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Radiozinc studies in experimental wound healing
Radiozinc Studies in Experimental Wound Heahng E D W I N D. S A V L O V , M . D . , W I L L I A M H. S T R A I N , P h . D . , A N D F E L I X H U E G I N , M . D . ,
University
of Rochester School of Medicine and Dentistry
S t r a i n , P o r i e s a n d H i n s h a w 1~ r e p o r t e d r e c e n t l y that the a d d i t i o n of m i l l i g r a m q u a n t i t i e s o f m e t h i o n i n e z i n c to s t a n d a r d r a t d i e t s c o n s i s t e n t l y p r o m o t e d t h e h e a l i n g of b u r n s a n d e x c i s e d wounds in r a t s . M i x t u r e s o f m e t h i o n ine and zinc oxide u s u a l l y p r o m o t e d h e a l i n g , but m e t h i o n i n e a l o n e , o r z i n c o x i d e a l 9 n e , gave variable results. T o e x a m i n e f u r t h e r th e r o l e o f z i n c in t h e h e a l i n g p r o c e s s , s t u d i e s h a v e b e e n m a d e in adult Sprague-Dawley rats with radiozinc-65, a g a m m a e m i t t e r w i t h a h a l f - l i f e o f 245 d a y s . Short term experiments with incised sutured a b d o m i n a l w o u n d s , and long t e r m s t u d i e s with excised full thickness skin wounds, have been c a r r i e d out. The w o r k has shown that r a d i o zinc l o c a l i z e s in the a c t i v e h e a l i n g a r e a a c c o r d i n g to a n i n t e r e s t i n g t i m e p a t t e r n but does not p e r s i s t in s i g n i f i c a n t a m o u n t s in scar t i s s u e . METHODS Short Term The procedure for incised abdominal w o u n d s d e v e l o p e d by S a v l o v a n d D u n p h y 8 w a s u s e d for the s h o r t t e r m e x p e r i m e n t s . E a c h o f 56 m a l e r a t s ( w e i g h i n g 4 0 0 - 4 5 0 g i n . ) w a s i n dividually etherized, and the h a i r of the a b d o m e n c l i p p e d . T o p a r a l l e l t h e s t u d i e s of B a l l o u ~ on t h e r e t e n t i o n o f r a d i o z i n c - 6 5 b y r a t s , e a c h a n i m a l was g i v e n by tail v e i n a p p r o x i m a t e l y 5 ~c o f z i n c - 6 5 c h l o r i d e c o n t a i n e d in 1 m l . o f i s o t o n i c s a l i n e . A 2 cm. v e r t i c a l i n c i s i o n was m a d e t h r o u g h the r i g h t a b d o m i n a l wall, and c l o s e d with i n t e r r u p t e d #B2 s t a i n l e s s s t e e l w i r e s u t u r e s . T h e r e w a s l i t t l e o r no b l e e d i n g . G r o u p s of four r a t s w e r e s a c r i f i c e d by e x s a n g u i n a t i o n at d a i l y i n t e r v a l s up to e i g h t F r o m the Dep art m e n t s of S u r g e r y and Radiology, the U n i v e r s i t y of R o c h e s t e r School of Medicine and Dentistry, Rochester 20, N e w York. Aided in p a r t by USPHS Grant C-3952. Submitted for publication S e p t e m b e r 19, 1961.
] S R -- Vol. 11, No. 3 - - IHgy, I962
d a y s , a n d t h e r e a f t e r on t h e 10th, 12th, 16th, 2 0 t h an d B0th d a y s . T h e s k i n w o u n d a n d u n d e r lying m u s c l e wound of e a c h rat was e x c i s e d a l o n g e a c h s i d e of t h e w o u n d i n c i s i o n . S a m p l e s of n o r m a l s k i n an d m u s c l e of a p p r o x i m a t e l y the s a m e w e i g h t w e r e e x c i s e d f r o m a d j a c e n t a r e a s of the left a b d o m i n a l wall. E a c h t i s s u e w a s w e i g h e d ( w e i g h t r a n g e 0.06 - 0 . 4 1 g r a m ) , and its radioactivity determined, without w a s h i n g , in a d e e p - w e l l g a m m a s c i n t i l l a t i o n c o u n t e r e q u i p p e d with an inch and a half sodium iodide crystal. S i m i l a r s t u d i e s w e r e m a d e by i n j e c t i n g by t a i l v e i n a p p r o x i m a t e l y 2 pc of s o d i u m i o d i d e - 1 3 1 , c o n t a i n e d i n 1 m l . of n o r m a l s a l i n e , i n t o e a c h o f 16 a d d i t i o n a l m a l e r a t s of the s a m e w e i g h t r a n g e . G r o u p s of f o u r r a t s w e r e s a c r i f i c e d daily for four days, and the t i s s u e s e x c i s e d and counted. Long Term T h e p r o c e d u r e d e v e l o p e d by S t r a i n e t a l . 9 f o r f u l l t h i c k n e s s w o u n d s on t h e b a c k s o f r a t s was u s e d f o r the long t e r m e x p e r i m e n t s . T w e l v e f e m a l e r a t s , old b r e e d e r s of w e i g h t range 300-400 gin., were anesthetized with ether. The back hair was clipped closely, and e a c h r a t i n j e c t e d by t a i l v e i n w i t h a p p r o x i m a t e l y 1 pc o f z i n c - 6 5 c h l o r i d e c o n t a i n e d i n 1 m l . of n o r m a l s a l i n e . Six r a t s w e r e u s e d as controls. Circles 3 cm. indiameter centering a b o u t t h e s p i n e and e x t e n d i n g f r o m t h e m i d lumbar to t h e m i d - t h o r a c i c region were s t a m p e d on t h e b a c k s of t h e o t h e r s i x r a t s . A full t h i c k n e s s skin e x c i s i o n was m a d e on e a c h e x p e r i m e n t a l r a t by n i c k i n g t h e c i r c l e w i t h a s c a l p e l and c u t t i n g a r o u n d t h e c i r c u m ference with curved scissors. The calculated a r e a of t h e s k i n r e m o v e d w a s 7.1 s q . c m . o r approximately 2 p e r c e n t of t h e t o t a l s k i n surface. Immediately a f t e r o p e r a t i o n , the e x c i s e d w o u n d a r e a e x p a n d e d to a b o u t 10.5 sq. c m . Although the wounded r a t s h e a l e d within 209
210
] S R - V o l . II, N o . 3 -- May, 1 9 6 2
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31 to 53 d a y s ( a v g . 42 d a y s ) , t h e y w e r c k e p t for I00 days before being killed t o allow complete redistribution of radiozinc after the a c t i v e h e a l i n g p r o c e s s was c o m p l e t e d . At a u t o p s y the r a t s w e r e c l i p p e d a g a i n and t i s s u e r a d i o a c t i v i t y was d e t e r m i n e d in s c a r t i s s u e , a d j a c e n t n o r m a l s k i n , a n d h a i r of t h e a n i m a l s that had b e e n o p e r a t e d upon, and in t h e s k i n an d h a i r o f t h e c o n t r o l s . The concentrations of r a d i o z i n c and of r a d i o i o d i n e in the t a b l e s and f i g u r e s a r e corrected f o r d e c a y and e x p r e s s e d as p e r c e n t of a d m i n i s t e r e d d o s e p e r g r a m o f t i s s u e . T h r o u g h the u s e of this s t a n d a r d p r o c e d u r e o f r e t e n t i o n v a l u e s , e r r o r s d u e to i n s t r u mentation become negligible.
