Transient estrogen exposure of female mice during early development permanently affects osteoclastogenesis in adulthood.

Estrogens modulate bone tissue turnover in both experimental animal models and postmenopausal women. Our previous studies have shown that exposure to diethylstilbestrol (DES) during the perinatal period increases peak bone mass in female mice in adulthood. We investigated whether developmental DES exposure can influence bone mass by affecting osteoclastogenesis. Female mice were injected with 100 microg/kg body weight DES from days 9-16 of gestation or, alternatively, pups received neonatal injections of 2 microg of DES from days 1-5 of life. Animals were weaned at 21 days of age and effects of estrogen on bone cells were evaluated in adulthood. A significant increase in bone mass in female mice was already observed at 2 months, with a maximal effect in older animals. Bone sections from DES-treated animals showed a significant decrease in osteoclast number and tartrate-resistant acid phosphatase (TRAP) enzymatic activity as compared with controls. To verify the importance of the estrogen surge at puberty in this event, a group of control and DES-treated mice were ovariectomized at 17 days to prevent puberty, and potential effect on osteoclastic cells was evaluated in adulthood. As expected, ovariectomy induced an increase of TRAP-positive cells. DES treatment blunted the ovariectomized-dependent increase of the total number of osteoclastic cells, suggesting a role of developmental DES exposure in the process of bone-cell imprinting. Our data indicate, for the first time, that transient changes in estrogen levels during development modulate bone turnover and osteoclastogenesis likely participating in bone-cell imprinting during early phases of bone development, and that this effect could be induced by direct alteration of bone microenvironment.

Enhanced nonsaturable calcium transport in the jejunum of rats during lactation, but not during pregnancy.

The lactating (L) rat loses in excess of 100 mg of calcium (Ca) per day to milk at peak lactation. Most of the Ca must be provided by increased intestinal absorption. In an effort to examine adaptation of intestinal calcium absorption during lactation, nonsaturable absorption from the small intestine of rats was calculated from the disappearance of Ca from in situ ligated loops of jejunum during the last week of pregnancy and throughout lactation and weaning. Efficiency of absorption is reflected by the slope of the regression line of Ca absorbed on Ca introduced into the loop. Absorption of Ca in the jejunum was markedly enhanced starting at 5 days of lactation and for the remainder of lactation. Two days after weaning, the efficiency ofjejunal Ca absorption decreased to the nonmated (NM) control level, while the lactation-associated intestinal hypertrophy persisted beyond 2 days postweaning. The percentages of water and Ca absorbed were positively and significantly correlated in both L and NM rats. In contrast to Ca, magnesium (Mg) and strontium (Sr) transport from ligated loops were not enhanced during lactation. Fifty millimolar glucose in the test solution increased the absorption of both water and Ca, but not Mg, from jejunal loops of NM rats. Glucose increased Ca absorption in NM rats up to the level seen in L rats. Glucose did not increase Ca absorption further in L rats, perhaps because of the greater availability of glucose to the intestine during lactation. We conclude that in rats the efficiency of nonsaturable Ca absorption from the jejunum is significantly increased during well established lactation, but not during pregnancy. The underlying mechanism appears to be specific for Ca, may be dependent on glucose, and is unrelated to intestinal hypertrophy.

Altered regulation of parathyroid hormone secretion by calcium in pregnant and lactating rats.

We have previously shown that the serum parathyroid hormone (PTH) concentration in lactating (L) rats is not suppressed by high serum Ca2+ to the same extent as in nonmated (NM) rats. To investigate further Ca2+ regulation of PTH secretion, parathyroid cells from NM rats and rats in late pregnancy and at peak lactation were dispersed and incubated for 2 h in medium containing 0.52-2.05 mM Ca2+. Medium PTH was assayed with a homologous immunoradiometric assay (IRMA). At the two highest Ca2+ levels (1.81 and 2.05 mM), medium PTH was significantly higher (p = 0.031) for cells from L rats than for cells from NM rats. In contrast, significantly less (p < 0.001) PTH was secreted for the L group versus the NM group at medium Ca2+ values of 1.27 and 1.46 mM. Estimated set points for L and NM groups were 1.17 mM and 1.35 mM, respectively, corresponding closely to the prevailing serum Ca2+ for these two groups. Consistent with the present in vitro data, high serum PTH (> 40 pg/ml) in L rats occurred only at serum Ca2+ values below 1.27 mM. Elevated serum PTH at lower serum Ca2+ levels was also seen in pregnant rats. Dispersed parathyroid cells from 20- to 21-day pregnant rats secreted significantly more PTH (p = 0.028) than cells from NM rats at all Ca2+ levels tested (1.1-1.6 mM). In conclusion, the relationship between extracellular Ca2+ and PTH secretion is altered in rats during late pregnancy and at peak lactation, perhaps as part of the adaptation to the demands for calcium for pre- and postnatal growth.

