Enzymes of white blood cells: A study of the acid and alkaline phosphatase, β-glucuronidase, amylase, and adenosinetriphosphatase activity of the leucocytes of man and the rabbit

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Enzymes of white blood cells: A study of the acid and alkaline phosphatase, β-glucuronidase, amylase, and adenosinetriphosphatase activity of the leucocytes of man and the rabbit

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ENZYMES OF WHITE BLOOD CELLS A Study o f th e Acid and A lk a lin e P h o sn h a ta s e ,

F.

E s t im a t io n o f A d e n o s in e t r ip h o s p h a t a s e A c t i v i t y

'

The a d e n o s in e t r i p h o s p h a t a s e a c t i v i t y o f th e l e u c o c y t e

; s u s p e n s io n s was d eterm ined by a m o d i f i c a t i o n o f th e method o f ! Du B o i s and P o t t e r (19 43) i n which t h e i n o r g a n i c phosphorus, l i b e r a t e d from th e a d e n o s in e tr ip h o s p h a t e by th e a c t i o n o f t h e enzyme, was measured by t h e method o f F is k e and Subbarow (1 9 2 5 ) | as s u b s e q u e n tly m o d ifie d by King ( 1 9 3 2 ) .

The c o lo u r o f t h e

| r e a c t i o n m ix tu re and t h a t o f th e c o n t r o l was read a g a i n s t a 1

i

b la n k i n t h e sp e c tr o p h o to m e te r a t 650 mi|. S o lu tio n s The v e r o n a l b u f f e r pH 7*2 was prepared as d e s c r ib e d i n I th e methods f o r am ylase ( p a g e 23) . j

T-he c a lc iu m s o l u t i o n 0 . 1 M. was prepared by d i s s o l v i n g 1 .2 3 grams o f Ca Cl^ i n 100 m l. o f water The s u b s t r a t e was prepared by d i s s o l v i n g 51*5 mgm. o f

!

a d e n o s in e tr ip h o s p h a t e o b t a in e d from Schwarz L a b o r a to r ie s I n c .

|

New York, i n 5 m l. o f w a t e r .

T h is c o n c e n t r a t i o n was a d j u s t e d

so t h a t t h e m o la r ity o f t h e s o l u t i o n w it h r e s p e c t t o adeno­ s i n e t r ip h o s p h a t e was 0 .0 1 5 M. volume

B e fo r e a d j u s t i n g t o th e f i n a l

t h e s o l u t i o n was n e u t r a l i z e d w ith 0 . 1 N HC1 or 0 . 1 N

NaOH u s in g

p h e n o lp h t h a le in as an i n d i c a t o r .

The f i n a l c o lo u r

was c a r e f u l l y d i s p e l l e d w it h 0 .0 1 N HC1. A

20% s o l u t i o n o f t r i c h l o r o a c e t i c a c id was p re p a r ed .

A

60%s o l u t i o n o f p e r c h l o r i c a c id was employed

A % s o l u t i o n o f ammonium molybdate was p rep ared . The r e d u c in g a g e n t was made by d i s s o l v i n g 200 mgm. o f .. I _____________________________________________________________________

with permission o f the copyright owner. Further reproduction prohibited without permission.

-261 , 2 ,4 - a m in o n a p h t h o ls u lf o n ic a c i d , 12 grams o f sodium m e ta b is u l p h i t e and 2 . 4 grams o f sodium s u l p h i t e i n w ater t o a f i n a l volume o f 100 ml. The p h osp h ate stan dard was prepared by adding 2 .1 9 4 grams o f KH2P04 t o 500 ml. o f w a te r . P roced u re To

each o f four c e n t r i f u g e tu b es was added; 0 . 5 m l. V e r o n a l b u f f e r pH 7*2 0 . 2 m l. S u b s t r a t e ( a d e n o s i n e t r i p h o s p h a t e 0 .0 1 5 M) 0 . 1 m l. CaCl2 0 . 0 1 M 0 . 1 m l. water

Three o f t h e s e tu b e s were d e s ig n a t e d as t e s t s and to t h e s e were added 0 . 1 ml. o f th e enzyme s o l u t i o n . tu b e was in c u b a te d as a c o n t r o l .

The rem aining

The t u b e s were in c u b a te d a t

37° C. f o r 15 m inu tes a f t e r w hich 1 ml. o f 20$ t r i c h l o r a c e t i c a c id was added t o each t u b e .

To t h e c o n t r o l tube was added

0 . 2 m l. o f th e enzyme s o l u t i o n . a t 2500 r . p . m . f o r 5 m in u te s .

The tu b e s were c e n t r i f u g e d The in o r g a n ic phosohorus was

d eterm in ed i n 1 m l. o f th e su p e rn a ta n t by adding 4 m l. o f w a t e r , 0 . 3 m l. p e r c h l o r i c a c id ( 6 0 $ ) , 0 . 4 m l. ammonium m olybd a t e ( 5 $ ) , and 0 . 2 ml. o f r e d u c in g a g e n t . mixed th o r o u g h ly a f t e r each a d d i t i o n .

T h ese r e a g e n t s were

A f t e r 30 m inutes th e

c o n t e n t s o f t h e tu b e s were read i n th e sp ectro p h o to m e ter at 650 iiji. a g a i n s t a b la n k c o n t a i n i n g 5 ml* water and th e r e a g e n t s f o r in o r g a n ic phosphorus as n oted ab ove. The p h osp hate stan dard was prepared by adding 2 ml. o f

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

jI -2 7 I I | a 1 :1 0 0 d i l u t i o n o f th e s t o c k s o l u t i o n t o 0 . 6 m l. o f o e r c h l o r i c a c id 60?:., 0 . 8 m l. o f ammonium m olybdate (5$)> and 0 . 4 m l.

i

| r e d u c in g a g e n t . | m l.

Water was added t o make a f i n a l volume o f 10

A f t e r 30 m inutes t h i s standard was rea d i n th e s p e c t r o -

{ photom eter a t 650 mji a g a i n s t t h e b la n k . I S t a n d a r d i z a t i o n o f th e A d e n o s in e tr ip h o s p h a te S o l u t i o n . The a d e n o s in e tr ip h o s p h a t e m o lecu le c o n t a i n s t h r e e p h o s i p h a te grouns a tta c h e d t o th e a d en o sin e r a d i c a l .

The enzyme

a d e n o s in e tr ip h o s p h a t a s e s p l i t s o f f t h e t e r m i n a l phosphate group l e a v i n g a d e n o s in e d ip h o s o h a te .

T h is compound may i n turn be

s p l i t t o adenosinem onophosphate or a d e n y l i c a c i d . I t has been shown by Lohman (1931) t h a t th e l a b i l e p h o s­ p h o ru s, or te r m in a l two phoso'nate groups o f t h e a d e n o s in e !

t r i p h o s o h a t e m o le c u le , may be s p l i t o f f by h y d r o l y s is w it h N-HC1 a t 100°C. f o r se v e n m in u te s.

F is k e (19 34) and Kerr

( 1 9 4 1 ) , how ever, recommended a lo n g e r p e r io d o f f i f t e e n m in u te s. [

B a i l e y (1 9 4 9 ) on r e i n v e s t i g a t i o n o f t h i s m atter r e p o r te d t h a t h y d r o l y s i s f o r n in e t o t e n m inutes at 100°C. w i t h N-HC1 s p l i t o f f t h e s e t e r m in a l grou p s.

T h is l a b i l e phosohorus i s 66?j o f

t h e v a l u e f o r t o t a l phosphorus i f th e p r e p a r a t io n i s p u r e .

In

order t o t e s t th e p u r i t y o f our sample o f a d e n o s in e t r ip h o s p h a t e , th e t o t a l p h osp horus, in o r g a n ic ph osp horus, 7-m inute ph osp horus, 1 0 -m in u te phosohorus as w e l l as th e phosphorus i n two b la n k s , one an ashed water b la n k and th e o th e r a 1 0-m inute N-HC1 b la n k , were d e ter m in e d .

The r e s u l t s are g iv e n i n T ab le 1.

The v a l u e s i n T ab le 1, c a l c u l a t e d from th e phosphorus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-2 8 -

T a b le 1 .

