AMERICAN
JOURNAL
Vol. 228, No. 6, June
OF PHYSIOLOGY 1975. Printed in U.S.A.
Effects of a diphosphonate in calcium-deprived
on calcium
rats
D. B. MORGAN, A. GASSER, U. LARGIADER, A. JUNG, Department of Pathophysiology, University of Berne, Berne, Switserland
MORGAN, D. B., A. GASSER, U. LARGIAD~R, A. JUNG, AND EL FLETSCEI. Effects of a diphosphonate on calcium metabolism in calcium1975.~Calcium &priued rats. ,4m. J- Physiol. 228(6) : 1750-l 756. metabolism was studied in growing rats, submitted to calcium deprivation of various intensity. A decreased intake resulted in decreased net absorption of calcium (V,,), no change in bone formation (V,+), and an increase in bone resorption (V, -)In animals given dichloromethylene diphosphonate (ClrMDP), a compound known to inhibit bone resorption, V, + was less than in the controls but again the same at all calcium intakes; V,, was less than in controls at high calcium intakes, but greater at medium intakes. When needed, that is as soon as V,, was below V,+, still increased as the calcium intake was reduced. The various VUkinetic parameters in rats receiving ClzMDP were indistinguishable from published data in parathyroidectomized (PTX) animals, yet blood calcium was low in PTX rats but normal in ClgMDPtreated rats. It appears that the rat has an efficient mechanism for increasing bone resorption which is not inhibited by ClzMDP and does not require parathyroid hormone. bone formation; bone phosphonic acids; diet;
resorption; calcium; serum calcium
intestinal
absorption;
IN THE RAT (3, 31) and in man (24, 26) have shown that a decrease in calcium intake has little effect on serum calcium concentration. Bone calcium deposition, although varying according to experimental conditions, does not change much (6, 31), but bone calcium resorption is greatly increased (7, 3 1, 33). Th is increase in bone resorption has been regarded as a key factor in serum calcium homeostasis during calcium deprivation. It seemed to us that further understanding of the interrelations between the various changes in calcium metabolism during calcium deprivation might be obtained from a study using diphosphonates. These synthetic compounds, characterized by a P-C -P bond, bind onto calcium phosphate crystals (17) and inhibit both their formation (8, 9, 12, 13) and dissolution (8, 10, 30). There is now much evidence from a variety of systems that several diphosphonates also inhibit the resorption of bone. The indirect evidence includes the prevention of hypercalcemia after parathyroid hormone (PTTH) administration (30), the prevention of osteoporosis induced by immobilization in rats (I 1, 20, 23), and the histological changes in the bones (32). The direct evidence comes from tissue culture experiments in which the diphosphonates slow down the resorption of calvaria when added to the culture medium (27, 28), and also from measureSTUDIES
metabolism
AKD
H. FLEISCH
ments of calcium balance and calcium kinetics in the intact rat (14). We have therefore tested the effect of the most potent diphosphonate (dichloromethylene diphosphonate, C&MDP) on the response to calcium deprivation in the growing rat. The results were compared with those reported in the literature for calcium deprivation in parathyroidectomized animals (31), since it has been suggested that the response of the latter animals to calcium deprivation is impaired in the absence of PTH. METHODS
AKD
MATERIALS
The general technique was that described by Milhaud et al. (2 1). The principle of the technique is the measurement of intake and excretion of stable calcium, and of the kinetics of injected 4;1Ca during a 72-h period. Female Wistar rats from our own breeding colony were given, from weaning, a commercial chow (no. 194, Nafag, Gossau) which contained 1.3 % calcium and 1 .O % P ( % dry wt). At the age of 54 & 1 days, they were weighed and put into single metabolic cages. From that time until the end of the experiment 10 days later, they were either given the same diet or were switched to one of four other diets containing different amounts of calcium and phosphate (Table 1). Uniformity of intake in an experimental group was achieved by giving all animals the same amount of food and reducing this daily ration during the equilibration period until none of the animals left more than 0.5 g of its ration. This uniformity was achieved within 3 days. In each experiment some of the animals were given, each morning, from the day they were put into the cages until the end of the experiment 10 days later, subcutaneous injections containing either 1.0 or 10 mg P as QMDP per kilogram body weight (Table 1). On the 7th day of each experiment, the animals were given a single injection into the tail vein of about 30 PCi of 45CaClz (Eidgeniissisches lnstitut ftir Reaktorforschung, Wtirenlingen, Switzerland; pH 6-7; sp act 30 &i/mg). A volume of the 4jCaCla solution equal to that injected was used as an internal standard for the 45Ca content of the various samples. Blood samples were taken from the tip of the tail 2, 4, 6, 24, 48, and 72 h after the injection of 4jCaClz+ Urine was collected for 72 h after the injection of 4”Ca. Feces were also collected for 72 h between markers of carmine red. Further details of the techniques are given in an earlier paper (14).
