





CREATINE COMBINATION THERAPY


skeletal muscle fibers 24, 25. The multitude of degenerative
processes that influence the aging process manifest in the
total reduction of slow oxidative muscle fibers (Type I
muscle) and fast glycolytic fibers (Type IIB muscle) 8, 26. It
is hypothesized that the specific combination of CoQ10
and creatine may attenuate the deterioration of both Type
I/IIB fibers and provide a therapeutic effect 16 27. In aging
studies, oxidative stress is a major factor in diminishing
integrity of functional tissue by destroying nucleic acids
coding for appropriate proteins needed to build those
tissues. When considering the amount of antioxidants a
normal individual would assimilate from diet alone, this
amount appears to be deficient in producing changes in the
progression of tissue oxidation. Antioxidant supplements
help regulate oxidative damage by reducing the unbridled
activity of reactive oxygen species in the cell. However,
most over-the-counter products have poor bioavailability
when taken in a standard-quality, generic form, thus their
effects go unnoticed in the organism. Despite the benefits
associated with CoQ10 supplementation, its slow and
ineffective absorption into the body due to its lipophilic
nature tends to be the major obstacle in getting access to
the nutrient through ingestion 28. This very problem has
been addressed by biochemist in their development of the
innovative Eufortyn compound (Scharper Company,
Milan, Italy), which is mainly comprised of CoQ10,
creatine, and ginseng extract. Eufortyn's unique
terclatrated structural composition makes it a highly
soluble, multicomposite entity while maintaining the
integrity of the CoQ10 moiety, thereby greatly increasing
absorption into the mucosa.

Unique Properties of Eufortyn�

Clatration (from the latin word, clatrum, i.e. cage) uses
mechanical energy to create a multicomposite substance;
that is, a combination of two or more chemical entities that
interact with each other without modifying their respective
physio-chemical properties, but end up with physical
characteristics that are unique to that multicomposite.
Clatration can be used when certain physical properties,
considered to be restrictive for pharmaceutical purposes,
need to be improved. According to Giorgio Bianchi, Ph.D,
a leading development expert of Qter�, a terclatrate (ie,
three-in-a-cage) designates a multicomposite arising from
delivering mechanical energy to three moieties:
1. A biologically active substance whose
physical properties need to be modified.
2. A pharmacologically inactive polymer
acting as a passive matrix that traps single moieties
of the active ingredient inside each of its loops,
thus preventing the trapped moieties from getting
in contact and interacting with each other.


3. A small molecule that acts as a catalyst for
the formation of the clatrate, enabling clatration to
occur at room temperature thus, keeping chemical
reactions from occurring between the components
of the multicomposite.
Qter� is a terclatrate multicomposite in which the active
moiety is coenzyme Qo1 (CoQo1), the inactive polymer is a
commonly used pharmaceutical excipient, and the catalyst
is a naturally occurring amino-acid. Native CoQo1 has poor
water solubility as a substance, forming a waxy and highly
electrostatic powder: impossible to be administered by
intravenous route, poorly absorbed by the GI mucosa when
orally administered, and troublesome in the industrial
pharmaceutical setting. Qter� is aimed at overcoming these
limitations, by trapping CoQio in the passive matrix;
electrostatic interactions between moieties are prevented,
and a finely dispersible, water soluble powder is obtained,
showing a fairly improved bio-availability profile in
humans, when compared to native CoQ1o.

