- Email: [email protected]
The connective tissue response to immobility
The ",onnective Tissue Response to Immobilit
Mucopolysaccharide Changes in Dog Tendon WAYNE I!. AKESON, M.I)., AND I)UANE F. L a V I O L E T T E , University o[ Washington School o[ Medicine
INTROI)UCTiON The atrophy of unmineralized connective tissue structures s e c o n d a r y to immobility has generally escaped serious attention. This is in contrast to the acute atrophy of bone which occurs subsequent to the removal of stress and motion and which is a phenomenon well recognized by physicians who treatconnnon fractures. The facts that are known about the connective tissue response to immobility can be covered in a few brief words. Brooke and Slack studied the saline soluble fraction of collagen in denervated rabbit limbs and noted it to be unchanged or reduced when compared to normal limbs, a Slack's work on the saline extractable fraction of coll a g e n was c o n f i r m e d by Peacock in the ligamentous structures about the knee of the dog.~Z One is forced to conclude that the rate of collagen formation is not accelerated in denervated or immobilized limbs and that changes in cross linkage between collagen fibers cannot be demonstrated wii~h techniques used to date. Neither Peacock using the dog knee nor Akeson using rat tail tendon was able to demonstrate significant changes in hydrothermal shrinkage characteristics in c o n n e c t i v e tissue from extremities that had been immobilized, z'~2 Radioactive sulfate uptake in the dcnervated rat limb was studied by Slack, who noted it to be reduced.3 Akeson demonstrdted decrease in total hexosamine and w a t e r concentration in connective tissue about the immobilized d o g knee. 1 Changes described in t h e latter two papers suggested mucopolysaccharide loss in the experimental limbs. Ilowever, the analyses
From the Division of Orthopedic Surgery, University of Washington, School of Medicine, Seattle, Washington 98105. Submitted for publication April 8, 1964. JSR - Vol. IV, No. 11 - November, 1964
described were done without prior mucopolysaccharide extraction and conclusions drawn were subject to alternative interpretations. The present work was undertaken t~ place the' connective tissue response to imr:~obility on a firmer basis with respect to the mucopolysaccharide changes. This paper describes the initial results on this problem. It supports and amplifies the previous findings described by the present author. I m p r o v e m e n t s in technique include mainly mucopolysaccharide extraction prior to analysis, which permits both hexosamine and uronic acid determination to be made and molar ratios to be established.
METIIOI)S The right knee joints of 36 dogs were immobilized by means of a t h r e a d e d wire in t h ~ manner previously described. The threaded wire passes from the mid-tibia into the proximal femur posterior to the knee joint, taking such a course that it remains within the skin fold behind the knee. The fixation was maintained for the period of time indicated in 24 of the animals. In 12 of the animals the wire was broken or an infection occun'ed, and the animals could not be included in the time-controlled series. S e v e n of the animals in the broken wire group had developed a contracture of the knee at the time theproblem was first noted and did not have an infection. These animals were pooled together in a group indicated in the tables as b r o k e n wirecontracture group. At the end of the time period indicated, the animals were killed with intravenous Nembutal and the quadriceps and patellar tendons were dissected free. The t i s s u e from the n o d a l weight-bearing control limb was pooled with that of the other animals of the same group. Similarly, tissue from the experimentally treated knees of 523
524
AKESON and LA VtOI~ET'FE
each group was pooled. In this manner, five groups were e s t a b l i s h e d covering the lime periods of one and a half weeks, four weeks, six to eight weeks, nine to t w e l v e weeks and a group which was not time-controlled from the animals in which fixation failed. The t i s s u e s were dried and defatted in s e v eral changes of a c e t o n e and e t h e r . The d,3., fat-free t i s s u e was finely ground in a ~'iley Mill and stored dry until ready for use. The t i s s u e was then s u s p e n d e d in 0.1 M a c c t a t e buffer ptl 5.5 ( 2 0 m l . per g r a m of dr>, t i s s u e ) c o n t a i n i n g 0.005 M c y s t e i n e IIC1 and 0 . 0 0 5 M d i s o d i u m v e r s e n a t e . Two rag. of c r y s t a l l i n e papain per gram of dry t i s s u e was added, and the mixture was incubated for 2.1 hours at 6 0 ° C . If complete solubilization did not occur, sodium hydroxide was added to 0.5 M and the mixture was s h a k e n at 4 ° C. for four hours. The solution was dialyzed against running tap water for 24 hours and then at 4 ° against d i s t i l l e d water for 24 hours. T h e dialyzed sample was d i g e s t e d with trypsin for 72 hours in a d i a l y s i s bag using 2.5 rag. of c r y s t a l l i n e trypsin per gram of sample. D i a l y s i s was performed a g a i n s t 0.1 M phosphate buffer pll 7.8 to 8.0 at 37 °. The d i a l y z e d sample was transferred to a tared beaker quantitatively with d i s t i l l e d water and cold t r i c h l o r a c e t i c acid was added to a final conccntration of 10%. T h e tric h l o r a c e t i c a c i d soluble material was d i a l y z e d a g a i n s t d i s t i l l e d water after filtering through a medium fritted disk. D i a l y s i s w a s continued at 4 ° C. against d i s t i l l e d water for two w e e k s . The contents of the d i a l y s i s bags at the end of this time were again transferred q u a n t i t a t i v e l y into a round evaporating flask and were e v a p o r a t e d to d r y n e s s at 35 ° C. in a rotary evaporator with a Welch Vacuum Pump. T h e dry m u c o p o l y s a c charide w ~ then transferred quantitatively into a volumetric flask of such s i z e that the final volume contained approximately 1 rag. of mucop o l y s a c c h a r i d e per ml. The m u c o p o l y s a c c h a r i d e extraction procedure so outlined is e s s e n t i a t l y t h a t which Schiller et al. u s e d for ~,t skin. , 3 llexosamine Analysis Aliquo~s of the e x t r a c t e d material were suitably diluted to contain approximately 200/zg. of h e x o s a m i n e per ml. One milliliter aliquots of this material were pipetted into t e s t tubes to which was added 1 ml. of 4 N tiC1. T h e s e tubes were s e a l e d and the material was hydrolyzed at ]18 °. T h e maximum hexosamine y i e l d 6n hyaluronic a c i d s t a n d a r d s was o b t a i n e d after two
JSR
-
V o L I V , No. 1 I -
November,
1964
hours' hydrolysis, that on chondroitin s u l f a t e s l a n d a r d s after two one-half h o u r s ' h y d r o l y s i s . The tendon extract showed a nmximum hexosamine y i e l d slightly later, however, so that a three hour hydrolysis time was u s e d for the data p r e s e n t e d here. T h e h y d r o l y s a t e was transferred quantitatively into an E rl e n m e y e r flask with d i s t i l l e d water. Approximately 5 ml. of toluene was added to reduce bumping d u r i n g e v a p o r a t l o n , and the material w a s evaporatcd to d r y n e s s over calcium chloride and sodium hydroxide with a vacuum pump. The dr3' malerial was made up to 10rot. volume and 1 m}. aliquots w e r e taken f o r an al y s i s ac cording to the El son-Morgan reaction 7 A c e t y l a t i o n was performed using r e d i s t i l l e d acetyl a c e t o n e stored in a deep freeze and made up to a 2% solution with 1 N sodium carbonate solution. A c e t y l a t i o n was carried out at 92 ° for 45 minutes in an oil-layered water bath, using 19 x 150 ram. g l a s s - s t o p p e r e d t e s t tubes. At the end of the a c e tylation steps, the tubes were cooled rapidly in running tap water to room temperature. Seven ml. of 95% alcohol was added, the contents thoroughly ,nixed and finally I ml. of E h r l i c h ' s reagent was added. T h e color was a l l o w e d to develop for one hour at room te,nperature and was then read on a Beckman Spectrophotometer at 530,ntt. Standards containing 20t~g. of glficosamine hydrochloride* were run s i m u l t a n e o u s l y in each s e r i e s . Blanks conlaining 1 ml. of water i n s t e a d o f h e x o s a m i n e were run with each s e r i e s . The quantity of unknown was determined by comparison of optical d e n s i t y (OD) of unknown with OD of standard after correction for the blank reading. Uronic Acid Uronic a c i d determinations were done by the carbazole m e t h o d of Dische. a O n e m i l l i I i t e r aliquots of unknown c o n t a i n l n g a p p r o x i m a t e l y 30 /2g. of u r o n i c acid were p i p e t t e d : . n t o 6 m l , of concentrated sulfuric a c i d which had been a l l o w e d to stand 20 minutes in an ice bath in a t e s t tube, 19 × 150 ram. in s.;ze. Mixing was performed gradually in the ice bath so a s to r e d u c e heat f o r m a t i o n . After tlle c o n t e n t s were thoroughly mixed, g l a s s stoppers were inserted and the t u b e s were placed in a boiling water bath for 20 minutes. At the end of that time, the tubes were cooled r a p i d l y with running tap water to room temperature. Two t e n t h s of a co. of 0.2% carbazole in absolute alcohol was added for color development. T h e carbazole had been *Nutritional Biochemical Corporation.
