15. 10. 1975
Specialia
zyten l~ oder eine E r h 6 h u n g bzw. E r n i e d r i g u n g des H i i m a t o k r i t s y o n B e d e u t u n g 6. Zu einer u m f a s s e n d e n Charakterisierung der Fliesseigenschaft geh6ren daher a u c h M e s s u n g e n d e r e n t s p r e c h e n d e n E i n z e l p a r a m e t e r 6. Ftir R o u t i n e m e s s u n g e n sowie als S u c h m e t h o d e tiber die e v e n t u e l l e B e d e u t u n g d e r Viskosit/it h a b e n sich die M e s s u n g e n der P l a s m a - u n d VollblutviskositS.t bew / i h r t 10. Z u r M e s s u n g d e r P l a s m a v i s k o s i ~ t sind seit l a n g e m K a p i l l a r v i s k o s i m e t e r a u s r e i c h e n d geeignet. Die Vollblutviskosit/~t b e d a r f j e d o c h w e g e n des s t r u k t u r v i s k 6 s e n V e r h a l t e n s des B l u t e s w e i t e r e r u n d a n d e r e r M e s s v o r a u s s e t z u n g e n 9,12. "Wesentlich ist h i e r b e i die Messung bei unterschiedlichen Schergraden. Besonders interessant
190
Plasma
7/ (sec)
180
\
11[
\ ".,,,..
"k
170
%.
\,
160 '
'
35 36
'
'
'
38 39 Temperatur
(skt.)
'
41 42~
23 22 21 20 19
115sec-1
10
19%
9 8
46 sec -1 i
7
i
ftir m e d i z i n i s c h e A u s s a g e n sind die S c h e r g r a d e u n t e r 100 sec -1, d a e t w a in d i e s e m B e r e i c h das B l u r v o n e i n e m N e w t o n s c t l e n F l i e s s v e r h a l t e n in ein p l a s t i s c h e s bzw. bei e x t r e m n i e d r i g e n S c h e r g r a d e n in ein p s e n d o p l a s t i s c h e s F l i e s s v e r h a l t e n tibergeht9,12. I d e a l ftir die M e s s u n g d e r Vollblutviskosit/~t w~re d a h e r ein Gergt, d a s bei e x t r e m n i e d r i g e n S c h e r g r a d e n , d . h . u n t e r 50 sec -1, o p t i m a l e M e s s b e d i n g u n g e n aufweist. Ftir Ger/ite, die m i t d e m P l a t t e - K e g e l - S y s t e m a r b e i t e n u n d d a m i t preislich ers c h w i n g l i c h sind, ist dies n a c h u n s e r e n U n t e r s u c h u n g e n nur beschr~nkt m6glich und verlangt zumindest Erfahrung in d e r M e s s t e c h n i k . Die E r g e b n i s s e b e i m K a p i l l a r v i s k o s i m e t e r zeigen w e g e n der gr6ssenordnungsm~tssig gleichen A b w e i c h u n g v o m E i c h w e r t bei drei u n t e r s c h i e d l i c h e n K a p i l l a r e n , dass d e r l i m i t i e r e n d e F a k t o r dieser M e s s k e t t e w a h r s c h e i n l i c h in der T e m p e r a t u r e i n s t e l l u n g u n d e v e n t u e l l e n T e m p e r a t u r k o n s t a n z liegt. Die B e d e u t u n g d e r T e m p e r a t u r a u f die Viskosit~it zeigen die w e i t e r e n U n t e r s u c h u n g e n . H i e r bei fiihren geringe T e m p e r a t u r e r l l 6 h u n g e n n i c h t n u r zu einer S e n k u n g d e r P l a s m a v i s k o s i t ~ t , s o n d e r n d a r f i b e r h i n a u s m u s s ein d i r e k t e r E i n f u s s a u f die E r y t h r o z y t e n a n g e n o m m e n w e r d e n , d a das V o l l b l u t p r o z e n t u a l eine f a s t d o p p e l t so grosse V e r m i n d e r u n g d e r V i s k o s i t ~ t gegentiber d e m P l a s m a zeigt. N u r d u r c h zus~tzliche Mess u n g e n der A g g r e g a t i o n u n d FlexibilitXt k 6 n n t e dieser E f f e k t nS~her d i f f e r e n z i e r t werden. O h n e E i n f l u s s a u f die F l i e s s e i g e n s c h a f t b l e i b t d a g e g e n eine V e r m i n d e r u n g d e r T h r o m b o z y t e n , o b w o h l d a m i t n i c h t a u s g e s c h l o s s e n ist, dass e x t r e m h o h e T h r o m b o z y t e n zahlen, wie bei d e r T h r o m b o p h i l i e , d o c h eine B e d e u t u n g a u f die V o l l b l u t v i s k o s i t X t h a b e n k 6 n n t e n .
