Measurement of skin protein breakdown in a rat model
Elena
Volpi1,4,
Marc G.
Jeschke2,4,
David N.
Herndon2,4, and
Robert R.
Wolfe2,3,4
Departments of 1 Internal Medicine, 2 Surgery, and
3 Anesthesiology, University of Texas Medical Branch, Galveston,
77555; and 4 Shriners Hospital, Galveston, Texas 77550
 |
ABSTRACT |
Whereas
skin protein synthesis can be measured with different approaches, no
method potentially applicable in humans is available for measurement of
skin protein breakdown. To that end, we measured mixed skin fractional
protein breakdown (FBR) in a rat model by use of a stable isotope
method (tracee release method) originally developed to measure muscle
protein breakdown. Skin mixed protein and collagen fractional synthesis
rates (FSR) were also measured. A primed continuous infusion of
L-[ring-2H5]phenylalanine
and
-[5,5,5-2H3]ketoisocaproate (KIC) was
given for 6 h. Arterial and skin phenylalanine and leucine free
enrichments were measured at plateau (5-6 h) and during the decay
that followed after the infusion was stopped. Skin FBR (%/h) was
0.260 ± 0.011 with phenylalanine and 0.201 ± 0.032 with
KIC/leucine [P = not significant (NS)]. Mixed skin
FSR (%/h) was 0.169 ± 0.055 with phenylalanine and 0.146 ± 0.020 with KIC/leucine (P = NS). Collagen FSR was
0.124 ± 0.023%/h (P = NS vs. mixed protein FSR).
The tracee release method is a sensitive method for measurement of skin
protein breakdown; however, given the high intersubject variability of
FSR, the calculation of skin net balance is not advisable.
collagen; proteolysis; protein synthesis
 |
INTRODUCTION |
THE
MEASUREMENT OF SKIN PROTEIN TURNOVER would be useful to assess
skin regeneration and wound healing as well as to test the effects of
different stimuli and pathophysiological conditions on skin metabolism.
However, although skin protein synthesis has been measured by use of
different methods (1, 2, 4, 8, 9, 11, 20), there are no
methods available for measurement of skin protein breakdown with the
exception of an arteriovenous rabbit ear model recently developed in
our laboratory (20). The rabbit ear model cannot be
applied when the arteriovenous catheterization of a specific skin
region is not possible, and it is therefore not applicable in humans.
We have recently described a method for measurement of muscle protein
fractional breakdown rate (FBR) by use of the precursor-product principle (19). This method requires the primed continuous
infusion of a stable isotope tracer of an essential amino acid and the measurement of its arterial and intracellular muscle enrichment at
plateau and during the decay that follows after stopping the infusion.
The FBR method requires sampling of the free amino acid pool of the
compartment in which protein breakdown takes place (19). In the case of skin tissue, protein breakdown takes place in both the
intracellular and the extracellular compartments (18).
Therefore, the pools into which breakdown releases amino acids are
comprised of both the intracellular and the extracellular free amino
acid pools. Thus the simple extraction of the free amino acids from the
skin tissue samples should be adequate for the measurement of skin FBR.
We measured skin protein FBR and skin mixed protein and collagen
fractional synthetic rates (FSRs) in rats by use of stable isotopes of
phenylalanine and
-ketoisocaproate (KIC).
 |
MATERIALS AND METHODS |
Animals.
The study was approved by the Institutional Animal Care and Use
Committee of the University of Texas Medical Branch, Galveston, TX.
Seven male Sprague-Dawley rats (350-375 g body wt) were housed in
wire-bottom cages in a temperature-controlled room with a 12:12-h light-dark cycle. Animals were fed a regular pellet diet (Harlan Diet
including 50% carbohydrate and 15% fat) and water ad libitum.
Infusion study.
On the day before the infusion study, catheters were implanted in the
carotid artery and the jugular vein under general anesthesia (pentobarbital sodium, 25 mg/kg) and analgesia (butorphanol, 0.2 mg).
