Vitamin Bronner,
Classical
phosphate
and
phosphorus with only
deficiency. the absence
situations.
The
rickets
leading
primarily proposed aggravates nutritional been
vitamin
Simple of th
expression
the
vitamin vitamin
of
level
differs whereas
in the two simple
without obvious of a nutritional defect
by rickets,
in
may
from
phosphate occur,
but
rickets, to both
conditions,
vtamin
structural vitamin
both
by simple
from experimental proteins common
changes,
metabolic
resulting reproducible
with
deficiency
alterations. D deficiency transport.
probably
It is that Simple
has always
J. Cli,z.Nutr. 29: 1307- 1314, 1976.
findings
of Clinical
deficiency,
of rickets
D deficiency
metabolic
unaccompanied
of problem
Journal
is a dual
features
differs markedly calcium-binding
of vitamin
and
a pre-existing
Simple vitamin D deficiency in the rat (6) leads to hypocalcemia, a drop in urinary calcium, a diminution in the rates of calcium The American
of the
D deficiency D-dependent
structural
D deficiency,
Am.
the rat
in
many
the regulatory function of bone, nutritional rickets is the result
expression
vitamin
rickets with
at tI)e bone
to profound
compromises that human
rare.
experimental D deficiency,
The title of this review may appear to be a tautology, as on histological and clinical grounds rickets and vitamin D deficiency have long been considered to be the same. Yet a close reading of the history of experimental rickets, especially in the rat, shows that most studies were aimed at reproducing the histological and gross anatomic features of human rickets and were not necessarily undertaken to study simple vitamin D deficiency (I, 2). Indeed, the first report of the symptoms and characteristic findings of simple vitamin D deficiency in the rat (3) showed that deficient animals develop hypocalcemia and not rickets. To produce the latter it is necessary to place animals from weaning on a high-calcium, low-phosphorus diet, deprived of vitamin D (4, 5). In other words, rickets in the rat is the result of a dual deficiency, of phosphorus and of vitamin D. The purpose of this review is to examine the relative importance of these two conditions in producing rickets in the rat and to speculate on how those findings apply to human rickets. Because the relevant literature is vast, citations will be illustrative, rather than encyclopedic. Experimental
ricket&’2
Ph.D.3
ABSTRACT
Definition
and
Nutrition
29: NOVEMBER
entry into and exit from bone, and a drop in bone ash, bone calcium, and bone phosphorus. Calcium absorption is diminished. Simple vitamin D deficiency does not lead to obvious histological changes in bone. Rats raised on a vitamin D-deficient diet, adequate in all other nutrients, will be almost indistinguishable in appearance from their controls and their body weights will be only a little lower than those of their controls (6-8). From the point of view of mechanism, one can ask whether the effects of simple vitamin D deficiency are mediated merely by lack of calcium. It is conceivable that the diminution of calcium absorption is the primary effect of simple vitamin D deficiency, all other changes being consequent to diminished input from the gut. This hypothesis seems improbable on both experimental and theoretical grounds. When vitamin D-replete animals, reared on an adequate calcium intake, are placed on a low From Dental 2
the
Department
Medicine,
The
of
University
experimental
Oral
Biology,
School
of
article
is
of Connecticut.
studies
on
which
this
based were aided by United States Public Health Service Research and Training grants, and by The University of Connecticut Research Foundation. This support is gratefully acknowleded. I also thank my colleagues, Drs. S. Hurwitz, T. S. Freund, E. Golub, and P. Cuisinier-Gleizes, for many fruitful discussions. Professor of Oral Biology, The University of Connecticut cut
Health
Center,
Farmington,
Connecti-
06032.
1976, pp. 1307-1314.
Printed
in
U.S.A.
1307
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
Felix
D deficiency
BRONNER
1308 TABLE Some
I parameters
of calcium
metabolism
Experim
s
entalgroup
% Ca
% P
PTX
Plasma Ca pooi’ (mg)
BoneCa deposition (v0., mg/day)
UrinaryCa excretion (vU, mg/day)
BoneCa clearance (day ‘)
UrinaryCa clearance (day ‘)
CD
+
0.5
0.5
No
0.61
59.2
0.20
97
C
-
0.5
0.5
No
0.40
47.2
0.16
118
RD
+
1.5
0.14
No
0.29
19.3
4.5
66
15.4 25.2
0.33 0.40
R
-
1.5
0.14
No
0.29
13.2
7.3
49
PTX
+
0.5
0.5
Yes
0.49
45.2
0.96
92
N
+
0.5
0.5
No
0.88
70.5
0.90
80
1.02
N2
+
when
net
=
20 mg
No
0.88
69.4
0.70
79
0.80
daily
Ca
N3
+
absorption
=
40mg
No
0.88
71.6
1.10
81
1.25
1
Based
on data
Values
for groups
values
were
from CD,
derived
output.
h Plasma
Clearance
=
Hurwitz C, RD.
for
net
calcium
deposition
et al. (Reference R, PTX,
and
absorption pool
=
plasma
N
equal
6: CD. derived
to
calcium
C, RD.
for
20 or 40 level
R) and
net absorption mg/Ca
x plasma
per
Sammon =
day.
volume.
et al. (Reference
30 mg ofCa/day. Net Plasma
1.96
= intake
absorption volume
I I: PTX,
In groups
=
0.035
N).
