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Cytological changes in the endocrine glands of alloxanized hypertensive diabetic rats Radford, Hugh William 1951

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CYTOLOGICAL CHANGES IN THE ENDOCRINE GLANDS OF ALLOXANIZED HYPERTENSIVE DIABETIC RATS BY HUGH WILLIAM RADFORD, B.A. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of BIOLOGY AND BOTANY We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree of MASTER OF ARTS Members o f the Department of B i o l o g y THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1951. ( i l l A b s t r a c t . Rats made d i a b e t i c w i t h a l l o x a n and f e d h i g h p r o t e i n and s a l t d i e t s showed h y p e r a c t i v i t y o f the p i t u i t a r y - a d r e n a l systems, as i n d i c a t e d by a d r e n a l hypertrophy, d e p l e t i o n o f ad r e n a l c h o l e s t e r o l and i n c r e a s e d p i t u i t a r y a c i d o p h i l s . T y p i c a l s i g n s "of h y p e r t e n s i o n such as a r t e r i o s c l e r o s i s , n e p h r o s c l e r o s i s , f a t t y degeneration o f the l i v e r and hemorrhages were common. I n s u l i n therapy and omission o f p r o t e i n from the d i e t d i d not l e s s e n the extent o f the p a t h o l o g i c a l c o n d i t i o n s . I t was con-cluded t h a t the animals were responding to a l l o x a n as an agent o f s t r e s s i n conformity w i t h the a d a p t a t i o n syndrome and t h a t the h i g h s a l t content o f the d i e t aggravated and s u s t a i n e d the r e s u l t i n g h y p e r t e n s i o n . The pathology o f a l l o x a n diabetes,which d i f f e r s from t h a t o f d i a b e t e s m e l l i t u s , i s changed to t h a t of the l a t t e r by the s a l t d i e t . Because of the s a l t c r a v i n g o f d i a b e t i c s , i t i s suggested t h a t the hyp e r t e n s i o n o f t e n a s s o c i a t e d w i t h d i a b e t e s i s l a r g e l y due to the s a l t Intake. ( i ) ACKNOWLEDGEMENTS The author expresses h i s thanks t o the f o l l o w i n g f a c u l t y members not o n l y f o r t h e i r a s s i s t a n c e and advice but a l s o f o r the p e r s o n a l i n t e r e s t they have shown i n the r e s e a r c h problem: Dr. A.H. Hutchinson. Dr. John A l l a r d y c e . Dr. W.C. Gibson, M.D. Dr. P. C o n s t a n t i n i d e s , M.D. I n a d d i t i o n , the author i s indebted to Dr. A.L. Chute f o r the t r e a t e d r a t s which he s u p p l i e d from the S i c k C h i l d r e n ' s H o s p i t a l i n Toronto. ( i i i ) TABLE OE CONTENTS PAGE Acknowledgements. ( i ) A b s t r a c t . ( i i ) T a b l e o f Contents. ( i i i ) I I n t r o d u c t i o n to the Problem. 1 I I H i s t o r i c a l Background. 2 A. A l l o x a n d i a b e t e s . 2 1. H i s t o r y . 2 2.. Chemistry. 2 3. A l l o x a n & s u l f h y d r a l compounds. 3 4, Pathology. 4 5.. Comparison w i t h o t h e r d i a b e t o g e n i c methods. 5 6. E f f e c t of a l l o x a n on humans. 5 B. Hypertension as a disease o f a d a p t a t i o n to s t r e s s . 6 1. The a d a p t a t i o n syndrome. 6 2. The c a t a b o l i c impulse. 7 3. A c t i v a t i o n of the p i t u i t a r y - a d r e n a l system. 8 C. M e t a b o l i c Changes Induced By P i t u i t a r y And Adrenal Hormone A d m i n i s t r a t i o n . 11 1. F u n c t i o n s of the a d r e n a l c o r t e x . 11 2. The p i t u i t a r y - a d r e n a l system. 13 D. Methods Of Studying Adrenal Cortex A c t i v i t y . 14 1. U r i n a r y e x c r e t i o n . 15 2. 1 7 - K e t o s t e r o i d s . 15 3. Assay of a d r e n a l blood. 15 4. Hormone content of the gland. 16 (iv) TABLE OF CONTENTS (cont'd) PAGE 5. A s s o c i a t e d m e t a b o l i c changes. I 6 (a) C h o l e s t e r o l & A s c o r b i c A c i d . 16 (b) B i o g e n e s i s of s t e r o i d hormones. 18 (c) E f f e c t o f A.C.T.H. on a d r e n a l c h o l e s -t e r o l & a s c o r b i c a c i d . 19 (d) Adrenal c h o l e s t e r o l & a s c o r b i c a c i d as 21 p r e c u r s o r s o f c o r t i c a l hormones. 6. The e f f e c t o f v a r i o u s s t r e s s e s on a d r e n a l 22 c h o l e s t e r o l and a s c o r b i o a c i d . E. The Chemical C y t o l o g y Of The P i t u i t a r y and 23 Adr e n a l s . 1.. The p i t u i t a r y gland. 24 2. The a d r e n a l s . 27 IT. H i s t o l o g y Of The Adr e n a l Gland. 31 I I I Methods. 34 IV R e s u l t s . 41 V D i s c u s s i o n s & C o n c l u s i o n s . 56 VI Summary. 60 1 I . I n t r o d u c t i o n T O The Problem: An experiment on a l l o x a n d i a b e t e s i n r a t s g i v e n h i g h s a l t and p r o t e i n d i e t s , c o n d u c t e d by Dr. Chute i n Toronto,pro-v i d e d the source o f experimental animals used i n t h i s problem. Dr. Chute's i n t e r e s t i n v o l v e d the h i g h d i e t a r y s a l t r e q u i r e -ments of d i a b e t i c human p a t i e n t s . D u r i n g the oourse o f the experiment, i t soon became apparent, however, t h a t the problem, though an e x c e e d i n g l y f r u i t f u l and i n t e r e s t i n g one, was a dup-l i c a t i o n o f one of S e l y e ' s many p r o j e c t s c o ncerning s t r e s s and the a d a p t a t i o n syndrome. P r e l i m i n a r y work c o n s i s t i n g o f h i s t o -l o g i c a l b r a i n s t u d i e s was done at t h i s u n i v e r s i t y by the author and Mr. W.A. R i v e r s . The f i n d i n g s of t h i s i n v e s t i g a t i o n w i l l be r e f e r r e d to i n connection w i t h the r e s u l t s o f t h i s t h e s i s . I t w i l l be shown by cytochemioal methods t h a t the animals were i n the t h r o e s o f the a d a p t a t i o n syndrome due to the a c t i o n of a l l o x a n as a d i r e c t or i n d i r e c t agent o f s t r e s s and t h a t the d i e t had a d i r e c t b e a r i n g on the s e v e r i t y of the accompanying p a t h o l o g i c a l c o n d i t i o n s . Because o f t h i s , the h y p e r t e n s i v e aspects o f the case w i l l r e c e i v e more emphasis than the d i a -b e t i o , s i n c e the a l l o x a n served d i r e o t l y as a v e h i c l e f o r the former • 2. I I . H i s t o r i c a l Background. A. A l l o x a n Diabetes. 1. H i s t o r y . A l l o x a n i s the u r e i d e o f mesoxalic a c i d . When i n -j e c t e d i n t o many l a b animals i t produces a . s e l e c t i v e n e c r o s i s of the b e t a c e l l s i n the I s l e t s o f Langerhans o f the pancreas. T h i s i s f o l l o w e d i n 24-48 hours by di a b e t e s m e l l i t u s . I t has been found t h a t most animals are susceptible-, w i t h the except-i o n o f the guinea p i g , duck, owl, chi c k e n , f r o g and toad. (3) Symptoms of d i a b e t e s m e l l i t u s are p o l y d i p s i a , p o l y u r e a , hyper-glycemia and g l u o o s u r l a . A s h o r t summarizing h i s t o r y o f a l l o x a n i s as f o l l o w s : 1838 - s y n t h e s i z e d by Woehler and L e l b i g . 1862 - d i s c o v e r e d i n I n t e s t i n a l mucus o f humans by L i e b i g . 1866 - found i n human u r i n e by Lang. 1931 - e x c r e t o r y r o u t e s t u d i e d by Cerecedo (14). 1937 - a c t i o n on sugars noted by Jacobs (42). 1943 - i s l e t n e c r o s i s d i s c o v e r e d by Dunn e t a l (27). 1943 - a l l o x a n d i a b e t e s i n r a b b i t s by B a i l e y ( 4 ) . 1943 - a l l o x a n d i a b e t e s i n r a t s by Dunn (60). 1943 - a l l o x a n d i a b e t e s i n dogs by Goldner (36). 2. Chemistry o f A l l o x a n . A l l o x a n , a pyrimidine-, i s white i n c o l o u r , water and a l c o h o l solible and i s a c i d i n s o l u t i o n . I t cannot be n e u t r a -l i z e d without I n a c t i v a t i n g i t . (Human bloo d plasma i n a c t i -v a tes it.\ I t s formula i s : a. HN CO I I CO CO HN CO It may act either as an oxidizer or a reducing agent. As an oxidizer i t i s i t s e l f reduced to dialuric acid whereas acting as a reducing agent i t i s ozldized to parabanic acid. In vitro, alloxan inhibits the conversion of glucose - 1 - P0 4 to glucose - 6 - P0 4. This i s the second step in the accepted metabolism of glycogen. This inhibition i s believed to be due to the action of alloxan as an enzyme destroyer. (48) Alloxan can be detected in the blood and in the pan-creas immediately after i t s intravenous injection but i t has a l l been destroyed or removed from the blood within five minutes of i t s injection. (47) The fate of alloxan in the body was thought by Cerecedo (14) to be a chemical"combination with sul-furic acid with a subsequent excretion i n the bile as ethereal sulfate. A small amount of alloxan i s converted to alloxantin and excreted i n the urine as murexlde. 3. Alloxan and Sulfhydral Compounds. Lab.es and Friesburger were the f i r s t to suggest that alloxan might react with sulfhydral compounds. (45) Leech and Bailey (47) discovered that non-gluoose reducing substances in the blood as well as glucose were markedly decreased after the injection of alloxan into rabbits. Since glutathione, a sulf-hydral compound, comprises most of the reducing substance of the blood, an interaction i s indicated. Glutathione remained below normal levels for 18-24 hours, at which time diabetes began to appear. The exact way in which alloxan produces i s l e t ne-crosis and diabetes i s not known. I t has been suggested that i s l e t o e l l destruction is a result of an action by the alloxan on cellular enzymes,possibly sulfhydral enzymes, whioh are known to react with alloxan.(46) Bruckmann and Wertheimer (11) suggest that alloxan may act in one of three ways: (i) By a selective accumulation of alloxan in toxic amounts in the beta c e l l s , ( i i ) By competing with a structurally similar compound for an enzyme with resulting disorganization of the c e l l metabolism and eventual destruction of the c e l l , ( i i i ) By a specific reaction of alloxan on the i s l e t c e l l , with or without an accumulation. Such a reaction would probably consist in the inactivation of an enzyme system intimately connected with insulin synthesis. That the i s l e t c e l l necrosis i s due to the direct action of the alloxan was shown neatly, by Gomori and Goldner (37), who temporarily ligated the arteries to one half of the dogs'pancreas, injected alloxan and then released the ligature five minutes later. This half remained normal. Hypophyseotomy does not affeot the destructive action, (5) (6) (31). A diet rich in lard increases a rat's sensitivity to alloxan, while protein decreases i t . 4. Pathology. The course of alloxan diabetes differs somewhat from naturally occurring diabetes in the following ways. (26) 5 (i) There i s a selective destruction of the B c e l l s i n the I s l e t s of Langerhans. ( i i ) There i s no e f f e c t on alpha or non-granular c e l l s of the pancreas. ( i i i ) F i b r o s i s or h y a l i n i z a t i o n i s missing when alloxan i s used as contrasted to that seen i n n a t u r a l l y oocurring diabetes. (iv) There i s acidosis, coma, cataracts and r e t i n a l hemor-rhage i n the experimental diabetes, (v) There i s temporary damage to the l i v e r , adrenals, p i t -u i t a r y and kidney. (vi) There i s no diabetic n e u r i t i s or a r t e r i o s c l e r o s i s . 5 . Comparison with other diabetogenic methods: There are three other commonly used methods for pro-ducing experimental diabetes. (i) Injeotion of anterior p i t u i t a r y extract (A.p.E.) ( i i ) Depanoreatization. ( i i i ) Injections of glucose. A.P.E. i s e f f e c t i v e on dogs and p a r t l y depancreatized rats and oats.. A previous hyperglycemia i s necessary and i n -jections must be repeated over several days or weeks. I t also acts on beta c e l l s . Depancreatization i s d i f f i c u l t since the gland i s often very d i f f u s e . Glucose administration i s effect-ive on cats only. I t s action i s very slow and again Involves the beta c e l l s . 6. E f f e c t of alloxan on humans: I t was hoped that hyperinsulinism due to tumours or 6 other causes could be treated with alloxan. Unfortunately, re-su l t s were poor, since alloxan i s too toxic to humans. Gonn (15) had no good r e s u l t s and found a f t e r removing the pancreas s u r g i o a l l y that no damage had been done to tumour c e l l s but instead the normal c e l l s had been affected. B. Hypertension As A Disease Of Adaptation To Stress. 