JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 1, 1990 Mary Ann Liebert, Inc., Publishers
Parathyroid Hormone Is Not Required for Normal Milk Composition or Secretion or Lactation-Associated Bone Loss in Normocalcemic Rats SANFORD C. GARNER, AGNA BOASS, and SVEIN U. TOVERUD
ABSTRACT
To determine if parathyroid hormone (PTH) is essential for lactation in rats, the parathyroid glands were removed surgically during the first week of lactation and the rats were given a diet containing a high calciumphosphorus ratio to maintain a normal serum calcium concentration. Lactating rats were placed on diet containing 1.2% calcium (Ca) and 0.8, 0.6, or 0.4% phosphorus (P) on day 2 postpartum (PP) and were parathyroidectomized (PTX) at 4-6 days PP. At 10 days PP serum Ca was 10.5 f 0.2 mg/dl (mean SEM) for PTX rats and 10.4 f 0.3 mg/dl in sham-operated lactating rats when the diet contained 0.6% P. When the diet P was 0.8%, the litters gained little or no weight and serum Ca fell to 6.9 * 0.6 mg/dl by day 10 PP in PTX rats compared with 10.2 f 0.2 mg/dl in sham rats. PTX rats fed the diet containing 1.2% Ca and 0.6% P maintained a normal serum Ca level until at least day 18 PP, but their serum P levels fell gradually from approximately 5 mg/dl at 10 days to 3 mg/dl at 18 days PP. In spite of this hypophosphatemia, the litters of PTX and sham rats had gained the same amount of weight by age 16 days, indicating equal milk production in the two groups. Milk Ca, P, and total solids were not significantly different between PTX and sham rats on day 11 PP. With the 1.2% Ca and 0.4% P diet, the femurs lost approximately 20% of dry bone mass in both PTX and sham rats 0) c 0.01) between 5 days PP, when PTX was carried out, and 20 days PP. We conclude that the absence of the parathyroid glands does not affect milk production, milk mineral composition, or the lactation-associated bone loss when normal serum Ca levels are maintained by diet. We suggest that PTH is not essential for lactation beyond its normal function of maintaining the serum Ca concentration.
*
INTRODUCTION
PTX in lactating but not in nonlactating rats, even in the presence of a high-calcium (1.2% Ca and 0.8% P) diet.'') When PTX was carried out prior to pregnancy, the mothers delivered small litters and had a marked tendency to eat their young.(5) More recent observations indicate that the need for parathyroid hormone appears to be increased during lactation; that is, lactating rats have increased circulating concentrations of immunoreactive parathyroid hormone (iPTH).(e,9) It is reasonable to assume that the cause of the failure of lactation in PTX rats is the inability to maintain a normal serum calcium level and to mobilize bone calcium for milk
ARATHYROID HORMONE is considered essential for
maintenance of lactation in the rat, primarily by maintaining normal serum calcium concentrations [see reviews by Toverud and Boass") and Garel(')]. Specific support for this contention includes the demonstration of (1) severe hypocalcemia 1 day after parathyroidectomy (PTX) of lactating rats accompanied by a reduction of the water con(2) a reduction in litter weight gain tent of the whether PTX was carried out before or during lactation,(+') and (3) a significant fall in serum Ca 3 h after
P
~~
~~
~
Department of Pharmacology and Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455. 69
GARNER ET AL.
70
production. However, it is conceivable that parathyroid hormone also affects milk secretion directly by an action on the mammary secretory epithelium. To test these hypotheses, we attempted to maintain normocalcemia in PTX rats by dietary means so that various parameters of lactational performance could be determined unhampered by the consequences of hypocalcemia. The purpose of the experiments was therefore twofold: (1) to manipulate the dietary calcium and phosphate concentrations so that normocalcemia could be maintained after PTX, and (2) in such a normocalcemic PTX model to determine litter weight gain, the pups' serum mineral concentrations, the concentration of various milk constituents, and the degree of lactation-associated bone loss. PTX was carried out early in lactation when the milk Ca drain is relatively low and the rats are less likely to develop severe hypocalcemia.
MATERIALS AND METHODS Rats and diets Pregnant rats (14 or 18 days of gestation) were obtained from Holtzman Co. (Madison, WI) along with nonmated controls of approximately the same age and weight. Upon arrival all rats were fed a basic diet(l") consisting of 75% whole wheat flour, 13qo casein, 5% cellulose (Alphacel), 4% corn oil, 2% salt mixture,(11) 1.3% vitamin D-free vitamin mixture (ICN Biochemicals), 5 IU cholecalciferol per g diet, and 0.8% CaC0, (prepared as jumbo pellets by ICN Biochemicals, Cleveland, OH). The final content of Ca, P, and magnesium in the diet were 0.4, 0.4, and 0.15%, respectively, by analysis.('") Litters were adjusted to 10 or 11 pups 2 days after birth, and the mothers were assigned to one of four diets, which varied from the basic diet only in the Ca and P content. Ca remained at 0.4% or was raised to 1.2% with additional CaCO,, whereas P either remained at 0.4% or was raised to 0.6 or 0.8% by the addition of KH,PO,. After 4-6 days of lactation, one-half of the rats had the parathyroid glands surgically removed under ether anesthesia; the other half underwent the same surgery without excision of the glands (sham-operated controls). Successful removal of all parathyroid tissue can be estimated at > 95% based on our previous experience and on the observation that all lactating rats placed on a diet containing 1.2% Ca and 0.8% P developed severe hypocalcemia. Intact nonlactating rats also served as controls.
aliquots were used immediately or stored at -20°C until analyses of Ca, P, and Mg were performed. Serum Ca and Mg were measured in a lanthanum chloride solution by atomic absorption spectrophotometer (Model 5100, Perkin-Elmer Corp., South Pasadena, CA) or by the semiautomated fluorometric titration procedure of Borle and Briggs.(I3)Inorganic P was determined colorimetrically.(14) In one experiment milk was collected manually 2-3 minutes after administration of 2 U oxytocin SC to PTX and sham-operated rats that had lactated for 11 days. The milk was dried at 95°C and then ashed at 600°C for 24 h. The ash was dissolved in 1 N HCl and heated in a boiling water bath to hydrolyze any pyrophosphate formed. Ca and P were determined as described for serum. To determine the weight of bone and bone ash, the left tibia and femur were dissected free of muscle and extracted with ethanol and ether. Weights were obtained of dry defatted bones and of bone ash after incineration for 24 h at 600°C.
