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Plasma arginine esterase activity in cystic fibrosis of the pancreas Chan, Katherine Yuet-Ha 1977

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PLASMA ARGININE ESTERASE ACTIVITY IN CYSTIC FIBROSIS OF THE PANCREAS by Katherine Yuet-Ha Chan B. Sc. (Honours), ]974 Un i v e r s i t y of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF , MASTER OF SCIENCE i n the Department of PATHOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1977 © c Catherine Yuet-Ha Chan, 1977 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of FATHDLf)& )/ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date (h/M J , ff 7J U s i n g a micro-method f o r 'the d e t e r m i n a t i o n of plasma a r g i n i n e e s t e r a s e a c t i v i t y , t h e v a l u e s f o r soy-bean t r y p s i n i n h i b i t o r ( S T I ) - i n h i b i t e d a r g i n i n e e s t e r a s e a c t i v i t y i n p a t i e n t s w i t h c y s t i c f i b r o s i s , o b l i g a t e h e t e r o z y g o s e s and age matched c o n t r o l i n d i v i d u a l s have been i n v e s t i g a t e d . The mean o f • S T I - i n h i b i t e d a c t i v i t y was l o w e s t f o r c y s t i c f i b r o s i s p a t i e n t s w h i l e t h e mean f o r normal c o n t r o l s was t h e h i g h e s t , The mean o f S T I -i n h i b i t e d a c t i v i t y f o r t h e h e t e r o z y g o t e s was midway, between t h e v a l u e s of t h e T ) a t i e n t s and t h e normal i n d i v -i d u a l s . The d e f i c i e n c y o f a r g i n i n e e s t e r a s e a c t i v i t y was s t a t i s t i c a l l y , s i g n i f i c a n t f o r b o t h c y s t i c f i b r o s i s . p a t i e n t s and h e t e r o z y g o t e s . By ion-exchange chromatography on DEAE- . Sephadex, a r g i n i n e estera.se a c t i v i t y i n c h l o r o f o r m -e l l a . g i c a c i d t r e a t e d plasma from c y s t i c f i b r o s i s p a t i e n t s and c o n t r o l s u b j e c t s has been r e s o l v e d i n t o two component f r a c t i o n s , ' The s m a l l e r peak of a c t i v i t y was e l u t e d a t a p p r o x i m a t e l y 0 . 0 2 Iv; NaCl w h i l e t h e b u l k o f t h e a c t i v i t y was. e l u t e d a t an NaCl c o n c e n t r a t i o n of 0.15 M. S i m i l a r e l u t i o n p a t t e r n s were o b t a i n e d i n b o t h c a s e s of c o n t r o l and c y s t i c f i b r o s i s p l asma, though a. r e d u c t i o n i n s i z e o f t h e two a c t i v i t y peaks was observed i n c y s t i c f i b r o s i s plasma. No iso-enzyme d e f i c i e n c y was d e t e c t u s i n g anion-exchange chromatography. Hence, t h e d e f i c i e n c y of a r g i n i n e e s t e r a s e a c t i v i t y i n t h e c y s t L f i b r o s i s plasma was a p p a r e n t l y e n t i r e l y q u a n t i t a t i v e r a t h e r t h a n q u a l i t a t i v e . I l l TABLE OF CONTENTS Page • I . INTRODUCTION ....... 1 A. H i s t o r y 1 B. G e n e t i c s 2 C. C l i n i c a l f e a t u r e s 3 D. P a t h o g e n e s i s • ... 4 E. The B a s i c d e f e c t i n c y s t i c f i b r o s i s 21 F. Plasma a r g i n i n e e s t e r a s e a c t i v i t y i n c y s t i c f i b r o s i s . 22 I I . MATERIALS AND METHODS 35 A. S u b j e c t s 35 B. B l o o d samples 37 C. Macro-assay of a r g i n i n e e s t e r a s e a c t i v i t y 38 D. M i c r o - a s s a y of a r g i n i n e e s t e r a s e a c t i v i t y 4 4 E. Assessment of t h e M i c r o - a s s a y t e c h n i q u e s 46 F. P r e p a r a t i o n o f columns 49 G. P r e p a r a t i o n o f Samples f o r chromatography and I s o e l e c t r o f o c u s i n g 51 H. DEAE-Sephadex Column chromatography 55 I . A n a l y s i s o f S l u t e d F r a c t i o n s 56 J . I s o e l e c t r o f o c u s i n g of Sephadex G - 5 0 e l u a t e . 64 K. Methods, of Data A n a l y s i s 75 I I I . RESULTS 79 A. Assessment o f t h e M i c r o - a s s a y t e c h n i q u e . 79 B. A c o m p a r i s o n o f t h e m i c r o - a s s a y w i t h t h e macro-assay o f a r g i n i n e e s t e r a s e a . c t i v i t y 86 C. Comparisons between a r g i n i n e e s t e r a s e a c t i v -i t i e s of t h e c y s t i c f i b r o s i s p a t i e n t s , c o n t r o l s u b j e c t s and h e t e r o z y g o t e s u s i n g t h e m i c r o -a s s a y . 91 D. C o r r e l a t i o n between t h e pla.sma a.rginine e s t -e r a s e a c t i v i t i e s o f c y s t i c f i b r o s i s p a t i e n t s and t h e i r p a r e n t s . 98 E. C o r r e l a t i o n between t h e plasma a r g i n i n e e s t -e r a s e a c t i v i t y and Shwa.chman s c o r e of t h e CF p a t i e n t s . 98 F. DEAE-Sepha.dex column chromatography of plasma f r o m CF p a t i e n t s and normal c o n t r o l s . 101 IV rage G. I s o e l e c t r o f o c u s i n g of Sephadex G-50 e l u a t e s . " 108 116 1^3 •IV. DISCUSSION V. CONCLUSION . . REFERENCES Ikb AFFSNDIX I 160 I I 165 K a l l i k r e i n a c t i v i t y i n plasma as measured, by Rao, Posner and Madler E f f e c t of storage on a.rginine esterase a c t i v i t y . • . 3? Day-to-day v a r i a t i o n of a r g i n i n e esterase a c t i v i t y . 88 Within-day v a r i a t i o n of a r g i n i n e esterase a c t i v i t y . 88 A comparison of the a r g i n i n e e s t e r -ase a c t i v i t i e s measured by the micro-A r g i n i n e esterase a c t i v i t y i n plasma.90 D i f f e r e n c e s of S T I - i n h i b i t e d a r g i n i n e esterase a c t i v i t i e s i n the plasma of c o n t r o l s u b j e c t s , p a t i e n t s w i t h c y s t i c f i b r o s i s and o b l i g a t e h e t e r ^ ozygotes. 96 C o r r e l a t i o n between the S T I - i n h i b i t e d a r g i n i n e esterase a c t i v i t i e s of CF p a t i e n t s and t h e i r parents. 99 C o r r e l a t i o n between S T I - i n h i b i t e d a r g i n i n e esterase a c t i v i t y and Shwachman scores of the CF p a t i e n t s . 100 H y d r o l y s i s of various a r g i n i n e and l y s i n e e s t e r s by plasma and t i s s u e e s t e r a s e s . 128 assay and macro-a.ssay. 89 A summary of t h e symptoms and s i g n s o f c y s t i c f i b r o s i s . K a l l i k r e i n - k i n i n system. E l u t i o n p r o f i l e of a r g i n i n e e s t e r a s e a c t i v i t y i n plasma from c o n t r o l s u b j e c t and c y s t i c f i b r o s i s p a t i e n t . Photograph and diagrammatic r e p r e s e n t a -t i o n of a r g i n i n e e s t e r a s e s e p a r a t e d by p o l y a c r y l a m i d e g e l e l e c t r o f o c u s i n g . H y d r o l y s i s of TAME by a r g i n i n e e s t e r a s e . An example o f one o f t h e methanol s t a n d a r d c u r v e s . An example o f a s t a n d a r d graph, o f B e n z o y l - a r g i n i n e . The r e a c t i o n s i n v o l v e d i n t h e d e t e c t i o n o f a r g i n i n e e s t e r a s e s w i t h t h e formazan system. E f f e c t o f c h l o r o f o r m on enzyme a c t i v i t y . E f f e c t of e l l a g i c a c i d c o n c e n t r a t i o n on enzyme a c t i v i t y . E f f e c t o f e l l a g i c a c i d i n c u b a t i o n t i m e on enzyme a c t i v i t y . E f f e c t o f soybean t r y p s i n i n h i b i t o r c o n c e n t r a t i o n on enzyme a c t i v i t y . The c o r r e l a t i o n between t h e a r g i n i n e e s t e r a s e a c t i v i t i e s measured by t h e m i c r o - a s s a y and t h e macro-assay. D i s t r i b u t i o n o f S T I - i n h i b i t e d a r g i n i n e e s t e r a s e a c t i v i t y i n t h e plasma samples. The mean and 95?> c o n f i d e n c e l i m i t s f o r each group. V l l Page Fi g u r e 16: D i s t r i b u t i o n of S T I - i n h i b i t e d a r g i n i n e esterase values i n the populations s t u d i e d . 97 17: D e s a l t i n g w i t h Sephadex G-50 column (K 9/15)• 102 13: DEAE-Sephadex chromatography of a c t i v a t e d plasma from a c o n t r o l s u b j e c t . 103 19: DEAE-Sephadex chromatography of a c t -i v a t e d plasma from a p a t i e n t with c y s t i c f i b r o s i s . . 105 20: D e s a l t i n g v/ith Serhadex G-50 column (K 15/30). * 106 21: DEAE-Sephadex chromatography of de-s a l t e d plasma sample from a c o n t r o l s u b j e c t . 107 22: DEAE-Sephadex chromatography of de-s a l t e d plasma, from a p a t i e n t w i t h c y s t i c f i b r o s i s . 109 23: Photograph of a r g i n i n e esterase separated by polyacrylamide g e l - i s o -e l e c t r o f o c u s i n g . I l l 2k: Photograph of a r g i n i n e esterase s t a i n i n g i n a c o n t r o l g e l s l a b . 112 25: . Photograph of a r g i n i n e esterase separated by d i s c g e l e l e c t r o p h o r e s i s u s i n g Buchler P o l y a n a l y s t . 114 26: Diagrammatic r e p r e s e n t a t i o n of F i g u r e 25. 115 27: Pathways of a c t i v a t i o n of plasma, pro-t e o l y t i c enzymes. 123 23: Diagram i l l u s t r a t i n g the p o i n t s of i n t e r a c t i o n of o^-macroglobulin'with the hemostatic, inflammatory and.com-plement pathways. 133 V I 1 1 ACKNOWLEDGEMENTS ' To Dr. D.A. A p p l e g a r t h , my s u p e r v i s o r , I w i s h ' t o e x t e n d my deepest g r a t i t u d e f o r h i s encouragement and g u i d a n c e t h r o u g h o u t t h e c o u r s e of t h i s s t u d y . I a l s o w i s h t o thank Dr. A.G.F. Dav i d s o n f o r h i s i n v a l u a b l e c r i t i c i s m s and a d v i c e . W i t h o u t t h e i r a s s i s t a n c e , t h i s work would have been i m p o s s i b l e . ' My s i n c e r e t h a n k s go t o Dr. W.H. Chase f o r h i s v a l u a b l e s u g g e s t i o n s a t t h e g r a d u a t e committee m e e t i n g s and c r i t i c i s m o f t h e o r i g i n a l m a n u s c r i p t . I would l i k e t o extend my thanks t o Dr. B. M o r r i s o n i n t h e Department of H e a l t h Care and E p i d e m i o l o g y f o r h e r a s s i s t a n c e w i t h s t a t i s t i c a l a n a l y s i s . I am a l s o g r a t e f u l t o Dr. L. Wong f o r h i s h e l p i n o b t a i n i n g t h e b l o o d samples used i n t h i s i n v e s t i g a t i o n and f o r h e l p f u l d i s c u s s i o n s . The t e c h n i c a l a s s i s t a n c e o f Dr. S, Wood i n t h e Department o f Medical- G e n e t i c s i s v e r y much a p p r e c i a t e d and I thank him f o r a l l o w i n g me t o use h i s equipment i n the s t u d y of i s o e l e c t r i c f o c u s i n g . I a l s o w i s h t o thank Mr. J . S c h o l e y f o r h e l p i n g w i t h t h e p r e p a r a t i o n o f t h e i s o e l e c t r i c - f o c u s i n g appa.ratus. i x The w r i t e r would l i k e t o e x p r e s s a g r e a t d e a l o f t h a n k s t o a l l t h e p e o p l e who d onated b l o o d f o r t h i s r e s e a r c h work. I would l i k e t o t a k e t h i s o p p o r t u n i t y t o thank t h e s t a f f o f the B i o c h e m i c a l D i s e a s e s L a b o r a t o r y a t t h e C h i l d r e n ' s H o s p i t a l f o r t h e i r c o o p e r a t i o n and a s s i s t a n c e , d u r i n g my work i n t h e l a b o r a t o r y . F i n a n c i a l s u p p o r t i n t h e f o r m of S t u d e n t s h i p f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada i s g r a t e f u l l y a cknowledged. I a l s o thank t h e C y s t i c F i b r o s i s F o u n d a t i o n f o r a g r a n t t o D r s . A p p l e g a r t h and D a v i d s o n w h i c h d e f r a y e d t h e c o s t o f some of t h e equipment and c h e m i c a l s u s e d i n t h i s s t u d y . 1 I . INTRODUCTION C y s t i c f i b r o s i s of t h e p a n c r e a s i s a g e n e r a l i z e d h e r e d i t a r y d i s o r d e r o f c h i l d r e n and a d o l e s c e n t s m a n i f e s t - ^ i n g p r i m a r i l y by d y s f u n c t i o n o f e x o c r i n e g l a n d s . The c l a s s i c f e a t u r e s a r e c h r o n i c b r o n c h i o l a r o b s t r u c t i o n and i n f e c t i o n of t h e l u n g s , s t e a t o r r h e a , a z o t o r r h e a , i n c r e a s e i n t h e s a l i n i t y o f sv/eat, m a l n u t r i t i o n and growt h f a i l u r e . The b a s i c d e f e c t remains unknown. A. HISTORY E a r l y r e p o r t s d e s c r i b e d v a r i o u s c l i n i c a l mani-f e s t a t i o n s of the d i s e a s e , but i t was n o t u n t i l 1 9 3 3 t h a t c y s t i c f i b r o s i s o f t h e p a n c r e a s was r e c o g n i z e d by Anderson as a s p e c i f i c d i s e a s e e n t i t y because o f t h e anatomic f i n d i n g of e x t e n s i v e p a n c r e a t i c I n v o l v e m e n t ( l ) . However, t h e r e c o g n i t i o n t h a t many o f t h e p a t h o l o g i c a l changes were produced by i n c r e a s e d v i s c o s i t y of mucous s e c r e t i o n s l e d t o t h e term m u c o v i s c i d o s i s ( 5 1 ) w h i c h i s s t i l l w i d e l y used i n Europe. S i n c e t h e d i s c o v e r y by d i Sant'-Agnese (41) i n 1 9 5 3 , "that i n c y s t i c f i b r o s i s t h e s a l i n i t y o f sweat i s i n c r e a s e d and t h a t t h e d i s e a s e i s a g e n e r a l i z e d e x o c r i n o p a t h y w i t h v a r i o u s a b n o r m a l i t i e s of e x o c r i n e s e c r e t i o n , a s h o r t e n e d form o f t h e o r i g i n a l name, c y s t i c f i b r o s i s , has become t h e most common name 2 used . B. GENETICS AND INCIDENCE C y s t i c f i b r o s i s i s t r a n s m i t t e d as an a u t o s o m a l r e c e s s i v e t r a i t ( 3 7 ) . A t b i r t h , b o t h sexes a r e e q u a l l y a f f e c t e d ( 1 3 2 ) , even though more males t h a n f e m a l e s s u r v i v e p a s t t h e age o f p u b e r t y ( 3 7 f 1 3 1 ) * P a r e n t s and o t h e r h e t e r o z y g o u s c a r r i e r s a r e u n a f f e c t e d . The d i s e a s e o c c u r s w i t h o u t r e g a r d t o m a t e r n a l age ( 3 7 ) > b i r t h o r d e r (14) o r season of t h e y e a r ( 9 6 ) . C y s t i c f i b r o s i s i s t h e most common o f a l l i n h e r i t e d d i s e a s e s i n C a u c a s i a n c h i l d r e n . Many e s t i m a t e s o f t h e i n c i d e n c e of c y s t i c f i b r o s i s have been made, w i t h f i g u r e s r a n g i n g from 1 i n 2 , 0 0 0 t o 1 i n 3 , 7 0 0 l i v e b i r t h s ( 3 7 , 81, 82, 1 0 5 , 1 3 3 ) . Thus, t h e f r e q u e n c y o f h e t e r o z y g o u s c a r r i e r s i n t h e C a u c a s i a n p o p u l a t i o n i s a p p r o x i m a t e l y 3 - 57° i m p l y i n g t h a t about 1 i n 5 ° 0 m a r r i a g e s has t h e p o t e n t i a l f o r p r o d u c i n g an a f f e c t e d c h i l d ( 1 0 2 ) . C y s t i c f i b r o s i s has a s t r i k i n g r a c i a l i n c i d e n c e . I t o c c u r s a p p r o x i m a t e l y e q u a l l y In a l l groups o f Caucas-i a n s , v e r y i n f r e q u e n t l y i n O r i e n t a l s , and i s r a r e i n Negroes (45). 3 C. CLINICAL FEATURES C y s t i c f i b r o s i s may p r e s e n t i n t h e n e o n a t a l p e r i o d as i n t e s t i n a l o b s t r u c t i o n by t h i c k e n e d meconium r e q u i r i n g s u r g i c a l r e l i e f . T h i s c o n d i t i o n i s c a l l e d meconium i l e u s and has an i n c i d e n c e of 10-15/« i n c y s t i c f i b r o s i s p a t i e n t s ( 4 8 ) . • D u r i n g t h e f i r s t y e a r of l i f e , most p a t i e n t s have f r e q u e n t l a r g e , f o u l s t o o l s , a l a r g e a p p e t i t e , poor w e i g h t g a i n and d e v e l o p a b d o m i n a l d i s t e n s i o n . S t e a t o r r -hea may be s e v e r e w i t h f e c a l f a t a v e r a g i n g 4 t o 5 t i m e s n o r m a l (102). Pulmonary symptoms of wheezing, c o u g h i n g and s t e a t o r o u s b r e a t h i n g may appear a t any t i m e a f t e r b i r t h , b u t most f r e q u e n t l y i n t h e f i r s t y e a r of l i f e (122). V i r a l i n f e c t i o n s a f f e c t i n g t h e l u n g s o f t e n d e v e l o p i n t o b a c t e r i a l pneumonia. The .common b a c t e r i a l i n v a d e r s a r e S t a p h y l o c o c c u s aureus (76) and Pseudomonas a e r u g i n o s a ( 4 7 ) . Pulmonary i n v o l v e m e n t l a t e r i n c h i l d h o o d may be s u g g e s t e d by b a r r e l - c h e s t d e f o r m i t i e s and d i g i t a l c l u b b i n g . C h r o n i c pulmonary d i s e a s e dominates t h e c l i n i c a l p i c t u r e and d e t e r m i n e s t h e f a t e of t h e p a t i e n t . I t i s p r e s e n t i n a l m o s t a l l p a t i e n t s a t some t i m e i n t h e c o u r s e of t h e i r d i s e a s e . Death may o c c u r as a r e s u l t 4 o f b r o n c h o - p n e u m o n i a , a s p h y x i a , c a r d i o r e s p i r a t o r y f a i l u r e a s s o c i a t e d w i t h c o r - p u l m o n a l e o r a c u t e p u l m o n a r y i n f e c t i o n o n t o p o f s e v e r e c h r o n i c l u n g d i s e a s e . A s u m m a r y o f t h e s y m p t o m s a n d s i g n s o f c y s t i c f i b r o s i s i s s h o w n i n F i g u r e 1. N o r m a l g r o w t h i s r e t a r d e d i n t h e c y s t i c f i b r o s i s p a t i e n t , w i t h b o d y w e i g h t - b e l o w t h a t e x p e c t e d f o r h e i g h t o r a g e , a n d e v e n l i n e a r g r o w t h i s s o m e t i m e s d e p r e s s e d ( 1 3 7 ) « T h e s e c h i l d r e n a r e t h e r e f o r e s m a l l a n d a p p e a r m a l n o u r i s h e d . T h e e x t r e m i t i e s a r e t h i n a n d t h e a b d o m e n , d i s t e n d e d . W i t h i m p r o v e d m e d i c a l t r e a t m e n t , m a n y c y s t i c f i b r o s i s p a t i e n t s h a v e s u r v i v e d t o a d u l t h o o d . T h e m e d i a n a g e o f d e a t h i n 1 9 7 0 w a s 15 y e a r s ( 4 6 ) , i n c o n t r a s t t o a m e d i a n a g e o f d e a t h o f 8 m o n t h s b e f o r e a n y t r e a t m e n t w a s a v a i l a b l e ( 1 4 5 ) . D. FATHOGENSIS C y s t i c f i b r o s i s i s p r o d u c e d b y a g e n e w i t h p l e i o t r o p i c e f f e c t s , i . e . t h e h o m o z y g o u s g e n e s t a t e m a n i f e s t s i t s e l f i n m u l t i p l e d e f e c t s o f s e e m i n g l y u n -r e l a t e d o r i g i n ( 1 0 2 ) . M o s t r e s e a r c h w o r k h a s c o n c e n t r a -t e d o n t h e e x o c r i n e s y s t e m , i n w h i c h t h e s e c r e t o r y f l u i d s h a v e m a n y a b n o r m a l p r o p e r t i e s w h i c h e x a c e r b a t e t h e c l i n i c a l p i c t u r e . N e v e r t h e l e s s , i t h a s b e e n Figure 1: A summary of the symptoms and signs of cystic fibrosis (66). PRIMARILY DUE TO PULMONARY LESION EARLY SYMPTOMS AND SIGNS OF LOW GRADE CHRONIC PULMONARY INFECTION Increased respiratory rate Dry, hacky, non-productive cough Prolonged expiration phase. Decreased activity MODERATE Change in personality—more introverted and irritable Decreased appetite—may still be good but not voracious Decreased exercise tclerance Scattered or localized wheezes, musical rhonchi, rales Signs of emphysema'-i-inereased A-P diameter diminished area of cardiac dullness depressed diaphraoms palpable liver bonier Failure to gain or grow or weight loss Repeated episodes of respiratory infection ADVANCED Listless. limitation of norma] activity, easily fatigued Marked doorcase in appetite associated with weight loss Growth failure—stuntinu Chronic, paroxysmal, productive cough often associated with vomiting Muscular weakness—flabby Increased respiratory rate, shortness of breath on exertion, orthopnea, dyspnea Noisy respiration—wheezing, bubbling, audible rales Cyanosis, digital clubbing Marked emphysema-marked increase in A-P diameter—barrel chest-—pigeon breast limited respiratory excursion of thoracic cage depressed diaphragms hyper-resonance over entire chest decreased exchange Diffuse and localizea rales and rhonchi . Fever, tachycardia, toxicity Rounded shoulders, forward position of head, poor posture Hemoptysis, signs of atelectasis, pneumothorax and lung abscess . Signs of cardiac failure—edema, enlarged tender liver, venous distention Visual impairment and eye ground changes PANCREATIC AND NUTRITIONAL Meconium ileus Poor weight gain despite voracious appetite Distended abdomen 3-4 bulky, greasy, floating, foul-smelling stools per day Rectal prolapse Palpable loops of bowel especially in right lower quadrant Cramps and excessive foul flatus Malnutrition, poor muscle tone, small flabby muscles, lack of subcutaneous fat Hypoproteinemia with generalized edema SIGNS OF HYPONATREMIA AND HYPOCHLOREMIA Loss of sodium and chloride by sweating Mild—forehead tastes salty Severe—muscle cramps, weakness, shock SIGNS OF BILIARY CIRRHOSIS AND PORTAL HYPERTENSION Firm, nodular liver—often palpable in midb'ne Splenomegaly Hypersplenism—decreased WBC and platelets, anemia Hematemesis and melena from esophageal varices 6 d i f f i c u l t t o f o c u s a t t e n t i o n on any one b i o c h e m i c a l a b n o r m a l i t y f o r i n v e s t i g a t i o n . The f o l l o w i n g i s a s u b j e c t i v e s e l e c t i o n of what I c o n s i d e r t o be some o f t h e r e l e v a n t r e s e a r c h done on c y s t i c f i b r o s i s . 1. Sweat Glands The i n c r e a s e d sodium and c h l o r i d e c o n c e n t r a t i o n s o f e c c r i n e sweat a r e t h e most c o n s t a n t and c l e a r l y d e f i n e d , a b n o r m a l i t i e s t h a t have been d e t e c t e d i n c y s t i c f i b r o s i s . I n 98fo o f p a t i e n t s t h e s e c r e t i o n of c h l o r i d e and sodium i s i n c r e a s e d two t o f i v e t i m e s above n o r m a l l e v e l s ( 4 l , 6 6 ) . Sweat p o t a s s i u m i s a l s o i n c r e a s e d , but 7 t o a l e s s e r degree t h a n sodium and c h l o r i d e ( 41 , 4 4 ) . T h i s c h a r a c t e r i s t i c f i n d i n g i s p r e s e n t a t b i r t h and t h r o u g h o u t l i f e and appears t o be u n r e l a t e d e i t h e r t o t h e s e v e r i t y o f t h e u n d e r l y i n g d i s e a s e o r t o t h e e x t e n t o f organ i n v o l v e m e n t . E x c e s s i v e s a l t l o s s i n sweat due t o sudden r i s e i n e n v i r o n m e n t a l t e m p e r a t u r e may l e a d t o s e v e r e sodium d e p l e t i o n and va.scular c o l l a p s e ( 4 l , 4 4 ) . Many s t u d i e s have been performed t o d e t e r m i n e the cause of e l e v a t e d s a l t c o n c e n t r a t i o n i n c y s t i c f i b r o s i s sweat. There appear t o be no m o r p h o l o g i c a l d i f f e r e n c e s between t h e sweat g l a n d s o f c y s t i c f i b r o s i s 7 p a t i e n t s and normal s u b j e c t s ( 5 9 , 103) . The sweat g l a n d i s of normal s i z e i n c y s t i c f i b r o s i s ( 7 ) . There a r e no d i f f e r e n c e s i n t h e r a t e s of s w e a t i n g ( 5 0 , 9 5 , 1 3 3 ) or i n c o n c e n t r a t i o n s of o r g a n i c c o n s t i t u e n t s (19, 5 7 ) . However, b o t h by d i r e c t m i c r o p u n c t u r e s t u d i e s ( 1 2 5 , 1 2 6 ) and i n d i r e c t c a l c u l a t i o n s ( 5 0 , 5 6 , 1 3 5 ) , t h e r e i s an i n d i c a t i o n t h a t t h e duct of t h e sweat g l a n d I n c y s t i c f i b r o s i s has a. g r e a t l y r e d u c e d c a p a c i t y t o r e -a b s o r b sodium. Sweat f r o m p a t i e n t s w i t h c y s t i c f i b r o s i s c o n t a i n s a h e a t - l a b i l e , n o n - d i a l y z a b l e f a c t o r w h i c h i n h i b i t s sodium r e a b s o r p t i o n i n t h e d u c t of t h e r a t p a r o t i d g l a n d ( 9 9 ) a n d i n t h e normal sweat g l a n d ( 7 3 ) . Whether t h i s f a c t o r produces t h e d e f e c t i n sodium r e a b s o r p t i o n i s unknown. 