An isotopic method for measurement of muscle protein

fractional breakdown rate in vivo

XIAO-JUN ZHANG, DAVID L. CHINKES, YOICHI SAKURAI, AND ROBERT R. WOLFE
Metabolism Unit, Shriners Burns Institute, Galveston; and Departments of Surgery and
Anesthesiology, The University of Texas Medical Branch, Galveston, Texas 77550

Zhang, Xiao-Jun, David L. Chinkes, Yoichi Sakurai, the cell from the arterial blood and release from protein
and Robert R. Wolfe. An isotopic method for measurement breakdown within the cell. Assuming that there is no label
of muscle protein fractional breakdown rate in vivo. Am. J. recycled from protein breakdown back to the MIF pool, then
Physiol. 270 (Endocrinol. Metab. 33): E759-E767, 1996.-We the arterial blood is the only source of tracer entering the MIF
have developed a novel method to measure the fractional pool, and both arterial blood and protein breakdown supply
breakdown rate (FBR) of muscle protein. This method in- tracee to the MIF pool (Fig. 1).
volves infusing isotope tracer to reach an isotopic equilibrium When an isotopically labeled amino acid is infused in a
and then observing its decay in the arterial blood and muscle tracer dose to achieve an isotopic equilibrium, the enrichment
intracellular pool. The calculation of FBR is based on the rate in the MIF pool is always lower than that in the arterial
at which tracee released from breakdown dilutes the intracel- blood. This is because the process of protein breakdown
lular enrichment using a modified precursor-product equa- continuously releases unlabeled amino acids to dilute the
tion. To test this method, L-[1,2J3C2]leucine and L-[ring- isotope enrichment in the MIF pool. The enrichment differ-
13C6]phenylalanine were infused into six dogs for ence between the arterial blood and the MIF pool reflects the
measurement of FBR and fractional synthesis rate (FSR), fractional contribution of tracee from these two pools. For
respectively. Leucine and phenylalanine kinetics in the hind- instance, if the arterial enrichment is 0.10 and the MIF
limb were measured simultaneously using the arteriovenous enrichment is 0.05, then the fractional contribution from
(A-V) balance method. The measured FBR (0.17 t 0.02%/h) these two sources is 50% each. After reaching an isotopic
and FSR (0.10 t 0.01%/h) were in agreement with the results equilibrium, if the tracer infusion is stopped, the decay curve
from the A-V balance method. In conclusion, our new method of the enrichment in the MIF pool is determined by the
provides a feasible approach for measurement of muscle arterial enrichment decay curve (which provides tracer and a
protein FBR. This method can be combined with the tracer part of tracee) and protein breakdown (which provides an-
incorporation method to measure both breakdown and synthe- other part of tracee). At a physiological steady state, since the
sis in the same infusion study. FBR of a tissue protein is constant and the decay curves in the
blood flow; arteriovenous balance; stable isotope; mass spec- arterial and MIF pools are measurable, FBR can be calcu-
trometry lated. The following is the equation to calculate FBR using
the tracee release method

E&l - EM&)
FBR = x (QM/T) (1)
THE DIRECT incorporation methods, known as
TRACER
P s”’ E*(t) dt - (1 + P) s” E,(t) dt
the “constant infusion method” and the “flooding dose t1 t1

method,” have been widely used to determine the Here P = EM/(EA - EM) at isotope plateau, EA and EM are
fractional synthesis rate (FSR) of a tissue protein. isotope enrichment in the arterial pool and MIF pool, respec-
These methods not only provide a feasible approach to tively; EM&> - E&) is the change of enrichment in the MIF
quantify tissue protein synthesis but also represent a pool from time 1 (tI) to time 2 (tz) after stopping the isotope
successful application of the precursor-product prin- infusion; JyE*(t )dt and J:EM(t )dt are areas under the decay
ciple to the measurement of substrate kinetics (9, 17, curves of arterial enrichment and MIF enrichment, respec-
20). However, there has been no corresponding method tively, from tl to tg Q&’ is the ratio of intracellular free
to determine the fractional breakdown rate (FBR). This tracee content versus protein-bound tracee content in the
deficiency in methodology is significant since, in most muscle.
cases, a complete understanding of protein metabolism If one ignores the variable Q&I’ in the above equation, then
depends on the knowledge of both synthesis and break- Eq. 1 is simply a precursor-product equation (23). It is
down. To this end, we have developed a novel method to necessary to include the variable P in this equation because
determine for the first time the FBR of muscle protein. the traditional precursor-product equation assumes that the
Our new method, which utilizes stable isotope tracers, product is only derived from one precursor pool. In this case
is an alternative application of the precursor-product the product is the MIF pool, which has the following two
principle, which is based on the rate at which tracee is precursor pools: the arterial blood and protein-bound amino
acids within the cell (see Fig. 1). Therefore, the relative
released from protein breakdown to dilute the intracel-
contribution of these two sources to the MIF pool needs to be
lular enrichment. This new method is referred to as the included in the calculation, which is accomplished by using
“tracee release method” in this communication. the variable of P. The P value, as defined above, is equal to the
ratio of fractional tracee from artery versus fractional tracee
METHODS from breakdown (see APPENDIXES A, B, and c), and 1 + P is the
ratio of total tracee from both artery and breakdown versus
Rationale and Equation of the Dacee Release Method fractional tracee from breakdown. After introduction of the
The muscle intracellular free (MIF) pool of an essential variable P, JFEA(t)dt in Eq. 1 is the amount of tracer entering
amino acid comes from two sources as follows: transport into the MIF pool from tl to t2 divided by fractional tracee from

0193-1849/96 $5.00 Copyright o 1996 the American Physiological Society E759

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E760 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN

To test the tracee release method, we used this method

along with the tracer incorporation method and the arteriove-
nous (A-V) balance method in the dog hindlimb. This enabled
MIF us to evaluate the validity of the tracee release method by
comparing FBR with FSR and further with the data derived
from the A-V balance method.

