RADIOIMMUNOASSAY AND RELATED PROCEDURES

IN MEDICINE V O L . I

PROCEEDINGS SERIES

RADIOIMMUNO ASSAY A N D R E L A T E D PROCEDURES

IN MEDICINE

P R O C E E D I N G S O F A S Y M P O S I U M O N R A D I O I M M U N O A S S A Y A N D R E L A T E D P R O C E D U R E S

IN C L I N I C A L M E D I C I N E A N D R E S E A R C H H E L D B Y T H E

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y IN I S T A N B U L , 10 - 14 S E P T E M B E R 1973

I n t w o v o l u m e s

VOL.I

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y V I E N N A , 1974 ~ ~

The following States are Members of the International Atomic Energy Agency:

AFGHANISTAN HAITI PAKISTAN ALBANIA HOLY SEE PANAMA ALGERIA HUNGARY PARAGUAY ARGENTINA ICELAND PERU AUSTRALIA INDIA PHILIPPINES AUSTRIA INDONESIA POLAND BANGLADESH IRAN PORTUGAL BELGIUM IRAQ ROMANIA BOLIVIA IRELAND SAUDI ARAB1A BRAZIL ISRAEL SENEGAL BULGARIA ITALY SIERRA LEONE BURMA IVORY COAST SINGAPORE BYELORUSSIAN SOVIET JAMAICA SOUTH AFRICA

SOCIALIST REPUBLIC JAPAN SPAIN CAMEROON JORDAN SRI LANKA C ANADA KENYA SUDAN CHILE KHMER REPUBLIC SWEDEN COLOMBIA KOREA, REPUBLIC OF SWITZERLAND COSTA RICA KUWAIT SYRIAN ARAB REPUBLIC CUBA LEBANON THAILAND CYPRUS LIBERIA TUNISIA CZECHOSLOVAK SOCIALIST LIBYAN ARAB REPUBLIC TURKEY

REPUBLIC LIECHTENSTEIN UGANDA DENMARK LUXEM BOURG UKRAINIAN SOVIET SOCIALIST DOM IN IC AN REPUBLIC MADAGASCAR REPUBLIC ECUADOR MALAYSIA UNION OF SOVIET SOCIALIST EGYPT, ARAB REPUBLIC OF MALI REPUBLICS EL SALVADOR / MEXICO UNITED KINGDOM OF GREAT ETHIOPIA MONACO BRITAIN AND NORTHERN FINLAND MONGOLIA IRELAND FRANCE MOROCCO UNITfcD STATES OF AMERICA GABON NETHERLANDS URUGUAY GERMAN DEMOCRATIC REPUBLIC NEW ZEALAND VENEZUELA GERM AN Y , FEDERAL REPUBLIC OF NIGER VIET-NAM GHANA NIGERIA YUGOSLAVIA GREECE NORWAY ZAIRE, REPUBLIC OF GUATEMALA ZAMBIA

The Agency's Statute was approved on 23 October 1956 by the Conference on die Statute of the IAEA held at United Nations Headquarters, New York; it entered into force on 29 July 1957. The Headquarters of the Agency are situated in Vienna. Its principal objecrive is "to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world".

Printed by the IAEA in Austria March 1974

C O R R I G E N D U M

R A D I O I M M U N O A S S A Y A N D R E L A T E D P R O C E D U R E S I N M E D I C I N E , V O L . I.

( S T I / P U B / 3 5 0 )

P a p e r I A E A - S M - 1 7 7 / 8 7 by B a d a w i et a l .

P a g e 417, l ine 7

F o r M R X 71/222 r e a d M R C 71/222

P a g e 417, l i n e s 8 - 1 1

T h e sentence should be as f o l l o w s :

It was also found that 1.0 m i l l i - a m p o u l e of the s e r u m Standard M R C 71/167 was equivalent to 0 .09 ng and 0. 9 pTJ of pi tui tary Standard M R C 71/222 with 95% f i d u c i a l l i m i t s at 0.06 - 0.14 and 0 .6 - 1 .4 , r e s p e c t i v e l y .

R A D I O I M M U N O A S S A Y A N D R E L A T E D P R O C E D U R E S IN M E D I C I N E I A E A , V I E N N A , 1974

S T I / P U B / 3 5 0

CONTENTS OF VOL. I

S T A N D A R D I Z A T I O N A N D C O N T R O L O F R E A G E N T S A N D P R O C E D U R E S (Session I)

Heterogenei ty of peptide h o r m o n e s : i ts i m p l i c a t i o n s for r a d i o i m m u n o a s s a y ( I A E A - S M - 1 7 7 / 2 1 3 ) 3 R o s a l y n S. Y a l o w D i s c u s s i o n * 21

Some p r o b l e m s of s p e c i f i c antibody product ion in r a d i o i m m u n o a s s a y techniques ( I A E A - S M - 1 7 7 / 5 ) 23 K a t a l i n M e r e t e y and L . T . K o c s ä r D i s c u s s i o n 29

A c o m p a r a t i v e a s s e s s m e n t of i m m u n i s a t i o n p r o c e d u r e s f o r r a d i o i m m u n o a s s a y ( I A E A - S M - 1 7 7 / 1 0 ) 31 Susan L a d e r , B . A . L . H u r n and G . C o u r t D i s c u s s i o n 44

P r o b l e m s of opt imizat ion of double antibody r a d i o i m m u n o a s s a y of H L H , H F S H and H G H : " h o o k " phenomenon, false negative patient values and l i n e a r i t y of va lues f r o m patient p l a s m a di lut ions ( I A E A - S M - 1 7 7 / 5 2 ) 45 E . W . J o e l , D . K . S c h 5 n b e r g , W . 11g and E . K e l l e r D i s c u s s i o n 57

P r e p a r a t i o n of human prote in h o r m o n e s for use i n r a d i o l i g a n d a s s a y s : s t r u c t u r e - f u n c t i o n considera t ions ( I A E A - S M - 1 7 7 / 2 0 9 ) . . . 59 L . E . R e i c h e r t , J r . D i s c u s s i o n 69

Standards and r e f e r e n c e reagents f o r r a d i o i m m u n o a s s a y of peptide h o r m o n e s and re la ted substances ( I A E A - S M - 1 7 7 / 2 0 5 ) . . . . 71 P . M a r y C o t e s D i s c u s s i o n 85

A S S A Y A U T O M A T I O N ; D A T A A N A L Y S I S . I M M U N O R A D I O M E T R I C A S S A Y S (Session II)