JSR
-
V o L IV, No. 11 - N o v e m b e r , 1964
recrys~allized twice from benzene. Standards and blanks were prepared in the same manner and run side by side with die unknowns. The standards contained 30/zg. of glucuronolactone.* The blanks contained 1 mI. of distilled water. Three standards and two b l a n k s were run with each group of unknowns. Color was read in a Beckman DU Spectrophotometer after one hour at 535m/1. The q u a n t i t y of unknown was determined by comparison of 01) of standard with 0I) of unknown after correction for the blank reading. Sul fate Method Aliquots of t h e extracted mucopolysaccharides containing approximately 1 rag. of sulfate were pipetted into test tubes. An equal amount of 12N formic acid was added to make the final concentration 6N. The tubes were s e a l e d and hydrolysis was carried out in a 100 ° oven for nine h o u r s . The hydrolysate was evaporated to dryness and diluted with water, so that the final dilution contained approximately 150/zg. of sulfate per ml. One ml. aliquots were set up in triplicate for each unknown. To the 1 ml.aliquot of the diluted hydrolysate was added l m l . of 25% IJ'ichloracetic a c i d (sulfate-free). After mixing, 5 nd. of 1% benzidine in absolute alcohol which was freshly prepared and filtered was added. After complete mixing, the contents of the t u ~ were poured onto a 13~chner type funnel which had a fritted disk of very fine porosity. T h e s e were allowed to stand overnight tat 4° C. as described by Muir. ~1 Quantitative transfer to the funnel was a c c o m p l i s h e d ~ t h 2 m l . of absolute alcohol. The precipitate was collected under slight suction the following day. It was washed twice with 5 ml. of a saturated solution of benzidine sulfate in acetone filtered before use. T h i s was followed with a single w a s h i n g with 5 ml. of acetone. The precipitate was dissolved with 3 ml. of 1N IIC1. The IIC1 was pulled through the funnel with light suction, and the funnel w a s washed once with 3 ml. of water. The dissolved benzidine and washings were collected in a filter f l a s k and the remainder of the reaction was run in this flask according to the method of I)ogson and Spencer. s One ml. of 0.15% sodium nitrite, prepared f r e s h l y , was added. After thorough mixing, 5 ml. 0.5% t h y m o l in 7.5% sodium hydroxide was added. After mixing, 240 ml. of distilled water was added for color dilution. The color was read immediately in a DU Spectrophotometer at 4 ~ m/z. *Nutritional Biochemical Corporation.
TISSUE RESPONSE TO IMMOBILITY
525
Nitrogen Analysis Nitrogen was detenniried by a micro Kjeldahl method. Chromatography Methods Chromatography of the unhydrolyzed mucopolysaccharides was performed according to the method of Kerby using W h a t m a n No. 1 filter paper. ~ An a l i q u o t of the final product was brought to d r y n e s s and it was dissolved in approximately 0.5 ml. of water. Ten microliter spots were applied to Whatman No. 1 paper three times with drying between applications. Ascending c h r o m a t o g r a p h y was done in o n e o f t w o solvent systems: 52 ml. of ethanol plus 48 ml. of 0.06 M phosphate buffer pll 6.6, or 37 ml. propanol plus 63 ml. of 0.06M phosphate buffer pll 6.6. After 40 hours, the chromatograms were dried in air and fixed in 20% formaldel~yde in ethanol for 15 minutes, immersed in ether and air dried. The chromatogram was immersed in 0.06% toluidine blue in 0.5% acetic acid. It was allowed to drain against the side of the pan and then was immersed in 2% acetic acid and was finally immersed in running tap water. The water was squeezed from the strip with a print roller against a glass plate, and the strip was dried at room tempe~ature. Chromatography of the hydrolyzed specimens was performed for qualitative estimation of hexosamine by the method of Kirk and l)yrbye. 1° The diluted hydrolysate from the hexosamine determination was evaporated to dryness in a beaker placed in a vacuum desiccator supplied with sodium hydroxide p e l l e t s and calcium chloride. The dry residue was dissolved in 200 /11. of distilled water. Ten gl. spots were applied to Whatman No. I filter paper. T h e s e were applied three times with drying between. Ascending chromatogTaphy was used with 2,6 tutidine:water (65:35) as solvent. Five/zl. of a 1% solution of glucosamine a n d galatosamine hydrochloride standards was used for comparison. The paper strips were placed in the chamber for 40 hours at room temperature and were then dried at room temperaturE. Color development was with 0.2% ninhydrin in dr), acetone using a pressurized glass atomizer. After drying, the cS~romatograms were stored in the dark ~[2 hours for maxhnum color development. l|exosamine separation was performed on a Gardell column using Dowex ~W-8X, 200 to 400 mesh, in the hydrogen form. s The 0.5 normal IiC1 effluent was collected with a fraction collector w i t h a drop-counting attachment s e t t o
JSR
526 AKESON and LA \IOLl+,l I lu c o l l e c t 0.5 ml. fractions. P e a k s were identified and q u a n t i t i e s of glucosanfine and g a l a c t o s a m l n e e s t i m a t e d on the b a s i s of the Elson Morgan reaction on individual t u b e s s o c o l l e c t e d . I I,SULTS Since m u c o p o l y s a c c b a r i d e s make up only 0,5% by dry weight of the c o n n e c t i v e t i s s u e studio(t, it w a s nece.~sary to p o o l the k n e e s of s e v e r a l animals in order to obtain q u a n t i t i e s of material a d e q u a t e for a n a l y s i s . For this r e a s o n , only one pair of p o o l e d s a m p l e s was a v a i l a b l e for analys i s at e a c h period and s t a t e m e n t s about the trend of c h a n g e s in relation to Lime are probably i n s e c u r e . N e v e r t h e l e s s , it is o b v i o u s that after four w e e k s a significant l o s s o f m u e o p o l y s a c charide had o c c u r r e d , and the l o s s p e r s i s t e d throughout the period of study. Comparison of data on hexosamine c o n t e n t and uronic acid content of m u c o p o l y s a c c h a r i d e s e x t r a c t e d from the c o n n e c t i v e t i s s u e of control m~d experimental k n e e s after varying p e r i o d s of immobility is given in T a b l e s | and 2. In the one and onehalf week group an initial i n c r e a s e in mucopolys a c c h a r l d e concentration o c c u r r e d . S u b s e q u e n t l y , b e x o s a m i n e and uronic a c i d c o n c e n t r a t i o n s were found to be reduced on the expprimental side. In addition, a s noted in T a b l e 3, e s t e r s u l f a t e v a l u e s were reduced in all experimental groups. T a b l e 3 l i s t s the a n t o u n t of hexosamine, uronic a c i d , s u l f a l e a n d m u c o p o l y s a c c h a r i d e nitrogen in terms of mieromoles per gram of d W tendon. T h e s e v a l u e s were o b t a i n e d on the materia| a n a l y z e d a f t e r extraction.Onewould e x p e c t chondroitin s u l f a t e g t o be p r e s e n t in T a b l e I.
