Collagen composition and turnover in ocular tissues of the rabbit

Exp,

Eyr Res. (1981)

Collagen

32, 737-745

Composition ROBERT

and Turnover the Rabbit E. LEE

AND PETER

in Ocular

Tissues

of

F. DAVISON*

Department of Fine Structure Research, Boston Biomedical Research Institute. Boston, Massachusetts 02114, U.S.A. and * Department of Ophthalmology, Harvard Medical School, Boston. Massachusetts 02114, U.S.A. (Received 25 July

1980, New

York)

The changes in the collagen composition of the rabbit sclera, cornea and eyelid with maturation were determined. Type I collagen was the major constituent. The cornea, sclera and eyelid contained about 2 X, 7 o/o and 25 o/0 type III respectively after birth, dropping to 0, 0 and 5 ‘$.b in the mature rabbit. The AB collagen in the sclera and eyelid decreased from about 2 Y& and 30x0 to near 0 “4 during the same time period. The AB collagen in the cornea, however, increased from about 5 56 at birth to about 9 0/0 at 4 years of age. A pulse of [3H]proline into the collagen of the cornea of newborn rabbits was used to demonstrate that the collagen on average has a half-life of 50 hr or less. k’py ~x~rds: collagen; cornea; turnover; connective tissue.

1. Introduction The major macromolecular component of the tough corneal-scleral covering of t#he eye is collagen [the human cornea is 70 o/0 collagen by dry weight (Hogan, Alvarado and Weddel, 1971)]. The collagen in the cornea and sclera is arranged in layers of parallel fibrils but with fibrils in adjacent layers at different angles (Hogan et al., 1971). The arrangement forms a meshwork which provides a strong shell for the eye. A young eye enlarges rapidly and since the collagen fibrils of the outer covering are essentially inextensible. production and degradation of collagen must occur. In this investigation the types of collagen of the maturing tissues of rabbit eyes were studied.

2. Materials

and Methods

New Zealand White rabbits were terminated by suffocation in carbon dioxide. The upper and lower eyelids were removed and the eyes were enucleated. The cornea was separated from the sclera by cutting around the cornea at the limbus. The retina was separated from the sclera and the eyelids and sclera were cleaned of as much muscle, fat, hair and choroid as possible. Descemet’s layer was stripped from the corneas of some recently killed rabbits and cows by placing the corneas in 002 M aqueous EDTA and teasing Descemet’s layer off with fine forceps. All operations were carried out at 4%. The tissues were minced and digested in pepsin (equal to @2 o/0of the wet weight of the tissue) in 0.05 M-W&C acid for two 48 hr periods at 4“C. During each period the tissue was homogenized twice. After each 48 hr digestion the suspension was centrifuged 10 min at 26000 x g and the supernatants were saved. After the second digestion the pellet was dialyzed against PO5 M-acetic acid, lyophilized and weighed. The amount of undigested collagens was determined by measuring the hydroxyproline content of the pellet (Switzer and Summer, 1971). The digest supernatants were pooled and an equal volume of 4 M-N&l was added. The collagens which precipitated were pelleted by centrifugation at 12OOOxg. dissolved in 0.05 M-acetic acid, dialyzed against @05 M-acetic acid at 4°C and lyophilized. Collagen types were separated on 6% polyacrylamide gel slabs, with and without 0014-4835/81/060737+09

$01.00/O

717

0

1981 Academic

Press Inc.

(London)

Limited

R. E. LEE

738

AIVI)

1’. F. DAVISOPI‘

mercaptoethanol, run in the sodium dodecyl sulfate electrophoresis buffer system described by Laemmli (1970). After staining the proteins with Coomassie Blue and destaining the gels (Davison, 1978), the protein tracks were scanned on a Joyce-Loebl microdensitometer. The areas under the curves of each collagen type were measured with an Ott planimeter and used to calculate the relative amounts of each collagen type recovered in the precipitate. Incorporation

of tritiated

proline

Proline-L-[2,3-3H] (NET-323, New England Nuclear) in 0.01 N-HCI was dried under a stream of dry nitrogen and redissolved in 914 M-NaCf, 901 M-phosphate, pH 7.4. Fifty&i of [3H Jproline in 50 ,uI of buffered saline were injected into the space between the closed eyelid and cornea of young rabbit pups. After 24 hr, in some of the pups, buffered saline containing cold L-proline (Sigma Chemical Co.) was injected into the same space. The rabbits were terminated at 24 hr intervals and the collagens were isolated. The lyophilized collagens were weighed, dissolved and aliquots were placed in scintillation vials, the vials were warmed to denature the collagens and Bray’s scintillation cocktail was added. The vials were counted in a Searle Mark III liquid scintillation counter.