A u t o m a t i o n of r a d i o i m m u n o a s s a y and other Saturation assay p r o c e d u r e s ( I A E A - S M - 1 7 7 / 2 0 6 ) 91 R . P . E k i n s D i s c u s s i o n 108

C a l c u l a t i o n of the r a d i o i m m u n o a s s a y Standard c u r v e by " s p l i n e funct ion" ( I A E A - S M - 1 7 7 / 7 1 ) 111 I. M a r s c h n e r , F . E r h a r d t and P . C . S c r i b a D i s c u s s i o n 120

R a p i d assay for l u t e i n i z i n g h o r m o n e and evaluation of data by new Computer p r o g r a m ( I A E A - S M - 1 7 7 / 8 0 ) 123 J . S p o n a

New developments i n the i m m u n o r a d i o m e t r i c assay ( I A E A - S M - 1 7 7 / 2 0 4 ) 131 G . M . A d d i s o n D i s c u s s i o n 147

P r o p e r t i e s of two-s i te i m m u n o r a d i o m e t r i c ( labelled antibody) assay Systems ( I A E A - S M - 1 7 7 / 2 1 ) 149 L . E . M . M i l e s , C . P . B i e b e r , L . F . E n g and D . A . L i p s c h i t z D i s c u s s i o n 163

S ta t i s t i ca l a n a l y s i s of r a d i o i m m u n o a s s a y s and i m m u n o ­r a d i o m e t r i c ( labelled antibody) a s s a y s : a g e n e r a l i z e d , weighted, i t e r a t i v e , l e a s t - S q u a r e s method for l o g i s t i c c u r v e f i t t ing ( I A E A - S M - 1 7 7 / 2 0 8 ) 165 D . R o d b a r d and D . M . H ü t t D i s c u s s i o n 189

G L Y C O P R O T E I N H O R M O N E S (Session III)

R a d i o i m m u n o a s s a y s of g l y c o p r o t e i n h o r m o n e s ( I A E A - S M - 1 7 7 / 2 0 1 ) . . 195 P . F r a n c h i m o n t , J . C . H e n d r i k , A i m e e - M a r g u e r i t e R e u t e r and H . G . B u r g e r D i s c u s s i o n 210

Dosage r a d i o i m m u n o l o g i q u e de l ' h o r m o n e thyreotrope dans l 'hypophyse et dans le sang du ra t : 6tude des v a r i a t i o n s de concentrat ion sous r e f f e t de differents t ra i tements ( I A E A - S M - 1 7 7 / 7 ) 213 L i g i a S i m i o n e s c u , V . S ä h l e a n u , M a r i a O p r e s c u et V i c t o r i a O r b e ^ t e a n u D i s c u s s i o n 219

R a d i o i n m u n o a n a l i s i s de t i r o t r o f i n a h u m a n a : r e l a c i o n m a t e r n o - f e t a l en l a a l tura ( I A E A - S M - 1 7 7 / 4 0 ) 221 M . P a r e d e s - S u ä r e z y J . V a r e a T e r ä n

I m m u n o l o g i c a l c r o s s - r e a c t i o n s of L H and H C G : contr ibut ions of the subunits and of the conformat ion of the native h o r m o n e ( I A E A - S M - 1 7 7 / 5 8 ) 237 H . S . J a c o b s , C a r o l i n e V a n t h u y n e and R . P . E k i n s D i s c u s s i o n 242

I m m u n o l o g i c a l identity of antibodies against u r i n a r y and pi tui tary f o l l i c l e - s t i m u l a t i n g hormone ( I A E A - S M - 1 7 7 / 9 1 ) 245 V . O l i v i e r i , G . R i c c i and P . D o n i n i D i s c u s s i o n 250

C o m p a r a i s o n de dif ferentes m £ t h o d e s de dosage r a d i o i m m u n o ­logique des gonadotrophines ( I A E A - S M - 1 7 7 / 6 ) 253 A i m e e - M a r g u e r i t e R e u t e r , J . C . H e n d r i c k , H . B e c k e r et P . F r a n c h i m o n t D i s c u s s i o n 264

D i a g n o s t i c use of a r a d i o r e c e p t o r assay f o r human c h o r i o n i c gonadotropin ( I A E A - S M - 1 7 7 / 8 1 ) 267 M . T a l a s and A . R . M i d g l e y , J r .

R a d i o i m m u n o a s s a y of human e r y t h r o p o i e t i n ( I A E A - S M - 1 7 7 / 1 9 ) 275 J . F . G a r c i a D i s c u s s i o n 286

P E P T I D E H O R M O N E S (Sessions I V and V)

S t a n d a r d i z a t i o n of r a d i o i m m u n o a s s a y technique f o r d e t e r m i n a t i o n of p l a s m a i n s u l i n and growth hormone ( I A E A - S M - 1 7 7 / 8 4 ) 291 O l g a Z a z u c o H i g a , I r a c e l i a T o r r e s d e T o l e d o e S o u z a , B . L . W a j c h e n b e r g , H e i d i P i n t o and R . R i b e i r o P i e r o n i

R a d i o i m m u n o l o g i c a l behaviour of endogenous and exogenous h u m a n growth hormone ( I A E A - S M - 1 7 7 / 9 ) 309 K . v o n W e r d e r , C . D e s a l m , S. G a l l e n b e r g e r , M . G o t t s m a n n and P . C . S c r i b a D i s c u s s i o n 321

D e g r a d a t i o n of l a b e l l e d h o r m o n e i n r a d i o i m m u n o a s s a y of A C T H ( I A E A - S M - 1 7 7 / 3 7 ) 323 E . V i r a s o r o , G . C o p i n s c h i and O . D . B r u n o D i s c u s s i o n 335

D o s a g e r a d i o i m m u n o l o g i q u e de 1 'hormone para thyroidienne p l a s m a t i q u e chez l ' h o m m e ( I A E A - S M - 1 7 7 / 6 4 ) 337 J . G u e r i s

A r a d i o i m m u n o a s s a y for human s e r u m p a r a t h y r o i d h o r m o n e : methods and c l i n i c a l evaluation ( I A E A - S M - 1 7 7 / 8 2 ) 353 R . B o u i l l o n , P . K o n i n c k x and P . D e M o o r

Dosage r a d i o i m m u n o l o g i q u e de l a calc i tonine humaine ( I A E A - S M - 1 7 7 / 8 ) 367 G . H e y n e n , P . F r a n c h i m o n t et S. G a s p a r D i s c u s s i o n 380

