Measurement of dermal collagen synthesis rate in vivo in humans

Wassim A. El-Harake1, Mikhail A. Furman1, Brian Cook2, K. Sreekumaran Nair3, Jayme Kukowski4, and Irwin G. Brodsky1

Departments of 1 Medicine, 2 Dermatology, and 4 Human Nutrition, University of Illinois at Chicago, Chicago, Illinois 60612; and 3 Department of Medicine and General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Accumulation of collagen produces organ dysfunction in many pathological conditions. We measured the fractional synthesis rate (FSR) of dermal collagen in five human volunteers from the increment of [13C]proline in detergent-soluble dermal collagen hydroxylated to hydroxyproline during a continuous infusion of L-[1-13C]proline. In these and eight other volunteers, we measured [13C]proline enrichment in skin aminoacyl-tRNA, skin tissue fluid amino acid, and plasma. The prolyl-[13C]tRNA enrichment was one-half that in tissue fluid proline and more than threefold less than in plasma. The FSR of dermal collagen was 0.076 ± 0.063%/h (mean ± SD), similar to previously reported rates for skeletal muscle contractile proteins and substantially slower than hepatically derived circulating proteins such as albumin or fibrinogen. We conclude that the FSR of human dermal collagen resembles that of other human proteins considered to display slow turnover. The current method for its measurement may be used to determine the regulation of collagen synthesis in other organs and disease states.

protein synthesis; connective tissue; metabolism; skin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALTERATIONS IN THE synthesis of collagen occur in many pathological states relevant to common human diseases. Evidence for altered collagen synthesis in such diseases has been derived from studies limited to those in nonhuman species or the in vitro circumstance. It is important to have reliable estimates of the rate of collagen synthesis in vivo in humans, as methods for measurement of collagen synthesis in animal models (11, 13, 16, 17, 22) or under in vitro conditions (4, 9, 11, 12) may not reflect actual in vivo rates in humans. A previous attempt to measure bone collagen synthesis in vivo in human subjects was reported in an abstract in 1992, which did not measure the obligatory precursor pool of collagen synthesis (21).

In the present paper, we report a method for in vivo measurement of the fractional synthesis rate (FSR) of collagen in human skin based on the precursor-product model. We have measured the incorporation of labeled proline into collagen as hydroxyproline. Because the presence of hydroxyproline in proteins other than collagen is quantitatively negligible, the measurement of its isotopic enrichment minimizes experimental error due to contamination of the product pool by noncollagen proteins. Furthermore, we have measured the fractional synthesis rate of collagen in vivo by using the isotopic enrichment of proline in prolyl-tRNA, the true precursor pool for collagen synthesis.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Study protocol. Thirteen healthy volunteers (10 males, 3 females) gave written informed consent as approved by the Institutional Review Board of the University of Illinois at Chicago. Eight were studied before perfection of methods for collagen hydrolysis and yielded samples suitable for estimation of precursor pool isotopic labeling. Samples from five subjects were used to measure dermal collagen FSR. For seven days before physiological testing, subjects were instructed in the consumption of a diet containing 1.5 g · kg-1 · day-1 protein, at least 160 mg/day of vitamin C, and enough caloric intake to meet energy needs determined by 3-day diet diaries (also used to ascertain food preferences) and Harris-Benedict estimates. Energy intake approximated 35 kcal/kg. Subjects were admitted for 24 h to the Clinical Research Center at the University of Illinois at Chicago and ate no food after 1900 on the day of admission.

Infusion protocol. After background sampling for L-[1-13C]proline enrichment in plasma, subjects underwent a 10-h primed infusion of L-[1-13C]proline (99%; Isotec, Miamisburg, OH) beginning at 0300 (15 µmol/kg bolus followed by 11.2 µmol · kg-1 · h-1 infusion). Elliptical biopsies of thigh skin measuring 20 × 7 mm (150-467 mg wet wt) were obtained subsequently at 0730 and 1300 (4.5 and 10 h). Arterialized blood samples, for measurement of [13C]proline enrichment, were obtained every 30 min after the first biopsy from a dorsal hand vein heated to 63°C (1). This produced blood samples with a 94% oxygen saturation. The skin samples were weighed, after removal of excess subcutaneous tissue, and were immediately frozen in liquid nitrogen to be stored at -80°C (Revco; Rheem, Ashville, NC).

Plasma preparation. Plasma (100 µl) was precipitated with an equal volume of acetone followed by chilling and centrifugation as previously decribed (9a). Supernatants, containing plasma amino acids, were dried and derivatized to tert-butyldimethylsilyl (TBDMS) derivatives using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (Pierce, Rockford, IL), as previously described (15).