N2 and minus
x body
N3
fecal weight.
or excretion/pool.
calcium intake, plasma calcium decreases marginally and hypocalcemia takes many weeks to achieve (9). Urinary calcium excretion drops, and the calcium deposition rate in bone is unchanged intially (see also Table I), the bone calcium resorption rate rising instead (10, 1 1). If one were to express the bone calcium deposition rate in animals on a low calcium intake as a fractional clearance of the plasma calcium pool it would barely diminish, whereas the fractional kidney clearance, i.e., urinary excretion expressed as a fraction of the plasma calcium pool, would drop (Fig. IC). However, as shown in Figure IA, yitamin D deficiency causes the relative bone and kidney clearances to go up. In other words, when clearances are calculated for conditions of equal net calcium absorption, vitamin D depletion has an effect opposite to that of calcium depletion. Clearly, then, the effect of vitamin D is not mediated nor can it be
confronted with an intraperitoneal calcium load (6). Is this diminished regulatory capacity parathyroid hormone-related? Au and Raisz (12) showed increased parathyroid gland activity in hypocalcemic, vitamin D-deficient animals. Recent measurements (J. Fischer and F. Bronner, unpublished data) of circulating parathyroid hormone levels in vitamin D-deficient rats have shown that these levels are about 5 times higher than in vitamin D-replete controls. Nevertheless, from what happens in parathyroidectomized rats as compared to euparathyroid controls it may be deduced (Fig. IB)that restoration of parathyroid function reduces the relative bone and kidney clearances. In other words, the direction ofchange is opposite to that in vitamin D deprivation. Instead, it is qualitatively similar to what happens when animals are shifted from high- to low-calcium diets (Fig. 1C). As reviewed in greater detail elsewhere in
restored by calcium alone. It follows that vitamin D deficiency must affect bone directly and not only because bone is deprived of calcium. One physiological expression of this effect of vitamin D deficiency is the alteration of the regulatory capacity of bone for plasma calcium homeostasis. This is evident from the fact that the animals are hypocalcemic and are less able to overcome a positive disturbance, as when
this Symposium, vitamin D is transformed in the body to an active metabolite, of which 1,25-dihydroxy-vitamin D3 is the best known example. This compound leads to the induction in the rat intestinal cell of the calciumbinding protein, an event that has recently been demonstrated in vitro (13). Moreover, induction of this compound is closely associated, both temporally and functionally, with increased calcium transport, both in vivo, in
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
D
Vit
#{176}
in the rat
VITAMIN
D DEFICIENCY
200
200
A LI
z
100
>
6-
PX
---BLOOD
TO BONE
-BLOOD
TO KIDNEY
INTACT
200
C LI
z 4
100 >
HIGH
Co
LOW
Ca
FIG. 1. The effect ofsimple vitamin D deficiency (A), restoration of parathyroid function (B), and lowering of calcium intake (C) on the relative clearances from blood to bone and blood to kidney in rats. Note that the directions are similar for lowering of net calcium abserption and restoration of parathyroid function, but that they differ from what happens as a result of vitamin D deficiency. The relative clearances were calculated from the data in Table I , with the values for net absorption the same (= 30 mg/day) for all for conditions in A and B, whereas C illustrates the effect when net absorption is lowered from 40 to 20 mg/day by dietary calcium restriction.
the chick and in the rat (Reference 14 and Bronner et al., unpublished observations), and in vitro, in the rat and in the chick (13, 15). If vitamin D is directly involved in the synthesis of a calcium-transporting molecule, it is not unreasonable to look for a similar mechanism in bone, if, as argued above, the metabolic function of bone is altered in vitamin D deficiency. As yet, no molecule has been identified in bone cells that is comparable to the intestinal calcium-binding protein (Wasserman, personal communication). This may be attributed in part to limited success in studying bone cells in vitro. DeLuca in this Symposium has argued on experimental grounds (16, 17) that in bone (but not in the intestine) 1,25-(OH)2-D3 cannot act unless parathyroid hormone is pres-
RICKETS
I309
ent. One explanation could be that bone cells have no receptors for vitamin D metabolites and all bone effects ofvitamin D are parathyroid hormone-mediated. In the light of Figure 1 this possibility seems remote. Alternatively it would mean bone cell response to vitamin D is enhanced by the action of parathyroid hormone on the bone cell. If, as is generally believed, the latter is mediated by the membrane-bound adenyl cyclase system (18, 19), one would look for vitamin D-parathyroid hormone interaction in the complicated cellular machinery of kinase-modulated protein synthesis. What happens in experimental rickets produced by a high-calcium, low-phosphorus diet, deficient in vitamin D? Here the question becomes one of the appropriate control, i.e., whether the experimental animal is compared with a vitamin D-replete animal on the same high-calcium and low-phosphorus diet, or whether it is compared with a vitamin Dreplete animal on normal calcium and phosphorus intake. Let us first look at the effect of phosphate deficiency in the presence of an adequate supply of vitamin. The immediate effect of dietary phosphate withdrawal is a marked hypophosphatemia (20) which, within 48 hr. is partly corrected. In other words, plasma phosphate exhibits a clear undershoot in rats placed on a low-phosphate diet. Nevertheless, the plasma phosphate thereafter remains low, its absolute level a positive function of phosphate intake (20). The hypophosphatemia is accompanied by a rise in plasma calcium. Although the two are related, there is no corresponding overshoot in plasma calcium. Urinary phosphate drops markedly, whereas urinary calcium rises, attaining levels more than one order of magnitude greater than in the phosphate-replete animal (6, 2 1 ; Table I). Bone calcium deposition and resorption diminish, with deposition dropping more than resorption (6). As a result the bone calcium balance becomes more negative (6). Fig. 2C shows what happens to the relative calcium clearances in phosphate deficiency. When net calcium absorption is held at the same value (30 mg/day) for the deficient and control animals, the bone calcium clearance of the phsophate-deficient group drops 28%,
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
100 LI
AND
BRONNER
1310
-
A
100-
1
6.