Statistical analysis Data are presented as the mean f SEM, and statistical significance of differences was determined using Student's two-tailed t-test. Multiple comparisons were made using the method of Scheffd.('"
Experimental protocols Protocol 1. Serum Ca and P and Litter Weights as Affected by the Dietary Ca/P Ratio: Groups of PTX and sham-operated lactating rats were fed diets containing 1.2% Ca and either 0.6 or 0.8% P. Serum Ca and P and litter weights were determined on days 10 and 18 of lactation. Protocol 2. Milk Composition in PTX Lactating Rats: Groups of PTX or sham-operated lactating rats fed diet containing 1.2% Ca and 0.6% P were compared with a group of intact lactating rats fed diet containing 0.4% Ca and 0.4% P. Serum Ca and P and milk total solids, Ca and P were determined on day 11 or 18 of lactation. Protocol 3. Serum Mineral Concentrations in Pups of PTX Rats: Groups of PTX or sham-operated lactating rats were given diet containing 1.2% Ca and 0.4% P. Litter weights and the concentration of Ca, P, and Mg in the serum of pups were determined at 16 days of age.
Protocol 4. Lactation-Associated Bone Loss in PTX and Sham-Operated Rats: Groups of lactating and nonlacAnalytic procedures tating rats were given a diet containing 1.2% Ca and 0.4% Mothers and litters were weighed two or three times per P. On day 5 of lactation one group of lactating rats was week. Unfasted rats were bled under ether anesthesia by bled for serum Ca and P determinations and then killed orbital sinus venipuncture,'") from the tail, or by heart for bone analyses. Two other groups of lactating rats were puncture during the terminal bIeeding of an experiment. parathyroidectomized or sham operated on day 5 of lactaAlso bled by heart puncture were 16-day-old pups, one tion. These groups were bled and killed for serum Ca and pup from each litter of the PTX and sham-operated P determinations and bone analyses on day 20 of lactation. mothers. After the serum was separated by centrifugation, Litter weights were obtained on days 5 and 16 of lactation.
PARATHYROID-FREELACTATING RATS
71
RESULTS Serum Ca and P concentrations and litter weights Lactating rats that were maintained on a diet containing 1.2% Ca and 0.6% P and parathyroidectomized during the first week of lactation had normal serum Ca values on both days 10 and 18 of lactation, and the values were not significantly different from those of the sham-operated controls (Table 1). The serum P values, which also were similar in the two groups, decreased markedly between days 10 and 18 of lactation. Based on the similarity of the litter weights between the two groups at both stages of lactation, it appears that milk production was not impaired in the PTX rats. The litter weights for the PTX rats were essentially the same as we reported earlier when a diet containing 0.4% Ca and 0.4% P was fed to intact rats.(161 The results were quite different when the phosphate content of the diet was 0.8%. By day 10 of lactation the serum Ca of the PTX rats was 6.9 mg/dl and significantly lower
than that of the sham-operated controls (Table 1). In addition, the weight of the litters of the PTX rats was far lower than that of the litters of the sham-operated rats. Over the 4 day period after parathyroidectomy 15 of the 80 pups died, three of the litters had an average weight loss of 60 g, and the remaining litters gained an average of only 32 g compared with an average weight gain of the litters of the sham-operated mothers of 87 g. Since it was obvious that this diet could not support lactation in PTX rats, the PTX group was terminated on day 1 1 of lactation. The shamoperated group had serum Ca and P values and litter weights on both 10 and 18 days of lactation that were essentially the same as for the groups on the 1.2% Ca and 0.6% P diet (Table 1). We were not successful in maintaining both serum Ca and P in PTX lactating rats in the range normally seen with diets having a Ca/P ratio closer to 1, that is, serum Ca in the range 9-11 mg/dl and serum P in the range 5-7 mg/dl, as shown for the control group in Table 2.
TABLE1. SERUM CALCIUM (Ca) AND PHOSPHORUS (P) CONCENTRATIONS AND LITTERWEIGHTS IN PTx AND SHAM-OPERATED LACTATING RATS~
Lactation day 10 Diet %Ca:%P
Group
No. of rats
1.2:0.6 1.2:0.6 1.2:0.8 1.2:0.8
PTX Sham PTXc Sham
8 3 7 3
Serum Ca (mg/dl)
10.5 f 10.4 f 6.9 f 10.2 f
Serum P (mg/dl)
0.2 0.3 0.6b 0.2
5.3 f 4.8 f 8.8 f 5.7 f
0.5 1.2 0.6b 0.4
Lactation day I8 Litter weight (g)
231 f 223 f 137 f 237 f
12 13 20b 14
Serum Ca (mgW
Serum P (mg/dl)
10.6 f 0.2 10.0 f 0.6
3.0 f 0.5 2.6 f 1.2
-
-
10.1
f
0.4
3.3
f
Litter weight (g)
421 338
f f
22 35
0.3
447
f
35
aOn day 2 of lactation the rats were switched from a diet containing 0.4% Ca and 0.4% P to the indicated diets. Parathyroidectomy (PTX) or a sham operation (Sham) was carried out 4 days later. The values are mean f SEM of three to eight rats. Although there were only three rats in each sham group, the serum calcium level for these rats is similar to that for a group of sham rats on 1.2% Ca and 0.6% P diet shown in Table 2. bp < 0.01 versus sham-operated group on the same diet. CBecause lactation could not be sustained on this diet, the mothers and their pups were killed on day 11 of lactation.
TABLE2. MILKCOMPOSITION IN PTX AND SHAM-OPERATED RATS~ ~
Milk Diet %Ca:%P
Total solids Group
(%)
Ca (mg/g of solids)
Serum P (mg/g of solids)
Ca (mg/dl)
P (mg/d[)
~~
0.4:0.4
Control 18 days
27.9
1.2:0.6
Sham 1 1 days
27.9
1.2:0.6
PTX 1 1 days
28.2 f 1.1
*
f
1.3
9.7 f 0.8
1.1
8.4
6.6
f
0.5
9.6
f
0.2
6.1 f 0.5
0.5
7.4 f 0.2
10.3 f 0.2
2.4 f 0.3
9.0 f 0.4
7.8 f 0.3
10.0 f 0.2
3.0 f 0.4
aThe data represent mean SEM for groups of six to seven rats milked either on day 11 or day 18 of lactation. The rats were placed on the designated diets on day 2 of lactation, and PTX or a sham operation was carried out on day 6. For the rats milked on day 11, litter weights on days 9, 12, and 16 were not significantly different between PTX and sham groups.
