194.
M E T A B O l I S M A N D D I S T R I B U T I O N OF V I T A M I W D
HECTOR DE LUCA
ERSITY O F WISCONSIN
. _ _ _ _ _U N-I V_
__.___._____-----------.----
The problem which I should l i k e t o discuss today has occupied t h e
i n t e r e s t of biochemists and n u t r i t i o n i s t s f o r some f o r t y years. This problem
simply s t a t e d i s how does Vitamin D work i n t h e body. This encompasses a
knowledge of where t h e vitamin goes i n t h e body, what i s i t s active form,
what i s t h e f i n a l phase of t h e Vitamin D molecule and how, most importantly,
does t h i s small amount of material bring about t h e dramatic p h y s i o l o g i c d
e f f e c t s that it i s known t o produce. There has been a great deal of
progress i n recent years on t h i s general problem through t h e advent of new
techniques and approaches, and it i s possible t h a t at some t i m e i n t h e near
future we w i l l be able t o explain the action of vitamins a t t h e molecular
level.
Now, i n contrast t o t h e water soluble vitamins, which we a l l know
now t o function i n t h e body as co-enzymes through metabolism of carbohydrates,
f a t s and proteins, the f a t soluble vitamins undoubtedly do not function i n
t h i s manner. A s a matter of f a c t many investigators have attempted t o rel a t e t h e f a t soluble vitamins i n such a capacity, and those have been miformly unsuccessful. Rather than t h i s , we have t o look t o very specialized
functions of t h e complex organisms f o r our answer t o how these f a t soluble
substances operate. For example, t h e r o l e of Vitamin K i s believed t o be
r e l a t e d t o t h e production of very special blood c l o t t i n g proteins, t h e
prothromin and so f o r t h . The function of Vitamin D i s only apparent i n
those animals which possess c a l c i f i e d endo-skeletons. O f course Vitamin A,
re well recognize, plays a very great role in directing t h e d i f f e r e n t i a t i o n
of t h e e p i t h e l i a l c e l l s . A t any rate, t h e present view held i n our laborat o r y i s t h a t t h e f a t soluble substances o r vitamins carry out their
metabolic functions primarily by e i t h e r inducing t h e synthesis of specific
functional proteins o r enzymes, o r by controlling t h e subcellular membrane
functions o r perhaps a colnbination of both. With t h i s general philosophy
i n mind, l e t us now t u r n t o Vitamin D and what we r e a l l y understand at the
present time concerning i t s mechanism of action.
Perhaps it would be best f o r me t o review f o r a moment some of
t h e w e l l known e f f e c t s of t h e vitamin at t h e physiologic l e v e l . All of you
are quite aware t h a t t h e primary defect i n Vitamin D deficiency i s a f a i l u r e
of c a l c i f i c a t i o n of newly formed bone o r bone which has been decalcified.
T h i s r e s u l t s i n t h e appearance of the deficiency disease which we recognize
as r i c k e t s i n t h e young and osteomalacia i n t h e adult. For many years since
i t s e a r l y discovery, because of t h i s function, many investigators have
attempted t o f i n d a r o l e of t h e vitamin i n t h e c a l c i f i c a t i o n process i t s e l f .
These attempts have been uniformly unsuccessful, and there i s now s u f f i c i e n t
evidence t o discount t h i s as a possible mechanism. As a matter of f a c t ,
much evidence i s accumulating quite t o t h e contrary.
The primary reason f o r t h e defective c a l c i f i c a t i o n t h a t occurs i s
t h a t there is a poor o r a very reduced supply of calcium and phosphorous t o
195.
t h e bones. This r e s u l t s i n i n s u f f i c i e n t mineral f o r the production of t h e
hydroxy a p a t i t e involved. It i s therefore evident, and it has been known
actually f o r many years, t h a t i n Vitamin D deficiency t h e r e i s a very marked
lowering of t h e calcium and phosphorous product of t h e blood. This uns u f f i c i e n t product f a l l s f a r below t h e s o l u b i l i t y product of t h e hydroxy
appetite and thus c a l c i f i c a t i o n f a l l s .
