Measurement of muscle protein fractional synthesis and
breakdown rates from a pulse tracer injection
Xiao-Jun
Zhang,
David L.
Chinkes, and
Robert R.
Wolfe
Metabolism Unit, Shriners Hospitals for Children, and
Departments of Surgery and Anesthesiology, The University of Texas
Medical Branch, Galveston, Texas 77550
 |
ABSTRACT |
We have developed a new
method to determine the fractional synthesis rate (FSR) and breakdown
rate (FBR) of muscle protein. This method involves a pulse tracer
injection and measurement of enrichment in the arterial blood and
muscle at three time points. The calculations of FSR and FBR are based
on the precursor-product principle. To test this method, we gave a
pulse injection of
L-[ring-13C6]phenylalanine
of 4-6 mg/kg in five rabbits. The measured FBR value (0.233 ± 0.060%/h) was almost identical (P = 0.35) to that (0.217 ± 0.078%/h) estimated from a leg arteriovenous balance model (Biolo G, Chinkes D, Zhang X-J, and Wolfe RR. J Parenter Enteral Nutr 16: 305-315, 1992). The measured FSR value
tended to be lower than that estimated from the leg model (0.125 ± 0.036 vs. 0.185 ± 0.086%/h; P = 0.14),
possibly because the new method measures only muscle FSR, whereas the
leg balance model also includes skin and bone contributions. The pulse
tracer injection did not affect muscle protein kinetics as measured by
leucine and phenylalanine kinetics in the leg. In another five rabbits,
we demonstrated that sampling could be reduced to either one or two
muscle biopsies when multiple pulse injections were used. This method
can be completed in 1 h with one muscle biopsy and has technical
advantages over currently used methods.
stable isotopes; gas chromatograph-mass spectrometer; arteriovenous
balance; rabbits
 |
INTRODUCTION |
THE METABOLIC STATUS OF
PROTEIN in a tissue is determined by the relative rates of
synthesis and breakdown. Changes in protein balance can be caused by
changes in synthesis or breakdown or both. Thus, to obtain a complete
knowledge of the protein metabolism in a tissue, it is necessary to
measure the rates of both synthesis and breakdown. The arteriovenous
(a-v) balance methods, with the use of either a two-compartment model
(3, 10) or a three-compartment model (5) for
calculations, are a common choice for measurement of protein kinetics
in a tissue because the rates of synthesis, breakdown, and net balance
can be obtained or inferred. A potential drawback of these methods is
that the a-v unit usually includes several tissues. For example, the
leg has been frequently used as an a-v unit to represent muscle protein
metabolism, but in some circumstances the metabolic contribution from
nonmuscle tissue (mainly skin) may be significant (7, 18).
The tracer incorporation methods are based on the rate of incorporation
of amino acid tracer into the target tissue protein. The tracer may be
given either as a constant infusion or as a bolus, either alone or with
a significant amount of tracee added (flooding dose method)
(11). These methods reflect protein metabolism in a
particular tissue, or even a particular protein, but do not provide a
measure of breakdown. Thus there is considerable information about the
regulation of protein synthesis in a variety of tissues such as muscle
(15), liver (2), and intestinal mucosa
(1), as well as specific proteins such as albumin and
fibrinogen (12), but few measurements of breakdown.
We previously described the measurement of protein breakdown by a
method that corresponds to fractional synthesis rate (FSR) and
therefore is called the fractional breakdown rate (FBR)
(25). This measurement requires infusion of a tracer to
achieve isotopic plateaus in the arterial blood and in the
intracellular free amino acid pool in tissue and then measurement of
decay in enrichment after the tracer infusion is stopped. Thus this FBR
measurement can be combined with the primed-constant infusion to obtain
both FSR and FBR simultaneously from the same tissue samples (9, 17). Whereas the above procedure provides a feasible approach to
measure tissue protein FSR and FBR, it requires at least 4-5 h to
complete, and usually three tissue biopsies are necessary. The goal of
the present experiment was to develop a new method to determine both
FSR and FBR of tissue protein within a shorter time frame (e.g., 1 h) and from only one biopsy. We selected the skeletal muscle as the
target tissue for development of this novel method because the protein
kinetics in muscle can be concomitantly determined by the
three-compartment a-v model (5), thereby providing
reference rates of synthesis and breakdown with which to evaluate the
validity of the new method.
 |
METHODS |
Rationale and Equations
Protein synthesis and breakdown are two opposing processes that
function simultaneously. The movement of amino acids from the muscle
intracellular free (MIF) pool into the protein-bound pool reflects
protein synthesis, and the movement of amino acids from the
protein-bound pool into the MIF pool represents protein breakdown if
the amino acid is not synthesized in the tissue. The measurements of
FSR and FBR are both based on the precursor-product principle except
that the definitions of precursor and product are reversed for the two
processes. For protein synthesis the MIF pool is a good approximation
of the precursor (i.e., aminoacyl-tRNA) (4) and the
product is the protein-bound pool. For protein breakdown the
protein-bound pool is one of the precursors (the other is the arterial
blood) and the MIF is the product pool. Thus the FSR is determined by
the rate of tracer incorporation from the MIF pool to the bound pool,
and the FBR is determined by the rate at which tracee release from the
bound pool dilutes the enrichment in the MIF pool.
The precursor-product principle commonly has been applied to the
movement of tracer from the MIF pool to the bound pool for determination of FSR. However, the information of FBR reflected by the
movement of tracee from the bound pool to the MIF pool has been
neglected. In fact, the rationale for the use of the precursor-product
principle to measure FBR is as solid as its use to measure FSR. The
intracellular protein breakdown is the only factor that causes
enrichment gradient between the arterial blood and MIF pool; again an
amino acid tracer is used that is not synthesized in the tissue.