1. The adaptation syndrome. The production of nephrosclerosis and hypertensionj i n the absence of sexual anomalies, i n experimental animals treated with D.C.A. l e d to the theory that a seleotive increase i n the endogenous mineral©- cortiooids might be the cause of corresponding syndromes i n man. Selye's concept of the diseases of adaptation was l a r g e l y based upon t h i s observation, since i t i s known that certain cortiooids are produced i n excess during adaptation and defence to any type of damaging agent. As previously mentioned i n the introduction, the pro-blem was seen to resemble one of Selye's stress experiments. Under the influence of st r e s s , occasioned by a variety of non-s p e c i f i c damaging agents, the organism responds with the general adaptation syndrome, whose manifestations are e s s e n t i a l l y i n -dependent of the s p e c i f i c nature of the stress employed and de-pend mainly on the degree and duration of the exposure. The syndrome evolves i n three d i s t i n c t phases, (1) the alarm re-action, (2) the stage of resistance, and (3) the stage of ex-haustion. Hypertension i s e s s e n t i a l l y a disease of the stage of resistance. 2. The c a t a b o l i c impulse. C e r t a i n types o f h y p e r t e n s i o n are caused by excessive exposure to s t r e s s , A v a r i e t y o f s t r e s s e s e l i c i t metabolio changes, the s o - c a l l e d * c a t a b o l i c impulse', which are prominent d u r i n g the shock phase o f the alarm r e a c t i o n . T h i s c a t a b o l i c impulse a p p a r e n t l y i n f l u e n c e s the a n t e r i o r p i t u i t a r y to produce an e x c e s s i v e amount o f A.C.T.H. The l a t t e r causes i n c r e a s e d c o r t i c o i d p r o d u c t i o n by the adrenal.:, r e s u l t i n g i n i n v o l u t i o n of the thymus and lymph nodes as w e l l as c e r t a i n metabolio changes,such as r e t e n t i o n of sodium, gluconeogenesis from p r o t e i n , and p r o d u c t i o n by the l i v e r o f a l p h a 2 g l o b u l i n ( hypertensinogen). D i r e c t l y or i n d i r e c t l y through t h e i r met-a b o l i c a c t i o n , the c o r t i c o i d s a l s o cause a r e n a l type of hyper-t e n s i o n w i t h n e p h r o s c l e r o s i s . (73) During s t r e s s , other f a c t o r s such as sympathetic s t i m u l a t i o n , l i b e r a t i o n o f a d r e n a l i n J , and p o s s i b l y even a d i s -charge o f p i t u i t a r y v a s o p r e s s i n may c o n t r i b u t e to the r a i s e d blood p r e s s u r e . I t has been found t h a t many e n t i r e l y u n r e l a t e d non-s p e c i f i c damaging agents produce the c a t a b o l i c impulse, which i s c h a r a c t e r i z e d by a breakdown of body p r o t e i n s and an i n -crease i n the c o n c e n t r a t i o n of p r o t e i n o a t a b o l i t e s and p r o -t e o l y t i c enzymes i n the b l o o d . T h i s i s accompanied by l o s s of weight and by h i s t o l o g i c a l evidence o f a g e n e r a l i z e d breakdown of c e l l s . There i s a l s o c h a r a c t e r i s t i c a d r e n a l - c o r t i c a l en-largement, g a s t r o i n t e s t i n a l u l c e r s , i n v o l u t i o n o f lymphatio organs, e t c . , at t h i s time. Prolonged s t r e s s l e a d s to hyper-8. t e n s i o n and n e p h r o s c l e r o s i s , e s p e c i a l l y i n animals s e n s i t i z e d by u n i l a t e r a l nephrectomy and h i g h sodium, h i g h p r o t e i n d i e t s . 3. A c t i v a t i o n o f the p i t u i t a r y - a d r e n a l system. Sayers and Sayers (62) b e l i e v e t h a t s t r e s s i n c r e a s e s the consumption or u t i l i z a t i o n o f c o r t i c o i d hormones by t i s s u e s and-that the r e s u l t i n g decrease i n t h e i r b l o o d - l e v e l causes a d i s c h a r g e o f c o t i o o t r o p h i n by the p i t u i t a r y and i s thus r e -sponsible f o r the c o r t i c a l enlargement. There i s an e x c e s s i v e p r o d u c t i o n o f c o r t i o o i d s a t t h i s time however, (73) (81) (82) so t h a t t h e r e must be an i n c r e a s e d u t i l i z a t i o n . C.N.H. Long b e l i e v e s t h a t the p i t u i t a r y - a d r e n a l system i s a c t i v a t e d by epinephrine (52) w h i l e Sayers and Sayers m a i n t a i n t h a t t h e r e i s an i n v e r s e r e l a t i o n s h i p between the b l o o d l e v e l s o f A.C.T.H. and A.C.H. t h a t determines the r e l a t i v e s e c r e t o r y a o t i v i t y of each gland. The l a t t e r workers r e g a r d the e f f e c t of epinephrine as due to i t s c a p a c i t y to i n c r e a s e the u t i l i z a t i o n o f c o r t i c a l hormones, thus r e d u c i n g t h e i r l e v e l s i n b l o o d t r a v e r s i n g the p i t u i t a r y . I n order to f u r t h e r h i s c o n t e n t i o n s , Long de v i s e d the f o l l o w i n g s e r i e s of experiments. A group of r a t s were hypophyseotomized and three days l a t e r homologous t r a n s p l a n t s of p i t u i t a r y t i s s u e from r a t s of the same s t r a i n were i n s e r t e d i n t o the a n t e r i o r chamber of the r i g h t eyes. The e o s i n o p h i l d e c l i n e i n the p e r i p h e r a l b l o o d was s e l e c t e d as an index of a d r e n a l c o r t i c a l a o t i v i t y and hence of A.C.T.H. r e l e a s e from the - g r a f t s . A subcutaneous i n j e c t i o n of e p i n e p h r i n e produced such a response, as d i d d i r e c t i n j e c t i o n 9. o f epinephrine i n t o the t r e a t e d eye chamber. The same minute dose g i v e n i n t o the chamber of the i n t a c t eye d i d not cause the r e a c t i o n . When the animals were s u b j e c t e d t o s t r e s s , they again responded by eo s i n o p e n i a . A f t e r removal of the eye con-t a i n i n g the t r a n s p l a n t , e p inephrine caused no response. The g r a f t s were assayed a f t e r removal and found to c o n t a i n A.C.T.H. T h i s s e t of experiments proved c o n c l u s i v e l y t h a t a d r e n a l i n e has a d i r e c t s t i m u l a t o r y e f f e c t on the p i t u i t a r y . E i t h e r hypophysectomy or adrenalectomy prevents s t r e s s from c a u s i n g h y p e r t e n s i o n or n e p h r o s c l e r o s i s , w h i l e e x t r a c t s of e i t h e r gland a d m i n i s t e r e d i n excess w i l l cause h y p e r t e n s i o n . The h y p e r t e n s i o n o f the shock phase-may--so deorease the blood supply o f the kidney t h a t r e n i n p r o d u c t i o n i s i n c r e a s e d as i t i s a f t e r the G o l d b l a t t clamp.(33) (34) S e l y e has shown f u r t h e r t h a t m i n e r a l o - o o r t i o o i d s such as D.C.A. cause h y a l i n i z a t i o n of i n d i v i d u a l g l o m e r u l i , thus d i m i n i s h i n g b l o o d supply i n the kidney a f t e r c o r t i c o i d overdosage. He b e l i e v e s t h a t such hya-l i n i z e d g l o m e r u l i may produce the p r e s s o r substance o f hyper-t e n s i o n . There i s a wealth o f da t a showing t h a t almost any type of n o n s p e c i f i c damage or s t r e s s causes e s s e n t i a l l y the same alarm r e a c t i o n symptoms. I t i s noteworthy, however, t h a t upon prolonged exposure to s t r e s s , d i s e a s e s o f a d a p t a t i o n are not always produced. D i f f e r e n t types of stress- may y i e l d d i f f e r e n t end r e s u l t s , e.g. exposure to c o l d tends t o produce n e p h r o s c l e r o s i s , w h i l e f o r c e d muscular e x e r c i s e or subcutaneous 10. i n j e c t i o n s of formaldehyde are more apt to cause h y p e r t e n s i o n . Y e t , i f the mechanism through which s t r e s s produces n e p h r o s c l e r -o s i s and h y p e r t e n s i o n i s the l i b e r a t i o n of m i n e r a l o - c o r t i c o i d s , i t i s d i f f i c u l t t o understand why the r e s u l t s o f s t r e s s are not i n v a r i a b l y the same. The excess p r o d u c t i o n o f c o r t i o o i d s i s a n o n s p e c i f i c e f f e c t of s t r e s s , but perhaps the c a t a b o l i c impulse i s more dependent upon the s p e c i f i c nature o f the damaging agent and i n t e r m e d i a t e metabolism p l a y s an important r o l e i n determining whether the excess m i n e r a l o - c o r t i c o i d s produced r e -s u l t i n n e p h r o s c l e r o s i s and h y p e r t e n s i o n . I t can r e a d i l y be shown t h a t A.P.E. or D.C.A. are made more damaging a f t e r sen-s i t i z a t i o n by d i e t s r i c h i n sodium and p r o t e i n or by p a r t i a l nephrectomy. Perhaps the s p e c i f i c m e t a b o l i c e f f e c t s of s t r e s s may s i m i l a r l y , s e n s i t i z e the organism to excess m i n e r a l o -c o r t i c o i d s . Recent p u b l i c a t i o n s i n d i c a t e t h a t the d i s e a s e s o f a d a p t a t i o n develop i n man i n e s s e n t i a l l y the same manner as i n experimental animals. I n c r e a s e d c o r t i c o i d e l i m i n a t i o n i n the u r i n e has been demonstrated by b i o a s s a y , (81) (82) i n c r e a s e d width and a c t i v i t y o f the a d r e n a l c o r t e x has been shown by h i s t o -l o g i c a l methods (Zamchek) and Increased r e n i n content o f the r e n a l v e i n has been proven i n h y p e r t e n s i v e humans. (39) I n r e -l a t i o n to the p r e v i o u s l y mentioned e f f e c t o f sodium, i t has been found t h a t low sodium d i e t s or treatment w i t h ammonium c h l o r i d e sometimes prove e f f e c t l v e i n d e p r e s s i n g the b l o o d p r e s s u r e (72) and t h a t d i e t s c o n s i s t i n g mainly of r i c e and f r u i t j u i c e s (43) l i k e w i s e tend to depress the blood p r e s s u r e i n h y p e r t e n s i v e 11 p a t i e n t s . I t i s b e l i e v e d t h a t the l a t t e r do not a c t by means of a p r e s s o r substance but r a t h e r by t h e i r low sodium and p r o t e i n content. M e t a b o l i c Changes Induced by A d m i n i s t r a t i n g A.C.T.H. & A.C.H. 1. F u n c t i o n s o f the a d r e n a l c o r t e x . At p r e s e n t , the f u n c t i o n s of the a d r e n a l c o r t e x appear to be the f o l l o w i n g : 1. R e g u l a t i o n o f e l e c t r o l y t e metabolism. 2. R e g u l a t i o n o f carbohydrate, p r o t e i n , and f a t metabolism. 3. R e g u l a t i o n o f lymphoid t i s s u e and c i r c u l a t i n g l e u k o c y t e s . 4. Androgenic f u n c t i o n . 5. R e g u l a t i o n o f p i g m e n t a t i o n . E l e c t r o l y t e - r e g u l a t i n g a d r e n a l s t e r o i d s , p a r t i c u l a r l y 11 - d e s o x y c o r t i o o s t e r o n e , are commonly r e f e r r e d to as sodium-r e t a i n i n g hormones, s i n c e t h i s i s t h e i r most conspicuous e f f e c t . The i n t e r r e l a t i o n o f carbohydrate, f a t and p r o t e i n metabolism, p a r t i c u l a r l y under f a s t i n g . c o n d i t i o n s , i s most e f f e c t i v e l y con-t r o l l e d by 11, 18 - o x y s t e r o i d s and to a l e s s e r extent by 11 -o x y s t e r o i d s . The 11, 17 - o x y s t e r o i d s are the most po t e n t i n promoting gluconeogenesls and i n causing atrophy of the thymus, l y m p h o l y s i s j and e o s i n o p e n i a . The androgenic e f f e c t appears to be caused by hormones resembling androsterone. The f a c t o r s r e -s p o n s i b l e f o r ohanges i n pigmentation have not been i d e n t i f i e d as y e t . I t has always been the custom to d i s c u s s the f u n c t i o n o f the a d r e n a l medulla s e p a r a t e l y from t h a t of the c o r t e x . 