D o s a g e r a d i o i m m u n o l o g i q u e de l a ca lc i tonine h u m a i n e : etude i m m u n o c h i m i q u e et c o m p a r a i s o n des resul ta ts avec ceux du dosage biologique ( I A E A - S M - 1 7 7 / 6 2 ) 381 M . S . M o u k h t a r , A . J u l l i e n n e , P . R i v a i l l e et G . M i l h a u d D i s c u s s i o n 397

Dosage r a d i o i m m u n o l o g i q u e de l a gastr ine — m i s e au point du dosage et resul ta ts c l in iques ( I A E A - S M - 1 7 7 / 6 5 ) 399 H . K a e s s et Br ig i t te M e r i a d e c D i s c u s s i o n 409

C o m p a r a t i v e evaluation of r a d i o i m m u n o a s s a y methods for h u m a n p r o l a c t i n u s i n g a n t i - o v i n e and a n t i - h u m a n p r o l a c t i n s e r a ( I A E A - S M - 1 7 7 / 8 7 ) 411 M . B a d a w i , S. B i l a , M . L ' H e r m i t e , F . R . P e r e z - L o p e z and C . R o b y n D i s c u s s i o n 421

R a d i o i m m u n o a s s a y of angiotensin II ( I A E A - S M - 1 7 7 / 2 3 ) 423 R . L . W o l f , C o r n e l i a E . G h e r m a n and M . M e n d l o w i t z D i s c u s s i o n 427

S i m p l i f i e d r a d i o i m m u n o a s s a y f o r p l a s m a angiotensin II ( I A E A - S M - 1 7 7 / 5 4 ) 429 J . N u s s b e r g e r , R . B e c k e r h o f f , W . V e t t e r , H . A r m b r u s t e r and W . S i e g e n t h a l e r

Dosage r a d i o i m m u n o l o g i q u e des Prostaglandines F a dans le p l a s m a ( I A E A - S M - 1 7 7 / 6 8 ) , 437 B . C h a r b o n n e l , A . S o u b r i e r et F . D r a y D i s c u s s i o n 450

I m m u n o l o g i c a l and b i o l o g i c a l s p e c i f i c i t y of a r a d i o i m m u n o a s s a y for Prostaglandins of the F group ( P G F ) ( I A E A - S M - 1 7 7 / 9 2 ) 451 C . P a t r o n o and G . C i a b a t t o n i D i s c u s s i o n 460

R a d i o i m m u n o a s s a y of the hypothalamic r e l e a s i n g h o r m o n e s , L H - R H and T R H , i n blood and u r i n e ( I A E A - S M - 1 7 7 / 2 4 ) 463 S . L . J e f f c o a t e , H . M . F r ä s e r , A . G u n n , D . T . H o l l a n d and N . W h i t e D i s c u s s i o n 467

C h a i r m e n of Sess ions and S e c r e t a r i a t of the S y m p o s i u m 471

IAEA-SM-177/9

RA DI Ol M MU NO LOGI CA L BEHAVIOUR OF ENDOGENOUS AND EXOGENOUS HUMAN GROWTH HORMONE*

K. von WERDER, C . DESALM, S. GALLENBERGER, M . GOTTSMANN, P .C. SCRIBA II. Medizinische Klinik der Universität München, Munich, Federal Republic of Germany

Abstract

RADIOIMMUNOLOGICAL BEHAVIOUR OF ENDOGENOUS AND EXOGENOUS HUMAN GROWTH HORMONE. The immunoreactivity of endogenous human growth hormone (HGH) and of exogenous HGH after

infusion into patients has been investigated. Four different HGH preparations gave identical Standard curves documenting immunoidentity. Complete immunoidentity could not be demonstrated for "big" HGH which was separated from regulär HGH by dextran gel filtration. The HGH immunoreactivity. which was eluted from the column ahead of "big" HGH, showed an even more pronounced change in immunoreactivity. Exogenous and endogenous human placental lactogen (HPL) showed the expected deviation from the HGH Standard curve, whereas bovine prolactin (BPr) and high endogenous immunoassayable HPr (3200 ng/ml) did not cross-react with the HGH antibody. Immunoidentity of HGH with the Standards was documented in 10 patients with acromegaly. This was true for samples from the jugular vein as well as from the periphery. Eight insulin tolerance tests (ITT) were performed in patients with normal HGH secretion. Samples at 30, 45, 60 and 90 min were diluted and the resulting slopes compared with the Standard curve. Whereas the slopes of the 30-, 45- and 60-min samples were parallel, the slopes of the 90-min samples deviated from the Standard curve. Exogenous HGH was infiised (2 mg for 30 min for each person) into five normal subjects (I) and five patients with HGH deficiency (II). HGH levels rose to 171. 6 ± 19. 8 ng/ml (I) and 192.4 ± 34. 8 ng/ml (II). HGH half-time of disappearance was 14.9 ± 3.9 min (I) and 19.1 ±6 min (II). There was no change of HGH immunoreactivity over a period of 2 hours.

In summary: (1) Cross-reactivity of HPL but not of HPr with HGH antibodies was demonstrated. (2) HGH in acromegaly and exogenous HGH after infusion have identical immunoreactivity. (3) The change in endogenous HGH immunoreactivity 90 min after Stimulation is apparently due to the secretion of a different HGH immunoreactivity that is most likely residing in a larger molecular weight fraction.

R a d i o i m m u n o a s s a y has become the method of choice for m e a s u r i n g peptide h o r m o n e s and other substances in b i o l o g i c a l f l u i d s s ince the development of the f i r s t r a d i o i m m u n o l o g i c a l d e t e r m i n a t i o n for i n s u l i n by Y a l o w and B e r s o n [ 1 ] . One of the c r i t e r i a f o r the v a l i d i t y of r a d i o i m m u n o ­a s s a y s i s the ident i ty of the i m m u n o r e a c t i v i t y of the Standard p r e p a r a t i o n and the unknown substance w h i c h i s m e a s u r e d i n the body f l u i d [2 , 3 ] . In the case of i m m u n o i d e n t i t y , the Standard curve and the d i l u t i o n c u r v e of the unknown w i l l r u n p a r a l l e l . A dev ia t ion of the d i l u t i o n curve f r o m the Standard c u r v e is e i ther due to a di f ferent antigen w i t h s i m i l a r , but not i d e n t i c a l , i m m u n o r e a c t i v i t y o r to c a t a b o l i c p r o c e s s e s in the body f l u i d changing the i m m u n o d e t e r m i n a n t s i te of the o r i g i n a l ant igen .