II Experimental Procedures
III AnimaZs. This study was approved by the Animal Care and
I Use Committee of the University of Texas Medical Branch.
4 + We used male mongrel adult dogs that had been fasted
Vein & Oxidation overnight (16 h).
Fig. 1. Schematic illustration of sources of essential amino acid in Isotopes. L-[1,2J3C2]leucine (Leu; 99.3% enriched) and L-[l-
muscle intracellular free (MIF) pool. Arterial blood and muscle 13C]Leu (99% enriched) were purchased from Tracer Technolo-
protein are the arterial blood pool and muscle protein-bound pool, gies (Somerville, MA). L- [ring-13Cg] phenylalanine (Phe; 99%
respectively. Arrows indicate movement of unlabeled (solid arrow) or enriched) and L- [ring-2HJ Phe (98% enriched) were purchased
labeled (dotted arrow) amino acid from one pool to another. from Cambridge Isotope Laboratories (Woburn, MA).
Design. After general anesthesia by intravenous injection
of pentobarbital sodium (initial dose of 30 mg/kg, followed by
breakdown, and (1 + P)JI:‘E&)dt is the amount of tracer bolus injection as necessary), a triple-lumen central venous
leaving the MIF pool from ti to t2 divided by fractional tracee catheter (Cook Central Venous catheter set; Cook, Blooming-
from breakdown. Therefore, the denominator of the equation ton, IN) was inserted into the jugular vein for infusion of
calculates the change in the tracer MIF pool size divided by tracers (the distal lumen), saline (the middle lumen), and
fractional tracee from breakdown, and the numerator is the blood withdrawal (the proximal lumen), and a silicon catheter
change in the tracer MIF pool size divided by the tracee MIF (Intramedicut C a th et er Kit; Sherwood, St. Louis, MO) was
pool size from ti to ta. The equation can be expressed as inserted into the carotid artery for arterial blood withdrawal
and arterial blood pressure monitoring. A 22-gauge Teflon
fractional tracee from breakdown/tracee MIF pool size (2) needle (Quick-Cath, Baxter Healthcare, Deerfield, IL) and a
16-gauge Teflon needle (iv catheter/needle unit; Becton-
Because the traditional precursor-product equation calcu-
Dickinson Vascular Access, Sandy, UT) were inserted into the
lates the rate of conversion of precursor to product divided by
femoral artery and vein at the level of the inguinal ligament
product pool size, Eq. 2 represents the rate of tracee release
of the leg for performance of the A-V balance method. The
from the protein-bound pool divided by the MIF pool size.
catheters and needles were placed through percutaneous
However, our goal is to calculate the rate of tracee release
from the protein-bound pool divided by the protein-bound incisions. After collection of a blood sample and a muscle
sample from a front leg for determination of background
pool size. Consequently, we have to multiply& 2 by the ratio
enrichment, the isotope infusion was started.
of the MIF pool size (QM) to protein-bound pool size (T). After
The experimental protocol is illustrated in Fig. 2. The
introducing Q&l?, Eq. 2 can be rearranged as
infusion of L-[1,2J3C2]Leu (0.3 umol*kg-l*min-l; prime: 18
fractional tracee from breakdown/T (3) umol/kg) and L- [ring- 13CG]Phe (0.1 umol=kg-l*min-l; prime:
4 umol/kg) was started at the same time. The Leu tracer was
which is exactly the definition of FBR. It should be kept in infused for 3 h for measurement of FBR, and the Phe tracer
mind that, in the tracee release method, the precursor is the was infused for 5.2 h for measurement of FSR. Meanwhile,
protein-bound amino acids and arterial amino acids, and the the rates of appearance (Ra> and disappearance (Rd) of Leu
product is the intracellular free amino acids. This precursor- and Phe in the limb were measured using the A-V balance
product definition is exactly the reverse of that in the tracer method.
incorporation method, in which the precursor is intracellular The A-V balance measurements were performed during the
free amino acids (precisely, tRNA charged amino acids), and 120- to 180-min and 250- to 310-min infusion periods. Be-
the product is protein. cause the infusion of Leu tracer was stopped at 180 min, Leu

0 lh 2h 3h 4h Sh
I I I I I I 1

L-[ ring-WG]-Phe primed constant infusion

1 1
Fig. 2. Experimental protocol. BF is blood flow measure- L-[ 1 ,2-l 3Cz]-Lsu primed constant infusion
ment, which includes infusion of indocyanine green for
25 min and 4 blood samplings from femoral vein and
jugular vein over the last 15 min. Arrows point to
sampling time. A-V, arteriovenous; Phe, phenylalanine; A-V
Leu, leucine. +
balance