3. Results The weight of the animals and their eyes, corneas and scleras are presented in Table I. In the 1-8 day rabbit the eye is rapidly growing, approximately doubling in mass over that period. This increase in weight is mirrored in the weights of the cornea and sclera. The collagen composition of the rabbit corneas, scleras and eyelids of different ages is presented in Table II and Fig. 1. The ratios of the collagen types were estimated by densitometry of the Coomassie Blue-stained gel. Type I collagen contributed the al and a2 chains and virtually all the p components as was shown by fractionation experiments (see Hong, Davison and Cannon, 1979). Type AB collagen contributed aA and aB chains in a consistent 1: 2 ratio, and type III collagen contributed a single a-chain whose migration was delayed to an appropriate position clear of other chains in the gel by the delayed application of mercaptoethanol (Sykes, Puddle, Francis and Smith, 1976). In all three tissues type I collagen accounts for most of the collagen, and type III collagen which is a minor component decreases as the animal matures. The AB collagen content of the sclera and eyelid also decreases from 2 days to 4 years but it increases in the cornea. Rabbit corneas stripped of Descemet’s layer and epithelium had about the same collagen composition as the whole cornea (Table II). Bovine Descemet’s layers that were pepsin-digested contained predominantly type I collagen and 12 o/0 AB collagen, similar to the 10 oh found in mature rabbit cornea. Some bands similar to those in the

TABLE

of

Number

animals 2 4 7 4

days days days months 2 years 4 years

I

4 4 2 1 3

1

Body

wt

72.1 g 81.5 g 174g 395 kg 43 kg &15 kg

wet

wt eye

337 mg 393 mg 590 mg 1.93 g 3.72 405

g g

wet cornea

wt (mg)

8.1 9.2 143 71.8 153 124

wet sclera

wt (mg)

26 33. I 54.5 306 521 502

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OF

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TABLE

Distribution

COLLAGEN

II

of collagen

types Type 111%

Cornea 2 days 4 days 7 days 14 days 4 months 2 years 4 years Sclera 2 days 4 days 7 days 14 days 4 months 2 years 4 years Eyelid 2 days 4 days 7 days 14 days 4 months 2 years 4 years Cornea minus Descemet’s layer (2 year rabbit) Calf Descemet’s layer Type IV 12.6 y0

739

Type AB %

93.0 930 934 944 938 91.9 91.8

2.2 2.0 1 .o 0.6 0 0 0

4.8 5.0 56 .50 6% 8.1 9.2

91.0 922 92.9 939 98.0 99.4 1090

7.2 6.2 53 53 1.5 Trace 0

1.8 1.6 1.X 68 0.5 0.6 0

72.1 696 685 786 80.9 94-7 947

24.8 27.X 28.3 192 17.0 49 53

3.1 2.6 3.2 2.2 2.1 0.4 0

91.5

0.6

7.9

746

05

12.3

Some gels showed an apparent preponderance of al (I) chains (particularly in salt-fractionated preparations, see Fig. 1) that could indicate the presence of the or1 (I) trimer. As noted previously (Davison et al., 1979) it can be at most only a minor component and was not included in the table above.

anterior lens capsule collagen (type IV) were also present and comprised up to 12 “lo of the collagen. A digest of four rabbit Descemet’s layers gave about the same distribution of collagens as those found in bovine Descemet’s layers although the number of layers used yielded less than the optimal amounts needed for reliable densitometry. This result makes it improbable that the increasing amount of AB collagen with maturation of the cornea is related to the relative thickening of the Descemet’s layer. The percentage of collagen digested in each tissue was determined by measuring the hydroxyproline content of the undigested tissue. The amount of collagen digested from the corneas varied from 75 y0 to 84 %, that of the scleras from 88 y0 to 99 y0 (the scleras of young animals being almost totally digested) and that of the eyelid from 80 9/o to 90 “‘. Descemet’s layer was essentially completely digested, with an insignificant amount of undigested material being recovered.

2 days O.?M SLID.

