Measurement of protein metabolism in epidermis and dermis
Xiao-Jun
Zhang1,2,
David L.
Chinkes1,2, and
Robert R.
Wolfe1,2,3
1 Metabolism Unit, Shriners Hospitals for Children;
and Departments of 2 Surgery and
3 Anesthesiology, University of Texas Medical Branch,
Galveston, Texas 77550
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ABSTRACT |
We found that, in the rabbit ear,
the dermal protein contains 75.5% of cutaneous phenylalanine and
97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and
2.1% of proline are in the epidermal protein. This finding led us to
develop two novel models that use phenylalanine and proline tracers and
the rabbit ear to quantify protein kinetics in the epidermis and
dermis. The four-pool model calculates the absolute rates of protein
kinetics and amino acid transport, and the two-pool model calculates
the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein
synthesis was 93.4 ± 37.6 mg · 100 g
1 · h
1, which was
10-fold greater than that of the dermal protein (9.3 ± 5.8 mg · 100 g
1 · h
1). These
synthetic rates were in agreement with those measured simultaneously by
the tracer incorporation method. Comparison of the four-pool and
two-pool models indicated that intracellular cycling of amino acids
accounted for 75 and 90% of protein kinetics in the dermis and
epidermis, respectively. We conclude that, in the skin, efficient
reutilization of amino acids from proteolysis for synthesis enables the
maintenance of protein mass in the postabsorptive state.
stable isotopes; gas chromatograph and mass spectrometer; arteriovenous balance; fractional synthesis rate; rabbits
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INTRODUCTION |
THE SKIN IS ONE OF
THE LARGEST TISSUES in the body. In normal circumstances, the
skin maintains a constant protein mass, whereas skeletal muscle may
have a significantly positive or negative protein balance
(28). The homeostasis of skin protein mass plays a crucial
role in preserving an intact barrier on the body surface. Once the
integrity of the skin barrier is destroyed, several problems ensue
immediately, such as loss of water and electrolytes, invasion of
microorganisms, and impairment of immune functions (11, 19, 20). Thus knowledge of skin protein metabolism is important in
understanding the maintenance and repair process of the skin barrier.
In our previous experiments (28-30), we investigated
protein metabolism in normal and scalded skin by use of a rabbit ear
model. This model has been the only approach to simultaneously quantify the in vivo rates of protein synthesis and breakdown as well as amino acid (AA) transport in the skin. However, this model is limited
by the consideration of the skin as a single pool. It is well known
that the epidermis and dermis have different structure, function, and
turnover rates (12, 17, 25). The dermis serves as a basis
for supporting and nourishing the epidermis, whereas the epidermis
exerts the barrier function of the skin. It is therefore important to
distinguish between the metabolic regulation of the epidermis and that
of the dermis under physiological and pathological conditions.
The goal of this study was to establish an approach for
quantitation of protein kinetics and AA transport in the epidermis and
dermis. This was accomplished by measurement of phenylalanine (Phe) and
proline (Pro) kinetics in rabbit ear skin. Because the epidermal and
dermal proteins contain different amounts of Phe and Pro, the measured
Phe and Pro kinetics are proportional to their contents in the
epidermis and dermis. Taking advantage of the different contents of Phe
and Pro in epidermal and dermal protein, we have developed models to
compute the respective protein and AA kinetics in the epidermis and
dermis. In addition, we modified a so-called heat treatment method
(14) for the separation of epidermis and dermis, which
allowed us to determine AA enrichment in the epidermal and dermal free
and protein-bound pools. To estimate the validity of the model-derived
values, we measured the fractional synthesis rate (FSR) of protein in
the epidermis and dermis by use of the tracer incorporation method.
Glossary Four-Pool Model
CAPhe |
Arterial Phe concentration
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CVPhe |
Venous Phe concentration
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CAPro |
Arterial Pro concentration
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CVPro |
Venous Pro concentration
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EAPhe |
Arterial Phe enrichment
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EVPhe |
Venous Phe enrichment
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EDPhe |
Dermal free Phe enrichment
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EEPhe |
Epidermal free Phe enrichment
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EAPro |
Arterial Pro enrichment
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EVPro |
Venous Pro enrichment
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EDPro |
Dermal free Pro enrichment
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EEPro |
Epidermal free Pro enrichment
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CDPhe |
Dermal protein-bound Phe content
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CEPhe |
Epidermal protein-bound Phe content
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CDPro |
Dermal protein-bound Pro content
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CEPro |
Epidermal protein-bound Pro content
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BF |
Blood flow rate in the ear skin
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FinPhe |
Arterial delivery of Phe to skin
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FoutPhe |
Venous exit of Phe from skin
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NBPhe |
Net balance of Phe across skin
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nbPhe |
Net balance of labeled Phe across skin
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FV,APhe |
Physiological shunting of Phe from artery to vein
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FD,APhe |
Transport of Phe from artery to dermis
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FV,DPhe |
Transport of Phe from dermis to vein
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FE,DPhe |
Transport of Phe from dermis to epidermis
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FD,EPhe |
Transport of Phe from epidermis to dermis
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FO,DPhe |
Irreversible loss of Phe from dermis (protein synthesis)
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FD,OPhe |
Production of Phe in dermis (protein breakdown)
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FO,EPhe |
Irreversible loss of Phe from epidermis (protein synthesis)
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FE,OPhe |
Production of Phe in epidermis (protein breakdown)
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FinPro |
Arterial delivery of Pro to skin
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FoutPro |
Venous exit of Pro from skin
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NBPro |
Net balance of Pro across skin
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nbPro |
Net balance of labeled Pro across skin
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FE,DPro |
Transport of Pro from dermis to epidermis
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FD,EPro |
Transport of Pro from epidermis to dermis
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FO,DPro |
Irreversible loss of Pro from dermis (protein synthesis)
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FO,EPro |
Irreversible loss of Pro from epidermis (protein synthesis)
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Two-Pool Model
NBEPhe |
Net balance of Phe across epidermis
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NBDPhe |
Net balance of Phe across dermis
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NBEPro |
Net balance of Pro across epidermis
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NBDPro |
Net balance of Pro across dermis
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RaEPhe |
Rate of appearance of Phe from epidermis to blood
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RaDPhe |
Rate of appearance of Phe from dermis to blood
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RdEPhe |
Rate of disappearance of Phe from blood to epidermis
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RdDPhe |
Rate of disappearance of Phe from blood to dermis
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METHODS |
Animals
We used male New Zealand White rabbits (Myrtle's Rabbitry,
Thompson Station, TN), 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]Phe
(99% enriched),
L-[ring-2H5]Phe (98%
enriched), L-[15N]Pro (98% enriched), and
L-[2H7]Pro (97-98%
enriched) were purchased from Cambridge Isotope Laboratories (Woburn,
MA). L-[ring-13C6]Phe
and L-[15N]Pro were used as tracers for
intravenous infusion.