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 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN E761

kinetics in the limb were measured only from the 120- to stantly boiling HCl and further processed to make the NAP
180-min period. In contrast, Phe kinetics were measured derivative (6).
from both the 120- to 180-min and 250- to 310-min periods. The muscle samples taken before the isotope infusion and
Each A-V balance measurement took 1 h. After a background at the end of L-[ring- 13C6]Phe infusion were processed for both
sample was drawn from the jugular vein, indocyanine green free and protein-bound Phe enrichment. After homogeniza-
(Becton-Di ck inson Microbiology System, Cockeysville, MD) tion and washing, the dry protein pellets were hydrolyzed
was infused into the femoral artery (0.125 mg/min) for 25 without internal standard, and the protein hydrolysate was
min. Four blood samples were simultaneously drawn from purified on a high-pressure liquid chromatography (HPLC)
the femoral vein and jugular vein over the last 15 min of dye system (LKB, Bromma, Sweden) to isolate Phe (6). The pure
infusion for measurement of the dye dilution. Another four Phe was cornbusted and the 13C-to-12C ratio of the resulting
blood samples were drawn over the following 35 min simulta- CO2 was determined by isotope ratio mass spectrometry
neously from the artery and femoral vein to measure the (Isogas, Middl ewich, Cheshire, England; see Ref. 6). To
tracer uptake. Between the two A-V balance measurements ensure a good chromatographic separation of Phe derivative
(180-250 min), FBR was measured by the tracer release on the gas chromatograph mass spectrometer (GC-MS), the
method. First, two muscle samples were taken at an interval muscle supernatant for free Phe enrichment was also loaded
of 10 min to determine plateau enrichment in the MIF pool, on the HPLC to purify Phe before the NAP derivative was
which was used for both the tracee release method and the made.
first A-V balance measurement. Thereafter, the infusion of The isotopic enrichment in the blood, muscle MIF pool, and
L-[1,2JCg]Leu was stopped; arterial blood was withdrawn at protein hydrolysate (with the internal standard) was deter-
5, 10, 20, 40, and 60 min; and muscle samples were taken at mined on a Hewlett-Packard 5985 GC-MS (Hewlett-Packard,
10, 20, 40, and 60 min. The muscle biopsies were taken Palo Alto, CA) with chemical ionization. Ions were selectively
through percutaneous incisions and followed the distal-to- monitored at mass-to-charge ratios of 218, 217, and 216 for
proximal sequence on the gastrocnemius (2 biopsies at isotope Leu and 256, 255, 251, and 250 for Phe (22). The isotopic
plateau) and biceps femoris (4 biopsies during the decay) of enrichment was expressed as tracer-to-tracee ratio after
the experimental limb. After completion of the second A-V correction for the contribution of the abundance of isotopom-
balance measurement, the final muscle sample was taken ers of lower weight to the apparent enrichment of isotopomers
from the biceps femoris, which was used both for measure- with larger weight (18).
ment of FSR and for the second A-V balance measurement. Cahdations. The FBR of muscle protein was calculated by
The muscle samples were immediately frozen in liquid nitro- Eq. 1. In this equation, the P value was calculated from the
gen and stored at -70°C. plateau enrichments in the arterial blood and MIF pool; the
Sample anaZysis. To determine the leg blood flow rate, decay curves were calculated by curve fitting the measured
blood samples were separated for serum, and indocyanine enrichment using an exponential fit (see APPENDIXES A, B, and
green concentration was measured on a spectrophotometer c); the ratio QM/T was calculated from MIF and protein-bound
(Spectronic 1001; B ausch & Lomb, Rochester, NY). To deter- Leu after normalization to 1 g wet muscle. The interstitial
mine Leu and Phe enrichments and concentrations in blood, 2 Leu was subtracted from the measured free Leu in the muscle
ml blood were transferred to a tube containing 2 ml of 15% acid extract by assuming that the interstitial water accounts
sulfosalicylic acid and 200 ul internal standard solution. The for 13% of the total water content in muscle and that the
internal standard solution contained 29.7 umol/l L-[ring- concentration in the interstitial fluid is between intracellular
2HF;]Phe and 69.5 umol/l L-[ lJ”C]Leu. After deproteinization, and blood concentrations.
the supernatant was processed to make the N-acetyl,n-propyl
ester (NAP) derivatives of the amino acids (22). muscle free Leu = C~JIF l VM,, + [(c,,, + CbloocJ21
Vi*tp*
l (4)
The muscle samples were processed to determine the
intracellular enrichments of Leu and Phe, intracellular free Here CMIF and Ct,10odare concentrations in the MIF pool and in
Leu pool size, protein-bound pool sizes of Leu and Phe, and the blood, respectively; VMIF and Vinter are intracellular and
the protein-bound Phe enrichment. Before processing, the interstitial water volumes, respectively. The fact that intersti-
frozen muscle samples were allowed to thaw. The visible fat tial water accounts for 13% of water content in muscle is the
and fascia tissues were then quickly removed with a scalpel, unpublished data from a previous study (11) in this labora-
and the pure muscle tissue was gently blotted with soft paper tory in which the interstitial water was measured on dog
to eliminate blood contamination. Internal standard solution muscle using the chloride method (4). We have also consid-
(50 ul), which contained 13.97 umol/l L-[1-l”C]Leu, was added ered the possible effect of interstitial Leu on the measured
to 50 mg muscle tissue. The samples were homogenized three intracellular enrichments. If we assume that the interstitial
times in 5% perchloric acid solution at 4°C. The supernatant enrichment is between blood and intracellular enrichments,
was pooled to make the NAP derivative (22), and the precipi- then the measured plateau enrichment of muscle free Leu
tate was washed three more times with 2% perchloric acid, overestimated the actual muscle intracellular Leu enrich-
two times with absolute alcohol, and one time with ethyl ment by -5%. During the 20-60 min of the decay period, the
ether to remove free amino acids and lipids. The resulting enrichment differences between arterial blood and the MIF
protein pellets were dried in an 80°C oven for 24 h to obtain pool were much smaller; hence the overestimation was much
the dry weight of the muscle. The weight differences between less than 5%. Because we do not know the real value of
wet tissue and dry tissue were recorded as total tissue water. interstitial enrichment and because this source of error is
Four aliquots of the dry protein pellets from each dog were minor, we did not attempt to correct the intracellular enrich-
used for measurement of protein-bound Leu and Phe pool ment.
sizes. One aliquot served as background, and an internal The equations for calculation of leg blood flow rate using
standard solution, which contained 13.32 umol/ml L-[l- the indocyanine green dilution method (12), for FSR using the
i3C]Leu and 5.17 umol/ml L-[2-1.5N]Phe, was added to the tracer incorporation method (ll), and for amino acid concen-
other three (1.5 ul internal standard solution to 1 mg dry tration or content using the internal standard method (22)
protein). The protein pellets were hydrolyzed in 6 N con- were described in previous publications.

