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UBC Theses and Dissertations

An investigation into the potential involvement of vasopressin in the regulation of amniotic fluid circulation Plath, Susan Marie 1976

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AN INVESTIGATION INTO THE POTENTIAL INVOLVEMENT OF VASOPRESSIN IN THE REGULATION OF AMNIOTIC FLUID CIRCULATION by SUSAN MARIE PLATH B.A., U n i v e r s i t y o f M i n n e s o t a , D u l u t h , 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f Z o o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 (3) Susan Marie P l a t h , 1976 In p r e s e n t i n g t h i s t h e s i s in p e r t i a l f u I f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Z o o l o g y  The U n i v e r s i t y o f B r i t i s h Co lumb ia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 30 Sep tember I976 i i .ABSTRACT The mechanisms which control the accumulation and c i r c u l a t i o n of the amniotic f l u i d are poorly understood. E a r l i e r studies had made the tentative suggestion that vasopressin, which i s important i n the control of water balance i n adults, might play a role i n the regulation of amniotic f l u i d turnover. In the work reported here, at-tempts were made to detect and measure the vasopressin l e v e l s . i n the amniotic f l u i d of guinea pigs, sheep and humans. Unconcentrated amniotic f l u i d samples from the guinea pig, sheep and human were assayed using the water-loaded, alcohol-anesthetized rat a n t i d i u r e t i c preparation. I f vasopressin i s present i n the f l u i d of these animals, i t would be at a concentration below the detectable l i m i t s of the assay, which were found to be 10 to 15 microunits/ml. Only 4 out of 36 experiments on guinea pig amniotic f l u i d gave even a.suggestioncof a response, with 3 of these being too small to be quantified accurately. No sheep or human samples produced an a n t i d i u r e s i s . An attempt was made to dehydrate the maternal guinea pigs, as i t . was thought t h a t . t h i s would create an osmotic stress i n the fetus which might r e s u l t i n increased output of vasopressin. In 29 experiments with f l u i d of fetuses whose mothers had been without water f o r 21+ or 1+8 hours p r i o r t o c o l l e c t i o n , 27 gave no r e s p o n s e . The r e s p o n s e s t o t h e t w o . i n j e c t i o n s t h a t d i d i n d i c a t e a c t i v i t y were t o o l o w . f o r a c c u r a t e q u a n t i f i c a t i o n . However, t h e s e e x p e r i -ments were n o t c o n s i d e r e d c o n c l u s i v e , a n d more e x t e n d e d i n v e s t i g a t i o n i s n e e d e d . S e v e n human a m n i o t i c f l u i d s a m p l e s were c o n c e n t r a t e d on CM-25 Sephadex columns, a l o n g w i t h t h r e e s t a n d a r d v a s o -p r e s s i n l o a d s . The s t a n d a r d s showed r e c o v e r i e s o f BQffo t o 90$ u n d e r o p t i m a l c o n d i t i o n s . An a n t i d i u r e t i c s u b s t a n c e was f o u n d i n f r a c t i o n s f r o m a l l o f t h e s e s a m p l e s , an d i t e l u t e d a t a s i m i l a r pH a n d m o l a r i t y t o t h e s t a n d a r d v a s o -p r e s s i n . T h i s m a t e r i a l m e a s u r e d a p p r o x i m a t e l y 200 m i c r o -u n i t s / m l , a n d was r e l a t i v e l y c o n s i s t e n t i n a l l o f t h e s a m p l e s t e s t e d . The a m n i o t i c f l u i d a n t i d i u r e t i c s u b s t a n c e (AFAS) a p -p e a r e d t o r e s p o n d w i t h a n a n t i d i u r e s i s s i m i l a r t o t h a t o f v a s o p r e s s i n on t h e a s s a y p r e p a r a t i o n . However, f u r t h e r work s u g g e s t e d t h a t t h e AFAS c o u l d n o t be i d e n t i f i e d a s v a s o p r e s s i n . A l t h o u g h t h e p a t t e r n o f a n t i d i u r e t i c r e -s p o n s e s were s i m i l a r , t h e r e were marked c h a n g e s i n t h e s e n s i t i v i t y o f t h e b i o a s s a y p r e p a r a t i o n t o t h e unknown m a t e r i a l d u r i n g p e r i o d s o f l i t t l e change i n t h e r e s p o n s e s t o v a s o p r e s s i n i t s e l f . A s i m i l a r c o n t r a s t was f o u n d b e -t w e e n d i f f e r e n t e x p e r i m e n t s . Sodium t h i o g l y c o l l a t e i n c u b a -t i o n a l s o f a i l e d t o d e a c t i v a t e t h e a c t i v e AFAS f r a c t i o n s , i v w h i l s t c o n t r o l v o l u m e s o f v a s o p r e s s i n l o s t a p p a r e n t l y a l l a c t i v i t y f o l l o w i n g t h i s p r o c e d u r e . The s t a b i l i t y c h a r a c -t e r i s t i c s were a l s o d i s s i m i l a r t o t h o s e o f v a s o p r e s s i n , a s t h e f r a c t i o n s r e m a i n e d a c t i v e f o r up t o f i v e weeks when s t o r e d a t n e u t r a l pH a t 4° C. Optimum pH c o n d i t i o n s f o r t h e s t o r a g e o f v a s o p r e s s i n a r e between 3.0 a n d 5.0. An a t t e m p t was made t o i d e n t i f y t h e AFAS a s a n g i o t e n -s i n I I . However, t h e r e s p o n s e p a t t e r n on t h e r e c o r d s o f t h e a n t i d i u r e t i c a s s a y were d i s s i m i l a r . F u r t h e r c o m p a r i s o n s w i t h c o m b i n a t i o n s o f v a s o p r e s s i n a n d a n g i o t e n s i n I I i n v a r y -i n g c o n c e n t r a t i o n s a l s o f a i l e d t o mimic t h e r e s p o n s e s t o t h e AFAS. I t was c o n c l u d e d t h a t t h e a n t i d i u r e t i c a c t i v i t y f o u n d i n t h e human a m n i o t i c f l u i d was n o t a t t r i b u t a b l e t o v a s o p r e s -s i n , a n g i o t e n s i n I I , o r t o a c o m b i n a t i o n o f t h e s e two h o r -mones, a t t h e l e v e l s t e s t e d . The n a t u r e o f t h e AFAS i s a t p r e s e n t unknown. F u r t h e r s t u d i e s a r e n e e d e d t o i d e n t i f y t h e unknown a n t i d i u r e t i c a g e n t e x t r a c t e d f r o m human a m n i o t i c f l u i d , a n d t h e p o s s i b l e mechanisms o f i t s r e l e a s e , a n d i t s p o t e n t i a l p h y s i o l o g i c a l f u n c t i o n s , a r e s t i l l unknown. S u p e r v i s o r V TABLE OF CONTENTS Page LIST OF TABLES i x LIST OF FIGURES x INTRODUCTION 1 A. The Amniotic F l u i d 1 1, Sources of the amniotic f l u i d ......... 2 2. C i r c u l a t i o n of the amniotic f l u i d ..... 5 B. The Amnion • 9 C. Control of the Accumulation and C i r c u l a t i o n of the Amniotic F l u i d ......... 11 D. The Role of Hormones i n Controlling Fetal Hydro-mineral Metabolism 14 E. Fetal Neurohypophysial Hormones and Kidney Function 16 1. Fetal neurohypophysial hormones 16 2. F e t a l kidney function 18s STATEMENT OF THE PROBLEM = 20 GENERAL MATERIALS AND METHODS 21 A. Sample Collection.Procedures 21 1. Guinea pigs 21 2. Sheep 21 3. Human 22 B. B i o l o g i c a l Assays 22 1, A n t i d i u r e t i c assay .................... 22 2. Vasopressor assay 25 v i Page C. I o n Exchange C h r o m a t o g r a p h y a n d A n a l y t i c a l M ethods 26 1 . I o n exchange c h r o m a t o g r a p h y 26 a . P r e c y c l i n g o f t h e g e l 26 b . B u f f e r s o l u t i o n ......... 26 c. P r e p a r a t i o n o f t h e i o n exchange columns . . . . . . . * . . . . . 27 d. P r e p a r a t i o n o f t h e sample 27 e. E l u t i o n o f b i o l o g i c a l a c t i v i t y ...... 2$ 2 . C o n d u c t i v i t y a n d pH measurements .... 28" 3 . C a l c u l a t i o n o f b i o l o g i c a l a c t i v i t y 28" D. Hormone P r e p a r a t i o n s 29 1 . V a s o p r e s s i n 29 2 . A n g i o t e n s i n I I 29 E . Sodium T h i o g l y c o l l a t e D e a c t i v a t i o n o f V a s o p r e s s i n • 30 SECTION I . COLLECTION AND ASSAY OF UNCONCENTRATED AMNIOTIC FLUID 31 INTRODUCTION 31 RESULTS 33 A. S t u d i e s o f U n t r e a t e d G u i n e a • P i g A m n i o t i c F l u i d 33 1 . Volume o f f l u i d 33 2 . pH 37 3 . C o l o r 37 4 . A n t i d i u r e t i c e f f e c t 37 B. A s s a y s o f F l u i d f r o m D e h y d r a t e d M a t e r n a l G u i n e a P i g s 40 v i i Page C. A s s a y o f Sheep A m n i o t i c F l u i d Samples 1+2 D. A s s a y o f U n t r e a t e d Human A m n i o t i c F l u i d 4 3 DISCUSSION 4 3 A. A n t i d i u r e t i c A s s a y • •• 4 3 B. U n t r e a t e d G u i n e a P i g A m n i o t i c F l u i d 4 4 C. D e h y d r a t i o n E x p e r i m e n t s 4 7 D. Sheep A m n i o t i c F l u i d 4 & E . Human U n t r e a t e d A m n i o t i c F l u i d 4 9 SECTION I I . CONCENTRATION OF ANTIDIURETIC MATERIAL FROM HUMAN AMNIOTIC FLUID ... 5 1 INTRODUCTION 5 1 RESULTS 5 3 A. E x p e r i m e n t s w i t h S t a n d a r d V a s o p r e s s i n 5 3 B. E x p e r i m e n t s w i t h Human A m n i o t i c F l u i d ....... 5 4 1 . The s e p a r a t i o n o f a n a m n i o t i c f l u i d a n t i d i u r e t i c s u b s t a n c e (AFAS) 5 4 2 . The p r o p e r t i e s o f t h e a m n i o t i c f l u i d a n t i d i u r e t i c s u b s t a n c e (AFAS) 5 9 a . D i s c r i m i n a t i o n b e t w e e n v a s o p r e s s i n a n d AFAS 5 9 hi Sodium t h i o g l y c o l l a t e d e a c t i v a t i o n •• 6 0 c. S t o r a g e c o n d i t i o n s o f s a m p l e s ....... 6 2 d. C o m p a r i s o n s w i t h a n g i o t e n s i n I I 6 3 DISCUSSION 6 7 A. C o n c e n t r a t i o n o f t h e F l u i d 6 7 B. The P r e s e n c e o f a n A n t i d i u r e t i c A c t i v i t y i n Human A m n i o t i c F l u i d 6 $ v i i i Page C. The Nature of the Amniotic F l u i d A n t i d i u r e t i c Substance • 69 1 . Comparison w i t h Vasopressin 69 2 . Comparison w i t h Angiotensin I I 72 3 . Comparisons w i t h combinations of vasopressin and a n g i o t e n s i n ............. 72 GENERAL DISCUSSION 74 A. Vasopressin i n the Amniotic F l u i d •• 74 1 . Consideration of the metabolism of vasopressin 74 2 . Vasopressinases and oxytocinases during pregnancy 76 3 . Considerations of the metabolism of an g i o t e n s i n • 77 4 . I n t e r - r e l a t i o n s h i p s between vasopressin and a n g i o t e n s i n 7$ B. Future Studies of Vasopressin and Other A c t i v e Agents i n Amniotic F l u i d SO 1. The co n f i r m a t i o n of the r e s u l t s by other methods $0 2 . The p u r i f i c a t i o n of the a c t i v e agent .... S3 3 . The p o s s i b l e a c t i o n of the AFAS i n the physiology o f . t h e . f e t u s S3 C• Summary • $5 1 . Unconcentrated amniotic f l u i d 6*5 2 . Concentration of human a m n i o t i c . f l u i d . . . . $5 3. The nature of the AFAS 36 REFERENCES 37 i x L I S T OF TABLES T a b l e Page I . GUINEA PIG AMNIOTIC FLUID COLLECTION AND ASSAY DATA 3 4 I I . ANTIDIURETIC ASSAY CONTROLS 3 9 I I I . DEHYDRATION EXPERIMENT COLLECTION AND ASSAY DATA 4 1 I V . OSMOTIC PRESSURES OF GUINEA PIG AMNIOTIC FLUID 4 2 V. COMPARISONS BETWEEN VASOPRESSIN AND AMNIOTIC FLUID ANTIDIURETIC SUBSTANCE (AFAS) 70 X L I S T OF FIGURES F i g u r e F a c i n g Page 1. Volumes o f g u i n e a p i g a m n i o t i c f l u i d f r o m m i d - t e r m t o t e r m 36 2 . C o n c e n t r a t i o n o f a f i v e u n i t s t a n d a r d v a s o p r e s s i n l o a d on a CM-25 Sephadex c olumn 55 3 . P o r t i o n o f a r a t a n t i d i u r e t i c assay-r e c o r d w h i c h shows t h e r e s p o n s e p a t t e r n o f s t a n d a r d v a s o p r e s s i n a n d a sample f r a c t i o n c o n t a i n i n g AFAS 57 4 . C o m p a r i s o n s o f t h e e l u t i o n p r o f i l e s o f t h e s e v e n human a m n i o t i c f l u i d s a m p l e s w i t h a one u n i t v a s o p r e s s i n . . s t a n d a r d r e c o v e r y c u r v e f r o m a CM-25 Sephadex column 5$ 5 . P o r t i o n o f a n a n t i d i u r e t i c a s s a y r e c o r d s h o w i n g s o d i u m t h i o g l y c o l l a t e d e a c t i v a t i o n a t t e m p t 6 l 6 . C o m p a r i s o n s o f t h e r e s p o n s e t o i n j e c t i o n s o f v a s o p r e s s i n , a n g i o -t e n s i n I I a n d t h e AFAS on a r a t a n t i d i u r e t i c a s s a y p r e p a r a t i o n 64-7. P o r t i o n o f a n a n t i d i u r e t i c a s s a y r e c o r d s h o w i n g c o m p a r i s o n s o f i n -j e c t i o n s o f v a s o p r e s s i n , a n g i o t e n -s i n I I a n d c o m b i n a t i o n s o f t h e s e two hormones a t d i f f e r i n g c o n c e n t r a t i o n s .. 66 ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k t h e f o l l o w i n g p e o p l e f o r t h e i r h e l p a n d s u p p o r t t h r o u g h o u t t h i s work. D r . A. M. P e r k s , f o r s u g g e s t i n g t h e p r o b l e m , f o r h i s g e n e r o u s s u p p o r t a n d g u i d a n c e , a n d f o r a s s i s t i n g i n t h e w r i t i n g o f t h i s t h e s i s . Dan R urak a n d M i s s D a v i d s o n , f o r t h e c o l l e c t i o n o f sample f l u i d s f r o m sheep, a n d humans, r e s p e c t i v e l y . J o a n M a r t i n , f o r g i v i n g f r e e l y o f h e r e x t e n s i v e t e c h n i c a l know-how a n d f o r g e n e r o u s f r i e n d l y a s s i s t a n c e i n many a r e a s . M a r g a r e t M c D o n a l d f o r m o r a l s u p p o r t a n d much a s s i s t a n c e i n t h e l a b . C o l i n P a r k i n s o n a n d R a c h e l l e B o u f f a r d f o r m o r a l s u p p o r t ; and, B i l l H o l t , who s p e n t u n a c c o u n t a b l e h o u r s a s s i s t i n g w i t h b o t h t h e t h e o r e t i c a l a n d t e c h n i c a l a s p e c t s o f t h i s work. To them a l l , I w o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s . 1 INTRODUCTION A. The A m n i o t i c F l u i d The a m n i o t i c f l u i d h a s l o n g b e e n t h o u g h t t o f u n c t i o n a s a p r o t e c t i v e c u s h i o n a g a i n s t m e c h a n i c a l s h o c k , a s a. t h e r m a l i n s u l a t o r , a n d a s a l i q u i d e n v i r o n m e n t w h i c h p r o -v i d e s t h e f e t u s w i t h room t o grow a n d change p o s i t i o n w h i l e p r e v e n t i n g d i s f i g u r i n g a d h e s i o n s o f d e v e l o p i n g t i s s u e . I t may a l s o f u n c t i o n i n l e s s o b v i o u s ways. The f l u i d may p l a y a r o l e i n f e t a l n o u r i s h m e n t a n d i n t h e r e g u l a t i o n o f f e t a l h y d r a t i o n a n d d e h y d r a t i o n ( R e y n o l d s , 1 9 7 2 ) . The a m n i o t i c f l u i d i s 9$ t o 99 p e r c e n t w a t e r a n d o n l y one t o two p e r c e n t s o l i d s , o f w h i c h h a l f a r e o r g a n i c s o l i d s ( B o n s -n e s , 1 9 6 6 ) . When c l e a r e d o f a l l i n s o l u b l e m a t e r i a l , n o r m a l a m n i o t i c f l u i d i s n e a r l y a s c l e a r a s w a t e r . I t h a s a s p e c i f i c g r a v i t y a n d v i s c o s i t y t h a t i s s l i g h t l y g r e a t e r , a n d a f r e e z i n g p o i n t t h a t i s s l i g h t l y l o w e r t h a n t h a t o f w a t e r ( B o n s n e s , 1 9 6 6 ) . The r a n g e o f n o r m a l l i m i t s f o r c o n -c e n t r a t i o n o f t h e components i n t h e a m n i o t i c f l u i d a r e f a r w i d e r t h a n t h o s e o f any o t h e r body f l u i d ( P l e n t l , 1 9 6 6 ) . The p r e s e n c e o f t h e a m n i o t i c f l u i d i s e s s e n t i a l f o r f e t a l s u r v i v a l . A l t h o u g h t h e f e t u s e s o f some s p e c i e s may c o n -t i n u e t o t e r m i f t h e f l u i d i s removed v e r y l a t e i n g e s t a -t i o n , i n most c a s e s , a m n i o t i c f l u i d w i l l n o t r e a c c u m u l a t e i f removed a n d f e t a l d e a t h w i l l r e s u l t ( A d o l p h , 1 9 6 7 ) . 2 1 . S o u r c e s o f t h e a m n i o t i c f l u i d A m n i o t i c f l u i d a c c u m u l a t i o n o c c u r s . e a r l y i n g e s t a t i o n , a c c o m p a n y i n g t h e f o r m a t i o n a n d g r o w t h o f t h e amnion, a n d i n t h e human i t i s f o u n d i n m e a s u r a b l e q u a n t i t i e s a t e i g h t weeks o f g e s t a t i o n , o r 0.19 o f t e r m ( w i t h 0 e q u a l t o c o n -c e p t i o n a n d 1 . 0 c o r r e s p o n d i n g t o t e r m ) ( K e r p e l - F r o n i u s , 1 9 7 0 ) . The f l u i d a c c u m u l a t e s a t a n a p p r o x i m a t e r a t e o f 25 mis/week i n t h e human, r e a c h i n g i t s maximum volume o f 500 t o 2 , 0 0 0 m i s a t 0 . 9 1 o f t e r m ( S a u n d e r s & Rhodes, 1 9 7 3 ) . The volume t h e n d i m i n i s h e s r a p i d l y , so t h a t by 0 . 9 9 o f t e r m i t i s l e s s t h a n 300 m i s ( S e e d s , 1 9 7 2 b ) . A l t h o u g h t h e o r i g i n o f t h e f l u i d r e m a i n s o b s c u r e , p o s -s i b l e s o u r c e s t h a t have b e e n s u g g e s t e d i n c l u d e a c t i v e s e c r e -t i o n by t h e amnion, p a s s i v e t r a n s u d a t i o n f r o m t h e m a t e r n a l c i r c u l a t i o n , p a s s i v e t r a n s u d a t i o n f r o m t h e f e t a l c i r c u l a t i o n , f e t a l u r i n e , s e c r e t i o n s o f t h e f e t a l r e s p i r a t o r y t r a c t , f e t a l s a l i v a a n d f e t a l s k i n ( R e y n o l d s , 1 9 7 2 ) . I n i t i a l a c c u m u l a t i o n o f t h e f l u i d must be e x t r a - f e t a l , due t o t h e f a c t t h a t t h e f l u i d i s p r e s e n t i n t h e a m n i o t i c s a c b e f o r e f e t a l t i s s u e s a r e f u n c t i o n a l . I n some c a s e s where t h e f e t u s i s a b s e n t o r r u d i m e n t a r y , a s m a l l amount o f f l u i d d o es a c c u m u l a t e ( B o u r n e , 1 9 6 2 ) . I t i s g e n e r a l l y a c c e p t e d a t t h e p r e s e n t t i m e t h a t t h e i n i t i a l a m n i o t i c f l u i d i s a d i a l y -s a t e o f m a t e r n a l serum. T h i s b e l i e f i s b a s e d p r i m a r i l y upon t h e s i m i l a r i t i e s o f c o m p o s i t i o n between t h e e a r l y a m n i o t i c 3 f l u i d a n d t h e components o f m a t e r n a l b l o o d w h i c h w o u l d be e x p e c t e d t o p e r m e a t e t h e f e t a l membranes. As f u r t h e r c o n -f i r m a t i o n o f t h i s , Behrman, e t a l . (1967) f o u n d t h a t i f monkey f e t u s e s were removed, t h e a m n i o t i c f l u i d c o n t i n u e d t o r e s e m b l e m a t e r n a l p l a s m a . The c o n t r i b u t i o n o f d i r e c t s e c r e t i o n f r o m t h e amnion i s somewhat more c o n t r o v e r s i a l . H i s t o l o g i c a l l y , t h e r e i s some e v i d e n c e t h a t t h e c e l l s o f t h e amnion may be s e c r e t o r y , a n d i t i s b e l i e v e d by some r e -s e a r c h e r s t h a t t h i s p l a y s a n i m p o r t a n t r o l e i n t h e e a r l y f l u i d a c c u m u l a t i o n ( A d o l p h , 1967; Behrman, e t a l , , 1967; S a u n d e r s & Rhodes, 1973). However, Hoyes (1975) h a s r e -v i e w e d t h e e v i d e n c e a n d c o n c l u d e d t h a t t h e r e i s l i t t l e e v i -d e n c e t h a t t h e a m n i o t i c c e l l s have s u f f i c i e n t s e c r e t o r y a c t i v i t y t o make an i m p o r t a n t c o n t r i b u t i o n t o t h e f l u i d v o l u m e . F e t a l c o n t r i b u t i o n s t o t h e a m n i o t i c f l u i d a r e c o n s i d e r e d by many w o r k e r s i n t h e f i e l d t o be l a r g e ( L i n d , e t a l . , 1972; L i n d & H y t t e n , 1972), a n d i n l a t e r f e t a l d e v e l o p m e n t , most o f t h e w a t e r i n t h e f l u i d i s t h o u g h t t o . o r i g i n a t e i n t h e f e t a l s y s t e m ( H u t c h i n s o n , e t a l . , 1959) . T h a t t h e f e t u s p l a y s a n . i m p o r t a n t r o l e , e v e n e a r l y i n d e v e l o p m e n t , i s s u g -g e s t e d by t h e f a c t t h a t t h e volume o f f l u i d i n e a r l y p r e g -n a n c y v a r i e s d i r e c t l y w i t h t h e s u r f a c e a r e a o f t h e f e t u s ( B o u r n e , 1962) . However, t h e r o l e o f t h e f e t u s i s u n d o u b t -e d l y more i m p o r t a n t l a t e r i n g e s t a t i o n . The p o s s i b l e f e t a l s o u r c e s o f a m n i o t i c f l u i d a r e t h e f e t a l k i d n e y s , l u n g a n d t r a c h e o b r o n c h i a l t r a c t , s a l i v a r y 4 g l a n d s a n d t h e s k i n . F e t a l m i c t u r a t i o n p r o b a b l y p l a y s t h e l a r g e s t r o l e ( A l e x a n d e r , e t a l . , 1 9 5 # ; Hoyes, 1 9 7 5 ; P i t k i n , e t a l . , 1 9 7 3 ) • A l t h o u g h k i d n e y f u n c t i o n i n t h e f e t u s w i l l be c o n s i d e r e d i n more d e t a i l i n a l a t e r s e c t i o n , some o f t h e l i n e s o f e v i d e n c e f o r a s i g n i f i c a n t c o n t r i b u t i o n o f t h e f e t a l k i d n e y s t o t h e f o r m a t i o n o f t h e a m n i o t i c f l u i d a r e : 1 ) a d e c r e a s e i n t o t a l s o l u t e c o n c e n t r a t i o n f r o m t h e t w e n t i e t h week o f g e s t a t i o n ( 0 . 5 o f t e r m ) t o t e r m . F e t a l u r i n e i s m a r k e d l y h y p o t o n i c ; 2 ) t h e c o n c e n t r a t i o n o f u r e a , u r i c a c i d a n d c r e a t i n i n e a r e s i g n i f i c a n t l y i n c r e a s e d i n t h e l a s t t r i m e s t e r , a l t h o u g h t h e p l a c e n t a l membranes a r e p e r m e a b l e t o t h e s e s m a l l m o l e c u -l a r w e i g h t s o l u t e s ; 3 ) t a u r i n e i n c r e a s e s , w h i l e o t h e r amino a c i d s d e c r e a s e . T a u r i n e i s a l s o p r e s e n t i n t h e f e t a l u r i n e ; 4) t h e r e i s a h i g h c o r r e l a t i o n between r e n a l a g e n e s i s a n d o l i g o h y d r a m i n o s i n l a t e p r e g n a n c y ; a n d 5 ) t h e f l u i d becomes more a c i d i c n e a r t o t e r m , a n d c l o s e r t o t h e pH o f f e t a l u r i n e ( S e e d s , 1 9 7 2 b ) . Human f e t a l . k i d n e y s have b e e n shown t o be a c t i v e a t t w e l v e weeks, o r 0 . 3 o f t e r m ( H u t c h i n s o n , e t a l . , 1 9 5 9 ) , a n d a m n i o t i c f l u i d i s a b s e n t a r o u n d f e t u s e s whose k i d n e y s do n o t d e v e l o p ( A d o l p h , 1 9 6 7 ) . I n t h e s h e e p , f e t a l u r i n e f l o w h a s b e e n d e t e r m i n e d t o be t w i c e t h a t o f t h e a d u l t ( K e r p e l - F r o n i u s , 1 9 7 0 ) , a n d f e t a l u r i n e i s t h o u g h t t o be o f s u f f i c i e n t q u a n t i -t y by 0 . 5 5 o f t e r m t o m o d i f y t h e f l u i d ( M e l l o r & S l a t e r , 1 9 7 1 ) . 