L-[ring-2H5]Phe and
L-[2H7]Pro were used to prepare
internal standard solutions for calculation of Phe and Pro
concentrations in the blood and tissue pools.
Rationale and Equations of the Models
Four-compartment model.
The design of the four-pool model was based on the structure and
function of the epidermis and dermis. Whereas the dermis has abundant
blood circulation, there are no blood vessels in the epidermis; the
nutritional supply to the epidermis depends on diffusion from the
dermis (17). The keratinocytes in the epidermis are packed
together tightly, indicating that the epidermal free AAs can be
regarded as an intracellular pool. In contrast, the dermal protein is
synthesized in the fibroblasts and deposited as the extracellular
matrix (25); thus the dermal free AAs can be divided into
intracellular and extracellular pools. When blood moves through the
skin circulation, AAs exchange with the interstitial fluid (i.e.,
extracellular compartment) of the dermis, from which the AAs are either
transported into the fibroblasts (i.e., intracellular compartment) or
diffuse through the basement membrane to nourish the epidermal cells
(keratinocytes). The AAs in the dermal intracellular pool are used for
synthesis of dermal protein. The process of dermal protein breakdown
releases AAs into the dermal intracellular compartment, from which a
portion is reincorporated into dermal protein and the reminder is
released into the dermal extracellular compartment and the venous
blood. The proteolysis of epidermal protein releases AAs into the
epidermal intracellular compartment, from which they are either used
for synthesis or transported into the dermal extracellular compartment.
A schematic illustration of the cutaneous protein and AA kinetics is
shown in Fig. 1.

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Fig. 1.
Schematic illustration of protein and amino acid
(AA) kinetics in the epidermis and dermis. This illustration is based
on the physiological structure of skin. Arrows indicate directions of
AA movements. AAs enter the dermal extracellular pool from arterial
blood and leave this pool via venous blood. In the dermal extracellular
pool, some of AAs exchange with the dermal intracellular pool. Because
there are no blood vessels in the epidermis, the epidermal
intracellular pool exchanges AAs with the dermal extracellular pool.
The movement of AAs out of the epidermal and dermal intracellular pools
(other than into the dermal extracellular pool) indicates incorporation
into protein, and movement into the intracellular pools (other than
from the dermal extracellular pool) indicates protein breakdown.
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In our model, we combined the dermal intra- and extracellular
free AA pools into a dermal free AA pool. This simplification ignores
the transport of AAs between the dermal intra- and extracellular compartments but does not affect the measurement of protein synthesis and breakdown in the dermis. The advantage of this simplification is
that it eliminates the necessity of distinguishing between AAs in the
dermal intra- and extracellular compartments. Figure 2A is an illustration of our
four-pool model for skin protein kinetics and AA transport.

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Fig. 2.
A: a 4-compartment model for protein
kinetics and AA transport in the epidermis and dermis. The 4 pools are
arterial and venous pools (pool A and pool V),
dermal fluid pool (pool D), and epidermal intracellular
fluid pool (pool E). Arrows indicate the movement of AA from
1 pool to another. Fin, rate of AA inflow into the skin
from the artery; Fout, exit from the skin via vein;
FD,A, rate of AA delivery from pool A to
pool D; FV,D, from pool D to
pool V; FV,A, from pool A to
pool V; FO,D and FO,E, rate of
incorporation of AA into dermal and epidermal protein (i.e., rate of
protein synthesis), respectively. FD,O and
FE,O, rates of AA release from proteolysis into pool
D and pool E (i.e., rate of protein breakdown),
respectively. The interstitial free AA pool in dermis (Fig. 1) is
eliminated for simplification of model calculation. B:
2-pool model for apparent protein kinetics. The 2 pools are arterial
(pool A) and venous pool (pool V). Arrows
indicate movements of AA from 1 pool to another. FO,A, rate
of disposal (Rd) of AA via protein synthesis, which is the
sum of disposal in the dermal protein (RdD) and epidermal
protein (RdE). FV,O, rate of appearance
(Ra) of AA into the venous blood from protein breakdown,
which is the sum of appearance from dermal protein (RaD)
and from epidermal protein (RaE).
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Epidermis and dermis had to be separated to measure AA enrichment
and concentration in the free and protein-bound pools. This was
performed on the ventral skin of the rabbit ear. Anatomically, the skin
on the ventral ear has a flat connection between epidermis and dermis
(12). When a partial thickness thermal injury is created
on the ear, the formation of blisters appears only on the ventral site
of the ear skin (29). This observation led us to use the
heat treatment method (14) for separation of epidermis and
dermis. This method was originally used to separate epidermis from
dermis in newborn hairless rats by immersing the skin in 55°C water
for 30 s and immediately cooling with ice-cold water. We reduced
the heat exposure to 2 s to minimize proteolysis, which would have
decreased the isotopic enrichment of the skin fluid. We found that, by
submerging the ventral ear skin in 65°C water for 2 s
immediately followed by cooling in ice-cold water, the epidermal sheet
can be easily peeled off from the dermis. Histologic observation showed
that this separation procedure generated pure epidermis and dermis
(data not presented here).
Ideally, if one AA is contained only in the epidermis and another AA is
only in the dermal protein, we could use these two AAs to reflect
protein kinetics in the epidermal and dermal protein, respectively.
However, such AAs do not exist. As an alternative, we used Pro and Phe
tracers to achieve this goal. This was because we found that the dermal
Pro accounted for 97.9% of total skin protein-bound Pro and epidermal
Pro accounted for only 2.1%. In contrast, the dermal protein contains
75.5% of total skin protein-bound Phe, whereas the remaining 24.5% is
in the epidermis. Thus a Pro tracer reflects dermal protein kinetics.
The use of Phe tracer to reflect epidermal protein kinetics was based
on the consideration that the epidermal protein has a much faster
turnover rate than the dermal protein, so that the 24.5% of epidermal
Phe should account for a large majority of Phe kinetics in the skin.
The distribution difference of Pro and Phe in the epidermal and dermal protein allowed us to solve the kinetic parameters in Fig.
2A with Pro and Phe tracers.
A glossary of symbols is provided above, and assumptions and
derivations of equations are described in the APPENDIX. The
following are equations used to calculate the kinetic parameters in the four-pool model (Fig. 2A). Theoretically, both Phe and Pro
kinetics can be calculated from the four-pool model. Because Phe and
Pro kinetic data can be converted to each other according to their respective concentrations in the four pools, we selected the Phe data
to represent protein and AA kinetics, and the Pro data were included
only when they were necessary for model calculations. The use of Phe
kinetics to reflect both epidermal and dermal protein kinetics does not
conflict with the notion that Phe kinetics reflect mainly epidermal
protein kinetics in the whole skin. This is because, in whole skin, Phe
kinetics represents mainly epidermal protein kinetics, but in the
dermis Phe kinetics reflect exclusively dermal protein kinetics
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(1)
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(2)
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(3)
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(4)
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(5)
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(6)
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(7)
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(8)
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(9)
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(10)
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(11)
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(12)
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(13)
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(14)
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(15)
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(16)
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(17)
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In the equations above, Fin, Fout,
FV,A, FD,A, FV,D, FD,E,
FE,D, FO,D, FO,E, FD,O,
and FE,O are defined in the four-pool model (Fig.