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E762 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN

The kinetics of Phe and Leu in the hindlimb were calcu- 0

lated from the modified A-V balance method using the 0
r
three-compartment model (5). The rationale and derivation of
equations of this model were described in detail in our
previous publication (5). The following are equations for Rd,
R,, and net balance (NB)
8
Rd = j[(EA x C,) - (E, x G,JI/E,] x BF (5)
NB = (C, - Cv) x BF (6) 4

R, = R, - NB (7) 13C-Leu Infusion

01 I I I
Here EA, Ev, and EM are enrichment in the arterial blood, -60 -20 20 60
venous blood, and MIF pool; CA and Cv are concentration in
the arterial and venous blood, respectively; and BF is blood Time (mln)
flow rate across the limb. Because Phe is neither synthesized Fig. 3. Plateau enrichment in arterial blood and decay curves in
nor degraded in the limb, Rd represents protein synthesis and arterial blood (0) and MIF amino acid pool (0) in 6 dogs: There was
synchronous decay of enrichments in arterial and muscle intracellu-
R, represents protein breakdown. Leu R, represents protein
lar pools during 20-60 min of the decay period.
breakdown because Leu is not synthetized in muscle, but Leu
Rd overestimates protein synthesis because it can be oxidized
in muscle. Therefore, the Phe kinetic data were converted to linemia. In the insulin-infused legs, the venous plasma
protein synthesis and breakdown, and Leu R, was converted insulin concentration was raised from the value of
to protein breakdown according to the measured content of 17.6 ? 2.3 uU/ml immediately after anesthesia to
each amino acid in leg muscle. 43.7 t 5.8 pU/ml after 3 h of insulin infusion, but the
To express results from the two methods in comparable insulin concentration in the contralateral legs de-
units, it was necessary to convert the A-V model data to FSR
and FBR. To accomplish this goal, we measured the percent- creased to 12.4 ? 1.6 uU/ml over the same time period.
age of dry protein in muscle and Phe and Leu content in the The arterial blood samples drawn between 145 and
dry protein (umol/g). Because we did not measure the total 180 min were at isotopic plateaus; after stopping
muscle mass in the limb, we used the factors that 68% of blood L- [ 1,2-13C2]Leu infusion, its enrichments in the arterial
flow in the leg supplies muscle (16) and 1 g of resting muscle pool and in the MIF pool both declined over the 60-min
receives 0.084 ml blood/min. The latter factor was calculated measurement period (Fig. 3). The decline of enrich-
from the data presented in Ref. 13 that, in dogs weighing 19.2 ments followed generally smooth curves. We further
kg, the average muscle mass was 8,455 g, and blood supply to smoothed the decay curves by using an exponential
the muscle was 713.2 ml/min. This was the intermediate curve fit (see APPENDIXES A, B, and c) to minimize the
value of muscle blood supply reported in the literature. The random variations. Table 1 shows the pool sizes of
other values were 0.092 (calculated from available data in unlabeled Leu in the MIF and protein-bound pools and
Ref. 15) and 0.061 (reported in Ref. 14). With the above
factors, we converted the R, and Rd data to FBR and FSR by the plateau enrichments in the arterial and MIF pools,
the calculation which were all required for calculation of QM/T and the
P value in Eq. 1. The values of FBR calculated from
FBR or FSR in %/h = (R, or Rd in umol l h-l l leg-l different time periods of decay, namely O-10 min,
lo-20 min, 20-40 min, 40-60 min, and 20-60 min, are
+ [(BF in ml=min-lleg-l X 68% presented in Table 2. The values during 20-60 min
+ 0.084 mlgl*minl) (8)
Table 1. Muscle intracellular free and protein-bound
X muscle protein content in % Leu and plateau enrichments in the arterial and
X Phe or Leu content in muscle protein in umol/g]] muscle intracellular pools

StatisticaL analysis. The data are expressed as means t Muscle Intracellular Pool Plateau Enrichment