2 days

4 days

8 days

I6 days

Rabblt

age

;r

;

FIG. 1. 6 “/b Polyacrylamide gel of collagens digwtecl by pepsin from corneas of different age r&hits. Mercaptoethanol was added approximately 1 hr after starting the gel to delay and resolve the a111 chains. The samples were adjusted so that approximately the same amount of aB chains was present in each. All the samples are of digests of whole tissue except the first,. which is the 0.7 M-Pl’aCI, pH 30 supernatant.

Incorporation

of [3H]proZine

into

collage~us

A single injection of [3H]proline into the space between the closed eyelid and cornea of l-day-old rabbit pups resulted in a reservoir of label that was probably retained for a relatively long period of time. This fact was demonstrated by the progressive uptake of label into the collagen of the cornea, sclera and eyelid (Fig. 2). The amount of label in each of these tissues reached a peak at about 3 days after birth and then dropped off. We infer that the [3H]proline is slowly absorbed and it is not until after

3

7 Days

14 after

injection

Fro. 2. Incorporation of label into the collagen from ocular tissues of rabbits injected with 50&i of [3H]proline into the space between the fused eyelid and cornea. Each point represents average of the counts obtained from six rabbits; the ranges of measured counts are indicated by the bars.

TURR’OVER

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the third day that the level ofremaining free [3H]proline is effectively diluted by proline from the circulation and local protein catabolism. The loss of Label in the collagen from the peak of radioactivity on the third day after injection to the 14th day is rapid, 3.1?,<) being left in the cornea and sclera and 41 y0 in the eyelid on the 14th day. This signifies a rapid turnover of collagen in these tissues. In a second set of experiments the [3H]proline was washed from the space between the cornea and eyelid after 24 hr by injecting and removing 100 ,~l of unlabeled proline at a concentration 100 times that of the original {3H]proline. A second injection of 100 ,~l of unlabeled proline was left in the space between the fused eyelids and cornea. This treatment gave a good pulse of [3H]proline, with the radioactivity in the collagen diminishing continuously after the ‘cold’ chase. Seventy-two hours after the chase 58 91~ of the label in the cornea had been catabolized (Fig. 3) but the semi-log plot is apparently non-linear.

3

.

8

I I

.

I 2

Cornea

I 3

I 4

Days FIG. 3. Incorporation of label into cornea1 collagen of rabbits injected with 50 gCi of [3H]proline at time zero into the space between the fused eyelid and cornea. After 24 hr the label was chased by washing and injecting excess unlabelled proline.

Attempts to determine the turnover of collagen in the eye by injecting [3H]proline int.0 the area between the cornea and closed eyelid in animals older than 5 or 6 days yielded very low rates of incorporation of label into collagen. Before the fifth or sixth day after birth the puncta of the nasolacrimal duct is closed, effectively sealing the r3H]proline into the space between the cornea and fused eyelids and allowing good incorporation of label into the collagen of the eye and eyelid. After the pun&a of the nasolacrimal duct opens the [3H]proline can be flushed almost immediately into the esophagal tract; the incorporation of label into the collagen of the eye and eyelid of these animals fell drastically and made measurements of turnover by this labeling procedure inefficient.

‘iti

K. E. LEE

ANI)

