Measurement of skin protein breakdown in a rat model

Elena Volpi1,4, Marc G. Jeschke2,4, David N. Herndon2,4, and Robert R. Wolfe2,3,4

Departments of 1 Internal Medicine, 2 Surgery, and 3 Anesthesiology, University of Texas Medical Branch, Galveston, 77555; and 4 Shriners Hospital, Galveston, Texas 77550


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whereas skin protein synthesis can be measured with different approaches, no method potentially applicable in humans is available for measurement of skin protein breakdown. To that end, we measured mixed skin fractional protein breakdown (FBR) in a rat model by use of a stable isotope method (tracee release method) originally developed to measure muscle protein breakdown. Skin mixed protein and collagen fractional synthesis rates (FSR) were also measured. A primed continuous infusion of L-[ring-2H5]phenylalanine and alpha -[5,5,5-2H3]ketoisocaproate (KIC) was given for 6 h. Arterial and skin phenylalanine and leucine free enrichments were measured at plateau (5-6 h) and during the decay that followed after the infusion was stopped. Skin FBR (%/h) was 0.260 ± 0.011 with phenylalanine and 0.201 ± 0.032 with KIC/leucine [P = not significant (NS)]. Mixed skin FSR (%/h) was 0.169 ± 0.055 with phenylalanine and 0.146 ± 0.020 with KIC/leucine (P = NS). Collagen FSR was 0.124 ± 0.023%/h (P = NS vs. mixed protein FSR). The tracee release method is a sensitive method for measurement of skin protein breakdown; however, given the high intersubject variability of FSR, the calculation of skin net balance is not advisable.

collagen; proteolysis; protein synthesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MEASUREMENT OF SKIN PROTEIN TURNOVER would be useful to assess skin regeneration and wound healing as well as to test the effects of different stimuli and pathophysiological conditions on skin metabolism. However, although skin protein synthesis has been measured by use of different methods (1, 2, 4, 8, 9, 11, 20), there are no methods available for measurement of skin protein breakdown with the exception of an arteriovenous rabbit ear model recently developed in our laboratory (20). The rabbit ear model cannot be applied when the arteriovenous catheterization of a specific skin region is not possible, and it is therefore not applicable in humans.

We have recently described a method for measurement of muscle protein fractional breakdown rate (FBR) by use of the precursor-product principle (19). This method requires the primed continuous infusion of a stable isotope tracer of an essential amino acid and the measurement of its arterial and intracellular muscle enrichment at plateau and during the decay that follows after stopping the infusion.

The FBR method requires sampling of the free amino acid pool of the compartment in which protein breakdown takes place (19). In the case of skin tissue, protein breakdown takes place in both the intracellular and the extracellular compartments (18). Therefore, the pools into which breakdown releases amino acids are comprised of both the intracellular and the extracellular free amino acid pools. Thus the simple extraction of the free amino acids from the skin tissue samples should be adequate for the measurement of skin FBR.

We measured skin protein FBR and skin mixed protein and collagen fractional synthetic rates (FSRs) in rats by use of stable isotopes of phenylalanine and alpha -ketoisocaproate (KIC).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The study was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, TX. Seven male Sprague-Dawley rats (350-375 g body wt) were housed in wire-bottom cages in a temperature-controlled room with a 12:12-h light-dark cycle. Animals were fed a regular pellet diet (Harlan Diet including 50% carbohydrate and 15% fat) and water ad libitum.

Infusion study. On the day before the infusion study, catheters were implanted in the carotid artery and the jugular vein under general anesthesia (pentobarbital sodium, 25 mg/kg) and analgesia (butorphanol, 0.2 mg). The animals were then housed overnight in Bollman cages and given a constant infusion of normal saline (2 ml/h). On the day of the study, after 200 µl of blood were drawn from the artery to measure the background enrichment of leucine and phenylalanine, a primed continuous infusion of alpha -[5,5,5-2H3]KIC (prime, 18 µmol; infusion rate, 0.15 µmol/min) and L-[ring-2H5]phenylalanine (prime, 10 µmol; infusion rate, 0.083 µmol/min) was started and was continued for 6 h (0-360 min; Cambridge Isotopes, Cambridge, MA). Phenylalanine infusion was performed in five of the seven rats receiving labeled KIC. Blood samples were drawn at 120, 300, 360, 375, 390, 420, and 480 min, and skin biopsies (~1.5 g tissue) were taken at 120, 360, 420, and 480 min (Fig. 1) using local anesthesia with lidocaine. Skin samples were immediately frozen in liquid nitrogen and were stored at -80°C until analysis. Blood samples were precipitated in 1 ml of 15% sulfosalicylic acid (SSA) and centrifuged, and the supernatant was stored at -20°C.