H u m a n g r o w t h h o r m o n e i s a Single-chain peptide w i t h a m o l e c u l a r weight of 21 500 [ 4 ] . It has been shown by s e v e r a l i n v e s t i g a t o r s that H G H -i m m u n o r e a c t i v i t y i s a l s o found i n a l a r g e r m o l e c u l a r weight f r a c t i o n i f p i t u i t a r y - o r s e r u m - H G H i s subjected to dext ran g e l f i l t r a t i o n [5 , 6, 7 ] . The ex i s tence of " b i g " h o r m o n e s , as have been d e m o n s t r a t e d for i n s u l i n [ 8 ] , p a r a t h y r o i d hormone [ 9 ] , g a s t r i n [ 1 0 ] , A C T H [11] and HGH [5 , 6, 7 ] , i s

* Supported by DFG/SFB 51.

309

310 Von WERDER et al.

not f u l l y e x p l a i n e d . T h e y can r e p r e s e n t p r o h o r m o n e s as has been shown f o r i n s u l i n [ 8 ] , h o r m o n e s bound covalent ly to prote ins [11] o r aggregated hormone c o m p l e x e s . S ince p r o h o r m o n e s should have a di f ferent t e r t i a r y s t r u c t u r e because they contain a d d i t i o n a l amino ac ids a d i f ference in i m m u n o r e a c t i v i t y c o m p a r e d wi th the f i n a l hormone could be encountered. The amino a c i d sequence and the i m m u n o r e a c t i v i t y of H G H i s different f r o m the other p i t u i t a r y h o r m o n e s except that human p r o l a c t i n ( H P r ) seems to have a s i m i l a r s t r u c t u r e [ 12] . E v e n m o r e r e l a t e d in b i o l o g i c a l a c t i v i t y , amino a c i d sequence and i m m u n o r e a c t i v i t y i s human p l a c e n t a l lactogen ( H P L ) [ 1 3 ] . T h u s , c r o s s - r e a c t i v i t y of H P L with H G H ant ibodies has been found [14, 1 5 ] .

T h e f o l l o w i n g study was des igned to invest igate the i m m u n o r e a c t i v i t y of v a r i o u s p i t u i t a r y H G H preparations before and af ter Separation into " b i g " and regulär H G H . T h e i m m u n o r e a c t i v i t y was i n v e s t i g a t e d by c o m p a r i n g the s lope of the r e s p e c t i v e d i l u t i o n c u r v e s w i t h the Standard c u r v e of a r e f e r e n c e p r e p a r a t i o n . T h i s method was a l so a p p l i e d to study the i m m u n o r e a c t i v i t y of endogenous s e r u m H G H . H G H of patients wi th a c r o m e g a l y as w e l l as endogenous H G H af ter m a x i m a l Stimulation i n patients w i t h n o r m a l pituitary funct ion w e r e i n v e s t i g a t e d . In addi t ion to the endogenous H G H exogenous H G H a f te r i n f u s i o n into human subjects was i n v e s t i g a t e d . T o determine the degree of c r o s s - r e a c t i v i t y of H P L and H P r wi th o u r H G H antibody, d i l u t i o n c u r v e s of endogenous and exogenous H P L and endogenous H P r w e r e c o m p a r e d wi th the H G H Standard c u r v e .

M A T E R I A L S A N D M E T H O D S

H G H was m e a s u r e d by a semiautomated double antibody r a d i o i m m u n o ­assay [ 1 6 ] . T h r e e d i f fe rent H G H p r e p a r a t i o n s w e r e used: Roos H G H [17] conta in ing 2 I U / m g p r o t e i n ( K a b i , Sweden, Lot N o . 19156), W i l h e l m i H G H (Societa R i c e r c h e I m p i a n t i N u c l e a r i , S a l u g g i a , Italy) and Raben H G H conta in ing 2 I U / m g p r o t e i n . A s r e f e r e n c e p r e p a r a t i o n , h ighly p u r i f i e d W i l h e l m i H G H f r o m the N a t i o n a l Institute of H e a l t h (batch N I H - G H - H S / 3 9 4 ) was u s e d . H G H Standards as w e l l as the s e r u m samples were di luted w i t h 0 . 1 3 M b o r a t e buf fer , pH 8 .4 , conta ining 0 .5% bovine a l b u m i n . It could be d e m o n s t r a t e d that the r e c o v e r y of exogenous H G H added to H G H - f r e e s e r u m was not s i g n i f i c a n t l y i n f l u e n c e d by a d i lu t ion up to 1:200 w i t h the assay b u f f e r . The graphs of the d i l u t i o n c u r v e s were made i n the fo l lowing way : the B / B 0 r a t i o of the highest s e r u m d i l u t i o n was used to d e t e r m i n e the H G H c o n c e n t r a t i o n . The H G H concentra t ion of this s a m p l e was r e a d f r o m the steepest and most accurate part of the Standard curve between 0.5 and 1.5 ng and was r e g a r d e d as the r e f e r e n c e c o n c e n t r a t i o n . F r o m th is r e f e r e n c e concent ra t ion the concentrat ions of the other d i lut ions w e r e ca lcu la ted and the ac tua l B / B 0 r a t i o was m a r k e d i n the c o - o r d i n a t e System. The r e s u l t i n g d i l u t i o n c u r v e s as w e l l as the Standard c u r v e s w e r e log i t t r a n s f o r m e d a c c o r d i n g to R o d b a r d [18] w h i c h made the Standard c u r v e l i n e a r f r o m 1 to 10 n g / m l .

I n s u l i n h y p o g l y c a e m i a tes ts w e r e p e r f o r m e d a c c o r d i n g to Greenwood et a l . [ 1 9 ] . T h e d iagnos is of act ive a c r o m e g a l y was c o n f i r m e d by glucose n o n - s u p p r e s s a b l e e levated H G H l e v e l s [ 2 0 ] . D e x t r a n ge l f i l t r a t i o n was p e r f o r m e d on a 3 c m X 110 c m c o l u m n of Sephadex G - 7 5 . The buffer used f o r the g e l f i l t r a t i o n and in the r a d i o i m m u n o a s s a y of the eluate was 0. 0 5 M

IAEA-SM-177/9 311

s o d i u m phosphate buffer wi th 0 . 1 % s o d i u m a z i d e , p H 7 . 5 . F o r r a d i o ­i m m u n o a s s a y 0 .5% bovine a l b u m i n was added to th is b u f f e r . The c o l u m n was sa tura ted wi th 5 m l of 5% a l b u m i n before c h r o m a t o g r a p h y . 1 2 5 I - H G H was l a b e l l e d a c c o r d i n g to Hunter and G r e e n w o o d [ 21] and p r e p u r i f i e d on a Sephadex G - 5 0 c o l u m n . The pooled p r o t e i n peak was then e luted on the G - 7 5 - c o l u m n and f r a c t i o n s of 10 m l were c o l l e c t e d . The r a d i o a c t i v e m a t e r i a l e l u t i n g at K D 0 . 8 (peak III) was used as t r a c e r f o r H G H r a d i o i m m u n o a s s a y . F o r t y jug of c l i n i c a l - g r a d e p i t u i t a r y H G H (Roos) and 2 m l of s e r u m f r o m an a c r o m e g a l i c patient w e r e used f o r Sephadex c h r o m a t o g r a p h y . F o r the r a d i o ­i m m u n o a s s a y of the c o l u m n eluate two di f ferent H G H ant ibodies — goat a n t i - H G H and g u i n e a - p i g a n t i - H G H s e r u m — were u s e d . T h e r e c o v e r y of the l a b e l l e d H G H ranged f r o m 90 to 100%, that of the n o n - l a b e l l e d H G H f r o m 80 to 90%.