P. F. D.4VISON

4. Discussion The predominant collagen in the cornea, sclera and eyelid of the rabbit is type 1. Maturation of these tissues involves shifts in the other collagen types that make up the minor components. These shifts we have assessed by densitometry of Coomassie blue-stained gels following electrophoresis. Claims for and against the linearity of dye absorption and protein content have been published, but. experiments made in the course of this study and some made previously in this laboratory (Davison: 1978) showed that under selected conditions and with fully destained gels we find on any one slab gel a consistent proportionality between protein loading and the densitometry of the stained bands. Therefore the method provides a useful preliminary survey of the collagen populations and the data in the tables appear to be reproducible to + 10 ‘!&. The reliability of the results depends upon the completeness of the solubilization of the collagen by pepsin and the recovery of the collagen by t,he salting out. On the average. SO;/, of the collagen of the cornea, sclera and eyelid was solubilized by the procedures used, so it is unlikely that the shifts in the collagen compositions noted here were significantly biased by differential extraction. To reinforce this presumption the undigested residues of the tissues were denatured by heating to 1OO’C for 1 min at pH 7.0 and were run on 6% polyacrylamide gels. The large fraction of each residue that was dissolved showed a similar distribution of collagen chains td the pepsinsolubilized material. The recovery of the solubilized collagen by salting out is more difficult to assess. Crystal and his co-workers (e.g. Steinmann, Martin, Baum and Crystal, 1979) have shown that a large fraction of newly synthesized collagen is degraded intracellularly, so the liberation oflabelled hydroxyproline by the cornea in the course of solubilization is not indicative of pepsin degradation of the collagen. On the other hand, from the non-dialyzable, labelled peptides and protein that are solubilized from the cornea more than 80 O/b is precipitated by 2 M-NaCl at pH 3. Much of the material not precipitated must be non-collagenous, so only a small fraction of the collagen can escape precipitation at this step. What does esca.pe may not be representative of the whole. so one or more of the minor collagen types may be depleted in the samples we analyzed. Our results show that type III collagen decreases with maturation in the rabbit cornea, sclera and eyelid. Similar results have been reported by other workers (Epstein, 1974; Shuttleworth and Forrest, 1975; Sykes, Puddle, Francis and Smith, 1976). The chick cornea contains type III collagen up to Hamburger-Hamilton embryological development stage 30 but no detectable type III collagen after this stage (Conrad, Dessau and Von der Mark, 1980; Trelstad and Kang, 1974). Our results with the rabbit cornea, sclera and eyelid are concordant with the generalization that type III collagen is enriched in juvenile tissues. There have been two reports of type 111 collagen in bovine cornea. Schmut (1977) did not give the percentage present and his data have been considered to be inconclusive (Conrad et al., 1980). P raus, Brettschneider and Adam (1979) reported that 20 y0 of the collagen of bovine corneas from animals of unspecified ages was type III. This result is at odds with that of Davison, Hong and Cannon (1979) who found no type III collagen in calf corneas freed of limbal tissue. Our present study is more consistent with the latter investigation, the corneas of rabbit eyes having less than 1 y0 type III collagen by the time the animals’ eyes open, a development stage comparable with the newborn calf.

TURNOVER

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743

The presence of relatively large amounts of AB collagen in bovine corneas has been shown by Davison et al. (1979) and is suggested by the studies of Freeman (1978) on rabbit corneas. The rabbit corneas in this study showed an increasing amount of AB collagen with maturation. It is known that Descemet’s layer of the cornea increases in thickness with age (Hogan et al., 1971; Prince, 1964) and that Descemet’s layer contains AB collagen (referred to as type VI in Davison and Cannon, 1977). Analysis showed, however, that the increase in AB collagen in the cornea could not be attrbuted to growth of this layer. The percentage of AB collagen in Descemet’s layer was only slightly higher than in the stroma. Our unpublished studies on sectioned bovine cornea1 stroma show a small gradient in AB content across the thickness of the stroma. Thus, overall we find AB collagen comprises between 7 y0 and 12 y0 of the collagen in the cornea. Other studies have associated relatively high concentrations of AB collagen with placental (Bailey, Sims, Duance and Light, 1979; Burgeson, El Adli, Kaitila and Hollister, 1976; Hong, Davison and Cannon, 1979; Sage and Bornstein, 1979) or juvenile tissues (Deyl, Macek and Adam, 1979). Our findings appear to be the first report of an increase in AB collagen content with age in any tissue. Descemet’s layer is usually referred to as a basal lamina (basement membrane) (e.g. Kefalides, 1970; Dehm and Kefalides, 1978; Kresina and Miller, 1979). Descemet’s layer is much thicker (3-12 pm in humans) than epithelial basal lamina (usually 50 nm). Basal lamina usually contain type IV collagen (Kefalides, 1970; Glanville, Rauter and Fietzek, 1979) although this term is applied to at least two a-chains and possibly several collagens (Crouch, Sage and Bornstein, 1980; Dixit and Kang, 1979). Several of these collagen chains were found in this study in Descemet’s layer, and we measured type IV collagen by the content of C and D bands in mercaptoethanolreduced samples (Dixit andKang, 1979). It is improbable that the paucity of type IV we found in Descemet’s layer is due to incomplete digestion of the material, but it, may be underestimated if the recovery of the C and D chains by salting out was incomplete or if some of the chains were degraded by the pepsin. Nevertheless, the large amounts of type I and AB collagens suggest that Descemet’s layer cannot be regarded as a typical basal lamina. Since the collagen populations in the stroma and Descemet’s layer differ only in the content of type IV chains in the latter, their marked difference in ultrastructural morphology may perhaps be ascribed to the non-collagen components. The rapid turnover of collagen in the scleral-cornea1 covering is presumably necessary to allow growth of an organ covered by an inextensible shell. This rapid turnover can be seen in the data on the degradation of labelled collagen in the cornea where 50 ‘/” of the pulse of [3H]proline was lost in less than 50 hr in the young rabbit pup. The rate of degradation of the labelled collagen is probably greater since some of the [3H]proline released by degradation of the labelled collagen may be reutilized. The measurement of turnover of rat skin collagen using [3H]proline alone underestimates degradation rate by about 50% (Jackson and Heininger, 1975). If this reincorporation applies to the cornea, the half-life of more than 50% of the cornea1 collagen in the young rabbit eye could be as low as 24 hr. The semi-log plot of the data is non-linear, however, so some of the fibrils survive substantially longer. Our estimates of turnover rate may be challenged on the grounds that pepsin digestion may degrade selectively nascent molecules so that they are not recovered by salting out. If this is so, the measured incorporation of proline into collagen after 24 hr may be too low. If this is true, collagen turnover may be even faster than we