Materials and Methods

The Eufortyn pilot was conducted in accordance with
the National Institutes of Health guidelines for the care and
use of laboratory animals. In an effort to examine the
efficacy of Eufortyn on the physical, cognitive and
biochemical aspects of an organism, the combined therapy
(Eufortyn) was fed for 6 weeks to male Fischer 344 x
Brown Norway rats obtained from the National Institute of
Aging colony (Harlan Sprague Dawley, Indianapolis, IN)
at 6, 19, and 27 months of age. The F344xBN is an
excellent model often used for aging research. The stock
and strain of these hybrid models have been characterized
under well-defined environmental and genetic conditions.
All animals were singly housed in a temperature
(20+2.50C) and light-controlled (12:12h light-dark cycle)
environment. The animals were provided with water, food
(ad libitum NIH31 pellets #7017 and a daily Eufortyn
pellet dosed at either 938 mg/tab or 375 mg/tab depending
on rodent weight at time of feeding), and were given two
week acclimation time before the experiments began.
Animals with documented pathology were not included in
the final analyses. All three age groups were randomly
selected into either control or experimental groups (C6,
C19, E19, C27, or E27) followed by a further separation
into one of four cohorts (Table 1), which were then
staggered throughout the treatment process to allow for
sufficient time to perform all necessary physical and
cognitive analysis prior to sacrifice and tissue extraction.
Eufortyn treatment began in line with pilot schedule (Fig.
2). No treatments were given to these animals so as to limit
confounding variables in tissue biochemistry analysis.
Animals were euthanized by decapitation using a dedicated


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
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DARYA VOROBYEVA


Groups Control Control Eufortyn Control Eufortyn

Age 6 19 19 27 27
(months)
Rats 7 8 8 7 7

Table 1: Eufortyn Pilot Animal Distribution. Randomized
selection of animals into experimental and control groups
followed by placement into cohorts C6, C19, E19, C27 or
E27.


Figure 2: Eufortyn Pilot Study Design Plan. Cohorts were
staggered so as to allow for sufficient assimilation,
treatment, physical study, and sacrifice time.

guillotine to avoid interference of anesthesia on
mitochondrial functions. All discomfort, distress, pain, or
injury was accounted for in this pilot study. Select muscle
gastrocnemiuss, plantaris, soleus, EDL, and quadriceps)
and organ tissue (heart, kidney, liver, and brain) were
extracted fresh, weighed, and flash-frozen in liquid
nitrogen followed by storage in -800C until analysis. These
methods were all consistent with the recommendation of
the Panel of Euthanasia of the American Veterinary
Medical Association �.

Grip strength-behavioral/functional testing

Five weeks into treatment, functionality analyses of the
forelimb grip were assessed to determine treatment effect
on retention of musculoskeletal performance of the three
age groups. An automated grip strength meter was used as
a standard measuring device for all animals (Fig. 3). The
mean force in grams was determined with a computerized
electronic pull strain gauge fitted directly to the grasping
ring. To normalize, the resulting measurement would then


Figure 3: Grip Strength Analysis. The grip strength apparatus
measured forelimb strength and is predictive of future physical
disability. Grip strength results were expressed as total grip
strength force (kg of force) and total force divided by body
weight (kg of force/kg body weight).


be divided by body mass. After 3 successful trials were
conducted, an average was taken to determine the final
outcome.

Permeability Transition Pore

In aging, progressive deregulation of the mitochondria
organelle leads to decreased life-sustaining functions such
as ATP production, intracellular Ca2+ buffering, and
regulation of cellular redox balance and apoptosis 29, 30
Additionally, increases in non-heme iron positively
correlate with age-related mRNA oxidative damage,
diminishing mitochondrial capacity to handle influxes of
Ca2 in skeletal muscle. This theory is central to the idea
that the pro-oxidant effects of non-heme iron influences
mitochondrial impermanence thus, triggering cellular
apoptosis and the overall dilemma of neuromuscular
degeneration 31. In a highly oxidized state, such as in
senescence, mitochondrial integrity is compromised by its
inability to tolerate membrane impermeable calcium
uptakes before opening and releasing cytotoxic factors.
This mitochondrial suicide is catalyzed by the permeability
transition pore (mPTP), a voltage-dependent, high-
conductance, non-specific passive pore that infiltrates the
mitochondrial matrix and the outer and inner mitochondrial
membranes. The opening of mPTP compromises
mitochondrial membrane potential and leads to a transition
in membrane permeability 32. The negative relationship
between Ca2+-retention capacity and iron contents implies
that higher iron content renders the mitochondria more
susceptible to Ca2+- induced mPTP opening and thus,
apoptosis 3. The mPTP of both control and Eufortyn


