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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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
CVPhe   Venous Phe concentration
CAPro   Arterial Pro concentration
CVPro   Venous Pro concentration
EAPhe   Arterial Phe enrichment
EVPhe   Venous Phe enrichment
EDPhe   Dermal free Phe enrichment
EEPhe   Epidermal free Phe enrichment
EAPro   Arterial Pro enrichment
EVPro   Venous Pro enrichment
EDPro   Dermal free Pro enrichment
EEPro   Epidermal free Pro enrichment
CDPhe   Dermal protein-bound Phe content
CEPhe   Epidermal protein-bound Phe content
CDPro   Dermal protein-bound Pro content
CEPro   Epidermal protein-bound Pro content
BF   Blood flow rate in the ear skin
FinPhe   Arterial delivery of Phe to skin
FoutPhe   Venous exit of Phe from skin
NBPhe   Net balance of Phe across skin
nbPhe   Net balance of labeled Phe across skin
FV,APhe   Physiological shunting of Phe from artery to vein
FD,APhe   Transport of Phe from artery to dermis
FV,DPhe   Transport of Phe from dermis to vein
FE,DPhe   Transport of Phe from dermis to epidermis
FD,EPhe   Transport of Phe from epidermis to dermis
FO,DPhe   Irreversible loss of Phe from dermis (protein synthesis)
FD,OPhe   Production of Phe in dermis (protein breakdown)
FO,EPhe   Irreversible loss of Phe from epidermis (protein synthesis)
FE,OPhe   Production of Phe in epidermis (protein breakdown)
FinPro   Arterial delivery of Pro to skin
FoutPro   Venous exit of Pro from skin
NBPro   Net balance of Pro across skin
nbPro   Net balance of labeled Pro across skin
FE,DPro   Transport of Pro from dermis to epidermis
FD,EPro   Transport of Pro from epidermis to dermis
FO,DPro   Irreversible loss of Pro from dermis (protein synthesis)
FO,EPro   Irreversible loss of Pro from epidermis (protein synthesis)

Two-Pool Model


NBEPhe   Net balance of Phe across epidermis
NBDPhe   Net balance of Phe across dermis
NBEPro   Net balance of Pro across epidermis
NBDPro   Net balance of Pro across dermis
RaEPhe   Rate of appearance of Phe from epidermis to blood
RaDPhe   Rate of appearance of Phe from dermis to blood
RdEPhe   Rate of disappearance of Phe from blood to epidermis
RdDPhe   Rate of disappearance of Phe from blood to dermis


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.


View larger version (16K):
[in this window]
[in a new window]
 
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.

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.


View larger version (13K):
[in this window]
[in a new window]
 
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).

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
F<SUB>in</SUB>Phe = C<SUB>A</SUB>Phe × BF (1)

F<SUB>out</SUB>Phe = C<SUB>V</SUB>Phe × BF (2)

NBPhe = F<SUB>in</SUB>Phe − F<SUB>out</SUB>Phe (3)

F<SUB>in</SUB>Pro = C<SUB>A</SUB>Pro × BF (4)

F<SUB>out</SUB>Pro = C<SUB>V</SUB>Pro × BF (5)

NBPro = F<SUB>in</SUB>Pro − F<SUB>out</SUB>Pro (6)

F<SUB>V,D</SUB>Phe = F<SUB>out</SUB>Phe × (E<SUB>A</SUB>Phe − E<SUB>V</SUB>Phe) (7)

÷ (E<SUB>A</SUB>Phe − E<SUB>D</SUB>Phe)

F<SUB>V,A</SUB>Phe = F<SUB>out</SUB>Phe − F<SUB>V,D</SUB>Phe (8)

F<SUB>D,A</SUB>Phe = F<SUB>in</SUB>Phe − F<SUB>V,A</SUB>Phe (9)

nbPhe = E<SUB>D</SUB>Phe × F<SUB>O,D</SUB>Phe + E<SUB>E</SUB>Phe × F<SUB>O,E</SUB>Phe (10)

nbPro = E<SUB>D</SUB>Pro × F<SUB>O,D</SUB>Pro + E<SUB>E</SUB>Pro × F<SUB>O,E</SUB>Pro (11)

F<SUB>O,D</SUB>Phe = [C<SUB>D</SUB>Phe × (nbPro × E<SUB>E</SUB>Phe × C<SUB>E</SUB>Phe − nbPhe <IT>×</IT> E<SUB>E</SUB>Pro × C<SUB>E</SUB>Pro)] (12)

÷ (E<SUB>D</SUB>Pro ×E<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe − E<SUB>D</SUB>Phe × E<SUB>E</SUB>Pro × C<SUB>E</SUB>Pro×C<SUB>D</SUB>Phe)

F<SUB>O,E</SUB>Phe = (nbPhe − E<SUB>D</SUB>Phe × F<SUB>O,D</SUB>Phe)/E<SUB>E</SUB>Phe (13)