2 . G l y c o p r o t e i n s and C a l c i u m G l y c o p r o t e i n m e t a b o l i s m i n c y s t i c f i b r o s i s has been t h e s u b j e c t of i n t e n s i v e i n v e s t i g a t i o n . A p p a r e n t i n c r e a s e d v i s c o s i t y of mucous s e c r e t i o n s i n c y s t i c f i b r o s i s organs and g l a n d s has s u g g e s t e d a d e f e c t i n t h i s a r e a o f m e t a b o l i s m t o many i n v e s t i g a t o r s . I n t h e d uodenal f l u i d and r e c t a l mucus of p a t i e n t s w i t h c y s t i c f i b r o s i s , t h e r e a r e g l y c o p r o t e i n s of abnormal c h e m i c a l s t r u c t u r e which a r e e a s i l y d e n a t u r e d and r e n d e r e d 3 i n s o l u b l e (39, 43, 120). The t o t a l amount o f g l y c o -p r o t e i n i s n o r m a l , b u t t h e c a r b o h y d r a t e m o i e t i e s c o n t a i n an i n c r e a s e i n f u c o s e and a d e c r e a s e i n • s i a l i c a c i d (39, 120). I t has been p o s t u l a t e d t h a t t h e r a t i o of f u c o s e t o s i a l i c a c i d may d e t e r m i n e g l y c o p r o -t e i n s o l u b i l i t y and v i s c o s i t y and may be a f a c t o r i n t h e p a t h o g e n s i s o f c y s t i c f i b r o s i s (93)• S i m i l a r comp-o s i t i o n a l changes i n g l y c o p r o t e i n a l s o o c c u r i n s a l i v a r y g l a n d (97) • Most r e c e n t l y , Vaze e t a l have d e m o n s t r a t e d a marked i n c r e a s e i n g l y c o p r o t e i n i n t h e p l a t e l e t s o f c y s t i c f i b r o s i s p a t i e n t s ( l 4 l ) . The s i a l i c a c i d - p r o t e i n r a t i o (1:50) i s much l o w e r i n t h e p a t i e n t s ' p l a t e l e t s when compared t o c o n t r o l s (1:15). However, t h e p r e s e n c e o f abnormal g l y c o p r o t e i n s i s n o t u n i q u e i n c y s t i c f i b r o s i s P a t i e n t s w i t h f u c o s i d o s i s a l s o e x h i b i t an a c c u m u l a t i o n o f f u c o s e c o n t a i n i n g g l y c o p r o t e i n s i n a l l t i s s u e s due t o t h e l a c k o f an a c i d l y s o s o m a l h y d r o l a s e , oC- f u c o s i d a s e (49). I n c r e a s e d c h l o r i d e and sodium c o n t e n t s o f sweat and mixed s a l i v a have been r e p o r t e d i n some p a t i e n t s w i t h t h i s d i s e a s e . G l y c o p r o t e i n s from t h e b r o n c h i a l t r e e o f c y s t i c f i b r o s i s p a t i e n t s have been s t u d i e d by L a m b l i n and c o -wor k e r s (35). T h e i r d a t a s u g g e s t t h a t t h e s e g l y c o p r o -o t e i n s a r e more a c i d i c and a ; f u c o s e - r i c h g l y c o p r o t e i n i s n o t f o u n d i n t h e c y s t i c f i b r o s i s sputum. Low l e v e l s of f u c o s e i n mucous g l y c o p r o t e i n s may be i m p o r t a n t because of a r e c e n t p r o m i s i n g f i n d i n g t h a t c y s t i c f i b r o s i s s e r a 3 do not p o s s e s s t h e p r o p e r t y f o r a d d i t i o n of -^H-fucose t o c e l l membrane s u r f a c e s ( 1 8 ) . The s u b m a x i l l a r y g l a n d o f c y s t i c f i b r o s i s p a t i e n t s s e c r e t e s a g l y c o p r o t e i n - r i c h s a l i v a w h i c h i s abnormal i n appearance and c o m p o s i t i o n as d e m o n s t r a t e d by Mandel e t a l and Chernic'k e t a l (16, 97). The f l u i d i s t u r b i d and c o n t a i n s a h i g h e r f u c o s e , s i a l i c a c i d , h exose, t o t a l p r o t e i n and c a l c i u m c o n t e n t (17). T h i s i s i n c o n t r a s t t o t h e c l e a r s e c r e t i o n i n u n a f f e c t e d p e r s o n s . C y s t i c f i b r o s i s s a l i v a c l e a r s w i t h t h e a d d i t i o n o f che-l a t i n g a g e n t s , w h i l e c a l c i u m added t o n o r m al s a l i v a w i l l i n c r e a s e i t s t u r b i d i t y (65). -An i n c r e a s e i n c a l c i u m l e v e l s o f many g l y c o p r o t e i n - r i c h b i o l o g i c f l u i d s i s i n d e e d one o f t h e most c o n s t a n t f i n d i n g s i n c y s t i c f i b r o s i s . An e f f e c t of d i v a l e n t i o n s on t h e s o l u b i l i t y o f g l y c o p r o t e i n s i s r e c o g n i z e d , a.nd Kwart and Sashoua s u g g e s t e d t h a t c a l c i u m might f o r m a l i n k between g l y c o -p r o t e i n s and amino a c i d s ( 8 4 ) . Svennerholm (139) and Lev et a l (88) a l s o s u g g e s t e d t h a t c a l c i u m might p l a y 10 an i m p o r t a n t p a r t i n d e c r e a s i n g t h e s o l u b i l i t y o f g l y c o p r o t e i n s i n c y s t i c f i b r o s i s . The p r e c i p i t a t i o n o f a r e l a t i v e l y i n s o l u b l e c a l c i u m - g l y c o p r o t e i n complex may e x p l a i n some of t h e p a t h o l o g i c a . l snd c l i n i c a l f i n d i n g s In. c y s t i c f i b r o s i s . E f f e c t s of c a l c i u m on the p e r m e a b i l i t y o f ' membranes a r e a l s o r e c o g n i z e d . An i n t e r e s t i n g t h e o r y has been proposed by G i b s o n and a s s o c i a t e s (60). T h i s c o n c e r n s t h e s u p p o s i t i o n t h a t mucus has a b i o l o g i c a l f u n c t i o n of i n h i b i t i n g p a s s i v e f l o w s o f water and s m a l l i o n s . The t h e o r y c l a i m s t h a t c y s t i c f i b r o s i s i s a d i s e a s e i n w h i c h mucus i s hyperpermeable t o wa t e r and f a i l s t o p e r f o r m t h i s f u n c t i o n a d e q u a t e l y , and t h a t t h i s f a i l u r e i s c a u s e d by an exce s s s e c r e t i o n o f c a l c i u m f rom e x o c r i n e g l a n d s . However, we s t i l l do not know v/hether t h e f u n d a -m e n t a l m o l e c u l a r a b n o r m a l i t y i n c y s t i c f i b r o s i s r e s i d e s w i t h i n t h e c e l l o r w i t h i n a c i r c u l a t i n g s u b s t a n c e a f f e c t i n g c e l l f u n c t i o n . 3 . P a n c r e a s P a t h o l o g i c a l changes i n t h e p a n c r e a s may be obs e r v e d d u r i n g f e t a l l i f e . A t f i r s t , s m a l l c a s t s of e o s i n o p h i l i c m a t e r i a l may be found i n t h e a c i n i w h i c h become i r r e g u l a r w i t h c e l l u l a r p r o l i f e r a t i o n . The c a s t s . 11 t h e n appear i n t h e d u c t s which become d i s t e n d e d and o c c l u d e d by f i b r o u s t i s s u e . C y s t f o r m a t i o n and a c i n a r a t r o p h y a r e f o l l o w e d by i n f i l t r a t i o n w i t h a d i p o s e t i s s u e . A f t e r 2 y e a r s of age t h e pancreas may be u n r e c o g n i z a b l e e x c e p t as i s l e t s c l u s t e r e d i n f i b r o u s o r a d i p o s e t i s s u e (2, 11). L o c a l i z e d f o c i o f b i l i a r y o b s t r u c t i o n and f i b r o s i s a r e common f i n d i n g s i n t h e l i v e r a t n e c r o p s y ( 6 6 ) . U n t r e a t e d p a n c r e a t i c d e f i c i e n c y r e s u l t s i n p r o g r e s s i v e m a l n u t r i t i o n , marked s t e a t o r r h e a and a z o t -o r r h e a , abnormal f a t i n t h e s t o o l s and d e f i c i e n c i e s o f l i p i d s o l u b l e v i t a m i n s . A p p r o x i m a t e l y 10-15/S o f p a t i e n t s do n o t d e v e l o p s i g n s o f p a n c r e a t i c d e f i c i e n c y . The s t e a t o r r h e a and a z o t o r r h e a of c y s t i c f i b r o s i s a r e due t o t h e d e f i c i e n c y of p a n c r e a t i c l i p a s e s , t r y p s i n , chymotryp-s i n and c a r b o x y p e p t i d a s e s (45, 93)• Hadorn and a s s o c i a t e s have s t u d i e d p a n c r e a t i c f u n c t i o n i n p a t i e n t s w i t h c y s t i c f i b r o s i s who do n o t have s t e a t o r r h e a ( 6 7 , 6 8 ) . Enzyme s e c r e t i o n i n r e s p o n s e t o p a n c r e o z y m i n was a l m o s t n o r m a l , b u t s e c r e t i n r e s p o n s e was much d e c r e a s e d . I n c o n t r a s t , c h i l d r e n w i t h p a n c r e a t i c i n s u f f i c i e n c y w i t h o u t c y s t i c f i b r o s i s had a more normal 12 secretion r e s p o n s e b u t d e f i c i e n t enzyme s e c r e t i o n ( 6 9 ) . I n 8 o f 10 p a t i e n t s v / i t h c y s t i c f i b r o s i s , t h e v i s c o s i t y o f duodenal j u i c e was i n c r e a s e d a f t e r s e c r e t i n s t i m u l a -t i o n . The b i c a r b o n a t e c o n c e n t r a t i o n d i d n o t r i s e , and c h l o r i d e c o n c e n t r a t i o n remained h i g h as t h e r a t e o f f l o w i n c r e a s e d . The same f i n d i n g s have been r e p o r t e d by R i c k ( 1 1 8 ) . Hadorn c o n c l u d e s t h a t d e c r e a s e d s e c r e t i n r e s p o n s e may c o n t r i b u t e t o the o b s t r u c t i v e p a t h o l o g y i n t h e p a n c r e a s and a l s o may i n d i c a t e a p r i m a r y a b n o r m a l i t y o f t h e d u c t c e l l s . 4. Lung C h r o n i c o b s t r u c t i v e l u n g d i s e a s e i s t h e most s e r i o u s a s p e c t of c y s t i c f i b r o s i s . Pulmonary d i f f i c u l t i e s i s s u e f r o m t h e i n a d e q u a t e removal o f mucous s e c r e t i o n s f r o m t h e t r a c h e o b r o n c h i a l t r e e and upper r e s p i r a t o r y t r a c t ( 4 0 ) . The mucous g l a n d s a r e d i s t e n d e d by a c c u m u l a t e d v i s c i d mucus (2) wh i c h may o b s t r u c t one o r more b r o n c h i (144) i n a p r o g r e s s i v e d i l a t a t i o n l e a d i n g t o b r o n c h i -e c t a s i s . P u l m o n a r y - f u n c t i o n s t u d i e s i n v a r i o u s l a b o r a -t o r i e s have c o n f i r m e d t h e c l i n i c a l f i n d i n g s of a i r w a y i n v o l v e m e n t . S e q u e n t i a l t e s t i n g o f a p a t i e n t v / i t h c y s t i c f i b r o s i s u s u a l l y shows i n c r e a s i n g o b s t r u c t i o n o f t h e a i r -ways as i n d i c a t e d by peak e x p i r a t o r y r a t e , mid-maximal 13 e x p i r a t o r y f l o w r a t e , and 1 s e c . f o r c e d e x p i r a t o r y volume. V i t a l c a p a c i t y d e c r e a s e s w i t h age and f u n c t i o n a l r e s i d u a l c a p a c i t y i s e l e v a t e d ( 8 ) . Pulmonary h y p e r t e n s i o n and c o r pulmonale a r e f r e q u e n t c o m p l i c a t i o n s ( 6 3 , 1 0 7 ) . The a n a l y s i s of b r o n c h i a l mucus, a l t h o u g h c o n t a m i n a t e d w i t h b a c t e r i a and i n f l a m m a t o r y e x u d a t e , shows a d e c r e a s e d c o n t e n t of sodium and c h l o r i d e i n c y s t i c f i b r o s i s p a t i e n t s ( 9 3 ) • The r a t i o o f f u c o s e t o s i a l i c a c i d i n t h e g l y c o p r o t e i n i s d e c r e a s e d ( 1 1 0 ) . These r e l a t i o n s h i p s a r e o p p o s i t e t o t h o s e o b s e r v e d f o r s a l i v a r y g l a n d s (41) and duodenal f l u i d ( 3 9 , 1 2 0 ) . S i n c e c h r o n i c pulmonary d i s e a s e i n c y s t i c f i b r o s i s p a t i e n t s i s p r o b a b l y t h e r e s u l t o f t h e p r e s e n c e of a b n o r m a l l y v i s c o u s mucus i n t h e r e s p i r a t o r y p a s s a g e s , i t c o u l d n a t u r a l l y be assumed t h a t t h e m u c o c i l i a r y f u n c -t i o n i n c y s t i c f i b r o s i s might be d i s t u r b e d . However, s t u d i e s by S a n c h i s e t a l ( 1 2 3 ) and Newhouse e t a l ( 1 0 9 ) i n d i c a t e d t h a t m u c o c i l i a r y c l e a r a n c e was n o r m al i n c y s t i c f i b r o s i s s u b j e c t s , as judged by t h e r e m o v a l of d e p o s i t e d l a b e l l e d a e r o s o l s f r o m t h e l u n g s . Most r e c e n t l y , Rossman and co-workers has shown t h a t c y s t i c f i b r o s i s serum i n h i b i t s m u c o c i l i a r y c l e a r a n c e when a p p l i e d t o n a s a l mucosa, p r e v i o u s l y t r e a t e d w i t h IgS t o 14 i n d u c e an i n f l a m m a t o r y r e s p o n s e and d i s r u p t t h e mucous h a r r i e r ( 1 2 1 ) . Normal serum has no e f f e c t on m u c o c i l i a r y a c t i v i t y under t h e same c o n d i t i o n s . 5• B l o o d E r y t h r o c y t e s The sodium and p o t a s s i u m c o n c e n t r a t i o n s and t h e morphology of e r y t h r o c y t e s a r e normal i n c y s t i c f i b r o s i s ( 4 , 9 3 , 9 4 ) . I n s t u d i e s of t h e sodium e f f l u x f r o m t h e s e c e l l s , B a l f e e t a l f o u n d a s m a l l d e c r e a s e i n o u a b a i n - , s e n s i t i v e sodium t r a n s p o r t and a.marked r e d u c t i o n i n t h e o u a b a i n - i n s e n s i t i v e t r a n s p o r t o f sodium ( 4 ) . T h i s l a t t e r was caused by d e f i c i e n c i e s i n b o t h th e e t h a c r y n i c a c i d -s e n s i t i v e and - i n s e n s i t i v e p o r t i o n s . P a r a d o x i c a l l y , t h e y n o t e d d e c r e a s e d o u a b a i n - s e n s i t i v e ( N a + / K + ) ATPase i n c y s t i c f i b r o s i s e r y t h r o c y t e membranes, whereas t h e oua-b a i n - i n s e n s i t i v e p o r t i o n was n o r m a l , Lapey and Gardner however, i n a s i m i l a r s t u d y , o b t a i n e d r a t h e r d i f f e r e n t r e s u l t s i n t h a t t h e r e was a d e c r e a s e i n t h e e t h a c r y n i c a c i d - s e n s i t i v e p o r t i o n of Na.+ e f f l u x i n t h e e r y t h r o c y t e s of. c y s t i c f i b r o s i s males and p o s t p u b e r t a l f e m a l e s or h e t e r o z y g o t e s ( 8 7 ) . H o r t o n et a l r e p o r t e d n o r m a l (Na /K ) and Mg ) ATPase a c t i v i t i e s b u t d e c r e a s e d 2 + (Ca ) ATPase i n c y s t i c f i b r o s i s homozygotes (75). The 15 degree of a b n o r m a l i t y was d i r e c t l y p r o p o r t i o n a l t o the . , c l i n i c a l s e v e r i t y of- d i s e a s e . C o l e and D i r k s o b s e r v e d n o r m al o u a b a i n - s e n s i t i v e .ATPa.se and d i m i n i s h e d ouabain-; 2+ i n s e n s i t i v e (Mg )-ATPase i n c y s t i c f i b r o s i s r e d b l o o d c e l l membranes ( 2 0 ) . L a t e r , C o l e and S e l l a s t u d i e d t h e e f f e c t o f s a l i v a f r om c h i l d r e n w i t h c y s t i c f i b r o s i s on an ATP h y d r o l y z i n g enzyme system. S i g n i f i c a n t d e c r e a s e i n t h e a c t i v i t i e s of t h e Ma +/K + a c t i v a t e d o u a b a i n - s e n s i -2+ t i v e component and a Ca - a c t i v a t e d component of ATPase was d e t e c t e d . They c o n c l u d e d t h a t t h e r e was a f a c t o r i n t h e s a l i v a of c y s t i c f i b r o s i s p a t i e n t s which i n t e r f e r e s w i t h a c t i v e c a t i o n t r a n s p o r t as a r e s u l t o f i n h i b i t i o n o f one or more components of t h e ATP h y d r o l y z i n g enzyme system. More r e c e n t l y , McEvoy e t a l demo n s t r a t e d normal 2+ 2+ 2+ l e v e l s of t h e Mg -dependent and Ca. +Mg -dependent ATPase a c t i v i t i e s i n t h e e r y t h r o c y t e membranes o f c y s t i c f i b r o s i s p a t i e n t s ( 1 0 3 ) . F e i g e t a l a l s o r e p o r t e d t h a t t h e t o t a l , o u a b a i n - s e n s i t i v e , and o u a b a i n - i n s e n s i t i v e + / + 2+ (Ma /K. ) ATPase and (Ca ) ATPase a c t i v i t i e s were i n d i s t i n g u i s h a b l e f r o m t h a t of normal c o n t r o l s u b j e c t s ( 5 2 ) . Hence, t h e y c o n c l u d e d t h a t i n t r i n s i c ATPase and monovalent c a t i o n t r a n s p o r t f u n c t i o n s a r e normal i n 16 c y s t i c f i b r o s i s r e d b l o o d c e l l s . Schraoyer and B a g l i a examined membrane a s s o c i a t e d ATPase a c t i v i t i e s i n human r e d b l o o d c e l l s f o l l o w i n g t h e a d d i t i o n o f spent f i b r o b l a s t c u l t u r e media from normal and c y s t i c f i b r o s i s c e l l l i n e s ( 1 2 4 ) . I n co m p a r i s o n t o nor m a l and unspent media, t h e c y s t i c f i b r o s i s media + / + +4-c o n s i s t e n t l y d e p r e s s e d t h e Na /K ATPase, Mg ATPase as w e l l as G a + + ATPase a c t i v i t i e s . However, t h e s e o b s e r v a -t i o n s have n ot been c o n f i r m e d by A p p l e g a r t h e t a l ( 3 ) . L e u k o c y t e s Danes- e t a l demonstrated metachromasia i n c u l t u r e d l e u k o c y t e s f r o m b o t h c y s t i c f i b r o s i s homozygotes and h e t e r o z y g o t e s upon s t a i n i n g w i t h t o l u i d i n e b l u e ( 3 5 ) . Danes and B e a m p r o p o s e d t h a t t h i s p r o b a b l y r e f l e c t s u p t a k e of h e p a r i n f r o m t h e cul t u r e - m e d i u m by p i n o c y t o s i s and n o t , as i n t h e g e n e t i c - m u c o p o l y s a c c h a r i d o s e s , s t o r e d m u c o p o l y s a c c h a r i d e ( 3 3 ) • T h i s h y p o t h e s i s has r e c e i v e d s u p p o r t from work o f R o b e r t s o n el; a l ( 1 1 9 ) . U s i n g ^ - % -h e p a r i n t h e y demonstrated an i n c r e a s e d u p t a k e o f h e p a r i n i n s h o r t - t e r m c y s t i c f i b r o s i s w h i t e b l o o d - c e l l c u l t u r e s w h i c h c o r r e s p o n d t o t h e o b s e r v a t i o n s made on t h e b a s i s of s t a i n i n g . 17 S t u d i e s o f s h o r t - t e r m l e u k o c y t e c u l t u r e s u s i n g Na.g S0^_ i n c o r p o r a t i o n i n t o glycosa.minoglycans (G.A.G.) have been done by C a n d i l l e t a l (15). The amount o f Mag SO^ i n c o r p o r a t i o n into.G.A.G. m a t e r i a l f o u n d i n b o t h the i n c u b a t i o n medium and t h e c e l l s f r om c y s t i c f i b r o s i s p a t i e n t s i s s i g n i f i c a n t l y i n c r e a s e d over n o r m a l s . However, t h e r e i s no m o r p h o l o g i c e v i d e n c e f o r a c c u m u l a t i o n o f G.A.G. i n v i v o ( 4 2 ) , and t h e i r u r i n a r y e x c r e t i o n was f o u n d t o be normal i n 2 7 p a t i e n t s w i t h c y s t i c f i b r o s i s (3D. Plasma and Serum ' Sera, from c y s t i c f i b r o s i s p a t i e n t s and h e t e r o -z y g o t e s c o n t a i n a f a c t o r which causes t h e d i s r u p t i o n o f no r m a l c i l i a r y b e a t i n r a b b i t t r a c h e a l e x p l a n t s (136) and th e t o t a l c e s s a t i o n o f t h e c i l i a r y b e a t of o y s t e r g i l l s (12). T h i s s u b s t a n c e i s a s s o c i a t e d w i t h a n o n d i a l y z a b l e , h e a t - l a b i l e , c a t i o n w i t h an a p p a r e n t m o l e c u l a r w e i g h t of 125,000 t o 180,000 d a l t o n s (13, 136). I n f a c t , t h e c y s t i c f i b r o s i s f a c t o r i s a s m a l l e r m o l e c u l e w i t h a mole-c u l a r w e i g h t between 1,000 and 10,000 d a l t o n s (23) whi c h i s bound t o p r o t e i n s or immuno g l o b u l i n s as d i s c u s s e d l a t e r . T h i s c i l i a r y d y s k i n e s i s f a c t o r i s presumed t o be s i m i l a r t o a s u b s t a n c e i n c y s t i c f i b r o s i s sweat (99) and 13 saliva. (100) which i n h i b i t s the rea.bsorption of sodium i n the rat parotid gland and human sweat gland ( 9 8 ) . Work on t h i s c i l i a r y dyskinesis factor has been hampered by the non-quantitative nature of the c i l i a r y e f f e c t and the seasonal and regional i n a c c e s s i b i l i t y of the c i l i a t e d tissue ( 10, 13). Furthermore, these bioassays do not distinguish.between an i n d i v i d u a l who i s homozygous and one who i s heterzygous f o r c y s t i c f i b r o s i s (10, 13, 102, 1 3 6 ) . Early attempts at f r a c t i o n a t i o n on gel f i l t r a -t i o n columns suggested that the a n t i c i l i a r y factor was absorbed to several serum proteins ( 1 3 6 ) . Bowman's group reported a consistent association of the fac t o r v/ith IgG-containing f r a c t i o n s ( 1 3 ) . Danes et a l l a t e r demonstrated that the c y s t i c f i b r o s i s factor a c t i v i t y i s class s p e c i f i c f o r human IgG, subclass f o r IgGl and IgG2. Interaction occurs with the constant region of heavy chains but not with K and )y- l i g h t chains ( 3 6 ) . By Isoelectrofocusing, Wilson et a l i d e n t i f i e d a c a t i o n i c protein which a. p i ( i s o e l e c t r i c point) of 8 . 4 l . Like the c i l i a r y dyskinesis f a c t o r , i t had a molecular weight ox less tha.n 10,000 daltons and a stoichiometric r e l a t i o n to IgG (147, 150) . Conover and co-workers (24) 19 have m o d i f i e d the o r i g i n a l r a b b i t t r a c h e a a s s a y of Spock et a l f o r a n t i c i l i a r y a c t i v i t y and c l a i m e d t h a t t h e i r method r e l i a b l y d i s t i n g u i s h e d normal c o n t r o l serum from t h a t of h e t e r o z y g o t e s and c y s t i c f i b r o s i s p a t i e n t s . However, t h i s t e s t d i d not d i s t i n g u i s h c l e a r l y between the h e t e r o z y g o t e and homozygote. The r e p o r t e d presence of a n t i c i l i a r y f a c t o r ( s ) i n such d i v e r s e t i s s u e as lymphocytes and f i b r o b l a s t s i s a d d i t i o n a l e v i d e n c e t h a t c y s t i c f i b r o s i s may n o t be s i m p l y a d i s o r d e r of e x o c r i n e g l a n d s . More l i k e l y , t h e f a c t o r ( s ) i s a normal c e l l u l a r or e x t r a c e l l u l a r c o n -s t i t u e n t which i s o v e r p r o d u c e d o r i n e f f e c t i v e l y c a t a b o l -i z e d i n c y s t i c f i b r o s i s (33)• 6. Complement S e v e r a l groups have a t t e m p t e d t o e s t a b l i s h a. r e l a t i o n between the c i l i a r y d y s k i n e s i s f a c t o r and one o f t h e complement components. Conover and h i s c o l l e a g u e s r e p o r t e d e l e v a t e d l e v e l s of the C3 component o f comple-ment i n p a t i e n t s w i t h c y s t i c f i b r o s i s and i n o b l i g a t e h e t e r o z y g o t e s (27). They a l s o o b s e r v e d p h y s i c o c h e m i c a l s i m i l a r i t i e s between the c y s t i c f i b r o s i s f a c t o r and C3a ( a n a p h y l a t o x i n ) , a fragment of C3 (26). P u r i f i e d C3a had a n t i c i l i a r y a c t i v i t y when bound t o IgG. M o reover, 20 t h e y showed t h a t t h e a n t i c i l i a r y a c t i v i t y i s i n a c t i v -a t e d by a n t i b o d y t o C3a ( 9 ) . Hence, c y s t i c f i b r o s i s may be a complement-caused or complement-mediated d i s -o r d e r , i n which a C 3 a - l i k e m o l e c u l e e i t h e r a l o n e or by c o m p l e x i n g w i t h IgG g i v e s r i s e t o the p h y s i o l o g i c a l and p a t h o l o g i c a l s t a t e of the d i s e a s e ( 2 5 ) . Once C3a ( o r CF f a c t o r ) i s complexed t o IgG, i t i s p r o t e c t e d f r o m i t s i n h i b i t o r ( s ) . Danes et a l showed t h a t t h e c y s t i c f i b r o s i s f a c t o r i s bound m o s t l y t o IgG^ (and I g G 2 ) ( 3 6 ) . M o l e n a a r e t a l a l s o demonstrated t h a t C3a. l i n k s t o IgG, and s u g g e s t e d t h a t t h e a c t i v i t y of C3a might be e v o l v e d on t h e s u r f a c e of an immune complex ( 1 0 6 ) . L a t e r , Conover et a l assumed t h a t a d e f e c t i n the a n a p h y l a t o x i n i n a c t i v a t o r ( A I ) or a n o t h e r enzyme s i m i l a r t o c a r b o x y -p e p t i d a s e 3 must o c c u r i n c y s t i c f i b r o s i s t o e x p l a i n t h e a c c u m u l a t i o n of C3a o r CF f a c t o r . However, a d e f e c t i n A l need n o t be p o s t u l a t e d i f C3a i s p r o t e c t e d f r o m A l when i t i s complexed t o IgG. The f i n d i n g s of Conover e t ( 2 7 ) has been c o n f i r m e d by Hann e t ajL ( 7 0 ) . b u t r e f u t e d by Lieberman ( 9 0 ) and S c a n l i n e t a l ( 3 3 ) . 21 E. THE BASIC DEFECT I K CYSTIC FIBROSIS What, t h e n , i s the b a s i c d e f e c t i n c y s t i c f i b r o s i s ? I s i t an i n b o r n e r r o r of g l y c o p r o t e i n meta-b o l i s m , as has been p o s t u l a t e d i n the p a s t ? What i s t h e r e l a t i o n of the e l e c t r o l y t e a b n o r m a l i t y t o t h e p h y s i c o c h e m i c a l and perhaps s t r u c t u r a l a b n o r m a l i t y o f mucous s e c r e t i o n s ? Where and how do t h e e l e c t r o l y t e s and g l y c o p r o t e i n s i n t e r a c t w i t h each o t h e r so as t o g i v e r i s e t o a d e f e c t p r e s e n t i n e x o c r i n e g l a n d s t h r o u g h o u t th e body? Does the c i l i a r y d y s k i n e s i s f a c t o r m o d i f y e x o c r i n e f u n c t i o n , g i v i n g r i s e t o symptoms, or i s i t j u s t a s e c o n d a r y m a n i f e s t a t i o n of t h e d i s e a s e ? A t p r e s e n t , no one h y p o t h e s i s can be made t o r e c o n c i l e and p l a c e i n t o a r a t i o n a l p a t t e r n a l l t h e d i v e r s e and o f t e n c o n f l i c t i n g o b s e r v a t i o n s so f a r advance As more i n s i g h t i s g a i n e d i n t o t h e b i o c h e m i c a l b a c k g r o u n d of c y s t i c f i b r o s i s , t h e p r o b a b i l i t y i n c r e a s e s t h a t the d i s e a s e i s g e n e t i c a l l y h e t e r o g e n e o u s . T h i s i s s u p p o r t e d by v/ork on c u l t u r e d c e l l metachromasia (34), serum-h e x o s a m i n i d a s e l e v e l s (29), and s u l p h a t e i n c o r p o r a t i o n of l e u k o c y t e s from p a t i e n t s w i t h c y s t i c f i b r o s i s .(15) • How-e v e r , t h e i d e n t i t y of the d e f e c t i v e gene(s) remains a c o mplete m y s t e r y . One hopes t h a t .from t h e v a r i e d 22 l a b o r a t o r y i n v e s t i g a t i o n s a u n i f i e d t h e o r y o f pathogen-e s i s of c y s t i c f i b r o s i s w i l l emerge but t h i s has n o t y e t happened. F. PLASMA ARGININE ESTERASE ACTIVITY IN CYSTIC FIBROSIS S e v e r a l t h e o r i e s f o r t h e p a t h o g e n e s i s o f abnormal e x o c r i n e s e c r e t i o n s i n p a t i e n t s w i t h c y s t i c f i b r o s i s o f t h e p a n c r e a s have been p r o p o s e d (45). Because of t h e p o t e n t i a l r o l e of b r a d y k i n i n i n t h e f u n c t i o n o f g l a n d u l a r t i s s u e s and i n t h e m e d i a t i o n of e l e c t r o l y t e t r a n s p o r t , an i n v e s t i g a t i o n of t h e k a l l i k r e i n -k i n i n system i n c y s t i c f i b r o s i s was u n d e r t a k e n by Lieberman and L i t t e n b e r g i n 1969 (92). F i g u r e 2 shows t h e pathway o f b r a d y k i n i n f o r m a t i o n . F a c t o r X I I (Hageman f a c t o r ) i s c o n v e r t e d by exposure t o a f o r e i g n s u r f a c e ( e . g . g l a s s o r c o l l a g e n ) t o a p r o t e l y t i c enzyme f a c t o r X I I a. A c t i v a t e d f a c t o r X I I , though p o s s e s s i n g p o t e n t c o a g u l a n t a c t i v i t y has r e l a t i v e l y low a c t i v i t y i n i n i t i a t i n g k i n i n f o r m a t i o n . F o r e f f i c i e n t c o n v e r s a t i o n o f p r e k a l l i k r e i n t o k a l l i k r e i n , f a c t o r X I I a must f i r s t be p r o t e o l y t i c a l l y s p l i t t o p o l y p e p t i d e f r a g m e n t s t h a t a r e more p o t e n t p r e k a l l i k r e i n a c t i v a t o r s . The m o l e c u l a r changes accompanying t h e c o n v e r s i o n o f human plasma p r e k a l l i k r e i n t o k a l l i k r e i n i s n o t y e t e l u c i d a t e d . 23 Hageman f a c t o r ( F a c t o r X I I ) G l a s s E l l a g i c a c i d K a o l i n A c t i v a t e d Hageman F a c t o r ( F a c t o r X l l a ) P l a s m i n P r e k a l l i k r e i n A c t i v a t o r • P r e k a l l i k r e i n X a l l i k r e i n -K i n i n o g e n - • ' I n h i b i t e d by-i n h i b i t o r s •> Bradykinin K i n i n a s e s ( c a r b o x y -p e p t i d a s e ) I n a c t i v e p r o d u c t s F i g u r e 2: K a l l i k r e i n - k i n i n s y s t e m . 