Age-related Changes in the Concentration of Hydroxypyridinium Crosslinks in Functionally Different Skeletal Muscles

Matrix Vol. 1211992, pp. 291-296 © 1992 by Gustav Fischer Verlag, Stuttgart

Age-related Changes in the Congentration of Hydroxypyridinium Crosslinks in Functionally Different Skeletal Muscles H. PALOKANGAS 1 , V. KOVANEN 1 ,A. DUNCAN 2 andS. P. ROBINS 2 1 2

Department of Health Sciences, University of]yvaskyla, Finland and The Rowett Research Institute, Aberdeen, Scotland.

Summary High-performance liquid chromatography methods were developed to measure the concentration of hydroxypyridinium crosslinks in the intramuscular collagen and tendinous parts of functionally different skeletal muscles at different ages. A significant increase in pyridinoline concentration took place during maturation reaching 0.32 ± 0.07 (moVmol collagen) in soleus (slow plantar flexor) and 0.28 ± 0.07 in plantaris (fast "mixed" plantar flexor) at the age of 4 months. In medial and lateral gastrocnemius (fast "mixed" plantar flexors) the pyridinoline concentrations (moVmol collagen) reached 0.24 ± 0.06 and 0.19 ± 0.04, respectively, similar to those in both the extensor digitorum longus (fast "mixed" dorsi flexor) and rectus femoris (fast "mixed" knee extensor) muscles, but higher than in the fast "mixed" dorsi flexor muscle, anterior tibialis (0.11 ± 0.05 moVmol). By comparison, pyridinoline concentrations of 0.33 moV mol collagen (± 0.10) was measured from longissimus dorsi, a slow-twitch back posture muscle. After maturation the most significant increase in pyridinoline concentration was measured in soleus and gastrocnemius muscles. No differences in the crosslinking between different parts of muscle belly were noticed at any time-point. However, significantly fewer pyridinoline crosslinks were found in tendinous parts of soleus, extensor digitorum longus and anterior tibialis than in intramuscular collagen. The concentration of pyridinoline crosslinks tended to be highest in slow-twitch postural muscles, soleus and longissimus dorsi, and generally higher in plantar flexors which are exposed to higher stretch than dorsal flexors. The reasons for the unexpectedly low concentrations of pyridinoline crosslinks in the tendinous parts of muscles remain to be clarified. Key words: collagen crosslinking, muscle, pyridinoline. Introduction

Crosslinks formed between lysine residues of neighbouring collagen molecules are known to be essential for maintaining proper structure and strength of collagen fibrils. These crosslinks are derived from the interaction of hydroxylysine with either lysine aldehyde to form aldimine bonds or with hydroxylysine aldehyde to form oxo-imine bonds (Bailey et al., 1974). The bifunctional crosslinks stabilizing the newly-formed collagen disappear during maturation and are thought to undergo spontanous struc-

tural modification into stable form of crosslinks (Robins et al.,1973). Pyridinoline, a fluorescent 3-hydroxypyridinium crosslink first isolated by Fujimoto and coworkers (Fujimoto et al., 1978), is one of the few well-characterized mature crosslinks. It appeared to be a trifunctional maturation product of oxo-imine bifunctional crosslink (Siegel et al., 1982), although the precise mechanism of formation and three-dimensional location is still uncertain (Robins, 1983; Wu and Eyre, 1984). A pyridinoline analogue, deoxypyridinoline, involving a lysine rather than hydroxylysine

