The renal clearance of hemoglobin in the dog

Citation preview

THE HEMAL CLEARANCE OF HEMOGLOBIN IN THE DOG

by J . VICTOR MGNKE 4# •

T h e s i s s u b m itte d t o t h e o f th e U n iv e rs ity fu lfillm e n t o f th e d e g re e o f

F a c u l t y o f t h e G ra d u a te S o h o o l o f M a ry la n d i n p a r t i a l t h e r e q u i r e m e n ts f o r D o c to r o f P h ilo s o p h y 1942

UMI Number: DP70173

All rights reserved INFORM ATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

UMI Dissertation Publishing

UMI DP70173 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code

ProQuest ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

ii

TABLE OF CONTENTS IN T R O D U C T IO N .................................................................................................................... 1 M E T H O D S ...................................................................................................................

2

EXPERIMENTAL OBSERVATIONS

6

.

D ISC U SSIO N ........................................................................................................................... 19 SUMMARY AND C O N C L U S I O N S ..................................................................................... 25 LITERATURE C I T E D ........................................................................................................ 27

96916

LIST OF TABLES TABLE I : TABLE l i t TABLE I l l s TABLE IV s TABLE V:

Summary o f E x p e rim e n t 8 Summary o f E x p e rim e n t 9

.

.

.

.

. •

Summary o f E x p e rim e n t 1 0

•

Summary o f A l l E x p e r im e n ts

.

.

•

.

•

. •

• .

.

.

Summary o f D e riv e d E x p e r im e n ta l C o n s ta n ts •

.

8

.

.

9

•

•

10

•

.

15

•

«

22

iv LIST OF FIGURES

FIGURE 1

..................................................................................................................................... 11

FIGURE 2

.....................................................................................................................................13

FIGURE 3

.....................................................................................................................................17

INTRODUCTION I n r e c e n t y e a r s , t h e u s e o f q u a n t i t a t i v e m e th o d s ( 2 4 , 2 2 ) i n t h e s tu d y o f r e n a l p h y s io lo g y h a s g r e a t l y e n la rg e d o u r u n d e rs ta n d in g o f t h e m ech an ism b y -which m o le c u le s s m a l l e r t h a n i n u l i n

(a p p a re n t

m o l e c u l a r -w e ig h t, 1 5 ,0 0 0 ( 3 ) ) a r e e l i m i n a t e d fro m t h e k id n e y *

T h ese

m e th o d s h a v e n o t a s y e t b e e n f u l l y a p p l i e d ( 7 ) t o a s t u d y o f t h e r e n a l e x c r e t i o n o f a m o le c u le a s l a r g e a s t h a t o f h e m o g lo b in (m o le c ­ u l a r w e i g h t , 6 8 ,8 0 0 )*

The n e e d f o r s u c h a q u a n t i t a t i v e s tu d y i s

i n d i c a t e d b y t h e d i s c r e p a n c y w h ic h e x i s t s b e tw e e n t h e m o dern c o n c e p ts o f r e n a l f u n o t i o n , w h ic h m a i n t a i n a s a m a jo r p r e m is e t h a t t h e g lo m e r u la r m em brane i s im p e rm e a b le t o c o l l o i d a l m o l e c u l e s , an d t h e f a c t , w e l l known c l i n i c a l l y a n d e x p e r i m e n t a l l y , t h a t h e m o g lo b in u r ia o c c u r s w hen f r e e h e m o g lo b in i s p r e s e n t i n t h e b lo o d s tre a m * The d a t a t o b e p r e s e n t e d a r e c o n c e rn e d w i t h a s t u d y o f t h e r e n a l e l i m i n a t i o n o f h e m o g lo b in i n t h e d o g , a n d w e re o b t a i n e d b y c o m p a rin g t h e e x c r e t i o n o f h e m o g lo b in w i t h t h e s im u lta n e o u s e x c r e t i o n o f c re a tin in e *

S in c e i n t h e d o g t h e r a t e o f e x c r e t i o n o f c r e a t i n i n e

i s known t o b e a m e a s u re o f t h e r a t e o f g lo m e r u la r f i l t r a t i o n

(2 3 ),

a d e t e r m i n a t i o n o f i t s e x c r e t i o n r a t e may b e u s e d a s a b a s i s o f c o m p a ris o n i n o b t a i n i n g i n f o r m a t i o n c o n c e r n i n g t h e e x c r e t i o n p a t t e r n o f a second s u b s ta n c e • I t i s g e n e r a lly conceded in th e l i t e r a t u r e

