2
508 Specialia 2XPERIENTIA 28/5 The mussel plasma does not contain fibrinogen, and in- cubation of a mixture of plasma and thrombin at 37 ~ was not seen to produce clots even after 1 h. Absence of fibri- nogen in the plasma confirms that the mussel is not in possession of a blood clotting mechanism comparable to vertebrates. The mussel plasma also does not contain any human agglutinins, since it was found unabIe to agglutinate wash- ed human red blood cells of A, B and O groups. g6ne ou d'agglutinines humaines, line activit6 des enzymes amylase, GOT et des phosphatases a 6t6 constat6. A. S. NARAIN4 Department o/Zoology, University o/Gorakhpur, Gorakhpur, U.P. (India), 23 March 1977. Rdsumd. Le sang de Lamellidens corrianus est alcalin et incolore. I1 contient peu de pigment respiratoire et de pro- teines. Le plasma contient peu de glucose et pas de fibro- It is a pleasure to acknowledge the stimulation received from Mr. M. P. SINGI~, Experimental Pathologist, King George's Medical College, Lucknow. The Effect of Alloxan Diabetes on Skin Collagen Metabolism The effects of alloxan induced diabetes on impaired wound healing was studied using granulation-and skin tissue in the attempt to elucidate possible alterations in the metabolism of collagen. Although it is well recognized that diabetes results in defects in various connective and vascular tissues 1, 2, the results reported here suggest that in the rate of formation and maturation of collagen may not be a significant factor. Materials and methods. Diabetes was induced in 2 groups of male Sprague-Dawley rats weighing 40-50 g and 100- 125 g by alloxan injection (120 mg/kg). Alloxan was administered either i.p. (40-50 g rats) 3 or by i.v.-injection (100-125 g rats)4. The presence of diabetes was indicated by marked glucosuria (> 0.5%), increased fluid intake and loss of body weight. Rats were maintained on a commercial stock diet through out the experimental period. The effect of treatment on amino acid uptake by gran- ulation and skin tissue was determined using a sponge implantation technique (40-50 g rats)5 and the in vitro skin biopsy method of UITTO (100--125 g rats) 6. In the implantation studies, polyvinyl sponges 1.5 • 0.5 • 0.3 cm) were inserted in the subcutaneous space along the dorsum 10 days after initiation of alloxan treatment. The sponges remained 6 days before removal and decapi- tation. The sponges were then cut into small pieces and incubated in phosphate-free Krebs-Ringer solution (3 ml, pH 7.6) containing 20 mM N-2-hydroxyethyl- piperazine-N1-2-ethane sulphonic acid (HEPES), 20 mM glucose and 0.5 /~Ci U-l*C-lysinet Incubations were carried out for 12 h at 37~ in a metabolic shaker. After incubation, the sponges were completed, homogeniz- ed and aliquots were taken for counting and DNA analysisL Dorsal skin biopsies (1 cm diameter, 100-150 mg) were taken 20 days after alloxan injection (100-125 g rats). The tissue was minced and incubated in the Krebs- HEPES solution (10 h, 37 ~ except U-14C-proline was added at 0.1 #Ci in place of radiolysine. The uptake of radioproline was linear for at least 18 h (Figure 1). Both tissues and sponges were dialyzed exhausively before radioactivity was determined by scintillation counting. The diameters of wounds after biopsy were also measur- ed daily until complete closure as an indirect index of wound healing (Figure 2). At the end of this period, a second biopsy was taken and total hydroxproline deter- mined*. Urinary hydroxyproline was monitored at various time intervals with rats receiving alloxaI1 injec- tion. In addition, skin samples were taken from these animals and collagen solubility in neutral NaC1 solution (1M) was determined 9. The extracted collagen was purified, electrophoresed on acrylamide gel, and the ratio of monomer (c~) to dimer (/3) collagen subunits was calculated after staining with amido black and scanning with a densitometer TM. Results. The uptake of radiolysine and radioproline as cpm into granulation tissue from sponge implants and skin biopsy material is indicated in Tables I and II, respectively. In both cases, alloxan treatment stimulated 24 '7~ 20 r "~- 12 N" Z fi lfl 14 IB 2Z 2fi 30h Time Fig. 1. The uptake of U-Cl~-proline into rat skin. Radioproline was present at p. 1 ~zCiin Krebs-HEPES solution (B mls) =. i j. W. CZERKAWSKI, Int. Review connect. Tissue Res. 1,307 (1963). 2 j. PEDERSON and S. OLSEN, Acta reed. seand. 771,551 (1962). 3 j. KAKOLEWSKIand 2. S. VALENSTEIN,J. comp. Phys. Psych. 68, 31 (1969). 4 j. VELEMINSKY, I. M. BURR and W. STAUFFACHER, 2urop. J. clin. Invest. 1,104 (1970). 5 j. F. WOESSNER and R. J. BOUCEK,Arch. Biochem. 93, 85 (1961). 6 j. UITTO, Biochim. biophys. Acta. 207, 438 (1970). 7 M. OGUR and G. ROSEN, Arch. Biochem. 25, 262 (1950). s j. F. WOESSNER, Arch. Biochem. Biophys. 93, 440 (1961). o D. S. JAcksoN and J. P. BENTLEY, J. biophys, biochem. Cytol. 7, 37 (1960). i0 R. B. RUCKER,H. E. PARKERand J. C. ROGLER,Biochem. Biophys. Res. Comm. 3d, 28 (1969).