6-
HIGHP
VITAMiN ---BLOOD
TO BONE
-BLOOD
TO 20U
LOSVP
D PRESENT
KiDtEY
4t B
D 3
I
too-
100-
2
4
8 1
c. LOWP
6-
HIGHP.D.
LOwiD-
DT
FIG. 2. The effect of vitamin D deficiency with normal mineral intake (A), of vitamin D deficiency in low phosphate intake (B), the effect of low phosphate intake alone (C), and the effect of low phosphate intake combined with vitamin D deficiency (D) on the relative clearances from blood to bone and blood to kidney in rats. Note that in phosphate deficiency, whether accompanied by vitamin D deficiency or not, the relative bone calcium clearance is diminished (B, C. D), whereas it is raised in simple vitamin deficiency (A). Vitamin D deficiency raises urinary clearance, whether or not phosphate deficiency exists in addition (A vs. B). In the presence of phosphate deficiency, however, the effect of vitamin D deficiency on the relative kidney and bone clearances is obscured, as C and D are nearly superimposable. The relative clearances were calculated from the data in Table I, with the values for net calcium absorption the same (= 30 mg/day) for all four groups of animals.
while the urinary calcium clearance increases by more than two orders of magnitude. The high urinary calcium excretion seems to reflect the inability of the body to utilize calcium, a byproduct of the body’s effort to “mine” the skeleton for the scarce phosphate. Histologically, phosphate deficiency (21) produces a picture reminiscent of classical rickets, i.e., widening of the epiphyseal plate, thinning out and loss of trabecular bone in the metaphyseal region, and narrowing of the cortical width in the diaphyseal region. The number of resorption cavities increases. Osteocyte number decreases, with the proportion of enlarged osteocytes increasing in phos-
The ciency Figure deficiency shown
overall similarity of phosphate defito rickets is brought out further in 2D, where the results of the dual of phosphate and vitamin D are in terms of the relative bone and
kidney clearances of calcium. Figure 2D indicates that the absence of both phosphorus and vitamin D leads to a 50% drop in the relative bone calcium clearance and a 100-fold rise in the relative kidney calcium clearance. Indeed, Figure 2D does not seem very different from Figure 2C, the effect of simple phosphorus deficiency. The two graphs are nearly superimposable, so that the imposition of vitamin D deficiency on phosphate deficiency does not change the direction or markedly the amount by which the two clearances are altered. In both situations, the system, because of the need for phosphorus, is flooded by calcium that is skeletal in origin, but at the same time there is less bone to help maintain plasma calcium. Moreover, in the case of the dual deficiency, the bone is probably abnormal. The result in both instances is greater reliance of the organism on renal regulation. Because, however, the capacity of the kidney to handle calcium is evidently limited-even though urinary calcium excretion is raised 100-fold in phosphate deficiency-hypercalcemia results. Hypercalcemia is absolute in pure phosphate deficiency; it is relative, but sometimes absolute, in the dual deficiency. Another way to analyze the effect of vitamin D deficiency is to compare its effects in Relative phosphate depletion of the bone mineral probably arises from the fact that the bone mineral laid down first is relatively rich in phosphate and that its low crystal size and higher solubility also favor its ready resorption (22).
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
phate deficiency. Phosphate deficiency leads to a 5% increase in the proportion of the trabecular surface covered by osteoclasts and a 200% increase in the proportion of the trabecular surface covered by osteoblasts. Compositional changes in bone-decrease in ash content, increase in water and hydroxyproline content, relative depletion in phosphate,4 (21)-are also consistent with the interpretation that the skeletal phosphate is being utilized to replenish diminished soft tissue stores.