Croup
-
Vol, IV, No, 11 - N o v e m b e r ,
1964
tendon structures of most mammals, l i o w e v e r , d e f i n i t i v e s t u d i e s on acid m u c o p o l y s a c c h a r i d e c o m p o s i t i o n of dog c o n n e c t i v e t i s s u e are p r e s ently unavailable, and s t a t e m e n t s about the p o l y s a c c h a r i d e composition of this t i s s u e cannot be made at p r e s e n t with c e r t a i n t y . Nitrogen molar ratios were noted to be three to ten times t h e o r e t i c a l values, which i n d i c a t e s the p r e s e n c e of protein impurity. One would, a n t i c i p a t e s u c h a degree ~of impurity in this t y p e of preparation s i n c e no s p e c i a l efforts were made to free the preparation from e x t r a n e o u s nitrogen beyond t r i c h l o r a c e t i c a c i d p r e c i p i t a t i o n and d i a l y s i s . Similarly, sulfate molar ratios are slightly higher than theoretical. A g a i n, molar r a t i o s gl"eater than one arc commonly o b s e r v e d in mueopolys a c c h a r i d e e x t r a c t s of this type. Chromatography of the m u c o p o l y s a c c h a r i d e s by the method of Kerby d i s c l o s e d at l e a s t two components, one with the ntobility of chondroitin s u l f a t e and a s e c o n d with the mobility of hyalutonic acid. There were no c o m p o n e n t s p r e s e n t with the mobility of heparin. Chromatography of thc acid h y d r o l y s a t e by the methods of Kirk and Dyrbye and by the m e t h o d s of J e a n l o z a n d Stroffyn la d e m o n s t r a t e d the p r e s e n c e of both g l u c o s a m i n e and g a l a c t o s a m i n e . Separation of g a l a c t o s a m i n e and g h t c o s a m i n e on a Gardel I column showed that g r e a t e r amounts of g a l a c t o s a mine than g l u c o s a m i n c are present in dog tendon. Of the t o t a l h e x o s a m i n c p r e s e n t , approximately 60 to 70 per cent is g a l a c t o s a m i n e with some variation b e t w e e n animals. T h e recovery of commercial* chondroitin sulfate and hyaluronic a c i d carried through the d i g e s t i o n and d i a l y s i s s t e p s d e s c r i b e d a b o v e *
Nutritional Biochemical Corporation
i l e x o s a m h l e C o n t e n t o[ M P S E x t r a c t e d / r o m C o n n e c t i v e T i s s u e o f Control a n d E x p e r i m e n t a l K n e e s
Period of h!}!n0bi/ity
No. of Animals
R5X
] ½ wks,
6
4,269
+0,425
+I 1. I
II 8(; R8X
4 wks.
3
(3. 457)* 2.118
-1.212
-36.4
R6C R6X
6-8 wks,
8
R7C I{7X
9-12 wks,
l~9(: i/9X
I~roken ~irc Group
R5 ( ]
rag. ltexosamine per l)iffereneet Gin. Dry Tendon ............................ 3.844
Per Cent Chan~e*/
-0.457
-12,3
7
3.731 274 3.131 1.942
-1. 189
--38.0
7
2.615 1.886
--0.729
--27,9
3.
C = Control X = Experimental (immobilized) Each group represents pooled quadrieeps and patellar tendon from the stated number of animals, * Aw~rage of the other control values. This sample was lost in dialysis. X--C.
Per Cent Change is calculated in this manner: ~ .
JSR
_
VoL IV, No, 11 Table 2.
Group
.
I ISSULr RESPONSE TO IMMOBILITY
November, 1964
~ '
527
Uronic A c i d Cotttent o / M P S E x t r a c t e d [rom Connective T i s s u e of Control and Experimental Knees
Period of No, of Immoi2i!!ty. . . . . . . . Animals
rag. Uronic Acid/ Gin. Dry Tendon
I)iHcrencei ..........
Per Cent Change*
ll 5C RSX
1½ wks.
6
3.149 3.524
+.375
+11.9
1t8(; llSX
4 wks.
3
( 3.055)* 2.039
--1.016
-33.3
It 6C 116X
6-8 w ks.
8
3.600 2.573
- t. 027
-28.5
117C R7X
9-12 wks.
7
2.910 1.680
- t. 230
--42.3
2.559 R9(: Breke n 7 -0.696 119X Wire Group 1.863 C = Control X = Expcrim~:nt.~] (immobilized). E;lch group represents pooled quadriceps and patellar tendon from the stated number of animals. * Awrrage of tim otl)cr control values. This sample was lost in dialysis. X -C X-C Per Cent change is calculated in this mannt;r: ~ ..
was 78 and 85 per cent respectively. The acid mucopolysaceharide used was reprecipitated in alcohol as tile calcium salt (two times) before use.