12
GARNER ET AL.
Evidence that normocalcemic PTX lactating rats maintained on the 1.2% Ca and 0.6% P diet were successfully parathyroidectomized was obtained in an experiment in which the serum Ca level fell from 9.75 f 0.21 mg/dl (n = 7) to 5.83 f 0.65 mg/dl3 days after the PTX rats were switched from the 1.2% Ca and 0.6% P diet to the 1.2% Ca and 0.8% P diet. Sham-operated lactating rats had only a small decrease in serum Ca from 10.33 f 0.19 mg/dl (n = 8) to 9.48 f 0.14 mg/dl in response to the diet change. Litter weight gain after the diet change in the PTX group was only 57% of that in the sham-operated group.
Milk composition Parathyroidectomized and sham-operated rats maintained on the 1.2% Ca and 0.6% P diet were milked on day 11 of lactation (5 days after surgery). There were no significant differences between the two groups in percentage total solids or in Ca and P concentrations (Table 2). As in the previous experiment (Table l), serum Ca and P concentrations and litter weights were not significantly different between the PTX and sham-operated groups. The values for total solids and Ca and P in the milk from the rats on the 0.4% Ca and 0.4% P diet were essentially the same as for the rats on the 1.2% Ca diet, even though the mean serum P value was approximately twice as high as for the rats on the higher Ca diet.
Serum mineral concentrations in the suckling pups Serum Ca, P, and Mg concentrations were essentially the same in pups of PTX and sham-operated mothers when determined at 16 days of age (Table 3).
Lactation-associated bone loss Results from preliminary experiments indicated that bone loss during lactation was greater when the phosphorus content of the high-calcium (1.2%) diet was reduced from 0.6 to 0.4%. We therefore tested the effect of PTX on bone loss in rats that were placed on the 1.2% Ca and 0.4% P diet on day 2 of lactation. When the PTX or
the sham operation was performed 3 days later, a third group of lactating rats was killed to provide baseline bone weights. There were no differences between PTX and sham-operated rats in serum Ca and P or in body weight or in litter weight (Table 4). As shown in Fig. 1, femur dry weight on day 20 of lactation was almost identical in PTX and sham-operated rats, and both groups of rats had femur weights that were approximately 20% lower than the 5 day baseline values. Nonlactating rats of approximately the same age and weight, that had been maintained on the same diet for the same length of time as the 20 day lactating rats, had a mean femur weight that was slightly higher (p < 0.025) than the mean for the 5 day lactating rats. Weight analysis of the tibiae also revealed a nonsignificant difference between PTX and sham-operated groups (not shown). However, the loss of tibia weight over the 15 day period appeared to be less than the loss of femur weight (13 versus 20%).
DISCUSSION The present results show that it is possible to maintain normal serum Ca levels in PTX lactating rats by proper manipulation of the Ca and P contents of the diet. A Ca content of 1.2% and a Ca/P ratio of 2: 1 or 3: 1 enabled the rats to maintain normocalcemia throughout lactation. A lower Ca/P ratio (1 3:1) led to severe hypocalcemia and lactation failure in PTX rats, but the parathyroid-intact rats were not adversely affected by this change in the Ca/P ratio. Parathyroid hormone is clearly needed to enable lactating rats to maintain normocalcemia in the face of a wide range of Ca and P intake levels. Although Fry et a1.(6'fed their PTX rats a diet with a Ca/P ratio of 2: 1 (1070 Ca and 0.5% P in a commercial diet), the rats remained severely hypocalcemic throughout lactation for reasons that are not clear. Perhaps the 20% higher Ca content in our diet is required to prevent hypocalcemia. Other differences in design, such as PTX having been carried out 8 weeks before mating by Fry et al., may also be relevant. Although it may be surprising that a minor change in the diet phosphate content can have such a dramatic effect on the serum Ca level, we have previously ~ h o w n ~ ' .that ' ~ ) the
TABLE3. SERUM MINERAL CONCENTRATIONS IN 16-DAY-OLD PUPS FROM PARATHYROIDECTOMIZED (PTX) AND SHAM-OPERATED MOTHERS^ Serum concentrations (rng/dl)
Group
P
Ca
Mg
Body weight (g)
PTX (10)
10.7 f 0.1
8.6
f
0.4
2.33
f
Sham (10)
10.8
8.7
f
0.2
2.29
* 0.06
f
0.1
0.08
40 f 0.9 42
f
2.0
aLactating rats were placed on diets containing 1.2% Ca and 0.4% P on day 2 of lactation and were subjected to PTX or a sham operation 4 days later. The values represent the mean f SEM for groups of 10 pups, 1 pup from each of 10 litters.
PARATHYROID-FREELACTATING RATS
TABLE4. SERUMCa AND P
AND
73
LITTERWEIGHTS FOR LACTATING RATSIN BONELoss EXPERIMENT^
Litter weight (g)
Body weight (g) Serum P (mg/dl)
Group and day of lactation
Serum Ca (mg/dI)
Intact, 5 day
11.0
f
0.3
2.17 f 0.25
309
PTX, 20 day
11.6
f
0.3
2.69
314 f 11
291
f
Sham, 20 day
11.5
f
0.3
2.31 f 0.15
309 f 9
290
f
f
0.21
Day 5 f
12
Day 20
-
Day 5
Day 16
119
f
9
-
7
122
&
3
382 f 4
6
117
f
5
354
f
12
aThe values represent the mean f SEM for groups of five to eight lactating rats for which bone weights are presented in Fig. 1. Serum Ca and P were determined on day 20 of lactation. See Fig. 1 for experimental details.
