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A reaction of the adrenals to vitamin D
128
BIOCI-I!NIICAET J3!OPHYSICAAGTA B~A 25 X84
A REACTION OF T H E A D R E N A L S TO VITAMIN D J. QUARTERMAN The Rozvett Research Inslitute, Bucksburn, Aberdeen (Great Britain)
(Received ApriI I4th, I964)
SUMMARY
When rats were given an intravenous injection of a water-miscible preparation of vitamin D a there was an increase in the concentration of an unidentified substance in the adrenal glands. This substance appeared as an ultraviolet-absorbing spot on a paper chromatogram after passage of the non-saponifiable extracts of the glands through MgO-kieselguhr columns. It was at its maximum concentration in the glands 3o rain after injection of the vitamin, had different chromatographic and spectroo scopic properties from vitamin D and had no anti-rachitic activity,
INTRODUCTION
There have recently been several reports on the relationship between the adrenal glands and vitamin D metabolism. RAOUL AND GOUNELLE1 found that 35 ~g of an intravenousiy injected dose of 4o/*g of vitamin D a was accumulated in the adrenals in 3o miD. The substance found was identified as vitamin D by its ultraviolet absorption and by its ability to induce healing in rachitic chicks. The recovery of this proportion of the injected dose implies a very great flow of blood through the adrenal glands. Other workers, however, using labelled vitamin D have found only a small percentage of the dose in the adrenals a few hours after administration but the concentration, compared with other tissues, was nevertheless high. Thus KODICZK, CRUICKS~ANK AND ASHBY2 found that 2o rain after an intracardial injection of 5o b~g I¢Clabelled vitamin D,, rat adrenals contained 1.3 % of the dose whereas the liver had 54.3 % and the blood Io.7 %. BLUMBERC,AESI, HURNI AND SC~ON~OLZERagave rhesus monkeys an oral dose of I.I8 mg l~C-labelled vitamin D~ in oil and found 24 h later that the activity in the adrenals was I3.38 counts/miD per mg dry tissue whereas that in the liver was 25.zo and in the blood 5.52. NORMAX AND DELUCA~ used ~ritiated vitamin D a. 2o rain after their rats had received 5oo I.U. of the labelied vitamin the activity in the adrenals was I I ooo counts/rain per mg drv tissue and in the !iver was !23oo, which means that as a percentage of the dose, the amount of the administered activity was o.Ii and 44.4 respectively for the two organs. All these workers point to the importance of the adrenals in -vitamin D metabolism by demonstrating a high concentration of the vitamin in the glands though RAOUL AND GOUN]CLLE'S1 observations differ considerably from the others in the degree of concentration. SALLIS AND HOLDSWORTI-[5 have evidence of another sort They found that some adrenal steroids and synthetic steroids enhanced the antiBiochim. Biophys. Acfa, 97 (I965) i28 I33
A REACTION OF ADRENALS TO VITAMIN D
12 9
rachitic effect of vitamin D when they were given to chicks along with the vitamin even though these steroids had no antirachitic effect themselves. When, however, the steroids were given daily for 3 days before the vitamin D was given, the anti-raehitic effect of the vitamin was reduced. They suggest that the vitamin m a y be converted to an active form by the adrenals and the steroids either stimulate the glands if given with the vitamin or cause some decrease in activity of the glands if given over a period of time before the vitamin is given. This paper presents some observations on the production of an unidentified substance b y adrenal glands in response to an injection of vitamin D. EXPERIMENTAL
Injection of vitamin D Pure vitamin D 2 or D a was dissolved in o.o 5 ml ethanol per mg, mixed with an equal volume of Tween 8o (polyoxyethylene sorbitan mono-oleate) and diluted with water. This solution was injected into the tail vein of rats, the ear vein of rabbits or the jugular vein of larger animals. Sham injections were made with the same solution but without vitamin .D. Mature hooded Norwegian rats were used in all the rat experiments.