Phosphorus A n a ly s i s o f A d e n o s in e t r ip h o s p h a t e ,

Phosphorus F r a c t i o n

T o t a l Phosphorus

Experiment #1

Experiment #2

Phosphorus

Phosphorus

3 7 .3

3 7 .6

I n o r g a n ic Phosphorus

2 .5

3 .1

7-m in u te Phosphorus

22.1

1 0 -m inute Phosphorus

2 2 .7

The v a l u e s are th e average o f t h r e e d e t e r m i n a t i o n s .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-

29 -

sta n d a r d , were c o r r e c t e d by s u b t r a c t in g th e v a l u e s o f th e ash ed w ater b la n k from th e t o t a l p h osp horus.

I n experim ent

# 2 th e 1 0 -m in u te phosphorus was c o r r e c t e d by s u b t r a c t i n g th e 10-m inute N-HC1 b la n k .

I t was c a l c u l a t e d t h a t th e sample o f

a d e n o s in e t r ip h o s p h a t e c o n t a in e d 3»41 mgm. o f phosphorus i n 25 mgm. o f t h e powder.

S in c e 1 . 4 mgm. o f phosphorus per

m i l l i l i t r e o f w ater r e s u l t s i n a s o l u t i o n w hich i s 0 .0 1 5 M. w it h r e s p e c t t o th e phosphorus; 51«5 mgm. o f t h e sample o f a d e n o s in e t r ip h o s p h a t e i n 5 m l. o f w ater g i v e s a s o l u t i o n w hich i s 0 .0 1 5 M. w i t h r e s p e c t t o phosphorus c o n t e n t . In ex p erim en t # 1 t h e r a t i o o f 7-m in u te phosphorus t o a d e n o s in e t r ip h o s p h a t e phosphorus was 56.3$» w h il e i n e x p e r i ­ ment # 2 t h e r a t i o o f 10 -m in u te phosphorus t o a d e n o s i n e t r i p h o s ­ p h a te phosphorus was 57»1$«

As t h e s e v a l u e s d i f f e r e d from

t h e t h e o r e t i c a l v a lu e o f 66%, i t may be con clu d ed e i t h e r t h a t ( a ) h y d r o l y s i s w it h N-HC1 a t 100°C. was n o t c o m p lete e v en a f t e r 10 m in u tes or (b ) t h a t t h e p r e p a r a tio n o f a d e n o s i n e t r i ­ p h osp hate was impure.

The maximum d e g r ee o f im p u r ity would

t h e n b e 9 /6 6 o r , a p p r o x im a te ly , 14%, R eco rd in g o f R e s u l t s Enzyme a c t i v i t y has b een r eco rd ed as th e number o f p g . o f phosphorus l i b e r a t e d per 100 ml. o f c e l l s u s p e n s io n per h o u r.

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STATISTICAL METHODS A s t a t i s t i c a l a n a l y s i s o f th e d ata o b ta in e d by th e f o r e ­ go in g methods forms a l a r g e p a r t o f t h i s stu d y .

S in c e t h e

v a l u e s f o r each enzyme were s u b j e c t e d t o a s im ila r a n a l y s i s , th e p ro ced u res employed are d e s c r ib e d t o g e t h e r i n t h i s s e c t i o n . R e f e r e n c e was made to ‘'An O u t lin e o f S t a t i s t i c a l Methods" by A rkin and C o lto n ( 1 9 3 9 ) • Sim ple C o r r e l a t i o n . Simple c o r r e l a t i o n d e f i n e s t h e d e g r e e o f r e l a t i o n s h i p betw een two s e t s o f d a ta grouped i n p a i r s .

When each v a lu e

o f one s e t o f t h e d a ta i s p l o t t e d g r a p h i c a l l y a g a i n s t i t s c o r -

I j

r esp o n d in g p a i r , th e tre n d o f th e p o i n t s ,

|

i s good, w i l l group about a s t r a i g h t l i n e .

; f I

may be d eterm in ed m a t h e m a tic a lly and i s c a l l e d th e l i n e o f ' ' ' r e g r e s s i o n . E xcep t i n t h e c a s e o f a p e r f e c t c o r r e l a t i o n , th e

i f th e c o r r e l a t i o n

p o i n t s w i l l have a s c a t t e r about t h i s l i n e .

T h is s t r a i g h t l i n e

The stan dard

e r r o r o f t h e e s t i m a t e o f one v a l u e , when o n ly th e v a lu e o f i t s p a ir i s known, i s a measure o f t h i s d eg ree o f s c a t t e r . T h e r e f o r e , th e d e g r ee o f t h i s r e l a t i o n s h i p may be e x p r e s s e d as th e stan d ard e r r o r o f e s t i m a t e d iv id e d by th e standard d e v i a t i o n o f th e p o i n t s about th e a r it h m e t i c mean. I f t h i s r e l a t i o n s h i p i s p e r f e c t , t h e standard e r r o r o f e s t i m a t e w i l l be e q u a l t o z e r o , w hich w i l l r e s u l t i n t h i s

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-

r a t i o b e in g e q u a l to z e r o .

31 -

The v a lu e a t th e o th e r end o f

t h e s c a l e i s t h a t o f a c o m p le te ly im p e r f e c t r e l a t i o n s h i p and t h e r a t i o would be eq u a l t o 1 .

T h is r a t i o i s u s u a l l y s u b t r a c ­

t e d from 1 t o g i v e a more e a s i l y a p p r e c ia t e d v a l u e .

T h er e ­

f o r e a p e r f e c t r e l a t i o n s h i p would r e s u l t i n a v a lu e o f 1, w h il e a c o m p le t e ly im p e r fe c t r e l a t i o n s h i p would be eq u a l to zero.

T h is tendancy may be e x p r e ss e d by t h e c o e f f i c i e n t o f

c o r r e l a t i o n which i s a s i m i la r measure o f th e d e g r ee o f a sso c ia tio n .

The form ula f o r th e c o e f f i c i e n t o f c o r r e l a t i o n

is:

where Sy i s e q u a l t o the standard e rro r o f e s t i m a t e o f y . y i s e q u a l to t h e stan dard d e v i a t i o n . T h is form ula

may be reduced a l g e b r a i c a l l y to t h e form u la; r=

E ^ x 500

03 ®

r - 0 8 7 1 0 05

500

1000

CALCULATED ( U N I T S / I 0 0 M L . )

P ig . 5.

R e l a t i o n betw een o b s e r v e d G lu cu ro n id a se A c t i v i t y

o f S u sp e n sio n s o f Human W hite C e l l s and G lu cu ro n id a se A c t i v i t y c a l c u l a t e d from t h e Polym orphonuclear L eu co cy te and Lymphocyte C e l l Count, u s in g th e M u l t ip le R e g r e s s io n E q u a tio n p r e s e n t e d i n th e T e x t .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

c o u n t i n 103 c e l l s p e r c u . mm. From t h i s e q u a t i o n i t c an he Ji |■ se e n t h a t , f o r th e d a ta p r e s e n t e d , t h e r a t i o o f th e enzyme I

a c t i v i t y i n t h e polym orphonuclear l e u c o c y t e t o t h a t i n th e

' ii j l

lym phocyte was 159*128, or ap p ro x im a te ly 5*4-.

The o b served

v a l u e o f g lu c u r o n id a s e a c t i v i t y was p l o t t e d a g a i n s t th e v a lu e c a l c u l a t e d from th e m u l t i p l e r e g r e s s i o n e q u a tio n i n F ig u r e 5.

The c o e f f i c i e n t o f m u l t i p l e c o r r e l a t i o n betw een th e g l u ­

c u r o n id a s e a c t i v i t y and t h e c e l l count f o r each o f t h e two c e l l i t y p e s (R1 .2 3 = 0*^7 * 0*0?) a g r e e s c l o s e l y w it h th e c o r r e l a t i o n b etw een g lu c u r o n id a s e a c t i v i t y and t h e t o t a l c e l l count (r = 0 . 8 6 ± 0 .0 5 ).