2OO
200
VITAMIN
D DEFICIENCY
Application
to human
disease
On the basis of the preceding considerations, one can visualize three possible situations in children: 1) a simple deficiency in vitamin D, resulting either from a nutritional deficiency or a metabolic defect leading to deficient production of the active vitamin D metabolite; 2) a phosphate deficiency either nutritional in origin or resulting from a defect
1311
RICKETS
in phosphate metabolism; 3) a combination of both deficiencies. Metabolic defects that lead to inadequate production of the active metabolite of vitamin D could result from inappropriate enzyme synthesis, presumed to be genetic in origin. A situation where administration of small amounts of 1,25-dihydroxy-vitamin D3 led to a dramatic improvement in a child diagnosed as having vitamin D-resistant rickets has been reported (25). Whether a given defect is at the level of hydroxylation of carbon I (as in Reference 25) or carbon 25 (none has yet been reported), one would predict that such defects, unaccompanied by deficient mineral intake, would lead to the defects in calcium metabolism characteristic of simple vitamin D deficiency. In the rat and presumably the child this would mean hypocalcemia, diminished bone turnover with a relative decrease in bone calcium resorption, an absolute hypocalcemia, but a relative increase in the fractional urinary calcium clearance. Calcium absorption would of course be diminished. However, the histological changes in bone would not be those of rickets. It is evident that simple nutritional deficiency of vitamin D in the human infant would fall into this category. The question arises whether simple vitamm D deficiency has ever been documented in man. Lapatsanis (personal communication) has seen a hypocalcemic infant without radiological signs of rickets, who has responded to daily doses of 1,000 U of vitamin D by growth resumption and normocalcemia. Moreover, he has been able retrospectively to classify cases of rickets in Greek infants into those that are predominantly hypocalcemic and do not exhibit radiological. signs of ricket and those predominantly hypophosphatemic with clear radiological signs of rickets. There is therefore reason to think that simple vitamin D deficiency does
The
older
literature
is difficult
the clinical manifestations of rickets ical. Moreover, the chemical signs confusingly.
For
example:
‘Usually
to analyze:
however,
are usually radiologof rickets are treated the
calcium
is in
the
normal range of 9.5- II mg per 100 cc. of serum, although at times it may be as low as 5 mg In normal infants, the serum inorganic phosphate is about 5 mg per 100 cc, but in rickets it is reduced and may be as low as 1 mg” (26).
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
the presence and in the absence of phosphate deficiency. A comparison of Figure 2 A and B shows that vitamin D deficiency superimposed on phosphate deficiency causes the relative bone clearance to drop and the relative kidney clearance to go up, whereas vitamin D deficiency in the presence of adequate phosphorus and calcium causes both clearances to go up. The only known molecular lesion that results from vitamin D deficiency is the absence of the vitamin D-dependent calciumbinding proteins from the intestine (7) and from kidney (8). This lesion is the same, whether vitamin D deficiency is imposed on animals on a normal mineral intake or one deficient in phosphorus (8, 23). Indeed, the absence of duodenal calcium-binding proteins has recently been proposed as a quantitative test of vitamin D deficiency in the rat (7) or as an assay for the chick (24). At the level of bone, however, it is apparent that vitamin D deficiency is expressed differently in the presence or absence of an adequate phosphorus supply. The major cause of rickets appears to be phosphorus deficiency, inasmuch as phosphorus deficiency alone can give rise to a syndrome in bone that cannot readily be distinguished from classical rickets. Vitamin D deficiency alone leads to a regulatory defect on the part of bone, as discussed above. Presumably the relative increase in bone destruction characteristic of the dual deficiency compromises the metabolic role of bone even further. However, not until the nature of the bone lesion caused by vitamin D becomes known will it be possible to understand in molecular terms how calcium or phosphorus deficiency modifies the expression of this lesion.
AND
1312
BRONN
2 of vitamin
TABLE Effect in
the
D deficiency
on phosphate
Parameter Body
metabolism
rat -D2
wt, g
146(1)
Plasma
calcium,
Plasma
calcium
mg/
10.3
100 ml
poo1, mg
(0.2)
0 67
243(18)
Calcium
intake,
Urinary
calcium,
mg/day mg/day
Urinary
calcium
clearance,
urinary
calcium
port defect by itself could be mild and asymptomatic. It would become symptomatic as a result of an environmentally caused vitamin D deficiency. Such deficiency would usually be the result of cultural practices. These might include industrial smoke pollution which filters out ultraviolet radiation,
:f:2)3
0.53
260(20)
11.5(1.0)
2.9(0.7)
21.7
4.3
day Relative
505
100
clearance Plasma
phosphorus,
Plasma
phosphorus
Phosphorus
mg/100 pool,
intake.
ml
2.4(0.1)
mg
Urinary
phosphorus,
mg/day
Urinary
phosphorus
clearance,
child-rearing practices such as infrequent exposure to the outdoors, use ofclothing that prevents exposure to ultraviolet light of any part of the body, poor dietary practices,
7.4(0.2)
0.12
0.42
l(#{216}
mg/day
I)
17(1)
167
mm D, the plasma phosphate was only 2.4 mg/100 ml in the vitamin D-deficient animals, whereas it was 7.4 in the controls. Both groups ingested similarly low quantities of phosphorus and their urinary phosphorus losses were similar. Clearly the presence of vitamin D exerted a significant effect on phosphate metabolism, including phosphate absorption (see also Reference 6). Therefore it does not seem unreasonable to propose that human nutritional rickets often results from a dual defect of phosphorus and vitamin D. Because nutritional deficiency of phosphorus intake is an unlikely situation, it seems more reasonable to postulate that human nutritional rickets arises in infants that have a low intake ofvitamin D and at the same time have some defect in phosphate transport, in the kidney (28) and/or in the intestine (29). Moreover, the phosphate trans-
prolonged nourished
095
mothers, breast feeding and
soby forth. relatively
poorly
day Relative
urinary
phosphorus
176
100
clearance
Ninety-two
(I
divided
weanling
into
two
phosphorus ing
diet
IU
plasma
calcium,
cages and
deficiency
differ
D2/kg
from
each
were
then
placed
Vitamin
the
were
of the vitamin protein. errors
of (P
numbers
the
means.
