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The effect of hypophysectomy and age on the stabilization of labile cross-links in collagen
Exp. Geront. Vol. 7, pp. 413~115. Pergamon Press 1972. Printed in Great Britain.
THE
EFFECT ON THE
OF
HYPOPHYSECTOMY
STABILIZATION
CROSS-LINKS
IN
AND
AGE
OF LABILE
COLLAGEN
L. DELBRIDGEand A. V. EVERrrT Department of Physiology,Universityof Sydney, Sydney,Australia (Received 12 June 1972)
INTRODUCTION
HYPOPnVSECVOMVretards the ageing of collagen fibres in rat tail tendon (Olsen and Everitt, 1965; Verz~r and Spichtin, 1966; Everitt et al. 1968; Everitt and Delbridge, 1972), probably, by inhibiting the rate of cross-link formation (Deyl et al., 1967; Everitt and Delbridge, 1972). Intermolecular cross-links of the lysine-derived aldehyde type have been shown to exist in two forms (1) an intermediate labile bond, which is converted by reduction to (2) a stable intermolecular cross-link (Bailey, 1967; Bailey, 1968). The ageing of collagen involves the progressive stabilization of the intermediate labile bond (Bailey, 1969). The aim of the present study was to investigate the nature of the inhibition of cross-link formation by hypophysectomy with respect to the lysine-derived aldehyde type of intermolecular cross-links. MATERIALS AND METHODS
Collagen fibres were obtained from the tail tendons of 22 hypophysectomized and 46 control male rats of the University of Sydney Wistar strain. Hypophysectomies were performed when rats were 45-65 days old, using either the parapharyngeal or the intra-aural technique. The effect of washing in acid phosphate was investigated. In the phosphate washed group, collagen fibres were removed from the tail, soaked in 0.5M NaH2PO4 at 2°C overnight and then placed on filter paper and allowed to dry at room temperature. In the unwashed group, collagen fibres were removed from the tail and placed on filter paper, allowed to dry at room temperature and then stored overnight at 2°C. Collagen age was measured by the breaking time test (Boros-Farkas and Everitt, 1967). Collagen fibres, ranging in weight from 1.5-2.5 mg per 10 cm length, were suspended under a load of 2g in a 7M urea solution at 40°C and the time to break measured. RESULTS (1) Control rats In Fig. 1 it can be seen that unwashed fibres from 19 control rats had a breaking time of about 30 min at 100 days rising to about 400 rain at 1000 days. After phosphate washing, collagen fibres from 35 control rats had lower breaking times than unwashed fibres. The breaking times increased from 0.3 min at 100 days to 400 min at 1000 days (Fig. 1). 413
414
L. DELBRIDGE AND A. V. EVERITT
1000'
CONTROL ~.~washed ,
Z
100" ~.A-~"
_Z m I-,
~
•
•
i
'~
i •
ii
J
•
*/ /
o z
|
l
unwasnea
/
. ~
~ 10-
" / HYPOX o~,
•
CONTROL Phosphate W a s h e d
•
*/. /
v Ill l"
" % °o * -
~'~ HYPOX Phosphate Washed
4 0.1. . 0
.
.
.
.
. . 500
.
.
.
. 1000
AGE IN DAYS
FIG. I. The breaking times in 7M urea at 40°C of phosphate (NaH2PO~) washed and unwashed collagen fibres from hypophysectomized and control rats. (2) Hypophysectomized rats Unwashed collagen fibres from 17 hypophysectomized rats had shorter breaking times than unwashed fibres from controls. The time to break increased from 10 min at 100 days to 60 min at 800 days (Fig. 1). Phosphate washed collagen fibres from 16 hypophysectomized rats broke in 1 min or less at all ages. The time to break increased from 0.3 min at 100 days to approximately 1 min at 800 days (Fig. 1). DISCUSSION Hypophysectomy inhibits the rate of cross-link formation in collagen (Everitt et al., 1968). It has been shown (Delbridge, Everitt and Steele, 1972) that soaking collagen fibres in acid phosphate breaks the labile form of the lysine--derived aldehyde type cross link and leaves the stable form intact. The breaking times of phosphate washed control collagen fibres show a steep rise from 500 to 1000 days (Fig. l). This would appear to represent the stabilization of the labile bond as the animal ages. Hypophysectomy inhibits the total rate of cross-link formation (that is labile plus stable plus any other type of bond present) by about half since the time to break a collagen fibre from an 800 day old hypophysectomized rat is the same as that from a control rat of 400 days (Fig. 1). However when the labile bonds are removed by soaking in acid phosphate, a different pattern emerges. There is no difference in the breaking times of phosphate washed fibres from control and hypophysectomized rats for the first 500 days, both remaining at less than 1 min (Fig. I.) At 500 days however, the breaking times of collagen fibres from control rats rise steeply to reach 80 rain by 900 days whilst the breaking time of collagen fibres from hypophysectomized rats fails to rise and remains less than 1 min even at 900 days. This would suggest that one effect of hypophysectomy is to inhibit the stabilization of the
THE EFFECTOF HYPOPHYSECTOMY
415
labile form of the lysine-derived aldehyde type of cross-link that occurs in the latter half of the lifespan of the animal. Acknowledgements--This work was supported in part by grants from the Consolidated Medical Research Fund of the University of Sydney and the University Research Grant. The National Health and Medical Research Council provided a B.Sc. (Med.) Scholarship.
