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Studies on the mode of action and the metabolism of vitamin D
STUDIES ON THE 1ViODE OF ACTION AND THE 1KETABOLISM OF VITAMIN D WALTEa tIE~AN~, M.D. CLEVELAND~ O H IO
tile present time our knowledge of the mode of action of U NTIL vitamin D has been extraordinarily meager and may be summed up almost entirely in the observation that vitamin D increases the diminished inorganic phosphate content of the blood in rickets and brings back to normal the diminished calcium content in spasmophilia. As soon as one raises the question how vitamin D brings about the regulation of inorganic phosphates and calcium in the blood, one enters a vast field of speculation. One of the hypotheses is the assumption that irradiated ergosterol stimulates an endocrine gland. The work of Erdheim, ~ Pappenheimer and Minor, ~ Ritter ~ and others seemed to indicate the importance of the parathyroid glands in infantile ric.kets, while the recent publications of Nitsehke 4 emphasized the connection between the lymphoid tissues (spleen, lymph nodes, and thymus) and the thyroid gland in regulating the phosphorus and calcium content of the blood. The results of all these studies are, however, far from conclusive, for the simple reason that it has never been possible to isolate any substances other than irradiated sterols that would regulate the inorganic phosphorus content of the blood or would cure rickets. The dose relationship between calcium and phosphate metabolism, as well as the discovery by Collip ~ of a hormone regulating calcium metabolism, has, however, made it at least plausible to assume the existence of a hormone regulating phosphorus metabolism. Two years ago 16 found that thyroxine elevates the inorganic phosphate content of the blood of healthy infants, an observation that has recently been confirmed by RietsehelJ I pointed out, however, that this does not mean that thyroxine can be regarded as the phosphateincreasing" substance when rickets is cured by vitamin D. As a matter of fact, I found later* that thyroxine does not alter the excretion of phosphorus and calcium through the feces and urine in the same manner as is observed when vitamin D is given. That is to say, up to the present time we do not know any substance that can be considered a phosphate hormone produced by an endocrine gland through the stimulating" effect of irradiated ergosterol. The importance of this question in understanding the physiologic action of vitamin D and the pathogenesis of riekeN, as well, induced me to approach the problem in another way. F r o m the Babies a~d Childrens H o s p i t a l and ti~e D e p a r t m e n t of P e di a t ri c s , School of Medicine, W e s t e r n tZeserve University. 480
HEYMANN:
ACTION
AND
METABOLISM
OF
VITAMIN
D
~I
Warkany ~ found that the administration of one large dose of vigantol to rabbits led to a definite increase in the inorganic phosphate content of the blood, an increase which, as I will show later, is not accompanied by a similar effect on the calcium content. My assumptiolL in undertaking the studies presented, was ~hat if vitamin D brought about this rise in inorganic phosphate in rabbits' blood by stimulating the production of a phosphate hormone, the chances were that this hypothetical substance might be found in their blood if it was taken at the peak of the vitamin D hyperphosphatemia.
~x
Y'~
~o.,,V~os{cro[ I(ZOo,~O~..Ler~ [~,EtL I~.it.t~.
//~ /
\
7
5"
~. 4X"
~. Chart l.--Curves showing response of inorganic 10hosphate and calcium in the blood of rabbits after a single dose of 2,0 c.c. of viosterol in oil (200,000 international units of vitamin I), or about 1.6 rug. of irradiated ergosterol).
The assumption that there was such a substance would be justified if it was found that the blood serum of these rabbits cured rachitic
rats by increasing the diminished blood phosphates, provided that it could be proved that the curative substance did not represent irradiated ergosterol itself. Warkany in his experiments used 100 mg. of irradiated ergosterol given as vigantol. The antirachitie preparations of today, however, are quite different from the vigantol of that time (1929). They contain less ergosterol and certainly fewer toxic by-products. It was necessary, therefore, to repeat Warkany's experiments, using modern ergosterol preparations. These results are now reported.
482
TIIE
JOURNAL
01~ PEDIATRICS
To rabbits weighing f r o m 3,000 to 4,000 gm. 20 c.e. of viosterol in oil was administered b y stomach tube. This is a large dose, representing 200,000. international units of v i t a m i n D ; however, it is equivalent to a p p r o x i m a t e l y 1.6 rag. of i r r a d i a t e d ergosterol in other words, a dose less t h a n one-fiftieth of t h a t used b y W a r k a n y . The blood of the rabbits was obtained f r o m the f e m o r a l vein, and duplicate phosphorus and calcium determinations were m a d e b y the t i t r i m e t r i e method of Samson ~~ using 1 e.e. of serum for each determination. Chart 1 shows that, f r o m five to ten d a y s a f t e r viosterol was given, the inorganic phosphate content of the blood increased a n d r e m a i n e d elevated for f r o m the t e n t h to the f o u r t e e n t h day, while no r e g u l a r and definite response was found in the calcium content of the blood.
Fig'. 1 . - - l = ~ o e n t g e n o g r a m s daily (right) intramuscular which the s e r u m w a s taken, (200,000 i n t e r n a t i o n a l u n i t s
of :Rat 5397 (see T a b l e I ) b e f o r e ( l e f t ) a n d a f t e r t e n i n j e c t i o n s of 0,6 e.c. of r a b b i t s e r u m . :Rabbit 446, f r o m h a d b e e n g i v e n , six d a y s p r e v i o u s l y , 20 e.e. of v i o s t e r o l of v i t a m i n I), or a b o u t 1.6 mY. of i r r a d i a t e d e r g o s t e r o l ) .