12 I t i s w e l l known t h a t the s e c r e t i o n of epinephrine i s under the c o n t r o l o f t h e autonomic nervous system whereas, the l i b e r a t i o n of c o r t i c a l hormones i s dependent upon p i t u i t a r y A.C.T.H. which i n t u r n . i s f u n c t i o n a l l y i n t e g r a t e d w i t h the autonomic nervous system. Now, by h i s t o l o g i c a l methods, i t i s p o s s i b l e to demonstrate t h a t the medullary hormone, e p i n e p h r i n e , a c t s as a s t i m u l a t o r of p i t u i t a r y A.C.T.H. s e c r e t i o n i n man. (58) P r o o f t h a t the a c t i o n i s a d i r e c t one was p r o v i d e d by Long. (See p r e v i o u s d i s c u s s i o n i n B-3). D.C.A. e x e r t s i t s e f f e c t by the r e n a l r e t e n t i o n o f sodium, c h l o r i d e and water, w i t h e x c r e t i o n of potassium, and upon the c o n c e n t r a t i o n of sodium and c h l o r i d e i n p e r s p i r a t i o n . (16) (38) E x c e s s i v e hydremia, edema, h y p e r t e n s i o n and c a r d i a c enlargement may be observed i n both lab. animals and p a t i e n t s t r e a t e d w i t h e x c e s s i v e doses of D.C.A. (38) (78) (61) (70) The h y p e r t e n s i o n a s s o c i a t e d w i t h D.C.A, i s enhanded by sodium c h l o r i d e a d m i n i s t r a t i o n , but i s not dependent s o l e l y on the i n c r e a s e i n b l o o d volume. (55) Recent s t u d i e s by Schroeder et a l . suggest t h a t D.C.A. i n j e c t e d i n t r a v e n o u s l y may e x e r t a d i r e c t p r e s s o r e f f e c t , p a r t i c u l a r l y i n h y p e r t e n s i v e p a t i e n t s . (35) E x c e s s i v e r e t e n t i o n of sodium and d e p l e t i o n o f potassium over l o n g p e r i o d s of time may cause tendon o o n t r a o t i o n s and f l a c c i d paralyses. I t i s a l s o p o s s i b l e t h a t sudden death occ-a s i o n a l l y r e p o r t e d i n D.C.A. t r e a t e d human p a t i e n t s may be r e -l a t e d to the d e t r i m e n t a l e f f e c t of a low serum potassium l e v e l on c a r d i a c conduction and c o n t r a c t i o n . (78) Symptoms of p o t a -ssium d e f i c i e n c y are enhanced by a d m i n i s t r a t i o n of g l u c o s e . 13. 2. The p i t u i t a r y - a d r e n a l system. I n p a t i e n t s w i t h an i n t a c t p i t u i t a r y - a d r e n a l system, s t r e s s causes a r a p i d d e p r e s s i o n of c i r c u l a t i n g e o s i n o p h i l s (30) P a t i e n t s w i t h Addison's d i s e a s e and p a n h y p o p i t u i t a r i s m f a i l to show t h i s change.(58) Because of these f a c t s , the measurement of c i r c u l a t i n g e o s i n o p h i l s can be used to i n d i c a t e the i n t e g r i t y of the p i t u i t a r y - a d r e n a l r e l a t i o n s h i p , abnormal A.C.T.H. pro-d u c t i o n , a b i l i t y t o meet a s t r e s s such as a major o p e r a t i o n , e t c . (76)--The a p p l i c a t i o n o f s t r e s s has been c o r r e l a t e d w i t h epinephrine p r o d u c t i o n i n e x p l a i n i n g t h i s e o s i n o p h i l i c a c t i o n . T h e r o l e of e p i n e p h r i n e and of the sympathetic nervous system i n the e a r l y response to s t r e s s was d e s c r i b e d some time ago by Cannon. (12) At the b e g i n n i n g of t h i s century, i t was noted t h a t repeated i n j e c t i o n s of epinephrine to r a b b i t s caused a d r e n a l enlargement. (2) Only recently. (1945), has the l i n k been e s t a b l i s h e d between the i n c r e a s e d a c t i v i t y of the a d r e n a l medulla and the s t i m u l a t i o n of the a d r e n a l c o r t e x under s t r e s s . Vogt (83) noted an a d r e n a l enlargement i n normal r a t s g i v e n epinephrine^, which was prevented by hypophyseotomy. Long et a l (50) showed t h a t a c r y s t a l of e p i n e p h r i n e p l a c e d d i r e c t l y on the p i t u i t a r y of a r a t l e d to a d r e n a l c o r t i c a l s t i m u l a t i o n , c h a r a c t -e r i s t i c o f t h a t induced by A.C.T.H. Recent s t u d i e s have shown t h a t epinephrine a d m i n i s t e r e d to e i t h e r dogs or humans r e s u l t s i n r a p i d - e o s i n o p e n i a (79) T h i s depends on the p i t u i t a r y - a d r e n a l combination being e n t i r e . (58) E p i n e p h r i n e a l s o causes i n c r e a s e d e x c r e t i o n of u r i n a r y hormones. Prolonged a d m i n i s t r a t i o n , however, does not g i v e r e s u l t s 14. i n the same-amount or r a t e as d i r e c t A.C.T.H. a d m i n i s t r a t i o n . T h i s suggests t h a t an e a r l y r e f r a c t o r i n e s s o f the p i t u i t a r y has been a t t a i n e d . I t i s l i k e l y t h a t the a c t i o n o f e p i n e p h r i n e i s l i m i t e d to the l i b e r a t i o n of preformed A.C.T.H., without o t h e r -wise s t i m u l a t i n g A.C.T.H. p r o d u c t i o n . Although t h e r e i s ample evidence t h a t epinephrine i s capable of i n d u c i n g i n c r e a s e d output of p i t u i t a r y A.C.T.H., i t i s by no means e s t a b l i s h e d t h a t i t i s the o n l y a c t i v a t o r . P r e l i m i n a r y evidence suggests t h a t a n e u r a l mechanism, l o c a t e d i n the hypothalamus i s a l s o a c t i v a t e d by epinephrine and i n t u r n s t i m u l a t e s the p i t u i t a r y . (41) The mechanism whereby A.C.T.H. s e c r e t i o n i s s u s t a i n e d f o r a prolonged p e r i o d would not appear to be based on the continuous a c t i o n o f e p i n e p h r i n e . D. Methods Of Stud y i n g Adrenal C o r t e x A c t i v i t y . I n the study of the f u n c t i o n of an endocrine gland i t i s important t o determine the circumstances under which an i n c r e a s e d supply o f i t s p a r t i c u l a r hormone i s made a v a i l a b l e . Both the circumstances t h a t cause an i n c r e a s e d s e c r e t i o n and the method of p r o d u c t i o n must be.considered. The mechanism of the b i o l o g i c a l s y n t h e s i s and r e l e a s e o f any hormone i s imp e r f e c t a t the present time. T h i s i s p a r t i c u l a r l y t r u e i n the case of the a d r e n a l corteac, whose c h a r a c t e r i s t i c hormone has o n l y r e -c e n t l y been s y n t h e s i z e d i n s m a l l amounts by a l a b o r i o u s t e c h n i c . The methods t h a t have been developed f o r the d e t e r -m i n a t i o n o f an i n c r e a s e d s e c r e t o r y a c t i v i t y o f the a d r e n a l c o r -t e x are as f o l l o w s : 15. 1. U r i n a r y E x c r e t i o n . C o r t i c a l hormones i n u r i n e are assayed e i t h e r by the a b i l i t y of the hormone to i n c r e a s e the l i v e r glycogen of r a t s and mice or by i t s c a p a c i t y to p r o t e c t adrenalectomized animals a g a i n s t the e f f e c t s o f exposure to c o l d . By u s i n g these t e c h n i -ques, i t has been c l e a r l y demonstrated t h a t t h e r e i s an i n -creased u r i n a r y e x c r e t i o n o f c o r t i n i n man a f t e r trauma, burns and i n f e c t i o n s . The l a r g e amounts of u r i n e r e q u i r e d l i m i t i t s use to man or l a r g e animals. 2. 17 - K e t o s t e r o i d s . The e x c r e t i o n o f 17 - k e t o s t e r o i d s has been suggested by some workers as an i n d i c a t i o n o f the c o r t i c a l a o t i v i t y i n man. (49) (53) However t h e r e are two f a c t s t h a t throw d i s -c r e d i t on t h i s method. F i r s t l y , the 17 - k e t o s t e r o i d l e v e l i n the u r i n e does not p a r a l l e l the l e v e l of c o r t i n . Secondly, the 17 - k e t o s t e r o i d e x c r e t i o n , a t l e a s t i n the male, i s a l s o com-p l i c a t e d by the f o r m a t i o n o f these substances from the t e s t i -c u l a r hormone. U n t i l methods are d e v i s e d f o r more s p e c i f i c d e t e r m i n a t i o n o f the k e t o s t e r o i d s w i t h C 11 s u b s t i t u t i o n , ^ t h e value of t h i s approach w i l l remain u n c e r t a i n . However t h i s method, along w i t h the f i r s t , has the advantage t h a t i t can be performed without the use o f o p e r a t i v e procedures. 3. Assay of a d r e n a l blood. Vbgt has shown r e c e n t l y t h a t the b l o o d plasma drawn from the a d r e n a l v e i n s of dogs c o n t a i n s s u f f i c i e n t q u a n t i t i e s of c o r t i c a l hormone to be determined by the Selye-Schenker 16 method o f assay. I n t h i s manner he has shown a d a i l y s e c r e t i o n i n dogs equal to 250 c.c.'s of commercial e x t r a c t . Even g r e a t e r outputs were ob t a i n e d by s p l a n c h n i c nerve s t i m u l a t i o n or by e p i n e p h r i n e . 4. Hormone Content o f the Gland. One o f the most w i d e l y used methods f o r the study o f v a r i a t i o n s i n endocrine a c t i v i t y i s the d e t e r m i n a t i o n o f the a c t u a l q u a n t i t i e s of hormone pr e s e n t i n the gland under d i f f -erent c o n d i t i o n s . One g e n e r a l d i f f i c u l t y o f t h i s method i s to decide whether a decreased content of hormone i n the gland i n d i c a t e s a g r e a t e r s e c r e t i o n or a decreased f o r m a t i o n , a matter t h a t i s u s u a l l y s e t t l e d by a c t u a l measurement of hormone r e l e a s e d i n t o the b l o o d . A s p e c i a l d i f f i c u l t y i n the case of the endocrine glands i s the s m a l l amount of hormone pr e s e n t a t any one time i n the g l a n d i t s e l f . Even by u s i n g b i o l o g i c a l t e c h n i c s i t i s not p o s s i b l e to d i r e c t l y measure changes i n hormone q u a n t i t y i n the adrenal oortex. 5. A s s o c i a t e d M e t a b o l i c Changes, (aj C h o l e s t e r o l and a s c o r b i c a c i d . Another approach to the problem i s , a v a i l a b l e however. I t may be assumed t h a t i n c r e a s e d hormone p r o d u c t i o n , p a r t i c u l a r l y i n a gland t h a t o n l y has a s m a l l r e s e r v e , w i l l be.accompanied by c h a r a c t e r i s t i c m e tabolic changes a s s o c i a t e d w i t h the syn-t h e s i s and r e l e a s e of the hormone. T h i s i s even more ev i d e n t when a p r e c u r s o r or an e s s e n t i a l c o n s t i t u e n t of the hormone i s p r e s e n t . Two i n s t a n c e s of t h i s are known. The f i r s t i s the 17. r e l a t i o n o f i o d i n e metabolism t o the t h y r o i d hormone and the second i s - t h e presence i n the a d r e n a l c o r t e x o f two chemical substances, both o f which appear to be s p e c i f i c a l l y r e l a t e d t o the form a t i o n o f the hormone and both o f whioh appear to r e f l e c t any changes i n the r a t e o f s e c r e t i o n . These two sub-stances are c h o l e s t e r o l and a s c o r b i c a c i d . The presence o f these two o r g a n i c compounds i s the o u t s t a n d i n g chemical c h a r a c t e r i s t i c o f the a d r e n a l c o r t e x . No othe r organ of the body except the corpus luteum approaches the organ i n i t s h i g h c o n c e n t r a t i o n s o f both these substances. Another c h a r a c t e r i s t i c of the a d r e n a l c h o l e s t e r o l i s t h a t approximately 90$ of i t i s pr e s e n t i n the gland i n e s t e r form. T h i s i s to be compared w i t h the 50$ o f the- e s t e r found i n the l i v e r and the 10$ found i n the b r a i n . I t i s g e n e r a l l y under-stood t h a t a h i g h p r o p o r t i o n of e s t e r i f i e d c h o l e s t e r o l i n d i c a t e s a high r a t e o f turnover of both the s t e r o i d and i t s a s s o c i a t e d f a t t y a c i d s . There has been c o n s i d e r a b l e s p e c u l a t i o n concerning the p o s s i b l e r e l a t i o n s h i p between the adrenals and the g e n e r a l c h o l e s t e r o l metabolism o f the body, although no d e f i n i t e a s s o c i a t i o n has y e t been shown. The f a c t t h a t the amount of c h o l e s t e r o l i n the adre n a l f l u c t u a t e d g r e a t l y under d i f f e r e n t circumstances was known. T h i s l a t t e r s u b j e c t has been reviewed by Sayers et a l (67) who have p o i n t e d out t h a t a l o w e r i n g o f . ad r e n a l c h o l e s t e r o l i s a s s o c i a t e d w i t h exposure o f the organism to a v a r i e t y o f s t r e s s e s . 