292

H. Palokangas et al.

residue in the helix, has later been identified (Ogawa et aI., 1982) and has been shown to be present primarily in bone collagen (Eyre et aI., 1984a). In tissues where hydroxylation of telopeptide lysine does not occur, such as skin, no oxo-imine intermediates and consequently no pyridinium crosslinks are formed. The aldimine forms of bifunctional crosslinks in these tissues are converted to non-reducible compounds such as histidino-hydroxylysinonorleucine (Yamauchi et aI., 1987). During maturation the mechanical stiffness and tensile strength of tissues increases, while the visco-elastic properties decrease. During ageing the mechanical stiffness increases further but the tensile strength slowly declines (Vogel, 1976). The influence of the known mature lysinebased collagen crosslinks on these changes remains controversial, although the increase in pyridinoline concentration has been reported to correlate well temporally with the changes in the mechanical properties of tissues during maturation (Moriguchi and Fujimoto, 1978). As a part of the parallel elastic component in muscle, intramuscular collagen affects the passive mechanical properties of muscles and is thought to contribute to the contractile properties of muscles through the functioning of the muscle spindle apparatus (Kovanen, 1989). Slow-twitch soleus muscles have been shown to contain more collagen and to be mechanically stiffer than the fast-twitch rectus femoris muscles, which can be considered as a specialization of muscles to their function; higher collagen concentration and mechanical stiffness could benefit the slow-twitch posture maintaining muscle adapted to continous static and/or eccentric actions (Kovanen et aI., 1980; Kovanen and Suominen, 1989). During maturation the concentration of soluble collagen in muscles decreases rapidly, while tensile strength and mechanical stiffness are increasing. During ageing the mechanical stiffness is increasing further especially in soleus muscle (Kovanen and Suominen, 1989). The purpose of the present work was to study variations with age in the concentrations of hydroxypyridinium crosslinks in intramuscular collagen from a wide range of rat skeletal muscles subjected to different functional demands.

Materials and Methods Materials Acetonitrile ('Hypersolv') and 2-mercaptoethanol was obtained from BDH Chemicals, Poole, Doset, UK. 0phthaldehyde (OPA), iodoacetamide and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) were purchased from Sigma Chemical Co., Poole, Dorset, UK. The ion pair agent used, heptafluorobutyric acid, was a product of Pierce Chemicals, Chester, UK.

Methods Preparation oftissues Male rats (Rowett Hooded strain) aged 1,2,3,4,6 and 12 months were used; 6 animals were used for each agegroup. The animals were killed by ether inhalation and left hindlimbs were removed for dissection. Musculus rectus femoris (MRF), medial and lateral gastrocnemius (MG, LG), plantaris (PL), musculus soleus (MS), extensor digitorum longus (EDL) and anterior tibialis (AT) were carefully removed with tendons. Also, for comparison a small muscle sample from the longissimus dorsi (LD), a back postural muscle, was prepared for analyses. After removing the adhering fat the available tendons were dissected from the tendon-muscle junction. Separately, the white tendinous insertion reaching over the muscle belly of some muscles (MS, EDL) was also dissected. Epimysium sheet covering the whole muscle was removed carefully with a scalpel, as well as the tendinous sheet which was occasionally present inside the muscle. To study different parts of muscle bellies, some muscles were also divided into proximal, medial and distal parts, and each section was analysed separately. The prepared samples were kept at - 20°C until analysed. For the analysis of collagen content by hydroxyproline, weighed samples of the freeze-dried tissues (0.2-0.5 g) were hydrolyzed in 6 M HCI (1 ml) at 110°C for 24 hours in sealed tubes.

Sample preparation for crosslink analyses Part of the hydrolysate was subjected to partition chromatography according to the method of Black et al. (1988) for the analysis of hydroxypyridinium crosslinks. Small columns (0.7 X 5 em) packed with 5 ml of a slurry (5% w/v) of CFl cellulose (Whatman, Maidstone, Kent, UK) in butan-1-01:acetic acid:water 4:1:1 (mobile phase) were washed with mobile phase (5 ml). The hydrolysate (0.5 ml) was mixed with glacial acetic acid (0.5 ml), CFlslurry (0.5 ml) and butan-1-o1 (2 ml) and applied to the column. The hydrolysis tube was washed with mobile phase (3 ml) and the washing applied to the column. The columns were then washed with a total of 15 ml (3 x 5 ml) of mobile phase and the washings were discarded. Finally, the crosslinks were eluted from the column with water (5 ml) into conical tubes from which the lower aqueous layer was carefully aspirated and freeze-dried. The freezedried samples were dissolved in an appropriate amount of loading buffer (1% HFBA) for analysis by HPLC. Recoveries of pyridinium crosslinks from the CF1 fractionation procedure were greater than 90% as determined for each tissue type by spiking the hydrolysates with standard pyridinoline.