Without protein breakdown the enrichment in the MIF pool will equal
that in the arterial blood. When the rate of inward transport from the
arterial blood into the MIF pool is constant, the greater the rate of
FBR, the lower the enrichment in the MIF pool, and vice versa. Thus the
enrichment difference between the arterial blood and MIF pool reflects
the rate of tracee movement from the bound pool to the MIF pool. Under
the physiological steady state, in which the concentrations of amino
acids in the blood and in the MIF pool are constant, the FBR of muscle
protein can be calculated from the following equation (25)
|
(1)
|
where P = EM/(EA
EM) at isotopic plateau, and EA and
EM are isotopic enrichments in the arterial and MIF pools,
respectively; EM(t2)
EM(t1) is the change of enrichment
in the MIF pool from time (t) t1 to
t2 after stopping the isotope infusion;

EA(t)dt and

EM(t)dt
are areas under the decay curves of arterial enrichment and MIF
enrichment, respectively, from t1 to
t2; and QM/T is the ratio of
intracellular free tracee content vs. protein-bound tracee content in
the muscle.
The derivation of Eq. 1 has been described in detail in our
previous work (25), and the following is a brief
explanation of this equation. Equation 1 is simply a
precursor-product equation (29) if one ignores the
variable QM/T. The variable P in this equation is necessary
because there are two precursor pools: the arterial blood and
protein-bound amino acids within the cell. Therefore, the relative
contribution of these two sources to product pool (i.e., MIF) needs to
be included in the calculation, which is accomplished by using
the variable P. The variable P can be calculated from isotope
enrichments in the arterial blood (EA) and MIF pool
(EM) at isotopic plateaus: P = EM/(EA
EM). The P variable,
as defined above, is equal to the ratio of fractional tracee from
artery vs. fractional tracee from breakdown, and 1 + P is the
ratio of total tracee from both artery and breakdown to fractional
tracee from breakdown. After introduction of the variable P, the
denominator of the equation calculates the change in the tracer MIF
pool size divided by fractional tracee from breakdown, and the
numerator is the change in the tracer MIF pool size divided by the
tracee MIF pool size from t1 to
t2. When the change in the tracer MIF pool size
is canceled out, the equation becomes (fractional tracee from
breakdown)/(tracee MIF pool size). However, the FSR calculates the rate
of tracer incorporation into protein vs. protein-bound pool size. To be
consistent with the unit of FSR, Eq. 1 has to be multiplied
by the ratio of QM/T. Then Eq. 1 can be
rearranged as (fractional tracee from breakdown)/T, which is exactly
the definition of FBR.
This original method requires infusion of tracer to reach isotopic
plateaus in the arterial blood and in the MIF pool and then observation
of decay after stopping the tracer infusion. If we eliminate P from
Eq. 1, we remove the requirement of isotopic plateaus. The
above equation holds for any two time points, so if we chose time
points t2 and t3 rather
than t1 and t2, then the
equation becomes
|
(2)
|
Thus we have two equations and two unknowns, i.e., FBR and P. If
we solve Eq. 2 for P and substitute it into Eq. 1, we obtain the equation
|
(3)
|
Therefore, a measurement at isotopic plateau is not required if
the arterial and intracellular enrichments are measured at three time
points. The detailed proof in mathematics is addressed in the
APPENDIX.
The FSR measures the rate of tracer incorporation from the MIF pool
into the protein-bound amino acid pool. According to the precursor-product principle, the equation for FSR (23) is
|
(4)
|
where EF and EB are the enrichments of
free and bound amino acid, respectively. Because of the difficulty and
impracticality of obtaining enrichment of the actual immediate
precursor (i.e., aminoacyl-tRNA), the free amino acid pool in muscle is
often used as an acceptable surrogate of the precursor for muscle
protein synthesis (4). Thus the nominator calculates the
increment of enrichment in the bound pool over time period
t, and the denominator is the average enrichment in the free pool.
Equation 3 indicates that if we administer a pulse injection
of an amino acid tracer that is not synthesized in the tissue and
measure the enrichment decay in the arterial blood and tissue MIF pool
at three time points and the ratio of the tracer amino acid in the free
and bound pools, we can calculate FBR. Equation 4 indicates
that if we measure the free and bound enrichment over the decay period,
we can calculate muscle protein FSR.
Experimental Procedures
Animal.
We used male New Zealand white rabbits (Myrtle's Rabbitry, Thompson
Station, TN), each weighing ~4.5 kg. This study was approved by the
Animal Care and Use Committee of The University of Texas Medical Branch
at Galveston.
Isotopes.
L-[ring-13C6]phenylalanine
(L-[ring-13C6]Phe;
99% enriched) and L-[1-13C]leucine
(L-[1-13C]Leu; 99% enriched) were purchased
from Cambridge Isotope Laboratories (Woburn, MA).
L-[ring-2H5]Phe (98%
enriched) and L-[1,2-13C2]Leu
(99.3% enriched) were purchased from Tracer Technologies (Somerville,
MA). L-[15N]Phe (99% enriched) was purchased
from Isotec (Miamisburg, OH).
Design.
There were two groups of five rabbits each. Group 1 was used
to establish the method for measuring FSR and FBR of muscle protein from a pulse injection of Phe tracer. The measured FSR and FBR values
were compared with the corresponding values estimated from the
three-pool leg model for validation. To assess whether the pulse tracer
injection affected muscle protein kinetics, we also used the three-pool
model to measure Phe and Leu kinetics in the leg before and after the
Phe tracer injection.
Group 2 was used to test the hypothesis that if three stable
isotopomers of Phe were injected at the same dose but at different times, muscle protein FSR and FBR could be measured from one muscle sample at 60 min after the first tracer injection. The rationale is as
follows:
L-[ring-13C6]Phe,
L-[ring-2H5]Phe, and
L-[15N]Phe are injected at 0, 30, and 55 min,
respectively. Because the three Phe tracers have similar metabolic fate
after being administered intravenously (13), at 60 min the
free enrichments of L-[15N]Phe,
L-[ring-2H5]Phe, and
L-[ring-13C6]Phe in
muscle represent 5, 30, and 60 min of decay, respectively. By the same
rationale, muscle protein FBR and FSR can also be measured from two
biopsies at 5 and 60 min or 30 and 60 min if two Phe tracers are
injected at 0 and 30 min or at 0 and 55 min.