Bone loss in PTX lactating rats 700 'I
550 500 450 400
350 0
NL
L
5
L PTX
L SHAM
20
Days of lactation FIG. 1. The effect of parathyroidectomy (PTX) on bone loss during lactation. Rats were parathyroidectomized after 5 days of lactation, having been fed a diet with 1.2% Ca and 0.4% P from 2 days after parturition. Nonlactating (NL) rats received the 1.2% Ca and 0.4% P diet for 3 weeks. The height of the bars and the vertical lines represent the mean value f SEM of defatted dry femora from each group. The number of rats per group is indicated on each bar. Femur ash weights (mg) were as follows: 364 f 18 (5 day), 287 f 6 (PTX), and 283 f 8 (sham). *p < 0.001 for 20 day lactating rats (PTX or sham) versus 5 day lactating rats (Schefffs contrast).
lactating rat is far more vulnerable to the loss of PTH and 1,25-dihydroxyvitamin D, [ 1,25-(OH),D3] than nonlactating rats. Under these circumstances the influx of Ca and P from the intestine has a much greater effect on the serum Ca and P concentrations in the lactating rat than in the nonlactating rat. A surprising finding was that normal milk production (based on litter weights) did not require maintenance of normal serum P levels as long as normal serum Ca levels were maintained. In rats given a diet with 1.2% Ca and 0.4% P, serum P levels were close to 2 mg/dl or only 3&40% of that of parathyroid-intact lactating rats maintained on our standard diet containing 0.4% Ca and 0.4% P. In spite of such hypophosphatemia litter weights were normal, suggesting a normal rate of milk production. Thus, galactopoiesis is clearly far more sensitive to changes in circulating Ca than in circulating P levels. The milk composition data are fully consistent with the litter weight data because no abnormalities were detected in the content of total solids, Ca, or P in PTX rats. In previous studies milk from hypocalcemic PTX rats had an increased total solids c ~ n t e n t . ' ~It, ~is' clear from the present data that the absence of parathyroid hormone does not affect the total solids content (or rather the water content) or the milk Ca content when a normal serum Ca level is maintained. The present data are therefore consistent with the conclusions from the earlier that it is the serum Ca level that determines the water content of the milk, not the presence or absence of the parathyroid glands per se. With regard to the milk P data, it is interesting to note that not only is the milk P content unaffected by the parathyroid status, but it also appears to be independent of the serum P level; the milk P content was the same despite a twofold difference in the serum P level. Mueller and showed that wide variations in dietary Ca and P contents have little influence on milk composition or milk production. However, serum Ca and P concentrations were not reported in this study. The most interesting observation of this study was the bone loss during lactation in the PTX rats, which was just as extensive as in the sham-operated rats. It has generally
GARNER ET AL.
14
been assumed that 1,25-(OH)2D, and parathyroid hormone are the main mediators of bone loss during lactation. However, Halloran and DeLuca('8) and Brommage and DeLuca(I9)have demonstrated clearly that vitamin D-deficient lactating rats [with undetectable serum 1,25-(OH),D3] lose just as much bone as do vitamin D-replete lactating rats, showing that 1,25-(OH),D3 is not essential for bone resorption. Brommage and D e L ~ c a " ~also ) reasoned that parathyroid hormone was probably not involved either, because they observed normal bone loss in lactating rats fed a high-calcium diet, which resulted in a mean serum Ca level of approximately 12 mg/dl, a level assumed to have suppressed PTH secretion. However, we have recently shown that circulating iPTH levels are not suppressed by hypercalcemia during lactation.(z0)This finding leads us to disagree with their reasoning, but in view of our present results we concur in their conclusion that PTH is not involved in normal lactational bone loss. If these major bone-resorbing hormones are not involved in the normal bone loss in lactation, what is the mechanism for the net increase in bone resorption during this period? Brommage and D e L ~ c a " ~observed ) normal lactational bone loss in ovariectomized rats as well as in adrenalectomized rats and could therefore rule out ovarian and adrenal hormones as the humoral signal to the bone cells. They were unable to prevent the bone loss by raising the Ca and P contents of the diets. However, we recently showed that feeding our basic diet with Ca and P increased to 1.6 and 1.4%. respectively, and providing drinking water with 0.5% Ca could eliminate the bone loss completely.(21)This observation shows that the bone loss is not an obligatory consequence of lactation, but it does not give us a clue to the identity of the stimulator of bone resorption when more phsyiologic Ca and P intakes are provided. A plausible, but still hypothetical, candidate is the PTHlike peptide (PTH-LP), which was first suggested by Brommage and D e L ~ c a " ~as) the bone-resorbing factor in lactation. The mRNA for PTH-LP is expressed only in actively secreting mammary glands, and an extract of mammary tissue has been shown to stimulate CAMPproduction by PTH-sensitive osteosarcoma cells. Thus, the peptide can exert a PTH-like effect on bone cells, but a direct connection between this factor and bone loss in lactation is not yet available. Although the PTH-LP has been described as a hypercalcemic factor (causing hypercalcemia of human malignancies), based on the present data the peptide is not able to prevent severe hypocalcemia in PTX lactating rats. This suggests that secretion of PTH-LP is not stimulated by hypocalcemia, as is that of PTH. However, our previous observation that bone loss can be prevented by manipulation of dietary Ca and PC2')is consistent with suppression of either the secretion or the effect of the peptide. Alternatively, one could suggest that the PTH-LP is produced by lactating mammary tissue but has no important function as an antihypocalcemic or bone-resorbing factor. Clearly, the physiologic function, if any, of PTH-LP in the lactating rat remains to be elucidated.
In summary, a dietary Ca/P ratio has been identified that will permit rats to maintain normocalcemia after parathyroidectomy during lactation. Under this regimen the rats produce a normal amount of milk with a normal composition (total solids, calcium, and phosphorus) despite severe hypophosphatemia. The parathyroidectomized lactating rats lose a normal amount of bone during weeks 2 and 3 of lactation. We conclude that parathyroid hormone is not directly essential for normal milk secretion or for maintaining lactation-associated bone loss. Its primary physiologic function in lactation, therefore, appears to be the same as in the nonlactating animal, namely, to prevent hypocalcemia.
ACKNOWLEDGMENTS The authors express their appreciation to Ms. Catherine Lane for technical assistance, to Ms. Gale Thompson for preparing the manuscript, and to Mr. Stefan Gottschalk for producing the figure. This work was supported in part by NIH grant HD12496.
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259. 18. Halloran BP, DeLuca HF 1980 Skeletal changes during pregnancy and lactation: The role of vitamin D. Endocrinology
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75 19. Brommage R, DeLuca H F 1985 Regulation of bone mineral loss during lactation. Am J Physiol 248:E182-187. 20. Garner SC, Boass A, Toverud SU 1989 Hypercalcemia fails to suppress elevated serum parathyroid hormone concentrations during lactation in rats. J Bone Min Res 4577-583. 21. Bruns ME, Boass A, Toverud SU 1987 Regulation by dietary calcium of vitamin D-dependent calcium-binding protein and active calcium transport in the small intestine of lactating rats. Endocrinology 121:278-283. 22. Thiede MA, Rodan GA 1988 Expression of a calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue. Science 242:278-280.
