REDUCING EARLY ATROPHY WITH LEUCINE DURING IMMOBILIZATION OF SKELETAL MUSCLE

Recovery from injury, illness, and/or disease is associated with periods of
skeletal muscle disuse. The inactivity associated with periods of disuse
results in a loss of skeletal muscle mass (1), leading to negative health
consequences including reductions in strength (1), the onset of insulin
resistance (2), a decline in basal metabolic rate (3), and body fat mass
accumulation (4). These conditions are associated with pre-mature physical
frailty (5), elevated health care costs (5), and an increased risk of mortality
(6). Muscle disuse forms a major health concern for the elderly who are already
at an increased risk for sarcopenia, or age related losses in skeletal muscle
mass and strength (7). Episodic periods of muscle disuse that are common in the
elderly are now thought to represent a period of *catabolic crisis* that
significantly accelerate the progression of sarcopenia (7). Currently, the
elderly (age 60 y or >) make up the fastest growing age group, with projections
to reach ~21% of the world*s population by the year 2050 (8). Population
studies demonstrate that sarcopenia affects >20% of people age 60-70, and ~50%
of people >75 (9). Direct healthcare costs attributable to sarcopenia in the
USA were ~$18.5 billion in the year 2000 (8). Clearly, it is critical to
identify strategies that can effectively offset the loss of muscle mass and
strength that occur in response to disuse in order to prevent an epidemic of
frailty among the elderly, a loss of independence, and to reduce the risk of
morbidity/mortality.
Disuse studies in humans have generally been conducted over periods exceeding
10 days in young healthy subjects (10). These studies have shown that 10-42
days of disuse leads to a rate of muscle loss of ~0.5-0.6% of total muscle mass
per day, with a decline in muscle strength between 0.3-4.2% per day (10).
However, the loss of muscle mass resulting from disuse is most rapid during the
early stages of inactivity (7), with substantial muscle loss occurring in as
little as 5 days of muscle disuse (11). This is a major problem for the elderly
since the loss of muscle mass and function resulting from 14 days of
immobilization cannot be regained with 4 weeks of aggressive resistance
exercise training (12). To put this loss into perspective, we have shown that
only 5 days of immobilization in elderly men results in a ~1.5% decline in
quadriceps muscle cross-sectional area (CSA) (13). Extrapolating this to a
whole body level, 5 days of bed-rest would result in *1 kg of muscle loss.
Thus, even with *80% recovery of lost muscle mass following this period of
disuse, *400 g of muscle would be lost following only two short periods of
illness or injury per year. This equates to 0.8% muscle loss per year,
contributing largely to the estimated 1-2% yearly muscle loss from age 50
onwards (14). Currently, little is known about the physiological mechanisms
that induce muscle atrophy in response to short-term disuse. Muscle mass is
determined by the balance between rates of muscle protein synthesis (MPS) and
muscle protein breakdown (MPB). When MPB exceeds MPS, the result is a negative
net protein balance and muscle loss. Anabolic stimulation by nutrients,
particularly amino acids, is a powerful modulator of skeletal muscle accretion
and is a fundamental process for maintenance of skeletal muscle mass. However,
declines in basal and postprandial MPS have been reported after 5-42 days of
bed rest and lower limb immobilization (10), however whether changes in MPB
also occur is unclear given the lack of in vivo assessments of dynamic MPB
during disuse. In the absence of direct measures of MPB rates, studies have
looked for evidence of an up-regulation of the ubiquitin-proteasome system
following disuse as a proxy of increased MPB (10), as this protein degradation
pathway and its component enzymes are activated in a catabolic state. However,
inconsistent molecular findings, together with a lack of data on dynamic MPB
rates, make it difficult to clearly assess the contribution of MPB to muscle
atrophy during disuse. The amino acid leucine is recognized as a unique
nutrient regulator of muscle protein metabolism as it serves to stimulate MPS,
and inhibit MPB by suppressing proteosomal degradation (15). Leucine appears to
inhibit muscle atrophy in animal models, in part, through down-regulation of
MPB (15). However there is no information available on the impact of leucine
supplementation on dynamic measures of MPS and MPB, markers of the
ubiquitin/proteasome pathway, and skeletal muscle mass during muscle disuse in
humans.