Days After Injection
Tabie. 1. Range of Weights and Radiozinc Retention of Wound Tissues in Rats
Days After Tissue * Injection
Tissue weights in grams
Per cent injected dose per gram tissue
Range Average
Range Average
Skin Skin w o u n d Muscle Muscle wound Blood
0.07-0.12 0.09 0.07-0.17 0.13 0.18-0.39 0 . 2 7 0.20-0.38 0.29 -
0.19-0,30 0.44-0.68 0.15-0.20 0.26-0.28 0.15-0.19
0.26 0.52 0.17 0.27 0.16
Skin Skin w o u n d Muscle Muscle wound Blood
0.10-0.18 0 . 1 4 0.12-0.15 0 . 1 4 0.16-0.25 0 . 2 1 0.22-0.33 0.26 -
0.19-0.29 0.24 0.53-0.65 0.60 0.21-0.31 0.25 0.34-0.45 0.38 0.14-0.17 0.16
Skin Sklnwound Muscle Muscle wound Blood
0.09-0.20 0 . 1 3 0.13-0.22 0 . 1 9 0.17-0.38 0.25 0.26-0.37 0 . 2 9 -
0.19-0.25 0.22 0.39-0.52 0.47 0.20-0.27 0.24. 0.34-0.44 0.39 0.14-0.19 0.16
Skin Skin w o u n d Muscle Muscle wound Blood
0.10-0.15 0.12 0.10-0.16 0.13 0.25-0.31 0 . 2 7 0.13-0.26 0 . 2 1 -
0.18-0.27 0.23 0.34-0.51 0.43 0.18-0.21 0.20 0.34-0.40 0.36 0.12-0.14 0.13
Skin Skinwound Muscle Musclewound Blood
0.13-0.16 0 . 1 4 0.10-0.14 0.12 0.19-0.36 0 . 2 5 0.25-0.41 0.33 -
0.17-0.22 0.33-0.39 0.18-0.25 0.27-0.38 0.12
0.19 0.36 0.22 0.32 0.12
Tissue weights in grams Range Average
Range Average
6
7
Skin 0.07-0.11 0.09 Skinwound 0.09-0.12 0 . 1 1 Muscle 0.21-0.27 0.24 Muscle wound 0.17-0.26 0 . 2 1 Blood -
0.18-0.24 0.22 0.24-0.49 0.36 0.21-0.26 0.24 0.23-0.28 0.25 0.10-0.13 0.11
Skin Skin w o u n d Muscle Musclewound Blood
0.07-0.13 0 . 1 1 0.09-0.13 0 . 1 1 0.1%0.29 0 . 2 4 0.20-0.23 0.22 -
0.13-0.17 0.20-0.29 0.20-0.30 0.24-0.32 0.10-0.13
0.15 0.23 0.25 0.27 0.12
10
Skin Skinwound Muscle Muscle wound Blood
0.09-0.17 0.13 0.08-0.13 0 . 1 1 0.22-0.35 0.30 0.16-0.20 0.19 -
0.10-0.13 0.16-0.20 0.17-0.21 0.21-0.31 0.07-0.09
0.11 0.18 0.20 0.25 0.08
12
Skin Skin w o u n d Muscle Muscle wound Blood
0.09-0.13 0 . 1 1 0.11-0.18 0.14 0.15-0.30 0 . 2 1 0.13-0.26 0.19
0.10-0.14 0.11"0.16 0.18-0.20 0.18-0.23 0.07-0.09
0.12 0.14 0.19 0.22
0.08
16
Skin 0.07-0.11 0.09 Skin w o u n d 0.08-0.15 0.12 Muscle 0.15-0.31 0.24 Musc[e wound 0.15-0.31 0.23 Blood -
0.06-0.14 0.11-0.13 0.17-0.20 0.11-0.18 0.05-0.06
0.10 0.12 0.18 0.14 0.06
20
Skin 0.06-0.15 0.10 Skin w o u n d 0.09-0.15 0.13 Muscle 0.11-0.29 0 . 2 0 Muscle wound 0.12-0.19 0 . 1 4 Blood -
0.06-0.15 0.10-0.11 0.16-0.20 0.16-0.23 0.05-0.06
0.12 0.10 0.18' 0.19 0.05
8
0.08-0.13 0.10 0.06-0.15 0.10 0.21-0.30 0.25 0.15-0.26 0.20 -
Per cent injected dose per gram tissue
Skin Skinwound Muscle Muscle wound Blood
RESULTS Short T e r m T h e range of tissue weights and per cent retention of the injected dose of radiozinc65 per g r a m of tissue are collected in Table I. T h e radioactivity data w e r e r e m a r k a b l y consistent and reproducible, as w a s s h o w n
Tissue *
-
-
0.14-0.26 0.31-0.38 0.19-0.26 0.28-0.34 O.10-0.14
0.20 0.34 0.22 0.32 0.11
"1 ml. blood used for all analyses.
by check experiments and by the good agreem e n t of the blood retention values with those reported by Ballou. 3 T h e r e w a s never any overlap in the zinc retention values for c o m parable w o u n d and n o r m a l tissues before the 10th day in the skin and the 6th day in the m u s c l e tissues. A definite pattern of preferential a c c u m u lation of radiozinc in the w o u n d tissues is evident during the first seven days of the short t e r m experiments (Figs. I, 2). T h e skin wound initially retained more than twice a s m u c h r a d i o z i n c as t h e n o r m a l s k i n , a n d s h o w e d a g r a d u a l d e c r e a s e f r o m t h e t h i r d to the fifth day. At this t i m e , the r a d i o z i n c l e v e l
]SR
Vol. ll, No. 5 -- May, 1962
- -
EXPERIMENTAL WOUND HEALING
RADIOZING LEVELS IN SKiN WOUNDS
o.1,
211
T a b l e 2. R e t e n t i o n of I T M by Incised Abdominal "Wounds Compared with Normal Tissues and Blood (Per Cent of Injected Dose Per Gram of Tissue; Each Value is the Average of 4 Rots)
0.6 0.5
0.4
Tissue ~
9---,....~ skin
0.2
~ %.~
0.1 .