The animals were then housed overnight in Bollman cages and given a
constant infusion of normal saline (2 ml/h). On the day of the study,
after 200 µl of blood were drawn from the artery to measure the
background enrichment of leucine and phenylalanine, a primed continuous
infusion of
-[5,5,5-2H3]KIC (prime, 18 µmol; infusion rate, 0.15 µmol/min) and
L-[ring-2H5]phenylalanine
(prime, 10 µmol; infusion rate, 0.083 µmol/min) was started and was
continued for 6 h (0-360 min; Cambridge Isotopes, Cambridge,
MA). Phenylalanine infusion was performed in five of the seven rats
receiving labeled KIC. Blood samples were drawn at 120, 300, 360, 375, 390, 420, and 480 min, and skin biopsies (~1.5 g tissue) were taken
at 120, 360, 420, and 480 min (Fig. 1)
using local anesthesia with lidocaine. Skin samples were immediately frozen in liquid nitrogen and were stored at
80°C until analysis. Blood samples were precipitated in 1 ml of 15% sulfosalicylic acid
(SSA) and centrifuged, and the supernatant was stored at
20°C.

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Fig. 1.
Study protocol. The day before the infusion study,
catheters were implanted in the carotid artery and the jugular vein
under general anesthesia and analgesia. The animals were then housed
overnight in Bollman cages and given a constant infusion of normal
saline (2 ml/h). On the day of the study, after 200 µl of blood were
drawn from the artery to measure the background enrichment of leucine
and phenylalanine, a primed continuous infusion of
-[5,5,5-2H3]ketoisocaproic acid and
L-[ring-2H5]phenylalanine
was started and continued for 6 h (0-360 min). Phenylalanine
infusion was performed in 5 of the 7 rats. Blood samples were drawn at
120, 300, 360, 375, 390, 420, and 480 min, and skin biopsies were taken
at 120, 360, 420, and 480 min.
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Analytical methods.
Approximately 50 mg of tissue were cut from each skin biopsy. An
internal standard solution containing
L-[2H10]leucine (122 µmol/l)
and
L-[ring-13C6]phenylalanine
(50 µmol/l, 2 µl/mg of skin) was added. SSA (15%, 450 µl) was immediately added to each sample, after which the sample was
ground and then centrifuged at 2,000 g for 15 min. The
supernatant was collected, and the pellet was resuspended in 15% SSA.
The process was repeated three times. The collected supernatant
containing free tissue amino acids was frozen for subsequent analysis,
and the pellet was washed once with distilled water and three times
with absolute ethanol. The pellet was dried overnight at 50°C and
then weighed and hydrolyzed in 2 ml of 6 N HCl at 110°C overnight.
The purification of collagen was performed using a modification of the
method in Refs. 7 and 12. Approximately 1.5 g of skin was
homogenized at 4°C in extraction buffer (10 ml/g skin) containing 150 mM NaCl, 50 mM Tris · HCL, 20 mM EDTA, 1.0 mM
phenylmethylsulfonyl fluoride (PMSF), 2.0 mM
N-ethylmaleimide (NEM) and 0.2 mM 2-aminopropionitrile, at pH 7.4. The samples were stirred vigorously and then centrifuged at
70,000 g for 25 min. The supernatant was filtered through
glass wool, and collagenous proteins were precipitated with the
addition of saturated ammonium sulfate. After centrifugation at 18,000 g for 30 min, the pellet was reconstituted in the extraction
buffer, and the solution was brought to 4.5 M with NaCl to precipitate all newly synthesized types of collagen and procollagen. The samples were centrifuged at 70,000 g for 25 min, and the pellet was
collected and reconstituted in column buffer containing 200 mM NaCl, 50 mM Tris · HCl, and 2 mM EDTA, at pH 7.6. After dialysis
(membrane cutoff 14.000-16.000 kDa) in column buffer, acidic
proteins were removed by DEAE cellulose chromatography as previously
described (7). Urea was added to the samples to achieve a
molarity of 1 mol/l. Collagen was separated by means of DEAE cellulose
chromatography with an increasing linear NaCl gradient. The samples
were divided into two aliquots, one for assessment of purity of the
fraction and one for the measurement of isotope incorporation rate.