Regulation of serum calcitriol by serum ionized calcium in rats during pregnancy and lactation.

Serum calcitriol concentrations in rats follow a biphasic pattern during reproduction, with elevated levels during late pregnancy, a decline after parturition, and a rise to even higher levels during peak lactation. We have previously shown that serum calcitriol in rats at peak lactation correlates significantly with, and appears to be regulated by, serum ionized Ca (Ca2+), with parathyroid hormone (PTH) serving a permissive role. We have extended this study by determining if serum calcitriol also correlates with serum Ca2+ during late pregnancy, when calcitriol levels are clearly elevated, and during early lactation, when only modest increases in serum calcitriol are observed. Analyses of data combined from nonmated, 21-day pregnant (P), and 1-day lactating rats (L) revealed a significant regression (p < 0.001) of calcitriol on Ca2+, but a nonsignificant regression (p = 0.34) of calcitriol on serum PTH. An even stronger correlation (p < 0.001) between calcitriol and Ca2+ was found for the combined data for 5-, 8-, and 14-day L rats. The partial correlation coefficient for calcitriol versus Ca2+, with PTH as the independent variable, was highly significant (p < 0.01) for the data from both combined groups. However, the coefficient for calcitriol versus PTH, with Ca2+ as the independent variable, was not significant (p > 0.05). Fetal weights (uterus and contents) correlated significantly with both maternal calcitriol and Ca2+ concentrations (p < 0.01), but not with maternal PTH levels. Litter weights for 14-day-old pups likewise correlated significantly with maternal calcitriol and Ca2+ (p < 0.001). We conclude that hypocalcemia, induced by the demands for Ca for fetal calcification and milk production, appears to be a controlling factor in serum calcitriol elevation in late pregnancy and throughout lactation, whereas PTH may be important for calcitriol synthesis without playing a direct regulatory role.

Duodenal active calcium transport in female rats increases with serum calcitriol concentrations, but reaches a plateau far below maximal calcitriol levels.

To determine the relationship between serum calcitriol concentration and duodenal active calcium (Ca) transport, a wide range of circulating calcitriol concentrations (18-950 pg/ml) was obtained by feeding nonmated, lactating, and weaned rats vitamin D-sufficient diets containing 0.04, 0.06, 0.1, or 0.4% Ca. Ca transport was measured in vitro with the everted gut sac technique using both the proximal (D-1) and distal (D-2) duodenal halves. The ratio of serosal [Ca]/mucosal [Ca] (S/M) as well as the amount of Ca transported was calculated. The S/M ratio correlated with the serum calcitriol concentration over the range 18-90 pg/ml with slopes for the regression lines of 0.066 +/- 0.010 (R2 = 0.64, n = 27) for D-1 and of 0.036 +/- 0.005 (R2 = 0.73, n = 24) for D-2. The regression lines are significantly different from zero (p < 0.001) and from each other (p < 0.01). For D-1, a plateau of the S/M ratio of 7-9 appeared to be reached at a calcitriol concentration of approximately 90 pg/ml, and the plateau was maintained over the range 90-900 pg/ml calcitriol. For D-2, a plateau of the S/M ratio of 4-6 appeared to be reached at 200-300 pg/ml calcitriol. Calculating the amount of Ca transported per 10 cm per hour revealed a pattern similar to that of the S/M ratio. When net Ca absorption was determined from balance studies over 4 days for rats on a 0.04% Ca diet, maximal absorption (mg/day) was already observed at a serum calcitriol concentration of 60-70 pg/ml (n = 14). We conclude that active Ca transport correlates with serum calcitriol concentrations, but that the transport capacity quickly reaches a maximal value, which is maintained over a 10-fold higher range of serum calcitriol concentrations.