0
.
.
.
.
.
.
.
.
.
.
.
II
I 2 3 4 5 6 7 8
I0
12
,
16
20
Blood
TIME IN DAYS
Fig. 1: "Radiozinc retention in ~ncised skin wounds and n o r m a l skin following injection of Zn65C12 intravenously at time of wounding. i n the s k i n w o u n d r e m a i n e d e s s e n t i a l l y c o n s t a n t u n t i l t h e s e v e n t h day, w h e n a r a p i d d e c r e a s e to a p p r o x i m a t e l y the s a m e a c t i v i t y as normal skin occurred. A similar, though less marked, p l a t e a u d e v e l o p e d w i t h the muscle wounds. T h e c o m p a r a t i v e studies with radioiodine131 are given in T a b l e 2. A l t h o u g h the radioactivity of the tissues w a s n e v e r c o m p a r a b l e to levels obtained after injection ofradiozinc, the w o u n d e d tissues w e r e u n i f o r m l y m o r e active than the n o r m a l . After four days, h o w ever, the activity w a s so l o w in both w o u n d e d and n o r m a l tissues that no differentiation could be m a d e . Long Term
T h e d a t a r e l a t i n g to the l o n g t e r m e x p e r i m e n t s w i t h r a d i o z i n c a r e g i v e n i n T a b l e 3. A f t e r 100 d a y s the h e a l e d c i c a t r i x c o n t a i n e d l i t t l e o r no a c t i v i t y c o m p a r e d w i t h a d j a c e n t n o r m a l s k i n w h e r e , i n p a r t , the r a d i o a c t i v i t y w a s d u e to h a i r r o o t s . I n b o t h c o n t r o l a n d e x p e r i m e n t a l a n i m a l s the r a d i o a c t i v i t y of the h a i r w a s r e l a t i v e l y h i g h , v a r i a b l e , a n d of a b o u t the s a m e m a g n i t u d e . DISCUSSION R e t e n t i o n f o r 12 d a y s of m e a s u r a b l y g r e a t e r a m o u n t s of r a d i o z i n c i n the h e a l i n g i n c i s e d wounds than in n o r m a l t i s s u e s is c o n s i s t e n t 0.5
~
0.4
~
0.3
~0.t. .
I
.
.
.
.
.
.
.
.
2 3 4 5 6 7 B
.
.
.
I0
II
12
16
2
0.07 0.12 0.009 0.021 -
0.03 0.08 0.004 0.02. " 0.02
3
0.04 0.06 0.001 0.009 0.015
0.02 0.05 0.008 0.012 0.008
w i t h a c t i v e p a r t i c i p a t i o n of z i n c i n t h e h e a l i n g process. 9176176 of the p l a t e a u s s h o w n i n F i g u r e s 1 a n d Z, f r o m the f i f t h to the s e v e n t h d a y , p o i n t s to a s p e c i a l s i t u a t i o n i n t h e h e a l ing w o u n d s a t t h i s t i m e . T h i s m a y m e a n t h e d e v e l o p m e n t of v a s c u l a r i t y i s g r e a t e s t a t t h i s p e r i o d . W e i b e r is s t u d i e d h e a l i n g w o u n d s i n r a b b i t s , g u i n e a p i g s a n d d o g s w i t h the a i d of 1131 , r a d i o a c t i v e i o d i n a t e d h u m a n s e r u m a l b u m i n , C r S l t a g g e d e r y t h r o c y t e s a n d P a2,all w i t h d a i l y i n j e c t i o n s ; he c o n c l u d e d t h a t v a s cularization progresses successively during the f i r s t five d a y s , the s o - c a l l e d lag p e r i o d . T h e r a d i o a c t i v i t y of the w o u n d a r e a s g r a d u a l l y d e c r e a s e d a f t e r the fifth d a y b u t s t i l l r e m a i n e d a b o u t 50 p e r c e n t h i g h e r t h a n i n t a c t t i s s u e a s l a t e a s the t h i r t e e n t h d a y . S i m i l a r l y , H a l e y , Z y d l o a n d W i l l i a m s o n 5 fou*/d w i t h e a r l y p o s t o p e r a t i v e p a t i e n t s t h a t r e t e n t i o n of r a d i o isotopes, administered by various routes, i n c r e a s e s to a m a x i m u m at f i v e to s e v e n days and then declines slowly. P l a t e a u f o r m a t i o n at the f i f t h to s e v e n t h d a y s i n the z i n c r e t e n t i o n c u r v e s m a y be associated with some particular enzyme s i t u a t i o n . D u n p h y 4 p o i n t e d out t h a t o n the s i x t h d a y of h e a l i n g t h e r e is a n i n t e r s e c t i o n Table 3. Radiozinr Retention by Scar Tissue of Healed Excised Wounds in Rats" Compared with Adjacent and Control Skin and Hair 100 Days after Injection (Per Cent of Injected Dose Per Gram of Tissue)
Skin Hair from Hair from Rat Scar Control Number Tissue Adjacent S k i n Wounded Control to Sear Rats Rats
RADIOZING LEVELS IN MUSCLEWOUNDS
0.2'
0
Skin Skin wound Muscle Muscle wound
Days After Injection 1
40
TIME IN DAYS Fig. 2. Radioz*nc retention in muscle wounds tnd normal muscle from s a m e rats as inFigure I.
