Direct measurement of the rate of collagen synthesis in skin

Direct measurement of the rate of collagen synthesis in skin

341 CLINICA CHIMICA ACTA DIRECT MEASUREMENT OF THE RATE OF COLLAGEN SYNTHESIS IN SKIN HARRY R. KEISER Experimental (Received AND Therape...

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341

CLINICA CHIMICA ACTA

DIRECT

MEASUREMENT

OF THE

RATE

OF COLLAGEN

SYNTHESIS

IN

SKIN

HARRY

R. KEISER

Experimental (Received

AND

Therapeutics

November

ALBERT

Branch,

S JOERDSMA

National

Heart Institute,

Bethesda,

Md. 20014 (U.S.A.)

4. 1968)

SUMMARY

A method for measuring collagen synthesis using punch biopsies of human and rat skin is presented. Minces of dermis are incubated with [14C]proline in KrebsRinger buffer for 3 h and the specific activity of radioactive proline determined on either whole dermis or a hot 5% TCA extract of dermis. The proline incorporation observed is shown to involve protein (collagen) synthesis. The method is practical, yielding significant and reproducible results. The level of proline incorporation correlates well with growth in man and animals. Preliminary findings in the skin of patients with scleroderma and other diseases are reported.

The unique occurrence of hydroxyproline (Hyp) in collagen has served as the basis for numerous studies of collagen metabolism in man112. Abnormalities in collagen metabolism have been detected in a variety of clinical disorders, either by alterations in the excretion rate of hydroxyproline peptides in urine, or by changes in the physicochemical state of collagen in human dermis, or both. Although it is apparent that changes in rates of both collagen synthesis and degradation contribute to results obtained, it has been difficult to assess these two variables independently with existing methods. In the case of urinary hydroxyproline, the relative contribution of various body collagens to urinary products is not known precisely though abnormalities have generally been related to changes in the metabolism of collagen in bone since the majority of body collagen exists in this locus. The extent to which changes in the status of collagen in tissue (e.g. a decreased level of acid-soluble collagen in the skin of patients with scleroderma3) reflect altered synthesis or degradation is unknown. Of possible approaches to selective study of collagen synthesis in man, incorporation of precursor amino acids into collagen during incubation of biopsy material seemed most feasible to us. Hydroxyproline cannot be used for this purpose since all the hydroxyproline in collagen arises via hydroxylation of proline (Pro) after the latter has been incorporated into the polypeptide chain+. Accordingly, proline was employed in these studies; the preliminary data to be presented indicate that it is possible to measure incorporation of radioactive proline into collagen of dermal C&z. Chim. Acta,

23 (1969) 341-346

342

KEISER, SJOERDSMA

minces in rat and man and that the rate of incorporation may serve as a suitable index of collagen synthesis ilz viva. METHODS

Priwiples In the following procedures, radioactive proline is incubated with minces of rat or human skin. Radioactive collagen is then prepared for analysis either by (I) multiple washings and dialysis to remove excess radioactive proline, or by (2) extraction into hot trichloracetic acid (TCA) followed by dialysis. The collagen so prepared is then hydrolyzed and its content of proline and hydroxyproline and radioactivity in proline measured. The ratio of Pro to Hyp affords an index of purity, the ratio in highly purified collagen being about 1.4 (ref. 5). Results are expressed as disint.1 min/ktmole of proline, i.e., the specific activity of proline. This provides an index of collagen synthesis in terms of total collagen present. Incorporated proline is also expressed in disint. /min/mg wet weight of dermis.

Rat skin and human skin were obtained either by punch biopsy or at autopsy if the latter was performed within 6 h of the time of death. Epidermis and subdermis were dissected away and the dermis was minced finely and weighed. From 25 to IOO mg of minced dermis was placed in IO ml of modified Krebs-Ringer buffer, pH 7.5, with IO mN glucose and 0.5 PC of L-[l*C]proline (u.1. New England Nuclear Corp., 206 mC/mmole). Samples were incubated in open beakers in a metabolic shaker at 37O under an atmosphere of 95% O,S~/~ CO,. Unless otherwise stated the period of incubation was for 3 h. At the end of the incubation the samples were I

.?

70

r

01

I

1

I

25

50

75

TISSUE

I loo

(mg)

Fig. 1. Proline incorporation into collagen as a function of the duration of incubation. Each point is the average of 3 determinations using rat skin and the hot TCA-extraction procedure. Fig. 2. Proline incorporation into collagen as a function of the amount of tissue. Each point is the average of at least a determinations on adult rat skin by the procedure employing dialysis only {see text).