Suppression of circulating calcitriol and duodenal active Ca transport by ketoconazole in pregnant rats.

Active Ca transport in the duodenum and the circulating level of calcitriol are elevated during pregnancy and lactation in the rat. Because calcitriol stimulates Ca transport in nonmated rats, we investigated its contribution to the increased transport during pregnancy and lactation. Rapid suppression of calcitriol from 28 +/- 3 to 8 +/- 0.4 pg/ml with the steroid hydroxylase inhibitor ketoconazole resulted in a 34% suppression of Ca transport in nonmated rats. At the end of pregnancy, when calcitriol concentration was suppressed from 64 +/- 7 to 12 +/- 2 pg/ml, the transport ratio decreased by 44%. Ca transport did not correlate with calcitriol levels between 40 and 80 pg/ml, suggesting a threshold level for maximal Ca transport stimulation. During lactation at even higher calcitriol levels, ketoconazole treatment again resulted in marked reduction in calcitriol from 124 +/- 1 to 71 +/- 12 pg/ml, but without any concurrent reduction in Ca transport in the duodenum. We conclude that in the vitamin D-replete rat the pregnancy-mediated, and probably also the lactation-mediated, increase in active Ca transport capacity is dependent on an increase in circulating calcitriol up to a certain threshold level.

Several anesthetics, but not diethyl ether, cause marked elevation of serum parathyroid hormone concentration in rats.

The effects of anesthetics on serum parathyroid hormone (PTH) concentrations were determined by a new homologous two-site immunoradiometric assay for rat PTH. Serum PTH concentrations (mean +/- SE) from ether-anesthetized rats (14.7 +/- 1.5 pg/ml, n = 22) were not significantly different from those of decapitated unanesthetized female rats (13.0 +/- 1.8 pg/ml, n = 21). Serum PTH concentrations in pg/ml (n = 4-14) for other anesthetics tested were: ketamine, 12.5 +/- 1.1; Na pentobarbital, 23.3 +/- 2.4; methoxyflurane (inhalation), 42.2 +/- 6.8; and xylazine combined with ketamine, 51.4 +/- 11.3 pg/ml. The latter two concentrations were significantly (p < 0.001) higher than the values for all other anesthetics and decapitation. Elevation of serum PTH induced by pentobarbital or ketamine + xylazine increased with time under anesthesia. Neither serum Ca2+ concentrations nor pH differed among any of the groups. We conclude that anesthesia induced by pentobarbital, methoxyflurane, or ketamine + xylazine in rats leads to a marked elevation of serum PTH levels that appears to be related to the duration of anesthesia and not due to any measurable fall in serum Ca2+.

Determination of bioactive rat parathyroid hormone (PTH) concentrations in vivo and in vitro by a 2-site homologous immunoradiometric assay.

A new homologous 2-site assay for rat parathyroid hormone (IRMA), developed by Immutopics, Inc., has been evaluated and compared with a bone cell cAMP bioassay. Circulating PTH for adult rats assayed with this IRMA are in the range 10-15 pg/ml, and of the same order of magnitude as published values for biologically active PTH. The standard curve for the IRMA was linear over the range 3.4-240 pg/ml of rPTH 1-34, and serum samples diluted in parallel with the standard curve. The within-assay and between-assay coefficients of variation ranged from 5.2% (n = 18) to 7.6% (n = 24) and 8.3% (n = 16) to 26.4% (n = 10), respectively. Serum PTH values (mean +/- S.E.) for parathyroidectomized rats were 3.5 +/- 0.6 pg/ml (n = 18) versus 10.3 +/- 1.4 pg/ml (n = 16) for intact non-mated rats. Calcium injections suppressed circulating PTH by 50%. Lactating rats had serum PTH levels 5-fold higher and vitamin D deficient rats 60-fold higher than non-mated controls. PTH secreted from parathyroid cells in vitro was in the range 60-490 pg/ml as determined by the IRMA. These values represented 86.0 +/- 9.0% of the comparable bioassay values, indicating that the IRMA detects only bioactive PTH.