REFERENCES

1. Deitrick JE. The effect of immobilization on metabolic and physiological
functions of normal men. Bulletin of the New York Academy of Medicine
1948;24(6):364-75.
2. Stuart CA, Shangraw RE, Prince MJ, Peters EJ, Wolfe RR. Bed-rest-induced
insulin resistance occurs primarily in muscle. Metabolism: clinical and
experimental 1988;37(8):802-6.
3. Haruna Y, Suzuki Y, Kawakubo K, Yanagibori R, Gunji A. Decremental reset in
basal metabolism during 20-days bed rest. Acta physiologica Scandinavica
Supplementum 1994;616:43-9.
4. Ferrando AA, Lane HW, Stuart CA, Davis-Street J, Wolfe RR. Prolonged bed
rest decreases skeletal muscle and whole body protein synthesis. The American
journal of physiology 1996;270(4 Pt 1):E627-33.
5. Strawbridge WJ, Shema SJ, Balfour JL, Higby HR, Kaplan GA. Antecedents of
frailty over three decades in an older cohort. The journals of gerontology
Series B, Psychological sciences and social sciences 1998;53(1):S9-16.
6. Metter EJ, Talbot LA, Schrager M, Conwit R. Skeletal muscle strength as a
predictor of all-cause mortality in healthy men. The journals of gerontology
Series A, Biological sciences and medical sciences 2002;57(10):B359-65.
7. English KL, Paddon-Jones D. Protecting muscle mass and function in older
adults during bed rest. Current opinion in clinical nutrition and metabolic
care 2010;13(1):34-9. doi: 10.1097/MCO.0b013e328333aa66.
8. Janssen I, Shepard DS, Katzmarzyk PT, Roubenoff R. The healthcare costs of
sarcopenia in the United States. Journal of the American Geriatrics Society
2004;52(1):80-5.
9. Berger MJ, Doherty TJ. Sarcopenia: prevalence, mechanisms, and functional
consequences. Interdisciplinary topics in gerontology 2010;37:94-114. doi:
10.1159/000319997.
10. Wall BT, Dirks ML, van Loon LJ. Skeletal muscle atrophy during
short-term disuse: implications for age-related sarcopenia. Ageing research
reviews 2013;12(4):898-906. doi: 10.1016/j.arr.2013.07.003.
11. Wall BT, Dirks ML, Snijders T, Senden JM, Dolmans J, van Loon LJ.
Substantial skeletal muscle loss occurs during only 5 days of disuse. Acta
Physiol (Oxf) 2014;210(3):600-11. doi: 10.1111/apha.12190.
12. Suetta C, Hvid LG, Justesen L, Christensen U, Neergaard K, Simonsen
L, Ortenblad N, Magnusson SP, Kjaer M, Aagaard P. Effects of aging on human
skeletal muscle after immobilization and retraining. J Appl Physiol
2009;107(4):1172-80. doi: 10.1152/japplphysiol.00290.2009.
13. Dirks ML, Wall BT, Nilwik R, Weerts DH, Verdijk LB, van Loon LJ.
Skeletal muscle disuse atrophy is not attenuated by dietary protein
supplementation in healthy older men. The Journal of nutrition
2014;144(8):1196-203. doi: 10.3945/jn.114.194217.
14. Nair KS. Aging muscle. The American journal of clinical nutrition
2005;81(5):953-63.
15. Dodd KM, Tee AR. Leucine and mTORC1: a complex relationship.
American journal of physiology Endocrinology and metabolism
2012;302(11):E1329-42. doi: 10.1152/ajpendo.00525.2011.

Primary Objectives:


1. Determine the impact of short-term (3 days) unilateral lower-limb
immobilization on *cumulative* rates of MPS in younger and older adults.
2. Determine whether leucine supplementation can prevent declines in cumulative
MPS and muscle mass in response to 3 days of immobilization in younger and
older adults.
3. Identify whether postabsorptive MPB is elevated and MPS is reduced following
3 days of immobilization-induced disuse in younger adults.
4. Determine whether leucine supplementation can reduce MPB, increase MPS, and
attenuate the loss of muscle mass during 3 days of immobilization in younger
adults.