24 Plasma k a l l i k r e i n i s a p r o t e o l y t i c and e s t e r -o l y t i c enzyme w i t h a r g i n i n e and l y s i n e e s t e r a s e a c t i v i t y . There a r e a l s o a number of t i s s u e k a l l i k r e i n s t h a t d i f f e r f r o m plasma k a l l i k r e i n i n b e h a v i o u r t o w a r d sub-s t r a t e s and i n h i b i t o r s , e l e c t r o p h o r e t i c m o b i l i t y and immunochemistry. Plasma k a l l i k r e i n a c t s on k i n i n o g e n s t o r e l e a s e b r a d y k i n i n . I t a l s o c a t a l y z e s a p o s i t i v e f e e d b a c k r e a c t i o n t h a t c o n v e r t s f a c t o r X I I a t o a more p o t e n t p r e k a l l i k r e i n a c t i v a t o r . The a c t i v i t y o f k a l l i -k r e i n i s i n h i b i t e d by t h r e e plasma p r o t e i n s - ^ - m a c r o -g l o b u l i n , C l e s t e r a s e i n a c t i v a t o r and, i n t h e p r e s e n c e of h e p a r i n , a n t i t h r o m b i n I I I ( 2 1 ) . These i n h i b i t o r s n o r m a l l y g u a r d a g a i n s t t h e p r o f o u n d e f f e c t s o f b r a d y k i n i n r e l e a s e on t h e o r g a n i s m . B r a d y k i n i n e x i s t s i n plasma i n v e r y l o w c o n cen-t r a t i o n ( l e s s t h a n 3 ng p e r m i l l i l i t e r as d e t e r m i n e d by r a d i o i m m u n o a s s a y ) . T h i s low l e v e l i s t h e r e s u l t o f t h e b a l a n c e s t r u c k between s i m u l t a n e o u s p r o c e s s e s o f p r o -d u c t i o n and i n a c t i v a t i o n . K i n i n a s e s a r e c a r b o x y p e p t i d -a s e s w h i c h d e s t r o y t h e plasma b r a d y k i n i n . K i n i n s have s t r i k i n g c a r d i o v a s c u l a r e f f e c t s , t h e most pronounced o f w h i c h i s t h e g e n e r a l i z e d d i l a t a t i o n of p e r i p h e r a l a r t e r -i o l e s . I n t r a v e n o u s a d m i n i s t r a t i o n o f b r a d y k i n i n i n man p r o d u c e s an i n c r e a s e i n c a r d i a c o u t p u t , an i n c r e a s e i n 25 s t r o k e f r e q u e n c y and volume, a d e c r e a s e i n s y s t e m i c a r t e r i a l b l o o d p r e s s u r e and t o t a l s y s t e m i c r e s i s t a n c e . K i n i n s a l s o i n c r e a s e s c a p i l l a r y p e r m e a b i l i t y , produce p a i n , and i n f l u e n c e m i g r a t i o n of l e u k o c y t e s (21). The g l a n d u l a r t i s s u e s i n which- th e k i n i n s were f i r s t d e t e c t e d a r e c h a r a c t e r i z e d by r a p i d a l t e r a t i o n s i n b l o o d f l o w (53» 73)• The k i n i n s a r e a l s o found to be . t h e m e d i a t o r s of e l e c t r o l y t e t r a n s p o r t i n some t i s s u e s (55)« Thus, a s t u d y of t h e k a . l l i k r e i n - b r a . d y k i n i n s y s t e m i s of i n t e r e s t i n t h e s e a r c h f o r t h e b a s i c d e f e c t i n c y s t i c f i b r o s i s . Lieberma.n and L i t t e n b e r g e v a l u a t e d t h e b r a d y -k i n i n - f o r m i n g system i n plasma, u r i n e , and s a l i v a . They f a i l e d t o y i e l d any e v i d e n c e f o r e x c e s s i v e f o r m a t i o n o r i m p a i r e d i n a c t i v a t i o n of b r a d y k i n i n i n c y s t i c f i b r o s i s (92) . However,' mixed s a l i v a , of p a t i e n t s w i t h c y s t i c f i b r o s i s was f o u n d t o c o n t a i n s i g n i f i c a n t l y h i g h e r k a l l i -k r e i n a c t i v i t y t h a n t h a t of normal c o n t r o l s . I t seems l i k e l y t h a t t h i s i n c r e a s e i n s a l i v a r y k a l l i k r e i n accom-p a n i e s t h e o v e r a l l I n c r e a s e of o r g a n i c c o n s t i t u e n t s shown t o e x i s t i n s a l i v a , o f p a t i e n t s and i t i s d o u b t f u l t h a t t h e i n c r e a s e d k a l l i k r e i n c o n t e n t of s a l i v a i s t h e abnorm-a l i t y u n d e r l y i n g t h e p a t h o g e n e s i s of c y s t i c f i b r o s i s (92). 26 The f i n d i n g s o f Lieberman and L i t t e n b e r g a r e i n c o n f l i c t w i t h o t h e r w o r k e r s ' o b s e r v a t i o n s as d i s c u s s e d below.. I n 19?2, Rao and N a d l e r began i n v e s t i g a t i o n s on c y s t i c f i b r o s i s i n o r d e r t o e x p l a i n t h e p r e s e n c e o f t h e c a t i o n i c f a c t o r i n h i b i t o r y t o sodium t r a n s p o r t i n t h e s a l i v a r y g l a n d of p a t i e n t s (113). The a c c u m u l a t i o n of t h i s m a c r o m o l e c u l a r f a c t o r may be due t o t h e d e f i c i e n c y of an enzyme or enzymes which n o r m a l l y h y d r o l y s e t h e f a c t o r t o an i n a c t i v e p r o d u c t . S i n c e t h e c a t i o n i c f a c t o r r e s e m b l e s p o l y l y s i n e w h i c h i s a l s o p o l y c a t i o n i c , I t v/ould be e x p e c t e d t o be h y d r o l y s e d by enzymes s i m i l a r t o t r y p -s i n . Rao and N a d l e r t r i e d t o compare t h e l e v e l o f t r y p -s i n - l i k e a c t i v i t y i n t h e s a l i v a , o f p a t i e n t s w i t h c y s t i c f i b r o s i s , t h e i r s i b l i n g s , and t h e i r p a r e n t s v/ith t h a t i n t h e c o n t r o l s u b j e c t s (113). T r y p s i n - l i k e a c t i v i t y v/as a s s a y e d by f o l l o w i n g t h e h y d r o l y s i s of c < - N - b e n z o y l - L - a r g i n i n e e t h y l e s t e r (BAEE). S i n c e e s t e r a s e s may c o n t r i b u t e t o t h e h y d r o l y s i s o f BAEE, t h e a c t i v i t y v/as a l s o a s s a y e d i n t h e p r e s e n c e o f e x c e s s soybean t r y p s i n i n h i b i t o r ( S T I ) . The d i f f e r e n c e s i n a c t i v i t y i n t h e p r e s e n c e and absence o f STI were t a k e n t o r e p r e s e n t t r y p s i n - l i k e a c t i v i t y . I n t h e s a l i v a o f c y s t i c f i b r o s i s p a t i e n t s , t h e enzyme a c t i v i t y was reduced 2? t o 1/10 of t h e c o n t r o l v a l u e . A l t h o u g h t h e t r y p s i n - l i k e a c t i v i t y i n t h e plasma of h e t e r o z y g o t e s was s i g n i f i c a n t l y d i f f e r e n t from t h a t i n e i t h e r c o n t r o l s or a f f e c t e d c h i l d r e n , t h e i n d i v i d u a l h e t e r o z y g o t e c o u l d n o t be . r e l i a b l y i d e n t i f i e d i n each c a s e . A t t e m p t s t o a s s a y t h e a c t i v i t y w i t h more s p e c i f i c s u b s t r a t e s such as BAA ( o( -N-b e n z o y l - L - a r g i n i n e amide) or 3APNA ( oC-N-benzoyl-DL-a r g i n i n e - p - n i t r o a n i l i d e ) were n o t s u c c e s s f u l , hence, t h e p r e s e n c e of t r y p s i n i t s e l f was' e x c l u d e d . The i n h i b i t i o n o f t r y p s i n - l i k e a c t i v i t y by S T I , t o g e t h e r w i t h t h e i n -a b i l i t y of s a l i v a r y e x t r a c t t o h y d r o l y s e BAA and BAPNA s u g g e s t e d t h a t t h e t r y p s i n - l i k e a c t i v i t y m i g h t be s i m i l a r i n a number of I t s p r o p e r t i e s t o t h o s e of k a l l i k r e i n s . S i n c e L ieberman and L i t t e n b e r g (92) have shown p r e v i o u s l y t h a t t h e t o t a l k a l l i k r e i n i n c y s t i c f i b r o s i s s a l i v a i s i n c r e a s e d i n p a r a l l e l w i t h p r o t e i n s e c r e t i o n , Rao and N a d l e r s u g g e s t e d t h a t t h e k a l l i k r e i n f r a c t i o n known t o be i n h i b i t e d by soybean t r y p s i n i n h i b i t o r c o u l d be d e f i c i e n t i n p a t i e n t s w i t h c y s t i c f i b r o s i s . S i n c e k a l l i k r e i n s a r e p r e s e n t i n human plasma, Rao and c o-workers t h e n extended t h e i r s t u d i e s t o I n c l u d e t h e d e t e r m i n a t i o n of k a l l i k r e i n a c t i v i t i e s i n t h e plasma of c o n t r o l s and p a t i e n t s w i t h c y s t i c f i b r o s i s ( 1 1 4 ) . They a s s a y e d k a l l i k r e i n a c t i v i t y as a r g i n i n e 2 8 e s t e r a s e a c t i v a t e d "by t r e a t i n g plasma w i t h c h l o r o f o r m and e l l a g i c a c i d . The r e s u l t s were e x p r e s s e d as m i c r o -moles of o C - N - ( p - t o l u e n e s u l f o n y ) - L - a r g i n i n e m e t h y l e s t e r . (TAME) u t i l i z e d p e r hour per ml"plasma. S i g n i f i c a n t d i f f e r e n c e s between t h e l e v e l s of t o t a l k a l l i k r e i n and S T I - i n h i b i t e d k a l l i k r e i n a c t i v i t y between the plasma o f c o n t r o l i n d i v i d u a l s and c y s t i c f i b r o s i s p a t i e n t s ( T a b l e 1) were f o u n d . M i x i n g plasma from c o n t r o l s and p a t i e n t s w i t h c y s t i c f i b r o s i s e i t h e r b e f o r e or a f t e r a c t i v a t i o n y i e l d e d t h e e x p e c t e d i n t e r m e d i a t e l e v e l s o f k a l l i k r e i n a c t i v i t y . T h i s r e s u l t e x c l u d e d t h e p o s s i b i l i t y t h a t an i n h i b i t o r was p r e s e n t , and t h a t t h e f a c t o r s needed t o a c t i v a t e p r e -k a l l i k r e i n were a b s e n t i n c y s t i c f i b r o s i s p l a s m a . The p r e s e n c e of t r y p s i n was e x c l u d e d s i n c e plasma f r o m con-t r o l s and c y s t i c f i b r o s i s p a t i e n t s d i d n o t h y d r o l y s e BAPNA. A l s o , i n i t i a l e x p e r i m e n t s showed t h a t t h e l e v e l s o f c a r b o x y p e p t i d a s e and k a l l i k r e i n i n h i b i t o r , tv/o o t h e r components of t h e k i n i n system, were n o t s i g n i f i c a n t l y d i f f e r e n t i n t h e c o n t r o l plasma compared t o t h a t i n p a t i e n t s w i t h c y s t i c f i b r o s i s . A l l t h e s e f i n d i n g s i n d i c a t e d t h a t the o b s e r v e d d e f i c i e n c y a p p e a r e d t o be r e s t r i c t e d t o k a l l i k r e i n s ( 1 1 4 ) . Table 1. Kallikrein activity in plasma as measured by Rao, Posner and Nadler. The results are expressed as micromoles of TAME utilized per hour per milliliter of plasma. (114) N Total activity STI-inhibited activity STI-resistant activity SAMPLE M ± S.D. Range M ± S.D. Range M + S.D. Range NORMAL ADULTS 15 43.3±17.7 20.2-73.5 30.2+15.3 12.9-69.1 13.L+13.1 1.1-51.6 NORMAL CHILDREN 20 41.2± 7.6 30.5-55.1 23.2± 6.2 14.1-35.6 17.9± 5.4 10.1-31.4 CYSTIC FIBROSIS (CF 1) 20 16.8± 6.9 2.4-27.6 9.6± 4.2 0.5-17.6 7.2± 4.0 1.9-17.2 CYSTIC FIBROSIS (CF2) 17 26.6±12.3 9.9-50.4 13.0± 6.9 3.4-29.5 13.3± 8.7 3.0-32.5 PARENTS 27 32.5±10.4 14.2-64.0 18.4± 8.4 6.1-47.2 14.8± 8.8 1.8-30.0 .ALL CONTROLS 35 42.1± 13.0 20.2-73.5 26.2±11.6 12.9-69.1 15.9± 9.8 1.1-51.6 .ALL CYSTIC FIBROSIS 37 .•21.3*10.9 2.4-50.4 11.2± 5.9 0.5-29.5 10.0± 7.2 1.9-32.5 30 I n 1974, Lieberman r e p o r t e d t h a t t h e f i n d i n g s o f Rao and c o - workers c o u l d n o t be r e p r o d u c e d i n h i s l a b o r a t o r y (91). However, Lieberman*s a r g i n i n e e s t e r a s e a s s a y d i f f e r e d from t h a t used by Rao e t a l i n t h r e e a s p e c t s : ( i ) Lieberman used BAEE as t h e s u b s t r a t e i n s t e a d of TAME, ( i i ) he used a b u f f e r o f pH 7.1 r a t h e r t h a n 7.6, ( i i i ) Rao e t a l i n c l u d e d 0.15M sodium c h l o r i d e i n t h e s u b s t r a t e m i x t u r e w h i l e Lieberman d i d n o t . Never-t h e l e s s , Lieberman c l a i m e d t h a t he o b t a i n e d i d e n t i c a l v a l u e s f o r a c t i v i t y w i t h the m o d i f i e d s u b s t r a t e a s ' w i t h t h e s u b s t r a t e used by Rao and a s s o c i a t e s when a s s a y i n g plasma a r g i n i n e e s t e r a s e . Rao and N a d l e r l a t e r c l a i m e d t o have q u a l i t a -t i v e and q u a n t a t i v e d i f f e r e n c e s i n a r g i n i n e e s t e r a s e a c t i v i t y i n t h e plasma from c o n t r o l s and c y s t i c f i b r o s i s p a t i e n t s ( i l l ) . U s i n g DEAE-Sephadex column chromato-g r a p h y , c h l o r o f o r m e l l a g i c a c i d t r e a t e d plasma f r o m c o n t r o l s u b j e c t s c o u l d be s e p a r a t e d i n t o two f r a c t i o n s o f a r g i n i n e e s t e r a s e a c t i v i t y ( F i g u r e 3) ( i l l ) . The f i r s t peak which c o m p r i s e d over 6<dfo of the t o t a l a c t -i v i t y was c o m p l e t e l y i n h i b i t e d by S T I . The second peak c o n s i s t e d of a r g i n i n e e s t e r a s e a c t i v i t y v/hich was a l m o s t t o t a l l y r e s i s t a n t t o i n h i b i t i o n o f S T I . I n the plasma o f a l l c y s t i c f i b r o s i s p a .tients t e s t e d , a 31 I •—• Assay without STI c e II o - o Assay with STI O 2 8 _ i \ CONTROL o E wi o 2.4 -E / TJ « 2.0 - / / O _> / O 1.6 / < / E 1.2 / O / . n \ / L E c 0.8 I / \ IO m CM 0.4 i / d " 1/ / k \ d < n urn i 0.3 M c o o c a> 0.2 M o o o 0.IM 10 20 30 40 50 60 Fraction Number E o \ o E «n _o a. a> o > E O \ E e IO m cu d d < 2.8 2.4 2.0 1.6 1.2 0.8 0.4 I — Assay without STI 31 o-o Assay with STI CYSTIC 0.3 M 0.2 M o o 0.1 M u c o o o o Z 10 20 30 40 50 60 Fraction Number F i g u r e 3: E l u t l o n p r o f i l e o f a r g i n i n e e s t e r a s e a c t i v i t y I n plasma from c o n t r o l s u b j e c t and c y s t i c f i b r o s i s p a t i e n t . 3-0 ml f r a c t i o n s were c o l l e c t e d and ass a y e d f o r a c t i v i t y . The dashed l i n e r e p r e s e n t s t h e NaCl g r a d i e n t . ( R e s u l t s o f Rao and N a d l e r a m > 32 a r e d u c t i o n of the s i z e of peak I v/as f o u n d . The s i z e o f peak I I was s i m i l a r t o t h a t o b s e r v e d i n c o n t r o l s . E l e c t r o f o c u s i n g on p o l y a c r y l a m i d e g e l s and subsequent s t a i n i n g f o r a r g i n i n e e s t e r a s e a c t i v i t y r e v e a l e d seven bands i n plasma samples from a l l t h e c o n t r o l s ( F i g u r e 4) ( i l l ) . I n plasma samples f r o m e l e v e n p a t i e n t s w i t h c y s t i c f i b r o s i s , o n l y f i v e bands were f o u n d . I n e i g h t samples, band D was m i s s i n g ; i n two s a m p l e s , band E was m i s s i n g ; and i n one sa.mple, band C was m i s s i n g . T h i s f i n d i n g may be e x p l a i n e d by g e n e t i c h e t e r o g e n e i t y o f t h e d i s e a s e . The p o s s i b i l i t y t h a t c y s t i c f i b r o s i s may not be a s i n g l e e n t i t y b u t r a t h e r a group of c l o s e l y r e l a t e d g e n e t i c a b n o r m a l i t i e s v / i t h s i m i l a r p a t h o l o g i c a l consequences ha.s been d i s -c u s s e d by many a u t h o r s ( 2 9 , 34, 3 3 ) . The p r e s e n t s t u d y was u n d e r t a k e n i n an a t t e m p t t o c o n f i r m and p o s s i b l y e x t e n d t h e o b s e r v a t i o n s made by Rao and c o - w o r k e r s . The a c t i v i t y of plasma a r g i n i n e e s t e r a s e i n c y s t i c f i b r o s i s was i n v e s t i g a t e d u t i l i z i n g e n z y m o l o g i c , c h r o m a t o g r a p h i c and i s o e l e c t r o f o c u s i n g t e c h n i q u e s . The g e n e r a l o b j e c t i v e was t o l o o k f o r q u a l -i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s I n a r g i n i n e e s t e r a s * a c t i v i t y i n plasma and f r a c t i o n s of plasma f r o m p a t i e n t s 33 Figure 4: Photograph and diagrammatic representation of asinine esterase separated by polyacrylamide gel electrofocusing. w i t h c y s t i c f i b r o s i s , p a r e n t s of p a t i e n t s and normal c o n t r o l s u b j e c t s . F a r t of t h i s work i s p u b l i s h e d i n t h e C l i n i c a C h i m i c a A c t a (Appendix I ) . 35 I I . MATERIALS AND METHODS •• . A. Subjects I. Cystic F i b r o s i s Patients Nineteen c l i n i c a l l y ascertained patients with c y s t i c f i b r o s i s (CF) were chosen f o r the study from i n d i v i d u a l s who attended the Cystic F i b r o s i s C l i n i c located at the Health Centre f o r Children, Vancouver General Hospital and from in-patients at the Children's. Hospital i n Vancouver. The diagnosis was confirmed i n a l l patients by the c l i n i c a l h i s t o r y and the presence of an elevated concentration of chloride i n the sweat (58). The severity of the disease was assessed using the Shwachman scoring system (130) which i s shown i n Appendix I I . The patients had Shwachman scores which ranged from 38 to 9 4 , and ages which ranged from 4 to 2 6 years. Ten of the patients were male and nine were female. Another group of 11 patients with CF were chosen f o r the column chromatographic and i s o e l e c t r o f o c u s i n g studies. These patients also attended the Cystic F i b r o s i s C l i n i c at Vancouver General Hospital or were in-patients of the Children's H o s p i t a l . They were also diagnosed by t h e i r c l i n i c a l h i s t o r i e s and elevated sweat ch l o r i d e t e s t s . The age of t h i s group ranged from 5 - 2 4 years. 36 2 . C o n t r o l S u b j e c t s F i f t y i n d i v i d u a l s who had no known f a m i l y h i s t o r y o r c l i n i c a l h i s t o r y i n d i c a t i v e o f c y s t i c f i b r o s i s were s t u d i e d and a s s a y e d f o r a r g i n i n e e s t e r a s e a c t i v i t i e s . They were d i v i d e d i n t o two g r o u p s - c h i l d r e n and a d u l t c o n t r o l s . The c o n t r o l c h i l d r e n group c o n s i s t e d o f 16 i n d i v i d u a l s who ranged i n age from 1 t o 15 y e a r s . They were s e l e c t e d f r om i n - p a t i e n t s a t t h e C h i l d r e n ' s H o s p i t a l i n Vancouver and o u t p a t i e n t s who a t t e n d e d t h e M e t a b o l i c I n v e s t i g a t i o n U n i t a t t h e C h i l d r e n ' s H o s p i t a l . They a l l had no known f a m i l y h i s t o r y o r c l i n i c a l h i s t o r y i n d i c a t i v e o f c y s t i c f i b r o s i s . The 3^ a d u l t c o n t r o l s u b j e c t s were chosen a t random f r o m t h e c l i n i c a l , l a b o r a t o r y and s e c r e t a r i a l s t a f f s of t h e C h i l d r e n ' s H o s p i t a l . They were a l l c l i n -i c a l l y h e a l t h y and ranged i n age from 16-42 y e a r s . A n o t h e r group o f 13 n o r m a l , h e a l t h y a d u l t s were s e l e c t e d f o r t h e c h r o m a t o g r a p h i c and i s o e l e c t r o f o c u s i n g s t u d i e s . They were s e l e c t e d f r o m t h e s t a f f o f C h i l d r e n ' s H o s p i t a l and ranged i n age from 20 t o 42 y e a r s . 3 . H e t e r o z y g o u s C a r r i e r s N i n e t e e n o b l i g a t e h e t e r o z y g o t e s were c h o s e n 37 among parents of c y s t i c f i b r o s i s children. Seventeen of the c a r r i e r s were female and two were male. The age of the heterozygous c a r r i e r s of c y s t i c f i b r o s i s ranged from 32 to 42 years. B, Blood Samples Informed consent was obtained from each subject before c o l l e c t i o n of blood. In the case of minors, t h e i r parents were informed of the reason f o r the study. Venous blood was co l l e c t e d from each subject . into a p l a s t i c syringe. For the micro-assay of arginine esterase a c t i v i t y , l.S ml of blood was immediately mixed with 0.2 mi of 3*3f° (w/v) sodium c i t r a t e i n a polypropy-lene tube. The blood was centrifuged at 3,000 r.p.m. f o r 15 min. at 4°C. The plasma samples were used immediately or stored at -20°G. A l l samples were handled i n p l a s t i c -ware and contact with glass surfaces was avoided. In the case of macro-assays of arginine esterase a c t i v i t y , studies on ion-exchange column chroma-tography and isoelectrofocusing, 9.0 ml of blood was c o l l e c t e d and mixed with 1.0 ml of 3-^7° (w/v) sodium c i t r a t e . Plasma v/as separated by centrifugation at 3,000 r.p.m. f o r 15 min. at 4°G and frozen i n polypropylene tubes at -20°C u n t i l use. 33 C. Macro-assay of A r g i n i n e E s t e r a s e A c t i v i t y 1 . Reagents (a) C h l o r o f o r m (b) E l l a g i c a c i d 0.1 mM - o b t a i n e d from A l d r i c h c h e m i c a l Co., I n c . , Milwaukee Wis. - 3.4 mg of e l l a g i c a c i d was suspended i n 0.10 M phosphate b u f f e r , pH 7.6, c o n t a i n i n g N a C l a t a c o n c e n t r a t i o n o f 0.15 M. ( c ) c C - N - ( p - t o l u e n e - 4 - s u l f o n y l ) - L - a r g i n i n e m e t h y l e s t e r (TAME) 0.015 M - o b t a i n e d from 3DH C h e m i c a l s L t d . , P o o l e , E n g l a n d . - 57.5 mg o f TAME v/as d i s s o l v e d i n 10.0 ml o f 0.10 M phosphate b u f f e r , pH 7.6 c o n t a i n i n g 0.15 M N a C l . (d) T r i c h l o r o a c e t i c a c i d (TCA), 10$ aqueous (e) Soybean T r y p s i n I n h i b i t o r (STI) - o b t a i n e d from Grand I s l a n d B i o l o g i c a l Co., Grand I s l a n d , New Y o r k . -10.0 mg of STI v/as d i s s o l v e d i n 1.0 ml o f n o r m a l s a l i n e . ( f ) P o t a s s i u m permanganate, 2^ 5 aqueous. (g) Sodium s u l f i t e , 10fo aqueous. (h) C h r o m o t r o p i c a c i d w o r k i n g r e a g e n t . 39 - I n a 1 - l i t e r v o l u m e t r i c f l a s k , 200 ml of c o l d water and 100 ml of 2fo aqueous c h r o m o t r o p i c a c i d ( B r i t i s h Drug Houses L t d . , P o o l e , E n g l a n d ) . - 600 ml of c o l d , c o n c e n t r a t e d s u l f u r i c a c i d was t h e n added. When c o o l e d t o f l a s k c a l i b r a t i o n t e m p e r a t u r e , t h e m i x t u r e was d i l u t e d t o 1000 ml w i t h w a t e r . The w o r k i n g r e a g e n t was k e p t i n t h e r e f r i g e r a t o r . ( i ) T r i c h l o r o a c e t i c a c i d , 57° aqueous ( f o r s t a n d a r d s and b l a n k s ) ( j ) M e t h a n o l - o b t a i n e d f r o m Matheson Colman and B e l l . M a n u f a c t u r -i n g C h e m i c a l s , Norwood, Ohio. - 2.02 ml o f methanol was added t o a 50 ml v o l u m e t r i c f l a s k and made up t o t h e mark w i t h 57° TCA. 0.10 ml of t h i s s o l u t i o n was d i l u t e d t o 50 ml w i t h 5% TCA, The f i n a l s o l u t i o n had a c o n c e n t r a t i o n o f 2.0 umoles/ml, 2. P r o c e d u r e The macro-assay o f a r g i n i n e e s t e r a s e a c t i v i t y was e s s e n t i a l l y t h e method d e s c r i b e d by Rao, P o s n e r and N a d l e r (114-). Measurement of e s t e r a s e a c t i v i t y was a c c o m p l i s h e d by q u a n t i t a t i o n o f t h e methanol r e l e a s e d i n 4 0 t h e h y d r o l y s i s of t h e s u b s t r a t e , TAME, u s i n g a. m o d i f i c a -t i o n of t h e p r o c e d u r e of S i e g e l m a n e t a l (134). The h y d r o l y s i s of TAME.by a r g i n i n e e s t e r a s e i s shown i n . F i g u r e 5« The methanol r e l e a s e d i s o x i d i z e d t o f o r m -a l d e h y d e by p o t a s s i u m permanganate. A f t e r t h e r e m o v a l o f e x c e s s permanganate by sodium s u l f i t e , t h e formaldehyde i s c o u p l e d w i t h c h r o m o t r o p i c a c i d t o y i e l d a p u r p l e -c o l o u r e d compound which a b s o r b s m a x i m a l l y a t 530 nm. A l l samples were h a n d l e d i n p o l y p r o p y l e n e con-t a i n e r s . The plasma was t r e a t e d w i t h an e q u a l volume o f . c o l d c h l o r o f o r m and mixed w i t h an e l e c t r i c r o t a t o r a t a p p r o x i m a t e l y 10 r.p.m. f o r 5 minutes a t 4°C. The m i x t u r e was t h e n c e n t r i f u g e d a t 2,000 r.p.m. f o r 3° m i n u t e s a t 4°C and t h e plasma was s e p a r a t e d . The a r g i n i n e e s t e r a s e i n t h e plasma f r a c t i o n was a c t i v a t e d by i n c u b a t i o n w i t h an e q u a l volume of 0.1 mM e l l a g i c a c i d s u s p e n s i o n a t 25°C f o r 15 m i n u t e s . 0.3 ml o f t h i s plasma was t r a n s f e r r e d t o t u b e s c o n t a i n i n g 0.? ml o f 0.015 M TAME i n 0.10 M phos-phate b u f f e r (pH 7.6) v / i t h NaCl a t a c o n c e n t r a t i o n o f 0.15 M. The r e a c t i o n m i x t u r e ( f i n a l volume, 1.0 ml) was i n c u b a t e d a t 37°C f o r 15 m i n u t e s , and t h e r e a c t i o n was t e r m i n a t e d by t h e a d d i t i o n of 1.0 ml o f 10fo t r i c h l o r a c e -t i c a c i d . The m i x t u r e was c e n t r i f u g e d a t 2,000 r.p.m. CH, NH. I C=NH I NH I CH, +HaO CHa CH, O..NHCH CO.OCH, NH2 C=NH I NH I -> (CH2), +CHsOH SO,.NH.CH I COOH CH, O-toluenesulphonyl-l-arginine methyl eater) (methyl alcohol) Figure 5: Hydrolysis of TAME by arginine esterase 42 f o r 10 minutes a t 4 C. 0.5 ml of s u p e r n a t a n t was added t o a 10-ml g r a d u a t e d t u b e . Other tubes c o n t a i n i n g e i t h e r 0.5 ml methanol s t a n d a r d o r 5?° TCA ( B l a n k ) were a l s o s e t up. 0.1 ml o f p o t a s s i u m permanganate was added t o each t u b e , mixed and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 min. 0.1 ml of sodium s u l f i t e v/as added and mixed u n t i l d e c o l -o u r i z e d . ( I f d e c o l o u r i z a t i o n was not c o m p l e t e , a d d i t i o n a l sodium s u l f i t e s o l u t i o n was added dro p w i s e u n t i l d e c o l o u r -i z e d . ) 4.0 ml c h r o m o t r o p i c a c i d w o r k i n g r e a g e n t was added and mixed w i t h a V o r t e x m i x e r . The m i x t u r e was b o i l e d f o r 15 minutes f o l l o w e d by c o o l i n g i n an i c e - b a t h . Water v/as added t o make t h e f i n a l volume t o 5»0 m l . The sample was t h e n r e a d a t 580 nm i n a 1-cm c u v e t t e on a G i l f o r d 2000 s p e c t r o p h o t o m e t e r . 3y r e f e r e n c e t o a s t a n d a r d methanol c u r v e , (an example i s g i v e n on F i g u r e 6) t h e mic r o m o l e s of e s t e r h y d r o l y z e d were c a l c u l a t e d and e x p r e s s e d as umoles of TAME u t i l i z e d p e r hour p e r ml o f plasma. The macro-assay f o r a r g i n i n e e s t e r a s e a c t i v i t y was a l s o performed i n t h e pr e s e n c e of soybean t r y p s i n i n h i b i t o r . 20 j u l of t h e STI s o l u t i o n was added t o 1.0 ml o f t h e c h l o r o f o r m t r e a t e d plasma t o g i v e a f i n a l con-Methanol concentration (umoles/ral.) Figure 6: An example of one of the methanol standard curves. 44 c e n t r a t i o n of 2 0 0 jug/ml o f plasma. The enzyme a c t i v i t y measured i n t h e p r e s e n c e of STI was e x p r e s s e d as S T I -r e s i s t a n t a r g i n i n e e s t e r a s e a c t i v i t y . The enzyme a c t i v i t y a s s a y e d w i t h o u t STI was e x p r e s s e d as t h e t o t a l a c t i v i t y . The d i f f e r e n c e i n a c t i v i t y I n t h e p r e s e n c e and absence o f STI was t a k e n t o r e p r e s e n t S T I - i n h i b i t e d a r g i n i n e e s t e r a s e a c t i v i t y . D. M i c r o - a s s a y of A r g i n i n e E s t e r a s e A c t i v i t y 1. Reagents Same as t h e r e a g e n t s used i n the macro-assay. 