(1 2 , 1 3 , 26) t h a t

h e m o g lo b in e s c a p e s i n t o t h e u r i n e v i a t h e g lo m e r u lu s , a l t h o u g h t h e m a n n er i n w h ic h i t d o e s so i s b y no m eans c l e a r *

Some a u t h o r s

a c c e p t t h e i d e a o f a t r a n s i e n t i n j u r y t o t h e g lo m e r u la r m embrane

2. ( 4 , 1 ) b u t m any p h y s i o l o g i c a l an d h i s t o l o g i c a l s t u d i e s g iv e n o e v id e n c e o f su ch i n j u r y (5 )*

W h ip p le a n d h i s c o w o rk e rs (1 4 ) h a v e im p lie d t h a t

a d y n am ic g lo m e r u la r m ech an ism i s r e s p o n s i b l e f o r t h e r e n a l t h r e s h o l d o f h e m o g lo b in n o te d b y th em a n d o t h e r s w hose w o rk t h e y q u o te *

The

p o s s i b i l i t y o f a d i f f e r e n t e x p l a n a t i o n f o r t h e phenom enon may b e draw n fro m t h e w o rk o f R ic h a r d s an d W a lk e r (2 0 ) who p o s t u l a t e d t h e e x i s t e n c e o f o c c a s i o n a l i r r e g u l a r d e f e c t s i n t h e g lo m e r u la r m em brane l a r g e e n o u g h t o a llo w t h e p a s s a g e o f w h o le p lasm a* A c o n tro v e rs y h a s e x is te d re g a rd in g th e r o le o f th e tu b u le s in t h e e x c r e t i o n o f h e m o g lo b in *

To many o b s e r v e r s t h e p r e s e n c e o f hemo­

g l o b i n w i t h i n t h e c e l l s o f t h e c o n v o lu te d t u b u l e s h a s i n d i c a t e d a phenom enon o f t u b u l a r e x c r e t i o n *

H o w ev er, t h e a l t e r n a t i v e , t h a t t h e

phenom enon i s e n t i r e l y o n e o f t u b u l a r r e c o v e r y , i s s u b s t a n t i a t e d by t h e r e c e n t o b s e r v a t i o n s o f L is o n (1 5 ) who d e s c r i b e s t h e im p e r m e a b i lit y o f t h e b a s a l m em brane o f t h e t u b u l a r c e l l s t o a n y l a r g e n e g a t i v e l y ch arg ed c o llo id s *

The a c t u a l m ech an ism o f r e c o v e r y i s s a i d by G e ra rd

(1 0 ) t o be one o f a t h r o c y t o s i s i n w h ic h t h e a p i c a l m em branes o f t h e t u b u l a r c e l l s p i c k u p e o l l o i d a l p a r t i c l e s fro m t h e t u b u l a r lu m in a an d s t o r e th e m i n m uch t h e same m an n er a s t h e r e t i c u l o e n d o t h e l i a l c e l l s p h a g o c y to s e a n d s t o r e p a r t i c u l a t e m a t t e r e ls e w h e r e i n t h e body* METHODS F em ale m o n g re l d o g s , t r a i n e d t o l i e q u i e t l y on a b o a rd w h il e b e in g c a t h e t e r ! z e d , w e re u s e d i n e a c h e x p e rim e n t* A p p ro x im a te ly 10 p e r c e n t s o l u t i o n s o f h e m o g lo b in w e re p r e p a r e d fro m t h e b lo o d o f c o lo n y d o g s t o w h ic h w as a d d ed 1 cc* o f s a t u r a t e d so d iu m c i t r a t e p e r 100 co*

The r e d c e l l s w e re s e p a r a t e d fro m t h e p la sm a

b y c e n t r i f u g a t i o n an d w a sh e d tw ic e w i t h i s o t o n i c s a l i n e s o l u t i o n #

The

p a c k e d r e d c e l l s o b t a i n e d a f t e r t h e f i n a l w a s h in g w e re s u sp e n d e d i n h a l f t h e i r volum e o f d i s t i l l e d w a t e r an d t h e n la k e d b y t h e a d d i t i o n o f o n e - t h i r d t h e i r volum e o f a n e s t h e s i a e t h e r #

A f t e r s h a k in g t h e m ix ­

t u r e v i g o r o u s l y s e v e r a l t i m e s d u r i n g a p e r i o d o f 15 m i n u t e s , t h e s u s ­ p e n s io n w as c e n t r i f u g e d #

The c l e a r b r i g h t r e d s u p e r n a t a n t s o l u t i o n

so o b t a i n e d w as f i l t e r e d t h r o u g h W hatman Ho# 50 f i l t e r p a p e r an d s t o r e d o v e r n i g h t a t 2° C .