T h e r e f o r e , i t may be co n clu d ed from th e d a ta p r e s e n ­

t e d t h a t t h e g lu c u r o n id a s e a c t i v i t y o f mixed l e u c o c y t e su sp e n ­ s i o n s was f a i r l y I

e v e n ly d i s t r i b u t e d b e tw e en t h e polymorphonu-

c l e a r l e u c o c y t e and t h e ly m p h ocyte. R a b b it W hite C e l l s

j

I n a s e r i e s o f 26 s u s p e n s io n s o f r a b b i t l e u c o c y t e s , t h e

| I ;

g lu c u r o n id a s e a c t i v i t y and t h e v a r io u s c e l l c o u n ts were d e t e r mined (T a b le 8 ) .

j

p r e s s e d i n u n i t s per 100 m l. c e l l s u s p e n s io n , was p l o t t e d

In F ig u r e 6 th e g lu c u r o n id a s e a c t i v i t y ,

ex-

a g a i n s t (a ) t h e polym orphonuclear l e u c o c y t e c e l l c o u n t, (b) t h e lym phocyte c e l l c o u n t, and ( c ) t h e t o t a l c e l l c o u n t .

The

r e g r e s s i o n e q u a t io n s and c o e f f i c i e n t s o f c o r r e l a t i o n (T a b le 9) were d eterm in ed i n each c a s e . There was a h ig h c o r r e l a t i o n betw een g lu c u r o n id a s e a c t i ­ v i t y and th e t o t a l c e l l count (r = 0 .6 9 ± 0 . 1 1 ) , as w e l l a s a h ig h c o r r e l a t i o n betw een g lu c u r o n id a s e a c t i v i t y and th e p o l y -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Table 8.

Method o f o b t a i n in g c e lls

Pe. Pe. Pe. Pe. Pe. Pe. Pe. P e. P e. Pe. Pe. Pe. Pe. Pe. Sp. Pe. Pe. Sp. Sp. Sp. Sp. Ac. Ac. Sp. Sp. Sp.

^-Glucuronidase of Suspensions of Rabbit White Cells.

C e l l Count ( 10^ c e l l s / c u . mm.) P olym orph onu clear leu co cy tes 1620 2300 3100 6190 3980 3330 1560 7450 3210 2100 4300 3260 3440 1580 2310 1210 2430 830 1570 1460 I 960 1040 1040 520 500 870

p -G lu c u r o n id a s e A c t i v i t y ( u n i t s per 100 m l. s u s p e n s io n )

Lymphocytes

160 150 340 77 0 760 88 0 150 480 710 660 430 580 470 140 1540 210 650 410 960 1060 . 800 540 530 850 1890 2230

A c . = A c a c ia method o f Spear ( 1 9 4 8 ) . Sp . = S p in n in g method o f B u t l e r and Cushman ( 1 9 4 0 ) . P e . = P e r i t o n e a l ex u d a te method o f de Haan ( 1 9 1 8 ) .

105 155 398 945 229 238 24 344 656 248 1081 301 362 66 267 94 114 49 209 256 186 95 100 58 95 124

-63 -

O o

.

POLYMORPH

UJ Q zo cr 3 U D O

.

LYMPHOCYTE

to


0 .6 9 * 0 .!l

r* -0 .0 4 * 0 2 C

iC E L L C O U N T , I O s C E L L S / CU. MM.

*O IOOO o

TOTAL C E LL CO U N T

a< #0° a z

o

cr 3

O 33 O

C EL L COUNT, IO*CELLS / CL. vv .

F ig . 6.

G lu c r o n id a se A c t i v i t y o f S u sp e n sio n s o f R abbit

W hite C e l l s p l o t t e d a g a i n s t Polym orphonuclear L eucocyte C e ll

Count, Lymphocyte C e l l Count, or T o t a l White C e l l

C ount,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

T a b le 9 .

C o r r e l a t i o n betw een [3-G lucuronidase A c t i v i t y and C e l l Count o f S u s p e n s io n s o f W h ite B lood C e l l s o f t h e R a b b it.

Type o f C o r r e l a t i o n

12

C o e ffic ie n t of C o r re la tio n

S .E . o f r .

C o e f f i c i e n t o f c o r r e l a t i o n b etw een (3 -g lu c u r o n id a se A c t i v i t y and t o t a l w h it e c e l l c o u n t.

0 .6 9

± 0 .1 1

C o e f f i c i e n t o f C o r r e l a t i o n betw een (3 -g lu c u r o n id a se a c t i v i t y and p olym orph onu clear l e u c o c y t e c e l l c o u n t .

0 .6 9

± 0 .1 1

r 13

C o e f f i c i e n t o f c o r r e l a t i o n betw een (3 -g lu c u r o n id a se a c t i v i t y and lym phocyte c e l l c o u n t .

-0 .0 4

± 0 .2 0

r 23

C o e f f i c i e n t o f c o r r e l a t i o n b etw een polym orphonuclear c e l l co u n t and lym phocyte c e l l c o u n t .

-0 .2 2

± 0 .1 9

r 1 2 .3

C o e f f i c i e n t o f p a r t i a l c o r r e l a t i o n betw een (3-glu cu r­ o n id a s e a c t i v i t y and polym orphonuclear l e u c o c y t e .

0 .9 6

± 0 .0 2

r 13.2

C o e f f i c i e n t o f p a r t i a l c o r r e l a t i o n betw een (3-glucur­ o n id a s e a c t i v i t y and lym phocyte c e l l c o u n t.

0 .2 2

± 0 .1 9

C o e f f i c i e n t o f m u l t i p l e c o r r e l a t i o n betw een (3-glu­ c u r o n id a s e a c t i v i t y and b o th polym orphonuclear l e u ­ c o c y t e c e l l c o u n t and lym phocyte c e l l c o u n t .

0 .7 0

±0.10

r 1 .2 3

-65m orphonuclear l e u c o c y t e c e l l count ( r ]_2 = 0 .6 9 * 0 . 1 1 ) .

The

c o r r e l a t i o n betw een g lu c u r o n id a s e a c t i v i t y and th e lym phocyte c e l l count (^13 = - 0 . 0 4 ± 0 .2 0 ) was n o t s i g n i f i c a n t .

A lso t h e

c o r r e l a t i o n betw een th e polym orphonuclear l e u c o c y t e c e l l count and t h e lym phocyte c e l l co u n t ( ^ 3 = - 0 . 2 2 ± 0 .1 9 ) was i n s i g ­ n ific a n t.

T h is l a t t e r o b s e r v a t i o n i n d i c a t e s t h a t th e s i g n i f i c a n t

c o r r e l a t i o n betw een th e polym orphonuclear l e u c o c y t e c e l l count and t h e g lu c u r o n id a s e a c t i v i t y r e p r e s e n t s a v a l i d a s s o c i a t i o n and i s n o t r e l a t e d t o a h ig h c o r r e l a t i o n b etw een th e two c e l l ty p es. The c o e f f i c i e n t s o f p a r t i a l c o r r e l a t i o n r e p e a te d th e p a t ­ t e r n s e t by t h e sim p le c o e f f i c i e n t s .

The c o e f f i c i e n t o f p a r ­

t i a l c o r r e l a t i o n betw een th e g lu c u r o n id a s e a c t i v i t y and t h e polym orphonuclear l e u c o c y t e c e l l count w it h th e lymphocyte c e l l count e x c lu d e d was h i g h l y s i g n i f i c a n t (r-|_2 .3 = O.9 6 * 0 . 0 2 ) , w h il e t h a t b etw een t h e enzyme a c t i v i t y and t h e lym phocyte c e l l cou n t w it h t h e polym orphonuclear l e u c o c y t e c e l l count ex clu d ed was n o t s i g n i f i c a n t ( ^ 3 2 = 0 .2 2 ± 0 . 1 9 ) .

These v a l u e s might

s u g g e s t t h a t t h e o o ly n o r p h o n u clea r l e u c o c y t e , r a th e r th a n t h e ly m p h ocyte, c o n ta in e d th e b u lk o f th e g lu c u r o n id a s e a c t i v i t y . The m u l t i p l e r e g r e s s i o n e q u a tio n , e x p r e s s in g t h e g l u c u r o n i ­ d a se a c t i v i t y i n term s o f th e c e l l count f o r each t y r e o f c e l l , i s found t o b e; Y = 0 .5 3 4 +

112 Xpoly> -f- 6 2 . 5 Xlymph>

where th e sym bols have th e same meaning as b e f o r e .

From t h i s

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-66e q u a t io n i t can be se en t h a t , on t h e a v e r a g e , th e r a t i o o f t h e enzyme a c t i v i t y i n t h e polym orphonuclear l e u c o c y t e t o t h a t i n t h e lym phocyte i s 1 1 2 : 6 2 . 5, or a p p roxim ately 9* 5 .