Analysis of adrenal glands Rats and rabbits were killed b y a blow on the head and larger animals with a humane killer. The glands were removed immediately and kept at o ° or below, or saponified at once b y heating at 70-80 ° for IO rain with 2 ml io % KOH in ethanolwater (i : I, v/v) or with 2 ml of the same solution per g tissue if tile weight of tissue was more than I g. About 2 mg pyrogallol were added before saponification. After heating the mixture was diluted once with water and extracted three times with two volumes of ether. The ether was removed b y distillation and the remaining solution, which contained water and ethanol, was evaporated in vacuo. The residue was made into a slurry with light petroleum (b.p. 40-60 °) containing 0.5 % (v/v) acetone and applied to a column of MgO-kieselguhr (50 : 5o) as described b y KODICEKAND ASI-IBY6. Their procedure for the subsequent chromatography of the eluate on paraffin-impregnated paper was followed except that the intermediate precipitation with digitonin was latterly omitted because this did not influence the results. Their Solvent B, n - p r o p a n o l - m e t h a n o l - w a t e r (15:82:3, v/v), gave the best definition and separation of ultraviolet-absorbing spots and was used in all the work described. The papers were prepared b y dipping 25 cm square sheets of Whatman No. 2 chromatography paper through 5 % (v/v) liquid paraffin in light petroleum, once in the direction of flow of the developing solvent and once in the opposite direction, drying for 15 min in air after each dipping. For tile estimation of substances on the paper chromatograms the following procedure was adopted. Where possible all the samples from one experiment were run on the same sheet of chromatography paper. Ten points, 2.5 cm apart were made on the starting line of each sheet of paper. Nothing was put on two of the points, on four were spotted known amounts of vitamin D, usually about 15, 3o, 45 and 6o/~g in ethanol and on the remaining four tile adrenal extracts were applied. The papers were allowed to equilibrate in the atmosphere of the developing solvent for at least t~iochim. Biophys. Acta, 97 (1965) 128-133
I3o
J. QUARTERS~AN
3 h and usually overnight before developing which usually took about 5 h. The papers were dried in air for 3o rain and then i ~ v a c u o . Spots which could be seen in u!traviolet light were outtined in pencil, cut out of the paper as a rectangle of a standard size and extracted with 5 ml ethanol. The blanks were necessary because the absorption in the ultraviolet due to residues from the solvents was appreciable and varied from sheet to sheet. The curative, radiographic assay with rats of vitamin D activity in the nonsaponifiable extract of rat adrenals was made using the method described by DALGARNO, HILL AND 13~cDONALD 7.
RESULTS
Rats were given Ioo b~g vitamin D a by injection into the tait vein and 30 rain later were killed and their adrenals removed and analysed for vitamin D a by paper chromatography. No vitamin D was detected in the adrenals of rats which were given vitamin D~ or D a or those which were sham-injected. In the animals given vitamin D 2 or D a an ultraviolet-absorbing substance was found with a mean RF of 0.40 compared with a mean R~ of o.5I for the vitamin. This material had an absorption maximum at 275 m/~ and a minimum at 262 m/~. The absorption curve is given in Fig. I with that of vitamin D for comparison.
\ \ \ \
\\
\
g_ t
<{
r 250
I 275
- -
WavelengLh (rn #) Fig. I. Absorption spectra of v i t a m i n D ( - - ) adrenals (. . . . ).
and the unidentified substance isolated from
The experiment was repeated with a sheep given 15 m g of the vitamin and the adrenals were dissected into medulla and cortex before saponification.The substance was found to be entirely in the cortex and not at all in the medulla. In the sheep and the goat the level of the substance was found to be m u c h greater in one gland, usually but not always, the left.This was not observed in the rat and other animals were not examined. The extracts from rat adrenals had no anti-rachiticactivity for rats, The Biochim. f~iophys, dora, 97 (i965) I 2 8 - I 3 3
A REACTION OF ADRENALS TO VITAMIN
D
I3I
ultraviolet-absorbing substance was detected in liver and ileum in small quantities 3o min after an intravenous iniection of vitamin D but not in spleen, kidney, duodenum or jejunum. Cl
,_c 100 E
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,f, otc-o
Om e4 -5 C ~o ~0.L_ co) otn
c~
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o~ ,oo
a °~
I
o
n
.
o
l
x
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30 ~ gO 120 Time f r o m injection of vi+.emin D to death (rain)
150
]Fig. 2. T h e c o n c e n t r a t i o n of t h e u n i d e n t i f i e d s u b s t a n c e in a d r e n a l s in r a t s ( × ) , s h e e p ( Q ) , g o a t s ( [] ), a r a b b i t ( * ) a n d a p i g ( O ), also of v i t a m i n D in r a t s f o u n d b y RAOIJL AND GOU~ELLE (~) * ,(ref. I). T h e line s h o w s t h e m e a n c h a n g e s in t h e rat. • F o r t h e s e d a t a t h e o r d i n a t e is p e r c e n t a g e r e c o v e r y of v i t a m i n D. • F o r t h e c a l c u l a t i o n of t h i s figure see t h e t e x t .