The effect of alloxan diabetes on skin collagen metabolism

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508 Specialia 2XPERIENTIA 28/5

The musse l p l a s m a does n o t con t a i n f ibr inogen, and in- c u b a t i o n of a m i x t u r e of p l a s m a a n d t h r o m b i n a t 37 ~ was no t seen to p roduce clots even a f te r 1 h. Absence of f ibri- nogen in t he p l a s m a conf i rms t h a t t he musse l is no t in possess ion of a b lood c lo t t i ng m e c h a n i s m c o m p a r a b l e to ve r t eb r a t e s .

The musse l p l a s m a also does no t c o n t a i n a n y h u m a n agglu t in ins , s ince i t was found unabIe to a g g l u t i n a t e wash- ed h u m a n red b lood cells of A, B and O groups.

g6ne ou d ' agg lu t i n ine s humaines , l i n e ac t iv i t6 des enzymes amylase , GOT et des p h o s p h a t a s e s a 6t6 consta t6 .

A. S. NARAIN4

Department o/Zoology, University o/Gorakhpur, Gorakhpur, U.P. (India), 23 March 1977.

Rdsumd. Le sang de Lamellidens corrianus est a l ca l in et incolore. I1 c o n t i e n t peu de p i g m e n t resp i ra to i re et de pro- te ines . Le p l a s m a c o n t i e n t peu de glucose et pas de f ibro-

It is a pleasure to acknowledge the stimulation received from Mr. M. P. SINGI~, Experimental Pathologist, King George's Medical College, Lucknow.

T h e Effect of A l l o x a n D i a b e t e s o n S k i n C o l l a g e n M e t a b o l i s m

The effects of a l loxan induced d iabe tes on impa i r ed w o u n d hea l ing was s tud ied us ing g r a n u l a t i o n - a n d skin t issue in t he a t t e m p t to e luc ida te poss ible a l t e r a t i ons in t he m e t a b o l i s m of collagen. A l t h o u g h i t is well recognized t h a t d i abe t e s resu l t s in defects in va r ious connec t ive a n d vascu l a r t i ssues 1, 2, t h e resu l t s r epo r t ed he re sugges t t h a t in t he r a t e of f o r m a t i o n and m a t u r a t i o n of col lagen m a y no t be a s ign i f i can t factor .

Materials and methods. Diabe te s was induced in 2 groups of ma le Sp rague -Dawley ra t s weighing 40-50 g and 100- 125 g b y a l loxan in jec t ion (120 mg/kg) . A l loxan was a d m i n i s t e r e d e i the r i.p. (40-50 g rats) 3 or b y i .v . - in jec t ion (100-125 g rats)4. The presence of d i abe t e s was ind ica t ed by m a r k e d g lucosur ia ( > 0.5%), increased f luid i n t a k e a n d loss of b o d y weight . R a t s were m a i n t a i n e d on a c o m m e r c i a l s tock d ie t t h r o u g h ou t t he e x p e r i m e n t a l period.