The

m u l t i p l e r e g r e s s i o n e q u a tio n shows t h a t t h e lymphocyte c o n t r i ­ b u ted c o n s i d e r a b l e g lu c u r o n id a s e a c t i v i t y t o th e r a b b i t mixed su sp en sio n s.

The low d e g r e e o f c o r r e l a t i o n found t o e x i s t

betw een t h e lym phocyte c e l l count and t h e enzyme a c t i v i t y must be due, i n p a r t , t o th e v e r y low v a lu e s f o r t h e lym phocyte c e l l c o u n ts o b ser v ed i n T ab le 8.

This was u n fo r tu n a te and

i l l u s t r a t e s t h e danger, s t r e s s e d i n S e c t i o n V, o f drawing c o n ­ c l u s i o n s from c o r r e l a t i o n d a ta a l o n e ,

^he m u lt ip le r e g r e s s i o n

e q u a t io n f o r t h e r a b b i t s u g g e s te d t h a t th e lynrohocyte, l i k e th e polym orphonuclear l e u c o c y t e , p la y e d an im portant r o l e i n th e g lu c u r o n id a s e a c t i v i t y o f r a b b i t w h ite c e l l s u s p e n s io n s , a lt h o u g h , p e r h a p s , th e e x t e n t o f i t s p a r t i c i p a t i o n (polym or­ p h on u clear : lym phocyte = 9 : 5 ) was n o t as g r e a t as fo r human l e u c o c y t e s u s p e n s io n s (polym orphonuclear : lymphocyte = 5 * 4 ) . The c a l c u l a t e d g lu c u r o n id a s e a c t i v i t y o b ta in e d from t h e mul­ t i p l e r e g r e s s i o n e q u a tio n was p l o t t e d a g a i n s t th e observed v a l u e i n F ig u r e 7*

The c o e f f i c i e n t o f m u l t i p l e c o r r e l a t i o n

b etw een th e g lu c u r o n id a s e a c t i v i t y and th e c e l l c o u n ts fo r e a ch o f th e two c e l l t y p e s (R1 .2 3 = 0*70 ± 0 .1 0 ) agreed c l o s e l y w i t h th e c o e f f i c i e n t o f c o r r e l a t i o n betw een g lu c u r o n id a s e a c t i v i t y and t h e t o t a l c e l l count (r = O.6 9 ± 0 . 1 1 ) .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-67-

*oao

z.

*0 0

to o

CALCULATED ( UNI TS / 1 0 0 ML.)

F i g . 7»

R e l a t i o n between observed G lu cu ro n id a se A c t i v i t y

o f S u sp e n sio n s o f R abbit W hite C e l l s and G lu cu ro n id a se A c t i v i t y c a l c u l a t e d from th e Polym orphonuclear L eu co cy te and Lymnhocyte C e l l Count, u s in g th e M u l t ip le R e g r e s s io n E q u ation p r e s e n te d i n th e T e x t .

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AMYLASE The am ylase a c t i v i t y o f a s e r i e s o f -white c e l l su sp e n ­ s i o n s o b t a in e d from r a b b i t s -was determ ined by th e

method o f

Somogyi (194-5), as d e s c r ib e d i n th e s e c t i o n on m ethods. E f f e c t o f Time on Enzyme A c t i v i t y ^he

e f f e c t o f tim e on enzyme a c t i v i t y was d eterm ined by

in c u b a tin g s e t s o f tu b es f o r 0 , 1, 2, 4, 8 and 18 hours r e s ­ p e c tiv e ly .

The a v e ra g e v a l u e s f o r am ylase a c t i v i t y , e x p r e s s e d

i n mgm. o f r e d u c in g sugar l i b e r a t e d per 100 m l. o f c e l l s u s ­ p e n s io n , a re found i n T ab le 1 0 .

These v a l u e s are p l o t t e d

a g a i n s t t h e tim e o f i n c u b a t io n i n F igu re 8 .

There was a l i n e a r

r e l a t i o n s h i p betw een th e enzyme a c t i v i t y and t h e tim e o f i n c u ­ b a t i o n d u rin g t h e f i r s t fo u r h o u rs, a f t e r w hich t h e r e was a g r a d u a l d e c r e a s e i n enzyme a c t i v i t y w it h t i m e . *

A second exp erim en t was s e t up, i n c u b a t in g t u b e s f o r 0 , 1, 2, 3 and 4 hours r e s p e c t i v e l y , i n o r d e r to c o n firm th e

l i n e a r r e l a t i o n s h i p t h a t occu rred between enzyme a c t i v i t y and tim e d u rin g a fo u r -h o u r i n c u b a t io n p e r io d .

The r e s u l t s o f

t h i s exp erim en t are g iv e n in T able 10, and t h e graph o b ta in e d by p l o t t i n g t h e s e v a lu e s a g a i n s t t h e tim e o f in c u b a t io n has been reproduced i n F ig u r e 8 .

T h is curve confirm ed t h e r e s u l t s

o f th e f i r s t exp erim en t i n t h a t th e r e was a l i n e a r r e l a t i o n s h i p







■■

'





Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Reproduced with permission of the copyright owner. Further reproduction prohibited without

T a b le 1 0 .

R e l a t i o n s h i p o f Enzyme A c t i v i t y t o t h e Time o f I n c u b a t i o n .

Experim ent # 1

C e l l Count 5340 c e l l s / c u . m m .

Time i n Hours

Amylase A c t i v i t y (mgm. g l u c o s e l i b e r a t e d per 100 m l. o f enzyme)

E xp erim ent # 2

C e l l Count 2540 c e l l s / c u . m m .

Time i n Hours

Amylase A c t i v i t y (mgm. g l u c o s e l i b e r a t e d per 100 m l. o f enzyme)

0

0

0

0

1

40

$

8

2

60

1

16

4

118

2

41

8

168

3

67

18

249

4

82

-70-

2 v.t

2 so

T1ML

(HOURS)

t

F ig . 8.

Time Course o f R e a c t io n fo r R ab bit L eucocyte A m ylase.

I

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

b etw een enzyme a c t i v i t y and t h e tim e o f in c u b a t io n during t h e f i r s t fou r h o u r s . Comparison o f W hite C e l l Count w it h Amylase A c t i v i t y The am ylase a c t i v i t y o f a s e r i e s o f r a b b i t le u c o c y t e s u s ­ p e n s io n s was compared w ith t h e t o t a l w h ite c e l l count i n T ab le 11.

The r e a c t i o n m ix tu r e was in c u b a te d f o r fo u r hours

i n each c a s e .

The wide range i n a c t i v i t y per 10^ c e l l s

( 1 - 32 mgm. g l u c o s e l i b e r a t e d p er 108 c e l l s ) i n d i c a t e s a poor c o r r e l a t i o n betw een enzyme a c t i v i t y and w h it e c e l l c o u n t. D e te r m in a t io n o f th e Source o f Amvlase A c t i v i t y in L e u c o c y te S u sp e n sio n s E xp erim ents were perform ed u sin g th e u n tr e a t e d c e l l s u s ­ p e n s i o n , c e l l s t h a t had been s e p a r a te d by c e n t r i f u g i n g and r e ­ suspended i n i s o t o n i c a a l i n e , n a l su sp en sio n .

and th e su p e rn a ta n t o f th e o r i g i

The r e s u l t s o f t h e s e ex p erim en ts are g iv e n

i n T a b le s 12 and 1 3 .

The v a l u e s o b ta in e d f o r t h e enzyme a c t i ­

v i t y i n t h e s e d e t e r m in a t io n s i n d i c a t e d t h a t t h e w h ite c e l l s c o n t a in e d l i t t l e ,

i f any, am ylase a c t i v i t y .

The g r e a t e r p a r t

o f t h e enzyme a c t i v i t y was found i n th e su p e rn a ta n t o f th e o r ig i n a l su sp en sio n .

The r e s u l t s o b ta in e d on c e l l s u s p e n s io n s

s e p a r a te d by th e de Haan (1 9 1 8 ) t e c h n iq u e were confirm ed by t e s t i n g s u s p e n s i o n s o b ta in e d by th e t e c h n iq u e s o f B u t l e r and Cushman (194-0) and Spear (194-8).

These ex p erim en ts i n d i c a t e d

t h a t th e l e u c o c y t e s o f t h e r a b b i t c o n ta in e d l i t t l e

am ylase

a c tiv ity .

.____________

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-7 2 1

T a b le 1 1 .