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Fig. 1.   Study protocol. The day before the infusion study, catheters were implanted in the carotid artery and the jugular vein under general anesthesia and analgesia. The animals were then housed overnight in Bollman cages and given a constant infusion of normal saline (2 ml/h). On the day of the study, after 200 µl of blood were drawn from the artery to measure the background enrichment of leucine and phenylalanine, a primed continuous infusion of alpha -[5,5,5-2H3]ketoisocaproic acid and L-[ring-2H5]phenylalanine was started and continued for 6 h (0-360 min). Phenylalanine infusion was performed in 5 of the 7 rats. Blood samples were drawn at 120, 300, 360, 375, 390, 420, and 480 min, and skin biopsies were taken at 120, 360, 420, and 480 min.

Analytical methods. Approximately 50 mg of tissue were cut from each skin biopsy. An internal standard solution containing L-[2H10]leucine (122 µmol/l) and L-[ring-13C6]phenylalanine (50 µmol/l, 2 µl/mg of skin) was added. SSA (15%, 450 µl) was immediately added to each sample, after which the sample was ground and then centrifuged at 2,000 g for 15 min. The supernatant was collected, and the pellet was resuspended in 15% SSA. The process was repeated three times. The collected supernatant containing free tissue amino acids was frozen for subsequent analysis, and the pellet was washed once with distilled water and three times with absolute ethanol. The pellet was dried overnight at 50°C and then weighed and hydrolyzed in 2 ml of 6 N HCl at 110°C overnight.

The purification of collagen was performed using a modification of the method in Refs. 7 and 12. Approximately 1.5 g of skin was homogenized at 4°C in extraction buffer (10 ml/g skin) containing 150 mM NaCl, 50 mM Tris · HCL, 20 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 2.0 mM N-ethylmaleimide (NEM) and 0.2 mM 2-aminopropionitrile, at pH 7.4. The samples were stirred vigorously and then centrifuged at 70,000 g for 25 min. The supernatant was filtered through glass wool, and collagenous proteins were precipitated with the addition of saturated ammonium sulfate. After centrifugation at 18,000 g for 30 min, the pellet was reconstituted in the extraction buffer, and the solution was brought to 4.5 M with NaCl to precipitate all newly synthesized types of collagen and procollagen. The samples were centrifuged at 70,000 g for 25 min, and the pellet was collected and reconstituted in column buffer containing 200 mM NaCl, 50 mM Tris · HCl, and 2 mM EDTA, at pH 7.6. After dialysis (membrane cutoff 14.000-16.000 kDa) in column buffer, acidic proteins were removed by DEAE cellulose chromatography as previously described (7). Urea was added to the samples to achieve a molarity of 1 mol/l. Collagen was separated by means of DEAE cellulose chromatography with an increasing linear NaCl gradient. The samples were divided into two aliquots, one for assessment of purity of the fraction and one for the measurement of isotope incorporation rate.

Collagen purity was assessed after dialysis in 0.15 M acetic acid (membrane cutoff 14.000-16.000 kDa) by SDS-PAGE (Mini-Protean apparatus, precast gradient gel 4-15%, Bio-Rad, Hercules, CA) using broad molecular mass standards (Bio-Rad). Two gels were loaded with the same amounts of the same samples. One of the gels was stained with Coomassie brilliant blue, whereas the other was blotted on nitrocellulose. A mixture of anti-body anti-collagen type I (Calbiochem, San Diego, CA), collagen type III (Calbiochem), collagen type IV (BioGenex Laboratories, San Ramon, CA), and collagen type VII (Sigma, St. Louis, MO) was used to identify collagen. Amino acid composition analysis of the extracted collagenous proteins was additionally performed on 10 µl of extracted sample after hydrolysis with an amino acid autoanalyzer (HP 3396A/6300AAA, Hewlett-Packard, Wilmington, DE).

The sample aliquots for the measurement of the isotope incorporation rates were dialyzed against distilled water (membrane cutoff 14.000-16.000 kDa), dried in a speed vacuum, and hydrolyzed in 2 ml of 6 N HCl at 110°C overnight.