1 2 3 4 5 6
0.005 0.004 0.001 0.001 0.007 0.004
0.018 0.029 0.053 0.029 0.014 0.024
0.030 0.016 0.008 0.009 0.017 0.013
1.18 0.82 2.09 0.70 1.66 1.45
0.70 2.03 1.14 1.88 1.25 1.59
Average
0.004
0.028
0.017
1.32
1.43
212
SAVLOV, STRAIN and HUEGIN
of the c u r v e showing the r i s i n g c o l l a g e n f o r m a t i o n with the c u r v e of the s h a r p l y d e clining hexosamine l e v e l of the wound. In c o n t r a s t to t h e z i n c r e t e n t i o n c u r v e s , t h e i o d i n e - 1 3 1 r e t e n t i o n s t u d i e s showy no l e v e l i n g o f f of t h e r a d i o a c t i v i t y at t h e f i f t h to s e v e n t h days. The concept that zinc may participate only in t he e a r l y p h a s e s o f h e a l i n g i s c o n s i s t e n t with the low r e t e n t i o n l e v e l of r a d i o z i n c in the s c a r t i s s u e of the e x c i s e d wound. T h e r e w a s a m p l e t i m e in I 0 0 d a y s f o r ~ e d i s t r i b u t i o n Of a n y r a d i o z i n c t h a t m a y h a v e p a r t i c i p a t e d in e a r l y p h a s e s of the h e a l i n g p r o c e s s . A l t e r n a t e l y , zinc m a y not h a v e b e e n r e q u i r e d d i r e c t l y f o r the f o r m a t i o n of the c o n n e c t i v e tissue of the scar. A l t h o u g h earlier w o r k i~ has s h o w n that minute a m o u n t s of dietary methionine zinc p r o m o t e the healing of excised w o u n d s in rats, the present studies s h o w nothing about possible participation 6f any a m i n o acid or a m i n o acid m o i e t y in the healing process. Evidence that nutritional zinc is important in growth and repair of tissue is abundant. Zinc supplementation of practical pig and poultry rations is well established for the prevention of deficiency state s and for optimal growth. In swine, zinc deficiency is characterized by poor appetite, d e c r e a s e d growth rate, inefficient food utilization and a severe skin disease calledparakeratosis, i Chickens exhibit m a r k e d bone and feather a b n o r m a l ities, d e r m a t o s e s and retarded growth w h e n fed diets in which the zinc content is low, or when zinc is not available. 2 B e c a u s e the zinc requirement of chickens appears to be greatest during the first few w e e k s of life, it s e e m s that active tissue growth depends on the presence of zinc in an available form. C a l c i u m and certain chelating agents have been s h o w n to be antagonists of zinc in these animals.t. 2 How zinc f u n c t i o n s in p r o m o t i n g g r o w t h a nd r e p a i r o f t i s s u e s is u n k n o w n . M a n y e n z y m e s c o n t a i n z i n c II but t h e r e d o e s n o t s e e m to be a d i f i c i e n c y o f c a r b o n i c a n h y d r a s e , f o r e x a m p l e , in the r e d c e l l s of zinc deficient chickens. Lewis, Hoekstra and Grummer6have shown that i n c r e a s i n g the z i n c c o n t e n t in t h e d i e t o f p i g s i n c r e a s e s i n v a r y i n g d e g r e e s the zinc c o n t e n t of h a i r a nd b o n e , p a r t i c u l a r l y , but d o e s n o t g i v e a p a r a l l e l i n c r e a s e in zinc c o n t e n t of the skin.
JSR
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Vol. II, No. 3 - May, I 9 6 2
T h e y s u g g e s t that the site of a c t i o n of zinc m a y w e l l be in the g a s t r o i n t e s t i n a l t r a c t . SUMMARY Studies of r a d i o z i n c r e t e n t i o n in e x p e r i m e n t a l wound h e a l i n g h a v e b e e n c a r r i e d out i n r a t s w i t h i n c i s e d a b d o m i n a l w o u n d s and excised back wounds. Apreferential accumul a t i o n of r a d i o a c t i v i t y was d e m o n s t r a t e d in t h e a c u t e h e a l i n g s t a g e of i n c i s e d w o u n d s , coinciding withthe development ofvascularity an d b i o c h e m i c a l a l t e r a t i o n s . M e a s u r e m e n t s m a d e on t h e s c a r c o n n e c t i v e t i s s u e o f t h e e x c i s e d w o u n d s a f t e r I00 d a y s s h o w e d v e r y l o w r e t e n t i o n of r a d i o z i n c c o m p a r e d to t h e s u r r o u n d i n g skin and h a i r . RE FERENCES 1. Anon.: Zinc deficiency and d i e t a r y c a l c i u m in swine. Nutr. Rev., 15:334-336, 1957. 2. Anon.: Zinc deficiency i n ch i ck en s. Nutr. Rev., 19:111-113, 1961. 3. Ballou, J. E.: Metabolism of ZnSSin the rat. H.W.-60062, Richland, Wash. H a n f o r d A t o m i c P r o d u ct Operation, A p r i l 17, 1959. 4. Dunphy, J. E.: On the nature and c a r e of wounds. Ann. Royal Coll. Surgeons, 26:69-87, 1960. 5 . Haley, H. B., Zydlo, S., and Williamson, M. B.: Deposition of radioisotopes in s u r g i c a l incisions in man. Surg. F o r u m , I0:316-318, 1960. 6. Lewis, P. K., J r . , Hoekstra, W. G., a n d G r u m m e t , R . H . : R e s t r i c t e d cal ci u m feeding v e r s u s zinc supplementation for the control o f p a r a k e r a t o s i s in swine. J. A n i m a l S c i . , 16:578-588, 1957. 7. O'DelI, B. L., Newberne, P. M., and Savage, J. E.: Significance of d i e t a r y zinc for the growing chicken. J. Nutrition, 65:503-523, 1958. 8. Savlov, E . D., and Dunphy, J. E.: The healing of the disrupted and r esu t u r ed w o u n d . Surgery, 36:362-370, 1954. 9. Strain, W. H., Dutton, A. M., Heyer, H. B., and Ramsey, G. H.: E x p e r i m e n t a l studies on the a c c e l e r a t i o n of burn and wound healing. Univ. of Rochester Reports, 1953. 10. Strain, %V. H., Pories, W. J., and Hinshaw, J. R.: Zinc studies in skin repair. Surg. Forum, 11:291-292, 1960. I I . Vallee, B. L.: Biochemistry, physiology and pathology of zinc. Physiol. Rev., 39:443-490, 1959. 12. Vallee, B. L., Hoch, F. L., and Hughes, %V. L.: Soluble zinc containing protein extracted from human leucocytes. Arch. Biochem., 48:347360, 1954. 13. Weiber, A.: Studies in vascularization of healing wounds with radioactive isotopes.Acts chir. scandinav., Supplementum, 237, 1959.