Hypothesis:
1. In both younger older adults, 3 days of disuse via unilateral lower-limb
immobilization will result in muscle atrophy and coincide with decreased rates
of *cumulative* MPS during disuse.
2. In both younger and older adults, leucine supplementation during 3 days of
disuse via unilateral lower-limb immobilization will attenuate the decline in
cumulative MPS and loss of muscle mass.
3. In younger adults, 3 days of disuse via unilateral lower-limb immobilization
will increase postabsorptive MPB rates and decreases postabsorptive MPS rates.
4. In younger adults, leucine supplementation during 3 days of disuse via
unilateral lower-limb immobilization will reduce the increase in postabsorptive
MPB rates and decline in postabsorptive MPS rates following immobilization.

Study Design
Twenty-four (12 men and 12 women) younger (18-35 years of age inclusive) and
twenty-four (12 men and 12 women) older (60-80 years of age inclusive) subjects
will be recruited to undergo 3 days of uni-lateral lower limb immobilization.
Subjects will be moderately active, but not currently engaged in a structured
exercise program. Exclusion criteria will include: lower limb and/or back
injuries, a history of thrombosis/cardiovascular disease, use of
anticoagulants, musculoskeletal/orthopedic disorders, structured resistance
exercise training, use of corticosteroids, use of protein supplements during
the study, presence of diabetes, pregnancy, hormone replacement therapy, third
generation oral contraceptives, and use of tobacco products. Inclusion criteria
will include: moderately active (see above), male or female age 18-35 or 60-80
years inclusive, and BMI not lower than 18.5 and not higher than 30.0.

Screening
When subjects respond to the advertisement, we will contact them by
e-mail/phone and briefly explain the study. We will also provide them with the
information brochure and the informed consent form which they can read and
discuss with family of friends before deciding whether or not to participate.
To assess whether subjects are eligible to participate in this study, we will
invite them for a screening visit at the University. Before we start the
screening, we will explain the entire experimental trial and answer any
potential questions. We will then ask them to read, fill out, and sign the
informed consent form. After signing the informed consent form, we will start
the screening by going through the medical questionnaire to assess their
general health, use of medication, and physical activity. The volunteer will be
instructed on how to use crutches and to not bear weight on the casted limb,
and to minimize muscle contractions of the upper leg. We will also assess
weight, height, and blood pressure. The medical questionnaire will be carefully
assessed by the responsible physician before subjects are allowed entry into
the study. This visit is expected to last 1.5 hours. After the screening the
test days (Test day #1, Test day #2 and Test day #3) will be scheduled if
applicable (informed consent signed + suitable based on inclusion/exclusion
criteria). It is aimed to book these Test days as soon as possible, depending
on the subjects availability. However scheduling will take into account that
subjects need the record their food intake for 3 days and wear the Actical
activity monitor for 3 days prior to Test day 1.


Diet and activity prior to testing
All subjects will consume a standardized dinner the evening before the onset of
immobilization (Test Day #2). In addition, the younger subjects will consume a
standardized dinner the evening before the acute amino acid 13C6 tracer
investigation (Test Day #3; Figure 3). This standardized dinner is an *Aviko
maaltijdpannetje* (Appendix D4.1) and will be purchased at a regular
supermarket in Maastricht. The expiration date from the manufacturer will be
checked. Meanwhile, the meals will be stored in an appropriate freezer of the
*dietary-kitchen* at the department of Human Biology. The precise composition
and preparation methods are described on the label of the product (Appendix
D4.1). The subjects will be instructed to store the meal in a freezer until
preparation and to prepare the meal themselves according to the instructions on
the label. All subjects will also be asked to record their food intake for 3
days immediately before the immobilization period and for 3 days during
immobilization period in a food diary that will be provided during the
screening (Appendix F2.1). In addition, all subjects will also be asked to fill
out an activity log and wear an Actical physical activity monitor (Philips) to
record their physical activity patterns for 3 days immediately before the
immobilization period and for 3 days during the immobilization period. Finally,
all subjects will be instructed to refrain from any sort of heavy physical
exercise and to keep their diet as constant as possible for the 3 days
immediately before and for 3 days during the immobilization period.