Skin homogenate preparation. With the use of a Polytron tissue grinder (Kinematika, Lucerne, Switzerland), each skin sample was homogenized in 50 mM cacodylate-1% SDS buffer (Sigma Chemicals, St. Louis, MO) at pH 5.6 (0.5 ml/100 mg of tissue). Homogenate was centrifuged at 1,000 g for 5 min, and the supernatant, containing the soluble protein fraction, was separated from insoluble proteins.

Measurement of L-[1-13C]proline enrichment in aminoacyl-tRNA and the tissue fluid amino acid pool. Total RNA from the skin homogenate devoid of SDS insoluble proteins was extracted and precipitated as previously described (6) using TRI-reagent (Molecular Research Center, Cincinnati, OH) such that a ratio of 4:1 (TRI-reagent-homogenate) was used. The RNA pellet was dissolved in diethyl pyrocarbonate-water, and the aminoacyl-tRNA complex was hydrolyzed by a 60-min incubation of the dissolved RNA pellet at 37°C with 2 µl of 0.12 M potassium hydroxide, pH 9 (Sigma Chemicals; see Ref. 10). RNA was repelleted by acidification of the incubation media with HCl. This was followed by centrifugation at 15,000 g, and supernatants, containing hydrolyzed amino acids, were dried in a vacuum concentrator.

Supernatants from RNA extracted homogenate were used for measurement of the isotopic enrichment in the tissue fluid amino acid pool, which includes ~85% intracellular fluid and 15% extracellular fluid. With use of disposable columns, slurried with cation-exchange resin AG50W-X8 (Bio-Rad, Richmond, CA), free amino acids were separated from components of TRI-reagent, eluted with 3 ml of 1 M NH4OH, and dried in a vacuum concentrator. Both tRNA-associated and tissue fluid amino acids were derivatized to TBDMS derivatives (30 µl final volume) in the same manner as plasma amino acids (200 µl final volume).

Gas chromatography-mass spectrometry analysis. Gas chromatography (GC)-mass spectrometry (MS) was performed with a Shimadzu GC-17A gas chromatograph and a QP-5000 mass selective detector controlled by a CLASS-5000 Chemstation (Shimadzu, Tokyo, Japan). Splitless injections of 1.0-µl samples from sealed vials with inserts (National Scientific, Lawrenceville, GA) were made via the AOC-17 automatic sampler fitted with a 10-µl Hamilton fixed needle syringe onto a 25 × 0.32 mm ID BP-5 (5% phenyl dimethylsiloxan) column, 0.5 µm film thickness (both SGE Scientific, Melbourne, Australia). Chromatographic conditions were as follows: initial temperature, 100°C, 5 min; 7.5°C/min from 100 to 280°C; 280°C, 1 min; injector temperature, 300°C; interface temperature, 300°C. Ultra-high-purity helium (99.999%; AGA, Hammond, IN) was used as a carrier gas at 31.6 ml/min total flow rate, and the column had an initial pressure of 5.0 kPa and pressure rate of 0.5 kPa/min from 5 to 16.5 kPa.

Derivatized plasma amino acids, amino acids hydrolyzed from tRNA, and tissue fluid amino acids underwent electron-impact GC-MS analysis for the estimation of [13C]proline enrichment. Selected ion monitoring of ions with mass-to-charge ratios 184, 258, 286, and 287 was performed (Fig. 1). The isotopic 13C-to-12C ratio was measured using the ratio of fragments 287 to 286.


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Fig. 1.   Chromatographic profile of amino acids hydrolyzed from tRNA and derivatized to n-tert-butyldimethylsilyl (tBDMS) derivatives. A: total ion chromatogram of amino acids. Proline retention time is 13.05 min. B: fragmentation profile of tBDMS proline under electron-impact ionization. C: selected ion monitoring profile of characteristic ions of tBDMS proline. D: fragmentation sites of tBDMS proline.