SE. The reproducibility of the data was assessed by the Dog No. Free Bound MIF Pool Arterial blood
coefficient of variation (CV). The paired t-test was used to test
the difference between values derived from different meth- 1 0.1769 113 0.0458 0.0953
2 0.1385 123 0.0522 0.1341
ods. P < 0.05 was considered statistically significant. 3 0.0860 115 0.0485 0.1256
4 0.1279 126 0.0664 0.1080
RESULTS 5 0.1626 127 0.0462 0.1057
6 0.1153 117 0.0423 0.1065
The body weight of the dogs was 19.8 2 0.4 kg. All of Mean
the animals maintained stable conditions during the k SE 0.1345 + 0.0134 12052 0.0502t0.0035 0.1125kO.0059
tracer infusion, as demonstrated by stable mean arte- Muscle intracellular free (MIF) and bound leucine (Leu; pmol/g wet
rial blood pressure, heart rate, and the absence of any muscle) stand for unlabeled Leu pool sizes, which were used to
calculate the ratio of the intracellular free trace content vs. the
visible response to muscle biopsy. Because of a concomi-
protein-bound tracer content in muscle (QM/T) in Eq. 1. Plateau
tant study in these dogs, insulin (0.1 or 0.2 mU kg-l. enrichments stand for L- [ l,2-13C2] Leu enrichments (tracer-to-tracee
min-l) was infused into the femoral artery of the ratio) at isotopic equilibrium in arterial blood and MIF pool, which
onoosite leg to achieve a local nhvsiological hvnerinsu- were used to calculate the P value in Ea. 1.

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 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN E763

Table 2. FBR measured by the tracee release method Table 3. FBR calculated from one enrichment decay
value in the MIFpool
Dog No.
Time Period,
Dog No.
min 1 2 3 4 5 6 Mean 5 SE Time Point,
min 1 2 3 4 5 6 Mean + SE
O-10 0.31 0.76 0.47 0.45 0.34 0.25 0.43+0.07*
10-20 0.18 0.24 0.25 0.22 0.17 0.13 0.2OkO.02 10 0.33 0.57 0.25 0.18 0.33 0.17 0.31t0.06
20-40 0.18 0.16 0.23 0.18 0.16 0.10 0.17+0.02 20 0.10 0.41 0.28 0.28 0.21 0.12 0.2320.05
40-60 0.24 0.13 0.15 0.14 0.20 0.09 0.16kO.02 40 0.32 0.15 0.29 0.10 0.18 0.14 0.20+0.04
20-60 0.21 0.15 0.19 0.16 0.18 0.10 0.17kO.02 60 0.18 0.23 0.17 0.21 0.19 0.10 0.18+0.02
Values for time period of 20-60 min are averages of 20- to 40-min Units are %/h. 10, 20, 40, and 60 min are time points of the single
and 40- to 60-min values. *P < 0.05 vs. value from time period of 20- MIF enrichment decay values used to calculate FBR.
to 60-min. FBR, fractional breakdown rate (%/h).

were the averages of 20- to 40 and 40- to 60-min periods
and were used as the real FBR (see DISCUSSION
explanation). The measured FBR was 0.17 * 0.02%/h,
which had a CV of 23%.
In each of the dogs, the decay of enrichment in the
arterial blood well follows the two-exponential curve,
and the decay curves in the arterial and MIF pools are
generally parallel during the 20- to 60-min period (see
Fig. 3). If this is the case, once the arterial decay curve
is known, the MIF enrichment decay curve could be
predicted from a single MIF enrichment decay value,
either from lo-, 20-, 40-, or 60-min muscle samples.
This hypothesis has been confirmed mathematically
(see APPENDIXES A, B, and c). The equation for prediction
of the MIF enrichment decay curve in APPENDIXES A, B,
and c shows that the FBR value can be calculated from
one MIF enrichment decay value. This equation also
shows that the decay curves in the MIF pool can be
drawn for various FBR values when the arterial decay
curve, P value, and QM/T in Eq. 1 are known. Figure 4 is
an example of predicted MIF enrichment decay curves
at various FBR values using the above theory. Because
the arterial decay curve can be obtained from the
arterial blood samples, the P value and QJI’ can be
obtained from the arterial blood samples and two
muscle samples taken before tracer infusion, and, at
isotopic plateau, the FBR value can be known from Fig.
4 as long as a MIF enrichment decay value is available.
Once again, this confirms that, during the decay period,
only one muscle sample is enough to determine the
FBR value. Using the above theory and the equations
in APPENDIXES A, B, and C, we have calculated the FBR
values from one MIF enrichment value during the
for decay period, and the results are presented in Table 3.
This single decay sample approach resulted in compa-
rable FBR values to those from the four muscle samples
during the decay period. Especially, the FBR values
derived from the 60-min muscle sample (0.18 t 0.02%/h;
CV of 25%) were almost identical to the values from
20-60 min of the decay period in Table 2.
The FSR was calculated from 5.2 ? 0.2 h of infusion
of L- [ring- 13CG]Phe. The muscle free Phe enrichment
was 0.0502 t 0.0046 and the rate of increase in
protein-bound Phe enrichment was 0.4958 X lOA 2
0.0189 X 10p4/h. The calculated FSR of 0.10 t 0.01%/h
(CV = 23%) was significantly lower (P < 0.01) than the
FBR.
Phe and Leu kinetic data measured by the A-V
balance method are presented in Table 4. The Phe
kinetic data measured in the 120- to HO-min and 250-
to 310-min infusion periods were comparable, indicat-
ing that the multiple sampling procedure of the tracee
release method did not affect amino acid and protein
kinetics in the limb. The measured Phe and Leu
contents were 240 ? 5 and 581 t 11 umol/g muscle
protein, respectively, and dry protein accounted for
20.7 t 0.2% of wet muscle. The FBR and FSR converted
from the A-V measurements, along with the FBR and
FSR data measured by the tracee release method and
tracer incorporation method, are presented in Table 5.
The average FSR values derived from the two ap-
proaches were identical (0.10%/h); the FBR values
derived from the tracee release method were higher
than those from the A-V approach, but the differences

- FBR=I%
----a FBR=2%
Fig. 4. Predicted decay curves of enrichment (tracer-to-
.. ... .. ... .. .. .. FBR=3% tracee ratio x 100) in MIF pool. Once plateau enrich-
ments in arterial blood and MIF pool and ratio of MIF
----- FBR=J% vs. bound tracee are known, decay curves in MIF pool
can be predicted from arterial enrichment decay curve
---- FBR=5% at various fractional breakdown rate (FBR) values (see
APPENDIXES A, B, and c for rationale). Accordingly, during
..-.I.- FBR=6%
the decay period, only 1 muscle sample is needed.