REFERENCES BAILEY, A. J. (1967) Biochem. J. 105, 34. BAILEY, A. J. (t968) Biochim. biophys. Acta 160, 447. BAILEY, A. J. (1969) Gerontologia 15, 65. BOROS-FARKAS, M. and EVERITT, A. V. (1967) Gerontologia 13, 37. DELBRIDGE, L., EVERITT, A. V. and STEELE, M. G. 0972) Connective Tissue Research, in press. DEYL, Z., EVERITT, A. V. and ROSMUS,J. (1967) Exp. Geront. 2, 249. EVERITT, A. V. and DELBRIDGE, L. (1972) Exp. Geront. 7, 45. EVERITT, A. V., OLSEN, G. G. and BURROWS, G. R. (1968)J. Gerontol. 23, 333. OLSEN, G. G. and EVERITT, A. V. (1965) Nature, Lond. 206, 307. VERZAR, F. and SPICHTIN,n . (1966) Gerontologia 12, 48. Summary--The breaking times of both unwashed and acid phosphate (NaH2PO4) washed collagen fibres removed from the tails of control and hypophysectomized rats were measured. The results suggest that one effect of hypophysectomy is to inhibit the conversion of the labile to the stable form of the lysine-derived aldehyde type of cross-link, a process which is normally seen to occur in the latter half of the lifespan of the rat. R6sum6--On a mesur6 les temps de rupture de collag6nes, non lav6s et lav6s au phosphate acide (NaH~PO4), pr~,lev6s sur les queues de rats t6moins et de rats qui avaient subi l'hypophysectomie. Les r6sultats sugg6rent qu'un des effets de l'hypophysectomie est d'inhiber la conversion de la forme instable ~t la forme stable de la liaison crois6e de type ald6hyde d6riv6e de la lysine, processus que l'on voit normalement se produire dans la deuxi6me moiti6 de la vie du rat. Zusammenfassung--Bei Kollagenfasern aus dem Schwanz yon normalen und hypophysektomierten Ratten wurde die Abbruchzeit yon nichtgewaschenen und in saurem Phosphat (NaH2PO4) gewaschenen Proben gemessen. Die Ergebnisse weisen daruaf hin, dab ein Effekt der Hypophysektomie in der Verhinderung des ~bergangs der labilen in die stabile Form der vom Lysin abgeleiteten Vernetzung vom Aldehydtyp besteht, ein Prozel], der normalerweise in der zweiten Lebensh~ilfte der Ratte beobachtet wird. Pe31oMe--ld3Mep~nocb BpeM~t pa3p~,iBa aeMbITbIX ri MblTblX B KHC.IIOMqbocd~axe (NaH2PO4) KOJ].rlaFeHOBblX BO.rlOKOH~ B31~ITblX143 XBOCTOB KOHTpO-rlbHblX KpblC H KpblC, HO]IBepFHyTblX FHHO~FIB3KTOMFIH. Pe3y.qbTaTbI noKa3blBalOT, tlTO O,/1HHMH3 3qbqbeKTOBFHHO~H33KTOMHH~B.q~IeTCflTOpMO~eHne npeBpatLteHH~ HeyCTOfi'-mBO~ B yCTO,~'~VIByrO qbopMy nn3vIno-npoH3BO~lHoro anbj/erH~Horo Trma nonepe,~HO~ CBII3H, npouecca, KOTOpbffIHOpMaYlbHO acrpe,JaeTc~ BO BTOpOfi nonoBnHe ;~H3HeHHOFO IlHKJIa rpbICbI.
