Chemical Studies on Preserved Blood

Citation preview

CHEMICAL STUDIES OH rffflSERVJEft;BLQpI). : ::

by

I r v i n g F ried m an

A d i s s e r t a t i o n s u b m itte d i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e d e g re e o f D o cto r o f P h ilo s o p h y , i n the D epartm ent o f C h e m istry , i n th e G ra d u a te C o lle g e o f th e S t a t e U n i v e r s i t y o f Iowa December 1948

ProQuest N um ber: 10984058

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10984058 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

WcoAVV i m

il x

C.O’yKX

ACKNOMDGEMHT

The w r i t e r w ish e s to e x p r e s s h i s g r a t i t u d e to D r. E. L. DeGowin who s u g g e s te d t h i s p ro b lem and a i d e d g r e a t l y i n i t s e x e c u t i o n , and to D r. H. A. M a t t l l l f o r much good a d v i c e .

54193

ill

TABLE OF OONTMMTS

Page Aeknowledgtaen t

ii

In tro d u c tio n

1

E x p e r im e n ta l

5

R e su lts

9

D is c u s s io n

34

Summary

36

B ib lio g r a p h y

38

A ppendix

41

1

INTRODUCTION T he manner l a which g lu c o s e and o t h e r s u g a r s such a s d e x t r o s e , f r u c t o s e , and s u c r o s e , a c t i n s t o r e d b lo o d t o d e la y h e m o ly sis i s n o t known#

T h is p ro b lem i s

o f g r e a t p r a c t i c a l and t h e o r e t i c a l im p o r ta n c e , e s p e c i a l l y s i n c e th e b lo o d bank i s becoming an e s s e n t i a l p a r t o f e v e ry l a r g e h o s p i t a l #

The u s e o f s t o r e d b lo o d i n war m edi­

c i n e n e e d s no e l a b o r a t i o n . The method o f a t t a c k i n g t h i s problem h a s u s u a l l y b ee n to s tu d y b lo o d m ix t u r e s s t o r e d a t 2 -4 ° C e n t i g r a d e . A n t i c o a g u l a n t s h ave o f n e c e s s i t y been i n c o r p o r a t e d i n th e d ilu e n ts.

C hem ical a n a l y s e s o r p h y s i o l o g i c a l t e s t s a r e

th e n p e rfo rm e d on t h e s t o r e d b lo o d i n a t t e m p t s to c o r r e ­ l a t e th e fin d in g © w ith the d i s i n t e g r a t i o n o f th e e r y t h r o ­ c y tes.

B r i e f l y , th e r e s u l t s o b t a i n e d so f a r a r e ;

(a )

The r e d c e l l l o s e s i t s norm al im p e r m e a b ility to c a t i o n s ( 1 ) , p o ta s s iu m d i f f u s i n g from t h e c e l l i n t o th e plasm a w h ile sodium p a s s e s i n th e o p p o s i t e d i r e c t i o n ( 2 ) .

The

exchange i s e q u im o la r a f t e r f i v e days (3) and ta k e s pia c© r e g a r d l e s s o f th e n a t u r e

o f th e p r e s e r v i n g medium,

(b)

The i n o r g a n i c p h o sp h o ru s c o n t e n t o f th e b lo o d r i s e s b u t i s s i g n i f i c a n t l y l e s s i n w e l l p r e s e r v e d b lo o d s ( 4 ,5 ) #

The

i n c r e a s e i s a t th e ex pen se o f the o r g a n ic a c i d - s o l u b l e p h o sp h o ru s f r a c t i o n i n th e c e l l s ,

(c )

In c re a s e s in l a c t i c

a c i d and e l e c t r i c a l c o n d u c t i v i t y ( 6 ) , i n u r i c a c i d ,

n o n -p ro te in n itr o g e n , c r e a t i n i n e , reduced g lu ta th io n e ( 7 ) , and d e c r e a s e s i n g lu c o s e (8* 9) h av e b ee n r e p o r t e d ,

(d)

O sm otic ch an g es o f th e e r y t h r o c y t e o c c u r and a r e somewhat d e p e n d e n t upon th e n a t u r e o f t h e p r e s e r v a t i v e m ix tu re * T hu s, i)©Gowin e t a l • (10) s t a t e t h a t i n c i t r a t e d b lo o d th e e r y t h r o c y t e s u n d e rg o p r o g r e s s i v e s w e l l i n g w h ile i n s u c r o s e s o l u t i o n s th e y s h r in k *

E r y t h r o c y t e s i n a 5*4 p e r c e n t

d e x t r o s e - c i t r a t e m ix t u r e u n d erg o r a p i d s w e l l i n g due to th e d i f f u s i o n o f d e x t r o s e an d w a te r i n t o t h e c e l l s *

(e)

The

p ro th ro m b in t i t e r o f the blood decrease© d u r in g s t o r a g e (1 1 ).