Collagen purity was assessed after dialysis in 0.15 M acetic acid
(membrane cutoff 14.000-16.000 kDa) by SDS-PAGE (Mini-Protean apparatus, precast gradient gel 4-15%, Bio-Rad, Hercules, CA) using broad molecular mass standards (Bio-Rad). Two gels were loaded
with the same amounts of the same samples. One of the gels was stained
with Coomassie brilliant blue, whereas the other was blotted on
nitrocellulose. A mixture of anti-body anti-collagen type I
(Calbiochem, San Diego, CA), collagen type III (Calbiochem), collagen
type IV (BioGenex Laboratories, San Ramon, CA), and collagen type VII
(Sigma, St. Louis, MO) was used to identify collagen. Amino acid
composition analysis of the extracted collagenous proteins was
additionally performed on 10 µl of extracted sample after hydrolysis
with an amino acid autoanalyzer (HP 3396A/6300AAA, Hewlett-Packard,
Wilmington, DE).
The sample aliquots for the measurement of the isotope incorporation
rates were dialyzed against distilled water (membrane cutoff
14.000-16.000 kDa), dried in a speed vacuum, and hydrolyzed in 2 ml of 6 N HCl at 110°C overnight.
Blood and tissue free amino acids, as well as amino acids derived from
the hydrolysis of collagen and mixed skin proteins, were purified by
means of cation exchange chromatography (17). Blood and
free tissue leucine and phenylalanine enrichments were determined on
their tert-butyldimethylsilyl (t-BDMS)
derivatives by gas chromatography-mass spectrometry (GC-MS; GC 8000 series, MD 800, Fisons Instruments, Manchester, UK) in the electron
impact mode. The enrichments of leucine and phenylalanine derived from the hydrolysis of collagen and mixed skin proteins were determined on
their t-BDMS derivatives in the electron impact mode by
GC-MS monitoring the ions 304 and 305 for leucine and 237 and 239 for phenylalanine with the use of the standard curve approach described by
Calder et al. (3) and modified by Patterson et al.
(10).
Calculations.
The FBR of mixed skin proteins was calculated with a method developed
for muscle and defined as the tracee release method (19).
This method enables the measurement of the rate of protein breakdown by
measuring the decay curves of arterial and tissue free amino acid
enrichment in the arterial and tissue free amino acid pools after
stopping the tracer infusion once the isotopic steady state is
achieved. At isotopic steady state, the enrichment in the tissue pool
is always lower than the enrichment in the arterial blood because of
tissue protein breakdown, which releases unlabeled amino acids. Once
the isotopic infusion is stopped, the enrichment decay in the free
tissue pool depends on the arterial enrichment decay, which provides
tracer and a part of tracee, and on the protein breakdown, which
provides another part of tracee (Fig. 2).
Therefore, the FBR can be calculated with the equation
|
(1)
|
where ES(t2)
ES(t1) is the change of enrichment in the free
tissue pool after stopping the isotopic infusion (time 360-480
min). P = ES/(EA
ES),
where EA and ES are the isotope enrichments in
the arterial and free tissue pools at isotopic plateau (EA,
300-360 min; ES, 360 min);
t1t2
EA(t)dt and
t1t2
ES(t)dt are the areas under the decay
curves of the arterial and the tissue enrichments, respectively. Qs/T
is the ratio of the intracellular free tracee content over the protein
bound tracee in the total skin. Q was calculated for each sample as the
measured tissue free amino acid concentration (Cs, nmol/ml)
multiplied by the measured tissue water content (ml/g wet tissue)
|
(2)
|
T was measured for each sample as the amino acid content of
mixed skin proteins per gram of tissue wet weight. The results are
presented in percent per hour, multiplying by the factors 60 (min/h)
and 100.