Amylase A c t i v i t y Compared w i t h C e l l C o n c e n t r a t io n *

I

C e l l Count ( c e l l s / c u . mm.)

Amylase A c t i v i t y (mgm. g l u c o s e l i b e r a t e d per 100 m l. o f enzyme)

Amylase A c t i v i t y (mgm. g l u c o s e l i b e r ­ a te d per 1 0 ° c e l l s )

5340

118

22

2770

12

6320

7

1

5610

115

20

2540

82

32

3390

65

19

i 860

28

15

■ 4

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

1 |

1

-7 3 -

T a b le 1 2 .

Amvlase A c t i v i t y o f W hite C e l l S u sp en sio n s S e p a r a ted by th e de Haan T e c h n iq u e .

C e l l Count ( c e l l s / c u . . mm.)

i I

Amylase A c t i v i t y (mgm. g l u c o s e l i b e r a t e d per 100 m l. o f enzyme) C e l l S u sp en sio n

Su pern atant

R esuspended C e l l s

3390

51

52

2

1860

28

37

3

-7 4 !

i I

T a b le 1 8 .

Amylase A c t i v i t y o f White C e l l S u sp en sio n s S en ara ted

by (a ) Butler-Cushm an T echnique, Cb) Snear T ech n iq u e.

E xperim ent

C e l l Count ( c e l l s / c u . mm.)

Amylase A c t i v i t y (mgm. g lu c o s e l i b e r a t e d per 100 ml. o f enzyme) F ir st S u pern atant

Second Supern atant

Resuspended C e lls

Serum

(a )

1290

98

3

■0

9640

(b)

4180

140

22

6

9230

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ADEH0 SIN BTRIPHO SPHATASE A d e n o sin e tr i-p h o so h a ta se A c t i v i t y o f Guinea P ig L iv er and B r a in The e x p e r im e n t a l p roced u re was checked by t e s t i n g th e a d e n o s i n e t r i p h o s p h a t a s e a c t i v i t y o f hom ogenized g u in e a p ig l i v e r and b r a i n .

Other w orkers r e p o r t a d e n o s in e t r ip h o s p h a t a s e

a c t i v i t y i n l i v e r and b r a i n (Du B o is and P o t t e r , 194-3, K a lck ar, 194-4).

The r e s u l t s o f t h e s e d e t e r m in a t io n s are found i n T ab le

14. A d e n o s in e t r io h o s u h a t a s e A c t i v i t y o f R a b b it W hite C e l l s T able 15 g i v e s th e v a l u e s o f a d e n o s in e tr ip h o s p h a t a s e a c t i ­ v i t y f o r r a b b i t w h ite c e l l s o b ta in e d by th e de Haan (191 8) tech n iq u e.

At low c e l l c o n c e n t r a t i o n s t h e r e was a n e g l i g a b l e

enzyme a c t i v i t y .

I f th e l e u c o c y t e s u s p e n s io n was c o n c e n tr a t e d

t o a c e l l c o u n t o f 2 0 0 ,0 0 0 c e l l s / c u . mm., a c t i v i t y was fo u n d . Saponin i n c r e a s e d th e a c t i v i t y o f th e enzyme t o a s l i g h t d e g r ee T h is e f f e c t was n o t as g r e a t as t h a t found f o r th e o th e r enzyme H o m og en izatio n d id n o t i n c r e a s e th e enzyme a c t i v i t y . E xperim ents i n t h i s la b o r a t o r y have dem onstrated t h a t 1010 w h it e c e l l s have a mean w e ig h t o f 4 grams. as a co n v ersio n f a c t o r , i t i s

U sin g t h i s v a lu e

seen t h a t a c e l l count o f 2 0 0 ,0 0 0

c e l l s per c u . mm. i s e q u i v a l e n t t o 80 m illig r a m s o f wet t i s s u e per m l.

Thus i t was o b ser v ed t h a t th e c o n c e n t r a t i o n o f ad en o-

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-7 6 T a b le 1 4 ,

A d e n o s in e t r ip h o s p h a t a s e A c t i v i t y o f Guinea P ig B r a in and L i v e r .

Enzyme C o n c e n tr a tio n

A d e n o s in e tr ip h o s p h a ta s e A c t i v i t y ( u g . Phosphorus l i b e r a t e d /1 0 0 m l. enzyme)

L iv e r

50

2 0 .0

B r a in

50

1 1 .4

L iv e r

100

2 4 .4

B r a in

100

1 9 .3

Enzyme Sou rce

i I

£ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-7 7 T a b le 15.

A d e n o s in e tr ip h o s p h a ta s e A c t i v i t y o f R a b b it W hite C e l l s 0

C e l l Count ( c e l l s / c u . mm.)

A d e n o s in e tr ip h o s p h a ta s e A c t i v i t y (u g . Phosphorus l i b e r a t e d / 1 0 0 m l. enzyme) Wo Saponin

Saponin

7 ,4 0 0

1 .0

8 6 ,6 6 6

2 .0

4 .0

2 0 0 ,0 0 0

1 4 .4

1 4 .8

2 0 0 ,5 3 3

9 .2

1 2 .1

I II

_______L_______ Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.

I ;

s i n e t r i p h o s p h a t a s e i n l e u c o c y t e p r e p a r a tio n s was o f comparable

i

v a lu e t o t h a t o f g u in ea p ig b r a i n .

j

S in c e low c e l l c o n c e n t r a -

i

| |

t i o n s f a i l e d t o g i v e m easurable a c t i v i t y by t h i s method, i t

i

was i m p o s s ib le t o do a s e r i e s o f enzyme measurements on th e

j

{

a d e n o s i n e t r i p h o s p h a t a s e a c t i v i t y o f mixed l e u c o c y t e p r e p a r a t i o n s .

i

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DISCUSSION P h o sp h a ta s e The d a t a p r e s e n t e d h e r e show t h a t f o r b o th man and th e r a b b i t t h e a c id p h o sp h a ta se a c t i v i t y was p r i n c i p a l l y i n th e ly m p h o cy te , a lth o u g h t h e r e was a l e s s e r c o n c e n t r a t i o n i n th e polym orph onu clear l e u c o c y t e .

In th e c a s e o f th e r a b b i t t h e s e

o b s e r v a t i o n s are i n agreement w it h th o s e o f Cram and R o s s i t e r (19 4 9).

The h i s t o c h e m i c a l o b s e r v a t i o n s o f a number o f workers

a l s o le n d su p p ort t o t h e f i n d i n g t h a t th e polym orphonuclear l e u c o c y t e c o n t a in e d l i t t l e a c id p h o sp h a ta se a c t i v i t y w h ile th e lym phocyte c o n ta in e d c o n s i d e r a b l y more (Gomori, 1941-b; W isl o c k i and Dempsey, 1946; W o lf, Kabat and Newman, 1943; R a b in o v i t c h , J u n q u eir a and Mendes, 1948; R a b in o v itc h and A n d reu cci, 1949). The human lym p h ocytes had more than t w i c e th e a c t i v i t y o f r a b b i t ly m p h o c y te s.

In o t h e r ex p erim en ts i t was shown th a t

t h e w h it e c e l l s o f th e r a t ana gu in ea p ig e x h i b i t e d an a c id p h o sp h a ta s e a c t i v i t y o f t h e same order as t h a t o f th e r a b b i t , w h i l e th e enzyme a c t i v i t y o f th e w h ite c e l l s o f t h e c a t , dog and p ig e o n was much l e s s . The a l k a l i n e p h o sp h a ta s e a c t i v i t y was c o n fin e d c h i e f l y to th e polym orphonuclear l e u c o c y t e as shown by th e d a ta p r e s e n te d f o r b o th man and t h e r a b b i t .

The lym phocytes were found to

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-80.

c o n t r i b u t e no a l k a l i n e p h o sp h a ta se a c t i v i t y t o mixed l e u c o c y t e

I su sp en sio n s, !

^ h ese f i n d i n g s are su pp orted by th e h i s t o c h e m i -

c a l o b s e r v a t i o n s o f a number o f w orkers u s i n g th e t e c h n iq u e s o f Gomori (1 9 3 9 ) and Takamatsu ( 1 9 3 9 ) .