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DIPHOSPHONATE
AND
CALCIUM
1751
METABOLISM
TABLE 1. Number of animuh in various treatment grou;bs in seven exfieriments, giving details of diets, calcium intake, and dose of Cl&fDP Diet
Expt
Calculation
CIMDP,
mg P/kg per day 1
20 121 54 48 178 164 47 9
16 13 15 20 18 22 11 r :
6 6
7 8
7
7
no. of animaIs
125
19
p, %
1.3 0.2 0.9 0.5 0.5 1.3 1.3 0.5 0.1
1 .o 0.3 0.5 0.35 0.35 1 .o 1 .o 0.35 0.3 Total
Ca Intake, mdday
0
Ca, %
178
10
4 4 4 34
of Variables
Balance study. The following variables can be calculated directly from the measurement of calcium in the food and the feces. V; dietary intake of calcium VF fecal output of calcium net absorption (V,, = Vi - V,). This is called Km Si by Sammon et al. (31). The total 45Ca in urine divided by the integral of the serum specific activity during the 72 h gives an estimate of V, urinary output of calcium From these variables it is possible to calculate VS calcium retention in the body, which is assumed to be entirely due to retention of calcium in the skeleton; calculated as the difierence between net absorption and urinary excretion. Va corresponds to the A of Aubert and Milhaud (2). Fluxes uf calcium in to and out of intestine. endogenous fecal calcium. The rate of excretion Vf of calcium from the body in the feces, calculated as the total amount of 45Ca in the feces divided by the integral of the serum specific activity. It corresponds to the Vndo o f Sammon et al. (3 1) and represents all the calcium secreted into the intestine, if this process occurs below the site of absorption (6). absorption of dietary calcium. The difference v,d between calcium intake and excretion of dietary calcium in the feces. Thus Vad = Vi - (VP V,). It corresponds to the V, of Aubert and Milhaud (2). a % percentage absorption of dietary calcium. ob% = Wad/Vi)
X
45Ca has been injected (i.e., the rapidly exchangeable pool) mass of calcium of compartment 2 (Le., the slowly E2 exchangeable pool) unidirectional flows of calcium between the ve pools El and ES. flow of calcium into bone, calculated as the loss v,+ of calcium from El out of the compartmental system that is not accounted for by the excretion of calcium in urine or feces. V,+ is taken as an estimate of the rate of bone calcium deposition. flow of calcium out of bone. The flow of calcium v3-out of the calcium pool, apparently not exchangeable within the period of experimentation into compartment I; calculated as the difierence between rate of entry of calcium into bone and rate of retention of dietary calcium in bone. V,is taken as an estimate of the rate of bone calcium resorption. All fluxes were calculated in milligrams per day, and exchangeable masses (pools) were calculated in milligrams. These variables were calculated on a comput& CDC 3600 with the program described by Richelle (29). RESULTS
The results In the figures
are illustrated in Figs. 1-8 and the abscissa is the dietary intake
in Table 2. of calcium,
I I Aubert et al.1961 * Sammon et al.1970 I Present series
4II __
I
01 i
q ,
200
I
150
I
I
100
50
b
Vi Cmg/day) *O
1
0
---
Controls
100
““Ca kinetics. The pattern of decrease of the specific activity of the plasma calcium according to time can be fitted reasonably well, between 2 and 72 h, to a two-termed exponential function. Milhaud et al. (21) suggested that this function could be solved in terms of a two-compartment model of calcium metabolism. The model used in this paper is the one used by those authors (21). The following variables have been derived by application of this model. El mass of calcium of compartment I, into which
100 Vi (mg /day) FIG. 1. A and B: relation between net absorption of calcium (V,,) and calcium intake (Vi) in A, untreated rats (Sammon et al. (31) and present study); and in B, rats given QMDP (present study) and rats after parathyroidectomy (PTX) (31). Interrupted line in this and Figs. 2, 4, 5, and 8 indicates relation in control rats in present series (drawn by eye). In this and subsequent figures, mg P/kg per day ClgMDP refers to P administered per kg per day as Cl2MDP.
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1752
MORGAN,
with the scale from higher values on the left to lower values on the right. This direction, although unconventional, was deliberately chosen to emphasize that what is done is to reduce the calcium intake. The results are given as means & SE in each treatment group for each experiment, except when the SE was too small to be included on the figures. Net Absorfhon
Table 2 shows was reduced. The one of the groups had no effect on in V,,. Secrthon
Figure IA shows for the control groups the relation between the net absorption of calcium and the dietary intake of calcium. The results are similar to those reported by Sammon et al. (31) and Aubert et al. (3). Figure LB shows this relation in the animals given ClaMDIP. The interrupted line is a line fitted by eye to the data in the control rats, and serves only as a general guide to the changes in the rats treated with ClzMDP. The animals treated with the larger dose of Cl2MDP had a smaller net absorption of calcium than the controls on the highest intakes of calcium (la3 % Ca diet), but had a greater net absorption on the intermediate intakes (0.5 % Ca diet). These differences were significant (P < 0.05) in the animals given the 0.5 % Ca diet when the ClzMDP-treated animals were compared with the control animals in the same experiment (Table 2). There was no significant difference at the very low intakes. The smaller dose of ClgMDP caused changes in the same direction but the differences were smaller and not significant. Figure 1B shows that these effects of ClgMDP re,;embled the reported effects of parathyroidectomy (PTX) (31) .
GASSER,
LARGIADkR,
JUNG,
AND
FLEISCH
that V,d decreased as the calcium intake larger dose of QMDP increased Vad in of animals given the 0.5 % Ca diet, but the other group in spite of an increase
of Calciuminto
Intestine
Figure 2A shows that in the control groups the endogenous fecal calcium decreases as the intake of calcium is diminished (31). F i g ure 2B shows that QMDP had no effect on this variable at the higher calcium intakes but decreased it significantly at the lower calcium intakes. This decrease of Vf was observed even when there was no effect of ClzMDP on the V,d. Urinary
Excretion
The excretion of calcium in the urine showed a tendency to decrease at the low intakes of calcium. However, since this excretion was small (1 mg/day), it does not affect significantly the retention of calcium, Vd = (V,, - V,), which therefore approximates V,,. Treatment with ClzMDP had no significant effect on the urinary excretion of calcium at any calcium intake. Plasma Calcium and Phosphate Figure 3 shows that in the control groups the plasma calcium did not change when the calcium intake was reduced.
TABLE 2. Measured and calculated variables of calcium metabolism in control rats and rats given ClJ-lDP (IO mg P/kg per day) according to calcium intake 1.3% Expt I
Vi
VF v
na
v
ad
a6) Vf vu VS Vo’ voEl E2
ve
7 I 7 I 7 I 7 1 7 I 7 1 7 I 7 I 7 I 7 1 7 I 7 1 7
Gas t 1 7
for
Ca Diet
03%
Controls
ClzMDP
178(8) 164(Z) 122(Z) 93 (6) 56.3(7.6) 71.2(5.4) 66.6(7.6) 79.1(4.9) 36 (2) W3) l&3(0.6) 7.8(0.6) l-5(0.2) 0.6(0.06) 54.9(7.6) 70.6(5.5) 53.9(3.2) 80.3(7.0) -0.9(6.9) 9.6(9.2) 21.3 (1.5) 24,7(4.2) 50.4(3.6) 56.4(6.3) 66.0(4.5) 82.1(11 l5) 9.97(0.28) 10.31(0.29)
163 (7) 166 (2) 122 (2) 103 (6) 41.2(6.8) 62.7(5.5) 52.2(7.0) 70.2(4.9) 31w 4-m) 9.0(0.9) 7.6(0.6) 1.4(0-Z) 1 .O(O* 17) 41.8(6.7) 61.7(5.6) 4I.6(6,5) 62.3(10.5) -0.3(5.2) 0.6 (13.8) 21.7(2.4) 23.0(7.6) 37.7(5.1) 62.3(11,2) 69.6(7.5) 113.3(23.7) 9.85(0.21) 10.33(0.3)
VaIues are means with k SE given the control rats in same experiment
Expt
4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7
Contrds
54.6(0.7) 46.3(0.3) 25.2(0.8) 23.7(1 .O) 29.4(0.7) 22.611 .O) 36.1(0.7) 27.0(0.8) 66 (2) 58(Z) 6.6(0.5) 4.4(0.2) 2.7(0.6) l.l(O.2) 26.7(0.7) 21.4 (l-0) 73.3(4.4) 63.4(5.7) 46.6(4.5) 41.9(4.8) 25.9(2.1) 24.3(2.9) 76.1(8.3) 64.6(6.2) 112.3(10.3) 91 l o (7.4) 10.62(0.18) 9.36(0.45)
in parentheses,
(P < 0.05).