J u s t b e f o r e i n j e c t i o n , t h e s o l u t i o n w as f i l t e r e d

a g a i n , an d i t s h e m o g lo b in c o n c e n t r a t i o n d e te r m in e d #

The am ount o f

s o l u t i o n i n j e c t e d d e p e n d e d u p o n t h e i n i t i a l p la sm a c o n c e n t r a t i o n d e s i r e d # I n j e c t i o n s w e re made i n t o t h e j u g u l a r v e i n a t a r a t e n o t e x c e e d in g 20 cc# p e r m in u te # An a d e q u a te d i u r e s i s w as o b ta i n e d b y g i v i n g t h e a n im a ls 3 0 0 t o 500 c c # o f t a p w a t e r th r o u g h a sto m a ch tu b e on t h e e v e n in g o f t h e d a y p r i o r t o t h e e x p e r im e n t , and a t i n t e r v a l s o f 90 t o 1 2 0 m in u te s b e f o r e a n d d u r i n g t h e e x p e r im e n t#

I n o rd e r t o m a in ta in an a l k a l i n e u r in e and

t h u s t o a v o id t h e f o r m a ti o n o f h e m o g lo b in c a s t s w h ic h l e a d t o r e n a l o b s t r u c t i o n i n a c i d u r i n e , 2 t o 6 gm# o f sodium b i c a r b o n a t e w e re a d d ed t o e a c h d o s e o f w a te r # A 10 p e r c e n t a q u e o u s s o l u t i o n o f c r e a t i n i n e w as i n j e c t e d , i n p a r t s u b c u ta n e o u s ly a n d i n p a r t i n t r a p e r i t o n e a l l y , i n a m o u n ts s u f f i ­ c i e n t t o p ro d u c e a n i n i t i a l p la sm a c o n c e n t r a t i o n o f 10 t o 15 mg# p e r 100 cc# o f p la sm a # One h o u r a f t e r t h e i n j e c t i o n o f c r e a t i n i n e , t h e a n i m a l s b l a d d e r w as w ash ed o u t w i t h warm i s o t o n i c s a l i n e s o l u t i o n #

A t t h e end o f e a c h

2 0 -m in u te p e r i o d t h e r e a f t e r , u r i n e s a m p le s w e re c o l l e c t e d th r o u g h a c u rv e d m e ta l c a t h e t e r i n t o a 20 cc# s y r in g e #

A t each c o lle c tio n , th e

4. volum e o f u r i n e w as n o te d a n d t h e b l a d d e r f l u s h e d w i t h t h r e e 14 o c . p o r t i o n s o f warm i s o t o n i o s a l i n e .

The u r i n e a n d s a l i n e w e re c o l l e c t e d

i n a v o lu m e tr io f l a s k a n d made u p t o a d e f i n i t e v o lu m e w i t h w a t e r . 15 o c . a l i q u o t s o f t h e d i l u t e d u r i n e w e re c e n t r i f u g e d i n s to p p e r e d t u b e s t o f r e e t h e s a m p le s o f a n y r e d c e l l s o r m u c u s. The h e m o g lo b in s o l u t i o n w as i n j e c t e d th ir d p e rio d .

a t t h e end o f t h e se c o n d o r

The d a t a o b t a i n e d d u r i n g t h e f i r s t p e r i o d s w e re u s e f u l

i n d e t e m i n i n g w h e th e r t h e i n j e c t i o n o f h e m o g lo b in h a d p ro d u c e d a n y n o t i c e a b l e c h a n g e i n t h e n o rm a l e x c r e t i o n o f c r e a t i n i n e .

The f i r s t

u r i n e p e r i o d c o n t a i n i n g h e m o g lo b in w as b e g u n 1 0 m in u te s a f t e r t h e end o f th e in j e c tio n .

I f t h e a n im a l u r i n a t e d s p o n ta n e o u s ly a t a n y tim e

d u r i n g t h e e x p e r im e n t , t h e b l a d d e r w as im m e d ia te ly f l u s h e d w i t h s a l i n e , euad a new p e r i o d w as b e g u n .