Baseline Testing - Test Day #1
Prior to the onset of the uni-lateral immobilization period, subjects will
report to the University for baseline testing. Subjects will arrive at the
laboratory at 07.30 am in the fasted state having not consumed anything (except
water) since 10.00 pm the previous evening. A baseline blood sample and 3
saliva samples will be obtained to measure background 2H enrichment in plasma
and body water. Subjects will then begin the deuterated water (deuterium oxide
or 2H2O) loading protocol which requires subjects to ingest 50mL of 70 APE
enriched deuterated water 8 times during waking hours, at: 0800, 0930, 1100,
1230, 1400, 1530, 1700, 1830 hr to obtain a plateau enrichment of 2H in body
water of ~1-2% (16). The subjects will only be required to remain at the
laboratory for the first five doses (at 0800, 0930, 1100, 1230, 1400); the
remainder can be ingested at home by the subject (1530, 1700, 1830). Following
this loading protocol, subjects will be required to ingest one 50mL dose of 70
APE enriched deuterated water each day upon waking to maintain 1-2 APE
enrichment in body water (see section 5.1.2). During this visit, we will also
assess whole muscle CSA of both the right and left upper leg via CT scan,
whole-body muscle mass by magnetic resonance imaging (MRI), and body
composition by dual-energy X-ray absorptiometry (DEXA). First, a single slice
CT scan (IDT 8000; Philips Medical Systems, Best, Netherlands) at 15 cm above
the base of the patella will be performed in the Academic Hospital Maastricht
(Department of Radiology) to determine cross sectional area (CSA) of the upper
leg muscle of both legs. While the subjects are supine with their legs extended
and their feet secured, a 3-mm thick axial image will be taken. The scanning
characteristics will be as follows: 120kV, 300mA, rotation time of 0.75 sec,
and a field of view of 500 mm. The exact scanning position will be measured and
marked with a semi-permanent marker for replication. Following the CT scans,
each participant will undergo a full body MRI scan with a 3 T MR system
(Achieva 3Tx; Philips Healthcare), using a radiofrequency transmit/receive body
coil with a receiver bandwidth of 31.25 kHz. Using a turbo spin echo sequence
with a proton flip angle of 90 degrees, proton density weighted scans will be
acquired. This will create an image characterizing the proton density of each
tissue type. The images will be acquired using a matrix size of 320-512 pixels
and a 55 cm field of view. Contiguous images of 10 mm thickness with no gap
will be taken in four to six sequences of 38-40 images for defined body regions
for each participant. Following the whole-body MRI, subjects will undergo a
DEXA scan. Subjects will be instructed to lie down on a table and stay
motionless for approximately 3 minutes during which the body scan takes place.
Performing the above mentioned tests will allow us to characterize the whole
muscle CSA of both upper legs and subjects* body composition. In case of an
unexpected medical finding, we will inform the subjects and their general
practitioner. If a subject does not want to receive this information, he or she
cannot participate in this study. This visit is expected to last 6.5 hours.

Onset of Immobilization * Test Day #2
The day after Test Day #1, subjects will arrive at the laboratory at 07.30 am
in the fasted state having not consumed anything (except water) since the
controlled meal (described above, section 3.2) finished no later than 10.00 pm
the previous evening. Subjects will first provide a saliva sample, thereafter a
blood sample will be obtained, and subsequently undergo a single biopsy from
the vastus lateralis of one leg to determine skeletal muscle protein-bound 2H
enrichment immediately prior to the onset of the immobilization period. This
biopsy will come from the free leg which will not be immobilized. The muscle
biopsy will be obtained from the middle region of the vastus lateralis (15 cm
above the patella and approximately 2 cm away from the fascia) by the
percutaneous needle biopsy technique (17). Muscle biopsies will be carefully
freed from any visible non-muscle tissue and immediately be frozen in liquid
nitrogen. Muscle biopsies will be stored at *80 ºC for subsequent analysis. The
leg chosen to undergo immobilization will be randomized. Following the biopsy,
the opposite leg will be immobilized with a cast from 10 cm above the ankle to
half way the upper leg (30 degree flexion of the knee-joint; see Figure 1). The
cast will remain on for 3 days. Next, the volunteer will be instructed on how
to use crutches and to not bear weight on the casted limb, and to minimize
muscle contractions of the upper leg. This protocol has been already
successfully used for previous immobilization studies (METC 09-3-011, METC
11-3-073, METC 12-3-012, METC 12-3-063, METC 13-3-023). A model of uni-lateral
lower limb immobilization will allow for comparison of an immobilized and
non-immobilized lower limb within the same research subject, allowing each
subject to serve as their own internal control (11, 13). Following the fitting
of the cast, transport home from the university will be arranged either by car
or taxi. This visit is expected to last 2 hours.