0.27& Ca Diet
Ca Diet ChMDP
52.2(0.9) 44.8(1.4) 17.5 (1.5)” 10.7(3.2)* 34.7(1.3)* 34.2(3.3)* 37.2 (1.2) 35.7(3.2)* n(2) 79 (3)* 2.5(0.5)* 1.6(0.3)* 2.9(0.4) 4,0(0.8)* 31.8 (1.6)* 30.1(2.8)* 35.4(3.4)’ 47.2(2.4)* 3.6 (3.9)* 17.1(1,8)* 17.5(2.3)* 27.2(1.3) 39.4(5.6)* 48.4(3.1)* 75.4(14.2)* 89.3(4.2) 9.65(0.3)* 9.42(0,23)
Expt
O.f%
con tro1s
ClzMDP
Expt
Controls
Ca Diet Cl&lDP
2
20.4(0.4)
19.2(0.5)
7
8.9(0.4)
7.6(0.7)
2
4.6(0.4)
Z.l(O.3)”
7
ZJ(O.3)
l.O(O.2)”
2
15.8(0.4)
17.1(0.4)
7
6.5(0.4)
6.5(0.4)
2
18.5(0.6)
17,8(0.6)
7
8.9(0.4)
7.4(0.6)
2
91@)
9w
7
97.5(l)
100(I)
2
2.7(0.4)
0.7(0.1)*
7
2.4(0.3)
0.9(0.2)”
2
l.l(O.2)
0.8(0.1)
7
0.5(0.08)
0.3(0.07)
2
14.7(0.3)
16.3(0.6)
7
6.0(0.4)
6.2(0.4)
2
77.5(12.4)
43,6(7.4)*
7
64.4(3.4)
39.8(4.5)
2
62.8(12.3)
27.3(7.9)”
7
58.4(3.3)
33.6(4.8)*
2
26.5(3.7)
25.2(4.7)
7
21.6 (2.3)
31.4(5.7)
2
84.1(12.5)
60.0(9.8)
7
69.7(5.2)
82.9(17.6)
2
115.4(16.6)
109.7(16.2)
7
110.2(8.2)
9.60(0.42)
7
2
10.08(0.22)
Symbols as explained in text. t Serum calcium in mg/lOO
* Values
are significantly
10.33(0.2) different
151.6 (26.5) 10.39(0.2) from
values
ml.
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DX:PHOSPHUNATE
AND
CALCIUM
1753
METABOLISM
The administration of QMDP (10 mg P/kg per day) had no significant effect on the serum calcium at the various intakes. Figure 3 also shows that the effect of ClzMDP is much different from the effects of PTX, which causes gross hypocalcemia at any calcium intake. Serum phosphate, which was only measured in the groups given the 0.5 % Ca diet, was less in the animals given
A
h ; 80+1 :s G 5 +o >
T *
I
I
-+
t + Sammon I Present
+B
-
f
&
I
60-
et
al 1970
series
40-
C12MDP (14).
I 200
I 150
1 100
1 50
1 0
Vi hg/day)
Figure
4A shows the relation between calcium intake and entry into bone. These results agree well with those reported by Sammon et al. (3 1). In our data there was no significant regression of V,+ on Vi (VO+ = 68.1 0.05 Vi; r = 0*18; 72 c 125). Figure 4B shows that C12MDP caused a decrease in V,+ at all calcium intakes compared with the controls, but with the larger dose of C12MDP there was no correlation between V,+ and Vi (r = 0.078; rt =
__-B
the rate of calcium
T
+ Sammon W Present
1
100
;
40-
3 -
E
______--------------________1_________11---
9 1
1
controls
1 200
i
I 150
P/kg / day CI,MDP (Sammon et aI 1970) 1 1 100 50
Vi hg
et al.1970 series
4. A and 23: relation between intake (Vi) A and B as in Fig. l
4
11
A lolmg * PTX
20-
I
4 ----n
+
FIG.
1
60-
9
cium
150
___-__
+I . u2
G cn
bone 1.
I 0
/ day)
formation
(VO+)
and cal-
34) V,+ was significantly less in the animals given the larger dose of ClzMDP than in the control animals (43.8 ZIZ 2.4 compared with 63.6 & 1.6; P < 0.05). Figure 4B also shows that the effect of PTX (31) was similar to the effect of the larger dose of C&MDP.
Vi(mg/day)
Flow of Calcium out of Bane
--- Controls * 1 m g P/kg /day * 10 1 o PTX
Cf2MDP
r
I 100
Q
1
Vi (mg/day)
2. A and B: relation between secretion of calcium and calcium intake (Vi). A and B as in Fig. 1.
into intestine
FIG.
(Vf)
PRESENT q Controls m CI2MDP
SERIES
SAMMON
IOmg
q ControIs El PTX
P/kg/day
et al 1970
Figure 5A shows that in the control groups, V,--, the rate of bone destruction, increased progressively as the calcium intake was diminished. This can be predicted from the results shown above, since V,is calculated as the difference between V,+ (Fig. 4A) and the retention of calcium, and the latter closely approximates V,, (Fig. 1A). Again, the values agree well with those reported by Sammon et al. (3 1). The linear regression of V, - on Vi in our data was = 63.1 - 0.376 Vi; r = 0.778; n = 125; P < 0.001. KFigure 5B shows the effect of ClzMDP on V,-- . In contrast to the control animals, where V,was about zero at high calcium intakes and increased progressively as intake was reduced, in the animals treated with the larger dose of ClzMDP there was no increase in V,-until the diet contained less than 0.5 % Ca (about 50 mg of calcium per day). As the intake was further decreased, there was a progressive increase in V, - The linear regression of V,on Vi in the rats given the larger dose of ClsMDP when Vi was less than 100 mg/day was V,= 38.5 - 0.557 Vi; r = 0.548; n = 23; P < 0.01. l
DIET CALCIUM
1.3
0.5
......:.... ........ ...... ........ .. ........... ................ ............ ...... .. ..h... .......... -3 ........ 0.5
:... . . . . . . . . . . . . . .. . .. .. . ..