5 The r o l e o f t h e f e t a l l u n g s and t r a c h e o b r o n c h i a l t r e e has not been shown as c o n v i n c i n g l y as t h a t o f t h e k i d n e y s . R eynolds (1953) was a b l e t o o b t a i n c o n s i d e r a b l e amounts o f f l u i d by c a n n u l a t i n g t h e f e t a l t r a c h e a , and t h e r a t e o f f l o w o f t h i s f l u i d i n t h e sheep has been d e t e r m i n e d t o be o f t h e o r d e r o f 1-2 mis/minute ( K e r p e l - F r o n i u s , 1970). However, i t i s g e n e r a l l y c o n c l u d e d t h a t t h e amount o f t r a c h e a l f l u i d , i f r e l e a s e d i n t o t h e a m n i o t i c p o o l , would not s i g n i f i c a n t l y add t o t h e volume ( B a t e s , 1963; Bourne, 1962; M e l l o r & S l a t e r , 1971; Saunders & Rhodes, 1973). F u r t h e r , t r a c h e a l f l u i d has been shown t o be q u i t e d i s s i m i l a r t o a m n i o t i c f l u i d i n com-p o s i t i o n , so i t p r o b a b l y does not p l a y a s i g n i f i c a n t r o l e i n t h e d e t e r m i n a t i o n o f t o t a l f l u i d volume ( B a t e s , 1963). I t has been s u g g e s t e d t h a t t h e f l u i d from t h e f e t a l l u n g s may f l o w i n t o t h e b u c c a l c a v i t y and t h e n be swa l l o w e d (Hoyes, 1975). I n t h e human, t h e s k i n o f t h e f e t u s k e r a t i n i z e s around 0.6 o f term. T h e r e . i s some e v i d e n c e t h a t t h e s k i n may be s e c r e t o r y between 0.25 and 0.45 o f term (Hoyes, 196$a, b ) . However, i t i s p r o b a b l y more i m p o r t a n t as an organ o f t r a n s -f e r a t t h i s e a r l y s t a g e i n development (Hoyes, 196Sa). 2. C i r c u l a t i o n o f t h e a m n i o t i c f l u i d The i d e a t h a t t h e a m n i o t i c f l u i d i s not a s t a t i c p o o l i s a r e l a t i v e l y r e c e n t one. Vosburgh (194$) was among t h e f i r s t t o show t h i s , when he de t e r m i n e d t h a t t h e t u r n o v e r r a t e f o r w a t e r was so r a p i d t h a t a l l o f t h e wa t e r i n t h e a m n i o t i c sac o f t h e human was r e p l e n i s h e d e v e r y t h r e e h o u r s (Saunders & Rhodes, 1973). The a m n i o t i c f l u i d t u r n o v e r r a t e i n t h e 6 g u i n e a p i g h a s b e e n shown t o be once e v e r y h o u r ( F l e x n e r & G e l l h o r n , 1942). When c o n s i d e r i n g t h e c i r c u l a t i o n o f t h e f l u i d , t h e r e a r e t h r e e compartments w h i c h a r e o f c o n c e r n ; t h e m a t e r n a l , t h e f e t a l a n d t h e a m n i o t i c f l u i d . E x c h a n g e s o c c u r between a l l t h r e e o f t h e s e compartments ( H u t c h i n s o n , e t a l . , 1959). Much o f t h e w a t e r t r a n s f e r between t h e a m n i o t i c f l u i d a n d t h e f e t u s i s v i a t h e u m b i l i c u s ( B o u r n e , 1 9 6 2 ) . The ex-change between m o t h e r a n d f l u i d i s i m p o r t a n t t h r o u g h o u t g e s t a t i o n ( H u t c h i n s o n , e t a l . , 1 9 5 9 ) . However, l a t e i n g e s t a t i o n , t h e f e t u s p l a y s a r o l e a s i n t e r m e d i a t e i n a b o u t 40 p e r c e n t o f t h i s f l u i d exchange ( B o u r n e , 1 9 6 2 ) . I n t h e human, w a t e r t u r n o v e r i s 350 t o 600 m i s / h o u r ( D a n f o r t h & H u l l , 195#), o r o n e - t h i r d t o o n e - h a l f o f t h e t o t a l f l u i d e a c h h o u r ( C a s t e l a z o - A y a l a , 1 9 7 2 ) . The r a t e o f exchange o f 100 m i s / h o u r a t 0 . 3 o f t e r m i n c r e a s e s t o 3 , 6 0 0 m i s / h o u r a t t e r m a n d i s p r o p o r t i o n a l t o t h e i n c r e a s e i n p l a c e n t a l w e i g h t ( H u t c h i n s o n , e t a l . , 1 9 5 9 ) . M a t e r n o - f e t a l w a t e r exchange c o r r e l a t e s w i t h f e t a l w e i g h t , w h i l e a m n i o t i c f l u i d e xchange shows c o r r e l a t i o n w i t h p l a c e n t a l w e i g h t ( L i l e y , 1 9 6 3 ) . The t o t a l q u a n t i t y o f s o l u t e r e l a t i v e t o w a t e r i n t h e s a c i s a m a j o r d e t e r m i n a n t o f f l u i d volume e x c h a n g e s ( M e l l o r & S l a t e r , 1 9 7 1 ) . E a c h component o f am-n i o t i c f l u i d i s e x c h a n g e d a t a r a t e i n d e p e n d e n t o f t h a t o f o t h e r components, a n d i t i s i n d e p e n d e n t o f c o n c e n t r a t i o n ( R e y n o l d s , 1 9 7 2 ) . 7 Recent evidence suggests t h a t two major pathways may e x i s t f o r amniotic f l u i d turnover i n the l a s t part of preg-nancy. The f i r s t of these pathways forms part of a c y c l e through which the f l u i d i s t r a n s p o r t e d from mother t o f e t u s across the placenta and back t o the mother through the amniotic f l u i d , and across the amniochorion (Hoyes, 1975). The second pathway i n v o l v e s only the f e t u s . F e t a l u r i n a -t i o n and d e g l u t t a t i o n , f o l l o w e d by a b s o r p t i o n i n the g a s t r o -i n t e s t i n a l t r a c t , play a s i g n i f i c a n t r o l e i n the second pathway ( J e f f c o a t e & S c o t t , 1959; Kerp e l - F r o n i u s , 1970). F e t a l swallowing has been shown to occur as e a r l y as 0.3 of term i n humans, and may amount f o r as much as 500 mis/day (Reynolds, 1972). The f e t a l stomach i s f r e q u e n t l y distended w i t h . f l u i d (Alexander, et a l . , 195&), and the presence of lanugo h a i r and epidermal squames i n the g a s t r o i n t e s t i n a l t r a c t support the importance of swallowing (Hoyes, 1975). However, human f e t a l swallowing accounts f o r only 1+ percent of water removed per hour (Reynolds, 1972), and even i f the f e t u s swallowed 20 mis/hour, t h i s would only account f o r 5 t o 10 percent of the amount of f l u i d removed per hour (Saunders & Rhodes, 1973). The term f e t u s has been found t o swallow 450 mis of f l u i d per day ( P l e n t l , 1966). Con-t r a r y t o t h i s evidence, Abramovich (1970) has suggested that f e t a l swallowing and v o i d i n g play only s m a l l r o l e s i n the c o n t r o l of amniotic f l u i d volume. 8 The t r a n s f e r r o u t e s o r s i t e s o f e xchange o f t h e f l u i d may change t h r o u g h o u t g e s t a t i o n a n d may even d i f f e r f o r e a c h c o n s t i t u e n t ( L i l e y , 1963). However, t h e m a j o r s i t e s t h a t have been s u g g e s t e d f o r exchange a r e t h e amnion, p a r -t i c u l a r l y t h e p l a c e n t a l p o r t i o n , t h e u m b i l i c a l c o r d a n d t h e f e t a l s k i n . The c e l l s i n t h e p l a c e n t a l p o r t i o n o f t h e a m n ion may have s e c r e t o r y a b i l i t y a n d p l a y a n a c t i v e , f u n d a m e n t a l r o l e i n f l u i d e xchange ( D a n f o r t h & H u l l , 195$) . H i g h c o u n t s o f i n j e c t e d i s o t o p e i n W h a rton's j e l l y , s u g g e s t e d t o H u t c h i -s o n , e_t a l . (1959) t h a t t h e u m b i l i c a l c o r d may be t h e m a j o r s i t e o f f l u i d e x c h a n g e d between f e t u s a n d t h e a m n i o t i c f l u i d . L a t e i n p r e g n a n c y , t h e a m n i o t i c e p i t h e l i u m may be i m -p o r t a n t i n f l u i d a b s o r p t i o n . E a r l y , t h e r e l a t i v e l y s i m p l e c e l l s c o u l d e a s i l y p e r m i t p a s s a g e o f t r a n s u d a t e . L a t e r i n d e v e l o p m e n t , f o r m a t i o n o f s u r f a c e m i c r o v i l l i a n d i n t e r c e l -l u l a r c a n a l s s u g g e s t t h a t movement o f m a t e r i a l i s h i g h l y s e l e c t i v e a n d s p e c i a l i z e d ( B o u r n e , 1962) . The complex s t r u c t u r e s u g g e s t s t h a t t h e membrane i s c a p a b l e o f t r a n s -m i t t i n g l a r g e q u a n t i t i e s o f f l u i d . The amnion i s f r e e l y p e r m e a b l e t o w a t e r i n v i t r o a n d i n v i v o , a n d f u n c t i o n s a s a l e a k y s e m i - p e r m e a b l e membrane ( S e e d s , 1965) . I t a l l o w s , most m o l e c u l e s w i t h a m o l e c u l a r w e i g h t l e s s t h a n 1000 t o c r o s s t h e membrane ( S e e d s , 1970b). The amnion p r o v i d e s a s i t e f o r exchange o f f l u i d between t h e m a t e r n a l s y s t e m a n d t h e a m n i o t i c f l u i d . However, t h e n e t t r a n s f e r o f w a t e r i s o u t o f t h e f l u i d ( R e y n o l d s , 1972) . C o n t r a r y t o most o t h e r 9 r e s e a r c h e r s i n t h e f i e l d , M e l l o r ( 1 9 7 0 ) a n d M e l l o r a n d S l a t e r ( 1 9 7 1 ) have c o n c l u d e d t h a t t h e amnion s e v e r e l y l i m i t s t h e p a s s a g e o f i o n s , a n d i s ev e n r e l a t i v e l y i m p e r m e a b l e t o w a t e r . The s t r u c t u r e o f t h e amnion w i l l be d e a l t w i t h i n d e t a i l i n a l a t e r s e c t i o n . F e t a l s k i n , p r i o r t o k e r a t i n i z a t i o n , may s e r v e a s a m a j o r pathway f o r w a t e r a n d s o l u t e exchange between t h e f e t u s a n d t h e a m n i o t i c f l u i d . A t 0 . 3 5 t o 0 . 4 0 o f t e r m , t h e s k i n o f human f e t u s e s h a s b e e n f o u n d t o be a s . p e r m e a b l e t o i s o t o p i c w a t e r a s t h e amnion ( P a r m l e y & S e e d s , 1 9 7 0 ) . L i n d a n d H y t t e n ( 1 9 7 2 ) have s u g g e s t e d t h a t i n t h e f i r s t h a l f o f g e s t a t i o n , t h e a m n i o t i c f l u i d h a s t h e c o m p o s i t i o n o f f e t a l e x t r a c e l l u l a r f l u i d a n d t h e amount i s c l o s e l y r e l a t e d t o f e t a l s i z e , so t h a t t h e l i m i t s o f t h e f e t u s a t t h i s s t a g e may be t h e amnion, n o t t h e s k i n . D u r i n g t h e s e c o n d h a l f o f g e s t a t i o n , t h e f e t a l s k i n becomes ' w a t e r p r o o f , a n d t h e a m n i o t i c f l u i d i s p r i m a r i l y t h e r e s u l t o f f e t a l m i c t u r a t i o n a n d s w a l l o w i n g . However, s w a l l o w i n g c a n n o t a c c o u n t f o r a l l o f t h e f l u i d removed. B. The Amnion As a n i n t e g r a l p a r t o f t h e f e t a l e n v i r o n m e n t , a l l mam-m a l s f o r m a n amnion e a r l y i n g e s t a t i o n . T r o p h o b l a s t c e l l s d i f f e r e n t i a t e i n t o a m n i o g e n i c c e l l s s e v e n t o e i g h t d a y s a f t e r f e r t i l i z a t i o n i n t h e human, a n d by t h e end o f t h e s e c o n d month o f g e s t a t i o n , t h e a m n i o t i c s a c h a s f i l l e d t h e 10 e m b r y o n i c v e s i c l e a n d a d h e r e s t o t h e s pongy l a y e r o f c e l l s s e p a r a t i n g i t f r o m t h e c h o r i o n (Thomsen & H i e r s c h e , 1 9 6 9 ) . E a r l y i n g e s t a t i o n , t h e amnion c o n s i s t s o f o n l y a n e p i t h e l i a l l a y e r a n d a n e x t r a e m b r y o n i c s o m a t o p l e u r e , b u t l a t e r i t d i f -f e r e n t i a t e s i n t o f i v e d i s t i n c t l a y e r s ( B o u r n e , 1 9 6 2 ) . The u p p e r m o s t l a y e r o f c u b o i d a l e p i t h e l i a l c e l l s r e s t s on a basement membrane c o n s i s t i n g o f r e t i c u l a r t i s s u e . The t h i r d , o r compact l a y e r , i s r e s p o n s i b l e f o r t h e s t r e n g t h o f t h e a mnion. U n d e r n e a t h t h e compact l a y e r i s a f i b r o b l a s t i c l a y e r , and, f i n a l l y , a s pongy l a y e r w h i c h e n a b l e s t h e amnion t o move c o m p a r a t i v e l y f r e e l y o v e r t h e f i x e d u n d e r l y i n g c h o r i o n ( S a u n d e r s & Rhodes, 1973; Thomsen & H i e r s c h e , 1 9 6 9 ) . I n t h e human, t h e e p i t h e l i a l l a y e r b e g i n s t o d i f f e r e n t i a t e i n t h e t h i r d month o f g e s t a t i o n , a n d t h e m a t u r e e p i t h e l i u m p o s s e s s e s numerous m i c r o v i l l i on b o t h s u r f a c e a n d l a t e r a l a s p e c t s o f t h e c e l l s (Hoyes, 1 9 7 5 ) . T h e s e c e l l s c o n t a i n many i n t r a -c e l l u l a r v a c u o l e s an d t h e r e i s a l s o a n e x t e n s i v e s y s t e m o f complex i n t e r c e l l u l a r c a n a l s ( Hoyes, 1 9 7 5 ; Thomsen & H i e r s c h e , 1 9 6 9 ) . The amnion has b e en f o u n d t o have no b l o o d v e s s e l s , l y m p h a t i c pathways, n e r v e o r m u s c l e f i b e r s i n most mammals (Thomsen & H i e r s c h e , 1 9 6 9 ) . I n t h e mouse a n d p r i m a t e , t h e a m n i o t i c c a v i t y a c t u a l l y a p p e a r s p r i o r t o t h e t i m e when t h e body o f t h e f e t u s h a s t a k e n d e f i n i t e . s h a p e a n d h a s become t h e embryo p r o p e r . The amnion i s c o n t i n u o u s w i t h t h e f e t a l s k i n , f o r m s t h e 11 c o v e r i n g o f t h e u m b i l i c a l c o r d , a n d i s c l o s e l y a s s o c i a t e d w i t h t h e p l a c e n t a b e f o r e i t r e c u r v e s t o f o r m t h e f r e e , r e f l e c t e d p a r t o f t h e membrane. The u l t r a s t r u c t u r e o f t h e r e f l e c t e d amnion r e v e a l s b a s i c a l l y a s i n g l e l a y e r o f s i m p l e n o n - c i l i a t e d c u b o i d a l e p i t h e l i a l c e l l s . P l a c e n t a l amnion, however, shows s t r u c t u r a l d i f f e r e n c e s i n t h a t t h e e p i t h e l i u m a p p e a r s t o be more c o l u m n a r a n d t h e r e f o r e more l i k e l y t o f a c i l i t a t e f l u i d t r a n s p o r t ( B o u r n e , 1962). C. C o n t r o l o f t h e A c c u m u l a t i o n a n d C i r c u l a t i o n o f t h e  A m n i o t i c F l u i d The f a c t o r s w h i c h c o n t r o l t h e r e g u l a t i o n o f a m n i o t i c f l u i d e xchange a r e p o o r l y u n d e r s t o o d a t t h i s t i m e . How-e v e r , t h e r e i s e v i d e n c e t h a t t h e s e c o n t r o l s e x i s t a n d p l a y a n i m p o r t a n t r o l e i n t h e p h y s i o l o g y o f t h e f e t u s . The f a c t t h a t t h e r e a r e e x t r e m e l y r a p i d t u r n o v e r r a t e s , b u t a r e l a t i v e l y c o n s t a n t volume s u g g e s t s a c a r e f u l c o n -t r o l , s i n c e any s m a l l v a r i a t i o n i n t h e r a t e o f p r o d u c t i o n o r d i s p o s a l o f t h e f l u i d w o u l d r a p i d l y a l t e r t h e t o t a l volume ( B o u r n e , 1962). F o r example, i f t h e r e was a change o f one p e r c e n t i n t h e t u r n o v e r r a t e f o r w a t e r , t h i s w o u l d r e s u l t i n a s i g n i f i c a n t a l t e r a t i o n i n volume on t h e o r d e r o f one l i t e r i n t e n d a y s ( S a u n d e r s & Rhodes, 1973)• I f t h e r e i s a d i s r u p t i o n i n s o l u t e t r a n s p o r t , t h e n p o l y -o r o l i g o - h y d r a m i n o s w i l l r e s u l t ( S c h r u e f e r , e t a l . , 1972). 12 D a n f o r t h a n d H u l l (1958") s u g g e s t e d t h a t t h e w a t e r exchange was n o t due t o d i f f u s i o n . A d o l p h (1967) f o u n d t h a t t h e t u r n o v e r i s due p a r t t o d i f f u s i o n , a n d p a r t t o b u l k f l o w . L i k e w i s e , A b r a m o v i c h (1970) a t t r i b u t e d e x c h a n g e s between f e t u s a n d a m n i o t i c f l u i d t o b u l k e x c h a n g e s a n d n o t t o f i n e l y b a l a n c e d e l e c t r o l y t e e x c h a n g e s , a s o c c u r a c r o s s t h e f e t o - p l a c e n t a l u n i t a n d m a t e r n a l t i s s u e s . The n e t t r a n s f e r o f w a t e r a c r o s s b i o l o g i c a l membranes must o c c u r by p a s s i v e mechanisms, a s no e v i d e n c e f o r a c t i v e t r a n s p o r t o f w a t e r h a s e v e r b e e n e s t a b l i s h e d ( S e e d s , 1973) . T h e r e may be w a t e r t r a n s f e r w h i c h i s l i n k e d t o t h e a c t i v e ex-change o f i o n s , b u t t h i s h a s n o t y e t b e e n shown t o e x i s t . However, t h e d r i v i n g f o r c e o f n e t f l u i d t r a n s f e r s between m o t h e r a n d a m n i o t i c f l u i d i s n o t o s m o t i c p r e s s u r e o r s o l -u t e g r a d i e n t s . C o l l o i d o s m o t i c p r e s s u r e , w i t h o u t o t h e r f o r c e s , w o u l d f a v o r movement o f w a t e r f r o m t h e f e t u s t o t h e mother a s w e l l . H y d r o s t a t i c p r e s s u r e g r a d i e n t s a l s o f a v o r movement t o w a r d t h e m o t h e r ( K e r p e l - F r o n i u s , 1970; S e e d s , 1965; S e e d s , 1972a). O s m o t i c g r a d i e n t s may be r e -s p o n s i b l e f o r m a i n t a i n i n g i n t r a u t e r i n e w a t e r , b u t o n l y i f t h e y a r e t e m p o r a r y a n d t o o s m a l l o r t o o i n t e r m i t t e n t t o measure w i t h c u r r e n t l y a v a i l a b l e t e c h n i q u e s ( S e e d s , 1965)» Indeed,, t h e r e may be s m a l l o s m o t i c g r a d i e n t s a c r o s s t h e membranes due t o d i f f e r i n g c o n c e n t r a t i o n s o f sodium, c h l o r i d e o r u r e a , b e c a u s e t h e s e m o l e c u l e s do n o t p e n e t r a t e t h e mem-b r a n e a s r a p i d l y a s w a t e r ( S e e d s , 1970) . 13 Net t r a n s f e r s o f w a t e r have been observed.? a c r o s s t h e amnion by i n d u c e d o s m o t i c g r a d i e n t s . The n e t w a t e r t r a n s f e r a c r o s s t h e human t e r m amnion, i n v i t r o , i n r e s p o n s e t o o s -m o t i c g r a d i e n t s , o c c u r s a t many t i m e s t h e r a t e e x p e c t e d by d i f f u s i o n a l o n e . T h i s i s a ' n o n d i f f u s i o n a l 1 o r ' b u l k f l o w ' p r o c e s s , b y w h i c h s o l v e n t w a t e r i s t r a n s f e r r e d a c r o s s p o r o u s membranes i n r e s p o n s e t o c h e m i c a l p o t e n t i a l g r a d i e n t s ( S e e d s , 1 9 6 7 ) . I f i n c r e a s e d f l u i d i s g i v e n t o t h e m o t h e r , t h e r e i s a n i n c r e a s e d volume o f d i l u t e a m n i o t i c f l u i d ( A d o l p h , 1 9 6 7 ) . I n f u s i o n s o f h y p e r t o n i c g l u c o s e o r m a n n i t o l i n t o t h e m o t h e r r e s u l t s i n prompt c h a n g e s i n t h e t o t a l o s m o t i c p r e s s u r e , e l e c t r o l y t e a n d p r o t e i n c o n c e n t r a t i o n s i n t h e f e t u s ( B a t -t a g l i a , e t a l . , I 9 6 0 ; B r u n s , e t _ a l . , 1 9 6 3). L i k e w i s e , i n -j e c t i o n o f s u c r o s e i n t o t h e a m n i o t i c f l u i d p r o d u c e d a r e -d u c t i o n i n f e t a l t o t a l body w a t e r , w i t h o u t a f a l l i n p l a c e n t a l t o t a l w a t e r , s u g g e s t i n g movement o f w a t e r f r o m f e t a l a n d m a t e r n a l i n t e r s t i t i a l s p a c e s i n t o t h e p l a c e n t a a n d f r o m t h e r e i n t o t h e a m n i o t i c f l u i d ( B r u n s , e t a l . , 1 9 6 4 ) . T h e s e o s m o t i c g r a d i e n t s c a n be m a i n t a i n e d f o r a n a p p r e c i a b l e t i m e on b o t h s i d e s o f t h e p l a c e n t a , a n d c h e m i c a l g r a d i e n t s b e -tween m o t h e r a n d f e t u s , o r f e t u s a n d a m n i o t i c f l u i d , c a n c a u s e c o n s i d e r a b l e s h i f t s o f w a t e r between f l u i d p o o l s . T h e s e s h i f t s o f f l u i d due t o i n d u c e d o s m o t i c g r a d i e n t s may be t o t h e e x t e n t t h a t t h e f e t u s may become d e h y d r a t e d o r o v e r h y d r a t e d ( K e r p e l - F r o n i u s , 1 9 7 0 ) . 14 The r e g u l a t i o n o f t h e s e d i f f e r e n t f l u i d e x c h a n g e s r e -ma i n s unknown. T h e r e i s no e v i d e n c e f o r a n e r v o u s o r h o r -monal r e g u l a t i o n . I t a p p e a r s l i k e l y t h a t t h e f e t u s e x e r t s a c o n s i d e r a b l e c o n t r o l on t h e r e g u l a t i o n , a s s e e n by t h e extr e m e v a l u e s f o r components o f t h e f l u i d f o u n d i n d i f f e r -e n t , b u t a d j a c e n t f e t u s e s ( A d o l p h , 1 9 6 7 ) . I t a l s o a p p e a r s t h a t t h e volume o f t h e f l u i d i s l e s s i m p o r t a n t t h a n t h e r e l a t i v e c o n c e n t r a t i o n o f s o l u t e s ( S e e d s , 1 9 7 2 a ) . When a m n i o t i c f l u i d was r e p l a c e d by d i s t i l l e d w a t e r i n monkeys, a . r a p i d r e a c c u m u l a t i o n o f t h e o r i g i n a l s o l u t e c o n c e n t r a t i o n o c c u r r e d o v e r IB t o 22 h o u r s w i t h a r e d u c t i o n i n t h e f l u i d volume ( S c h r u e f e r , e t a l . , 1 9 7 2 ) . M e l l o r a n d S l a t e r (1971) have p r o p o s e d t h e e x i s t e n c e o f a c h l o r i d e pump i n t h e amnion o r f e t a l s k i n , by w h i c h c h l o r i d e i s a c t i v e l y pumped i n t o t h e f l u i d ; however, t h i s w o u l d p r o b a b l y n o t be o f s u f f i c i e n t a c t i v i t y t o a f f e c t t h e t o t a l v olume. D. The R o l e o f Hormones i n C o n t r o l l i n g F e t a l H y d r o - m i n e r a l  M e t a b o l i s m Many f o r m s o f m e t a b o l i s m , i n c l u d i n g w a t e r a n d i o n b a l a n c e , a r e c o n t r o l l e d by hormones i n t h e a d u l t mammal. However, t h e r e h a s b e e n l i t t l e i n v e s t i g a t i o n i n t o t h e h o r -monal . c o n t r o l o f f e t a l m e t a b o l i s m . I n t h e s h e e p , t h e f e t u s may c o n t r o l t h e l e v e l o f i o n s i n a l l a n t o i c f l u i d by s e c r e -t i o n o f c o r t i c o s t e r o i d s by t h e f e t a l a d r e n a l g l a n d ( C o m l i n e , e t a l . , 1 9 7 0 ) . A l e x a n d e r a n d W i l l i a m s (1963) f o u n d t h a t 15 a l l a n t o i c f l u i d volume may be c o n t r o l l e d by m a t e r n a l s e x s t e r o i d s e c r e t i o n , e v e n t h o u g h a m n i o t i c f l u i d volume was n o t a l t e r e d by m a t e r n a l o v a r i e c t o m y . A l t h o u g h t h e e v i d e n c e i s l i m i t e d , i t seems r e a s o n a b l e t o s u s p e c t t h a t m a t e r n a l o r f e t a l hormones may p l a y a r o l e i n t h e w a t e r a n d i o n m e t a b o l i s m o f t h e a m n i o t i c f l u i d . T h i s c o n t r o l , however, c a n n o t f o l l o w t h e c l a s s i c a l p a t t e r n f o r h o r m o n a l a c t i o n , s i n c e t h e membrane i s a v a s -c u l a r ( B o u r n e , 1 9 6 2 ) . One p o s s i b l e pathway f o r t h e h o r m o n a l c o n t r o l o f t h e membrane • w o u l d be v i a t h e f e t a l u r i n e . As w i l l be s e e n i n a l a t e r s e c t i o n , f e t a l u r i n e f l o w i s h i g h , a n d t h e hormone c o u l d be t r a n s p o r t e d i n t o t h e a m n i o t i c p o o l , where i t c o u l d a c c u m u l a t e a n d p e r h a p s e x e r t a n e f f e c t on t h e a m n i o n . A l t h o u g h l e s s l i k e l y , t h e hormone may come f r o m t h e m o t h e r . T h i s s m a l l p e p t i d e hormone may be c a p a b l e o f p e n e -t r a t i n g t h e i n t r a u t e r i n e c o n t e n t s ( V i z s o l y i & P e r k s , 1 9 7 4 ) . A t p r e s e n t , t h e most l i k e l y hormone t o be i n v o l v e d i n t h e r e g u l a t i o n o f f e t a l f l u i d s i s v a s o p r e s s i n . T h i s hormone p l a y s a s i g n i f i c a n t r o l e i n t h e a d u l t i n t h e m a i n t e n a n c e o f w a t e r b a l a n c e , b y a c t i n g on t h e c e l l s o f t h e d i s t a l t u b u l e s a n d c o l l e c t i n g d u c t s o f t h e k i d n e y t o c a u s e w a t e r r e a b s o r p -t i o n . P r e l i m i n a r y work w i t h t h i s hormone h a s shown t h a t v a s o p r e s s i n h a d no e f f e c t on t h e r a t e o f d i f f u s i o n o f s o d i u m a c r o s s f e t a l s k i n ( L i n d , e t a l . , 1 9 7 2 ) . A l s o , a d d i t i o n o f UP t o 1 , 0 0 0 mU/ml o f v a s o p r e s s i n h a d no m e a s u r a b l e e f f e c t on w a t e r t r a n s f e r a c r o s s t h e t e r m human amnion t a k e n a f t e r d e l i v e r y ( S e e d s , 1 9 6 7 ) . 