THE
EFFECT ON THE
OF
HYPOPHYSECTOMY
STABILIZATION
CROSS-LINKS
IN
AND
AGE
OF LABILE
COLLAGEN
L. DELBRIDGEand A. V. EVERrrT Department of Physiology,Universityof Sydney, Sydney,Australia (Received 12 June 1972)
INTRODUCTION
HYPOPnVSECVOMVretards the ageing of collagen fibres in rat tail tendon (Olsen and Everitt, 1965; Verz~r and Spichtin, 1966; Everitt et al. 1968; Everitt and Delbridge, 1972), probably, by inhibiting the rate of cross-link formation (Deyl et al., 1967; Everitt and Delbridge, 1972). Intermolecular cross-links of the lysine-derived aldehyde type have been shown to exist in two forms (1) an intermediate labile bond, which is converted by reduction to (2) a stable intermolecular cross-link (Bailey, 1967; Bailey, 1968). The ageing of collagen involves the progressive stabilization of the intermediate labile bond (Bailey, 1969). The aim of the present study was to investigate the nature of the inhibition of cross-link formation by hypophysectomy with respect to the lysine-derived aldehyde type of intermolecular cross-links. MATERIALS AND METHODS
Collagen fibres were obtained from the tail tendons of 22 hypophysectomized and 46 control male rats of the University of Sydney Wistar strain. Hypophysectomies were performed when rats were 45-65 days old, using either the parapharyngeal or the intra-aural technique. The effect of washing in acid phosphate was investigated. In the phosphate washed group, collagen fibres were removed from the tail, soaked in 0.5M NaH2PO4 at 2°C overnight and then placed on filter paper and allowed to dry at room temperature. In the unwashed group, collagen fibres were removed from the tail and placed on filter paper, allowed to dry at room temperature and then stored overnight at 2°C. Collagen age was measured by the breaking time test (Boros-Farkas and Everitt, 1967). Collagen fibres, ranging in weight from 1.5-2.5 mg per 10 cm length, were suspended under a load of 2g in a 7M urea solution at 40°C and the time to break measured. RESULTS (1) Control rats In Fig. 1 it can be seen that unwashed fibres from 19 control rats had a breaking time of about 30 min at 100 days rising to about 400 rain at 1000 days. After phosphate washing, collagen fibres from 35 control rats had lower breaking times than unwashed fibres. The breaking times increased from 0.3 min at 100 days to 400 min at 1000 days (Fig. 1). 413
414
L. DELBRIDGE AND A. V. EVERITT
1000'
CONTROL ~.~washed ,
Z
100" ~.A-~"
_Z m I-,
~
•
•
i
'~
i •
ii
J
•
*/ /
o z
|
l
unwasnea
/
. ~
~ 10-
" / HYPOX o~,
•
CONTROL Phosphate W a s h e d
•
*/. /
v Ill l"
" % °o * -
~'~ HYPOX Phosphate Washed
4 0.1. . 0
.
.
.
.
. . 500
.
.
.
. 1000
AGE IN DAYS
FIG. I. The breaking times in 7M urea at 40°C of phosphate (NaH2PO~) washed and unwashed collagen fibres from hypophysectomized and control rats. (2) Hypophysectomized rats Unwashed collagen fibres from 17 hypophysectomized rats had shorter breaking times than unwashed fibres from controls. The time to break increased from 10 min at 100 days to 60 min at 800 days (Fig. 1). Phosphate washed collagen fibres from 16 hypophysectomized rats broke in 1 min or less at all ages. The time to break increased from 0.3 min at 100 days to approximately 1 min at 800 days (Fig. 1). DISCUSSION Hypophysectomy inhibits the rate of cross-link formation in collagen (Everitt et al., 1968). It has been shown (Delbridge, Everitt and Steele, 1972) that soaking collagen fibres in acid phosphate breaks the labile form of the lysine--derived aldehyde type cross link and leaves the stable form intact. The breaking times of phosphate washed control collagen fibres show a steep rise from 500 to 1000 days (Fig. l). This would appear to represent the stabilization of the labile bond as the animal ages. Hypophysectomy inhibits the total rate of cross-link formation (that is labile plus stable plus any other type of bond present) by about half since the time to break a collagen fibre from an 800 day old hypophysectomized rat is the same as that from a control rat of 400 days (Fig. 1). However when the labile bonds are removed by soaking in acid phosphate, a different pattern emerges. There is no difference in the breaking times of phosphate washed fibres from control and hypophysectomized rats for the first 500 days, both remaining at less than 1 min (Fig. I.) At 500 days however, the breaking times of collagen fibres from control rats rise steeply to reach 80 rain by 900 days whilst the breaking time of collagen fibres from hypophysectomized rats fails to rise and remains less than 1 min even at 900 days. This would suggest that one effect of hypophysectomy is to inhibit the stabilization of the
THE EFFECTOF HYPOPHYSECTOMY
415
labile form of the lysine-derived aldehyde type of cross-link that occurs in the latter half of the lifespan of the animal. Acknowledgements--This work was supported in part by grants from the Consolidated Medical Research Fund of the University of Sydney and the University Research Grant. The National Health and Medical Research Council provided a B.Sc. (Med.) Scholarship.