The effect of t r e a t m e n t on a m i n o acid u p t a k e b y gran- u l a t i on a n d sk in t i ssue was d e t e r m i n e d us ing a sponge i m p l a n t a t i o n t e c h n i q u e (40-50 g ra ts )5 a n d t he in v i t ro sk in b i o p s y m e t h o d of UITTO (100--125 g ra ts) 6. I n t he i m p l a n t a t i o n s tudies , po lyv iny l sponges 1.5 • 0.5 • 0.3 cm) were inse r ted in t he s u b c u t a n e o u s space a long t h e d o r s u m 10 days a f te r i n i t i a t i o n of a l loxan t r e a t m e n t . The sponges r e m a i n e d 6 days before r e m o v a l and decapi- t a t ion . The sponges were t h e n cu t in to smal l pieces a n d i n c u b a t e d in phospha t e - f r ee K r e b s - R i n g e r so lu t ion (3 ml, p H 7.6) c o n t a i n i n g 20 m M N - 2 - h y d r o x y e t h y l - p ipe raz ine -N1-2-e thane su lphon ic acid (HEPES), 20 m M

glucose and 0.5 /~Ci U- l*C- lys ine t I n c u b a t i o n s were car r ied ou t for 12 h a t 37~ in a me tabo l i c shaker . Af te r i ncuba t ion , t h e sponges were comple ted , homogeniz - ed and a l iquo ts were t a k e n for coun t ing a n d DNA ana lys i sL Dorsa l sk in biopsies (1 cm d iameter , 100-150 mg) were taken 20 days after alloxan injection (100-125 g rats). The tissue was minced and incubated in the Krebs- H E P E S so lu t ion (10 h, 37 ~ excep t U-14C-proline was added a t 0.1 #Ci in place of radiolys ine . The u p t a k e of r ad iopro l ine was l inear for a t l eas t 18 h (Figure 1). B o t h t i ssues and sponges were d ia lyzed exhaus ive ly before r a d i o a c t i v i t y was d e t e r m i n e d b y sc in t i l l a t ion count ing .

The d i a m e t e r s of w o u n d s a f t e r b iopsy were also measur - ed da i ly un t i l comple t e closure as a n ind i rec t index of w o u n d hea l ing (Figure 2). A t t he end of th i s period, a second b iopsy was t a k e n and t o t a l h y d r o x p r o l i n e deter - mined*. U r i n a r y h y d r o x y p r o l i n e w a s m o n i t o r e d a t va r ious t ime i n t e rva l s w i t h r a t s rece iv ing alloxaI1 injec- t ion . I n addi t ion , sk in samples were t a k e n f rom these an ima l s a n d col lagen so lub i l i ty in n e u t r a l NaC1 solu t ion (1M) was d e t e r m i n e d 9. The e x t r a c t e d col lagen was purif ied, e lec t rophoresed on ac ry l amide gel, and t he r a t io of m o n o m e r (c~) to d imer (/3) col lagen s u b u n i t s was ca lcu la ted a f te r s t a i n i n g w i t h amido b l ack a n d scann ing w i t h a d e n s i t o m e t e r TM.

Results. The u p t a k e of r ad io lys ine a n d rad iopro l ine as c p m in to g r a n u l a t i o n t i ssue f rom sponge i m p l a n t s and skin b iopsy m a t e r i a l is i nd ica t ed in Tab les I and I I , respect ively . I n b o t h cases, a l l oxan t r e a t m e n t s t i m u l a t e d

24

'7~ 20

r

"~- 12 N "

Z fi lfl 14 IB 2Z 2fi 30h Time

Fig. 1. The uptake of U-Cl~-proline into rat skin. Radioproline was present at p. 1 ~zCi in Krebs-HEPES solution (B mls) =.

i j . W. CZERKAWSKI, Int. Review connect. Tissue Res. 1,307 (1963). 2 j . PEDERSON and S. OLSEN, Acta reed. seand. 771,551 (1962). 3 j. KAKOLEWSKI and 2. S. VALENSTEIN, J. comp. Phys. Psych. 68, 31 (1969).