T h e H G H used f o r i n f u s i o n was the same Roos p r e p a r a t i o n as that used for c h r o m a t o g r a p h y ( G e r m a n K a b i , L t d . , M u n i c h ) . 2 m g of t h i s p r e p a r a t i o n i n 250 m l of sa l ine w e r e in fused for 30 m i n into f ive subjects w i t h n o r m a l H G H s e c r e t i o n and f i v e patients w i t h H G H d e f i c i e n c y . B l o o d s a m p l e s f o r r a d i o i m m u n o a s s a y w e r e taken unt i l 3 hours a f ter the s tar t of the i n f u s i o n . The b i o l o g i c a l a c t i v i t y of the H G H was v e r i f i e d by m e a s u r i n g the i n c r e a s e i n c o n c e n t r a t i o n of f r e e fatty a c i d s i n s e r u m and by i t s effect upon g l u c o s e -s t i m u l a t e d i n s u l i n s e c r e t i o n [ 22] .

R E S U L T S

T h e d i l u t i o n c u r v e s (= Standard curves) of the three H G H p r e p a r a t i o n s and the N I H r e f e r e n c e H G H were shown to be i d e n t i c a l w h i c h demonstra tes i m m u n o i d e n t i t y of the Standard p r e p a r a t i o n s . The d i l u t i o n c u r v e of the T S H p r e p a r a t i o n (NIH, b i o l o g i c a l a c t i v i t y 3 .5 I U / m g prote in) shows an i n h i b i t i o n p a r a l l e l to the H G H Standard c u r v e . T h i s i s due to contaminat ion of the T S H wi th H G H ( F i g . 1). F r o m the T S H i n h i b i t i o n c u r v e i t can be deducted that 100 nU of the T S H contain 1 ng of H G H . T h e r e i s p a r t i a l

B / B o - i007.

95 r

90

HGH

HPL

10 n g / m l or jjU/ml

25 5 10 20 100 200 500 1000 5 000 10 000 20000 25000

FIG. 1. HGH Standard curve and inhibition curves of TSH, LH BPr and HPL with the HGH antibody.

312 Von WERDER et al.

B/ B o-100(7.)

95,

90

80

70

60

50

40

30

20

2.5

ng/ml HGH

20 10

FIG. 2. Dilution curve of a serum containing 5. 6 ng HGH/ml and 3200 ng HPr/ml (o) showing parallelism to the HGH Standard curve (•). Deviating dilution curves of eight serum samples from pregnant women ( f ± SE).

•100(%)

95h

90 h

70" 60-50-40-30-20-

10-

5- 25

ng/ml HGH

10 15 20

FIG. 3. HGH Standard curve (o patients (± SE).

•o). The points ( •) result from serum dilutions of ten acromegalic

IAEA-SM-177/9 313

HGH ng/ml B / ß o - 100(7, ] 7 0 f 9 0 r

FIG. 4. Left: HGH response to insulin hypoglycaemia in eight subjects (• • ). Right: dilution curve of all 30-, 45- and 60-min samples •*) and the 90-min samples (•— — —•) compared with the Standard curve ( ) (± SE).

c r o s s - r e a c t i v i t y w i t h H P L and no c r o s s - r e a c t i v i t y w i t h L H (3. 7 N I H - L H - S j / m g protein) and bovine p r o l a c t i n ( B P r ) . C r o s s - r e a c t i v i t y of endogenous H P L wi th the H G H antibody was d e m o n s t r a t e d by the devia t ion of the d i l u t i o n c u r v e s of eight s e r u m s a m p l e s f r o m pregnant women w i t h a mean H P L concentra t ion of 12.3 ± 2 .3 jug/ml ± SD ( F i g . 2). The s e r u m of a patient w i t h a p r o l a c t i n - p r o d u c i n g adenoma conta ining 5 .6 ng H G H / m l and high endogenous i m m u n o a s s a y a b l e human p r o l a c t i n (3200 ng/ml) showed no deviat ion f r o m the Standard c u r v e ( f ig . 2). The d i l u t i o n c u r v e s of 10 s e r u m s a m p l e s f r o m patients wi th ac t ive a c r o m e g a l y a lso d id not deviate f r o m the s lope of the Standard d i lu t ions ( F i g . 3). The H G H concentra t ion ranged f r o m 14 n g / m l to 200 n g / m l . In three patients s a m p l e s w e r e a l s o taken f r o m the j u g u l a r v e i n where the H G H c o n c e n t r a t i o n was 1.1 to 2 .4 t i m e s h i g h e r than i n the c u b i t a l v e i n . These findings demonstrate i m m u n o i d e n t i t y between H G H f r o m a c r o m e g a l i c s ' s e r u m and H G H Standards.

T h e H G H r e s p o n s e to i n s u l i n - i n d u c e d h y p o g l y c a e m i a of eight non-obese subjects w i t h n o r m a l p i t u i t a r y function i s depicted i n the lef t part of F i g . 4 . These i n s u l i n h y p o g l y c a e m i a tes ts w e r e used for o u r d i l u t i o n studies s ince they showed a r a t h e r h igh r i s e of H G H c o m p a r e d wi th that of 54 subjects who had a m e a n m a x i m a l H G H r i s e to 39. 9 ± 3. 0 n g / m l ± SE r a n g i n g f r o m 16.5 to 92 .0 n g / m l . The s e r u m s a m p l e s of the eight subjects w e r e di luted and the r e s u l t i n g d i l u t i o n c u r v e s of the 30- 45- and 6 0 - m i n s a m p l e s were shown to be p a r a l l e l to the Standard c u r v e . Only the d i l u t i o n c u r v e s of the 9 0 - m i n s a m p l e deviated s i g n i f i c a n t l y f r o m the Standard c u r v e and the other d i l u t i o n c u r v e s ( F i g . 4).