See f o r exam ple, t h e

o b s e r v a t i o n s o f Gomori ( 1 9 4 1 - a ) ; Kabat and F u rth (1941)*, W is i

l o c k i and Dempsey (1 9 4 6 ) and W a c h ste in ( 1 9 4 6 ) . I t was n oted t h a t , i n t h e m u l t i p l e r e g r e s s i o n e q u a t io n s !

f o r th e a l k a l i n e p h o sp h a ta se a c t i v i t y o f t h e w h ite c e l l s o f b o th man and t h e r a b b i t , t h e term w hich i n v o l v e d t h e lympho­ c y t e c e l l c o u n t was n e g a t i v e .

T h is would s u g g e s t t h a t t h e

lym ph ocyte c o n t a in e d some i n h i b i t o r o f a l k a l i n e p h osp h ata se a c tiv ity .

S in c e th e c o e f f i c i e n t o f p a r t i a l c o r r e l a t i o n betw een

t h e a l k a l i n e p h o sp h a ta se a c t i v i t y and t h e lym phocyte c e l l c o u n t w it h t h e polym orphonuclear l e u c o c y t e c e l l count e x c lu d e d was a l s o n e g a t i v e , t h i s might len d su p p ort t o th e p r o p o s a l t h a t t h e r e i s an a l k a l i n e p h o sp h a ta se i n h i b i t o r i n t h e lympho­ cy te.

It is

s e e n , however, t h a t t h e c o e f f i c i e n t o f p a r t i a l

c o r r e l a t i o n betw een t h e a l k a l i n e p h o sp h a ta s e a c t i v i t y and t h e lym phocyte c e l l count w i t h t h e polym orphonuclear l e u c o c y t e c e l l count e x c lu d e d was n o t s t a t i s t i c a l l y fore, i f

sig n ific a n t.

There­

su ch a h y p o t h e t i c a l i n h i b i t o r d o e s e x i s t i n th e

lym p h ocyte, i t s c o n c e n t r a t i o n would be s u b j e c t to a h ig h a n im a l- t o - a n im a l v a r i a t i o n . A stu dy o f t h e a l k a l i n e p h o so h a ta se a c t i v i t y o f th e whole b lo o d o f p a t i e n t s w i t h leu k em ia a l s o p o i n t s t o th e polym or­ p h o n u clea r l e u c o c y t e and o th e r g r a n u lo c y t e s as a so u rce o f

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-8 1 enzyme a c t i v i t y .

Umeno (1 9 3 1 ) and Iw a tsu ru and Nanjo (1939)

r e p o r t e d an i n c r e a s e i n t h e b lo o d a l k a l i n e p h o sp h a ta se i n p a t i e n t s w i t h m yeloid le u k e m ia .

They a l s o ob served t h a t , as

t h e w h ite c e l l count d e c r e a se d f o l l o w i n g r a d i a t i o n t h e r a p y , th e c o n c e n t r a t i o n o f t h e b loo d a l k a l i n e p h o sp h a ta se a l s o decreased.

In p a t i e n t s w i t h e o s i n o p h i l i c leu kem ia and i n

an im a ls w i t h e x p e r im e n ta l e o s i n o p h i l i a th e b lo o d a l k a l i n e p h o sp h a ta se a l s o i n c r e a s e d ,

s u g g e s t in g t h a t th e e o s i n o p h i l ,

l i k e th e polym orphonuclear l e u c o c y t e , i s r i c h i n a l k a l i n e p h osp h atase

(Iw a tsu r u , Minami, and N anjo, 1 9 3 9 ) .

a l k a l i n e p h o sp h a ta se o f a p a t i e n t w it h ly m p h a tic

The b lo o d leukem ia

was unchanged,w hich would i n d i c a t e t h a t t h e lym phocyte was r e l a t i v e l y poor i n t h i s enzyme (Iw a tsu r u and Minami, 1 9 3 4 ) . A lb e r s (1 9 3 9 ) r e p o r te d t h a t t h e r e was no change i n th e a l k a l i n e p h o sp h a ta se l e v e l s o f the serum o f p a t i e n t s w it h e i t h e r m yeloid or lym p h atic le u k e m ia .

When r a d i a t i o n th e r a p y was a d m in is t e r e d ,

r e s u l t i n g i n a d s c r e a s e d v /h ite c e l l c o u n t, t h e serum of p a t i e n t s w i t h m yeloid leu kem ia showed an e l e v a t i o n i n a l k a ­ l i n e p h o sp h a ta se a c t i v i t y , t h e r e b e in g no such change in th e a l k a l i n e p h o sp h a ta s e a c t i v i t y o f t h e serum o f o a t i e n t s w it h lym p h atic le u k e m ia .

T h is a g a in s u g g e s te d t h a t th e lym phocyte

c o n ta in e d a l e s s e r c o n c e n t r a t i o n o f a l k a l i n e p h o sp h a ta se than d id th e polym orphonuclear l e u c o c y t e . The a c t i v i t y o f th e a l k a l i n e o h o s p h a ta se i n th e polymor­ ph onu clear l e u c o c y t e o f th e r a b b i t was a p p r o x im a te ly e ig h t t im e s t h a t o f th e polym orphonuclear l e u c o c y t e o f man.

Other

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-82e x p e r im e n ts have dem onstrated c o n s id e r a b le s p e c i e s - t o - s p e c i e s I

v a r i a t i o n i n th e a l k a l i n e p h o sp h a ta se a c t i v i t y o f th e p o l y ! morphonuclear l e u c o c y t e s .

I t was found th a t th e c o n c e n t r a t i o n

o f t h e enzyme was h ig h i n th e polym orphonuclear l e u c o c y t e s I i o f th e r a b b i t , 9 r a t and guin ea n ' i g , w h ile i t was a lm o st a b sen t i.

;

i n t h e polym orphonuclear l e u c o c y t e s o f th e c a t , dog and p ig e o n .

; I

In s p i t e o f t h e d i f f e r e n c e s i n th e r e l a t i v e c o n c e n t r a t i o n s o f a c id and a l k a l i n e o h o sn h a ta se a c t i v i t y i n v a r io u s s p e c i e s ,

!

I

t h e g e n e r a l i z a t i o n rem ains t h a t th e polym ornhonuclear l e u c o c y t e i s r i c h i n a l k a l i n e o h o s r h a ta s e a c t i v i t y and poor i n a c id p h o sp h a ta se a c t i v i t y , w h ile t h e lym phocyte i s poor i n a l k a l i n e

j

I |

p h o sp h a ta se a c t i v i t y and r i c h i n a c id p h o sp h a tase a c t i v i t y . ft-G lu cu ron id ase

;

The d a ta p r e s e n t e d show t h a t fo r b o th man and t h e r a b b i t

!

| i

t h e ft-g lu c u r o n id a se a c t i v i t y o f mixed w h ite c e l l s u s p e n s io n

|

was i n b o th th e polym orphonuclear l e u c o c y t e and i n t h e lympho-

!

cy te.

T h is would support t h e o b s e r v a t i o n o f Fishman, Sp rin ger

and B r u n e t t i (19 48) t h a t t h e r e was no c o r r e l a t i o n o f th e b u f f y c o a t g lu c u r o n id a s e a c t i v i t y and th e p e r c e n ta g e o f p o l y ­ morphonuclear l e u c o c y t e s and lym phocytes i n th e b lo o d .

These

w orkers used t h e b u f f y c o a t r e c o v e r e d from a g iv e n volume o f b lo o d , and e s t im a t e d th e p e r c e n ta g e o f each type o f w h it e c e l l from c o u n ts done on a sample o f whole b lo o d .

In t h i s study

w h it e c e l l c o u n ts were done on t h e c e l l s u s p e n s io n s a f t e r th e y had been se p a r a te d and any clumped c e l l s had been rem oved.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

I t was a l s o found t h a t th e e x t r a c t i o n o f t h e enzyme from th e l e u c o c y t e s by sa p o n in ( R o s s i t e r and Wong, 1 9 5 0 -a ) was more j e f f i c i e n t th a n t h e f r e e z i n g - t h a w in g method used by Fishm an. i T h ese v a r i a t i o n s i n te c h n iq u e might texp la in , i n p a r t , th e j l fin d in g o f a c l o s e r c o r r e l a t i o n betw een th e w h ite c e l l c o u n ts I and |3-g'iu cu ron id ase a c t i v i t y i n th e p r e s e n t stu d y d a ta o f Fishman e t a l . j

As

than th e

(1 9 43) might s u g g e s t ,

e a r l y as 1914 Sera r e p o r t e d th e p r e s e n c e o f an enzyme

' ! i n s p l e e n w hich would h y d r o ly s e th e g lu c u r o n id e l i n k a g e . i | T h is work was expanded by Oshima (1 934 ) and by T a l a l a y , F i s h ­ man and H uggins ( 1 9 4 6 ) , who s tu d ie d th e d i s t r i b u t i o n o f [3- g l u ­ c u r o n id a s e i n t h e anim al body.