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Fig. 2.
Model of skin amino acid kinetics. Sources of essential
amino acids in skin tissue free pool. Arterial blood and skin proteins
are the arterial blood pool and skin protein-bound pool, respectively.
Solid arrows indicate movement of unlabeled essential amino acids, and
dotted arrows indicate movement of labeled amino acids from one pool to
another. However, during KIC infusion, labeled leucine can also appear
from transamination within the skin cells into the skin tissue free
pool (not depicted).
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|
The FSR of collagen and mixed skin proteins were calculated by the
precursor-product model. The increment of the enrichment in the
product, i.e., the difference of protein bound amino acid enrichment
between the first and the second skin biopsy (
EProtein), was divided by the average free tissue amino acid tracer-to-tracee ratio in the first and second skin biopsy
|
(3)
|
where EPrecursor(1) and EPrecursor(2)
are enrichments of the labeled amino acids in the precursor pool at the
time of the two sequential skin biopsies, and t is the time
interval between the two sequential biopsies. The results are presented
in percent per hour multiplying by the factors 60 (min/h) and 100.
Statistical analysis.
The comparisons between skin protein turnover parameters were carried
out using the two-tail paired t-test. Differences were considered significant at P < 0.05.
 |
RESULTS |
Blood phenylalanine and leucine enrichments were at steady state
during the last hour of isotope infusion, i.e., the enrichment values
at t = 300 were not significantly different from those at t = 360 (Fig. 3). The
tissue water was 69 ± 1% (by wt). Tissue free phenylalanine and
leucine enrichments were at steady state from t = 120 to 360 min. The values of the plateau enrichments of blood and tissue
phenylalanine and leucine are reported in Table
1. Blood and tissue free
phenylalanine and leucine enrichments decayed in a synchronous
fashion, following a single exponential curve, after the isotope
infusion was stopped (Fig. 3). The blood-to-free tissue enrichment
ratio was significantly higher for phenylalanine than for leucine
(1.65 ± 0.14 vs. 1.33 ± 0.04, P = 0.027),
indicating that some KIC was converted to leucine in the skin cells.

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Fig. 3.
Leucine and phenylalanine enrichments in the arterial
blood and in the skin tissue free pool. Tissue free phenylalanine and
leucine enrichments were at steady state from t = 120 to t = 360 min from the start of the isotope infusion.
Blood and tissue free phenylalanine and leucine enrichments decayed in
a synchronous fashion after the isotope infusion was stopped.
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Table 1.
Plateau enrichments of free phenylalanine and leucine in arterial blood
and skin tissue, skin free phenylalanine and leucine
concentrations, and protein bound phenylalanine and leucine
content in mixed skin proteins
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|
Tissue phenylalanine and leucine concentrations did not change over
time. The average values of tissue phenylalanine and leucine concentrations and the measured mixed skin protein bound phenylalanine and leucine contents are reported in Table 1. These values were used to
calculate the ratio of the intracellular free tracee content over the
protein bound tracee in the skin (Qs/T in Eq. 1).
Skin FBR, mixed skin protein FSR, and collagen FSR are reported in
Table 2. Skin FBR measured with
phenylalanine as tracer was similar to that measured with leucine. Skin
protein FSRs were calculated using both skin free amino acid enrichment
and blood free amino acid enrichment as precursor pool enrichment. The
collagen fraction separated from skin tissue was pure, as assessed by
SDS-PAGE and Western blot (Fig. 4). The
analysis of the amino acid content of the extracted collagen fraction
confirmed that the amino acid composition of the proteins contained in
the fraction was similar to that of collagenous proteins (data not
shown). Mixed skin FSR measured with phenylalanine was not different
from that measured with leucine. A power analysis indicated that, to be
able to detect a significant difference, with
= 0.05 and
= 0.8, between the FSRs measured with the two different
tracers, we would have had to study more than 300 animals. All the FSRs
were significantly lower when the blood enrichment was used as
precursor, compared with the value when the tissue enrichment was used.