16 However, V i z s o l y i a n d P e r k s (1974) a n d H o l t (1975) have shown t h a t v a s o p r e s s i n may be a b l e t o e x e r t a n e f f e c t on t h e amnion, i n v i t r o , c a u s i n g i t t o t r a n s p o r t w a t e r f r o m t h e m a t e r n a l s i d e t o t h e f e t a l s i d e , i n o p p o s i t i o n t o e x i s t -i n g g r a d i e n t s . T h i s may be a n i n d i c a t i o n t h a t t h i s hormone c o u l d a c t a s a r e g u l a t o r o f f l u i d e xchange t h r o u g h i t s a c t i o n on t h e a m nion. E . F e t a l N e u r o h y p o p h y s i a l Hormones a n d K i d n e y F u n c t i o n T h e r e i s a n e l e v a t i o n i n c i r c u l a t i n g m a t e r n a l v a s o p r e s -s i n l e v e l s d u r i n g p r e g n a n c y . W h i l s t t h e p l a s m a v a s o p r e s s i n l e v e l s o f n o n p r e g n a n t human f e m a l e s a r e 1 2 . 5 m i c r o u n i t s / m l , p r e g n a n t women show l e v e l s o f 3$ m i c r o u n i t s / m l , a n d d u r i n g l a b o r , t h e v a l u e s r i s e a s h i g h a s 42 m i c r o u n i t s / m l . D e s p i t e t h e e l e v a t e d l e v e l s , i t i s u n l i k e l y t h a t t h e m a t e r n a l h o r -mones a f f e c t t h e a m nion. T h e r e i s l i t t l e e v i d e n c e a v a i l a b l e , b u t t h e r e s u l t s s u g g e s t t h a t v a s o p r e s s i n does n o t c r o s s t h e p l a c e n t a l b a r r i e r , e i t h e r due t o i m p e r m e a b i l i t y , o r t o p o s -s i b l e d e s t r u c t i o n ( C h e r n o f f & Grabowsky, 1971 ; Skowsky, et a l . , 1 9 7 3 ) . F u r t h e r , p r e l i m i n a r y e x p e r i m e n t s o f V i z s o l y i a n d P e r k s (1974) s u g g e s t e d t h a t v a s o p r e s s i n d i d n o t s t i m u -l a t e t h e m a t e r n a l s i d e o f t h e amnion o f t h e g u i n e a p i g , i n  v i t r o . 1 . F e t a l n e u r o h y p o p h y s i a l hormones I t h a s b e en e s t a b l i s h e d t h a t t h e h y p o t h a l a m i c - n e u r o -h y p o p h y s i a l s y s t e m d e v e l o p s e a r l y i n g e s t a t i o n , w i t h v a s o -p r e s s i n b e i n g p r e s e n t b y 0 . 3 o f t e r m i n t h e g u i n e a p i g 17 ( B u r t o n & F o r s l i n g , 1972) . I n t h e s e a l , t h i s hormone i s p r e s e n t a t 0.19 o f t e r m , t h e e a r l i e s t s t a g e i n v e s t i g a t e d t o d a t e ( P e r k s & V i z s o l y i , 1973) . I n t h e human, hormones o c c u r i n t h e h y p o t h a l a m u s a t 0.5 o f t e r m , a n d i n t h e p i t u -i t a r y a t 0 .57 o f t e r m ( B e n i r s c h k e & McKay, 1953) . The amount i n c r e a s e s s t e a d i l y t o 0.85 o f t e r m . N e u r o s e c r e t o r y m a t e r i a l h a s b e e n f o u n d i n a l l f e t a l mammals s t u d i e d , ex-c e p t t h e r a t ( P e r k s & V i z s o l y i , 1973) . However, t h e a c -t i v i t y , c a l c u l a t e d p e r m i l l i g r a m d r y t i s s u e , o f t h e p o s t e r -i o r l o b e s o f newborn i n f a n t s c o n t a i n e d o n l y 20 p e r c e n t o f t h e n e u r o h y p o p h y s i a l p r i n c i p l e s f o u n d i n t h e g l a n d s o f a d u l t s ( H e l l e r & Z a i m i s , 1949) . More r e c e n t work i n t h e s h e e p h a s f o u n d t h a t f e t a l v a s o p r e s s i n l e v e l s f r o m l y o p h y -l i z e d t i s s u e s were a s h i g h a s t h o s e o f t h e a d u l t , o r h i g h e r , a t b i r t h ( V i z s o l y i & P e r k s , 1976) . The n e u r o h y p o p h y s i s o f f e t u s e s have a m a r k e d l y h i g h e r v a s o p r e s s i n t o o x y t o c i n r a t i o t h a n a d u l t s , . w i t h r a t i o s i n human f e t u s e s o f 15 a t 0.58 o f t e r m , 6 a t 0.70 o f t e r m , a n d n e a r i n g u n i t y , w h i c h i s t h e a d u l t r a t i o , a t t e r m ( D i c k e r , 1 9 6 l ) . The f e t u s has a l s o b e e n shown t o be a b l e t o r e l e a s e t h i s n e u r o h y p o p h y s i a l s t o r e o f v a s o p r e s s i n i n r e s p o n s e t o o s m o t i c s t i m u l i o r ' s t r e s s ' c o n d i t i o n s ( C h a r d , e t a l . , 1971; H o p p e n s t e i n , e t . a l . , 1968; Skowsky, e t a l . , 1973) . B o t h o s m o r e c e p t o r s a n d volume r e c e p t o r s h a v e b e e n f o u n d t o be f u n c t i o n a l a t b i r t h (Skowsky, e t a l . , 1973) . H o p p e n s t e i n , e t a l . (1968) f o u n d e l e v a t e d l e v e l s o f v a s o p r e s s i n i n i n f a n t s 18 a t b i r t h f o l l o w i n g n o r m a l v a g i n a l d e l i v e r y , b u t n o t a f t e r C a e s a r i a n b i r t h s ; t h e y c o n c l u d e d t h a t t h i s was a n endogenous r e l e a s e due t o f e t a l s t r e s s . V e r y h i g h l e v e l s o f v a s o p r e s s i n were a l s o f o u n d by C h a r d , et_ a l . (1971) i n u m b i l i c a l p l a s m a a t d e l i v e r y , l e v e l s a v e r a g i n g 550 m i c r o u n i t s / m l f o r a r t e r i a l a n d $0 m i c r o u n i t s / m l f o r v e n o u s b l o o d , w h i c h a r e among t h e h i g h e s t v a s o p r e s s i n l e v e l s f o u n d i n human p h y s i o l o g y . They a t t r i b u t e d t h i s t o f e t a l s o u r c e s . However, a t a n e v e n e a r l i e r s t a g e o f g e s t a t i o n , Skowsky, e t a l . (1973) f o u n d t h a t s h e e p f e t u s e s ( 0 . 8 2 t o 0.8*6* o f t e r m ) showed c i r c u l a t i n g l e v e l s o f v a s o p r e s s i n t h a t were a l m o s t f o r t y t i m e s g r e a t e r t h a n t h o s e o f t h e m o t h e r . T h e s e f i n d i n g s were c o n f i r m e d i n r h e s u s monkeys by t h e same r e s e a r c h e r s . 2 . F e t a l k i d n e y f u n c t i o n The f e t a l k i d n e y s b e g i n t o f u n c t i o n e a r l y i n g e s t a t i o n , a n d by t h e end o f t h e f i r s t t r i m e s t e r , c e r t a i n t u b u l a r s e c -r e t o r y a c t i v i t i e s a n d g l o m e r u l a r f i l t r a t i o n a r e w e l l u n d e r way; t h i r t y p e r c e n t o f g l o m e r u l i a r e m a t u r e by t h e f i f t h month o f g e s t a t i o n ( B a r n e s , 1 9 6 8 ) . The f e t a l k i d n e y p r o -d u c e s l a r g e amounts o f d i l u t e u r i n e , a n d t h e f e t u s h a s b e e n compared t o a n a d u l t w i t h d i a b e t e s i n s i p i d u s ( S e e d s , 1 9 6 5 ) . The u r i n e p r o d u c t i o n p e r k i l o g r a m o f body w e i g h t i s much more r a p i d i n . t h e m i d t e r m f e t u s . t h a n i n t h e a d u l t u n d e r b a s a l c o n d i t i o n s ( B a r n e s , 1 9 6 8 ) . U r i n e f o r m a t i o n i n t h e human b e g i n s i n t h e t h i r d month a n d i n c r e a s e s i n volume t o t e r m ( V e r n i e r & S m i t h , 1 9 6 8 ) . I n t h e s h e e p , u r i n e f l o w i s 19 m a x i m a l a t 0 . 8 o f t e r m a n d d e c l i n e s t o w a r d b i r t h , w i t h t h e f e t a l s h e e p m e t a n e p h r o s p r o d u c i n g h i g h e r u r i n e f l o w p e r u n i t body w e i g h t t h a n b a s a l f l o w i n t h e a d u l t . T h i s i s p o s s i b l y due t o t h e h i g h f i l t r a t i o n r a t e a n d l o w c a p a c i t y f o r t u b u l a r w a t e r r e a b s o r p t i o n ( A l e x a n d e r , e t a l . , 1 9 5 8 ) . T u b u l a r w a t e r r e a b s o r p t i o n does n o t e x c e e d 66 p e r c e n t u n t i l 0 . 8 9 o f t e r m , 8 0 . t o 90 p e r c e n t f r o m 0 . 9 0 t o 0 . 9 7 o f g e s t a t i o n , a n d e l e c -t r o l y t e r e a b s o r p t i o n i s h i g h e r t h a n t h a t o f w a t e r , s h o w i n g t h a t t h e m e t a n e p h r i c t u b u l e s o f t h e s h e e p a r e c a p a b l e o f d o i n g . o s m o t i c work e a r l y i n d e v e l o p m e n t ( A l e x a n d e r , e t a l . , 1 9 5 8 ) . The f a c t t h a t v a s o p r e s s i n does n o t seem t o a c t on t h e f e t a l k i d n e y may be i n l a r g e p a r t r e s p o n s i b l e f o r t h e v e r y h i g h u r i n e f l o w b e f o r e b i r t h , a s t h e k i d n e y b e f o r e b i r t h i s t h o u g h t t o be r e l a t i v e l y i n s e n s i t i v e t o v a s o p r e s s i n (McCance & Widdowson, 1 9 5 3 ) . The p e r i n a t a l k i d n e y i s n o t a s e f f i c i e n t a s t h e a d u l t , a n d b o t h t h e g l o m e r u l a r a n d t u b u -l a r f u n c t i o n s a r e low, so t h e newborn c a n n o t c o n c e n t r a t e u r i n e a s w e l l a s t h e a d u l t ( K l e i n m a n , 1 9 7 0 ) . However, t h e r e may be o t h e r f a c t o r s a f f e c t i n g t h e f e t a l k i d n e y ' s a b i l i t y t o c o n c e n t r a t e u r i n e ; t h e s e i n c l u d e : 1) t h e m a t u r a t i o n a l l e v e l o f t h e r e n a l t u b u l e s , 2) t h e a b i l i t y o f t h e t a r g e t s i t e s t o r e s p o n d t o hormone, 3 ) u r e a e x c r e t i o n , 4) s o l u t e l o a d a n d 5) t h e f l o w t h r o u g h t h e v a s a r e c t a ( K l e i n m a n , 1 9 7 0 ) . I t h a s b e e n s u g g e s t e d t h a t t h e l o w r e n a l c o n c e n t r a t i n g a b i l i t y o f t h e newborn c o u l d be a t t r i b u t e d t o a n a t o m i c a l 20 i m m a t u r i t y o f t h e t a r g e t o r g a n , o r i n s e n s i t i v i t y t o v a s o -p r e s s i n , r a t h e r t h a n t o a l a c k o f t h e hormone o r a n immature o r h y p o f u n c t i o n a l h y p o t h a l a m i c - n e u r o h y p o p h y s i s ( K e r p e l -F r o n i u s , 1970; Skowsky, e t a l . , 1973; V e r n i e r & S m i t h , 1 9 6 8 ) . T h i s l a r g e volume o f u r i n e f l o w c o u l d be c a p a b l e o f c a r r y i n g v a s o p r e s s i n f r o m t h e f e t a l c i r c u l a t i o n i n t o t h e a m n i o t i c s a c . I n t h i s way, i t w o u l d be a b l e t o a c c u m u l a t e a n d r e a c h t h e amnion, on w h i c h i t may e x e r t a c o n t r o l a n d p a r t i c i p a t e i n t h e r e g u l a t i o n o f a m n i o t i c f l u i d t u r n o v e r . STATEMENT OF THE PROBLEM S i n c e e a r l i e r work h a d s u g g e s t e d t h a t v a s o p r e s s i n m i g h t be c a p a b l e o f c a u s i n g w a t e r movement t h r o u g h t h e amnion, i t was i m p o r t a n t t o d e t e r m i n e w h e t h e r a t r u e p h y s i o l o g i c a l s y s t e m e x i s t e d . Good e v i d e n c e f o r a p h y s i o l o g i c a l f u n c t i o n f o r v a s o p r e s s i n i n t h e c o n t r o l o f f e t a l w a t e r exchange c o u l d be o b t a i n e d i f t h e hormone c o u l d be d e t e c t e d above t h r e s h o l d l e v e l s i n t h e a m n i o t i c f l u i d . To d a t e , t h e r e i s no l i t e r -a t u r e r e p o r t i n g v a s o p r e s s i n i n a m n i o t i c f l u i d , a n d t h e work r e p o r t e d h e r e a t t e m p t e d t o d e t e c t i t i n t h e f l u i d s o f g u i n e a p i g s , s h e e p and humans. 21 GENERAL MATERIALS AND METHODS A. Sample C o l l e c t i o n P r o c e d u r e s 1 . G u i n e a p i g s P r e g n a n t g u i n e a p i g s , b etween m i d t e r m a n d t e r m were a n e s t h e t i z e d w i t h e t h e r , a n d a m i d l i n e a b d o m i n a l i n c i s i o n was made. The u t e r i n e h o r n s were removed a n d f e t u s e s , i n t h e i r a m n i o t i c s a c s , were f r e e d by c u t t i n g t h r o u g h t h e u t e r -i n e w a l l a n d y o l k s a c . The s u r f a c e o f t h e a m n ion was q u i c k l y washed w i t h d i s t i l l e d w a t e r an d a 27 gauge n e e d l e was i n -s e r t e d a n d t h e f l u i d drawn i n t o t h e a t t a c h e d s y r i n g e . T h i s method a l l o w e d c o l l e c t i o n o f most, b u t n o t a l l , o f t h e f l u i d a r o u n d t h e f e t u s . The r e s i d u a l f l u i d was e s t i m a t e d t o be l e s s t h a n 0 . 2 o f a m l . O n l y a few s a m p l e s c o n t a i n e d b l o o d c o n t a m i n a t i o n , a n d t h e s e were d i s c a r d e d . The s a m p l e s were i m m e d i a t e l y p u t i n t o l a b e l l e d p l a s t i c v i a l s a n d s t o r e d i n t h e deep f r e e z e ( - 1 6 ° C) u n t i l a s s a y e d . I n c a s e s where t h e amount o f f l u i d p e r f e t u s was l e s s t h a n one m l , t h e f l u i d s f r o m s e v e r a l f e t u s e s were p o o l e d . I n f i v e c a s e s , s a m p l e s were c o l l e c t e d f r o m f e t u s e s whose m o t h e r s h a d b e en a l l o w e d no w a t e r f o r 24 t o 46* h o u r s p r i o r t o c o l l e c t i o n . 2 . Sheep A m n i o t i c f l u i d f r o m p r e g n a n t s h e e p was c o l l e c t e d by Mr. Dan Rurak a t O x f o r d U n i v e r s i t y . The f l u i d was t a k e n i n . F e b r u a r y , 1974, f r o m i n d w e l l i n g c a t h e t e r s i n t o t h e am-n i o t i c s a c s o f l a t e t e r m f e t u s e s . U n t r e a t e d , c o n t r o l 22 s a m p l e s were o b t a i n e d t o g e t h e r w i t h s a m p l e s d u r i n g a n d f o l l o w i n g t h e i n f u s i o n o f v a s o p r e s s i n a t 10 mU/minute i n -t o t h e a m n i o t i c s a c . One sample was t a k e n a t d e l i v e r y . The s a m p l e s were i m m e d i a t e l y f r o z e n a n d t r a n s p o r t e d on d r y i c e . 3. Human Human sa m p l e s were t a k e n f r o m t e r m p r e g n a n c i e s a t V a n -c o u v e r G e n e r a l H o s p i t a l by M i s s D a v i d s o n . A n e e d l e a t t a c h e d t o . a 50-ml s y r i n g e was i n t r o d u c e d v a g i n a l l y i n t o t h e a m n i o t i c sa c j u s t p r i o r t o d e l i v e r y , a n d t h e sample a s p i r a t e d . O n l y t h o s e s a m p l e s w i t h no v i s i b l e b l o o d c o n t a i m i n a t i o n were u s e d . I n one c a s e , human sample number 7, t h e f l u i d was o b t a i n e d a t 36 weeks ( 0 . 9 o f t e r m ) by a b d o m i n a l p u n c t u r e . T h e s e s a m p l e s were i m m e d i a t e l y f r o z e n a n d t r a n s p o r t e d on d r y i c e . They were k e p t f r o z e n a t - 1 6 ° C u n t i l a s s a y e d . B. B i o l o g i c a l A s s a y s 1. A n t i d i u r e t i c a s s a y A n t i d i u r e t i c a c t i v i t y was d e t e r m i n e d by a m o d i f i c a t i o n o f t h e w a t e r - l o a d e d , a l c o h o l - a n e s t h e t i z e d r a t a s s a y o f J e f f e r s , et_ a l _ . (1931) a s m o d i f i e d by Sawyer ( 1 9 6 1 ) , Rurak a n d P e r k s (1974)> a n d Rurak ( p e r s o n a l c o m m u n i c a t i o n ) . Female a l b i n o r a t s , w e i g h i n g 150 t o 200 grams were s t a r v e d f o r 12 t o 1&\ h o u r s p r i o r t o t h e a s s a y , b u t a l l o w e d f r e e a c c e s s t o water.. I n t r a p e r i t o n e a l i n j e c t i o n s o f 12$ EtOH a t . 5 $ o f body w e i g h t was g i v e n a s a n a n e s t h e t i c , f o l -l o w e d by o r a l i n t u b a t i o n o f a n e q u a l volume o f d i s t i l l e d 23 w a t e r . S u r g e r y was commenced a f t e r t h i r t y m i n u t e s t o a n h o u r . The a n i m a l was p l a c e d i n a s u p i n e p o s i t i o n a n d t h e i n n e r l e f t l e g was s h a v e d . A s u p e r f i c i a l i n c i s i o n was made an d t h e s a p h e n o u s v e i n was c a r e f u l l y s e p a r a t e d f r o m t h e a r t e r y . The l o w e r p o r t i o n o f t h e v e i n was t i e d o f f w i t h a s u t u r e . W i t h t h e a i d o f a d i s s e c t i n g m i c r o s c o p e , t h e v e i n was p u n c t u r e d w i t h a n e n t o m o l o g i c a l p i n a n d c a n n u l a t e d w i t h t h e b e v e l e d end o f PE 10 I n t e r m e d i c t u b i n g . The t u b i n g was a t t a c h e d t o a one ml s y r i n g e c o n t a i n i n g 0 . 9 % N a C l s a l i n e , w h i c h was i n j e c t e d i n t o t h e v e i n t o i n s u r e t h a t t h e c a n n u -l a t i o n was c o m p l e t e . The c a n n u l a was s e c u r e d w i t h two s u t u r e s . T h i s was f o l l o w e d b y c a n n u l a t i o n o f t h e b l a d d e r . A h a l f - i n c h m i d - v e n t r a l i n c i s i o n was made t h r o u g h t h e s k i n a n d t h e a b d o m i n a l m u s c l e , a n d t h e b l a d d e r was e a s e d t h r o u g h t h e i n c i s i o n . A s m a l l h o l e was c u t i n a r e l a t i v e l y a v a s -c u l a r a r e a . The f l a r e d end o f a PE 200 I n t e r m e d i c c a n n u l a was i n s e r t e d t h r o u g h t h e c u t , a n d t i e d i n t o p l a c e w i t h a l i g a t u r e c l o s e t o t h e u r e t h r a l e x i t . The a b d o m i n a l i n c i s i o n was s u t u r e d . X y l o c a i n e (2% h y d r o c h l o r i d e , A s t r a Pharmaceu-t i c a l , M i s s i s s a u g , O n t a r i o ) was a p p l i e d t o t h e i n c i s i o n s i f t h e r a t a p p e a r e d t o be l i g h t l y a n e s t h e t i z e d . The p r e p a r e d r a t was t h e n h y d r a t e d a t a r a t e o f 5 m i s / h o u r t h r o u g h . t h e s a p h e n o u s v e i n c a n n u l a u s i n g a n LKB p e r i -s t a l t i c pump. The i n f u s i o n s o l u t i o n c o n t a i n e d 2% o r 4% EtOH ( t o m a i n t a i n a n e t h e s i a ) , 3«34 grams d e x t r o s e / 2 0 0 m i s , and 6 6 . 7 m i s 0 . 9 % N a C l s a l i n e / 2 0 0 m i s . Oxygen was blown 24 o v e r t h e n o s e a n d mouth o f t h e a n i m a l d u r i n g t h e a s s a y i f t h e b r e a t h i n g was l a b o r e d . The a s s a y was s t a r t e d when t h e r a t was p r o d u c i n g a c o n s t a n t , r e g u l a r f l o w o f u r i n e o f a p p r o x i m a t e l y 0 . 5 t o 1 . 0 m l / m i n u t e . T h i s was u s u a l l y 30 t o 90 m i n u t e s a f t e r t h e s u r g e r y . I n j e c t i o n s were g i v e n v i a t h e s a p h e n o u s v e i n c a n n u l a , a n d were between 0 . 0 1 a n d 0 . 9 m i s . The i n j e c t i o n s were g i v e n a t 30 t o 60 m i n u t e i n t e r v a l s , a l l o w i n g t h e r e s t -i n g f l o w t o s t a b i l i z e f o l l o w i n g a r e s p o n s e . R e s p o n s e s t o i n j e c t i o n s were r e c o r d e d on a smoked Kymograph drum b y _ t h e f o l l o w i n g method: t h e c a n n u l a t e d b l a d d e r was c o n n e c t e d t o a d r o p c o u n t e r (B. 150 d r o p t u b e , B. 151 e l e c t r o n i c d r o p r e c o r d i n g u n i t a n d B. 152/3 Gaddum d r o p t i m e r ; C. F. P a l m e r ) . The d r o p c o u n t e r i n c o r p o r a t e d a w r i t i n g l e v e r , w h i c h r e s t e d a g a i n s t a smoked Kymograph drum (C. F. P a l m e r ) . The w r i t i n g l e v e r was a c t i v a t e d , a n d began t o r i s e s l o w l y when a d r o p o f u r i n e b r i d g e d t h e two e l e c -t r o d e s o f t h e d r o p c o u n t e r . As s o o n a s t h e d r o p f e l l f r o m t h e e l e c t r o d e s , t h e w r i t i n g l e v e r f e l l r a p i d l y b a c k t o t h e b a s e l i n e . I n t h i s way, t h e sweep o f t h e l e v e r was d i r e c t l y p r o p o r t i o n a l t o t h e l e n g t h o f t i m e t h a t t h e u r i n e d r o p l e t s p a n n e d t h e e l e c t r o d e s ; t h e r e f o r e , a d e c r e a s e i n u r i n e f l o w r e g i s t e r e d on t h e smoked drum a s a r i s e i n t h e s t r o k e s r e -c o r d e d . A p p r o x i m a t e l y one o u t o f t h r e e a n i m a l s t h a t were p r e -p a r e d p r o d u c e d a u r i n e f l o w w h i c h met t h e r e q u i r e m e n t s o f t h e a s s a y . 25 2. V a s o p r e s s o r a s s a y The r a t v a s o p r e s s o r a s s a y was c a r r i e d o u t a c c o r d i n g t o t h e method o f L a n d g r e b e , M a c a u l a y a n d W a r i n g (1946), a s m o d i f i e d by D e k a n s k i (1952) a n d d e s c r i b e d by Sawyer ( 1 9 6 l ) . R a t s w e i g h i n g 250 t o 300 grams were a n e s t h e t i z e d by s u b c u t a n e o u s i n j e c t i o n o f u r e t h a n e (175 mg/100 grams body w e i g h t ; M a t h e s o n , Coleman a n d B e l l C o . ) . A t t h e same t i m e , a n a d r e n e r g i c b l o c k i n g a g e n t , p h e n o x y b e n z a m i n e h y d r o c h l o r i d e ( D i b e n z y l e n e ; S m i t h , K l i n e a n d F r e n c h C o . ) , was i n j e c t e d t o l o w e r t h e b l o o d p r e s s u r e a n d t o b l o c k p r e s s o r r e s p o n s e s t o h y p e r t e n s i v e a g e n t s , s u c h a s a d r e n a l i n , n o r - a d r e n a l i n a n d h i s t a m i n e ( D e k a n s k i , 1952). The a n i m a l s were r e a d y f o r s u r g e r y 30 t o 45 m i n u t e s a f t e r t h e i n j e c t i o n o f t h e a n e s t h e t i c . The s k i n on t h e t h r o a t was c u t a n d t h e r i g h t j u g u l a r v e i n was l o c a t e d a n d f r e e d f r o m c o n n e c t i v e t i s s u e . Two l i g a t u r e s were p l a c e d a r o u n d t h e v e i n , a n d t h e one d i s t a l t o t h e h e a r t was t i e d . The v e i n was p u n c t u r e d w i t h a 22 gauge n e e d l e , t h e n e e d l e w i t h d r a w n a n d a c a n n u l a (PE 10 I n t e r m e d i c , C l a y Adams Co.) was i n s e r t e d t h r o u g h t h e p u n c t u r e . The c a n n u l a was s e c u r e d i n p l a c e by t y i n g b o t h l i g a t u r e s . The c a r o t i d a r t e r y was c a n n u l a t e d i n t h e same manner d e s c r i b e d f o r t h e j u g u l a r v e i n e x c e p t t h a t PE 50 I n t e r m e d i c t u b i n g was u s e d . To p r e v e n t t h e f o r m a t i o n o f . b l o o d c l o t s d u r i n g t h e a s s a y , 0.5 mg/100 grams body w e i g h t o f h e p a r i n ( H e p a r i n 26 sodium, Connaught M e d i c a l R e s e a r c h L a b o r a t o r i e s , T o r o n t o ; L o t . 