University
of Rochester School of Medicine and Dentistry
S t r a i n , P o r i e s a n d H i n s h a w 1~ r e p o r t e d r e c e n t l y that the a d d i t i o n of m i l l i g r a m q u a n t i t i e s o f m e t h i o n i n e z i n c to s t a n d a r d r a t d i e t s c o n s i s t e n t l y p r o m o t e d t h e h e a l i n g of b u r n s a n d e x c i s e d wounds in r a t s . M i x t u r e s o f m e t h i o n ine and zinc oxide u s u a l l y p r o m o t e d h e a l i n g , but m e t h i o n i n e a l o n e , o r z i n c o x i d e a l 9 n e , gave variable results. T o e x a m i n e f u r t h e r th e r o l e o f z i n c in t h e h e a l i n g p r o c e s s , s t u d i e s h a v e b e e n m a d e in adult Sprague-Dawley rats with radiozinc-65, a g a m m a e m i t t e r w i t h a h a l f - l i f e o f 245 d a y s . Short term experiments with incised sutured a b d o m i n a l w o u n d s , and long t e r m s t u d i e s with excised full thickness skin wounds, have been c a r r i e d out. The w o r k has shown that r a d i o zinc l o c a l i z e s in the a c t i v e h e a l i n g a r e a a c c o r d i n g to a n i n t e r e s t i n g t i m e p a t t e r n but does not p e r s i s t in s i g n i f i c a n t a m o u n t s in scar t i s s u e . METHODS Short Term The procedure for incised abdominal w o u n d s d e v e l o p e d by S a v l o v a n d D u n p h y 8 w a s u s e d for the s h o r t t e r m e x p e r i m e n t s . E a c h o f 56 m a l e r a t s ( w e i g h i n g 4 0 0 - 4 5 0 g i n . ) w a s i n dividually etherized, and the h a i r of the a b d o m e n c l i p p e d . T o p a r a l l e l t h e s t u d i e s of B a l l o u ~ on t h e r e t e n t i o n o f r a d i o z i n c - 6 5 b y r a t s , e a c h a n i m a l was g i v e n by tail v e i n a p p r o x i m a t e l y 5 ~c o f z i n c - 6 5 c h l o r i d e c o n t a i n e d in 1 m l . o f i s o t o n i c s a l i n e . A 2 cm. v e r t i c a l i n c i s i o n was m a d e t h r o u g h the r i g h t a b d o m i n a l wall, and c l o s e d with i n t e r r u p t e d #B2 s t a i n l e s s s t e e l w i r e s u t u r e s . T h e r e w a s l i t t l e o r no b l e e d i n g . G r o u p s of four r a t s w e r e s a c r i f i c e d by e x s a n g u i n a t i o n at d a i l y i n t e r v a l s up to e i g h t F r o m the Dep art m e n t s of S u r g e r y and Radiology, the U n i v e r s i t y of R o c h e s t e r School of Medicine and Dentistry, Rochester 20, N e w York. Aided in p a r t by USPHS Grant C-3952. Submitted for publication S e p t e m b e r 19, 1961.
] S R -- Vol. 11, No. 3 - - IHgy, I962
d a y s , a n d t h e r e a f t e r on t h e 10th, 12th, 16th, 2 0 t h an d B0th d a y s . T h e s k i n w o u n d a n d u n d e r lying m u s c l e wound of e a c h rat was e x c i s e d a l o n g e a c h s i d e of t h e w o u n d i n c i s i o n . S a m p l e s of n o r m a l s k i n an d m u s c l e of a p p r o x i m a t e l y the s a m e w e i g h t w e r e e x c i s e d f r o m a d j a c e n t a r e a s of the left a b d o m i n a l wall. E a c h t i s s u e w a s w e i g h e d ( w e i g h t r a n g e 0.06 - 0 . 4 1 g r a m ) , and its radioactivity determined, without w a s h i n g , in a d e e p - w e l l g a m m a s c i n t i l l a t i o n c o u n t e r e q u i p p e d with an inch and a half sodium iodide crystal. S i m i l a r s t u d i e s w e r e m a d e by i n j e c t i n g by t a i l v e i n a p p r o x i m a t e l y 2 pc of s o d i u m i o d i d e - 1 3 1 , c o n t a i n e d i n 1 m l . of n o r m a l s a l i n e , i n t o e a c h o f 16 a d d i t i o n a l m a l e r a t s of the s a m e w e i g h t r a n g e . G r o u p s of f o u r r a t s w e r e s a c r i f i c e d daily for four days, and the t i s s u e s e x c i s e d and counted. Long Term T h e p r o c e d u r e d e v e l o p e d by S t r a i n e t a l . 9 f o r f u l l t h i c k n e s s w o u n d s on t h e b a c k s o f r a t s was u s e d f o r the long t e r m e x p e r i m e n t s . T w e l v e f e m a l e r a t s , old b r e e d e r s of w e i g h t range 300-400 gin., were anesthetized with ether. The back hair was clipped closely, and e a c h r a t i n j e c t e d by t a i l v e i n w i t h a p p r o x i m a t e l y 1 pc o f z i n c - 6 5 c h l o r i d e c o n t a i n e d i n 1 m l . of n o r m a l s a l i n e . Six r a t s w e r e u s e d as controls. Circles 3 cm. indiameter centering a b o u t t h e s p i n e and e x t e n d i n g f r o m t h e m i d lumbar to t h e m i d - t h o r a c i c region were s t a m p e d on t h e b a c k s of t h e o t h e r s i x r a t s . A full t h i c k n e s s skin e x c i s i o n was m a d e on e a c h e x p e r i m e n t a l r a t by n i c k i n g t h e c i r c l e w i t h a s c a l p e l and c u t t i n g a r o u n d t h e c i r c u m ference with curved scissors. The calculated a r e a of t h e s k i n r e m o v e d w a s 7.1 s q . c m . o r approximately 2 p e r c e n t of t h e t o t a l s k i n surface. Immediately a f t e r o p e r a t i o n , the e x c i s e d w o u n d a r e a e x p a n d e d to a b o u t 10.5 sq. c m . Although the wounded r a t s h e a l e d within 209
210
] S R - V o l . II, N o . 3 -- May, 1 9 6 2
SAVLOV, STRAIN and HUEGIN
31 to 53 d a y s ( a v g . 42 d a y s ) , t h e y w e r c k e p t for I00 days before being killed t o allow complete redistribution of radiozinc after the a c t i v e h e a l i n g p r o c e s s was c o m p l e t e d . At a u t o p s y the r a t s w e r e c l i p p e d a g a i n and t i s s u e r a d i o a c t i v i t y was d e t e r m i n e d in s c a r t i s s u e , a d j a c e n t n o r m a l s k i n , a n d h a i r of t h e a n i m a l s that had b e e n o p e r a t e d upon, and in t h e s k i n an d h a i r o f t h e c o n t r o l s . The concentrations of r a d i o z i n c and of r a d i o i o d i n e in the t a b l e s and f i g u r e s a r e corrected f o r d e c a y and e x p r e s s e d as p e r c e n t of a d m i n i s t e r e d d o s e p e r g r a m o f t i s s u e . T h r o u g h the u s e of this s t a n d a r d p r o c e d u r e o f r e t e n t i o n v a l u e s , e r r o r s d u e to i n s t r u mentation become negligible.