The ch e m ic al ch a n g es and o t h e r p h a s e s o f t h e sub­

j e c t have been e x h a u s t i v e l y re v ie w e d by DeGowin (12). The i n c r e a s e i n i n o r g a n i c p h o sp h o ru s w i t h s im u l­ ta n e o u s d e c r e a s e of o r g a n i c p h o sp h o ru s compounds shows some a p p a r e n t r e l a t i o n t o h e m o ly s is .

In w ell p re se rv e d b lo o d s,

a f t e r lo n g p e r i o d s o f s t o r a g e , th e low I n o r g a n ic p h o sp h o ru s c o n t e n t and h ig h c e l l o r g a n i c p h o sp h o ru s c o n t e n t a r e due to

th e p e r s i s t e n c e o f th e o r g a n ic compounds p a r t i c i p a t i n g

in th e g l y c o l y t i c c y c le *

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

may e x i s t betw een g l y c o l y s i s and h e m o ly sis s i n c e th e o r­ g a n ic a c i d - s o l u b l e ph o sp h o ru s compounds i n th e c e l l a r e i n t i m a t e l y c o n n e c te d w ith c a r b o h y d r a te m e ta b o lism .

In -

d ee d , i t a p p e a r s p o s s i b l e t h a t th e i n t e g r i t y o f th e c e l l m ig h t depend on g l y c o l y s i s i t s e l f .

T h is seems p l a u s i b l e

when i t i s r e c a l l e d t h a t th e e r y t h r o c y t e show© b u t s l i g h t r e s p i r a t o r y a c t i v i t y and t h a t the e n e rg y n e c e s s a r y f o r i t s

3

m a in te n a n c e m u st be d e r iv e d l a r g e l y by th e breakdow n o f g lu c o s e t o l a c t i c a c id *

somewhat t h e same I d e a b a s e d on

e x p e r i m e n ta l f i n d i n g s was e x p r e s s e d by M a iz e ls ( 1 3 ) , who s t a t e s t h a t "an a g e n t ( g lu c o s e ) w hich k e e p s c e l l c h e m is tr y norm al w i l l a l s o a s s i s t c e l l p r e s e r v a t i o n and t h a t t h e m ain­ te n a n c e o f a norm al p h y s i o l o g i c a l c y c le o f m e ta b o lism w i t h i n th e e r y t h r o c y t e s h o u ld h e l p to s u s t a i n i t s n u t r i ­ t i o n and to a i d i t s s u r v i v a l * " I f t h i s h y p o t h e s i s be t r u e , t h a t c e l l u l a r p r e ­ s e r v a t i o n depends on g l y c o l y s i s , th e n i t s h o u ld be p o s s i b l e t o d e m o n s tr a te d i f f e r e n c e s i n g l y c o l y s i s betw een b lo o d s which a r e w e ll p r e s e r v e d and t h o s e i n w hich a p p r e c i a b l e c e l l breakdown h as o c c u r r e d . I t a l s o becomes im p o r ta n t to d e te rm in e th e e f f e c t , i f a n y , o f such f a c t o r s a s d i l u t i o n o f th e blood a n d t h e a c t i o n of th e d i l u e n t i n v a r y in g c o n c e n t r a t i o n s on hemo­ ly sis*

The d a t a o f DeGowin @t a l* (14) l e a d to th e con­

c l u s i o n t h a t th e

f i n a l c o n c e n t r a t i o n o f g lu c o s e i n th e

b lo o d m ix tu r e i s th e most I m p o r ta n t f a c t o r co n c e rn e d i n c e l l p r e s e r v a t i o n and t h a t d i l u t i o n p l a y s b u t l i t t l e p a r t , w h ile D e n s te d t e t a l . (15) s t r e s s t h a t the d e g re e o f d i ­ l u t i o n and th e c o n c e n t r a t i o n o f th e d i l u e n t a r e th e

im­

p o r t a n t f a c t o r s c o n c ern ed r a t h e r th a n th e f i n a l c o n c e n tr a ­ t i o n o f g lu c o se* T h is t h e s i s a t t e m p t s to answer th e p ro b lem s s t a t e d above by s tu d y i n g i n some d e t a i l th e g l y c o l y t i c