F<SUB>D,O</SUB>Phe = [C<SUB>D</SUB>Phe × C<SUB>D</SUB>Phe × NBPro − C<SUB>E</SUB>Pro <IT>×</IT> C<SUB>D</SUB>Phe × NBPhe + F<SUB>O,D</SUB>Phe × (C<SUB>E</SUB>Pro ×C<SUB>D</SUB>Phe − C<SUB>D</SUB>Phe − C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe)]/(C<SUB>E</SUB>Pro × C<SUB>D</SUB>Phe − C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe) (14)

F<SUB>E,O</SUB>Phe = −NBPhe + F<SUB>O,E</SUB>Phe − F<SUB>D,O</SUB>Phe + F<SUB>O,D</SUB>Phe (15)

F<SUB>E,D</SUB>Phe = F<SUB>E,O</SUB>Phe × E<SUB>E</SUB>Phe/(E<SUB>D</SUB>Phe − E<SUB>E</SUB>Phe) (16)

F<SUB>D,E</SUB>Phe = F<SUB>E,D</SUB>Phe + F<SUB>E,O</SUB>Phe − F<SUB>O,E</SUB>Phe (17)
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
R<SUB>d</SUB>Phe = (E<SUB>A</SUB>Phe × F<SUB>in</SUB>Phe − E<SUB>V</SUB>Phe  (18)

× F<SUB>out</SUB>Phe)/E<SUB>A</SUB>Phe

R<SUB>d</SUB>Pro = (E<SUB>A</SUB>Pro × F<SUB>in</SUB>Pro − E<SUB>V</SUB>Pro × F<SUB>out</SUB>Pro)/E<SUB>A</SUB>Phe (19)

R<SUB>a</SUB>Phe = R<SUB>d</SUB>Phe −NBPhe (20)

R<SUB>a</SUB>Pro = R<SUB>d</SUB>Pro − NBPro (21)

R<SUB>aE</SUB>Phe = (C<SUB>E</SUB>Phe × C<SUB>D</SUB>Phe × R<SUB>a</SUB>Pro

− C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe × R<SUB>a</SUB>Phe) (22)

÷ (C<SUB>D</SUB>Phe × C<SUB>E</SUB>Pro − C<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro)

R<SUB>aD</SUB>Phe = R<SUB>a</SUB>Phe − R<SUB>aE</SUB>Phe (23)

NB<SUB>E</SUB>Phe = (C<SUB>E</SUB>Phe × C<SUB>D</SUB>Phe × NBPro − C<SUB>D</SUB>Pro <IT>×</IT> C<SUB>E</SUB>Phe × NBPhe) (24)

÷ (C<SUB>D</SUB>Phe × C<SUB>E</SUB>Pro − C<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro)

NB<SUB>D</SUB>Phe = NBPhe − NB<SUB>E</SUB>Phe (25)

Rd<SUB>E</SUB>Phe = R<SUB>aE</SUB>Phe − NB<SUB>E</SUB>Phe (26)

Rd<SUB>D</SUB>Phe = Ra<SUB>D</SUB>Phe − NB<SUB>D</SUB>Phe (27)
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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Enrichment and concentration of phenylalanine and proline

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.


View larger version (15K):
[in this window]
[in a new window]
 
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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Protein kinetics in the epidermis and dermis

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   FSR of dermal and epidermal protein



View larger version (14K):
[in this window]
[in a new window]
 
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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 Delta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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
F<SUB>in</SUB>Phe = C<SUB>A</SUB>Phe × BF (A1)

F<SUB>out</SUB>Phe = C<SUB>V</SUB>Phe × BF (A2)

NBPhe = F<SUB>in</SUB>Phe − F<SUB>out</SUB>Phe (A3)

F<SUB>in</SUB>Pro = C<SUB>A</SUB>Pro × BF (A4)

F<SUB>out</SUB>Pro = C<SUB>V</SUB>Pro × BF (A5)

NBPro = F<SUB>in</SUB>Pro − F<SUB>out</SUB>Pro (A6)

F<SUB>V,D</SUB>Phe = F<SUB>out</SUB>Phe  (A7)

× (E<SUB>A</SUB>Phe − E<SUB>V</SUB>Phe)/(E<SUB>A</SUB>Phe − E<SUB>D</SUB>Phe)

F<SUB>V,A</SUB>Phe = F<SUB>out</SUB>Phe − F<SUB>V,D</SUB>Phe (A8)