The anesthetic and surgical procedures were described in our previous
publications (24, 27, 28). In brief, after an overnight
fast with free access to water, the rabbits were anesthetized with
ketamine and xylazine. Catheters were placed in the right jugular vein
and left carotid artery. The venous line was used for infusion of
anesthetics and saline and also for the primed-constant infusion of
isotopes (group 1). The arterial line was for collection of
arterial blood and monitoring of heart rate and mean arterial blood
pressure. The femoral vein on the left leg was exposed via a
groin incision, and a catheter was inserted into the left
femoral vein for pulse injection of the Phe tracers (groups
1 and 2) and withdrawal of venous blood drawing from
the leg (group 1). In group 1 the left femoral
artery was exposed at the inguinal level, and a 1.5-RB flow probe
(Transonics Systems, Ithaca, NY) was placed on the artery for
measurement of blood flow rate on a small animal blood flowmeter (T106;
Transonic Systems). A tracheal tube was placed via tracheotomy in both
groups. The tracheal tube was placed in an open hood, which was
connected to an oxygen line so that the rabbit inhaled oxygen-enriched
room air.
The isotopic infusion protocols in the two groups are illustrated in
Fig. 1. In group 1 (Fig.
1A), after collection of a blood sample and a muscle
specimen from the left leg for measurement of background enrichment, a
primed-constant infusion of L-[1-13C]Leu
(rate 0.35 µmol · kg
1 · min
1; prime
21 µmol/kg) and L-[15N]Phe (rate 0.15 µmol · kg
1 · min
1; prime
6 µmol/kg) was started. During the basal period of 180 min, five
pairs of simultaneous arterial and femoral venous blood (0.25 ml each)
were collected. At 180 min a muscle specimen was taken from the
adductor muscle of the left leg, and
L-[ring-13C6]Phe
(4-6 mg/kg) in 3 ml of 0.45% saline was injected into the left
femoral vein within 20 s and the line was flushed with 2 ml of
saline. The selection of the dose for the pulse injection was based on
our pilot studies, in which varying amounts of
L-[ring-13C6]Phe
ranging from 4-12 mg/kg were injected. We found that a dose of 4 mg/kg was minimal for measurement of a significant change in enrichment
in the muscle protein-bound pool over a 60-min period. Over the
following 90 min after the pulse tracer injection, another five pairs
of simultaneous a-v blood were collected. The blood flow rate was
recorded from the blood flowmeter at each a-v blood collection.
Additional arterial blood (0.25 ml each) and muscle samples (~70 mg
each from the adductor muscle of the right leg) were taken at 5, 10, 30, 60, and 90 min (see details in Fig. 1A).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Experimental protocols in group 1 (A) and group 2 (B); X indicates
sampling of blood or muscle. In group 1, during the 180 min
of the basal period, 5 pairs of arterial (A) and femoral venous (V)
blood samples were collected at 120, 135, 150, 165, and 180 min. After
a muscle sample was taken from the adductor muscle of the left leg, the
pulse tracer injection was performed. Over the following 90 min,
another 5 pairs of arterial and venous blood samples were collected at
10, 20, 30, 60, and 90 min after the pulse tracer injection. Additional
arterial blood and muscle samples were taken at 5, 10, 30, 30, 60, and
90 min. In group 2, only the 120 min of the injection period
are illustrated, and the 3-h basal period without isotope infusion is
not included. Thus 0 min in group 2 is equal to 180 min in
group 1.
L-[ring-13C6]phenylalanine
(L-[ring-13C6]Phe),
L-[ring-2H5]phenylalanine
(L-[ring-2H5]Phe), and
L-[15N]phenylalanine
(L-[15N]Phe) were injected at 0, 30, and 55 min of the injection period. Arterial blood was taken at 5, 10, 15, 30, 35, 40, 45, 60, 65, 70, 85, and 115 min after the injection of
L-[ring-13C6]Phe.
Muscle samples were taken at 5, 30, and 60 min.
|
|
In group 2 (Fig. 1B), the background blood and
muscle samples were collected after completion of the surgical
procedures. During the basal period of 180 min, only the anesthetics
and saline were infused into the jugular vein to match the treatment of
group 1.
L-[ring-13C6]Phe (5.56 mg/kg),
L-[ring-2H5]Phe (5.53 mg/kg), and L-[15N]Phe (5.40 mg/kg) in 3 ml
of 0.45% saline were injected into the femoral venous catheter at
time 0 and 30 and 55 min after the end of the basal period,
respectively. The three doses of Phe tracer were each 32.5 µmol/kg.
Frequent arterial blood samples (0.25 ml each; see Fig. 1B)
were taken over a 115-min time period to measure the enrichment decay
in arterial blood of each tracer for 60 min. Adductor muscle samples
from the right leg were taken at 5, 30, and 60 min (~70 mg for 5- and
30-min samples and ~200 mg for 60-min sample). The three muscle
biopsies allowed us to measure enrichment decay in the MIF pool from
L-[ring-13C6]Phe at 5, 30, and 60 min; from
L-[ring-13C6]Phe and
L-[ring-2H5]Phe at 5 and 60 min; from
L-[ring-13C6]Phe and
L-[15N]Phe at 30 and 60 min; and from
L-[ring-13C6]Phe,
L-[ring-13C6]Phe, and
L-[15N]Phe using one biopsy at 60 min.
Therefore, we were able to calculate muscle FBR and FSR from either one
or two muscle biopsies as well as from three muscle biopsies. At the
end of the sampling for kinetic measurements, additional blood was
taken from the arterial line to measure blood gas and hematocrit.
After collection, the muscle samples were either gently blotted or
quickly washed in ice-cold saline to remove visible blood. The 60-min
muscle sample in group 2 was processed differently because from this single muscle biopsy the following information was
obtained: enrichments of three Phe tracers, content of unlabeled Phe,
and percentage of dry protein in the sample. To conveniently accomplish
these analyses, we took a relatively large muscle sample (~200 mg)
and cut the muscle into two pieces. One piece was washed in ice-cold
saline for measurement of Phe enrichment; the other piece was gently
blotted only, for measurement of free Phe content and percent protein
in muscle. This was because washing in saline may change free amino
acid content and water content in the muscle. The muscle samples were
immediately frozen in liquid nitrogen and stored at
80°C for later
processing. The blood samples were processed immediately after
collection as described in Sample analysis.
Heart rate, mean arterial blood pressure, and rectal temperature were
maintained stable by adjustment of the doses of anesthetics, saline,
and heating lamps. These vital signs were monitored through the
experiments and recorded every 30 min.
Sample analysis.