Days After Injection
Tabie. 1. Range of Weights and Radiozinc Retention of Wound Tissues in Rats
Days After Tissue * Injection
Tissue weights in grams
Per cent injected dose per gram tissue
Range Average
Range Average
Skin Skin w o u n d Muscle Muscle wound Blood
0.07-0.12 0.09 0.07-0.17 0.13 0.18-0.39 0 . 2 7 0.20-0.38 0.29 -
0.19-0,30 0.44-0.68 0.15-0.20 0.26-0.28 0.15-0.19
0.26 0.52 0.17 0.27 0.16
Skin Skin w o u n d Muscle Muscle wound Blood
0.10-0.18 0 . 1 4 0.12-0.15 0 . 1 4 0.16-0.25 0 . 2 1 0.22-0.33 0.26 -
0.19-0.29 0.24 0.53-0.65 0.60 0.21-0.31 0.25 0.34-0.45 0.38 0.14-0.17 0.16
Skin Sklnwound Muscle Muscle wound Blood
0.09-0.20 0 . 1 3 0.13-0.22 0 . 1 9 0.17-0.38 0.25 0.26-0.37 0 . 2 9 -
0.19-0.25 0.22 0.39-0.52 0.47 0.20-0.27 0.24. 0.34-0.44 0.39 0.14-0.19 0.16
Skin Skin w o u n d Muscle Muscle wound Blood
0.10-0.15 0.12 0.10-0.16 0.13 0.25-0.31 0 . 2 7 0.13-0.26 0 . 2 1 -
0.18-0.27 0.23 0.34-0.51 0.43 0.18-0.21 0.20 0.34-0.40 0.36 0.12-0.14 0.13
Skin Skinwound Muscle Musclewound Blood
0.13-0.16 0 . 1 4 0.10-0.14 0.12 0.19-0.36 0 . 2 5 0.25-0.41 0.33 -
0.17-0.22 0.33-0.39 0.18-0.25 0.27-0.38 0.12
0.19 0.36 0.22 0.32 0.12
Tissue weights in grams Range Average
Range Average
6
7
Skin 0.07-0.11 0.09 Skinwound 0.09-0.12 0 . 1 1 Muscle 0.21-0.27 0.24 Muscle wound 0.17-0.26 0 . 2 1 Blood -
0.18-0.24 0.22 0.24-0.49 0.36 0.21-0.26 0.24 0.23-0.28 0.25 0.10-0.13 0.11
Skin Skin w o u n d Muscle Musclewound Blood
0.07-0.13 0 . 1 1 0.09-0.13 0 . 1 1 0.1%0.29 0 . 2 4 0.20-0.23 0.22 -
0.13-0.17 0.20-0.29 0.20-0.30 0.24-0.32 0.10-0.13
0.15 0.23 0.25 0.27 0.12
10
Skin Skinwound Muscle Muscle wound Blood
0.09-0.17 0.13 0.08-0.13 0 . 1 1 0.22-0.35 0.30 0.16-0.20 0.19 -
0.10-0.13 0.16-0.20 0.17-0.21 0.21-0.31 0.07-0.09
0.11 0.18 0.20 0.25 0.08
12
Skin Skin w o u n d Muscle Muscle wound Blood
0.09-0.13 0 . 1 1 0.11-0.18 0.14 0.15-0.30 0 . 2 1 0.13-0.26 0.19
0.10-0.14 0.11"0.16 0.18-0.20 0.18-0.23 0.07-0.09
0.12 0.14 0.19 0.22
0.08
16
Skin 0.07-0.11 0.09 Skin w o u n d 0.08-0.15 0.12 Muscle 0.15-0.31 0.24 Musc[e wound 0.15-0.31 0.23 Blood -
0.06-0.14 0.11-0.13 0.17-0.20 0.11-0.18 0.05-0.06
0.10 0.12 0.18 0.14 0.06
20
Skin 0.06-0.15 0.10 Skin w o u n d 0.09-0.15 0.13 Muscle 0.11-0.29 0 . 2 0 Muscle wound 0.12-0.19 0 . 1 4 Blood -
0.06-0.15 0.10-0.11 0.16-0.20 0.16-0.23 0.05-0.06
0.12 0.10 0.18' 0.19 0.05
8
0.08-0.13 0.10 0.06-0.15 0.10 0.21-0.30 0.25 0.15-0.26 0.20 -
Per cent injected dose per gram tissue
Skin Skinwound Muscle Muscle wound Blood
RESULTS Short T e r m T h e range of tissue weights and per cent retention of the injected dose of radiozinc65 per g r a m of tissue are collected in Table I. T h e radioactivity data w e r e r e m a r k a b l y consistent and reproducible, as w a s s h o w n
Tissue *
-
-
0.14-0.26 0.31-0.38 0.19-0.26 0.28-0.34 O.10-0.14
0.20 0.34 0.22 0.32 0.11
"1 ml. blood used for all analyses.
by check experiments and by the good agreem e n t of the blood retention values with those reported by Ballou. 3 T h e r e w a s never any overlap in the zinc retention values for c o m parable w o u n d and n o r m a l tissues before the 10th day in the skin and the 6th day in the m u s c l e tissues. A definite pattern of preferential a c c u m u lation of radiozinc in the w o u n d tissues is evident during the first seven days of the short t e r m experiments (Figs. I, 2). T h e skin wound initially retained more than twice a s m u c h r a d i o z i n c as t h e n o r m a l s k i n , a n d s h o w e d a g r a d u a l d e c r e a s e f r o m t h e t h i r d to the fifth day. At this t i m e , the r a d i o z i n c l e v e l
]SR
Vol. ll, No. 5 -- May, 1962
- -
EXPERIMENTAL WOUND HEALING
RADIOZING LEVELS IN SKiN WOUNDS
o.1,
211
T a b l e 2. R e t e n t i o n of I T M by Incised Abdominal "Wounds Compared with Normal Tissues and Blood (Per Cent of Injected Dose Per Gram of Tissue; Each Value is the Average of 4 Rots)
0.6 0.5
0.4
Tissue ~
9---,....~ skin
0.2
~ %.~
0.1 .