2. P r o c e d u r e A r g i n i n e e s t e r a s e a c t i v i t y i n t h e plasma samples were measured u s i n g t e c h n i q u e s m o d i f i e d f r o m t h e m a cro-assay, w i t h most volumes s c a l e d down t o a p p r o x i m a t e l y o n e - f i f t h . The p l a s m a , i n p o l y p r o p y l e n e t u b e s , was t r e a t e d w i t h an e q u a l volume of c o l d c h l o r o f o r m and mixed u s i n g an e l e c t r i c r o t a t o r a t a p p r o x i m a t e l y 10 r.p.m. f o r 5 m i n u t e s a t 4°C. The m i x t u r e was t h e n c e n t r i f u g e d a t 2,000 r.p.m. f o r 30 m i n u t e s a t 4°C, a f t e r w h i c h t h e plasma wa.s s e p a r a t e d , and i n c u b a t e d w i t h an e q u a l volume o f 0.1 mM e l l a g i c a c i d s u s p e n s i o n a t 25°C f o r 15 m i n u t e s . 50 jil o f t h e a c t i v a t e d plasma was t h e n added t o a t u b e c o n -t a i n i n g 150 u l of 0.10 M phosphate b u f f e r , pK 7.6 w i t h 45 0.15 ^ NaCl and 0.015 M TAME. . The r e a c t i o n mixture.was t e r m i n a t e d by t h e a d d i t i o n of 200 jxl of 10% t r i c h l o r o a c e -t i c a c i d , and t h e m i x t u r e c e n t r i f u g e d a t 2,000 r.p.m. f o r 10 m i n u t e s a t 4°C. Methanol i n t h e s u p e r n a t a n t was as s a y e d u s i n g t h e method of S i e g e l m a n e t a l (134) mod-i f i e d by u s i n g o n e - f i f t h of t h e r e p o r t e d volumes o f t h e samples and r e a g e n t s . 100 jxl of s u p e r n a t a n t was t r a n s f e r r e d t o a 5-ml g r a d u a t e d t u b e . 20 jxl of p o t a s s i u m permanganate was added t o each t u b e , which was t h e n mixed and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 m i n u t e s . 20 jxl o f sodium s u l f i t e was added and t h e s o l u t i o n was mixed u n t i l i t d e c o l o u r i z e d . 0.8 ml c h r o m o t r o p i c a c i d w o r k i n g r e a g e n t was added and mixed v/ith a V o r t e x m i x e r . The m i x t u r e was b o i l e d f o r 15 m i n u t e s and t h e n c o o l e d i n an i c e - b a t h . Water v/as added t o make t h e f i n a l volume 1.0 ml and t h e a b s o r b a n c e a t 580 nm v/as r e c o r d e d . The e f f e c t o f STI on t o t a l a r g i n i n e e s t e r a s e a c t i v i t y was a l s o s t u d i e d . b jxl o f t h e STI s o l u t i o n was added t o 0.2 ml o f t h e c h l o r o f o r m t r e a t e d plasma t o g i v e a f i n a l c o n c e n t r a t i o n of 200 jug/ml. The r e s t o f t h e p r o -c e d u r e v/as t h e same as mentioned above f o r m i c r o - a s s a y . The enzyme a c t i v i t y v/as e x p r e s s e d as S T I - i n h i b i t e d 46 a r g i n i n e e s t e r a s e a c t i v i t y ( t h e d i f f e r e n c e between t h e v a l u e s o b t a i n e d by a s s a y i n g i n t h e p r e s e n c e and absence of S T I ) . One u n i t o f a r g i n i n e e s t e r a s e a c t i v i t y was d e f i n e d as t h e number of micromoles o f TAME h y d r o l y z e d per- hour p e r ml pla.sma. E. Assessment o f t h e M i c r o - a s s a y Technique The m i c r o - a s s a y t e c h n i q u e v/as v a l i d a t e d by s t u d y i n g t h e e f f e c t s of d i f f e r e n t c o n c e n t r a t i o n s o f e l l a g i c a c i d and S T I , and v a r y i n g t h e i n c u b a t i o n t i m e s f o r e l l a g i c a c i d and c h l o r o f o r m . 1. E f f e c t of S t o r a g e on Enzyme A c t i v i t y Plasma samples were o b t a i n e d f rom 2 f e m a l e c o n t r o l s u b j e c t s A and B (ages 21 and 26 r e s p e c t i v e l y ) and s t o r e d i n 0.5 ml a l i q u o t s a t -20°C. The m i c r o - a s s a y o f a r g i n i n e e s t e r a s e a c t i v i t y was performed on Day 1 ( t h e day when t h e b l o o d samples were c o l l e c t e d ) , and a l s o on t h e 3rd, 4 t h , 8 t h , and 10th days a f t e r t h e s t o r a g e o f plasma samples. 2. E f f e c t of C h l o r o f o r m on Enzyme A c t i v i t y 18. ml o f b i o o d from a normal s u b j e c t was added t o a p o l y p r o p y l e n e t u b e c o n t a i n i n g 2.0 ml o f 3.3?* sodium c i t r a t e . The plasma v/as s e p a r a t e d by c e n t r i f u g a t i o n a t 47 3,000 r.p.m. f o r 15 minutes a t 4°C, and d i v i d e d i n t o a l i q u o t s of 0.5 m l . An e q u a l volume o f c o l d c h l o r o f o r m • was added t o t h e plasma and t h e r e s u l t i n g d i s p e r s i o n mixed a t 4°C u s i n g an e l e c t r i c r o t a t o r a t " a p p r o x i m a t e l y 10 r.p.m. f o r v a r i o u s p e r i o d s of ti m e up t o 15 m i n u t e s . The m i x t u r e was c e n t r i f u g e d a t 2,000 r.p.m. f o r 5 minutes a t 4°C, t h e plasma s e p a r a t e d and a r g i n i n e e s t e r a s e a c t i v i t y a s s a y e d as i n d i c a t e d p r e v i o u s l y . 3. E f f e c t of E l l a g i c A c i d on Enzyme A c t i v i t y ( i ) C o n c e n t r a t i o n of E l l a g i c A c i d E l l a g i c a c i d s u s p e n s i o n s were p r e p a r e d i n 5 d i f f e r e n t c o n c e n t r a t i o n s - 0.025 mM, 0.05 mMf 0.1 mM, 0.2 mM and 0.5 mM u s i n g 0.10 M phosphate b u f f e r , pH 7.6, c o n t a i n i n g N a C l a t a c o n c e n t r a t i o n o f 0.15 M» To each 0.2 ml a l i q u o t o f t h e C h l o r o f o r m - t r e a t e d plasma, an e q u a l volume o f t h e e l l a g i c a c i d s o l u t i o n was added. . The samples were t h e n i n c u b a t e d a t 25°C f o r 15 min. I n c u b a t i o n was a l s o c a r r i e d out i n t h e absence o f e l l a g i c a c i d . A r g i n -i n e e s t e r a s e a c t i v i t y was measured as s t a t e d i n t h e p r o -c e d ure f o r m i c r o - a s s a y . ' ( i i ) I n c u b a t i o n w i t h E l l a g i c A c i d • An e q u a l volume o f 0.1 mM e l l a g i c a c i d s u s p e n s i o n 48 was added t o 1.0 ml of c h l o r o f o r m - t r e a t e d plasma, and t h e m i x t u r e i n c u b a t e d a t 25°G. A t v a r i o u s t i m e s , 5° p i of t h e m i x t u r e was t r a n s f e r r e d t o a tube s i t t i n g i n an i c e b a t h and c o n t a i n i n g 150 u l of 0.10 M phosphate b u f f e r , pH 7.6 w i t h 6.15 M NaCl and 0.015 M TAME. The m i c r o - a s s a y o f a r g i n i n e e s t e r a s e a c t i v i t y was t h e n p e r f o r m e d . 4. E f f e c t o f Soybean T r y p s i n I n h i b i t o r on Enzyme A c t i v i t y S T I s o l u t i o n was p r e p a r e d i n v a r i o u s c o n c e n -t r a t i o n s - 0, 2.5, 5.0, 7.5, 10.0 and 15.0 jug/ml o f normal s a l i n e . 4 p i o f t h e STI s o l u t i o n w i t h d i f f e r e n t c o n c en-t r a t i o n s was added t o each 0.2 ml c h l o r o f o r m - t r e a t e d p lasma, and t h e s o l u t i o n s a l l o w e d t o s t a n d a t room temp-e r a t u r e f o r 10 m i n u t e s . E l l a g i c a c i d v/as t h e n added and t h e a r g i n i n e e s t e r a s e a s s a y was pe r f o r m e d . 5« Day-to-day V a r i a t i o n o f Enzyme A c t i v i t y B l o o d was c o l l e c t e d f r o m a n o r m a l s u b j e c t (male, age 30) on t h r e e s e p a r a t e days. B l o o d was a l s o t a k e n 7 and 11 days l a t e r . A r g i n i n e e s t e r a s e a c t i v i t y o f a l l t h e . plasma samples was d e t e r m i n e d on t h e 11th day. 6. V/ith i n - d a y V a r i a t i o n o f Enzyme A c t i v i t y B l o o d samples v/ere o b t a i n e d f r o m a n o r m a l 49 s u b j e c t (male, "age 25) a t d i f f e r e n t t i m e s on t h e same day. B l o o d was t a k e n a t 8:00 a.m. ( f a s t i n g b l o o d sample) 11:00 a.m. ( b e f o r e l u n c h ) , 2:00 p.m. ( a f t e r l u n c h ) , and 4:30 p.m. F. Prena.ra.tion of Columns Sephadex G-50 ( f i n e , p a r t i c l e s i z e 20-80 m i c r o n s ) DEAE-Sephadex (A -50, t o t a l c a p a c i t y 3.5 ± 0.5 meq/g) and Sephadex columns, K 9/15 ( o f i n t e r n a l d i a m e t e r 0.9cm) and K 15/30 ( o f i n t e r n a l d i a m e t e r of 1.5 cm) were p u r -c h a s e d from Pharmacia (Canada) L t d . , D o r v a l , Quebec. 1. P r e p a r a t i o n .of Sephadex C—50 Columns Sephadex G-50 was a l l o w e d t o s w e l l i n exce s s 5 mM phosphate b u f f e r , pH 8.0 and l e f t t o s t a n d f o r a t l e a s t l | hours on a b o i l i n g w a t e r - b a t h . Two columns w i t h d i f f e r e n t s i z e s (K 9/15 and K 15/30) were u s e d , and f i l l e d w i t h t h e phosphate b u f f e r . The c o o l e d s w o l l e n g e l was t h e n poured c a r e f u l l y i n t o t h e column down a g l a s s r o d . W h i l e t h e column was a l l o w e d t o f l o w f r e e l y , t h e a d d i t i o n of s l u r r y was c o n t i n u e d u n t i l a bed h e i g h t of 13 cm was o b t a i n e d f o r t h e K 9/15 column; i n t h e case of th e K 15/30 column, a bed h e i g h t o f 25 cm. A b u f f e r r e s e r v o i r was c o n n e c t e d t o t h e column. The g e l bed 50. s t a b i l i z e d and equilibrated with 5 column volumes of buffer at 4°C. The t o t a l bed volume of the K 9/15 column was 9.5 ml while the bed volume of the K 15/30 column was ca. 40 ml. Each gram of the o r i g i n a l dry Sephadex G-50 occupied a packed volume of ca 10 ml. With a pressure head (distance between the top of the buffer r e s e r v o i r and the top of the column packing) of 25 cm of b u f f e r , the flow rate was about 35-40 ml/hour f o r both columns. 2. Preparation of DEAE-Senhadex Column DEAE-Sephadex A-50 v/as mixed v/ith excess ^raM pho.sphate buffer, pH 8 . 0 . The mixture was s t i r r e d at i n t e r v a l s and allowed to s e t t l e and swell i n a b o i l i n g water-bath f o r l i hours. The supernatant was aspirated and the buffer replaced. The suspension was thoroughly s t i r r e d and an a l i q u o t , removed by Pasteur pipette, i n t r o -duced under the buffer surface i n a Sephadex column (K 9/15) and the s l u r r y expelled. While the column was allowed to flow f r e e l y , the addition of s l u r r y was con-tinued u n t i l a bed height of 13-14 cm v/as obtained. The prepared column was attached to a r e s e r v o i r of buffer and allowed to run overnight at 4°C. Columns were not r e -used i n t h i s work. A new column was prepared f o r each 51 experiment. The t o t a l hed volume of such a column was 9*5 ml. Using a head of 30 cm of eluant, the flow rate varied between 24 and 36 ml/hour from one column to another. G. Preparation of Samples f o r Chromatography and  Isoelectrofocusing An equal volume of cold chloroform v/as added to 5»0 ml plasma. The dispersion was mixed with an e l e c t r i c rotator at ca. 10 r.p.m. f o r 5 minutes at 4°cl A f t e r centrifugation at 2,000 r.p.m. f o r 3° minutes, 3.0 ml of plasma was removed and treated with an equal amount of 0.1 mM e l l a g i c acid suspended i n d i s t i l l e d water. The mixture was incubated at 25°C f o r 15 minutes and then c h i l l e d to 4°C i n an ice-bath. 1.06 g. of s o l i d ammonium su l f a t e was added under s t i r r i n g to an ammonium su l f a t e concentration equivalent to 257° saturation at 4°C. After 15 minutes, the mixture was centrifuged at 3,500 r.p.m. f o r 15 minutes and the p r e c i p i t a t e discard-ed. 2.33 g. of s o l i d ammonium su l f a t e was added to the supernatant under s t i r r i n g (557» saturation) and the mixture centrifuged at 150,000 r.p.m. f o r 20 mins. at 4°C. The p r e c i p i t a t e was dissolved in about 1.5 ml of 52 d i s t i l l e d w a t e r , and d e s a l t e d on a column o f Sephadex G-50 e q u i l i b r a t e d w i t h 5mM phosphate b u f f e r , pH 8.0 a t 4°C. 1.0 ml f r a c t i o n s were c o l l e c t e d and a b s o r b a n c e s a t 230 nm (A280) o f t h e Sephadex G--50 f r a c t i o n s were r e c o r d e d . The f r a c t i o n s i n c l u d e d I n t h e A230 peak were p o o l e d . The t o t a l volumes ranged from 7.6-9.0 m l . 0.2 ml o f t h i s p o o l e d e l u a t e was removed f o r a r g i n i n e e s t e r a s e a s s a y and a n o t h e r 0.1 ml was used f o r p o l y a c r y -l a m i d e g e l e l e c t r o f o c u s i n g a n a l y s e s . The r e s t o f t h e Sephadex G-50 e l u a t e was used f o r f u r t h e r c h r o m a t o g r a p h i c s t u d i e s . D e s a l t i n g o f t h e s o l u t i o n s w i t h Sephadex G-50 columns was per f o r m e d on 24 a c t i v a t e d plasma samples -13 samples from normal i n d i v i d u a l s and 11 f r o m C F p a t i e n t s . 5 normal and 3 C F plasma samples were d e s a l t e d on a column o f s i z e K 9/l5» and t h e n a n a l y z e d by DEAE-Sephadex column chromatography. A n o t h e r 8 plasma samples f r o m normal c o n t r o l s and 8 from C F p a t i e n t s were d e s a l t e d on a Sephadex G-50 column (K 15/3°). The e l u a t e s c o l l e c t e d f r o m t h i s l a r g e r column were chromatographed on DEAE-Sephadex. The d e s a l t i n g a b i l i t i e s o f t h e two columns (K 9/15 and K 15/30)' of Sephadex G-50 were a s s e s s e d . 53 The c o n c e n t r a t i o n of p r o t e i n i n t h e e l u t e d f r a c t i o n s was measured by r e a d i n g absorbance a t 230 nm (A2 3 0 ) , w i t h a G i l f o r d 2000 s p e c t r o p h o t o m e t e r . The amount of ammonium i o n i n t h e f r a c t i o n s was a s s a y e d by means of a c o m m e r c i a l t e s t d e s i g n e d t o measure ammonium w i t h a l k a l i n e p h e n o l - h y p o c h l o r i t e r e a g e n t s ( d e t a i l s o f t e s t a r e shown b e l o w ) . Measurement o f Ammonium i o n i n t h e E l u t e d F r a c t i o n s 1. Reagents (a) P h e n o l c o l o u r r e a g e n t - o b t a i n e d from H y l a n d , D i v . T r a v e n o l Lab. I n c . , C o s t a Mesa, C a l i f o r n i a . - c o n t a i n e d sodium n i t r o f e r r i c y a n i d e 0.57* (w/v) and p h e n o l . (b) A l k a l i - K y p o c h l o r i t e r e a g e n t - o b t a i n e d from H y l a n d , D i v . T r a v e n o l Lab. I n c . , C o s t a Mesa, C a l i f o r n i a . - c o n t a i n e d sodium h y p o c h l o r i t e 1.57° ( w / v ) , and sodium h y d r o x i d e 17.97' (w / v ) . ( c ) N i t r o g e n S t a n d a r d - o b t a i n e d from H y l a n d , D i v . T r a v e n o l Lab., I n c . C o s t a Mesa, C a l i f o r n i a . 54 - c o n t a i n e d ammonium s u l f a t e 70.7 mg/dl, and s u l f u r i c a c i d 13 M. • (d) Ammonium s u l f a t e s t a n d a r d - o b t a i n e d from F i s h e r S c i e n t i f i c Co., F a i r Lawn, N.J. - 1 M s o l u t i o n was p r e p a r e d by d i s s o l v i n g 1.32 g (NH^) 2S0^ i n 10 ml of d e i o n i z e d w a t e r . -. 1> 2, 3, 5 and 10 mM s o l u t i o n s were p r e p a r e d by d i l u t i o n o f t h e 1M s t o c k . 2. P r o c e d u r e The e l u t e d f r a c t i o n s were d i l u t e d 100 t i m e s by a d d i n g 20 u l of t h e f r a c t i o n s t o 2.0 ml o f d e i o n i z e d w a t e r . I n t o t h e tu b e s f o r b l a n k , s t a n d a r d , c o n t r o l and d u p l i c a t e t e s t s , 200 p i o f d e i o n i z e d w a t e r was added. 20 u l o f t h e samples were t r a n s f e r r e d t o each tube and t h e tube mixed. 1.0 ml Phe n o l r e a g e n t was added f o l l o w e d by 1.0 ml A l k a l i n e - H y p o c h l o r i t e r e a g e n t and t h e tube mixed i m m e d i a t e l y u s i n g a V o r t e x m i x e r . A l l tub e s were i n c u b a t e d a t 37°C f o r 15 mins. 5«0 ml o f d e i o n i z e d water was t h e n added t o each t u b e and a b s o r b -ance a t 560 nm was r e c o r d e d . 3y r e f e r e n c e t o t h e s t a n d a r d c u r v e , t h e c o n c e n t r a t i o n of ammonium s u l f a t e i n each e l u t e d f r a c t i o n c o u l d be c a l c u l a t e d . 55 H. DEAE-Sephadex Column Chromatography DEAE-Sephadex chromatography v/as p e r f o r m e d on 13 normal c o n t r o l and 11 CF plasma a t 4°C. The Sephadex G-50 e l u a t e of t h e ammonium s u l p h a t e p r e c i p i -t a t e was a p p l i e d t o t h e DEAE-Sephadex column i n p o r t i o n s , "by p i p e t t e , u n t i l a l l had been added. D u r i n g t h i s t i m e , t h e column was open b u t s p e c i a l c a r e was t a k e n t o ensure t h a t t h e t o p of t h e packed g e l n e v e r became d r y . The column was t h e n a t t a c h e d t o a g r a d i e n t system of two i d e n t i c a l 250 ml c o n i c a l f l a s k s c o n n e c t e d by p o l y e t h y l e n e t u b i n g . One o f t h e s e , t h e m i x i n g v e s s e l , v/as s t i r r e d m a g n e t i c a l l y . From t h i s l a t t e r v e s s e l a tube c a r r i e d t h e e l u t i n g f l u i d t o t h e column. The g r a d i e n t system c o n s i s t e d o f 200 ml o f 5 mM phosphate b u f f e r , pH 8.0 i n t h e m i x i n g v e s s e l and 200 ml o f 5 mM phosphate b u f f e r , pH 8.0 c o n t a i n i n g 0.6(1 N a C l i n t h e donor v e s s e l . The column was a l l o w e d t o f l o w f r e e l y under a head o f ca 30 cm o f e l u t i n g s o l u t i o n . 2.0 ml f r a c t i o n s were c o l l e c t e d by a LKB U l t r o r a c 7000 f r a c t i o n c o l l e c t o r . A t t h e c o m p l e t i o n o f t h e N a C l G r a d i e n t , t h e g e l bed shov/ed a JOfo s h r i n k a g e . 56 I . A n a l y s i s of E l u t e d F r a c t i o n s A r g i n i n e esterase a c t i v i t i e s i n the e l u t e d f r a c t i o n s were determined u s i n g a modified micro-method with TAME as the s u b s t r a t e . For the chromatography of 5 normal and 3 G F plasma samples, the enzyme a c t i v i t y of the samples and f r a c t i o n s from Sephadex column was a l s o measured s p e c t r o p h o t o m e t r i c a l l y u s i n g 3AEE as s u b s t r a t e . The amount of p r o t e i n i n a l l e l u t e d f r a c t i o n s was estim-ated by reading the absorbance at 280 nm on a G i l f o r d 2000 spectrophotometer. The co n c e n t r a t i o n of c h l o r i d e i n the e l u t e d f r a c t i o n s was measured u s i n g a Corning c h l o r i d e meter 9 2 ' 0 M. • 1• A r g i n i n e esterase assay u s i n g TAME ( i ) Reagents (a) G^-N-(p-toluenesulfonyl)-L-arginine methyl e s t e r (TAME) 0.033M -obtained from BDH Chemical L t d . , Poole, England. - 128 mg of TAME was d i s s o l v e d i n 10.0 ml of 0.10 M phosphate b u f f e r , pH 7 « 6 c o n t a i n i n g 0 . 1 5 M NaCl. (b) Soybean T r y p s i n I n h i b i t o r (STI) - obtained from. Worthington Biochemical Corp., Freehold, N.J. 57 - 20.0 ng of STI was dissolved i n 1.0 ml of 0.10 M phosphate buffer, pH 7.6 containing 0.I5M -NaCl. ( c ) T r i c h l o r o a c e t i c acid (TCA), 205' aqueous. (d) T r i c h l o r o a c e t i c acid (TCA), kOf? aqueous. ( i i ) Procedure 200 jxl of the DEAE-Sephadex fr a c t i o n s and 100 u l of the Sephadex G-50 eluates were used f o r the arginine esterase assay. 100 jxl of the TAME s o l u t i o n was added to each tube which was then incubated at 37°C for 15 min. 100 u l of 20 fo TCA was added to terminate the reaction. The t o t a l volume of the f i n a l mixture was 400 u l . At the end of incubation, the reaction mixtures of the DEAE-Sephadex f r a c t i o n s contained: 200 u l of eluted f r a c t i o n 100 u l of substrate solution (.033 M TAME i n 0.10 M phosphate buffer, pH 7.6 containing 0.15 M NaCl). 100 p i of 20;» TCA . The contents of the reaction mixtures of Sephadex G-50 eluates at the end of the incubation were: 100 p i of Sephadex G-50 eluate 100 u l of substrate solution solution (TAME) 100 jxl of 5.mM phosphate buffer, pH 8.0 53 100 u l of 20fo TCA The c o n t e n t s of r e a c t i o n m i x t u r e s of t h e c h l o r o f o r m -e l l a g i c a c i d t r e a t e d plasma samples a t t h e end o f i n c u b a t i o n c o n s i s t e d o f : 50 jul of a c t i v a t e d plasma 100 jul o f s u b s t r a t e s o l u t i o n (TAME) 150 jul o f 5 mM phosphate b u f f e r , pH 8.0 100 j u l o f 20fo TCA The c o n t e n t s o f t h e r e a g e n t b l a n k s were: 200 jxl of 5 mM phosphate b u f f e r pH 3.0 100 u l o f s u b s t r a t e s o l u t i o n (TAME) 100 jxl o f 20fo TCA The m i x t u r e s were th e n c e n t r i f u g e d a t 2,000 r.p.m. f o r 10 mins. a t 4°C and methanol i n t h e s u p e r -n a t a n t a s s a y e d i n e x a c t l y t h e same p r o c e d u r e a s " i n d i c a t e d i n p a r t (D) f o r t h e m i c r o - a s s a y of a r g i n i n e e s t e r a s e a c t i v i t y . The above enzyme a s s a y v/as a l s o p e r f o r m e d i n th e p r e s e n c e o f S T I . 5° / J l o f t h e STI s o l u t i o n was added t o an a p p r o p r i a t e amount of e l u a t e . The m i x t u r e was a l l o w e d t o s t a n d a t room te m p e r a t u r e f o r 15 m i n s . 100 p i of t h e s u b s t r a t e s o l u t i o n (.0.33 M TAME .in 0.10 M 59 phosphate b u f f e r , pH 7.6 c o n t a i n i n g 0.15-M NaCl) was added and t h e m i x t u r e i n c u b a t e d a t 37°C f o r 15 m i n s . A t t h e end of t h e r e a c t i o n , 50 jxl of kOfo TCA was added. The f i n a l volume of t h e m i x t u r e v/as 400 u l . A t t h e end of i n c u b a t i o n s , the r e a c t i o n m i x t u r e s o f t h e DEAE-Sephadex f r a c t i o n s c o n t a i n e d : 200 u l o f e l u t e d f r a c t i o n 50 jxl of STI s o l u t i o n 100 jxl o f s u b s t r a t e s o l u t i o n (TAME) 50 u l of kOfa TCA The r e a c t i o n m i x t u r e s of Sephadex G-50 e l u a t e s a t t h e end o f i n c u b a t i o n c o n s i s t e d o f : 100 jxl o f Sephadex G-50 e l u a t e 50 jxl o f STI s o l u t i o n 100 jal o f 5 mM. phosphate b u f f e r , pH 8 .0 100 jal o f s u b s t r a t e s o l u t i o n (TAME) 50 JUl Of UrOfo TCA The r e a c t i o n m i x t u r e s of c h l o r o f o r m - e l l a g i c a c i d t r e a t e d plasmas a t t h e end o f i n c u b a t i o n c o n s i s t e d o f : 50 jxl of a c t i v a t e d plasma 50 u l o f STI s o l u t i o n 150 jxl o f 5 mM phosphate b u f f e r , pH 8.0 100 pi o f s u b s t r a t e s o l u t i o n (TAME) 50 jxl of 40f 0 TCA 6 0 The re s t of the assay was the same as the procedure f o r measurement of arginine esterase a c t i v i t y in the absence of STI. 2. Arginine Esterase Assay Using 3AEE ( i ) Reagents (a) 0(-N-benzoyl-L-arginine ethyl ester (BAEE) 5mM - obtained from Sigma Chemical Co., St. Louis, Mo. - 1 7 . 4 mg of BAEE was dissolved i n 1 0 . 0 ml of water (b) Soybean Trypsin Inhibitor (STI) - obtained from Worthington Biochemical Corp., Freehold, N.J. - 1 0 . 0 mg was dissolved i n 1 . 0 ml of normal s a l i n e . (c) Phosphate buffer, pH 7 . 6 - 0 . 1 0 M sodium phosphate, pH 7 - 6 containing 0 . 1 5 M NaCl. . (d) o(-N-Benzoyl-L-arginine 5mM - obtained from Sigma Chemical Co., St. Louis, Mo.• - 1 3 . 9 mg was dissolved i n 1 0 . 0 ml of phosphate buffer, pH 7 - 6 ( i i ) Procedure Arginine esterase a c t i v i t y was assayed by 6l following the hydrolysis of 3AEE (113). The reaction mixture of DEAE-Sephadex eluted f r a c t i o n s i n a. cuvette contained 100 jxl of the eluted . f r a c t i o n and 800 jxl of 0.10 M phosphate buffer, pH 7.6 containing 0.15 M NaCl. The cuvette v/as kept at 37°C and the reaction i n i t i a t e d by the addition of 100 jxl of 5 mM BAEE, also at 37°C. The increase in absorbance at 253 nm (A253) was followed against a blank not containing the eluate (900 jxl of phosphate buffer and 100 ^ul of 3AEE s o l u t i o n ) . In the case of Sephadex G-50 eluates, the reaction mixture contained: 50 jxl of eluate 850 jxl of 0.10 M phosphate buffer, pH 7.6 containing 0.15 M NaCl. 100 jxl of BAEE solut i o n '//hen assaying the enzyme a c t i v i t y in the chloroform-eLlagic acid treated plasma, the reaction mixture contained: 50 yl of activated plasma 850 jxl of phosphate buffer, pK 7.6 100 p.1 of BAEE .solution A series of Benzoyl-arginine stardards were prepared and absorbances at 253 nm recorded. The standard graph i s shown i n Figure 7, the change in .1 . 2 . 3 . 4 Benzcyl-arginine concentration (mM) Figure 7: An example of a standard graph of Benzoyl-arginine. 63 a b s o r b a n c e per min. was c o r r e c t e d , t o p r o v i d e u n i t s i n d i c a t i n g t h e number of micromoles of BAEE u t i l i z e d p e r hour p e r ml o f plasma o r e l u a t e . The e f f e c t of STI was e v a l u a t e d by f i r s t m i x i n g t h e e l u a t e or plasma sample w i t h 100 ^ul o f STI s o l u t i o n . 