After collection, the blood samples were transferred to tubes with 1 ml
of 7.5% sulfosalicylic acid for deproteinization. In group
1, 50 µmol of the internal standard solution, which contained L-[1,2-13C2]Leu (70 µmol/l) and
L-[ring-2H5]Phe (30 µmol/l), were added to each tube for calculation of Phe and Leu
concentrations in the blood. The exact amounts of the internal standard
solution and blood were obtained by weighing on a scale. After
centrifugation, the supernatant was processed to make the
t-butyldimethylsilyl (TBDMS) derivatives of amino acids
(16).
To measure both free Phe enrichment and content in the muscle, a tissue
internal standard solution, which contained
L-[ring-2H5]Phe at 6 µmol/l, was added to ~30 mg of muscle (1 µl for each mg of
tissue). After homogenization in 10% perchloric acid, three times at
4°C, the pooled supernatant was processed for the TMDMS derivatives
(16). The 60-min muscle samples in group 2 were processed as follows. The piece of muscle that had been washed in
ice-cold saline was processed without the internal standard solution
for measurement of enrichments of the three Phe tracers. The piece of
muscle that had been gently blotted, but not washed, was cut into two
aliquots (~30 mg each): one was processed without the internal
standard solution for measurement of
L-[ring-2H5]Phe
enrichment from injected
L-[ring-2H5]Phe; the
other was added with the tissue internal standard solution (1 µl for
each mg of tissue). The enrichment difference of
L-[ring-2H5]Phe was
used to calculate the content of unlabeled Phe in muscle.
The protein precipitates were washed thoroughly to remove free amino
acids and fat and were dried in an oven at 80°C overnight to obtain
the dry protein pellets (25). The percentage of dry protein in the muscle was calculated from the wet and dry weights. The
dry protein pellets were hydrolyzed and then processed for the
N-acetyl, n-propyl ester (NAP) derivatives of
amino acids (23).
The isotopic enrichments in the blood and muscle supernatant were
determined on a Hewlett-Packard 5980/5989B gas chromatograph-mass spectrometer (GC-MS); ions were selectively monitored at mass-to-charge (m/z) ratios of 234, 235, 239, and 240 for Phe
enrichment and at m/z ratios of 302, 303, and 304 for Leu enrichment. Isotopic enrichments were expressed as mole percent
excess for the three-pool model and for the tracer incorporation method
and as tracer-to-tracee ratio for the enrichment decay in the arterial
blood and MIF pool and for the internal standard method. The
enrichments were corrected for the contribution of the abundance of
isotopomers of lower weight to the apparent enrichment of isotopomers
with larger weight. A skew correction factor was also used to calculate
L-[ring-13C6]Phe
enrichment in the blood and muscle supernatant (21). L-[ring-13C6]Phe
enrichment in the muscle protein hydrolysate was measured on a gas
chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS;
Finnigan, MAT, Bremen, Germany). The measured
13CO2 enrichment was converted to Phe
enrichment by multiplying by 14/6 to account for the dilution of 6 labeled carbons with the total 14 carbons in the derivatized Phe.
Calculations.
FBR was calculated by Eq. 3. In this equation,
QM/T is the ratio of free to bound Phe in muscle. In
practice, we measured the amount (in µmol) of free Phe in the piece
of muscle that was homogenized and normalized to micromoles of free Phe
per gram of muscle. In our previous study (26), we
reported that 1 g of dry muscle protein contains 250 µmol Phe.
The content of protein-bound Phe in 1 g of muscle was therefore
calculated by [(250 µmol/g) × (%dry protein in muscle)].
Because both the free and bound Phe contents were expressed in
micromoles per gram of muscle, QM/T is equal to free Phe
content divided by bound Phe content. FSR was calculated from Eq. 4, in which the free enrichment in muscle was used as a surrogate
of precursor enrichment.
Leu and Phe kinetics in the leg were calculated from a
three-compartment model that we published previously (5).
The following are equations for calculating the rate of disappearance
(Rd), rate of appearance (Ra), and net balance
(NB)
|
(5)
|
|
(6)
|
|
(7)
|
Here EA, EV, and EM are
enrichment in the arterial blood, venous blood, and MIF pool,
respectively; CA and CV are concentration in
the arterial blood and venous blood, respectively; and BF is blood flow
rate in the femoral artery. Because Phe is neither synthesized nor
degraded in the limb, the Rd represents protein synthesis
and the Ra represents protein breakdown. Because Leu is not
synthesized in muscle, its endogenous Ra comes exclusively from breakdown. However, because Leu can be oxidized in muscle, its
Rd is the sum of incorporation into protein (synthesis) and oxidation.
To convert Phe Rd and Ra calculated from the
three-pool model to FSR and FBR, we need to know the muscle mass in the
hindlimb and the amount of Phe in leg muscle protein, which are
calculated by the following equations
|
(8)
|
|
(9)
|
Here BF is the blood flow rate in the femoral artery. The value
of 0.0783 ± 0.0385 ml · g
1 · min
1 is that
1 g of leg adductor muscle receives 0.0783 ml of blood per min,
which we measured in five rabbits using the microsphere technique
(unpublished data). In Eq. 9 the assumed value that 1 g
of muscle protein contains 250 µmol Phe was calculated from the
internal standard method in our previous experiment (26); the percent protein was calculated from the wet weight and dry weight
of muscle samples. Knowing the Phe amount in leg muscle protein, we
converted Phe Rd and Ra to FSR and FBR
|
(10)
|
|
(11)
|
Here, FSRC and FBRC are converted from
Rd and Ra of Phe, which are distinguished from
FSR and FBR measured from the pulse tracer injection.
Statistical analysis.
Data are expressed as means ± SD. Differences between two groups
or between FSR and FBR were evaluated using the Student's t-test. P < 0.05 was considered
statistically significant.
 |
RESULTS |
The general characteristics of the rabbits in the two groups are
presented in Table 1. In group
1, after 2 h of primed-constant infusion, the
enrichments of L-[15N]Phe and
L-[1-13C]Leu in the arterial blood reached
plateaus (Fig. 2A). The
arterial Phe concentration of unlabeled Phe plus
L-[15N]Phe was constant during the
120-270 min, although the pulse tracer injection caused the
concentration of
L-[ring-13C6]Phe to
transiently increase and fall (Fig. 2B). The isotope enrichments in the arterial blood, femoral venous blood, and muscle free pool and the concentrations of Phe and Leu in the arterial and
femoral venous blood are presented in Table
2. These data, along with the
blood flow rates in the femoral artery (see Table 1),
were used to calculate Leu and Phe kinetics using Eqs.