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
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CREATINE COMBINATION THERAPY


180
160 - lcium Injection :
140 I
120
100
80 -
60
40 PTP Ope-
20
0
0:00:00 0:02:53 0:05:46 0:08:38 0:11:31 0:14:24

Figure 4: Example of mitochondrial permeability transition
pore. Mitochondria were energized with succinate and a known
amount of Ca2+ (10 uM Ca2+; Calcium Green 5N) was added
stepwise after each minute, reflective of fluorescence pulses. The
increase of fluorescence is directly linked to an increase of extra-
mitochondrial Ca2+ and the reduction in fluorescence pulses
shows the uptake of calcium by the mitochondria. The total
content of calcium intake prior to PTP is used as data for PTP.
The control data points (blue) reach maximum capacity
prematurely as indicated by the expulsion of mitochondrial
contents whereas the Eufortyn treated mitochondria (red)
indicate imperviousness to additional Ca2+ at the same point,
opening substantially later in the injection process.


samples were infused with Calcium Green - 5N and the
uptakes were measured using micro plate reader with
automatic injectors (Fig. 4).

Non-heme iron assay

In homeostatic biological organisms, free-radical formation
is a natural, continuous phenomenon and is typically kept
in check by endogenously occurring antioxidants localized
in specific areas of the skeletal muscle 9. Despite the
unremitting scavenging of free-radicals by antioxidants,
oxidant production in aging may surpass the capability of
antioxidants to fully shield oxidants from incurring
extensive oxidative damage on the muscle cell 2. Iron is
widely recognized as a powerful pro-oxidant that catalyzes
the formation of free radicals within cells. Despite a natural
tendency to form free-radicals, iron may inflict further
oxidation creating a summation effect of oxidation 31
Conducting the non-heme assay provided insight into non-
heme iron accumulation in muscle tissue with normal aging
as well as non-heme iron status in Eufortyn-treated muscle
tissue. Gastrocnemius muscle non-heme iron content was
measured after performing an iron assay described by Xu
et al.


Food Intake


26-
20-
16-

5,1.


Fo
25-
20


10-


I9
19


Age
(months)
od Intake Control
SEufortyn


Age
(months)


i2
27


Figure 5: Food Intake Trends. Aging effect on food intake of
control diet age-cohort animals (left panel). Eufortyn-effect on
food intake of age-match groups.

RNA and DNA oxidation measurement using HPLC-
ECD

Previously, studies have observed a strong correlation
between state of oxidation and mitochondrial levels of iron.
It is hypothesized that the antioxidant properties of
Eufortyn may have mediated some of the effects of non-
heme mitochondrial iron on vulnerable nucleic acids
resulting in greater longevity of the organelle in an aging
organism. Investigation of the oxidation status of muscle
tissue established the intracellular role of Eufortyn on pro-
oxidant regulation. Total RNA and DNA oxidation levels
and RNA/DNA ratios of plantaris muscles were analyzed
using a novel HPLC-ECD method 34

Results

Eufortyn supplementation during the 6-weeks showed a
significant increased in body weight between 6 and 19
months of age, but did not show a further increase at 27
months of age. No adverse changes in body weight were
experienced by any of the groups. Food intake increased in
relation to age-cohorts; both 19-month and 27-month old
animals consumed greater amounts of food as compared to
6-month old controls. There was no significant difference
in food intake between Eufortyn groups and age-matched
control groups in the animals at 19 and 27 months of age
(Fig. 5). Muscle weight in the gastrocnemius, plantaris,


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5


B






DARYA VOROBYEVA


soleus, extensor digitorum longus (EDL), and quadriceps
muscle of the 19-month and 27-month old animals
progressively decreased in contrast with the 6-month old
rats (Fig. 6). No significant difference was seen in all
muscles weights when comparing age-matched controls







Gastrocnenius


0 19 27
Age
monthss)


EDL


with Eufortyn groups (Fig. 7). Heart weight, kidney weight
and liver weight of the 19-month and 27-month old rats
gradually increased with age as compared to the 6-month
old animals. In contrast, brain weight did not change with
age.