I n t h e absence of Vitamin D t h e r e i s always a lowering of t h e
serum calcium concentration and i n t h e case of some of t h e special diets
there is a reduction i n t h e serum phosphate concentration. If one were t o
examine closely t h e data with regard t o ricketogenic diets, and i f one were
t o multiply t h e calcium times t h e phosphorous concentration i n the minus D
a n i m a l s , you would see t h a t t h e value f a l l s f a r below t h e number t h a t i s
necessary f o r normal mineralization t o occur. The addition of Vitamin D
t o t h e diet, of course brings t h e calcium phosphorus product back. So t h a t
t h e next question t h a t should be asked with regard t o how Vitamin D works
is, how does t h i s vitamin bring about t h e elevation of serum calcium and
secondarily perhaps t h e serum phosphate concentration, which i n t u r n
p r o m t e s normal bone mineralization?
It i s w e l l established through t h e work of many investigators
over a period of many years t h a t Vitamin D has a primary e f f e c t i n stimulating
t h e transport of calcium across t h e i n t e s t i n a l membrane, thereby increasing
t h e net absorption of t h a t mineral element. Phosphate i s also absorbed
secondarily t o calcium, and so Vitamin D has a secondary e f f e c t i n t h e
t r a n s p o r t of phosphate across t h a t i n t e s t i n a l membrane. A t t h e kidney s i t e
t h e r e i s sone question r e a l l y as t o what does Vitamin D do here, but recent
evidence suggests t h a t t h e vitamin stimulates t h e renal tubular absorption
of calcium and perhaps secondarily of phosphates. Perhaps t h e most allowable of e f f e c t s i s what takes place at t h e bone s i t e . It w a s at f i r s t
supposed t h a t t h e vitamin's function w a s i n t h e actual deposition of mineral
s a l t s i n bone. Through t h e use of radio-isotopic calcium and phosphate it
can be demnstrated t h a t i n contrast t o t h i s b e l i e f t h e vitamin actually
stimlates t h e mobilization of bone mineral and actually stimulates then t h e
solution of bone mineral and placing of that mineral i n t o t h e blood serum.
These t h r e e processes then all function t o elevate the serum calcium and
secondarily serum phosphate concentration which of course goes back t o
providing adequate n u t r i t i o n t o t h e bone so t h a t normal c a l c i f i c a t i o n can
occur.
With these sites of action a t t h e physiologic l e v e l well i n
mind, l e t us now turn t o the question of where does t h e Vitamin D molecule
go and how does it metabolize i n the body. In order t o investigate the
fate of the Vitamin D molecule and where it goes, it w a s necessary t o
produce a radioactive Vitamin D of s u f f i c i e n t radioactivity so t h a t a very
small concentration of the vitamin could be given. You w i l l r e c a l l t h a t
.025 micrograms of Vitamin D represents one International Unit, and t h e
rat responds maximally t o ten International Units. It i s c l e a r then t h a t
t h e radioactivity necessary f o r the detection of t h e vitamin o r its
metabolites would have t o be very g r e a t indeed, and thus f o r lrany years
t h i s problem r e a l l y l a i d unexplored except f o r t h e work of Protochek
i n which milligram doses of t h e vitamin w e r e given t o r a t s . O f course,
e were
t h e l o c a l i z a t i o n under those conditions i s subject t o question. W
able t o produce a labeled Vitamin D by a technique such t h a t t h e
provitamins, which are the ergosterol and 7-dehydrocholesterol, were exposed
t o tritium gas. The t r i t i u m molecules exchanged with the hydrogens on t h e
196.
p a r t i c u l a r active s i t e s of t h e molecule. Then a f t e r two weeks of exposure
these were r e c r y s t a l l i z e d t o remove t h e saturated materials t h a t are produced
by t h e tritium exposure. This i s of course t h e greatest danger i n t h i s
technique. Then t h e provitamins are irradiated t o produce Vitamin D, and
of course t h i s i s another p u r i f i c a t i o n process because only t h e s t e r o l s t h a t
have t h e correct delta f i v e delta seven double bonds w i l l be activated t o
Vitamin D. The unconverted vitamins o r provitamins w e r e removed by rec r y s t a l l i z a t i o n ; t h e tachysterol was removed from t h e maleic anhydride, and
f i n a l l y Vitamins D2 and D3 w e r e c r y s t a l l i z e d out as a dinitrobenzoate.