The concentration of this unidentified substance in the adrenals varied with the -time between injection and death of the animal and also with the species of animal. Fig. 2 shows results from experiments with 27 rats, 7 sheep, 4 goats, 3 rabbits and 2 pigs. In the rats there was a rapid increase in concentration which reached a maximum 3o min after injection followed by a rapid decrease. In the figure the ab:sorbancy of the unknown substance is assumed to be the same as that of vitamin D so that the ordinate is the concentration of the substance as a percentage of the injected vitamin D. These results can then be compared directly with those of RAOUL AND GOUNELLE1 who claimed a high recovery of the vitamin from the adrenals. With a larger injection than IOO/zg of vitamin D the concentration of the substance in the :adrenals was not increased. The substance appeared on the chromatograms of extracts from 5 out of IO rats which had had a sham injection or no injection at all but the spot produced was then of low intensity, having a mean value of 6.8 ~ 3-9 units .on the ordinate of Fig. 2. The value for the control in each experiment was subtracted from the concentration of the substance in the adrenals of rats which had received a n injection of vitamin D. Similarly values of 2 and 3 units were obtained from two uninjected sheep and were subtracted from the values obtained from experimental animals. With the sheep there was a maximum between 35 and 8o rain after injection of a dose of 15 mg of vitamin D. The goats were given the same dose and showed similar .changes while the rabbit was given 2.5 mg and the pig I3 mg. The concentrations observed in all these animals were very small compared with those from t h e rats. The actual concentration of the substance was about Io times as high at its maximum Biochim. Biophys. Acta, 97 (1965) 128-133
I32
j° 9 UAP~TE~
in t h e r a t t h a n in a n y of the o t h e r animals. I n t h e larger animals only one level or: injection (about 0. 4 m g p e r kg live wt 0 was used for each species. A few eolour reactions were o b s e r v e d w i t h this s u b s t a n c e on c h r o m a t o g r a p h y paper. The s u b s t a n c e a p p e a r e d d a r k in u l t r a v i o l e t light, gave a similar b u t d a r k e r r e a c t i o n t h a n v i t a m i n D with t h e SbC1 s r e a g e n t a n d a fainter r e a c t i o n t h a n the v i t a m i n w i t h 12 in light p e t r o l e u m . There was a s t r o n g r e a c t i o n with Schiff base, D N P - h y d r a z i n e a n d t e t r a z o l i u m reagent, a faint r e a c t i o n w i t h the PO~rER--SmBE_V, r e a g e n t s a n d none w i t h p H indicators. Thus t h e s u b s t a n c e has reducing properties, p r o b a b l y a r e a c t i v e c a r b o x y l group a n d a n e u t r a ! reaction. DISCUSSION The changes in c o n c e n t r a t i o n of the unidentified s u b s t a n c e p r o d u c e d in response to an injection of v i t a m i n D h a v e been s t u d i e d in t h e a d r e n a l gland, The s u b s t a n c e has been f o u n d in liver, k i d n e y and ileum as well as a d r e n a l g l a n d b u t was n o t s t u d i e d in these larger organs because of t h e difficulty of purification a n d the poor q u a l i t y of t h e c h r o m a t o g r a m s of e x t r a c t s from these organs. There were indications t h a t similar changes occurred in these organs as occurred in the adrenals. SALLIS AXD HOLDSWORTI-I5 h a v e s u g g e s t e d t h a t the a d r e n a l s or their secretions h a v e a function in controlling the conversion of v i t a m i n D to an active form b u t this effect need no-t. of course, be confined to t h e glands themselves. The large a m o u n t s of v i t a m i n D f o u n d in