S u m m a r y . T h e c a p i l l a r y v i s c o s i m e t e r (Viscotimer) t e s t e d w i t h d i f f e r e n t U b b e l o h d e - v i s c o s i m e t e r s is s u i t a b l e for m e a s u r e m e n t s of p l a s m a v i s c o s i t y ; t h e l i m i t t i n g f a c t o r of t h e m e a s u r i n g m e t h o d is t h e e x a c t a d j u s t m e n t of t h e t e m p e r a t u r e . W h e n t e s t i n g t h e plate-cone-viscosim e t e r (Wells-Brookfield Microviscosimeter), we a c h i e v e d scientifically a d e q u a t e r e s u l t s o n l y w h e n u s i n g s h e a r r a t e s 230, 115 a n d 46 sec 1. A n influence of d i f f e r e n t t h r o m b o c y t e - c o n c e n t r a t i o n s o n t h e v i s c o s i t y of w h o l e b l o o d a n d p l a s m a could n o t b e d e t e c t e d . T h e flow c a p a c i t y of whole b l o o d a n d p l a s m a is d i s t i n c t l y d e p e n d e n t o n t h e temperature. H. LEONHARDT u n d I. STELTER
Vollblut
25 24
1181
Medizinische K l i n i k und Polihlinik im K l i n i k u m Steglitz der Freien Universitiit Berlin, Hindenburgdamm 30, D-7 Berlin 45 (Bundesrepublik Deutschland, B R D ) , 7d. M a i 1975.
i
3; 16 3'7 38 39 4'0 41 42 ~ Temperatur
Fig. 2, a @ b. Verhalten der Vollblut- ntld Plasmaviscosittit bei physiologisehen Temperatur~inderungen.
10 A. M. EHRLY, Habilitationsschrift, Frankfurt/Main (1969). 11 A. M. EHRLY und H. E. MOLLER, Aeta haemat. 36, 323 (1966). 12 H. SCttMIDT-ScHoNBEIN, in Zelluldre Physiologie der Mikrozirkulation (Springer-Verlag, Berlin, Heidelberg, New York 1974), p. 1-18.
Collagen in Aging Muscles Collagen of m a m m a l s a c c u m u l a t e s r a p i d l y d u r i n g g r o w t h in o r g a n s r i c h in c o n n e c t i v e tissue a t a r a t e e x c e e d i n g t h a t of some o t h e r p r o t e i n s 1. A c c o r d i n g t o FISHER a n d I~AMSEY 2, ]7IELANDER 3, a n d GUTH 4, t h e r e is a r e l a t i v e increase in collagenous p r o t e i n s a n d a decrease in m y o s i n a n d t o t a l p r o t e i n s in m u s c u l a r a t r o p h y of v a r i o u s types. H o w e v e r , t h e d i f f e r e n t f r a c t i o n s of collagen a n d t h e i r q u a n t i t a t i v e c h a n g e s d u r i n g aging w i t h reference
to slow, fast, a n d c a r d i a c muscles in t h e r a t h a v e n o t b e e n r e p o r t e d so far, a n d h e n c e t h e p r e s e n t s t u d y .
1 D. 2 E. E. 4 L.
A. LOWTHER, Int. Rev. connect. Tissue Res. 1, 63 (1963). FlSllER and V. W. RAMSEY, Am. J. Physiol. 143, 571 (1946). HELANDER, Nature Lond. 182, 1035 (1958). GUTH, Neurosci. Res. Progr. Bull. 7, 1 (1969).
1182
EXPERIENTIA31/10
Specialia
M a t e r i a l s a n d methods. A l b i n o r a t s of w i s t a r s t r a i n a g e d 10, 15 a n d 20 m o n t h s , w e r e m a i n t a i n e d a t 28 -4- 2 ~ on a commercial diet. The animals were decapitated; the red, white and cardiac muscles were removed and weighed immediately. The water and protein content ~ were estimated. Tissues from 2 animals were pooled for each estimation. The salt soluble, acid soluble, and insoluble c o l l a g e n w a s e x t r a c t e d a n d p u r i f i e d a c c o r d i n g t o JACKs o n ~, a n d h y d r o l y z e d i n 6 N HC1 f o r 3 h a t 50 y~. T h e a m o u n t of h y d r o x y p r o l i n e p r e s e n t i n e a c h f r a c t i o n w a s t h e n e s t i m a t e d b y t h e c o l o u r r e a c t i o n o f STEGMANN a s m o d i f i e d b y WOESSNER 7. T h e t i s s u e s o m a t i c i n d e x s
(.e, wt o,
=c,o
wet wt. of the animal
)
• 100
was calculated.
The ratios of salt soluble to acid soluble and acid soluble to insoluble collagen in aging muscles of rat Ratio
Age in months
Salt soluble Acid soluble
ACid soluble Insoluble
R e s u l t s . A u n i f o r m d e c r e a s e , in t i s s u e s o m a t i c i n d e x ( F i g u r e 1) w a t e r c o n t e n t a n d t o t a l p r o t e i n s ( F i g u r e 2) is
10
15
20
Red 0.4748 White 0.2866 Heart 0.4039
0.3890 0.1512 0:2536
0.3174 0.1503 0:1656
Red 0.8807 White 0.7610 Heart 0.7968
0.8211 0.7987 0.5297
0.6086 0.6109 0.6432
Each value represents the average of 4 observations in case of 10 and 20 months and 2 observations in case of 15 months.
•
3
s e e n w i t h a d v a n c e i n a g e in all t h e 3 m u s c l e s . H o w e v e r , when compared to heart and white muscles, the red m u s c l e c o n t a i n s m o r e p r o t e i n s ( F i g u r e 2) a n d c o l l a g e n ( F i g u r e 3). T h e t o t a l c o l l a g e n l e v e l e x p r e s s e d a s p e r c e n t c o l l a g e n w i t h r e f e r e n c e t o t o t a l p r o t e i n s i n c r e a s e s i n all t h e 3 m u s c l e s w i t h a d v a n c e i n a g e , b u t t h e r a t e of i n c r e a s e v a r i e s w i t h t h e t y p e of m u s c l e ( F i g u r e 3), w h e r e a s s a l t s o l u b l e c o l l a g e n d e c r e a s e s p r o g r e s s i v e l y i n all 3 m u s c l e s in contrast to acid soluble and insoluble collagen (Figure
....... .L.2 o
. . . . .