Address reprint requests to: Dr. S.U. Toverud Dental Research Center, CB# 7455 University of North Carolina Chapel Hi", NC 27599 Received for publication April 3, 1989; in revised form July 3, 1989;accepted July 5, 1989.
Parathyroid Hormone Is Not Required for Normal Milk Composition or Secretion or Lactation-Associated Bone Loss in Normocalcemic Rats SANFORD C. GARNER, AGNA BOASS, and SVEIN U. TOVERUD
ABSTRACT
To determine if parathyroid hormone (PTH) is essential for lactation in rats, the parathyroid glands were removed surgically during the first week of lactation and the rats were given a diet containing a high calciumphosphorus ratio to maintain a normal serum calcium concentration. Lactating rats were placed on diet containing 1.2% calcium (Ca) and 0.8, 0.6, or 0.4% phosphorus (P) on day 2 postpartum (PP) and were parathyroidectomized (PTX) at 4-6 days PP. At 10 days PP serum Ca was 10.5 f 0.2 mg/dl (mean SEM) for PTX rats and 10.4 f 0.3 mg/dl in sham-operated lactating rats when the diet contained 0.6% P. When the diet P was 0.8%, the litters gained little or no weight and serum Ca fell to 6.9 * 0.6 mg/dl by day 10 PP in PTX rats compared with 10.2 f 0.2 mg/dl in sham rats. PTX rats fed the diet containing 1.2% Ca and 0.6% P maintained a normal serum Ca level until at least day 18 PP, but their serum P levels fell gradually from approximately 5 mg/dl at 10 days to 3 mg/dl at 18 days PP. In spite of this hypophosphatemia, the litters of PTX and sham rats had gained the same amount of weight by age 16 days, indicating equal milk production in the two groups. Milk Ca, P, and total solids were not significantly different between PTX and sham rats on day 11 PP. With the 1.2% Ca and 0.4% P diet, the femurs lost approximately 20% of dry bone mass in both PTX and sham rats 0) c 0.01) between 5 days PP, when PTX was carried out, and 20 days PP. We conclude that the absence of the parathyroid glands does not affect milk production, milk mineral composition, or the lactation-associated bone loss when normal serum Ca levels are maintained by diet. We suggest that PTH is not essential for lactation beyond its normal function of maintaining the serum Ca concentration.
*
INTRODUCTION
PTX in lactating but not in nonlactating rats, even in the presence of a high-calcium (1.2% Ca and 0.8% P) diet.'') When PTX was carried out prior to pregnancy, the mothers delivered small litters and had a marked tendency to eat their young.(5) More recent observations indicate that the need for parathyroid hormone appears to be increased during lactation; that is, lactating rats have increased circulating concentrations of immunoreactive parathyroid hormone (iPTH).(e,9) It is reasonable to assume that the cause of the failure of lactation in PTX rats is the inability to maintain a normal serum calcium level and to mobilize bone calcium for milk
ARATHYROID HORMONE is considered essential for
maintenance of lactation in the rat, primarily by maintaining normal serum calcium concentrations [see reviews by Toverud and Boass") and Garel(')]. Specific support for this contention includes the demonstration of (1) severe hypocalcemia 1 day after parathyroidectomy (PTX) of lactating rats accompanied by a reduction of the water con(2) a reduction in litter weight gain tent of the whether PTX was carried out before or during lactation,(+') and (3) a significant fall in serum Ca 3 h after
P
~~
~~
~
Department of Pharmacology and Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455. 69
GARNER ET AL.
70
production. However, it is conceivable that parathyroid hormone also affects milk secretion directly by an action on the mammary secretory epithelium. To test these hypotheses, we attempted to maintain normocalcemia in PTX rats by dietary means so that various parameters of lactational performance could be determined unhampered by the consequences of hypocalcemia. The purpose of the experiments was therefore twofold: (1) to manipulate the dietary calcium and phosphate concentrations so that normocalcemia could be maintained after PTX, and (2) in such a normocalcemic PTX model to determine litter weight gain, the pups' serum mineral concentrations, the concentration of various milk constituents, and the degree of lactation-associated bone loss. PTX was carried out early in lactation when the milk Ca drain is relatively low and the rats are less likely to develop severe hypocalcemia.
MATERIALS AND METHODS Rats and diets Pregnant rats (14 or 18 days of gestation) were obtained from Holtzman Co. (Madison, WI) along with nonmated controls of approximately the same age and weight. Upon arrival all rats were fed a basic diet(l") consisting of 75% whole wheat flour, 13qo casein, 5% cellulose (Alphacel), 4% corn oil, 2% salt mixture,(11) 1.3% vitamin D-free vitamin mixture (ICN Biochemicals), 5 IU cholecalciferol per g diet, and 0.8% CaC0, (prepared as jumbo pellets by ICN Biochemicals, Cleveland, OH). The final content of Ca, P, and magnesium in the diet were 0.4, 0.4, and 0.15%, respectively, by analysis.('") Litters were adjusted to 10 or 11 pups 2 days after birth, and the mothers were assigned to one of four diets, which varied from the basic diet only in the Ca and P content. Ca remained at 0.4% or was raised to 1.2% with additional CaCO,, whereas P either remained at 0.4% or was raised to 0.6 or 0.8% by the addition of KH,PO,. After 4-6 days of lactation, one-half of the rats had the parathyroid glands surgically removed under ether anesthesia; the other half underwent the same surgery without excision of the glands (sham-operated controls). Successful removal of all parathyroid tissue can be estimated at > 95% based on our previous experience and on the observation that all lactating rats placed on a diet containing 1.2% Ca and 0.8% P developed severe hypocalcemia. Intact nonlactating rats also served as controls.
aliquots were used immediately or stored at -20°C until analyses of Ca, P, and Mg were performed. Serum Ca and Mg were measured in a lanthanum chloride solution by atomic absorption spectrophotometer (Model 5100, Perkin-Elmer Corp., South Pasadena, CA) or by the semiautomated fluorometric titration procedure of Borle and Briggs.(I3)Inorganic P was determined colorimetrically.(14) In one experiment milk was collected manually 2-3 minutes after administration of 2 U oxytocin SC to PTX and sham-operated rats that had lactated for 11 days. The milk was dried at 95°C and then ashed at 600°C for 24 h. The ash was dissolved in 1 N HCl and heated in a boiling water bath to hydrolyze any pyrophosphate formed. Ca and P were determined as described for serum. To determine the weight of bone and bone ash, the left tibia and femur were dissected free of muscle and extracted with ethanol and ether. Weights were obtained of dry defatted bones and of bone ash after incineration for 24 h at 600°C.