D e x t r a n ge l f i l t r a t i o n of l a b e l l e d growth hormone r e v e a l e d three r a d i o -act ive peaks ( F i g . 5). The f i r s t peak bound only p o o r l y to the ant ibody. A second peak eluted at K D 0 .45 and the t h i r d and l a r g e s t r a d i o a c t i v e peak eluted at K D 0. 8, r e p r e s e n t i n g the l a b e l l e d r e g u l ä r H G H . D e x t r a n gel

314 Von WERDER et al.

0.0 O.S 1.0 1.5 2.0 2.S

RECHROMATOGRAPHY

"BIGM-HGH

0.0 u.S 1.0 1.S 2.0 2.5 K0

RECHROMATOGRAPHY

"LITTLE" HGH

0.0 O.S 1.0 1.S 2.0 2.5 K0

FIG. 5. Chromatography of 1 2 5 I - H G H on Sephadex G-75 (top). Three distinct peaks can be seen, the first eluting at K D 0.18 (peak I), the second eluting at K D 0.45 (peak II) and the third eluting at Kp 0. 8 (peak III) representing regulär HGH, used as tracer for radioimmunoassay. Pituitary HGH (middle) and acromegalic serum HGH (bottom) elutes in a similar pattern. The immunoreactivity in the area under peak I is small in relation to the radioactivity, but was found consistently.

IAEA-SM-177/9 315

316 Von WERDER et al.

I.PEAK II. PEAK ID.PEAK

HGH calcuiated (ng/ml)

FIG. 7. Discrepancy of measured and calcuiated concentration of HGH immunoreactivity of peak I and peak II. Peak III shows good agreement between measured and calcuiated results. Two different antibodies were used: goat anti-HGH (•) and guinea-pig anti-HGH (•).

2mg HGH 2mg HGH

t (min) t(min)

FIG. 8. HGH levels in subjects with normal pituitary function (o o) and patients with HGH deficiency (• •) after infusion of 2 mg of pituitary HGH.

IAEA-SM-177/9 317

5-15 minutes after HGH- infusion

20-30 minutes after HGH-infusion

45-95 minutes after HGH-infusion

E 6 ~ai c

4» E c 3 o

~ 2

§ 0

! i

J / ' . y

°o */\

0 /

OO/

Jr A

/ i

• A

/

•

• • C ,

}

0

j V OO '

*' a

o

a

f 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5

HGH concentration calcuiated (ng/ml)

FIG. 9. Good agreement between measured and calcuiated HGH concentrations in the different dilutions of serum samples taken after infusion with 2 mg of pituitary HGH in subjects with and without endogenous HGH.

f i l t r a t i o n of c l i n i c a l - g r a d e p i t u i t a r y growth h o r m o n e and endogenous H G H f r o m a patient w i t h a c r o m e g a l y and e x t r e m e l y e levated H G H l e v e l s (3100 ng/ml) a l s o showed t h r e e peaks ( F i g . 5). The homogenei ty of i m m u n o -r e a c t i v e f r a c t i o n s obtained under peak II and peak III was c o n f i r m e d by r e c h r o m a t o g r a p h y ( F i g . 6). If the d i l u t i o n s of the f r a c t i o n s conta in ing peak I E w e r e a n a l y s e d for t h e i r H G H c o n c e n t r a t i o n the m e a s u r e d H G H concen­t r a t i o n was found to be i d e n t i c a l w i t h the c a l c u i a t e d H G H content ( F i g . 7). The d i l u t i o n c u r v e of the i m m u n o r e a c t i v i t y of peak II showed a devia t ion f r o m the Standard d i l u t i o n c u r v e r e s u l t i n g i n d i s c r e p a n c y between m e a s u r e d and c a l c u i a t e d H G H concent ra t ions i n the di f ferent d i l u t i o n s . T h i s was even m o r e pronounced for the i m m u n o r e a c t i v i t y of peak I. The i m m u n o r e a c t i v i t y of the l a r g e r m o l e c u l a r weight f r a c t i o n s was d e m o n s t r a t e d to be dif ferent f r o m that of the H G H Standard w i t h two di f ferent ant ibodies ( F i g . 7).

The i n f u s i o n of 2 m g of H G H into pat ients w i t h r e g u l ä r H G H s e c r e t i o n r e s u l t e d i n a H G H r i s e of 1 71. 6 ± 19.8 n g / m l . The H G H l e v e l i n patients wi th endogenous H G H d e f i c i e n c y r o s e a f ter i n f u s i o n to 192.4 ± 34.8 n g / m l ( F i g . 8) . T h e d i f f e rence in h a l f - t i m e of the d i sappearance f r o m the s e r u m of the H G H i m m u n o r e a c t i v i t y between the two groups was not s i g n i f i c a n t : 14 .9 ± 3 .9 m i n in subjects w i t h n o r m a l H G H s e c r e t i o n and 19.1 ± 6 m i n in patients without endogenous H G H s e c r e t i o n . The d i l u t i o n c u r v e s of the s e r u m s a m p l e s of both groups taken u n t i l 2 h o u r s af ter the s tar t of the i n f u s i o n w e r e p a r a l l e l to the d i l u t i o n of the in fused H G H Standard. T h e r e was t h e r e f o r e no d i f f e r e n c e between m e a s u r e d and c a l c u i a t e d H G H concen­t r a t i o n i n the s e r u m d i l u t i o n s ( F i g . 9).

D I S C U S S I O N

S p e c i f i c i t y is e s s e n t i a l f o r r a d i o i m m u n o a s s a y of h o r m o n e s . It r e l i e s on an ant igenic determinant p a r t — i n the case of peptide h o r m o n e s a c e r t a i n

CO

CO

I-HOH-Peafc 1 ^I-HOH-Ptfak H 125IZHGH-Peak_m

HGH l n 9 ' m l ) HGH ( n 9 , m , ) HGH { n * ' m "

FIG. 10. Standard curves of "big-big" (• •), "big" (o o) and "little" ( a £ ) HGH with different 1 2 5 I - H G H tracers. Only "big-big" HGH inhibits 1 2 5 I-HGH-peak I binding to the antibody. The inhibition curves of the different HGH fractions are superimposable only when 1 2 5 I-HGH-peak III, i .e . the purified labelled "little" HGH, is used.