==
1 ~ 0 0
R
1;
15 '
4).
20'
D i s c u s s i o n . T h e d e c r e a s e in t h e t i s s u e s o m a t i c i n d e x of the muscles probably indicate the progressive senile muscular atrophy with increase in age, The present r e s u l t , of i n c r e a s e i n t o t a l c o l l a g e n c o n t e n t i n t h e h e a r t m u s c l e , s u p p o r t s t h e v i e w of SOBEI~ e t a12. T h e s a l t soluble collagen seems to be the form in which collagen
Age in months
180
160 "6
~
140
5 E. LAYNE,in Methods in Emymology (Eds. S. P. COLOWICKand N. O. KAPLAN;Academic Press, New York 1957), vol. 3, p. 454. 6 I). S. JACKSON, in Methods o/Biochemical Analysis (Ed. D. GLICK; Interseience Publishers, New York, London, Sydney 1967), vol. 15. 7 j . F. WOESSNER, Arch. Biochem. 93, 440 (1961).
R
120
8 I~. SATYANARAYANA, V. R . SELVARAJAN, V. CHANDRASEKI~ARAN,
100
"'~ H
I
T
I
10
15
20
R. RAMA~URTm and K. S. SWAMI, Curr. Sci. 44, 127 (1975). 9 H. SOBEL,H. THOMAS and R. ]Y[ASSERMAN, Proc. Soc. exp. Biol. Med. 116, 918 (1964).
.~ 16 %
Age in months
"a 12
28
R .~
20
g~
~ ~Os
12
.9o
-=
~,~~
K.\
Ri
A Ra
g
~..--..~..,,-.--
.c
s o salt soluble a = acid soluble i = insoluble
4
"~. Wi ~"-. Wa
"~Hi
. . . . . . . "* Ha
8
0
A.
o2,oRs
=...~ .......
10
15
20
Age in months
Fig. 1-3. Tissue somatic index (Figure 1), total protein content (Figure 2), and percentage total collagen with reference to total proteins (Figure 3) in red, white and cardiac muscles of 10-, 15- and 20-month-old albino rats. R, red (soleus); W, white (extensor digitoruln longus); H, cardiac muscle.
0 (D
~5
1.0
1.5
2.0
2.5
30
-*Hs
3.5
Tissue somatic index
Fig. 4. Tissue somatic index versus percentage salt soluble, acid soluble and insoluble collagen with reference to total proteins in red, white and cardiac muscles of albino rats.
15. 10. 1975
Specialia
a p p e a r s extracellularly, w h i c h is t h e p r e c u r s o r of b o t h acid soluble a n d m a t u r e f o r m 1~ A u n i f o r m decrease in t h e salt e x t r a c t a b l e collagen, w h i c h is g e n e r a l l y a c c e p t e d as t h e n e w l y s y n t h e s i z e d material, could b e e i t h e r due t o decrease in t h e r a t e of s y n t h e s i s w i t h a c o n s t a n t t u r n over, or increase in t h e t u r n o v e r r a t e w i t h a c o n s t a n t r a t e of synthesis, or v a r i a t i o n s in t h e c o n v e r s i o n r a t e s of salt soluble to acid soluble collagen d u r i n g aging. W h e n t h e ratios of salt soluble to acid soluble, a n d acid soluble t o insoluble collagen (Table) are c o m p a r e d , it is seen t h a t t h e r e is a c c u m u l a t i o n of m o r e m a t u r e or insoluble collagen w h i c h p r o b a b l y indicates t h e o c c u r r e n c e of cross linkages d u r i n g t h e m a t u r a t i o n of fibres. The q u a n t i t a t i v e v a r i a t i o n s in salt soluble, acid soluble and insoluble collagen p r o b a b l y m a y h a v e physiological i m p l i c a t i o n s d u r i n g aging. H o w e v e r , t h e a c c u m u l a t i o n of m o r e m a t u r e f o r m of collagen in muscle w i t h a d v a n c e in age is d i s a d v a n t a g e o us t o t h e organism, since it affects t h e c o n t r a c t i l i t y a n d muscle mechanics, a l t h o u g h it c o n t r i b u t e s s t r e n g t h a n d s u p p o r t to t h e tissue *~ The p r e s e n t s t u d y is a p r e l i m i n a r y r e p o r t on t h e c h a r a c t e r i z a t i o n of m e t a b o l i s m of collagen d u r i n g aging.
1183
Summary. T h e salt, acid a n d insoluble collagen fract i o n s were e s t i m a t e d in red, w h i t e a n d cardiac muscles of 10-, 15- a n d 2 0 - m o n t h - o l d albino rats. The t o t a l collagen level w i t h reference t o t o t a l p r o t e i n s is m o r e in r e d t h a n in w h i t e a n d cardiac muscle. A c c u m u l a t i o n of m o r e of insoluble collagen a n d decrease in salt e x t r a c t a b l e collagen is seen in all t h r e e muscles w i t h aging. S. MOHAN a n d E. RADttA 11
Department o/ Zoology, Bangalore University, Bangalore 560001 (India), 21 March 1975.