Blood and tissue free amino acids, as well as amino acids derived from the hydrolysis of collagen and mixed skin proteins, were purified by means of cation exchange chromatography (17). Blood and free tissue leucine and phenylalanine enrichments were determined on their tert-butyldimethylsilyl (t-BDMS) derivatives by gas chromatography-mass spectrometry (GC-MS; GC 8000 series, MD 800, Fisons Instruments, Manchester, UK) in the electron impact mode. The enrichments of leucine and phenylalanine derived from the hydrolysis of collagen and mixed skin proteins were determined on their t-BDMS derivatives in the electron impact mode by GC-MS monitoring the ions 304 and 305 for leucine and 237 and 239 for phenylalanine with the use of the standard curve approach described by Calder et al. (3) and modified by Patterson et al. (10).

Calculations. The FBR of mixed skin proteins was calculated with a method developed for muscle and defined as the tracee release method (19). This method enables the measurement of the rate of protein breakdown by measuring the decay curves of arterial and tissue free amino acid enrichment in the arterial and tissue free amino acid pools after stopping the tracer infusion once the isotopic steady state is achieved. At isotopic steady state, the enrichment in the tissue pool is always lower than the enrichment in the arterial blood because of tissue protein breakdown, which releases unlabeled amino acids. Once the isotopic infusion is stopped, the enrichment decay in the free tissue pool depends on the arterial enrichment decay, which provides tracer and a part of tracee, and on the protein breakdown, which provides another part of tracee (Fig. 2). Therefore, the FBR can be calculated with the equation
FBR<IT>=</IT><FR><NU>E<SUB>s(<IT>t2</IT>)</SUB><IT>−</IT>E<SUB>s(<IT>t1</IT>)</SUB></NU><DE>P<IT> ∫</IT><SUP>t<IT>2</IT></SUP><SUB>t<IT>1</IT></SUB> E<SUB>A</SUB>(<IT>t</IT>)d<IT>t−</IT>(<IT>1+</IT>P)<IT> ∫</IT><SUP>t<IT>2</IT></SUP><SUB>t<IT>1</IT></SUB> Es(<IT>t</IT>)d<IT>t</IT></DE></FR><IT>·</IT><FR><NU>Qs</NU><DE>T</DE></FR><IT>·60·100</IT> (1)
where ES(t2) - ES(t1) is the change of enrichment in the free tissue pool after stopping the isotopic infusion (time 360-480 min). P = ES/(EA - ES), where EA and ES are the isotope enrichments in the arterial and free tissue pools at isotopic plateau (EA, 300-360 min; ES, 360 min); int t1t2 EA(t)dt and int t1t2 ES(t)dt are the areas under the decay curves of the arterial and the tissue enrichments, respectively. Qs/T is the ratio of the intracellular free tracee content over the protein bound tracee in the total skin. Q was calculated for each sample as the measured tissue free amino acid concentration (Cs, nmol/ml) multiplied by the measured tissue water content (ml/g wet tissue)
Q<IT>=</IT>C<SUB>s</SUB><IT>·</IT><FR><NU>(tissue wet weight<IT>−</IT>tissue dry weight)</NU><DE>tissue wet weight</DE></FR> (2)
T was measured for each sample as the amino acid content of mixed skin proteins per gram of tissue wet weight. The results are presented in percent per hour, multiplying by the factors 60 (min/h) and 100. 


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Fig. 2.   Model of skin amino acid kinetics. Sources of essential amino acids in skin tissue free pool. Arterial blood and skin proteins are the arterial blood pool and skin protein-bound pool, respectively. Solid arrows indicate movement of unlabeled essential amino acids, and dotted arrows indicate movement of labeled amino acids from one pool to another. However, during KIC infusion, labeled leucine can also appear from transamination within the skin cells into the skin tissue free pool (not depicted).

The FSR of collagen and mixed skin proteins were calculated by the precursor-product model. The increment of the enrichment in the product, i.e., the difference of protein bound amino acid enrichment between the first and the second skin biopsy (Delta EProtein), was divided by the average free tissue amino acid tracer-to-tracee ratio in the first and second skin biopsy
FSR<IT>=</IT>(<IT>&Dgr;</IT>E<SUB>Protein</SUB><IT>/&Dgr;t</IT>)<IT>/</IT> (3)

[(E<SUB>Precursor(<IT>1</IT>)</SUB><IT>+</IT>E<SUB>Precursor(<IT>2</IT>)</SUB>)<IT>/2</IT>]<IT>·60·100</IT>
where EPrecursor(1) and EPrecursor(2) are enrichments of the labeled amino acids in the precursor pool at the time of the two sequential skin biopsies, and t is the time interval between the two sequential biopsies. The results are presented in percent per hour multiplying by the factors 60 (min/h) and 100.