18. The h i g h c o n c e n t r a t i o n o f a s c o r b i c a c i d i n the a d r e n a l s f u r n i s h e d the f i r s t source f o r i t s i s o l a t i o n i n pure form. F l u c t u a t i o n s are a g a i n present under v a r i o u s c i r -cumstances and the r e l a t i o n of the v i t a m i n to the n a t u r a l r e -s i s t a n c e of the organism has a l s o been suggested. (56) I t has been r e p o r t e d t h a t the a d r e n a l c o n t a i n s w a t e r - s o l u b l e s t e r o i d s i n which the s t e r o i d i s a s s o c i a t e d w i t h a s c o r b i c a c i d . (86) Compounds o f t h i s type whioh possess the b i o l o g i c a l a c t i v i t y o f the c o r t i c a l hormones have been i s o l a t e d . (b) B i o g e n e s i s of t h e s t e r o i d hormones. The i n t r o d u c t i o n of i s o t o p e s t u d i e s has g i v e n new i n f o r m a t i o n concerning an o l d b e l i e f . The demonstration t h a t c h o l e s t e r o l had a s i m i l a r chemical s t r u c t u r e to many b i o l o g -i c a l l y important substances had l e d to the h y p o t h e s i s t h a t c h o l e s t e r o l might undergo t r a n s f o r m a t i o n i n the body i n t o such r e l a t e d substances as the s t e r o i d hormones and b i l e a c i d s . R e c e n t l y i t has been shown by use of c h o l e s t e r o l tagged w i t h deuterium t h a t t h i s substance i s a c t u a l l y converted i n t o c h o l i c a c i d (8) and t h a t i n women t h i s i s f o l l o w e d by the e x c r e t i o n of p r e g n a n e d i o l c o n t a i n i n g the i s o t o p e i n the u r i n e . (9) T h i s l a s t e xperiment i s of p a r t i c u l a r i n t e r e s t i n r e l a t i o n to s i m i l a r t r a n s f o r m a t i o n s i n the adrenal c o r t e x , s i n c e p r e g n a n e d i o l i s the u r i n a r y e x c r e t i o n product of progesterone, a substance o f v e r y s i m i l a r composition to the a d r e n a l c o r t i c a l s t e r o i d s . R i t t e n b e r g has r e p o r t e d experiments on mice i n which a constant deuterium content of 1.5 atom per c e n t was maintained. 19. A f t e r 60 days the c h o l e s t e r o l i n these mice showed a r e l a t i o n between deuterium and hydrogen content which was h a l f t h a t found i n body f l u i d s . I t was concluded t h a t a t l e a s t 22 hydrogen atoms i n the c h o l e s t e r o l molecule are exchangeable w i t h the deuterium of the body f l u i d s i n some step of i t s b i o -g e n e s i s . The f a c t was i n t e r p r e t e d to mean t h a t c h o l e s t e r o l i s s y n t h e s i z e d i n the body from s m a l l e r molecules. The p o s s i b i l i t y o f hydrogen-deuterium exchange r e a c t i o n s has been e l i m i n a t e d , s i n c e t h e amount of deuterium pres e n t i n the c h o l e s t e r o l i s g r e a t e r than c o u l d thus be accounted f o r . A l l these f i n d i n g s suggest t h a t c h o l e s t e r o l i s f i r s t b u i l t up from one, two or three carbon compounds and subsequently degraded to the s t e r o i d hormones. A comparison of the m o l e c u l a r s t r u c t u r e of p r e g -nenolone to t h a t of any of the s t e r o i d hormones shows a p o s s i b l e r e l a t i o n s h i p i n b i o g e n e s i s . I f the hormones are d e r i v e d from c h o l e s t e r o l , t h i s compound would'appear t o be the l o g i c a l p a r ent substance of a l l s t e r o i d hormones, as i t d i f f e r s from c h o l e s t e r o l o n l y i n i t s s i d e c h a i n . P h a r m a c o l o g i c a l l y , p r e -gnenolone a l s o occupies a r a t h e r unique p o s i t i o n i n t h a t i t i s the o n l y compound known to possess a l l the independent s t e r o i d hormone a c t i o n s . I t appears t h a t the s p e c i a l i z a t i o n f o r a c e r t a i n p h a r m a c o l o g i c a l a c t i o n occurs g r a d u a l l y and always at the expense of other p r o p e r t i e s o f the parent compound. (o) E f f e c t of A.C.T.H. on adrenal c h o l e s t e r o l and a s c o r b i c a c i d . . S t u d i e s w i t h A.C.T.H. have proved t h a t t h i s p i t u i t a r y 20 hormone caused d e p l e t i o n o f both c h o l e s t e r o l and a s c o r b i c a c i d i n the adre n a l c o r t e x . (51) I n the r a t , a s i n g l e i n j e c t i o n o f 1-4 mg./100 g r . i s f o l l o w e d by a r a p i d f a l l i n a d r e n a l a s c o r b i c a c i d and a slower f a l l i n a d r e n a l c h o l e s t e r o l . (63) (64) (65) (66) (67) Some twenty minutes a f t e r the i n j e c t i o n , the a d r e n a l a s c o r b i c a c i d has been reduced by.30$ and i n an hour by 60$. I t then begins to r i s e again and by the n i n t h hour; has re t u r n e d to normal. The c h o l e s t e r o l , on the other hand, does not reach i t s maximum f a l l u n t i l the t h i r d hour and the r e t u r n t o normal i s prolonged u n t i l 24 hours a f t e r the i n j e c t i o n . These e f f e c t s o f A.C.T.H. upon the composition o f the gl a n d are accompanied by d e f i n i t e i n d i c a t i o n s of an i n -creased output o f the c o r t i c a l hormones themselves. Both i n -creased l i v e r glycogen (67) and maximum lymphopenia (25) re a c h t h e i r h e i g h t between the 6 t h and 9th hours. Both these e f f e c t s are s p e c i f i c a d r e n a l c o r t i c a l hormone r e s u l t s . The guinea p i g , l i k e man, i s unable t o s y n t h e s i z e a s c o r b i c a c i d . I t has been found t h a t a d m i n i s t r a t i o n o f A.C.T.H. to t h i s animal when on a non-ascorbic a c i d d i e t causes a f a l l i n a s c o r b i c a c i d and c h o l e s t e r o l as i n the pre v i o u s l y . m e n t i o n e d r a t s but t h a t the r e f o r m a t i o n o f a s c o r b i c a c i d i n the gland i s r e t a r d e d . (64) T h i s slow r e t u r n o f a s c o r b i c a c i d i s b e l i e v e d to be due to a withdrawal o f the v i t a m i n from the b l o o d and a subsequent storage i n the c e l l s of the g l a n d . I t should be emphasized t h a t comparable changes i n c h o l e s t e r o l a s c o r b i c a c i d l e v e l s of other t i s s u e s do not occur 21. w i t h A.C.T.H. a d m i n i s t r a t i o n . Since A.C.T.H. has no known p h y s i o l o g i c a l e f f e c t other than s t i m u l a t i o n o f the a d r e n a l c o r t e x , we may conclude t h a t the changes i n a d r e n a l c h o l e s t e r o l and a s c o r b i c a c i d f o l l o w i n g i t s i n j e c t i o n are i n d i c a t i v e o f an i n c r e a s e d r a t e o f form a t i o n and r e l e a s e o f the hormone from the gla n d . (d) Adrenal c h o l e s t e r o l and a s c o r b i c a c i d as p r e c u r s o r s Of c o r t i c a l hormones. The problem o f whether o r n o t a s c o r b i c a c i d and c h o l e s t e r o l combine to form c o r t i c a l hormones i s s t i l l r e -l a t i v e l y new. The evidence i s strengthened by the f a c t t h a t c h o l e s t e r o l i s known to be c o n v e r t i b l e to progesterone i n man. The a s s o c i a t i o n o f v i t a m i n C wi t h the c o r t i c a l hormones was f i r s t suggested by Zwemer.and Lowenstein.(86) These workers have i s o l a t e d from the a d r e n a l a water s o l u b l e s t e r o i d i n which they b e l i e v e t h a t the v i t a m i n C i s a t t a c h e d to r i n g D of the s t e r o i d n u c l e u s , a p p a r e n t l y by carbon-to-carbon l i n k a g e . The compound has a h i g h degree of i n s t a b i l i t y i n water and breaks down i n an a c i d medium to form a s t e r o i d , w i t h o u t c o r t i c a l a c t i v i t y , p l u s v i t a m i n C. T h i s important c l a i m c l a r i f i e s the a l t e r a t i o n s observed i n the ad r e n a l c o r t e x a f t e r A.C.T.H. i n j e c t i o n s . I t would a l s o account f o r the r a p i d and e a r l y d e p l e t i o n of a s c o r b i c a c i d i n response to s t i m u l a t i o n o f the gland, s i n c e t h i s may r e p r e s e n t the d i s c h a r g e o f preformed hormone which i s f o l l o w e d by a slower change i n c h o l e s t e r o l content as new q u a n t i t i e s o f hormone are formed. 22. The e f f e c t s of v a r i o u s s t r e s s e s such as hemorrhage, burns, c o l d , muscle trauma and p a i n f u l s t i m u l a t i o n of p e r i -p h e r a l nerves have been shown to p a r a l l e l the e f f e c t s of A.C.T.H. on the a d r e n a l s . Thus the f a l l i n g and subsequent r i s i n g o f both a s c o r b i c a c i d and c h o l e s t e r o l proceeds at a v e r y s i m i l a r r a t e i n the two types o f experiments. (67) (57) (38) I t i s now g e n e r a l l y accepted t h a t one of the e f f e c t s of s t r e s s i s an i n c r e a s e d demand f o r c o r t i c a l hormones and t h a t the i n i t i a t i o n o f the g r a t i f i c a t i o n o f t h i s need i s an i n c r e a s e d A.C.T.H. out-put . Evidence f o r t h i s l a t t e r f a c t can be o b tained from a com-p a r i s o n of the e f f e c t s of s t r e s s on normal and hypophysecto-mized animals. (51) (80) These experiments a l s o I l l u s t r a t e the r a p i d i t y of the a d r e n a l response, s i n c e changes can be e a s i l y d i s c e r n e d w i t h i n h a l f an hour a f t e r the s t r e s s has been a p p l i e d . S i n c e the agents t h a t cause d e p l e t i o n of a d r e n a l a s c o r b i c a c i d and c h o l e s t e r o l are so d i v e r s i f i e d , the q u e s t i o n of what common denominator i s possessed by such s t i m u l i a r i s e s . That th e a l t e r a t i o n s i n a d r e n a l composition depend on apre-l i m i n a r y a c t i v a t i o n o f the p i t u i t a r y has been mentioned p r e -v i o u s l y . Three mechanisms may c o n t r o l the. s e c r e t o r y a c t i v i t y o f the a n t e r i o r p i t u i t a r y i n the l i b e r a t i o n of A.C.T.H. These are ( i ) e x c i t a t i o n of a nervous s e c r e t o r y mechanism ( i i ) changes i n composition o f the blood t r a v e r s i n g the blood, causing s e c r e t i o n ( i i i ) the l e v e l o f c o r t i c a l hormones i n the b l o o d. The l a t t e r view i s h e l d by Sayers and Sayers (68) who found £3. t h a t a p r e v i o u s a d m i n i s t r a t i o n of c o r t i c a l hormones to r a t s exposed to c o l d f o r one hour prevented the u s u a l f a l l i n a d r e n a l a s c o r b i c a c i d . The q u e s t i o n o f a d i r e c t nervous c o n t r o l of a n t e r i o r lobe s e c r e t i o n i s a debatable one a t the moment, but t h e r e i s d e f i n i t e l y one type of nervous a c t i v i t y a s s o c i a t e d w i t h a l l o f the v a r i o u s s t r e s s e s . T h i s i s an e x c i t a t i o n of the sym-p a t h e t i c nervous system and the r e l e a s e of i t s s p e c i f i c hormone, epin e p h r i n e . Long (51) has i n v e s t i g a t e d the r e s u l t s of i n j e c t i o n s of epinephrine on the a d r e n a l and has found t h a t there i s a f a l l of both a s c o r b i c a c i d and c h o l e s t e r o l i n i n t a c t but not i n hypophysectomized r a t s . ( E p i n e p h r i n e s t i l l r e t a i n s i t s g l y c o g e n o l y t i c e f f e c t i n hypophysectomized animals). These experiments would i n d i c a t e t h a t the e f f e c t of epinephrine on the a d r e n a l c o n s t i t u e n t s i s a l s o mediated by the p i t u i t a r y . (84) I f the a c t i v a t i o n of the a d r e n a l depends on r e l e a s e of epine-p h r i n e , then animals w i t h complete a d r e n a l d e n e r v a t i o n or de-m e d u l l a t i o n should have d i f f i c u l t y i n meeting s t r e s s . T h i s may be so, as the work o f Wyman suggests (85) although such animals are not v e r y s e n s i t i v e to s t r e s s . I t i s not to be i m p l i e d t h a t the p i t u i t a r y i s i n n e r v a t e d by the sympathetic nervous system but t h a t the e p i n e p h r i n e a c t s as i t does on s k e l e t a l muscle, by g i v i n g a s t r i k i n g m e t a b o l i c e f f e c t . E. The Chemical Cytology Of The P i t u i t a r y and A d r e n a l s . The i d e a l g o als of chemical c y t o l o g y are s i m i l a r to those of b i o c h e m i s t r y - to a s c e r t a i n q u a l i t a t i v e l y andquantit-24. , a t i v e l y the events o f the v a r i o u s m e t a b o l i c p r o c e s s e s . Cytology possesses the advantage of p e r m i t t i n g exact l o c a t i o n of v a r i o u s m e t a b o l i c substances. Even n o n - s p e c i f i c methods may y i e l d v a l i d r e s u l t s i f m o r p h o l o g i c a l changes can be r e l a t e d to p h y s i o l o g i c a l s t a t e s . 