Plantaris


Soius


(mo 6) 19
Age Ag.
months. (month.)


27


Figure 6: Control Animal Muscle Tissue Weights. Aging effect on various muscle groups of control diet age-cohorts.


Gastrocnenlus mConol
Bjtfrtyn








(mnalnl)


Plantrts iCaord
lEuraftyn


o.oIl

"�"
~; O

-m,
C .M


Soleus icorm
iEul3ftn


Ag(
If tha)


EDL Canti
EIthtyn


IL
I, 27
Age
(morth.)


-I


0-0


Bs"


Quadriceps Control
mEulortyn


Age
month. )


Figure 7: Treatment Effect On Muscle Tissue. Eufortyn effect on various muscle groups consisting primarily from Type II skeletal
muscle tissue. Supplementation with Eufortyn did not affect muscle tissue weights in either cohort.






University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6


Quadriceps
I"
b


0.-

6 19 27
Age
monthsl


* o


a,


Ss
-.a
- Cl C-
-|�.. a






CREATINE COMBINATION THERAPY


Grip Strength Analysis


Grip strength results were expressed as total grip strength
force (kg of force) and total force divided by body weight
(kg of force/kg body weight) (Fig. 8). Control cohorts
expressed diminished grip strength capabilities with age
when the kg force/kg body weight were normalized with
body weight of the animal. The Eufortyn treated cohorts
did not exhibit significantly


Ag (Non-nonnazed


Odd Distance


E looo-
1000
o
5 760-
2 O
0
I-.


controls.


Day


Figure 9: Distance in maze measurement

Odd duration





30-
0
I-�
20
Day

Figure 10: Duration in maze measurement


Figure 8: Grip Strength Analysis. Absolute grip strength force
did not change with age (Fig 8A upper Panel). In contrast, grip
strength expressed by body weight showed a decline in the 19-
month and 27-month old animals as compared to the 6-month old
controls (Fig 8A lower Panel). There was a marked increase
(12%) in total grip strength force for the 19-month old Eufortyn
group as compared to their age-matched controls after 4-weeks
of treatment with Eufortyn (Fig 8B upper panel). In addition,
forces normalized by body weight showed a similar (14%)
increase in the 19-month old Eufortyn treated group (Fig 8B
lower panel). The older 27-month old cohorts exhibited no
comparable difference due to Eufortyn treatment.


improved strength, however, there was a slight
improvement (12%) in total grip strength for the 19-month
old Eufortyn cohort compared to their control counterparts.
Eufortyn produced no distinguishable effects for the 27-
month old cohorts in terms of grip strength.


Morris Water Maze Spatial Discrimination

Initial (day 1) and terminal learning performance (day 3)
using the distance measure were determined. A significant
effect of both day (p < 0.001) and age (p = 0.02) was seen
in that all groups improved between initial and terminal
learning and this effect was larger in the younger versus
older animals (Fig. 9). Though, there was no significance
observed in either aging or treatment effect, there was a
promising trend observed for the initial (day 1) trial of the


Odd Velocity
27.S.
ao


o
17.a
15.0
12.5 .


o i 2
Day


0c1g
...E19
--0C27
* E27


3 4


Figure 11: Velocity in maze measurement

experiment; the Eufortyn groups achieved a shorter
distance to the platform. This indicated that the Eufortyn
groups experienced improved cognitive performance
relative to the
Over the course of the experiment, the learning curve of
the Eufortyn and control groups began to coincide and
finally no significant difference in duration was seen on the
final day of water maze. The similar pattern was observed
for the distance traveled by each cohort and group (Fig.
10). The Eufortyn groups started off with an advantage in
that they had to travel the shortest distance to reach the
hidden platform as compared to the control, however at the
final stage, all groups tended to travel the same small
distance resulting from the inevitable assimilation to the
water maze in general. The velocity at which the rats
traversed to the hidden platform was consistently greatly in
the Eufortyn groups than the control groups (Fig. 11). The


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
7


N-1bn~d gaw -M
Nowumr-1.