Hydroxypyridinium Crosslinks in Skeletal Muscles Sample preparation for hydroxyproline analyses

Hydroxyproline analyses were carried out on a second portion of the hydrolysate using an OPAlFMOC-CIderivatization system similar to that described previously (Teerlink et al., 1989). After neutralization of the hydrolysate with 3 M NaOH and an appropriate dilution with water, a portion (0.8ml) was buffered to pH9.0 by the addition of 0.8 M borate buffer (0.1 ml). Derivatization of the primary amino acids was achieved by the addition of 100 J-li of o-phthaldehyde (50 mg OPA, 26 J-li mercaptoethanol/ml acetonitrile). O-phthaldehyde was allowed to react for 30 s after which time 100-J-l1 iodoacetamide (140 mg/ml acetonitrile) was added. After a further 30 s, 300 J-li of FMOC-CI in acetone (1.29 mg/ml) was added and the sample was immediately diluted to 5 ml with 0.8 M borate buffer, pH 6.2. Suitable dilutions were made and the samples were analyzed by the HPLC system reported below. High-performance liquid chromatography

The HPLC analyses were performed by a two-pump gradient system incorporating Gilson Model 302 pumps, a Gilson 231 autosampler, a Shimadzu RF 530 fluorometer and a Shimadzu C-R3A integrator. For both analyses Hypersil ODS columns (5 J-l, 250 x 4,6 mm) and sample loops of 100-J-l1 volume were used. The flow rate used was 1.0mlJmin. For hydroxyproline analysis, solvent A comprised 50 mM sodium acetate buffer, pH 4.3, made 30% with respect to acetonitrile, and solvent B was 50% acetonitrile in the sodium acetate buffer. A linear gradient from 2% to 18% solvent B over 10 min was used: hydroxyproline eluted at 6.0 min and was detected by the fluorescence emission of the FMOC-derivate at 313 nm (excit 260 nm). The crosslink samples in 1% HFBA were run by HPLC using an acetonitrile gradient similar to that reported previously (Eyre et al., 1984a) and the natural fluorescence of hydroxypyridinium crosslinks (em 400 nm, excit 295 nm) was used for the detection. The preparation of the standards used for the quantitation of crosslinks has been reported ealier (Black et al., 1988). Statistical methods

The statistical significance of the differences was determined using Student's t-test (unpaired).

Results

The age-related changes in the concentration of pyridinoline crosslinks in muscle belly, tendinous insertion and tendons are summarized in Fig. 1. For all of the muscle

293

types, the concentrations of pyridinoline in the central, tendon-free portions of the muscle were significantly increased up to 3 months of age. With advancing age the concentration of pyridinoline generally increased in the muscles, most significantly in MS and MG. At the age of 4 months the concentration of pyridinoline was highest in MS, PL and LD without significant differences between these muscles. On the other hand, MRF, EDL and AT formed a group of three muscles with the lowest values for pyridinoline. Significantly lower concentrations of pyridinoline were found in the tendons and tendinous insertions compared to the intramuscular collagen of MS, EDL and AT at the age of 4 months. The concentrations of pyridinoline were higher in the tendons of MS compared to the tendons of EDL and AT. In EDL, the concentration of pyridinoline was much lower in the tendinous insertions than in the tendons. By contrast, similar concentrations of pyridinoline were present in the tendons and tendinous insertions of MS, both of which increased linearly after the age of 4 months. Very low concentrations of the pyridinoline analogue, deoxypyridinoline, were found in all the tissues studied « 0.05 mol/mol collagen). Although the small amounts present made it difficult to quantify this crosslink accurately, the concentrations of deoxypyridinoline appeared to parallel those of pyridinoline (results not shown). The highest concentration of intramuscular collagen was found in MS and EDL muscles, 34.5 and 38.2 J-lg/mg d. w., respectively. The lowest collagen concentrations were found in PL, LD and medial part of MG. A significant increase (p <0.005) in the collagen concentration up to the age of 12 months was found only in MS (results not shown).

Discussion

This study of functionally different skeletal muscles, their tendons and tendon insertion has been facilitated by newlydeveloped HPLC techniques to measure the concentrations of collagen and of mature collagen crosslinks. For measurement of collagen concentration, the OPA and FMOC-CI derivatization system (Einarsson, 1985; Teerlink et al., 1989) avoided any interference from other amino compounds that may occur with colorimetric extraction procedures. The HPLC method is particularly suitable for muscle samples where the relative amounts of hydroxyproline are small. Indeed, analysis of standard mixtures showed that hydroxyproline could be measured in the presence of over 1000-fold molar excess of a-amino acids. For hydroxypyridinium crosslink measurements, the preliminary partition chromatography step (Black et al., 1988) allowed accurate and reproducible analyses. Both methods were quick and reliable and made possible the analysis of very small biological samples. The concentration and structural characteristics of colla-

H. Palokangas et a1.

294

mol/ mol collagen

05

mol/mol collagen

0.5

A. 0.4

04

03

03

0.2

0.2

0.1

01 2

8 6 4 Age. months

10

12

0.4

0.3

0,2

0.2

0.1

0.1 0

2

8 6 4 Age. months

10

12

mol/mol collagen E.