Many were r e c r y s t a l l i z e d several times t o constant specific a c t i v i t y and
then they were chromatographed i n at least five d i f f e r e n t systems t o show
radio-chemic a1 p u r i t y .
This j u s t represents one such technique, and i n a l l cases w e used
t h e f i r s t phase paper chromatography, absorption column chromatography, e t c
and i n a l l cases t h e r a d i o a c t i v i t y corresponded exactly w i t h t h e appearance
of t h e Vitamin D.
.,
When we look a t the properties of t h i s vitamin, o r these vitamin
preparations, w e notice t h a t we end up with a Vitamin D3 which has a
thousand disintegrations per minute p e r International Unit of t h e vitamin -i n t h e case of t h e IE. I n t h e case of t h e D2 it i s somewhat lower. These
preparations a l l have f u l l biological a c t i v i t y when assayed f o r curing
r i c k e t s i n t h e chicken and i n the rat, and of course t h e radiochemical
p u r i t y has already been discussed.
Now, I can't r e a l l y overemphasize t h e need f o r exhaustive proof
of p u r i t y when t h e Wilsbach o r t h e tritium gas exposure technique i s used.
This i s one of t h e prime dangers and there are many examples i n t h e literat u r e of p i t f a l l s which have trapped many investigators. A t any rate, with
these vitamin preparations it w a s then possible t o proceed with a study of
t h e t i s s u e localization of t h e vitamin. It was s t i l l necessary, even with
t h e high specific a c t i v i t y we obtained, t o give 500 International Units of
Vitamin D t o allow f o r t h e convenient detection of radioactivity. You must
remember that t h i s i s at l e a s t twenty times i n excess of t h e need of t h e r a t
f o r maximal response. So the d i s t r i b u t i o n of t h e r a d i o a c t i v i t y followed
t h e expected pattern, except t h a t we had a f a i r amount of t h e vitamin
appearing i n t h e muscle as f a r as t o t a l amount i s concerned, and a f e w other
unexpected places. When the r e s u l t s are expressed on a t i s s u e weight basis
we can r e a d i l y see t h a t the l i v e r , t h e kidneys and the bone have a very
high concentration except t h a t there i s a tremendous amunt of i n e r t
material which contributes t o t h e weight there, so t h a t t h e bone would have
very high o r f a i r l y high specific a c t i v i t y . The i n t e s t i n e , of course, which
e have t h e
i s a t a r g e t organ has considerable concentration of Vitamin D. W
Vitamin appearing o r r a d i o a c t i v i t y appearing i n t h e f a t pad and other surp r i s i n g places.
Recently we have been able t o use much lower doses of Vitamin D
with some synthetic Vitamin D we have been able t o work up, and the l o c a l i zation of t h e Vitamin then becomes very s p e c i f i c . It i s then found only i n
t h e i n t e s t i n e , bone, kidney, l i v e r and blood. It i s not found i n t h e muscle
o r i n t h e adrenals o r i n t h e f a t pad as indicated. So there i s a specific
l o c a l i z a t i o n of t h e vitamin i n t h e t a r g e t organs and i n t h e primary storage
organ, which i s t h e l i v e r .
197.
The next step was t o determine whether the vitamin w a s active as
such o r whether it was converted t o active form. T h i s required t h e extract i o n of the vitamin from the t i s s u e s . The radioactivity was found t o be
quantitatively extracted from the protein material by a methanol chloroform
procedure. When we examined the amount of radioactivity distributed i n the
chloroform o r i n the aqueous phase, t h e following d i s t r i b u t i o n w a s found.
A fair concentration of t h e radioactivity w a s always i n the aqueous phase.
When t h a t aqueous phase was t e s t e d biologically, it w a s found t o be comp l e t e l y inactive and the aqueous phase material w a s t o t a l l y unable t o cure
r i c k e t s i n rats. The chloroform soluble material s t i l l retained considerable biological a c t i v i t y .