::. . . . . .. . . .. . . . . . . . . . . . 4.4. n.... . . . . n.... .. .. . .. . . . . . . ‘I
Ii
(%)
FIG. 3. Serum calcium at 3 calcium intakes in left, control rats given C12MDP (10 mg P/kg per day) in present series combined) and right, control rats and parathyroidectomized rats .(3 1).
ExchangeablePools
0.1
rats and (all data (PTX)
The reduction in calcium intake had no influence on the rapidly exchangeable pool, increased somewhat the slowly exchangeable pool, and increased the flow between them. ClgMDP had no consistent effect on any of these variables (Table 2), and these results will not be discussed.
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1754
MORGAN,
T i I
GASSER,
BALANCE
E
80
f#
LARGIADGR,
STUDIES
Sammon
a Present
L
, 150
I 100
0.3
60
0.2 %
20
eta1 1970 series
0.1 : 0
0 ::
, 50
, 0
Vi (mg/day)
60
8
1
--- Controls 0 PTX (Sammon
I 150
et al 1970)
I 100 Vi (mglday)
2’
I 50
4’
0’,=
0
FIG. 5. A and & relation between bone resorption (V.-) and calcium intake (Vi). A and B as in Fig. 1. Interrupted line in B is calculated linear regression of V,on Vi in control rats. Continuous oblique line in B is calculated linear regression of V,on Vi in rats given larger dose of CIZMDP with a calcium intake less than 100 q/day. DISCUSSION
Calcium Deprivation
STUDlES
Vad
100 Vi
5 200
FLEISCH
0.4
Vad
200
-zoJ
AND
PERFUSION
40 t
JUNG,
in Control Animals
When the calcium intake is reduced to the extent that it was in these experiments, there is inevitably some reduction in the net absorption of calcium. The relation between V,, and calcium intake for the whole range of calcium intakes agrees well with the relation given by Sammon et al. (31), who performed experiments similar to the ones reported here, and with the relation in the data of Aubert et al. (3), in which variation in intake was due to spontaneous variation between the animals in their intake of food of constant composition. The explanation of the observed relation between net absorption or dietary absorption of calcium and calcium intake is not known. But as Fig. 6 shows, the relation is such that the percentage of ingested calcium which is absorbed (cu Yo) increases as the intake is decreased. One interpretation of these results is that they indicate an increase in the efficiency of calcium absorption as the intake of calcium is diminished, and that this is due to a compensatory adaptation in the intestinal calcium transport mechanism (19, 35). However, another explanation of these findings is that the characteristics of the absorption mechanism are such that the smaller the load of calcium presented to the intestine, the greater will be the proportion of that load which will be
0L
0 Calcium
0.6 input
0.4
0.2 (mg
0 per
0 hour)
FIG. 6. Left: absorption of dietary calcium (Vad, in mg/day) and percentage absorption of dietary calcium (&) according to calcium intake in control group of the present study. Right: net calcium absorption and percentage absorption of cakium according to rate of perfusion of calcium through isolated segments of proximal small intestine in the rat in vitro (5, 22).
absorbed. Support for this explanation comes from experiments in which the load of calcium presented to the perfused intestine was suddenly reduced (5, 22) (Fig. 6). There was an increase in the proportion of the calcium load which was absorbed, and it is unlikely that this increase was due to adaptation in the gut absorption mechanism. In these experiments V,+ did not change significantly despite a drastic reduction of calcium intake. These results are in agreement with those of Sammon et al. (3 1) and between V,+ and Cohn et al. (7), who found a relation calcium intake, which was, however, not statistically significant. Aubert and Milhaud (2) concluded that there was an inverse relation between V,+ and calcium intake, but in their experiments the variation in Vi between animals was due to spontaneous variation in the intake of food, so that their results are not comparable with ours. The results of StauEer et al, (33), which show a direct relation between both variables, are also not comparable, because they are based on a morphological analysis of the shaft Finally, in man, short-term reduction in calcium intake is without effect on V,+ (26). It is still debated whether V,+ can be taken as a measure of bone formation rate. Formally V,+ is the rate of entry into bone of calcium which does not significantly reenter the compartment El for at least 3 days. If V,+ represents bone formation, as assumed here, V,represents bone destruction, since it is calculated as the difference between VS and V,+. The rate of bone destruction increases progressively as the calcium intake is reduced (Fig. 5A), in agreement with observations made in rats (3, 7, 31, 33) and in man (24, 26). In the growing animal there is a need for large amounts of calcium. When the calcium intake is adequate, this calcium is obtained from the diet; when intake of calcium is reduced, the supply of calcium is obtained by an increased resorption of bone. These relationships are outlined schematically in Fig. 7. E$ects of CI&fDP
and Comparison with Parathyroidecfomy
It is interesting that the effects of ClaMDP on the several variables are strikingly similar to the results obtained after parathyroidectomy (1, 18, 3 1). Thus ClzMDP and PTX lead to a decrease in V na at higher calcium intakes, and an
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DIPHOSPHONATE
AND
CALCIUM
1755
METABOLISM
increase in V,, at medium calcium intakes (Fig. lB), a decrease in V,+ at all calcium intakes (Fig. 4-B), and a decrease in V,-at all intakes (Fig. 5B). These effects are summarized in Figure 8. On the other hand, the effects are different on serum calcium and phosphate, since PTX causes gross hypocalcemia whereas ClzMDP does not, even with the smallest calcium intake, and ClzMDP causes low serum phosphate (14) whereas PTX does not These results raise the possibility that fluxes other than L, V,+, and V,---, such as those in the kidney (25), may have a role in setting the plasma calcium. The finding that the results of PTX and QMDP administration are not identical suggests also that ClgMDP does not act by blocking the effects of PTH. The performed experiments also allow some conclusions concerning the regulation of the various flows. It appears that in each of the experimental conditions (control, PTX, QMDP) V,+ is constant at all calcium intakes. This is made possible through an increase in V,when the intake of calcium and, therefore, V,, is diminished (Fig. 7). ln the presence of QMDP or PTX, V,+ is smaller and V, -therefore increases only at lower calcium intakes, when the V,, decreases below V,+ But at these low intakes V,increases with a slope at least as great as that in intact rats (Fig. 8). This is surprising since it is known that ClzMDP inhibits resorption, and since it is generally thought that the resorption induced by lack of calcium is due to PTH. The factors that control this PTH-independent and CLMDP-resistant increase in bone resorption are unknown. One mechanism might be an increased production of one of the metabolites of vitamin D, which have been shown to increase bone resorption (34). l
60-l
hm
JOURNAL
Vol. 228, No. 6, June
OF PHYSIOLOGY 1975. Printed in U.S.A.