0.5 0.4

0

0.2

0.2

0

0.5 0.4

~

... 0

2

8 6 4 Age. months

10

12

01 0

05

mol/mol collagen G.

0,4

03

0,3

02

0,2

0.1

0.1 2

6 8 4 Age. months

10

12

12

8 6 4 Age. months

2

10

12

mol/mol collagen F.

0,4 03

0,1

0

0,5

0,3

10

mol/mol collagen D

0.4

0.3

0

8 6 4 Age.months

2

0.5

mol/mol collagen C.

0.5

B

.~ ,

"

0

2

6 8 4 Age. months

10

12

10

12

mol/mol collagen H.

2

6 8 4 Age. months

gen has profound effects on the passive mechanical properties of tissues. Among other regulatory factors, mechanical stimulus may exert important effects on the properties of collagen fibrils. The few studies concerning the effects of exercise or immobilization on the properties of collagen in different tissues are restricted mainly to the measurement of collagen concentration (Woo et al.,1980). Skeletal muscles adapted to different functional demands offer an excellent opportunity to study the relationship between the properties of muscle collagen and muscle activity. According to

Fig. 1. The age-related changes in the concentrations of pyridinoline crosslinks in the muscle belly, tendinous insertion and tendons of the functionally different skeletal muscles. (A.) m. soleus. (B.) m. gastrocnemius, medial. (C.) m. gastrocnemius, lateral. (D.) plantaris. (E.) anterior tibialis. (F.) extensior digitorum longus. (G.) m. rectus femoris. (H.) longissimus dorsi. The symbols used; 0 - 0 muscle belly, 0--0 tendon, l::,--l::, tendinous insertion. The results expressed as mean ± SO of 6 animals/group. Statistical significance of the differences between successive timepoints are indicated (* p < 0.05; n p < 0.01; *n p < 0.001).

the present results the concentrations of pyridinoline in muscles are not solely related to the muscle fibre composition, but also to the type of loading. The highest concentration of pyridinoline crosslinks found in MS, PL and LD could be related to their function either as a slow-twitch antigravity posture-maintaining muscle or as a plantar flexor. MS and PL muscles are plantar flexors, which are generally known to be under heavier mechanical stretch than the dorsiflexors, AT and EDL. In the slow-twitch MS in particular the stretch reflex is of great importance for the

Hydroxypyridinium Crosslinks in Skeletal Muscles muscular activity (Vrbova, 1979) and the muscle is known to have an important role in the rat as an antigravity muscle (Kugelberg, 1976). It can be envisaged that the more heavily stabilized collagen structures would be a greater benefit in the posture-maintaining muscles adapted to continous static and/or eccentric actions (Kovanen, 1989). On the other hand, less crosslinked, more flexible collagen could be preferred in the fast-twitch dorsiflexors (EDL and AT), which are prepared for more dynamic actions. The results of this study also indicated significant differences in the collagen concentration between different muscles and between different compartments of the muscletendon complex in rat. In agreement with earlier findings (Kovanen et aI., 1984; Kovanen and Suominen, 1989), the highest collagen concentrations were found in MS and EDL muscles. As the lowest collagen concentrations were found in PL, LD and medial part of MG, the collagen concentration of muscles seems not to be related to the muscle function as directly as the concentration of pyridinoline crosslinks. In contrast to our results, no differences in the concentrations of pyridinoline in different types of rabbit muscles was found by ]ha (1982) using conventional ion-exchange chromatography. In our hands, however, this method has given significant differences between rat skeletal muscles in pyridinoline concentrations (unpublished results). Comparisons with the results of ]ha are also difficult because the latter report contains no detailed information about the muscles studied. The crosslink concentrations in muscle reported by]ha were, however, at similar levels to those reported here (0.2-0.4 mol/mol collagen). In general, however, the relatively few reports of pyridinoline crosslink concentrations in muscle tissues (Jha, 1982; Eyre et aI., 1984a) have shown considerable variations. This may be due in part to a failure to take into account the variability, demonstrated in this study, between functionally different muscles. In addition, it is particularly important to remove the epimyseal sheath and tendinous insertions of the muscles which may confound the results. As increased loading of muscle is known to cause increased rates of collagen synthesis (Kovanen et aI., 1980; Kovanen and Suominen, 1989) and turnover (Laurent et aI., 1978; Palmer et aI., 1982), this factor should be considered as an additional source of variability in mature crosslink concentrations. Thus, a higher rate of turnover associated with increased loading could result in a lower proportion of mature crosslinks. Evidence in support of this hypothesis has been obtained from experiments with the tendons of growing chickens undergoing intense physical exercise (Curwin et aI., 1988). Alternatively, changes in the loading of tendons or ligaments may cause a change in the proportion of pyridinium crosslink precursors relative to the aldimine type bonds (Eyre et aI., 1984 b), possibly associated with changes in the activity of the enzyme responsible for hydroxylation of telopeptide lysine residues.