C&z. Chit% Acta, 23 (1969) 341-346

COLLAGEN SYNTHESIS IN SKIN

343

either chilled or TCA was added. Chilled samples were then centrifuged in a refrigerated centrifuge at 30000 6 for 15 min and the supernatant discarded. The pellet was washed twice with IO ml of buffer, resuspended in 5 ml 0.15 M acetic acid and dialysed for at least 21 h against 2 changes of 200 volumes of 0.15 M acetic acid at 4”. To achieve higher specificity, other samples were treated with cold 5% TCA to precipitate proteins, the precipitates washed twice with IO ml cold 50/d TCA, and the collagen contained therein extracted twice with a total of IO ml hot (100”) 5% TCA for 60 min. The “hot” TCA extracts were then dialyzed as above. In both procedures, the contents of dialysis bags were washed into tubes and hydrolyzed for 20 h in 6 N hydrochloric acid at 120~. Specific radioactivities of proline were determined by the method of Peterkofsky and Prockops. Counting of radioactivity was performed in a Packard Series #3375 scintillation counter with automatic external standardization. Counting efficiency was 70 to 87% with from o to 3% quenching. Samples were counted for up to 60 min to achieve greater than 95Oi;i confidence levels. RESULTS

Preliminary

observations

Only minces of fresh dermis incorporated radioactive proline into collagen. No incorporation could be demonstrated in homogenates of either human or rat dermis. The incorporation of radioactive proline into collagen was directly related to the duration of incubation (Fig. I) and to the amount of tissue used (Fig. 2). Proline incorporation increased with increasing concentrations of glucose in the incubation medium (Table I). The glucose concentration used in subsequent studies was IO mM because higher concentrations yielded significantly higher blank values. Freezing and thawing destroyed more than 90% of the activity and skin so treated serves as a convenient blank sample (Table I). Preincubation for 15 min with Puromycin at IO-~ M was shown repeatedly to produce more than 84% (average 95 %) inhibition of proline incorporation into both human and rat dermal collagen. Skin samples with the highest activity also showed the greatest percentage inhibition with both freezing-thawing and Puromycin. No significant radioactivity could be found in collagen hydroxyproline from either human or rat dermis even when all of the known requirements for proline hydroxylation were included in the incubation medium’. Addition of cc,cr-dipyridyl (IO-~ M), an iron chelator and known TABLE

I

EVALUATION

OF FACTORS

Variable Glucose:

AFFECTING -

PROLINE

INCORPORATION

INTO

COLLAGEN

Prolilae ~~~o~~o~ation as 0/0 ofco&oE* IO mM 50

mM

100

mM

Freeze-thaw Puromycin : IO-~M a,a-Dipyridyl: IO-~M * Control values were taken as ~00% ; all values represent the average of at least 3 determinations. The control incubation mixture is described in the text and was IO mM with respect to glucose. Cl&. Chim. Acta,

23 (1969) 341-346

KEISER, SJOERDSMA

344 inhibitor of proline hydroxylase, proline incorporation (Table I).

to the standard

incubation

mixture

failed to alter

Activity of rat and human dermis Proline incorporation into collagen of dermis from 14 rats of varying

ages was

studied to evaluate the methods presented (Table II). Highest levels of incorporation occurred in dermal samples from newborn (704 disint./min/pmole Pro or 8.8 disint.1 min/mg tissue) and young rats (324 disint./min/pmole Pro or 6.4 disint./min/mg tissue). Proline incorporation decreased near the end of the first month of life and remained at a low level thereafter. The relative purity of the final collagen in which proline incorporation is measured determines the specificity of the assay for collagen synthesis. The Pro-Hyp ratio was 5.8 in the skins of rats I to 9 days old, decreasing to 1.6 in adult animals. Thus, the high levels of proline incorporation noted in newborn rats might be due to a large amount of non-collagen protein synthesis. However, the Pro-Hyp ratio in hot TCA soluble collagen was 1.3-1.4 regardless of the age of the animal (Table II) and proline incorporation into this more purified collagen usually accounted for more than 909/b of that found in whole dermis. Thus, while significant amounts of non-collagen proteins are apparently present in the skin of young rats, the vast majority of proline incorporation observed is occurring in collagen. It was also observed that the rate of proline incorporation related to the proline content of the skin and paralleled the observed

was inversely rate of growth

of the animals. Proline incorporation was measured in 24 patients (Table III). It was relatively high in the skin of a 6-year old child (69 disint./min//rmole Pro) and was at a low level by age 21 years or older, averaging 4.5 disint./min/qole Pro in adult subjects. TABLE

II

Number of animals

47

days

3 2 4 5

I-9

‘3-17 22-27 339-120

Total collagen disint. lmin disint. Imix Pro-Hyp

Hot TCA-soluble collagen disint./min disint./min Pro-Hyp

pM proline

ratio

piI4 proline

mg tissue

ratio

8.8 6.4 0.4

5.8 3.8 2.3

7.0 5.7

0.2

1.6

696 302 25 6

I.3 1.4 1.4 1.4

rng

704 324 40 5

tissue

0.2 0.1

* Proline incorporated is expressed as disint./pmole proline in the final assay mixture disint./min/mg wet weight of dermis used in incubation. Samples contained I .o to 13.9 ,uM and 35 to 3500 net counts/mix T.4BLE

and as proline

III

INCORPORATION

OF

RADIOACTIVE

Condition

NO.

Normal growth Active acromegaly Cushing’s syndrome Scleroderma Miscellaneous* * Includes

PROLINE

I

2 I

I I3 7

INTO

Age (Yr.)