10 F. ~V[AROTTSINEX, in Advances in Gerontological Research (Ed. L. BERNARD STREHLER; Academic Press, New York 1964), vol. 1, p. 165. it Acknowledgment. We acknowledge our thanks to Dr. A. R. I~ASTURIBAI for the facilities and encouragement and to Dr. R. V. KRISHNAMOORTHY for valuable suggestions and C.S.I.R. for financial assistance.
S t u d i e s on A g e i n g of Collagen by P e r c h l o r a t e Reactions T e n d o n fibres are a biological o b j e c t of n e a r l y p u r e collagen w h i c h is of p l a s m a origin a n d has well d e f i n e d age changes. I s o m e t r i c t e n s i o n of t e n d o n fibres increases w i t h t h e age of t h e animal. W e s t u d i e d d i f f e r e n t f a c t o r s w h i c h influence t h e s e age c h a n g e s ~-a. In t h e following ~) Soluble collagen Insoluble collagen
0.600[ O.200r ~
,
0,00t
o~o} ~1761"/76 \ 20 3
m~
0.000 520
540
560
580
600nrn
Time
20
0.600r
/
Soluble collagen Insoluble collagen
~ ~
~176176
g 10
~176176 .t/
\
=o 0.30oi-/
.~,-.. k
o0 o t /
\\
~u 0.100t 0 10 2g $0 40rain
OOO0'.
.
520
. 540
.
560
.58(I
60Onto
Time
20 o) ~
0.600[ 0s001 J o.~ooI ~~ 0,0oI-
~_ ; 10 5
Soluble collagen Insoluble collagen
\
e x p e r i m e n t s , we use s o d i u m p e r c h l o r a t e t o s t i m u l a t e isolated c o l l a g e n - t e n d o n - f i b r e s to i s o m e t r i c t e n s i o n p r o d u c t i o n u n d e r d i f f e r e n t conditions, s u c h as age, influence of a l d e h y d e , etc. a n d c h a n g e s of t h e fibres weight. Methods. I s o m e t r i c t e n s i o n is p r o d u c e d b y i m m e r s i n g t e n d o n fibres in 5 3~r NaC104 solution (signed PC). Tail t e n d o n s of r a t s are used w h i c h are 50 m m long, h a v e a d i a m e t e r of ~ 0.15 m m a n d a w e i g h t of +_ 4-5 mg. T h e y can be fixed in an electric t e n s i m e t e r a t low t e n s i o n a n d i m m e r s e d in d i f f e r e n t solutions. T e n s i o n c h a n g e s a t cons t a n t l e n g t h are m e a s u r e d . T h e y are a n a l y z e d a n d t h e i r c o n t e n t of soluble and insoluble collagen m e a s u r e d b y p h o t o m e t r i c a b s o r p t i o n (NEUMANN a n d LOGAN4). W e used an Elco p h o t o m e t e r a n d r e g i s t e r e d b e t w e e n 520-620 n m ; 562 n m is used as t h e value for h y d r o x y p r o l i n (i.e. collagen) c o n t e n t . The e x c h a n g e of d i f f e r e n t solutions, such as of p h y s i o l o g i c a l NaC1 (0.85%), or solutions of f o r m a l d e h y d e a n d o t h e r s u b s t a n c e s , is d e s c r i b e d in t h e text. Experiments. C o l l a g e n - t e n d o n - f i b r e s were i m m e r s e d in 5 M NaC104. A n increase of t e n s i o n s t a r t s i m m e d i a t e l y a n d b e c o m e s m a x i m a l in a b o u t 1 min. I t t h e n r e l a x e s again. F i b r e s of y o u n g a n d old a n i m a l s b e h a v e d i f f e r e n t l y ; t h e i r t e n s i o n decreases in y o u n g in a s h o r t e r t i m e t h a n in old a n i m a l s t e n d o n . C o n t i n u o u s i m m e r s i o n in P C will r e s u l t a f t e r a few m i n u t e s in a d e f i n i t i v e d i s a p p e a r a n c e of t e n s i o n c a p a c i t y in t h e r e l a x e d state. If, h o w e v e r , t h e fibre, after it h a s b e e n i m m e r s e d in P C a n d p r o d u c e d m a x i m a l t e n s i o n , is i m m e d i a t e l y t r a n s f e r r e d i n t o p h y s i o logical NaCI (0.85%), t h e n t h e t e n s i o n decreases a g a i n t o a b o u t t h e original v a l u e a n d t e n s i o n a n d r e l a x a t i o n c a n n o w be r e p e a t e d several times. W e generally u s e d 4 t e n s i o n t e s t s for e a c h e x p e r i m e n t . F i g u r e s 1 a - c s h o w
,, \
.~- 0.2001-x
ol 0 0 ~ ~ ~ , 1
0 10 20 50 40rain Time
0.000/ , 520
, 540
, 560
580
60Ohm
Iqg. 1. Tension in 5 1~'[ NaCIO4 and photometric absorption of soluble and insoluble collagen in a) 4-month-old, b) 10-month-old arid c) 25inorlth-old r~tt~.
1 A. MEYER and F. VERZkR, Gerontologia 3, 184 (1959). 2 F. VERZkR, Int. Rev. connect. Tissue Res. 2, 245 (1964). 3 17. VERZ.•R, E. STRITTMATTER-ACKERSCHOTT and P. MOSER, Gerontologia 19, 129 (1973). &~'~.~F..NAU~IANNand M. A. LOGAN,J. biol. Cheln. 184, 299 (1950).