2A) and further described in the Glossary. BF
stands for blood flow rate in the ear skin. NB and nb stand for net
balance of total (labeled + unlabeled) and labeled Phe or Pro,
respectively. CD and CE represent contents of
Phe or Pro in the dermal and epidermal protein. FO,D and
FO,E are the measures of protein synthesis, which are
expressed as the rates of Phe incorporation into dermal and epidermal
protein, respectively. FD,O and FE,O are the
measures of protein breakdown, which are expressed as the rates of Phe release from dermal and epidermal protein, respectively.
Two-compartment model.
The two-pool model calculates the apparent rate of disposal
(Rd) of AAs in the dermal or epidermal protein by use of
the blood-derived AAs. The rate of appearance (Ra) is the
rate of AA release, from dermal or epidermal protein breakdown, into
the venous blood. The protein kinetics measured by the two-pool model
do not include AAs that are released from breakdown and reused for
synthesis without ever appearing in the blood. In other words, the
kinetic data from the two-pool model underestimate the absolute rates of synthesis and breakdown by the amount of intracellular AA cycling.
The assumptions and derivations of the equations are presented in the
APPENDIX. The principle of the calculations was originally described by Galim et al. (6). Because we used two tracers to measure respective protein kinetics in the epidermis and dermis, new
equations are necessary to achieve the goal. The schematic illustration
of the two-pool model is presented in Fig. 2B, and the
formulas are described below. Phe and Pro kinetics in the epidermis and
dermis can be interconverted on the basis of their tissue concentration
difference. For clarity, we have presented only the Phe kinetics to
represent protein kinetics and used the Pro data only where necessary
for model calculations
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(18)
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(19)
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(20)
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(21)
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(22)
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(23)
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(24)
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(25)
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(26)
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(27)
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In Eqs. 18-27, Rd reflects
incorporation of arterial Phe or Pro into protein (i.e., apparent
synthesis); Ra reflects apparent breakdown, which releases
AAs into venous blood. BF stands for blood flow rate in the ear skin.
NB is the net balance of Phe or Pro in the skin. The subscripted small
capital D and E to the right of Ra,
Rd, and NB indicate dermis and epidermis, respectively, and
are distinguished from whole skin Ra, Rd, and NB.
Experimental Design
Five rabbits were studied in the postabsorptive state under
general anesthesia. The anesthetic and surgical procedures were described in detail in our previous publications (28, 30). In brief, after an overnight fast with free access to water, the rabbits were anesthetized with ketamine and xylazine. Catheters were
inserted into the right femoral artery and vein through a groin
incision. The arterial line was used for blood collection and
monitoring of blood pressure and heart rate; the venous line was for
infusion. A tracheal tube was placed via tracheotomy. The central ear
artery was dissected from the base of the left ear, and a flow probe
(1RB; Transonic Systems, Ithaca, NY) was placed on the artery to
measure blood flow rate on an ultrasonic small animal blood flowmeter
(model T106, Transonic Systems).
After collection of a blood sample and a skin specimen from the
incision on the groin for background measurements, priming doses of
L-[ring-13C6]Phe (6 µmol/kg) and L-[15N]Pro (8 µmol/kg) were
injected intravenously, which were followed immediately by a continuous
infusion of
L-[ring-13C6]Phe
(0.15 µmolkg
1min
1) and
L-[15N]Pro (0.2 µmolkg
1min
1). Blood samples were
collected from the arterial line at 60 and 120 min to check the
isotopic enrichment. Between 180 and 240 min, four simultaneous
arterial and ear-venous blood samples were collected every 15 min. The
blood flow rate in the ear was recorded at each arteriovenous (a-v)
sampling. At 240 min, a skin sample was taken from the ventral side of
the left ear. At the end of the experiment, the ear was cut off at the
skin fold between ear base and auricle to weigh the ear. The weight of
the ear was multiplied by 0.78 to get the weight of ear skin; the value
of 0.78 was derived from dissection of 10 ears in our previous
experiment (28). The measured blood flow rates in the ear
in milliliters per minute per ear were then converted to the unit of
milliliters per 100 grams per minute.
The blood samples were immediately transferred to tubes containing
sulfosalicylic acid and the internal standard solution and kept in an
ice-water bath until the end of the infusion study (30).
The skin samples were immediately frozen in liquid nitrogen and stored
at
80°C for later processing.
During the isotope infusion, mean arterial blood pressure, heart rate,
rectal temperature, and ear blood flow rate were monitored continuously
and were maintained relatively constant by adjusting the dose of
anesthetic, infusion rate of saline, and heating lamps. The surface
temperature of the left ear was maintained at 37°C using a heating
lamp. The vital signs and ear blood flow rate were recorded every 30 min.
Blood Preparation
Immediately after collection of blood samples, 250 µl of blood
were transferred to a tube containing 1 ml of 7.5% sulfosalicylic acid
and 75 µl of an internal standard solution (28, 30). The
internal standard solution contained 30 µmol/l of
L-[ring-2H5]Phe and 60 µmol/l of L-[2H7]Pro.
After deproteinization the supernatant was processed to make the
N-acetyl, n-propyl ester (NAP) derivatives for
measurement of Phe and Pro enrichment in the blood (26).
Separation of Epidermis and Dermis
Skin samples from the ventral side of the experimental ears were
cut into strips of ~0.5 × 5 cm2. Each time, one
strip of skin was submerged in 65°C saline for 2 s and then
immediately put in ice-cold saline for 2 s. The epidermal sheet
was peeled off from the dermis with a fine forceps at 4°C.
Skin Sample Processing
The samples of epidermis, dermis, and whole skin of ~30 mg
each were homogenized in 10% perchloric acid three times at 4°C. The
supernatant was pooled and evenly divided into two parts. One part was
processed to make the NAP derivatives for measurement of Pro
enrichment, and the other part was processed for
t-butyldimethylsilyl (TBDMS) derivatives for measurement of
Phe (5). The protein pellets were washed thoroughly to
remove free AAs and lipids (28). The samples were dried in
an oven at 80°C overnight and hydrolyzed in 6 N HCl at 110°C for
24 h. The hydrolysate was dried and processed for the NAP
derivatives (28). These samples were used for analysis of
AA enrichment in the protein-bound pools.