3 4 9 - 1 ) , d i s s o l v e d i n 0 . 9 $ NaCI s o l u t i o n , was i n j e c t e d i n t r a v e n o u s l y . I n j e c t i o n s o f t h e s t a n d a r d a n d t h e unknown s o l u t i o n s were g i v e n t h r o u g h t h e j u g u l a r c a n n u l a a n d washed i n w i t h 0 . 2 ml o f 0 . 9 $ NaCI. The b l o o d p r e s s u r e was r e c o r d e d f r o m t h e c a r o t i d c a n n u l a by means o f a S t a t h a m 23 AA t r a n s d u c e r , c o n n e c t e d , t o a Beckman d y n o g r a p h r e c o r d e r . C. I o n Exchange C h r o m a t o g r a p h y a n d A n a l y t i c a l Methods 1. I o n exchange c h r o m a t o g r a p h y a ) P r e c y c l i n g o f t h e g e l F o l l o w i n g t h e m a n u f a c t u r e r ' s d i r e c t i o n s f o r u s e , t w e n t y grams o f CM-25 Sephadex ( P h a r m a c i a F i n e C h e m i c a l s AB, U p p s a l a , Sweden; L o t 4 6 4 ) were s u s p e n d e d i n e x c e s s 0 . 5 M ammonium h y d r o x i d e a n d a l l o w e d t o s w e l l f o r two d a ys a t room t e m p e r a t u r e . T h i s amount was s u f f i c i e n t f o r t h e b u i l d i n g o f two columns, a n d t h e e x t e n d e d s w e l l i n g t i m e a l l o w e d f o r t h e c o u n t e r i o n t o be c h a n g e d f r o m s o d i u m t o ammonium. A f t e r s e d i m e n t a t i o n a n d d e c a n t a t i o n , t h e g e l was r e s u s p e n d e d i n 0 . 0 2 M ammonium a c e t a t e b u f f e r , pH 5 . T h i s was r e p e a t e d s i x t o t e n t i m e s u n t i l t h e pH o f t h e d e c a n t e d f l u i d was 5 a n d t h e c o n d u c t i v i t y m e a s u r e d t h e same a s f o r a 0 . 0 2 M s o l u t i o n . b) B u f f e r . s o l u t i o n . Ammonium a c e t a t e b u f f e r was u s e d a n d p r e p a r e d i n t h e f o l l o w i n g manner. A s t o c k s o l u t i o n o f 0 . 2 M a c e t i c a c i d 27 was t i t r a t e d t o pH 5 w i t h ammonium h y d r o x i d e a n d d i l u t e d w i t h d i s t i l l e d w a t e r t o t h e r e q u i r e d m o l a r i t y (0.02 M), u s i n g t h e measurements o f e l e c t r i c a l c o n d u c t i v i t y a s a r a p i d a n d c o n v e n i e n t method t o d e t e r m i n e c o n c e n t r a t i o n . c ) P r e p a r a t i o n o f t h e i o n exchange columns T w e n t y - f i v e ml b u r e t t e s (1.0 cm i n d i a m e t e r ) were c u t t o be no l o n g e r t h a n 1$ cm, a n d t h e c u t end was f i r e - p o l i s h e d . The b u r e t t e was f i l l e d w i t h t h e s t a r t i n g b u f f e r , t h e b o t t o m 0.0$ cm was p a c k e d w i t h g l a s s w o o l , a n d a few g l a s s beads were p l a c e d on t o p . Then t h e CM Sephadex g e l s l u r r y was s t i r r e d i n t o t h e column f r o m a f u n n e l w i t h t h e a i d o f a n e l e c t r i c m i x e r . The p a c k e d column was washed w i t h 15 t o . 2 0 t i m e s i t s v o i d volume w i t h s t a r t i n g b u f f e r , u n t i l t h e c o n d u c t i v i t y . o f t h e e l u a t e was i d e n t i c a l t o t h a t o f t h e s t a r t i n g b u f f e r . P r e p a r e d columns were s t o r e d i n t h e c o l d room (4° C) u n t i l u s e d . d) P r e p a r a t i o n o f t h e sample . A l l human s a m p l e s were thawed by i m m e r s i o n i n warm w a t e r . They were t h e n spun i n a r e f r i g e r a t e d c e n t r i -f u g e . (4° C) a t 5,000 rpm f o r 30 m i n u t e s t o remove a n y c y t o -l o g i c a l c o n t a m i n a t i o n . A f t e r c a r e f u l d e c a n t a t i o n , t h e c o n d u c t i v i t y a n d pH o f t h e s a m p l e s were a d j u s t e d w i t h d i s -t i l l e d w a t e r a n d a c e t i c a c i d t o be i d e n t i c a l w i t h t h a t o f t h e . s t a r t i n g , b u f f e r . The sample was t h e n l o a d e d o n t o t h e c o l u m n . a t a f l o w r a t e n o t e x c e e d i n g 20 m i s / h o u r . F o r t h e l o a d i n g a n d e l u t i n g , a B u c h l e r D e k a s t a l t i c pump ( F o r t L e e , 28 N. J . ) was u s e d . F i f t y m i s o f t h e sample were g e n e r a l l y d i l u t e d so t h a t a p p r o x i m a t e l y 300 m i s were l o a d e d o n t o t h e col u m n . e) E l u t i o n o f b i o l o g i c a l a c t i v i t y A t t h e c o m p l e t i o n o f t h e l o a d i n g p r o c e d u r e , t h e column was washed w i t h s t a r t i n g b u f f e r f o r 3 0 m i n u t e s . A f t e r t h e w a s h i n g , a g r a d i e n t m i x e r was a t t a c h e d t o t h e column. A b e a k e r c o n t a i n i n g e x c e s s 0 . 4 M ammonium a c e t a t e b u f f e r , pH 8 , was c o n n e c t e d t o a B u c h l e r m u l t i - c h a n n e l pump, e m p t y i n g i n t o a 100 ml m i x i n g chamber o f s t a r t i n g b u f f e r , pH 5 , 0 , 0 2 M, w h i c h was m a g n i t i c a l l y s t i r r e d t h r o u g h o u t t h e e l u t i o n . A s e c o n d c h a n n e l o f t h e pump c o n n e c t e d t h e m i x i n g chamber t o t h e column and s o l u t i o n s were f e d t h r o u g h an 18 gauge n e e d l e a t t a c h e d t o t h e t o p o f t h e column w i t h a c o r k . The pump was s t a r t e d a t a r a t e o f 10 m i s / h o u r , a n d t h e . e l u a t e . w a s c o l l e c t e d by a n LKB f r a c t i o n c o l l e c t o r i n 2 ml f r a c t i o n s . The t u b e s c o n t a i n i n g t h e e l u a t e s were c o v e r e d w i t h P a r a f i l m a n d s t o r e d a t 4 ° C u n t i l t h e y were a s s a y e d . 2 . C o n d u c t i v i t y a n d pH measurements The pH was m e a s u r e d w i t h a n I n s t r u m e n t L a b o r a t o r y I n c . pH/mV E l e c t r o m e t e r , M o d e l 245 t y p e pH m e t e r . The c o n d u c -t i v i t y was me a s u r e d by means o f a R a d i o m e t e r t y p e CDM 2d c o n d u c t i v i t y m e t e r . 3.. C a l c u l a t i o n o f b i o l o g i c a l a c t i v i t y A n t i d i u r e t i c r e s p o n s e s were q u a n t i f i e d b y m e a s u r i n g t h e a r e a o f t h e r e s p o n s e on t h e kymograph r e c o r d , a n d 29 e x p r e s s i n g t h e v a l u e a s a p e r c e n t a g e o f a p a r a l l e l , s t a n d -a r d p e r i o d o f r e s t i n g f l o w . The measurement o f a r e a was made by a g r a v i m e t r i c method, w h i c h u s e d p a p e r o f a u n i f o r m t h i c k n e s s . The p e r c e n t d e c r e a s e i n u r i n e f l o w was c a l c u -l a t e d , a n d l o g dose r e s p o n s e c u r v e s were u s e d t o o b t a i n t h e a c t i v i t y i n r e l a t i o n t o t h e v a s o p r e s s i n s t a n d a r d r e -s p o n s e s . D. Hormone P r e p a r a t i o n s 1. V a s o p r e s s i n The v a s o p r e s s i n u s e d i n t h e p r e s e n t s t u d i e s a s a s t a n d -a r d , c o n s i s t e d o f P i t r e s s i n , a n a r g i n i n e / l y s i n e v a s o p r e s s i n p r e p a r a t i o n ( P i t r e s s i n , P a r k e D a v i s Co., L o t s Ck 217, E j 112 a n d Ga 103). . U n l e s s o t h e r w i s e s t a t e d , t h i s w i l l be r e f e r -r e d , t o a s v a s o p r e s s i n . T h i s i s a n aq u e o u s s o l u t i o n a t a c o n c e n t r a t i o n o f 20 p r e s s o r u n i t s / m l . S t a n d a r d s o l u t i o n f o r t h e a n t i d i u r e t i c a s s a y was d i l u t e d 1:40,000 w i t h 0.9$ N a C I . s a l i n e . . F o r t h e v a s o p r e s s o r a s s a y , a 1:400 d i l u t i o n was u s e d . F r e s h s t a n d a r d s were made f o r e a c h a s s a y . 2. A n g i o t e n s i n I I F o r t h e a s s a y s i n w h i c h t h e r e s p o n s e o f a n g i o t e n s i n w a s . t e s t e d , ( A s p \ I l e ^ ) - a n g i o t e n s i n I I ( S p e c t r u m M e d i c a l I n d u s t r i e s , I n c . , L.A., C a l i f o r n i a ; L o t number 208009) was u s e d . One mg o f t h i s d r y . p r e p a r a t i o n was d i s s o l v e d i n one l i t e r o f 0.9$ NaCI s a l i n e . T h i s was u s e d a s a s t o c k s o l u -t i o n , a n d s t o r e d i n i n d i v i d u a l s y r i n g e s i n t h e deep f r e e z e 30 ( - 1 6 ° C ) , When u s e d i n t h e a n t i d i u r e t i c a s s a y , i t was thawed s l o w l y a n d f u r t h e r d i l u t e d w i t h s a l i n e t o t h e n e c e s -s a r y c o n c e n t r a t i o n . E . Sodium T h i o g l y c o l l a t e D e a c t i v a t i o n o f V a s o p r e s s i n I n a c t i v a t i o n o f a c t i v e a n t i d i u r e t i c f r a c t i o n s w i t h s o d i u m t h i o g l y c o l l a t e was c a r r i e d o u t a c c o r d i n g t o t h e method o f Vog t ( 1 9 5 3 ) . A f r e s h s o l u t i o n o f 0 . 1 M sodium t h i o g l y c o l l a t e was made by m i x i n g e q u a l v o l u m e s o f 0 . 2 M s o d i u m b i c a r b o n a t e a n d 0 . 2 M t h i o g l y c o l l i c a c i d . T h i s was a d d e d t o t h e e x t r a c t e d f r a c t i o n s i n a r a t i o by volume o f 1 p a r t o f . s o d i u m t h i o g l y c o l l a t e t o 9 p a r t s o f e l u a n t ; t h e f i n a l i n c u b a t i o n c o n c e n t r a t i o n was 0 . 0 1 M s o d i u m t h i o -g l y c o l l a t e . The i n c u b a t e was a d j u s t e d t o pH 7 a n d a l l o w e d t o s t a n d . f o r a t l e a s t two h o u r s a t room t e m p e r a t u r e . Con-t r o l i n a c t i v a t i o n s were c a r r i e d o u t on t h e v a s o p r e s s i n s t a n d a r d i n a l l c a s e s . 31 SECTION I COLLECTION AND ASSAY OF UNCONCENTRATED AMNIOTIC FLUID INTRODUCTION There have been no controls found to date f o r the regu-l a t i o n of amniotic f l u i d turnover, and the f l u i d accumulates throughout gestation against a l l known ex i s t i n g gradients. Vasopressin has been suggested as a possible hormonal i n -fluence, and i t has been discussed how t h i s hormone may be able to a f f e c t the membrane, to help regulate the passage of water. Therefore, i t i s important to see i f vasopressin can be detected i n the amniotic f l u i d . Although there are several assays available f o r the detection of vasopressin, the a n t i d i u r e t i c assay was chosen fo r t h i s work, due to i t s high s e n s i t i v i t y . The dose of vasopressin needed to increase blood pressure i s 100 time larger than that needed to cause an a n t i d i u r e s i s (Thorn, 1958). Rats under ethanol anesthesia, with a constant water load, are the most sensitive of the a n t i d i u r e t i c assay prep-arations, with a s e n s i t i v i t y of 0.001 to 0.005 mU/ml (Dicker, 196l). . Ethanol anesthesia was chosen because i t w i l l allow a water d i u r e s i s , unlike many other anesthetic agents, which stimulate the neurohypophysis or i n t e r f e r e with renal func-t i o n (Walker, 1967). It has also been shown that alcohol prevents endogenous vasopressin release (Ginsburg & Brown, 1957). This inve s t i g a t i o n was undertaken to determine whether measurable amounts of vasopressin could be detected i n the 32 u n t r e a t e d a m n i o t i c f l u i d o f g u i n e a p i g s , s h e e p a n d humans. I n t h e g u i n e a p i g , a m n i o t i c f l u i d was c o l l e c t e d f r o m v a r i o u s s t a g e s o f g e s t a t i o n between m i d - t e r m a n d b i r t h , t o s e e i f t h e r e . w e r e any v a r i a t i o n s c o r r e l a t e d w i t h t h e age o f t h e f e t u s . I t was t h o u g h t t h a t t h e l e v e l s o f v a s o p r e s s i n i n t h e a m n i o t i c f l u i d m i g h t be h i g h enough t o be d e c t e c t a b l e w i t h o u t e x t r a c t i o n o r c o n c e n t r a t i o n . T h i s was b a s e d on t h e f i n d i n g s o f Ames ( 1 9 5 3 ) , who f o u n d l e v e l s o f v a s o p r e s s i n i n t h e u r i n e o f newborn i n f a n t s w h i c h , a t 1 .25 mU/ml, were a l m o s t a t p r e s s o r l e v e l s . T h i s w o u l d be 100 t i m e h i g h e r t h a n t h e t h r e s h o l d f o r t h e a n t i d i u r e t i c a s s a y . F u r t h e r , A l e x a n d e r a n d N i x o n (1961) d e t e c t e d c o n s i d e r a b l e amounts o f a n t i d i u r e t i c m a t e r i a l i n t h e u r i n e o f i n f a n t s l e s s t h a n t h r e e d a y s o l d , who h a d h a d f l u i d w i t h h e l d f o r e i g h t h o u r s . T h e s e i n f a n t s , however, r e s p o n d e d p o o r l y , i f a t a l l , t o exogenous v a s o p r e s s i n . I t h a s a l s o b e e n shown t h a t t h e f e t u s r e s p o n d s t o h y p e r t o n i c s a l i n e w i t h an i n c r e a s e i n endogenous v a s o p r e s s i n r e l e a s e (Skowsky, e t a l . , 1 9 7 3 ) . T h e r e f o r e , s e v e r a l a t t e m p t s were made t o i n d u c e v a s o p r e s s i n o u t p u t f r o m t h e f e t u s by de-h y d r a t i n g t h e mo t h e r f o r 24 o r 1+8 h o u r s . I t was h o p e d t h a t t h i s c o u l d c r e a t e a n o s m o t i c s t r e s s w i t h i n t h e f e t u s , w h i c h w o u l d r e s u l t i n a n i n c r e a s e d o u t p u t o f v a s o p r e s s i n . T h i s m i g h t r e s u l t i n m e a s u r a b l e q u a n t i t i e s o f t h e hormone i n t h e a m n i o t i c f l u i d , i f t h e l e v e l s i n t h e . u n t r e a t e d s a m p l e s were n o t s u f f i c i e n t l y h i g h f o r d e t e c t i o n . 33 Amniotic fluid samples from sheep, which had been ob-tained under varying conditions were also tested. There were routine samples, collected from indwelling catheters, along with samples taken during and after infusion of vaso-pressin into the amniotic sac. A limited number of unconcentrated amniotic fluid samples from near-term human fetuses were also assayed for the possible detection of vasopressin. RESULTS A. Studies of Untreated Guinea Pig Amniotic Fluid The data pertaining to the collection of guinea pig amniotic fluid samples at various stages of gestation from mid-term to term, including the volumes and characteristics of the samples, are shown in Table I. Fetal guinea pig ages were estimated according to Draper (1920) based on length and weight, and the ages are expressed as a propor-tion of term with 0 equalling conception and 1.0 being term. 1. Volume of fluid The volumes of the fluids collected from guinea pigs are shown graphically in Figure 1. Anbapproximately bell-shaped curve can be estimated from the data in the second half of gestation. The points from the fetuses of 0.75 and 0.78 of term were not included on the graph because these samples were apparently anomalous by more than one criterion (see later). 34 TABLE I GUINEA P I G AMNIOTIC FLUID COLLECTION AND ASSAY DATA Age Volume pH C o l o r .51 .54 .54 .55 ,59 .65 .66 .68 .69 .70 .75 .78 1.0 1.75 2.5 3.0 3.2 3.0 2.5 2.0 2.5 0.5 2.0 3,5 4.0 5.0 4.5 4.0 5.0 4.5 4,5 5.0 4.5 5.0 4,5 4.0 4.0 2.0 1.0 1,5 1.0 1.0 — • c l e a r — c l e a r 9.5 c l e a r 9.6 c l e a r 9.7 c l e a r 9.8 c l e a r 9.7 c l e a r 9.6 c l e a r 9.8 c l e a r — y e l l o w — y e l l o w 9.8 c l e a r 9.6 p a l e y e l l o w 9.6 p a l e y e l l o w 9.6 p a l e y e l l o w 9.3 9~6 " 9.3 y e l l o w 9*4 y e l l o w 9.4 — 9.6 — 9.2 : 9.7 — y e l l o w p a l e y e l l o w p a l e y e l l o w p a l e y e l l o w p a l e y e l l o w Number o f i n j e c t i o n s 1 2 R e s u l t Lp-H/ml) C o n c l u s i o n ' 1 2 1 1 4 1 2 3 1 <10 <10 <15 <10 <10 <io <15 <10 <10 <io n i l n i l n i l n i l n i l n i l n i l n i l n i l n i l 1 - s u g g e s t i o n t r a c e 1- r e s p o n s e 35l*J/ml 2- <10 n i l s u g g e s t i o n t r a c e 1 - s u g g e s t i o n t r a c e 1- <15 n i l <20 n i l <20 n i l 35 T a b l e I ( c o n t i n u e d ) ^ Number o f R e s u l t 2 Age Volume pH C o l o r i n j e c t i o n s (uU/ml) C o n c l u s i o n . 8 9 1 . 0 — c l e a r 1 < 2 0 n i l 3 . 0 9 . 0 c l e a r 2 . 5 8 . 7 v e r y p a l e y e l l o w 2 . 0 8 . 1 c l e a r . 8 9 4«5 — p a l e y e l l o w 3 . 0 7 . 9 p a l e y e l l o w 4 < 1 0 n i l . 9 2 1 .5 — — 3 < 1 0 n i l 3 . 0 — 5 < 1 0 n i l . 9 2 3 — — — 1 < 1 5 n i l • — — — 3 < 1 5 n i l . 9 7 3 — . — 2 <15 n i l . 9 9 1 . 0 — c l e a r 1 < 2 0 n i l 2 . 0 8 . 9 c l e a r 1.5 — p a l e y e l l o w 1 < 2 0 n i l . 5 0 -. 9 9 2 . 0 — 1 < 1 0 n i l Age i s shown a s a p r o p o r t i o n o f t e r m , w i t h 0 e q u a l t o c o n -c e p t i o n a n d 1 e q u a l t o t e r m . ' n i l ' i n d i c a t e s t h a t t h e q u a n t i t y o f v a s o p r e s s i n was b e l o w t h e t h r e s h o l d o f t h e a s s a y w h i c h i s shown i n t h e R e s u l t c o lumn. T h e s e s a m p l e s were a s s a y e d f r e s h , w i t h o u t h a v i n g b e e n s t o r e d . 36 F i g u r e 1 . V o l u m e s o f g u i n e a p i g a m n i o t i c f l u i d f r o m m i d - t e r m t o t e r m . E a c h p o i n t r e p r e s e n t s "the f l u i d f r o m one f e t u s , e x c e p t w h e r e numbers i n b r a c k e t s s p e c i f y o t h e r w i s e . 3 M (s i U J ) 3 lAI n 1 O A 37 2 . £ H The pH o f t h e f l u i d was n o t a l t e r e d a f t e r c o l l e c t i o n , a n d t h e s a m p l e s were s t o r e d a n d a s s a y e d a t t h e i r o r i g i n a l pH's. .These v a l u e s a r e shown i n T a b l e I . The r a n g e i s f r o m 7 . 9 t o 9 . 8 , w i t h a t r e n d t o w a r d s t h e f l u i d b e c o m i n g l e s s b a s i c n e a r e r t o t e r m . 3 . C o l o r The c o l o r o f t h e g u i n e a p i g a m n i o t i c f l u i d s a m p l e s a r e shown i n T a b l e I . T h e r e d i d n o t a p p e a r t o be a n y c o r -r e l a t i o n between c o l o r o f f l u i d a n d age o f f e t u s . 4 . A n t i d i u r e t i c e f f e c t U n c o n c e n t r a t e d g u i n e a p i g a m n i o t i c f l u i d s a m p l e s were a s s a y e d , a n d t h e number o f s a m p l e s t e s t e d f r o m e a c h s t a g e o f g e s t a t i o n a r e i n d i c a t e d i n T a b l e I . The volumes i n j e c t e d d u r i n g t h e a s s a y were u s u a l l y between 0 . 4 a n d 0 . 6 m i s , w i t h s a m p l e s between 0 . 1 a n d 0 . 9 b e i n g g i v e n on o c c a s i o n . T a b l e I shows t h a t i n 36 i n j e c t i o n s o f u n t r e a t e d f l u i d , 32 gave no r e s p o n s e , a n d v a s o p r e s s i n , i f p r e s e n t , w o u l d be a t a c o n c e n t r a t i o n l e s s t h a n 10 t o 1$ m i c r o u n i t s / m l . P r e p -a r a t i o n s were n o t u s e d f o r a s s a y i f t h e y d i d n o t r e s p o n d w i t h a c l e a r a n t i d i u r e t i c e f f e c t t o a t l e a s t 2$ m i c r o u n i t s . The a v e r a g e s e n s i t i v i t y o f t h e a s s a y t o v a s o p r e s s i n s t a n d -a r d ( P i t r e s s i n , P a r k e D a v i s Co.) was a b o u t t e n m i c r o u n i t s p e r i n j e c t i o n , w i t h some p r e p a r a t i o n s r e s p o n d i n g t o f i v e m i c r o u n i t s w i t h a c l e a r a n t i d i u r e s i s . No r e s p o n s e s were 3$ obtained from doses lower than f i v e microunits. In general, the s e n s i t i v i t y of each assay appeared to increase as the assay progressed, with the s e n s i t i v i t y often doubling a f t e r four or f i v e hours. On three occasions, there were suggestions of a re-sponse to injec t i o n s of the untreated guinea pig f l u i d which were too small to quantify accurately, and i n one case a clear response of approximately 35 microunits/ml was observed. Upon repeated i n j e c t i o n s of these active samples, no further responses could be obtained. It should be pointed out that these active samples were unusual i n a further way. The volumes of amniotic f l u i d were unusu-a l l y low when compared with those suggested by the general curve f o r amniotic f l u i d volumes given i n Figure 1 , and i t i s possible that a natural concentration of the f l u i d had occurred. Most of the samples used i n t h i s part of the i n v e s t i -gation had been frozen p r i o r to assay. Therefore, several attempts were made to assay f l u i d which had been f r e s h l y obtained from guinea pigs. The re s u l t s of these attempts are included i n Table I. In ten experiments, there was no in d i c a t i o n of any response to the fresh samples of amniotic f l u i d from fetuses of 0 . 9 2 or 0 . 9 7 of term. Table II shows the re s u l t s of.various controls which were run on the a n t i d i u r e t i c assay. There did not seem to 39 TABLE I I ANTIDIURETIC ASSAY CONTROLS C o n t r o l i n j e c t i o n R e s u l t (uU/ml) C o n c l u s i o n A. Volume 0.4 m i s 0.( 0.5 m i s 0.< 0.6 m i s 0.< 0.8 m i s 0.< f> NaCI s a l i n e $ NaCI s a l i n e fo NaCI s a l i n e $ NaCI s a l i n e 2 x 20 uU v a s o p r e s s i n s t a n d a r d i n 0.5 m i s 0.9$ NaCI s a l i n e 10 uU v a s o p r e s s i n s t a n d -a r d i n 0.8 m i s 0.9$ NaCI s a l i n e < 1 0 < 1 0 < 1 0 < 1 0 20 10 no d e t e c t a b l e e f f e c t no d e t e c t a b l e e f f e c t no d e t e c t a b l e e f f e c t no d e t e c t a b l e e f f e c t no change i n a n t i d i u r e s i s no change i n a n t i d i u r e s i s B. I n h i b i t i o n o r A u g m e n t a t i o n 3 x 20 JJ.U v a s o p r e s s i n s t a n d a r d i n 0.5 m i s a m n i o t i c f l u i d 20 20 uU v a s o p r e s s i n s t a n d -a r d i n 0.5 m i s a m n i o t i c 20 f l u i d ( i n c u b a t e d h r s ) no i n h i b i t i o n o r a u g m e n t a t i o n by-f l u i d no i n h i b i t i o n o r a u g m e n t a t i o n by f l u i d 40 be a n y a n t i d i u r e t i c r e s p o n s e t o c o n t r o l i n j e c t i o n s o f 0 . 9 % N a C l s a l i n e i n a n y o f t h e v o l u m e s u s e d , n o r was t h e r e a n y augmented r e s p o n s e e f f e c t when a known amount o f v a s o p r e s s i n was a d d e d t o t h e u n c o n e e n t r a t e d s a m p l e s . Volume e f f e c t s were a l s o t e s t e d by i n j e c t i n g known amounts o f s t a n d a r d v a s o p r e s s i n i n v o l u m e s o f s a l i n e e q u a l t o t h e amount o f t h e unknown sample i n j e c t i o n s . T h i s was n e c e s s a r y a s t h e volume o f s t a n d a r d r o u t i n e l y i n j e c t e d was 0 . 0 1 t o 0 . 