During Immobilization
During the 3 days of immobilization, all subjects will be checked upon
regularly by a member of the research team. These check-ups will serve to
answer any questions/concerns from the participant and to check compliance with
the protocol. All subjects are at liberty to seek contact with the researcher
and/or physician to discuss any possible problems related to the immobilization
or participation in the study.
During immobilization, subjects will continue to ingest 2H2O (50 ml serving 1 x
day in the morning) to *maintain* the steady-state enrichment in body water and
provide a saliva sample for the determination of 2H enrichment in body water
(in the evening). The subjects will provided with plastic salivette vials that
contain a cotton swab used to collect the saliva sample. The subject will chew
and suck on the cotton swab for 30-60 seconds, until the swab is saturated with
saliva. Subsequently, they will place the wet swab back inside the salivette
vial and store it in their freezer until the next visit to the University.
During immobilization, subjects will receive either a leucine or carbohydrate
(maltodextrine) supplement in a double-blinded manner to ingest with each main
meal (breakfast, lunch, dinner). Each supplement serving will amount to 5.0 g
leucine or carbohydrate for a total of 15 g per day.


End of Immobilization * Test Day #3

Older Subjects
In the morning, after 3 days (or 72 hours) of immobilization, subjects will
come to the university for the post immobilization visit. First, the cast will
be removed, however the volunteer will not be allowed to place any weight on
the leg. Instead, the subject will be transported in a wheelchair to the CT
scanner where a single slice CT scan will be performed as described above.
Thereafter, subjects will be transported by wheelchair to the laboratory where
they will immediately undergo 2 skeletal muscle biopsies; one from the
immobilized and non-immobilized leg respectively. Thus, the biopsy from the leg
exposed to immobilization will be collected while the subject is still in the
immobilized state (i.e. before they have performed any weight bearing muscle
contractions). The two biopsies will allow for the determination of cumulative
(over 3 days) measures of muscle protein synthesis rates in both the
immobilized and non-immobilized leg based on the change in muscle protein-bound
2H enrichment from the first biopsy taken before the onset of immobilization.
We will also take a blood and saliva sample to measure 2H enrichment in plasma
and body water as described above. In total the elderly subjects will have 3
muscle biopsies and 3 blood samples (3 x 8 mL = 24 mL total). This visit is
expected to last 2 hours.

Younger Subjects
In the morning, after 3 days (or 72 hours) of immobilization, subjects will
come to the university for the post immobilization visit. First, the cast will
be removed, however the volunteer will not be allowed to place any weight on
the leg. Instead, the subject will be transported in a wheelchair to the CT
scanner where a single slice CT scan will be performed as described above.
Thereafter, subjects will be transported by wheelchair to the laboratory where
they will undergo a primed-constant infusion of L-[ring-13C6] phenylalanine for
5-hours. 2 h after the start of the infusion subject will undergo 2 skeletal
muscle biopsies; one from the immobilized and non-immobilized leg respectively.
Thus, the biopsy from the leg exposed to immobilization will be collected while
the subject is still in the immobilized state (i.e. before they have performed
any weight bearing muscle contractions). The two biopsies will allow for the
determination of cumulative (over 3 days) measures of muscle protein synthesis
rates in both the immobilized and non-immobilized leg based on the change in
muscle protein-bound 2H enrichment from the first biopsy taken before the onset
of immobilization. We will also take a blood and saliva sample to measure 2H
enrichment in plasma and body water as described above. In young subjects,
these two biopsies will also serve for the assessment of intracellular free and
protein-bound L-[ring-13C6] phenylalanine and baseline 3,3-D2 phenylalanine
enrichment in muscle to measure MPB using the tracee-release method (18) (See
Figure 3). Immediately after these first two biopsies subjects will undergo a
primed-constant infusion of 3,3-D2 phenylalanine for 2-hours, after which this
infusion will be stopped to assess the decay in 3,3-D2 phenylalanine in blood
and muscle (intracellular free amino acid pool). For this purpose, 40 min after
the 3,3-D2 phenylalanine infusion is stopped, a second set of biopsies will be
collected from each leg (figure 3) to assess intracellular free and
protein-bound L-[ring-13C6] phenylalanine and 3,3-D2 phenylalanine enrichment
in muscle. At 60min after stopping the 3,3-D2 phenylalanine infusion, the final
two biopsies will be obtained, again one from each leg to measure the dilution
in intracellular free 3,3-D2 phenylalanine enrichment in muscle and
enrichements in [ring-13C6] phenylalanine. Blood samples will be collected
every hour for the first 4-hours to measure L-[ring-13C6] phenylalanine and
3,3-D2 phenylalanine enrichment in blood plasma. During the decay period, blood
samples will be collected every 10 minutes to assess the decay in 3,3-D2
phenylalanine in blood (18). In total the young subjects will have 7 muscle
biopsies and 13 blood samples (13 x 8 mL = 104 mL total). Note that the second
blood sample in the 72-hour immobilization trial will also serve as the first
blood sample in the acute FBR trial. This visit is expected to last 7 hours.