The e x p e r im e n ts w e re c o n tin u e d u n t i l g r o s s

h e m o g lo b in u r ia c o u ld no l o n g e r b e d e t e o t e d . 8 c c . s a m p le s o f b lo o d w e re t a k e n b e f o r e t h e f i r s t p e r i o d , a t t h e m id p o in t o f a l t e r n a t e p e r i o d s , a n d a f t e r t h e l a s t p e r i o d .

One sam p le

w as t a k e n j u s t p r i o r t o t h e i n j e c t i o n o f h e m o g lo b in an d a n o t h e r 5 m in u te s a f t e r t h e i n j e c t i o n .

The s a m p le s

w e re a d d e d t o

s a t u r a t e d sodium c i t r a t e a n d c e n t r i f u g e d . A f t e r n o t i n g

0 .1 o c . o f th e h e m a to c rit,

t h e p la sm a w as im m e d ia te ly t r a n s f e r r e d t o a n o t h e r v e s s e l .

P ro te in -fre e

f i l t r a t e s f o r t h e d e t e r m i n a t i o n o f t o t a l c h ro m o g e n ic m a t e r i a l i n t h e a n a l y s i s o f c r e a t i n i n e w e re p r e p a r e d by a d d in g 1 c c . o f p la sm a t o 1 2 c c . o f H a l a 's r e a g e n t ( 1 1 ) .

2 o c . o f p la sm a w e re a d d ed t o

12 cc*

o f M a la * s r e a g e n t f o r t h e d e t e r m i n a t i o n o f t o t a l n o n - c r e a t i n i n e chrom o­ g e n ic m a t e r i a l .

The p r o t e i n p r e c i p i t a t e s w e re s e p a r a t e d by c e n t r i f u g a ­

t i o n an d a l i q u o t p o r t i o n s o f t h e s u p e r n a t a n t f l u i d w e re u s e d f o r a n a ly s is .

P la s m a a n d u r i n e c r e a t i n i n e d e t e r m i n a t i o n s w e re c a r r i e d

5. o u t a c c o r d i n g t o t h e m e th o d o f S h a n n o n , J o l l i f e , a n d S m ith (2 3 )* The h e m o g lo b in c o n c e n t r a t i o n s i n b o t h p la sm a a n d u r i n e w e re d e ­ t e r m in e d e o l o r i m e t r i c a l l y a f t e r c o n v e r t i n g t h e h e m o g lo b in t o o y a n m e th e m o g lo b in a s d i r e c t e d b y E v e ly n a n d S a l t e r ( 8 ) * A l l c o l o r i m e t r i c m e a s u re m e n ts w e re m ade on t h e E v e ly n p h o t o e l e c t r i c c o lo rim e te r.

C are w as t a k e n t o i n s u r e t h a t a p p r o p r i a t e b l a n k s w e re

u sed in e a c h c o lo r im e tr ic d e te rm in a tio n * C r e a t i n i n e an d h e m o g lo b in p h o t o c o l o r i m e t r i c c a l i b r a t i o n c u r v e s r e l a t i n g t h e c o n c e n t r a t i o n o f t h e s e s u b s ta n c e s t o g a lv a n o m e te r d e f l e c ­ t i o n w e re c o n s t r u c t e d w i t h t h e u s e o f c a r e f u l l y p r e p a r e d s t a n d a r d s o lu tio n s *

The c r e a t i n i n e s t a n d a r d w as p r e p a r e d b y t h e c r e a t i n i n e z i n c

c h l o r i d e m e th o d ( 9 ) , a n d t h e h e m o g lo b in s t a n d a r d b y t h e s p e c t r o p h o t o m e t r i c m eth o d o f D ra b k in an d A u s t i n ( 6 ) . V a lu e s f o r t h e p la s m a c o n c e n t r a t i o n s o f b o th c r e a t i n i n e an d hemo­ g l o b i n w e re p l o t t e d a g a i n s t t h e tim e o f w ith d r a w in g t h e r e s p e c t i v e b lo o d s a m p le s fro m t h e a n im a l*

By i n t e r p o l a t i o n fro m t h i s g r a p h , a

v a lu e w as o b t a i n e d f o r t h e p la sm a c o n c e n t r a t i o n o f e a c h s u b s ta n c e a t t h e m id p o in t o f e a c h p e r i o d , w h ic h w as t a k e n a s t h e a v e r a g e c o n c e n t r a ­ t i o n o f t h e s u b s ta n c e f o r t h a t p a r t i c u l a r p e r io d *

The u r i n a r y

e x c r e t i o n o f b o t h h e m o g lo b in an d c r e a t i n i n e i n e a c h u r i n e sam p le w as e x p r e s s e d a s mg* e x c r e t e d p e r m in u te an d g ra p h s w e re made r e l a t i n g t h e r a t e o f e x c r e t i o n o f e a c h s u b s ta n c e t o t h e p la sm a c o n c e n t r a t i o n d u r i n g t h e same p e r i o d .