When the chloroform phase w a s then chromatographed on a long
t h i s p a r t i c u l a r one i s an i n t e s t i n a l mucosa extract
s i l i c i c acid colunn
we detected as many as three metabolites of Vitamin D i n t h i s chloroform
phase. The Vitamin D comes off i n t h i s chromatogram, and it naturally at
the 500 International U n i t dosage l e v e l r e a l l y contributed most of t h e
radioactivity t o t h e t i s s u e . However, when t h e metabolites, which are i n dicated by Peak 1 and t h a t l i t t l e shoulder Peak 2 and Peak 3, o r Peak 4,
which i s t h e Peak at tube 60, o r so, were biologically active; t h a t is,
when given back t o rats these materials were able t o cure r i c k e t s . Now,
exactly how biologically active i s an open question, since we can only
estimate t h e quantity of materid on the basis of the radioactivity present.
But t h i s l a s t Eetabolite here has biological a c t i v i t y almost equivalent t o
t h e parent vitamin.
--
-0
In a chromatogram of a kidney extract of a rat given 500 I n t e r national Units of the vitamin, the sane p6ttern i s observed. In t h e case
of liver there i s a f a i r concentration of t h e Tube 60 o r Peak 4 material,
and i n the l i v e r preparation t h e Peak 4 material i s extremely biologically
active. Exactly how active we are not certain, but c e r t a i n l y it i s very
close t o t h a t of t h e parent vitamin.
Finally, .the serum chromatogram showed t h e same s o r t of d i s tribution, and again these products were biologically active. The important
point t o be gathered from these data i s t h a t the vitamin i s converted t o
biologically active metabolites. Whether the vitamin i s active as such i n
the t i s s u e s o r whether one of t h e metabolites i s the active form, cannot
be answered at t h e present time. The f a c t t h a t we have had t o give more
Vitamin D than needed means t h a t there may w e l l be a l o t of nonspecific
localization of t h e vitamin i n these t i s s u e s and it may not r e a l l y function.
So t h i s question must remain open. The important point here i s t h a t the
vitamin is i n fact converted t o biologically active metabolites.
The next question is, where does t h e vitamin go i n t h e c e l l ?
That is, what i s t h e subcellular location of t h e vitamin? When t h e animals
that have been given 500 International U n i t s of t h e radioactive vitamin are
then k i l l e d and the t i s s u e s homogenized and t h e c e l l f r a c t i o n s separated by
d i f f e r e n t i a l centrifugation i n t o the nuclei debris fraction, the mitochond r i a l , t h e microsomal and the cytoplasmic fractions, the following r e s u l t s
w e r e obtained. If one looks at t o t a l radioactivity, one i s impressed by
the f a c t t h a t these three c e l l fractions, the nuclear debris, the mitochond r i a l and t h e microsomal fractions, contained about equal t o t a l mounts of
the radioactivity. The cytoplasmic fraction, t h a t i s the supernatent
198.
f r a c t i o n f r o m t h e centrifugations, had no r a d i o a c t i v i t y o r very l i t t l e
a c t i v i t y a t a l l . If one expresses the r e s u l t s on t h e basis of radioactivity
p e r u n i t weight of tissue, t h a t i s t h e disintegrations per minute per hundred
milligrams of the t i s s u e , one obtains a very s t r i k i n g p i c t u r e on t h e l o c a l i zation of t h e radioactivity. The microsomal f r a c t i o n which of course repres e n t s t h e endoplasmic reticulum of t h e c e l l and perhaps the c e l l membranes
contains t h e highest concentration of t h e vitamin or t h e radioactivity,
followed next by t h e mitochondria, at l e a s t i n t h e case of t h e t w o t a r g e t
organs, t h e i n t e s t i n e and t h e kidney. When one then determines i n these
two membranous fractions, t h a t i s t h e microsomal and mitochondrial. fraction,
when one determines t h e neture of the r a d i o a c t i v i t y there, we f i n d that i n
the microsome and t h e mitochondria t h e r a d i o a c t i v i t y i s almost exclusively
in t h e form of Vitamin D. I n subsequent work it was found t h a t t h e
metabolites are found i n t h e nuclear f r a c t i o n o r nuclear debris f r a c t i o n .