Effects of a diphosphonate in calcium-deprived
on calcium
rats
D. B. MORGAN, A. GASSER, U. LARGIADER, A. JUNG, Department of Pathophysiology, University of Berne, Berne, Switserland
MORGAN, D. B., A. GASSER, U. LARGIAD~R, A. JUNG, AND EL FLETSCEI. Effects of a diphosphonate on calcium metabolism in calcium1975.~Calcium &priued rats. ,4m. J- Physiol. 228(6) : 1750-l 756. metabolism was studied in growing rats, submitted to calcium deprivation of various intensity. A decreased intake resulted in decreased net absorption of calcium (V,,), no change in bone formation (V,+), and an increase in bone resorption (V, -)In animals given dichloromethylene diphosphonate (ClrMDP), a compound known to inhibit bone resorption, V, + was less than in the controls but again the same at all calcium intakes; V,, was less than in controls at high calcium intakes, but greater at medium intakes. When needed, that is as soon as V,, was below V,+, still increased as the calcium intake was reduced. The various VUkinetic parameters in rats receiving ClzMDP were indistinguishable from published data in parathyroidectomized (PTX) animals, yet blood calcium was low in PTX rats but normal in ClgMDPtreated rats. It appears that the rat has an efficient mechanism for increasing bone resorption which is not inhibited by ClzMDP and does not require parathyroid hormone. bone formation; bone phosphonic acids; diet;
resorption; calcium; serum calcium
intestinal
absorption;
IN THE RAT (3, 31) and in man (24, 26) have shown that a decrease in calcium intake has little effect on serum calcium concentration. Bone calcium deposition, although varying according to experimental conditions, does not change much (6, 31), but bone calcium resorption is greatly increased (7, 3 1, 33). Th is increase in bone resorption has been regarded as a key factor in serum calcium homeostasis during calcium deprivation. It seemed to us that further understanding of the interrelations between the various changes in calcium metabolism during calcium deprivation might be obtained from a study using diphosphonates. These synthetic compounds, characterized by a P-C -P bond, bind onto calcium phosphate crystals (17) and inhibit both their formation (8, 9, 12, 13) and dissolution (8, 10, 30). There is now much evidence from a variety of systems that several diphosphonates also inhibit the resorption of bone. The indirect evidence includes the prevention of hypercalcemia after parathyroid hormone (PTTH) administration (30), the prevention of osteoporosis induced by immobilization in rats (I 1, 20, 23), and the histological changes in the bones (32). The direct evidence comes from tissue culture experiments in which the diphosphonates slow down the resorption of calvaria when added to the culture medium (27, 28), and also from measureSTUDIES
metabolism
AKD
H. FLEISCH
ments of calcium balance and calcium kinetics in the intact rat (14). We have therefore tested the effect of the most potent diphosphonate (dichloromethylene diphosphonate, C&MDP) on the response to calcium deprivation in the growing rat. The results were compared with those reported in the literature for calcium deprivation in parathyroidectomized animals (31), since it has been suggested that the response of the latter animals to calcium deprivation is impaired in the absence of PTH. METHODS
AKD
MATERIALS
The general technique was that described by Milhaud et al. (2 1). The principle of the technique is the measurement of intake and excretion of stable calcium, and of the kinetics of injected 4;1Ca during a 72-h period. Female Wistar rats from our own breeding colony were given, from weaning, a commercial chow (no. 194, Nafag, Gossau) which contained 1.3 % calcium and 1 .O % P ( % dry wt). At the age of 54 & 1 days, they were weighed and put into single metabolic cages. From that time until the end of the experiment 10 days later, they were either given the same diet or were switched to one of four other diets containing different amounts of calcium and phosphate (Table 1). Uniformity of intake in an experimental group was achieved by giving all animals the same amount of food and reducing this daily ration during the equilibration period until none of the animals left more than 0.5 g of its ration. This uniformity was achieved within 3 days. In each experiment some of the animals were given, each morning, from the day they were put into the cages until the end of the experiment 10 days later, subcutaneous injections containing either 1.0 or 10 mg P as QMDP per kilogram body weight (Table 1). On the 7th day of each experiment, the animals were given a single injection into the tail vein of about 30 PCi of 45CaClz (Eidgeniissisches lnstitut ftir Reaktorforschung, Wtirenlingen, Switzerland; pH 6-7; sp act 30 &i/mg). A volume of the 4jCaCla solution equal to that injected was used as an internal standard for the 45Ca content of the various samples. Blood samples were taken from the tip of the tail 2, 4, 6, 24, 48, and 72 h after the injection of 4jCaClz+ Urine was collected for 72 h after the injection of 4”Ca. Feces were also collected for 72 h between markers of carmine red. Further details of the techniques are given in an earlier paper (14).