Specialia
zyten l~ oder eine E r h 6 h u n g bzw. E r n i e d r i g u n g des H i i m a t o k r i t s y o n B e d e u t u n g 6. Zu einer u m f a s s e n d e n Charakterisierung der Fliesseigenschaft geh6ren daher a u c h M e s s u n g e n d e r e n t s p r e c h e n d e n E i n z e l p a r a m e t e r 6. Ftir R o u t i n e m e s s u n g e n sowie als S u c h m e t h o d e tiber die e v e n t u e l l e B e d e u t u n g d e r Viskosit/it h a b e n sich die M e s s u n g e n der P l a s m a - u n d VollblutviskositS.t bew / i h r t 10. Z u r M e s s u n g d e r P l a s m a v i s k o s i ~ t sind seit l a n g e m K a p i l l a r v i s k o s i m e t e r a u s r e i c h e n d geeignet. Die Vollblutviskosit/~t b e d a r f j e d o c h w e g e n des s t r u k t u r v i s k 6 s e n V e r h a l t e n s des B l u t e s w e i t e r e r u n d a n d e r e r M e s s v o r a u s s e t z u n g e n 9,12. "Wesentlich ist h i e r b e i die Messung bei unterschiedlichen Schergraden. Besonders interessant
190
Plasma
7/ (sec)
180
\
11[
\ ".,,,..
"k
170
%.
\,
160 '
'
35 36
'
'
'
38 39 Temperatur
(skt.)
'
41 42~
23 22 21 20 19
115sec-1
10
19%
9 8
46 sec -1 i
7
i
ftir m e d i z i n i s c h e A u s s a g e n sind die S c h e r g r a d e u n t e r 100 sec -1, d a e t w a in d i e s e m B e r e i c h das B l u r v o n e i n e m N e w t o n s c t l e n F l i e s s v e r h a l t e n in ein p l a s t i s c h e s bzw. bei e x t r e m n i e d r i g e n S c h e r g r a d e n in ein p s e n d o p l a s t i s c h e s F l i e s s v e r h a l t e n tibergeht9,12. I d e a l ftir die M e s s u n g d e r Vollblutviskosit/~t w~re d a h e r ein Gergt, d a s bei e x t r e m n i e d r i g e n S c h e r g r a d e n , d . h . u n t e r 50 sec -1, o p t i m a l e M e s s b e d i n g u n g e n aufweist. Ftir Ger/ite, die m i t d e m P l a t t e - K e g e l - S y s t e m a r b e i t e n u n d d a m i t preislich ers c h w i n g l i c h sind, ist dies n a c h u n s e r e n U n t e r s u c h u n g e n nur beschr~nkt m6glich und verlangt zumindest Erfahrung in d e r M e s s t e c h n i k . Die E r g e b n i s s e b e i m K a p i l l a r v i s k o s i m e t e r zeigen w e g e n der gr6ssenordnungsm~tssig gleichen A b w e i c h u n g v o m E i c h w e r t bei drei u n t e r s c h i e d l i c h e n K a p i l l a r e n , dass d e r l i m i t i e r e n d e F a k t o r dieser M e s s k e t t e w a h r s c h e i n l i c h in der T e m p e r a t u r e i n s t e l l u n g u n d e v e n t u e l l e n T e m p e r a t u r k o n s t a n z liegt. Die B e d e u t u n g d e r T e m p e r a t u r a u f die Viskosit~it zeigen die w e i t e r e n U n t e r s u c h u n g e n . H i e r bei fiihren geringe T e m p e r a t u r e r l l 6 h u n g e n n i c h t n u r zu einer S e n k u n g d e r P l a s m a v i s k o s i t ~ t , s o n d e r n d a r f i b e r h i n a u s m u s s ein d i r e k t e r E i n f u s s a u f die E r y t h r o z y t e n a n g e n o m m e n w e r d e n , d a das V o l l b l u t p r o z e n t u a l eine f a s t d o p p e l t so grosse V e r m i n d e r u n g d e r V i s k o s i t ~ t gegentiber d e m P l a s m a zeigt. N u r d u r c h zus~tzliche Mess u n g e n der A g g r e g a t i o n u n d FlexibilitXt k 6 n n t e dieser E f f e k t nS~her d i f f e r e n z i e r t werden. O h n e E i n f l u s s a u f die F l i e s s e i g e n s c h a f t b l e i b t d a g e g e n eine V e r m i n d e r u n g d e r T h r o m b o z y t e n , o b w o h l d a m i t n i c h t a u s g e s c h l o s s e n ist, dass e x t r e m h o h e T h r o m b o z y t e n zahlen, wie bei d e r T h r o m b o p h i l i e , d o c h eine B e d e u t u n g a u f die V o l l b l u t v i s k o s i t X t h a b e n k 6 n n t e n .