Skin FBR measured with phenylalanine was not different from mixed skin
protein FSR measured with the same tracer, whereas it was slightly but
significantly higher (P < 0.05) than mixed skin
protein FSR measured with KIC/leucine. However, skin FBR measured with
leucine was not different from mixed skin protein and collagen FSRs
measured with the same tracer.

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Fig. 4.
SDS-PAGE and Western blot analysis of isolated skin
collagen fractions. The isolated collagen fractions were tested for
purity by SDS-PAGE analysis. Because the isolated fractions contained
any collagen type, we identified the collagen bands by Western blot
using a mixture of antibodies of anti-collagen type I and type III,
collagen type IV, and collagen type VII.
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 |
DISCUSSION |
Skin protein breakdown rate.
We applied the tracee release method (19), originally
developed for measurement of muscle protein breakdown, to measure skin
protein breakdown rate. Phenylalanine was the reference tracer used to
measure skin FBR, because it is an essential amino acid and, therefore,
its appearance in the tissue free pool is due solely to transport from
blood and release from proteolysis (Fig. 2). Also, the proportional
contribution of phenylalanine to keratin and to collagen is about the
same, so its appearance should reflect both dermal and epidermal
protein breakdown. An important assumption of the tracee release
method, essential for its correct application, is that the enrichment
of the pool in which unlabeled amino acids are released by protein
breakdown is measured (19). In the skin, the pool into
which the tracee is released is comprised of both the intracellular and
extracellular free amino acid pools, because proteolysis takes place
both intracellularly and extracellularly (18). Because we
measured free phenylalanine enrichment in the total tissue water, which
includes both intracellular and extracellular water, the assumption
that the enrichment of the pool in which protein breakdown releases
unlabeled amino acids is measured was satisfied. The FBR value measured
in this experiment is similar to the value obtained in the rabbit with
the ear arteriovenous model after conversion to the same units
(20). Specifically, Zhang et al. (20) found
that, in the normal rabbit, skin breakdown rate was ~21 mg of skin
protein · h
1 · ear
1.
Because the ear of the rabbit contained ~5.5 g of protein, the fractional breakdown rate was ~0.38%/h, which is similar to the 0.26%/h found in our experiment, especially when we consider that we
used two different species and different methodological approaches. From these results, we feel confident that the tracee release method is
valid when applied in the rat skin for measurement of mixed protein breakdown.
To assess the sensitivity of the FBR method, we used the phenylalanine
data to calculate the smallest difference that can be detected between
two treatments by use of a t-test with
= 0.05 and
= 0.8. The coefficient of variation of the FBR measurement with phenylalanine as tracer was 9.5% with n = 5. We
found that we can detect a 20% difference between two groups of five
rats each, a 17% difference with six rats in each group, or a 15.5% difference with seven rats per group. Compared with the arteriovenous model (20), which can detect a 22.5% difference with
n = 5, we found that both methods have similarly high sensitivities.
Precursor pool for protein synthesis.
In this study, not only did we measure skin protein breakdown, but we
also measured skin protein FSR to obtain a measure of skin protein
turnover and net balance. A potential problem with the measurement of
FSR in the skin is the measurement of the precursor pool enrichment.