The vitamin c e r t a i n l y g e t s t o t h e membranous portions of t h e c e l l . It seems
t o g e t t o t h e mitochondrial and microsomal portions. The metabolites seem
t o go predominantly t o t h e nuclear f r a c t i o n o r t h e debris f r a c t i o n .
The next question t h a t one could ask is, i f t h e vitamin does get
t h e subcellular f r a c t i o n s does it r e a l l y have any e f f e c t o r has any e f f e c t
been demonstrated here? It has been shown i n previous work t h a t t h e vitamin
has really a very marked e f f e c t on t h e structure and function of a t l e a s t
t h e mitochondrial membrane. Through t h e study of c i t r a t e metabolism by mitochondria and t h e e f f e c t s of Vitamin 13 it w a s found o r suggested t h a t t h e
vitamin i n some way a f f e c t s t h e penetration of c i t r a t e i n t o t h e mitochondria.
This suggested t h a t perhaps t h e mitochondria from Vitamin D d e f i c i e n t animals
were damwed due t o c i t r a t e molecules r e s u l t i n g i n a very much more rapid
oxidation. Upon examination of i s o l a t e d mitochondria by the electron microscope, this hypothesis proved t o be t r u e . These preparations represent
mitochondria i s o l a t e d from Vitamin D deficient rats, t i b i a mitochondria
i s o l a t e d from Vitamin D deficient rats under t h e b e s t conditions t h a t were
available at t h a t t i m e . The mitochondria i s swollen, and many damaged
preparations o r forms can be seen. The mitochondria i s o l a t e d i n p a r a l l e l
from rats fed Vitamin D are q u i t e i n t a c t with very l i t t l e evidence of
damage.
Since t h e basic physiologic action of t h e vitamin i s t o stimulate
t h e transport of calcium and phosphorous across c e l l u l a r membranes, t h i s
represented a c e l l u l a r menibrane t h a t i s probably affected by Vitamin D. We
b.egan t o question whether the mitochondria might now show very marked
effects of Vitamin D on i t s transport of calcium in and out of the mitochondrial membrane. This also proved t o be t h e case w i t h regard t o t h e
transport of calcium out of t h e mitochmdria. Calcium i s bound by i s o l a t e d
mitochondria i n an active fashion dependent upon aetabolic energy. This
process i s t o t a l l y unaffected by Vitamin D. The mitochondria fromVitamin
D f e d rats and rats not receiving Vitamin D both took up calcium rapidly at
equal rates and t o t h e same general o r t o t a l .mount. If now t h e active uptake of calcium i s stopped by t h e addition of hexokinase, glucose which
wipes out t h e ATP which i s used f o r t h e transport of t h i s calcium, w e can
measure t h e flux of calcium back out of t h e mitochondria. Under these cond i t i o n s t h e mitochondria from rats f e d Vitamin D w i l l release calcium m c h
m r e rapidly. This release i s not due t o damage of t h e mitochondria. You
will r e c a l l t h a t t h e minus D mitochondria axe t h e damaged ones, and f u r t h e r
more, i f m r e ATP i s added t o these mitochondriathey w i l l again bind
199.
calcium. So we have a subcellular membrane system which does respond t o
e know t h e Vitamin D i s t h e r e .
Vitamin D with regard t o calcium transport. W
Whether t h e actual Vitamin D t h a t i s present i n these membranes i s t h e
functional point of t h e vitamin remains t o be established. There are two
alternative hypotheses t h a t could explain t h e results: t h e vitamin i t s e l f
could be active i n these membranes i n functioning i n a calcium transport
system, o r it may be t h a t t h e metabolites of Vitamin D may influence t h e
nucleus t o produce some p r o t e i n o r some other material, such as RNA which
w i l l then compose t h e subcellular membranes and w i l l thereby function i n
calcium transport.
These alternatives o r these p o s s i b i l i t i e s can be sorted out i n
t h e future and use of these techniques and systems may eventually give t h e
answer t o how t h i s vitamin operates.