S u m m a r y . T h e c a p i l l a r y v i s c o s i m e t e r (Viscotimer) t e s t e d w i t h d i f f e r e n t U b b e l o h d e - v i s c o s i m e t e r s is s u i t a b l e for m e a s u r e m e n t s of p l a s m a v i s c o s i t y ; t h e l i m i t t i n g f a c t o r of t h e m e a s u r i n g m e t h o d is t h e e x a c t a d j u s t m e n t of t h e t e m p e r a t u r e . W h e n t e s t i n g t h e plate-cone-viscosim e t e r (Wells-Brookfield Microviscosimeter), we a c h i e v e d scientifically a d e q u a t e r e s u l t s o n l y w h e n u s i n g s h e a r r a t e s 230, 115 a n d 46 sec 1. A n influence of d i f f e r e n t t h r o m b o c y t e - c o n c e n t r a t i o n s o n t h e v i s c o s i t y of w h o l e b l o o d a n d p l a s m a could n o t b e d e t e c t e d . T h e flow c a p a c i t y of whole b l o o d a n d p l a s m a is d i s t i n c t l y d e p e n d e n t o n t h e temperature. H. LEONHARDT u n d I. STELTER
Vollblut
25 24
1181
Medizinische K l i n i k und Polihlinik im K l i n i k u m Steglitz der Freien Universitiit Berlin, Hindenburgdamm 30, D-7 Berlin 45 (Bundesrepublik Deutschland, B R D ) , 7d. M a i 1975.
i
3; 16 3'7 38 39 4'0 41 42 ~ Temperatur
Fig. 2, a @ b. Verhalten der Vollblut- ntld Plasmaviscosittit bei physiologisehen Temperatur~inderungen.
10 A. M. EHRLY, Habilitationsschrift, Frankfurt/Main (1969). 11 A. M. EHRLY und H. E. MOLLER, Aeta haemat. 36, 323 (1966). 12 H. SCttMIDT-ScHoNBEIN, in Zelluldre Physiologie der Mikrozirkulation (Springer-Verlag, Berlin, Heidelberg, New York 1974), p. 1-18.
Collagen in Aging Muscles Collagen of m a m m a l s a c c u m u l a t e s r a p i d l y d u r i n g g r o w t h in o r g a n s r i c h in c o n n e c t i v e tissue a t a r a t e e x c e e d i n g t h a t of some o t h e r p r o t e i n s 1. A c c o r d i n g t o FISHER a n d I~AMSEY 2, ]7IELANDER 3, a n d GUTH 4, t h e r e is a r e l a t i v e increase in collagenous p r o t e i n s a n d a decrease in m y o s i n a n d t o t a l p r o t e i n s in m u s c u l a r a t r o p h y of v a r i o u s types. H o w e v e r , t h e d i f f e r e n t f r a c t i o n s of collagen a n d t h e i r q u a n t i t a t i v e c h a n g e s d u r i n g aging w i t h reference
to slow, fast, a n d c a r d i a c muscles in t h e r a t h a v e n o t b e e n r e p o r t e d so far, a n d h e n c e t h e p r e s e n t s t u d y .
1 D. 2 E. E. 4 L.
A. LOWTHER, Int. Rev. connect. Tissue Res. 1, 63 (1963). FlSllER and V. W. RAMSEY, Am. J. Physiol. 143, 571 (1946). HELANDER, Nature Lond. 182, 1035 (1958). GUTH, Neurosci. Res. Progr. Bull. 7, 1 (1969).
1182
EXPERIENTIA31/10
Specialia
M a t e r i a l s a n d methods. A l b i n o r a t s of w i s t a r s t r a i n a g e d 10, 15 a n d 20 m o n t h s , w e r e m a i n t a i n e d a t 28 -4- 2 ~ on a commercial diet. The animals were decapitated; the red, white and cardiac muscles were removed and weighed immediately. The water and protein content ~ were estimated. Tissues from 2 animals were pooled for each estimation. The salt soluble, acid soluble, and insoluble c o l l a g e n w a s e x t r a c t e d a n d p u r i f i e d a c c o r d i n g t o JACKs o n ~, a n d h y d r o l y z e d i n 6 N HC1 f o r 3 h a t 50 y~. T h e a m o u n t of h y d r o x y p r o l i n e p r e s e n t i n e a c h f r a c t i o n w a s t h e n e s t i m a t e d b y t h e c o l o u r r e a c t i o n o f STEGMANN a s m o d i f i e d b y WOESSNER 7. T h e t i s s u e s o m a t i c i n d e x s
(.e, wt o,
=c,o
wet wt. of the animal
)
• 100
was calculated.
The ratios of salt soluble to acid soluble and acid soluble to insoluble collagen in aging muscles of rat Ratio
Age in months
Salt soluble Acid soluble
ACid soluble Insoluble
R e s u l t s . A u n i f o r m d e c r e a s e , in t i s s u e s o m a t i c i n d e x ( F i g u r e 1) w a t e r c o n t e n t a n d t o t a l p r o t e i n s ( F i g u r e 2) is
10
15
20
Red 0.4748 White 0.2866 Heart 0.4039
0.3890 0.1512 0:2536
0.3174 0.1503 0:1656
Red 0.8807 White 0.7610 Heart 0.7968
0.8211 0.7987 0.5297
0.6086 0.6109 0.6432
Each value represents the average of 4 observations in case of 10 and 20 months and 2 observations in case of 15 months.
•
3
s e e n w i t h a d v a n c e i n a g e in all t h e 3 m u s c l e s . H o w e v e r , when compared to heart and white muscles, the red m u s c l e c o n t a i n s m o r e p r o t e i n s ( F i g u r e 2) a n d c o l l a g e n ( F i g u r e 3). T h e t o t a l c o l l a g e n l e v e l e x p r e s s e d a s p e r c e n t c o l l a g e n w i t h r e f e r e n c e t o t o t a l p r o t e i n s i n c r e a s e s i n all t h e 3 m u s c l e s w i t h a d v a n c e i n a g e , b u t t h e r a t e of i n c r e a s e v a r i e s w i t h t h e t y p e of m u s c l e ( F i g u r e 3), w h e r e a s s a l t s o l u b l e c o l l a g e n d e c r e a s e s p r o g r e s s i v e l y i n all 3 m u s c l e s in contrast to acid soluble and insoluble collagen (Figure
....... .L.2 o
. . . . .