4 j . VELEMINSKY, I. M. BURR and W. STAUFFACHER, 2urop. J. clin. Invest. 1,104 (1970).

5 j . F. WOESSNER and R. J. BOUCEK, Arch. Biochem. 93, 85 (1961). 6 j . UITTO, Biochim. biophys. Acta. 207, 438 (1970). 7 M. OGUR and G. ROSEN, Arch. Biochem. 25, 262 (1950). s j . F. WOESSNER, Arch. Biochem. Biophys. 93, 440 (1961). o D. S. JAcksoN and J. P. BENTLEY, J. biophys, biochem. Cytol. 7,

37 (1960). i0 R. B. RUCKER, H. E. PARKER and J. C. ROGLER, Biochem. Biophys.

Res. Comm. 3d, 28 (1969).

15.5. 1972 Specialia 509

t h e i n c o r p o r a t i o n of t h e a m i n o a c i d s t e s t e d , a l t h o u g h n o t s i g n i f i c a n t l y . T h e i n c o r p o r a t i o n of p r o l i n e or l y s i n e w a s n o t t a k e n as a d i r e c t m e a s u r e of c o l l a g e n s y n t h e s i s , b u t t h e f a c t t h a t i n c o r p o r a t i o n i n t o t i s s u e w a s n o t de - c r e a s e d w i t h a l l o x a n t r e a t m e n t i n d i c a t e d a t l e a s t u n - i m p a i r e d p o t e n t i a l for s y n t h e s i s . T o t a l c o l l a g e n as m e a s u r - ed b 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 e r m g of s k i n t i s s u e w a s n o t a l t e r e d a f t e r a l l o x a n t r e a t m e n t ( T a b l e I I ) .

T h e t i m e s r e q u i r e d for w o u n d c l o s u r e f r o m b i o p s i e s a r e g i v e n in F i g u r e 2. N o d i f f e r e n c e s w e r e n o t e d b e t - w e e n g r o u p s , a l t h o u g h t h e w o u n d s of a l l o x a n - t r e a t e d r a t s a p p e a r e d m o r e i n f l a m e d t h r o u g h o u t t h e h e a l i n g p r o c e s s a n d s k i n t h i c k n e s s w a s r e d u c e d . A l l o x a n t r e a t - m e n t h a d n o a f f e c t o n c o l l a g e n s o l u b i l i t y o r t h e a p p a r e n t d e g r e e of c r o s s l i n k i n g as m e a s u r e d b y t h e r a t i o of e t o fl c o l l a g e n s ( T a b l e I I I ) . L i k e w i s e h y d r o x y p r o l i n e e x c r e t i o n r a t e s w e r e n o t s i g n i f i c a n t l y a l t e r e d ( T a b l e IV) .

Discussion. I n s u l i n a p p e a r s t o a c c e l e r a t e p r i m a r y w o u n d h e a l i n g . C o n v e r s e l y , d i a b e t e s o f t e n r e s u l t s in de - c r e a s e d v a s c u l a r i z a t i o n a n d r e t a r d e d w o u n d h e a l i n g L D e f e c t i v e c o l l a g e n m e t a b o l i s m a t l e a s t d u r i n g t h e o n s e t of a l l o x a n d i a b e t e s d o e s n o t a p p e a r a s i g n i f i c a n t f a c t o r . Of p a r t i c u l a r s i g n i f i c a n c e is t h e o b s e r v a t i o n t h e u r i n a r y h y d r o x y p r o l i n e leve ls , c o l l a g e n s o l u b i l i t y a n d s u b u n i t d i s t r i b u t i o n a r e n o t a l t e r e d s i g n i f i c a n t l y w i t h t r e a t m e n t . A d e c r e a s e in s a l t s o l u b l e c o l l a g e n s o m e t i m e s i n d i c a t e s r a p i d d e g r a d a t i o n of n e w l y f o r m e d co l l agen , a n d a n in- c r e a s e o f t e n r e p r e s e n t s i m p a i r e d m a t u r a t i o n n , ~2

T h e s u g g e s t i o n t h a t t h e r a t e of m a t u r e c o l l a g e n f ib r i l f o r m a t i o n in g r a n u l a t i o n t i s s u e f r o m a l l o x i n a n i m a l s is t h e s a m e of c o n t r o l r a t s h a s a l so b e e n o f f e r e d b y CATAN- ZARo-GUIMARAES 13, w h o u s e d h i s t o c h e m i c a l t e c h n i q u e s . W i t h r e s p e c t to sk in , t h e s t u d i e s r e p o r t e d h e r e a l so col- l a b o r a t e s u c h f i n d i n g s .