0.10 M phosphate b u f f e r , pH 7.6 c o n t a i n i n g 0.15 K NaCl v/as added t o make a f i n a l volume of 900 p i . T h i s m i x t u r e i n a. c u v e t t e was a l l o w e d t o s t a n d a t 37°C f o r 10 mins. 100 jil of BAEE s o l u t i o n v/as t h e n added t o i n i t i a t e t h e r e a c t i o n . The enz y m a t i c r e a c t i o n was r e c o r d e d i n t h e G i l f o r d s p e c t r o p h o t o m e t e r a t 37°C. The r e a c t i o n m i x t u r e s of DEAE-Sephadex elu.ted f r a c t i o n s c o n t a i n e d : 100 \xl o f e l u t e d f r a c t i o n 100 p i o f STI s o l u t i o n 700 p i o f phosphate b u f f e r , pH 7.6 100 p i o f BAEE S o l u t i o n The r e a c t i o n m i x t u r e s of Sephadex G-50 e l u a t e s c o n s i s t e d o f : 50 p i of e l u a t e 100 p i of STI s o l u t i o n 750 p i of phosphate b u f f e r , pH 7.6 64 1 0 0jul of.BASE s o l u t i o n The r e a c t i o n m i x t u r e s of c h l o r o f o r m - e l l a g i e a c i d t r e a t e d plasmas c o n s i s t e d o f : 50 p.l o f a c t i v a t e d plasma 100 j u l o f STI s o l u t i o n s 7 5 0jul o f phosphate b u f f e r , pH 7.6 100 j u l o f BAEE s o l u t i o n J . I s o e l e c t r o f o c u s i n g of Sephadex G - 5 0 E l u a t e P o l y a c r y l a m i d e g e l i s o e l e c t r o f o c u s i n g (PAG-IEF) o f t h e Sephadex G-50 e l u a t e o f a c t i v a t e d plasma was c a r r i e d out i n a d i s c g e l e l e c t r o p h o r e s i s a p p a r a t u s ( B u c h l e r P o l y a n a l y s t ) and i n Brinkman t h i n l a y e r IEF d o u b l e chamber. I s o e l e c t r i c f o c u s i n g i s a p r o c e d u r e d e v e l o p e d by V e s t e r b e r g and Svensson (143) t o s e p a r a t e p r o t e i n s (and o t h e r ampholytes) a c c o r d i n g t o t h e i r d i f f e r e n t i s o e l e c t r i c p o i n t s . I n p r i n c i p l e , t h e method i s q u i t e s i m i l a r t o e l e c t r o p h o r e s i s b u t d i f f e r s f r o m i t i n t h a t i t i s n o t c a r r i e d out a t a s p e c i f i c pH v a l u e . A g r a d i e n t of pH i s u sed and t h i s i s a c c o m p l i s h e d by u s i n g s y n t h e t i c p o l y a m i n o - p o l y c a r b o x y l i c a c i d s w h i c h p o s s e s s s u b t l y d i f f e r e n t i s o e l e c t r i c p o i n t s . I n an e l e c t r i c f i e l d t h e s e c a r r i e r ampholytes (one t r a d e name 65 o f w h i c h i s 'Ampholines') q u i c k l y form a g r a d i e n t o f pH from anode t o c a t h o d e . On t h i s g r a d i e n t a m i x t u r e of p r o t e i n s can be s e p a r a t e d . The added p r o t e i n s move towards t h e anode or cathode a c c o r d i n g t o t h e i r n e t charge b u t e v e n t u a l l y become s t a t i o n a r y a t a p o i n t i n th e pH g r a d i e n t e q u a l t o t h e i r i s o e l e c t r i c p o i n t . I n t h i s i n v e s t i g a t i o n , i s o e l e c t r i c f o c u s i n g was p e r f o r m e d u s i n g b o t h v e r t i c a l g e l t u b e s ( B u c h l e r P o l y a n a l y s t ) and f l a t g e l s l a b s (by Brinkman t h i n l a y e r I S ? d o u b l e chamber). The t e c h n i q u e s used f o r d e t e c t i n g p r o t e i n and a r g i n i n e e s t e r a s e a c t i v i t y were s i m i l a r f o r g e l t u b e s and g e l s l a b s . 1. Reagents (a) Ampholines pH 5 - 8 , kQfo (w/v) - o b t a i n e d f r o m L K B - P r o d u c k t e r , B r omnia, Sweden. (b) P o l y a c r y l a m i d e s t o c k s o l u t i o n - a c r y l a m i d e and N, N ' - m e t h y l e n e - b i s a c r y l a m i d e were p u r c h a s e d f r o m Eastman Kodak Co., R o c h e s t e r , New Y o r k . - 33 «° g of a c r y l a m i d e and 1.0 g o f m e t h y l e n e - b i s a -c r y l a m i d e were d i s s o l v e d i n 100 ml o f d i s t i l l e d w a t e r . (c) N N N ' N ' - t e t r a m e t h y l - e t h y l e n e - d i a m i n e (TEMED) % (w/v) 66 - o b t a i n e d f rom Eastman Kodak Co., R o c h e s t e r , New Y o r k . (d) Ammonium p e r s u l f a t e 10fo (w/v) (e) E l e c t r o d e s o l u t i o n s f o r g e l s l a b : anode: 0 . 2 M s u l f u r i c a c i d c a t h o d e : 0 . 3 M e t h y l e n e d i a m i n e ( f ) E l e c t r o d e s o l u t i o n s f o r d i s c g e l t u b e s : anode: 0,2fo s u l f u r i c a c i d c a t h o d e : O.kfo d i e t h a n o l a m i n e (g) S u c r o s e kOyo (w/v) (h) Ampholyte s o l u t i o n , 0.75% -O .38 ml o f hOfo (w/v) Ampholine was d i l u t e d t o 20 ml w i t h d i s t i l l e d w a t e r . ( i ) S o l u t i o n s f o r d e t e c t i o n o f P r o t e i n : F i x i n g s o l u t i o n : \2fo (w/v) t r i c h l o r o a c e t i c a c i d , 5fo (w/v) s u l f o s a l i c y l i c a c i d , i n e t h a n o l - w a t e r - a c e t i c a c i d ( 6 : 1 3 s l » by volume) S t a i n i n g s o l u t i o n : 0 .lfo (w/v) Coomassie b r i l l i a n t b l u e G ( o b t a i n e d f r o m Sigma C h e m i c a l Co.) i n e t h a n o l - w a t e r -a c e t i c a c i d ( 9 : 9 : 2 , by volume) D e s t a i n i n g s o l u t i o n : e t h a n o l - w a t e r - a c e t i c a c i d 6? (6:13:1, by volume) P r e s e r v i n g s o l u t i o n : a c e t i c a c i d Reagents f o r d e t e c t i o n of a r g i n i n e e s t e r a s e a c t i v i t y : ( i ) B - n i c o t i n a m d e - a d e n i n e d i n u c l e o t i d e (NAD +) - o b t a i n e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , Mo. P r o d u c t no. N - 7 2 5 4 . ( i i ) P h e n a z i n e m e t h o s u l f a t e (PMS) - o b t a i n e d from Sigma C h e m i c a l Co., S t . L o u i s , Mo.. . . ( i i i ) N i t r o B l u e T e t r a z o l i u m (N3T) - o b t a i n e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , Mo. ( i v ) Y e a s t _ a l c o h o l dehydrogenase (ADH) - o b t a i n e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , Mo. P r o d u c t no. A-70II. (v) N-oC-benozoyl-L-arginine e t h y l e s t e r (BAEE) - o b t a i n e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , Mo. ( v i ) C o b a l t c h l o r i d e (Co C l 2 ) - o b t a i n e d from M a l l i n c k r o d t C h e m i c a l Works, S t . L o u i s , Mo. ( v i i ) T r i s - E D T A - b o r a t e b u f f e r , 0.08 M, pH 9 . 2 68 - T r i s (Hydroxy m e t h y l ) aminomethane (TRAM) and D i s o d i u m e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) were o b t a i n e d f rom F i s h e r S c i e n t i f i c Co., F a i r Lawn, New J e r s e y . -9.69 g', o f THAM and 1.86 g of EDTA were d i s s o l v e d i n 600 ml o f d i s t i l l e d w a t e r . 0.08 M B o r i c a c i d v/as added d r o p s i s e u n t i l pH o f t h e s o l u t i o n became 9-2. D i s t i l l e d w a t e r v/as t h e n added t o make t h e f i n a l volume of 1000 m l . ( v i i i ) M e t h a n o l - a c e t i c a c i d - w a t e r , 5 s i * 5 °y volume. ( i x ) A c e t i c a c i d 7?o. 1• E l e c t r o f o c u s i n g U s i n g Brinkman T h i n - L a y e r IEF Chamber ( i ) P r e p a r a t i o n of P o l y a c r y l a m i d e G e l P l a t e s " The FAG p o l y m e r i z a t i o n chamber c o n s i s t e d o f : (a) Frame w i t h l o c k i n g screws (b) P l a s t i c p l a t e w i t h r u b b e r s p a c e r s t r i p s ( c ) Two 140 x 200 mm g l a s s p l a t e s ( t h e t h i n one was 1 mm t h i c k and used as c a r r i e r p l a t e , t h e t h i c k one (3 mm) v/as used t o r e i n f o r c e t h e c a r r i e r p l a t e ) . The g e l s o l u t i o n c o n t a i n e d t h e f o l l o w i n g : .• (a) .0.9 ml• kOfo ampholyte (pH'5-3) (b) 7.5 ml p o l y a c r y l a m i d e s t o c k s o l u t i o n ( c ) 0.25 ml 5 % TEMED ' -(d) 41.1 ml d i s t i l l e d water ($) 0.25 ml 10fo ammonium p e r s u l f a t e The m i x t u r e c o n t a i n i n g t h e r e a g e n t s ( a ) - ( d ) was i n t r o -duced i n a. vacuum f l a s k (125 ml) and mixed by magnetic s t i r r e r . The s o l u t i o n was degassed f o r about 3 minutes d u r i n g s t i r r i n g . Ammonium p e r s u l f a t e was t h e n added and t h e m i x t u r e was s t i r r e d f o r a few seconds. T h i s s o l u t i o n r e s u l t s i n a g e l w i t h T=5/* and C=y%, c o n t a i n i n g 0.75?S (w/v) ampholyte.* The g e l s o l u t i o n was t r a n s f e r r e d i n t o t h e p o l y -m e r i z a t i o n chamber by a p i p e t t e . Once t h e chamber was p r o p e r l y f i l l e d , t h e p o l y m e r i z a t i o n s o l u t i o n was o v e r -l a i d w i t h d i s t i l l e d w a ter (3-5 mm) t o o b t a i n a s t r a i g h t edge, and i t was l e f t o v e r n i g h t a t 4°C. When t h e g e l s l a b was t o be used, t h e PAG l a y e r , t o g e t h e r w i t h a g l a s s p l a t e was removed and p l a c e d i n t o t h e p r e - c o o l e d (5°C) t h i n - l a y e r IEF chamber. "fi> T = grams o f a c r y l a m i d e + grams o f b i s a c r y l a m i d e p e r 100 ml ,of s o l u t i o n fo C = 100 t i m e s grams o f b i o a c r y l a m i d e / l O O ml s o l u t i o n / T (Nomenclature a c c o r d i n g t o V e s t e r b e r g and N i c e (142)) 70 ( i i ) I s o e l e c t r i c F o c u s i n g The e l e c t r o d e s o l u t i o n s c o n s i s t e d o f 0.2M s u l f u r i c a c i d f o r t h e anode and 0.3 M e t h y l e n e d i a m i n e f o r t h e c a t h o d e . F i l t e r paper s t r i p s i m p regnated w i t h t h e a p p r o p r i a t e e l e c t r o d e s o l u t i o n were p l a c e d on b o t h ends (200 mm s i d e ) o f t h e g e l - f r e e g l a s s p l a t e and pushed a g a i n s t t h e g e l t o ensure c o n t a c t . The p l a t i n u m r i b b o n e l e c t r o d e s were t h e n p l a c e d on t h e o u t e r edge o f t h e paper s t r i p s . The g e l v/as p r e f o c u s e d f o r 1 hour a t 10 mA t o remove t h e r e s i d u a l ammonium p e r s u l f a t e . A f t e r . p r e f o c u s i n g , 20 u l o f t h e Sephadex G-50 e l u a t e s were p i p e t t e d onto 5 x 10 mm pads o f Whatman no. 3 MM chromatography p a p e r , which were p l a c e d on t h e g e l i n a l i n e p a r a l l e l t o t h e anode and a t a d i s t a n c e o f a p p r o x i m a t e l y 1.5 cm from t h e a n o d i c end o f t h e g e l . E l e c t r o f o c u s i n g was performed a t 4°C s t a r t i n g w i t h a v o l t a g e o f 100 v o l t s . The v o l t a g e was i n c r e a s e d b y 100 v o l t s e v e r y 15 m i n u t e s u n t i l t h e f i n a l v o l t a g e o f 800 v o l t s was a t t a i n e d . The t o t a l t i m e used f o r e l e c t r o -f o c u s i n g v/as about 4 h o u r s . 2. Tube E l e c t r o f o c u s i n g U s i n g D i s c G e l E l e c t r o p h o r e s i s  A p p a r a t u s 71 ( i ) P r e p a r a t i o n of P o l y a c r y l a m i d e G e l s C o m p o s i t i o n o f t h e g e l s o l u t i o n v/as • •' '; s i m i l a r t o t h e one used i n p o l y a c r y l a m i d e g e l s l a b s . 1:.2 ml o f t h e g e l mixture'was i n t r o d u c e d i n t o t h e e l e c t r o p h o r e s i s tube (75 mm l o n g w i t h i n t e r n a l d i a m e t e r o f 5 mm) c o n t a i n i n g 50 jxl of kOfo s u c r o s e a t t h e bottom t o ensure a f l a t s u r f a c e . The end of t h e e l e c t r o p h o r e s i s t u b e was c l o s e d v/ith a r u b b e r tube cap. The l e n g t h o f t h e g e l was c a . 65 mm. D i s t i l l e d w a ter was l a i d on t o p of t h e g e l s u r f a c e t o o b t a i n a s t r a i g h t edge. The g e l s were l e f t o v e r n i g h t a t h°C t o a l l o w complete p o l y m e r i z a -t i o n . When t h e g e l s were t o be used, t h e wa t e r was removed and r e p l a c e d by a l a y e r of 0.75?^ a m p h o l i n e . The tu b e s v/ere mounted i n a d i s c g e l e l e c t r o p h o r e s i s a p p a r a t u s ( B u c h l e r P o l y a n a l y s t ) . ( i i ) I s o e l e c t r o f o c u s i n g The anode compartment c o n t a i n e d 600 ml o f 0.2% KgSO^ w h i l e t h e cathode compartment c o n t a i n e d 200 ml o f O.kfo d i e t h a n o l a m i n e . The whole a p p a r a t u s was c o o l e d by p a s s i n g r u n n i n g water a t a t e m p e r a t u r e o f 4°C t h r o u g h t h e o u t e r j a c k e t of t h e column. The g e l s were p r e - r u n a t 200V f o r .1 hour. Ammonium p e r s u l f a t e v/as removed from t h e g e l by t h i s p r e f o c u s i n g . 72 A f t e r the p r e - r u n , a. m i x t u r e o f 50 Jul o f Sephadex G-50 e l u a t e of t h e a c t i v a t e d plasma and 50 j u l o f kOfo s u c r o s e was i n t r o d u c e d under t h e l a t e r of a m p h o l i n e . E l e c t r o f o c u s i n g v/as p e r f o r m e d a t 4°C s t a r t i n g w i t h t h e v o l t a g e o f 200 v o l t s . The v o l t a g e was i n c r e a s e d b y 200 v o l t s e v e r y 15 minutes u n t i l t h e f i n a l v o l t a g e o f 800 v o l t s was a t t a i n e d . F o c u s i n g was c o n t i n u e d f o r 3-4 h o u r s , by which t i m e , t h e c u r r e n t dropped t o c a 5 tnA. F o l l o w i n g t h i s , each g e l was d i s p l a c e d from i t s tube by p a s s i n g a s m a l l amount of water between the g e l and t h e i n s i d e s u r f a c e of t h e tube by means of a l o n g - t i p p e d s y r i n g e . 3• D e t e c t i o n of P r o t e i n i n P o l y a c r y l a m i d e G e l s The g e l s were f i x e d o v e r n i g h t i n a s o l u t i o n of 12$ TCA, and 5$ s u l f o s a l i c y l i c a c i d , i n e t h a n o l - w a t e r -a c e t i c a c i d (6:13:1). T h i s removes t h e a m p h olytes and p r e c i p i t a t e s t h e p r o t e i n s which were s t a i n e d s u b s e q u e n t l y w i t h 0.1$. Coomassie b r i l l i a n t b l u e G i n e t h a n o l - w a t e r -a c e t i c a c i d (9:9:2) f o r ijr h o u r s . S u c c e s s i v e d e s t a i n i n g washes o f e t h a n o l - w a t e r - a c e t i c a c i d (6:13:1) were t h e n p e r f o r m e d u n t i l t h e g e l background was c l e a r . The g e l s v/ere s t o r e d i n 5$ a c e t i c a c i d . 73 k. D e t e c t i o n of A r g i n i n e E s t e r a s e A c t i v i t y i n  P o l y a c r y l a m i d e G e l s T h i s method was m o d i f i e d f rom t h e t e c h n i q u e of F u j i m o t o and a s s o c i a t e s ( 5 ^ ) . D e t e c t i o n of a r g i n i n e e s t e r a s e a c t i v i t y i s based on t h e f o r m a t i o n of t e t r a z o -l i u m s a l t by t h e e l e c t r o n r e l e a s e d w i t h a l c o h o l dehydro-genase f r o m e t h a n o l which i s l i b e r a t e d f r om 3AEE by t h e a c t -i v i t y of a r g i n i n e e s t e r a s e , as i l l u s t r a t e d i n F i g u r e 8. Two d i f f e r e n t c o n c e n t r a t i o n s o f t h e s t a i n i n g s o l u t i o n were u s e d . The f i r s t one ( s o l u t i o n A) was e x a c t l y t h e same c o n c e n t r a t i o n as used by Rao and N a d l e r (111), w h i l e t h e second one ( s o l u t i o n 3) was t h r e e t i m e s t h e p r e v i o u s c o n c e n t r a t i o n f o r a l l t h e components e x c e p t a l c o h o l dehydrogenase which was t e n t i m e s more c o n c e n -t r a t e d . The c o m p o s i t i o n s o f t h e s t a i n i n g s o l u t i o n s were: S o l u t i o n A S o l u t i o n B (a) NAD 100 mg 200 mg (b) PMS 10 mg 20 mg ( c ) NBT 30 mg 60 mg (d) ADH 5 mg 33 TRg (e) BASS 250 mg 500 mg ( f ) o l g 300 mg 600 mg (g) T r i s - E D T A - b o r a t e b u f f e r 150 ml 100 ml 74 ARGININE ESTERASES (KALLIKREIN) F i g u r e 8 : The r e a c t i o n s i n v o l v e d i n t h e d e t e c t i o n o f a r g i n i n e e s t e r a s e s w i t h t h e f o r m a z a n s y s t e m . 75 The g e l s were immersed i n t h e a r g i n i n e e s t e r a s e s t a i n i n g s o l u t i o n and kep t i n t h e dark a t 37°C f o r l i h o u r s . The g e l s were t h e n f i x e d i n m e t h a n o l - a c e t i c a.cid-water (5*1 :5) o v e r n i g h t and de-s t a i n e d i n 7/5 a c e t i c a c i d u n t i l t h e bands were c l e a r l y v i s i b l e on a p a l e background. K. Methods of Data A n a l y s i s 3 1. C o e f f i c i e n t o f v a r i a t i o n = x 100% x S = S t a n d a r d d e v i a t i o n x = mean . 2. 95<> c o n f i d e n c e l i m i t = x ±(t .05, n - l ) y = -n = number o f o b s e r v a t i o n s from t h e sample I f t h e 95'f° c o n f i d e n c e l i m i t s of one group do n o t i n c l u d e t h e mean o f a n o t h e r g r o u p , t h o s e two groups a r e l i k e l y t o be s i g n i f i c a n t l y d i f f e r e n t . 3. T e s t f o r normal d i s t r i b u t i o n of t h e enzyme a c t i v i t i e s i n each p o p u l a t i o n : ( i ) C h i - s q u a r e t e s t , . -v-2 (n. - e. ) ^  c h i - s q u a r e = X = ) 1 1 1 = 1 * i 76 K = number, o f c l a s s e s m - o b s e r v e d f r e q u e n c i -es = e x p e c t e d f r e q u e n c y N u l l h y p o t h e s i s : The o b s e r v e d d i s t r i b u t i o n c o n s t i t u t e s a sample from a p o p u l a t i o n h a v i n g t h e t h e o r e t i c a l (normal) d i s t r i b u t i o n . 2 I n g e n e r a l , a l a r g e ^ v a l u e i n d i c a t e s a poor f i t w h i l e ~v~ 2 a s m a l l jL v a l u e i n d i c a t e s a good f i t . A CHIPROB i s t h e p r o b a b i l i t y of o b t a i n i n g a X v a l u e as l a r g e as t h e one c a l c u l a t e d f o r t h e sample. I f CHIPROB ^ 0 . 5 , t h e n u l l h y p o t h e s i s i s n o t r e j e c t e d . However, i f CHIPROB <C0.05, t h e n u l l h y p o t h e s i s i s r e j e c t e d w i t h t h e c o n c l u s i o n t h a t t h e o b s e r v e d d i s t r i b u t i o n i s n o t n o r m a l l y d i s t r i b u t e d . ( i i ) Kolmogorov-Smirnov t e s t T h i s goodness o f f i t t e s t i s based on c u m u l a t i v e f r e q u e n c i e s as f o l l o w s : D = max S n (x) - F (x) S (x) •= IT . j / \ K i n N and F ( x ) = — K = number of o b s e r v a t i o n s 4 x i n t h e o b s e r v e d d i s -t r i b u t i o n K i = number of o b s e r v a t i o n s 4 x i n t h e t h e o r e t i c a l d i s t r i b u t i o n . 77 N = t o t a l number of o b s e r v a t i o n s i n t h e o b s e r v e d d i s t r i b u t i o n D i s t h e maximum d e v i a t i o n which can be used a l o n g w i t h a c r i t i c a l v a l u e f r o m s t a n d a r d Kolmogorov-Smirnov t a b l e s t o d e t e r m i n e whether o r n o t t h e observed and t h e o r e t i c a l ( normal) d i s t r i b u t i o n s d i f f e r s i g n i f i c a n t l y . Hence, f o r D<KS (.05) t h e r e i s no s i g n i f i c a n t d i f f e r e n c e s between t h e two d i s t r i b u t i o n s a t a l e v e l o f s i g n i f i c a n c e o f O.05. F o r D > KS (.05) t h e observed d i s t r i b u t i o n i s s i g n i f i c a n t l y d i f f e r e n t f r o m a n o r m a l d i s t r i b u t i o n . 4. S t u d e n t ' s t - t e s t ( i ) V a l u e s assuming o n l y t h e n o r m a l i t y o f t h e p a r e n t p o p u l a t i o n : X, - Xp t = x c I 2 2 Jnl . N 2 x. = a r i t h m e t i c means o f t h e sample i 2 s i = e s t i m a t e d v a r i a n c e o f sample i n i = number of o b s e r v a t i o n from sample i PROS ( t ) . = p r o b a b i l i t y o f o b t a i n i n g a v a l u e of t whose l v a l u e ! i s g r e a t e r t h a n o r e q u a l t o l v a l u e ! 78 o f t h e one c a l c u l a t e d , g i v e n t h a t t h e two p o p u l a t i o n means a r e e q u a l . I f PR03 ( t ) <.05, i t i s c o n c l u d e d t h a t t h e sample means a r e s i g n i f i c a n t l y d i f f e r e n t . DF ( t ) = degrees of freedom 2 2 z ( S l + '6Z >- n 1 ° 1 o 2 2 n2 n ] _ - x n2 ( i i i ) V a l u e s assuming t h e p o p u l a t i o n v a r i a n c e s a r e e q u a l t k l n l + n2 n ^ n 2 ( n ^ + n 2 -2) L ( n 1 - l ) + ( n 2 - l ) S 2 PR03 ( t ) = s i m i l a r t o ( i ) above DF ( t ) = n 1 + n 2 - 2 5. C o r r e l a t i o n C o e f f i c i e n t ( r ) between two v a r i a b l e s , x and y. ^ (x - x) • (y - y) E( -v2 / ->2 x) /_^(y - y) 79 I I I . RESULTS A. Assessment of t h e M i c r o - A s s a y Technique 1. E f f e c t of C h l o r o f o r m Treatment o f plasma w i t h c h l o r o f o r m t o remove t h e a r g i n i n e e s t e r a s e i n h i b i t o r was t h e major s o u r c e o f v a r i a b i l i t y i n t h e a s s a y . The e f f e c t o f c h l o r o f o r m r o t a t i o n t i m e on a r g i n i n e e s t e r a s e a c t i v i t y i s shown i n F i g u r e 9* The enzyme a c t i v i t y r e a c h e d a s t e a d y l e v e l a t between 2 and 10 m i n u t e s of m i x i n g w i t h t h e e l e c t r i c r o t a t o r . Enzyme a c t i v i t y began t o f a l l as t h e t i m e o f r o t a t i o n was l o n g e r t h a n 10 rains. S i n c e 5-minutes was n e a r t h e c e n t e r o f t h e p l a t e a u on t h e g r a p h , i t was chosen f o r t h e plasma a r g i n i n e e s t e r a s e a s s a y . 2. E f f e c t of E l l a g i c A c i d C o n c e n t r a t i o n E a r l y work had been done u s i n g e l l a g i c a c i d i n s o l u t i o n i n d i l u t e sodium h y d r o x i d e . V e r y low a r g i n i n e e s t e r a s e a c t i v i t y was o b t a i n e d f o r the plasma f r o m normal c o n t r o l s . L a t e r , e l l a g i c a c i d was p r e p a r e d i n s u s p e n s i o n i n 0.10 M phosphate b u f f e r , pH 7.6 c o n t a i n i n g 0.15 M o f N a C l . W i t h t h i s t e c h n i q u e , more r e p r o d u c t i b l e r e s u l t s were o b t a i n e d . 8 0 0.61 0.4 o CO < 0.2 e o - © A i » I I I — J _ ! L 4 8 J2 Time of Rotation (minutes) 16 Figure 9: EFFECT O F C H L O R O F O R M O N E N Z Y M E ACTIVITY 31 A s t u d y of t h e e f f e c t of e l l a g i c a c i d concen-t r a t i o n on a r g i n i n e e s t e r a s e v/as p e r f o r m e d . R e s u l t s v/ere p l o t t e d on a. graph p r e s e n t e d on F i g u r e 10. I n t h e absence of e l l a g i c a c i d , the enzyme a . c t i v i t y v/as v e r y low. -With the a d d i t i o n o f e l l a g i c a c i d s u s p e n s i o n t o a c t i v a t e t h e e s t e r a s e , h i g h e r a c t i v i t y was r e c o r d e d . The amount o f e l l a g i c a c i d p r e s e n t d i d n o t a f f e c t the enzyme a c t i v i t y . S i n c e Rao and co-workers used 0,1 mM I n t h e i r a s s a y , 0.1 mM v/as chosen f o r t h e micro-method o f a r g i n i n e e s t e r a s e a s s a y . 3• E f f e c t of E l l a g i c A c i d I n c u b a t i o n Time The t i m e c o u r s e o f a c t i v a t i o n f o l l o w i n g t h e a d d i t i o n of e l l a g i c a c i d t o plasma o b t a i n e d f r o m a normal s u b j e c t i s shown on F i g u r e 11. Maximal a c t i v a t i o n o f t h e a r g i n i n e e s t e r a s e was a c h i e v e d a f t e r 5-10 m i n s . o f expos-u r e t o e l l a g i c a c i d . T h e r e a f t e r , a g r a d u a l decay o f a r g i n i n e e s t e r a s e a c t i v i t y was o b s e r v e d . 15 m i n u t e s o f i n c u b a t i o n v/ith e l l a g i c a c i d was used because Rao e t a l used t h e same i n c u b a t i o n time i n t h e i r o r i g i n a l a s s a y . 82 0.6r 0.4 0 0.21 0.1 0.2 0.3 0.4 0.5 Concentration of Ellagic Acid Solution (mM) Figure 10; EFFECT O F E L L A G I C A C I D C O N C E N T R A T I O N O N E N Z Y M E ACTIVITY 8 3 Figure 11; EFFECT O F E L L A G I C A C I D I N C U B A T I O N TIME O N E N Z Y M E ACTIVITY 84 4• E f f e c t of Soybean T r y p s i n I n h i b i t o r C o n c e n t r a t i o n A s t u d y of t h e e f f e c t of STI c o n c e n t r a t i o n on a r g i n i n e e s t e r a s e a . c t i v i t y v/as p e r f ormed and r e s u l t s a r e p r e s e n t e d on F i g u r e 12, Enzyme a c t i v i t y wa.s t h e h i g h e s t i n t h e absence of S T I . W i t h . t h e a d d i t i o n of S T I , enzyme a c t i v i t y began t o d e c l i n e . As t h e c o n c e n t r a t i o n of STI added r e a c h e d 100 ug/ml plasma, t h e enzyme a c t i v i t y began t o l e v e l o f f . I n c r e a s e i n STI t o c o n c e n t r a t i o n h i g h e r t h a n 100 3.1 g/m 1 plasma d i d n o t have any s i g n i f i c a n t e f f e c t on enzyme a . c t i v i t y . In t h e a r g i n i n e e s t e r a s e a s s a y , 200 jj.g o f STI v/as added per ml o f c h l o r o f o r m t r e a t e d plasma. 5. R e n r o d u c t i b i l i t y o f t h e M i c r o - A s s a y . The micro-method f o r a r g i n i n e e s t e r a s e a c t i v i t y o f a plasma sample o b t a i n e d f r o m a n o r m a l a d u l t was p e r -formed 10 t i m e s on t h e same day. Enzyme a c t i v i t i e s measured from t h e t e n r u n s were 60.1, 6o.5» 60.8, 61.4, 62.4, 63.8, 64.1, 66.?, 67.2, and 67.4 u n i t s . The mean v a l u e o b t a i n e d was 63.4 w i t h a. s t a n d a r d d e v i a t i o n o f 2-85. The c o e f f i c i e n t of v a r i a t i o n of t h e m i c r o - a s s a y was + 4.5$. The m i c r o - a s s a y of a r g i n i n e e s t e r a s e a c t i v i t y was a l s o c a r r i e d out on two plasma samples on d i f f e r e n t 0.3 r •3-JL ± ± A. 0 100 200 300 Soybean Trypsin Inhibitor Concentration (jig/ml) Figure 12; EFFECT O F S O Y B E A N TRYPSIN INHIBITOR C O N C E N T R A T I O N O N E N Z Y M E A C T I V I T Y 36 d a y s . R e s u l t s a r e p r e s e n t e d on T a b l e 2. The enzyme a c t i v i t y u n i t s were r e p r o d u c i b l e a.t + 10 or + 17?°. r e s p e c t i v e l y over a t e n day s t o r a g e p e r i o d . B l o o d was t a k e n from the same normal . s u b j e c t on t h r e e s e p a r a t e days. The a r g i n i n e e s t e r a s e a c t i v i t y o f t h e plasma samples was a s s a y e d and t h e r e s u l t s were p r e s e n t e d i n T a b l e 3« L i t t l e v a r i a t i o n o f t h e enzyme a c t i v i t y was d e t e c t e d f o r plasma samples c o l l e c t e d on d i f f e r e n t days. B l o o d samples were a l s o c o l l e c t e d f r o m an a d u l t s u b j e c t a t d i f f e r e n t t i m e s on t h e same day. R e s u l t s o b t a i n e d f o r t h e enzyme a s s a y o f t h e f o u r plasma samples a r e shown i n T a b l e 4. T h i s experiment i n d i c a t e d t h a t t h e method was n o t s u b j e c t t o g r o s s v a r i a t i o n s f o r t h e t i m e of s a m p l i n g . B. A Comparison of t h e M i c r o - A s s a y W i t h t h e Macro-Assay  of A r g i n i n e E s t e r a s e A c t i v i t y The a.rginine e s t e r a s e a c t i v i t i e s o f 26 plasma samples ( f r o m b o t h c o n t r o l s and p a t i e n t s ) v/ere measured u s i n g b o t h t h e macro-and m i c r o - a s s a y s . The a c t u a l v a l u e s of S T I - i n h i b i t e d enzyme a c t i v i t y o f a l l samples a s s a y e d were p r e s e n t e d i n T a b l e 5- The c o r r e l a t i o n between the T a b l e 1: E f f e c t o f s t o r a g e on a r g i n i n e e s t e r a s e a c t i v i t y . Day o f enzyme a s s a y U n i t s o f S T I - i n h i b i t e d enzyme a c t i v i t y A B 1 26.8 37.1 3 26.6 36.2 4 26.3 8 27.8 44.8 10 38.3 36.5 Mean 29.2 38.6 S.D. 5.1 4.1 C o e f f i c i e n t o f v a r i a t i o n 17.4$ 10.6$ i 0 Table 3: Day-to-day v a r i a t i o n of arginine esterase a c t i v i t y . Day of blood c o l l e c t i o n Units of enzyme a c t i v i t y 1 36.0 7 35.6 I 11 1 33.0 Mean 34.9 Standard de v i a t i o n 1.6 C o e f f i c i e n t of V a r i a t i o n ± 4 . 7 % ! 1 1 Table 4: Within-day v a r i a t i o n of arginine esterase a c t i v i t y . Time of blood c o l l e c t i o n Units of enzyme a c t i v i t y 8:00a.m. i 14.8 • | 11:00a.m. 18.9 ! ! 