Statistical analysis. The comparisons between skin protein turnover parameters were carried out using the two-tail paired t-test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Blood phenylalanine and leucine enrichments were at steady state during the last hour of isotope infusion, i.e., the enrichment values at t = 300 were not significantly different from those at t = 360 (Fig. 3). The tissue water was 69 ± 1% (by wt). Tissue free phenylalanine and leucine enrichments were at steady state from t = 120 to 360 min. The values of the plateau enrichments of blood and tissue phenylalanine and leucine are reported in Table 1. Blood and tissue free phenylalanine and leucine enrichments decayed in a synchronous fashion, following a single exponential curve, after the isotope infusion was stopped (Fig. 3). The blood-to-free tissue enrichment ratio was significantly higher for phenylalanine than for leucine (1.65 ± 0.14 vs. 1.33 ± 0.04, P = 0.027), indicating that some KIC was converted to leucine in the skin cells.


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Fig. 3.   Leucine and phenylalanine enrichments in the arterial blood and in the skin tissue free pool. Tissue free phenylalanine and leucine enrichments were at steady state from t = 120 to t = 360 min from the start of the isotope infusion. Blood and tissue free phenylalanine and leucine enrichments decayed in a synchronous fashion after the isotope infusion was stopped.


                              
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Table 1.   Plateau enrichments of free phenylalanine and leucine in arterial blood and skin tissue, skin free phenylalanine and leucine concentrations, and protein bound phenylalanine and leucine content in mixed skin proteins

Tissue phenylalanine and leucine concentrations did not change over time. The average values of tissue phenylalanine and leucine concentrations and the measured mixed skin protein bound phenylalanine and leucine contents are reported in Table 1. These values were used to calculate the ratio of the intracellular free tracee content over the protein bound tracee in the skin (Qs/T in Eq. 1).

Skin FBR, mixed skin protein FSR, and collagen FSR are reported in Table 2. Skin FBR measured with phenylalanine as tracer was similar to that measured with leucine. Skin protein FSRs were calculated using both skin free amino acid enrichment and blood free amino acid enrichment as precursor pool enrichment. The collagen fraction separated from skin tissue was pure, as assessed by SDS-PAGE and Western blot (Fig. 4). The analysis of the amino acid content of the extracted collagen fraction confirmed that the amino acid composition of the proteins contained in the fraction was similar to that of collagenous proteins (data not shown). Mixed skin FSR measured with phenylalanine was not different from that measured with leucine. A power analysis indicated that, to be able to detect a significant difference, with alpha  = 0.05 and beta  = 0.8, between the FSRs measured with the two different tracers, we would have had to study more than 300 animals. All the FSRs were significantly lower when the blood enrichment was used as precursor, compared with the value when the tissue enrichment was used. Skin FBR measured with phenylalanine was not different from mixed skin protein FSR measured with the same tracer, whereas it was slightly but significantly higher (P < 0.05) than mixed skin protein FSR measured with KIC/leucine. However, skin FBR measured with leucine was not different from mixed skin protein and collagen FSRs measured with the same tracer.

                              
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Table 2.   Skin FBR and skin FSR of mixed skin proteins and skin collagen