5-7. Because the calculated Phe kinetics during the injection
period did not include
L-[ring-13C6]Phe, the
contribution of
L-[ring-13C6]Phe could
be estimated. After the pulse injection,
L-[ring-13C6]Phe could
incorporate into protein at a rate proportionate to its enrichment in
the MIF pool. Thus Phe Rd was the sum of Rd
from the three-pool model and Rd × 8.4 ± 2.5%,
where 8.4 ± 2.5% was the average
L-[ring-13C6]Phe
enrichment in the MIF pool over the 90-min time period. Because the
pulse injection should not affect Phe Ra calculated from
the three-pool model, Phe Ra did not need correction for L-[ring-13C6]Phe
kinetics. The values of Rd, Ra, and net balance
of Phe and Leu before and after the pulse tracer injection are
presented in Table 3.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
A: arterial enrichments of
L-[15N]Phe and
L-[1-13C]leucine (Leu) reached isotopic
plateaus from 120 to 270 min of the arteriovenous (a-v) sampling
period. This indicates that the pulse injection of
L-[ring-13C6]Phe did
not have a significant effect on the isotopic steady state of
L-[15N]Phe or
L-[1-13C]Leu. B: the concentration
of unlabeled Phe plus L-[15N]Phe in the
arterial blood was constant from 120 to 270 min of a-v sampling period.
The change in concentration of total Phe (i.e., sum of unlabeled plus
15N- or 13C6-labeled Phe) was due
to the pulse injection of
L-[ring-13C6]Phe.
|
|
To calculate FBR, we measured the enrichment of
L-[ring-13C6]Phe in
the arterial blood and in the MIF pool as well as the ratio of free Phe
to protein-bound Phe (i.e., QM/T in Eq. 4) in
muscle. These data from group 1 are presented in Table
4. The FBR values calculated from five
combinations of three sampling times, namely 5, 30, and 60 min, 10, 30, and 60 min, 5, 30, and 90 min, 10, 30, and 90 min, and 30, 60, and 90 min, are presented in Table 5. To assess
whether the variability of the FBR values from the five
measurements was due to an interindividual or intermeasurement variation, we compared the standard deviations between rabbits and
within rabbits. The standard deviation within rabbits calculated from
the five measurements (0.0279 ± 0.0136) was significantly (P < 0.05) smaller than that of measurements between
the five rabbits (0.0498 ± 0.0074), indicating that the
measurements of the FBR value were consistent within rabbits. We
selected the 5, 30, and 60-min data as a representative measurement.
In group 2, the arterial enrichments of the three Phe
tracers followed the same decay pattern (Fig.
3A), confirming that they have
a similar metabolic fate in the body (13). The decay curve of L-[ring-13C6]Phe
in the MIF pool was either measured from the
L-[ring-13C6]Phe
tracer using the three muscle samples (5, 30, and 60 min) or taken from
L-[15N]Phe and/or
L-[ring-2H5]Phe
enrichments using two or one muscle biopsy (Fig. 3B). For example, when the 60-min muscle sample was used for FBR, the enrichment decay in the MIF pool at 5 min was taken from
L-[15N]Phe, which was injected at 55 min
(i.e., 5 min before the 60-min biopsy); the enrichment decay at 30 min
was taken from
L-[ring-2H5]Phe, which
was injected at 30 min (i.e., 30 min before the 60-min biopsy); and the
enrichment decay at 60 min was measured directly from
L-[ring-13C6]Phe
tracer, which was injected at 0 min (i.e., 60 min before the 60-min
biopsy). The same rationale was used to determine the decay curves when
two muscle biopsies were taken at 5 and 60 min or at 30 and 60 min. The
dry protein in muscle was 21.7 ± 0.6%, the free and bound Phe
contents in muscle were 0.1059 ± 0.0194 and 54.27 ± 1.60 µmol/g, respectively, and the QM/T ratio was 526 ± 96. The calculated FBR values with three muscle samples (5, 30, and 60 min), two muscle samples (5 and 60 min or 30 and 60 min), and one
muscle sample (60 min) were almost identical (P > 0.05; Fig. 4).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
A: enrichment decay curves of
L-[ring-13C6]Phe,
L-[ring-2H5]Phe, and
L-[15N]Phe in the arterial blood after the
pulse injection of these 3 tracers. The comparable decay curves
indicate that these 3 Phe tracers have the same metabolic fate after
intravenous injection. B: the
L-[ring-13C6]Phe
enrichment decay in the muscle intracellular free pool is expressed in
4 ways. The curve of 3 biopsies represents values of
L-[ring-13C6]Phe
enrichment at 5-, 30-, and 60-min muscle samples; the curve of 2 biopsies A represents values from
L-[ring-13C6]Phe
enrichment at 5 and 60 min and
L-[ring-2H5]Phe at 30 min; the curve of 2 biopsies B represents values from
L-[ring-13C6]Phe
enrichment at 30 and 60 min and from
L-[15N]Phe at 5 min; and the curve of 1 biopsy represents
L-[ring-13C6]Phe
enrichment at 60 min; and the values at 5 and 30 min were the
enrichments of L-[15N]Phe and
L-[ring-13C6]Phe,
respectively.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
The value of muscle protein fractional breakdown
rate (FBR) in group 2 calculated from 3 muscle biopsies at
5, 30, and 60 min was almost identical (P > 0.05) with
values from 2 muscle biopsies at 5 and 60 min or at 30 and 60 min and
from 1 muscle biopsy at 60 min.
|
|
The values of muscle protein FSR, calculated from the pulse injection
of L-[ring-13C6]Phe,
are presented in Table 6. In group
1, the FSR was calculated from 5 to 60 min after the Phe tracer
injection; in group 2, the FSR was calculated from 0 to 60 min. Because in group 2 we measured the FSR value from a
60-min muscle sample, we used the blood background sample to estimate
the background enrichment of
L-[ring-13C6]Phe in
the muscle. The FSR values were not significantly different between the
groups (P > 0.05). For each rabbit, the value of FBR was consistently greater than the value of FSR, indicating a negative balance of muscle protein.