744

R. E. LEE

ANL) I’. F. DAVISON

have estimated. In an earlier study Coleman, Herrmann and Hess (1965) report a very slow turnover of embryonic chick cornea1 stroma, but the label was not chased from the tissue so the turnover rate must be underestimated. Collagen has a half-life of about 4 weeks in young rat or mouse skin (Gerber, Gerber and Altman, 1960; Ohuchi and Tsurufuji, 1970; Klein and Chandrarajan,, 1977). Collagen is almost inert in tendon (Neuberger, Perrone and Slack, 1951) and intestine (Klein and Chandrarajan, 1977) while having a half-life of 50 days in muscle and 30 days in liver (Gerber et al., 1960). The half-life of 24-58 hr estimated for cornea1 collagen in 2-day-old rabbit pups in this study is the shortest obtained for any tissue except the periodontal ligaments (Sodek, 1976). The turnover rates in the different collagen types in the cornea, sclera and eyelid will be presented in a subsequent paper. ACKNOWLEDGMENTS We are grateful for the excellent technical supported by N.1.H grant EY 2213.

assistance

of Sue Ingram.

This study was

REFERENCES Bailey, A. J., Sims, T. J., Duance, V. C. and Light, N. D. (1979). Partial characterization of a second basement membrane collagen in human placenta. FEBS Letter8 99, 361-5. Brown, R. A., Shuttleworth, C. A. and Weiss, J. B. (1978). Three new a-chains of collagen from a non-basement membrane source. Biochem. Biophya. Res. Commun. 80, 866-72. Burgeson, R. E., El Adli, F. A., Kaitila, I. I. and Hollister, D. W. (1976). Fetal membrane collagens: identification of two new collagen alpha chains. Proc. Natn. Acud. Sci. U.S.A. 73, 2579-83. Coleman, J. R., Herrmann, H. and Hess, B. (1965). Biosynthesis of collagen and non-collagen protein during development of the chick cornea. J. Cell Biol. 25, 69-78. Conrad, G. W., Dessau, W. and von der Mark, K. (1980). Synthesis of type III collagen by fibroblasts from the embryonic chick cornea. J. Cell Biol. 84, 501-12. Crouch, E., Sage, H. and Born&in, P. (1980). Structural basis for apparent heterogeneity of collagens in human basement membranes: Type IV procollagen contains two distinct chains. Proc. Natl. Acud. Sci. U.S.A. 77, 745-9. Davison, P. F. (1978). Bovine tendons. Aging and collagen crosslinking. J. Biol. Chem. 253, 563541. Davison, P. F. and Cannon, D. J. (1977). Heterogeneity of collagens from basement membranes of lens and cornea. Exp. Eye Res. 25, 12937. Davison, P. F., Hong, B.-S. and Cannon, D. J. (1979). Q uantitative analysis of the collagens in the bovine cornea. Exp. Eye Res. 29, 97-107. Dehm, P. and Kefalides, N. A. (1978). The collagenous component of lens basement membrane. J. Biol. Chem. 253, 6686-6. Deyl, Z., Macek, K. and Adam, M. (1979). Collagen aA and aB chains constitute two separate molecular species. Biochem. Biophys. Res. Commun. 89, 627-34. Dixit, S. N. and Kang, A. H. (1979). Anterior lens capsule collagens: cyanogen bromide peptides of the C chain. Biochemistry 18, 568692. Epstein, E. H., Jr (1974). [al(III)], H uman skin collagen. J. Biol. Chem. 249, 3225-31. Etherington, D. J. (1977). Stability of rat skin collagen during recovery from under-nutrition. Biochem. J. 168, 579-81. Freeman, I. L. (1978). Collagen polymorphism in mature rabbit cornea. Invest. OphthaZmoE. 17, 171-7. Gerber, G., Gerber, G. and Altman, K. I. (1960). Studies on the metabolism of tissue proteins. J. Biol. Chem. 235, 2653-6. Glanville, R. W., Rauter, A. and Fietzek, P. P. (1979). Isolation and characterization of a native placental basement-membrane collagen and its component c( chains. Eur. J. Biochem. 95, 383-9.