295

Recent studies of calf muscle tendons have suggested a correlation between pyridinium crosslink concentration and thermal stability of the tissue (Horgan et aI., 1990). Clearly, this finding would favour a mechanism for pyridinoline formation involving linkage between microfibrils (Eyre, 1980) rather than the intramicrofibrillar mechanism proposed for the formation of this crosslink in cartilage. Other crosslinks, such as the Ehrlich chromogen proposed by Scott et ai. (1981) may also influence the mechanical properties of the tissue (Horgan et aI., 1990). The presence of other crosslinks may explain differences in the crosslink concentrations that could not be attributed to mechanical loading effects, such as the lower amounts of pyridinoline in tendons and tendinous insertions of EDL and AT compared to the medial portions of the muscles. The function of rat MS as a posture maintaining static muscle is closely related to the weight development of the animals on the basis of the changes in muscle fiber composition (Kugelberg, 1976). The present study shows that after maturation when the weight of animals is known to be still increasing, significant increases in both collagen concentration and the concentration of pyridinoline crosslinks takes place only in MS. Accordingly, the metabolism of collagen also appears to be involved to the adaptation of soleus muscle to the functional demands put by the increased body weight to the posture maintenance. The results of this study indicate real differences in both the structure and concentration of intramuscular collagen in different skeletal muscles. Although the precise role of hydroxypyridinium crosslinks in the mechanical properties of tissues is still uncertain, the increase in pyridinoline concentration in intramuscular collagen seems to correlate well with the increase during maturation in the mechanical stiffness and tensile strength of muscles (Kovanen, 1989). Both the high concentration of collagen and the extensive crosslinking of collagen by pyridinoline in MS also fit the earlier results of higher tensile strength and mechanical stiffness of MS muscles (Kovanen et aI., 1984; Kovanen and Suominen, 1988). Generally, the collagen crosslinking was found in this study to be better related to the muscle function compared to collagen concentration. Further studies are needed to clarify the basic reason for the differences in the collagen concentration and collagen crosslinking of different compartments of muscle-tendon complex in rat.

References Bailey, A. j., Robins, S. P. and Balian, G.: Biological significance of the intermolecular crosslinks of collagen. Nature 251 :

105-109,1974.

Black, D., Duncan,A. and Robins,S.P.: Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography.

Anal. Biochem. 169: 197-203,1988.