Measurement of [13C]hydroxyproline enrichment in dermal collagen. Pellets of skin proteins, formed after RNA extraction, were triple washed with absolute ethanol-diethyl ether (50:50) and centrifuged on each occasion at 15,000 g for 20 min. Supernatants were discarded, and samples were dried. Dry pellets (~10 mg) were transferred into sealed culture tubes (Fisher Scientific, Glenlake, IL), flushed with nitrogen, and hydrolyzed in 2.0 ml of 0.05 M HCl in the presence of ion-exchange resin AG50W-X8 (100 mg) at 110°C in a heated block (Fisher Scientific) for 24 h as previously described (19), which is a proton attack enhancement method that reduces formation of proline and hydroxyproline hydantoins. The amino acids in solution were combined with those sequestered in the resin after the latter were eluted with 1 M NH4OH. The quantity of amino acids eluted from the resin was approximately eightfold greater than the quantity in solution after hydrolysis. The abundance of hydroxyproline obtained in these skin samples ranged from 400 to 1,200 nmol. [We have noted that abundances <250 nmol will not produce sufficient signal strength in isotope ratio mass spectrometry (IRMS) after reaction with ninhydrin to produce reliable measurements of isotope incorporation.] Hydrolysates were filtered through 0.22-µm syringe filters (Whatman, Clifton, NJ), taken to dryness, and reconstituted in the mobile phase used for HPLC separation.

HPLC separation. Hydroxyproline was isolated by HPLC and ultraviolet (UV) absorbance detection (Waters, Division of Millipore, Milford, MA) without derivatization using ligand exchange chromatography in a modification of a method previously described (8). Briefly, a 100 × 4.6 mm ID Partisil-5 silica gel analytic column (Whatman) was modified by percolation with 0.02 M cupric sulfate in 1.0 M ammonia (Sigma Chemicals). Percolation continued until blue eluate appeared.

The mobile phase was composed of water-acetonitrile (35:65, vol/vol), 0.35 mol/l ammonia, and 5 × 10-6 mol/l of cupric sulfate. Each component of the mobile phase was degassed with helium before mixing and pumping. Isocratic HPLC separation of the amino acids was performed under the following operating conditions: flow rate 0.7 ml/min; pressure 700 psi; UV detection at 210 nm. The column temperature was maintained at 30°C using a 330 Column Heater (Alltech Associates, Deerfield, IL).

L-Hydroxyproline fractions were collected and dried for IRMS. Purity of the hydroxyproline fraction was confirmed in 100-µl aliquots by GC-MS analysis of TBDMS derivatives.

IRMS analysis of hydroxyproline. Isotopic enrichment of hydroxyproline was determined using GC-IRMS (Delta Plus; Finnegan MAT, San Jose, CA) of CO2 liberated by reaction of hydroxyproline with ninhydrin as has been reported in studies of other amino acids (2). Samples were dissolved with 0.1 M HCl, transferred to 1-ml autosampler vials, dried, and sealed. After evacuation, 0.2 ml of saturated nynhydrin were added, and vials were heated for 1 h at 100°C. Samples were allowed to cool to room temperature, and 100 µl of gaseous phase was separated isothermally at 50°C on a Poraplot Q capillary micropacked 27 m x 0.32 mm ID column (Chrompack, Bridgewater, NJ) interfaced with the IRMS. The isotope ratio of the CO2 (13C/12C) was measured in the mass spectrometer.

Calculations. Collagen synthesis was measured as the incorporation of proline as hydroxyproline into the detergent-soluble collagen fraction of total dermal collagen. Net fractional dermal collagen synthesis rate (FSR) is described as (24)
FSR = <FR><NU>E<SUB>[<SUP>13</SUP>C]Hyp(<IT>t</IT><SUB>2</SUB>)</SUB> − E<SUB>[<SUP>13</SUP>C]Hyp(<IT>t</IT><SUB>1</SUB>)</SUB></NU><DE><LIM><OP>∫</OP></LIM> E<SUB>[<SUP>13</SUP>C]Pro-tRNA</SUB> d<IT>t</IT></DE></FR> × <FR><NU>[Hyp] + [Pro]</NU><DE>[Hyp]</DE></FR> × 100 (1)
where t2 represents the biopsy taken after 10 h of isotope infusion and t1 represents a time point 5.5 h earlier . The ratio [Hyp] + [Pro]/[Hyp] is a factor correcting for the proportion of proline incorporated into collagen as hydroxyproline. Measurements for the ratio were derived from the content of proline ([Pro]) and hydroxyproline ([Hyp]) in the mature, detergent-insoluble collagen fraction. This equation, under steady-state conditions of isotopic enrichment (E) between the earlier and later biopsy time points simplifies to
FSR = <FR><NU>E<SUB>[<SUP>13</SUP>C]Hyp(<IT>t</IT><SUB>2</SUB>)</SUB> − E<SUB>[<SUP>13</SUP>C]Hyp(<IT>t</IT><SUB>1</SUB>)</SUB></NU><DE>E<SUB>[<SUP>13</SUP>C]Pro-tRNA-ss</SUB> × &Dgr;<IT>t</IT></DE></FR> × <FR><NU>[Hyp] + [Pro]</NU><DE>[Hyp]</DE></FR> × 100 (2)
where E([13C]Pro-tRNA-ss) represents the steady-state isotopic enrichment of L-[1-13C]proline associated with tRNA.