I f it were t r u e t h a t this h y p e r p h o s p h a t e m i e response was due to the p r o d u e t i o n of a p h o s p h a t e hormone, the shape of the curves in C h a r t 1 would indicate t h a t its greatest concentration in the blood was on the fifth to the t e n t h d a y a f t e r the a d m i n i s t r a t i o n of v i t a m i n D, beeause the g r e a t e s t stimulation of tile p h o s p h a t e m e t a b o l i s m occurred at that time. Consequently, I t o o k the blood of tlle r a b b i t s on the fifth to t e n t h d a y a f t e r the viosterol h a d been given and injected daily, for ten consecutive days, 0.6 e.e. of the serum i n t r a m u s c u l a r l y in r a t s which had been for three weeks on a rickets-producing diet (Steenhoek No. 2965). The results of these five experiments, r e c o r d e d in Table I, show t h a t the r a b b i t s e r u m possessed high antiraehitie p o t e n c y in each of the
:~IEY~[ANN:
ACTION
AND lVIE:TABOLISIK O F V I T A M I N
48,~
D
thirteen r a t s used. The r o e n t g e n o g r a p h i c findings in all the r a t s w e r e without exception the same as those s h o w n in Fig. 1 (R.at 5397). flYABLE I ~EALING OF RACHITIC RATS BY INTRAIvIUSCULAg INJECTION OF 0.6 C.C. OF S E R U M OBTAINED ~ R O M ]~ABJ31TS GIVEN 200~000 INTERNATIONAL UNITS OF VITAMIN D BY ~r (20 C.C. OF VIOSTEROL) FIVE TO TEN DAYS PREVIOUSLY
I~AT
X-RAY HEALING AFTER EIGHT TEN DAYS DAYS
TREATMENT
NO,
5392 5393 5396
Rabbit 445 on 11/3/34 received c.c. o f v i o s t e r o l . Oi~ 1 1 / 1 3 / 3 4 b l o o d w a s t a k e n a n d 0.6 c.c. this serum injected daffy in r~s.
20 its of the
§ -l+ ++
+++ +++ +++
5397 5399 5401
Rabbit 446 on 10/31/3~ received c.c. o f v i o s t e r o l . On _11/5/34 b l o o d w a s t a k e n a n d 0.6 c.c. this serum injected daffy in rats.
20 ~ts of the
++ ++ ++
+++ +++ +++
5452 5453 5459
Rabbits 447 and 448 on 12/22/34 e a c h r e c e i v e d 20 c.c. o f v i o s t e r o l . 0 ~ 1 2 / 2 7 / 3 4 t h e i r bloo,d w a s t a k e n a n d 0.6 e.e. o f t h i s m i x e d se~ rum injected daily in the rats.
++ ++ +++
5533 553~
Rabbits 458 and 457 on 2/t9/35 e a c h r e c e i v e d 20 e.e. o f v i o s t e r o l . O n 2/25/35 t h e i r b l o o d w a s t a k e n a n d 0.6 c.c. o f t h i s m i x e d s e r u m injec'ted daily in the rats.
++ ++
§
5636 5637
R a b b i t s 468 a n d 469 on 4 / 1 5 / 3 5 e a c h r e c e i v e d 20 c.c. o f v i o s t e r o l . On 4/20/35 their blood was taken a n d 0.6 c.c. o f t h i s m i x e d s e r u m injected daily in the rats.
++ ++
+++i ++++l
§
6.37
]2.4
5.15
18.6
10.3
14.6
5.5
]4.4
32.2
14,0
5.43
13.6
5.1
15.0
3.99
--f
7.0
1 5 , 0 ] 4~.94
+++ ++++
+++
-
Averages
SEgUM OF RABBITS OB~ RATS +: P Ca , P Ca ~2.~ ~. % M,. % ~G.
-
11.2
i
5.0
16.9
*Throughou~ these studies the rats were killed ten days after treatment had been s t a r t e d so t h a t t h e i r b l o o d w a s o b t a i n e d f o r d e t e r m i n a t i o n o f p h o s p h o r u s a n d c a l c i u m on t h e s a m e d a y o n w h i c h t h e l a s t r o e n t g e n o g r a m s w e r e t a k e n . Phosphorus and c a l c i u m c o n t e n t s of b l o o d s e r u m a r e g i v e n in m i l l i g r a m s p e r h u n d r e d c u b i c c e n t i m e t e r s . $ S e r u m f o r d e t e r m i n a t i o n w a s lost.
Several control e x p e r i m e n t s m u s t be r e p o r t e d here before any further discussion can take place. I t was first n e c e s s a r y to p r o v e t h a t r a t s k e p t u n d e r the same conditions as were the e x p e r i m e n t a l animals did not show a n y healing without t r e a t m e n t ; f u r t h e r m o r e , t h a t the s e r u m of r a b b i t s which h a d n e v e r received viosterol did not cure the rachitie condition; and, finally, t h a t the c u r a t i v e effect of the serum of those rabbits which h a d received the large v i t a m i n dose was not due to the increased phosphate e o n t e n t - - a question t h a t could be decided by i n j e c t i n g aqueous solution of Na2HP Q in the same concentration as was p r e s e n t in the s e r u m .
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THE JOURNAL OF PEDIATRICS
The results from the group of control experiments were entirely negative and are presented in Table II. TABLE I I CONTROL EXPERIMENTS
RAT NO.
TREAT~[ENT
X-RAY HEALING AFTEIr EIGHT TEN DAYS DAYS
SERUl~[ 0]~~ I~ATS* P Ca ~G. e/v MG. v/~
5388 5394 5395
None
0 0 0
0 0 0
3.77
14.0
5398 5400 5402
None
0 0 0
0 0 0
5.71
13.0
5380 5382 5383
Received daily i n t r a m u s c u l a r i n j e c t i o n s of 0.6 c.c. of s e r u m f r o m healthy t~abbit 450 which died not receive viosterol.
0 0 0
O 0 0
4.72
13.0
5385 5389 5391
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of s e r u m f r o m healthy R a b b i t 448 which had not received viosterol.
O 0 0
0 0 0
4.72
11.8
5448 5455
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of s e r u m f r o m healthy l~abbit 449 which did not receive viosterol.
0 0
0 0
5.]5
10.4
5649 5648
Received dMly i n t r a m u s c u l a r injections of 0.5 c.c. of serum f r o m i n f a n t ill with spasmophilia (whose serum P was 1.7 rag. % and Ca 7.4 mg. % ) .
0 0
0 0
4.1
10.2
5457 5462
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of N a f H P O ~ solution containi n g 15 rag. % P.
0 0
0 0
2.0
1J.6
4.31
] 2.0
Averages *S'ee footnote 1, Table I.