HUMAN

DERMAL

Mean i

6.15

disint.lmin/pM

Proline

69311 13

49

50 28-65 21-59

SEM

COLLAGEN

IO

4.8 & 0.6 3.9 i 0.5

9. 9, 7, 6, 5, 5, 4. 4, 4. 3. 3. 2, 2 5, 5, 5, 4. 4, 2. 2

normal subject and patients (No.) with: inactive acromegaly (2), lupus erythematosus

(I), myositis ossificans progressiva (I), rheumatoid arthritis (I) and hypertension (I). C&z. Chim. Acta, 23 (1969)

341-346

COLLAGEN

SYNTHESIS

345

IN SKIN

In a patient with active acromegaly, aged 49 years, the proline incorporation (13 disint./min/,umole) was 2.5 times greater than that in other adults and slightly higher than that of a 15 year old boy (Table II). High levels of proline incorporation were noted also in one patient with Cushing’s syndrome, and three patients with scleroderma. In all human subjects the Pro-Hyp ratio was 1.3 to 1.5 and was unaltered by hot TCA extraction of the collagen. Also, the amount of proline incorporation into human dermal collagen was similar using the two methods of assay. DISCUSSION

Incorporation of radioactive proline by dermal minces appears to be an active process requiring fresh tissue. Inhibition of this incorporation by Puromycin is strong evidence that the reaction requires protein synthesis. Inhibition by freezing and thawing and the inability to find active proline incorporation in homogenates plus the stimulation of incorporation by increasing glucose concentration all suggest that intact cells, presumably fibroblasts, are required. Hot 5% TCA selectively extracts collagen and not other proteinss. The demonstration that the Pro-Hyp ratio of the hot TCA-extractable material is the same as that of purified collagen is evidence that the proline incorporation observed is into collagen. The finding of major agreement between proline incorporation determined on whole dermis and on hot TCA-soluble collagen in the skin of young rats in spite of the large reduction of Pro-Hyp ratio on TCA extraction, is a further indication that the proline incorporation observed is attributable to collagen synthesis. The problem of the specificity of proline incorporation into collagen in this method cannot be answered precisely since we could not detect any conversion of incorporated proline into hydroxyproline. However this is not surprising since the amount of radioactivity incorporated in this system is small, representing 0.005 to 0.15% of the total radioactivity in the incubation mixture. Nimni et aL9 could not detect any radioactive proline or hydroxyproline in citrate-soluble or insoluble fractions of rabbit skin collagen even by using 6 times more radioactivity and I g of tissue. The feasibility of this method for measuring collagen synthesis is adequately demonstrated by the number of counts obtained, the range of values in adults, and the relatively large changes seen during growth. The method is reliable, yielding reproducible results on serial biopsies from the same person or animal. The good correlation between increased levels of proline incorporation and active growth seen in young rats, children and an adult with acromegaly further validates the method as an indicator of collagen synthesis rate. Our findings in patients with various diseases are highly preliminary. The proline incorporation in dermis from patients with scleroderma was highest in those with very active disease. There was also greater proline incorporation into affected forearm skin when compared to unaffected gluteal skin from the same patient in some patients with scleroderma. These findings suggest that active scleroderma has an increased rate of collagen synthesis. However, the differences were not sufficiently consistent nor of significant magnitude to prove this. The increased level of proline incorporation found in one patient with Cushing’s syndrome is opposite to reported corticosteroid effects on collagenlo. C&z. Chim.

Acta, 23 (969) 341-346

KEISER,

346

SJOERDSMA

The method should be applicable in further studies of dermal collagen synthesis in a variety of clinical conditions. It can be applied also to other tissues, especially those rich in collagen. However, one must be careful to ascertain that the proline incorporation observed is into collagen protein. This can be resolved by use of hot TCA extraction

and subsequent

determination

reported should be a useful tool in further of collagen in man.

of Pro-Hyp

ratio.

Thus the method

studies of the physiology

and pathology

REFERENCES I A. SJOERDSMA, S. UDENFRIEND, H. KEISER AND E. C. LEROY, Ann. Internal Med., 63 (1965) 672. 2 D. J. PROCKOP AND K. I. KIVIRIKKO, Ann. Internal Med., 66 (1967) 1234. 3 E. D. HARRIS AND A. SJOERDSMA, Lance& ii (1966) 707. 4 B. PETERKOFSKY AND S. UDENFR~END, J. Bid. Chem., 238 (1963) 3966. 5 R. FLEISCHMAJER AND L. FISHMAN, Nature, 205 (1965) 264. 6 B. PETERKOFSKY AND D. I. PROCKOP, Anal. B&hem., 4 (1962) 400. 7 J. J. HUTTOX, A. L. TAP~EL AND S. UDENFRIEND, A&z.‘BioCh~m. Biophys., 118 (1967) 231. 8 S. M. FITCH, M. L. R. HARKNESS AND R. D. HARKNESS, Nature, 176 (1955) 163. 9 M. E. NIMNI, E. DEGUIA AND L. A. BAVETTA, Biochem. J,, 102 (1967) 143. IO Q. T. SMITH, Biochem. Pharmacol., 16 (1967) 2171.

Clin. Chim. Acta. 23 (1969) 341-346