0 6 m i s , o r a b o u t o n e - t e n t h o f t h e volume o f t h e unknown sample i n -j e c t i o n s . I t a l s o a p p e a r e d t h a t t h e a m n i o t i c f l u i d h a d no i n h i b i t o r y e f f e c t on s t a n d a r d v a s o p r e s s i n . No n o t i c e a b l e d e c r e a s e i n t h e r e s p o n s e t o t h e s t a n d a r d was o b s e r v e d when i t was m i x e d w i t h a m n i o t i c f l u i d a n d a l l o w e d t o i n c u b a t e f o r up t o one a n d a h a l f h o u r s . B. A s s a y s o f F l u i d f r o m D e h y d r a t e d M a t e r n a l G u i n e a P i g s The a m n i o t i c f l u i d s a m p l e s f r o m f e t u s e s whose m o t h e r s ha d b e e n d e h y d r a t e d f o r 24 o r 4$ h o u r s were a s s a y e d , a n d i n 2 9 . e x p e r i m e n t s , 27 n e g a t i v e r e s p o n s e s were o b t a i n e d . Two i n j e c t i o n s f r o m a . f e t u s o f 0 . 5 4 o f t e r m , whose m o t h e r h a d b e e n d e h y d r a t e d f o r 24 h o u r s , however, gave a s l i g h t s u g -g e s t i o n o f a r e s p o n s e . The r e s p o n s e was n o t r e p e a t a b l e , n o r was i t l a r g e enough t o be q u a n t i f i e d a c c u r a t e l y . The r e s u l t s o f t h e s e e x p e r i m e n t s , t o g e t h e r w i t h t h e c o l l e c t i o n d a t a , a r e summarized i n T a b l e I I I . 41 TABLE I I I DEHYDRATION EXPERIMENT COLLECTION AND ASSAY DATA A v e r a g e Age v o l u m e / f e t u s C o l o r 2 4-hour d e h y d r a t i o n . 5 4 2 . 0 c l e a r . 5 0 - . 9 9 — Number o f R e s u l t i n j e c t i o n s (uU/ml) C o n c l u s i o n ' 2 - s u g g e s -t i o n 4-<15 < 1 0 trace.. n i l n i l 48-hour d e h y d r a t i o n . 5 2 1.5 c l e a r 5 .78 3 . 0 v e r y y e l l o w 9 . 9 0 2 . 2 5 c l e a r 7 <15 < 1 0 < 1 0 n i l n i l n i l Age i s shown a s a p r o p o r t i o n o f t e r m , w i t h 0 e q u a l t o c o n -c e p t i o n a n d 1 e q u a l t o t e r m . ' n i l ' i n d i c a t e s t h a t t h e q u a n t i t y o f v a s o p r e s s i n was b e l o w t h e t h r e s h o l d o f t h e a s s a y w h i c h i s shown i n t h e R e s u l t c olumn. 42 T h e r e i s some doubt a s t o w h e t h e r , i n f a c t , t h e s e f e t u s e s were d e h y d r a t e d , a s t h e o s m o t i c p r e s s u r e s a n d v o l -umes o f a m n i o t i c f l u i d were s i m i l a r t o t h o s e o f c o n t r o l s o f t h e same s t a g e o f g e s t a t i o n . The o s m o t i c p r e s s u r e s o f t h e s e f e t u s e s a n d c o n t r o l s a r e shown i n T a b l e I V . TABLE I V OSMOTIC PRESSURES OF GUINEA PIG AMNIOTIC FLUID P r o p o r t i o n o f t e r m mOsM/L 2 4-hour d e h y d r a t i o n 0 . 5 4 285 c o n t r o l 0 . 5 5 290 4 8-hour d e h y d r a t i o n 0 . 9 0 288 c o n t r o l 0 . 9 0 287 C. A s s a y o f Sheep A m n i o t i c F l u i d Samples The r e s p o n s e s t o t h e s h e e p s a m p l e s i n d i c a t e d no r e -s p o n s e i n 12 e x p e r i m e n t s . Sample v o l u m e s o f 0 . 5 t o 0 . 6 m i s were u s e d . F o u r o r f i v e i n j e c t i o n s were made o f e a c h t y p e o f f l u i d s ample: t h e s e t y p e s were 1) r o u t i n e s a m p l e s , 2) t h o s e o b t a i n e d d u r i n g , a n d i m m e d i a t e l y f o l l o w i n g , v a s o p r e s -s i n i n f u s i o n a t 10 mU/minute, an d 3 ) one sample a t d e l i v e r y . I f a n y hormone was p r e s e n t , i t w o u l d have b e e n b e l o w t h e t h r e s h o l d o f t h e a s s a y , w h i c h was 10 t o 15 m i c r o u n i t s / m l . The pH o f t h e s e s h e e p s a m p l e s were m e a s u r e d , a n d i t was f o u n d t h a t t h e y h a d n o t b e e n a c i d i f i e d p r i o r t o b e i n g s h i p p e d f r o m E n g l a n d . The pH o f a r o u t i n e sample was 8 . 6 , 43 o f a sample d u r i n g t h e v a s o p r e s s i n i n f u s i o n , 8.5, a n d t h e pH o f t h e sample o b t a i n e d a t d e l i v e r y was 8.1. T h e r e f o r e , i t was p o s s i b l e t h a t t h e s e s a m p l e s c o u l d h a v e l o s t a l l v a s o p r e s s i n a c t i v i t y i n t h i s a l k a l i n e e n v i r o n m e n t , a n d t h e r e s u l t s must be i n t e r p r e t e d w i t h c a u t i o n . D. A s s a y o f U n t r e a t e d Human A m n i o t i c F l u i d T h r e e s a m p l e s o f human t e r m a m n i o t i c f l u i d were a s -s a y e d . T h e r e was no r e s p o n s e t o i n j e c t i o n s o f t h e s e s a m p l e s , w h i c h were g i v e n i n 0.5 t o 0.6 ml v o l u m e s . The pH o f t h e s e s a m p l e s were between 7«0 a n d 7«4, a n d were u n a d j u s t e d p r i o r t o a s s a y . DISCUSSION A. A n t i d i u r e t i c A s s a y A b i o l o g i c a l a s s a y s h o u l d be p r e c i s e , s p e c i f i c , s e n -s i t i v e , s i m p l e a n d r e l i a b l e ; however, no v a s o p r e s s i n a s s a y p r e s e n t l y a v a i l a b l e f i t s a l l o f t h e s e c r i t e r i a . The e t h a n o l a n e s t h e t i z e d r a t a s s a y i s h i g h l y s e n s i t i v e a n d s p e c i f i c , however i t i s t e c h n i c a l l y d i f f i c u l t a n d r e p e a t e d a s s a y s a r e n e c e s s a r y i f p r e c i s i o n i s r e q u i r e d (Sawyer, 196l). T h i s a s s a y shows r e l a t i v e l y good s p e c i f i c i t y f o r v a s o p r e s s i n , a n d t o g e t h e r w i t h i t s h i g h s e n s i t i v i t y , i t i s t h e a s s a y o f c h o i c e f o r body f l u i d s ( S m i t h , 1970). The t e c h n i q u e o f c o n s t a n t w a t e r l o a d i n g d i d n o t i m p r o v e t h e s e n s i t i v i t y o f 44 t h e a s s a y , b u t a l l o w e d t h e r a t p r e p a r a t i o n t o be u s e d f o r a l o n g e r t i m e ( S m i t h , 1970). The t h r e s h o l d d ose f o r t h i s a s s a y a p p e a r s t o depend on t h e m o d i f i c a t i o n s u s e d , a n d v a r i e s f r o m one t o t e n m i c r o u n i t s / i n j e c t i o n , a c c o r d i n g t o t h e i n -v e s t i g a t o r s c o n c e r n e d . The change i n u r i n e volume w o u l d a p p e a r , a p r i o r i , t o be t h e most v a l i d measure o f a n t i d i -u r e s i s ; o t h e r methods u s e d , s u c h a s c h a n g e s i n s p e c i f i c g r a v i t y , o s m o l a l i t y o r c o n d u c t i v i t y o f u r i n e a r e n o t a s r e l i a b l e o r s e n s i t i v e (Sawyer, 196l). D i c k e r (1953) f o u n d t h a t n e i t h e r i n t r a v e n o u s i n j e c t i o n s o f 0.9$ NaCI, n o r ex-t e r n a l d i s t u r b a n c e s s u c h a s n o i s e s o f v a r i o u s i n t e n s i t y , s q u e e z i n g , p r o d d i n g o r e v e n c u t t i n g o f f t h e t i p o f t h e r a t ' s t a i l h a d a n y s i g n i f i c a n t a n t i d i u r e t i c e f f e c t . B. U n t r e a t e d G u i n e a P i g A m n i o t i c F l u i d The r e s u l t s o f t h i s s e c t i o n seemed t o i n d i c a t e t h a t i f a n y v a s o p r e s s i n was i n . t h e a m n i o t i c f l u i d o f t h e g u i n e a p i g , i t w o u l d be a t a c o n c e n t r a t i o n b e l o w t h e d e t e c t a b l e l i m i t s o f t h e a s s a y (5 t o 10 m i c r o u n i t s / m l ) . One o f t h e l i m i t a t i o n s o f t h e a s s a y o f t h e u n t r e a t e d f l u i d m i g h t h a v e b e e n t h a t i t was n e c e s s a r y t o i n j e c t r e l a t i v e l y l a r g e v o l -umes o f t h e s a m p l e s . A l t h o u g h J e f f e r s , e t a l . (1931) s u g -g e s t e d t h a t i n j e c t i o n s s h o u l d n o t e x c e e d 0.5 m l / d o s e , Bon-j o u r a n d M a l v i n (1970) f o u n d t h a t w i t h i n j e c t i o n v olumes g r e a t e r t h a n 0.2$ m i s , s p u r i o u s r e s u l t s were o b t a i n e d . 45 Likewise, Dettelbach (1958) advised that i n j e c t i o n volumes were best kept between 0.05 and 0 . 2 mis, although a l i m i t e d number of 0.5 ml inj e c t i o n s could be given. So, many of the sample in j e c t i o n s were probably of too great a volume fo r optimal assay conditions. However, control i n j e c t i o n s of saline of equal volumes produced no apparent effect on the preparation. Neither was a volume eff e c t observed when vasopressin standard was added to a volume of saline equal to those of the sample i n j e c t i o n s . There may also have been complications from i n h i b i -t i n g or degrading substances which may be found i n unex-t r a c t e d . f l u i d . However, to date, the enzymes oxytocinase and yasopressinase have only been found to occur i n the amniotic f l u i d of primates (Rosenbloom, 1975). Further, Smith and Perks (unpublished observation) found that vaso-pressin which had been added to amniotic f l u i d of guinea pigs.retained i t s a c t i v i t y for as long as 12 hours when unfrozen. • Likewise,, i n these experiments, when standard vasopressin was mixed with amniotic f l u i d f o r as long as one and a half hours p r i o r to i n j e c t i o n , there was found to be no i n h i b i t i o n or augmentation of the response. It does not seem l i k e l y that enzymatic degradation was the cause.for the lack of an a n t i d i u r e t i c response. However, aqueous solutions of vasopressin have been found to lose considerable a c t i v i t y i f l e f t at room temperature f o r 24 46 h o u r s , a n d o n l y 10 p e r c e n t o f t h e o r i g i n a l a c t i v i t y i s l e f t a f t e r 72 h o u r s ( D e t t l e b a c h , 195$). I t i s u n l i k e l y t h a t e n z y m a t i c d e g r a d a t i o n was r e s p o n s i -b l e f o r t h e l a c k o f a c t i v i t y shown by t h e s a m p l e s . However, t h e s a m p l e s were s t o r e d i n t h e deep f r e e z e p r i o r t o a s s a y w i t h o u t h a v i n g b een a c i d i f i e d . V a s o p r e s s i n h a s a n o p t i m a l s t o r a g e pH o f 3.0 t o 5.0, and t h e pH o f t h e g u i n e a p i g a m n i o t i c f l u i d was b a s i c , b e i n g between 7.9 a n d 9.$. T h i s may have been r e s p o n s i b l e f o r a l o s s o f a c t i v i t y , i f t h e r e were t h r e s h o l d l e v e l s o f v a s o p r e s s i n i n t h e f l u i d i n i t i a l l y . T h i s was n o t i n d i c a t e d by t h e f a c t t h a t t h o s e s a m p l e s w h i c h were a s s a y e d a l m o s t i m m e d i a t e l y a f t e r c o l l e c t i o n d i d n o t show a n a n t i d i u r e t i c r e s p o n s e when t e s t e d . A r e f r a c t o r y phenomenon may have o c c u r r e d i n t h e few s a m p l e s w h i c h d i d show a r e s p o n s e . T h e s e r e s p o n s e s were a l w a y s e l i c i t e d by t h e f i r s t i n j e c t i o n o f t h e sample, w i t h s u b s e q u e n t i n j e c t i o n s g i v i n g no a n t i d i u r e s i s . T h i s seems t o be a v e r y d i f f e r e n t r e s p o n s e p a t t e r n t h a n t h a t shown by t h e v a s o p r e s s i n s t a n d a r d s . The a s s a y became i n c r e a s i n g l y more s e n s i t i v e t o t h e s t a n d a r d , a n d l e s s s e n s i t i v e t o t h o s e few s a m p l e s t h a t d i d r e s p o n d . I t may be a r g u e d t h a t t h e r e s p o n s e p a t t e r n o f t h e a m n i o t i c f l u i d s a m p l e s may be a t -t r i b u t e d t o a n a t u r a l l o s s o f a c t i v i t y a s t h e f l u i d was k e p t a t . r o o m t e m p e r a t u r e d u r i n g t h e a s s a y . However, t h e l e n g t h o f t i m e t h a t t h e s e s a m p l e s were l e f t a t room t e m p e r a t u r e was n o t c o n s i d e r a b l e (1 t o 5 h o u r s ) . Adamsons, e t a l . (1958) r e p o r t e d t h a t o n l y o n e - h a l f o f t h e a c t i v i t y o f h i g h l y p u r i f i e d v a s o p r e s s i n was l o s t i n 12 d a y s a t room t e m p e r a t u r e i n a n e u t r a l pH s o l u t i o n . F u r t h e r , t h e v a s o -p r e s s i n s t a n d a r d s were a l s o k e p t a t room t e m p e r a t u r e a n d d i d n o t show a s i m i l a r l o s s o f a c t i v i t y . T h e r e may a l s o have been a c o r r e l a t i o n b e t w e e n t h e f a c t s t h a t t h e s a m p l e s t h a t d i d show a c t i v i t y , 0 . 7 5 a n d O . 7 8 o f t e r m , were f r o m m o t h e r s who a p p e a r e d t o be a b n o r m a l i n . t e r m s o f t h e volume o f f l u i d a r o u n d t h e i r f e t u s e s . I t w o u l d a p p e a r f r o m t h e d a t a shown i n F i g u r e 1 t h a t t h e v o l -umes o f f l u i d f r o m t h e s e f e t u s e s s h o u l d h a v e b e e n h i g h e r . T h e r e may p e r h a p s h a ve b e e n some t y p e o f n a t u r a l ' s t r e s s ' w h i c h c a u s e d t h e d e h y d r a t i o n , l o w e r f l u i d v o l u m e s , a n d i n -c r e a s e d v a s o p r e s s i n l e v e l s . T h i s i s , however, s p e c u l a t i o n , a s t h e a c t i v i t y shown by t h e s e s a m p l e s was n o t d e m o n s t r a t e d t o be v a s o p r e s s i n . C. D e h y d r a t i o n E x p e r i m e n t s A c o m p a r i s o n o f t h e o s m o t i c p r e s s u r e s a n d v o l u m e s o f a m n i o t i c f l u i d b e t w e e n t h e c o n t r o l f e t u s e s a n d t h o s e whose m o t h e r s h a d b e e n d e h y d r a t e d i n d i c a t e t h a t t h e w a t e r l o s s was n o t f r o m t h e a m n i o t i c f l u i d . T h e r e was no d e c r e a s e i n t o t a l volume, n o r i n c r e a s e i n c o n c e n t r a t i o n t o i n d i c a t e t h a t any d e h y d r a t i o n h a d o c c u r r e d . The w a t e r l o s s may h a v e b e e n a b s o r b e d by t h e m o t h e r , who ' b u f f e r e d ' t h e f e t u s e s , 4 $ o r t h e f e t a l b o d y w a t e r compartment may h a v e a b s o r b e d t h e l o s s w i t h o u t d r a w i n g on t h e a m n i o t i c f l u i d . T h e r e was no i n d i c a t i o n o f a volume o r c o n c e n t r a t i o n d e b t f o r t h e f l u i d i t s e l f . F u r t h e r s t u d i e s w i t h d e h y d r a t i o n a t t e m p t s t o i n d u c e v a s o p r e s s i n r e l e a s e f r o m t h e f e t u s i n t o t h e a m n i o t i c f l u i d p o o l w i l l r e q u i r e more d i r e c t a n d e f f e c t i v e methods o f d e -h y d r a t i n g t h e f e t u s i t s e l f . I f t h e a m n i o t i c f l u i d does,, i n f a c t , s e r v e a s a r e s e r v e p o o l f o r t h e f e t u s , one w o u l d ex-p e c t t o f i n d t h a t i t w o u l d have been a f f e c t e d i f t h e f e t a l t i s s u e s became d e h y d r a t e d . T h i s a r e a n e e d s more e x t e n d e d i n v e s t i g a t i o n . However, i n t h e p r e s e n t s t u d y , i t was d e -c i d e d t o p u r s u e t h e s e a r c h f o r v a s o p r e s s i n by c o n c e n t r a t i o n o f t h e f l u i d r a t h e r t h a n t h e i n d u c e m e n t o f t h e hormone by means o f a n o s m o t i c s t r e s s . D. Sheep A m n i o t i c F l u i d The i n a b i l i t y t o d e t e c t a n y m e a s u r a b l e l e v e l s o f a c t i v i t y i n t h e s h e e p a m n i o t i c f l u i d s a m p l e s , e v e n t h o s e i n t o w h i c h v a s o p r e s s i n h a d b e e n i n f u s e d , seems t o i n d i c a t e t h a t t h e r e may be a n . i n h i b i t i n g o r d e a c t i v a t i n g s u b s t a n c e p r e s e n t i n t h e f l u i d . A l t e r n a t i v e l y , t h e s t o r a g e a n d t r a n s p o r t a t i o n c o n d i t i o n s were n o t o p t i m a l f o r v a s o p r e s s i n s t a b i l i t y , a n d may have r e s u l t e d i n t h e . l o s s o f any i n i t i a l a c t i v i t y . E n z y m a t i c d e g r a d a t i o n may have b e e n i n v o l v e d . However, a s p r e v i o u s l y d i s c u s s e d , v a s o p r e s s i n a s e h a s o n l y b e e n demon-s t r a t e d i n t h e a m n i o t i c f l u i d o f p r i m a t e s , t o d a t e . P e r h a p s 49 more i m p o r t a n t , t h e f l u i d s were t a k e n a n d f r o z e n one y e a r p r i o r t o t h e t i m e o f a s s a y , a n d t h e pH was u n a d j u s t e d when t h e s a m p l e s were s t o r e d . A l l o f t h e s a m p l e s h a d a pH b e -tween $.0 a n d 9.0, w h i c h i s f a r f r o m o p t i m a l f o r t h e m a i n -t e n a n c e o f v a s o p r e s s i n ' s a c t i v i t y . T h e s e s a m p l e s h a d a l s o b e e n t r a n s p o r t e d f r o m E n g l a n d , a n d v a r i a t i o n s i n s t o r a g e c o n d i t i o n s a n d t h e l e n g t h o f t i m e t h e y were s t o r e d may f e a s i b l y be c o n s i d e r e d s u f f i c i e n t t o d e s t r o y any o f t h e h o r m o n a l a c t i v i t y w h i c h may h a v e b e e n p r e s e n t . E . Human U n t r e a t e d A m n i o t i c F l u i d T h e r e was no a c t i v i t y f o u n d i n t h e few s a m p l e s o f human a m n i o t i c f l u i d w h i c h were a s s a y e d p r i o r t o c o n c e n -t r a t i o n . T h i s may be due t o s t o r a g e c o n d i t i o n s , f o r t h e sa m p l e s were k e p t a t t h e i r i n i t i a l pH o f 7.0 t o 7.4. How-e v e r , t h e s a m p l e s were f r o z e n a n d were o n l y s t o r e d f o r s h o r t p e r i o d s o f t i m e (one t o t h r e e w e e k s ) . E n z y m a t i c d e g r a d a t i o n a l s o does n o t a p p e a r t o be a l i k e l y e x p l a n a -t i o n , a s t h e r e was no d i m i n i s h m e n t o f r e s p o n s e when s t a n d -a r d v a s o p r e s s i n was i n c u b a t e d w i t h t h e f l u i d f o r up t o a n h o u r a n d a h a l f p r i o r t o i n j e c t i o n . The l a c k o f a c t i v i t y i s most l i k e l y due t o t h e l e v e l s o f t h e hormone b e i n g s u b -t h r e s h o l d t o t h e a s s a y . E a r l y e v i d e n c e i n d i c a t e d t h a t p e r i n a t a l u r i n e c o n t a i n e d h i g h l e v e l s o f v a s o p r e s s i n ( A l e x a n d e r & N i x o n , 196l; Ames, 50 1953). In addition, f e t a l urine i s known to be a major contributor to the amniotic f l u i d . Therefore, i t was thought that t h i s hormone might accumulate i n the f l u i d , and be present i n s u f f i c i e n t quantity to be detected without con-centration procedures. However, t h i s i n v e s t i g a t i o n f a i l e d to detect vasopressin i n the amniotic f l u i d of the guinea pig, sheep or human, at l e v e l s above 10 microunits/ml. It was therefore decided to attempt to concentrate the f l u i d i n order to detect any possible low quantities of a c t i v i t y which may be present. 51 SECTION I I CONCENTRATION OF ANTIDIURETIC MATERIAL FROM HUMAN AMNIOTIC FLUID INTRODUCTION The s t u d y o f v a s o p r e s s i n i n body f l u i d s h a s g e n e r a l l y b e e n c o m p l i c a t e d b y t h e n e c e s s i t y o f e s t a b l i s h i n g s a t i s -f a c t o r y t e c h n i q u e s f o r t h e e x t r a c t i o n and c o n c e n t r a t i o n o f t h e a c t i v e p r i n c i p l e . E x t r a c t i o n p r o c e d u r e s a r e n e c e s s a r y b e c a u s e body f l u i d s u s u a l l y c o n t a i n s u b s t a n c e s w h i c h may i n t e r f e r e w i t h most o f t h e a s s a y s w h i c h a r e u s e d f o r t h e d e t e c t i o n o f v a s o p r e s s s i n (Sawyer, 1 9 6 l ) . The n e c e s s i t y f o r a d e q u a t e c o n c e n t r a t i o n t e c h n i q u e s i n t h e s t u d y o f v a s o -p r e s s i n i s due t o t h e e x t r e m e l y l o w c i r c u l a t i n g l e v e l s o f t h e hormone. As l a t e a s 1 9 6 l , Sawyer r e p o r t e d t h a t t h e p h y s i o l o g i c . c o n c e n t r a t i o n o f v a s o p r e s s i n i n b l o o d was b e -l o w t h e r a n g e o f a v a i l a b l e a s s a y methods, e x c e p t u n d e r c o n d i t i o n s w h i c h e l e v a t e t h e c i r c u l a t o r y l e v e l s ( e . g . , a n e s t h e s i a , h e morrhage, e x c i t e m e n t , d e h y d r a t i o n o r d r u g s ) . More r e c e n t l y , however, w i t h t h e d e v e l o p m e n t o f i m p r o v e d e x t r a c t i o n a n d c o n c e n t r a t i o n t e c h n i q u e s a n d more s e n s i t i v e a s s a y methods, i t h a s b e e n p o s s i b l e t o d e t e c t a n d measure t h e s m a l l amounts o f v a s o p r e s s i n t h a t a r e b e l i e v e d t o be p h y s i o l o g i c a l . I t i s g e n e r a l l y a g r e e d t h a t t h e amount o f s y s t e m i c v a s o p r e s s i n i n a d u l t humans i s i n p i c o g r a m , o r - 1 2 10 M, q u a n t i t i e s — w i t h v a l u e s o f l e s s t h a n one t o f i v e 52 m i c r o u n i t s / m l b e i n g r e p o r t e d i n n o r m a l l y h y d r a t e d s u b j e c t s ( W a l k e r , 1 9 6 7 ) . I n t h e f e t u s , however, Skowsky, e t a l . (1973) f o u n d t h a t c i r c u l a t i n g l e v e l s o f v a s o p r e s s i n a t t h e t i m e o f d e l i v e r y were h i g h e r t h a n t h o s e p r e s e n t i n t h e m o t h e r . The f e t a l l e v e l s o f v a s o p r e s s i n i n t h e s e ex-p e r i m e n t s were a l m o s t h i g h enough t o c a u s e a p r e s s o r e f f e c t . I o n - e x c h a n g e g e l s have been u s e d t o e x t r a c t v a s o p r e s s i n f r o m b o t h b l o o d a n d u r i n e . V a s o p r e s s i n c a n be removed by columns o f c a t i o n - e x c h a n g e g e l o r c a r b o x y m e t h y l c e l l u l o s e p r e p a r e d i n . a c i d f o r m , a n d c a n be r e c o v e r e d b y e l u t i o n w i t h i n c r e a s i n g g r a d i e n t s o f pH a n d i o n i c s t r e n g t h ( S m i t h , 1 9 7 0 ) . A l t h o u g h s e v e r a l t y p e s o f g e l s have b e e n u s e d , CM-25 S e p h a -dex ( P h a r m a c i a ) was c h o s e n f o r t h i s i n v e s t i g a t i o n . T h i s g e l a l l o w s f o r s e p a r a t i o n o f t h e hormone b o t h on t h e b a s i s o f s i z e a n d i o n i c c h a r g e . As no a c t i v i t y c o u l d be d e t e c t e d w i t h i n t h e l i m i t s o f t h e a n t i d i u r e t i c a s s a y i n u n t r e a t e d g u i n e a p i g a m n i o t i c f l u i d s a m p l e s , i t was c o n s i d e r e d n e c e s s a r y t o employ a method o f c o n c e n t r a t i o n . I n t h i s s e c t i o n o f t h e i n v e s t i -g a t i o n , human a m n i o t i c f l u i d s a m p l e s were c o n c e n t r a t e d a n d a s s a y e d w i t h t h e r a t a n t i d i u r e t i c a s s a y i n a n a t t e m p t t o measure any a n t i d i u r e t i c a c t i v i t y w h i c h may have b e e n e l u t e d f r o m t h e c olumn. I t was f o u n d t o be n e c e s s a r y t o change t h e s o u r c e o f a m n i o t i c f l u i d a t t h i s p o i n t i n t h e i n v e s t i -g a t i o n , a s t h e amount o f f l u i d w h i c h , c o u l d be o b t a i n e d f r o m s m a l l r o d e n t s , s u c h a s t h e g u i n e a p i g , were s m a l l , a n d 53 large quantities were needed f o r the concentration experi-ments. Therefore, human amniotic f l u i d , which could