AGO 01M-ftodl





DARYA VOROBYEVA


SSM


to-
Gno-
50.
d'~
40.


200]
E 0



ii



1~1 1001
Z


SSM


t-I
40]

L
10


iControt
Eufortyn


-r-


Age
(months)


10
Age
(months)


Figure 12: SSM and IFM Calcium Retention. The calcium retention capacity of the subsarcolemmal mitochondria
(SSM) decreased significantly with age in the 19- and 27-month old animals compared to the 6-month old control
(Fig 12A lower panel). One-way ANOVA revealed a significant age effect for SSM (Fig 12A lower panel; p< 0.05).
Eufortyn treated mitochondrial demonstrated a marked increase in the calcium retention capacity of the SSM
isolated from the 19-month old rats (66%) and a modest increase in the 27-month old rats (19%) (Fig 12B upper
panel). IFM mitochondria did not exhibit any significant trend.


19-month Eufortyn treated rats appeared to be fastest
initially, but eventually leveled off in speed with the 27-
month Eufortyn treated rats. Nevertheless, the control
groups consistently lagged behind their age-matched
Eufortyn groups throughout the course of the experiment.

Permeability transition pore (PTP) opening

The maximal calcium loading capacity of isolated
mitochondria was determined by using membrane
impermeable fluorescent probe, Calcium Green-5N.The
calcium uptakes of subsarcolemmal (SSM) and
intermyofibrillar (IFM) mitochondria were measured by
using the microplate reader with automatic injectors. In
control 19 and 27-month old rats as compared to the 6-
month olds, the SSM endured more calcium additions
before pore opening and expulsion of cell-death markers
(Fig. 12). In other words, with age the mitochondria uptake
less calcium additions (in nmol of calcium/ mg of
mitochondrial protein) for the opening of the permeability
transition pore and release of cytotoxic factors such as the
pro-apoptotic proteins cytochrome c and apoptosis-
inducing factor.

Eufortyn and non-heme iron

The total non-heme iron content of gastrocnemius


muscle in control cohorts showed significant iron
accumulation as part of a normal aging effect (Fig. 13).
Eufortyn treatment of age-matched cohorts showed no
distinction at the 6 and 19-months, but a significant (54%,
p< 0.05) mitigation in iron content was observed in the 27-
month old senescent rats.

Eufortyn and oxidative stress

Oxidized DNA was assessed by the levels of oxidative
products 8-oxo-7,8-2'-deoxyguanosine from control and
Eufortyn rats at 6, 19, and 27 months of age. One-way
ANOVA showed no significant age effect for the DNA
oxidative damage (Fig. 14 left panel). However, DNA
oxidative damage in both 19- and 27-month old rats was
attenuated by 31% and 24% individually in the Eufortyn
groups as compared to age-matched controls (Fig. 14 right
panel). Two-way ANOVA indicated a significant treatment
effect (p < 0.10), suggesting that Eufortyn effectively
mitigates the DNA oxidative damage in the gastrocnemius
muscle.