==
1 ~ 0 0
R
1;
15 '
4).
20'
D i s c u s s i o n . T h e d e c r e a s e in t h e t i s s u e s o m a t i c i n d e x of the muscles probably indicate the progressive senile muscular atrophy with increase in age, The present r e s u l t , of i n c r e a s e i n t o t a l c o l l a g e n c o n t e n t i n t h e h e a r t m u s c l e , s u p p o r t s t h e v i e w of SOBEI~ e t a12. T h e s a l t soluble collagen seems to be the form in which collagen
Age in months
180
160 "6
~
140
5 E. LAYNE,in Methods in Emymology (Eds. S. P. COLOWICKand N. O. KAPLAN;Academic Press, New York 1957), vol. 3, p. 454. 6 I). S. JACKSON, in Methods o/Biochemical Analysis (Ed. D. GLICK; Interseience Publishers, New York, London, Sydney 1967), vol. 15. 7 j . F. WOESSNER, Arch. Biochem. 93, 440 (1961).
R
120
8 I~. SATYANARAYANA, V. R . SELVARAJAN, V. CHANDRASEKI~ARAN,
100
"'~ H
I
T
I
10
15
20
R. RAMA~URTm and K. S. SWAMI, Curr. Sci. 44, 127 (1975). 9 H. SOBEL,H. THOMAS and R. ]Y[ASSERMAN, Proc. Soc. exp. Biol. Med. 116, 918 (1964).
.~ 16 %
Age in months
"a 12
28
R .~
20
g~
~ ~Os
12
.9o
-=
~,~~
K.\
Ri
A Ra
g
~..--..~..,,-.--
.c
s o salt soluble a = acid soluble i = insoluble
4
"~. Wi ~"-. Wa
"~Hi
. . . . . . . "* Ha
8
0
A.
o2,oRs
=...~ .......
10
15
20
Age in months
Fig. 1-3. Tissue somatic index (Figure 1), total protein content (Figure 2), and percentage total collagen with reference to total proteins (Figure 3) in red, white and cardiac muscles of 10-, 15- and 20-month-old albino rats. R, red (soleus); W, white (extensor digitoruln longus); H, cardiac muscle.
0 (D
~5
1.0
1.5
2.0
2.5
30
-*Hs
3.5
Tissue somatic index
Fig. 4. Tissue somatic index versus percentage salt soluble, acid soluble and insoluble collagen with reference to total proteins in red, white and cardiac muscles of albino rats.
15. 10. 1975
Specialia
a p p e a r s extracellularly, w h i c h is t h e p r e c u r s o r of b o t h acid soluble a n d m a t u r e f o r m 1~ A u n i f o r m decrease in t h e salt e x t r a c t a b l e collagen, w h i c h is g e n e r a l l y a c c e p t e d as t h e n e w l y s y n t h e s i z e d material, could b e e i t h e r due t o decrease in t h e r a t e of s y n t h e s i s w i t h a c o n s t a n t t u r n over, or increase in t h e t u r n o v e r r a t e w i t h a c o n s t a n t r a t e of synthesis, or v a r i a t i o n s in t h e c o n v e r s i o n r a t e s of salt soluble to acid soluble collagen d u r i n g aging. W h e n t h e ratios of salt soluble to acid soluble, a n d acid soluble t o insoluble collagen (Table) are c o m p a r e d , it is seen t h a t t h e r e is a c c u m u l a t i o n of m o r e m a t u r e or insoluble collagen w h i c h p r o b a b l y indicates t h e o c c u r r e n c e of cross linkages d u r i n g t h e m a t u r a t i o n of fibres. The q u a n t i t a t i v e v a r i a t i o n s in salt soluble, acid soluble and insoluble collagen p r o b a b l y m a y h a v e physiological i m p l i c a t i o n s d u r i n g aging. H o w e v e r , t h e a c c u m u l a t i o n of m o r e m a t u r e f o r m of collagen in muscle w i t h a d v a n c e in age is d i s a d v a n t a g e o us t o t h e organism, since it affects t h e c o n t r a c t i l i t y a n d muscle mechanics, a l t h o u g h it c o n t r i b u t e s s t r e n g t h a n d s u p p o r t to t h e tissue *~ The p r e s e n t s t u d y is a p r e l i m i n a r y r e p o r t on t h e c h a r a c t e r i z a t i o n of m e t a b o l i s m of collagen d u r i n g aging.
1183
Summary. T h e salt, acid a n d insoluble collagen fract i o n s were e s t i m a t e d in red, w h i t e a n d cardiac muscles of 10-, 15- a n d 2 0 - m o n t h - o l d albino rats. The t o t a l collagen level w i t h reference t o t o t a l p r o t e i n s is m o r e in r e d t h a n in w h i t e a n d cardiac muscle. A c c u m u l a t i o n of m o r e of insoluble collagen a n d decrease in salt e x t r a c t a b l e collagen is seen in all t h r e e muscles w i t h aging. S. MOHAN a n d E. RADttA 11
Department o/ Zoology, Bangalore University, Bangalore 560001 (India), 21 March 1975.