Contents of Phe and Pro in Skin Proteins
To determine the contents of Phe and Pro in epidermal and dermal
protein, we separated epidermis and dermis from the ventral side of the
ear skin with the modified heat treatment method. The samples of
epidermis and dermis were cut into small pieces and washed twice in
ice-cold distilled water and kept in ice-cold water overnight to
eliminate free AAs. After centrifugation, the supernatant was
discarded, and the tissue samples were washed in absolute alcohol and
ether to remove lipids and were then dried in an oven at 80°C. An
internal standard solution was added to the dry tissue at the ratio of
1 µl of the solution to 1 mg of dry tissue; the internal
standard solution contained
L-[ring-13C6]Phe at 9.21 µmol/ml
and L-[15N]Pro at 28.80 µmol/ml (for
epidermis) or at 96.61 µmol/ml (for dermis). The samples were
hydrolyzed in 6 N HCl at 110°C and processed for the NAP derivatives
(26). The dry tissue weight, amount of internal standard
solution added, and isotopic enrichment were used to calculate the
content of these two tracer AAs in the epidermal and dermal protein.
Measurement of Isotopic Enrichment
The isotopic enrichment in the blood and in the skin protein
hydrolysate for AA content was measured on a Hewlett-Packard 5985 gas
chromatograph-mass spectrometer (GC-MS) (Hewelett-Packard, Palo Alto,
CA) with chemical ionization. Ions were selectively monitored at
mass-to-charge (m/z) ratios of 250, 251, 255, and 256 for
Phe and at m/z ratios of 200, 201, and 207 for Pro. The isotopic enrichment in the skin supernatant prepared as the NAP derivatives was determined on a Hewlett-Packard 5980/5989B
GC-MS; ions were selectively monitored at m/z
ratios of 200, 201, and 207 for Pro. The isotopic enrichment in the
skin supernatant prepared as the TBDMS derivatives was determined on a
Fison MD 800 GC-MS (Beverly, MA); ions were selectively monitored at
m/z ratios of 234, 235, 239, and 240 for Phe.
L-[ring-13C6]Phe and
L-[15N]Pro enrichment in the skin protein
hydrolysate was measured on a gas chromatograph-combustion-isotope ratio mass spectrometer (Finnigan). The measured
13CO2 enrichment was converted to Phe
enrichment by multiplying 14/6 to account for the dilution of
the labeled carbons with the carbons in other positions of the
derivatized Phe. The measured 15N enrichment represented
the Pro enrichment, because each molecule of
L-[15N]Pro has only one nitrogen and that
nitrogen was labeled. The enrichment of Phe and Pro was used to
calculate the FSR of skin proteins by the tracer incorporation method.
The isotopic enrichment was expressed as mole percent excess (MPE)
after correction for the contribution of the abundance of isotopomers
of lower weight to the apparent enrichment of isotopomers with larger
weight and also a skew correction factor to calculate L-[ring-13C6]Phe enrichment
(21).
Measurement of Hydroxyproline Concentration
Some of the Pro incorporated into dermal collagen is converted
to hydroxyproline (HPro) via posttranslational modification on the
collagen peptide (1, 9). Thus the Pro a-v balance should
include the release of HPro, and the content of Pro in the dermal
protein-bound pool should include HPro. To this end, we measured HPro
in the a-v blood and in the dermal protein hydrolysate. This was
completed by measuring the peak sizes of Pro and HPro in the NAP
derivative on a 5890A gas chromatograph (Hewelett-Packard) equipped
with an sp-2330 column (Supelco, Bellefonte, PA). The oven temperature
was set at 160-400°C at 8.0°C/min with an injection temperature of 250°C. The measured ratio of Pro peak size to HPro peak size from the gas chromatograph was used to calculate the concentration of HPro. Because the concentration of Pro was measurable using the internal standard method, the concentration of HPro equals
Pro concentration divided by the peak size ratio of Pro to HPro. The
net release of HPro is equal to venous HPro concentration minus
arterial HPro concentration. The value of HPro net release was added to
the venous Pro concentration to calculate the net balance of Pro across
the skin. The Pro content in the dermal protein-bound pool also
included HPro.
Calculations of FSR
The FSR of skin proteins was calculated for the tracer
incorporation method (26). FSR = [(Et1
Et0)/Ep
(t1
t0)] × (t1
t0), where (Et1
Et0) is the
increment of enrichment in the protein-bound pool between two sampling
times, and Ep (t1
t0) is the average precursor enrichment between
time 0 and time 1.
The FSR of skin proteins was also converted from the rate of synthesis
measured from the four-pool model. We used FSRC to distinguish it from FSR measured directly by the tracer incorporation method. FSRC = (rate of synthesis in µmol
Phe · 100 g
1 · h
1)/(µmol
protein-bound Phe in 100 g of tissue).
Statistics
Data are expressed as means ± SD. Student's
t-test was used to test the difference between two
variables. A P value < 0.05 was considered
statistically significant.
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RESULTS |
We measured Phe and Pro contents in epidermal and dermal protein.
Six pieces of skin were taken from the ventral side of the ear. After
separation using the modified heat treatment method, the wet epidermis
and dermis accounted for 15 ± 2 and 85 ± 2% of the wet
skin. After a thorough washing and complete drying, the dry epidermis
and dermis were 2.64 ± 0.53 and 22.86 ± 1.09% of wet skin
weight, respectively. By use of the internal standard method, 1 g
of dry epidermal protein contained 242 ± 25 µmol of Phe and
262 ± 67 µmol of Pro, 1 g of dermal protein contained 86 ± 5 µmol of Phe and 1,094 ± 15 µmol of Pro. In the
dermal protein hydrolysate samples, the ratio of peak area of Pro to
HPro was 3.9 ± 0.4, meaning that the content of HPro was 283 ± 30 µmol/g. The total content of Pro and HPro was then 1,379 ± 35 µmol/g. Thus the dermal Pro (including Hpro) accounted for
97.9% of total skin protein-bound Pro, and epidermal Pro accounted for
only 2.1% of the total. In contrast, the dermal protein contained
75.5% of total skin protein-bound Phe, and the remaining 24.5% was in the epidermis.
The mean arterial blood pressure, heart rate, and rectal temperature
were relatively constant in the rabbits (data not presented here).
During the 180-240 min of the isotope infusion, isotopic equilibrium was achieved for both Phe and Pro. The recorded blood flow
rate in the ear skin was 8.5 ± 0.5 ml · 100 g
1 · min
1. The isotopic enrichment
and concentration values used to calculate the parameters in the
four-pool model are presented in Table 1.
The HPro concentrations in the arterial and ear-venous blood were
0.0110 ± 0.0020 and 0.0126 ± 0.0032 µmol/ml
(P < 0.01 by paired t-test,
n = 18), resulting in a net increase of 0.0016 µmol/ml in the venous blood. Thus the Pro concentration in the venous
blood was increased by 0.0016 µmol/ml to account for net release of HPro.
The calculated protein kinetics and Phe transport in the epidermis and
dermis according to the four-pool model are presented in Fig.
3. Whereas the protein mass of epidermis
and dermis was essentially maintained in these rabbits, the epidermis
had a much faster turnover rate than the dermis. Expressed as Phe
kinetics, the absolute rate of Phe incorporation into epidermal protein was 22.6 ± 9.1 µmol · 100 g
1 · h
1, which was 28-fold greater
than that of the dermal protein (0.8 ± 0.5 µmol · 100 g
1 · h
1). Because 1 g of dry
epidermal protein contains 242 µmol of Phe and 1 g of dry dermal
protein contains only 86 µmol of Phe, the protein synthetic rates
converted from the Phe incorporation rates were 93 and 9.3 mg · 100 g
1 · h
1 for
epidermis and dermis, respectively. Thus the epidermal protein had a
fast synthetic rate that was 10-fold greater than the dermal protein.