the liver, k i d n e y a n d i n t e s t i n e couid be i n v o l v e d in processes such as storage, d e g r a d a t i o n to i n a c t i v e p r o d u c t s a n d e x c r e t i o n as well as a c t i v a t i o n . KoDIcE~: 9 has shown t h a t in the liver 5o % of the r a d i o a c t i v i t y d e r i v e d from an oral dose of labelled v i t a m i n D is not biologically a c t i v e a n d is p r e s u m a b l y in the form of b r e a k d o w n p r o d u c t s a n d the t y p e s of b r e a k down p r o d u c t s are different from those which a p p e a r in the intestine. NORh~A~ Axn. DELUCA ~ f o u n d a b o u t ~ % a n d KODICEK9 a b o u t 2 % of the r a d i o a c t i v e label in t h e urine after 24 h b u t t h e l a t t e r showed t h a t none of this was biologically a c t i v e v i t a m i n D. KODtCUK et al. 2 h a v e shown t h a t 2. 5 % ~f i n j e c t e d v i t a m i n D can be f o u n d in t h e g u t c o n t e n t s 5 h a f t e r t h e injection. This m a y , in p a r t , be due ~o d e s q u a m a t i o n of the i n t e s t i n a l e p i t h e l i u m i m m e d i a t e l y after d e a t h (BADAWV et al. a° . This p a p e r describes t h e f o r m a t i o n , in response to v i t a m i n D dosing, of a subs t a n c e which is no~ v i t a m i n D. There is no evidence w h e t h e r or noz it is d e r i v e d from v i t a m i n D b u t comparisons w i t h the d a t a of o t h e r workers who used simiIar dosage levels show some similarities in the w a y the c o n c e n t r a t i o n changes with time. i n t h e a d r e n a l s there is a v e r y ciose correspondence in t i m e a n d c o n c e n t r a t i o n I as e s t i m a t e d b y u l t r a v i o l e t a b s o r p t i o n / w i t h RAOUL AND GOUNELL'S ~ d a t a (Fig. 2) a l t h o u g h there are differences in t h e s p e c t r a l a n d biological p r o p e r t i e s of t h e substances concerned. Using i n t r a c a r d i a l doses of t h e v i t a m i n , [
97 (1965) i28 I33
A REACTION OF ADRENALS TO VITAMIN D
I33
r o u t e of a d m i n i s t r a t i o n . NORMAN AND DELUCA ¢ h a v e s h o w n t h e m a x i m u m l e v e l s of r a d i o a c t i v i t y i n l i v e r a n d i n t e s t i n e o c c u r a t a l o n g e r i n t e r v a l a f t e r a n o r a l d o s e of t h e v i t a m i n , n a m e l y a t a b o u t 4 h. I t is p o s s i b l e , t h e r e f o r e , i n v i e w of all t h e s i m i l a r i t i e s i n b e h a v i o u r , t h e r e is s o m e r e l a t i o n s h i p b e t w e e n t h e u n i d e n t i f i e d s u b s t a n c e i n t h e a d r e n a l s d e s c r i b e d in t h i s p a p e r a n d t h e r e c o g n i s e d p h y s i o l o g i c a l f u n c t i o n s of v i t a m i n D.
REFERENCES I Y. RAOUL AND J.-C. GOUNELLE, Compl. Rend., 247 (1958) 161. 2 E. I~ODICEK, E. M. CRUICKSHANKANn D. R. ASHBY, Biochem. J., 76 (196o) 159. 3 A. BLUMBERG, 1-I. AEBI, H. HUR?¢I AND G. SCHbNHOLZER, Helv. Physiol. Pharmacol. Acta, 18 (196o) 56 . 4 A. W. NORMAN AND H. F. DELUCA, Biochemistry, 2 (1963) 116o. 5 J- D. SALLIS AXD E. S. I-IOLDSWORTH,Am. J. Physiol., 203 (1962) 506. 6 E. ItODICEK AND D. R. ASHBY, Biochem. J., 57 (1954) xii a n d xiii. 7 A. C. DALC-ARNO, R. HILL AND I. MCDONALD, Bri~. or. Nutr., 16 (1962) 91. 8 C. C. PORTER AND R. H. SILBER, jr. Biol. Chem., 185 (195 o) 2Ol. 9 E. KODICEK, Ciba Found. Symp. Bone Struct. Metc~b., Bu~terworth, London, 1956, p. 161. IO A. ]V[. BADAWY, t~. M. CAMPBELL, D. P. CUTHBERTSON, B. F. FELL AND W. S. MACI
Nutv., 12 (I958) 367. Biochim. Biophys. Acta, 97 (1965) I28-133
BIOCI-I!NIICAET J3!OPHYSICAAGTA B~A 25 X84
A REACTION OF T H E A D R E N A L S TO VITAMIN D J. QUARTERMAN The Rozvett Research Inslitute, Bucksburn, Aberdeen (Great Britain)
(Received ApriI I4th, I964)
SUMMARY