DISCUSSION q]~c results presented here clearly confirm previous indications that a major part of the conneclive tissue response to innuobility cons i s t s of a decrease in c e m e n t of acid mucopolysaccharides. The c h a n g e is evident by four weeks and continues through the study period of 12 weeks. An initial increase in the acid mucopolysaccharide content noted in this study at one and one-half weeks probably represents an artefact of the method ofimmobilization used, namely internal fixation with a threaded wire. The increased production of acid mucopolysacc h a r | d e s in wound healing is well documented, s The internal fixation used here is attended with an unavoidable in flammatory response during the period of wound healing and the increased mucopolysaccharide levels noted in the early period are probably a par: of this response. The possible mechanisms for development of joint contracture have been summarized previously. ~ From all evidence to date, mainly the saline extractable collagen data and the data on hydrothennal shrinkage c h a r a c t e r i s t i c s of collagen in the immobilized extremity, collagen plays a su~risingly passive role in the connective tissue respofise to i m m o b i l i t y if one excludes problems of direct, trauma to joints. There are no p u b l i s h e d data on changes in noncoliagenous p r o t e i n in t h e immobilized
- 2% 2
extremity. The lesser mucopolysaccharide content which is demonstrated here could cause interference with the buffering or lubricating mechanism between collagen bundles. Such an explanation provides an attractive hypothesis for speculation. Ilowever, the exact mechanism of such a change at a molecular level is uncertain, and considering the lack of knowledge of normal connective tissue physiology at this level such a supposition would have little concrete s u p p o r t at present. T h e r e f o r e , the implications of tile work just presented with respect to physical changes in c o n n e c t i v e t i s s u e following immobility must remain uncertain for the moment. The loss of mucopolysaccharide which has been described could simply repres e n t an incidental change r e s u l t i n g f r o m a generally reduced rate of metabolism. Reduced overall metabolism in tile limb m i ~ t be reflected in decreased synthesis of a group of molecules whose turnover rate is rapid and might have no relationship to joint stiffness itself. On the other hand, the change may be responsible for interference with collagen-protein-mucopolysacchar|de interactions w h i c h are the basis for normal p h y s i c a l c h a r a c t e r i s t i c s of the connective tissue. Evidence presented here on nmcopolysacchar|de content of immobilized connective tissue of the dog knee is e n t i r e l y quantitative in nature. There have b e e n no publications on qualitative changes in this state. To b e t t e r understand the phenomenon, it would be useful to know whether the mucopolysaccharides were uniformly reduced in amount or whether specific
528 AKESON and LA VIOLEq~FE
JSR - VoL IV, No. I 1 - November, 1964
Table 3. 31icromoles o / I l e x o s a m b m , Uro~lic Acid, Sial/ate and Nitrogen fit 31PS Extracted from Connecth.~e T i s s u e of Control and Experimental Knees* Group
Period of Immobility
R5C R5 X R8C ~ llgX
I½ wks. 4 wks.
116C ll6X R7C II 7X
6-8 wks. 9-12 wks.
ll9C l19X
I:~roken l~ireContractu Group e
tlexosamine
Uronic Acid ~
Sulfate
Nitrogen
21,45 23.83 (18.59) 11.82
16.22 l 8.15 (15.74) 10.50
38.33 36.53
241.40 170,69
20.82 18.27 17.48 10.84
18.54 13.25 l 4.99 8.65
62.88 23.63 30.61 20.82
202.83 209.26 97.13 94.27
14.60
I3.18
28.63
67.85
10.53
9.60
26.65
57.14
C = Control X = Experimental * Vatucs exprcssed as #moles/gin. dry tendon. Carbazole method. ~; Average values of other control ~ >ups. This sample was lost in dialysis. o n e s were i n v o l v e d to a greate~ d e g r e e than o t h e r s . T h e fact that t h e normal p a t t e r n of mucop o l y s a c c h a r i d c in dog c o n n e c t i v e t i s s u e h a s not been d e s c r i b e d has d e l a y e d p r o g r e s s ou t h i s a s p e c t of the problem. T h e c h a r a c t e r i z a t i o n o f normal dog a c i d m u c o p o l y s a c c h a r i d e s and fl-act i o n a t i o n of n m c o p o l y s a c c h a r i d e s of experintentally immobilized c o n n e c t i v e t i s s u e are in p r o g r e s s in the a u t h o r ' s l a b o r a t o r y at the p r e s e n t time and will be r e p o r t e d later.
SUMMARY Tendon m u c o p o l y s a c c h a r i d e s were e x t r a c t e d f r o m e o n t r o | and inmmbilized k n e e j o i n t s of d o g s o v e r the time i n t e r v a l s of o n e and or~e-half to 12 w e e k s . Molar r a t i o s o f h e x o s a m i n e , uronic a c i d , s u l f a t e and n i t r o g e n w e r e e s t a b l i s h e d for the m u c o p o l y s a c c h a r i d e so e x t r a c t e d . T o t a l acid mucopolysaccbarides were found to be r e d u c e d in the e x p e r i m e n t a l limbs in the four, six to eight, and nine to t w e l v e week g r o u p s . T h e reduction in m u c o p o l y s a c c h a r i d e was 20 to 40 per c e n t .
REFERENCES I. Akeson, W.Ii.: An experimental study of joint stiffness. J. Bone & Joint Surg., 43-A:10221034, I96l. 2. Akeson, V¢.ll.: Relationship between the aging phenomena in connective tissue response to immobility: A thermodynamic approach. Surg. Forum, I4:438, 1963.
3. Brooke, J.W., and Slack, H.G.B.: Metabolism of connective tissue in limb atrophy in the rabbit. Ann. Itheumat. Dis., 18: 129, 1959. 4. l)ische, Z.: A new specific color reaction of hexuronic acids. J. Biol. Chem., 167:189, 1947. 5. Dogson, K.S., and Spencer, B.: Studies on sulphatases. 5. The determination of inorganic sulphate in the study of sulphatases. Biochem.
J., 55:436, 1953. 6. Edwards, L.C., and Dunphy, J.E.: Wound healing. I. Injury a n d normal repair. New England J. Med., 259:224-233, 1958, 7. Elson, L.A., and Morgan, W.T.J.: A colorimetrie method for the determination of glucosamine and chondrosamine. B i o e h e m . J., 27:1824, I933. 8. Gardell, S.: Separation on I)owex 50 ion exchange resin of glueosamine and galactosamine and their quantitative s e p a r a t i o n . Acta chem. scandinav., 7:207, 1953. 9. Kerby, G.P.: Excretion of acid mucopolysaccharides in human u r i n e . J. Clin. Invest., 34: 1 I68, 1954. lO. Kirk, J.E., and Dyrbye, M.: Mucopo|ysaccharides of human arterial tissue. 1I. Analysis of total isolated mucopolysaccharide m a t e r i a l . J. Gerontol., 12:23, 1957. I1. Muir, lI.: The nature of the link between protein and carbohydrate of a chondroitin sulfate eom~ plex from hyaline cartilage. Bioehem. J., 69: t95, 1958. I2. Peacock, E.E.: Comparison of eollagenous tissue surrounding normal and immobilized joints. Surg. Forum, 14:440, 1963. 13. Schiller, S., Slover, G.A., a n d Dorfman, A.:A method for the separation of acid mueopotysaccharides: Its application to the isolation of heparin from the skin of rats. J. Biol. Chem., 236:983, 1961. 14. Stoffyn, P.J., and Jeanloz, I/.W.: Identification of amino sugars by p a p e r chromatography. Arch. Biochem. Biophys., 52:373, 1954.