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DIPHOSPHONATE
AND
CALCIUM
1751
METABOLISM
TABLE 1. Number of animuh in various treatment grou;bs in seven exfieriments, giving details of diets, calcium intake, and dose of Cl&fDP Diet
Expt
Calculation
CIMDP,
mg P/kg per day 1
20 121 54 48 178 164 47 9
16 13 15 20 18 22 11 r :
6 6
7 8
7
7
no. of animaIs
125
19
p, %
1.3 0.2 0.9 0.5 0.5 1.3 1.3 0.5 0.1
1 .o 0.3 0.5 0.35 0.35 1 .o 1 .o 0.35 0.3 Total
Ca Intake, mdday
0
Ca, %
178
10
4 4 4 34
of Variables
Balance study. The following variables can be calculated directly from the measurement of calcium in the food and the feces. V; dietary intake of calcium VF fecal output of calcium net absorption (V,, = Vi - V,). This is called Km Si by Sammon et al. (31). The total 45Ca in urine divided by the integral of the serum specific activity during the 72 h gives an estimate of V, urinary output of calcium From these variables it is possible to calculate VS calcium retention in the body, which is assumed to be entirely due to retention of calcium in the skeleton; calculated as the difierence between net absorption and urinary excretion. Va corresponds to the A of Aubert and Milhaud (2). Fluxes uf calcium in to and out of intestine. endogenous fecal calcium. The rate of excretion Vf of calcium from the body in the feces, calculated as the total amount of 45Ca in the feces divided by the integral of the serum specific activity. It corresponds to the Vndo o f Sammon et al. (3 1) and represents all the calcium secreted into the intestine, if this process occurs below the site of absorption (6). absorption of dietary calcium. The difference v,d between calcium intake and excretion of dietary calcium in the feces. Thus Vad = Vi - (VP V,). It corresponds to the V, of Aubert and Milhaud (2). a % percentage absorption of dietary calcium. ob% = Wad/Vi)
X
45Ca has been injected (i.e., the rapidly exchangeable pool) mass of calcium of compartment 2 (Le., the slowly E2 exchangeable pool) unidirectional flows of calcium between the ve pools El and ES. flow of calcium into bone, calculated as the loss v,+ of calcium from El out of the compartmental system that is not accounted for by the excretion of calcium in urine or feces. V,+ is taken as an estimate of the rate of bone calcium deposition. flow of calcium out of bone. The flow of calcium v3-out of the calcium pool, apparently not exchangeable within the period of experimentation into compartment I; calculated as the difierence between rate of entry of calcium into bone and rate of retention of dietary calcium in bone. V,is taken as an estimate of the rate of bone calcium resorption. All fluxes were calculated in milligrams per day, and exchangeable masses (pools) were calculated in milligrams. These variables were calculated on a comput& CDC 3600 with the program described by Richelle (29). RESULTS
The results In the figures
are illustrated in Figs. 1-8 and the abscissa is the dietary intake
in Table 2. of calcium,
I I Aubert et al.1961 * Sammon et al.1970 I Present series
4II __
I
01 i
q ,
200
I
150
I
I
100
50
b
Vi Cmg/day) *O
1
0
---
Controls
100
““Ca kinetics. The pattern of decrease of the specific activity of the plasma calcium according to time can be fitted reasonably well, between 2 and 72 h, to a two-termed exponential function. Milhaud et al. (21) suggested that this function could be solved in terms of a two-compartment model of calcium metabolism. The model used in this paper is the one used by those authors (21). The following variables have been derived by application of this model. El mass of calcium of compartment I, into which
100 Vi (mg /day) FIG. 1. A and B: relation between net absorption of calcium (V,,) and calcium intake (Vi) in A, untreated rats (Sammon et al. (31) and present study); and in B, rats given QMDP (present study) and rats after parathyroidectomy (PTX) (31). Interrupted line in this and Figs. 2, 4, 5, and 8 indicates relation in control rats in present series (drawn by eye). In this and subsequent figures, mg P/kg per day ClgMDP refers to P administered per kg per day as Cl2MDP.
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1752
MORGAN,
with the scale from higher values on the left to lower values on the right. This direction, although unconventional, was deliberately chosen to emphasize that what is done is to reduce the calcium intake. The results are given as means & SE in each treatment group for each experiment, except when the SE was too small to be included on the figures. Net Absorfhon
Table 2 shows was reduced. The one of the groups had no effect on in V,,. Secrthon
Figure IA shows for the control groups the relation between the net absorption of calcium and the dietary intake of calcium. The results are similar to those reported by Sammon et al. (31) and Aubert et al. (3). Figure LB shows this relation in the animals given ClaMDIP. The interrupted line is a line fitted by eye to the data in the control rats, and serves only as a general guide to the changes in the rats treated with ClzMDP. The animals treated with the larger dose of Cl2MDP had a smaller net absorption of calcium than the controls on the highest intakes of calcium (la3 % Ca diet), but had a greater net absorption on the intermediate intakes (0.5 % Ca diet). These differences were significant (P < 0.05) in the animals given the 0.5 % Ca diet when the ClzMDP-treated animals were compared with the control animals in the same experiment (Table 2). There was no significant difference at the very low intakes. The smaller dose of ClgMDP caused changes in the same direction but the differences were smaller and not significant. Figure 1B shows that these effects of ClgMDP re,;embled the reported effects of parathyroidectomy (PTX) (31) .
GASSER,
LARGIADkR,
JUNG,
AND
FLEISCH
that V,d decreased as the calcium intake larger dose of QMDP increased Vad in of animals given the 0.5 % Ca diet, but the other group in spite of an increase
of Calciuminto
Intestine
Figure 2A shows that in the control groups the endogenous fecal calcium decreases as the intake of calcium is diminished (31). F i g ure 2B shows that QMDP had no effect on this variable at the higher calcium intakes but decreased it significantly at the lower calcium intakes. This decrease of Vf was observed even when there was no effect of ClzMDP on the V,d. Urinary
Excretion
The excretion of calcium in the urine showed a tendency to decrease at the low intakes of calcium. However, since this excretion was small (1 mg/day), it does not affect significantly the retention of calcium, Vd = (V,, - V,), which therefore approximates V,,. Treatment with ClzMDP had no significant effect on the urinary excretion of calcium at any calcium intake. Plasma Calcium and Phosphate Figure 3 shows that in the control groups the plasma calcium did not change when the calcium intake was reduced.
TABLE 2. Measured and calculated variables of calcium metabolism in control rats and rats given ClJ-lDP (IO mg P/kg per day) according to calcium intake 1.3% Expt I
Vi
VF v
na
v
ad
a6) Vf vu VS Vo’ voEl E2
ve
7 I 7 I 7 I 7 1 7 I 7 1 7 I 7 I 7 I 7 1 7 I 7 1 7
Gas t 1 7
for
Ca Diet
03%
Controls
ClzMDP
178(8) 164(Z) 122(Z) 93 (6) 56.3(7.6) 71.2(5.4) 66.6(7.6) 79.1(4.9) 36 (2) W3) l&3(0.6) 7.8(0.6) l-5(0.2) 0.6(0.06) 54.9(7.6) 70.6(5.5) 53.9(3.2) 80.3(7.0) -0.9(6.9) 9.6(9.2) 21.3 (1.5) 24,7(4.2) 50.4(3.6) 56.4(6.3) 66.0(4.5) 82.1(11 l5) 9.97(0.28) 10.31(0.29)
163 (7) 166 (2) 122 (2) 103 (6) 41.2(6.8) 62.7(5.5) 52.2(7.0) 70.2(4.9) 31w 4-m) 9.0(0.9) 7.6(0.6) 1.4(0-Z) 1 .O(O* 17) 41.8(6.7) 61.7(5.6) 4I.6(6,5) 62.3(10.5) -0.3(5.2) 0.6 (13.8) 21.7(2.4) 23.0(7.6) 37.7(5.1) 62.3(11,2) 69.6(7.5) 113.3(23.7) 9.85(0.21) 10.33(0.3)
VaIues are means with k SE given the control rats in same experiment
Expt
4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7 4 7
Contrds
54.6(0.7) 46.3(0.3) 25.2(0.8) 23.7(1 .O) 29.4(0.7) 22.611 .O) 36.1(0.7) 27.0(0.8) 66 (2) 58(Z) 6.6(0.5) 4.4(0.2) 2.7(0.6) l.l(O.2) 26.7(0.7) 21.4 (l-0) 73.3(4.4) 63.4(5.7) 46.6(4.5) 41.9(4.8) 25.9(2.1) 24.3(2.9) 76.1(8.3) 64.6(6.2) 112.3(10.3) 91 l o (7.4) 10.62(0.18) 9.36(0.45)
in parentheses,
(P < 0.05).