Returning to the results of the five experiments recorded in Table I, the conclusion consequently must be drawn that the serums of rabbits which had received, five to ten days previously, approximately 1.6 rag. of irradiated ergosterol (200,000 international units of vitamin D) possessed very high antirachitic potencies. The question naturally arises at once whether or not this was due, partly or entirely, to the presence of vitamin D in the blood. It must, however, be kept in mind that the presence of irradiated ergosterol would not exclude the assumption that there might be a second substance produced by, and present with, vitamin D in the same serum. The data recorded in Table III clearly answer these questions. Experiments I and II show that the rickets-curing substance can be partly removed from the serum by simple extraction with ether and, furthermore, that it is soluble in oil. Experiments III and IV demonstrate that this substance gradually loses its antirachitic potency if it is overirradiated with ultraviolet light. Finally, Experiments V and VI
IIEY~/[ANN:
ACTION AND IViETABOLISM OF VITAMIN D
485
show that the ultrafiltrate, ~ used in even l a r g e r doses than was the original serum, does not contain any rickets-curlng substance. TABLE III EXPERIMENTS PKOVINO TttAT THERE IS ~]'O I~ACHITIC :HEALING SUBSTANCE OT~IER THAN VITAMIN D IN TIIE SEBU1VI OF ~ABBITS ~IVEN 200,000 INTERNATIONAL UNITS OF VITA1V[IN D BY 1ViOUTI-I (20 C.C. OF VIOSTEROL) FIVE TO TEN DAYS PREVIOUSLY vs OF EXPERIMENT
TREATltIENT
st Ca 1viG. v~
F r o m R a b b i t s 468 a n d 469 ~ 15 c.c. of serum w a s e x t r a c t e d 8 t i m e s w i t h 15 c.c. of ether. A f t e r e v a p o r a t i o n of e~her, r e s i d u e was dissolved i n 5 c.c. of wesson oil; 0.2 c.c. of t h i s oil ( e q u a l to 0.6 e.c. of o r i g i n a l s e r u m ) was i n j e c t e d .daily i n t r a m u s c u l a r l y i n t h e rats.
11.4
II
A f t e r e x t r a c t i o n 8 t i m e s w i t h ether, 0.6 c.c. o f serum f r o m l ~ a b b i t s 468 a n d 469 ~" w a s d a i l y i n j e c t e d i n t r a m u s cularly.
12.0
III
S e r u m f r o m R a b b i t s 447 a n d 448 ~ w a s i r r a d i a t e d 12 hr. w i t h a t t a n a u mercury v a p o r q u a r t z l a m p a% 46 cm. d i s t a n c e ; 0.6 c.c. of t hi s s e r u m w a s daily injected intramuscularly in the rats.
12.0
IV
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 0.6 e.c. of s e r u m f r o m R a b b i t 458:': i r r a d i a t e d f o r 26 hr. as d e s c r i b e d in Experiment III.
11.4
V
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 0.6 c.c. of u ] t r a f i l t r a t e f r o m s e rum of Rabb i t s 447 a n d 448. "~
10.8
VI
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 1 c.c. of u l t r a f i l t r a t e f r o m s e rum of Rabb i t s 457 a n d 458. ~
9.8
*The a n t i r a c h i t i c potency of the u n t r e a t e d s e r u m is recorded in Table I. ~See footnote 1, Table I. $Serum for d e t e r m i n a t i o n was lost.
These results prove that the curative p o t e n c y of this serum was due to a substance t h a t had all the qualities possessed also b y i r r a d i a t e d ergosterol: namely, it was soluble in ether and in oil; its antirachitic effectiveness was destructible by overirradiation ;11 and, because of its colloidal state in the lipoid fraction of serum, it was not ultrafiltrable. That e v e r i r r a d i a t i o n with ultraviolet light completely d e s t r o y e d the antirachitic value of the serum makes e x t r e m e l y unlikely the possibility that a second substance was present, as do also the negative *Filters were m a d e w i t h an 8 per cent collodion solution in g l a c i a l acetic acid. U l t r a f l l t r a t e s w e r e free from proteins, as shown by the s u l p h o s a l i c y l i e a c i d test.
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THE
JOURNAL
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results obtained w i t h serum ultrafiltrates. The conclusion must therefore be d r a w n that v i t a m i n D itself produced the healing of the rachitic bone lesion a n d t h a t , under the conditions described, it does not lead to the production of another substance capable of healing rickets or of increasing the p h o s p h a t e content of the blood. H a v i n g ascertained that the serum of rabbits to which 200,000 international units of vitamin D had been given contained, five to ten days later, considerable a m o u n t s of irradiated ergosterol, I t h o u g h t it w o r t h while to attempt to learn h o w l o n g after its administration vitamin D circulates in vivo in the blood. Such e x p e r i m e n t s have not, so far as I k n o w , been p e r f o r m e d or reported in the literature up to the present time and should reveal the fate of v i t a m i n D w i t h i n the body in a more physiologic m a n n e r than do e x p e r i m e n t s in vitro. The same rabbits were used for this group of experiments as for the previous group. Their blood w a s taken at intervals of one, three, six, TABLE I V SHOWING
OF TIME TIIAT VITA]YIIN :D IS ~OUND IN ]:~L00D OF ]~ABBITS ADMINISTEATION OF SINGLE :DosE
LENGTH
I RAB-
DATE 2 0 C.C.
BIT NO.
VIOSTEROL WAS
GIVEN 445
12/
DATE BLOOD WAS TAKEN
LENGTH OF 2K-RAY TItlE BEHEALING T-WEEN AFTER RAT VIOSTEROL ADMINISTRA- NO. p C~t TION AND EIGtIT TEN5[G. % MG, % TAKING OF DAYS DAYS BLOOD
6/34 12/12/34
12/27/34
2/23/35 448
12/22/34
12/28/34
2/23/35 457
2/19/35
2/ 5/35 3/ 0/35
RABBIT SEI~UM
4.8 4.8
I wk.
6.7
3 wk.
5.1
11 w k .
4.1 7.8
1 wk.
5.2
9 wk.
4.2 4.3
I wk.
4.8
6 wk.
5/ 3/35
11 w k .
*See f o o t n o t e 1, T a b l e I. j'Serum for determination
was
lost.