Three days of immobilization via a full leg cast
One leg will be immobilized at a 30 degree knee joint angle of flexion for 3
days by means of leg cast (see Figure 1). The leg to be immobilized will be
randomized and balanced in the study between left and right. Prophylactic
medication to prevent deep vein thromboses (DVT) is not warranted when a single
joint (in this study the knee joint) is immobilized (21). However, to further
minimize the risk of DVT development all subjects will perform daily exercises
to activate the calf muscle pump. The time needed to perform these exercises is
5 minutes. Exercises will be performed 3 times a day (i.e. morning, afternoon
and evening) for the 3 days of the immobilization period. Subjects will be
provided with crutches because they are not allowed to bear weight on their
immobilized leg. Full instructions will be given on the correct use of
crutches. Although subjects will be provided with and instructed on the use of
crutches, subjects are most likely to experience a large degree of physical
impairment during the 3 days of immobilization. Activities like driving a car
and/or bicycle, and sport activities will be prohibited. Other activities might
require assistance (e.g. travelling, climbing stairs). Walking is only
permitted with crutches, without weight bearing on the immobilized leg.
Overall, it is important to note that for the duration of the immobilization
period physical mobility will be severely hampered for all subjects.
Following the removal of the cast, subjects are likely to experience some of
the effects of a reduction in muscle mass and strength of their immobilized
leg. Normal mobility and activities of daily living will no longer be impaired,
but sports performance may be somewhat below normal level for 2-14 days.
Throughout the immobilization and recovery period the researcher will establish
regular contact with the participating subjects to discuss any problems or
queries. During the recovery period all subjects are also at liberty to seek
contact with the researcher, physiotherapist and/or physician to discuss any
possible problems related to the immobilization period. Given the short
duration (3 days) of the recovery period, we expect muscle mass and function to
restore itself entirely within a few weeks after cast removal.

Leucine
During immobilization, subjects will receive bottles containing either leucine
or carbohydrate (maltodextrine) in a double-blinded manner to ingest with each
main meal (breakfast, lunch, dinner). Subject will be instructed to add water
to the bottle and ingest the supplement. Each supplement serving will amount to
5.0 g leucine or carbohydrate for a total of 3 bottles or 15 g per day.