Statistical analysis. Descriptive statistics are provided for each of the measurements reported (mean ± SD). Comparison of isotopic enrichment values among precursor pools was performed using a one-way analysis of variance with post hoc comparisons between groups made using the Newman-Keuls multiple comparison test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Precursor pools. The isotopic enrichment of [13C]proline in the true amino acid precursor pool for collagen synthesis (i.e., proline-tRNA; Table 1) was 4.47 ± 4.00 atom percent excess (APE) among the five subjects for whom collagen FSR was calculated. The magnitude of the enrichment in the tRNA pool was corroborated by a similar value in eight other subjects (5.20 ± 1.43 APE) receiving L-[1-13C]proline at an identical infusion rate. Enrichment in the skin tissue fluid amino acid pool was 69% higher (P < 0.05). Enrichment in both of these pools was substantially less than in the plasma pool of proline, which yielded a value more than threefold that in the tRNA pool (P < 0.01).

Dermal collagen synthesis rate. The net FSR of dermal detergent-soluble collagen was 0.076 ± 0.063%/h (Table 2). We calculated an estimated FSR using isotopic enrichment of [13C]proline in the tissue fluid amino acid pool to represent a surrogate precursor and found that it underestimated FSR by 50%.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study represents the first report, to our knowledge, of the measurement in vivo of human dermal collagen synthesis rate. This report provides four new types of information. It outlines the analytic methods for measuring collagen synthesis using a stable isotopically labeled proline precursor and a hydroxyproline product within collagen. It describes the differences in isotopic labeling among three proline pools that could be considered to represent the precursor pools for collagen synthesis, namely the plasma proline, skin tissue fluid proline, and skin prolyl-tRNA pools. Of these, prolyl-tRNA is the immediate precursor of collagen synthesis. Third, the report presents the mean value for the fractional synthesis of a newly synthesized pool of dermal collagen, the SDS soluble portion, among a sample of young healthy volunteers. Finally, the study provides an estimate of the interindividual variability of a dermal collagen synthesis measurement.

                              
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Table 1.   Isotopic enrichment of [13C]proline in amino acid precursor pools

The data indicate that the net FSR of human dermal collagen is approximately similar to that of muscle proteins (3), less than one-fifth the rate of albumin synthesis (7), and 12-fold lower than the the rate of synthesis of fibrinogen (7). This finding places dermal collagen in the category of slow production proteins like skeletal muscle myosin heavy chain (3), a characteristic not unexpected for a structural protein. It is important to note the possibility that, as dermal thickness varies by anatomical region, some variation in dermal collagen synthesis rate may occur with variation in the anatomical site of the skin biopsy.

                              
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Table 2.   Isotopic enrichment of [13C]hydroxyproline in collagen and FSR

The FSR of immature, detergent-soluble human dermal collagen is lower than that obtained in rats (0.17%/h) either when collagen precursors (procollagen, precollagen) are extracted for measurement of isotope incorporation (13) or when more aggressive acetic acid solubilization is used for collagen extraction (0.13%/h; see Ref. 16). As acetic acid extraction would potentially extract some collagen in a more mature, cross-linked state than SDS, thereby providing a lower estimate of synthesis rate, one can infer from our data that dermal collagen synthesis rate in humans is likely slower than that of rats.

The interindividual variability of the dermal collagen FSR measurement is somewhat higher than that reported in human skeletal muscle measured using labeled leucine infusions (3). There are two elements of metabolic physiology that would be expected to contribute to the increased variability. First, although use of a proline tracer is appropriate given its hydroxylation to a product contained almost exclusively in collagen, it is a nonessential amino acid that is produced by both de novo synthesis and protein degradation. The two means of proline production may contribute to interindividual variability in dilution of the tracer in precursor pools. We have previously noted an increased variability of alanine production rate compared with that of leucine among the same individuals (5). Second, the characteristic of collagen as a protein that matures by chemical cross-linking makes it possible that interindividual variability in the rate of cross-link formation could contribute to variability in the age (and isotopic enrichment) of the collagen pool extracted with SDS.