Mucopolysaccharide Changes in Dog Tendon WAYNE I!. AKESON, M.I)., AND I)UANE F. L a V I O L E T T E , University o[ Washington School o[ Medicine
INTROI)UCTiON The atrophy of unmineralized connective tissue structures s e c o n d a r y to immobility has generally escaped serious attention. This is in contrast to the acute atrophy of bone which occurs subsequent to the removal of stress and motion and which is a phenomenon well recognized by physicians who treatconnnon fractures. The facts that are known about the connective tissue response to immobility can be covered in a few brief words. Brooke and Slack studied the saline soluble fraction of collagen in denervated rabbit limbs and noted it to be unchanged or reduced when compared to normal limbs, a Slack's work on the saline extractable fraction of coll a g e n was c o n f i r m e d by Peacock in the ligamentous structures about the knee of the dog.~Z One is forced to conclude that the rate of collagen formation is not accelerated in denervated or immobilized limbs and that changes in cross linkage between collagen fibers cannot be demonstrated wii~h techniques used to date. Neither Peacock using the dog knee nor Akeson using rat tail tendon was able to demonstrate significant changes in hydrothermal shrinkage characteristics in c o n n e c t i v e tissue from extremities that had been immobilized, z'~2 Radioactive sulfate uptake in the dcnervated rat limb was studied by Slack, who noted it to be reduced.3 Akeson demonstrdted decrease in total hexosamine and w a t e r concentration in connective tissue about the immobilized d o g knee. 1 Changes described in t h e latter two papers suggested mucopolysaccharide loss in the experimental limbs. Ilowever, the analyses
From the Division of Orthopedic Surgery, University of Washington, School of Medicine, Seattle, Washington 98105. Submitted for publication April 8, 1964. JSR - Vol. IV, No. 11 - November, 1964
described were done without prior mucopolysaccharide extraction and conclusions drawn were subject to alternative interpretations. The present work was undertaken t~ place the' connective tissue response to imr:~obility on a firmer basis with respect to the mucopolysaccharide changes. This paper describes the initial results on this problem. It supports and amplifies the previous findings described by the present author. I m p r o v e m e n t s in technique include mainly mucopolysaccharide extraction prior to analysis, which permits both hexosamine and uronic acid determination to be made and molar ratios to be established.
METIIOI)S The right knee joints of 36 dogs were immobilized by means of a t h r e a d e d wire in t h ~ manner previously described. The threaded wire passes from the mid-tibia into the proximal femur posterior to the knee joint, taking such a course that it remains within the skin fold behind the knee. The fixation was maintained for the period of time indicated in 24 of the animals. In 12 of the animals the wire was broken or an infection occun'ed, and the animals could not be included in the time-controlled series. S e v e n of the animals in the broken wire group had developed a contracture of the knee at the time theproblem was first noted and did not have an infection. These animals were pooled together in a group indicated in the tables as b r o k e n wirecontracture group. At the end of the time period indicated, the animals were killed with intravenous Nembutal and the quadriceps and patellar tendons were dissected free. The t i s s u e from the n o d a l weight-bearing control limb was pooled with that of the other animals of the same group. Similarly, tissue from the experimentally treated knees of 523
524
AKESON and LA VtOI~ET'FE
each group was pooled. In this manner, five groups were e s t a b l i s h e d covering the lime periods of one and a half weeks, four weeks, six to eight weeks, nine to t w e l v e weeks and a group which was not time-controlled from the animals in which fixation failed. The t i s s u e s were dried and defatted in s e v eral changes of a c e t o n e and e t h e r . The d,3., fat-free t i s s u e was finely ground in a ~'iley Mill and stored dry until ready for use. The t i s s u e was then s u s p e n d e d in 0.1 M a c c t a t e buffer ptl 5.5 ( 2 0 m l . per g r a m of dr>, t i s s u e ) c o n t a i n i n g 0.005 M c y s t e i n e IIC1 and 0 . 0 0 5 M d i s o d i u m v e r s e n a t e . Two rag. of c r y s t a l l i n e papain per gram of dry t i s s u e was added, and the mixture was incubated for 2.1 hours at 6 0 ° C . If complete solubilization did not occur, sodium hydroxide was added to 0.5 M and the mixture was s h a k e n at 4 ° C. for four hours. The solution was dialyzed against running tap water for 24 hours and then at 4 ° against d i s t i l l e d water for 24 hours. T h e dialyzed sample was d i g e s t e d with trypsin for 72 hours in a d i a l y s i s bag using 2.5 rag. of c r y s t a l l i n e trypsin per gram of sample. D i a l y s i s was performed a g a i n s t 0.1 M phosphate buffer pll 7.8 to 8.0 at 37 °. The d i a l y z e d sample was transferred to a tared beaker quantitatively with d i s t i l l e d water and cold t r i c h l o r a c e t i c acid was added to a final conccntration of 10%. T h e tric h l o r a c e t i c a c i d soluble material was d i a l y z e d a g a i n s t d i s t i l l e d water after filtering through a medium fritted disk. D i a l y s i s w a s continued at 4 ° C. against d i s t i l l e d water for two w e e k s . The contents of the d i a l y s i s bags at the end of this time were again transferred q u a n t i t a t i v e l y into a round evaporating flask and were e v a p o r a t e d to d r y n e s s at 35 ° C. in a rotary evaporator with a Welch Vacuum Pump. T h e dry m u c o p o l y s a c charide w ~ then transferred quantitatively into a volumetric flask of such s i z e that the final volume contained approximately 1 rag. of mucop o l y s a c c h a r i d e per ml. The m u c o p o l y s a c c h a r i d e extraction procedure so outlined is e s s e n t i a t l y t h a t which Schiller et al. u s e d for ~,t skin. , 3 llexosamine Analysis Aliquo~s of the e x t r a c t e d material were suitably diluted to contain approximately 200/zg. of h e x o s a m i n e per ml. One milliliter aliquots of this material were pipetted into t e s t tubes to which was added 1 ml. of 4 N tiC1. T h e s e tubes were s e a l e d and the material was hydrolyzed at ]18 °. T h e maximum hexosamine y i e l d 6n hyaluronic a c i d s t a n d a r d s was o b t a i n e d after two
JSR
-
V o L I V , No. 1 I -
November,
1964
hours' hydrolysis, that on chondroitin s u l f a t e s l a n d a r d s after two one-half h o u r s ' h y d r o l y s i s . The tendon extract showed a nmximum hexosamine y i e l d slightly later, however, so that a three hour hydrolysis time was u s e d for the data p r e s e n t e d here. T h e h y d r o l y s a t e was transferred quantitatively into an E rl e n m e y e r flask with d i s t i l l e d water. Approximately 5 ml. of toluene was added to reduce bumping d u r i n g e v a p o r a t l o n , and the material w a s evaporatcd to d r y n e s s over calcium chloride and sodium hydroxide with a vacuum pump. The dr3' malerial was made up to 10rot. volume and 1 m}. aliquots w e r e taken f o r an al y s i s ac cording to the El son-Morgan reaction 7 A c e t y l a t i o n was performed using r e d i s t i l l e d acetyl a c e t o n e stored in a deep freeze and made up to a 2% solution with 1 N sodium carbonate solution. A c e t y l a t i o n was carried out at 92 ° for 45 minutes in an oil-layered water bath, using 19 x 150 ram. g l a s s - s t o p p e r e d t e s t tubes. At the end of the a c e tylation steps, the tubes were cooled rapidly in running tap water to room temperature. Seven ml. of 95% alcohol was added, the contents thoroughly ,nixed and finally I ml. of E h r l i c h ' s reagent was added. T h e color was a l l o w e d to develop for one hour at room te,nperature and was then read on a Beckman Spectrophotometer at 530,ntt. Standards containing 20t~g. of glficosamine hydrochloride* were run s i m u l t a n e o u s l y in each s e r i e s . Blanks conlaining 1 ml. of water i n s t e a d o f h e x o s a m i n e were run with each s e r i e s . The quantity of unknown was determined by comparison of optical d e n s i t y (OD) of unknown with OD of standard after correction for the blank reading. Uronic Acid Uronic a c i d determinations were done by the carbazole m e t h o d of Dische. a O n e m i l l i I i t e r aliquots of unknown c o n t a i n l n g a p p r o x i m a t e l y 30 /2g. of u r o n i c acid were p i p e t t e d : . n t o 6 m l , of concentrated sulfuric a c i d which had been a l l o w e d to stand 20 minutes in an ice bath in a t e s t tube, 19 × 150 ram. in s.;ze. Mixing was performed gradually in the ice bath so a s to r e d u c e heat f o r m a t i o n . After tlle c o n t e n t s were thoroughly mixed, g l a s s stoppers were inserted and the t u b e s were placed in a boiling water bath for 20 minutes. At the end of that time, the tubes were cooled r a p i d l y with running tap water to room temperature. Two t e n t h s of a co. of 0.2% carbazole in absolute alcohol was added for color development. T h e carbazole had been *Nutritional Biochemical Corporation.