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|
Fig. 3.
Measured rates of Phe kinetics in the 4 compartments in
µmol · 100 g wet
skin 1 · h 1. There was no
significant positive or negative balance in pool D and
pool E, indicating maintenance of protein mass in the
epidermis and dermis. Rate of epidermal protein turnover was much
faster than that of dermal protein. Rates of AA transport between
pool E and pool D were comparable with those of
transport from pool A to pool D and
from pool D to pool V, indicating active
movements of AAs between epidermis and dermis.
|
|
The rates of protein synthesis and breakdown in the epidermis and
dermis calculated from the two-pool model were lower than the
corresponding values calculated from the four-pool model. This was
because the data from the two-pool model did not include the rates of
intracellular cycling. Table 2 presents
the rates of protein synthesis, breakdown, net balance, and
intracellular cycling calculated from the four-pool and two-pool models
and expressed as Phe kinetics. The rate of cycling was the difference between the rates of synthesis and breakdown calculated by the four-pool and two-pool models.
The enrichment data of Phe and Pro in the skin free and protein-bound
pools and the FSR values of epidermal, dermal, and whole skin protein
calculated from the tracer incorporation method are presented in Table
3. The enrichment values of Phe and Pro
in the free pool in epidermis were consistently much lower than the corresponding values in the free pool in dermis, and the values of
whole skin were intermediate. The epidermal FSR calculated by the
tracer incorporation method was greater than that of dermis when both
Phe and Pro tracers were used, which was in agreement with the
synthesis rates derived from the four-pool model. Because the rates of
protein synthesis in the epidermis and dermis using the four-pool model
were calculated from both Phe and Pro tracers, the FSR values measured
from the Phe and Pro tracers with the tracer incorporation method were
averaged. The average FSR values from the tracer incorporation method
were 103 ± 22 and 1.5 ± 0.5%/day for epidermal and dermal
protein, respectively. The rates of protein synthesis in epidermis and
dermis in Fig. 3 were converted to FSRC values according to
the determination that there are 639 µmol of epidermal Phe and 1,966 µmol of dermal Phe in 100 g of wet skin. The FSRC
values converted from the four-pool model data were 85 ± 34%/day
for epidermis and 1.0 ± 0.7%/day for dermis, which were
comparable with the averaged FSR values from the tracer incorporation
method (Fig. 4).

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|
Fig. 4.
Fractional synthesis rates (FSR) of epidermal and dermal
protein. Converted FSR (FSRC) values derived from the
4-pool model were comparable with the corresponding FSR values measured
by the tracer incorporation method. Model-derived FSRC
values were calculated from Phe and Pro tracers. FSR values from the
tracer incorporation method were averages of FSR values from Phe and
Pro tracers to be comparable with the model-derived FSRC
values.
|
|
 |
DISCUSSION |
The present experiment successfully measured protein and AA
kinetics in the epidermis and dermis. Epidermal protein was shown to
have a fast turnover rate, which was 10-fold that of dermal protein. In
the postabsorptive state, AA recycling provided over 75 and 90% of AAs
for protein synthesis in the dermis and epidermis, respectively. The
efficiency in reutilization of AAs from proteolysis for synthesis
likely plays an important role in the maintenance of skin homeostasis.
The rapid AA transport between epidermis and dermis, being comparable
with the rate of AA transport between blood and dermis, ensured
sufficient nutritional supply to the avascular epidermis.
The validity of the four-pool model for measurement of protein kinetics
in the epidermis and dermis is supported not only by the FSR values
measured from the tracer incorporation method (Fig. 4) but also by
published data. Using the flooding dose method, Harmon and Park
(7) reported an FSR of epidermal protein of 61%/day in
hairless mice. Their results were subject to underestimation, because
the epidermal samples that they harvested with a dermatome contained
~20% of dermis. Thus the epidermal FSRC of 85%/day that we obtained from the four-pool model is comparable with their value,
both showing very fast turnover rates. For dermal collagen synthesis,
El-Harake et al. (4) reported that the immature, detergent-soluble collagen FSR in human skin was 0.038 ± 0.030%/h when skin free Pro enrichment was used as the precursor. Our
model-derived dermal protein FSRC value was 1.0 ± 0.7%/day (0.042 ± 0.029/h), which is slightly greater than the
human dermal collagen value but smaller than the collagen FSR
(0.17%/h) in rats (15, 24). These small differences are
probably due to differences in species and experimental conditions. The
rate of protein turnover in rabbits appears to be between the human and
rat values. For example, the FSR of muscle protein in rabbits was
0.079 ± 0.095%/h (28), which is greater than the
human value of 0.055%/h (averaged from 80 human muscle measurements,
unpublished data from this laboratory) and smaller than the
value of 0.20%/h in rats (16). In the present experiment,
the four-pool model measured the dermal protein (including noncollagen
components) in anesthetized rabbits with stress response, which differs
from dermal collagen measured in conscious patients (4) or
rats (15, 24). Thus the dermal protein kinetic data measured by the four-pool model are considered reasonable. The comparisons of the synthesis rates between the four-pool and two-pool models indicate that the intracellular recycling of AAs in the dermis
accounted for 75% of protein synthesis. This is also consistent with
the finding that, in fibroblasts, ~90% of Pro for collagen synthesis
is derived from collagen degradation (see Ref. 13).
In comparison with the four-pool model, the two-pool model measures the
rate of protein synthesis by using the blood-derived (as opposed to the
proteolysis-derived) AAs for protein synthesis and the rate of
releasing AAs from proteolysis into venous blood (as opposed to reuse
for protein). If the end points of an experiment are the efficiency of
using blood-derived AAs for synthesis and the net protein balance in
the skin, the two-pool model is sufficient. This is because only an
increased incorporation of blood-derived AAs into protein could lead to
an anabolic response if the rate of AA release from proteolysis to the
venous blood is not proportionally increased. Any increase in AA
recycling will increase the absolute rate of protein turnover but will
not improve the net balance, since 100% reutilization would yield a
zero balance.