0
.
.
.
.
.
.
.
.
.
.
.
II
I 2 3 4 5 6 7 8
I0
12
,
16
20
Blood
TIME IN DAYS
Fig. 1: "Radiozinc retention in ~ncised skin wounds and n o r m a l skin following injection of Zn65C12 intravenously at time of wounding. i n the s k i n w o u n d r e m a i n e d e s s e n t i a l l y c o n s t a n t u n t i l t h e s e v e n t h day, w h e n a r a p i d d e c r e a s e to a p p r o x i m a t e l y the s a m e a c t i v i t y as normal skin occurred. A similar, though less marked, p l a t e a u d e v e l o p e d w i t h the muscle wounds. T h e c o m p a r a t i v e studies with radioiodine131 are given in T a b l e 2. A l t h o u g h the radioactivity of the tissues w a s n e v e r c o m p a r a b l e to levels obtained after injection ofradiozinc, the w o u n d e d tissues w e r e u n i f o r m l y m o r e active than the n o r m a l . After four days, h o w ever, the activity w a s so l o w in both w o u n d e d and n o r m a l tissues that no differentiation could be m a d e . Long Term
T h e d a t a r e l a t i n g to the l o n g t e r m e x p e r i m e n t s w i t h r a d i o z i n c a r e g i v e n i n T a b l e 3. A f t e r 100 d a y s the h e a l e d c i c a t r i x c o n t a i n e d l i t t l e o r no a c t i v i t y c o m p a r e d w i t h a d j a c e n t n o r m a l s k i n w h e r e , i n p a r t , the r a d i o a c t i v i t y w a s d u e to h a i r r o o t s . I n b o t h c o n t r o l a n d e x p e r i m e n t a l a n i m a l s the r a d i o a c t i v i t y of the h a i r w a s r e l a t i v e l y h i g h , v a r i a b l e , a n d of a b o u t the s a m e m a g n i t u d e . DISCUSSION R e t e n t i o n f o r 12 d a y s of m e a s u r a b l y g r e a t e r a m o u n t s of r a d i o z i n c i n the h e a l i n g i n c i s e d wounds than in n o r m a l t i s s u e s is c o n s i s t e n t 0.5
~
0.4
~
0.3
~0.t. .
I
.
.
.
.
.
.
.
.
2 3 4 5 6 7 B
.
.
.
I0
II
12
16
2
0.07 0.12 0.009 0.021 -
0.03 0.08 0.004 0.02. " 0.02
3
0.04 0.06 0.001 0.009 0.015
0.02 0.05 0.008 0.012 0.008
w i t h a c t i v e p a r t i c i p a t i o n of z i n c i n t h e h e a l i n g process. 9176176 of the p l a t e a u s s h o w n i n F i g u r e s 1 a n d Z, f r o m the f i f t h to the s e v e n t h d a y , p o i n t s to a s p e c i a l s i t u a t i o n i n t h e h e a l ing w o u n d s a t t h i s t i m e . T h i s m a y m e a n t h e d e v e l o p m e n t of v a s c u l a r i t y i s g r e a t e s t a t t h i s p e r i o d . W e i b e r is s t u d i e d h e a l i n g w o u n d s i n r a b b i t s , g u i n e a p i g s a n d d o g s w i t h the a i d of 1131 , r a d i o a c t i v e i o d i n a t e d h u m a n s e r u m a l b u m i n , C r S l t a g g e d e r y t h r o c y t e s a n d P a2,all w i t h d a i l y i n j e c t i o n s ; he c o n c l u d e d t h a t v a s cularization progresses successively during the f i r s t five d a y s , the s o - c a l l e d lag p e r i o d . T h e r a d i o a c t i v i t y of the w o u n d a r e a s g r a d u a l l y d e c r e a s e d a f t e r the fifth d a y b u t s t i l l r e m a i n e d a b o u t 50 p e r c e n t h i g h e r t h a n i n t a c t t i s s u e a s l a t e a s the t h i r t e e n t h d a y . S i m i l a r l y , H a l e y , Z y d l o a n d W i l l i a m s o n 5 fou*/d w i t h e a r l y p o s t o p e r a t i v e p a t i e n t s t h a t r e t e n t i o n of r a d i o isotopes, administered by various routes, i n c r e a s e s to a m a x i m u m at f i v e to s e v e n days and then declines slowly. P l a t e a u f o r m a t i o n at the f i f t h to s e v e n t h d a y s i n the z i n c r e t e n t i o n c u r v e s m a y be associated with some particular enzyme s i t u a t i o n . D u n p h y 4 p o i n t e d out t h a t o n the s i x t h d a y of h e a l i n g t h e r e is a n i n t e r s e c t i o n Table 3. Radiozinr Retention by Scar Tissue of Healed Excised Wounds in Rats" Compared with Adjacent and Control Skin and Hair 100 Days after Injection (Per Cent of Injected Dose Per Gram of Tissue)
Skin Hair from Hair from Rat Scar Control Number Tissue Adjacent S k i n Wounded Control to Sear Rats Rats
RADIOZING LEVELS IN MUSCLEWOUNDS
0.2'
0
Skin Skin wound Muscle Muscle wound
Days After Injection 1
40
TIME IN DAYS Fig. 2. Radioz*nc retention in muscle wounds tnd normal muscle from s a m e rats as inFigure I.