AFTER
EAT S E R U M ~"
~) MG,
Ca
MG. %
5397 5399 5401
+ + ++ ++
+++ +++ +++
5.5
14.4
5446 5447 5456
+ + ++ + +
++ + +++ + ++
4.9
13.0
5540 5541
O 0
+ q
3.9
14.2
5452 5453 5459
+ + ++ ++
++ + +++ +++
5.4
13.6
5537 5539
0 0
+ ? +
2.3
13.6
5533 5534
++ ++
+ + + +++
4.0
5608 5609 i 5610
0 0 0
+ § + + +
5684 5696 5698
0 0 0
+ ? 0 i 0
8.5
10.4
HEY~[ANN:
ACTIO,N AND
:~ETABOLISM
O l ,~ V I T A M I N
D
487
nine, and eleven weeks following the a d m i n i s t r a t i o n of 200,000 intern a t i o n a l units of v i t a m i n D (20 e.c. of viosterol in oil, c o r r e s p o n d i n g a p p r o x i m a t e l y to 1.6 rag. of i r r a d i a t e d ergosterol), and was tested on rachitie r a t s in the m a n n e r a l r e a d y described. The e x p e r i m e n t s s u m m a r i z e d in Table I V show that, u n d e r the conditions prevailing, the s e r u m of r a b b i t s which h a d received viosterol was able to establish healing in raehitic r a t s in ten days, even if the blood was t a k e n as long as f r o m two to two and a h a l f months a f t e r the a d m i n i s t r a t i o n of the vitamin. The first distinct decrease in the potency of the s e r u m was noticed a f t e r six to nine weeks, and t r a c e s cbuld be detected in one instance (Rabbit 445) a f t e r eleven weeks. ]n other words, r a b b i t s weighing f r o m 3,000 to 4,000 gin. needed f r o m two and a half to three months t o d e s t r o y or excrete 1.6 rag. of irr a d i a t e d ergosterol (200,000 i n t e r n a t i o n a l units). COMMENT
One finds in the l i t e r a t u r e no conclusive evidence w h a t e v e r to supp o r t the view t h a t there m i g h t exist a h o r m o n a l r e g u l a t i o n of the phosphate metabolism, as is k n o w n to be the case for the calcium content o f the blood. The studies p r e s e n t e d here, in an a t t e m p t to approach the p r o b l e m in a new way, have p r o v e d entirely negative. It must consequently be considered wiser to a b a n d o n a hypothesis t h a t cannot be confirmed. I t is better, I think, to see in i r r a d i a t e d sterols themselves the only substances the lack of which leads to rachitic h y p o p h o s p h a t e m i a and the presence of which elevates the inorganic phosphorus in the blood. I n which tissue v i t a m i n D has its point of a t t a c k and b y which chemical m e c h a n i s m it increases the diminished inorganic p h o s p h a t e s in the blood of rachitic subjects m u s t still be considered unknown. So f a r as the low p h o s p h o r u s rickets in infants i s concerned, I f o u n d 1~ w e i g h t y evidence t h a t suggests t h a t the r e g u l a t i o n of the intestinal absorption of p h o s p h a t e s does not explain the situation satisfactorily. F u r t h e r studies on this subject are necessary and are in progress. The f a c t t h a t it has now been shown t h a t v i t a m i n D was circulating in the blood for at least two to three months a f t e r the a d m i n i s t r a t i o n of a single dose of a p p r o x i m a t e l y 1.6 rag. of i r r a d i a t e d ergosterol in o volves interesting new problems. I t is superfluous to emphasize the clinical value of these observations to p e d i a t r i c i a n s who use v i t a m i n D more or less r o u t i n e l y as an a g e n t f o r p r e v e n t i n g rickets in infants. I t is k n o w n t h a t d a m a g e can be done b y o v e r d o s a g e of v i t a m i n D, 13 and it is now shown t h r o u g h these studies t h a t continuous administration m a y easily lead to an undesirable a c c u m u l a t i o n because of the a p p a r e n t l y low r a t e of consumption. The question w h e t h e r the b o d y destroys or excretes v i t a m i n D cannot yet be decided. The w o r k of Coppens and Metz, 1~ who r e p o r t e d
488
THE
JOURNAL
0]~~ P E D I A T R I C S
t h a t vitamin D is destroyed in vitro by blood and lung tissue, makes the assumption likely t h a t it m a y be d e s t r o y e d in vivo. The possibility that i r r a d i a t e d ergosterol may also be excreted t h r o u g h bile or t h r o u g h the intestinal wall must, however, be kept in mind. Studies on these problems are in progress. SUMMARY
1. The oral administration to rabbits of 20 c.c. of viosterol in oil (200,000 international units of vitamin D, equivalent to a p p r o x i m a t e l y 1.6 mg. of i r r a d i a t e d ergosterol) led to an increase in the inorganic phosphorus content of the blood without c o r r e s p o n d i n g l y affecting the blood calcium content. This action took place about five days after the viosterol had been given and lasted until the tenth to the fifteenth day. 2. I n order to learn w h e t h e r or not this action might be due to the stimulating effect of vitamin D upon an endocrine gland, thus leading to the p r o d u c t i o n of a phosphate hormone, the serum of these rabbits was tested on rachitic rats. The r a b b i t s ' blood was obtained five days after the viosterol had been given and was f o u n d to cure r achitic rats in f r o m eight to ten days if 0.6 c.c. of the serum was daily injected intramuscularly. 3. No evidence was found, however, to j u s t i f y the assumption that a n y substance other t h a n irradiated ergosterol was present in this serum or was even p a r t l y responsible for its curative potency, because the rickets-curing substance of this serum was soluble in ether and in oil, was destroyed completely by overirradiation with ultraviolet rays, and was f o u n d not to be ultrafiltrable. 4. W h e n viosterol was given b y m o u t h to rabbits in a single dose corresponding to a p p r o x i m a t e l y 1.6 rag. of irradiated ergosterol, detectable amounts of vitamin D were f o u n d to be circulating in the blood for f r o m two to three months. RE~ERENCES
1. Erdheim, J. : Raehitis und Epithe]kSrperehen, Denksehr. d. k. Akad. der Wissensch. ~r Klasse, Wien, 1914, p. 90. 2. Pappenheimer, A. M., and Minor, 5. : J. 3/i. t~esearch 42: 391, 1920-21. 3. ]~itter, C.: ~'rankfurt. Ztsehr. f. Path. 24: 137, 1921. 4. Nitsehke, A." Klin. ~Vchnschr. 12: 1793, 1933. 5. Co]lip, J . B . : Ann. Clin. 1Vied. 4: 219, 1925-26. 6. iqeymann, W., and ~aier, E. : Ztschr. f. Kinderh. 55: 512, 1933. 7. I~ietsehel: Discussion of paper by Nitschke, Verhandl. d. deutseh. Gese]lseh. f. Kinderh., ~onatsehr. f. Kinderh. 62: 37, 1934. 8. Heymann, W. : Unpublished data. 9. Wgrkany, if.: Klin. Wchnsehr. 9: 63, 1930. 10. Samson, K.: Klin. Wchnsehr. 10: 1766, 1931. 11. Hess, A . F . : Ztschr. f. Kinderh. 39: 395, 1925. 12. tteymann, W.: Ztsehr. f. Kinderh. 45: 239,, 1927-28; 46: 575, 584, ]928; 13. Pfunnenstiel, W.: 3/iiinchen. reed. Wehnsehr. 75" 1113, 1928. 14. Coppens~ P. A., and Me%z, G.A.: Biochem. Ztschr. 266: 169, 1933.