The finding that Phe and Pro have different distributions in epidermal
and dermal protein can be a useful tool in estimating metabolic
contributions from epidermal and dermal proteins. Because 97.9% of
skin Pro is in the dermal protein, the whole skin FSR value measured
from Pro tracer mainly represents dermal protein FSR. In contrast,
75.5% of skin Phe is in the dermis; the whole skin FSR value measured
from Phe tracer represents dermal protein to a lesser extent than does
the Pro tracer. As the rates of Phe incorporation into epidermal and
dermal protein (Fig. 3) and the contents of Phe and Pro in the
epidermal and dermal protein are known, the rate of Phe incorporation
into skin proteins could be calculated as (22.6 × 24.5%) + (0.8 × 75.5%) = 6.141 (µmol · 100 g
1 · h
1). This means that
90% of the Phe tracer was incorporated into epidermal protein and only
10% was incorporated into dermal protein. In contrast, the rate of Pro
incorporation into skin protein was (22.6 × 2.1%) + (0.8 × 97.9%) = 1.2578 (µmol · 100 g
1 · h
1). Thus 38% of Pro tracer
was incorporated into epidermal protein, and 62% was incorporated into
dermal protein. Thus, when the Phe tracer was used to measure whole
skin FSR, the value largely reflected epidermal protein synthesis.
Because the rate of Phe incorporation into epidermal protein was found
to be 28-fold greater than that of dermis (Fig. 3), the Phe tracer
generated a high value of whole skin FSR. In contrast, when the Pro
tracer was used for skin FSR, a mixture of epidermal protein synthesis
(38%) and dermal protein synthesis (62%) was reflected. Consequently,
the FSR value was much lower when the Pro tracer was used. When both
Phe and Pro tracers are used, the measured whole skin FSR values enable
us to estimate the respective FSR values in the epidermal and dermal protein. Taking the rabbit ear skin as an example (see Table 3), the
whole skin FSR values measured from the Phe and Pro tracers were 8.1 and 1.6%/day, respectively. If we use EFSR and
DFSR to represent epidermal and dermal protein FSR and
TPhe and TPro to represent total skin Phe and
Pro, we will have the following two equations: (24.5% × TPhe × EFSR) + (75.5% × TPhe × DFSR) = TPhe × 8.1 (%/day); and (2.1% × TPro × EFSR) + (97.9% × TPro × DFSR) = 1.6 (%/day). In these two equations, 24.5 and
75.5% are the percentage contents of Phe in the epidermal and dermal
protein, and 2.1 and 97.9% are the percentage contents of Pro in the
epidermal and dermal protein. TPhe and TPro can
be canceled out from the equations. Solving these two equations, we get
EFSR = 30%/day and DFSR = 1%/day.
Whereas these estimated FSR values are lower than the corresponding
average FSR values (epidermis 103%/day; dermis 1.5%/day) that were
directly measured from the tracer incorporation method, the calculation
above indicates that the whole skin FSR values measured from both Phe
and Pro tracers provide useful information with regard to FSR in the
epidermis and dermis. Even if the percentage contents of Phe and Pro in
the whole skin are not known, the changes of whole skin FSR measured
from Phe and Pro tracers allow us to estimate whether the experimental
perturbation primarily affects epidermal or dermal protein synthesis or
both. This provides a reasonable approach when it is not feasible to
distinguish epidermal and dermal synthesis directly. This could be
potentially useful for human studies in which only a very small biopsy
is available.
The measurement of skin protein metabolism has been hindered by several
major limitations. One is the uncertainty of the true precursor (i.e.,
aminoacyl-tRNA) for skin protein synthesis. To our knowledge, the skin
aminoacyl-tRNA enrichment has been measured only by El-Harake et al.
(4). They reported that, in human skin, the prolyl-tRNA
enrichment was ~50% of the free Pro enrichment. However, they did
not distinguish between the prolyl-tRNA enrichments in dermis and
epidermis. Cutaneous collagen and keratin are synthesized in
fibroblasts and keratinocytes, respectively. Thus it is necessary to
obtain their respective aminoacyl-tRNA enrichments for calculations of
dermal collagen and epidermal keratin synthesis. This issue remains
unresolved. In most published experiments, the skin free enrichment has
been used as a surrogate of precursor for skin protein synthesis. This
may have underestimated the skin protein turnover rate. Nevertheless,
as long as the relationship between skin fluid and tRNA enrichment
remains constant, this approach still gives valuable information. When
two AA tracers are used for skin protein measurement, as in the present
experiment, an additional issue is that, if they have a different
relationship between tissue fluid and tRNA enrichment, the use of
tissue fluid as precursor enrichment could result in inconsistent FSR
values. It is likely that possible compartmentalization of Pro pools
(18) explains why the FSR values in epidermal and dermal
protein measured from Phe and Pro tracers were different (see Table 3),
although both showed that epidermal FSR was much greater than dermal
FSR. In the present experiment, we also measured skin protein FSR from a leucine tracer. The average FSR values were 72%/day for epidermis and 2.9%/day for dermis, which were close to the values of 55%/day for epidermis and 2.6%/day for dermis measured by the Phe tracer. Another major limitation is that the skin is a highly heterogeneous tissue. In this experiment, we assumed that the skin is composed of
epidermal and dermal proteins. This assumption is still a
simplification of the skin structure, because the dermis is composed of
different types of collagen and elastin and epidermis is composed of a
large family of keratin polypeptides (25). Thus our models
and the tracer incorporation method all measure protein kinetics in
mixed dermal and epidermal proteins.
In our a-v models, we have assumed that neither Phe nor Pro is
synthesized or degraded in the skin. This is well established to be
true in the case of Phe. Pro, on the other hand, is traditionally considered to be a nonessential AA because it can be synthesized from
ornithine and glutamate. However, our data showed that the rate of net
Pro balance in the skin was consistent with that of Phe: neither was
significantly different from zero (Table 1). The only way that this
result could be obtained if Pro was synthesized and/or metabolized in
skin is if the rate of biosynthesis equaled that of degradation. It is
more likely that Pro is neither produced nor metabolized in skin. The
metabolic pathways of Pro are closely related to
1-pyrroline-5-carboxylate (P5C), the obligate
intermediate in the interconversions of Pro, ornithine, and glutamate
(1-3, 8-10, 22, 23, 27). The degradation of Pro
to P5C is catalyzed by the mitochondrial enzyme Pro oxidase. This
enzyme is localized in the liver, kidney, heart, lung, and small
intestine but is not detectable in muscle and adipose tissue (8,
27) or in fibroblasts (22). These findings support
the notion that Pro is not degraded in the skin, meaning that the
possibility of equal rates of Pro biosynthesis and degradation can be
excluded. Furthermore, biosynthesis of Pro from its immediate precursor
P5C is catalyzed by the cytosolic enzyme P5C reductase, with either
glutamate or ornithine as precursor. Although this enzyme may reside in
almost all cells (8), the biosynthesis of Pro in the skin
has been shown to constitute a minor contribution to collagen synthesis (2, 3, 13). In particular, in fibroblasts, P5C reductase is sensitive to feedback inhibition, with 50% inhibition at a Pro
concentration of 200 nmol/ml (23). In the ear skin of
rabbits, the concentration of free Pro was 426 ± 75 nmol/ml
(350-504 nmol/ml). Therefore, even if P5C reductase exists in the
skin, it would have been inhibited by the high Pro concentration. To
assess the extent of Pro synthesis in the skin, we infused
[2H3]glutamate into three rabbits. The
arterial glutamate enrichment plateau was 2.5-3.5%, but the
enrichment of Pro was not detectable in the arterial blood (reflection
of whole body Pro biosynthesis) or ear-venous blood (reflection of skin
Pro biosynthesis) (unpublished data in this laboratory). The
above discussion supports the notion that Pro biosynthesis constitutes
no more than a minor contribution to dermal collagen synthesis. Thus,
in this animal model, it is acceptable to use a Pro tracer in
conjunction with Phe tracer to quantify protein kinetics in the skin.