The v o lu m e s o f p la sm a c l e a r e d o f c r e a t i n i n e

a n d h e m o g lo b in p e r m in u te d u r i n g e a c h p e r i o d w e re c a l c u l a t e d b y d i v i d ­ i n g t h e mg* o f e a c h s u b s ta n c e e x c r e t e d p e r m in u te b y t h e c o r r e s p o n d i n g p l a a a a c o n c e n t r a t i o n i n te r m s o f mg* p e r 1 0 0 c c * , an d m u l t i p l y i n g b y 100.

F i n a l l y , t h e a b s o l u t e c l e a r a n c e o f h e m o g lo b in and t h e h e m o g lo b in /

6* c r e a t i n i n e c l e a r a n c e r a t i o w e re e a c h p l o t t e d a g a i n s t t h e h e m o g lo b in p la sm a c o n c e n t r a t i o n * EXPERIMENTAL OBSERVATIONS A t o t a l o f s e v e n te e n i n d i v i d u a l e x p e r im e n ts w e re c a r r i e d o u t on f i v e a n im a ls *

I n i t i a l p la sm a c o n c e n t r a t i o n s o f h e m o g lo b in , f o l l o w i n g

i t s i n j e c t i o n , ra n g e d fro m 200 t o 1 3 0 0 mg* p e r 1 0 0 cc* o f p la sm a * The c o n d i t i o n o f t h e a n im a ls d u r i n g t h e c o u r s e o f e a c h e x p e r im e n t re m a in e d n o rm a l e x c e p t i n e x p e r im e n ts 13 an d 1 7 , i n b o th o f w h ic h t h e a n im a ls becam e r a t h e r l i s t l e s s f o l l o w i n g t h e i n j e c t i o n o f h e m o g lo b in , and f o r s e v e r a l h o u r s t h e r e a f t e r showed a n e l e v a t i o n o f t e m p e r a t u r e t o ab o u t 42° C.

I t i s p o s s i b l e t h a t t h e h e m o g lo b in s o l u t i o n i n j e c t e d i n

t h e s e i n s t a n c e s c o n ta i n e d some r e d c e l l stro m a * I n v ie w o f c o m p le te r e p o r t s on t h e h i s t o l o g i c a l f i n d i n g s i n d o g s w h ic h h a d r e c e i v e d m u l t i p l e i n j e c t i o n s o f h e m o g lo b in ( 5 , 1 7 ) , i t w as n o t deem ed n e c e s s a r y t o a u to p s y a l l o f t h e a n im a ls s t u d i e d i n t h e s e e x p e r im e n ts *

H ow ever, dog 3 8 -2 8 4 , w h ic h had r e c e i v e d a t o t a l o f

13*67 gm. o f h e m o g lo b in i n t h r e e s e p a r a t e i n j e c t i o n s , w as k i l l e d 15 d a y s a f t e r t h e l a s t i n j e c t i o n a n d a u t o p s i e d im m e d ia te ly * a b n o r m a l i t i e s w e re fo u n d i n a n y o rg a n *

No g r o s s

M o d e ra te d e p o s i t s o f i r o n -

c o n t a i n i n g p i g n e n t w e re o b s e r v e d h i s t o l o g i c a l l y i n t h e l i v e r an d s p le e n * H e m a to x y lin and e o s i n s t a i n s o f t h e k i d n e y showed no p a t h o l o g i c a l c h a n g e s , a n d s p e c i a l s t a i n i n g o f t h e b a s e m e n t m em branes o f t h e g lo m e r­ u l a r c a p i l l a r i e s re v e a le d no a b n o rm a litie s o f th e s e s tr u c tu r e s *

No

i r o n - c o n t a i n i n g p ig m e n t w as fo u n d i n t h e e p i t h e l i u m o f a n y o f t h e tu b u le s *