They a l s o found a h ig h enzyme

a c t i v i t y i n th e s p l e e n and t h i s organ has b een a sou rce o f many |

p a r t i a l l y p u r i f i e d enzyme e x t r a c t s (Fishman, 1939; Fishman and T a l a l a y , 1947; Graham, 1946; M i l l s , 1 9 4 8 ) .

Oshima (193 4)

j

showed t h a t th e thymus was r i c h i n th e enzyme, a lth o u g h F i s h -

!

man and A nlyan (1 947 ) c o u ld f i n d l i t t l e

!

v i t y i n lymph n o d e s .

{3 -g lu cu ron id ase a c t i -

From t h e s e o b s e r v a t i o n s i t

might be

e x p e c t e d t h a t th e lym phocyte would be much p oorer i n th e enzyme th a n t h e oolym orp hon uclear l e u c o c y t e . H is t o c h e m i c a l e v id e n c e o f {3-g lu cu ro n id a se a c t i v i t y i s o r o v id e d by F ried en w a ld and Becker (194 8) who dem onstrated enzyme a c t i v i t y i n th e c y to o la s m o f c e l l s o f th e

s p l e e n , lym­

p h a t i c n o d u le s and bone marrow. R o s s i t e r and Wong ( 1 9 5 0 - a and b) have commented upon th e f u n c t i o n o f {3-glu cu ron id ase i n w h ite c e l l s .

I t has been p o s t u -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-8 4 -

l a t e d t h a t {3-glu cu ron id a se has t o do w it h m e ta b o lic c o n j u g a t io n and, p o s s i b l y , d e t o x i c a t i o n (Fishm an, 1940; Fishman, 1947 and Oshima, 1 9 3 4 ) .

T h is i s an a t t r a c t i v e th e o r y s i n c e t h e a c t i o n

o f t h e polym orphonuclear l e u c o c y t e s , w hich a g g r e g a te a t th e s i t e o f i n f e c t i o n , might be a c co m p lish ed , i n D art, by th e c o n j u g a t i o n and su bseq uent i n a c t i v a t i o n o f t o x i c s u b s t a n c e s . Fishman b a sed h i s th e o r y on th e o b s e r v a t io n t h a t th e i n j e c t i o n o f m enthol i n t o dogs and mice caused an i n c r e a s e i n th e p - g l u c u r o n id a s e a c t i v i t y o f th e l i v e r ,

s p le e n and k id n e y , w it h no

change i n t h e u te r u s (Fishm an, 1 9 4 0 ).

I f o e s t r o g e n s were

a d m in is t e r e d , however, to o v a r ie c to m iz e d m ice , -the (3 -g lu c u r o n id a se a c t i v i t y o f th e u t e r u s was in c r e a s e d , w it h no change i n t h e l i v e r 3 - g l u c u r o n i d a s e (Fishm an, 1947; Fishman and F i s h ­ man, 1 9 4 4 ) .

T h ese o b s e r v a t i o n s were i n t e r p r e t e d as e v id e n c e

t h a t m enthol and o e s t r o g e n s were c o n ju g a te d as g lu c u r o n id e s by t h e a c t i o n o f t h e enzyme (3 -glu cu ron id ase and e lim in a t e d from t h e b od y. T h is t h e o r y has b een c r i t i c i z e d by s e v e r a l workers ( L e w y , 1943; L e w y , Kerr and C am pbell, 1948; Karunairatnam, Kerr and L evvy, 1949; Karunairatnam and L e w y , 1 9 4 9 ) .

Masamune (1934)

and M i l l s and P a u l (1 9 4 9 ) found t h a t th e c h i e f f u n c t i o n o f th e enzyme appeared t o be h y d r o l y t i c .

L e w y (1948) and L e w y

and S t o r e y ( 1 9 4 9 ) , as a r e s u l t o f i n v i t r o s t u d i e s , r e p o r t e d t h a t t h e s y n t h e s i s o f g lu c u r o n id e s was brough t about by a d i f f e r e n t enzyme sy stem from t h a t r e s p o n s i b l e f o r th e h y d r o ly ­ s i s of b io s y n th e tic g lu cu ro n id es.

T h is was confirm ed by t h e

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

i n v i v o s t u d i e s by Karunairatnam , Kerr and L e w y (194-9).

Other

w orkers who have su pp orted t h e s e o b s e r v a t io n s in c lu d e De Meio and A r n o lt ( 1 9 4 4 ) , Hemingway, Pryde and W i ll i a m s , (193 5) L i p s c h i t z and Beuding (193 9) and Karunairatnam and L e w y ( 1 9 4 9 ) . The l a t t e r w orkers p o s t u l a t e d t h a t th e d e g r e e o f c e l l u l a r p r o ­ l i f e r a t i o n i s r b l a t e d to (3-glucuronidase a c t i v i t y .

Thus t i s s u e s

i n a s t a t e o f r e g e n e r a t i o n or p r o l i f e r a t i o n e x h i b i t h ig h (3-glu­ c u r o n id a s e a c t i v i t y (Kerr and L e w y , 1947; K err, L e w y and C am pbell, 1947; L e w y , Kerr and Campbell, 1 9 4 3 ) .

The in c r e a s e d

u t e r i n e (3- g l u c u r o n i d a s e a c t i v i t y a f t e r th e a d m i n is t r a t i o n o f o e s t r o g e n s m ight be e x p la in e d i n terms o f c e l l p r o l i f e r a t i o n r a t h e r th an by an i n c r e a s e i n m e ta b o lic c o n j u g a t i o n . S in c e h y d r o l y s i s o f g lu c u r o n id e s seems to be a major e f f e c t o f (3 -g lu c u r o n id a s e , i t s p r e se n c e i n t h e w h ite c e l l might be con cern ed w it h th e f u n c t i o n a l h y d r o l y s i s o f su b s ta n c e s c o n ­ t a i n i n g g lu c u r o n ic a c id and n orm a lly p r e s e n t i n t i s s u e s . Examples o f such s u b s t a n c e s are h y a lu r o n ic a c id and c h o n d r o it in s u lp h u r ic a c i d .

I n t h i s way th e enzyme might p la y i t s p a r t

by way o f th e l e u c o c y t e s i n th e p r o c e s s o f t i s s u e in fla m m a tio n , or th e r e a c t i o n o f t h e t i s s u e t o damage. Amylase The d a t a p r e s e n t e d show t h a t f o r washed r a b b it w h ite c e l l s u s p e n s io n s th e am ylase a c t i v i t y was v e ry low .

The g r e a te r

p a r t o f t h e enzyme a c t i v i t y was found i n t h e su p e rn a ta n t f l u i d o b ta in e d by s u c c e s s i v e w a sh in g s o f th e l e u c o c y t e s w it h i s o t o n i c sa lin e .

The v a l u e s o b ta in e d f o r serum am ylase were ap p rox im a tely

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

1000 t i m e s h i g h e r t h a n t h e v a l u e s o b t a i n e d f o r t h e l e u c o c y t e suspensions.

I t v.'as c o n c l u d e d t h a t under t h e c o n d i t i o n s o f

t h e t e s t t h e r e was no a m y lase a c t i v i t y i n washed e o l y n o r p h o n u c l e a r le u c o c y te s of th e r a b b i t .