10 F. ~V[AROTTSINEX, in Advances in Gerontological Research (Ed. L. BERNARD STREHLER; Academic Press, New York 1964), vol. 1, p. 165. it Acknowledgment. We acknowledge our thanks to Dr. A. R. I~ASTURIBAI for the facilities and encouragement and to Dr. R. V. KRISHNAMOORTHY for valuable suggestions and C.S.I.R. for financial assistance.
S t u d i e s on A g e i n g of Collagen by P e r c h l o r a t e Reactions T e n d o n fibres are a biological o b j e c t of n e a r l y p u r e collagen w h i c h is of p l a s m a origin a n d has well d e f i n e d age changes. I s o m e t r i c t e n s i o n of t e n d o n fibres increases w i t h t h e age of t h e animal. W e s t u d i e d d i f f e r e n t f a c t o r s w h i c h influence t h e s e age c h a n g e s ~-a. In t h e following ~) Soluble collagen Insoluble collagen
0.600[ O.200r ~
,
0,00t
o~o} ~1761"/76 \ 20 3
m~
0.000 520
540
560
580
600nrn
Time
20
0.600r
/
Soluble collagen Insoluble collagen
~ ~
~176176
g 10
~176176 .t/
\
=o 0.30oi-/
.~,-.. k
o0 o t /
\\
~u 0.100t 0 10 2g $0 40rain
OOO0'.
.
520
. 540
.
560
.58(I
60Onto
Time
20 o) ~
0.600[ 0s001 J o.~ooI ~~ 0,0oI-
~_ ; 10 5
Soluble collagen Insoluble collagen
\
e x p e r i m e n t s , we use s o d i u m p e r c h l o r a t e t o s t i m u l a t e isolated c o l l a g e n - t e n d o n - f i b r e s to i s o m e t r i c t e n s i o n p r o d u c t i o n u n d e r d i f f e r e n t conditions, s u c h as age, influence of a l d e h y d e , etc. a n d c h a n g e s of t h e fibres weight. Methods. I s o m e t r i c t e n s i o n is p r o d u c e d b y i m m e r s i n g t e n d o n fibres in 5 3~r NaC104 solution (signed PC). Tail t e n d o n s of r a t s are used w h i c h are 50 m m long, h a v e a d i a m e t e r of ~ 0.15 m m a n d a w e i g h t of +_ 4-5 mg. T h e y can be fixed in an electric t e n s i m e t e r a t low t e n s i o n a n d i m m e r s e d in d i f f e r e n t solutions. T e n s i o n c h a n g e s a t cons t a n t l e n g t h are m e a s u r e d . T h e y are a n a l y z e d a n d t h e i r c o n t e n t of soluble and insoluble collagen m e a s u r e d b y p h o t o m e t r i c a b s o r p t i o n (NEUMANN a n d LOGAN4). W e used an Elco p h o t o m e t e r a n d r e g i s t e r e d b e t w e e n 520-620 n m ; 562 n m is used as t h e value for h y d r o x y p r o l i n (i.e. collagen) c o n t e n t . The e x c h a n g e of d i f f e r e n t solutions, such as of p h y s i o l o g i c a l NaC1 (0.85%), or solutions of f o r m a l d e h y d e a n d o t h e r s u b s t a n c e s , is d e s c r i b e d in t h e text. Experiments. C o l l a g e n - t e n d o n - f i b r e s were i m m e r s e d in 5 M NaC104. A n increase of t e n s i o n s t a r t s i m m e d i a t e l y a n d b e c o m e s m a x i m a l in a b o u t 1 min. I t t h e n r e l a x e s again. F i b r e s of y o u n g a n d old a n i m a l s b e h a v e d i f f e r e n t l y ; t h e i r t e n s i o n decreases in y o u n g in a s h o r t e r t i m e t h a n in old a n i m a l s t e n d o n . C o n t i n u o u s i m m e r s i o n in P C will r e s u l t a f t e r a few m i n u t e s in a d e f i n i t i v e d i s a p p e a r a n c e of t e n s i o n c a p a c i t y in t h e r e l a x e d state. If, h o w e v e r , t h e fibre, after it h a s b e e n i m m e r s e d in P C a n d p r o d u c e d m a x i m a l t e n s i o n , is i m m e d i a t e l y t r a n s f e r r e d i n t o p h y s i o logical NaCI (0.85%), t h e n t h e t e n s i o n decreases a g a i n t o a b o u t t h e original v a l u e a n d t e n s i o n a n d r e l a x a t i o n c a n n o w be r e p e a t e d several times. W e generally u s e d 4 t e n s i o n t e s t s for e a c h e x p e r i m e n t . F i g u r e s 1 a - c s h o w
,, \
.~- 0.2001-x
ol 0 0 ~ ~ ~ , 1
0 10 20 50 40rain Time
0.000/ , 520
, 540
, 560
580
60Ohm
Iqg. 1. Tension in 5 1~'[ NaCIO4 and photometric absorption of soluble and insoluble collagen in a) 4-month-old, b) 10-month-old arid c) 25inorlth-old r~tt~.
1 A. MEYER and F. VERZkR, Gerontologia 3, 184 (1959). 2 F. VERZkR, Int. Rev. connect. Tissue Res. 2, 245 (1964). 3 17. VERZ.•R, E. STRITTMATTER-ACKERSCHOTT and P. MOSER, Gerontologia 19, 129 (1973). &~'~.~F..NAU~IANNand M. A. LOGAN,J. biol. Cheln. 184, 299 (1950).