Conclusion

As a whole, these results have advanced our
understanding of Eufortyn's effectiveness on intracellular
energy regulation in skeletal muscle mitochondria by way


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
8






CREATINE COMBINATION THERAPY


03
v,�
E
0 0M
Z =


g.L-


Age
(Months)


Eufortyn

T


Control
=Eufortyn


19
Age
(Months)


Figure 13: Non-Heme Iron Aging and Eufortyn Effect. A substantial aging effect depicted by total non-heme iron content
measured in gastrocnemius muscle tissue of ad-libitum fed 6, 19, and 27-month old rats (left panel) normalized to micrograms of iron
per gram of wet tissue weight (p< 0.05 )by Tukey's Multiple Comparison Test, n= 7-8 per group. Total non-heme iron content
measured in gastrocnemius muscle tissue of 19 and 27-month old ad-libitum and Eufortyn fed rats (right panel) normalized to
micrograms of iron per gram of wet tissue weight. With respect to 19-month old control group, Eufotyn treatment showed no effect,
but in the 27-month old age Eufortyn treated animals show a remarkable suppression of non-heme iron (54%) as compared to control.
(p< 0.05), n =7-8 per group.


19 27
Age
;months


mContol
Eutartyn


Age
(months)


Figure 14: Effects of aging and eufortyn on DNA oxidation levels in control and eufortyn-treated gastrocnemius muscle. Levels
of oxidized DNA were assessed by examining levels of oxidative products 8-oxo-7.8-2'-deoxyguanosine using HPLC-ECD. One way
ANOVA exhibited no significant difference in DNA oxidative damage between control cohorts (Fig 14 left panel), but oxidative
damage in both 19 and 27 month old rats was diminished by 31% and 24% respectively in the Eufortyn groups as compared to the
control cohort matches (Fig 14 right Panel). Two-way ANOVA suggested a significant treatment effect (p< 0.10), indicating that
Eufortyn successfully lowered DNA oxidative damage in the gastrocnemius muscle.


of physical and cognitive improvements of the aging
organism. Our results demonstrated the treatment's role in
altering biomarkers specific to mitochondrial bioenergetics
and skeletal muscle aging. All tissue weights appeared
unaffected by Eufortyn treatment when compared with
age-matched controls indicating that the diet utilized has no
negative effects on organ weight and is safe for
consumption. Moreover, Eufortyn had no negative effects
on body weight, food intake and muscle weights in this


study. These findings establish the safety of Eufortyn as a
supplement. The distance measurements delineate that the
Eufortyn groups had a higher initial performance capacity
and were therefore able to exhibit outstanding performance
compared to the control groups. Resultantly, Eufortyn may
play a role in delaying cognitive deterioration with age by
extending its protective properties beyond the blood brain
barrier. The beginning trials of the water maze experiment
in which the Eufortyn groups display a distinct lead over


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
9






DARYA VOROBYEVA


their control counterparts support the notion that the
Eufortyn treatment boosts mental acuity. The velocity
(cm/s) measure confirms the two previous measures of
distance and duration in corroborating the positive effect of
the treatment on cognition and therefore, facilitation of
learning, habituation, and application of problem solving
strategy in the 19 and 27-month old animal models.
In the PTP analysis of the Eufortyn treated groups, the
calcium retention capacity increased by 66% in the 19-
month old rats and 19% in the 27-month old rats
establishing sufficient evidence that Eufortyn prevented
mitochondrial-mediated apoptosis and reduced inclinations
toward cell death in vivo; this is a remarkable result
pointing out the treatment's preventative features in
delaying mitochondrial-mediated apoptosis and reducing
the susceptibility to cell death in vivo.
The non-heme iron assay results provided a positively
correlating trend between the aging rats and non-heme iron
concentration found in gastrocnemius muscle tissue. The
typical increase of iron seen in aging skeletal muscle has
been substantially suppressed by supplementation of
Eufortyn just over a six week period providing strong
evidence that Eufortyn helped diminish non-heme iron
levels in aging muscle.
Nucleic acid oxidation results propose that the treatment
is clearly more promising in the middle-aged animals than
in the 27-month old rats; 27-month olds failed to benefit as
much as 19-month olds because of irreversible damage.
The intervention with Eufortyn needs to be initiated at an
earlier age than 27-month old to see optimal effects.
Considering all the trends, it is imperative for future
clinical trials to take place. Further, in-depth studies will
work to validate Eufortyn as an effective therapy in
delaying aging effects while improving the strength,
fatigue resistance, and independence of the elderly
population.

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