tile present time our knowledge of the mode of action of U NTIL vitamin D has been extraordinarily meager and may be summed up almost entirely in the observation that vitamin D increases the diminished inorganic phosphate content of the blood in rickets and brings back to normal the diminished calcium content in spasmophilia. As soon as one raises the question how vitamin D brings about the regulation of inorganic phosphates and calcium in the blood, one enters a vast field of speculation. One of the hypotheses is the assumption that irradiated ergosterol stimulates an endocrine gland. The work of Erdheim, ~ Pappenheimer and Minor, ~ Ritter ~ and others seemed to indicate the importance of the parathyroid glands in infantile ric.kets, while the recent publications of Nitsehke 4 emphasized the connection between the lymphoid tissues (spleen, lymph nodes, and thymus) and the thyroid gland in regulating the phosphorus and calcium content of the blood. The results of all these studies are, however, far from conclusive, for the simple reason that it has never been possible to isolate any substances other than irradiated sterols that would regulate the inorganic phosphorus content of the blood or would cure rickets. The dose relationship between calcium and phosphate metabolism, as well as the discovery by Collip ~ of a hormone regulating calcium metabolism, has, however, made it at least plausible to assume the existence of a hormone regulating phosphorus metabolism. Two years ago 16 found that thyroxine elevates the inorganic phosphate content of the blood of healthy infants, an observation that has recently been confirmed by RietsehelJ I pointed out, however, that this does not mean that thyroxine can be regarded as the phosphateincreasing" substance when rickets is cured by vitamin D. As a matter of fact, I found later* that thyroxine does not alter the excretion of phosphorus and calcium through the feces and urine in the same manner as is observed when vitamin D is given. That is to say, up to the present time we do not know any substance that can be considered a phosphate hormone produced by an endocrine gland through the stimulating" effect of irradiated ergosterol. The importance of this question in understanding the physiologic action of vitamin D and the pathogenesis of riekeN, as well, induced me to approach the problem in another way. F r o m the Babies a~d Childrens H o s p i t a l and ti~e D e p a r t m e n t of P e di a t ri c s , School of Medicine, W e s t e r n tZeserve University. 480
HEYMANN:
ACTION
AND
METABOLISM
OF
VITAMIN
D
~I
Warkany ~ found that the administration of one large dose of vigantol to rabbits led to a definite increase in the inorganic phosphate content of the blood, an increase which, as I will show later, is not accompanied by a similar effect on the calcium content. My assumptiolL in undertaking the studies presented, was ~hat if vitamin D brought about this rise in inorganic phosphate in rabbits' blood by stimulating the production of a phosphate hormone, the chances were that this hypothetical substance might be found in their blood if it was taken at the peak of the vitamin D hyperphosphatemia.
~x
Y'~
~o.,,V~os{cro[ I(ZOo,~O~..Ler~ [~,EtL I~.it.t~.
//~ /
\
7
5"
~. 4X"
~. Chart l.--Curves showing response of inorganic 10hosphate and calcium in the blood of rabbits after a single dose of 2,0 c.c. of viosterol in oil (200,000 international units of vitamin I), or about 1.6 rug. of irradiated ergosterol).
The assumption that there was such a substance would be justified if it was found that the blood serum of these rabbits cured rachitic
rats by increasing the diminished blood phosphates, provided that it could be proved that the curative substance did not represent irradiated ergosterol itself. Warkany in his experiments used 100 mg. of irradiated ergosterol given as vigantol. The antirachitie preparations of today, however, are quite different from the vigantol of that time (1929). They contain less ergosterol and certainly fewer toxic by-products. It was necessary, therefore, to repeat Warkany's experiments, using modern ergosterol preparations. These results are now reported.
482
TIIE
JOURNAL
01~ PEDIATRICS
To rabbits weighing f r o m 3,000 to 4,000 gm. 20 c.e. of viosterol in oil was administered b y stomach tube. This is a large dose, representing 200,000. international units of v i t a m i n D ; however, it is equivalent to a p p r o x i m a t e l y 1.6 rag. of i r r a d i a t e d ergosterol in other words, a dose less t h a n one-fiftieth of t h a t used b y W a r k a n y . The blood of the rabbits was obtained f r o m the f e m o r a l vein, and duplicate phosphorus and calcium determinations were m a d e b y the t i t r i m e t r i e method of Samson ~~ using 1 e.e. of serum for each determination. Chart 1 shows that, f r o m five to ten d a y s a f t e r viosterol was given, the inorganic phosphate content of the blood increased a n d r e m a i n e d elevated for f r o m the t e n t h to the f o u r t e e n t h day, while no r e g u l a r and definite response was found in the calcium content of the blood.
Fig'. 1 . - - l = ~ o e n t g e n o g r a m s daily (right) intramuscular which the s e r u m w a s taken, (200,000 i n t e r n a t i o n a l u n i t s
of :Rat 5397 (see T a b l e I ) b e f o r e ( l e f t ) a n d a f t e r t e n i n j e c t i o n s of 0,6 e.c. of r a b b i t s e r u m . :Rabbit 446, f r o m h a d b e e n g i v e n , six d a y s p r e v i o u s l y , 20 e.e. of v i o s t e r o l of v i t a m i n I), or a b o u t 1.6 mY. of i r r a d i a t e d e r g o s t e r o l ) .
I f it were t r u e t h a t this h y p e r p h o s p h a t e m i e response was due to the p r o d u e t i o n of a p h o s p h a t e hormone, the shape of the curves in C h a r t 1 would indicate t h a t its greatest concentration in the blood was on the fifth to the t e n t h d a y a f t e r the a d m i n i s t r a t i o n of v i t a m i n D, beeause the g r e a t e s t stimulation of tile p h o s p h a t e m e t a b o l i s m occurred at that time. Consequently, I t o o k the blood of tlle r a b b i t s on the fifth to t e n t h d a y a f t e r the viosterol h a d been given and injected daily, for ten consecutive days, 0.6 e.e. of the serum i n t r a m u s c u l a r l y in r a t s which had been for three weeks on a rickets-producing diet (Steenhoek No. 2965). The results of these five experiments, r e c o r d e d in Table I, show t h a t the r a b b i t s e r u m possessed high antiraehitie p o t e n c y in each of the
:~IEY~[ANN:
ACTION
AND lVIE:TABOLISIK O F V I T A M I N
48,~
D
thirteen r a t s used. The r o e n t g e n o g r a p h i c findings in all the r a t s w e r e without exception the same as those s h o w n in Fig. 1 (R.at 5397). flYABLE I ~EALING OF RACHITIC RATS BY INTRAIvIUSCULAg INJECTION OF 0.6 C.C. OF S E R U M OBTAINED ~ R O M ]~ABJ31TS GIVEN 200~000 INTERNATIONAL UNITS OF VITAMIN D BY ~r (20 C.C. OF VIOSTEROL) FIVE TO TEN DAYS PREVIOUSLY
I~AT
X-RAY HEALING AFTER EIGHT TEN DAYS DAYS
TREATMENT
NO,
5392 5393 5396
Rabbit 445 on 11/3/34 received c.c. o f v i o s t e r o l . Oi~ 1 1 / 1 3 / 3 4 b l o o d w a s t a k e n a n d 0.6 c.c. this serum injected daffy in r~s.