2:00p.m. 17.8 | 4:30p.m. 13.5 Mean 16.3 Standard deviation 2.5 C o e f f i c i e n t of v a r i a t i o n i — ± 15.5% Table 5- A comparison of the arginine esterase a c t i v i t i e s measured by the micro-assay and macro-assay. Sample S T I - i n h i b i t e d arginine esterase a c t i v i t y No. (pinoles tame hydrolysed /hr. /ml plasma) Micro assay Macroassay 1 50.6 !•. • 32.8 2 50.1 ! 15-6 5 22.9 22.1 4 48.0 46.9 5 56.8 51.2 6 19-8 20.3 •7 60.2 61.8 8 10.9 8.0 .9 49.0 29.9 10 25.5 17.6 11 14.7 11.2 12 4.0.6 51.2 13 11.2 8.5 • 14 25-0 20.8 15 95-0 101.5 16 61.8 86.4 17 42.6 49-5 13 51.5 54.4 19 11.8 9.3 20 8.0 9.1 21 55.5 21.9 22 18.6 17-5 23 1 1 . 2 ' 6.9 24 25.0 16.8 25 28.8 19.2 26 15-8 9-9 Mean 51 -8 29.1 S. D. 20.5 24-5 Variance 411.5 592.2 90 Table 6: ARGININE ESTERASE ACTIVITY IN PLASMA The r e s u l t s are expressed as micromoles of TAME u t i l i z e d per hour per ml. of plasma. SAMPLE NO. TOTAL ACTIVITY STI-INHIBITED ACTIVITY STI-RESISTANT ACTIVITY 1 Mean + S.D. Mean ± S.D. Mean ± S.D. (A) CONTROLS 50 53.8 + 15.8 48.2 ± 14.8 5.6 ± 3.9 Adults 34 54.7 + 14.0 50.1 ± 13.7 4.9 ± 3.1 o* adults 16 50.4 + 11.0 46.9 ± 10.0 3.5 ± 2.2 adults 18 58.5 + 15.5 52.4 ± 14.9 6.1 ± 3.3 C h i l d r e n 16 51.7 + 19.5 44.8 ± 18.2 7.0 ± 5.1 cf c h i l d r e n 7... 43.6 + 19.9 37.3 ± 19.5 6.4 + 6.5 _o_ c h i l d r e n 9 58.0 + 17.7 50.7 ± 16.0 7.4 ± 4.0 (B) CYSTIC FIBROSIS 19 34.4 + 12.0 30.4 ± 10.6 3.9 ± 2.9 I. Grouped according to sex : O* pat i e n t s 10 32.8 + 9.3 29.6 ± 8.8 3.2 ± 2.5 o_ p a t i e n t s 9 36.2 + 14.9 31.5 ± 12.8 4.7 ± 3.2 II. Grouped according to age : Ages < 16 11 30.8 + 9.6 27.6 ± 8.2 3.3 ±3.1 Ages £ 16 8 39.3 + 14.5 34.5 ± 12.7 4.8 ±2.5 (C) Heterozygotes 19 43.1 + 14.0 37.1 ± 15.0 6.0 ± 5.0 Fathers of CF 2 34.6 + 3.6 31.6 ± 3.2 2.9 ± 0.4 Mothers of CF 17 44.1 + 14.4 38.9 ± 17.0 6.4 ± 5.2 91 enzyme a c t i v i t y measured by b o t h a s s a y s i s shown g r a p h -i c a l l y i n F i g u r e 13. The c o r r e l a t i o n c o e f f i c i e n t between t h e two a s s a y s v/as c a l c u l a t e d , and t h e s i g n i f i c a n c e of each c o r r e l a t i o n c o e f f i c i e n t v/as a s s e s s e d by a S t u d e n t ' s t - t e s t . The l e v e l s o f enzyme a c t i v i t y o b t a i n e d u s i n g t h e m i c r o a s s a y were f o u n d t o be s i g n i f i c a n t l y c o r r e l a t e d to t h e l e v e l s measured by t h e macro-assay. The c o r r e l a -t i o n c o e f f i c i e n t between t h e two methods was O.947. C. Comparisons Between A r g i n i n e E s t e r a s e A c t i v i t i e s ' o f  t h e C y s t i c F i b r o s i s P a t i e n t s , C o n t r o l S u b j e c t s and  H e t e r o z y g o t e s U s i n g t h e M i c r o - A s s a y The i n d i v i d u a l v a l u e s f o r a r g i n i n e e s t e r a s e a c t i v i t y o b t a i n e d f r o m 50 c o n t r o l s u b j e c t s , 19 p a t i e n t s v/Ith c y s t i c f i b r o s i s and 19 o b l i g a t e h e t e r o z y g o t e s were e x p r e s s e d as t o t a l a c t i v i t y , S T I - i n h i b i t e d a c t i v i t y and S T I - r e s i s t a n t a c t i v i t y . The means and s t a n d a r d d e v i a -t i o n s o f t h e a r g i n i n e e s t e r a s e a c t i v i t i e s i n t h e plasma o f p a t i e n t s w i t h c y s t i c f i b r o s i s , t h e i r p a r e n t s ( o b l i g a t e h e t e r o z y g o t e s ) and n o r m a l c o n t r o l s a r e shown i n Ta.ble 6. The a c t u a l v a l u e s of S T I - i n h i b i t e d a c t i v i t y o f a l l i n d i v i d u a l s measured a r e p r e s e n t e d i n F i g u r e 14. The Figure 13: The correlation between the arginine esterase a c t i v i t i e s measured by the micro-assay and the macro-assay. SO L_ 70, 60 50 9 40 e e % 3 Cm e o 9 30 20 0 CF Children CF children. control adults I Adult control Hetero-zygote Figure 14; D i s t r i b u t i o n of STI-inhibited arginine esterase a c t i v i t y i n the plasma samples. (The bars represent the means of each group.) o d i f f e r e n c e s i n S T I - i n h i b i t e d a r g i n i n e e s t e r a s e l e v e l s between t h e groups a r e shown g r a p h i c a l l y i n F i g u r e 15. To o b t a i n s l i g h t l y more a c c u r a c y i n comparing group means, S t u d e n t ' s " t - " v a l u e s were c a l c u l a t e d and p r e s e n t e d i n T a b l e 7. The g a u s s i a n d i s t r i b u t i o n o f enzyme a c t i v -i t i e s w i t h i n each p o p u l a t i o n i s shown i n F i g u r e 16. I t was c o n f i r m e d u s i n g t h e C h i - s q u a r e t e s t and Kolmogorov Smirnov t e s t f o r goodness o f f i t . The s i g n i f i c a n t d i f f e r ences f o u n d u s i n g S t u d e n t ' s t - t e s t ( T a b l e 7) c o n f i r m t h e d i f f e r e n c e s t h a t can be seen v i s u a l l y ( t h e 95$ c o n f i d e n c e l i m i t s of one group do n o t I n c l u d e t h e mean o f a n o t h e r group ( F i g u r e 15))• The mean l e v e l of S T I - i n h i b i t e d a r g i n i n e e s t e r -ase a c t i v i t y i n t h e p a t i e n t s w i t h c y s t i c f i b r o s i s was a p p r o x i m a t e l y t w o - t h i r d s of t h e mean l e v e l o f a c t i v i t y i n age-matched c o n t r o l s u b j e c t s , v / h i l e t h e mean h e t e r -o z y g o t e a c t i v i t y was midway between t h a t o f normal con-t r o l s and p a t i e n t s . However, t h e v a l u e s were s c a t t e r e d o v e r a wide range f o r each group. The a c t i v i t y i n CF p a t i e n t s v/as s i g n i f i c a n t l y l o w e r t h a n t h a t o f c o n t r o l groups i n b o t h c h i l d r e n and a d u l t s (p < 0.03). The d i f f e r e n c e between t h e a c t i v i t i e s i n o b l i g a t e h e t e r -o x y g o t e s and t h e a c t i v i t i e s i n c o n t r o l s u b j e c t s was a l s o 60 I— 50 40 ! — 30 20 10 CF Children C o n t r o l Chi"! di'er CF A d u l t s C o n t r o l H e t c r -A d u l t e z y g o t e s F i g u r e 15: The mesn(©) u.r.c 95:,'- c o n f i d e n c e l i m i t e d ) f c r c-ch TABLE 7 Differences of STI-inhibited arginine esterase activities in the plasma of control subjects, patients with cystic fibrosis and obligate heterozygotes. The " t " values were obtained by using the Student "t" test. SUBJECT C.F.TChildren Control Children AGE A - 15 1-15 N O . 11 16 STI-INHIBITED ARGININE ESTERASE ACTIVITY (ymoles of TAME hydrolyzed/hour/ml of plasma) MEAN ± S.D. 27.6 ± 8.2 44.8 ± 18.2 RANGE OF ACTUAL VALUES 13.5 4 12.2 42.2 82.2 PROBABILITY (t) <0.003* CF."Adults Control Adults 16 - 26 16-26 8 21 34.5 ± 12.7 47.6 ±12.9 18.9 26.8 52.5 71.6 <0.028* Obligate Heterozygotes 32 - 42 Control Adults 27-46 19 13 37.1 ± 15.0 53.4 ± 12.6 6.4 30.0 65.3 81.2 <0.002* C.F.tAdults 16-26 Obligate Heterozygotes 32 - 42 8 19 34.5 ± 12.7 37.1 ± 15.0 18.9 6.4 52.5 65.3 <0.65** •difference significant **difference not significant tp.F, = cystic fibrosis 5-i 15H 10H (A) CYSTIC FIBROSIS PATIENTS Mean (Adults and Children) (B) CONTROL SUBJECTS (Adults and Children) Mean \C) OBLIGATE HETEROZYGOTES Mean T—™l 1 1~—I 1 T — T r 5 15 25 35 45 55 65 75 85 95 STI INHIBITED A R G I N I N E ESTERASE ACTIVITY ( ^moles T A M E h y d r o l y z e d / h r / m l plasma) Figure 16: Distribution of STI-inhibited arginine esterase values i n the peculations studied. 93 s i g n i f i c a n t ( p < 0.002). However, t h e d i f f e r e n c e between t h e a c t i v i t i e s of p a t i e n t s w i t h CF and t h e i r p a r e n t s was n o t s i g n i f i c a n t . A r g i n i n e e s t e r a s e a c t i v i t y v/as u n r e l a t e d t o sex or age of t h e i n d i v i d u a l t e s t e d . D, C o r r e l a t i o n Between t h e Plasma. A r g i n i n e E s t e r a s e  A c t i v i t i e s of C y s t i c F i b r o s i s P a t i e n t s , and T h e i r  P a r e n t s Plasma a r g i n i n e estera.se a c t i v i t i e s o f 8 p a i r s o f CF p a t i e n t - m o t h e r v a l u e s a r e p r e s e n t e d i n T a b l e 3. The c o r r e l a t i o n c o e f f i c i e n t v/as c a l c u l a t e d t o be 0.773 ( s i g n i f i c a n t a t t h e 0.05 l e v e l ) . Hence, t h e r e seems t o be s l i g h t c o r r e l a t i o n between t h e enzyme l e v e l s o f t h e CF p a t i e n t s and p a r e n t s i n t h e same f a m i l y . I f t h e p a t i e n t has a below-average enzyme a c t i v i t y , h i s mother I s l i k e l y t o have a. low l e v e l o f enzyme a c t i v i t y and v i s e v e r s a . E. C o r r e l a t i o n Between t h e Plasma A r g i n i n e Estera.se  A c t i v i t y and Shwachman Score o f t h e CF P a t i e n t s T a b l e 9 shov/s the c o r r e l a t i o n between a r g i n i n e e s t e r a s e a c t i v i t y and Shwachman s c o r e of t h e CF p a t i e n t s . The Shwachman s c o r e s f o r t h e p a t i e n t s v a i r e d f r o m 33 t o . 9 4 -while t h e range of enzyme a c t i v i t i e s was 13«5 _52.5 u n i t s . There was no o b v i o u s c o r . r e l a . t i o n between a r g i n i n e e s t e r a s e a . c t i v i t y and Shwachman s c o r e of t h e CF p a t i e n t . oo Table 8: Correlation between the STI-inhibited arginine esterase ac t i v i t i e s of CF patients and their parents. Family no. Patients with CF Parents of CF patients Sex Age Units of ' I Sex | Age j Units of enzyme i i enzyme i a c t i v i t y j j i i a c t i v i t y j i 1 • i i 1 F r 1 3 . 5 i l * I 30 8.0 I 2 16 • 28.1 F 38 2 2 . 4 i 3 ; F 16 19.9 F 36 32.0 4- M 11 31.4 F i 36 5 2 . 2 5 M 13 27.9 42 33.3 6 M 6 21.8 36 32.0 7 M 13 i 28.8 42 40.4 . 8 M I15 ! 1 26.2 3? 38 4 2 . 2 100 Table 9: • Correlation' between S T l - i n h i b i t e d a r g i n i n e esterase a c t i v i t y .and Shwachman scores of the CF p a t i e n t s . P a t i e n t Sex ! Age | i Units of arginine esterase a c t i v i t y Shwachman score i N. H. F I 16 | 28.1 i 86 D. F. . M 15 . 42.2 62 X.'F. F . 19 18.9 81 D. 3. ' F 16 52.5 94 R. E. Ll 26 30.1 71 J . C. 15 19.9 67 J . s . . M .. 10 21.2 81 A. W. M 11 31.4 68 " : 3. G-. M 14 27.9 . 6 4 P. U. M 6 21.8 89 JJ. S. F 15 35-2 89 . J . M. M 12 28.8 78 W. D. M " 26.2 46 J . M. ' F 4 35-2 58 K. 3. F 24 46.1. 74 M. E. M .17 19-5 85 M. P. • F 17 55-9 78 C. D. F 7 15-5 72 G. V. lVi 19 |- . • • . 46.7 I I . 7 5 101 Hence, t h e enzyme a c t i v i t y was n o t r e l a t e d t o t h e s e v e r i t y o f t h e d i s e a s e . F. PEAS-Sephadex Column Chromatography o f Plasma From  C y s t i c F i b r o s i s P a t i e n t s and Normal C o n t r o l s . F o r t h e ammonium s u l f a t e p r e c i p i t a t e s o b t a i n e d f r o m 5 normal c o n t r o l and 3 c y s t i c f i b r o s i s plasma s a m p l e s , d e s a l t i n g was p e r f o r m e d on a Sephadex G-50 column u s i n g column s i z e K 9/l5» Incomplete s e p a r a t i o n o f t h e p r o t e i n and ammonium s u l f a t e peaks can be seen i n F i g u r e 17. A t a t i m e when t h i s p r o b lem was n o t a p p r e c i a t e d , t h e p a r t i a l l y d e s a l t e d p r o t e i n peak wa.s p o o l e d and chromatographed on DEAE-Sephadex.- . A r g i n i n e e s t e r a s e a c t i v i t y was a s s a y e d u s i n g b o t h TAME and BAEE as s u b s t r a t e s and i n t h e absence and p r e s e n c e o f S T I . An e l u t i o n p a t t e r n f o r c o n t r o l p l a s m a i s shown i n F i g u r e 18. A l a r g e unadsorbed peak o f a r g i n i n e e s t e r a s e emerged b e f o r e t h e a p p l i c a t i o n o f t h e g r a d i e n t . T h i s peak o f enzyme a c t i v i t y was i n c o m p l e t e l y i n h i b i t e d by S T I . N e g l i g i b l e amounts o f enzyme a c t i v i t y v/as d e t e c t e d a f t e r t h e a p p l i c a t i o n o f an N a C l g r a d i e n t . S i m i l a r r e s u l t s were o b s e r v e d f o r t h e Sephadex G-50 e l u a t e o f t h e a c t i v a t e d plasma from CF p a t i e n t s . An example o f t h e e l u t i o n p r o f i l e o f CF plasma. I s shown i n 102 Tube number ( 1.0 ml f r a c t i o n s ) Figure 17: Desalting with Sephadex G-50 column (K9/15). (Fractions nos. Q-13 were pooled and chromatographed on DEAE-Sephadex.) 103 3.0 2.0 1.0 1° 15 10 .15M H .10M —I .05M o U a u c. o u 10 15 20 25 Tube number (2.0 ml fr a c t i o n s ) 30 Figure 18: DEAE-Sephadex chromatography of activa t e d plasma from a control subject (F, age 37, plasma arginine esterase a c t i v i t y = 35.8 units) © Assay without STI o Assay with STI 104 F i g u r e 19. The peak of enzymatic a c t i v i t y was not ab s o r b e d on DEAE-Sephadex, and v e r y l i t t l e enzyme a c t i v i t y . w a s d e t e c t e d a f t e r t h e a p p l i c a t i o n o f t h e NaCl g r a d i e n t . The c h r o m a t o g r a p h i c t e c h n i q u e was t h e n improved u s i n g a Pharmacia column s i z e of K 15/30 f o r Sephadex G-50. Complete s e p a r a t i o n of t h e p r o t e i n peak f r o m t h e ammonium s u l f a t e peak v/as a c h i e v e d as shown i n F i g u r e 20. Recovery of a r g i n i n e e s t e r a s e a c t i v i t y i n t h e Sephadex G-50 e l u a t e was ca 60$. The p o o l e d e l u a t e was t h e n a p p l i e d on a DEAE-Sephadex column and r e s u l t s of t h e no r m a l c o n t r o l and c y s t i c f i b r o s i s plasma a r e shown i n F i g u r e s 21 and 22 r e s p e c t i v e l y . C h l o r o f o r m - e l l a g i c a c i d t r e a t e d plasma f r o m n o r m a l i n d i v i d u a l s c o u l d be s e p a r a t e d u s i n g DEAE Sephadex i n t o two f r a c t i o n s by t h e i r a r g i n i n e e s t e r a s e a c t i v i t i e s ( F i g u r e 21), The f i r s t peak ( I ) w h i c h c o m p r i s e d a p p r o x -i m a t e l y kOfo o f t h e t o t a l a c t i v i t y emerged i m m e d i a t e l y a f t e r t h e a p p l i c a t i o n of the NaCl g r a d i e n t . Peak I I emerged a t a h i g h e r i o n i c s t r e n g t h (ca. 0.15 M NaCl) and c o n s t i t u t e d c a . 6.0$ o f t h e a r g i n i n e e s t e r a s e a . c t i v i t y . The peak, f r a c t i o n s were a l s o a s s a y e d i n t h e p r e s e n c e o f S T I . Peak I was found t o be c o m p l e t e l y i n h i b i t e d by STI 4.0i 3.or 2.01 1.0 II ll u 15 0.4M 10 0.3M c 10.2M « o c_> r-i CJ JO.IM 10 20 30 40 Tube number (2.0 ml fractions) 50 60 70 80 Figure 19: DEAE-Sephadex chromatography of activated plasma from a patient with cystic fibrosis (F, age 16, plasma arginine esterase activity = 32.6 units) ® Assay without STI o Assay with STI 106 25 i— — , 5.0 10 20 30 40 Tube number (1.0 ml f r a c t i o n s ) 50 Figure 20: Desalting with Sephadex G-50 column (K15/30) (Fractions nos.. 10-16 were pooled and chromatographed on DEAE-Sephadex.) 0.3M 0.2M 0.1M 20 30 40 Tube number (2.0 ml fractions) Figure 21: DEAE-Sephadex chromatography of desalted plasma sample from a control subject (F, age 20, plasma arginine esterase activity = 77.4 units) Assay without STI o Assay with STI 108 w h i l e peak I I was a l m o s t t o t a l l y r e s i s t a n t t o i n h i b i t i o n by S T I . T h i s p a t t e r n of d i s t r i b u t i o n of a c t i v i t i e s was • c o n s i s t e n t l y o b s e r v e d i n a l l plasma samples from c o n t r o l s . An example of t h e DEAE-Sephadex column chroma-t o g r a p h y f o r c y s t i c f i b r o s i s plasma i s p r e s e n t e d i n F i g u r e 22. A s i m i l a r e l u t i o n p a t t e r n was o b t a i n e d as f o r normal plasma. The major peak ( I I ) emerged a t an i o n i c s t r e n g t h o f 0.15 M N a C l and was a l m o s t t o t a l l y r e s i s t a n t t o i n h i b i t i o n by S T I . The minor peak ( I ) emerged a t t h e b e g i n n i n g of t h e NaCl g r a d i e n t and was a l m o s t c o m p l e t e l y i n h i b i t e d by STI. F o r b o t h normal and CF plasma, chromatography on DEAE-Sephadex r e s u l t e d i n t h r e e p r o t e i n peaks as measured by A280 ( F i g u r e s 21 and 2 2 ) . The f i r s t u n a dsorbed peak emerged w i t h t h e v o i d volume. A f t e r t h e b e g i n n i n g o f t h e NaCl g r a d i e n t , t h e second peak emerged, f o l l o w e d by a l a r g e t h i r d peak whose a s c e n d i n g l i m b c o n t a i n e d most o f t h e a r g i n i n e e s t e r a s e a c t i v i t y . G. I s o e l e c t r o f o c u s i n g of Sephadex-G50 E l u a t e s A n a l y s i s o f Sepha.dex G-50 e l u a t e s by p o l y a c r y l -a m i d e - g e l IEF i n t h e Erinkma.n t h i n - l a y e r IEF d o u b l e 109 0 10 20 30 AO 50 60 Tube number (2.0ml f r a c t i o n s ) Figure 22: DEAE-Sephadex chromatography of desalted plasma from a patient with c y s t i c f i b r o s i s (F, age 16, plasma arginine esterase a c t i v i t y = 42.2units). • Assay without STI o Assay with STI 110 chamber was r a t h e r u n s u c c e s s f u l . Upon c o m p l e t i o n o f f o c u s i n g , t h e g e l s l a b was immersed i n a r g i n i n e e s t e r a s e s t a i n i n g s o l u t i o n A and i n c u b a t e d a t 3?°C i n t h e d a r k f o r 1-2 h o u r s . No r e a c t i o n o c c u r r e d ho m a t t e r how long; t h e g e l s l a b was k e p t i n the s t a i n i n g s o l u t i o n . S o l u t i o n B was t h e n p r e p a r e d by i n c r e a s i n g t h e c o n c e n t r a t i o n s o f . a l l t h e components of t h e s t a i n i n g s o l u t i o n . V/hen a f o c u s e d g e l s l a b was i n c u b a t e d a t 37°C i n s t a i n i n g s o l u t i o n B, f o u r d i s t i n c t hands of a p p a r e n t a r g i n i n e e s t e r a s e a c t i v i t y , a ppeared. The c a t h o d i c end o f t h e gel was d a r k l y s t a i n e d as shown i n F i g u r e 24. P r o t e i n s t a i n i n g by Cooma.ssie b r i l l i a n t b l u e G r e s u l t e d i n 9, bands ( F i g u r e 23). S i n c e t h e a r g i n i n e e s t e r a s e bands s p r e a d a l l a c r o s s t h e g e l , an ex p e r i m e n t v/as performed w h i c h c o n s i s -t e d o f f o c u s i n g a g e l s l a b w i t h o u t t h e a d d i t i o n of any plasma samples e x c e p t hemoglobin (used as t h e i n d i c a t o r ) . A s i m i l a r p a t t e r n was o b t a i n e d t o t h a t shown i n F i g u r e 24. F o u r d i s t i n c t bands o f s o - c a l l e d " a r g i n i n e e s t e r a s e a c t i v i t y " a p p a r e n t l y a r t e f a c t s , v/ere o b s e r v e d . F u r t h e r work, on i s o e l e c t r o f o c u s i n g was c a r r i e d out u s i n g v e r t i c a l g e l tubes i n a B u c h l e r P o l y a n a l y s t . Sephadex G-50 e l u a t e s of a c t i v a t e d plasma, from 5 n o r m a l I l l a r g i n i n e e s t e r a s e K r o t e i n s t a i n i n g s t a i n i n g Figure 23: Photograph of arginine esterase separated by poly-acrylamide gel-isoelectrofocusing. N = Normal plasma Figure 24: Photograph of arginine esterase staining i n a control gel slab. 113 c o n t r o l s and 5 CF p a t i e n t s were a n a l y z e d . A r g i n i n e e s t e r a s e s t a i n i n g was c a r r i e d out u s i n g b o t h s o l u t i o n s A and B. No enzyme a c t i v i t y was d e t e c t e d i n t h e g e l s f o r i n c u b a t i o n v/ith s o l u t i o n A. F i g u r e 25 shows, t h e r e s u l t s o b t a i n e d v/ith s o l u t i o n B. A d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e photograph i s shown i n F i g u r e 26. I n t h e b l a n k g e l w i t h t h e a p p l i c a t i o n o f no p r o t e i n sample, k bands appeared.. A l l g e l s e x h i b i t e d i n t e n s e s t a i n i n g a t t h e upper ( c a t h o d i c ) end. The g e l s v / i t h t h e a p p l i c a t i o n o f Sephadex G-50 e l u a t e s r e v e a l e d 9-11 bands o f a r g i n i n e e s t e r a s e a c t i v i t y . The p a t t e r n o f a c t i v i t y f o r t h e bands d i f f e r e d f o r a l l 10 samples s t u d i e d . There was no c o n s i s t e n t p a t t e r n o b s e r v e d f o r n o r m a l c o n t r o l s amples. Kence, i t was d i f f i c u l t t o d e t e c t any d i f f e r e n c e i n t h e enzyme a c t i v i t y p a t t e r n s o f t h e CF plasmas and t h e c o n t r o l s . 114 B N N . N CF CF CF CF Figure 25: Photograph of arginine esterase separated by d i s c g e l electrophoresis using Buchler Polyanalyst. B - Blank N = Control plasma CF = Cystic f i b r o s i s plasma N N N N CP CF Figure 26: Diagrammatic representation of Figure 25. B = Blank N = Normal CF = Cystic fibrosis 116 IV. DISCUSSION The p r e s e n t s t u d y u s i n g a m i c r o a n a l y t i c a l t e c h n i q u e c o n f i r m s t h e o b s e r v a t i o n (114) of Rao and c o -wor k e r s t h a t t h e r e i s a d e f i c i e n c y of S T I - i n h i b i t e d a r g i n i n e e s t e r a s e a . c t i v i t y i n plasma of p a t i e n t s w i t h c y s t i c f i b r o s i s . The advantage of emp l o y i n g a. m i c r o -a s s a y i s t o en a b l e i n v e s t i g a t o r s t o work on a r g i n i n e e s t e r a s e a c t i v i t y w i t h o u t r e q u i r i n g u n a c c e p t a b l y l a r g e volumes o f b l o o d from p e d i a t r i c p a t i e n t s . The w r i t e r o b t a i n e d i d e n t i c a l v a l u e s f o r a r g i n i n e e s t e r a s e a c t i v i t y w i t h t h e micro-method as w i t h t h e macro-method used by Rao e t a l when a s s a y i n g plasma a r g i n i n e e s t e r a s e . I n most f a m i l i e s , p a r e n t s ( o b l i g a t e h e t e r o z y -g o t e s ) have l e v e l s of enzyme a c t i v i t y i n t e r m e d i a t e between t h a t of t h e i r c h i l d r e n and c o n t r o l s u b j e c t s . The d i f f e r e n c e i n t h e mean plasma a r g i n i n e e s t e r a s e a c t i v i t y between t h e c o n t r o l s u b j e c t s and p a t i e n t s w i t h c y s t i c f i b r o s i s i s s t a t i s t i c a l l y s i g n i f i c a n t . The mean enzyme a c t i v i t y i n t h e plasma, of t h e h e t e r o z y g o t e s i s a l s o s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e normal c o n t r o l s . However, we have been u n a b l e t o d e t e c t the s i g n i f i c a n t d i f f e r e n c e between t h e mea.n v a l u e s o f p a t i e n t s and. c a r r i e r s r e p o r t e d by Rao e t a l ( 1 1 4 ) . 117 There i s no s i g n i f i c a n t c o r r e l a t i o n o f t h e a r g i n i n e e s t e r a s e l e v e l s w i t h sex or age of t h e c o n t r o l s u b j e c t s , CF p a t i e n t s or h e t e r o z y g o t e s s t u d i e d . . The v a l u e s o f S T I - i n h i b i t e d a r g i n i n e . e s t e r a s e a c t i v i t y . f o r . p a t i e n t s w i t h c y s t i c f i b r o s i s c o u l d n o t be r e l a t e d t o t h e s e v e r i t y of t h e d i s e a s e as a s s e s s e d by Shwachman s c o r i n g . The w r i t e r was u n a b l e t o r e p r o d u c e t h e work o f Rao and N a d l e r (111) on DEAE-Sephadex column chromato-graphy and polyac r y l a . m i d e g e l e l e c t r o f o c u s i n g . Rao and N a d l e r r e p o r t e d t h a t the a r g i n i n e e s t e r a s e a c t i v i t y , c o u l d be r e s o l v e d i n t o ' t w o f r a c t i o n s by DEAE-Sephadex column chromatography as shown i n F i g u r e J. The b u l k of th e a.rginine e s t e r a s e a c t i v i t y i s e l u t e d a t a p p r o x i m a t e l y 0.02M NaCl a.nd i s c o m p l e t e l y i n h i b i t e d by S T I . A s m a l l e r peak i s e l u t e d a t about 0..13M NaCl and c o n t a i n s a r g i n i n e e s t e r a s e a c t i v i t y w h i c h i s a l m o s t t o t a l l y r e s i s t a n t t o i n h i b i t i o n by S T I , I n plasma of CF p a t i e n t s , t h e f i r s t , m a jor a c t i v i t y peak v/as re d u c e d t o a p p r o x i m a t e l y 30$ o f t h e c o r r e s p o n d i n g f r a c t i o n i n c o n t r o l p lasma. In c o n t r a s t , t h e second peak of a r g i n i n e estera.se a c t i v i t y d i d n o t show any s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s between c o n t r o l and CF plasma samples. 