IAEA-SM-177/9 319

amino acid sequence — which i s unique for the hormone to be measured. The structure of H G H has been revised r e c e n t l y [4] . A c c o r d i n g to L i et al . it is a Single-chain peptide with 190 amino acids, two disulphide bridges and a molecular weight of 21 500. B o v i n e and human prolactin ( B P r , H P r ) seem to be very similar to H G H [ 12 ] . It is therefore surprising that H G H anti­bodies do not cross-react with B P r and H P r . The fact that there i s no cross-reactivity i s proven by the failure of B P r to inhibit 1 2 5 I - H G H binding to the antibody and by identity of the slope of the serum dilution curve prepared with H G H in the presence of a high concentration of H P r with the slope of the H G H Standard curve. If the H P r were to react with the H G H antibody the serum dilution would deviate from the Standard slope and the measurement of H G H would not be possible since one of the basic criteria for the validity of radioimmunoassays — identity of the dilution curves of Standard and unknown — i s not fulfilled.

H P L i s a peptide hormone with 190 amino acids, two disulphide b r i d g e s and a molecular weight of 21 000 [ 13] . S ince 160 of the 190 amino-acid residues of H P L occupy positions that are i d e n t i c a l to those in H G H it i s not surprising that both hormones have similar ant igenic determinants. B e c a u s e of the resulting cross-reactivity H G H measurements during pregnancy will remain difficult. T h i s has been demonstrated before and is again pointed out by our results which show inhibition of 1 2 5 I - H G H binding to the H G H anti­body by H P L Standards ( F i g . l ) and non-parallelism of the H G H Standard curves and the serum dilution curves for pregnant women. S ince the other pituitary hormones have no structural similarity to H G H , cross-reactivity of these hormones with the H G H antibody should not occur. The displacement of 1 2 5 I - H G H by the T S H Standard must be due to contamination of this preparation, as is demonstrated by the parallel TSH-inhibition curve ( F i g . l ) and the absence of interference from elevated endogenous T S H in the H G H immunoassay (unpublished data). A c c o r d i n g to these findings the only material in non-pregnant women that reacts with the H G H antibody should be H G H . U n l e s s the H G H i s a l t e r e d , dilution curves of serum containing H G H and Standards should be parallel. T h e immunoidentity of four different H G H preparations was documented in th i s manner. In contrast to Greenwood et al . [ 1 4 ] , who described one acromegalic patient with circulating H G H immunologically different from the Standard preparation, we found identical H G H immunoreactivity in all our patients ( F i g . 3). When subjects with normal H G H secretion were investigated we found that individuals with very pronounced H G H responses to insulin hypoglycaemia showed a change in immunoreactivity 90 min after insulin was given ( F i g . 4 ) . L a n d o n et a l . [23] also found a change in A C T H immunoreactivity after maximal Stimulation of its secretion. These findings could be explained by two different mechanisms:

1. C a t a b o l i s m of the circulating antigenic peptide gives r i s e to fragments of the antigen which only have partial immunoreactivity.

2. A f t e r maximal Stimulation the pituitary secretes precursor hormones which differ immunologically from the original hormone.

H e t e r o g e n e i t y in molecular size of peptide hormones has been described [ 7-11 ] . H G H immunoreactivity has been shown to be present in various molecular fractions [5 , 6, 7, 2 4 ] . O u r r e s u l t s demonstrating H G H immuno­reactivity eluting in a less retarded peak from the Sephadex column are in

320 Von WERDER et al.

good agreement wi th these of Görden et a l . [ 6J and G o o d m a n et a l . [ 7 j , who found the l e s s r e t a r d e d " b i g " H G H (peak II, F i g . 5) i n p i t u i t a r y H G H , in s e r u m of a c r o m e g a l i c s and in s e r u m of subjects wi th n o r m a l H G H s e c r e t i o n . They a l so found i m m u n o r e a c t i v i t y be ing e luted before " b i g " H G H i n some of t h e i r s a m p l e s . In our studies s igni f i cant amounts of i m m u n o r e a c t i v e m a t e r i a l w e r e a lways e luted just ahead of a l b u m i n (peak I, F i g . 5), w h i c h has a l so been demonst ra ted by B e r s o n and Y a l o w [ 24 ] . A c c o r d i n g to Goodman et a l . [7] " b i g " and regulär H G H have i d e n t i c a l i m m u n o r e a c t i v i t y [ 7] . B y us ing the r e c e p t o r a s s a y G o r d o n et a l . [6] showed i n p r e l i m i n a r y s tudies a l o w e r af f in i ty to the r e c e p t o r of " b i g " c o m p a r e d w i t h r e g u l ä r H G H . O u r studies demonstra te w i t h two di f ferent a n t i s e r a to H G H that " b i g " (peak II) and m o r e pronounced " b i g - b i g " (peak I) reac t d i f f e r e n t l y w i t h the H G H - a n t i b o d i e s , c o m p a r e d to r e g u l ä r H G H (peak III). S ince the d i f f e r e n c e i n i m m u n o r e a c t i v i t y between " b i g " and r e g u l ä r H G H i s not so obvious and w i l l d i f f e r depending on the antibody used, t h i s may e x p l a i n the d i s c r e p a n c y between our r e s u l t s and those of Goodman et a l . The i m m u n o r e a c t i v i t y of " b i g - b i g " H G H has not been inves t iga ted by these a u t h o r s . Of i n t e r e s t i s the f inding that s e r u m of a c r o m e g a l i c s contain l e s s " b i g " H G H than s e r u m of patients a f ter i n s u l i n h y p o g l y c a e m i a [ 6 ] , One could a s s u m e , t h e r e f o r e , that the change in H G H i m m u n o r e a c t i v i t y du r i n g i n s u l i n h y p o g l y c a e m i a i s due to the i n c r e a s e of " b i g " and, maybe , " b i g - b i g " H G H i n the s e r u m . " B i g " f o r m s of H G H a r e able to cause better i n h i b i t i o n of 1 2 5 I - H G H peak II than " l i t t l e " H G H w h i c h causes a change i n the s lope of the i n h i b i t i o n c u r v e ( F i g . 10). The c o n v e r s i o n of in fused H G H into l a r g e r i m m u n o r e a c t i v e m o l e -cu les i n the c i r c u l a t i o n i s i m p r o b a b l e s ince no i m m u n o l o g i c a l changes i n c i r c u l a t i n g H G H can be o b s e r v e d o v e r a p e r i o d of 2 h o u r s a f t e r i n f u s i o n ( F i g . 9 ) .

R E F E R E N C E S

[1] YALOW. R.S. . BERSON. S .A. . J. Clin. Invest. 39 (1960) 1157.