The a b s e n c e o f s u c h p ig n e n t may be r e l a t e d t o a m o d e ra te

d e g r e e o f a n e m ia p ro d u c e d i n t h e a n im a l b y t h e f r e q u e n t w ith d r a w a l o f b lo o d s a m p le s*

7* I n T a b le s I , I I , a n d I I I , an d i n F i g . 1 , a r e r e c o r d e d t h e d e t a i l e d d a t a o b t a i n e d i n t h r e e c h a r a c t e r i s t i c e x p e r im e n ts i n w h ic h t h e i n i t i a l p la s m a c o n c e n t r a t i o n o f h e m o g lo b in w as r e l a t i v e l y lo w (1 9 0 m g. p e r c e n t ) , m edium (4 5 0 mg* p e r c e n t ) , a n d h i g h (1 3 2 0 m g. p e r c e n t ) . The d a t a o n t h e e x c r e t i o n o f c r e a t i n i n e i n t h e s e e x p e r im e n ts a r e a n a ly z e d i n c o lu m n s 3 a n d 4 o f t h e t a b l e s an d i n t h e g r a p h s o f s e r i e s B in F ig . 1 .

I n t h e g r a p h s i t w i l l b e n o te d t h a t t h e r e l a t i o n s h i p

b e tw e e n t h e p la s m a c o n c e n t r a t i o n o f c r e a t i n i n e a n d t h e r a t e o f i t s u r i n a r y e x c r e t i o n p e r m in u te i s a l i n e a r o n e*

The o r i g i n o f t h i s

g ra p h i s a t z e r o , i n d i c a t i n g t h a t t h e e x c r e t i o n o f c r e a t i n i n e i s r e * l a t e d o n l y t o t h e r a t e o f g lo m e r u la r f i l t r a t i o n an d i s n o t m o d if ie d b y any tu b u la r a c tiv ity *

The volum e o f p la s m a , c o n t a i n i n g t h e num ber o f

mg* o f c r e a t i n i n e e x c r e t e d p e r m i n u te , d e s i g n a t e s t h e volum e o f p la s m a w a t e r f i l t e r e d th r o u g h t h e g lo m e ru lu s d u r i n g e a c h m i n u t e .

S u ch

c l e a r a n c e v a l u e s a r e l i s t e d f o r e a c h p e r i o d i n colum n 5 o f t h e t a b l e s . V a r i a t i o n s i n t h e r a t e o f c r e a t i n i n e c l e a r a n c e o b s e r v e d fro m p e r i o d t o p e r i o d a r e a t t r i b u t e d t o c h a n g e s i n g lo m e r u la r f i l t r a t i o n p r e s s u r e an d a r e b e l i e v e d t o b e s y s te m ic i n o r i g i n * S i m i l a r t r e a t m e n t o f t h e d a t a on t h e p la sm a c o n c e n t r a t i o n a n d t h e r a t e o f e x c r e t i o n o f h e m o g lo b in i s

shown i n co lu m n s 6 an d 7 o f

t h e f i r s t t h r e e t a b l e s an d i n t h e g r a p h s o f s e r i e s A i n F i g . 1 . R e f e r e n c e t o t h e g ra p h s w i l l show t h a t i n e a c h e x p e r im e n t t h e r e w as a d e f i n i t e p la s m a c o n c e n t r a t i o n o f h e m o g lo b in b e lo w w h ic h n o n e e s c a p e d fro m t h e k i d n e y .

T h is c r i t i c a l v a lu e o f p la s m a c o n c e n t r a t i o n i s d e s i g ­

n a t e d a s t h e r e n a l t h r e s h o l d o f h e m o g lo b in an d a b o v e t h i s l e v e l t h e r a t e o f h e m o g lo b in e x c r e t i o n w as p r o p o r t i o n a l t o t h e p la sm a c o n c e n t r a ­ tio n .

S in c e t h e e s t a b l i s h m e n t o f t h e s e f a c t s i s i m p o r t a n t , a l l o f

TABLE I

Summary o f E xperim ent 8 . P e rio d

1

1

L ength of p e rio d

Plasm a

U rin e

C learan o e

2

3

4

5

mg ./m in .

o o ./m in .

Dog 3 8 -2 2 9 .