1. The p i t u i t a r y gland. C o n s i d e r a b l e e f f o r t has been made to c o r r e l a t e the c y t o l o g i c a l appearance of the c e l l types w i t h d i f f e r e n t p h y s i o -l o g i c a l s t a t e s . (1) (74) Two k i n d s of g r a n u l a r c e l l s have been d e s c r i b e d , the a c i d o p h i l s and b a s o p h i l s , and one nongranular type, the chromophobes. I n humans, these are i n the p e r c e n t r a t i o of 37:11:52 r e s p e c t i v e l y . (54) The number may change w i t h age, sex, sexual m a t u r i t y , e t c . The c e l l s are c l a s s i f i e d by t h e i r s t a i n i n g behaviour when s t a i n e d by one o f the t r i a c i d - procedures. Thus b a s o p h i l s s t a i n w i t h a n i l i n b l u e , a c i d o p h i l s w i t h a c i d f u c h s i n or azocarmine and chromophobes r e j e c t these dyes. The c e l l types were f i r s t r e c o g n i z e d by Schonemann (71) a f t e r s t a i n i n g w i t h e i t h e r eosin-methylene b l u e or hematoxylin and e o s i n . R e c e n t l y (1945) the b a s o p h i l i c c e l l s o f the a n t e r i o r l o b e have been i n v e s t i g a t e d by D e s c l i n (24) and Dempsey and W i s l o c k i (23) who found t h a t d i g e s t i o n o f s e c t i o n s w i t h r i b -onuclease destroyed the c y t o p l a s m i c b a s o p h i l i a . Thus the baso-p h i l s a p p a r e n t l y c o n t a i n a c o n s i d e r a b l e q u a n t i t y o f c y t o p l a s m i c r i b o n u c l e o p r o t e i n . I n many other l o c a t i o n s such a c o n c e n t r a t i o n o f r i b o n u c l e o p r o t e i n occurs i n c e l l s engaged i n r a p i d p r o t e i n s y n t h e s i s . Consequently i t seems l i k e l y t h a t the p i t u i t a r y b a s o p h i l s are engaged i n r a p i d p r o t e i n f o r m a t i o n . 25. Although the chemical natures o f most o f the p r o t e i n hormones of the a n t e r i o r p i t u i t a r y are not known i n d e t a i l , i t has been e s t a b l i s h e d t h a t s e v e r a l o f them c o n t a i n a h i g h percentage of carbohydrate r e s i d u e s such as glucosamine and t h e r e f o r e may be c l a s s i f i e d as g l y c o p r o t e i n s . Attempts have been made r e c e n t l y to l o c a t e the s i t e o f hormone p r o d u c t i o n by i d e n t i f y i n g the carbohydrate r e s i d u e of these g l y c o p r o t e i n s . Catchpole (13) u t i l i z e d p e r i o d i c a c i d to f r e e the carbohydrate which was then s t a i n e d w i t h S c h i f f ' s l e u c o f u c h s i n reagent. By t h i s procedure some o f the c e l l s o f the a n t e r i o r p i t u i t a r y were d i f f e r e n t i a l l y s t a i n e d . S i m i l a r l y , JJempsey has succeeded i n impregnating such carbohydrates w i t h s i l v e r a f t e r a l k a l i n e h y d r o l y s i s and o x i d a t i o n . (21) Such methods have o n l y r e c e n t l y been i n t r o d u c e d and holdngreat promise f o r f u t u r e work. There has been a l a c k of agreement r e g a r d i n g the s p e c i f i c c e l l type i n the a n t e r i o r hypophysis which i s r e -s p o n s i b l e f o r the s e c r e t i o n of A.C.T.H. (1) (75) Post mortems from A d d i s o n ' s - d i s e a s e and from t e r m i n a l stages of t o t a l adre-nalectomy r e v e a l d i m i n u a t i o n and degeneration o f both a c i d o -p h i l s and b a s o p h i l s . (44) Crooke and R u s s e l l (17) s t a t e that the constant f e a t u r e s i n p a t h o l o g i c a l a d r e n a l i n s u f f i c i e n c y are the extreme d e c l i n e i n the number of b a s o p h i l i c c e l l s , a v a r i a b l e r e d u c t i o n i n the number o f a c i d o p h i l s , the presence o f a s e r i e s of abnormal b a s o p h i l i c c e l l s and l a r g e chromophobes. The p o s s i -b i l i t y t h a t such a d i s e a s e might be primary i n the hypophysis or i n the a d r e n a l s w i t h o p p o s i t e appearances i n the c y t o l o g y of 26 the hypophysis must always be c o n s i d e r e d . Reese e t a l (59), s t u d y i n g p i t u i t a r i e s of b i l a t e r a l l y adrenalectomized r a t s found d i m i n u a t i o n i n number and s i z e of a c i d o p h i l s c o r r e l a t e d w i t h a p r o g r e s s i v e l o s s of g r a n u l e s and r e g r e s s i v e changes i n the G o l g i apparatus. The q u e s t i o n may be r a i s e d as to whether the changes were a r e s u l t o f a m o d i f i c a t i o n i n p i t u i t a r y -a d r e n a l r e l a t i o n s h i p or of abnormal metabolism of the body as a whole, (69) . Conversely, Heinbecker and R o l f (40) i n s t u d i e s on the dog found t h a t f o l l o w i n g hypophysectomy, p r o g r e s s i v e atrophy o f the a d r e n a l c o r t e x o c c u r r e d . S t u d i e s were a l s o made on "puncture" dogs i n which the i n f u n d i b u l a r stem and the f i b e r s running c a u d a l l y from the p a i r e d p a r a v e n t r i c u l a r n u c l e i i n the hypothalamus were severed. Under such c o n d i t i o n s the a c i d o p h i l s are maintained, the b a s o p h i l s decrease i n number, the a d r e n a l c o r t e x remains normal, w h i l e the gonads and t h y r o i d s atrophy. T h i s suggests t h a t the a c i d o p h i l s may be the source o f A.C.T.H. w h i l e the b a s o p h i l s are the source o f 1T.S.H. and t h y r o t r o p h i n . However A l b r i g h t and E l r i c k (1) a t t r i b u t e o n l y E.S.H. to the b a s o p h i l s . Daughaday e t a l (18) c o r r e l a t e the a c i d o p h i l s w i t h A.C.T.H. p r o d u c t i o n because o f i n c r e a s e d a d r e n a l f u n c t i o n i n acromegaly, a c o n d i t i o n u s u a l l y caused by a c i d o p h i l tumours o f the hypophysis. The response to u n i l a t e r a l adrenalectomy i s compen-s a t o r y hypertrophy of the remaining gland. S i n c e t h i s does not occur i n hypophysectomized animals i t appears to be dependent 27 upon an i n c r e a s e d A.C.T.H. p r o d u c t i o n . Under the former con-d i t i o n s there i s a concomitant r i s e i n a c i d o p h i l s . (1) (29) Hypertrophy i s I n c r e a s e d by s t r e s s , which a l s o i n c r e a s e s the a c i d o p h i l s . 2. The a d r e n a l s . . A r e l a t i v e l y l a r g e number of cytochemical components have been d e t e c t e d i n the a d r e n a l s . On gross examination the gland does not appear to s t a i n h e a v i l y w i t h b a s i c dyes but on m i c r o s c o p i c examination i t can be seen that, the l i p i d d r o p l e t s of the c e l l s compress the cytoplasm i n t o t h i n , h e a v i l y baso-p h i l i c s t r a n d s . T h i s cytoplasmic b a s o p h i l i a i s more i n t e n s e i n the p e r i p h e r a l zones o f the gland than i n the r e t i c u l a r i s , where the p r o t o p l a s m i c strands are d i s t i n c t l y e o s i n o p h i l i c . T h i s o b s e r v a t i o n suggests the presence of n u c l e o p r o t e i n s and o f r a p i d p r o t e i n s y n t h e s i s . The a d r e n a l c o r t e x c o n t a i n s c o n s i d e r a b l e amounts o f a l k a l i n e phosphatase. A f ^ e r hypophysectomy, phosphatase r a p i d l y d isappears from .the parenchyma but i n c r e a s e s i n the endothelium f o r a time b e f o r e g r a d u a l l y d e c r e a s i n g . Elftman (28) has de-s c r i b e d sex d i f f e r e n c e s i n the d i s t r i b u t i o n of phosphatase i n the a d r e n a l s of mice, and has shown t h a t i n j e c t i o n s of sex hor-mones modify i t s l o c a t i o n . A s c o r b i c a c i d has been demonstrated i n the a d r e n a l c o r t e x by v i r t u e of i t s r e d u c i n g power i n a c i d s o l u t i o n s (32)-. T h i s i s important from a p h y s i o l o g i c a l viewpoint i n t h a t i t i s l i n k e d m e t a b o l i c a l l y w i t h the p r o d u c t i o n of c o r t i c a l hormone. (51) 28. The l i p i d content of the a d r e n a l c o r t e x has r e c e i v e d i n t e n s i v e study. The s u d a n o p h i l i c and b i r e f r i n g e n t p r o p e r t i e s of the l i p i d d r o p l e t s have l e d to the r e v e l a t i o n t h a t a h i g h c o n c e n t r a t i o n of c h o l e s t e r o l i s 'present i n the a d r e n a l c e l l s . More r e c e n t l y , the i d e n t i f i c a t i o n of the a d r e n a l hormones as s t e r o i d s has s t i m u l a t e d i n t e r e s t i n l o c a t i n g the a c t i v e com-pounds i n the a d r e n a l gland. I n 1940, Bennett (7) showed t h a t l i p i d s l o c a t e d i n the outer l a y e r of the f a s c i c u l a t a i n the c a t ' s a d r e n a l c o r t e x e x h i b i t e d a number o f r e a c t i o n s s i m i l a r t o those demonstrated by a c t i v e hormones i n chemical experiments. These were ( i ) acetone s o l u b i l i t y , ( i i ) r e a c t i v i t y w i t h semi-c a r b a z i d e and phenylhydrazine, i . e . ketone r e a c t i o n s , ( i i i ) r e d u c t i o n of a l k a l i n e s i l v e r s a l t s , and (iv) f o r m a t i o n o f , d i g i t o n i d e s . The l i p i d s i n other r e g i o n s do not e x h i b i t t h i s a r r a y o f r e a c t i o n s . L a t e r , (1943), i n attempting to l o c a t e the s t e r o i d s o f the ovary, Dempsey and B a s s e t t (20) d e f i n e d the f o l l o w i n g h i s t o o h e m i c a l c h a r a c t e r i z a t i o n s of k e t o s t e r o i d s : ( i ) acetone s o l u b i l i t y , ( i i ) r e a c t i v i t y w i t h c a r b o n y l reagents, ( i i i ) a u t o f l u o r e s c o n c e , ( i v ) b i r e f r i r g e n c e , and (v) r e a c t i v i t y w i t h concentrated s u l f u r i c a c i d as i n the Liebermann - Burchardt r e a c t i o n . Each of these r e a c t i o n s depends upon a d i f f e r e n t p h y s i c a l or chemical p r o p e r t y o f the s t e r o i d molecule. No s i n g l e one o f them i s s p e c i f i c f o r k e t o s t e r o i d s , but no substance other than k e t o s t e r o i d s i s known to e x h i b i t a l l f i v e r e a c t i o n s . The most e x t e n s i v e r e s e a r c h o f t h i s n ature has been made on the r a t by Deane and Greep (19). In the r a t , both the glomerulosa 29 and o u t e r f a s e i c u l a t a r e a c t p o s i t i v e l y w i t h the e n t i r e s e r i e s of k e t o s t e r o i d procedures. A f t e r hypophysectomy, the k e t -o s t e r o i d s o f the f a s e i c u l a t a disappear w h i l e those o f the glomerulosa remain. C o n d i t i o n s l e a d i n g t o a d r e n a l s t i m u l a t i o n (exposure t o c o l d , noxious agents, c e r t a i n v i t a m i n d e f i c i e n c i e s ) cause augmented k e t o s t e r o i d r e a c t i o n s i n the f a s e i c u l a t a hut do not a l t e r t he glomerulosa. I n j e c t i o n s of c o r t i c o s t e r o n e cause a d i s u s e atrophy and subsequent disappearance o f the k e t o s t e r o i d s o f the f a s e i c u l a t a . On the other hand, the glomerulosa becomes a t r o p h i c and d e p l e t e d o f k e t o s t e r o i d s a f t e r i n j e c t i o n s o f d e s o x y c o r t i c o s t e r o n e i n t o e i t h e r i n t a c t or hypophysectomized r a t s . S i n c e a f t e r hypophystectomy c a r -bohydrate metabolism i s markedly d e f i c i e n t but s a l t r e g u l a t i o n i s r e l a t i v e l y unimpaired, these experiments p o i n t towards a separate s i t e of o r i g i n o f these two p r i n c i p l e s . The evidence supports the hypothesis t h a t d e s o x y c o r t i o o s t e r o n e - l i k e com-pounds which r e g u l a t e e l e c t r o l y t e balance are formed independ-e n t l y o f the p i t u i t a r y i n the zona glomerulosa, and the 11 -oxygenated s t e r o i d s which r e g u l a t e carbohydrate metabolism are formed under the i n f l u e n c e of the p i t u i t a r y g l a n d i n the zona f a s e i c u l a t a . I n a d d i t i o n to the s t e r o i d s , other l i p i d s occur i n the a d r e n a l c o r t e x . T h i s m a t e r i a l i s mostly n e u t r a l f a t w i t h some f a t t y a c i d s . The r e t i c u l a r i s a l s o c o n t a i n s v a r i a b l e q u a n t i t i e s of brownish y e l l o w pigment which i s not r e a d i l y e x t r a c t a b l e from the t i s s u e s . L i t t l e i s known concerning the nature of these cytochromes. 