When rats were given an intravenous injection of a water-miscible preparation of vitamin D a there was an increase in the concentration of an unidentified substance in the adrenal glands. This substance appeared as an ultraviolet-absorbing spot on a paper chromatogram after passage of the non-saponifiable extracts of the glands through MgO-kieselguhr columns. It was at its maximum concentration in the glands 3o rain after injection of the vitamin, had different chromatographic and spectroo scopic properties from vitamin D and had no anti-rachitic activity,
INTRODUCTION
There have recently been several reports on the relationship between the adrenal glands and vitamin D metabolism. RAOUL AND GOUNELLE1 found that 35 ~g of an intravenousiy injected dose of 4o/*g of vitamin D a was accumulated in the adrenals in 3o miD. The substance found was identified as vitamin D by its ultraviolet absorption and by its ability to induce healing in rachitic chicks. The recovery of this proportion of the injected dose implies a very great flow of blood through the adrenal glands. Other workers, however, using labelled vitamin D have found only a small percentage of the dose in the adrenals a few hours after administration but the concentration, compared with other tissues, was nevertheless high. Thus KODICZK, CRUICKS~ANK AND ASHBY2 found that 2o rain after an intracardial injection of 5o b~g I¢Clabelled vitamin D,, rat adrenals contained 1.3 % of the dose whereas the liver had 54.3 % and the blood Io.7 %. BLUMBERC,AESI, HURNI AND SC~ON~OLZERagave rhesus monkeys an oral dose of I.I8 mg l~C-labelled vitamin D~ in oil and found 24 h later that the activity in the adrenals was I3.38 counts/miD per mg dry tissue whereas that in the liver was 25.zo and in the blood 5.52. NORMAX AND DELUCA~ used ~ritiated vitamin D a. 2o rain after their rats had received 5oo I.U. of the labelied vitamin the activity in the adrenals was I I ooo counts/rain per mg drv tissue and in the !iver was !23oo, which means that as a percentage of the dose, the amount of the administered activity was o.Ii and 44.4 respectively for the two organs. All these workers point to the importance of the adrenals in -vitamin D metabolism by demonstrating a high concentration of the vitamin in the glands though RAOUL AND GOUN]CLLE'S1 observations differ considerably from the others in the degree of concentration. SALLIS AND HOLDSWORTI-[5 have evidence of another sort They found that some adrenal steroids and synthetic steroids enhanced the antiBiochim. Biophys. Acfa, 97 (I965) i28 I33
A REACTION OF ADRENALS TO VITAMIN D
12 9
rachitic effect of vitamin D when they were given to chicks along with the vitamin even though these steroids had no antirachitic effect themselves. When, however, the steroids were given daily for 3 days before the vitamin D was given, the anti-raehitic effect of the vitamin was reduced. They suggest that the vitamin m a y be converted to an active form by the adrenals and the steroids either stimulate the glands if given with the vitamin or cause some decrease in activity of the glands if given over a period of time before the vitamin is given. This paper presents some observations on the production of an unidentified substance b y adrenal glands in response to an injection of vitamin D. EXPERIMENTAL
Injection of vitamin D Pure vitamin D 2 or D a was dissolved in o.o 5 ml ethanol per mg, mixed with an equal volume of Tween 8o (polyoxyethylene sorbitan mono-oleate) and diluted with water. This solution was injected into the tail vein of rats, the ear vein of rabbits or the jugular vein of larger animals. Sham injections were made with the same solution but without vitamin .D. Mature hooded Norwegian rats were used in all the rat experiments.