Curwin, S. L., Vailas, A. C. and Wood,).: Immature tendon adap-

296

H. Palokangas et al.

tation to strenuous exercise. J. Appl. Physiol. 65: 2297 - 2301, 1988. Einarsson, S.: Selective determination of secondary amino acids using precolumn derivatization with 9-fluorenylmethylchloroformate and reversed-phase high-performance liquid chromatography.]. Chromatogr. 348: 213-220, 1985. Eyre, D. R.: Collagen: molecular diversity in the body's protein scaffold. Science 207: 1315 -1322, 1980. Eyre, D. R., Koob, T.]. and Van Ness, K. P.: Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography. Anal. Biochem. 137: 380-388, 1984. Eyre,D.R., Paz, M.A. and Gallop,M.A.: Cross-linking in collagen and elastin. Ann. Rev. Biochem. 53: 717-748, 1984. Fujimoto, D., Moriguchi, T., Ishida, T. and Hayashi, H.: The structure of pyridinoline, a collagen crosslink. Biochem. Biophys. Res. Commun. 84: 52-57, 1978. Horgan,D.j., King,N.L., Kurth,L.B. and Kuypers,R.: Collagen crosslinks and their relationship to the thermal properties of calf tendons. Arch. Biochem. Biophys. 281: 21-26, 1990. Jha, M.: Age-related changes in pyridinoline content of rabbit collagen. Exp. Gerontal. 17: 7-9, 1982. Kovanen, V.: Effects of ageing and physical training on rat skeletal muscle. Acta Physiol. Scand. 135 (supplementum 577),1989. Kovanen, V. and Suominen, H.: Age- and training-related changes in collagen metabolism of rat skeletal muscle. Eur. ]. Appl. Physiol. 58: 765 -771,1989. Kovanen, V., Suominen, H. and Heikkinen, E.: Connective tissue of "fast" and "slow" skeletal muscle in rats - effects of endurance training. Acta Physiol. Scand. 108: 173 -180, 1980. Kovanen, V., Suominen, H. and Heikkinen, E.: Mechanical properties of fast and slow skeletal muscle with special reference to collagen and endurance training.]. Biomech. 17: 725-735, 1984. Kugelberg, E.: Adaptive transformation of rat soleus motor units during growth.]. Neural. Sci. 27: 269-289, 1976. Laurent,G.j., Sparrow, M.P., Bates,P.c. and Millward, D.].: Turnover of muscle protein in the fowl. Changes in rates of protein synthesis and breakdown during hypertrophy of the anterior and posterior latissimus dorsi muscles. Biochem. ]. 176: 419-427, 1978. Moriguchi, T. and Fujimoto, D.: Age-related changes in the content of the collagen crosslink, pyridinoline. ]. Biochem. 84: 933-935,1978. Ogawa, T., Ono, T., Tsuda, M. and Kawanashi, X.: A novel fluor in insoluble collagen: a crosslinking moiety in collagen

molecule. Biochem. Biophys. Res. Commun. 107: 1252-1257, 1982. Palmer,R.M., Reeds,P.]., Lobley,G.E. and Smith,R.H.: The effect of intermittent changes in tension on protein and collagen synthesis in isolated rabbit muscles. Biochem.]. 198: 491-498, 1981. Robins, S. P.: Crosslinking of collagen: isolation, structural characterization and glycosylation of pyridinoline. Biochem.]. 215: 167-173,1983. Robins, S. P., Shimokomaki, M. and Bailey, A.].: The chemistry of collagen crosslinks: age-related changes in the reducible components of intact bovine collagen fibres. Biochem. ]. 131: 771-780, 1973. Scott,]. E., Hughes, E. W. and Shuttleworth, A.: A collagen-associated Ehrlich chromogen: a pyrrolic cross-link? Biosci. Rep. 1: 611-618,1981. Siegel, R. c., Fu,J. c., Uto, N., Horiuchi, K. and Fujimoto, D.: Collagen crosslinking: Iysyl oxidase dependent synthesis of pyridinoline in vitro: confirmation that pyridinoline is derived of collagen. Biochem. Biophys. Res. Commun. 108: 1546-1550,1982. Teerlink, T., Tavenier, P. and Netelenbos,]. C.: Selective determination of hydroxyproline in urine by high-performance liquid chromatography using precolumn derivatization. Clin. Chim. Acta 183: 309-316, 1989. Vogel, H. G.: Tensile strength, relaxation and mechanical recovery in rat skin as influenced by maturation and age. ]. Med. 7: 177-188,1976. Vrbova, G.: Influence of activity on some characteristic properties of slow and fast mammalian muscles. Exerc. Sport Sci. Rev. 7: 181-213,1979. Woo, S. L., Ritter, M. A., Amiel, D., Sanders, T. M., Gomez, M. A. and Kvei, S. c.: The biomechanical and biochemical properties of swine tendons - long term effects of exercise on the digital extensors. Connect. Tissue. Res. 7: 177-183,1980. Wu,].-J., Eyre, D. R.: Identification of hydroxypyridinium crosslinking sites in type II collagen of bovine articular cartilage. Biochemistry 23: 1850-1857, 1984. Yamauchi, M., London, R. E., Guenat, c., Hashimoto, F. and Mechanic, G.L.: Structure and formation of a stable histidinebased trifunctional cross-link in skin collagen. ]. BioI. Chem. 262: 11428-11434,1987. Dr. H. Palokangas, Dept. of Anatomy, Univ. of Oulu, Kajaanintie 52A, 90220 Oulu, Finland.