To evaluate the validity of the FSR and FBR values from the pulse
tracer injection, we converted the rates of synthesis and breakdown
from the three-pool model to FSRC and FBRC
using Eqs. 8-11. The total amount of Phe in leg muscle
was 11,330 ± 1,656 µmol. The values of FSRC and
FBRC were 0.185 ± 0.086%/h and 0.217 ± 0.078%/h, respectively, which were not significantly
(P = 0.14 and 0.35 by one-tail paired
t-test) different from the values of FSR (0.125 ± 0.036%/h) and FBR (0.233 ± 0.060%/h) measured from the pulse
injection method.
 |
DISCUSSION |
Our goal was to develop a stable isotope method to determine
muscle protein FSR and FBR within the shortest time period and using
the least number of muscle biopsies. The results demonstrate that by
the pulse Phe tracer injection and measurement of enrichment decay at
three time points, muscle protein FSR and FBR can be determined over a
60-min time frame. Furthermore, when two or three Phe tracers were used
for multiple pulse injections, the number of muscle biopsies could be
reduced to two or even one. Thus the pulse tracer injection method
satisfactorily achieves our goal.
To evaluate the validity of the pulse injection method, we converted
the Rd and Ra of Phe, measured from the
three-compartment leg model, to FSRC and FBRC.
The FBR values from these two independent methods were almost identical
(0.233 ± 0.060%/h vs. 0.217 ± 0.078%/h; P = 0.35). The FSR value from the pulse injection method (0.125 ± 0.036%/h) tended to be lower than that converted from Phe
Rd (0.185 ± 0.086%/h; P = 0.14).
This is likely because the pulse injection method directly measures the
protein metabolism in the adductor muscle, whereas the three-pool model
measures protein metabolism in the total leg, including not only muscle
but also skin, bone, and so forth. Thus the three-pool model may
overestimate muscle protein synthesis compared with breakdown. In
humans or dogs, nonmuscle tissue (mainly skin) is estimated to be
10-15% of total leg protein kinetics (6, 7).
However, in some species the contribution may increase. Preedy and
Garlick (18) reported that the rat hemicorpus contains
39% by weight of nonmuscle tissue. Because skin has a faster protein
synthesis rate than muscle, the metabolic contribution by the nonmuscle
tissue could be a considerable portion of the measured protein kinetics
in the hemicorpus (18, 19). In our previous experiment
(24), we estimated that the limb skin accounted for
26-42% of the total limb tracer uptake in the rabbit. Using the
primed-constant infusion method, we reported that the FSR of ear skin
was 0.30%/h (28). Because the skin is able to maintain
its protein mass in the postabsorptive state (24), its FSR
equals its FBR. If the leg skin has a similar protein turnover rate to
that of the ear skin, the FBR in leg skin (~0.30%/h) should be close
to the FBR in leg muscle (0.23%/h; see Table 5). Therefore, the
inclusion of skin protein breakdown should have little import on the
FBRC values converted from the three-pool model; hence, the
FBR and FBRC values measured from these two independent
methods were close. In contrast, because the synthesis rate of skin
protein is greater than that of muscle protein, the inclusion of skin
protein synthesis could cause overestimation of the FSRC
value converted from the three-pool model. Thus we would expect the FSR
from muscle tissue to be slightly lower than synthesis calculated from
the balance method.
The pulse Phe tracer injection did not affect protein metabolism in the
muscle, which was supported by the almost identical values of Leu and
Phe kinetics in the leg before and after the pulse injection. It has
been reported that injection of a flooding dose of Phe tracer (50 mg/kg) doubled muscle protein FSR (22). To avoid this
problem, the doses of
L-[ring-13C6]Phe we
used for pulse injections were only 10% of the flooding dose
previously reported. On average, these doses of Phe tracer increased
Phe concentrations in the MIF pool by 10% during the 60-min
measurement period. With this approach we avoided stimulating muscle
protein synthesis with the doses of Phe tracers.
In group 1, the leg blood flow rate in the injection period
was ~13% lower than in the basal period (see Table 1). This was most
likely due to the frequent blood sampling during the injection period
(see Fig. 1A), although the volume of blood for each sample was minimized to 0.25 ml. The hematocrit value (33.9 ± 0.6%; see Table 1) measured at the end of the experiments
indicated that the volume of blood withdrawn was acceptable. In both
human and rabbit experiments in our laboratory, we found that a 20%
reduction of leg blood flow rate by mechanical clamping of the femoral
artery had no detectable effects on the rates of protein synthesis and breakdown in leg muscle measured by both the leg a-v model and the
tracer incorporation method (unpublished data). Thus a 13% reduction
of leg blood flow rate should not have a significant impact on muscle
protein kinetics in the leg.
The pulse tracer injection method has certain advantages over the
currently used primed-constant infusion or flooding dose method to
measure muscle protein FSR. Because the enrichment in the muscle free
pool is a good approximation of the true precursor (4,
20), the primed-constant infusion method commonly has been
selected for measurement of muscle FSR. However, this method may
require several hours of stable isotope tracer use to achieve sufficient enrichment in the product for accurate measurement. Even
after a successful prime, it usually takes 1 h or more to reach an
isotopic plateau in intracellular enrichment, and at least 2-3 h
more may be required to increase the enrichment in the protein-bound
pool to the level that can be accurately determined. Thus the minimal
time to complete the measurement in muscle may be 3-4 h. Depending
on the experiment, it may be difficult to maintain a physiological
steady state over that time period, particularly in small animals. This
is one reason that the flooding dose method has been an option of
methods in small animals. However, the use of the flooding dose method
is subject to the risk of stimulating muscle protein synthesis because
of the large amount of amino acid injected (20), and this
large bolus may obscure the treatment effect, particularly if the
treatment involves amino acids (14). The pulse injection
method combines the advantages of the primed-constant infusion (no
effect on synthesis) and flooding dose (relatively short time interval)
methods while limiting the disadvantages of the other methods.