0.27& Ca Diet
Ca Diet ChMDP
52.2(0.9) 44.8(1.4) 17.5 (1.5)” 10.7(3.2)* 34.7(1.3)* 34.2(3.3)* 37.2 (1.2) 35.7(3.2)* n(2) 79 (3)* 2.5(0.5)* 1.6(0.3)* 2.9(0.4) 4,0(0.8)* 31.8 (1.6)* 30.1(2.8)* 35.4(3.4)’ 47.2(2.4)* 3.6 (3.9)* 17.1(1,8)* 17.5(2.3)* 27.2(1.3) 39.4(5.6)* 48.4(3.1)* 75.4(14.2)* 89.3(4.2) 9.65(0.3)* 9.42(0,23)
Expt
O.f%
con tro1s
ClzMDP
Expt
Controls
Ca Diet Cl&lDP
2
20.4(0.4)
19.2(0.5)
7
8.9(0.4)
7.6(0.7)
2
4.6(0.4)
Z.l(O.3)”
7
ZJ(O.3)
l.O(O.2)”
2
15.8(0.4)
17.1(0.4)
7
6.5(0.4)
6.5(0.4)
2
18.5(0.6)
17,8(0.6)
7
8.9(0.4)
7.4(0.6)
2
91@)
9w
7
97.5(l)
100(I)
2
2.7(0.4)
0.7(0.1)*
7
2.4(0.3)
0.9(0.2)”
2
l.l(O.2)
0.8(0.1)
7
0.5(0.08)
0.3(0.07)
2
14.7(0.3)
16.3(0.6)
7
6.0(0.4)
6.2(0.4)
2
77.5(12.4)
43,6(7.4)*
7
64.4(3.4)
39.8(4.5)
2
62.8(12.3)
27.3(7.9)”
7
58.4(3.3)
33.6(4.8)*
2
26.5(3.7)
25.2(4.7)
7
21.6 (2.3)
31.4(5.7)
2
84.1(12.5)
60.0(9.8)
7
69.7(5.2)
82.9(17.6)
2
115.4(16.6)
109.7(16.2)
7
110.2(8.2)
9.60(0.42)
7
2
10.08(0.22)
Symbols as explained in text. t Serum calcium in mg/lOO
* Values
are significantly
10.33(0.2) different
151.6 (26.5) 10.39(0.2) from
values
ml.
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DX:PHOSPHUNATE
AND
CALCIUM
1753
METABOLISM
The administration of QMDP (10 mg P/kg per day) had no significant effect on the serum calcium at the various intakes. Figure 3 also shows that the effect of ClzMDP is much different from the effects of PTX, which causes gross hypocalcemia at any calcium intake. Serum phosphate, which was only measured in the groups given the 0.5 % Ca diet, was less in the animals given
A
h ; 80+1 :s G 5 +o >
T *
I
I
-+
t + Sammon I Present
+B
-
f
&
I
60-
et
al 1970
series
40-
C12MDP (14).
I 200
I 150
1 100
1 50
1 0
Vi hg/day)
Figure
4A shows the relation between calcium intake and entry into bone. These results agree well with those reported by Sammon et al. (3 1). In our data there was no significant regression of V,+ on Vi (VO+ = 68.1 0.05 Vi; r = 0*18; 72 c 125). Figure 4B shows that C12MDP caused a decrease in V,+ at all calcium intakes compared with the controls, but with the larger dose of C12MDP there was no correlation between V,+ and Vi (r = 0.078; rt =
__-B
the rate of calcium
T
+ Sammon W Present
1
100
;
40-
3 -
E
______--------------________1_________11---
9 1
1
controls
1 200
i
I 150
P/kg / day CI,MDP (Sammon et aI 1970) 1 1 100 50
Vi hg
et al.1970 series
4. A and 23: relation between intake (Vi) A and B as in Fig. l
4
11
A lolmg * PTX
20-
I
4 ----n
+
FIG.
1
60-
9
cium
150
___-__
+I . u2
G cn
bone 1.
I 0
/ day)
formation
(VO+)
and cal-
34) V,+ was significantly less in the animals given the larger dose of ClzMDP than in the control animals (43.8 ZIZ 2.4 compared with 63.6 & 1.6; P < 0.05). Figure 4B also shows that the effect of PTX (31) was similar to the effect of the larger dose of C&MDP.
Vi(mg/day)
Flow of Calcium out of Bane
--- Controls * 1 m g P/kg /day * 10 1 o PTX
Cf2MDP
r
I 100
Q
1
Vi (mg/day)
2. A and B: relation between secretion of calcium and calcium intake (Vi). A and B as in Fig. 1.
into intestine
FIG.
(Vf)
PRESENT q Controls m CI2MDP
SERIES
SAMMON
IOmg
q ControIs El PTX
P/kg/day
et al 1970
Figure 5A shows that in the control groups, V,--, the rate of bone destruction, increased progressively as the calcium intake was diminished. This can be predicted from the results shown above, since V,is calculated as the difference between V,+ (Fig. 4A) and the retention of calcium, and the latter closely approximates V,, (Fig. 1A). Again, the values agree well with those reported by Sammon et al. (3 1). The linear regression of V, - on Vi in our data was = 63.1 - 0.376 Vi; r = 0.778; n = 125; P < 0.001. KFigure 5B shows the effect of ClzMDP on V,-- . In contrast to the control animals, where V,was about zero at high calcium intakes and increased progressively as intake was reduced, in the animals treated with the larger dose of ClzMDP there was no increase in V,-until the diet contained less than 0.5 % Ca (about 50 mg of calcium per day). As the intake was further decreased, there was a progressive increase in V, - The linear regression of V,on Vi in the rats given the larger dose of ClsMDP when Vi was less than 100 mg/day was V,= 38.5 - 0.557 Vi; r = 0.548; n = 23; P < 0.01. l
DIET CALCIUM
1.3
0.5
......:.... ........ ...... ........ .. ........... ................ ............ ...... .. ..h... .......... -3 ........ 0.5
:... . . . . . . . . . . . . . .. . .. .. . ..