be obtained i n 50-ml quantities, was used f o r the remainder of the study. After the eluants had been assayed, i t was essential to show that any extracted material which was active was physiochemically s i m i l a r to highly p u r i f i e d or synthetic vasopressin, and to demonstrate that the material showed changes i n response to a variety of appropriate physiologi-c a l circumstances (Chard, 1973). Sodium t h i o g l y c o l l a t e deactivation was used as a c r i t e r i o n f o r the i d e n t i f i c a t i o n of the eluted a c t i v i t y as vasopressin. The h a l f - l i f e of oxytocin i n 20 mM t h i o g l y c o l l a t e i s 10 minutes at 20°, and i s s i m i l a r f o r vasopressin (Martin & Schild, 1962). RESULTS A. Experiments with Standard Vasopressin In three experiments, a standard vasopressin prepara-tion.was applied to a CM-Sephadex column (1 x 13 cm), and various.gradient systems were tested f o r a suitable elution p r o f i l e . A standard vasopressin preparation ( P i t r e s s i n , Parke Davis Co.), of one or f i v e units, was run through the column to determine the e l u t i o n p r o f i l e and degree of concentration which could be obtained by t h i s method. Although the ac-t i v i t y of these standards were much higher than those 54 e x p e c t e d t o be e n c o u n t e r e d i n t h e f l u i d s , i t was n e c e s s a r y t o u s e d o s a g e s s u f f i c i e n t t o be d e t e c t a b l e by p r e s s o r a s s a y . I t w o u l d h a v e b e e n t o o t i m e - c o n s u m i n g t o t e s t a l l f r a c t i o n s by t h e a n t i d i u r e t i c method. One m i l l i l i t e r f r a c t i o n s were c o l l e c t e d f r o m t h e s e s t a n d a r d c o l u m n s , a n d t h e s e were a s -s a y e d u s i n g t h e r a t v a s o p r e s s o r a s s a y , t o d e t e r m i n e t h e p o s i t i o n o f e l u t e d a c t i v i t y . C o n d u c t i v i t y ( a s a c o n v e n i e n t a p p r o x i m a t i o n o f m o l a r i t y ) , a n d pH, were t h e n t e s t e d i n t h e d i f f e r e n t f r a c t i o n s so t h a t t h e a p p r o x i m a t e p o s i t i o n o f t h e a c t i v i t y c o u l d be a s s e s s e d p r i o r t o a n t i d i u r e t i c a s s a y . The r e c o v e r y f r o m t h e CM-Sephadex column was good, w i t h a b o u t 90$ o f t h e v a s o p r e s s i n b e i n g r e c o v e r e d u n d e r o p t i m a l c o n d i t i o n s . The c o n c e n t r a t i o n f a c t o r f o r t h e s t a n d -a r d v a s o p r e s s i n was 25 t i m e s , w i t h a n i n i t i a l l o a d i n g volume o f 100.mis, t h e a c t i v i t y was c o n c e n t r a t e d i n t o f o u r 1-ml f r a c t i o n s . The r e c o v e r y c u r v e f o r t h e f i v e u n i t s t a n d a r d i s g i v e n i n F i g u r e 2. The a c t i v i t y was e l u t e d between pH 1.0 a n d 7.5, a n d between m o l a r i t y o f 0.1$ and 0.22. B. E x p e r i m e n t s w i t h Human A m n i o t i c F l u i d 1. The s e p a r a t i o n o f a n a m n i o t i c f l u i d a n t i d i u r e t i c  s u b s t a n c e (AFAS) S e v e n human a m n i o t i c f l u i d s a m p l e s , t a k e n c l o s e t o d e l i v e r y , were e x t r a c t e d on CM-Sephadex, a s d e s c r i b e d i n t h e p r e v i o u s e x p e r i m e n t s . The r a t a n t i d i u r e t i c a s s a y was 55 F i g u r e 2. C o n c e n t r a t i o n o f a f i v e u n i t s t a n d a r d v a s o p r e s s i n l o a d on a CM-25 Sephadex column (1 x 13 cm). A c t i v i t y i s e x p r e s s e d a s mU/ml, a s d e t e r m i n e d on t h e r a t v a s o p r e s s o r a s s a y p r e p a r a t i o n . E a c h t u b e c o n t a i n e d one m i l l i l i t e r o f e l u a t e . E l u t i o n was c a r r i e d o u t w i t h a m o l a r i t y g r a d i e n t o f 0.02 t o 0.4 M, a n d a pH g r a d i e n t o f 5.0 t o S.O. 10 20 TUBE _ j — ^ 40 50 60 NUMBER ^ 56 used f o r the d e t e c t i o n of any a c t i v e m a t e r i a l . A n t i d i u r e t i c a c t i v i t y was detected i n a l l of the preparations,, and the l e v e l s of a c t i v i t y from a l l of the samples were s i m i l a r i n magnitude. The responses obtained on the kymograph record appeared.similar t o those of the standard vasopressin i n -j e c t i o n s . An example from human sample #7 i s shown i n Figure 3. The e l u t i o n p r o f i l e s of the amniotic f l u i d a n t i d i u r e t i c substance (AFAS) f o r each of the seven samples are shown i n Figure 4, along w i t h the recovery.graph of the one u n i t vasopressin standard ( F i g . 4, #8). Although the f r a c t i o n numbers are not the same i n a l l cases, t h i s i s due t o the f a c t t h a t the pump was run at speeds v a r y i n g from 10 t o 14 mis/hour, and the pH gradient v a r i e d between 5«0 t o 8.0 and 5.0 to 8.4. However, as can be seen on Figure 4, the ac-t i v i t y of a l l of the samples came o f f at a s i m i l a r pH and m o l a r i t y . The.average t o t a l a c t i v i t y from each human sample was 807 m i c r o u n i t s , w i t h a concent r a t i o n of approximately 200 microunits/ml. This would i n d i c a t e a concen t r a t i o n f a c t o r of at l e a s t 30 times, as unconeentrated human samples had an a c t i v i t y of l e s s than 10 microunits/ml, and some of the concentrated samples had a c t i v i t i e s measuring as high as 300 microunits/ml. I f these values are e x t r a p o l a t e d back, to determine the concent r a t i o n of the AFAS i n unconcentrated amniotic f l u i d , values of 8 t o 10 microunits/ml are obtained. 57 Figure 3• Portion of a rat a n t i d i u r e t i c assay record which shows the response pattern of standard vasopressin and a sample f r a c t i o n containing AFAS. The samples were from human sample #7, and i n j e c t i o n volumes were 0.1 ml. Standard vasopressin responses to 15 and 20 micro-unit i n j e c t i o n s are indicated f o r comparison. 57 A 5$ F i g u r e 4» C o m p a r i s o n o f t h e e l u t i o n p r o f i l e s o f t h e s e v e n human a m n i o t i c f l u i d s a m p l e s w i t h a one u n i t v a s o -p r e s s i n s t a n d a r d r e c o v e r y c u r v e f r o m a 1 x 13 cm CM-25 Sephadex column. A c t i v i t y i s e x p r e s s e d a s m i c r o u n i t s / m l a s d e t e r -m i n e d on t h e r a t a n t i d i u r e t i c a s s a y f o r t h e human s a m p l e s , a n d a s mU/ml f o r t h e v a s o p r e s s i n s t a n d a r d ( # 8 ) , a s d e t e r m i n e d on t h e r a t v a s o p r e s s o r a s s a y . E l u t i o n was c a r r i e d o u t w i t h a m o l a r i t y g r a d i e n t o f . 0 . 0 2 t o 0 . 4 M, a n d a pH g r a d i e n t o f 5 . 0 t o 3 . 0 . E a c h t u b e o f human sample c o n t a i n e d two m i l l i l i t e r s o f e l u a t e , . a n d e a c h t u b e o f s t a n d a r d v a s o p r e s s i n c o n t a i n e d one m i l l i l i t e r o f e l u a t e . 58A T U B E N U M B E R 59 T h i s v a l u e i s v e r y c l o s e t o t h e t h r e s h o l d o f t h e a s s a y , a n d i n most c a s e s w o u l d p r o b a b l y n o t be d e t e c t e d . However, i t i s n e a r enough t o d e t e c t a b l e l e v e l s t h a t i t may show a s u g g e s t i o n o f a r e s p o n s e on t h e more s e n s i t i v e a s s a y p r e p -a r a t i o n s . ( T h e r e were no d i f f e r e n c e s f o u n d i n t h e r e s u l t s f r o m t h e sample w h i c h h a d b e e n o b t a i n e d a t 36 weeks o f g e s t a t i o n by a b d o m i n a l p u n c t u r e . The t o t a l q u a n t i t y a n d c o n c e n t r a t i o n o f AFAS was s i m i l a r i n m a g n i t u d e t o t h o s e s a m p l e s w h i c h h a d b e en o b t a i n e d a t t e r m , by t h e v a g i n a l r o u t e . 2 . The p r o p e r t i e s o f t h e a m n i o t i c f l u i d a n t i d i u r e t i c  s u b s t a n c e (AFAS)  a ) D i s c r i m i n a t i o n b etween v a s o p r e s s i n a n d AFAS I n a l l o f t h e p r e p a r a t i o n s u s e d f o r a s s a y , t h e v a s o p r e s s i n s t a n d a r d s showed c o n s i s t e n t , d o s e - d e p e n d e n t r e s p o n s e s . However, u n l i k e v a s o p r e s s i n , t h e AFAS v a r i e d c o n s i d e r a b l y i n t h e r e s p o n s e s w h i c h were e l i c i t e d t o r e -p e a t e d i n j e c t i o n s o f a c o n s t a n t volume. V a r i a t i o n s were o b s e r v e d b o t h w i t h i n a n d between a s s a y s . I n $0 e x p e r i m e n t s , a n e g a t i v e r e s p o n s e was o b t a i n e d 24 t i m e s f r o m f r a c t i o n s w h i c h showed m e a s u r a b l e a c t i v i t y a t o t h e r t i m e s . T h e s e n e g a t i v e s do n o t i n c l u d e a n y l a c k o f r e s p o n s e o b t a i n e d a t a t i m e when t h e a c t i v i t y a p p e a r e d t o d i m i n i s h , b u t o n l y t h o s e e l i c i t e d p r i o r t o a d e f i n i t i v e r e s p o n s e . D oses o f 60 v a s o p r e s s i n i n t h e n o r m a l , a b o v e - t h r e s h o l d r a n g e s n e v e r showed s p o r a d i c a n d u n e x p e c t e d f a i l u r e s t o p r o d u c e a r e s p o n s e , a s shown by t h e unknown p r e p a r a t i o n s . b) Sodium t h i o g l y c o l l a t e d e a c t i v a t i o n I n two s e p a r a t e e x p e r i m e n t s , a n a t t e m p t was made t o i d e n t i f y t h e AFAS a s v a s o p r e s s i n , by i n c u b a t i n g i t w i t h 0.01 M s o d i u m t h i o g l y c o l l a t e . T h i s r e a g e n t i s known t o i n -a c t i v a t e n e u r o h y p o p h y s i a l p r i n c i p l e s . The a n t i d i u r e t i c s u b s t a n c e , w h i c h h a d been c o n c e n t r a t e d f r o m human a m n i o t i c f l u i d , was n o t i n a c t i v a t e d b y t h i s method, a l t h o u g h c o n t r o l v o l u m e s o f v a s o p r e s s i n ( P i t r e s s i n , P a r k e D a v i s C o . ) , w h i c h were r u n s i m u l t a n e o u s l y , were d e g r a d e d . I n t h e f i r s t o f t h e two d e g r a d a t i o n a t t e m p t s , t h e sample a n d c o n t r o l were i n c u b a t e d f o r two h o u r s a t room t e m p e r a t u r e . The r e s u l t s o f . t h e a s s a y o f t h e a c t i v i t y a r e shown i n F i g u r e 5. The a m n i o t i c f l u i d sample r e t a i n e d a l l o f i t s o r i g i n a l a c t i v i t y , w i t h t h e f r a c t i o n h a y i n g a n a v e r a g e a c t i v i t y o f 21$ m i c r o -u n i t s / m l p r i o r t o t h e t h i o g l y c o l l a t e i n c u b a t i o n , a n d 214 m i c r o u n i t s / m l f o l l o w i n g t h e e x p e r i m e n t . The v a s o p r e s s i n c o n t r o l showed a l o s s o f a t l e a s t p e r c e n t o f i t s a n t i -d i u r e t i c a c t i v i t y . The a s s a y showed a c l e a r a n t i d i u r e s i s i n r e s p o n s e t o a 10 m i c r o u n i t i n j e c t i o n o f s t a n d a r d p r i o r t o i n a c t i v a t i o n , a n d t h e $0 m i c r o u n i t i n j e c t i o n o f v a s o -p r e s s i n , w h i c h h a d b e e n i n c u b a t e d w i t h s o d i u m t h i o g l y c o l l a t e , r e s u l t e d i n a s u b t h r e s h o l d r e s p o n s e . 61 F i g u r e 5 . P o r t i o n o f . a n a n t i d i u r e t i c a s s a y r e c o r d s h o w i n g s o d i u m t h i o g l y c o l l a t e d e a c t i v a t i o n a t t e m p t . R e s p o n s e s t o f r a c t i o n #33, f r o m human sample #6, i s shown p r i o r t o , a n d f o l l o w i n g , a t w o - h o u r i n c u b a t i o n w i t h s o d i u m t h i o g l y c o l l a t e . S t a n d a r d v a s o p r e s s i n was a l s o i n c u b a t e d f o r two h o u r s , a n d t h e f i n a l r e s p o n s e t o a n i n j e c t i o n of 50 m i c r o u n i t s , shows a l o s s o f a t l e a s t 80fo o f a c t i v i t y . A l l human sample i n j e c t i o n s were 0 .1 ml i n v o l u m e . I n j e c t i o n s o f f r a c t i o n s #32 a n d #34 were n o t a p a r t o f t h e d e a c t i v a t i o n e x p e r i m e n t . 6IA 0. > < .2 tn CO *-CO + 3 Q. =- > o < CM CO Q. > < CO CO 62 When t h e s e c o n d d e g r a d a t i o n a t t e m p t w i t h s o d i u m t h i o -g l y c o l l a t e was done, t h e m i x t u r e s were i n c u b a t e d f o r two d a y s , a f t e r w h i c h t h e y were i n j e c t e d i n t o a new a n t i d i u r e t i c a s s a y p r e p a r a t i o n . The new p r e p a r a t i o n was t e s t e d , a n d i t was f o u n d t o be s e n s i t i v e t o a n i n j e c t i o n o f t h e u n t r e a t e d f r a c t i o n . The v a s o p r e s s i n c o n t r o l was c o m p l e t e l y d e a c t i -v a t e d f o l l o w i n g t h i s e x t e n s i v e i n c u b a t i o n p e r i o d . However, t h e a m n i o t i c f l u i d sample showed a s l i g h t i n c r e a s e i n a c -t i v i t y . The a c t i v i t y o f t h e f r a c t i o n p r i o r t o i n c u b a t i o n a v e r a g e d 257 m i c r o u n i t s / m l , w h i l e a f t e r t r e a t m e n t w i t h s o d i u m t h i o g l y c o l l a t e i t was 269 m i c r o u n i t s / m l . I t was c o n c l u d e d f r o m t h e s e e x p e r i m e n t s t h a t t h e r e was no e v i d e n c e f o r t h e d e s t r u c t i o n o f t h e AFAS by s o d i u m t h i o g l y c o l l a t e . c ) S t o r a g e c o n d i t i o n s o f s a m p l e s F u r t h e r e v i d e n c e i n d i c a t i n g t h a t t h e a n t i d i u r e t i c s u b s t a n c e was n o t v a s o p r e s s i n , comes f r o m a c o n s i d e r a t i o n o f t h e s t o r a g e c o n d i t i o n s o f t h e s a m p l e s . . The a c t i v e f r a c -t i o n s were e l u t e d f r o m t h e col u m n a t pH 6 . 8 t o 7 . 2 , and were s t o r e d a t 4 ° C, w i t h o u t a l t e r a t i o n o f t h e pH. O p t i m a l s t o r a g e c o n d i t i o n s f o r v a s o p r e s s i n l i e between pH 3 . 0 a n d 5 . 0 , w i t h a maximum breakdown o f t h e hormone o c c u r r i n g a t pH 7 . 5 . The AFAS c o n t i n u e d t o show a c t i v i t y a f t e r f i v e weeks o f s t o r a g e w i t h n o r m a l v a l u e s i n s i x c a s e s , a n d o n l y a s l i g h t r e d u c t i o n i n sample #2 (20$ d e c r e a s e ) . T h i s s u g -g e s t s t h a t t h e s u b s t a n c e was n o t v a s o p r e s s i n . 63 d) C o m p a r i s o n s w i t h a n g i o t e n s i n I I As t h e a n t i d i u r e t i c m a t e r i a l d i d n o t a p p e a r t o be v a s o p r e s s i n , i t seemed p o s s i b l e t h a t i t m i g h t be a n g i o -t e n s i n I I , w h i c h C a r r e t e r o , e t a l . (1971) s u g g e s t e d may-e x i s t i n human a m n i o t i c f l u i d . T h i s hormone i s o f a s i m i -l a r s i z e a n d i d e n t i c a l c h a r g e t o v a s o p r e s s i n , a n d c o u l d be e x p e c t e d t o r u n i n a s i m i l a r manner on a n i o n - e x c h a n g e c o l u m n . A n g i o t e n s i n I I h a s b o t h p r e s s o r a n d a n t i d i u r e t i c p r o p e r t i e s (Bock, 1963; B o u c h e r , e t a l , , 1961; H e a l y , e t a l . , 1965). S t a n d a r d a n g i o t e n s i n I I was, t h e r e f o r e , i n j e c t e d i n t o . t h e r a t a n t i d i u r e t i c a s s a y p r e p a r a t i o n t o a s s e s s i t s e f f e c t , a n d t o compare i t w i t h t h a t o f t h e AFAS w h i c h had b e e n c o n c e n t r a t e d f r o m t h e s a m p l e s . The a n g i o t e n s i n s t a n d -a r d , was t e s t e d o v e r a 1,000-fold r a n g e , f r o m 0.0002 t o 0.2 micrograms/0.1 ml i n j e c t i o n . S i n c e t h e n a t u r e o f t h e a n t i -d i u r e t i c r e s p o n s e t o a n g i o t e n s i n may be a f u n c t i o n o f d o s e , t h i s w i de r a n g e o f dose l e v e l s were t e s t e d . I t i n c l u d e d h i g h e r l e v e l s t h a n a r e n e c e s s a r y f o r a p r e s s o r e f f e c t , a s w e l l a s c o n s i d e r a b l y l o w e r d o s e s . A l l o f t h e d o s e s above t h r e s h o l d gave a s i m i l a r t y p e o f r e s p o n s e , w h i c h were n o t a b l y d i s s i m i l a r f r o m t h o s e o f t h e AFAS, a s w e l l a s t h e s t a n d a r d v a s o p r e s s i n . F i g u r e 6 shows a c o m p a r i s o n o f t y p i c a l r e -s p o n s e s t o i n j e c t i o n s o f v a s o p r e s s i n , a n g i o t e n s i n I I , a n d t h e AFAS. The m a j o r d i f f e r e n c e i n t h e a p p e a r a n c e o f t h e r e s p o n s e s , a s shown on t h e s e kymograph r e c o r d s , i s t h a t 64 F i g u r e 6. C o m p a r i s o n . o f t h e r e s p o n s e s t o i n j e c t i o n s o f v a s o p r e s s i n , a n g i o t e n s i n I I a n d t h e AFAS on a r a t a n t i d i u r e t i c a s s a y p r e p a r a t i o n . A r r o w s show p o i n t o f i n j e c t i o n . a . 20 m i c r o u n i t s o f v a s o p r e s s i n . b . 0 , 2 m i c r o g r a m s o f a n g i o t e n s i n I I . c. 0 . 1 m i l l i l i t e r o f f r a c t i o n #34 f r o m human sample #4. 65 t h e v a s o p r e s s i n a n d AFAS r o s e s l o w l y a n d r e t u r n e d t o r e s t -i n g f l o w a f t e r a h i g h r e s p o n s e . I n c o n t r a s t , t h e r e s p o n s e t o i n j e c t i o n s o f a n g i o t e n s i n r o s e v e r y q u i c k l y a n d t h e n a p p e a r e d t o c a u s e t h e p r e p a r a t i o n t o become d i u r e t i c . As i t d i d n o t a p p e a r t h a t t h e a n t i d i u r e t i c a c t i v i t y i n t h e s a m p l e s c o u l d be a t t r i b u t e d t o a n g i o t e n s i n I I , i t was c o n s i d e r e d p o s s i b l e t h a t t h e a c t i v i t y m i g h t be due t o s y n e r g i s t i c o r o t h e r i n t e r a c t i o n s between t h e e f f e c t s o f a n g i o t e n s i n a n d v a s o p r e s s i n . M i x t u r e s o f b o t h hormones, i n w h i c h e a c h i n d i v i d u a l a g e n t was c l o s e t o , b u t below, t h r e s h o l d , c o n t i n u e d t o be s u b t h r e s h o l d when i n j e c t e d t o -g e t h e r . C o m b i n a t i o n s were a l s o g i v e n w h i c h i n c l u d e d d e -t e c t a b l e l e v e l s o f one hormone and s u b t h r e s h o l d l e v e l s o f t h e o t h e r . I n a l l o f t h e s e c a s e s , t h e r e s p o n s e o b t a i n e d r e s e m b l e d t h a t o f t h e hormone w h i c h was i n d e t e c t a b l e q u a n t i -t i e s a l o n e . I t was c o n c l u d e d t h a t t h e r e d i d n o t seem t o be a n y s y n e r g i s t i c e f f e c t b etween t h e s e two hormones, an d no s u c h e f f e c t c o u l d a c c o u n t f o r . t h e a n t i d i u r e t i c a c t i v i t y f o u n d i n human a m n i o t i c f l u i d . F i g u r e 7 shows t h e r e s p o n s e s t o some o f t h e c o m b i n a t i o n s w h i c h were t e s t e d . The a p p a r -e n t d i f f e r e n c e s i n t h e r e s p o n s e s t o t h e i n j e c t i o n s o f 10 a n d 15 m i c r o u n i t s o f v a s o p r e s s i n a r e most l i k e l y a t t r i b u -t a b l e t o t h e i n c r e a s i n g s e n s i t i v i t y o f t h e a s s a y , w h i c h was a g e n e r a l phenomenon o b s e r v e d . 66 Figure 7. P o r t i o n of an a n t i d i u r e t i c assay record showing comparisons of i n j e c t i o n s of vasopressin, angio-t e n s i n I I and combinations of these two hormones at d i f f e r i n g c oncentrations. AVP = vasopressin. ANG = a n g i o t e n s i n I I . UA 67 DISCUSSION A. C o n c e n t r a t i o n o f t h e F l u i d The method o f c o n c e n t r a t i n g t h e f l u i d w h i c h was u s e d i n t h i s i n v e s t i g a t i o n a p p e a r e d t o have met C h a r d ' s (1973) c r i t e r i a . T h e s e i n c l u d e : 1) s p e e d a n d s i m p l i c i t y o f t h e method, 2) c o n c e n t r a t i o n o f t h e hormone i n t o a s m a l l e r volume t o i m p r o v e t h e s e n s i t i v i t y o f t h e s u b s e q u e n t a s s a y , 3) a v o i d a n c e o f n o n - s p e c i f i c i n h i b i t o r s o r p r o t e o l y t i c enzymes, w h i c h m i g h t be c o n c e n t r a t e d w i t h t h e hormone, a n d 4) a r e p r o d u c i b l e r e c o v e r y o f g r e a t e r t h a n $0 p e r c e n t . I t has a l w a y s been e s s e n t i a l t o e x t r a c t v a s o p r e s s i n f r o m b l o o d s a m p l e s p r i o r t o a s s a y . However, a m n i o t i c f l u i d i s s i m p l i e r i n i t s c o m p o s i t i o n , a n d i t s c o n s t i t u t i o n i s c l o s e r t o t h a t o f u r i n e . Due t o t h i s r e s e m b l a n c e , t h e t e c h n i q u e s u s e d f o r t h e e x t r a c t i o n o f v a s o p r e s s i n f r o m u r i n e were c o n s i d e r e d more s u i t a b l e f o r t h e c o n c e n t r a t i o n o f v a s o p r e s s i n f r o m t h e a m n i o t i c f l u i d . The p r o c e s s was n o t u n d u l y l o n g o r c o m p l i -c a t e d . I t c o n c e n t r a t e d t h e hormone i n t o a s m a l l e r volume, a n d . i t h a d a r e p r o d u c i b l e r e c o v e r y o f much g r e a t e r t h a n 50 p e r c e n t . As CM-Sephadex s e p a r a t i o n i s b a s e d on b o t h t h e s i z e a n d t h e c h a r g e o f t h e m o l e c u l e , i t p r o b a b l y e l i m i n a t e d p r o t e o l y t i c enzymes a n d some i n h i b i t o r s . A good c o n c e n t r a -t i o n f a c t o r ( a p p r o x i m a t e l y 25 t i m e s ) was o b t a i n e d f r o m t h e method u s e d i n t h i s s t u d y , w i t h a n i n i t i a l c o n c e n t r a t i o n o f 10 mU/ml o f v a s o p r e s s i n b e i n g r e d u c e d f r o m 100 m i s t o 4 m i s , w h i c h h a d a c o n c e n t r a t i o n o f 250 mU/ml. 68 One o f t h e l i m i t a t i o n s o f t h e r a t a n t i d i u r e t i c assay-i s t h a t o n l y a s m a l l number o f s a m p l e s c a n be a s s a y e d on e a c h p r e p a r a t i o n . I t w o u l d n o t have b e e n f e a s i b l e t o t e s t e a c h f r a c t i o n b y t h e a n t i d i u r e t i c method, so r e l a t i v e l y h i g h . c o n c e n t r a t i o n s o f s t a n d a r d v a s o p r e s s i n were l o a d e d on t h e columns a n d t h e i r e l u t i o n p r o f i l e d e t e r m i n e d . I n t h i s way, t h e m o l a r i t y a n d pH c h a r a c t e r i s t i c s o f s a m p l e s s h o w i n g a c t i v i t y on t h e r a t v a s o p r e s s o r a s s a y were d e t e r m i n e d . As v a s o p r e s s i n e x h i b i t s b o t h v a s o p r e s s o r a n d a n t i d i u r e t i c a c t i v i t y , t h e a m n i o t i c f l u i d f r a c t i o n s w h i c h were e l u t e d i n t h e same p o s i t i o n a s t h e v a s o p r e s s o r - a c t i v e s t a n d a r d f r a c t i o n s , were s u b s e q u e n t l y a s s a y e d f o r a n t i d i u r e t i c a c -t i v i t y . B. The P r e s e n c e o f a n A n t i d i u r e t i c A c t i v i t y i n Human Am-n i o t i c F l u i d A s u b s t a n c e c o n t a i n i n g a n t i d i u r e t i c a c t i v i t y was e l u t e d f r o m human a m n i o t i c f l u i d on i o n - e x c h a n g e Sephadex c o l u m n s . The a c t i v i t y w h i c h .was o b t a i n e d was c o n s i s t e n t a n d r e p r o -d u c i b l e f r o m sample t o sample, i n . t e r m s o f c h r o m a t o g r a p h i c m o b i l i t y a n d a p p r o x i m a t e q u a n t i t y . However, i t c o u l d n o t be i d e n t i f i e d . C a r r e t e r o , e t a l . (1971) d i s c o v e r e d a p r e s -s o r p o l y p e p t i d e i n human a m n i o t i c f l u i d w h i c h t h e y i d e n t i -f i e d a s a n g i o t e n s i n , o r one o f i t s a n a l o g u e s , o r a f r a g m e n t o f t h a t p o l y p e p t i d e . However, t h e i s o l a t e d s u b s t a n c e d i d n o t have a. peak s i m i l a r t o s t a n d a r d a n g i o t e n s i n I o r I I when s e p a r a t e d on Sephadex, n o r a s i m i l a r R „ when 69 c h r o m a t o g r a p h e d on p a p e r . A l t h o u g h t h e s e r e s e a r c h e r s c o n -c l u d e d t h a t t h e i r p o l y p e p t i d e was a n g i o t e n s i n , i t may p e r -h a p s have been t h e same s u b s t a n c e w h i c h was f o u n d t o be a c t i v e i n t h e s a m p l e s o f t h e p r e s e n t s t u d y . P r e s s o r a n d a n t i d i u r e t i c p r o p e r t i e s a r e n o t n e c e s s a r i l y l i n k e d ; however, s o m e . p e p t i d e s , i n c l u d i n g v a s o p r e s s i n a n d a n g i o t e n s i n I I , do p o s s e s s b o t h a c t i v i t i e s . As t h e AFAS was n o t i d e n t i f i e d , a n a p p r o p r i a t e s t a n d -a r d , f o r .the q u a n t i f i c a t i o n o f t h e d a t a was u n a v a i l a b l e . I n c o n s e q u e n c e , t h e a c t i v i t y h a s b e e n e x p r e s s e d i n t e r m s o f i t s . . a n t i d i u r e t i c , v a s o p r e s s i n - e q u i v a l e n t . T h e r e f o r e , t h e n u m e r i c a l v a l u e s a s s i g n e d a r e o n l y m e a n i n g f u l i n t e r m s o f t h e i r v a s o p r e s s i n - e q u i v a l e n t a c t i v i t y , and must be r e g a r d e d a s no more t h a n . a p p r o x i m a t e i n d i c a t i o n s o f t h e m a g n i t u d e o f t h e a c t i v i t y . G. The N a t u r e o f . t h e A m n i o t i c F l u i d A n t i d i u r e t i c S u b s t a n c e 1. C o m p a r i s o n t o . V a s o p r e s s i n The c h a r a c t e r i s t i c s o f t h e AFAS were compared t o t h o s e o f v a s o p r e s s i n i n T a b l e V. The Sephadex c h r o m a t o g r a p h y e l u t i o n p r o f i l e s u g g e s t s t h a t t h e AFAS may be v e r y s i m i l a r i n p h y s i c o - c h e m i c a l p r o p e r t i e s t o v a s o p r e s s i n , a s b o t h s u b s t a n c e s were e l u t e d i n a s i m i l a r p o s i t i o n (See F i g . 