REFERENCES BAILEY, A. J. (1967) Biochem. J. 105, 34. BAILEY, A. J. (t968) Biochim. biophys. Acta 160, 447. BAILEY, A. J. (1969) Gerontologia 15, 65. BOROS-FARKAS, M. and EVERITT, A. V. (1967) Gerontologia 13, 37. DELBRIDGE, L., EVERITT, A. V. and STEELE, M. G. 0972) Connective Tissue Research, in press. DEYL, Z., EVERITT, A. V. and ROSMUS,J. (1967) Exp. Geront. 2, 249. EVERITT, A. V. and DELBRIDGE, L. (1972) Exp. Geront. 7, 45. EVERITT, A. V., OLSEN, G. G. and BURROWS, G. R. (1968)J. Gerontol. 23, 333. OLSEN, G. G. and EVERITT, A. V. (1965) Nature, Lond. 206, 307. VERZAR, F. and SPICHTIN,n . (1966) Gerontologia 12, 48. Summary--The breaking times of both unwashed and acid phosphate (NaH2PO4) washed collagen fibres removed from the tails of control and hypophysectomized rats were measured. The results suggest that one effect of hypophysectomy is to inhibit the conversion of the labile to the stable form of the lysine-derived aldehyde type of cross-link, a process which is normally seen to occur in the latter half of the lifespan of the rat. R6sum6--On a mesur6 les temps de rupture de collag6nes, non lav6s et lav6s au phosphate acide (NaH~PO4), pr~,lev6s sur les queues de rats t6moins et de rats qui avaient subi l'hypophysectomie. Les r6sultats sugg6rent qu'un des effets de l'hypophysectomie est d'inhiber la conversion de la forme instable ~t la forme stable de la liaison crois6e de type ald6hyde d6riv6e de la lysine, processus que l'on voit normalement se produire dans la deuxi6me moiti6 de la vie du rat. Zusammenfassung--Bei Kollagenfasern aus dem Schwanz yon normalen und hypophysektomierten Ratten wurde die Abbruchzeit yon nichtgewaschenen und in saurem Phosphat (NaH2PO4) gewaschenen Proben gemessen. Die Ergebnisse weisen daruaf hin, dab ein Effekt der Hypophysektomie in der Verhinderung des ~bergangs der labilen in die stabile Form der vom Lysin abgeleiteten Vernetzung vom Aldehydtyp besteht, ein Prozel], der normalerweise in der zweiten Lebensh~ilfte der Ratte beobachtet wird. Pe31oMe--ld3Mep~nocb BpeM~t pa3p~,iBa aeMbITbIX ri MblTblX B KHC.IIOMqbocd~axe (NaH2PO4) KOJ].rlaFeHOBblX BO.rlOKOH~ B31~ITblX143 XBOCTOB KOHTpO-rlbHblX KpblC H KpblC, HO]IBepFHyTblX FHHO~FIB3KTOMFIH. Pe3y.qbTaTbI noKa3blBalOT, tlTO O,/1HHMH3 3qbqbeKTOBFHHO~H33KTOMHH~B.q~IeTCflTOpMO~eHne npeBpatLteHH~ HeyCTOfi'-mBO~ B yCTO,~'~VIByrO qbopMy nn3vIno-npoH3BO~lHoro anbj/erH~Horo Trma nonepe,~HO~ CBII3H, npouecca, KOTOpbffIHOpMaYlbHO acrpe,JaeTc~ BO BTOpOfi nonoBnHe ;~H3HeHHOFO IlHKJIa rpbICbI.