To summarize, Vitamin D i s i n f a c t converted t o biologically
active metabolites. It o r i t s metabolites l o c a l i z e s p e c i f i c a l l y i n the
t a r g e t t i s s u e s and storage organs of t h a t vitamin. Its metebolites o r t h e
vitamin itself i s found i n t h e membranous containing c e l l f r a c t i o n s . It i s
not found i n t h e cytoplasm. Finally, the vitamin has marked effects on
subcellular membrane s t r u c t u r e and function and t h e i r a b i l i t y t o transport
calcium.
DR. KASTELIC: Thank you, D r . De Luca. Since our time
is moving along I think we should m v e d i r e c t l y i n t o t h e d i s cussion and we welcome now any questions t h a t may be directed
e i t h e r t o Dr. Kummerow o r D r . De Luca. I would l i k e t o ask D r .
De Luca one question, and I don't expect an answer. There i s a
marked difference i n t h e s e n s i t i v i t y of animals t o Vitamin D
deficiency. You might compare t h e rat, which i s r e l a t i v e l y i n sensitive, w i t h t h e chick, which i s r e l a t i v e l y sensitive. Is
t h e r e any explanation t h a t might be derived from t h e comments you
made about t h i s postulated function t h a t might be offered i n explanation of t h i s species difference?
DR. DE LUCA:
I am r e a l l y pleased t h a t you brought up
I
think
t h a t t h i s i s a long standing b e l i e f
since
t h a t question,
among s c i e n t i s t s , t h a t there i s a species difference with regard
t o whether r i c k e t s can be produced or not. I t h i n k one wants t o
d i f f e r e n t i a t e between Vitamin D deficiency and r i c k e t s . If we
are speaking of Vitamin D deficiency, then I say t h a t there i s
very l i t t l e species difference between t h e chicken and t h e r a t .
I n t h e s i t u a t i o n where t h e diet i s normal i n calcium
and phosphate r a t s do not develop r i c k e t s but instead they
acquire a very low serum calcium concentration and retarded
growth. The bone appears normal t o all outward appearances.
There may be s l i g h t h i s t o l o g i c a l differences but t h e c l a s s i c a l
c h a r a c t e r i s t i c s of r i c k e t s do not *pear.
That d i e t contains
.45% calcium and .3$ phosphorous. The s e r u m calcium concentrat i o n i n t h e Vitamin D deficient rats i s down t o 6.4, and when
199.
calcium. So we have a subcellular membrane system which does respond t o
e know t h e Vitamin D i s t h e r e .
Vitamin D with regard t o calcium transport. W
Whether t h e actual Vitamin D t h a t i s present i n these membranes i s t h e
functional point of t h e vitamin remains t o be established. There are two
alternative hypotheses t h a t could explain t h e results: t h e vitamin i t s e l f
could be active i n these membranes i n functioning i n a calcium transport
system, o r it may be t h a t t h e metabolites of Vitamin D may influence t h e
nucleus t o produce some p r o t e i n o r some other material, such as RNA which
w i l l then compose t h e subcellular membranes and w i l l thereby function i n
calcium transport.
These alternatives o r these p o s s i b i l i t i e s can be sorted out i n
t h e future and use of these techniques and systems may eventually give t h e
answer t o how t h i s vitamin operates.
To summarize, Vitamin D i s i n f a c t converted t o biologically
active metabolites. It o r i t s metabolites l o c a l i z e s p e c i f i c a l l y i n the
t a r g e t t i s s u e s and storage organs of t h a t vitamin. Its metebolites o r t h e
vitamin itself i s found i n t h e membranous containing c e l l f r a c t i o n s . It i s
not found i n t h e cytoplasm. Finally, the vitamin has marked effects on
subcellular membrane s t r u c t u r e and function and t h e i r a b i l i t y t o transport
calcium.
DR. KASTELIC: Thank you, D r . De Luca. Since our time
is moving along I think we should m v e d i r e c t l y i n t o t h e d i s cussion and we welcome now any questions t h a t may be directed
e i t h e r t o Dr. Kummerow o r D r . De Luca. I would l i k e t o ask D r .