Classical
phosphate
and
phosphorus with only
deficiency. the absence
situations.
The
rickets
leading
primarily proposed aggravates nutritional been
vitamin
Simple of th
expression
the
vitamin vitamin
of
level
differs whereas
in the two simple
without obvious of a nutritional defect
by rickets,
in
may
from
phosphate occur,
but
rickets, to both
conditions,
vtamin
structural vitamin
both
by simple
from experimental proteins common
changes,
metabolic
resulting reproducible
with
deficiency
alterations. D deficiency transport.
probably
It is that Simple
has always
J. Cli,z.Nutr. 29: 1307- 1314, 1976.
findings
of Clinical
deficiency,
of rickets
D deficiency
metabolic
unaccompanied
of problem
Journal
is a dual
features
differs markedly calcium-binding
of vitamin
and
a pre-existing
Simple vitamin D deficiency in the rat (6) leads to hypocalcemia, a drop in urinary calcium, a diminution in the rates of calcium The American
of the
D deficiency D-dependent
structural
D deficiency,
Am.
the rat
in
many
the regulatory function of bone, nutritional rickets is the result
expression
vitamin
rickets with
at tI)e bone
to profound
compromises that human
rare.
experimental D deficiency,
The title of this review may appear to be a tautology, as on histological and clinical grounds rickets and vitamin D deficiency have long been considered to be the same. Yet a close reading of the history of experimental rickets, especially in the rat, shows that most studies were aimed at reproducing the histological and gross anatomic features of human rickets and were not necessarily undertaken to study simple vitamin D deficiency (I, 2). Indeed, the first report of the symptoms and characteristic findings of simple vitamin D deficiency in the rat (3) showed that deficient animals develop hypocalcemia and not rickets. To produce the latter it is necessary to place animals from weaning on a high-calcium, low-phosphorus diet, deprived of vitamin D (4, 5). In other words, rickets in the rat is the result of a dual deficiency, of phosphorus and of vitamin D. The purpose of this review is to examine the relative importance of these two conditions in producing rickets in the rat and to speculate on how those findings apply to human rickets. Because the relevant literature is vast, citations will be illustrative, rather than encyclopedic. Experimental
ricket&’2
Ph.D.3
ABSTRACT
Definition
and
Nutrition
29: NOVEMBER
entry into and exit from bone, and a drop in bone ash, bone calcium, and bone phosphorus. Calcium absorption is diminished. Simple vitamin D deficiency does not lead to obvious histological changes in bone. Rats raised on a vitamin D-deficient diet, adequate in all other nutrients, will be almost indistinguishable in appearance from their controls and their body weights will be only a little lower than those of their controls (6-8). From the point of view of mechanism, one can ask whether the effects of simple vitamin D deficiency are mediated merely by lack of calcium. It is conceivable that the diminution of calcium absorption is the primary effect of simple vitamin D deficiency, all other changes being consequent to diminished input from the gut. This hypothesis seems improbable on both experimental and theoretical grounds. When vitamin D-replete animals, reared on an adequate calcium intake, are placed on a low From Dental 2
the
Department
Medicine,
The
of
University
experimental
Oral
Biology,
School
of
article
is
of Connecticut.
studies
on
which
this
based were aided by United States Public Health Service Research and Training grants, and by The University of Connecticut Research Foundation. This support is gratefully acknowleded. I also thank my colleagues, Drs. S. Hurwitz, T. S. Freund, E. Golub, and P. Cuisinier-Gleizes, for many fruitful discussions. Professor of Oral Biology, The University of Connecticut cut
Health
Center,
Farmington,
Connecti-
06032.
1976, pp. 1307-1314.
Printed
in
U.S.A.
1307
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1307/4649727 by University of Rhode Island user on 08 December 2018
Felix
D deficiency
BRONNER
1308 TABLE Some
I parameters
of calcium
metabolism
Experim
s
entalgroup
% Ca
% P
PTX
Plasma Ca pooi’ (mg)
BoneCa deposition (v0., mg/day)
UrinaryCa excretion (vU, mg/day)
BoneCa clearance (day ‘)
UrinaryCa clearance (day ‘)
CD
+
0.5
0.5
No
0.61
59.2
0.20
97
C
-
0.5
0.5
No
0.40
47.2
0.16
118
RD
+
1.5
0.14
No
0.29
19.3
4.5
66
15.4 25.2
0.33 0.40
R
-
1.5
0.14
No
0.29
13.2
7.3
49
PTX
+
0.5
0.5
Yes
0.49
45.2
0.96
92
N
+
0.5
0.5
No
0.88
70.5
0.90
80
1.02
N2
+
when
net
=
20 mg
No
0.88
69.4
0.70
79
0.80
daily
Ca
N3
+
absorption
=
40mg
No
0.88
71.6
1.10
81
1.25
1
Based
on data
Values
for groups
values
were
from CD,
derived
output.
h Plasma
Clearance
=
Hurwitz C, RD.
for
net
calcium
deposition
et al. (Reference R, PTX,
and
absorption pool
=
plasma
N
equal
6: CD. derived
to
calcium
C, RD.
for
20 or 40 level
R) and
net absorption mg/Ca
x plasma
per
Sammon =
day.
volume.
et al. (Reference
30 mg ofCa/day. Net Plasma
1.96
= intake
absorption volume
I I: PTX,
In groups
=
0.035
N).