F<SUB>D,A</SUB>Phe = F<SUB>in</SUB>Phe − F<SUB>V,A</SUB>Phe (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.
nbPhe = E<SUB>D</SUB>Phe × F<SUB>O,D</SUB>Phe + E<SUB>E</SUB>Phe × F<SUB>O,E</SUB>Pro (A10)

nbPro = E<SUB>D</SUB>Pro × F<SUB>O,D</SUB>Pro + E<SUB>E</SUB>Pro × F<SUB>O,E</SUB>Pro (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.
F<SUB>O,D</SUB>Phe/C<SUB>D</SUB>Phe = F<SUB>O,D</SUB>Pro/C<SUB>D</SUB>Pro
and
F<SUB>O,E</SUB>Phe/C<SUB>E</SUB>Phe = F<SUB>O,E</SUB>Pro/C<SUB>E</SUB>Pro
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


F<SUB>O,D</SUB>Phe = [C<SUB>D</SUB>Phe × (nbPro × E<SUB>E</SUB>Phe × C<SUB>E</SUB>Phe − nbPhe (A12)

× E<SUB>E</SUB>Pro × C<SUB>E</SUB>Pro)]/(E<SUB>D</SUB>Pro ×E<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro

× C<SUB>E</SUB>Phe − E<SUB>D</SUB>Phe × E<SUB>E</SUB>Pro × C<SUB>E</SUB>Pro × C<SUB>D</SUB>Phe)

F<SUB>O,E</SUB>Phe = (nbPhe − E<SUB>D</SUB>Phe × F<SUB>O,D</SUB>Phe)/E<SUB>E</SUB>Phe (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.
NBPhe = F<SUB>O,D</SUB>Phe − F<SUB>D,O</SUB>Phe + F<SUB>O,E</SUB>Phe − F<SUB>E,O</SUB>Phe
and
NBPro = F<SUB>O,D</SUB>Pro − F<SUB>D,O</SUB>Pro + F<SUB>O,E</SUB>Pro − F<SUB>E,O</SUB>Pro
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
F<SUB>D,O</SUB>Phe = [C<SUB>D</SUB>Phe × C<SUB>D</SUB>Phe <IT>×</IT> NBPro − C<SUB>E</SUB>Pro × C<SUB>D</SUB>Phe  (A14)

× NBPhe + F<SUB>O,D</SUB>Phe × (C<SUB>E</SUB>Pro ×C<SUB>D</SUB>Phe − C<SUB>D</SUB>Phe

− C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe)]/(C<SUB>E</SUB>Pro  × C<SUB>D</SUB>Phe − C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe)

F<SUB>E,O</SUB>Phe = −NBPhe + F<SUB>O,E</SUB>Phe − F<SUB>D,O</SUB>Phe + F<SUB>O,D</SUB>Phe (A15)

F<SUB>E,D</SUB>Phe = F<SUB>E,O</SUB>Phe × E<SUB>E</SUB>Phe/(E<SUB>D</SUB>Phe − E<SUB>E</SUB>Phe) (A16)

F<SUB>D,E</SUB>Phe = F<SUB>E,D</SUB>Phe + F<SUB>E,O</SUB>Phe − F<SUB>O,E</SUB>Phe (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
RdPhe = (E<SUB>A</SUB>Phe × F<SUB>in</SUB>Phe  (A18)

− E<SUB>V</SUB>Phe × F<SUB>out</SUB>Phe)/E<SUB>A</SUB>Phe

RdPro = (E<SUB>A</SUB>Pro × F<SUB>in</SUB>Pro − E<SUB>V</SUB>Pro × F<SUB>out</SUB>Pro)/E<SUB>A</SUB>Pro (A19)

RaPhe = RdPhe − NBPhe (A20)

RaPro = RdPro − NBPro (A21)

Ra<SUB>E</SUB>Phe = (C<SUB>E</SUB>Phe × C<SUB>D</SUB>Phe × RaPro  (A22)

− C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe × RaPhe)

÷ (C<SUB>D</SUB>Phe × C<SUB>E</SUB>Pro − C<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro)

Ra<SUB>D</SUB>Phe = RaPhe − Ra<SUB>E</SUB>Phe (A23)

NB<SUB>E</SUB>Phe = (C<SUB>E</SUB>Phe × C<SUB>D</SUB>Phe × NBPro (A24)

− C<SUB>D</SUB>Pro × C<SUB>E</SUB>Phe × NBPhe)

÷ (C<SUB>D</SUB>Phe × C<SUB>E</SUB>Pro − C<SUB>E</SUB>Phe × C<SUB>D</SUB>Pro)

NB<SUB>D</SUB>Phe = NBPhe − NB<SUB>E</SUB>Phe (A25)

Rd<SUB>E</SUB>Phe = Ra<SUB>E</SUB>Phe − NB<SUB>E</SUB>Phe (A26)

Rd<SUB>D</SUB>Phe = Ra<SUB>D</SUB>Phe − NB<SUB>D</SUB>Phe (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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
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 Delta 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 Delta 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




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
284/6/E1191    most recent
00460.2002v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Zhang, X.-J.
Articles by Wolfe, R. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Zhang, X.-J.
Articles by Wolfe, R. R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.