We conclude that our newly described models provide reasonable values
with respect to protein and AA kinetics in the epidermis and dermis.
The results indicate that the skin protein maintains a balance between
synthesis and breakdown in the postabsorptive state, thereby
maintaining skin barrier integrity. Epidermal protein turns over
rapidly, which is likely essential for the repair process. There is
also rapid AA transport between epidermis and dermis, which suggests
that a healthy dermal base is essential for normal protein metabolism
in the epidermis. Our new models of skin protein metabolism may be used
to study skin protein metabolism in physiological and pathological
conditions. Furthermore, our results indicate that it is possible to
distinguish between dermal and epidermal protein synthesis even when it
is not feasible to physically separate the tissues for direct analysis.
 |
APPENDIX |
Four-Compartment Model
Assumptions.
To solve the parameters in the four-pool model (Fig. 2), we assume that
1) the values of protein synthesis and breakdown in the
dermis and epidermis measured from the Pro and Phe tracers are
proportional to the contents of Pro and Phe in the dermal and epidermal
protein; 2) both dermis and epidermis are composed of a
single free AA pool and a protein-bound AA pool, and free AAs in the
dermis and epidermis are precursors for protein synthesis; 3) there are no de novo synthesis and degradation of Phe and
Pro in the skin, so that their appearance and disappearance in the dermis and epidermis represent protein breakdown and synthesis, respectively; and 4) there is no label recycle from the
protein-bound pool to the free pool in either epidermis or dermis.
Calculation of Phe transport between pools.
Because we used both Pro and Phe tracers, the protein and AA kinetics
could be expressed by either Pro or Phe. To simplify the calculation,
we used Phe data to represent protein and AA kinetics; the Pro data
were included only when they were necessary to solve the parameters of
Phe kinetics
|
(A1)
|
|
(A2)
|
|
(A3)
|
|
(A4)
|
|
(A5)
|
|
(A6)
|
|
(A7)
|
|
(A8)
|
|
(A9)
|
Calculation of protein synthesis.
We can calculate net tracer uptake across the tissue bed by multiplying
the a-v tracer concentration difference by the tissue blood flow. We
will denote net Phe tracer uptake across the tissue bed as nbPhe and
net Pro tracer uptake across the tissue bed as nbPro. On the basis of
assumption 3, the only pathways of irreversible loss of Phe
and Pro across skin are incorporation into bound dermis and bound
epidermis, i.e.
|
(A10)
|
|
(A11)
|
where FO,DPhe and FO,DPro are the rates
of incorporation of Phe and Pro into dermis bound protein,
FO,EPhe and FO,EPro are the rates of
incorporation of Phe and Pro into epidermis bound protein,
EDPhe and EDPro are the precursor enrichments
of dermis bound Phe and Pro, and EEPhe and
EEPro are the precursor enrichments of epidermis bound Phe
and Pro (assumption 2).
According to assumption 1, the incorporation of Phe
and Pro into protein occurs in proportion to their respective contents in the protein, i.e.
and
where CDPhe and CDPro are the dermis
bound contents of Phe and Pro and CEPhe and
CEPro are the epidermis bound contents of Phe and Pro. If
the four equations above are solved for the four unknowns
(FO,DPhe, FO,DPro, FO,EPhe, and
FO,EPro), then one obtains
|
(A12)
|
|
(A13)
|
Calculation of protein breakdown.
We can calculate net tracee uptake across the tissue bed by multiplying
the a-v tracee concentration difference by the tissue blood flow rate.
We will denote net Phe uptake across the tissue bed as NBPhe and net
Pro uptake across the tissue bed as NBPro. According to
assumption 3, the only pathways of production of Phe and Pro
across skin are release from bound dermis and bound epidermis, i.e.
and
where FD,OPhe and FD,OPro are the rates
of release of Phe and Pro from the dermis protein-bound pool and
FE,OPhe and FE,OPro are the rates of release of
Phe and Pro from the epidermis protein-bound pool.
According to assumption 1, the release of Phe and Pro
from protein breakdown occurs in proportion to their respective
contents in protein, i.e., FD,OPhe/CDPhe = FD,OPro/CDPro, and
FE,OPhe/CEPhe = FE,OPro/CEPro. If the four equations above are
solved for the four unknowns (FD,OPhe, FD,OPro,
FE,OPhe, and FE,OPro), then one obtains
|
(A14)
|
|
(A15)
|
|
(A16)
|
|
(A17)
|
In Eqs. A14-A17, CEPhe,
CDPhe, CEPro, and CDPro are
constant values, and the values of isotopic enrichment can be measured
after separation of epidermis and dermis. Thus all the kinetic values in Fig. 2 can be solved.
Two-Compartment Model
Assumptions.
The assumptions of the two-pool model are that 1) the Fick
Principle holds; 2) the release of Phe and Pro from dermis
and epidermis is proportional to the relative amount of Phe and Pro in
dermal and epidermal protein; 3) there is no label recycle from the protein-bound pool to the free pool in either epidermis or
dermis. On the basis of assumption 1, the total release of Phe or Pro from skin is equal to the sum of release from dermis and
epidermis. On the basis of assumption 2, the rate of release of Phe from dermis divided by the rate of release of Pro from dermis is
equal to the concentration of Phe in dermal protein divided by the
concentration of Pro in dermal protein, and the rate of release of Phe
from epidermis divided by the rate of release of Pro from epidermis is
equal to the concentration of Phe in epidermal protein divided by the
concentration of Pro in epidermal protein. The total rates of release
of Phe and Pro from skin are computed using the traditional two-pool
model equations. The concentrations of Phe and Pro in dermis and
epidermis are constant values. To calculate the rate of release of Phe
from dermal protein breakdown, four equations are required. There are
four equations and four unknowns, so the unknowns can be determined.
The incorporation of blood-derived Phe and Pro into epidermal and
dermal protein and the net balance of Phe and Pro across epidermis and
dermis can be calculated using the same assumptions as above. The
formulas are described below.
Calculations.
The calculation of FinPhe, FoutPhe, NBPhe,
FinPro, FoutPro, and NBPro are described in
Eqs. A1-A6 for the four-pool model
|
(A18)
|
|
(A19)
|
|
(A20)
|
|
(A21)
|
|
(A22)
|
|
(A23)
|
|
(A24)
|
|
(A25)
|
|
(A26)
|
|
(A27)
|
 |
ACKNOWLEDGEMENTS |
We are grateful to Yunxia Lin, Guy Jones, and Zhiping Dong for
technical assistance. We also thank the staff at the Electron Microscope Laboratory of Shriners Hospitals for Children for
preparation of the histological slides of epidermis and dermis
separated by the modified heat treatment method, and the staff at the
Animal Resource Center of The University of Texas Medical Branch for professional care of experimental animals.
 |
FOOTNOTES |
This work was sponsored by Grants 8630 and 8490 from the Shriners Hospital.