4). The p a t t e r n o f t h e a n t i d i u r e t i c r e s p o n s e , a s s e e n on t h e . kymograph r e c o r d s i n F i g s . 3 a n d .6 were i n d i s t i n g u i s h a b l e . However, t h e s t a n d a r d v a s o p r e s s i n i n j e c t i o n s were c o n s i s t e n t TABLE V COMPARISONS BETWEEN VASOPRESSIN AND AMNIOTIC FLUID ANTIDIURETIC SUSTANCE (AFAS) C h a r a c t e r i s t i c I o n - e x c h a n g e c h r o m a t o g r a p h y p r o f i l e P a t t e r n o f a n t i d i u r e t i c r e s p o n s e C o n s i s t e n c y o f a n t i d i u r e t i c r e s p o n s e S e n s i t i v i t y o f a s s a y t o s u b s t a n c e Sodium t h i o g l y c o l l a t e i n c u b a t i o n S t a b i l i t y c h a r a c t e r i s t i c s V a s o p r e s s i n E l u t e d between pH 6.$ a n d 7.2, and 0.1$ a n d 0.22 M Slow r i s e a n d f a l l C o n s i s t e n t i n n o r m a l dose r a n g e s A s s a y became i n c r e a s -i n g l y s e n s i t i v e t o r e p e a t e d i n j e c t i o n s A l l a c t i v i t y i n -a c t i v a t e d a f t e r 2 h o u r s S t a b l e a t pH 3 AFAS E l u t e d between pH 7.0 a n d $.0, a n d 0.1$ a n d 0.26 M S l o w r i s e a n d f a l l I n c o n s i s t e n t i n n o r m a l dose r a n g e s A s s a y became a p p a r e n t l y l e s s s e n s i t i v e t o r e -p e a t e d i n j e c t i o n s * A c t i v i t y c o n s t a n t , o r h i g h e r , a f t e r 2 d a y s S t a b l e f o r up t o 5. weeks a t n e u t r a l pH * T h i s r e s p o n s e was o b t a i n e d f r o m u n c o n c e n t r a t e d f l u i d o n l y , w h i c h may o r may n o t c o n t a i n m a t e r i a l i d e n t i c a l t o t h e AFAS. 71 i n g i v i n g a n a n t i d i u r e s i s when t h e y were a b o v e t h e n o r m a l t h r e s h o l d s f o r t h e a s s a y s . T h i s was n o t t h e c a s e f o r t h e a c t i v e AFAS f r a c t i o n s . N e a r l y o n e - t h i r d o f t h e i n j e c t i o n s o f AFAS s a m p l e s gave n e g a t i v e r e s p o n s e s , e v e n when t h e same f r a c t i o n s gave c l e a r e f f e c t s i n l a t e r e x p e r i m e n t s . T h e r e a p p e a r e d t o be g r o s s d i f f e r e n c e s i n t h e s e n s i t i v i t y t o t h e unknown m a t e r i a l i n d i f f e r e n t p r e p a r a t i o n s , a n d t h i s was n o t s e e n i n t h e c a s e o f v a s o p r e s s i n . F u r t h e r e v i d e n c e i n d i c a t i n g t h a t t h e AFAS m a t e r i a l was n o t . i d e n t i c a l t o v a s o p r e s s i n was shown i n t h e s o d i u m t h i o g l y c o l l a t e d e a c t i v a t i o n e x p e r i m e n t s . I f t h e a c t i v i t y o f a s u b s t a n c e i s n o t l o s t f o l l o w i n g i n c u b a t i o n w i t h s o d i u m t h i o g l y c o l l a t e , t h e n i t most l i k e l y i s n o t a n e u r o h y p o p h y s i a l p e p t i d e (Sawyer, 1966) . T h i s t e s t i s n o t n e c e s s a r i l y s p e c i -f i c f o r p o s t e r i o r p i t u i t a r y p e p t i d e s , f o r i f i n a c t i v a t i o n d oes o c c u r , t h e m a t e r i a l may be a d i f f e r e n t d i s u l f i d e -c o n t a i n i n g s u b s t a n c e . However, i t i s g e n e r a l l y a c c e p t e d a s a r e a s o n a b l e t e s t f o r v a s o p r e s s i n by most w o r k e r s i n t h e f i e l d . I t w o u l d a p p e a r f r o m t h e r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s w i t h s o d i u m t h i o g l y c o l l a t e , t h a t t h e AFAS does n o t c o n t a i n a d i s u l f i d e l i n k a g e w h i c h i s e s s e n t i a l f o r i t s a c t i v i t y . A s e c o n d c r i t e r i o n f o r t h e i d e n t i f i c a t i o n o f t h e a n t i -d i u r e t i c a c t i v i t y w i t h v a s o p r e s s i n i s t h e s t a b i l i t y c h a r a c -t e r i s t i c s o f t h i s hormone. I t has b e e n f o u n d t h a t s t o r a g e 72 a t pH 3 g i v e s optimum s t a b i l i t y ( T h o r n , 1 9 5 8 ) . The s t a b i l -i t y o f n e u r o h y p o p h y s i a l hormones changes w i t h t e m p e r a t u r e , h y d r o g e n - i o n c o n c e n t r a t i o n a n d b u f f e r , a n d v a s o p r e s s i n i s n o t a b l y l e s s s t a b l e when h i g h l y p u r i f i e d ( S m i t h , 1 9 7 0 ) . The a c t i v e f r a c t i o n s o f . t h e c o n c e n t r a t e d human a m n i o t i c f l u i d were k e p t a t pH 7*0 t o 7»4, a n d r e m a i n e d s t a b l e f o r up t o f i v e weeks when s t o r e d a t 4 ° C. T h i s i n d i c a t e s t h a t t h e m a t e r i a l was p r o b a b l y t o o s t a b l e t o be v a s o p r e s s i n . 2.. C o m p a r i s o n w i t h A n g i o t e n s i n I I The a c t i v i t y w h i c h was f o u n d i n t h e human a m n i o t i c f l u i d d i d n o t a p p e a r t o be a n g i o t e n s i n I I , a l t h o u g h b o t h r e n i n .and a n g i o t e n s i n I I have been f o u n d t o o c c u r i n r e l a -t i v e l y h i g h c o n c e n t r a t i o n s i n t h e b l o o d o f t h e f e t u s a n d may o c c u r i n t h e . a m n i o t i c f l u i d (Brown, e t a l . , 1964; C a r r e t e r o , e t a l . , 1971 ; P i p k i n , e t a l . , 1 9 7 4 ) * F u r t h e r , t h i s hormone may have b e e n e x p e c t e d t o r u n on a n i o n -exchange column i n a manner s i m i l a r t o . v a s o p r e s s i n a n d t h e AFAS, b a s e d on i t s s i z e a n d c h a r g e . However, b a s e d on t h e d i s s i m i l a r i t y b etween t h e r e s p o n s e s o f a n g i o t e n s i n a n d t h e AFAS on t h e r a t a n t i d i u r e t i c p r e p a r a t i o n , i t seemed l i k e l y t h a t a n g i o t e n s i n was n o t r e s p o n s i b l e f o r t h i s a c t i v i t y . 3. C o m p a r i s o n s w i t h c o m b i n a t i o n s o f v a s o p r e s s i n a n d  a n g i o t e n s i n The a c t i v i t y o f t h e AFAS c o u l d n o t be a t t r i b u t e d t o a c o m b i n a t i o n e f f e c t between a n g i o t e n s i n I I a n d v a s o p r e s s i n , 73 based, on t h e e x p e r i m e n t s w h i c h were done i n t h i s i n v e s t i -g a t i o n , N e i t h e r hormone seemed t o i n f l u e n c e t h e r e s p o n s e o f t h e o t h e r when i n j e c t e d t o g e t h e r . I f t h e r e i s a n i n t e r -a c t i o n between t h e s e two p e p t i d e s , i t was n o t e v i d e n c e d i n r e s p o n s e t o any o f t h e c o n c e n t r a t i o n s w h i c h were t e s t e d t o g e t h e r i n t h i s s t u d y . As t h e AFAS c o u l d n o t be i d e n t i f i e d a s v a s o p r e s s i n , a n g i o t e n s i n I I , o r a c o m b i n a t i o n e f f e c t o f t h e s e two h o r -mones, i t must be c o n c l u d e d t h a t i t s a n t i d i u r e t i c a c t i v i t y i s a t t r i b u t a b l e t o a n u n t e s t e d , o r unknown s u b s t a n c e . 74 GENERAL DISCUSSION A. V a s o p r e s s i n i n t h e A m n i o t i c F l u i d A l t h o u g h i t h a s b e en a r g u e d t h a t t h e r e i s a r e a s o n a b l e p o s s i b i l i t y . t h a t v a s o p r e s s i n c o u l d e x i s t i n t h e a m n i o t i c f l u i d ( V i z s o l y i & P e r k s , 1 9 7 4 ) , t h i s hormone c o u l d n o t be i d e n t i f i e d i n t h e a m n i o t i c f l u i d o f g u i n e a p i g s , s h e e p an d humans. T h i s f i n d i n g i s . c o n f i r m e d by t h e i m m u n o l o g i c a l s t u d i e s o f F i s h e r , e t a l . , who were n o t a b l e t o d e t e c t a n y v a s o p r e s s i n i n t h e f l u i d o f humans o r s h e e p d u r i n g t h e l a s t t r i m e s t e r o f p r e g n a n c y ( p e r s o n a l c o m m u n i c a t i o n , A p r i l , 1 9 7 5 ) . No t e s t s have b e e n done, t o d a t e , on human a m n i o t i c f l u i d p r i o r t o t h e t h i r d t r i m e s t e r . A l l o f t h e s t u d i e s r e p o r t e d have, u s e d f l u i d f r o m n e a r t e r m p r e g n a n c i e s ; a t t h i s t i m e , t h e r e i s a r a p i d l o s s o f f l u i d , w h i c h may i n f l u e n c e , o r be i n f l u e n c e d by, t h e a b s e n c e o r p r e s e n c e o f a c o n t r o l l i n g hormone. 1. C o n s i d e r a t i o n o f t h e m e t a b o l i s m o f v a s o p r e s s i n I f t h e v a s o p r e s s i n i n t h e a m n i o t i c f l u i d was a t a c o n -c e n t r a t i o n t h a t was b e l o w t h e d e t e c t a b l e l i m i t s o f t h e a s s a y , t h e q u e s t i o n r e m a i n s a s t o what becomes o f t h e h i g h h o r m o n a l l e v e l s w h i c h h ave been r e p o r t e d t o e x i s t i n t h e f e t a l h y p o -t h a l a m i c - n e u r o h y p o p h y s i a l s y s t e m , a s w e l l a s i n t h e b l o o d o f t h e f e t u s . ( H o p p e n s t e i n , e t . a l . , 1 9 6 $ ; P e r k s & V i z s o l y i , 1973; V i z s o l y i & P e r k s , 1 9 7 6 ) . P e r h a p s t h e v a s o p r e s s i n p u t o u t by t h e f e t a l p o s t e r i o r p i t u i t a r y d u r i n g g e s t a t i o n i s 75 m e t a b o l i z e d i n t h e f e t a l k i d n e y o r l i v e r , i n a manner s i m i l a r t o t h a t o f t h e a d u l t . I t may a l s o be t h e c a s e t h a t i t N r a p i d l y d i f f u s e s t h r o u g h t h e e x t r a e m b r y o n i c membranes, o r i t may be i n a c t i v a t e d o r d e n a t u r e d by t h e s e membranes. I n t h e a d u l t , t e n p e r c e n t o f i n j e c t e d v a s o p r e s s i n i s e x c r e t e d i n t h e u r i n e , w i t h f i f t y p e r c e n t c l e a r e d by t h e k i d n e y a n d f o r t y p e r c e n t by t h e l i v e r ( D i c k e r , 1 9 6 l ) . The h a l f - l i f e o f v a s o p r e s s i n i n a d u l t humans i s o n l y t e n t o t w e n t y m i n u t e s ( L a u s o n , i 9 6 0 ) . The p e r i n a t a l m e t a b o l i s m o f v a s o p r e s s i n a p p e a r s t o be s i m i l a r , w i t h t e n p e r c e n t o f i n j e c t e d h o r -mone b e i n g e x c r e t e d ( H e l l e r , 1 9 5 2 ) . However, t h e r e i s l i t -t l e o r no i n f o r m a t i o n a b o u t t h e r o l e o f t h e k i d n e y a n d l i v e r i n t h e m e t a b o l i s m o f f e t a l hormones. Skowsky, e t a l . (1973) f o u n d t h a t l a b e l l e d v a s o p r e s s i n was c l e a r e d f r o m t h e c i r c u -l a t i o n o f f e t a l lambs an d r h e s u s monkeys a t a g r e a t e r r a t e t h a n i n t h e a d u l t s . I n t h e human f e t u s , t h e p l a c e n t a may be o f i m p o r t a n c e i n c l e a r i n g t h i s hormone f r o m t h e c i r c u -l a t i o n , a s i t c o n t a i n s h i g h l e v e l s o f e n z y m a t i c a c t i v i t y ( C h a l l i s , et a l . , 1 9 7 6 ) . T h e r e may a l s o be c i r c u l a t i n g l e v e l s o f v a s o p r e s s i n a s e i n p r i m a t e n e o n a t a l p l a s m a (Skowsky, e t a l . , 1 9 7 3 ) , D e s p i t e t h e s e c o n s i d e r a t i o n s , h i g h l e v e l s o f v a s o p r e s s i n do a p p e a r i n t h e u r i n e o f t h e n e o n a t e ( A l e x -a n d e r & N i x o n , 1 9 6 l; Ames, 1 9 5 3 ) . I t seems l i k e l y t h a t t h e s e hormones w o u l d r e a c h t h e a m n i o t i c f l u i d . 76 2 . V a s o p r e s s i n a s e s a n d o x y t o c i n a s e s d u r i n g p r e g n a n c y V a s o p r e s s i n c a n b e . e n z y m a t i c a l l y d e g r a d e d by e i t h e r v a s o p r e s s i n a s e o r o x y t o c i n a s e . O x y t o c i n a s e l e v e l s i n t h e m a t e r n a l c i r c u l a t i o n i n c r e a s e d r a m a t i c a l l y d u r i n g p r e g n a n c y , i n c r e a s i n g 60 t o 1 , 0 0 0 f o l d ( C a l d e y r o - B a r c i a , I 9 6 0 ; R o s e n -bloom, 1 9 7 5 ) . V a s o p r e s s i n a s e h a s o n l y b e e n f o u n d i n p r i m a t e serum d u r i n g p r e g n a n c y (Rosenbloom, 1 9 7 5 ) . I t h a s b een s u g g e s t e d t h a t t h e s e e l e v a t e d enzyme l e v e l s o f t h e m other p l a y o n l y .a s m a l l r o l e , q u a n t i t a t i v e l y , i n t h e c l e a r a n c e o f n e u r o h y p o p h y s i a l hormones f r o m t h e m a t e r n a l c i r c u l a t i o n ( L a u s o n , 1 9 6 7 ) . The r o l e o f t h e s e e l e v a t e d l e v e l s r e m a i n s unknown. H i g h l e v e l s o f v a s o p r e s s i n a s e have a l s o b e e n f o u n d i n t h e b l o o d o f n e w l y b o r n i n f a n t s ( H o p p e n s t e i n , e t a l . , 1 9 6 8 ; Skowsky, e t a l . , 1 9 7 3 ) . Of more d i r e c t i n t e r e s t t o t h i s s t u d y , i s t h e f a c t t h a t o x y t o c i n a s e h a s b e e n f o u n d i n t h e a m n i o t i c f l u i d , a n d K l e i n e r a n d B r o u e t - Y a g e r (1973) a t t i b u t e i t s o r i g i n t o f e t a l t i s s u e s . Rosenbloom, e t a l . (1975) have a l s o f o u n d v e r y l o w l e v e l s o f v a s o p r e s s i n a s e i n t h e a m n i o t i c f l u i d o f humans a t t e r m . However, i t has n o t y e t b een d e m o n s t r a t e d i n t h e f l u i d s o f o t h e r mammals. As t h e .presence o f a n enzyme i s g e n e r a l l y h e l d t o be e v i d e n c e f o r . t h e p h y s i o l o g i c a l . o c c u r r e n c e o f i t s s u b s t r a t e , t h i s w o u l d s u g g e s t t h a t v a s o p r e s s i n m i g h t be p r e s e n t i n t h e a m n i o t i c f l u i d o f a t l e a s t p r i m a t e s . 77 3• C o n s i d e r a t i o n s o f t h e m e t a b o l i s m o f a n g i o t e n s i n .. . The a t t e m p t t o c l a r i f y t h e r o l e o f v a s o p r e s s i n i n t h e f e t u s may,be c o m p l i c a t e d by a n g i o t e n s i n . A n g i o t e n s i n may a c t t h r o u g h i t s c o n n e c t i o n t o a l d o s t e r o n e s e c r e t i o n . How-e v e r , i t may a l s o have a n i n d e p e n d e n t a c t i o n on t h e r e n a l s y s t e m (Bock, 1 9 6 3 ; Mouw, e t a l . , 1 9 7 1 ) . A n g i o t e n s i n I I an d v a s o p r e s s i n a r e so s i m i l a r i n p h y s i c a l c h a r a c t e r i s t i c s a n d p h y s i o l o g i c a l e f f e c t s , t h a t complex s e p a r a t i o n a n d c o n -c e n t r a t i o n p r o c e d u r e s f o r a n g i o t e n s i n have f a i l e d t o remove a l l t r a c e s o f , v a s o p r e s s i n f r o m t h e p r e p a r a t i o n s ( B o u c h e r , e t a l . , 1 9 7 1 ) . H i g h c o n c e n t r a t i o n s o f r e n i n - l i k e enzyme have been f o u n d i n a l l , s p e c i m e n s o f human a m n i o t i c f l u i d t e s t e d by Brown, e t a l . ( 1 9 6 4 ) , a n d a n a n g i o t e n s i n - l i k e p r e s s o r m a t e r -i a l r a p i d l y a p p e a r e d i n i n c u b a t i o n m i x t u r e s . I n s e v e r a l o f t h e s a m p l e s i n v e s t i g a t e d by t h e s e r e s e a r c h e r s , t h e r e n i n c o n c e n t r a t i o n s were among t h e h i g h e s t f o u n d i n a n y b i o l o g i -c a l f l u i d , a n d t h e o r i g i n o f t h e m a t e r i a l was t h o u g h t t o be t h e m a t e r n a l p l a s m a , o r t h e c e l l s o f t h e p l a c e n t a a n d d e c i d u a , o r t h e f e t a l u r i n e . P l a s m a r e n i n l e v e l s a r e e l e v a t e d i n t h e mother d u r i n g . t h e . t h i r d t r i m e s t e r o f p r e g n a n c y , a n d a r e even h i g h e r d u r i n g l a b o r ( G e e l h o e d & V a n d e r , 1 9 6 $ ) . T h e s e w o r k e r s h ave shown t h a t t h e f e t a l k i d n e y s p r o d u c e r e n i n , a s t h e c o n c e n t r a t i o n i n t h e u m b i l i c a l a r t e r y i s ' h i g h e r t h a n t h a t o f t h e u m b i l i c a l v e i n . R e n i n - l i k e m a t e r i a l h as a l s o b e e n f o u n d i n t h e p l a c e n t a e a n d u t e r i n e e x t r a c t s o f t h e 78 c a t , dog a n d human. R e n i n i s c o n t a i n e d i n t h e k i d n e y s o f f e t u s e s . b o r n f r o m b o t h n o r m a l a n d n e p h r e c t o m i z e d . a n i m a l s , w h i c h . i n d i c a t e s t h a t t h e m a t e r i a l i s . o f f e t a l o r i g i n ( H o d a r i , 1970). Human f e t u s e s o f a l l a g e s have b e e n o b s e r v e d t o h a v e g r a n u l a t e d j u x t a g l o m e r u l a r c e l l s , i n d i c a t i n g t h a t t h e r e may be a c t i v e r e n i n f o r m a t i o n ( H o d a r i , 1970). T o g e t h e r w i t h t h e s e h i g h l e v e l s o f r e n i n a n d a n g i o t e n s i n , a l d o s t e r o n e s e c r e t i o n i s e l e v a t e d i n t h e m o t h e r by 0'.37 o f t e r m ; s o , i n p r e g n a n c y , r e n i n , r e n i n s u b s t r a t e , a n d p l a s m a a n g i o t e n -s i n a s e s a r e a l l i n c r e a s e d (Mulrow, 1971)* 4. I n t e r - r e l a t i o n s h i p s between v a s o p r e s s i n a n d a n g i o -t e n s i n T h e r e seems t o be a c o n s i d e r a b l e c o n t r o v e r s y o v e r t h e r o l e o f a n g i o t e n s i n I I i n t h e r e l e a s e o f v a s o p r e s s i n . Bon-j o u r a n d M a l v i n (1970) r e p o r t e d t h a t a n g i o t e n s i n h a d a n e f f e c t on v a s o p r e s s i n r e l e a s e . However, S h a r e , e t a l . (1972), Shade a n d S h a r e (1975) a n d C a d n a p a p h o r n c h a i , e t a l . (1975) have a r g u e d t h a t a n g i o t e n s i n does n o t have a n e f f e c t on t h e r e l e a s e o f v a s o p r e s s i n , s t i m u l a t o r y o r s u p p r e s i v e , n o r does i t a l t e r r e n a l w a t e r f l o w . A l t h o u g h t h e r e seems t o be d i s a g r e e m e n t , i t does seem l i k e l y t h a t t h e r e w o u l d be a n i n t e r r e l a t i o n s h i p between t h e a c t i o n o f t h e s e two hormones. I n r e a d j u s t m e n t s r e q u i r e d by o s m o t i c s t r e s s , t h e r e i s c o n c u r r e n t a c t i v a t i o n o f a t l e a s t two s y s t e m s : t h e 79 h y p o t h a l a m i c - n e u r p h y p h y s i a l a n d t h e a d e n o h y p o p h y s i a l -a d r e n o c o r t i c a l a x e s . The two s y s t e m s a r e a c t i v a t e d s i m -u l t a n e o u s l y , a n d i t seems j u s t i f i a b l e t o assume t h a t a n i n t e g r a t i o n o f t h e r e s p o n s e s l i e s i n t h e h y p o t h a l a m u s . The a d r e n o c o r t i c a l i n f l u e n c e s on w a t e r e x c r e t i o n may be a n t a g o n i s t i c o r com p l e m e n t a r y t o v a s o p r e s s i n , d e p e n d i n g upon w h e t h e r sodium i s b e i n g r e t a i n e d o r e x c r e t e d (Hayes & C o l l e r , 1952). F u r t h e r , a l d o s t e r o n e h a s b e e n f o u n d t o have a ' p e r m i s s i v e e f f e c t ' i n e n h a n c i n g v a s o p r e s s i n a c t i o n (Hays & L e v i n e , 1974)* T h e r e i s a l s o r e c e n t d i r e c t e v i d e n c e t h a t a n g i o t e n s i n I I r e l e a s e s v a s o p r e s s i n by c e n t r a l mechanisms; t h i s was shown by i n j e c t i o n o f a n g i o t e n s i n i n t o t h e v e n -t r i c u l a r c e r e b r o s p i n a l f l u i d . The s u b s e q u e n t r e l e a s e o f v a s o p r e s s i n a p p e a r e d t o be dose d e p e n d e n t ( K e i l , _et a l . , 1975). A l l c o n c e n t r a t i o n s o f a n g i o t e n s i n i n j e c t e d i n t o t h e c a r o t i d a r t e r y o r i n t r a v e n o u s l y r e s u l t e d i n a s i g n i f i -c a n t r e l e a s e o f v a s o p r e s s i n (Mouw, j 3 t a J L . , 1971). T h e s e w o r k e r s c o n c l u d e d t h a t some a r e a o f t h e b r a i n i s r e s p o n -s i v e t o l o c a l c o n c e n t r a t i o n s o f a n g i o t e n s i n , a n d t h e p e p t i d e r e s u l t s i n s t i m u l a t i o n o f v a s o p r e s s i n r e l e a s e (Mouw, e t a l . , 1 9 7 D . The r e n a l r e s p o n s e s t o a n g i o t e n s i n v a r y w i t h t h e d o s e . Some d o s e a g e s c a u s e d i u r e s i s , w h i l s t o t h e r s c a u s e a n t i d i -u r e s i s ( H e a l y , _ e t _ a l . , 1965). A n g i o t e n s i n i s s h o r t - l i v e d , Bo w i t h a h a l f - l i f e o f o n l y a few m i n u t e s , o r l e s s (Mulrow, 1971). T h i s may e x p l a i n t h e v e r y r a p i d d e c l i n e i n t h e r e s p o n s e when an a n t i d i u r e s i s was o b s e r v e d i n t h e e x p e r i -ments i n t h i s s t u d y . N e i t h e r t h e v a s o p r e s s i n s t a n d a r d , n o r t h e f r a c t i o n s c o n t a i n i n g t h e AFAS showed t h i s s h o r t - l i v e d a n t i d i u r e t i c r e s p o n s e . However, t h e pH c o n d i t i o n s d u r i n g sample s t o r a g e were c l o s e t o optimum f o r t h e l i b e r a t i o n o f a n g i o t e n s i n f r o m r e n i n ( B o u c h e r , e t a l . , 1967)» B. F u t u r e S t u d i e s o f V a s o p r e s s i n and O t h e r A c t i v e A g e n t s  i n . A m n i o t i c F l u i d F u t u r e work s h o u l d c o n s i d e r a number o f f a c e t s s u g -g e s t e d by t h i s i n v e s t i g a t i o n , a n d t h e s e a r e c o n s i d e r e d below.. 