B a r n e s (1940) r e p o r t e d a

s l i g h t a m y l a s e a c t i v i t y i n t h e lymphocyte and a more marked a c t i v i t y i n th e polymorphonuclear le u c o c y te , of th e r a b b i t . He o b t a i n e d h i s c e l l s by t h e he Haan t e c h n i q u e and, i f he f a i l e d t o wash them, h i s r e s u l t s a r e i n reported h ere,

accordance

w ith those

( b i l l s t a t t e r , Bamann and R a h d ev a ld (1 9 3 0 ) showed

t h a t t h e l e u c o c y t e s o f t h e h o r s e c o n t a i n e d am ylase a c t i v i t y . R o b e r t - L e v y and fvnbe ( 1 ? £’: ?) a l s o r e p o r t e d t h a t t h e e o s i n o p h i l s o f man c o n t a i n e d a m y la s e a c t i v i t y . whether th e c e l l p r e p : r a t i o n or n o t .

u s e d

'I.'here i s no’ record, as t o by t h e s e w o r k e r s was washed

I f t h e n e u t r o p h i l c o n c e n t r a t i o n was i n c r e a s e d i n t h e i r

p r e p a r a t i o n s , an i n c r e a s e i n enzyme a c t i v i t y was o b s e r v e d . B e r n a r d a n t Rose n (1941) and M0r r i ?

found t h a t t h e

a m y l a s e a c t i v i t y o f whole b l o o d was l e s s

-.ban t h a t o f p l a s m a .

The f i n d i n g s r e p o r t e d i n t h i s t h e s i s on t h e enzyne a c t i v i t y o f w h i t e c e l l s u s p e n s i o n s o n t a i n e d by t h e de Haan ( 191 ?) t e c h n i q u e are s im ila r.

The

a m

y l a s e

activ ity

o f

the w hite c e l l s u s p e n s io n s

was l e s s t h a n t h e combined a c t i v i t i e s o f t h e su p e rrv -'t an t ( ' h o i 0 12 , p a g e 73 ) •

v; a s k i n g s and t h e r e s u s p e n d e d w h i t e c e l l s

li v e r s and Hi H i sr. ( 1917 ) r e p o r t e d an i n c r e a s e d d i a s t a t i c a c t i v i t y i n t h e b l o o d o f p a t i e n t s w i t h d i a b e t e s ana n e p h r i t i s . P o p p e r and P i o t k e ( 1 9 4 1 ) , S o r k i n ( 1 9 4 3 ) , and Polowe (1 ?4 6 ) d e m o n s t r a t e d an i n c r e a s e d b lo o d n n v l n s e j_-n c e r t a i n d i s e a . s e s o f

*

-

-

-

.

.

.

.

.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-37the p an creas. c ip a tio n of

S o rk in (194-3) s t a t e d t h a t t h e r e was no p a r t i ­ am ylase i n in t e r m e d ia r y ca rb o h y d r a te m etab o lism .

T h is i s a c o n f ir m a t io n o f th e r e p o r t s o f C o r i, C ori and Schmidt ( 1 9 3 9 ) , Cori and C ori ( 1 9 4 1 ) , and O sten , H erbert and Holmes ( 1 9 3 9 ) . I t appears t h a t t h e r e i s l i t t l e am ylase a c t i v i t y a s s o c i ­ a te d w it h t h e .polym orphonuclear l e u c o c y t e s o f th e r a b b i t . Thus any f u n c t i o n a s s o c i a t e d w i t h amylase a c t i v i t y would not be found i n th e polym orphonuclear l e u c o c y t e s .

As has been

n o te d p r e v i o u s l y , t h e enzyme am ylase p la y s no r o l e i n i n t e r ­ m ediary carb oh yd rate m eta b o lism . A d e n o s in e tr ip h o s p h a ta s e The d a ta p r e s e n t e d show t h a t t h e r e was a low c o n c e n t r a t i o n o f a d e n o s in e t r ip h o s p h a t a s e a c t i v i t y i n r a b b i t polym orphonuclear l e u c o c y t e s u s p e n s io n s w i t h low c e l l c o u n t s .

I f such s u s p e n s io n s

were c o n c e n t r a t e d , a m easurable enzyme a c t i v i t y could be o b ta in e d , w h ich was o f th e order o f th e a c t i v i t y o f g u in ea p ig b r a in homog en ates.

B eca u se o f t h e s e low enzyme c o n c e n t r a t i o n s th e data

c o u ld n ot be a n a ly z e d s t a t i s t i c a l l y as i n th e c a se o f th e p h o s­ p h a t a s e s or B - g l u c u r o n i d a s e s . Du B o is and P o t t e r (1 9 4 3 ) found a d e n o s in e t r ip h o s p h a t a s e in sp lee n .

T h is work was f o l lo w e d by t h e o b s e r v a t i o n s o f

M e is t e r (19 49) on t h e enzyme a c t i v i t y o f normal and p a t h o l o g ic serum.

B ecause th e enzyme i s w id e ly d i s t r i b u t e d in t h e v a r io u s

t i s s u e s o f th e body i t s p r e s e n c e i n th e w h it e b lo o d c e l l s might be e x p e c t e d .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-8 8 -

CONCLTJSIONS 1.

Three methods have b een used t o o b t a in s u s p e n s io n s o f l e u c o c y t e s from th e r a b b i t .

Two o f t h e s e methods were

• s u it a b le f o r o b t a i n in g s u sp e n sio n s o f w h ite c e l l s from man.

The a c t i v i t y o f a c id and a l k a l i n e p h o sp h a ta s e , (3-

g l u c u r o n i d a s e , am ylase and ad e n o sin e tr ir> h o sn h a ta se i n t h e s e s u s p e n s io n s was determ in ed . 2.

For a c i d and a l k a l i n e p h o sn h a tase and (3 -g lu c u r o n id a se, th e c o r r e l a t i o n c o e f f i c i e n t betw een th e enzyme a c t i v i t y and b o t h th e lym phocyte c e l l count and t h e polymorphonu­ c l e a r l e u c o c y t e c e l l count was d e ter m in e d .

By s t a t i s t i ­

c a l methods i t was p o s s i b l e t o a s s e s s t h e c o n t r i b u t i o n t o t h e t o t a l enzyme a c t i v i t y made by each o f th e two major c e l l t y p e s . 3.

For b o t h man and th e r a b b i t , th e a c id p h o sp h atase a c t i v i t y was found t o be c h i e f l y i n th e ly m p h o cy te s, a lth o u g h th e polym orphonuclear l e u c o c y t e s c o n ta in e d l e s s e r c o n c e n tr a ­ t i o n s o f t h i s enzyme a l s o .

4.

The a c i d p h o sp h a ta se a c t i v i t y o f th e human lym phocytes was more than t w ic e t h a t o f th e r a b b i t lym p h ocy tes.

5.

The a l k a l i n e p h o sp h a ta se a c t i v i t y was c o n fin e d t o th e morphonuclear l e u c o c y t e s i n b oth s p e c i e s , w i t h l i t t l e

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

p o ly ­

a c t i v i t y in th e lymphocytes.

6.

The a l k a l i n e p h o s p h a t a s e a c t i v i t y o f t h e r a b b i t p o l y m o r ­ p h o n u c l e a r l e u c o c y t e vys e i g h t t i m e s t h a t of t h e

poly­

m o r p h o n u c l e a r l e u c o c y t e s o f man. 7.

The C - - g I n c u ro n i d a s e a c t i v i t y was found t o be f a i r l y e v e n l y d i s t r i b u t e d betvteen t h e two c e l l t y p e s .

For man

t h e r e - t i o o f t h e enzyme a c t i v i t y i n t h e polymorphonu­ c l e a r l e u c o c y t e t o t h a t i n t h e ly m p h o c y t e was fou n d t o b e 5*4, w h i l e f o r t h Q r a b b i t i t was ? : 5 « o.

The a m y la s e a c t i v i t y

of r n u o l l polym orphonuclear le u c o c v te

s u s p e n s i o n s was l i n e a r w i t h tim e d u r i n g t h e f i r s t f o u r hours of th e r e a c t i o n . 9.

The a m y la s e a c t i v i t y o f r a b b i t p o l y m o r p h o n u c l e a r l e u c o c y t e s u s p e n s i o n s v n s found t o oe c h i e f l y i n t h e s u p e r n a t a n t f l u i d , w ith only a s l i g h t degree of a c t i v i t y

i n v.:usneb

leucocytes. 10.

The a h e p o s i n p t r i r h o s p h a t a s e a c t i v i t y o f v,-ib b it r o l v m o r '^ h o n u c l e c r l e u c o c y t e s u s p o n s i o n s was v e r y low in suspensions w i t h a lov c e l l c o u n t,

i f the c ^ I l s

e r e co ac e n t r a t e d ,

enzyme a c t i v i t y c o u l d he bemuas t r a t e d . 11.

The enzyme

ictiv itv

f fp guinea

u.g.

of c o n c e n t r a t e d l e u c o c y t e s u s p e n s i o n s

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

-9 0 -

;

i

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