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Fig. 4.   SDS-PAGE and Western blot analysis of isolated skin collagen fractions. The isolated collagen fractions were tested for purity by SDS-PAGE analysis. Because the isolated fractions contained any collagen type, we identified the collagen bands by Western blot using a mixture of antibodies of anti-collagen type I and type III, collagen type IV, and collagen type VII.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Skin protein breakdown rate. We applied the tracee release method (19), originally developed for measurement of muscle protein breakdown, to measure skin protein breakdown rate. Phenylalanine was the reference tracer used to measure skin FBR, because it is an essential amino acid and, therefore, its appearance in the tissue free pool is due solely to transport from blood and release from proteolysis (Fig. 2). Also, the proportional contribution of phenylalanine to keratin and to collagen is about the same, so its appearance should reflect both dermal and epidermal protein breakdown. An important assumption of the tracee release method, essential for its correct application, is that the enrichment of the pool in which unlabeled amino acids are released by protein breakdown is measured (19). In the skin, the pool into which the tracee is released is comprised of both the intracellular and extracellular free amino acid pools, because proteolysis takes place both intracellularly and extracellularly (18). Because we measured free phenylalanine enrichment in the total tissue water, which includes both intracellular and extracellular water, the assumption that the enrichment of the pool in which protein breakdown releases unlabeled amino acids is measured was satisfied. The FBR value measured in this experiment is similar to the value obtained in the rabbit with the ear arteriovenous model after conversion to the same units (20). Specifically, Zhang et al. (20) found that, in the normal rabbit, skin breakdown rate was ~21 mg of skin protein · h-1 · ear-1. Because the ear of the rabbit contained ~5.5 g of protein, the fractional breakdown rate was ~0.38%/h, which is similar to the 0.26%/h found in our experiment, especially when we consider that we used two different species and different methodological approaches. From these results, we feel confident that the tracee release method is valid when applied in the rat skin for measurement of mixed protein breakdown.

To assess the sensitivity of the FBR method, we used the phenylalanine data to calculate the smallest difference that can be detected between two treatments by use of a t-test with alpha  = 0.05 and beta  = 0.8. The coefficient of variation of the FBR measurement with phenylalanine as tracer was 9.5% with n = 5. We found that we can detect a 20% difference between two groups of five rats each, a 17% difference with six rats in each group, or a 15.5% difference with seven rats per group. Compared with the arteriovenous model (20), which can detect a 22.5% difference with n = 5, we found that both methods have similarly high sensitivities.

Precursor pool for protein synthesis. In this study, not only did we measure skin protein breakdown, but we also measured skin protein FSR to obtain a measure of skin protein turnover and net balance. A potential problem with the measurement of FSR in the skin is the measurement of the precursor pool enrichment. The infusion of an amino acid tracer such as [2H5]phenylalanine produces a gradient of tracer enrichment between blood and tissue, which is due to proteolysis. In tissues where most of the free amino acid pools are intracellular and proteolysis takes place only intracellularly, such as muscle, the measurement of the free amino acid enrichment in a tissue sample yields values close to those of the true precursor. In the skin, however, the tracer is diluted by proteolysis in both the intracellular and the extracellular space (18), and a significant portion of the tissue free amino acids is extracellular. Thus it is expected that the tracer enrichment in a skin sample that includes a significant amount of extracellular amino acids will lead to an overestimation of the true precursor. The measurement of the amino acyl-tRNA enrichment could solve this problem (6), but the large amount of tissue required (~500 mg) prevents a routine use of this method in humans or in the present experiment, where large amounts of skin were used for other analyses. We tried to circumvent the problem by infusing labeled KIC and measuring leucine tissue enrichment, which should give a more accurate estimate of the precursor pool for protein synthesis, because KIC is intracellularly converted to leucine (14). This method has led to the successful estimation of the enrichment of the precursor for muscle protein synthesis (5). Although a study showed that leucine is transaminated to KIC in human skin fibroblasts (15), it was unknown whether this method would also be valid for the measurement of skin protein synthesis in vivo.

We addressed this problem by using different methods. First, we compared skin FSR and FBR calculated with the use of either tracer. In the case of a significant contribution of skin cells to labeled leucine production during labeled KIC infusion, both the FSR and the FBR should yield a significantly lower value than values measured with labeled phenylalanine. Although FBR calculated with leucine was 22% lower than that calculated with phenylalanine, this difference was not significant. Thus it does not appear that the conversion of KIC to leucine in the rat skin is extensive. Second, we calculated mixed skin protein FSR with either the tissue free enrichments or the blood enrichments as precursor. If significant amounts of KIC were converted to leucine in the skin, the difference between the FSR calculated with either skin or blood leucine enrichment should be significantly lower than the difference between the FSR calculated with skin or blood phenylalanine enrichments. We found that the FSR values calculated with blood leucine enrichment were 22% lower than those calculated with skin leucine enrichment, whereas the difference in FSR using blood or skin as precursor was significantly higher (39%) with the use of phenylalanine. These data indicate that there is some advantage in the use of KIC as tracer to measure skin protein FSR, although the estimation of the precursor pool enrichment in the skin seems not to be so accurate as it is in the muscle. Further studies are required for a specific assessment of the accuracy of the KIC-leucine method in the skin.