Statistical analysis Data are presented as the mean f SEM, and statistical significance of differences was determined using Student's two-tailed t-test. Multiple comparisons were made using the method of Scheffd.('"
Experimental protocols Protocol 1. Serum Ca and P and Litter Weights as Affected by the Dietary Ca/P Ratio: Groups of PTX and sham-operated lactating rats were fed diets containing 1.2% Ca and either 0.6 or 0.8% P. Serum Ca and P and litter weights were determined on days 10 and 18 of lactation. Protocol 2. Milk Composition in PTX Lactating Rats: Groups of PTX or sham-operated lactating rats fed diet containing 1.2% Ca and 0.6% P were compared with a group of intact lactating rats fed diet containing 0.4% Ca and 0.4% P. Serum Ca and P and milk total solids, Ca and P were determined on day 11 or 18 of lactation. Protocol 3. Serum Mineral Concentrations in Pups of PTX Rats: Groups of PTX or sham-operated lactating rats were given diet containing 1.2% Ca and 0.4% P. Litter weights and the concentration of Ca, P, and Mg in the serum of pups were determined at 16 days of age.
Protocol 4. Lactation-Associated Bone Loss in PTX and Sham-Operated Rats: Groups of lactating and nonlacAnalytic procedures tating rats were given a diet containing 1.2% Ca and 0.4% Mothers and litters were weighed two or three times per P. On day 5 of lactation one group of lactating rats was week. Unfasted rats were bled under ether anesthesia by bled for serum Ca and P determinations and then killed orbital sinus venipuncture,'") from the tail, or by heart for bone analyses. Two other groups of lactating rats were puncture during the terminal bIeeding of an experiment. parathyroidectomized or sham operated on day 5 of lactaAlso bled by heart puncture were 16-day-old pups, one tion. These groups were bled and killed for serum Ca and pup from each litter of the PTX and sham-operated P determinations and bone analyses on day 20 of lactation. mothers. After the serum was separated by centrifugation, Litter weights were obtained on days 5 and 16 of lactation.
PARATHYROID-FREELACTATING RATS
71
RESULTS Serum Ca and P concentrations and litter weights Lactating rats that were maintained on a diet containing 1.2% Ca and 0.6% P and parathyroidectomized during the first week of lactation had normal serum Ca values on both days 10 and 18 of lactation, and the values were not significantly different from those of the sham-operated controls (Table 1). The serum P values, which also were similar in the two groups, decreased markedly between days 10 and 18 of lactation. Based on the similarity of the litter weights between the two groups at both stages of lactation, it appears that milk production was not impaired in the PTX rats. The litter weights for the PTX rats were essentially the same as we reported earlier when a diet containing 0.4% Ca and 0.4% P was fed to intact rats.(161 The results were quite different when the phosphate content of the diet was 0.8%. By day 10 of lactation the serum Ca of the PTX rats was 6.9 mg/dl and significantly lower
than that of the sham-operated controls (Table 1). In addition, the weight of the litters of the PTX rats was far lower than that of the litters of the sham-operated rats. Over the 4 day period after parathyroidectomy 15 of the 80 pups died, three of the litters had an average weight loss of 60 g, and the remaining litters gained an average of only 32 g compared with an average weight gain of the litters of the sham-operated mothers of 87 g. Since it was obvious that this diet could not support lactation in PTX rats, the PTX group was terminated on day 1 1 of lactation. The shamoperated group had serum Ca and P values and litter weights on both 10 and 18 days of lactation that were essentially the same as for the groups on the 1.2% Ca and 0.6% P diet (Table 1). We were not successful in maintaining both serum Ca and P in PTX lactating rats in the range normally seen with diets having a Ca/P ratio closer to 1, that is, serum Ca in the range 9-11 mg/dl and serum P in the range 5-7 mg/dl, as shown for the control group in Table 2.
TABLE1. SERUM CALCIUM (Ca) AND PHOSPHORUS (P) CONCENTRATIONS AND LITTERWEIGHTS IN PTx AND SHAM-OPERATED LACTATING RATS~
Lactation day 10 Diet %Ca:%P
Group
No. of rats
1.2:0.6 1.2:0.6 1.2:0.8 1.2:0.8
PTX Sham PTXc Sham
8 3 7 3
Serum Ca (mg/dl)
10.5 f 10.4 f 6.9 f 10.2 f
Serum P (mg/dl)
0.2 0.3 0.6b 0.2
5.3 f 4.8 f 8.8 f 5.7 f
0.5 1.2 0.6b 0.4
Lactation day I8 Litter weight (g)
231 f 223 f 137 f 237 f
12 13 20b 14
Serum Ca (mgW
Serum P (mg/dl)
10.6 f 0.2 10.0 f 0.6
3.0 f 0.5 2.6 f 1.2
-
-
10.1
f
0.4
3.3
f
Litter weight (g)
421 338
f f
22 35
0.3
447
f
35
aOn day 2 of lactation the rats were switched from a diet containing 0.4% Ca and 0.4% P to the indicated diets. Parathyroidectomy (PTX) or a sham operation (Sham) was carried out 4 days later. The values are mean f SEM of three to eight rats. Although there were only three rats in each sham group, the serum calcium level for these rats is similar to that for a group of sham rats on 1.2% Ca and 0.6% P diet shown in Table 2. bp < 0.01 versus sham-operated group on the same diet. CBecause lactation could not be sustained on this diet, the mothers and their pups were killed on day 11 of lactation.
TABLE2. MILKCOMPOSITION IN PTX AND SHAM-OPERATED RATS~ ~
Milk Diet %Ca:%P
Total solids Group
(%)
Ca (mg/g of solids)
Serum P (mg/g of solids)
Ca (mg/dl)
P (mg/d[)
~~
0.4:0.4
Control 18 days
27.9
1.2:0.6
Sham 1 1 days
27.9
1.2:0.6
PTX 1 1 days
28.2 f 1.1
*
f
1.3
9.7 f 0.8
1.1
8.4
6.6
f
0.5
9.6
f
0.2
6.1 f 0.5
0.5
7.4 f 0.2
10.3 f 0.2
2.4 f 0.3
9.0 f 0.4
7.8 f 0.3
10.0 f 0.2
3.0 f 0.4
aThe data represent mean SEM for groups of six to seven rats milked either on day 11 or day 18 of lactation. The rats were placed on the designated diets on day 2 of lactation, and PTX or a sham operation was carried out on day 6. For the rats milked on day 11, litter weights on days 9, 12, and 16 were not significantly different between PTX and sham groups.