TURNOVEROFOCULAR~'OLLAGEN

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Hogan, M. J., Alvarado, J. A. and Weddel, J. E. (1971). Histology of the Human Eye. Pp. 687. W. B. Saunders Co., Philadelphia. Hong, B.-S., Davison, P. F. and Cannon, D. J. (1979). Isolation and characterization of a distinct type of collagen from bovine fetal membranes and other tissues. Biochemistr?y 18, 4278-82. Jackson, S. H. and Heininger, J. A. (1975). Proline recycling during collagen metabolism as determined by concurrent iaO,- and 3H-labeling. B&him. Biophys. Acta 381, 359-67. Kefalides, N. A. (1970). Chemical properties of basement membranes. Znt. Rev. Exp. I’&. 10, l-39. Klein, L. R. and Chandrarajan, J. (1977). Collagen degradation in rat skin but not in intestine during rapid growth : Effect on collagentypes I and III from skin. Proc. N&Z. Acad. f&-i. IJ.8.A. 74, 14369. Kresina, T. F. and Miller, E. J. (1979). Isolation and characterization of basement membrane collagen from human placental tissue. Evidence for the presence of two genetically distinct collagen chains. Biochemistry 18, 3089-97. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 68&5. Neuberger, A., Perrone, J. C. and Slack, H. G. B. (1951). Th e relative metabolic inertia of tendon collagen in the rat. B&hem. J. 49, 199204. Ohuchi, K. and Tsurufuji, S. (1970). Degradation and turnover of collagen in the mouse skin and the effect of whole body x-irradiation. Biochim. Biophys. Acta 208, 475-81. Praus, R., Brettschneider, I. and Adam, M. (1979). Heterogeneity of the bovine cornea1 co]lagen. Exp. Eye Res. 29. 46%77. Prince, J. H. (1964). Dimensions of the eye, cornea, trabecular region, sclera. In The Rc&it in Eye Research (Ed. Prince, J. H.). Pp. 86-139. Thomas, Springfield, Illinois. Sage, H. and Bornstein, P. (1979). Characterization of a novel collagen chain in human placenta and its relation to AB collagen. Biochemistry 18, 3815-22. Schmut, 0. (1977). The identification of type III collagen in calf and bovine cornea and srlera. Exp. Eye Res. 25, 505-9. Shuttleworth. C. A. and Forrest, L. (1975). Changes in guinea-pig dermal collagen during development. Eur. J. Biochem. 55, 391-5. Sodek, J. (1976). A new approach to assessing collagen turnover by using a micro-assay. Biochem. J. 160, 243-6. Steinmann, B. U., Martin, G. R., Baum, B. I. and Crystal, R. G. (1979). Synthesis and degradation of collagen by skin fibroblasts from controls and from patients with osteogenesis imperfecta. FEBS Letters 101, 269-72. Switzer, B. R. and Summer, G. K. (1971). Improved method for hydroxyproline analysis in tissue hydrolyzates. Anal. Biochem. 39, 487-91. Sykes, B., Puddle, B., Francis, M. and Smith, R. (1976). The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem. Biophys. Ilcx /!ommun. 72. 1472-80. Trelstad, R. L. and Kang, A. H. (1974). Collagen heterogeneity in the avian eye: lens. vitreous body, cornea, and sclera. Exp. Eye Res. 18, 39ri-406.