JSR
-
V o L IV, No. 11 - N o v e m b e r , 1964
recrys~allized twice from benzene. Standards and blanks were prepared in the same manner and run side by side with die unknowns. The standards contained 30/zg. of glucuronolactone.* The blanks contained 1 mI. of distilled water. Three standards and two b l a n k s were run with each group of unknowns. Color was read in a Beckman DU Spectrophotometer after one hour at 535m/1. The q u a n t i t y of unknown was determined by comparison of 01) of standard with 0I) of unknown after correction for the blank reading. Sul fate Method Aliquots of t h e extracted mucopolysaccharides containing approximately 1 rag. of sulfate were pipetted into test tubes. An equal amount of 12N formic acid was added to make the final concentration 6N. The tubes were s e a l e d and hydrolysis was carried out in a 100 ° oven for nine h o u r s . The hydrolysate was evaporated to dryness and diluted with water, so that the final dilution contained approximately 150/zg. of sulfate per ml. One ml. aliquots were set up in triplicate for each unknown. To the 1 ml.aliquot of the diluted hydrolysate was added l m l . of 25% IJ'ichloracetic a c i d (sulfate-free). After mixing, 5 nd. of 1% benzidine in absolute alcohol which was freshly prepared and filtered was added. After complete mixing, the contents of the t u ~ were poured onto a 13~chner type funnel which had a fritted disk of very fine porosity. T h e s e were allowed to stand overnight tat 4° C. as described by Muir. ~1 Quantitative transfer to the funnel was a c c o m p l i s h e d ~ t h 2 m l . of absolute alcohol. The precipitate was collected under slight suction the following day. It was washed twice with 5 ml. of a saturated solution of benzidine sulfate in acetone filtered before use. T h i s was followed with a single w a s h i n g with 5 ml. of acetone. The precipitate was dissolved with 3 ml. of 1N IIC1. The IIC1 was pulled through the funnel with light suction, and the funnel w a s washed once with 3 ml. of water. The dissolved benzidine and washings were collected in a filter f l a s k and the remainder of the reaction was run in this flask according to the method of I)ogson and Spencer. s One ml. of 0.15% sodium nitrite, prepared f r e s h l y , was added. After thorough mixing, 5 ml. 0.5% t h y m o l in 7.5% sodium hydroxide was added. After mixing, 240 ml. of distilled water was added for color dilution. The color was read immediately in a DU Spectrophotometer at 4 ~ m/z. *Nutritional Biochemical Corporation.
TISSUE RESPONSE TO IMMOBILITY
525
Nitrogen Analysis Nitrogen was detenniried by a micro Kjeldahl method. Chromatography Methods Chromatography of the unhydrolyzed mucopolysaccharides was performed according to the method of Kerby using W h a t m a n No. 1 filter paper. ~ An a l i q u o t of the final product was brought to d r y n e s s and it was dissolved in approximately 0.5 ml. of water. Ten microliter spots were applied to Whatman No. 1 paper three times with drying between applications. Ascending c h r o m a t o g r a p h y was done in o n e o f t w o solvent systems: 52 ml. of ethanol plus 48 ml. of 0.06 M phosphate buffer pll 6.6, or 37 ml. propanol plus 63 ml. of 0.06M phosphate buffer pll 6.6. After 40 hours, the chromatograms were dried in air and fixed in 20% formaldel~yde in ethanol for 15 minutes, immersed in ether and air dried. The chromatogram was immersed in 0.06% toluidine blue in 0.5% acetic acid. It was allowed to drain against the side of the pan and then was immersed in 2% acetic acid and was finally immersed in running tap water. The water was squeezed from the strip with a print roller against a glass plate, and the strip was dried at room tempe~ature. Chromatography of the hydrolyzed specimens was performed for qualitative estimation of hexosamine by the method of Kirk and l)yrbye. 1° The diluted hydrolysate from the hexosamine determination was evaporated to dryness in a beaker placed in a vacuum desiccator supplied with sodium hydroxide p e l l e t s and calcium chloride. The dry residue was dissolved in 200 /11. of distilled water. Ten gl. spots were applied to Whatman No. I filter paper. T h e s e were applied three times with drying between. Ascending chromatogTaphy was used with 2,6 tutidine:water (65:35) as solvent. Five/zl. of a 1% solution of glucosamine a n d galatosamine hydrochloride standards was used for comparison. The paper strips were placed in the chamber for 40 hours at room temperature and were then dried at room temperaturE. Color development was with 0.2% ninhydrin in dr), acetone using a pressurized glass atomizer. After drying, the cS~romatograms were stored in the dark ~[2 hours for maxhnum color development. l|exosamine separation was performed on a Gardell column using Dowex ~W-8X, 200 to 400 mesh, in the hydrogen form. s The 0.5 normal IiC1 effluent was collected with a fraction collector w i t h a drop-counting attachment s e t t o
JSR
526 AKESON and LA \IOLl+,l I lu c o l l e c t 0.5 ml. fractions. P e a k s were identified and q u a n t i t i e s of glucosanfine and g a l a c t o s a m l n e e s t i m a t e d on the b a s i s of the Elson Morgan reaction on individual t u b e s s o c o l l e c t e d . I I,SULTS Since m u c o p o l y s a c c b a r i d e s make up only 0,5% by dry weight of the c o n n e c t i v e t i s s u e studio(t, it w a s nece.~sary to p o o l the k n e e s of s e v e r a l animals in order to obtain q u a n t i t i e s of material a d e q u a t e for a n a l y s i s . For this r e a s o n , only one pair of p o o l e d s a m p l e s was a v a i l a b l e for analys i s at e a c h period and s t a t e m e n t s about the trend of c h a n g e s in relation to Lime are probably i n s e c u r e . N e v e r t h e l e s s , it is o b v i o u s that after four w e e k s a significant l o s s o f m u e o p o l y s a c charide had o c c u r r e d , and the l o s s p e r s i s t e d throughout the period of study. Comparison of data on hexosamine c o n t e n t and uronic acid content of m u c o p o l y s a c c h a r i d e s e x t r a c t e d from the c o n n e c t i v e t i s s u e of control m~d experimental k n e e s after varying p e r i o d s of immobility is given in T a b l e s | and 2. In the one and onehalf week group an initial i n c r e a s e in mucopolys a c c h a r l d e concentration o c c u r r e d . S u b s e q u e n t l y , b e x o s a m i n e and uronic a c i d c o n c e n t r a t i o n s were found to be reduced on the expprimental side. In addition, a s noted in T a b l e 3, e s t e r s u l f a t e v a l u e s were reduced in all experimental groups. T a b l e 3 l i s t s the a n t o u n t of hexosamine, uronic a c i d , s u l f a l e a n d m u c o p o l y s a c c h a r i d e nitrogen in terms of mieromoles per gram of d W tendon. T h e s e v a l u e s were o b t a i n e d on the materia| a n a l y z e d a f t e r extraction.Onewould e x p e c t chondroitin s u l f a t e g t o be p r e s e n t in T a b l e I.