N2 and minus
x body
N3
fecal weight.
or excretion/pool.
calcium intake, plasma calcium decreases marginally and hypocalcemia takes many weeks to achieve (9). Urinary calcium excretion drops, and the calcium deposition rate in bone is unchanged intially (see also Table I), the bone calcium resorption rate rising instead (10, 1 1). If one were to express the bone calcium deposition rate in animals on a low calcium intake as a fractional clearance of the plasma calcium pool it would barely diminish, whereas the fractional kidney clearance, i.e., urinary excretion expressed as a fraction of the plasma calcium pool, would drop (Fig. IC). However, as shown in Figure IA, yitamin D deficiency causes the relative bone and kidney clearances to go up. In other words, when clearances are calculated for conditions of equal net calcium absorption, vitamin D depletion has an effect opposite to that of calcium depletion. Clearly, then, the effect of vitamin D is not mediated nor can it be
confronted with an intraperitoneal calcium load (6). Is this diminished regulatory capacity parathyroid hormone-related? Au and Raisz (12) showed increased parathyroid gland activity in hypocalcemic, vitamin D-deficient animals. Recent measurements (J. Fischer and F. Bronner, unpublished data) of circulating parathyroid hormone levels in vitamin D-deficient rats have shown that these levels are about 5 times higher than in vitamin D-replete controls. Nevertheless, from what happens in parathyroidectomized rats as compared to euparathyroid controls it may be deduced (Fig. IB)that restoration of parathyroid function reduces the relative bone and kidney clearances. In other words, the direction ofchange is opposite to that in vitamin D deprivation. Instead, it is qualitatively similar to what happens when animals are shifted from high- to low-calcium diets (Fig. 1C). As reviewed in greater detail elsewhere in
restored by calcium alone. It follows that vitamin D deficiency must affect bone directly and not only because bone is deprived of calcium. One physiological expression of this effect of vitamin D deficiency is the alteration of the regulatory capacity of bone for plasma calcium homeostasis. This is evident from the fact that the animals are hypocalcemic and are less able to overcome a positive disturbance, as when
this Symposium, vitamin D is transformed in the body to an active metabolite, of which 1,25-dihydroxy-vitamin D3 is the best known example. This compound leads to the induction in the rat intestinal cell of the calciumbinding protein, an event that has recently been demonstrated in vitro (13). Moreover, induction of this compound is closely associated, both temporally and functionally, with increased calcium transport, both in vivo, in
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D
Vit
#{176}
in the rat
VITAMIN
D DEFICIENCY
200
200
A LI
z
100
>
6-
PX
---BLOOD
TO BONE
-BLOOD
TO KIDNEY
INTACT
200
C LI
z 4
100 >
HIGH
Co
LOW
Ca
FIG. 1. The effect ofsimple vitamin D deficiency (A), restoration of parathyroid function (B), and lowering of calcium intake (C) on the relative clearances from blood to bone and blood to kidney in rats. Note that the directions are similar for lowering of net calcium abserption and restoration of parathyroid function, but that they differ from what happens as a result of vitamin D deficiency. The relative clearances were calculated from the data in Table I , with the values for net absorption the same (= 30 mg/day) for all for conditions in A and B, whereas C illustrates the effect when net absorption is lowered from 40 to 20 mg/day by dietary calcium restriction.
the chick and in the rat (Reference 14 and Bronner et al., unpublished observations), and in vitro, in the rat and in the chick (13, 15). If vitamin D is directly involved in the synthesis of a calcium-transporting molecule, it is not unreasonable to look for a similar mechanism in bone, if, as argued above, the metabolic function of bone is altered in vitamin D deficiency. As yet, no molecule has been identified in bone cells that is comparable to the intestinal calcium-binding protein (Wasserman, personal communication). This may be attributed in part to limited success in studying bone cells in vitro. DeLuca in this Symposium has argued on experimental grounds (16, 17) that in bone (but not in the intestine) 1,25-(OH)2-D3 cannot act unless parathyroid hormone is pres-
RICKETS
I309
ent. One explanation could be that bone cells have no receptors for vitamin D metabolites and all bone effects ofvitamin D are parathyroid hormone-mediated. In the light of Figure 1 this possibility seems remote. Alternatively it would mean bone cell response to vitamin D is enhanced by the action of parathyroid hormone on the bone cell. If, as is generally believed, the latter is mediated by the membrane-bound adenyl cyclase system (18, 19), one would look for vitamin D-parathyroid hormone interaction in the complicated cellular machinery of kinase-modulated protein synthesis. What happens in experimental rickets produced by a high-calcium, low-phosphorus diet, deficient in vitamin D? Here the question becomes one of the appropriate control, i.e., whether the experimental animal is compared with a vitamin D-replete animal on the same high-calcium and low-phosphorus diet, or whether it is compared with a vitamin Dreplete animal on normal calcium and phosphorus intake. Let us first look at the effect of phosphate deficiency in the presence of an adequate supply of vitamin. The immediate effect of dietary phosphate withdrawal is a marked hypophosphatemia (20) which, within 48 hr. is partly corrected. In other words, plasma phosphate exhibits a clear undershoot in rats placed on a low-phosphate diet. Nevertheless, the plasma phosphate thereafter remains low, its absolute level a positive function of phosphate intake (20). The hypophosphatemia is accompanied by a rise in plasma calcium. Although the two are related, there is no corresponding overshoot in plasma calcium. Urinary phosphate drops markedly, whereas urinary calcium rises, attaining levels more than one order of magnitude greater than in the phosphate-replete animal (6, 2 1 ; Table I). Bone calcium deposition and resorption diminish, with deposition dropping more than resorption (6). As a result the bone calcium balance becomes more negative (6). Fig. 2C shows what happens to the relative calcium clearances in phosphate deficiency. When net calcium absorption is held at the same value (30 mg/day) for the deficient and control animals, the bone calcium clearance of the phsophate-deficient group drops 28%,
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100 LI
AND
BRONNER
1310
-
A
100-
1
6.