Table I. Effect of alloxan on the uptake of U-14C-lysine into sponge implant granulation tissue

Treatment cpm]sponge [xg DNA]sponge cpm//zg DNA

Alloxan 4338-t-1370 583=123 7.40=2.1

Control 31564-1156 708=150 4.46=1.21

Average of 5 samples -4- S.E.M.

Table III. The effect of alloxan on the solubility and distribution of and/~ collagens in NaC1 extracts

Treatment mg Hypro o:[fl Ratio extracted/100 mg tissue

Alloxan 0.33=0.07 2.0=t=0.2

Control 0.34:E0.06 1.8:~0.2

Average of 6 samples ~ S.E.M.

Table II. Effect of alloxan on the uptake of U-14C-proIine and the hydroxyproline content of skin biopsies

Trea tment Days~ cpm/100 mg mg HyPro/100 mg of tissue of tissue

Alloxan 20 1428• -- 45 -- 4.4=0.3

Control 20 1170~178 45 -- 4 .010.2

Days after initial alloxan injection.

i _

r

1.0

Z 6 li la 1B 2Z

Days(I)

Z 6 18 14 15 ZZ

Days(II)

Fig. 2. Diameters of open wound areas after biopsy of dorsum skin. Biopsies were taken 20 and 45 days after the initial alloxan treatment. Blood glucose levels at 30 days were > 200 mg 100 ml. aUoxan and < 100 mg/100 ml, controls. Each point represents the average from 4-6 rats (alloxan, broken line; control, solid line).

Table IV. The Effect of alloxan on the excretion of hydroxyproline

Treatment Days ~ ~zg Hypro excretedlday

Alloxan 25 361= 32 33 374=25

Cotxtrol 25 406 = 27 33 410-t-25

Days after initial s.c. injection. Average of 5 samples.

Rdsumd. O n a 6 t u d i 6 chez d e s r a t s r e n d u s d i a b 6 t i q u e s p a r l ' a l l o x a n e le m 6 t a b o l i s m e d u co l l agbne de la p e a u e t la g u 6 r i s o n d e s b t e s su r e s . Le m 6 t a b o l i s m e d u co l l ag~ne f u t 6 v a l u 6 p a r la l y s i n e et la p r o l i n e d a n s de s b i o p s i e s de la p e a u , p a r la so lub i l i t 6 d u co l l agbne , p a r l ' e x c r 6 t i o n j o u r n a l i ~ r e de I ' h y d r o x y p r o l i n e . e t p a r le deg r6 a p p a r e n t de <~crosslinking~> d u c o l l a g ~ n e C h e z les d i a b 6 t i q u e s , o n o b s e r v e s o u v e n t u n e d 6 p r e s s i o n et u n e v a s e u l a r i s a t i o n de la p e a u . m a i s la v i t e s s e de g u 6 r i s o n de s b l e s s u r e s e t de f o r m a t i o n de s f ib r i l l es d u co l l ag~ne a p a r u n o r m a l e

P. YEP, S. GULLANDER a n d R . B . RUCKER

Department oi Nutrition, University o/Cali[ornia, Davis (Cali/ornia. 95676, USA), 17 October 1971.

11 L MIKKONEN, K. LAMPIAHO and E. KULONEN, Acta. Endocr., Copenh. 51, 23 (1966).

[2 D. J. PROCKOP and K. I. KIVIRIKKO, Ann. Int. Med. 66, 1243 (1967). 13 S. A . CATANZARDO-GuIMARAES, Experientia 24, 1168 (1968).