20 its of the
§ -l+ ++
+++ +++ +++
5397 5399 5401
Rabbit 446 on 10/31/3~ received c.c. o f v i o s t e r o l . On _11/5/34 b l o o d w a s t a k e n a n d 0.6 c.c. this serum injected daffy in rats.
20 ~ts of the
++ ++ ++
+++ +++ +++
5452 5453 5459
Rabbits 447 and 448 on 12/22/34 e a c h r e c e i v e d 20 c.c. o f v i o s t e r o l . 0 ~ 1 2 / 2 7 / 3 4 t h e i r bloo,d w a s t a k e n a n d 0.6 e.e. o f t h i s m i x e d se~ rum injected daily in the rats.
++ ++ +++
5533 553~
Rabbits 458 and 457 on 2/t9/35 e a c h r e c e i v e d 20 e.e. o f v i o s t e r o l . O n 2/25/35 t h e i r b l o o d w a s t a k e n a n d 0.6 c.c. o f t h i s m i x e d s e r u m injec'ted daily in the rats.
++ ++
§
5636 5637
R a b b i t s 468 a n d 469 on 4 / 1 5 / 3 5 e a c h r e c e i v e d 20 c.c. o f v i o s t e r o l . On 4/20/35 their blood was taken a n d 0.6 c.c. o f t h i s m i x e d s e r u m injected daily in the rats.
++ ++
+++i ++++l
§
6.37
]2.4
5.15
18.6
10.3
14.6
5.5
]4.4
32.2
14,0
5.43
13.6
5.1
15.0
3.99
--f
7.0
1 5 , 0 ] 4~.94
+++ ++++
+++
-
Averages
SEgUM OF RABBITS OB~ RATS +: P Ca , P Ca ~2.~ ~. % M,. % ~G.
-
11.2
i
5.0
16.9
*Throughou~ these studies the rats were killed ten days after treatment had been s t a r t e d so t h a t t h e i r b l o o d w a s o b t a i n e d f o r d e t e r m i n a t i o n o f p h o s p h o r u s a n d c a l c i u m on t h e s a m e d a y o n w h i c h t h e l a s t r o e n t g e n o g r a m s w e r e t a k e n . Phosphorus and c a l c i u m c o n t e n t s of b l o o d s e r u m a r e g i v e n in m i l l i g r a m s p e r h u n d r e d c u b i c c e n t i m e t e r s . $ S e r u m f o r d e t e r m i n a t i o n w a s lost.
Several control e x p e r i m e n t s m u s t be r e p o r t e d here before any further discussion can take place. I t was first n e c e s s a r y to p r o v e t h a t r a t s k e p t u n d e r the same conditions as were the e x p e r i m e n t a l animals did not show a n y healing without t r e a t m e n t ; f u r t h e r m o r e , t h a t the s e r u m of r a b b i t s which h a d n e v e r received viosterol did not cure the rachitie condition; and, finally, t h a t the c u r a t i v e effect of the serum of those rabbits which h a d received the large v i t a m i n dose was not due to the increased phosphate e o n t e n t - - a question t h a t could be decided by i n j e c t i n g aqueous solution of Na2HP Q in the same concentration as was p r e s e n t in the s e r u m .
484
THE JOURNAL OF PEDIATRICS
The results from the group of control experiments were entirely negative and are presented in Table II. TABLE I I CONTROL EXPERIMENTS
RAT NO.
TREAT~[ENT
X-RAY HEALING AFTEIr EIGHT TEN DAYS DAYS
SERUl~[ 0]~~ I~ATS* P Ca ~G. e/v MG. v/~
5388 5394 5395
None
0 0 0
0 0 0
3.77
14.0
5398 5400 5402
None
0 0 0
0 0 0
5.71
13.0
5380 5382 5383
Received daily i n t r a m u s c u l a r i n j e c t i o n s of 0.6 c.c. of s e r u m f r o m healthy t~abbit 450 which died not receive viosterol.
0 0 0
O 0 0
4.72
13.0
5385 5389 5391
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of s e r u m f r o m healthy R a b b i t 448 which had not received viosterol.
O 0 0
0 0 0
4.72
11.8
5448 5455
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of s e r u m f r o m healthy l~abbit 449 which did not receive viosterol.
0 0
0 0
5.]5
10.4
5649 5648
Received dMly i n t r a m u s c u l a r injections of 0.5 c.c. of serum f r o m i n f a n t ill with spasmophilia (whose serum P was 1.7 rag. % and Ca 7.4 mg. % ) .
0 0
0 0
4.1
10.2
5457 5462
Received daily i n t r a m u s c u l a r injections of 0.6 c.c. of N a f H P O ~ solution containi n g 15 rag. % P.
0 0
0 0
2.0
1J.6
4.31
] 2.0
Averages *S'ee footnote 1, Table I.