1.0 f I I I 1 I 1
0 IO 20 30 40 50 60
Time (min)

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E764 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN

Table 4. Leu and Phe kinetics in the hindlimb study the enrichment gradient (at the end of the tracer
measured by the A-V balance method infusion) between muscle free and bound pools was
measured to be an average of 273:1, indicating that any
Phe Tracer
tracer recycling from proteolysis would be negligible in
Leu Tracer 1 2 relation to the measured intracellular enrichment.
Rd 181.5 i 28.1 65.5 2 17.3 i53.1k7.8 This assumption is shared by the A-V balance method
NB -49.0127.6 -24.429.0 -24.4k4.6 (12) and the t racer incorporation method (9, 17). The
RI 230.5~41.6 89.75 18.4 77.5 + 12.0 defined precursor pool (muscle protein and arterial
Data are means 2 SE in umol h- l leg-l. Rd, rate of disappearance;
l l
blood) and product pool (the MIF amino acid pool) for
NB, net balance; R,, rate of appearance; 1 and 2, first (120-180 min) this method are both easily accessible and were actu-
and second (250-310 min) arteriovenous (A-V) balance measurement ally measured in this study. This is an important
from L- [ring- l:Cs]phenylalanine (Phe) infusion, respectively; Leu
kinetic data were obtained only from the first (120-180 min) A-V
advantage because this method measures true precur-
measurement because, thereafter, L-[1,2-13C2]Leu infusion was sor (i.e., protein-bound amino acids) and does not
stopped for decay measurement. require any assumptions, such as those required when
measuring FSR, and a surrogate for the aminoacyl-
were not statistically significant (P > 0.05). Accord- tRNA enrichment is used (2 1).
ingly, the values of NB of muscle protein calculated Practically, the tracee release method involves infu-
from the direct approach (-0.06 t 0.01%/h) were sion of tracer to reach an isotopic equilibrium and then
slightly greater than those calculated from the A-V observation of its decay in the arterial and MIF pools.
balance measurement (-0.04 t 0.01 or -0.05 2 O.Ol%/ The isotopic equilibrium is crucial to ensure both
h), but the differences were not statistically significant reliable plateau enrichment values and reliable decay
(P > 0.05). curves, which are required for determination of the P
value and decay parameters in Eq. 1. In this study, 3 h
DISCUSSION primed constant infusion of Leu tracer was sufficient to
The present study was designed to develop and reach isotopic equilibrium, and 60 min of decay was
validate insofar as is possible the tracee release method sufficient to obtain smooth decay curves. The FBR
for measurement of FBR in muscle. As defined by Eq. 3, values calculated from the O-10 min and lo-20 min of
the muscle protein FBR calculates the rate of release of the decay periods were much higher than those from
free amino acids from proteolysis versus total muscle the later periods, and the values calculated from the
protein. This indicates that the tracee release method 20-40 min and 40-60 min of the decay periods were
is the complement of the tracer incorporation method, very close (see Table 2). This is because the tracer is
because the muscle protein FSR calculates the rate of infused into the body from the venous catheter, and the
incorporation of free amino acids into protein versus arterial enrichment is instantly affected by the tracer
total muscle protein. Accordingly, the difference be- infusion rate. When the tracer infusion rate changes or
tween FBR and FSR is exactly the NB. stops, it takes a certain time for the change to be
The theory of the tracee release method is simple. completely reflected in the MIF pool. In other words, at
The major assumption is that the arterial blood is the the moment the Leu tracer was stopped for decay
only source of tracer entering the MIF pool, meaning measurements, the isotopic equilibrium was violated.
that there is no significant tracer recycling from prote- There was a time delay for the MIF pool to have the
olysis. This is a reasonable assumption, because in this synchronous decay of enrichment as in the arterial

Table 5. FBR and FSR from the direct approach and the A-V approach (% I h)

Dog No.