Analysis of adrenal glands Rats and rabbits were killed b y a blow on the head and larger animals with a humane killer. The glands were removed immediately and kept at o ° or below, or saponified at once b y heating at 70-80 ° for IO rain with 2 ml io % KOH in ethanolwater (i : I, v/v) or with 2 ml of the same solution per g tissue if tile weight of tissue was more than I g. About 2 mg pyrogallol were added before saponification. After heating the mixture was diluted once with water and extracted three times with two volumes of ether. The ether was removed b y distillation and the remaining solution, which contained water and ethanol, was evaporated in vacuo. The residue was made into a slurry with light petroleum (b.p. 40-60 °) containing 0.5 % (v/v) acetone and applied to a column of MgO-kieselguhr (50 : 5o) as described b y KODICEKAND ASI-IBY6. Their procedure for the subsequent chromatography of the eluate on paraffin-impregnated paper was followed except that the intermediate precipitation with digitonin was latterly omitted because this did not influence the results. Their Solvent B, n - p r o p a n o l - m e t h a n o l - w a t e r (15:82:3, v/v), gave the best definition and separation of ultraviolet-absorbing spots and was used in all the work described. The papers were prepared b y dipping 25 cm square sheets of Whatman No. 2 chromatography paper through 5 % (v/v) liquid paraffin in light petroleum, once in the direction of flow of the developing solvent and once in the opposite direction, drying for 15 min in air after each dipping. For tile estimation of substances on the paper chromatograms the following procedure was adopted. Where possible all the samples from one experiment were run on the same sheet of chromatography paper. Ten points, 2.5 cm apart were made on the starting line of each sheet of paper. Nothing was put on two of the points, on four were spotted known amounts of vitamin D, usually about 15, 3o, 45 and 6o/~g in ethanol and on the remaining four tile adrenal extracts were applied. The papers were allowed to equilibrate in the atmosphere of the developing solvent for at least t~iochim. Biophys. Acta, 97 (1965) 128-133
I3o
J. QUARTERS~AN
3 h and usually overnight before developing which usually took about 5 h. The papers were dried in air for 3o rain and then i ~ v a c u o . Spots which could be seen in u!traviolet light were outtined in pencil, cut out of the paper as a rectangle of a standard size and extracted with 5 ml ethanol. The blanks were necessary because the absorption in the ultraviolet due to residues from the solvents was appreciable and varied from sheet to sheet. The curative, radiographic assay with rats of vitamin D activity in the nonsaponifiable extract of rat adrenals was made using the method described by DALGARNO, HILL AND 13~cDONALD 7.
RESULTS
Rats were given Ioo b~g vitamin D a by injection into the tait vein and 30 rain later were killed and their adrenals removed and analysed for vitamin D a by paper chromatography. No vitamin D was detected in the adrenals of rats which were given vitamin D~ or D a or those which were sham-injected. In the animals given vitamin D 2 or D a an ultraviolet-absorbing substance was found with a mean RF of 0.40 compared with a mean R~ of o.5I for the vitamin. This material had an absorption maximum at 275 m/~ and a minimum at 262 m/~. The absorption curve is given in Fig. I with that of vitamin D for comparison.
\ \ \ \
\\
\
g_ t
<{
r 250
I 275
- -
WavelengLh (rn #) Fig. I. Absorption spectra of v i t a m i n D ( - - ) adrenals (. . . . ).
and the unidentified substance isolated from
The experiment was repeated with a sheep given 15 m g of the vitamin and the adrenals were dissected into medulla and cortex before saponification.The substance was found to be entirely in the cortex and not at all in the medulla. In the sheep and the goat the level of the substance was found to be m u c h greater in one gland, usually but not always, the left.This was not observed in the rat and other animals were not examined. The extracts from rat adrenals had no anti-rachiticactivity for rats, The Biochim. f~iophys, dora, 97 (i965) I 2 8 - I 3 3
A REACTION OF ADRENALS TO VITAMIN
D
I3I
ultraviolet-absorbing substance was detected in liver and ileum in small quantities 3o min after an intravenous iniection of vitamin D but not in spleen, kidney, duodenum or jejunum. Cl
,_c 100 E
%- C O o
,f, otc-o
Om e4 -5 C ~o ~0.L_ co) otn
c~
g~ O C ~
o~ ,oo
a °~
I
o
n
.
o
l
x
I"
30 ~ gO 120 Time f r o m injection of vi+.emin D to death (rain)
150
]Fig. 2. T h e c o n c e n t r a t i o n of t h e u n i d e n t i f i e d s u b s t a n c e in a d r e n a l s in r a t s ( × ) , s h e e p ( Q ) , g o a t s ( [] ), a r a b b i t ( * ) a n d a p i g ( O ), also of v i t a m i n D in r a t s f o u n d b y RAOIJL AND GOU~ELLE (~) * ,(ref. I). T h e line s h o w s t h e m e a n c h a n g e s in t h e rat. • F o r t h e s e d a t a t h e o r d i n a t e is p e r c e n t a g e r e c o v e r y of v i t a m i n D. • F o r t h e c a l c u l a t i o n of t h i s figure see t h e t e x t .