Returning to the results of the five experiments recorded in Table I, the conclusion consequently must be drawn that the serums of rabbits which had received, five to ten days previously, approximately 1.6 rag. of irradiated ergosterol (200,000 international units of vitamin D) possessed very high antirachitic potencies. The question naturally arises at once whether or not this was due, partly or entirely, to the presence of vitamin D in the blood. It must, however, be kept in mind that the presence of irradiated ergosterol would not exclude the assumption that there might be a second substance produced by, and present with, vitamin D in the same serum. The data recorded in Table III clearly answer these questions. Experiments I and II show that the rickets-curing substance can be partly removed from the serum by simple extraction with ether and, furthermore, that it is soluble in oil. Experiments III and IV demonstrate that this substance gradually loses its antirachitic potency if it is overirradiated with ultraviolet light. Finally, Experiments V and VI
IIEY~/[ANN:
ACTION AND IViETABOLISM OF VITAMIN D
485
show that the ultrafiltrate, ~ used in even l a r g e r doses than was the original serum, does not contain any rickets-curlng substance. TABLE III EXPERIMENTS PKOVINO TttAT THERE IS ~]'O I~ACHITIC :HEALING SUBSTANCE OT~IER THAN VITAMIN D IN TIIE SEBU1VI OF ~ABBITS ~IVEN 200,000 INTERNATIONAL UNITS OF VITA1V[IN D BY 1ViOUTI-I (20 C.C. OF VIOSTEROL) FIVE TO TEN DAYS PREVIOUSLY vs OF EXPERIMENT
TREATltIENT
st Ca 1viG. v~
F r o m R a b b i t s 468 a n d 469 ~ 15 c.c. of serum w a s e x t r a c t e d 8 t i m e s w i t h 15 c.c. of ether. A f t e r e v a p o r a t i o n of e~her, r e s i d u e was dissolved i n 5 c.c. of wesson oil; 0.2 c.c. of t h i s oil ( e q u a l to 0.6 e.c. of o r i g i n a l s e r u m ) was i n j e c t e d .daily i n t r a m u s c u l a r l y i n t h e rats.
11.4
II
A f t e r e x t r a c t i o n 8 t i m e s w i t h ether, 0.6 c.c. o f serum f r o m l ~ a b b i t s 468 a n d 469 ~" w a s d a i l y i n j e c t e d i n t r a m u s cularly.
12.0
III
S e r u m f r o m R a b b i t s 447 a n d 448 ~ w a s i r r a d i a t e d 12 hr. w i t h a t t a n a u mercury v a p o r q u a r t z l a m p a% 46 cm. d i s t a n c e ; 0.6 c.c. of t hi s s e r u m w a s daily injected intramuscularly in the rats.
12.0
IV
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 0.6 e.c. of s e r u m f r o m R a b b i t 458:': i r r a d i a t e d f o r 26 hr. as d e s c r i b e d in Experiment III.
11.4
V
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 0.6 c.c. of u ] t r a f i l t r a t e f r o m s e rum of Rabb i t s 447 a n d 448. "~
10.8
VI
D a i l y i n t r a m u s c u l a r i n j e c t i o n s of 1 c.c. of u l t r a f i l t r a t e f r o m s e rum of Rabb i t s 457 a n d 458. ~
9.8
*The a n t i r a c h i t i c potency of the u n t r e a t e d s e r u m is recorded in Table I. ~See footnote 1, Table I. $Serum for d e t e r m i n a t i o n was lost.
These results prove that the curative p o t e n c y of this serum was due to a substance t h a t had all the qualities possessed also b y i r r a d i a t e d ergosterol: namely, it was soluble in ether and in oil; its antirachitic effectiveness was destructible by overirradiation ;11 and, because of its colloidal state in the lipoid fraction of serum, it was not ultrafiltrable. That e v e r i r r a d i a t i o n with ultraviolet light completely d e s t r o y e d the antirachitic value of the serum makes e x t r e m e l y unlikely the possibility that a second substance was present, as do also the negative *Filters were m a d e w i t h an 8 per cent collodion solution in g l a c i a l acetic acid. U l t r a f l l t r a t e s w e r e free from proteins, as shown by the s u l p h o s a l i c y l i e a c i d test.
486
THE
JOURNAL
OF P E D I A T R I C S
results obtained w i t h serum ultrafiltrates. The conclusion must therefore be d r a w n that v i t a m i n D itself produced the healing of the rachitic bone lesion a n d t h a t , under the conditions described, it does not lead to the production of another substance capable of healing rickets or of increasing the p h o s p h a t e content of the blood. H a v i n g ascertained that the serum of rabbits to which 200,000 international units of vitamin D had been given contained, five to ten days later, considerable a m o u n t s of irradiated ergosterol, I t h o u g h t it w o r t h while to attempt to learn h o w l o n g after its administration vitamin D circulates in vivo in the blood. Such e x p e r i m e n t s have not, so far as I k n o w , been p e r f o r m e d or reported in the literature up to the present time and should reveal the fate of v i t a m i n D w i t h i n the body in a more physiologic m a n n e r than do e x p e r i m e n t s in vitro. The same rabbits were used for this group of experiments as for the previous group. Their blood w a s taken at intervals of one, three, six, TABLE I V SHOWING
OF TIME TIIAT VITA]YIIN :D IS ~OUND IN ]:~L00D OF ]~ABBITS ADMINISTEATION OF SINGLE :DosE
LENGTH
I RAB-
DATE 2 0 C.C.
BIT NO.
VIOSTEROL WAS
GIVEN 445
12/
DATE BLOOD WAS TAKEN
LENGTH OF 2K-RAY TItlE BEHEALING T-WEEN AFTER RAT VIOSTEROL ADMINISTRA- NO. p C~t TION AND EIGtIT TEN5[G. % MG, % TAKING OF DAYS DAYS BLOOD
6/34 12/12/34
12/27/34
2/23/35 448
12/22/34
12/28/34
2/23/35 457
2/19/35
2/ 5/35 3/ 0/35
RABBIT SEI~UM
4.8 4.8
I wk.
6.7
3 wk.
5.1
11 w k .
4.1 7.8
1 wk.
5.2
9 wk.
4.2 4.3
I wk.
4.8
6 wk.
5/ 3/35
11 w k .
*See f o o t n o t e 1, T a b l e I. j'Serum for determination
was
lost.