1 2 3 4 5 6 Mean + SE

Direct approach
FBR 0.21 0.15 0.19 0.16 0.18 0.10 0.17 kO.02"'
FSR 0.13 0.11 0.13 0.08 0.09 0.08 0.10 + 0.02
NB (FSR - FBR) 0.08 -0.04 -0.05 -0.08 -0.07 -0.02 -0.06+ 0.01
A-V approach
FBR Phe (1) 0.16 0.16 0.19 0.10 0.16 0.08 0.14+0.02"i
FBR Phc 12) 0.18 0.16 0.19 0.10 0.14 0.09 0.14+0.02:':
FBR Leu ( 1) 0.14 0.19 0.19 0.15 0.16 0.08 0.15+0.02+
FSR Phe ( 1) 0.10 0.12 0.18 0.08 0.08 0.05 0.10 * 0.02
FSR Phe (2) 0.13 0.09 0.12 0.08 0.09 0.07 0.10 + 0.01
NB Pherll 0.05 -0.04 - 0.01 -0.02 -0.08 -0.03 -0.042 0.01
NB Phc 121 0.05 -0.07 - 0.07 -0.02 -0.05 -0.02 -0.05 20.01
Direct approach represents values derived from tracee release method (for FBR) and tracer incorporation method [for fractional synthetic
rate (FSR)] using L-[1,2J3C2]Leu and L-[ringJ3Cs]Phe, re s p ectively; A-V approach, converted from A-V balance data; FBRpho (I), FSRphe (11,
NBphe (I,, FBRphc Q), FSRphe Q), NBphe (2, are values converted from Phe kinetics from the first (120-180 min; 1) and second (250-310 min; 2)
A-V balance measurements; FBRL~~ ( I), values converted from Leu R, from the first (120-180 min) A-V balance measurement. Thereafter,
L-[1,2-‘%2]Leu tracer infusion was stopped for decay measurement. ‘i’P < 0.01 and 4P < 0.05 compared with corresponding FSR values.

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 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN E765

pool. On the other hand, because the studies were to assume that the A-V system reflects only muscle
performed in the physiological steady state, which was metabolism. We have previously determined that the
not affected by the stop of the tracer infusion, the FBR contribution of skin and other tissues is minor but not
should be a constant value. Therefore, we used the 20- negligible (7). Consequently, there is some inherent
to 60-min decay period for calculation of FBR. We did inaccuracy in the comparison of the two methods, since
not extend the sampling beyond 60 min because what- the FBR and FSR represent exclusively muscle. Consid-
ever benefit would have been attained from additional ering these limitations, the values from the two ap-
data would have been offset by the difficulty in accu- proaches correspond well. The values of FSR from the
rately detecting low levels of enrichment. A-V approach were almost identical to those from the
Whereas the four muscle samples in the 60 min of the well-established tracer incorporation method (see Table
decay period enabled us to draw a smooth decay curve 5). This agreement is similar to our previous findings
(see Fig. 3), the multiple muscle biopsies are theoreti- and supports the A-V approach for measurement of
cally not necessary and usually too aggressive in a muscle protein synthesis. For FBR, the values derived
human subject. As illustrated in Fig. 1, the arterial from the A-V approach (0.14-0.15%/h) were lower than
blood is the only source of the tracer entering the MIF those from the tracee release method (0.17%/h). Accord-
pool. During the decay period, because the total rate of ingly, the NB (FSR - FBR) from the direct approach
tracee entering the MIF pool from the arterial blood (-0.06 t 0.01%/h) was larger than that from the A-V
and proteolysis remains constant, the rate of decline of approach (-0.04 to -0.05%/h). In the postabsorptive
the MIF enrichment is closely related to the rate of state, the skeletal muscle is known to provide amino
decline of the arterial enrichment. The relationship acids for splanchnic protein synthesis, whereas more
between the two decay curves lays the basis of predict- limited data suggest that skin is essentially preserved
ing the MIF enrichment decay curves as shown in Fig. (7, 8). Therefore, it is reasonable that the direct ap-
4. Whereas the whole 60-min decay curve in the MIF proach would indicate a more negative balance than
pool can be predicted from any single MIF enrichment the A-V approach, since only the A-V results would be
value between 10 and 60 min of the decay period, the dampened by the contribution from skin metabolism.
60-min value appears superior to the others, which is In summary, the tracee release method is successful
shown by almost identical values of FBR and CV in in measuring muscle protein FBR with satisfactory
comparison with the values derived from the 20- to reproducibility and precision. This method directly
60-min period of decay. This single decay sample ap- analyzes pure muscle tissue, which is superior to the
proach is an important advantage in the application of currently used methods in reflecting muscle protein
the tracee release method because both FBR and FSR breakdown. The tracee release method can be combined
can be measured from a total of three muscle biopsies, easily with the tracer incorporation method to measure
namely a background sample, a plateau sample, and a both muscle protein synthesis and breakdown in one
decay sample. infusion study. This combined application represents a
The optimal way in which to validate a new method is complete methodology for the measurement of muscle
to compare with a standard method or a known value. protein metabolism. Theoretically, this method can be
However, in this case, it is impossible to find such a applied to other tissues of the body so long as a few
standard, since the tracee release method is the first biopsies can be obtained.
direct measurement of muscle FBR. Some available
methods, such as excretion of 3-methylhistidine (2) or
APPENDIX A
the ratio of 3-methylhistidine to creatinine (19), are not
suitable because they provide markers rather than Definition of Variable P
quantitative values of muscle protein breakdown. The
Assuming that, at the physiological and isotopic steady
A-V balance method used in this study is the only
state, Pa and PB are the rates of tracee entering the MIF pool
previously published method with which to quantify
from artery and proteolysis, respectively, and EA and EM are
muscle protein breakdown other than the method to be enrichments in the arterial pool and MIF pool, respectively,
tested. Our A-V balance method is different from then PA x EA is the rate of tracer entering the MIF pool.
traditional models. The traditional A-V balance meth- According to the assumption that the arterial blood is the only
ods (3, 10) reflect only the tracee kinetics between the source of tracer entering the MIF pool, then
bloodstream and tissue intracellular free pool. Any
amino acids released by protein breakdown that are P, x E, = EM x (PA + PB) (Al)
subsequently reincorporated into protein, without ever
By rearranging Eq. Al, we have
entering the blood, are missed by those methods. Our
modified A-V model includes tissue biopsy data, en- PA x EA = (PA x EhlI) + (PB x Ehl) Ma
abling the model to further include tracee kinetics
between the intracellular free pool and protein-bound PA x (EA - EM) = PB x E, (A3)
pool, thereby enabling an estimation of total protein
E,/(E, - E,) = PA/PB (A41
breakdown. However, direct comparison with FBR is
difficult, because the A-V model used in this study Therefore, P = EM/(EA - EM) means that P is equal to the
represents total kinetics in the limb. To compare the ratio of fractional tracee from artery versus the fractional
limb kinetic data with FBR and FSR, it was necessary tracee from breakdown.