The risks involved in participating in this experiment are minimal. If the
subjects are interested, they will receive their own results from the tests.
The subjects will receive reimbursement for their time investment and burden of
the study. Besides this, there is no additional benefit for participating in
this study.
The incision made for obtaining the muscle biopsy will be performed under local
anesthetic by an experienced physician. Within our research group we have
extensive experience with taking muscle biopsies. There is a small risk of
infection and the muscle biopsy canlead to minor hematoma. During the blood
draw there is a small risk of fainting or haematoma. These risks are minimized
by using trained and experienced personnel for taking the muscle biopsies and
blood draws. Adequate pressure will be applied after the blood draw and
biopsies to minimize the risk of developing a hematoma.
The Aviko meals are normal food products and have been cleared for human
consumption. There are no complications associated with the procedure of a
single slice lower limb CT scan. The level of radiation emitted is very low;
approximately 0.053 mSv per CT scan. In this study, subjects will undergo two
CT scans (one from each leg both before and after the immobilization period)
and will therefore be subjected to 0.106 mSV of radiation. These scans are
routinely performed in studies by our group (e.g. see MEC 06-3-062, 11-3-073,
12-3-012). The DEXA scan will be used to assess body composition. Subjects will
be exposed to 0.001 mSv of radiation from the DEXA scan. For comparison, the
average person living in The Netherlands is exposed to ~2.5 mSv/year in
background radiation. For comparison, the average person living in The
Netherlands is exposed to ~2.5 mSv/year in background radiation. The MRI scan
is non-invasive, though subject can feel somewhat unpleasant as the scanner
will make some noise when making the images, thereby, the subjects will be
lying on a table in a small *tunnel*.
In terms of time investment to visit the University, the screening visit will
take 1.5 hours of the subject*s time. The baseline testing (Test Day #1) will
take ~2 hours of the subject*s time. The onset of immobilization visit (Test
Day #2) will take ~2 hours. Lastly, the end of immobilization visit (Test Day
#3) will take ~2 hours for the older subjects and ~7 hours for the younger
subjects. An additional burden will be the requirement for subjects to fill in
the food diaries and wear the Actical physical activity monitor (Philips) both
for 3 days before and (see appendix F2.1 and F2.2) during the immobilization
intervention (6 days in total). However, the greatest inconvenience in terms of
time and also mobility will be the 3 days of one-legged immobilization. One leg
will be immobilized for 3 days by means of a full leg cast at a 30 degree angle
of flexion (see Figure 1). In a healthy population deep vein thrombosis (DVT)
will develop in 1 out every 1000 individuals a year (51). After surgery and/or
bone fracture, immobilization of two or more joints will increase the risk of
developing DVT. The incidence of DVT occurrence during limb immobilization
following surgery and/or bone fracture is estimated between ~0.2 and ~17% (21,
52-54). However, in the present study prophylactic medication to prevent DVT is
not warranted as only a single joint is immobilized (21, 52) particularly for a
time period of only 3 days. However, to further minimize the risk of DVT
development, subjects with any history of thrombosis will be excluded (please
see exclusion criteria; section 4.3) and all subjects will perform daily
exercises to activate the calf muscle pump. The time needed to perform these
exercises is 5 minutes. Exercises will be performed 3 times a day (i.e.
morning, afternoon and evening) for the 3 day immobilization period. Finally,
throughout the immobilization period all subjects will be monitored closely by
the research team, who will establish regular contact with the subjects, to
identify early signs of DVT development and act accordingly. Thus, in this
study the overall risk of DVT is minimal (<0.1%). The leg cast will cause a
large degree of physical impairment. Subjects will be provided with crutches as
they are not allowed to bear any weight on their immobilized leg. Full
instructions will be given on the correct use of crutches. To minimize the risk
of any injury due to this loss of physical mobility, activities like driving a
car and/or bicycle, and sport activities will be prohibited for the 3 day
immobilization period. Other activities might require assistance (e.g.
travelling, climbing stairs). Walking is only permitted with crutches, without
weight bearing on the immobilized leg. During the first days/week after removal
of the leg cast subjects may encounter the effects of the reduced muscle mass
and strength of their immobilized leg. Normal mobility and activities of daily
living will no longer be impaired, but sports performance might be somewhat
below normal level in the first week after the study. However, since only
healthy subjects will participate in this study, muscle function will most
likely completely restore itself within a few weeks after cast removal (55)
Indeed, it has been shown that even after 3 weeks of leg immobilization in
young individuals, the resumption of spontaneous activity for 2 weeks results
in the recovery of 90% of muscle strength and 95% of muscle fibre size (55). A
final follow-up phone call or e-mail will be performed 6 weeks after cast
removal to confirm that full recovery has taken place. If this is not the case,
further follow up will be performed, subjects will receive individual advice
from a sport physician and/or physiotherapist on a short recovery training
program to restore muscle mass and strength, or referral to the general
practitioner will take place when appropriate.
As discussed above, the leucine supplement is widely accepted for scientific
and clinical purpose. No major risks are associated with the use of leucine,
even for longer periods or when using higher dosages than in the current study,
in healthy subjects (34, 42, 43, 47).