De Luca one question, and I don't expect an answer. There i s a
marked difference i n t h e s e n s i t i v i t y of animals t o Vitamin D
deficiency. You might compare t h e rat, which i s r e l a t i v e l y i n sensitive, w i t h t h e chick, which i s r e l a t i v e l y sensitive. Is
t h e r e any explanation t h a t might be derived from t h e comments you
made about t h i s postulated function t h a t might be offered i n explanation of t h i s species difference?
DR. DE LUCA:
I am r e a l l y pleased t h a t you brought up
I
think
t h a t t h i s i s a long standing b e l i e f
since
t h a t question,
among s c i e n t i s t s , t h a t there i s a species difference with regard
t o whether r i c k e t s can be produced or not. I t h i n k one wants t o
d i f f e r e n t i a t e between Vitamin D deficiency and r i c k e t s . If we
are speaking of Vitamin D deficiency, then I say t h a t there i s
very l i t t l e species difference between t h e chicken and t h e r a t .
I n t h e s i t u a t i o n where t h e diet i s normal i n calcium
and phosphate r a t s do not develop r i c k e t s but instead they
acquire a very low serum calcium concentration and retarded
growth. The bone appears normal t o all outward appearances.
There may be s l i g h t h i s t o l o g i c a l differences but t h e c l a s s i c a l
c h a r a c t e r i s t i c s of r i c k e t s do not *pear.
That d i e t contains
.45% calcium and .3$ phosphorous. The s e r u m calcium concentrat i o n i n t h e Vitamin D deficient rats i s down t o 6.4, and when
200.
Vitamin D i s given t h e calcium will immediately shoot up t o 10.3.
Those rats are d e f i c i e n t i n t h e i r parathyroid hormone. Their
parathyroids do not function normally with regard t o calcium
metabolism, and i f one takes great pains he can actually have
these rats d i e of tetany due t o Vitamin D deficiency. Now i n a
d i e t t h a t produces rickets, t h e r e i s a very s m a l l change i n
serum calcium due t o Vitamin D. The g r e a t e s t change occurs i n
serum phosphorous, and t h i s i s t h e reason f o r the statement t h a t
r i c k e t s i n rats i s r e a l l y aphosphate deficiency. The chicken
of course requires only t h a t Vitamin D be l e f t out of the d i e t
and r i c k e t s w i l l ensue, and I think t h i s i s the besis for t h e
statement.
DR. KASTELIC: Thank you, D r . De Luca.
f o r one more question. Yes, Dr. Kummerow?
W
e hme time
DR, KUMMEROW: Hector, did you t r y C-14 any at a l l ?
We used both C-14 labeled l i n o l e i c acid and tritium l a b e l
l i n o l e i c acid and we found s p e c i f i c a c t i v i t y t h e same i n both
cases. If you are worried about producing tritium l d e l
Vitamin D so t h a t you do not get an exchange of tritium, I
wondered i f you could make sure about t h a t i n your own mind i f
you used a carbon 14 label, whether it would work t h e same way.
DR. DE LUCA: We don't have any carbon 14 labeled
Vitamin D at t h e present t i m e . W e have some low s p e c i f i c
a c t i v i t y material ordered, and we are synthesizing some. Now,
with regard t o worrying about t h e change of tritium, t h e f a c t
t h a t t h e metabolites t h a t we have and t h e f a c t t h a t we recover
the vitamin f r o m t h e t i s s u e s with t h e same s p e c i f i c a c t i v i t y ,
t h e same biological a c t i v i t y as you would expect, would tend t o
disregard o r tend t o make t h i s worry minimal. However, there i s
a p o s s i b i l i t y t h a t we be missing a metabolite i n which a change
has occurred. Carbon 14 labeled material would be very useful.
DR. WTELIC: Thank you. I want t o thank W. Kummerow
and D r . De Luca f o r their presentations t h i s d t e r n o o n on behalf
of those assembled here. I would l i k e t o ask Dr. I b t y t o take
t h e podium.
DR, M y r y : I think t h i s completes t h e very excellent
program arranged by t h e Committee on Mztrition, and we w i l l now
have a f e w minutes recess.
(Recess)
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