The infusion of an amino acid tracer such as [2H5]phenylalanine produces a gradient of
tracer enrichment between blood and tissue, which is due to
proteolysis. In tissues where most of the free amino acid pools are
intracellular and proteolysis takes place only intracellularly, such as
muscle, the measurement of the free amino acid enrichment in a tissue
sample yields values close to those of the true precursor. In the skin,
however, the tracer is diluted by proteolysis in both the intracellular
and the extracellular space (18), and a significant
portion of the tissue free amino acids is extracellular. Thus it is
expected that the tracer enrichment in a skin sample that includes a
significant amount of extracellular amino acids will lead to an
overestimation of the true precursor. The measurement of the amino
acyl-tRNA enrichment could solve this problem (6), but the
large amount of tissue required (~500 mg) prevents a routine use of
this method in humans or in the present experiment, where large amounts
of skin were used for other analyses. We tried to circumvent the problem by infusing labeled KIC and measuring leucine tissue
enrichment, which should give a more accurate estimate of the precursor
pool for protein synthesis, because KIC is intracellularly converted to
leucine (14). This method has led to the successful
estimation of the enrichment of the precursor for muscle protein
synthesis (5). Although a study showed that leucine is
transaminated to KIC in human skin fibroblasts (15), it
was unknown whether this method would also be valid for the measurement
of skin protein synthesis in vivo.
We addressed this problem by using different methods. First, we
compared skin FSR and FBR calculated with the use of either tracer. In
the case of a significant contribution of skin cells to labeled leucine
production during labeled KIC infusion, both the FSR and the FBR should
yield a significantly lower value than values measured with labeled
phenylalanine. Although FBR calculated with leucine was 22% lower than
that calculated with phenylalanine, this difference was not
significant. Thus it does not appear that the conversion of KIC to
leucine in the rat skin is extensive. Second, we calculated mixed skin
protein FSR with either the tissue free enrichments or the blood
enrichments as precursor. If significant amounts of KIC were converted
to leucine in the skin, the difference between the FSR calculated with
either skin or blood leucine enrichment should be significantly lower
than the difference between the FSR calculated with skin or blood
phenylalanine enrichments. We found that the FSR values calculated with
blood leucine enrichment were 22% lower than those calculated with
skin leucine enrichment, whereas the difference in FSR using blood or
skin as precursor was significantly higher (39%) with the use of
phenylalanine. These data indicate that there is some advantage in the
use of KIC as tracer to measure skin protein FSR, although the
estimation of the precursor pool enrichment in the skin seems not to be
so accurate as it is in the muscle. Further studies are required for a
specific assessment of the accuracy of the KIC-leucine method in the skin.
Skin protein synthesis.
The potential limitations of the quantification of FSR notwithstanding,
the results of mixed skin protein FSR were lower, but not
significantly, than the FBR measured with the same tracer. Because
previous data in normal skin obtained with the arteriovenous balance
showed that skin protein synthesis and breakdown rates are similar in a
variety of circumstances (20), this indicates that we
overestimated the precursor pool enrichment with both tracers, thereby
underestimating the true FSR value by 28 and 35% with the use of
KIC/leucine and phenylalanine, respectively. Thus skin protein net
balance (calculated by subtraction of the FBR from the FSR) would be
underestimated because FBR is not underestimated.
Because collagen comprises ~90% of skin proteins (16),
we also purified skin collagen to evaluate the contribution of its synthesis rate to mixed skin protein FSR. We did not measure the synthesis rates of each different collagen type, because it is well
known that 95% of the skin collagens are comprised of types I and III,
which have very similar turnover rates (7, 13). Although
we could not use phenylalanine as tracer because of its low content in
collagen, we found that the collagen FSR measured with KIC/leucine was
not statistically different from the mixed skin FSR. This result was
not due to impurity of the extracted collagen, because we confirmed the
purity of each sample by amino acid composition analysis, SDS-PAGE, and
Western blot. The mixed skin protein FSR is the average FSR of soluble
(newly synthesized) collagens, insoluble (older) collagens, and
epidermal proteins. Although epidermal proteins have a higher turnover
rate compared with that of the collagens, due to the small size of
their pool they did not significantly influence the overall (mixed)
skin protein FSR. However, we cannot exclude the possibility
that the contribution of epidermal proteins to mixed skin protein FSR
may increase in specific pathological conditions. It is interesting to
note that the collagen and mixed protein FSR values obtained in this
study were not statistically different from the collagen FSR values
(0.076 ± 0.063%/h, mean ± SD) obtained in humans by El-Harake et al. (6) with a different methodological
approach, i.e., proline as tracer and prolyl-tRNA enrichment as
precursor. Although the lack of a significant difference between the
data from El-Harake et al. and the data from the present study might be
due to a lack of power, the absolute values of FSR are strikingly similar despite the different species studied and the different methodological approach used.