30. The methods f o r i d e n t i f y i n g s p e c i f i c l i p i d s i n t i s s u e s e c t i o n s l e a v e much to be d e s i r e d . The c h a r a c t e r i s t i c s o l u -b i l i t i e s o f l i p i d s i n non-aqueous s o l v e n t s are used f r e q u e n t l y , but s i n c e l i p i d s enter i n t o both l o o s e and f i r m combinations w i t h p r o t e i n s t h e i r s o l u b i l i t i e s are not e n t i r e l y t r u s t w o r t h y i n d i c e s . They may be c h a r a c t e r i s t i c a l l y s t a i n e d w i t h Sudan dyes - compounds p o s s e s s i n g no p o l a r groups which s t a i n by v i r t u e of t h e i r s o l u t i o n i n t o the l i p i d o f the t i s s u e . The f i r s t o f these o i l s o l u b l e dyes was Sudan I I I , i n t r o d u c e d by Daddi i n 1896. Sudan IV, was l a t e r i n t r o d u c e d by M i c h a e l i s i n 1901 and has s i n c e been the most popular o f the o i l s o l u b l e dyes. Some l i p i d s possess a c t i v e groups which a i d i n t h e i r d e t e c t i o n and i d e n t i f i c a t i o n . Such compounds as p h o s p h o l i p i n s and s t e r o i d s have such d i s t i n g u i s h i n g f e a t u r e s . The s t e r o i d s e x h i b i t a number o f c h a r a c t e r i s t i c f e a t u r e s . (21) Many of the b i o l o g i c a l l y a c t i v e compounds c o n t a i n ketone groups and r e a c t w i t h c a r b o n y l reagents such as l e u c o f u c h s i n and p h e n y l h y d r a z i n e . The s t e r o i d s form s p h e r o c r y s t a l s which are s t r o n g l y b i r e f r i n g e n t . When t r e a t e d w i t h co n c e n t r a t e d s u l f u r i c a c i d together w i t h de-h y d r a t i n g agents suoh as a c e t i c anhydride, a c h a r a c t e r i s t i c p l a y of c o l o u r s r e s u l t s . T h i s phenomenon depends on the u n s a t u r a t e d nature of t h e i r p o l y c y c l i c r i n g s t r u c t u r e s . I n S c h u l t z ' s method f o r d etermining c h o l e s t e r o l , the Liebermann - Burchardt s t e r o l r e a c t i o n has been adapted t o c y t o l o g y . When i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t , some s t e r o i d s f l u o r e s c e , u s u a l l y a s t a b l e 31. green c o l o u r . Under p o l a r i z e d l i g h t , the l o n g rhomboidal c r y s t a l s o f c h o l e s t e r o l l i g h t up and are e x t i n g u i s h e d a l t e r -n a t e l y once i n each 90° of r o t a t i o n . F i n a l l y , s t e r o i d s are r e a d i l y s o l u b l e i n acetone or a l c o h o l and i t can, t h e r e f o r e , be determined i f each o f the above phenomena i s d e s t r o y e d by d i s s o l v i n g out the l i p i d s . Wo one t e s t i s s p e c i f i c f o r s t e r o i d s , but no other substance d i s p l a y s the e n t i r e b a t t e r y o f r e a c t i o n s . (7) (20) (22) F. H i s t o l o g y Of The A d r e n a l Gland. The a d r e n a l s were f i r s t d e s c r i b e d by E u s t a c h i u s i n 1856. More than 300 years l a t e r , V u l p i a n d i s c o v e r e d t h a t the c e l l s o f the medulla d i f f e r i n t h e i r s t a i n i n g a b i l i t y from those o f the c o r t e x . He noted t h a t i f a s l i c e a c r o s s an a d r e n a l i s immersed i n f e r r i c c h l o r i d e s o l u t i o n , the medulla takes a g r e e n i s h t i n g e , w h ile the c o r t e x does not. Subsequently, i n 1865, Henle d i s c o v e r e d t h a t c e r t a i n g r a n u l e s i n the medulla gave a r u s t c o l o u r e d p r e c i p i t a t e w i t h d i l u t e potassium b i c h -romate, the so c a l l e d c h r o m a f f i n r e a c t i o n . The h i s t o l o g i c a l s t r u c t u r e of the a d r e n a l medulla i s q u i t e d i f f e r e n t from t h a t o f the c o r t e x . D i f f e r e n t i a t i o n i s not d i f f i c u l t , although c e l l cords of the c o r t e x may r e a c h deep i n t o the medulla and v i c e v e r s a . The medullary c e l l i s p o l y -gonal and measures 18 - 30 microns, w i t h a v e s i c u l a r nucleus about 7 microns i n diameter. One of the most c h a r a c t e r i s t i c f e a t u r e s of the medullary c e l l s i s t h e i r c h r o m a f f i n i t y , which i s a good index o f t h e i r a d r e n a l i n e content. 32. The c o r t e x c o n s i s t s o f three s t r u c t u r a l l y d i f f e r e n t l a y e r s . The e x t e r n a l zone, immediately under the connective t i s s u e capsule, i s c a l l e d the zona glomerulosa. I t c o n t a i n s s m a l l , i r r e g u l a r l y arranged c e l l s t h a t c o n t a i n scanty cytoplasm. The n u c l e i are s m a l l but r i c h i n chromatin. The next l a y e r i s the zona f a s c i c u l a t a , which c o n s i s t s of r e g u l a r rows of l a r g e p o l y g o n a l c e l l s w i t h v e s i c u l a r n u c l e i . The c e l l columns are arranged r a d i a l l y , w i t h s i n u s o i d s between them. The w a l l s o f these s i n u s o i d s are studded w i t h r e t i c u l o - e n d o t h e l i a l c e l l s . The zona r e t i c u l a r i s , the innermost l a y e r of the. c o r t e x , con-s i s t s of i r r e g u l a r strands which form a network o f smal l c e l l s w i t h dark n u c l e i . Many melanin and iron-pigment c o n t a i n i n g phagocytes are found i n t h i s zone t Although t h e r e i s c o n s i d -e r a b l e v a r i a t i o n , the zona f a s c i c u l a t a i s always the widest l a y e r . Because of the numerous l i p i d g r a n u l e s , the c o r t i c a l c e l l s , e s p e c i a l l y i n the f a s c i c u l a t a , have a v a c u o l i z e d c y t o -plasm and are c a l l e d spongiocytes. Around the c e n t r a l v e i n , the c o r t e x i s i n v a g i n a t e d towards the medulla. The r e g e n e r a t i o n o f t h e c o r t e x i s b e l i e v e d by some to occur from the outer s u r f a c e inwards, but t h i s view i s not unanimously accepted. I s l e t s o f lymphatic t i s s u e are o f t e n found w i t h i n the a d r e n a l s , e s p e c i a l l y under c e r t a i n p a t h o l o g i o a l c o n d i t i o n s such as Addison's d i s e a s e . The a d r e n a l a r t e r i e s enter the gland at v a r i o u s 33. p o i n t s on i t s s u r f a c e and the v e i n emerges at the hilum. A l l of the,blood flows t h r u the c o r t e x before r e a c h i n g the medulla, so t h a t the l a t t e r i s e x c l u s i v e l y s u p p l i e d w i t h venous b l o o d , maximally s a t u r a t e d w i t h the m e t a b o l i t e s of the c o r t e x . There are important muscular s p h i n c t e r s i n the w a l l of the c e n t r a l v e i n . T h e i r p e r i o d i c c o n t r a c t i o n and r e l a x a t i o n h e l p s to c o l l e c t and r e l e a s e the hormone s a t u r a t e d blood i n accordance w i t h the requirements o f the organism. 34. III. Methods. As mentioned p r e v i o u s l y , the r a t s were t r e a t e d i n -i t i a l l y by Dr. Chute i n Toronto. A l l animals, i n c l u d i n g the c o n t r o l s , r e c e i v e d a d i e t o f known p r o t e i n and s a l t content. The r a t s are i d e n t i f i e d as f o l l o w s : ( f o r example PS 3 B 11). PS B 40$ p r o t e i n , 10$ s a l t d i e t . B a subgroup i n the experiment. 11 s the number o f the experiment. 3 a the number o f the r a t i n the sub-group. The subgroups are as f o l l o w s : A - A l l o x a n i z e d d i a b e t i c , weighed d i e t o n l y . B = A l l o x a n i z e d d i a b e t i c , weighed d i e t p l u s i n s u l i n . C = Normal c o n t r o l , ad l i b d i e t . D - A l l o x a n i z e d d i a b e t i c , ad l i b d i e t . E = A l l o x a n i z e d d i a b e t i c , ad l i b d i e t p l u s i n s u l i n . F = A l l o x a n i z e d , not d i a b e t i c , ad l i b d i e t . G = A l l o x a n i z e d d i a b e t i c , weighed p r o t e i n and s a l t d i e t p l u s ad l i b normal d i e t . CO r . A l l o x a n i z e d i n t r a v e n o u s l y w i t h kidney p e d i c l e s clamped f o r 10 minutes b e f o r e and twenty minutes a f t e r the i n j e c t i o n , w i t h hot s a l i n e packs a p p l i e d to the abdomen. From the above subgroups i t can be seen t h a t v a r i o u s methods were used to ensure t h a t the r a t s i n g e s t e d the given s a l t and p r o t e i n . The CO group was used to determine whether or not the kidneys were a f f e c t e d d i r e c t l y by the a l l o x a n . 35. The animals were p l a c e d on t e s t a t approximately two months age and kept t h e r e f o r 3 - 4 months b e f o r e b e i n g s a c r i f i c e d or dying spontaneously. The l i v e r s , kidneys and h e a r t were examined i n the Toronto l a b o r a t o r y , w h i l e the a d r e n a l s , p i t u i t a r i e s and b r a i n s were s t u d i e d a t the U n i v e r s i t y o f B r i t i s h Columbia. The a u t o p s i e s performed here f o l l o w e d t h i s procedure: the hypophysis was removed by r a i s i n g the c a l v a r i u m , l i f t i n g the b r a i n upward and backward to expose the o p t i c t r a c t s and adjacent areas; dura surrounding the gland was c a r e f u l l y r e -moved and the g l a n d was f r e e d from the c r a n i a l v a u l t . Both p i t u i t a r i e s and adrenals were examined g r o s s l y f o r s i g n s of hemorrhage, degeneration, e t c . The a d r e n a l s were weighed to f i n d s i g n s of h y p e r p l a s i a , h y p o p l a s i a , atrophy, hypertrophy, e t c . The a d r e n a l s were examined m i c r o s c o p i c a l l y u s i n g t h r e e types o f s t a i n s . 1. General s t a i n s such as hematoxylin and e o s i n were used to show t i s s u e changes, hemorrhages, and the number o f m i t o s i s . 2. Trichrome c o n n e c t i v e t i s s u e s t a i n s were used t o show connective t i s s u e i n f i l t r a t i o n and t h i c k e n i n g of w a l l s of b l o o d v e s s e l s . 3. The most important s t a i n s used were the f a t s t a i n s , the s t a i n of choice being Sudan IV. I t was used to s t a i n the c h o l e s t e r o l o f the a d r e n a l c o r t e x to i n -d i c a t e the m e t a b o l i c s t a t e o f the hormone-producing 36 c e l l s . The method, adapted from L i l l i e , used the s a t u r a t e d i s o p r o p a n o l method f o r Sudan IV. The a d r e n a l s were s e c t i o n e d on a f r e e z i n g microtome a t 15 microns, s t a i n e d i n Sudan IV and hematoxylin and mounted i n Apathe's Gum Syrup. The p i t u i t a r i e s were s t a i n e d w i t h hematoxylin and e o s i n as a g e n e r a l s t a i n . S e c t i o n s were a l s o s t a i n e d by the B r i s e n o - C a s t r e j o n method f o r d i f f e r e n t i a l c e l l a n a l y s i s . (10) Azocarmine s t a i n i n g o f a c i d o p h i l g r a n u l e s i s e x c e l l e n t f o r sharp d i f f e r e n t i a t i o n . P r e s t a i n i n g o f the n u c l e a r membranes by a short immersion i n alum hematoxylin b r i n g s out the n u c l e i s h a r p l y , making c o u n t i n g much e a s i e r . The complete procedure f o r the r a t hypophysis i s as f o l l o w s : 1. F i x i n Zenker-formol, 6 - IS hours. Potassium bichromate 25 gr. Me r c u r i c b i c h l o r i d e 50 g r. Rin g e r ' s s o l u t i o n 1000 ml. Add 1 c c . n e u t r a l f o r m a l i n 10 ml. s o l u t i o n b e f o r e u s i n g . 2. Wash 6 - 1 2 hours i n running water. 