4

a c t i v i t y o f the r e d c e l l when p l a c e d i n v a r i o u s m il ie u s * S t u d i e s w ere a l s o p e rfo rm e d t o t e s t th e e f f e c t t h a t d i l u ­ t i o n o f th e b lo o d and c o n c e n t r a t i o n o f g lu c o s e p l a y i n © e ll p r e s e r v a t i o n *

ix P E ru 'M M m C o l l e c t i o n of Blood The p r o c e d u r e f o llo w e d f o r c o l l e c t i n g and a p p o r ­ t i o n i n g th e b lo o d I s i n g e n e r a l th e same a s t h a t d e s c r i b e d by DeGowin ©t a l .

(10)*

su c h m o d i f i c a t i o n s a s have been

made w i l l b© i n d i c a t e d u n d e r d i s c u s s i o n o f th e p a r t i c u l a r e x p e rim e n ts *

E rle n ia e y e r f l a s k s of p y re x g l a s s o f from

1000 to 15QQ ec* c a p a c i t y were a u to c la v e d w ith a p p r o p r i a t e am ounts o f th e p r e s e r v a t i v e to b e s tu d ie d *

I n l a t e r ex­

p e r im e n ts U pjohn b o t t l e s o f 1S00 cc* c a p a c i t y were used* ft hen g lu c o s e s o l u t i o n s were to be combined w ith o t h e r c h e m ic a ls , th e fo rm e r were h e a te d s e p a r a t e l y to a v o id c a ­ ram el i z a t io n and l a t e r combined w ith th e l a t t e r a s e p t i e a l l y * fthen a s u c r o s e s o l u t i o n was em ployed i t was p a s s e d th ro u g h a 3 e i t z f i l t e r i n s t e a d o f b e in g a u t o c l a v e d , s i n c e i t became a p p a r e n t t h a t a u t o c l a v i n g c a u se s p a r t i a l h y d r o l y s i s o f t h e s u c ro s e *

U s u a l l y , b lo o d was drawn in to t h e f l a s k s , con­

t a i n i n g lOOcc* o f 5 ,2 p e r c e n t sodium c i t r a t e s o l u t i o n , from th e median b a s i l i c v e in o f norm al h e a l t h y d o n ors i n 500 cc# q u a n t i t i e s *

The b lo o d was mixed w ith th e p r e s e r ­

v a t i v e by g entl© r o t a t i o n o r s h a k in g o f the r e c e i v i n g fla sk s*

A f t e r th o ro u g h m ix in g l a r g e a l i q u o t s were t r a n s ­

f e r r e d to two o r more JSrlenmeyer f l a s k s o f flora 250 to 500 cc* c a p a c ity *

Th© o t h e r d e s i r e d s o l u t i o n s were ad d ed ,

and a f t e r m ix in g , th e whole was a p p o r tio n e d i n t o

s m a ll

6

E rle n m e y e r f l a s k s c l o s e d w ith c o t t o n p lu g s*

The e n t i r e l o t

was s t o r e d a t 2 - 4 ° C e n tig r a d e u n d e r I d e n t i c a l c o n d i t i o n s * and when an a n a l y s i s was t o he mad© th e e n t i r e c o n t e n t s o f one sm a ll f l a s k was used*

T h is p r o c e d u r e I n s u r e d a u n if o rm

d i s t r i b u t i o n o f b lo o d and p r e s e r v a t i v e th r o u g h o u t th e ex­ p e r im e n t and o b v i a t e d th© d i f f i c u l t i e s a t t e n d a n t on samp-* l i n g th e same f l a s k r e p e a te d l y * Me th o d s C e l l volume was m easured by th e Van A lle n hema­ t o c r i t m ethod. L a c t i c a c i d was d e te rm in e d by th e m ethod o f B a r­ k e r and S um m erv ille (17) which was fo un d s a t i s f a c t o r y *

,ith

s u c r o s e - b l o o d m ix tu re * a drop o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d was ad d ed to on© cc* o f th e somogyi f i l t r a t e and h e a t e d i n a b o i l i n g w a te r b a t h f o r f i v e m in u te s i n o r d e r to h y d ro ­ ly s e th e su cro se.