Our results indicate that collagen is the major contributor to mixed
skin protein FSR and, in turn, that mixed skin protein FSR is a good
indicator of skin collagen synthesis in normal skin. This is important,
because the isolation of collagen requires large amounts of skin
(150-1,000 mg according to the method used), which prevents the
routine measurement of skin collagen synthesis in humans. On the other
hand, mixed skin protein FSR, as well as FBR, requires <15-30 mg,
and these methods are therefore more easily applicable in human
studies. For example, a 4-mm punch biopsy of the skin of the back (~4
mm deep) could yield >50 mg of tissue, which is more than sufficient
for the measurement of FSR and FBR.
In conclusion, we found that the tracee release method is a reliable
means for measurement of skin protein breakdown. The small amount of
skin required to measure skin FBR makes this technique applicable in
humans. However, although FSR can be reasonably measured by use of free
tissue enrichment as precursor, it is probably not accurate enough to
be compared quantitatively with FBR to measure skin net balance. The
between-subject coefficient of variation for skin FSR was 36% with
KIC/leucine and 65% with phenylalanine in our study. The high
variability was not limited to the present study, because the
coefficient of variation was 83% also in the study by El-Harake et al.
(6). We do not have an explanation for this high
variability. One possibility is that skin is not homogeneous, so the
synthetic rates may differ in specific regions. Whereas skin for this
study was taken from the same general area, variations in FSR could
have reflected the actual differences in the synthetic activities of
the tissue samples. It is also possible that the high intersubject
variability might be due to the uncertainty of the precursor
enrichment. However, this explanation does not apply to the El-Harake
et al. study where the aminoacyl-tRNA enrichment was measured. The
other possibility is that skin FSR varies according to specific stimuli
that do not affect FBR. This could be supported by the observation
that, despite a high coefficient of variation of skin FSR, the
coefficient of variation of skin FBR in our study was 10% with
phenylalanine. Further studies are required to assess the validity of
these methods in humans and in pathological conditions such as burn
injuries and wound healing.
 |
ACKNOWLEDGEMENTS |
This study was supported by Shriners Grant No. 8490.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: E. Volpi, 815 Market St., Galveston, TX 77550 (E-mail:
evolpi{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.
Received 8 December 1999; accepted in final form 23 May 2000.
 |
REFERENCES |
1.
Baracos, VE,
Brun-Bellut J,
and
Marie M.
Tissue protein synthesis in lactating and dry goats.
Br J Nutr
66:
451-465,
1991[ISI][Medline].
2.
Breuille, D,
Arnal M,
Rambourdin F,
Bayle G,
Levieux D,
and
Obled C.
Sustained modifications of protein metabolism in various tissues in a rat model of long-lasting sepsis.
Clin Sci (Colch)
94:
413-423,
1998[ISI][Medline].
3.
Calder, AG,
Anderson SE,
Grant I,
McNurlan MA,
and
Garlick PJ.
The determination of low d5-phenylalanine enrichment (0.002-0.09 atom percent excess), after conversion to phenylethylamine, in relation to protein turnover studies by gas chromatography/electron ionization mass spectrometry.
Rapid Commun Mass Spectrom
6:
421-424,
1992[ISI][Medline].
4.