The concentration of this unidentified substance in the adrenals varied with the -time between injection and death of the animal and also with the species of animal. Fig. 2 shows results from experiments with 27 rats, 7 sheep, 4 goats, 3 rabbits and 2 pigs. In the rats there was a rapid increase in concentration which reached a maximum 3o min after injection followed by a rapid decrease. In the figure the ab:sorbancy of the unknown substance is assumed to be the same as that of vitamin D so that the ordinate is the concentration of the substance as a percentage of the injected vitamin D. These results can then be compared directly with those of RAOUL AND GOUNELLE1 who claimed a high recovery of the vitamin from the adrenals. With a larger injection than IOO/zg of vitamin D the concentration of the substance in the :adrenals was not increased. The substance appeared on the chromatograms of extracts from 5 out of IO rats which had had a sham injection or no injection at all but the spot produced was then of low intensity, having a mean value of 6.8 ~ 3-9 units .on the ordinate of Fig. 2. The value for the control in each experiment was subtracted from the concentration of the substance in the adrenals of rats which had received a n injection of vitamin D. Similarly values of 2 and 3 units were obtained from two uninjected sheep and were subtracted from the values obtained from experimental animals. With the sheep there was a maximum between 35 and 8o rain after injection of a dose of 15 mg of vitamin D. The goats were given the same dose and showed similar .changes while the rabbit was given 2.5 mg and the pig I3 mg. The concentrations observed in all these animals were very small compared with those from t h e rats. The actual concentration of the substance was about Io times as high at its maximum Biochim. Biophys. Acta, 97 (1965) 128-133
I32
j° 9 UAP~TE~
in t h e r a t t h a n in a n y of the o t h e r animals. I n t h e larger animals only one level or: injection (about 0. 4 m g p e r kg live wt 0 was used for each species. A few eolour reactions were o b s e r v e d w i t h this s u b s t a n c e on c h r o m a t o g r a p h y paper. The s u b s t a n c e a p p e a r e d d a r k in u l t r a v i o l e t light, gave a similar b u t d a r k e r r e a c t i o n t h a n v i t a m i n D with t h e SbC1 s r e a g e n t a n d a fainter r e a c t i o n t h a n the v i t a m i n w i t h 12 in light p e t r o l e u m . There was a s t r o n g r e a c t i o n with Schiff base, D N P - h y d r a z i n e a n d t e t r a z o l i u m reagent, a faint r e a c t i o n w i t h the PO~rER--SmBE_V, r e a g e n t s a n d none w i t h p H indicators. Thus t h e s u b s t a n c e has reducing properties, p r o b a b l y a r e a c t i v e c a r b o x y l group a n d a n e u t r a ! reaction. DISCUSSION The changes in c o n c e n t r a t i o n of the unidentified s u b s t a n c e p r o d u c e d in response to an injection of v i t a m i n D h a v e been s t u d i e d in t h e a d r e n a l gland, The s u b s t a n c e has been f o u n d in liver, k i d n e y and ileum as well as a d r e n a l g l a n d b u t was n o t s t u d i e d in these larger organs because of t h e difficulty of purification a n d the poor q u a l i t y of t h e c h r o m a t o g r a m s of e x t r a c t s from these organs. There were indications t h a t similar changes occurred in these organs as occurred in the adrenals. SALLIS AXD HOLDSWORTI-I5 h a v e s u g g e s t e d t h a t the a d r e n a l s or their secretions h a v e a function in controlling the conversion of v i t a m i n D to an active form b u t this effect need