C r e a tin in e

m in .

mg./lO O

1 5 .7 5

8 .8 0

7.61

W eights 1 2 .0 K ilo s Hemoglobin

8 6 .4

Plasm a 6 m g./lOO --

U rin e

C le a ra n c e

H b ./C r. c le a r a n c e ra tio

7

8

9

m g ./m in .

c o ./m in

—

- -

U rin e flo w

10 c o ./m in .

—

0 .7 6

1 .6 6 gm» o f hem oglobin d is s o lv e d i n 45 c c . w a te r i n j e c t e d in tr a v e n o u s ly (138 mg . / k g . ) 2

26.25

7 .4 0

5 .6 0

7 5 .5

—

—

3

23.25

7.18

4 .8 3

67.3

148

2 .1 5

4

1 6 .2 5

6 .4 0

3 .8 7

6 0.5

112

5

26.25

5 .0 0

3 .7 8

7 5 .5

6

23.75

3 .6 8

2 .9 9

7

22.25

3 .2 0

3 .1 4

—

0 .4 6

1 .4 5

0 .0216

0 .9 5

1 .0 1

0 .9 0

0.0149

1*26

92

0 .5 6

0 .6 1

0 .0 0 8 0

2 .8 8

8 1 .3

81

0 .4 0

0 .5 0

0.0061

2 .9 1

9 8 .0

78

0 .0 6

0 .0 8

0.0008

2 .9 4

mm mm

00

«

TABLE II

Summary o f E x p erim en t 9 .

1

1 2 3

Dog 3 8 -2 2 9 .

C r e a tin in e

W eight: 11*3 K ilo s U rin e flo w

C learan o e

H b ./C r. c le a ra n o e ra tio

7

8

9

10

mg •/m in .

c o ./m in .

Hemoglobin

L ength of p e rio d

Plasm a

U rin e

C learan o e

Plasm a

U rin e

2

3

4

5

6

min*

mg ./L 0 0

mg ./m in .

o o ./m in .

mg . / l 00

22*25 24 .0 0 15.25

10 .8 7 9.15 8.4 7

7 .6 4 6 .6 3 5 .2 4

70 .3 7 7 .6 6 1 .8

c o ./m in .

M

— —

— —

—

—

1 .6 8 2 .2 9 2 .7 5

2*59 gm• o f hem oglobin d is s o lv e d i n 32 o c . w a te r i n j e c t e d in tr a v e n o u s ly (229 mg . / e g . ) 4 5

6 7 8 9 10 11 12 13 14

1 2 .0 0 21.75 2 1 .0 0 1 9 .5 0 1 7 ,0 0 22.75 22.25 21 .0 0 2 4 .0 0 27.75 2 2 .5 0

8 .7 5 8 .3 4 6 .6 0 5 .6 6 5 .1 8 4 .8 0 3 .7 5 3 .3 0 2 .8 8 2 .6 0 2 .3 0

6 .7 5 2 .8 8 3 .9 0 3 .7 6 3 .2 6 3 .0 4 2 .1 4 1 .9 1 1 .9 7 1 .7 4 1 .5 3

77.1 3 4 .6 5 9 .1 6 6 .4 6 3 .0 6 3 .4 57 .1 5 7 .8 6 8 .4 6 7 .0 66 .5

434 390 325 282 257 236 166 148 130 115 100

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

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

0 .0 2 8 9 0 .0 2 8 9 0 .0 2 5 4 0 .0 2 3 4 0 .0 2 2 4 0 .0 2 2 4 0 .0 1 1 3 0 .0 0 8 2 0 .0053 0 .0 0 3 8 0 .0 0 3 0

3 .7 5 2 .1 2 3 .1 9 4 .6 0 4 .5 6 4 .7 0 0 .9 0 0 .3 3 0 .4 2 4 .0 5 5 .6 0

to

TABLE I I I

Summary o f E xperim ent 1 0 . L ength of p e rio d

min 1

2

C r e a tin in e Plasm a

1 8 .5 0 26.75

m £ ./.0 0 15 .5 5 13.05

4

C le a ra n c e

Plasm a

U rin e

C le a ra n c e

H b ./C r. c le a ra n c e ra tio

5

6

7

8

9

m g./m in*

o c ./m in .

m g ./m in . o c ./m in . 9.05 6.1 7

W eig h t: 1 1 .2 K ilo s

hem oglobin

U rin e

3

1

Dog 3 8 -2 2 9 .

5 8 .2 4 7 .3

m g./lO O —

—

—

U rin e flo w

10 oc • / n i n .

*•