Charters, Y,
and
Grimble RF.
Effect of recombinant human tumour necrosis factor alpha on protein synthesis in liver, skeletal muscle and skin of rats.
Biochem J
258:
493-497,
1989[ISI][Medline].
5.
Chinkes, D,
Klein S,
Zhang XJ,
and
Wolfe RR.
Infusion of labeled KIC is more accurate than labeled leucine to determine human muscle protein synthesis.
Am J Physiol Endocrinol Metab
270:
E67-E71,
1996[Abstract/Free Full Text].
6.
El-Harake, WA,
Furman MA,
Cook B,
Nair KS,
Kukowski J,
and
Brodsky IG.
Measurement of dermal collagen synthesis rate in vivo in humans.
Am J Physiol Endocrinol Metab
274:
E586-E591,
1998[Abstract/Free Full Text].
7.
Miller, EJ,
and
Rhodes RK.
Preparation and characterization of the different types of collagen.
Methods Enzymol
82:
33-64,
1982[ISI][Medline].
8.
Nash, JE,
Rocha HJ,
Buchan V,
Calder GA,
Milne E,
Quirke JF,
and
Lobley GE.
The effect of acute and chronic administration of the
-agonist, cimaterol, on protein synthesis in ovine skin and muscle.
Br J Nutr
71:
501-513,
1994[ISI][Medline].
9.
Patterson, BW,
Nguyen T,
Pierre E,
Herndon DN,
and
Wolfe RR.
Urea and protein metabolism in burned children: effect of dietary protein intake.
Metabolism
46:
573-578,
1997[ISI][Medline].
10.
Patterson, BW,
Zhang XJ,
Chen Y,
Klein S,
and
Wolfe RR.
Measurement of very low stable isotope enrichments by gas chromatography/mass spectrometry: application to measurement of muscle protein synthesis.
Metabolism
46:
943-948,
1997[ISI][Medline].
11.
Preedy, VR,
McNurlan MA,
and
Garlick PJ.
Protein synthesis in skin and bone of the young rat.
Br J Nutr
49:
517-523,
1983[ISI][Medline].
12.
Robins, SP.
Metabolism of rabbit skin collagen. Differences in the apparent turnover rates of type-I- and type-III-collagen precursors determined by constant intravenous infusion of labelled amino acids.
Biochem J
181:
75-82,
1979[ISI][Medline].
13.
Rucklidge, GJ,
Milne G,
McGaw BA,
Milne E,
and
Robins SP.
Turnover rates of different collagen types measured by isotope ratio mass spectrometry.
Biochim Biophys Acta
1156:
57-61,
1992[ISI][Medline].
14.
Schwenk, WF,
Beaufrere B,
and
Haymond MW.
Use of reciprocal pool specific activities to model leucine metabolism in humans.
Am J Physiol Endocrinol Metab
249:
E646-E650,
1985[Abstract/Free Full Text].
15.
Wendel, U,
and
Langenbeck U.
Intracellular levels and metabolism of leucine and
-ketoisocaproate in normal and maple syrup urine disease fibroblasts.
Biochem Med
31:
294-302,
1984[ISI][Medline].
16.
Widdowson, EM,
and
Dickerson JWT
The effect of growth and function on the chemical composition of soft tissues.
Biochem J
77:
30-43,
1960[ISI].
17.
Wolfe, RR.
Appendix A: Laboratory Methods.
In: Radioactive and Stable Isotope Tracers in Biomedicine. Principle and Practice of Kinetic Analysis, edited by Wolfe RR. New York: Wiley-Liss, 1992, p. 417-438.
18.
Woolley, DE.
Mammalian collagenases.
In: Extracellular Matrix Biochemistry, edited by Piez KA,
and Reddi AH. New York: Elsevier, 1984, p. 119-157.
19.
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].
20.
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].
Am J Physiol Endocrinol Metab 279(4):E900-E906
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