113 I n my s t u d i e s , i o n exchange chromatography u s i n g DEAE-Sephadex was c a r r i e d out f o r c h l o r o f o r m -e l l a g i c a.cid t r e a t e d plasma samples o b t a i n e d f r o m 8 p a t i e n t s w i t h CF and 8 c o n t r o l s u b j e c t s . The a c t i v a t e d samples were p r e c i p i t a t e d w i t h S 5 ' 7 ° ammonium s u l f a t e . The p r e c i p i t a t e s were t h e n d e s a l t e d on a column (K 15/ 30) of Sephadex G-50. Chromatography of t h e Sephadex G-50 e l u a t e s was p e r f o r m e d on DEAE-Sephadex columns. A r g i n i n e e s t e r a s e a c t i v i t y was r e s o l v e d i n t o two compon-ent f r a c t i o n s . The minor peak I emerged a t t h e b e g i n n i n g o f t h e g r a d i e n t (0.02M NaCl) w h i l e t h e major peak I I was e l u t e d a t an NaCl c o n c e n t r a t i o n o f 0.15M. S i m i l a r e l u t i o n p a t t e r n s were o b t a i n e d i n b o t h c a s e s o f c o n t r o l and CF plasma, though a r e d u c t i o n i n s i z e o f t h e two a c t i v i t y peaks v/as o b s e r v e d i n CF plasma. No i s o -enzyme d e f i c i e n c y was d e t e c t e d u s i n g anion-exchange-chromatography. Hence, t h e d e f i c i e n c y o f a r g i n i n e e s t e r - . ' a se a c t i v i t y i n t h e CF plasma was a p p a r e n t l y e n t i r e l y q u a n t i t a t i v e b u t not q u a l i t a t i v e . I n i t i a l e x p e r i m e n t a t i o n of DEAE-Sephadex column chromatography was performed on plasma, samples d e s a l t e d on a Sephadex G-50 column ( K 9/15)• A r g i n i n e e s t e r a s e a c t i v i t y v/as e l u t e d w i t h t h e v o i d volume b e f o r e 119 t h e a p p l i c a t i o n of t h e NaCl g r a d i e n t . Next DEAE-c e l l u l o s e chromatography was t r i e d . S i m i l a r r e s u l t s were o b s e r v e d f o r plasma samples o b t a i n e d f rom 3 c o n t r o l s u b j e c t s and 3 CF. p a t i e n t s . A l m o s t a l l o f t h e enzyme a c t i v i t y emerged b e f o r e t h e appearance o f t h e : NaCl g r a d i e n t . T h e r e f o r e , i t seemed l o g i c a l t o t r y C M - c e l l u l o s e column chromatography. R e s u l t s o f t h i s c a t i o n exchange chromatography gave a s t r a n g e pH p r o f i l e . The pH of t h e e l u a t e dropped from 6.0 t o 5.0 as t h e a r g i n i n e e s t e r a s e peak emerged, and r o s e when a l l t h e p r o t e i n v/as e l u t e d f r o m t h e column. A t t h i s p o i n t , t h e problem o f i n c o m p l e t e d e s a l t i n g by t h e Sephadex G-50 column became a p p a r e n t . A l a r g e r column o f Sephadex G-50 ( w i t h volume i n c r e a s e d f o u r f o l d ) was u s e d and t h i s t i m e t h e d e s a l t i n g a b i l i t y of t h i s column was c h e c k e d by m e a s u r i n g t h e ammonium s u l f a t e c o n c e n t r a t i o n i n t h e e l u t e d f r a c t i o n s . DEAE-Sephadex column chromatography was t h e n r e p e a t e d and y i e l d e d t h e r e s u l t s d i s c u s s e d above. My f i n d i n g s f o r c o m p l e t e l y d e s a l t e d p r o t e i n f r a c t i o n s of plasma s u b j e c t e d t o DEAE-Sephadex chroma-t o g r a p h y do n o t agree v/ith t h o s e o f Rao and N a d l e r . T h e i r DEAE-Sephadex chromatography gave e l u t i o n p r o f i l e s w h i c h seemed t o be r a t h e r s i m i l a r t o t h e e l u t i o n 120 p a t t e r n s obtained i n my work when the p r o t e i n samples were i n c o m p l e t e l y . d e s a l t e d . I t i s tempting t o speculate t h a t one could a.c count f o r the r e s u l t s of Rao and Nadler by incomplete d e s a l t i n g . This would be impossible t o prove. E l e c t r o f o c u s i n g on polyacrylamide g e l s and sub-sequent s t a i n i n g f o r a r g i n i n e esterase a c t i v i t y r e v ealed 9-11 bands i n plasma, samples from c o n t r o l s and CF p a t i e n t s . Since f o u r bands were a r t i f a . c t s , about 5-7 bands represented the a r g i n i n e esterase a c t i v i t y . The a c t i v i t y p a t t e r n was r a t h e r v a r i a b l e i n a l l the c o n t r o l samples s t u d i e d ; t h e r e f o r e , i t was d i f f i c u l t to det e c t any d i f f e r e n c e i n the a r g i n i n e esterase a c t i v i t y p a t t e r n s of plasma samples from the CF p a t i e n t s and the c o n t r o l s . According to Rao and Nadler (111), s i x a c t i v i t y bands co u l d be detected i n c o n t r o l samples, whereas f i v e bands were detected i n CF samples. However, the w r i t e r was unable t o confirm t h e i r o b s e r v a t i o n s . In t h i s i n v e s t i g a t i o n , the plasma a r g i n i n e esterase a c t i v i t y has been determined u s i n g a micro-method. The mean of S T I - i n h i b i t e d a c t i v i t y i s lowest f o r CF p a t i e n t s while the mean f o r normal c o n t r o l s i s the h i g h e s t . The mean of S T I - i n h i b i t e d a c t i v i t y f o r the heterozygotes i s midway between the values of the 121 p a t i e n t s and t h e normal i n d i v i d u a l s . The d e f i c i e n c y of a r g i n i n e e s t e r a s e a c t i v i t y i s s t a t i s t i c a l l y s i g n i f i c a n t f o r b o t h c y s t i c f i b r o s i s p a t i e n t s and h e t e r o z y g o t e s . However, no q u a l i t a t i v e d i f f e r e n c e c o u l d be d e monstrated by ion-exchange chromatography on•DEAE-Sephadex or i s o -e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l s . N ext, I w i l l d i s c u s s a r g i n i n e e s t e r a s e s i n g e n e r a l b e f o r e r e t u r n i n g t o t h e r e l e v a n c e of t h i s work i n the whole f i e l d . A r g i n i n e e s t e r a s e s a r e a group of enzymes e x h i b i t i n g p r o t e o l y t i c a c t i v i t y i n v o l v e d i n t h e h y d r o l y s i s of p e p t i d e bonds d e r i v e d f r om t h e c a r b o x y l groups of a r g i n i n e . T r y p s i n , c h y m o t r y p s i n , k a l l i k r e i n , t h r o m b i n , p l a s m l n , and carboxypeptida.se 3, a r e a l l p r e s e n t i n plasma and p o s s e s s a r g i n i n e e s t e r a s e a c t i v i t y . A l l t h e s e enzymes w i l l be examined t o d e t e r m i n e w h i c h of t h e v a r i o u s a r g i n i n e e s t e r a s e s i s most l i k e l y t o be i n v o l v e d i n c y s t i c f i b r o s i s and i n t h i s i n v e s t i g a t i o n . The p o s s i b i l i t y t h a t t r y p s i n i s t h e a r g i n i n e e s t e r a s e d e f i c i e n t i n c y s t i c f i b r o s i s i s u n l i k e l y because Rao e t a l were u n a b l e t o a s s a y th e a r g i n i n e e s t e r a s e a c t i v i t y u s i n g more s p e c i f i c . s u b s t r a t e s such as oc-N-ben-z o y l - L - a r g i n i n e amide (BAA) or o c - N - b e n z o y l - D L - a r g i n i n e -p - n i t r o a . n i l i d e (BAPNA) (113, 114). A l s o , plasma, t r y p s i n a c t i v i t y s h o u l d be c o m p l e t e l y i n h i b i t e d by soybean 122 t r y p s i n i n h i b i t o r ( 1 3 4 ) . I n t h i s work, t h e a r g i n i n e e s t e r a s e , t h a t i s d e f i c i e n t i n c y s t i c f i b r o s i s i s o n l y ,.. p a r t i a l l y i n h i b i t e d by S T I . Hence, t h e a r g i n i n e e s t e r a s e s t u d i e d i n t h i s i n v e s t i g a t i o n i s n o t t r y p s i n . C h y m o t r y p s i n , an e n d o p e p t i d a s e , a l s o a c t s on amides and e s t e r s o f s u s c e p t i b l e amino a c i d s . I t p r e -f e r e n t i a l l y c a t a l y z e s t h e h y d r o l y s i s of bonds o f l e u c y l , m e t h i o n y l , a s p a r a g i n y l and g l u t a m y l r e s i d u e s . S i n c e TAME i s .not h y d r o l y z e d by c h y m o t r y p s i n ( 1 3 4 ) , t h e p o s s i b i l i t y of t h e a r g i n i n e e s t e r a s e measured i n t h i s work b e i n g c h y m o t r y p s i n i s e x t r e m e l y u n l i k e l y . I n t h e p r e s e n t s t u d y , a r g i n i n e e s t e r a s e a c t i v i t y was measured a f t e r a c t i v a t i o n w i t h e l l a g i c a c i d ( 4 , 4 ' , 5» 5 ' i 6 , 6* - h e x a h y d r o x y - d i p h e n i c a c i d 2 , 6 : 2 ' 6 ' - d e l a c t o n e ) . R a t n o f f and Crum r e p o r t e d t h a t e l l a g i c a c i d a c t i v a t e s Hageman f a c t o r a t c o n c e n t r a t i o n s as low as 1 0 " 8 M ( 1 1 6 ) . The c h e m i c a l n a t u r e of i t s a c t i o n seems t o be r e l a t e d t o t h e pr e s e n c e o f o - d i h y d r o x y l groups I n t h e e l l a g i c a c i d m o l e c u l e . Once a c t i v a t e d , Hageman f a c t o r i s a b l e t o t r i g g e r f o u r p r o t e o l y t i c enzyme systems i n plasma, t h e c o a g u l a t i o n , f i b r i n o l y t i c , complement a.nd k i n i n - f o r m i n g pathways ( F i g u r e 2 7 ) ( 2 1 ) . Among t h e enzymes d i r e c t l y o r i n d i r e c t l y a c t i v a t e d by 123 INITIATION KININ FORMATION ——— — — 1 r — — * —— Figure 27: Pathways of activation of plasma proteolytic enzymes (21).. •. 124 •activated' Hageman fa c t o r , thrombin, plasrain, carbo-xypeptidase B and k a l l i k r e i n exhibit arginine esterase a c t i v i t y and they are a.ll t h e o r e t i c a l l y possible can-didates to represent the a.rginine esterase a c t i v i t y measured i n my studies and those of others discussed i n t h i s section. Thrombin catalyzes the transformation of fibrinogen to f i b r i n . . I t i s generated from i t s i n a c t i v e zymogen, prothrombin, and i s not i t s e l f present i n normal plasma. Its formation requires calcium and thromboplastin (146). Glover et a l have worked on thrombin and reported that thrombin could be inactivated by l-chloro-3-"tosylamido-7-a'mino-2-heptanone (TLCK) which complexes with thombin and alkylates a c r u c i a l h i s t i d i n e residue i n the active s i t e of thrombin (62). Rao and Nadler suggested that the arginine esterase that they measured using TAME as a substrate did not represent thrombin because of the i n a b i l i t y of TLCK to inactivate arginine esterase a c t i v i t y (112). The present study has not re-examined t h i s . Plasrnin also exists i n plasma as an i n a c t i v e precursor, plasminogen. Plasminogen, can be converted to plasrnin-by the d i r e c t action of thrombin or i n d i r e c t l y by 125 a c t i v a t e d Hageman f a c t o r ( F i g u r e 27). One of t h e major b i o l o g i c a l r o l e s of p l a s r n i n i s t o d i g e s t f i b r i n o g e n o r f i b r i n t o fr a g m e n t s known as f i b r i n d e g r a d a t i o n p r o d u c t s ( 2 1 ) . Groskopf et a l have shown t h a t TLCK c o u l d i n h i b i t t h e p r o t e o l y t i c a c t i v i t y of p l a s r n i n by a s t o i c h i o m e t r i c i r r e v e r s i b l e r e a c t i o n a t t h e a c t i v e s i t e of p l a s r n i n (64). Hence, the i n a b i l i t y o f TLCK t o i n h i b i t t h e a r g i n i n e e s t e r a s e a . c t i v i t y d e m o n s t r a t e d by Rao and N a d l e r (112) a l s o e x c l u d e s t h e p o s s i b i l i t y t h a t t h e a r g i n i n e e s t e r a s e measured by., t h e h y d r o l y s i s of TAME may r e p r e s e n t plasrnin. A g a i n , t h i s has n o t been v e r i f i e d i n t h i s p r e s e n t s t u d y . Carboxypeptida.se B i s formed from an ina c t i v e zymogen p r e c u r s o r , p r o c a r b o x y p e p t i d a s e by t h e a.ction of t r y p s i n . Ca.rboxypeptida.se B can d e s t r o y plasma brady-k i n i n ( t h i s i s d i s c u s s e d i n t h e " I n t r o d u c t i o n " q.v.) by r e m o v i n g t h e C - t e r m i n a l a r g i n i n e ( 2 1 ) . Carboxypep-t i d a s e B e x h i b i t s a n a p h y l a t b x i n (C3a) i n h i b i t o r a c t i v i t y (30). Hence, serum c a r b o x y p e p t i d a s e 3 i n a c t i v a t e s the complement d e r i v e d anaphylatoxins C3a and C5a, as w e l l a.s b r a d y k i n i n . C a r b o x y p e p t i d a s e B presumably s e r v e s as t h e major r e g u l a t o r y enzyme.of c i r c u l a t i n g v a s o a c t i v e p e p t i d e s . Conover e t el r e p o r t e d t h a t i n normal serum 126 t r e a t e d w i t h a c a r b o x y p e p t i d a s e i n h i b i t o r , a n t i - c i l i a r y . . a c t i v i t y d e v e l o p s . T h i s b i o l o g i c p r o p e r t y of CF serum i s a b o l i s h e d by t r e a t m e n t w i t h c a r b o x y p e p t i d a s e 3 (27). They p o s t u l a t e d t h a t a g e n e t i c d e f i c i e n c y of serum c a r b o x y p e p t i d a s e i n CF p a t i e n t s may a c c o u n t f o r t h e e x p r e s s i o n of a f a c t o r i n serum from c y s t i c f i b r o s i s homozygotes and h e t e r o z y g o t e s which i n d u c e s c i l i a r y d y s k i n e s i s . U n f o r t u n a t e l y , Lieberma.n has shown r e c e n t l y t h a t c a r b o x y p e p t i d a s e B - l i k e a c t i v i t y i s normal i n CF serum (39) • T h i s s t u d y . h a s no i n f o r m a t i o n on t h i s p o i n t . E a r l y s t u d i e s by Rao e t a l s u g g e s t e d t h a t t h e a r g i n i n e e s t e r a s e ' a c t i v i t y measured by them m i g h t , r e p r e s e n t plasma k a l l i k r e i n ( 1 1 4 ) . K a l l i k r e i n i s a plasma p r o t e o l y t i c and e s t e r o l y t i c enzyme t h a t a c t s on s p e c i f i c ^ - g l o b u l i n s u b s t r a t e s c a l l e d k i n i n o g e n s t o r e l e a s e b r a d y k i n i n ( F i g u r e 2 ) , The e s t e r o l y t i c a b i l i t y o f k a l l i k r e i n can be i n h i b i t e d by soybean t r y p s i n i n h i b i t o r as w e l l as by 0 6 , - m a c r o g l o b u l i n ( 2 1 , 80). Colman e t a l p r o v i d e d e v i d e n c e t h a t t h e a r g i n i n e e s t e r -ase a c t i v i t y i n plasma, a f t e r t h e a d d i t i o n of k a o l i n o r e l l a g i c a c i d was p r o b a b l y i d e n t i c a . l v/i t h plasma k a l l i -k r e i n (22). The e v i d e n c e was as f o l l o w s : ( i ) p u r i f i -c a t i o n d a t a and s i m i l a r i t i e s i n e s t e r a s e a c t i v i t y between 1 2 7 p u r i f i e d k a l l i k r e i n and k a o l i n - a c t i v a t e d e s t e r a s e ; ( i i ) e v i d e n c e t h a t t h e a c t i v i t y cannot be a s c r i b e d t o o t h e r known plasma o r t i s s u e a r g i n i n e e s t e r a s e s ; ( i i i ) t h e r e q u i r e m e n t of Hageman f a c t o r f o r t h e a c t i v a t i o n of., a r g i n i n e e s t e r a s e a c t i v i t y ; and ( i v ) s i m i l a r i t i e s i n t h e b e h a v i o u r of t h e plasma enzyme and p u r i f i e d k a l l i -k r e i n toward i n h i b i t o r s ( 2 2 ) . Colman and c o - w o r k e r s a l s o compared t h e k a o l i n - a c t i v a t e d plasma a r g i n i n e e s t e r a s e w i t h p u r i f i e d plasma p r e p a r a t i o n s of human t h r o m b i n , human p l a s r n i n , b o v i n e t r y p s i n and u r i n a r y k a l l i k r e i n as i n d i c a t e d i n T a b l e 10 ( 2 2 ) . V e r y l i t t l e d i f f e r e n c e was n o t e d I n t h e s u b s t r a t e r a t i o s between t h e k a o l i n - a c t i v a t e d a r g i n i n e e s t e r a s e a c t i v i t y and t h e p u r i f i e d k a l l i k r e i n p r e p a r a t i o n . Hence, t h e a r g i n i n e e s t e r a s e a c t i v i t y measured i n t h i s work seems v e r y l i k e l y t o r e p r e s e n t plasma k a l l i k r e i n a c t i v i t y . Rao and a s s o c i a t e s n e x t r e p o r t e d a d e f i c i e n c y o f a r g i n i n e e s t e r a s e a . c t i v i t y i n t h e plasma o f CF p a t i e n t s (114) and t h i s t i m e c a l l e d i t a. k a l l i k r e i n d e f i c i e n c y w i t h o u t e q u i v o c a t i o n . T h e i r f i n d i n g s have been c o n f i r m e d by my p r e s e n t s t u d y , as w e l l as by W i l s o n and Fudenberg ( 1 4 3 ) , u s i n g TAME as a s u b s t r a t e . However, Lieberman (91) c o u l d n o t r e p r o d u c e t h e work of 123 T a b l e 1 0 : H y d r o l y s i s o f v a r i o u s a r g i n i n e and l y s i n e e s t e r s by p l a s m a and t i s s u e e s t e r a s e s . T o s y l - a r g i n i n e m e t h y l e s t e r B e n z o y l - a r g i n i n e m e t h y l e s t e r A c e t y l - a r g i n i n e m e t h y l e s t e r T o s y l - l y s i n e m e t h y l e s t e r A c e t y l - l y s i n e m e t h y l e s t e r Enzyme — i . — — — — — — S u b s t r a t e / TAME R a t i o : ; BAME AAME TIME AIME K a o l i n a c t i v a t e d a r g i n i n e e s t e r a s e 0 .89 0. 66 0 .22 0. 15 Thrombin • 1 .00 1. 30 1 •7° 0. 40 P l a s m i n 1 .01 0 1 .48 2. 35 T r y p s i n 0 .18 • 0. 51 1 .00 0. 73 K a l l i k r e i n 0 .81 0 .07 0 .25 0. 23 TAKE = BAME = AAME = TIME = ALME = 129 Rao et a l . Ke found t h a t the l e v e l of a r g i n i n e -e s t e r a s e a c t i v i t y i n CF plasma was normal when he used BAEE as a s u b s t r a t e . Lieberman and L i t t e n b e r g even r e p o r t e d an i n c r e a s e d k a l l i k r e i n c o n t e n t of s a l i v a f r om p a t i e n t s w i t h CF u s i n g b i o l o g i c a l a s s a y s (92). I n o t h e r works, Talamo et a l c o u l d n o t f i n d any ' s e v e r e ' a b n o r m a l i t y of t h e plasma k a l l i k r e i n - k i n i n s y stem i n c y s t i c f i b r o s i s u s i n g b i o c h e m i c a l and immunochemical methods ( 1 4 0 ) . There i s o b v i o u s l y c o n f u s i o n i n t h i s f i e l d b u t i t s t i l l seems l i k e l y t h a t the a.rginine e s t e r -ase a c t i v i t y measured i n my s t u d y and i n t h o s e o f Rao and N a d l e r i s k a l l i k r e i n . .. A f t e r t h e e x p e r i m e n t a l work i n t h i s t h e s i s was c o m p l e t e d , i d e a s c o n c e r n i n g the p a t h o g e n e s i s o f c y s t i c f i b r o s i s began t o c e n t e r around th e r o l e o f oCg-macro-g l o b u l i n as an i n h i b i t o r o f a r g i n i n e e s t e r a s e a c t i v i t y . T h i s i s coming about because W i l s o n e t a l showed e a r l i e r t h a t sera, from CF p a t i e n t s and c a r r i e r s were d e f i c i e n t .. i n a p r o t e i n of t h e ^ - g l o b u l i n f r a c t i o n and s p e c u l a t e d t h a t t h e d e f i c i e n c y might be r e s p o n s i b l e f o r t h e accumula t i o n of CF f a c t o r ( s ) i n serum (1^7, 150). L a t e r , u s i n g m u l t i p l e c h r o m a t o g r a p h i c t e c h n i q u e s , g e l f i l t r a t i o n , and s t a r c h b l o c k e l e c t r o p h o r e s i s , W i l s o n and Fudenberg documented t h e absence of a p r o t e i n f r a c t i o n w i t h a p i • 130 of 5.5 i n plasma from CF p a t i e n t s (148). They produced evidence t h a t t h i s v/as a p r o t e i n normally d e r i v e d from oc^-macroglobulin as a r e s u l t of i n t e r a c t i o n of ^-macro-g l o b u l i n ' w i t h a r g i n i n e esterase p r o t e i n s . • .They found a •• normal c o n c e n t r a t i o n of i n t a c t Alpha .-.-macrbglobulin ( oc 2 M) i n c y s t i c f i b r o s i s . plasma but a s l i g h t l y reduced b i n d i n g c a p a c i t y of the cc^ r-.T from CF p a t i e n t s f o r t r y p s i n and suggested that the d e f i c i e n t p r o t e i n may be a p r o -t e o l y t i c d e r i v a t i v e of cc„ ( l 4 8 ) . oCg-macroglobulin i s a plasma i n h i b i t o r which appears to p a r t i c i p a t e i n the r e g u l a t i o n of s e v e r a l . c i r c u l a t i n g enzyme systems, s i n c e i t forms complexes w i t h plasrnin (72), thrombin (36) and k a l l i k r e i n (71). I t s i n t e r a c t i o n w i t h the hemostatic inflammatory and comple-ment pathways i s shown i n Figure 23. In a d d i t i o n , OC^  M has been found to bind t r y p s i n , chymotrypsin, cathepsins and other endopeptidases (6, 72,). K a r p e i has shown th a t the subunit s t r u c t u r e of oCg M i s a l t e r e d by i n t e r a c t i o n w ith the p r o t e o l y t i c enzymes i t i n h i b i t s (72). cc^ M normally c o n s i s t s of f o u r subunit chains of molecular weight approximately 185,000 each but upon complex form-a t i o n with t r y p s i n , plasrnin, thrombin, k a l l i k r e i n and p o s s i b l y other plasma protea.ses, i t i s p r o t e o l y t i c a l l y 131 cleaved at the same region i n each subunit chain, r e s u l t i n g i n the production of a single protein with a molecular weight approximately half that of the. subunit . chain (35,000). ( 7 2 ) . Interaction of arginine esterase with oCg !•: may involve p r o t e o l y t i c cleavage at a single arginine or l y s i n e residue i n oC, M, located within a peptide loop formed by intrachain d i s u l f i d e bridging ( 7 2 ) . A covalent bond i s then formed between the enzyme and oc^  M, involving an acyl enzyme or tetrahedral intermediate, i n which a serine residue at the active s i t e of the enzyme forms an ester bond with the carboxyl of an arginine or l y s i n e residue at the active s i t e of cc, M. In t h i s reaction, the act i v e s i t e of the enzyme i s blocked, and an enzyme - r^2 macroglobulin complex i s formed ( 7 2 , 143). This complex retains i t s e s t e r o l y t i c a c t i v i t y towards low molecular weight substrates but not p r o t e o l y t i c a c t i v i t y towards pro t e i n . The other product of the reaction i s a smaller protein with a. molecular weight of approximately 85,000 as mentioned e a r l i e r . Wilson and Fudenberg reported a decreased l e v e l of the p r o t e o l y t i c fragments from cC>. M i n plasma from CF homozygotes and obligate heterozygote c a r r i e r s (148). 132 P o s s i b l e mechanisms s u g g e s t e d by W i l s o n and Fudenberg a r e t h e f o l l o w i n g : (a) Plasma p r o t e a s e s i n g e n e r a l may n o t be a c t i v a t e d t o normal l e v e l s because of an a b n o r m a l i t y In Hageman ' f a c t o r or i n t h e a c t i v a t i o n of Hageman f a c t o r . B e f o r e a c t i v a t i o n of Hageman f a c t o r by g l a s s o r e l l a g i c a.cid, a l m o s t a l l plasma a r g i n i n e e s t e r a s e a c t i v i t y i s i n an i n a c t i v e o r proenzyme form and w i l l n o t i n t e r a c t w i t h cx^ M (72, 79). A t t h i s t i m e the p r o t e o l y t i c d e r i v a t i v e s of cc> M cannot be o b s e r v e d by i s o e l e c t r o f o c u s i n g of whole plasma (148) When Hageman f a c t o r i s a c t i v a t e d , i t can i n i t i a t e t h e c o n v e r s i o n o f the p r o t e a s e s t o a c t i v e forms c a p a b l e o f c o m p l e x i n g t o oCg M (77). They can at t h i s t i m e a l s o i n t e r a c t w i t h t h e systems t h a t gen-e r a t e k i n i n and a n a p h y l a t o x i n ( F i g u r e 28) (79, 115). (b) A s p e c i f i c p r o t e a s e may be abnormal s t r u c t u r a l l y o r re m a i n p a r t i a l l y i n i t s proenzyme form ( e . g . K a l l i -k r e i n ) ( c ) There may be a g e n e t i c a l l y d e t e r m i n e d a b n o r m a l i t y i n t h e s t r u c t u r e of c< 2 M, d e c r e a s i n g i t s b i n d i n g a f f i n i t y f o r a l l pla.sma. p r o t e a s e s . (d) The a c t i v e s i t e by which oc„ M b i n d s t o p r o t e a s e s 133 Plasminogen activator 0( 2M Plasmin CIS -y c i s Kallikrein Koninogen > Bradykinin -> Plasminogen •>C3a \ \ \ Anaphylatoxin Inactivator / (Carbcixypeptidase) Figure 28: Diagram i l l u s t r a t i n g the points of i n t e r a c t i o n of ^ • ^ ^ c r c g l o b u l i n with the hemostatic, inflammatory and complement pathways.(Modified from Wilson and Fudenberg(148)) ^ Activation • Inactivation 134 may be blocked by an i n h i b i t o r . (e) The active s i t e of a l l the proteases by which they bind to C < 2 M may be blocked, i n h i b i t i n g t h e i r . a b i l i t y to in t e r a c t with oCg M (143).. P o s s i b i l i t i e s (c) and (d) seem rather a t t r a c t -ive. A s t r u c t u r a l abnormality i n , or a blockage of, the binding s i t e of oc2 M could decrease the i n h i b i t i o n of k a l l i k r e i n , plasrnin, and other plasma proteases. f?C2 M i s thought to be the major i n h i b i t o r of plasrnin (79) • Fla.smin, i f not e f f e c t i v e l y i n h i b i t e d , could part-i c i p a t e In increased a c t i v a t i o n of k a l l i k r e i n and thus increase the production of kinins, (Figures 2, 2?) . This series of events should at f i r s t glance cause an increase i n k a l l i k r e i n a c t i v i t y . However, i n my invest-i g a t i o n , a decrease i n arginine esterase was observed. According to Wilson and Fudenberg, t h i s could be explained by the f a c t that the increased production of plasrnin might be enough to i n h i b i t Hageman fact o r formation (148). Unfortunately, no evidence can be found to support t h i s hypothesis that increased plasrnin v/ould i n h i b i t Hageman factor a c t i v a t i o n . This i n turn would i n h i b i t p r e k a l l i k r e i n and hence k a l l i k r e i n . A c o r r e l a t i o n between decreased arginine 135 esterase a c t i v i t y and a decrease' observed i n the cC, M fragment ba.nds by i s o e l e c t r i c focusing was demonstrated (143). Wilson and Fudenberg suggested that t h i s concom-i t a n t decrease i n the concentration of cC, M.fragments. and arginine esterase a c t i v i t y or k a l l i k r e i n a c t i v i t y i n C F might be explained by assuming (I) hyperconversion of p r e k a l l i k r e i n to k a l l i k r e i n , brought about by plasmin, with subsequent I n h i b i t i o n of k a l l i k r e i n by i n h i b i t o r s present In the plasma; ( i i ) feedback i n h i b i t i o n of Kageman factor a c t i v a t i o n by uninhibited plasmin; or ( i i i ) a concurrent defect i n k a l l i k r e i n , or another arginine esterase, but not plasmin (148). Additional support f o r the r o l e of oCg-macro-gl o b u l i n i n the pathogenesis of arginine esterase d e f i c -iency comes from the work by Shapira, Rao, Wessel and Nadler (129). Using immunological methods, they demon- . strated that the arginine esterase a c t i v i t y present i n normal plasma and d e f i c i e n t i n c y s t i c f i b r o s i s patients was bound to oc? M- l i k e molecule.. Pooled plasma from normal individuals was activated by chloroform-ellagic• acid treatment and pre c i p i t a t e d by ammonium sul f a t e at 50;'J saturation. Since the arginine . esterase a c t i v i t y •could be i n h i b i t e d by.STI, the plasma, p r e c i p i t a t e was 136 p u r i f i e d by a f f i n i t y chromatography u s i n g STI cova.l-. e n t l y bound t o Sepharose 43. T h i s p u r i f i e d p r o t e i n f r a c t i o n was r e s o l v e d by i s o e l e c t r i c f o c u s i n g on p o l y -a c r y l a m i d e g e l s . - P r o t e i n s " w i t h most o f . t h e a r g i n i n e e s t e r a s e a c t i v i t y had a p i range of 5-5- T h i s f r a c t i o n was t h e one which i s d e c r e a s e d i n t h e pla.sma o f p a t i e n t s w i t h CF. I m m u n o e l e c t r o p h o r e s i s w i t h a c t i v a t e d pla.sma. samples from c o n t r o l s and p a t i e n t s w i t h CF was preformed, u s i n g t h e a n t i s e r u m . I n c o n t r o l s , f o u r p r e c i p i t i n a r c s w i t h a r g i n i n e e s t e r a s e a c t i v i t y were o b s e r v e d whereas o n l y t h r e e were seen i n plasma from CF p a t i e n t s . One a r c w i t h e n z y m a t i c a c t i v i t y wa.s f o u n d t o be a n t i g e n i c a l l y a.n oC ?