::. . . . . .. . . .. . . . . . . . . . . . 4.4. n.... . . . . n.... .. .. . .. . . . . . . ‘I
Ii
(%)
FIG. 3. Serum calcium at 3 calcium intakes in left, control rats given C12MDP (10 mg P/kg per day) in present series combined) and right, control rats and parathyroidectomized rats .(3 1).
ExchangeablePools
0.1
rats and (all data (PTX)
The reduction in calcium intake had no influence on the rapidly exchangeable pool, increased somewhat the slowly exchangeable pool, and increased the flow between them. ClgMDP had no consistent effect on any of these variables (Table 2), and these results will not be discussed.
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1754
MORGAN,
T i I
GASSER,
BALANCE
E
80
f#
LARGIADGR,
STUDIES
Sammon
a Present
L
, 150
I 100
0.3
60
0.2 %
20
eta1 1970 series
0.1 : 0
0 ::
, 50
, 0
Vi (mg/day)
60
8
1
--- Controls 0 PTX (Sammon
I 150
et al 1970)
I 100 Vi (mglday)
2’
I 50
4’
0’,=
0
FIG. 5. A and & relation between bone resorption (V.-) and calcium intake (Vi). A and B as in Fig. 1. Interrupted line in B is calculated linear regression of V,on Vi in control rats. Continuous oblique line in B is calculated linear regression of V,on Vi in rats given larger dose of CIZMDP with a calcium intake less than 100 q/day. DISCUSSION
Calcium Deprivation
STUDlES
Vad
100 Vi
5 200
FLEISCH
0.4
Vad
200
-zoJ
AND
PERFUSION
40 t
JUNG,
in Control Animals
When the calcium intake is reduced to the extent that it was in these experiments, there is inevitably some reduction in the net absorption of calcium. The relation between V,, and calcium intake for the whole range of calcium intakes agrees well with the relation given by Sammon et al. (31), who performed experiments similar to the ones reported here, and with the relation in the data of Aubert et al. (3), in which variation in intake was due to spontaneous variation between the animals in their intake of food of constant composition. The explanation of the observed relation between net absorption or dietary absorption of calcium and calcium intake is not known. But as Fig. 6 shows, the relation is such that the percentage of ingested calcium which is absorbed (cu Yo) increases as the intake is decreased. One interpretation of these results is that they indicate an increase in the efficiency of calcium absorption as the intake of calcium is diminished, and that this is due to a compensatory adaptation in the intestinal calcium transport mechanism (19, 35). However, another explanation of these findings is that the characteristics of the absorption mechanism are such that the smaller the load of calcium presented to the intestine, the greater will be the proportion of that load which will be
0L
0 Calcium
0.6 input
0.4
0.2 (mg
0 per
0 hour)
FIG. 6. Left: absorption of dietary calcium (Vad, in mg/day) and percentage absorption of dietary calcium (&) according to calcium intake in control group of the present study. Right: net calcium absorption and percentage absorption of cakium according to rate of perfusion of calcium through isolated segments of proximal small intestine in the rat in vitro (5, 22).
absorbed. Support for this explanation comes from experiments in which the load of calcium presented to the perfused intestine was suddenly reduced (5, 22) (Fig. 6). There was an increase in the proportion of the calcium load which was absorbed, and it is unlikely that this increase was due to adaptation in the gut absorption mechanism. In these experiments V,+ did not change significantly despite a drastic reduction of calcium intake. These results are in agreement with those of Sammon et al. (3 1) and between V,+ and Cohn et al. (7), who found a relation calcium intake, which was, however, not statistically significant. Aubert and Milhaud (2) concluded that there was an inverse relation between V,+ and calcium intake, but in their experiments the variation in Vi between animals was due to spontaneous variation in the intake of food, so that their results are not comparable with ours. The results of StauEer et al, (33), which show a direct relation between both variables, are also not comparable, because they are based on a morphological analysis of the shaft Finally, in man, short-term reduction in calcium intake is without effect on V,+ (26). It is still debated whether V,+ can be taken as a measure of bone formation rate. Formally V,+ is the rate of entry into bone of calcium which does not significantly reenter the compartment El for at least 3 days. If V,+ represents bone formation, as assumed here, V,represents bone destruction, since it is calculated as the difference between VS and V,+. The rate of bone destruction increases progressively as the calcium intake is reduced (Fig. 5A), in agreement with observations made in rats (3, 7, 31, 33) and in man (24, 26). In the growing animal there is a need for large amounts of calcium. When the calcium intake is adequate, this calcium is obtained from the diet; when intake of calcium is reduced, the supply of calcium is obtained by an increased resorption of bone. These relationships are outlined schematically in Fig. 7. E$ects of CI&fDP
and Comparison with Parathyroidecfomy
It is interesting that the effects of ClaMDP on the several variables are strikingly similar to the results obtained after parathyroidectomy (1, 18, 3 1). Thus ClzMDP and PTX lead to a decrease in V na at higher calcium intakes, and an
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DIPHOSPHONATE
AND
CALCIUM
1755
METABOLISM
increase in V,, at medium calcium intakes (Fig. lB), a decrease in V,+ at all calcium intakes (Fig. 4-B), and a decrease in V,-at all intakes (Fig. 5B). These effects are summarized in Figure 8. On the other hand, the effects are different on serum calcium and phosphate, since PTX causes gross hypocalcemia whereas ClzMDP does not, even with the smallest calcium intake, and ClzMDP causes low serum phosphate (14) whereas PTX does not These results raise the possibility that fluxes other than L, V,+, and V,---, such as those in the kidney (25), may have a role in setting the plasma calcium. The finding that the results of PTX and QMDP administration are not identical suggests also that ClgMDP does not act by blocking the effects of PTH. The performed experiments also allow some conclusions concerning the regulation of the various flows. It appears that in each of the experimental conditions (control, PTX, QMDP) V,+ is constant at all calcium intakes. This is made possible through an increase in V,when the intake of calcium and, therefore, V,, is diminished (Fig. 7). ln the presence of QMDP or PTX, V,+ is smaller and V, -therefore increases only at lower calcium intakes, when the V,, decreases below V,+ But at these low intakes V,increases with a slope at least as great as that in intact rats (Fig. 8). This is surprising since it is known that ClzMDP inhibits resorption, and since it is generally thought that the resorption induced by lack of calcium is due to PTH. The factors that control this PTH-independent and CLMDP-resistant increase in bone resorption are unknown. One mechanism might be an increased production of one of the metabolites of vitamin D, which have been shown to increase bone resorption (34). l
60-l
hm