The p r o c e d u r e from h e r e i s c a r r i e d o u t

i n th e u s u a l m anner. Hemoglobin was d e te rm in e d by th e method o f

;u ( 1 8 ) .

I n o r g a n i c ph o sp h o ru s and o r g a n i c - a c i d s o l u b l e p h o sp h o ru s were d e t e m i n e d by t h e method o f F is k e and su b ­ barrow ( 1 9 ) .

The h y d ro ly sa b l© f r a c t i o n s of th e o r g a n ic a c i d -

s o l u b l e p h o sp h o ru s w ere d e te rm in e d by th e method o f Lohman (SO) w ith p h o sp h o ru s d e t e r m i n a t i o n s made a f t e r Q and 150 m in u te s ’ h y d r o l y s i s \«ith N HgS04 i n a b o i l i n g w a te r bath* The p h o sp h o ru s l i b e r a t e d a f t e r 8 m in u te s1 h y d r o l y s i s r e p r e -

7

s e n t s th e p h o sp h o ru s t h a t I s e a s i l y s p l i t from a d e n o s in e t r i p h o s p h a t e , b e in g t f c o - t h i r d s o f th e p h o sp h o ru s i n aden o­ s in e trip h o sp h a te #

The p h o sp h o ru s l i b e r a t e d d u r in g 7 to

150 m in u te s o f a c i d h y d r o l y s i s i s , a c c o r d i n g t o G u est and R a p o p o rt (8)L')t d e r i v e d p a r t l y from h e x o s e d ip h o s p h a te and t o some e x t e n t from hexosemonophossphat© and from r i b o s e p h o s p h a te o f a d e n y l i c ac id #

The p h o sp h o ru s r e m a in in g unhy­

d r o l y s e d a f t e r t h i s tim e c o n s i s t s l a r g e l y o f d ip h o s p h o g ly c e r a t e , a lth o u g h s m a ll am ounts o f o t h e r o r g a n i c a c i d - s o l u b l e compounds a r e p r e s e n t# I n s e v e r a l i n s t a n c e s when th e c o n c e n t r a t i o n o f g lu c o s e was one p e r c e n t o r l e s s , g lu c o s e was d e te rm in e d by th e S h a f f e r and somogyi method (33) on a soraogyi f i l t r a t e ; o t h e r w i s e , g lu c o s e was d e te rm in e d by t h e g r a v i m e t r i c method o f A l l i h n (33) on a Somogyi f i l t r a t e *

{See A ppendix f o r a

d i s c u s s i o n o f th e A l l i h n m e th o d .) I n o r d e r to compare a p a r t i c u l a r b lo o d c o n s t i t u ­ e n t , a s f o r example i n o r g a n i c p h o s p h o ru s , I n a b lo o d which was d i l u t e d to d i f f e r e n t e x t e n t s , i t was assumed t h a t th© blood as c o l l e c t e d from the don or c o n ta in e d 15 gm. o f hemo­ g l o b i n p e r 100 c c .

The t o t a l hem oglobin c o n t e n t of th e

v a r i o u s l y d i l u t e d sam ples was d e te rm in e d , and th e v a lu e so o b ta in e d d i v i d e d in to 15 gave th© amount of d i l u t i o n *

T h is

f a c t o r m u l t i p l i e d by th e v a lu e o f th e p a r t i c u l a r c o n s t i t u e n t u n d e r c o n s i d e r a t i o n g av e a r e s u l t i n term s of 15 gm. o f hemoglobin*

T h is c a l c u l a t i o n may have in v o lv e d a s l i g h t

s

c o n s t a n t e r r o r , b u t th e c o n s t i t u e n t s compared were alw ays from t h e same o r i g i n a l b lo o d .

9

BBSUhTS The c o m p o s itio n of th e b lo o d m i x t u r e s u s e d i n t h i s e x p e r im e n t and t h e p r o g r e s s o f h e m o ly s is a r e shown i n T able X,

I t i s se e n t h a t h e m o ly sis p ro c e e d e d f a s t e s t i n

c i t r a t e and s u c r o s e b lo o d s b o th o f w h ich had a v e r y low i n i t i a l g lu c o s e c o n te n t*

Th© b l o o d - d e x t r o s e * - c i t r a t e mix­

t u r e s showed l e a s t h e m o ly s is , a lth o u g h some d i f f e r e n c e ap ­ p e a re d betw een th e t h r e e p e r c e n t and 0*6 p e r c e n t g lu c o s e b lo o d s*

The fo rm e r i s th e B o u s -T u rn e r m ix tu re a s m o d if ie d

by DeGowin e t a l* (16) and i s u s e d r o u t i n e l y i n some b lo o d banks* The p e r i o d d u r in g which h e m o ly s is took p l a c e m ost r a p i d l y i n t h e s u c r o s e and c i t r a t e b lo o d s (10 to 20 d ay s) c o i n c id e d w ith th e i n t e r v a l a t which m ost o f the g lu c o s e d i s a p p e a r e d ( T a b le s X and XX).