Skin protein synthesis. The potential limitations of the quantification of FSR notwithstanding, the results of mixed skin protein FSR were lower, but not significantly, than the FBR measured with the same tracer. Because previous data in normal skin obtained with the arteriovenous balance showed that skin protein synthesis and breakdown rates are similar in a variety of circumstances (20), this indicates that we overestimated the precursor pool enrichment with both tracers, thereby underestimating the true FSR value by 28 and 35% with the use of KIC/leucine and phenylalanine, respectively. Thus skin protein net balance (calculated by subtraction of the FBR from the FSR) would be underestimated because FBR is not underestimated.

Because collagen comprises ~90% of skin proteins (16), we also purified skin collagen to evaluate the contribution of its synthesis rate to mixed skin protein FSR. We did not measure the synthesis rates of each different collagen type, because it is well known that 95% of the skin collagens are comprised of types I and III, which have very similar turnover rates (7, 13). Although we could not use phenylalanine as tracer because of its low content in collagen, we found that the collagen FSR measured with KIC/leucine was not statistically different from the mixed skin FSR. This result was not due to impurity of the extracted collagen, because we confirmed the purity of each sample by amino acid composition analysis, SDS-PAGE, and Western blot. The mixed skin protein FSR is the average FSR of soluble (newly synthesized) collagens, insoluble (older) collagens, and epidermal proteins. Although epidermal proteins have a higher turnover rate compared with that of the collagens, due to the small size of their pool they did not significantly influence the overall (mixed) skin protein FSR. However, we cannot exclude the possibility that the contribution of epidermal proteins to mixed skin protein FSR may increase in specific pathological conditions. It is interesting to note that the collagen and mixed protein FSR values obtained in this study were not statistically different from the collagen FSR values (0.076 ± 0.063%/h, mean ± SD) obtained in humans by El-Harake et al. (6) with a different methodological approach, i.e., proline as tracer and prolyl-tRNA enrichment as precursor. Although the lack of a significant difference between the data from El-Harake et al. and the data from the present study might be due to a lack of power, the absolute values of FSR are strikingly similar despite the different species studied and the different methodological approach used.

Our results indicate that collagen is the major contributor to mixed skin protein FSR and, in turn, that mixed skin protein FSR is a good indicator of skin collagen synthesis in normal skin. This is important, because the isolation of collagen requires large amounts of skin (150-1,000 mg according to the method used), which prevents the routine measurement of skin collagen synthesis in humans. On the other hand, mixed skin protein FSR, as well as FBR, requires <15-30 mg, and these methods are therefore more easily applicable in human studies. For example, a 4-mm punch biopsy of the skin of the back (~4 mm deep) could yield >50 mg of tissue, which is more than sufficient for the measurement of FSR and FBR.

In conclusion, we found that the tracee release method is a reliable means for measurement of skin protein breakdown. The small amount of skin required to measure skin FBR makes this technique applicable in humans. However, although FSR can be reasonably measured by use of free tissue enrichment as precursor, it is probably not accurate enough to be compared quantitatively with FBR to measure skin net balance. The between-subject coefficient of variation for skin FSR was 36% with KIC/leucine and 65% with phenylalanine in our study. The high variability was not limited to the present study, because the coefficient of variation was 83% also in the study by El-Harake et al. (6). We do not have an explanation for this high variability. One possibility is that skin is not homogeneous, so the synthetic rates may differ in specific regions. Whereas skin for this study was taken from the same general area, variations in FSR could have reflected the actual differences in the synthetic activities of the tissue samples. It is also possible that the high intersubject variability might be due to the uncertainty of the precursor enrichment. However, this explanation does not apply to the El-Harake et al. study where the aminoacyl-tRNA enrichment was measured. The other possibility is that skin FSR varies according to specific stimuli that do not affect FBR. This could be supported by the observation that, despite a high coefficient of variation of skin FSR, the coefficient of variation of skin FBR in our study was 10% with phenylalanine. Further studies are required to assess the validity of these methods in humans and in pathological conditions such as burn injuries and wound healing.


    ACKNOWLEDGEMENTS

This study was supported by Shriners Grant No. 8490.


    FOOTNOTES

Address for reprint requests and other correspondence: E. Volpi, 815 Market St., Galveston, TX 77550 (E-mail: evolpi{at}utmb.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 December 1999; accepted in final form 23 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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