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E766 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN

APPENDIX B If the equation for FBR (Eq. 1) is rearranged, we obtain

Smoothing Enrichment Curves
FBR x (T/&,) x [P r” E,(t) dt - (1 + P) r’” E,(t) dt]
Jtl -- Jtl
Variability in the measurement of enrichment will cause (C3)
variability in the calculated FBR values. To minimize this = E& - E,(t,)
source of variability, we smoothed the enrichment curves. The
decay of the Leu enrichment after the tracer infusion was If we take the derivative of both sides of the above equatior
stopped could be well described by a two-exponential decay with respect to t, then we obtain
curve
FBR x (T/&J x [P x EA(t) - (1 + P)E,(t >:] = E,(t) (C4
Y(t) = A exp(K x t) + B exp(L x t) (Bl)
The above equation can be written
Y(t) is the enrichment at time t, and A, B, K, and L are
constants. To obtain the model parameters A, B, K, and L, we I&(t) = X x E,(t) + Y(t)
used our program EZFit, which gives the same results as
SAAM and MLAB but does not require initial parameter where X = -(l + P) X FBR X (T/&M) (i.e., a constant) and
estimates from the user (9a). We then obtained the area Y(t) = E*(t) X P X FBR X (T/Q& = [A exp(K X t) +
under the curve using Y( t >. B exp(L X t)] X P X FBR X (T/&M), so the general solution of
EM (i.e., Eq. C2) must have the form
APPENDIX C
E,(t) = F exp(X X t) + G exp(K X t) + H exp(L x t) CC61
Prediction of Decay Curve in the Muscle
Intracellular Pool where F, G, and H are constants with values to be determined
below.
In practice, the arterial enrichment [E*(t)] can be well
described by the sum of two exponentials, i.e. E,(t) = F X X X exp(X X t) + G X K
(C7)
X exp(K X t) + H X L X exp(L X t)
E,(t) = A exp(K x t) + B exp(L x t) (Cl>
Also, according to Eq. C6
In this case, one can rearrange the FBR equation (see text for
this equation) into the following equation (see below for the E,(O) = F + G + H CC@
derivation of this equation)
By substituting Eqs. C6 and C7 into Eq. C5 and comparing
P P x FBR x (T/QM) x A the coefficients of the three exponentials, we obtain the
E,(t) = l+p(A + B> - following three equations
K + (1 + P) x FBR x (T.QM)

P x FBR x (T/&J x B exp(Xt):FxX=FxX (W

-
I + (1 + P) x FBR x (T/Q& 1 exp(Kt): G X K = G X X + A X P X FBR X (T/&,) (ClO)
l exp[--(1 + P) x FBR X (T/&J X tI exp(Lt): H X L = H x X + B x P x FBR x (T/&,) (Cll)
W)
P x FBR x U’lQM)x A Solving the three equations C8, C10, and Cl1 for F, G, and H
+ K + (1 + P) x FBR x U’IQ,) yields

l exp(K x t) G = [A x P x FBR x (T/Q,)]/(K - X) (cm

P x FBR x (T/Q& x B
+ H = [B x P x FBR x (T/Q,)]/(L - X>
L + (1 + P) x FBR x (T/Q& exp(L* t’ (C13)

F = E,(O) - [A x P x FBR x WQ,)lW - x>

Here EM(t) is the enrichment in the muscle intracellular free (C14)
(MIF) pool at time t; in the expression P = EM/(EA - EM), EM - [B x P x FBR x (T/Q,)I/(L - X)
and EA are enrichments in the MIF pool and arterial blood,
respectively, at isotopic plateau (see METHODS for details); If we substitute Eqs. C12, C13, and C14 into Eq. C6, we
FBR is a constant value in the physiological steady state; and obtain the general solution of EM, i.e., Eq. C2.
T/QM is a constant. From Eq. Cl, we can estimate A, B, K, and
L. Then, in Eq. C2, there is only one variable [i.e., EM(t)] and
one unknown constant (i.e., FBR). Apparently, for every value We thank Dr. Yaoqing Zheng and Yanping Sun for technical
of EM(t), there is one FBR value and vice versa. Therefore, the assistance.
FBR value can be known from only one EM value at the decay This work was supported by Grant 15849 from Shriners Hospital.
time point t, and also the decay curves in the MIF pool can be This work was presented in abstract form at the Experimental
predicted (see Fig. 4 in text) at varying FBR values during the Biology meeting in Atlanta, GA, April g-13,1995.
decay period when the A, B, K, L (solved from the arterial Address for reprint requests: R. R. Wolfe, 815 Market St., Shriners
decay curve in Eq. Cl), P, and QM/T are known. The following Burns Institute, Galveston, TX 77555-1220.
is the derivation of Eg. C2. Received 28 June 1995; accepted in final form 13 December 1995.

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 ISOTOPIC METHOD FOR MUSCLE PROTEIN FRACTIONAL BREAKDOWN E767
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