6-
HIGHP
VITAMiN ---BLOOD
TO BONE
-BLOOD
TO 20U
LOSVP
D PRESENT
KiDtEY
4t B
D 3
I
too-
100-
2
4
8 1
c. LOWP
6-
HIGHP.D.
LOwiD-
DT
FIG. 2. The effect of vitamin D deficiency with normal mineral intake (A), of vitamin D deficiency in low phosphate intake (B), the effect of low phosphate intake alone (C), and the effect of low phosphate intake combined with vitamin D deficiency (D) on the relative clearances from blood to bone and blood to kidney in rats. Note that in phosphate deficiency, whether accompanied by vitamin D deficiency or not, the relative bone calcium clearance is diminished (B, C. D), whereas it is raised in simple vitamin deficiency (A). Vitamin D deficiency raises urinary clearance, whether or not phosphate deficiency exists in addition (A vs. B). In the presence of phosphate deficiency, however, the effect of vitamin D deficiency on the relative kidney and bone clearances is obscured, as C and D are nearly superimposable. The relative clearances were calculated from the data in Table I, with the values for net calcium absorption the same (= 30 mg/day) for all four groups of animals.
while the urinary calcium clearance increases by more than two orders of magnitude. The high urinary calcium excretion seems to reflect the inability of the body to utilize calcium, a byproduct of the body’s effort to “mine” the skeleton for the scarce phosphate. Histologically, phosphate deficiency (21) produces a picture reminiscent of classical rickets, i.e., widening of the epiphyseal plate, thinning out and loss of trabecular bone in the metaphyseal region, and narrowing of the cortical width in the diaphyseal region. The number of resorption cavities increases. Osteocyte number decreases, with the proportion of enlarged osteocytes increasing in phos-
The ciency Figure deficiency shown
overall similarity of phosphate defito rickets is brought out further in 2D, where the results of the dual of phosphate and vitamin D are in terms of the relative bone and
kidney clearances of calcium. Figure 2D indicates that the absence of both phosphorus and vitamin D leads to a 50% drop in the relative bone calcium clearance and a 100-fold rise in the relative kidney calcium clearance. Indeed, Figure 2D does not seem very different from Figure 2C, the effect of simple phosphorus deficiency. The two graphs are nearly superimposable, so that the imposition of vitamin D deficiency on phosphate deficiency does not change the direction or markedly the amount by which the two clearances are altered. In both situations, the system, because of the need for phosphorus, is flooded by calcium that is skeletal in origin, but at the same time there is less bone to help maintain plasma calcium. Moreover, in the case of the dual deficiency, the bone is probably abnormal. The result in both instances is greater reliance of the organism on renal regulation. Because, however, the capacity of the kidney to handle calcium is evidently limited-even though urinary calcium excretion is raised 100-fold in phosphate deficiency-hypercalcemia results. Hypercalcemia is absolute in pure phosphate deficiency; it is relative, but sometimes absolute, in the dual deficiency. Another way to analyze the effect of vitamin D deficiency is to compare its effects in Relative phosphate depletion of the bone mineral probably arises from the fact that the bone mineral laid down first is relatively rich in phosphate and that its low crystal size and higher solubility also favor its ready resorption (22).
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phate deficiency. Phosphate deficiency leads to a 5% increase in the proportion of the trabecular surface covered by osteoclasts and a 200% increase in the proportion of the trabecular surface covered by osteoblasts. Compositional changes in bone-decrease in ash content, increase in water and hydroxyproline content, relative depletion in phosphate,4 (21)-are also consistent with the interpretation that the skeletal phosphate is being utilized to replenish diminished soft tissue stores.