Preliminary data from other investigators examining bone collagen synthesis in human knees surgically removed for knee replacement indicate that the FSR (0.24-0.43%/h) is higher than that reported in the present study when a proline flooding method is used in an attempt to equilibrate plasma and tRNA pool isotopic enrichment (21). Values of bone collagen FSR were fourfold lower (0.034-0.061%/h) when plasma alanine enrichment was used to represent the precursor pool enrichment during a constant infusion of labeled alanine. As plasma alanine isotopic enrichment was used for the estimate of the precursor pool enrichment in the FSR calculation and might overestimate the true enrichment of the intracellular precursor, the investigators were concerned about the possibility of underestimating FSR. Although the FSR calculation derived from the studies using alanine infusion are similar to those we obtained, it is important to recognize that accuracy with the alanine infusion method could be potentially compromised by alanine in the protein hydrolysate that is derived from contamination by noncollagen protein. This may combine with the recognized potential inaccuracy in using plasma isotopic enrichment to estimate the intracellular precursor pool enrichment.

There are distinct advantages of the present method for determining fractional collagen synthesis rate compared with other possible methods. First, our measurement of the isotopic enrichment of hydroxyproline, present in few noncollagen proteins, minimizes the possibility of error in the FSR measurement due to contamination of the sample by noncollagen proteins. Exceptions may occur in conditions that result in complement deposition into the dermis as the C1q component of complement is another protein that may contain hydroxyproline. Second, the measurement of the proline isotopic enrichment in the prolyl-tRNA pool enhances confidence that isotopic enrichment of the true precursor of collagen synthesis was used in the calculation of FSR, obviating the need to extrapolate the precursor enrichment from plasma measurements. This approach is particularly important given our findings that there are large disparities between the plasma enrichment of proline and those of proline in the tRNA and tissue fluid amino acid pools. The latter finding is not surprising given that proline may be diluted intracellularly by both protein degradation and de novo synthesis (10, 18). Third, the choice of SDS-cacodylate as a less vigorous agent for solubilization of collagen than acetic acid, another common agent for collagen extraction, avoided solubilization of mature collagen. Inclusion of mature, acid-soluble collagen with the newly synthesized pool would have provided an underestimate of the synthesis rate of new collagen. However, the SDS-cacodylate solubilization approach provides the theoretical limitation that a portion of newly synthesized collagen may not enter the mature pool and that an increased synthesis rate of SDS-cacodylate soluble collagen may not always be accompanied by accumulation of mature collagen.

A substantial dilution of proline enrichment in prolyl-tRNA compared with other pools is intriguing. To the extent that intracellular partitioning of proline resembles that of skeletal muscle leucine in which unlabeled leucine diluting leucyl-[13C]tRNA is derived from endogenous protein breakdown rather than an extracellular (i.e., plasma) source (14), the current data suggest a high rate of recycling of proline for collagen synthesis.

We conclude that the fractional synthesis of dermal collagen occurs at rates that are measurable in human subjects over a several hour infusion of stable isotopically labeled proline. Rates are similar to the slow turnover proteins of skeletal muscle and less than abundant fast turnover hepatically derived proteins such as fibrinogen. With appropriate measurement of amino acid isotopic enrichment in tissue rather than plasma precursor pools, the method that we have described may be used to study collagen synthesis in vivo in many human tissues and in many fibrotic human diseases.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of G. C. Ford in providing technical expertise for gas chromatography-isotope ratio mass spectrometry analyses. We appreciate the technical assistance of M. Wagner, the expert nursing care of our subjects provided by the nurses of the University of Illinois at Chicago Clinical Research Center, and the suggestions of A. Furman regarding methods of protein hydrolysis. We are indebted to P. Storrs and B. Jackson for clinical skill and effort in obtaining skin biopsies from our subjects.

    FOOTNOTES

This study was supported by a grant from the University of Illinois at Chicago Campus Research Board and by General Clinical Research Center Grant RR-00585 to the Mayo Foundation.

Current addresses: W. A. El-Harake: Jones Clinic, 110 Ridge Rd., Munster, IN, 46321; and B. Cook, Dept. of Dermatology, Northwestern Univ. School of Medicine, 222 E. Superior, Suite 240, Chicago, IL.

Address for reprint requests: I. G. Brodsky, Univ. of Illinois at Chicago, Section of Endocrinology and Metabolism (M/C 640), 1819 W. Polk St., Chicago, IL 60612-7333.

Received 28 June 1996; accepted in final form 18 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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