AFTER
EAT S E R U M ~"
~) MG,
Ca
MG. %
5397 5399 5401
+ + ++ ++
+++ +++ +++
5.5
14.4
5446 5447 5456
+ + ++ + +
++ + +++ + ++
4.9
13.0
5540 5541
O 0
+ q
3.9
14.2
5452 5453 5459
+ + ++ ++
++ + +++ +++
5.4
13.6
5537 5539
0 0
+ ? +
2.3
13.6
5533 5534
++ ++
+ + + +++
4.0
5608 5609 i 5610
0 0 0
+ § + + +
5684 5696 5698
0 0 0
+ ? 0 i 0
8.5
10.4
HEY~[ANN:
ACTIO,N AND
:~ETABOLISM
O l ,~ V I T A M I N
D
487
nine, and eleven weeks following the a d m i n i s t r a t i o n of 200,000 intern a t i o n a l units of v i t a m i n D (20 e.c. of viosterol in oil, c o r r e s p o n d i n g a p p r o x i m a t e l y to 1.6 rag. of i r r a d i a t e d ergosterol), and was tested on rachitie r a t s in the m a n n e r a l r e a d y described. The e x p e r i m e n t s s u m m a r i z e d in Table I V show that, u n d e r the conditions prevailing, the s e r u m of r a b b i t s which h a d received viosterol was able to establish healing in raehitic r a t s in ten days, even if the blood was t a k e n as long as f r o m two to two and a h a l f months a f t e r the a d m i n i s t r a t i o n of the vitamin. The first distinct decrease in the potency of the s e r u m was noticed a f t e r six to nine weeks, and t r a c e s cbuld be detected in one instance (Rabbit 445) a f t e r eleven weeks. ]n other words, r a b b i t s weighing f r o m 3,000 to 4,000 gin. needed f r o m two and a half to three months t o d e s t r o y or excrete 1.6 rag. of irr a d i a t e d ergosterol (200,000 i n t e r n a t i o n a l units). COMMENT
One finds in the l i t e r a t u r e no conclusive evidence w h a t e v e r to supp o r t the view t h a t there m i g h t exist a h o r m o n a l r e g u l a t i o n of the phosphate metabolism, as is k n o w n to be the case for the calcium content o f the blood. The studies p r e s e n t e d here, in an a t t e m p t to approach the p r o b l e m in a new way, have p r o v e d entirely negative. It must consequently be considered wiser to a b a n d o n a hypothesis t h a t cannot be confirmed. I t is better, I think, to see in i r r a d i a t e d sterols themselves the only substances the lack of which leads to rachitic h y p o p h o s p h a t e m i a and the presence of which elevates the inorganic phosphorus in the blood. I n which tissue v i t a m i n D has its point of a t t a c k and b y which chemical m e c h a n i s m it increases the diminished inorganic p h o s p h a t e s in the blood of rachitic subjects m u s t still be considered unknown. So f a r as the low p h o s p h o r u s rickets in infants i s concerned, I f o u n d 1~ w e i g h t y evidence t h a t suggests t h a t the r e g u l a t i o n of the intestinal absorption of p h o s p h a t e s does not explain the situation satisfactorily. F u r t h e r studies on this subject are necessary and are in progress. The f a c t t h a t it has now been shown t h a t v i t a m i n D was circulating in the blood for at least two to three months a f t e r the a d m i n i s t r a t i o n of a single dose of a p p r o x i m a t e l y 1.6 rag. of i r r a d i a t e d ergosterol in o volves interesting new problems. I t is superfluous to emphasize the clinical value of these observations to p e d i a t r i c i a n s who use v i t a m i n D more or less r o u t i n e l y as an a g e n t f o r p r e v e n t i n g rickets in infants. I t is k n o w n t h a t d a m a g e can be done b y o v e r d o s a g e of v i t a m i n D, 13 and it is now shown t h r o u g h these studies t h a t continuous administration m a y easily lead to an undesirable a c c u m u l a t i o n because of the a p p a r e n t l y low r a t e of consumption. The question w h e t h e r the b o d y destroys or excretes v i t a m i n D cannot yet be decided. The w o r k of Coppens and Metz, 1~ who r e p o r t e d
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t h a t vitamin D is destroyed in vitro by blood and lung tissue, makes the assumption likely t h a t it m a y be d e s t r o y e d in vivo. The possibility that i r r a d i a t e d ergosterol may also be excreted t h r o u g h bile or t h r o u g h the intestinal wall must, however, be kept in mind. Studies on these problems are in progress. SUMMARY
1. The oral administration to rabbits of 20 c.c. of viosterol in oil (200,000 international units of vitamin D, equivalent to a p p r o x i m a t e l y 1.6 mg. of i r r a d i a t e d ergosterol) led to an increase in the inorganic phosphorus content of the blood without c o r r e s p o n d i n g l y affecting the blood calcium content. This action took place about five days after the viosterol had been given and lasted until the tenth to the fifteenth day. 2. I n order to learn w h e t h e r or not this action might be due to the stimulating effect of vitamin D upon an endocrine gland, thus leading to the p r o d u c t i o n of a phosphate hormone, the serum of these rabbits was tested on rachitic rats. The r a b b i t s ' blood was obtained five days after the viosterol had been given and was f o u n d to cure r achitic rats in f r o m eight to ten days if 0.6 c.c. of the serum was daily injected intramuscularly. 3. No evidence was found, however, to j u s t i f y the assumption that a n y substance other t h a n irradiated ergosterol was present in this serum or was even p a r t l y responsible for its curative potency, because the rickets-curing substance of this serum was soluble in ether and in oil, was destroyed completely by overirradiation with ultraviolet rays, and was f o u n d not to be ultrafiltrable. 4. W h e n viosterol was given b y m o u t h to rabbits in a single dose corresponding to a p p r o x i m a t e l y 1.6 rag. of irradiated ergosterol, detectable amounts of vitamin D were f o u n d to be circulating in the blood for f r o m two to three months. RE~ERENCES
1. Erdheim, J. : Raehitis und Epithe]kSrperehen, Denksehr. d. k. Akad. der Wissensch. ~r Klasse, Wien, 1914, p. 90. 2. Pappenheimer, A. M., and Minor, 5. : J. 3/i. t~esearch 42: 391, 1920-21. 3. ]~itter, C.: ~'rankfurt. Ztsehr. f. Path. 24: 137, 1921. 4. Nitsehke, A." Klin. ~Vchnschr. 12: 1793, 1933. 5. Co]lip, J . B . : Ann. Clin. 1Vied. 4: 219, 1925-26. 6. iqeymann, W., and ~aier, E. : Ztschr. f. Kinderh. 55: 512, 1933. 7. I~ietsehel: Discussion of paper by Nitschke, Verhandl. d. deutseh. Gese]lseh. f. Kinderh., ~onatsehr. f. Kinderh. 62: 37, 1934. 8. Heymann, W. : Unpublished data. 9. Wgrkany, if.: Klin. Wchnsehr. 9: 63, 1930. 10. Samson, K.: Klin. Wchnsehr. 10: 1766, 1931. 11. Hess, A . F . : Ztschr. f. Kinderh. 39: 395, 1925. 12. tteymann, W.: Ztsehr. f. Kinderh. 45: 239,, 1927-28; 46: 575, 584, ]928; 13. Pfunnenstiel, W.: 3/iiinchen. reed. Wehnsehr. 75" 1113, 1928. 14. Coppens~ P. A., and Me%z, G.A.: Biochem. Ztschr. 266: 169, 1933.