-ma.croglobulin. On I m m u n o e l e c t r o p h o r e s i s , t h i s oCg M p r e c i p i t i n band c o r r e s p o n d e d t o t h e band a b s e n t i n plasma of CF p a t i e n t s . The presumed i m p l i c a t i o n of t h e above work i s t h a t t h e a r g i n i n e e s t e r a s e band d e f i c i e n t i n c y s t i c f i b r o s i s plasma c o n t a i n e d oc^ M d e t e r m i n a n t s . oCg M l e v e l s measured i m m u n o l o g i c a l l y were s i m i l a r i n plasma, from c o n t r o l s ' a n d CF p a t i e n t s . The work o f Shapira. e t a l i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t i n c y s t i c f i b r o s i s t h e r e i s an absence of an cC, M -a r g i n i n e e s t e r a s e complex and t h a t t h i s r e f l e c t s a m o l e c u l a r d e f e c t i n e i t h e r of t h e two components. T h i s 137 i s . t h e same h y p o t h e s i s as t h a t s u g g e s t e d by W i l s o n and Fudenberg ( 1 4 3 ) . More r e c e n t s t u d i e s by S h a p i r a e t a l have shown, th a t ' t h e CC, M m o l e c u l e i s a l t e r e d i n p a t i e n t s w i t h c y s t i c f i b r o s i s and o b l i g a t e h e t e r o z y g o t e s ( 1 2 3 ) . T h i s work c o n s i s t e d of an. i n v e s t i g a t i o n o f t h e b i n d i n g c a p a c i t y of i s o t o p i c a l l y l a b e l l e d p a p a i n and i s o t o p i c a l l y l a b e l l e d b o v i n e t r y p s i n t o o:'2 M. They showed t h a t t h e amount o f b i n d i n g i n normal c o n t r o l s i s g r e a t e r t h a n t h a t o f CF p a t i e n t s (127). B i n d i n g i n o b l i g a t e h e t e r o z y g o t e s i s i n t e r m e d i a r y between t h e normal and CF l e v e l s . They c o n c l u d e t h a t t h e p r i m a r y d e f e c t i n c y s t i c f i b r o s i s i s l i k e l y t o r e s i d e i n t h e Xg-macroglobulin. m o l e c u l e . D e f i c i e n c y i n plasma a r g i n i n e e s t e r a s e a c t i v i t y r e p o r t e d i n t h i s s t u d y would t h e n be a s e c o n d a r y e f f e c t caused by a d e c r e a s e d b i n d i n g a f f i n i t y of oC> ^ from CF p a t i e n t s f o r t h e enzyme. A d e c r e a s e d b i n d i n g a f f i n i t y o f cC, M t o a r g i n i n e e s t e r a s e can l e a d t o a d e f i c i e n c y of S T I - i n h i b i t e d a r g i n i n e e s t e r a s e a c t i v i t y due t o t h e f o l l o w i n g h y p o t h -eses proposed by the w r i t e r : ( i ) P o s i t i v e f e e d b a c k o f a r g i n i n e e s t e r a s e ( o r k a l l i k r e i n ) s y n t h e s i s may be d i m i n i s h e d by t h e l a c k of b i n d i n g of t h e enzyme t o o<9 M. 138 ( i i ) Decreased b i n d i n g of oc2. K t o a r g i n i n e e s t e r a s e f a c i l i t a t e s t h e i n t e r a c t i o n of a r g i n i n e e s t e r a s e w i t h o t h e r plasma i n h i b i t o r by a d i f f e r e n t mechanism.- U n l i k e t h e cC M - a r g i n l n e e s t e r a s e b i n d i n g w h i c h r e t a i n s t h e enzyme a c t i v i t y , t h e . i n t e r a c t i o n o f a r g i n i n e e s t e r a s e w i t h o t h e r i n h i b i t o r s may r e s u l t i n t h e f o r m a t i o n o f an e n z y m e - i n h i b i t o r complex w i t h no a r g i n i n e e s t e r a s e a c t i v i t y . T h e r e f o r e , t o t a l and S T I - i n h i b i t e d a r g i n i n e e s t e r a s e a c t i v i t i e s d e c r e a s e i n c y s t i c f i b r o s i s as demonstrated by t h e p r e s e n t i n v e s t i -g a t i o n . I n t h e a n n u a l m e e t i n g df the C y s t i c F i b r o s i s C l u b i n A p r i l , 1977. S h a p i r a r e p o r t e d t h e r e s u l t s o f h i s most r e c e n t work on t h e i n t e r a c t i o n o f c6>-roa.croglobulin w i t h t r y p s i n (151) . He s u g g e s t e d t h e f o l l o w i n g sequence o f e v e n t s f o r t h e i n t e r a c t i o n . ( i ) CCgf'i p l u s t r y p s i n forms an c ^ M - t r y p s i n complex i n l e s s t h a n 30 s e c o n d s . T h i s complex has no a c t i v i t y t o t h e h i g h m o l e c u l a r w e i g h t s u b s t r a t e s such as c a s e i n , and no s u s c e p t i b i l i t y t o i n h i b i t i o n by h i g h mole-c u l a r w e i g h t i n h i b i t o r s such as S T I . ( i i ) Over a p e r i o d o f 3°-o0 m i n u t e s , t h i s oCgM-trypsin 139 complex undergoes a c o n f o r m a t i o n a l change i n which i t r e g a i n s some a c t i v i t y t o h i g h m o l e c u l a r weight s u b s t r a t e s and some s u s c e p t i b i l i t y t o i n h i b i t i o n by h i g h m o l e c u l a r w e i g h t i n h i b i t o r s , ( i i i ) T h i s a l t e r e d complex then decomposes s l o w l y , t o OC^'L; fragments and t r y p s i n . I n my work, t h e b u l k o f a r g i n i n e e s t e r a s e a c t i v i t y as measured by the e n z y m o l o g i c a l a s s a y i s i n h i b i t e d by S T I , i n b o t h cases of c o n t r o l and c y s t i c f i b r o s i s plasma. I f t h e above mechanism o f i n t e r a c t i o n between oCgM and t r y p s i n i s t r u e and can be a p p l i e d t o a r g i n i n e estera.se, t h e w r i t e r would s p e c u l a t e t h a t a l l t h r e e s t e p s would t a k e p l a c e i n c y s t i c f i b r o s i s p a t i e n t s and normal c o n t r o l s . . However, Shapira. and N a d l e r s u g g e s t e d t h a t t h e second s t e p i n t h e proposed mechanism does n o t o c c u r i n c y s t i c f i b r o s i s (151). T h i s cannot be t r u e because N a d l e r * s e a r l i e r work i n a r g i n i n e e s t e r a s e showed tha.t about 70?^  t h e t o t a l a r g i n i n e e s t e r a s e a c t i v i t y of b o t h c o n t r o l and c y s t i c f i b r o s i s plasma i s i n h i b i t e d by S T I . These f i n d i n g s a r e s i m i l a r t o t h e d a t a of t h e p r e s e n t s t u d y . T h e r e f o r e , t h e second s t e p i n . Sha.pira.'s s u g g e s t e d mechanism s h o u l d o c c u r i n c y s t i c f i b r o s i s . Work on.column chromatography s u g g e s t s t h a t t h e r e i s no d e f i c i e n c y of an isoenzyme o f a.rginine e s t e r a s e . - T h i s . i s . c o n s i s t e n t v/ith a. p r i m a r y d e f e c t e l s e w h e r e which l e a d s t o a secondary d e f i c i e n c y o f a r g i n i n e e s t e r a s e a c t i v i t y i n c y s t i c f i b r o s i s . My chromatography f i n d i n g s a r e d i f f e r e n t f r om t h o s e o f Rao and N a d l e r i n t h e f o l l o w i n g a s p e c t s : ( i ) I n t h i s s t u d y , two peaks of a r g i n i n e e s t e r a s e . a c t i v i t y , a r e o b s e r v e d w i t h t h e major peak e l u t e d a t an NaCl c o n c e n t r a t i o n o f 0.15 M.. In Rao and N a d l e r * s work, two peaks o f enzyme a c t i v i t y a r e de m o n s t r a t e d •with the major peak e l u t e d a t a p p r o x i m a t e l y 0.02 M . N a C l . ( i i ) The major a c t i v i t y peak i n my s t u d y i s n o t i n h i b i t e d by S T I . The major peak of a r g i n i n e e s t e r a s e a c t i v i t y i n t h e work o f Rao and N a d l e r i s i n h i b i t e d by S T I . The d i s c r e p a n c y of my o b s e r v a t i o n s and t h o s e o f Rao a.nd N a d l e r may be a c c o u n t e d f o r by t h e f o l l o w i n g a s s u m p t i o n s : ( i ) I n c o m p l e t e d e s a l t i n g of Rao and Na.dler's column chromatography work might cause t h e b u l k o f a r g i n i n e e s t e r a s e a c t i v i t y t o be e l u t e d a t t h e b e g i n n i n g o f 141 t h e NaCl g r a d i e n t . ( i i ) The i n t e r a c t i o n s of cc, M a.nd a r g i n i n e e s t e r a s e may he ti m e dependent. .Longer time used i n t h e m a n i p u l a t i o n of t h e column chromatography e x p e r i -ments would e n a b l e t h e i n t e r a c t i o n s o f oc, M and a r g i n i n e . e s t e r a s e t o ta.ke p l a c e i n a l o n g e r p e r i o d o f t i m e b e f o r e p r o t e i n p r e c i p i t a t i o n . S i n c e cc, M and STI b o t h r e a c t w i t h a r g i n i n e e s t e r a s e s t h r o u g h t h e same mechanisms ( ? 2 ) . l o n g e r t i m e a l l o w e d f o r t h e i n t e r a c t i o n s o f oc^ M and a r g i n i n e e s t e r a s e would make t h e m o l e c u l e more r e s i s t a n t t o S T I . T h i s may g i v e r i s e t o t h e d i f f e r e n t degrees of i n h i b i t i o n o f a r g i n i n e e s t e r a s e a c t i v i t y by STI i n t h e chromatography r e s u l t s o f Rao and N a d l e r , and i n t h i s s t u d y . ( i i i ) I f S h a p i r a ' s s u g g e s t e d mechanism of t h e i n t e r -a c t i o n o f Kg M w i t h t r y p s i n can be a p p l i e d i n g e n e r a l t o o t h e r p r o t e a s e s i n c l u d i n g a r g i n i n e e s t e r a s e , t h e n the time t a k e n f o r t h e a.mmonium s u l p h a t e p r e c i p i t a t i o n s t e p i s v e r y c r i t i c a l . P r e sumably, t h e i n t e r a c t i o n between c<2 M and a r g i n i n e e s t e r a s e s t o p s once p r e c i p i t a t i o n o c c u r s . I f t h e t i m i n g o f S h a p i r a ' s proposed mechanism i s 142 c o r r e c t , t h e p r e c i p i t a t i o n . s t e p i n my column work would t a k e p l a c e i n t h e second s t e p when t h e cc> M-enzyme complex b e g i n s t o r e g a i n some s u s c e p t i b i l i t y t o i n h i b i t i o n by S T I . Hence, i n my DEAS-chromato-'. gra.phy d a t a , the b u l k of a r g i n i n e e s t e r a s e a c t i v i t y , i s r e s i s t a n t t o S T I . The b i o l o g i c a l r o l e of t h e DO, M - a r g i n i n e e s t e r a s e complex i s n o t y e t c l e a r . C e r t a i n l y , f u r t h e r r e s e a r c h i s r e q u i r e d t o e x p l o r e t h e r e l a t i o n s h i p o f a r g i n i n e e s t e r a s e d e f i c i e n c y t o oC g - m a c r o g l o b u l i n and t o e l u c i d a t e t h e i n v o l v e m e n t o f co, M i n t h e p a t h o p h y s i o l o g y o f c y s t i c f i b r o s i s . However, i t may w e l l be t h a t an a b n o r m a l i t y i n oc 2 M - a r g i n i n e e s t e r a s e i n t e r a c t i o n s c o u l d e x p l a i n t h e p r e s e n c e of CF f a c t o r ( s ) i n c y s t i c f i b r o s i s . 143 V. CONCLUSION The r e s u l t s of t h i s i n v e s t i g a t i o n have shown t h a t t h e r e i s a s i g n i f i c a n t r e d u c t i o n i n t h e l e v e l o f a r g i n i n e e s t e r a s e a c t i v i t y i n t h e plasma, of p a t i e n t s w i t h c y s t i c f i b r o s i s r e l a t i v e t o t h a t of c o n t r o l s u b j e c t s . A l t h o u g h as a group, o b l i g a t e h e t e r o z y g o t e s have a r g i n i n e e s t e r a s e a c t i v i t i e s hither t h a n t h o s e of p a t i e n t s , t h e d e t e c t i o n of I n d i v i d u a l h e t e r o z y g o u s c a r r i e r s i s n o t p o s s i b l e . A r g i n i n e e s t e r a s e a c t i v i t y i n c h l o r o f o r m -e l l a g i c a c i d t r e a t e d pla.sma from CF. p a t i e n t s and c o n t r o l s u b j e c t s has been r e s o l v e d i n t o two component f r a c t i o n s by i o n exchange chromatography on DEAS-Sephadex. 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M c i n t o s h . B a l t i m o r e , M a r y l a n d , F r e n c h - B r a y , i960, page 27. 140. Talarno, R . C , R.W, Colma.n and A . M i l u n s k y : The plasma, k a l l i k r e i n - k i n i n system i n c y s t i c f i b r o s i s . P e d i a t . Res., 6: 430/170(1972). 141. Vaze, D., K.M. K u t t y , R.K. Chandra and S . I . H y c h e v i a h : G l y c o p r o t e i n s i n t h e s a l i v a and p l a t e l e t s of c y s t i c f i b r o s i s p a t i e n t s . A b s t r a c t s of m e d i c a l s e s s i o n s . V I I I n t e r -n a t i o n a l C y s t i c F i b r o s i s C o n g r e s s , 1976,0. 6. 153 142. V e r t e r b e r g , L. and G. N i s e : U r i n a r y p r o t e i n s s t u d i e d by use of i s o e l e c t i c f o c u s i n g . I . T u b u l a r m a l f u n c t i o n i n a s s o c i a t i o n w i t h exposure t o cadmium. C l i n . Chem. 19: 1179, 1973-143. V e r t e r b e r g , ' 0. and H. Svensson: I s o e l e c t r i c f r a c t i o n a t i o n , a n a l y s i s , and c h a r a c t e r -i z a t i o n of ampholytes In n a t u r a l pH g r a d i e n t s . A c t a Chem. Scand., 20; 820, 1966. 144. V/aring, V.'.V/., C H . Br u n t and B.C. H i l m a n : Mucoid i m p a c t i o n of b r o n c h i i n c y s t i c f i b r o s i s / P e d i a t r i c s , 3_9_: 166, 1967. 145. Warwick, W.J. and R.S. Fogue: The p r o g n o s i s of c h i l d r e n w i t h c y s t i c f i b r o s i s b a sed on re a s o n e d approaches t o t h e r a p y : p a s t , •present, and f u t u r e . J . Asthma Res,, 5: 277, 1963. . 146. 'White, A., P. H a n d l e r and S.L. S m i t h . P r i n c i p l e s o f B i o c h e m i s t r y . 4th E d i t i o n . McGraw-H i l l Book Co., Mew York. 1 9 6 8 . pr>. 724-734. 147. W i l s o n , G.3.: S t u d i e s on t h e c y s t i c f i b r o s i s serum f a c t o r and on the n a t u r e o f t h e c i l i a r y b e a t of C o l p i d i u m s t r i a t u m . Ph. D. T h e s i s . U n i v e r s i t y o f C a l i f o r n i a , Los A n g e l e s . D i s . A b s t . I n t e r n . , 35s 479, 1974. 148. W i l s o n , G.B. and H.H. Fudenberg: S t u d i e s on c y s t i c f i b r o s i s u s i n g i s o e l e c t r i c f o c u s i n g I I . P e d i a t . Res., 10: 87, 1976. ' .. 149. W i l s o n , G.B. and T.L. Jahn: Decreased r a t e o f c y t o l y s i s of C o l p i d i u m s t r i a t u m by c y s t i c f i b r o s i s serum. I . L i f e S c i . , . 11' 551, 1974. 150. 'Wilson, G.B., T.L. Jahn and J.R. F o n s e c a : Demon-s t r a t i o n of serum p r o t e i n d i f f e r e n c e s i n c y s t i c f i b r o s i s by i s o e l e c t r i c f o c u s i n g i n t h i n - l a y e r p o l y a c r y l a m i d e g e l s . C l i n . Chim. A c t a , 49_: 79, 1973-159 151. S h a p i r a , S, L e c t u r e on " I n t e r a c t i o n of CK?-macro-g l o b u l i n and t r y p s i n " . 1 8 t h Annual M e e t i n g of C y s t i c F i b r o s i s F o u n d a t i o n , San F r a n c i s c o , A p r i l , . 1 9 7 7 . APPENDIX I Reprinted from Clinica Chimica Acta, 74 (1977) 71—75 © Elsevier/North-Holland Biomedical Press CCA 8202 PLASMA ARGININE ESTERASE ACTIVITY IN CYSTIC FIBROSIS OF THE PANCREAS KATHERINE Y.H. CHAN, DEREK A. APPLEGARTH * and A. GEORGE F. DAVIDSON Department of Paediatrics, The University of British Columbia and Biochemical Diseases Laboratory, Children's Hispital, Vancouver, B.C. (Canada) (Received June 26th, 1976) Summary Using a micro-method for the determination of plasma arginine esterase ac-tivity, we have investigated the values for soybean trypsin inhibitor (STI)-inhib-ited arginine esterase activity in patients with cystic fibrosis, obligate heterozy-gotes and age matched control individuals. The mean of STI-inhibited activity is lowest for cystic fibrosis patients while the mean for normal controls is the highest. The mean of STI-inhibited activity for the heterozygotes is midway be-tween the values of the patients and the normal individuals. The deficiency of arginine esterase activity was statistically significant for both cystic fibrosis pa-tients and heterozygotes. Introduction A deficiency in plasma arginine esterase activity in patients with cystic fi-brosis of the pancreas was reported in 1972 [1]. This deficiency was measured by using as substrate tosylarginine methyl ester (TAME). The work was extend-ed in 1974, using as a substrate a-Af-benzoyl-L-arginine ethyl ester (BAEE) [2]. In another paper in 1974 [3] this work was reinvestigated. BAEE was used as substrate and the deficiency of arginine esterase activity was not confirmed. We have modified the original method of Rao et al. [1], to allow for determination of arginine esterase activity in micro samples of blood and here report the re-sults of our measurements in patients with cystic fibrosis. We used TAME as substrate and our results substantially confirm those of Rao et al. A recent report has demonstrated deficient proteolytic cleavage of a2-macro-globulin in cystic fibrosis plasma [4]. Since a2-macroglobulin appears to be in-volved in control of the arginine esterase system [5], it seems to be relevant to * Reprint request should be addressed to Dr. Derek A. Applegarth, Biochemical Diseases Laboratory, Children's Hospital, 250 West 59th Avenue, Vancouver, B.C., Canada. 72 report our confirmation of the deficiency of plasma arginine esterase activity in patients with cystic fibrosis at this time. Materials and methods Blood samples were obtained from 19 patients with cystic fibrosis, 19 obli-gate heterozygotes, 34 normal, healthy adults and 16 hospitalized control children who were age matched for the cystic fibrosis patients. Cystic fibrosis patients were assessed for the severity of their disease, using the Shwachman Scoring System [6]. Arginine esterase activity in the plasma samples was measured using tech-niques modified from those of Rao et al. [1], with most volumes scaled down to approximately one-fifth. All samples were handled in polypropylene con-tainers. 1.8 ml blood was mixed with 0.2 ml 3.8% sodium citrate. The plasma was separated by centrifugation at 3000 r.p.m. for 15 min at 4°C and used im-mediately or stored at —20° C. An equal amount of cold chloroform was added to the plasma and mixed using an electric rotator at approximately 10 r.p.m. for 5 min at 4°C. The mixture was then centrifuged at 2000 r.p.m. for 30 min at 4°C, after which the plasma was separated, and incubated with an equal vol-IA| EFFECT OF CHLOROFORM O N ENZYME ACTIVITY |C| EFFECT OF ELLAGIC ACID CONCENTRATION 0.4 0.2 < o.: O N ENZYME ACTIVITY •r -4 8 13 16 Time of Rotation (minutes) |B) EFFECT OF SOYBEAN TRYPSIN INHIBITOR 0.3 r CONCENTRATION O N ENZYME ACTIVITY 0.2 0.1 0.2 0.3 0.4 0.5 Concentration of Ellagic Acid Solution (mM) |D) _ EFFECT OF ELLAGIC ACID INCUBATION TIME O N ENZYME ACTIVITY 0.4 o CO to < 0.2 0 100 . 200 300 Soybean Trypsin Inhibitor Concentration l/ig/ml) 10 20 Incubation Time (minutes) 30 Fig. 1. Investigations of the parameters of the micro-assay for plasma arginine esterase activity. (The ar-rows indicate the values used in the micro-assay). 1 ume of 0.1 mM ellagic acid (Aldrich Chemical Co. Inc.) suspended in 0.10 M phosphate buffer, pH 7.6, containing NaCl at a concentration of 0.15 M, at 25°C, for 15 min. 50 of the activated plasma was then added to a tube con-taining 150 ul of 0.10 M phosphate buffer pH 7.6 with 0.15 M NaCl and 0.015 M TAME. The reaction mixture was incubated at 37°C for 15 min. The reac-tion was terminated by the addition of 200 iA of 10% trichloroacetic acid, and the mixture centrifuged at 2000 r.p.m. for 10 min at 4°C. Methanol in the su-pernatant was assayed according to Siegelman et al. [7] modified by using one-fifth of the reported volumes of the samples and reagents. Micro-assay for arginine esterase activity was also performed in the presence of soybean trypsin inhibitor (STI) (Grand Island Biological Co). 10.0 mg of STI was dissolved in 1.0 ml of normal saline and 4 jul of the STI solution was added to 0.2 ml of the chloroform treated plasma to give a final concentration 200 /ug/ml. The enzyme activity was expressed as STI-inhibited arginine esterase ac-tivity which was the difference between the values obtained by assaying in the presence and absence of STI. The micro-assay technique was validated by assessing the effects of different concentrations of ellagic acid and STI. The effects of varying incubation times for ellagic acid and chloroform on arginine esterase activity were studied. The macro-method of Rao et al. [1] for arginine esterase activity was repeated for 26 plasma samples which had already been assayed using our micro-assay. A calibration plot was constructed to provide a correlation coefficient between the two assays. Results of the micro-assays on the various control subjects, pa-tients with cystic fibrosis and obligate heterozygotes were assessed using the Student " r " test. The Gaussian distribution of STI inhibited arginine esterase activity in each population was confirmed by both the Kolmogorov-Smirnov test and the chi-square test. T A B L E I DIFFERENCES OF STI-INHIBITED ARGININE ESTERASE ACTIVITIES IN THE PLASMA OF CON-T R O L SUBJECTS. PATIENTS WITH CYSTIC FIBROSIS (CK.) AND OBLIGATE HETEROZYGOTES T H E P VALUES WERE OBTAINED BY USING THE STUDENT "t" TEST Subject Age (years) No. STI-inhibited arginine esterase activity (Mmol of TAME hydrolyzed/h/ml of plasma) Mean ± S.D. Range of actual values Probability (P) C F . children 4—15 11 27.6 + 8.2 Control children 1-15 16 44.8 + 18.2 C F . adults 16—26 8 34.5 + 12.7 Control adults 16—26 21 47.6 + 12.9 Obligate heterozygotes 32-^2 19 37.1 + 15.0 Control adults 27-46 13 53.4 + 12.6 C F . adults 16—26 8 34.5 + 12.7 Obligate heterozygotes 32-42 19 37.1 + 15.0 13.5-42.2 12.2—82.2 18.9-52.5 26.8— 71.6 6.4-65.3 30.0-81.2 18.9— 52.5 6.4-65.3 <0.003 * <0.028 * <0.002 * <0.65 * * * Difference significant. * * Difference not significant. 74 10 3 10 5 -_ (A) CYSTIC FIBROSIS P M«an »TIENTS ^dutfs and Childrtm) -|t>| CONN! OL SUBJECTS (Adulti and Children) Mean -IQ OBLIGATE METE Mean iOZYGOTES U 1 1 1 1 | , , 1 • 5 1} 25 35 45 55 65 75 85 95 STI INHIBITED ARGININE ESTERASE ACTIVITY ( pmoies TAME hyHrolyzed/hr/ml plasma) Fig. 2. Distribution of STI inhibited arginine esterase values in the populations studied. Results . . „ The results of the investigations of the method parameters are shown in Fig. 1. The comparison of the actual results of the micro-method with the macro-method, using a linear regression plot showed a correlation coefficient of 0.947. The values of STI inhibited arginine esterase activity in plasma for the various populations are shown in Table land Fig. 2. The difference in the mean plasma arginine esterase activity between the control subjects and patients with cystic fibrosis was statistically significant. The mean enzyme activity in the plasma of the heterozygotes was also significantly different from that of the normal controls. There was no significant difference between the mean values of patients and carriers. The actual values of STI inhibited arginine esterase ac-tivity for patients with cystic fibrosis could not be related to the severity of the disease as assessed by Shwachman scoring. Discussion It can be seen that our results confirm the original work of Rao et al. [1], showing deficient arginine esterase activity in cystic fibrosis. It seems notewor-thy that not only is the deficiency in arginine esterase activity statistically sig-nificant for both patients and heterozygotes, but the mean heterozygote activ-ity is midway between normal control and patient values. Our findings take on greater significance in view of the demonstration of de-ficient proteolytic cleavage of a2-macroglobulin in cystic fibrosis plasma [4] and the suggestion that a2-macroglobulin is involved in the control of plasma arginine esterase activity. Although much more work remains to be done on the 164 75 relationships between a2-macroglobulin and arginine esterase activities, the like-lihood of a functional correlation between these two areas must be seriously considered and evaluated with respect to the pathogenesis of cystic fibrosis. The micro-method which we have described for the estimation of arginine esterase activity should aid other investigations in confirming and extending these results without requiring unacceptably large volumes of blood from pedi-atric oatients. i  p i  Acknowledgements ' The authors wish to thank Dr. Brenda Fraser for her assistance with statisti-cal analysis and Dr. Lawrence Wong for help in obtaining the blood samples used in this investigation and for helpful discussions. References 1 Rao, G.J.S., Posner. L .A. and Nadler. H.L. (1972) Science 177, 610 2 Rao, G.J.S. and Nadler, H.L. (1974) Pediatr. Res. 8, 684 3 Lieberman, J. (1974) Am. Rev. Resp. Dis. 109, 399 4 Wilson, G.B. and Fudenberg, H.H. (1976) Pediatr. Res. 10, 87 5 Harpel, P.C. (1973) J. Exp. Med. 138, 508 6 Shwachman, H. and Kulczycki, L .L . (1958) Am. 3. Dis. Child. 96, 6 7 Siegelman, A.M. , Carlson, A.S. and Robertson, T. (1962) Arch. Biochem. Biophys. 97, 159 165 . ™ ^ L I I _ : shwachman scoring system of c l i n i c a l evaluation of patients with c y s t i c f i b r o s i s (130). : CRADLN'C Eicellcnf i (68-100) i POINTS 25 C E N E R A L A C T I V I T Y Full normal activity; plays ball, goes to school regu-larly, eta Good (71-S5) Mild (OO-/0; i Mcderato " (41-55) ; Severe *•: (-;ocr I UclO'r-) £0 15 10 L e d a endurance end tires fct end of day; good rcKool c'rtendanoe May rest voluntarily dur-ing roe liay; ares easily after exertion; fair school Attendance P H Y S I C A L E X A M I N A T I O N Normal; no cough; pubs and respirntions normal; clear lungs; good posture Resting p u l » cad reap inborn norma]; rare coughing or clear-ing of throat; no clubbing; clear lungs; minimiJ emphysema N U T R I T I O N X-RAY FINDINCS Maintains weight and height st above 25th percentile; well-formed stools, almost Donna]; good muscle mass and te&a Clear Sung fields Weight and Height at approxi-mately 15th to 20th percentile; stools slightly abnormal; fair muscle tone and mass Roma teacher, (byrpvtic after short vnlk; rests a great deal Orthopneic, confined bed or clulr to Occasional cough, perhaps in morning upon ruing; respira-tions slightly elevated; mild em-physema; coarse breath sounds; rarely localized rales; early clubbing Frequent cough, usasHy pro-ductive; chest retraction; mod-erate emphysema; may have chest deformity; rales usually present; clubbing 2 to 3+ Severe coughing spells; tachyp-nea with tachycardia and exten-sive pulmonary changes; may show signj of right-sided cardi-ac failure; clubbing 3 to 4 + Weigbt tnd height abovo 3rd percentile; stools usually ab-normal, large and poorly form-ed; yefy little, if any abdominal distension; poor muscle tone with reduced muscle mass Weigh! cod fcsteht fceJosr 3rd percentile; poorly formed, buTJry, fatty, offensive stools; flabby muscles end reduced mass; ab-dominal distension, mild to moderate Malnutrition narked; Itrxe pro-tuberant abdomen; rectal pro-lapse: large, foul, frequent, fatty movements Minimal cccectuatkm ct b r o n c h o v a s c u l a r mark-ings; early emphysema Mild emphysema with patchy atelectasis; tacnassa Dron-cbovascuLir markings Moderatst eavpbyjerrja; wide-spread areas of atelectasis with super-imposed areas of infection; mininvd brcncliial' ectasia t • 1 Extensive dhanRest with pul- ! inonxry obstructive plienom- ; tna arid infection; lobar \ atelectasis and bronchiectasij . 

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