2OO
200
VITAMIN
D DEFICIENCY
Application
to human
disease
On the basis of the preceding considerations, one can visualize three possible situations in children: 1) a simple deficiency in vitamin D, resulting either from a nutritional deficiency or a metabolic defect leading to deficient production of the active vitamin D metabolite; 2) a phosphate deficiency either nutritional in origin or resulting from a defect
1311
RICKETS
in phosphate metabolism; 3) a combination of both deficiencies. Metabolic defects that lead to inadequate production of the active metabolite of vitamin D could result from inappropriate enzyme synthesis, presumed to be genetic in origin. A situation where administration of small amounts of 1,25-dihydroxy-vitamin D3 led to a dramatic improvement in a child diagnosed as having vitamin D-resistant rickets has been reported (25). Whether a given defect is at the level of hydroxylation of carbon I (as in Reference 25) or carbon 25 (none has yet been reported), one would predict that such defects, unaccompanied by deficient mineral intake, would lead to the defects in calcium metabolism characteristic of simple vitamin D deficiency. In the rat and presumably the child this would mean hypocalcemia, diminished bone turnover with a relative decrease in bone calcium resorption, an absolute hypocalcemia, but a relative increase in the fractional urinary calcium clearance. Calcium absorption would of course be diminished. However, the histological changes in bone would not be those of rickets. It is evident that simple nutritional deficiency of vitamin D in the human infant would fall into this category. The question arises whether simple vitamm D deficiency has ever been documented in man. Lapatsanis (personal communication) has seen a hypocalcemic infant without radiological signs of rickets, who has responded to daily doses of 1,000 U of vitamin D by growth resumption and normocalcemia. Moreover, he has been able retrospectively to classify cases of rickets in Greek infants into those that are predominantly hypocalcemic and do not exhibit radiological. signs of ricket and those predominantly hypophosphatemic with clear radiological signs of rickets. There is therefore reason to think that simple vitamin D deficiency does
The
older
literature
is difficult
the clinical manifestations of rickets ical. Moreover, the chemical signs confusingly.
For
example:
‘Usually
to analyze:
however,
are usually radiologof rickets are treated the
calcium
is in
the
normal range of 9.5- II mg per 100 cc. of serum, although at times it may be as low as 5 mg In normal infants, the serum inorganic phosphate is about 5 mg per 100 cc, but in rickets it is reduced and may be as low as 1 mg” (26).
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the presence and in the absence of phosphate deficiency. A comparison of Figure 2 A and B shows that vitamin D deficiency superimposed on phosphate deficiency causes the relative bone clearance to drop and the relative kidney clearance to go up, whereas vitamin D deficiency in the presence of adequate phosphorus and calcium causes both clearances to go up. The only known molecular lesion that results from vitamin D deficiency is the absence of the vitamin D-dependent calciumbinding proteins from the intestine (7) and from kidney (8). This lesion is the same, whether vitamin D deficiency is imposed on animals on a normal mineral intake or one deficient in phosphorus (8, 23). Indeed, the absence of duodenal calcium-binding proteins has recently been proposed as a quantitative test of vitamin D deficiency in the rat (7) or as an assay for the chick (24). At the level of bone, however, it is apparent that vitamin D deficiency is expressed differently in the presence or absence of an adequate phosphorus supply. The major cause of rickets appears to be phosphorus deficiency, inasmuch as phosphorus deficiency alone can give rise to a syndrome in bone that cannot readily be distinguished from classical rickets. Vitamin D deficiency alone leads to a regulatory defect on the part of bone, as discussed above. Presumably the relative increase in bone destruction characteristic of the dual deficiency compromises the metabolic role of bone even further. However, not until the nature of the bone lesion caused by vitamin D becomes known will it be possible to understand in molecular terms how calcium or phosphorus deficiency modifies the expression of this lesion.
AND
1312
BRONN
2 of vitamin
TABLE Effect in
the
D deficiency
on phosphate
Parameter Body
metabolism
rat -D2
wt, g
146(1)
Plasma
calcium,
Plasma
calcium
mg/
10.3
100 ml
poo1, mg
(0.2)
0 67
243(18)
Calcium
intake,
Urinary
calcium,
mg/day mg/day
Urinary
calcium
clearance,
urinary
calcium
port defect by itself could be mild and asymptomatic. It would become symptomatic as a result of an environmentally caused vitamin D deficiency. Such deficiency would usually be the result of cultural practices. These might include industrial smoke pollution which filters out ultraviolet radiation,
:f:2)3
0.53
260(20)
11.5(1.0)
2.9(0.7)
21.7
4.3
day Relative
505
100
clearance Plasma
phosphorus,
Plasma
phosphorus
Phosphorus
mg/100 pool,
intake.
ml
2.4(0.1)
mg
Urinary
phosphorus,
mg/day
Urinary
phosphorus
clearance,
child-rearing practices such as infrequent exposure to the outdoors, use ofclothing that prevents exposure to ultraviolet light of any part of the body, poor dietary practices,
7.4(0.2)
0.12
0.42
l(#{216}
mg/day
I)
17(1)
167
mm D, the plasma phosphate was only 2.4 mg/100 ml in the vitamin D-deficient animals, whereas it was 7.4 in the controls. Both groups ingested similarly low quantities of phosphorus and their urinary phosphorus losses were similar. Clearly the presence of vitamin D exerted a significant effect on phosphate metabolism, including phosphate absorption (see also Reference 6). Therefore it does not seem unreasonable to propose that human nutritional rickets often results from a dual defect of phosphorus and vitamin D. Because nutritional deficiency of phosphorus intake is an unlikely situation, it seems more reasonable to postulate that human nutritional rickets arises in infants that have a low intake ofvitamin D and at the same time have some defect in phosphate transport, in the kidney (28) and/or in the intestine (29). Moreover, the phosphate trans-
prolonged nourished
095
mothers, breast feeding and
soby forth. relatively
poorly
day Relative
urinary
phosphorus
176
100
clearance
Ninety-two
(I
divided
weanling
into
two
phosphorus ing
diet
IU
plasma
calcium,
cages and
deficiency
differ
D2/kg
from
each
were
then
placed
Vitamin
the
were
of the vitamin protein. errors
of (P
numbers
the
means.