1 2 3 4 5 6
0.005 0.004 0.001 0.001 0.007 0.004
0.018 0.029 0.053 0.029 0.014 0.024
0.030 0.016 0.008 0.009 0.017 0.013
1.18 0.82 2.09 0.70 1.66 1.45
0.70 2.03 1.14 1.88 1.25 1.59
Average
0.004
0.028
0.017
1.32
1.43
212
SAVLOV, STRAIN and HUEGIN
of the c u r v e showing the r i s i n g c o l l a g e n f o r m a t i o n with the c u r v e of the s h a r p l y d e clining hexosamine l e v e l of the wound. In c o n t r a s t to t h e z i n c r e t e n t i o n c u r v e s , t h e i o d i n e - 1 3 1 r e t e n t i o n s t u d i e s showy no l e v e l i n g o f f of t h e r a d i o a c t i v i t y at t h e f i f t h to s e v e n t h days. The concept that zinc may participate only in t he e a r l y p h a s e s o f h e a l i n g i s c o n s i s t e n t with the low r e t e n t i o n l e v e l of r a d i o z i n c in the s c a r t i s s u e of the e x c i s e d wound. T h e r e w a s a m p l e t i m e in I 0 0 d a y s f o r ~ e d i s t r i b u t i o n Of a n y r a d i o z i n c t h a t m a y h a v e p a r t i c i p a t e d in e a r l y p h a s e s of the h e a l i n g p r o c e s s . A l t e r n a t e l y , zinc m a y not h a v e b e e n r e q u i r e d d i r e c t l y f o r the f o r m a t i o n of the c o n n e c t i v e tissue of the scar. A l t h o u g h earlier w o r k i~ has s h o w n that minute a m o u n t s of dietary methionine zinc p r o m o t e the healing of excised w o u n d s in rats, the present studies s h o w nothing about possible participation 6f any a m i n o acid or a m i n o acid m o i e t y in the healing process. Evidence that nutritional zinc is important in growth and repair of tissue is abundant. Zinc supplementation of practical pig and poultry rations is well established for the prevention of deficiency state s and for optimal growth. In swine, zinc deficiency is characterized by poor appetite, d e c r e a s e d growth rate, inefficient food utilization and a severe skin disease calledparakeratosis, i Chickens exhibit m a r k e d bone and feather a b n o r m a l ities, d e r m a t o s e s and retarded growth w h e n fed diets in which the zinc content is low, or when zinc is not available. 2 B e c a u s e the zinc requirement of chickens appears to be greatest during the first few w e e k s of life, it s e e m s that active tissue growth depends on the presence of zinc in an available form. C a l c i u m and certain chelating agents have been s h o w n to be antagonists of zinc in these animals.t. 2 How zinc f u n c t i o n s in p r o m o t i n g g r o w t h a nd r e p a i r o f t i s s u e s is u n k n o w n . M a n y e n z y m e s c o n t a i n z i n c II but t h e r e d o e s n o t s e e m to be a d i f i c i e n c y o f c a r b o n i c a n h y d r a s e , f o r e x a m p l e , in the r e d c e l l s of zinc deficient chickens. Lewis, Hoekstra and Grummer6have shown that i n c r e a s i n g the z i n c c o n t e n t in t h e d i e t o f p i g s i n c r e a s e s i n v a r y i n g d e g r e e s the zinc c o n t e n t of h a i r a nd b o n e , p a r t i c u l a r l y , but d o e s n o t g i v e a p a r a l l e l i n c r e a s e in zinc c o n t e n t of the skin.
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Vol. II, No. 3 - May, I 9 6 2
T h e y s u g g e s t that the site of a c t i o n of zinc m a y w e l l be in the g a s t r o i n t e s t i n a l t r a c t . SUMMARY Studies of r a d i o z i n c r e t e n t i o n in e x p e r i m e n t a l wound h e a l i n g h a v e b e e n c a r r i e d out i n r a t s w i t h i n c i s e d a b d o m i n a l w o u n d s and excised back wounds. Apreferential accumul a t i o n of r a d i o a c t i v i t y was d e m o n s t r a t e d in t h e a c u t e h e a l i n g s t a g e of i n c i s e d w o u n d s , coinciding withthe development ofvascularity an d b i o c h e m i c a l a l t e r a t i o n s . M e a s u r e m e n t s m a d e on t h e s c a r c o n n e c t i v e t i s s u e o f t h e e x c i s e d w o u n d s a f t e r I00 d a y s s h o w e d v e r y l o w r e t e n t i o n of r a d i o z i n c c o m p a r e d to t h e s u r r o u n d i n g skin and h a i r . RE FERENCES 1. Anon.: Zinc deficiency and d i e t a r y c a l c i u m in swine. Nutr. Rev., 15:334-336, 1957. 2. Anon.: Zinc deficiency i n ch i ck en s. Nutr. Rev., 19:111-113, 1961. 3. Ballou, J. E.: Metabolism of ZnSSin the rat. H.W.-60062, Richland, Wash. H a n f o r d A t o m i c P r o d u ct Operation, A p r i l 17, 1959. 4. Dunphy, J. E.: On the nature and c a r e of wounds. Ann. Royal Coll. Surgeons, 26:69-87, 1960. 5 . Haley, H. B., Zydlo, S., and Williamson, M. B.: Deposition of radioisotopes in s u r g i c a l incisions in man. Surg. F o r u m , I0:316-318, 1960. 6. Lewis, P. K., J r . , Hoekstra, W. G., a n d G r u m m e t , R . H . : R e s t r i c t e d cal ci u m feeding v e r s u s zinc supplementation for the control o f p a r a k e r a t o s i s in swine. J. A n i m a l S c i . , 16:578-588, 1957. 7. O'DelI, B. L., Newberne, P. M., and Savage, J. E.: Significance of d i e t a r y zinc for the growing chicken. J. Nutrition, 65:503-523, 1958. 8. Savlov, E . D., and Dunphy, J. E.: The healing of the disrupted and r esu t u r ed w o u n d . Surgery, 36:362-370, 1954. 9. Strain, W. H., Dutton, A. M., Heyer, H. B., and Ramsey, G. H.: E x p e r i m e n t a l studies on the a c c e l e r a t i o n of burn and wound healing. Univ. of Rochester Reports, 1953. 10. Strain, %V. H., Pories, W. J., and Hinshaw, J. R.: Zinc studies in skin repair. Surg. Forum, 11:291-292, 1960. I I . Vallee, B. L.: Biochemistry, physiology and pathology of zinc. Physiol. Rev., 39:443-490, 1959. 12. Vallee, B. L., Hoch, F. L., and Hughes, %V. L.: Soluble zinc containing protein extracted from human leucocytes. Arch. Biochem., 48:347360, 1954. 13. Weiber, A.: Studies in vascularization of healing wounds with radioactive isotopes.Acts chir. scandinav., Supplementum, 237, 1959.