Croup
-
Vol, IV, No, 11 - N o v e m b e r ,
1964
tendon structures of most mammals, l i o w e v e r , d e f i n i t i v e s t u d i e s on acid m u c o p o l y s a c c h a r i d e c o m p o s i t i o n of dog c o n n e c t i v e t i s s u e are p r e s ently unavailable, and s t a t e m e n t s about the p o l y s a c c h a r i d e composition of this t i s s u e cannot be made at p r e s e n t with c e r t a i n t y . Nitrogen molar ratios were noted to be three to ten times t h e o r e t i c a l values, which i n d i c a t e s the p r e s e n c e of protein impurity. One would, a n t i c i p a t e s u c h a degree ~of impurity in this t y p e of preparation s i n c e no s p e c i a l efforts were made to free the preparation from e x t r a n e o u s nitrogen beyond t r i c h l o r a c e t i c a c i d p r e c i p i t a t i o n and d i a l y s i s . Similarly, sulfate molar ratios are slightly higher than theoretical. A g a i n, molar r a t i o s gl"eater than one arc commonly o b s e r v e d in mueopolys a c c h a r i d e e x t r a c t s of this type. Chromatography of the m u c o p o l y s a c c h a r i d e s by the method of Kerby d i s c l o s e d at l e a s t two components, one with the ntobility of chondroitin s u l f a t e and a s e c o n d with the mobility of hyalutonic acid. There were no c o m p o n e n t s p r e s e n t with the mobility of heparin. Chromatography of thc acid h y d r o l y s a t e by the methods of Kirk and Dyrbye and by the m e t h o d s of J e a n l o z a n d Stroffyn la d e m o n s t r a t e d the p r e s e n c e of both g l u c o s a m i n e and g a l a c t o s a m i n e . Separation of g a l a c t o s a m i n e and g h t c o s a m i n e on a Gardel I column showed that g r e a t e r amounts of g a l a c t o s a mine than g l u c o s a m i n c are present in dog tendon. Of the t o t a l h e x o s a m i n c p r e s e n t , approximately 60 to 70 per cent is g a l a c t o s a m i n e with some variation b e t w e e n animals. T h e recovery of commercial* chondroitin sulfate and hyaluronic a c i d carried through the d i g e s t i o n and d i a l y s i s s t e p s d e s c r i b e d a b o v e *
Nutritional Biochemical Corporation
i l e x o s a m h l e C o n t e n t o[ M P S E x t r a c t e d / r o m C o n n e c t i v e T i s s u e o f Control a n d E x p e r i m e n t a l K n e e s
Period of h!}!n0bi/ity
No. of Animals
R5X
] ½ wks,
6
4,269
+0,425
+I 1. I
II 8(; R8X
4 wks.
3
(3. 457)* 2.118
-1.212
-36.4
R6C R6X
6-8 wks,
8
R7C I{7X
9-12 wks,
l~9(: i/9X
I~roken ~irc Group
R5 ( ]
rag. ltexosamine per l)iffereneet Gin. Dry Tendon ............................ 3.844
Per Cent Chan~e*/
-0.457
-12,3
7
3.731 274 3.131 1.942
-1. 189
--38.0
7
2.615 1.886
--0.729
--27,9
3.
C = Control X = Experimental (immobilized) Each group represents pooled quadrieeps and patellar tendon from the stated number of animals, * Aw~rage of the other control values. This sample was lost in dialysis. X--C.
Per Cent Change is calculated in this manner: ~ .
JSR
_
VoL IV, No, 11 Table 2.
Group
.
I ISSULr RESPONSE TO IMMOBILITY
November, 1964
~ '
527
Uronic A c i d Cotttent o / M P S E x t r a c t e d [rom Connective T i s s u e of Control and Experimental Knees
Period of No, of Immoi2i!!ty. . . . . . . . Animals
rag. Uronic Acid/ Gin. Dry Tendon
I)iHcrencei ..........
Per Cent Change*
ll 5C RSX
1½ wks.
6
3.149 3.524
+.375
+11.9
1t8(; llSX
4 wks.
3
( 3.055)* 2.039
--1.016
-33.3
It 6C 116X
6-8 w ks.
8
3.600 2.573
- t. 027
-28.5
117C R7X
9-12 wks.
7
2.910 1.680
- t. 230
--42.3
2.559 R9(: Breke n 7 -0.696 119X Wire Group 1.863 C = Control X = Expcrim~:nt.~] (immobilized). E;lch group represents pooled quadriceps and patellar tendon from the stated number of animals. * Awrrage of tim otl)cr control values. This sample was lost in dialysis. X -C X-C Per Cent change is calculated in this mannt;r: ~ ..
was 78 and 85 per cent respectively. The acid mucopolysaceharide used was reprecipitated in alcohol as tile calcium salt (two times) before use.