In th is in te rv a l, a lso ,

t h e r e was a r a p i d l i b e r a t i o n o f i n o r g a n i c p h o sp h o ru s due to th e breakdown o f o r g a n i c a c i d - s o l u b l e p h o sp h o ru s compounds p r e s e n t i n th e r e d c e l l ( F ig u r e 1)*

I t appears th a t in

th e c i t r a t e b lo o d a l l th e o r g a n ic a c i d - s o l u b l e ph osph oru s compounds can t r i b u t e to th© r i s e r a th © r th a n any one f r a c ­ t i o n , show ing t h a t the e n t i r e g l y c o l y t i c system i s a f f e c t e d . F r a c t i o n a t i o n o f th e o r g a n ic a c i d - s o l u b l e compounds o f th e s u c r o s e b lo o d c o u ld n o t be c a r r i e d o u t b e c a u s e o f th e c h a r r i n g o f s u c r o s e d u rin g a c i d h y d r o l y s i s .

However, th©

c h a n g e s i n t h i s f r a c t i o n p r o b a b ly p a r a l l e l th o s e i n t h e

10

ta b le I H a te s o f H em o ly sis i n Human Blood s t o r e d a t 2° 0 . i n V a r io u s P r e s e r v a t i v e M ix tu re s V a lu e s E x p re s s e d a s P e r c e n t a g e o f E r y t h r o c y t e s Hemolysed E x p e rim e n t No* 1A

SOAl, AS

SGBl, B2

10

10

23

25

2

2

2

5

13

-

-

3

I jI . S

1 2 *5 :1

3 .1 :1

I n i t i a l G o n centra* t i o n o f Sodium C i t r a t e ( p e r c e n t ) 0 .2 5

0*25

0 .2 6

0 ,5 3

I n i t i a l C o n c e n tra ­ t i o n o f G lu co se ( p e r c e n t)

0*029

0 .0 5

0*58

B lo od M ix tu re (by volum e) Blood Sodium C i t r a t e 3*2% s o l u t i o n G lu co se 5#4% so lu tio n S u c r o s e 1 0 .3 # so lu tio n D i l u t i o n o f Blood

20C1, 02

20 d1 , 02

13 1 :1 * 5

3*18

P e r C ent o f C e l l s Hemolysed Days o f s t o r a g e 0

0*00 0,00

0*00 0*00

0,00 0.00

0,00 0.00

10 20 30

0 .1 0 0 .1 2 0 .1 3 0 ,4 8 0 .3 1 0 ,5 6

0 ,5 5 0 .5 1 1 .3 1 1 .6 5 1 .3 8 1 .5 4

0 .5 0 0 ,2 3 1 ,9 9 1 .3 7 4 ,9 2 2 .9 4

0 .1 7 0 .1 2 0 .3 9 0 . 4 8 0 .6 3 0*80

* Th© d o u b le l e t t e r s r e f e r t o t h e f a c t t h a t t h e e x p e rim e n t was done i n d u p l i c a t e .

IX

T a b le IX G lu co se C o n te n ts o f Blood M ix tu r e s a t V a r io u s I n t e r v a l s o f S t o r a g e ; A ls o , L a c t i c Acid I n c r e a s e a f t e r 30 Days o f s t o r a g e

Blood M ix tu re

G lu co se C o n c e n t r a t i o n 1*' (mg . *) 0 Days 10 Days 20 Days 50 Days

4 7 .5 20Bl (S u c ro s e ) 2001 ( C i t r a t e ) 9 2 .8 20A1 ( 3 , 0 $ 2930 G lu co se) 611 20Dl ( 0 , 6 ^ G lu co se)