no-t. of course, be confined to t h e glands themselves. The large a m o u n t s of v i t a m i n D f o u n d in the liver, k i d n e y a n d i n t e s t i n e couid be i n v o l v e d in processes such as storage, d e g r a d a t i o n to i n a c t i v e p r o d u c t s a n d e x c r e t i o n as well as a c t i v a t i o n . KoDIcE~: 9 has shown t h a t in the liver 5o % of the r a d i o a c t i v i t y d e r i v e d from an oral dose of labelled v i t a m i n D is not biologically a c t i v e a n d is p r e s u m a b l y in the form of b r e a k d o w n p r o d u c t s a n d the t y p e s of b r e a k down p r o d u c t s are different from those which a p p e a r in the intestine. NORh~A~ Axn. DELUCA ~ f o u n d a b o u t ~ % a n d KODICEK9 a b o u t 2 % of the r a d i o a c t i v e label in t h e urine after 24 h b u t t h e l a t t e r showed t h a t none of this was biologically a c t i v e v i t a m i n D. KODtCUK et al. 2 h a v e shown t h a t 2. 5 % ~f i n j e c t e d v i t a m i n D can be f o u n d in t h e g u t c o n t e n t s 5 h a f t e r t h e injection. This m a y , in p a r t , be due ~o d e s q u a m a t i o n of the i n t e s t i n a l e p i t h e l i u m i m m e d i a t e l y after d e a t h (BADAWV et al. a° . This p a p e r describes t h e f o r m a t i o n , in response to v i t a m i n D dosing, of a subs t a n c e which is no~ v i t a m i n D. There is no evidence w h e t h e r or noz it is d e r i v e d from v i t a m i n D b u t comparisons w i t h the d a t a of o t h e r workers who used simiIar dosage levels show some similarities in the w a y the c o n c e n t r a t i o n changes with time. i n t h e a d r e n a l s there is a v e r y ciose correspondence in t i m e a n d c o n c e n t r a t i o n I as e s t i m a t e d b y u l t r a v i o l e t a b s o r p t i o n / w i t h RAOUL AND GOUNELL'S ~ d a t a (Fig. 2) a l t h o u g h there are differences in t h e s p e c t r a l a n d biological p r o p e r t i e s of t h e substances concerned. Using i n t r a c a r d i a l doses of t h e v i t a m i n , [
97 (1965) i28 I33
A REACTION OF ADRENALS TO VITAMIN D
I33
r o u t e of a d m i n i s t r a t i o n . NORMAN AND DELUCA ¢ h a v e s h o w n t h e m a x i m u m l e v e l s of r a d i o a c t i v i t y i n l i v e r a n d i n t e s t i n e o c c u r a t a l o n g e r i n t e r v a l a f t e r a n o r a l d o s e of t h e v i t a m i n , n a m e l y a t a b o u t 4 h. I t is p o s s i b l e , t h e r e f o r e , i n v i e w of all t h e s i m i l a r i t i e s i n b e h a v i o u r , t h e r e is s o m e r e l a t i o n s h i p b e t w e e n t h e u n i d e n t i f i e d s u b s t a n c e i n t h e a d r e n a l s d e s c r i b e d in t h i s p a p e r a n d t h e r e c o g n i s e d p h y s i o l o g i c a l f u n c t i o n s of v i t a m i n D.
REFERENCES I Y. RAOUL AND J.-C. GOUNELLE, Compl. Rend., 247 (1958) 161. 2 E. I~ODICEK, E. M. CRUICKSHANKANn D. R. ASHBY, Biochem. J., 76 (196o) 159. 3 A. BLUMBERG, 1-I. AEBI, H. HUR?¢I AND G. SCHbNHOLZER, Helv. Physiol. Pharmacol. Acta, 18 (196o) 56 . 4 A. W. NORMAN AND H. F. DELUCA, Biochemistry, 2 (1963) 116o. 5 J- D. SALLIS AXD E. S. I-IOLDSWORTH,Am. J. Physiol., 203 (1962) 506. 6 E. ItODICEK AND D. R. ASHBY, Biochem. J., 57 (1954) xii a n d xiii. 7 A. C. DALC-ARNO, R. HILL AND I. MCDONALD, Bri~. or. Nutr., 16 (1962) 91. 8 C. C. PORTER AND R. H. SILBER, jr. Biol. Chem., 185 (195 o) 2Ol. 9 E. KODICEK, Ciba Found. Symp. Bone Struct. Metc~b., Bu~terworth, London, 1956, p. 161. IO A. ]V[. BADAWY, t~. M. CAMPBELL, D. P. CUTHBERTSON, B. F. FELL AND W. S. MACI
Nutv., 12 (I958) 367. Biochim. Biophys. Acta, 97 (1965) I28-133