DISCUSSION q]~c results presented here clearly confirm previous indications that a major part of the conneclive tissue response to innuobility cons i s t s of a decrease in c e m e n t of acid mucopolysaccharides. The c h a n g e is evident by four weeks and continues through the study period of 12 weeks. An initial increase in the acid mucopolysaccharide content noted in this study at one and one-half weeks probably represents an artefact of the method ofimmobilization used, namely internal fixation with a threaded wire. The increased production of acid mucopolysacc h a r | d e s in wound healing is well documented, s The internal fixation used here is attended with an unavoidable in flammatory response during the period of wound healing and the increased mucopolysaccharide levels noted in the early period are probably a par: of this response. The possible mechanisms for development of joint contracture have been summarized previously. ~ From all evidence to date, mainly the saline extractable collagen data and the data on hydrothennal shrinkage c h a r a c t e r i s t i c s of collagen in the immobilized extremity, collagen plays a su~risingly passive role in the connective tissue respofise to i m m o b i l i t y if one excludes problems of direct, trauma to joints. There are no p u b l i s h e d data on changes in noncoliagenous p r o t e i n in t h e immobilized
- 2% 2
extremity. The lesser mucopolysaccharide content which is demonstrated here could cause interference with the buffering or lubricating mechanism between collagen bundles. Such an explanation provides an attractive hypothesis for speculation. Ilowever, the exact mechanism of such a change at a molecular level is uncertain, and considering the lack of knowledge of normal connective tissue physiology at this level such a supposition would have little concrete s u p p o r t at present. T h e r e f o r e , the implications of tile work just presented with respect to physical changes in c o n n e c t i v e t i s s u e following immobility must remain uncertain for the moment. The loss of mucopolysaccharide which has been described could simply repres e n t an incidental change r e s u l t i n g f r o m a generally reduced rate of metabolism. Reduced overall metabolism in tile limb m i ~ t be reflected in decreased synthesis of a group of molecules whose turnover rate is rapid and might have no relationship to joint stiffness itself. On the other hand, the change may be responsible for interference with collagen-protein-mucopolysacchar|de interactions w h i c h are the basis for normal p h y s i c a l c h a r a c t e r i s t i c s of the connective tissue. Evidence presented here on nmcopolysacchar|de content of immobilized connective tissue of the dog knee is e n t i r e l y quantitative in nature. There have b e e n no publications on qualitative changes in this state. To b e t t e r understand the phenomenon, it would be useful to know whether the mucopolysaccharides were uniformly reduced in amount or whether specific
528 AKESON and LA VIOLEq~FE
JSR - VoL IV, No. I 1 - November, 1964
Table 3. 31icromoles o / I l e x o s a m b m , Uro~lic Acid, Sial/ate and Nitrogen fit 31PS Extracted from Connecth.~e T i s s u e of Control and Experimental Knees* Group
Period of Immobility
R5C R5 X R8C ~ llgX
I½ wks. 4 wks.
116C ll6X R7C II 7X
6-8 wks. 9-12 wks.
ll9C l19X
I:~roken l~ireContractu Group e
tlexosamine
Uronic Acid ~
Sulfate
Nitrogen
21,45 23.83 (18.59) 11.82
16.22 l 8.15 (15.74) 10.50
38.33 36.53
241.40 170,69
20.82 18.27 17.48 10.84
18.54 13.25 l 4.99 8.65
62.88 23.63 30.61 20.82
202.83 209.26 97.13 94.27
14.60
I3.18
28.63
67.85
10.53
9.60
26.65
57.14
C = Control X = Experimental * Vatucs exprcssed as #moles/gin. dry tendon. Carbazole method. ~; Average values of other control ~ >ups. This sample was lost in dialysis. o n e s were i n v o l v e d to a greate~ d e g r e e than o t h e r s . T h e fact that t h e normal p a t t e r n of mucop o l y s a c c h a r i d c in dog c o n n e c t i v e t i s s u e h a s not been d e s c r i b e d has d e l a y e d p r o g r e s s ou t h i s a s p e c t of the problem. T h e c h a r a c t e r i z a t i o n o f normal dog a c i d m u c o p o l y s a c c h a r i d e s and fl-act i o n a t i o n of n m c o p o l y s a c c h a r i d e s of experintentally immobilized c o n n e c t i v e t i s s u e are in p r o g r e s s in the a u t h o r ' s l a b o r a t o r y at the p r e s e n t time and will be r e p o r t e d later.
SUMMARY Tendon m u c o p o l y s a c c h a r i d e s were e x t r a c t e d f r o m e o n t r o | and inmmbilized k n e e j o i n t s of d o g s o v e r the time i n t e r v a l s of o n e and or~e-half to 12 w e e k s . Molar r a t i o s o f h e x o s a m i n e , uronic a c i d , s u l f a t e and n i t r o g e n w e r e e s t a b l i s h e d for the m u c o p o l y s a c c h a r i d e so e x t r a c t e d . T o t a l acid mucopolysaccbarides were found to be r e d u c e d in the e x p e r i m e n t a l limbs in the four, six to eight, and nine to t w e l v e week g r o u p s . T h e reduction in m u c o p o l y s a c c h a r i d e was 20 to 40 per c e n t .
REFERENCES I. Akeson, W.Ii.: An experimental study of joint stiffness. J. Bone & Joint Surg., 43-A:10221034, I96l. 2. Akeson, V¢.ll.: Relationship between the aging phenomena in connective tissue response to immobility: A thermodynamic approach. Surg. Forum, I4:438, 1963.
3. Brooke, J.W., and Slack, H.G.B.: Metabolism of connective tissue in limb atrophy in the rabbit. Ann. Itheumat. Dis., 18: 129, 1959. 4. l)ische, Z.: A new specific color reaction of hexuronic acids. J. Biol. Chem., 167:189, 1947. 5. Dogson, K.S., and Spencer, B.: Studies on sulphatases. 5. The determination of inorganic sulphate in the study of sulphatases. Biochem.
J., 55:436, 1953. 6. Edwards, L.C., and Dunphy, J.E.: Wound healing. I. Injury a n d normal repair. New England J. Med., 259:224-233, 1958, 7. Elson, L.A., and Morgan, W.T.J.: A colorimetrie method for the determination of glucosamine and chondrosamine. B i o e h e m . J., 27:1824, I933. 8. Gardell, S.: Separation on I)owex 50 ion exchange resin of glueosamine and galactosamine and their quantitative s e p a r a t i o n . Acta chem. scandinav., 7:207, 1953. 9. Kerby, G.P.: Excretion of acid mucopolysaccharides in human u r i n e . J. Clin. Invest., 34: 1 I68, 1954. lO. Kirk, J.E., and Dyrbye, M.: Mucopo|ysaccharides of human arterial tissue. 1I. Analysis of total isolated mucopolysaccharide m a t e r i a l . J. Gerontol., 12:23, 1957. I1. Muir, lI.: The nature of the link between protein and carbohydrate of a chondroitin sulfate eom~ plex from hyaline cartilage. Bioehem. J., 69: t95, 1958. I2. Peacock, E.E.: Comparison of eollagenous tissue surrounding normal and immobilized joints. Surg. Forum, 14:440, 1963. 13. Schiller, S., Slover, G.A., a n d Dorfman, A.:A method for the separation of acid mueopotysaccharides: Its application to the isolation of heparin from the skin of rats. J. Biol. Chem., 236:983, 1961. 14. Stoffyn, P.J., and Jeanloz, I/.W.: Identification of amino sugars by p a p e r chromatography. Arch. Biochem. Biophys., 52:373, 1954.