L a c t i c A cid In c r e a s e (mg. fJ) A f t e r 30 Days

3 .2 1 7 .3 2980

3 7 2980

0 0 2980

6 5 .3 1 0 2 .0 1 0 0 .0

542

492

455

17Q.0

* G luco se d e te rm in e d v o l u m e t r l e a l l y by t h e S h a f f e r and Somogyi method (22) on a S o aog yi f i l t r a t e (27)

12 F ig u re 1 Changes ^ ^ f ^ p h o r u e F r a c t i o n s C u r in g S t o r a g e i u V arious P r e s e r v a t i v e M ix tu r e s INORGANIC

PHOSPHORUS

140

-

0.6 % GLUCOSE

0

20

0RGAHIG A C ID -S O L U B L E

PHOSPHORUS

PY RO PH O SPH A TE

"

.20

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30

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4.0

15

I DAYS

20

0

30

OF S T O R A G E

'E A S IL Y " HYOROLYSABLE

10

DAYS

20

OF

S T O RAG E

" DIFFICULTLY "MYDftOLYS ABLE

PHOSPHORUS

JO

PHOSPHORUS

!6.0 - 9

1C 0SE.

0

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SO

c i t r a t e b lo o d a s ju dg ed from th© more r a p i d d e c r e a s e i n t h e o r g a n i c a c i d - s o l u b l e and p y r o p h o s p h a te f r a c t i o n s i n t h i s b lo o d m ix tu r e th a n i n th e c i t r a t e m ix tu re * The i n o r g a n i c p h o sp h o ru s l i b e r a t e d re m a in s m o s tly i n th e c e l l s w here i t s c o n c e n t r a t i o n may r i s e a s much a s 30- t o 4 0 - f o ld * ma*

T h ere i s o n ly a slow le a k a g e i n t o th e p l a s ­

B a lp e r n (24) f i r s t showed t h a t a t low t e m p e r a tu r e s th e

c e l l membrane i s r e l a t i v e l y im perm eable to i n o r g a n i c p h o s­ p h a t e , b u t h e r e x p e rim e n ts were o f o n ly a few hours* d u ra ­ tio n *

Our e x p e r im e n ts d e m o n s tr a te t h a t so l o n g a s i t r e ­

m ain s i n t a c t and i n th e c o l d , th e c e l l m a i n t a i n s i t s im per­ m e a b i l i t y to i n o r g a n i c p h o s p h a te f o r l o n g p e r i o d s o f time# i n c o n t r a s t to t h i s i s th e b e h a v io r o f th e phos­ p h o ru s compounds i n th e g l u c o s e - c i t r a t e b lo o d s*

T here i s

a much slo w e r r i s e i n c e i l i n o r g a n i c p h o s p h a te due to th e more g r a d u a l breakdown o f c e l l o r g a n i c a c i d - s o l u b l e p h o s­ p ho rus*

R h e re as o n ly a b o u t o n e - t h i r d o f the o r g a n i c a c i d -

s o l u b l e p h o sp h o ru s i s b ro k e n down in th e two g lu c o s e b lo o d s o v e r t h r e e - f o u r t h s o f t h e s e compounds a r e s p l i t i n th e s u c r o s e and c i t r a t e bloods*

L ess s p l i t t i n g o c c u rs i n th e

" d i f f i c u l t l y ” h y d r o ly s a b l e f r a c t i o n th a n i n th e " e a s i l y ” h y d ro ly sa b le fra c tio n *

The fo rm e r f r a c t i o n c o n s i s t s l a r g e ­

l y o f p h o s p h o g ly c e r ic a c i d which Hay and R obison (25) con­ s i d e r r e l a t i v e l y r e s i s t a n t to p h o s p h a ta s e a c t i v i t y * No s i g n i f i c a n t d i f f e r e n c e s a p p e a r i n th e g ly c o ­ l y t i c r a t e s o f th e v a r i o u s b lo od m ix tu r e s ( T a b le XXI)*

14

T a b le I I I G l y c o l y t i c R a te s o f Blood M ix tu r e s f o r V a rio u s I n te r v a ls o f S to ra g e The V a lu e s Are Given a s mg* % I n c r e a s e i n L a c t i c A cid p e r 15 gm. o f H emoglobin

Blood M ix tu re

G l y c o l y t i c R ate 10 Days 20 Days 30 Days

2QB1 S0C1 2QA1 gODl

(S u cro se) (C itra te ) ( 3 . 0 ^ G lucose) ( 0 . 6 $ G lu co se)