12
GARNER ET AL.
Evidence that normocalcemic PTX lactating rats maintained on the 1.2% Ca and 0.6% P diet were successfully parathyroidectomized was obtained in an experiment in which the serum Ca level fell from 9.75 f 0.21 mg/dl (n = 7) to 5.83 f 0.65 mg/dl3 days after the PTX rats were switched from the 1.2% Ca and 0.6% P diet to the 1.2% Ca and 0.8% P diet. Sham-operated lactating rats had only a small decrease in serum Ca from 10.33 f 0.19 mg/dl (n = 8) to 9.48 f 0.14 mg/dl in response to the diet change. Litter weight gain after the diet change in the PTX group was only 57% of that in the sham-operated group.
Milk composition Parathyroidectomized and sham-operated rats maintained on the 1.2% Ca and 0.6% P diet were milked on day 11 of lactation (5 days after surgery). There were no significant differences between the two groups in percentage total solids or in Ca and P concentrations (Table 2). As in the previous experiment (Table l), serum Ca and P concentrations and litter weights were not significantly different between the PTX and sham-operated groups. The values for total solids and Ca and P in the milk from the rats on the 0.4% Ca and 0.4% P diet were essentially the same as for the rats on the 1.2% Ca diet, even though the mean serum P value was approximately twice as high as for the rats on the higher Ca diet.
Serum mineral concentrations in the suckling pups Serum Ca, P, and Mg concentrations were essentially the same in pups of PTX and sham-operated mothers when determined at 16 days of age (Table 3).
Lactation-associated bone loss Results from preliminary experiments indicated that bone loss during lactation was greater when the phosphorus content of the high-calcium (1.2%) diet was reduced from 0.6 to 0.4%. We therefore tested the effect of PTX on bone loss in rats that were placed on the 1.2% Ca and 0.4% P diet on day 2 of lactation. When the PTX or
the sham operation was performed 3 days later, a third group of lactating rats was killed to provide baseline bone weights. There were no differences between PTX and sham-operated rats in serum Ca and P or in body weight or in litter weight (Table 4). As shown in Fig. 1, femur dry weight on day 20 of lactation was almost identical in PTX and sham-operated rats, and both groups of rats had femur weights that were approximately 20% lower than the 5 day baseline values. Nonlactating rats of approximately the same age and weight, that had been maintained on the same diet for the same length of time as the 20 day lactating rats, had a mean femur weight that was slightly higher (p < 0.025) than the mean for the 5 day lactating rats. Weight analysis of the tibiae also revealed a nonsignificant difference between PTX and sham-operated groups (not shown). However, the loss of tibia weight over the 15 day period appeared to be less than the loss of femur weight (13 versus 20%).
DISCUSSION The present results show that it is possible to maintain normal serum Ca levels in PTX lactating rats by proper manipulation of the Ca and P contents of the diet. A Ca content of 1.2% and a Ca/P ratio of 2: 1 or 3: 1 enabled the rats to maintain normocalcemia throughout lactation. A lower Ca/P ratio (1 3:1) led to severe hypocalcemia and lactation failure in PTX rats, but the parathyroid-intact rats were not adversely affected by this change in the Ca/P ratio. Parathyroid hormone is clearly needed to enable lactating rats to maintain normocalcemia in the face of a wide range of Ca and P intake levels. Although Fry et a1.(6'fed their PTX rats a diet with a Ca/P ratio of 2: 1 (1070 Ca and 0.5% P in a commercial diet), the rats remained severely hypocalcemic throughout lactation for reasons that are not clear. Perhaps the 20% higher Ca content in our diet is required to prevent hypocalcemia. Other differences in design, such as PTX having been carried out 8 weeks before mating by Fry et al., may also be relevant. Although it may be surprising that a minor change in the diet phosphate content can have such a dramatic effect on the serum Ca level, we have previously ~ h o w n ~ ' .that ' ~ ) the
TABLE3. SERUM MINERAL CONCENTRATIONS IN 16-DAY-OLD PUPS FROM PARATHYROIDECTOMIZED (PTX) AND SHAM-OPERATED MOTHERS^ Serum concentrations (rng/dl)
Group
P
Ca
Mg
Body weight (g)
PTX (10)
10.7 f 0.1
8.6
f
0.4
2.33
f
Sham (10)
10.8
8.7
f
0.2
2.29
* 0.06
f
0.1
0.08
40 f 0.9 42
f
2.0
aLactating rats were placed on diets containing 1.2% Ca and 0.4% P on day 2 of lactation and were subjected to PTX or a sham operation 4 days later. The values represent the mean f SEM for groups of 10 pups, 1 pup from each of 10 litters.
PARATHYROID-FREELACTATING RATS
TABLE4. SERUMCa AND P
AND
73
LITTERWEIGHTS FOR LACTATING RATSIN BONELoss EXPERIMENT^
Litter weight (g)
Body weight (g) Serum P (mg/dl)
Group and day of lactation
Serum Ca (mg/dI)
Intact, 5 day
11.0
f
0.3
2.17 f 0.25
309
PTX, 20 day
11.6
f
0.3
2.69
314 f 11
291
f
Sham, 20 day
11.5
f
0.3
2.31 f 0.15
309 f 9
290
f
f
0.21
Day 5 f
12
Day 20
-
Day 5
Day 16
119
f
9
-
7
122
&
3
382 f 4
6
117
f
5
354
f
12
aThe values represent the mean f SEM for groups of five to eight lactating rats for which bone weights are presented in Fig. 1. Serum Ca and P were determined on day 20 of lactation. See Fig. 1 for experimental details.