[2] BERSON, S . A . , YALOW. R.S. . Clinical Endocrinology II (ASTWOOD, E .B . , CASSIDY. C. E. , Eds). Grüne and Stratton, New York (1968) 699.

[3] BERSON. S .A. , YALOW, R.S. . in Protein and Polypeptide Hormones (MARGOULIES, M . . GREENWOOD, F. C. . Eds), Int. Congr. Series No. 241, Excerpta Medica Foundation, Amsterdam (1971) 38.

[4] LI, C . H . . DIXON, J.S. , Arch. Biochem. Biophys. 146 (1971) 233. [5] BALA, R . M . . FERGUSON. K . A . . BECK. J . C , Endocrinology 87 (1970) 506. [6] GÖRDEN, P. , HENDRICKS, C M . , ROTH, J. , J. Clin. Endocrinol. Metab. 36 (1973) 178. [7] GOODMAN, A . D . , TANENBAUM, R. , RABINOWITZ, D. , J. Clin. Endocrinol. Metab. 35 (1972) 868. [8] ROTH, J. , GORDON. P. . PASTAN, J. , Proc. Natl. Acad. Sei. U.S . 61 (1968) 138. [9] BERSON, S.A. , YALOW, R.S. . Am. J. Med. 50 (1971) 623.

[10] YALOW, R.S. , BERSON, S . A . , Gastroenterology 60 (1971) 203. [11] YALOW, R.S. , BERSON. S . A . , J. Clin. Endocrinol. Metab. 36 (1973) 415. [12] FRIESEN, H. , et al. . in Growth and Growth Hormone (PECILE, A. . MÜLLER, E. E. . Eds), Int. Congr.

Series No. 244, Excerpta Medica Foundation, Amsterdam (1972) 244. [13] LI, C . H . , DIXON, J.S. , CHUNG, D. , Science 173 (1971) 56. [14] GREENWOOD, F. C. , HUNTER, W . M . , KLOPPER, A. . Br. Med. J. 1 (1964) 22. [15] VARMA, S.K. , et al. , J. Clin. Endocrinol. Metab. 32 (1971) 328. [16] BOTTERMANN, P. , ERMLER, R. , HENNER, J. , Horm. Met. Res. 3 (1971) 55. [17] ROOS, P., FEVOLD, H.R. , GEMZELL. C A . , Biochim. Biophys. Acta 74 (1963) 525. [18] RODBARD, D. . RAYFRORD, P.L. , ROSS, G . T . , in Statistics in Endocrinology (McARTHUR, J.W. .

COLTAR, Th. , Eds), The MIT-Press, Boston (1970). [19] GREENWOOD, F. C. , LANDON. J. , STAMP, T . C . B . , J. Clin. Invest. 45 (1966) 429.

IAEA-SM-177/9 321

[20] VON WERDER. K. . BOTTERMANN. P. . HARTMANN. P. . SCHWARZ, K. . Med. Klin. (Munich) 67 (1972) 398.

[21] GREENWOOD, F. C. . HUNTER. W . M . . GLOVER, J. S. , Biochem. J. 89 (1963) 114. [22] VON WERDER. K. , et al . . Acta Endocrinol. , Suppl. 173 (1973) 99. [23] LANDON. J. . GIRARD, J. , GREENWOOD, F. C. . in Protein and Polypeptide Hormones (MARGOUUES. M . ,

Ed.). Int. Congr. Series 161, Excerpta Medica Foundation, Amsterdam (1968) 29. [24] BERSON. S. A. , YALOW , R.S. , Proceedings of the XI Reunion of French-Speaking Endocrinologists,

Paris. Masson et Cie (1971) pp 105-135.

D I S C U S S I O N

R o s a l y n S. Y A L O W : Y o u r l a r g e r growth hormone (GH) components s e e m to be m u c h m o r e stable than the " b i g " G H of other w o r k e r s . A r e there any p a r t i c u l a r methods which help i n m a i n t a i n i n g the s t a b i l i t y of the " b i g " f o r m ?

F r o h m a n and F r i e s e n c a r r i e d out independent b iosynthet i c s tudies to e s t a b l i s h the p r e c u r s o r nature of the two l a r g e r h o r m o n a l f o r m s , and a l so noted a u s u a l l y r a p i d appearance of l a b e l l e d amino ac ids i n the " l i t t l e " f o r m , again s u g g e s t i n g l a c k of s t a b i l i t y of the " b i g g e r " f o r m s , as c o m p a r e d to the " b i g g e r " f o r m s of other h o r m o n e s , s u c h as i n s u l i n or g a s t r i n .

T h e q u e s t i o n of s t a b i l i t y i s i m p o r t a n t in c o n s i d e r i n g whether the " b i g g e r " f o r m s r e s u l t f r o m covalent bonding.

K . v o n W E R D E R : We have, i n fact , seen v e r y l i t t l e c o n v e r s i o n of " b i g " into " l i t t l e " H G H , even af ter s torage of the " b i g " H G H p r e p a r a t i o n f o r four w e e k s . T h e s e r e s u l t s a r e , h o w e v e r , i n contras t to the f indings of Goodman et a l . 1 and Görden et a l . 2 . O u r methodology d i f f e r s f r o m t h e i r s i n that we use 0. 05 M phosphate buffer f o r e lu t ion , and per f o r m our g e l f i l t r a t i o n at 24°C.

S ince G o o d m a n et a l . r e c e n t l y r e p o r t e d at Chicago that there i s a l a b i l e " b i g " H G H , w h i c h i s conver t ib le into " l i t t l e " H G H by 6 M u r e a , as w e l l as b y f r e e z i n g and thawing the " b i g " H G H p r e p a r a t i o n , and that there i s i n a d d i t i o n a stable f o r m of the " b i g " h o r m o n e w h i c h i s not so c o n v e r t e d , i t might be that we a r e dea l ing m a i n l y w i t h the l a t t e r . O u r s e r u m s a m p l e s w e r e f r o z e n and thawed at leas t once and so m o s t of the c o n v e r t i b l e " b i g " H G H may have been c o n v e r t e d . The " b i g " H G H r e m a i n i n g was there fore i n the s table f o r m . We b e l i e v e , as suggested by G o o d m a n , that we are d e a l i n g w i t h two f o r m s of " b i g " H G H , one of w h i c h i s not cova lent ly bound to another p r o t e i n , w h i l e the other i s . The l a t t e r i s the stable f o r m and, i n our o p i n i o n , a t r u e p r e c u r s o r h o r m o n e .

1 See Ref. [7] of the paper. 2 See Ref. [6] of the paper.