3. Dehydrate i n a l c o h o l s : 30, 50, 70, 80, 95% - | hour each. 100$ - 2 changes - 1 hour each. 4. C l e a r : (a) a b s o l u t e a l c o h o l - oedarwood o i l (equal p a r t s ) , 1 hour. . (b) oedarwood o i l , 1 hour. 37. (c) x ylene, 15 min. 5. I n f i l t r a t e i n hard p a r a f f i n (56 - 58° 0 ) . (a) 1 change i n t o p a r a f f i n (5 min.). (b) 3 ohanges hour each). (c) embed. 6. S e c t i o n a t 4 microns. 7. Remove p a r a f f i n w i t h ;xylene, 2 changes, 3 min. each. 8. Two changes a b s o l u t e a l c o h o l , 3 min. each. 9. A l c o h o l , 95$, 3 min. 10. D i s t i l l e d water, 3 min* 11. Lugol's s o l u t i o n (4 I : 6K I : 100 HgO), 3 min. 12. Sodium t h i o s u l f a t e , 0.5$ aqueous, 3 min. 13. D i s t i l l e d water, 3 min. 14. Alum hematoxylin, 30 sec. 15. Tap water wash. 16. D i s t i l l e d water, 3 min. 17. A l c o h o l , 80$ 3 min. 18. A n i l i n a l c o h o l , 15 min. a n i l i n e o i l 1 ml. 95$ a l c o h o l 1000 ml. 19. S t a i n i n azocarmine G a t 60°C, 45 min. azocarmine G 1 g r . D i s t i l l e d water 100 ml. Warm and a l l o w to c o o l to room temperature. F i l t e r w i t h f i l t e r paper ( a l l day). Add g l a c i a l a c e t i c a c i d , 4 ml., to f i l t r a t e . P l a c e i n oven 1 hour before u s i n g . 38. 30. Rinse i n d i s t i l l e d water. 21. D i f f e r e n t i a t e i n a n i l i n a l c o h o l ( s o l u t i o n 18) 2 - 3 min. 22. Wash i n a c i d a l c o h o l , g - 1 min. G l a c i a l a c e t i c a c i d 10 ml. A l c o h o l , 95$ 1000 ml. 23. Phosphotungstic a c i d , 5$, 1 hour. 24. Dehydrate i n a l c o h o l s : A l c o h o l 70$, 2 min. A l c o h o l 95$, 2 min., not l o n g e r . . Absolute a l c o h o l , 2 min. or more. 25. C o u n t e r s t a i n i n a c i d green s o l u t i o n , 5 min. A c i d green 0.1 gr. Orange G 0.5 g r . r Clove O i l 100 ml. Use f r e s h s t a i n f o r each 20 s l i d e s . 26. l y l e n e , 1 min. 27. Two changes xylene, 30 min. each. 28. Mount. A f t e r the above s t a i n i n g , alpha c e l l g r a n u l e s are p u r p l i s h r e d , beta c e l l granules are l i g h t green, n u c l e a r membranes are w e l l d e f i n e d , m i t o c h o n d r i a are orange-red, red b l o o d c e l l s are b r i l l i a n t orange, G o l g i apparatus shows as a n e g a t i v e image, chromophobes show l i t t l e or no cytoplasm, which i s c o l o u r l e s s to p a l e green. The c o u n t i n g method was m o d i f i e d from Rasmussen, 39. 1922. The r a t p i t u i t a r y i s s e c t i o n e d i n a h o r i z o n t a l p l a n e . A l l the c e l l s are counted i n every f i f t h f i e l d i n each of t hree h o r i z o n t a l s e c t i o n s at e q u i d i s t a n t l e v e l s i n the g l and. Most v a r i a t i o n i n c e l l percentage e x i s t s between the c e n t r a l and p e r i p h e r a l r e g i o n s , which are both sampled adequately by a center s e c t i o n . About-1200-1400 c e l l s can be counted thus, u s i n g one c e n t r a l s e c t i o n of the p i t u i t a r y g l a n d . The use of v a r i o u s c o l o u r f i l t e r s f a c i l i t a t e s c o u n t i n g . The type of f i x a t i o n of the hypophysis markedly i n -f l u e n c e s the percentage o f a c i d o p h i l s , so i t i s important to c o n s i d e r t h i s f a c t o r i n comparing the c e l l counts o f d i f f e r e n t i n v e s t i g a t o r s . F i x a t i o n by d i f f e r e n t techniques can be d i v i d e d i n t o 3 groups: those c o n t a i n i n g mercuric b i c h l o r i d e , without a c e t i c a c i d (Zenker - f o r m o l ) ; those without mercuric b i c h l o r i d e but w i t h a c e t i c a c i d { B o u i n ' s ) ; and those which c o n t a i n osmic a c i d . Each has i t s own advantages and disadvantages. The Zenker - formol type g i v e s e x c e l l e n t c o l o u r d i f f e r e n t i a t i o n i n subsequent s t a i n i n g ^ I t can be c r i t i c i z e d a g a i n s t i t s use i n c e l l counts i n t h a t i t p r e s e r v e s m i t o c h o n d r i a , which' are d i f f i -c u l t to d i s t i n g u i s h from a c i d o p h i l g r a n u l e s . B o u i n T s g i v e s the l e a s t c e l l u l a r d i s t o r t i o n but does not p r o v i d e as sharp t i n c t o r i a l d i f f e r e n t i a t i o n . The a c e t i c a c i d c o n t a i n e d t h e r e i n d i s s o l v e s out m i t o c h o n d r i a from the scene. Osmic a c i d methods are e x c e l l e n t f o r c y t o l o g i c a l d e t a i l , e s p e c i a l l y f o r G o l g i apparatus, but are o f l i t t l e use i n d i f f e r e n t i a l counts because of v a r i a b i l i t y and t i s s u e damage. 40. The type o f f i x a t i o n seems to be fundamental i n determining the approximate c o n t r o l v a l u e as r e p o r t e d by d i f f e r e n t i n v e s t i g a t o r s . Thus Wolfe, u s i n g Regaud's found 32-40$ alpha c e l l s , as d i d P f e i f f e r , F i n e r t y and B r i s e n o , usingzenker - f o r m o l ; F i n e r t y , Meyer and Marvin, u s i n g Bouins, found 24 - 27$. 41. IV R e s u l t s : Rat No. Adrenal Wt. i n mgm. Gross Observations. PS 3 A 11 27 7 41 9 32 12 31 13 34 14 20 16 22 17 29 18 28 Average 50i PS 2 B 11 34 3 27 4 5 52 Very f a t . 7 54 P i t u i t a r y hemorrhage. •8 10 11 12 13 14 Average PS 1 C 11 2 35 42 25 27 31 56  58 34 16 42. Rat. No. Ad r e n a l Wt. i n mgm. Gross O b s e r v a t i o n s . 3 39 4 20 5 39 6 17 Average 27 PS 2 D 11 35 4 45 Small hemorrhage. 5 6 30 7 52 8 43 Dark d i s c o l o u r a t i o n . 10 38 S p o t t y d i s c o l o u r a t i o n . 11 40 12 24 Average 38 PS 1 T 11 31 3 33 4 40 6 50 7 44 8 37 9 51 Average 38 PS 1 0 11 35 2 33 3 38 43 Rat. No. A d r e n a l Wt. i n mgm. Gross O b s e r v a t i o n s . 5 36 6 49 , Average 58 S I D 10 £0 5 £5 Dark d i s c o l o u r a t i o n . 5 £4 Hemorrhage. 9 44 12 50 15 50 Average 28 S 2 E 7 55 5 45 Average 40 C 0 10 50 12 30 15 40 25 56 Hemorrhage. 29 41 32 Dark d i s c o l o u r a t i o n . 46 42 Average - 59-2. Small hemorrhages were fre q u e n t both i n the p i t u i t a r i e s and a d r e n a l s of a l l experimental subgroups except the c o n t r o l s . (Fig, 4) A more common occurance was the engorgement of the 44 b l o o d sinuses of these glands to about twice t h e i r normal width. ( F i g . 5). 3. S e v e r a l c l a s s i c a l examples of severe a r t e r i o s c l e r o s i s were seen. Two examples o f e n d a r t e r i t i s o b l i t e r a n s are shown,, one i n the a d r e n a l c o r t e x ( F i g . 1) and the other i n an un-r e l a t e d p a r t o f the body, the s a l i v a r y g l a n d ( F i g , 2). P r o l i f e r a t i o n of the media w i t h complete disappearance of the e l a s t i c a i n t e r n a was common i n the b r a i n . ( F i g . 3) 4. M i t o t i c f i g u r e s were s u r p r i s i n g l y r a r e . Chromatin g r a n u l e s , which some authors r e f e r t o as prochromosomes, were abun-dant i n both glands. ( F i g . 8 ) . 5. A l l experimental animals except the c o n t r o l s and groups F & A showed severe d e p l e t i o n o f c h o l e s t e r o l i n the a d r e n a l s . F i g . 6, 7 and 8) Groups F & A had o n l y a r e l a t i v e l y s m a l l drop i n c h o l e s t e r o l . I t i s important to note t h a t t h e r e was no apparent d i f f e r e n c e i n the extent of t h i s decrease when comparing a l l o x a n i z e d animals w i t h those t h a t had been a l l o x a n i z e d and given i n s u l i n therapy. The CO group a l s o showed the same drop i n c h o l e s t e r o l content. C e l l s i n which the c h o l e s t e r o l was i n the p r o c e s s o f b e i n g u t i l i z e d more q u i c k l y than i n the normals were e a s i l y d i s c e r n a b l e due to the f a c t t h a t as the c h o l e s t e r o l was removed from i t s s p h e r i c a l g l o b u l e there was l e f t behind a c h a r a c t e r i s t i c c r e s c e n t shape ( F i g . 7 and 8) before the c h o l e s t e r o l d i s -appeared completely. 6. The f o l l o w i n g t a b l e shows the percent r a t i o of the t h r e e 45. c e l l types i n the p i t u i t a r y as shown by the B r i s e n o - C a s t r e j o n method. (See F i g . 9) % R a t i o . Rat. No. alpha beta chrom. PS 3 A 11 47 6 47 9 46 7 47 PS 3 B 11 52 7 41 7 67 6 27 8 54 7 39 PS 1 0 11 41 7 52 3 40 7 53 4 40 7 53 PS 2 D 11 50 7 43 4 54 6 40 PS 1 F 11 43 6 51 3 45 6 49 PS 2 G 11 55 6 39 6 53 7 40 S 1 D 10 56 6 38 3 52 7 41 6 56 7 37 13 55 6 39 S 5 E 7 57 6 37 GO 10 46 7 47 15 70 6 24 23 83 4 13 46. 7. A comparison of the p e r c e n t i n c r e a s e i n a d r e n a l weight w i t h the p e r c e n t i n c r e a s e i n p i t u i t a r y a c i d o p h i l s i s g i v e n i n the f o l l o w i n g t a b l e : % I n c r e a s e i n % I n c r e a s e i n Group Adr e n a l Wt. P i t . a c i d o p h i l s PS - A 11.1 15 PS - B 40.7 45 PS - D 40.7 30 PS - V 40.7 10 PS - G 40.7 35 S - B 3.7 35 S - E 48.1 43 CO 44.4 65 47. F i g . 1. E n d a r t e r i t i s o b l i t e r a n s i n an a r t e r y o f the adr e n a l c o r t e x o f r a t PS 12 A 11. Note the s i z e o f the lumen, which i s almost n o n - e x i s t e n t , x 1000. 48. F i g . 2. E n d a r t e r i t i s obliterans i n the sali v a r y gland of r at S 9 D 10, showing the laminated nature of the oocluding tissue, x 1000. 49. F i g . 3. P r o l i f e r a t i o n o f t h e m e d i a i n t h r e e a r t e r i e s o f t h e c e r e b r a l c o r t e x o f r a t S 3 D 10. N o t e t h e d i s a p p e a r a n c e o f t h e e l a s t i c a i n t e r n a , x 4 4 0 . 50. Fig. 4 . Large internal hemorrhage in the adrenal cortex of rat PS IS A 11, The hemorrhage was situated across the junction of the zona faseiculata and the zona reticularis. x 800. 51. mm % r Fig. 5. A low power view of the adrenal cortex of rat PS 5 D 11 showing the greatly expanded blood sinuses. Note the relative distribution of blood to the three layers. (Mallory-Heidenhain rapid connective tissue stain), x 180. 52. Fig. 6. Low power view of the adrenal cortex of rat PS 4 0 11 stained with Sudan IV in isopropanol to show the normal distribution of l i p i d material, x 400. 53. Fig. 7. Greatly magnified view of nearly exhausted adrenal cortex of rat PS 5 G 11 stained with Sudan IT. Note the typical crescent shaped structure formed as cholesterol i s ut i l i z e d from the globule in which i t i s stored, x 3 5 0 0 . 54. F i g . 8. A s i m i l a r c o n d i t i o n t o t h a t seen i n F i g . 7, but s l i g h t l y more severe. Note the t y p i c a l c r e s c e n t i h the upper centre of the photograph. Chromatin m a t e r i a l of the n u c l e i i s a l s o c l e a r l y v i s i b l e i n many o f the c e l l s , x 2500. Rat CO 10. 55. A t y p i c a l b a s o p h i l c e l l w i t h p l e n t i f u l l i g h t l y s t a i n e d cytoplasm i s seen near the c e n t r e . The numerous a c i d o p h i l s have darker s t a i n e d cytoplasm w h i l e the chromophobes have l i t t l e or none. The a o i d o p h i l count i s h i g h , the % r a t i o of a l p h as: betas: chromophobes • 55: 6: 39, x, 1700. 