Address for reprint requests and other correspondence:
R. R. Wolfe, Shriners Hospitals for Children, 815 Market
St., Galveston, TX 77550-1220 (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.
First published February 25, 2003;10.1152/ajpendo.00460.2002
Received 24 October 2002; accepted in final form 21 February 2003.
 |
REFERENCES |
1.
Adams, E,
and
Frank L.
Metabolism of proline and the hydroxyprolines.
Annu Rev Biochem
49:
1005-1061,
1980[ISI][Medline].
2.
Ball, RO,
Atkinson JL,
and
Bayley HS.
Proline as an essential amino acid for the young pig.
Br J Nutr
55:
659-668,
1986[ISI][Medline].
3.
Berthold, HK,
Reeds PJ,
and
Klein PD.
Isotopic evidence for the differential regulation of arginine and proline synthesis in man.
Metabolism
44:
466-473,
1995[ISI][Medline].
4.
El-Harake, WA,
Furman MA,
Cook B,
Nair KS,
Kukowski J,
and
Brodsky IG.
Measurement of dermal collagen synthesis rate in vivo in human.
Am J Physiol Endocrinol Metab
274:
E586-E591,
1998[Abstract/Free Full Text].
5.
Ferrando, AA,
Tipton KD,
Bamman MM,
and
Wolfe RR.
Resistance exercise maintains skeletal muscle protein synthesis during bed rest.
J Appl Physiol
82:
807-810,
1997[Abstract/Free Full Text].
6.
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].
7.
Harmon, CS,
and
Park JH.
Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.
Biochem J
247:
525-530,
1987[ISI][Medline].
8.
Herzfeld, A,
Mezl VA,
and
Knox WE.
Enzymes metabolizing
1-pyrroline-5-carboxylate in rat tissues.
Biochem J
166:
95-103,
1977[ISI][Medline].
9.
Jaksic, T,
Wagner DA,
Burke JF,
and
Young VR.
Plasma proline kinetics and the regulation of proline synthesis in man.
Metabolism
36:
1040-1046,
1987[ISI][Medline].
10.
Jaksic, T,
Wagner DA,
Burke JF,
and
Young VR.
Proline metabolism in adult male burned patients and healthy control subjects.
Am J Clin Nutr
54:
408-413,
1991[Abstract].
11.
Johnson, C.
Pathological manifestations of burn injury.
In: Burn Care and Rehabilitation: Principles and Practice, edited by Richard RL,
and Staley MJ.. Philadelphia, PA: Davis, 1994, p. 29-33.
12.
Joseph, J,
and
Towsend EJ.
The healing of defects in immobile skin in rabbits.
Br J Surg
48:
557-564,
1961[ISI].
13.
Karna, E,
Miltyk W,
Wolczynski S,
and
Palka JA.
The potential mechanism for glutamine-induced collagen biosynthesis in cultured human skin fibroblasts.
Comp Biochem Physiol B
130:
23-32,
2001[ISI][Medline].
14.
Marrs, JM,
and
Voorhees JJ.
A method for bioassay of an epidermal chalone-like inhibitor.
J Invest Dermatol
56:
174-181,
1971[ISI][Medline].
15.
Molnar, JA,
Alpert N,
Burke JF,
and
Young VR.
Synthesis and degradation rates of collagen in vivo in whole skin of rats, studied with 18O labelling.
Biochem J
240:
431-435,
1986[ISI][Medline].
16.
Obled, C,
and
Arnal M.
Contribution of skin to whole-body protein synthesis in rats at different stages of maturity.
J Nutr
122:
2167-2173,
1992[ISI][Medline].
17.
Odland, GF.
Structure of the skin.
In: Physiology, Biochemistry, and Molecular biology of The Skin (2nd ed.), edited by Goldsmith LA.. New York: Oxford Univ. Press, 1991, vol. 1, p. 3-63.
18.
Opsahl, WP,
and
Ehrhart LA.
Compartmentalization of proline pools and apparent rate of collagen and non-collagen protein synthesis in arterial smooth muscle cells in culture.
Biochem J
243:
137-144,
1987[ISI][Medline].
19.
Phipps, A.
Evidence-based management of patients with burns.
J Wound Care
7:
299-302,
1998[Medline].
20.
Ramzy, PI,
Barret JP,
and
Herndon DN.
Thermal injury.
Crit Care Clin
15:
333-352,
1999[ISI][Medline].
21.
Rosenblatt, J,
Chinkes D,
Wolfe M,
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.
Semon, BA,
and
Phang JM.
Accumulation of pyrroline 5-carboxylic acid in conditioned medium of cultured fibroblast: stimulatory effects of serum, insulin, and IGF-1.
In Vitro Cell Dev Biol
27A:
665-669,
1991[ISI].
23.
Valle, D,
Blaese RM,
and
Phang JM.
Increased sensitivity of lymphocyte
1-pyrroline-5-carboxylate reductase to inhibition by proline with transformation.
Nature
253:
214-216,
1975[ISI][Medline].
24.
Volpi, E,
Jeschke MG,
Herndon DN,
and
Wolfe RR.
Measurement of skin protein breakdown in a rat model.
Am J Physiol Endocrinol Metab
279:
E900-E907,
2000[Abstract/Free Full Text].
25.
Wenstrup, RJ,
Murad S,
and
Pinnell SR.
Dermal macromolecules and their metabolism.
In: Physiology, Biochemistry, and Molecular Biology of The Skin (2nd ed.), edited by Goldsmith LA.. New York: Oxford Univ. Press, 1991, vol. 1, p. 481-508.
26.
Wolfe, RR.
Radioactive and Stable Isotopes Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. New York: Wiley-Liss, 1992.
27.
Wu, G,
Davis PK,
Flynn NE,
Knabe DA,
and
Davidson JT.
Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs.
J Nutr
127:
2342-2349,
1997[Abstract/Free Full Text].
28.
Zhang, X-J,
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].
29.
Zhang, X-J,
Chinkes DL,
Wolf SE,
and
Wolfe RR.
Insulin but not growth hormone stimulates protein anabolism in skin wound and muscle.
Am J Physiol Endocrinol Metab
276:
E712-E720,
1999[Abstract/Free Full Text].
30.
Zhang, X-J,
Sakurai Y,
and
Wolfe RR.
An animal model for measurement protein metabolism in the skin.
Surgery
119:
326-332,
1996[ISI][Medline].
Am J Physiol Endocrinol Metab 284(6):E1191-E1201
0193-1849/03 $5.00
Copyright © 2003 the American Physiological Society
Copyright © 2003 by the American Physiological Society.