The pulse tracer injection method has advantages over the
original FBR technique (25). Most importantly, the pulse
tracer method does not require an isotope plateau and therefore can be performed without tracer infusion. In addition, by use of two or three
Phe tracers, the pulse tracer method can be used with only two or even
one biopsy. The use of multiple stable isotopomers of an amino acid to
reduce the number of tissue biopsies is based on the fact that these
isotopomers have similar metabolic fates (13). This
concept has been used previously. For example, Dudley et al.
(8) reported a method of staged infusion of six stable isotopomers (2 isotopomers of Leu and 4 isotopomers of Phe) to measure
the FSR of lactase phlorizin hydrolase from frequent blood samples and
one tissue (intestinal mucosa) biopsy in pigs. FSR values measured from
the multiple-tracer, single-sample approach compared well with values
measured from the conventional isotope infusion approach with tissue
samples collected at timed intervals during the infusion
(8). In the present experiment, the similar curves of Phe
enrichments in the arterial blood and muscle free amino acid pool (Fig.
3, A and B) support the validity of the timed
pulse injections of the Phe tracers as an alternative to multiple
muscle biopsies.
We have expressed the enrichment as mole percent excess for
calculation of FSR and tracer-to-tracee ratio for FBR. The protein breakdown releases unlabeled Phe into the MIF pool. Thus the enrichment decay was expressed as tracer-to-tracee ratio, which reflects dilution
of tracer in the MIF pool by the unlabeled Phe released from
proteolysis. For the same reason, the value of QM/T, the ratio of free vs. bound Phe in muscle, was also referred to as unlabeled Phe. In contrast, the movement of amino acids from the MIF
pool to protein-bound pool included both labeled and unlabeled Phe.
Accordingly, the enrichment values for FSR calculation were expressed as mole percent excess, which accounts for not only L-[ring-13C6]Phe but
also L-[15N]Phe in group 1 and
all three Phe tracers injected in group 2. To be
consistent with the FSR calculation, the enrichment used for
the three-pool model was also expressed as mole percent excess, which
was derived from L-[15N]Phe infusion.
In summary, the pulse tracer injection method is satisfactory in
measurement of muscle protein FSR and FBR. This method requires only
1 h and one or two muscle biopsies for both FSR and FBR. The dose
of tracer does not stimulate muscle protein synthesis. Thus this new
method has advantages over both the currently used primed-constant
infusion and the flooding dose methods. The principle of this method is
applicable to tissues other than muscle.
 |
APPENDIX |
We have previously presented a method of calculating
protein FBR in muscle. The original method required the infusion of a labeled amino acid and the measurement of arterial amino acid enrichment and intracellular free amino acid enrichment during isotopic
steady state and during isotopic nonsteady state. The formula used to
calculate FBR is
|
(A1)
|
where EM(t) is the intracellular
free enrichment at time t,
EA(t) is the arterial enrichment at
time t, QM/T is the ratio of
intracellular free content to protein bound content in muscle, and
P = EM/(EA
EM), where
EM and EA are the intracellular and arterial
enrichments, respectively, at isotopic steady state.
If we eliminate P from the above equation, we can remove the
requirement of obtaining steady-state measurements. The above equation
holds for any two time points, so if we choose time points t2 and t3 rather than
t1 and t2, then the
equation becomes
|
(A2)
|
Thus we have two equations and two unknowns, i.e., FBR
and P. If we solve Eq. A2 for P and substitute it
into Eq. A1, we obtain the equation
|
(A3)
|
Therefore, a measurement at isotopic steady
state is not required if the arterial and intracellular enrichments are
measured at three time points.
Note that if we define
and
then Eq. A3 can be written
Proof
If we rearrange the denominator in Eq. A2, we
get
|
(A4)
|
Rearranging Eq. A4 further yields
|
(A5)
|
so
|
(A6)
|
and
|
(A7)
|
If we substitute Eq. A7 into
Eq. A1, then
|
(A8)
|
so
and thus
so
thus
Hence we get Eq. A3 as desired
 |
ACKNOWLEDGEMENTS |
We are grateful to Yunxia Lin, Zhinpin Wu, Guy Jones, and Zhiping
Dong for technical assistance. We also thank the staff of the Animal
Resource Center at The Univ. of Texas Medical Branch for professional
care of experimental animals.
 |
FOOTNOTES |
This work was supported by Shriners Hospitals for Children Grants 8630 and 8490. Address for reprint requests and other correspondence: R. Wolfe, Shriners Hospitals for Children, 815 Market Street, Galveston, TX 77550 (E-mail:rwolfe{at}utmb.edu).
Address for reprint requests and other correspondence:
R. R. Wolfe, Shriners Hospitals for Children, 815 Market
St., Galveston, TX 77550 (E-mail:
rwolfe{at}utmb.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 11, 2002;10.1152/ajpendo.00053.2002
Received 6 February 2002; accepted in final form 3 June 2002.
 |
REFERENCES |
1.
Adegoke, OAJ,
McBurney MI,
and
Baracos VE.
Jejunal mucosal protein synthesis: validation of luminal flooding dose method and effect of luminal osmolarity.
Am J Physiol Gastrointest Liver Physiol
276:
G14-G20,
1999[Abstract/Free Full Text].
2.
Barle, H,
Nyberg B,
Ramel S,
Essen P,
McNurlan MA,
Wernerman J,
and
Garlick PJ.
Inhibition of liver protein synthesis during laparoscopic surgery.
Am J Physiol Endocrinol Metab
277:
E591-E596,
1999[Abstract/Free Full Text].
3.
Barrett, EJ,
Revkin JH,
Young LH,
Zaret BL,
Jacob R,
and
Gelfand RA.
An isotopic method for measurement of muscle protein synthesis and degradation in vivo.
Biochem J
245:
223-228,
1987[ISI][Medline].
4.
Baumann, PQ,
Stirewalt WS,
O'Rouke BD,
Howard D,
and
Nair KS.
Precursor pools for protein synthesis: a stable isotope study in a swine model.
Am J Physiol Endocrinol Metab
267:
E203-E209,
1994[Abstract/Free Full Text].
5.
Biolo, G,
Chinkes D,
Zhang XJ,
and
Wolfe RR.
A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle.
J Parenter Enteral Nutr
16:
305-315,
1992[Abstract].
6.
Biolo, G,
Fleming RYD,
Maggi SP,
and
Wolfe RR.
Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle.
Am J Physiol Endocrinol Metab
268:
E75-E84,
1995[Abstract/Free Full Text].
7.
Biolo, G,
Gastaldelli A,
Zhang XJ,
and
Wolfe RR.
Protein synthesis and breakdown in skin and muscle: a leg model of amino acid kinetics.
Am J Physiol Endocrinol Metab
267:
E467-E474,
1994[Abstract/Free Full Text].
8.
Dudley, MA,
Burrin DG,
Wykes LJ,
Toffolo G,
Cobelli C,
Nichols BL,
Rosenberger J,
Jahoor F,
and
Reeds PJ.
Protein kinetics determined in vivo with a multiple-tracer, single-sample protocol: application to lactase synthesis.
Am J Physiol Gastrointest Liver Physiol
274:
G591-G598,
1998[Abstract/Free Full Text].
9.
Ferrando, AA,
Stuart CA,
Sheffield-Moore M,
and
Wolfe RR.
Inactivity amplifies the catabolic response of skeletal muscle to cortisol.
J Clin Endocrinol Metab
84:
3515-3521,
1999[Abstract/Free Full Text].
10.
Galim, EB,
Hruska K,
Bier DM,
Matthews DE,
and
Haymond MW.
Branched-chain amino acid nitrogen transfer to alanine in vivo in dogs. Direct isotopic determination with [15N]leucine.
J Clin Invest
66:
1295-1304,
1980[ISI][Medline].
11.
Garlick, PJ,
McNurlan MA,
Essen P,
and
Wernerman J.
Measurement of tissue protein synthesis rates in vivo: a critical analysis of contrasting methods.
Am J Physiol Endocrinol Metab
266:
E287-E397,
1994[Abstract/Free Full Text].
12.
Imoberdorf, R,
Garlick PJ,
McNurlan MA,
Casella GA,
Peheim E,
Turgay M,
Bartsch P,
and
Ballmer PE.
Enhanced synthesis of albumin and fibrinogen at high altitude.
J Appl Physiol
90:
528-537,
2001[Abstract/Free Full Text].
13.
Krempf, M,
Hoerr RA,
Marks L,
and
Young VR.
Phenylalanine flux in adult men: estimates with different tracers and route of administration.
Metabolism
39:
560-562,
1990[ISI][Medline].
14.
McNurlan, MA,
Essen P,
Milne E,
Vinnars E,
Garlic PJ,
and
Wernerman J.
Temporal responses of protein synthesis in human skeletal muscle to feeding.
Br J Nutr
69:
117-126,
1993[ISI][Medline].
15.
Nair, KS,
Halliday D,
and
Griggs RC.
Leucine incorporation into mixed skeletal muscle protein in humans.
Am J Physiol Endocrinol Metab
254:
E208-E213,
1988[Abstract/Free Full Text].
16.
Phillips, SM,
Tipton KD,
Aarsland A,
Wolf SE,
and
Wolfe RR.
Mixed muscle protein synthesis and breakdown after resistance exercise in humans.
Am J Physiol Endocrinol Metab
273:
E99-E107,
1997[Abstract/Free Full Text].
17.
Phillips, SM,
Tipton KD,
Ferrando AA,
and
Wolfe RR.
Resistance training reduces the acute exercise-induced increase in muscle protein turnover.
Am J Physiol Endocrinol Metab
276:
E118-E124,
1999[Abstract/Free Full Text].
18.
Preedy, VR,
and
Garlick PJ.
Rates of protein synthesis in skin and bone, and their importance in the assessment of protein degradation in the perfused rat hemicorpus.
Biochem J
194:
373-376,
1981[ISI][Medline].
19.
Preedy, VR,
McNurlan MA,
and
Garlick PJ.
Protein synthesis in skin and bone of the young rat.
J Nutr
49:
517-523,
1983.
20.
Rennie, M,
Smith K,
and
Watt PW.
Measurement of human tissue protein synthesis: an optimal approach.
Am J Physiol Endocrinol Metab
266:
E298-E307,
1994[Abstract/Free Full Text].
21.
Rosenblatt, J,
Chinkes D,
Wolfe MH,
and
Wolfe RR.
Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects.
Am J Physiol Endocrinol Metab
263:
E584-E596,
1992[Abstract/Free Full Text].
22.
Smith, K,
Reynolds N,
Downie S,
Patel A,
and
Rennine MJ.
Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein.
Am J Physiol Endocrinol Metab
275:
E73-E78,
1998[Abstract/Free Full Text].
23.
Wolfe, RR.
Radioactive and Stable Isotope Tracers in Biomedicine. Principle and Practice of Kinetic Analysis. New York: Wiley-Liss, 1992.
24.
Zhang, XJ,
Chinkes DL,
Doyle D, Jr,
and
Wolfe RR.
Metabolism of skin and muscle protein is regulated differently in response to nutrition.
Am J Physiol Endocrinol Metab
274:
E484-E492,
1998[Abstract/Free Full Text].
25.
Zhang, XJ,
Chinkes DL,
Sakurai Y,
and
Wolfe RR.
An isotopic method for measurement of muscle protein fractional breakdown rate in vivo.
Am J Physiol Endocrinol Metab
270:
E759-E767,
1996[Abstract/Free Full Text].
26.
Zhang, XJ,
Cortiella J,
Doyle D, Jr,
and
Wolfe RR.
Ketamine anesthesia causes greater muscle catabolism in rabbits than does propofol.
J Nutr Biochem
8:
133-139,
1997[ISI].
27.
Zhang, XJ,
Irtun O,
Zheng Y,
and
Wolfe RR.
Methysergide reduces nonnutritive blood flow in normal and scalded skin.
Am J Physiol Endocrinol Metab
278:
E452-E461,
2000[Abstract/Free Full Text].
28.
Zhang, XJ,
Sakurai Y,
and
Wolfe RR.
An animal model for measurement of protein metabolism in the skin.
Surgery
119:
326-332,
1996[ISI][Medline].
29.
Zilversmit, DB.
The design and analysis of isotope experiments.
Am J Med
29:
832-848,
1960[ISI][Medline].
Am J Physiol Endocrinol Metab 283(4):E753-E764
0193-1849/02 $5.00
Copyright © 2002 the American Physiological Society