56. D i s c u s s i o n and C o n c l u s i o n s : I t i s apparent from the r e s u l t s o b t a i n e d t h a t the p i t u i t a r y - a d r e n a l systems o f the experimental animals were i n a s t a t e o f h y p e r a c t i v i t y . I n many types o f experiments, as i n u n i l a t e r a l adrenalectomy, the p i t u i t a r y r e t u r n s to normal when the remaining adr e n a l r e g a i n s the ne c e s s a r y l e v e l o f c o r t i c a l f u n c t i o n t h r u hypertrophy. I n the case w i t h which we are con-cerned, however, th e r e i s a s u s t a i n e d d e p l e t i o n o f ad r e n a l c h o l e s t e r o l and a oonstant i n c r e a s e d p i t u i t a r y a c i d o p h i l count. T h e r e f o r e , the demand f o r c o r t i c a l hormones must be c o n t i n -u o u s l y h i g h . At t h i s p o i n t , t h r e e f a c t s should be noted, (i) t h a t a l l animals, i n c l u d i n g the c o n t r o l s , r e c e i v e d a h i g h p r o t e i n , h i g h s a l t d i e t , ( i i ) t h a t a l l o x a n i z e d animals r e c e i v e d o n l y one dose of a l l o x a n and th a t such a dose i s completely de-s t r o y e d i n the body f i v e minutes a f t e r i t s i n j e c t i o n , and ( i i i ) t h a t t h e r e was no d i f f e r e n c e i n the r e s u l t s o b t a i n e d f o r a l l -o x anized animals when i n s u l i n was g i v e n t o remedy the d i a b e t e s . I t can be seen then t h a t the d i a b e t e s i s not a f a c t o r i n the p a t h o l o g i c a l c o n d i t i o n s w i t h which we are concerned. S i m i l a r l y , the d i e t alone i s not a s u f f i c i e n t s t i m u l u s . I t t h e r e f o r e seems l o g i c a l t h a t the a l l o x a n i s the i n i t i a l agent of s t r e s s , sub-sequently a i d e d and abe t t e d by the d i e t a r y p r o t e i n and s a l t . That such a s m a l l f a c t o r as a s i n g l e dose o f a l l o x a n a c t i n g over such a short p e r i o d o f time c o u l d have such a g r e a t e f f e c t i s made p l a u s i b l e by two f a c t s . One i s t h a t d u r i n g t h a t s h o r t time, the a l l o x a n i s capable o f d e s t r o y i n g the i n s u l i n - p r o d u c i n g b e t a c e l l s o f the i s l e t s o f Langerhans. The other i s t h a t 57 h y p e r t e n s i o n , comparable t o what i s produced i n these animals, has been produced by a former student a t t h i s u n i v e r s i t y u s i n g o n l y a s i n g l e s m a l l dose o f d e s o x y c o r t i c o s t e r o n e a c e t a t e . (70) The nature o f the a c t i o n o f a l l o x a n i n the a d a p t a t i o n syndrome i s p r o b a b l y the same as t h a t o f the host o f other n o n - s p e c i f i c agents o f s t r e s s . But i t i s tempting to s p e c u l a t e t h a t a l l o x a n c o u l d a t t a c k the adrenals d i r e c t l y i n a s i m i l a r manner t o t h a t i n which i t d e s t r o y s i s l e t t i s s u e . S i n c e the adre n a l gland has one o f the h i g h e s t g l u t a t h i o n e contents o f any t i s s u e i n the body and because o f the f a c t t h a t a l l o x a n removes g l u t a -t h i o n e from the bloo d and a t t a c k s s u l f h y d r a l s i n g e n e r a l , such a d i r e c t a c t i o n i s a d e f i n i t e p o s s i b i l i t y . I f t h i s was the case, the problem would be almost i d e n t i c a l w i t h the f a m i l i a r a d renalectomy-salt experiments t h a t are a l s o concerned w i t h s t r e s s . The e f f e c t o f the d i e t on the experimental animals r e c e i v e s some p o s s i b l e c l a r i f i c a t i o n by the r e s u l t s i n groups PS-A, and S-D & 33. The method o f s u p p l y i n g the s a l t and pro-t e i n i n the d i e t o f group A d i d not gi v e the animals as much of these two components and as much a d r e n a l hypertrophy as d i d the more a c c u r a t e methods used i n the l a t e r experiments, e s p e c i a l l y group G. There i s a d i s c r e p a n c y between A & B t h a t i s d i f f i c u l t to e x p l a i n . There i s a p o s s i b i l i t y t h a t the z i n c c o n tained i n the i n s u l i n c o u l d have oaused an a l l e r g i c or p o i s o n i n g a c t i o n as i t o c c a s i o n a l l y does i n some human p a t i e n t s . On the other hand, the low v a l u e s obtained i n group 3?, i n which the a l l o x a n f a i l e d to produce d i a b e t e s , are p r o b a b l y o n l y 58. due to a h i g h l e v e l o f c i r c u l a t i n g sulfhydrals i n the animals at the time o f i n j e c t i o n o f the a l l o x a n . Groups S-D and S-E, i n which s a l t was added but not p r o t e i n , gave readings p a r a l l e l w i t h those obtained w i t h p r o t e i n i n c l u d e d . The more important f a c t o r then i s p r o b a b l y the s a l t . Since i t i s an e s t a b l i s h e d f a c t t h a t t h e r e i s an i n -creased supply and demand f o r c o r t i c a l hormones d u r i n g s t r e s s , and s i n c e i t has been confirmed that there i s a s u s t a i n e d over-p r o d u c t i o n o f these hormones i n t h i s experiment, then t h e r e should be an i n c r e a s e d amount of c i r c u l a t i n g D.C.A. type hor-mones. The a c t i o n of these s a l t r e t a i n i n g hormones on the h i g h s a l t content coming i n t o the body must cause a c r i t i c a l i n c r e a s e i n the Na CI content o f the b l o o d . T h i s r i s e i s com-p a r a b l e to t h a t seen i n the r e s i s t a n c e phase o f the a d a p t a t i o n syndrome. I f t h i s sequence o f events i s prolonged, the hor-mone produci n g mechanism becomes exhausted and, as Selye p o i n t s out, there may be a l a c k o f these hormones when t e r m i n a t i o n o f l i f e o c c u r s . T h i s exhaustion phase was e v i d e n t , s i n c e some o f the animals showed a complete absence of a d r e n a l c h o l e s t e r o l and d i e d a spontaneous death. The method by which the s a l t and m i n e r a l o - c o r t i c o i d s produce the h y p e r t e n s i o n may i n v o l v e one or both of the two f o l l o w i n g p aths. The h i g h sodium l e v e l i n the blood might e x e r t a d i r e c t a c t i o n on the w a l l s of the a r t e r i e s due to the tendancy of sodium to cause s u s t a i n e d muscular c o n t r a c t i o n t h e r e i n . Again, the m i n e r a l o - o o r t i c o i d s c o u l d cause a n e p h r i t i c 59. type o f hy p e r t e n s i o n by t h e i r h y a l i n i z i n g a c t i o n on g l o m e r u l i . There are important d i f f e r e n c e s between the u s u a l pathology o f a l l o x a n d i a b e t e s and t h a t seen when s a l t i s added to the d i e t . The c o n d i t i o n s seen i n the l a t t e r show an i n -t e r e s t i n g and remarkable p a r a l l e l i s m t o t y p i c a l , n a t u r a l l y o c c u r r i n g d i a b e t e s m e l l i t u s . Thus one f i n d s a r t e r i o s c l e r o s i s , f a t t y degeneration of the l i v e r , n e p h r o s c l e r o s i s and f i b r o s i s i n both d i a b e t e s m e l l i t u s and i n the a l l o x a n i z e d animals having the s a l t d i e t i n t h i s experiment, but not i n a l l o x a n i z e d animals alone. Dr. Chute has made the c l i n i c a l o b s e r v a t i o n t h a t d i a -b e t i c c h i l d r e n consume abnormally l a r g e q u a n t i t i e s o f s a l t . From t h i s f a c t and from the r e s u l t s o f t h i s t h e s i s i t appears l i k e l y t h a t the o n l y reason t h a t a l l o x a n d i a b e t e s d i f f e r s from di a b e t e s m e l l i t u s p a t h o l o g i c a l l y i s t h a t l a b animals kept a f t e r a l l o x a n i z a t i o n do not no r m a l l y get the chance to i n g e s t e x t r a s a l t as would human d i a b e t i c s . I n other words, the reason t h a t a l l o x a n d i a b e t e s i s not the same as di a b e t e s m e l l i t u s l i e s i n the f a c t t h a t the lab. animals are f e d a b e t t e r d i e t than t h e i r human c o u n t e r p a r t s . Conversely, the c o m p l i c a t i o n s o f human di a b e t e s must be due t o a secondary h y p e r t e n s i o n caused l a r g e l y by the c r a v i n g o f s a l t . 60. Summary. The p i t u i t a r i e s and a d r e n a l s o f a l l o x a n i z e d d i a b e t i c r a t s f e d h i g h p r o t e i n and s a l t d i e t s showed t h a t the p i t u i t a r y -a d r e n a l systems of these animals were i n a s t a t e o f hyper-a c t i v i t y , as shown by a d r e n a l hypertrophy, d e p l e t e d a d r e n a l c h o l e s t e r o l and i n c r e a s e d p i t u i t a r y a c i d o p h i l s . The use of i n s u l i n to remedy the di a b e t e s and the omission o f p r o t e i n from the d i e t d i d not a l t e r the extent o f t h i s response. The animals showed t y p i c a l s i g n s o f hy p e r t e n s i o n such as a r t e r i o s c l e r o s i s , n e p h r o s c l e r o s i s , f a t t y degeneration o f the l i v e r and hemorrhages. I t was concluded t h a t the animals were responding t o a l l o x a n as an agent of s t r e s s i n conformity w i t h the a d a p t a t i o n syndrome and t h a t the hig h s a l t content o f the d i e t aggravated and sus-t a i n e d the r e s u l t i n g h y p e r t e n s i o n . Normally, the pathology o f a l l o x a n d i a b e t e s d i f f e r s from t h a t o f di a b e t e s m e l l i t u s . However on the s a l t d i e t the a l l o x a n type of d i a b e t e s was the same as the n a t u r a l l y o c c u r r i n g v a r i e t y . S i n c e Dr. Chute has observed t h a t d i a b e t i c s consume abnormally l a r g e q u a n t i t i e s of s a l t , i t i s proposed t h a t the o n l y reason t h a t a l l o x a n d i a b e t e s does not resemble d i a b e t e s m e l l i t u s p a t h o l o g i c a l l y i s that l a b animals are u s u a l l y kept on r e g u l a t i o n foods t h a t do not permit the i n g e s t i o n o f excess s a l t , a d o u b t f u l l u x u r y enjoyed by t h e i r human c o u n t e r p a r t s . Conversely, the h y p e r t e n s i o n t h a t so o f t e n c o m p l i c a t e s human d i a b e t e s i s p r o -b a b l y l a r g e l y due to the s a l t i n t a k e . 61. References: 1. A l b r i g h t , E. & E l r i e k , H; An Attempt To C l a s s i f y Hormone D i s o r d e r s Of The Hypophysis. Trans. Assoc. Am. P h y s i c i a n s . , 61, 42, 1948. 2. Babes, U, & Joesco, U.; Compt. rend.soc. b i o l . 65, 67. 3. B a i l e y , C.C.; A l l o x a n D i a b e t e s . V i t . & Hor. 7, 365, 1949. 4. B a i l e y , C C . & B a i l e y , O.T..; The P r o d u c t i o n Of Diabetes M e l l i t u s I n Rabbits With A l l o x a n . J.A.M.A., 122, 1165,1943. 5. B a i l e y , CC. & Lecompte, P.M.; A l l o x a n Diabetes I n Hypo-physectomized Rats. M e d . C l i n i c s o f N.A., 31, 427, 1947. 6. B a i l e y , C C et a l ; The E f f e c t o f A l l o x a n On Hypophy-sectomized R a t s . P.S.E.B.M. 66, 271, 1947. 7. Bennett, H.S.; S t e r o i d P r o p e r t i e s Of A d r e n a l L i p i d s . Am. J . Anat., 67, 151. 8. B l o c h , K. et a l ; B i o l o g i c a l Conversion Of C h o l e s t e r o l To C h o l i c A c i d . J . B i o l . Chem. 149, 511, 1943. 9. B l o c h , K.; B i o l o g i c a l Conversion Of C h o l e s t e r o l To Pre -g n a n e d i o l . J . B i o l . Chem., 157, 661, 1945. 10. B r i s e n o - C a s t r e j o n , B. & E i n e r t y , J . 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P.S.E.B.M. 58, 232, 1945. 64. 38. H a r k i n s , H.N. & Long, C.N.H.; M e t a b o l i c Changes I n Shock A f t e r Burns. Am. J . P h y s i o l . , 144, 661, 1945. 39. Haynes, F.W. & Dexter, L.; Renin, Hypertensinogen & Hypertensinase C o n c e n t r a t i o n s Of The Blood Of Dogs D u r i n g The Development Of Hyp e r t e n s i o n By Cons trite t i o n Of The Renal A r t e r y . Am. J . P h y s i o l . , 150, 190,-1947. 40. Heinbecker, P. & R o l f , D.; Experimental O b e i s i t y I n The Dog. Am. J . P h y s i o l . 141, 549, 1944. 41. Hune, D.; J . C l i n . I n v e s t i g a t i o n s 28, 790; A b s t r a c t . 42. Jacobs, H.R.; Hypoglycemic A c t i o n Of A l l o x a n . P.S.E.B.M., 37, 407, 1937. 43. Kempner, W.; N. Carolina"Med. J . 6, 61, 117; A b s t r a c t . 44. Kraus, E . J . ; Path. Anat. u A l l g . P ath. 78, 283, 1927; A b s t r a c t . 45. Labes, R. & F r i e s b u r g e r , H,; Arch. E x p t l . Path. Pharm. 156, 226, 1930. A b s t r a c t . 46. Lazarow, A.; P r o t e c t i v e E f f e c t Of G l u t a t h i o n e & C y s t e i n e A g a i n s t A l l o x a n D i a b e t e s I n The Rat. P.S.E.B.M., 61, 441, 1946. 47. Leech, R.S.; Blood A l l o x a n & Blood G l u t a t h i o n e I n R a b b i t s I n j e c t e d W i th A l l o x a n . J.B. Ghem., 157, 525, 1945. 48. Lehmann, H.; F a c t o r s I n f l u e n c i n g The Formation Of The Robison E s t e r . Biochem. J . , 33, 1241, 1939. 49. Lieberman, S. & D o b r i n e r , K.; S t e r o i d E x c r e t i o n I n H e a l t h & Diseas e . Rec. Prog. I n Hor. Res. 3, 91, 1948. 50. Long, G.N.H. & F r y , E.G.; E f f e c t Of Ep i n e p h r i n e On A d r e n a l C h o l e s t e r o l & A s c o r b i c A c i d . P.S.E.B.M., 59, 67, 1945. 65. 51. Long, C.N.H.; The R e l a t i o n Of C h o s t e r o l & A s c o r b i c A c i d To The S e c r e t i o n s Of The Adr e n a l Cortex. Rec. Prog. I n Hor. Res., 1, 99, 1947. 52. 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