1 0 0 .0 77*0 8 7 .3 8 8 .0

54*3 3 8 .6 6 4 .0 67*7

0 0 51*8 6 6 .0

15

F o r th e f i r s t t e n days o f s t o r a g e , th e r a t e o f g l y c o l y ­ s i s i s a p p r o x im a te l y e q u a l i n a l l b lo o d s d e s p i t e th e w ide d i f f e r e n c e i n g lu c o s e c o n c e n t r a t i o n in th® m i x t u r e s , and t h e c o n s i d e r a b l e h e m o ly s is which o c c u r r e d i n th e c i t r a t e and s u c r o s e b loods* R e l a t i o n Between G lu co se C o n c e n tr a t io n and P r e s e r v a t i o n S in c e i t a p p e a r e d from th e p r e c e d i n g e x p e rim e n t t h a t d i l u t i o n and th e i n i t i a l g lu c o s e c o n c e n t r a t i o n m ig h t b o th be f a c t o r s i n f l u e n c i n g the p r o g r e s s of h e m o ly s is , th e e f f e c t o f v a r i a t i o n s in c o n c e n t r a t i o n o f g lu c o s e a t a con­ s t a n t d i l u t i o n o f b lo o d was n e x t i n v e s t i g a t e d . The o b s e r v a t i o n s a r e shown in T a b le IV#

The modi­

f i e d R o u s -T u rn e r m ix tu r e (SIB) was a l s o u s e d a s a b a s i s f o r com parison*

E x c e p tin g th© R o u s-T u rn er m i x t u r e , th e d e g re e

o f h e m o ly s is i n th e v a r i o u s b lo o d m i x t u r e s i s a b o u t th e same d e s p i t e th e wide v a r i a n c e i n the g lu c o s e c o n c e n tr a ­ t i o n s used*

I n th e m o d if ie d R o u s-T u rn er m i x t u r e th e amount

o f h e m o ly s is i s n e g l i g i b l e ; s i n c e i t s c o n c e n t r a t i o n o f g lu c o s e (3*12 p e r c e n t) i s p r a c t i c a l l y e q u a l to t h a t o f b lo o d 21F (3*39 p e r c e n t) th e im p o r ta n t e f f e c t o f d i l u t i o n o f the b lo o d on h e m o ly s is becomes a p p a r e n t*

whether th e

f a c t o r o f d i l u t i o n i s s o l e l y a c c o u n ta b le f o r th e d i f f e r ­ e n c e s i s , h ow ever, open to some q u e s t i o n . a p p e a r i n t h e above e x p e rim en ts#

Two v a r i a b l e s

The c o n c e n t r a t i o n of

c i t r a t e i© o n e - h a l f a s g r e a t * i n th e R o u s-T u rn er m ix tu r e

T a b le XV H em olysis In B l o o d - D e x t r o s e - C i t r a t e M ix tu re s A f t e r S to r a g e f o r 30 Days a t 2° C. D i l u t i o n o f Blood Was H eld C o n s ta n t W hile C o n c e n t r a t i o n o f D e x tro s e Was V a r ie d 21A 2lB Blood M ix tu re (by volume) B lo od 8*5 10 Sodium C i t r a t e 1 .7 2 D e x tr o s e 3.3% 1 D e x tr o s e 5.4% 13 D e x tro se 7.0% -i D e x tro s e 11•0% D e x tr o s e 22.0% D e x tr o s e 33*0% D e x tr o s e 44.0% D ilu tio n o f Blood

21C

21D

21K

2lF

210

8 .3 1 .7

8*3 1 .7

8 .3 1 .7

8 .3 1 .7

8 .3 1 .7

1 -

•» 1 -

* 1 -

1 1

3 , 1 : 1 1 : 2 . 5 3 . 1 : 1 3 . 1 : 1 3 . 1 : 1 3 . 1 : 1 3 . 1:1

C o n c e n tra tio n o f Sodium C i­ tr a te (per c e n t)

0 .4 9

0 .2 3

0 .4 9

0 .4 9

0 .4 9

0 .4 9 i0 . 4 9

C o n c e n tr a t io n o f G lu co se (p e r c e n t)

0 .5 8

5 .1 2

0 .7 6

1 .2 0

2 ,2 4

3 .5 9 -£.51

P e r C ent o f C e l l s Hemo­ l y s e d in 30 Days

0 .5 3

0 .0 3

0 .4 0

0 .4 0

0 .3 2

0 .4 0