Bone loss in PTX lactating rats 700 'I
550 500 450 400
350 0
NL
L
5
L PTX
L SHAM
20
Days of lactation FIG. 1. The effect of parathyroidectomy (PTX) on bone loss during lactation. Rats were parathyroidectomized after 5 days of lactation, having been fed a diet with 1.2% Ca and 0.4% P from 2 days after parturition. Nonlactating (NL) rats received the 1.2% Ca and 0.4% P diet for 3 weeks. The height of the bars and the vertical lines represent the mean value f SEM of defatted dry femora from each group. The number of rats per group is indicated on each bar. Femur ash weights (mg) were as follows: 364 f 18 (5 day), 287 f 6 (PTX), and 283 f 8 (sham). *p < 0.001 for 20 day lactating rats (PTX or sham) versus 5 day lactating rats (Schefffs contrast).
lactating rat is far more vulnerable to the loss of PTH and 1,25-dihydroxyvitamin D, [ 1,25-(OH),D3] than nonlactating rats. Under these circumstances the influx of Ca and P from the intestine has a much greater effect on the serum Ca and P concentrations in the lactating rat than in the nonlactating rat. A surprising finding was that normal milk production (based on litter weights) did not require maintenance of normal serum P levels as long as normal serum Ca levels were maintained. In rats given a diet with 1.2% Ca and 0.4% P, serum P levels were close to 2 mg/dl or only 3&40% of that of parathyroid-intact lactating rats maintained on our standard diet containing 0.4% Ca and 0.4% P. In spite of such hypophosphatemia litter weights were normal, suggesting a normal rate of milk production. Thus, galactopoiesis is clearly far more sensitive to changes in circulating Ca than in circulating P levels. The milk composition data are fully consistent with the litter weight data because no abnormalities were detected in the content of total solids, Ca, or P in PTX rats. In previous studies milk from hypocalcemic PTX rats had an increased total solids c ~ n t e n t . ' ~It, ~is' clear from the present data that the absence of parathyroid hormone does not affect the total solids content (or rather the water content) or the milk Ca content when a normal serum Ca level is maintained. The present data are therefore consistent with the conclusions from the earlier that it is the serum Ca level that determines the water content of the milk, not the presence or absence of the parathyroid glands per se. With regard to the milk P data, it is interesting to note that not only is the milk P content unaffected by the parathyroid status, but it also appears to be independent of the serum P level; the milk P content was the same despite a twofold difference in the serum P level. Mueller and showed that wide variations in dietary Ca and P contents have little influence on milk composition or milk production. However, serum Ca and P concentrations were not reported in this study. The most interesting observation of this study was the bone loss during lactation in the PTX rats, which was just as extensive as in the sham-operated rats. It has generally
GARNER ET AL.
14
been assumed that 1,25-(OH)2D, and parathyroid hormone are the main mediators of bone loss during lactation. However, Halloran and DeLuca('8) and Brommage and DeLuca(I9)have demonstrated clearly that vitamin D-deficient lactating rats [with undetectable serum 1,25-(OH),D3] lose just as much bone as do vitamin D-replete lactating rats, showing that 1,25-(OH),D3 is not essential for bone resorption. Brommage and D e L ~ c a " ~also ) reasoned that parathyroid hormone was probably not involved either, because they observed normal bone loss in lactating rats fed a high-calcium diet, which resulted in a mean serum Ca level of approximately 12 mg/dl, a level assumed to have suppressed PTH secretion. However, we have recently shown that circulating iPTH levels are not suppressed by hypercalcemia during lactation.(z0)This finding leads us to disagree with their reasoning, but in view of our present results we concur in their conclusion that PTH is not involved in normal lactational bone loss. If these major bone-resorbing hormones are not involved in the normal bone loss in lactation, what is the mechanism for the net increase in bone resorption during this period? Brommage and D e L ~ c a " ~observed ) normal lactational bone loss in ovariectomized rats as well as in adrenalectomized rats and could therefore rule out ovarian and adrenal hormones as the humoral signal to the bone cells. They were unable to prevent the bone loss by raising the Ca and P contents of the diets. However, we recently showed that feeding our basic diet with Ca and P increased to 1.6 and 1.4%. respectively, and providing drinking water with 0.5% Ca could eliminate the bone loss completely.(21)This observation shows that the bone loss is not an obligatory consequence of lactation, but it does not give us a clue to the identity of the stimulator of bone resorption when more phsyiologic Ca and P intakes are provided. A plausible, but still hypothetical, candidate is the PTHlike peptide (PTH-LP), which was first suggested by Brommage and D e L ~ c a " ~as) the bone-resorbing factor in lactation. The mRNA for PTH-LP is expressed only in actively secreting mammary glands, and an extract of mammary tissue has been shown to stimulate CAMPproduction by PTH-sensitive osteosarcoma cells. Thus, the peptide can exert a PTH-like effect on bone cells, but a direct connection between this factor and bone loss in lactation is not yet available. Although the PTH-LP has been described as a hypercalcemic factor (causing hypercalcemia of human malignancies), based on the present data the peptide is not able to prevent severe hypocalcemia in PTX lactating rats. This suggests that secretion of PTH-LP is not stimulated by hypocalcemia, as is that of PTH. However, our previous observation that bone loss can be prevented by manipulation of dietary Ca and PC2')is consistent with suppression of either the secretion or the effect of the peptide. Alternatively, one could suggest that the PTH-LP is produced by lactating mammary tissue but has no important function as an antihypocalcemic or bone-resorbing factor. Clearly, the physiologic function, if any, of PTH-LP in the lactating rat remains to be elucidated.
In summary, a dietary Ca/P ratio has been identified that will permit rats to maintain normocalcemia after parathyroidectomy during lactation. Under this regimen the rats produce a normal amount of milk with a normal composition (total solids, calcium, and phosphorus) despite severe hypophosphatemia. The parathyroidectomized lactating rats lose a normal amount of bone during weeks 2 and 3 of lactation. We conclude that parathyroid hormone is not directly essential for normal milk secretion or for maintaining lactation-associated bone loss. Its primary physiologic function in lactation, therefore, appears to be the same as in the nonlactating animal, namely, to prevent hypocalcemia.
ACKNOWLEDGMENTS The authors express their appreciation to Ms. Catherine Lane for technical assistance, to Ms. Gale Thompson for preparing the manuscript, and to Mr. Stefan Gottschalk for producing the figure. This work was supported in part by NIH grant HD12496.
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Address reprint requests to: Dr. S.U. Toverud Dental Research Center, CB# 7455 University of North Carolina Chapel Hi", NC 27599 Received for publication April 3, 1989; in revised form July 3, 1989;accepted July 5, 1989.