1. The c o n f i r m a t i o n o f t h e r e s u l t s by o t h e r methods .The work r e p o r t e d h e r e u t i l i z e d t h e a n t i d i u r e t i c b i o -a s s a y f o r t h e d e t e c t i o n o f v a s o p r e s s i n i n a m n i o t i c f l u i d . However, t h i s method s h o u l d be s u p p l e m e n t e d by o t h e r methods, a n d t h e p o s s i b l e a s s e t s o f d i f f e r e n t t e c h n i q u e s a r e i m p o r t -a n t i n c o n s i d e r i n g t h e f u t u r e d i r e c t i o n o f t h e s e i n v e s t i -g a t i o n s . The a n t i d i u r e t i c a s s a y was c h o s e n b e c a u s e o f i t s h i g h s e n s i t i v i t y a n d r e l a t i v e l y good s p e c i f i c i t y . However, i t must be o b s e r v e d t h a t t h e r e a r e i n h e r e n t m e t h o d o l o g i c a l e r r o r s i n v o l v e d i n t h i s b i o a s s a y , e s p e c i a l l y when d e a l i n g w i t h l o w l e v e l s o f v a s o p r e s s i n (Skowsky, e t a l . , 1973)* Si I n h i b i t i o n o f d i u r e s i s c a n be c a u s e d by o s m o t i c , haemo-dynamic an d n e r v o u s s t i m u l i , so c o n c l u s i o n s r e g a r d i n g t h e r e l a t i o n b etween d i u r e t i c i n h i b i t i o n a n d v a s o p r e s s i n a c -t i v i t y i n p l a s m a must be v i e w e d w i t h c a u t i o n . A c t u a l d i -u r e s i s c a n be m o d i f i e d by c h a n g e s i n f i l t r a t i o n r a t e , i n t r a -r e n a l haemodynamics an d e l e c t r o l y t e r e a b s o r p t i o n f r o m t h e r e n a l t u b u l e s , t h e l a t t e r b e i n g u n d e r t h e c o n t r o l o f t h e a d r e n a l c o r t e x ( B u c h b o r n , 1957)• C u t t i n g , t e a r i n g , c r u s h i n g , b u r n i n g , d i s s e c t i n g , h a n d -l i n g , m a n i p u l a t i n g a n d s u t u r i n g l i v i n g t i s s u e c o n s t i t e ' s t r e s s e s ' a n d w i l l r e s u l t i n endogenous r e l e a s e o f v a s o -p r e s s i n , a s w i l l h emorrhage, a n e s t h e s i a , s t a r v a t i o n , p a i n , f e a r a n d a n x i e t y (Moore, 1957). E t h a n o l a n e s t h e t i c was u s e d i n t h i s s t u d y t o b l o c k endogenous r e l e a s e o f v a s o p r e s s i n , w h i c h may be c a u s e d by t h e above f a c t o r s . However, a l c o h o l w i l l o n l y b l o c k t h e r e l e a s e o f n e u r o h y p o p h y s i a l hormones i f a d m i n i s t e r e d p r i o r t o t h e r e l e a s i n g s t i m u l u s (Kleeman, e t a l . , 1955) . F u r t h e r , T a t a a n d B u z a l k o v (1966) f o u n d t h a t a l c o h o l was o n l y e f f e c t i v e i n p r e v e n t i n g endogenous v a s o -p r e s s i n r e l e a s e t h a t was i n d u c e d by a n i n c r e a s e i n o s m o t i c p r e s s u r e o f t h e b l o o d . I t h a d r e l a t i v e l y l i t t l e e f f e c t i n t h e c a s e s o f r e l e a s e due t o p a i n a n d h e m o r r h a g e , e v e n i n d e e p l y a n e s t h e t i z e d a n i m a l s ( G i n s b u r g & Brown, 1956) . A n o t h e r l i m i t a t i o n o f t h i s a s s a y was t h a t s e v e r a l p r e p a r a t i o n s were e s s e n t i a l f o r t h e a s s a y i n g o f e a c h sample 8 2 ( F o r s l i n g , e t a l . , 1 9 6 8 ) . T h i s i s due b o t h t o t h e r e l a -t i v e l y l a r g e v a r i a t i o n i n s e n s i t i v i t y i n d i f f e r e n t a n i m a l s , a n d t o t h e f a c t t h a t o n l y a few s a m p l e s c a n be a s s a y e d i n a s i n g l e t e s t a n i m a l ( M i l l e r & Moses, 1 9 7 1 ) . I n t h e p r e s e n t e x p e r i m e n t s , i t was f o u n d t h a t a maximum o f 1 $ i n j e c t i o n s c o u l d b e . g i v e n p e r p r e p a r a t i o n , a n d u s u a l l y many l e s s were p o s s i b l e . B e c a u s e o f t h i s l i m i t a t i o n , s t r i c t f o u r - p o i n t b i o a s s a y s were n o t p o s s i b l e f o r t h e a c c u r a t e q u a n t i f i c a t i o n o f t h e d a t a . I f v a s o p r e s s i n i s t o be l o o k e d f o r i n t h e f u t u r e , i t w o u l d p r o b a b l y be b e s t t o u s e t h e r a d i o i m m u n o a s s a y t e c h -n i q u e ( R I A ) . P r i o r t o t h e s t a r t o f t h e p r e s e n t s t u d y , t h e r e p o r t e d r e s u l t s o f t h e RIA h a d n o t shown good c o r r e l a -t i o n w i t h b i o a s s a y v a l u e s . Due t o t h e l o w m o l e c u l a r w e i g h t o f v a s o p r e s s i n , i t h a d b e e n d i f f i c u l t t o o b t a i n s p e c i f i c a n t i b o d i e s a t h i g h t i t e r ( B e r d e , 1 9 7 0 ) . However, more r e c e n t work h a s sh o w n . t h a t t h e RIA i s b e c o m i n g more s e n s i -t i v e a n d . s p e c i f i c (Hays & L e v i n e , 1 9 7 4 ) * Skowsky, e t a l . ( 1 9 7 4 ) f o u n d a RIA s e n s i t i v i t y o f 0 . 3 t o 0 . 5 m i c r o u n i t s / m l , a n d t h e method a l l o w e d f o r t h e measurement o f v a s o p r e s s i n i n t h e maternal, a n d f e t a l b l o o d e v e n i n t h e p r e s e n c e o f e l e v a t e d v a s o p r e s s i n a s e . The RIA i s now a s s e n s i t i v e a s t h e b i o a s s a y , o r more s o , more s p e c i f i c a n d p r e c i s e , e a s i e r t o p e r f o r m a n d h a s a l o w a s s a y v a r i a t i o n ( C h a r d , 1 9 7 3 ; H u s a i n , e t a l . , 1 9 7 3 ) . S3 2. The p u r i f i c a t i o n o f t h e a c t i v e a g e n t The a n t i d i u r e t i c s u b s t a n c e , w h i c h was c o n c e n t r a t e d f r o m human a m n i o t i c f l u i d , was n o t i d e n t i f i e d i n t h i s work. However, i t h a d a c h r o m a t o g r a p h i c m o b i l i t y on CM-Sephadex w h i c h was s i m i l a r t o t h a t o f v a s o p r e s s i n . T h i s s u g g e s t s t h a t b o t h i t s i o n i c c h a r g e a n d i t s m o l e c u l a r w e i g h t a r e c l o s e t o t h o s e o f t h a t p e p t i d e . F u r t h e r , t h e l a c k o f de-s t r u c t i o n by s o d i u m t h i o g l y c o l l a t e s u g g e s t s t h a t i t d i f f e r s f r o m n e u r o h y p o p h y s i a l p e p t i d e s i n t h a t a d i s u l f i d e b r i d g e , i f p r e s e n t , i s n o t n e c e s s a r y f o r t h e a c t i v i t y o f t h e AFAS. N e v e r t h e l e s s , t h e n a t u r e o f t h e s u b s t a n c e r e m a i n s unknown, and i t s p u r i f i c a t i o n a n d i d e n t i f i c a t i o n n e e d s more e x t e n s i v e i n v e s t i g a t i o n . 3• The p o s s i b l e a c t i o n o f t h e AFAS i n t h e p h y s i o l o g y  o f t h e f e t u s As d i s c u s s e d i n t h e g e n e r a l i n t r o d u c t i o n , t h e r e t e n -t i o n o f w a t e r i n t h e a m n i o t i c s a c i s d i f f i c u l t t o e x p l a i n . A l l o f t h e g r a d i e n t s p r e s e n t , i n c l u d i n g o s m o t i c , c o l l o i d o s m o t i c a n d h y d r o s t a t i c , t e n d t o f a v o r t h e p a s s a g e o f w a t e r f r o m t h e a m n i o t i c compartment t o t h e m o t h e r ( S e e d s , 1973). V a s o p r e s s i n , o r a n o t h e r a n t i d i u r e t i c s u b s t a n c e , may be f o u n d t o have a r o l e i n a l t e r i n g t h e p e r m e a b i l i t y o f t h e a m n i o n . T h i s w o u l d . h e l p . t o e x p l a i n how t h e a m n i o t i c f l u i d i s m a i n t a i n e d d u r i n g p r e g n a n c y . The c o n t r o l o f t h e accumu-l a t i o n , t u r n o v e r a n d s u b s e q u e n t d i m i n i s h m e n t o f t h e f l u i d may a l s o be a n i m p o r t a n t f a c t o r i n t h e i n i a t i o n o f b i r t h . $4 The s o u r c e o f t h e v a s o p r e s s i n , o r o t h e r a n t i d i u r e t i c m a t e r i a l , may be f e t a l o r m a t e r n a l . The r o u t e w h i c h may be i m p o r t a n t f r o m t h e f e t u s t o t h e a m n i o t i c f l u i d , v i a t h e f e t a l k i d n e y s h a s b e e n d i s c u s s e d p r e v i o u s l y . O x y t o c i n h a s b e e n f o u n d t o c r o s s t h e p l a c e n t a i n s h e e p ( N o o d l e , 1 9 6 4 ) , a n d v a s o p r e s s i n may a l s o be t r a n s f e r r e d f r o m m o t h e r t o f e t u s . H i g h l e v e l s o f v a s o p r e s s i n i n t h e f e t a l c i r c u l a t i o n may a l s o e x e r t a n i n f l u e n c e on w a t e r movement t h r o u g h t h e y o l k s a c o f t h e g u i n e a p i g , s i n c e u n l i k e t h e amnion, t h e y o l k s a c i s i n c o n t a c t w i t h t h e f e t a l c i r c u l a t o r y s y s t e m . The y o l k s a c may p l a y a more i m p o r t a n t r o l e i n t h e r e g u l a -t i o n o f t h e volume a n d c o m p o s i t i o n o f t h e a m n i o t i c f l u i d o f the.mouse t h a n d o e s t h e amnion ( R e n f r e e , e t a l . , 1 9 7 5 ) . T h i s must.be l e f t f o r f u t u r e i n v e s t i g a t i o n . A l t h o u g h t h e c o n c e n t r a t i o n o f any p o s s i b l e v a s o p r e s s i n c o u l d n o t h a v e been above 10 m i c r o u n i t s / m l i n t h i s s t u d y , i t may be p o s s i b l e t h a t i t e x e r t s i t s e f f e c t on t h e f e t a l m e m b r a n e s . s y n e r g i s t i c a l l y w i t h a n o t h e r hormone, o r hormones. A n g i o t e n s i n o r p r o l a c t i n ( H o l t , 1975)> f o r example, may c o m b i n e . t o a c t on t h e membranes i n t h e r e g u l a t i o n o f f l u i d e x c h a n g e . I n a d d i t i o n , t h e s e n s i t i v i t y o f f e t a l t i s s u e s t o v a s o -p r e s s i n - l i k e m a t e r i a l s i s unknown i n t h e i n t a c t a n i m a l , a n d i t i s p o s s i b l e ..that s m a l l l e v e l s o f a n t i d i u r e t i c m a t e r i a l , u n d e t e c t a b l e by t h e c l a s s i c a l a s s a y , a r e a b l e . . t o have a n a c t i o n i n t h e n a t u r a l p h y s i o l o g i c a l e n v i r o n m e n t . $5 T h e . p r e s e n c e o f t h e a n t i d i u r e t i c m a t e r i a l , AFAS, i n t h e a m n i o t i c f l u i d . d o e s n o t p r o v e t h a t i t h a s a n y t r u e p h y s i o l o g i c a l r o l e . However, t h e p r e s e n c e o f a n a n t i d i -u r e t i c s u b s t a n c e , w i t h some p r o p e r t i e s i n common w i t h v a s o -p r e s s i n , i n a f l u i d w h i c h c o n t a c t s f e t a l t i s s u e s w h i c h a r e known t o r e s p o n d t o v a s o p r e s s i n i t s e l f , s u g g e s t s t h a t a p o t e n t i a l r o l e f o r t h i s m a t e r i a l c o u l d e x i s t , a n d s h o u l d be i n v e s t i g a t e d c a r e f u l l y i n f u t u r e work. C. Summary 1. . U n c o n c e n t r a t e d a m n i o t i c f l u i d A m n i o t i c f l u i d s a m p l e s f r o m t h e g u i n e a p i g , s h e e p a n d human were t e s t e d on t h e r a t a n t i d i u r e t i c a s s a y . I n a l m o s t a l l o f t h e e x p e r i m e n t s , t h e c o n c e n t r a t i o n o f v a s o p r e s s i n i n t h e . s a m p l e s , i f a n y , was f o u n d t o be b e l o w 10 m i c r o u n i t s / m l , w h i c h was t h e t h r e s h o l d o f t h e a s s a y . F o u r i n j e c t i o n s o f g u i n e a p i g f l u i d e l i c i t e d a s u g g e s t i o n o f a r e s p o n s e when i n j e c t e d i n t o t h e . b i o a s s a y p r e p a r a t i o n . However, t h e s e r e s p o n s e s c o u l d n o t be a c c u r a t e l y q u a n t i f i e d . 2 . . C o n c e n t r a t i o n o f human a m n i o t i c f l u i d A s u b s t a n c e w i t h a n t i d i u r e t i c a c t i v i t y was c o n c e n t r a t e d f r o m seven, human a m n i o t i c f l u i d s a m p l e s . o n a CM-25 Sephadex column. T h e , a m n i o t i c f l u i d a n t i d i u r e t i c s u b s t a n c e (AFAS) h a d a n e l u t i o n p r o f i l e s i m i l a r t o t h a t o f s t a n d a r d v a s o -p r e s s i n . The a c t i v i t y o f e a c h sample was r e l a t i v e l y 86 c o n s i s t e n t w i t h the 'others, and measured approximately 200 microunits/ml of vasopressin-equivalent a c t i v i t y , on the r a t a n t i d i u r e t i c assay. 3. The nature of the AFAS Although the concentrated a n t i d i u r e t i c a c t i v i t y had a s i m i l a r e l u t i o n p r o f i l e t o that of standard vasopressin, and showed a response p a t t e r n i n d i s t i n g u i s h a b l e from t h a t peptide when i n j e c t e d i n t o the r a t a n t i d i u r e t i c p r e p a r a t i o n , i t could.not be i d e n t i f i e d as vasopressin. I t d i f f e r e d from vasopressin i n s t a b i l i t y c h a r a c t e r i s t i c s , consistency of response when assayed, and i t was r e s i s t a n t to i n a c t i -v a t i o n when incubated w i t h sodium t h i o g l y c o l l a t e . A t t e m p t s t o i d e n t i f y the a c t i v e agent i n the f l u i d as a n g i o t e n s i n I I , or as a combination of a n g i o t e n s i n I I and vasopressin, proved negative. This was based p r i m a r i l y upon the contrast between the response patterns found during the r a t a n t i d i u r e t i c assay. I t was concluded that the ac-t i v i t y was a t t r i b u t a b l e t o some unknown substance. $7 REFERENCES Ab r a m o v i c h , . D . R. 1 9 7 0 . . F e t a l f a c t o r s i n f l u e n c i n g t h e volume and c o m p o s i t i o n o f l i q u o r a m n i i . _J. O b s t e t . Gynaec, B r i t . Cmmwth. 77: $ 6 5 - 3 7 7 . Adamsons, K., J r . , S. L. E n g e l , an d H. B. v a n Dyke. 1 9 5 $ . The s t a b i l i t y o f n a t u r a l a n d s y n t h e t i c n e u r o h y p o p h y s i a l hormones i n v i t r o . E n d o c r i n o l o g y . 6 3 : 6 7 9 - 6 $ 7 . A d o l p h , E . F. 1 9 6 7 . Ontogeny o f volume r e g u l a t i o n s i n e m b r y o n i c e x t r a c e l l u l a r f l u i d s . Q u a r t . Rev. B i o l . 4 2 : 1 - 3 9 . A l e x a n d e r , D. P. a n d D. A. N i x o n . 1 9 6 1 . The f o e t a l k i d n e y . B r i t . Med. B u l l . 17: 1 12-117 . A l e x a n d e r , D. P., D. A. N i x o n , W. F. Widdas and F. X. W o h l -z o g e n . 195S. R e n a l f u n c t i o n i n t h e s h e e p f o e t u s . J . P h y s i o l . 1 4 0 : 1 4 - 2 2 . A l e x a n d e r , ~ D . P., D. A. N i x o n , ¥ . F. Widdas and F. X. W o h l -z o g e n . 1 9 5 9 . G e s t a t i o n a l v a r i a t i o n s i n t h e c o m p o s i t i o n o f t h e f o e t a l f l u i d s a n d f o e t a l u r i n e i n t h e s h e e p . J . P h y s i o l . 1 4 0 : 1 - 1 3 . A l e x a n d e r G . a n d D. W i l l i a m s . 1 9 6 $ . H o r m o n a l c o n t r o l o f a m n i o t i c a n d a l l a n t o i c f l u i d volume i n o v a r i e c t o m i z e d s h e e p . _J. E n d o c r i n o l . 4 1 : 4 7 7 - 4 $ 5 . Ames, R. G. 1 9 5 3 . U r i n a r y w a t e r e x c r e t i o n a n d n e u r o -h y p o p h y s i a l f u n c t i o n i n f u l l t e r m a n d p r e m a t u r e i n f a n t s s h o r t l y a f t e r b i r t h . P e d i a t r i c s . 1 2 : 2 7 2 - 2 $ l . Ames, R. G. a n d H. B. v a n Dyke. 1 9 5 1 . T h i o g l y c o l l a t e i n -a c t i v a t i o n o f p o s t e r i o r p i t u i t a r y a n t i d i u r e t i c p r i n -c i p l e s a s d e t e r m i n e d i n t h e r a t . P r o c . S o c . Exp. B i o l . Med. 76: 5 7 6 - 5 7 $ . Ames, R. G. a n d H. B. v a n Dyke. 1 9 5 2 . A n t i d i u r e t i c hormone i n t h e serum o r p l a s m a o f r a t s . E n d o c r i n o l o g y . 5 0 : 3 5 0 - 3 6 0 . B a r a t z , R.. A. and.R. C. Ingraham, . 1 9 5 9 . S e n s i t i v e b i p a s s a y method f o r m e a s u r i n g a n t i d i u r e t i c hormone i n mammalian p l a s m a . P r o c . S o c . E x £ . B i o l . Med. 1 0 0 : 2 9 6 - 2 9 9 . BB B a r n e s , A. C. 1 9 6 8 . r F u n c t i o n a l d i f f e r e n t i a t i o n o f t h e k i d n e y . I n : " I n t r a u t e r i n e D e v e l o p m e n t " (A. C. B a r n e s , e d . ) . L e a & F e b i g e r , P h i l a d e l p h i a , pp. 2 4 5 - 2 5 2 . B a t e s , H. R. 1 9 6 3 . C o m p a r i s o n o f p h a r y n g e a l a n d a m n i o t i c f l u i d s i n t h e g u i n e a p i g f e t u s . Am. _J. O b s t e t . Gynec. 8 5 : 4 8 4 - 4 8 6 . B a t t a g l i a , F., H. P r y s t o w s k y , C. S m i s s o n , A. H e l l e g e r s a n d P. B r u n s . i 9 6 0 . F e t a l b l o o d s t u d i e s . X I I I : The e f f e c t o f t h e a d m i n i s t r a t i o n o f f l u i d s I V t o . m o t h e r s upon t h e c o n c e n t r a t i o n s o f w a t e r a n d e l e c t r o l y t e s i n p l a s m a o f human f e t u s e s . P e d i a t r i c s . 2 5 : 2 - 1 0 . Behrman, R..E., J . T. P a r e r a n d C. W. de L a n n o y . 1 9 6 7 . P l a c e n t a l g r o w t h a n d t h e f o r m a t i o n o f a m n i o t i c f l u i d . N a t u r e . ,214: 6 7 8 - 6 8 0 . B e n i r s c h k e , K. a n d D. G. McKay. 1 9 5 3 . The a n t i d i u r e t i c hormone i n f e t u s a n d i n f a n t . O b s t e t . G y n e c o l . 1: 6 3 8 - 6 4 9 . B e r d e , B. . 1 9 7 0 . V a s o p r e s s i n a n d o x y t o c i n . I n : " A s s a y o f P r o t e i n a n d . P o l y p e p t i d e Hormones" (H. v a n Cauwenberge a n d P. F r a n c h i m o n t , e d s . ) . Pergamon P r e s s , O x f o r d , pp. 1 2 8 - 1 4 0 . . Bock, K. D. 1 9 6 3 . Some e x p e r i m e n t s a n d c o n s i d e r a t i o n s on t h e c i r c u l a t o r y a n d r e n a l e f f e c t s o f a n g i o t e n s i n . I n : "Hormones a n d t h e K i d n e y " (P. C." W i l l i a m s , e d . ) . A c a d e m i c . P r e s s , London, pp. 3 1 7 - 3 2 1 . B o n j o u r , J , P. a n d R. L. M a l v i n . 1 9 7 0 a . S t i m u l a t i o n o f ADH r e l e a s e by t h e r e n i n - a n g i o t e n s i n s y s t e m . Am. _J. P h y s i o l . . 218: 1 5 5 5 - 1 5 5 9 . . B o n j o u r , J . P. a n d R. L. M a l v i n . . 1 9 7 0 b . P l a s m a c o n c e n t r a -t i o n s o f ADH i n c o n s c i o u s a n d a n e s t h e t i z e d d o g s . Am. J . P h y s i o l . 21$: 1 1 2 8 - 1 1 3 2 . B o n s n e s , R. W. 1 9 6 6 . C o m p o s i t i o n o f a m n i o t i c f l u i d . C l i n . O b s t e t . Gynec. 9 : 4 4 0 - 4 4 $ . B o u c h e r , R., P. Birp.n a n d J . G e n e s t . 1 9 6 1 . . P r o c e d u r e f o r i s o l a t i o n a n d d e t e r m i n a t i o n o f human b l o o d a n g i o t e n s i n . Can. Biochem.. P h y s i o l . 3 9 : 5 8 1 - 5 9 0 . B o u c h e r , R., J..Menard an d J . G e n e s t . . 1 9 6 7 . A m i c r o m e t h o d f o r measurement o f r e n i n i n t h e p l a s m a a n d k i d n e y o f r a t s . Can. J . P h y s i o l . P h a r m a c o l . 4 5 : 8 8 1 - 8 9 0 . 89 B o u r n e , G. 1962. The c i r c u l a t i o n o f a m n i o t i c f l u i d . I n : "The Human Amnion a n d C h o r i o n 1 ' (G. B o u r n e , e d . ) . Y e a r b o o k M e d i c a l P u b l i s h e r s , I n c . , C h i c a g o , pp. 135-141* B o y l a n , J. W., E . P. C o l b o u r n a n d R. A. McCance. 195$. R e n a l f u n c t i o n i n the' f o e t a l a n d new-born g u i n e a - p i g . J. P h y s i o l . 141: 323-331. Brown, J. J., D. L. D a v i e s , P. B. Doak, A. F. L e v e r , J. I . S. R o b e r t s o n , a n d M. 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B u j a k , A. A. H o d a r i , C. P. H o d g k i n s o n an d F. M. Bumpus.. 1971. I d e n t i f i c a t i o n o f a p r e s s o r p o l y p e p t i d e i n human a m n i o t i c f l u i d . Am. J_. O b s t e t . G y n e c o l . I l l : 1075-1032. 90 C a s t e l a z o - A y a l a , L., S. Karchmer a n d V. S h o r - P i n s k e r . 1 9 7 2 . The b i o c h e m i s t r y o f a m n i o t i c f l u i d d u r i n g n o r m a l p r e g -n a n c y : C o r r e l a t i o n w i t h m a t e r n a l a n d f e t a l b l o o d . I n : " P h y s i o l o g i c a l B i o c h e m i s t r y o f t h e F e t u s " (A. A. H o c f a r i a n d F. M a r i o t i a , e d s . ) . C h a r l e s C. Thomas, S p r i n g f i e l d , 1 1 1 . , pp. 3 2 - 5 3 . C e s s i o n , G. 1 9 7 0 . R e n i n . I n : " A s s a y o f P r o t e i n a n d P o l y -p e p t i d e Hormones" (H. v a n Cauwenberge a n d P. F r a n c h i -mont, e d s . ) . Pergamon P r e s s , O x f o r d , pp. 1 9 3 - 2 0 5 . C h a l l i s , J . R. G., J . J . R o b i n s o n , D. W. Rurak a n d G. D. T h o r b u r n . 1 9 7 6 . The d e v e l o p m e n t o f e n d o c r i n e f u n c t i o n i n t h e human f e t u s . I n : "The B i o l o g y o f Human F e t a l G rowth" ( D . F . R o b e r t s a n d A. M. Thomson, e d s . ) . T a y l o r . a n d . F r a n c i s , London, pp. 1 4 9 - 1 9 4 . C h a r d , T. 1 9 7 3 . The r a d i o i m m u n o a s s a y o f o x y t o c i n a n d v a s o p r e s s i n . _J. E n d o c r i n o l . 5 $ : 1 4 3 - 1 6 0 . C h a r d , T., C. N. Hudson, C. R. W. Edwards a n d N. R. H. Boyd. 1971* R e l e a s e o f o x y t o c i n a n d v a s o p r e s s i n by t h e human f o e t u s . d u r i n g l a b o u r . N a t u r e . 234: 3 5 2 - 3 5 3 . C h e r n o f f , N. and.C. T. G r a b o w s k i . 1 9 7 1 . R e s p o n s e s o f t h e r a t f o e t u s t o m a t e r n a l i n j e c t i o n s o f a d r e n a l i n e a n d v a s o p r e s s i n . B r i t . J . P h a r m a c o l . 4 3 : 2 7 0 - 2 7 $ . 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