Parathyroidectomy abolishes the increase of renal 25-hydroxyvitamin D-1 alpha-hydroxylase in lactating rats.

Serum ionized calcium (Ca), but not inorganic phosphorus or immunoreactive parathyroid hormone, negatively correlates with renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) and serum 1,25-dihydroxyvitamin D in intact lactating rats. The present study tested the hypothesis that the presumed stimulation of renal 1 alpha-hydroxylase by hypocalcemia requires the presence of intact parathyroid glands. Lactating and nonlactating rats were surgically parathyroidectomized (PTX) or sham-operated (sham) at 9-10 days of lactation. Later (24 h) the rats were bled, nephrectomized, and killed. In lactating PTX rats, serum ionized Ca decreased to 50% of the level of sham rats, and serum 1,25-dihydroxyvitamin D fell to 37 +/- 5.0 pg/ml compared with 82 +/- 13.0 pg/ml for sham lactating rats but was still 2.5 times the value for nonlactating PTX rats (15 +/- 0.8 pg/ml). In contrast to the still elevated serum 1,25-dihydroxyvitamin D concentration in lactating PTX rats, renal 1 alpha-hydroxylase was suppressed to the same low level as in nonlactating PTX rats, suggesting the existence of extrarenal synthesis of 1,25-dihydroxyvitamin D in lactation. A curvilinear relationship was revealed between serum ionized Ca and renal 1 alpha-hydroxylase in sham lactating and nonlactating rats (r2 = 0.71, P < 0.0001). However, in PTX rats, decreasing ionized Ca did not lead to any increase in 1 alpha-hydroxylase above the low baseline values seen at ionized Ca concentrations between 1.3 and 1.5 mM. We therefore conclude that intact parathyroid glands are required for hypocalcemia to activate renal 1 alpha-hydroxylase in female rats.

Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs.

The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.

Pregnancy- and lactation-induced changes in active intestinal calcium transport in rats.

A time course study of active Ca transport in the duodenum and the terminal ileum was conducted using the everted gut sac technique during the last week of pregnancy and throughout lactation. A triphasic pattern was revealed in the proximal duodenum: a marked rise between 18 and 20 days of pregnancy, a plateau maintained during the last 3 days of pregnancy and the first 2-3 days of lactation, and a fall by day 4 of lactation. The late-pregnancy rise was significant also when expressed as milligrams Ca transported relative to tissue weight, indicating that intestinal hypertrophy was not the cause of the increase. The ratio of serosal to mucosal Ca concentration remained low until approximately day 11 of lactation, when it rose toward a new peak. There was no active Ca transport in the ileum until the third week of lactation. Serum prolactin levels increased 10-fold between 18 and 20 days of pregnancy and remained high until at least day 7 of lactation, but did not correlate significantly with duodenal Ca transport. Injected rat prolactin did not result in a precocious rise in Ca transport in pregnant rats. The fluctuations in duodenal Ca transport during lactation were reflected by a small, but statistically significant, decrease in net fractional Ca absorption at 6-9 days compared with either 2-4 days or 13-16 days. We suggest that duodenal active Ca transport plays only a small role in total intestinal Ca absorption in the lactating rat except when dietary Ca is greatly restricted.

Intensity of lactation modulates renal 1 alpha-hydroxylase and serum 1,25(OH)2D in rats.

Renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity and serum 1,25-dihydroxyvitamin D [1,25(OH)2D] concentration were measured in lactating rats suckling litters of 3, 6, or 12 pups to determine the effect of increasing lactational intensity on the biosynthesis of 1,25(OH)2D. Serum Ca2+, total Ca, Pi, and immunoreactive parathyroid hormone were also determined. The average daily litter weight gain for each litter size was calculated from the gain over the last 4-6 days of each of three experiments and was used as an index of lactational intensity. Highly significant correlation coefficients were found between 1 alpha-hydroxylase and average daily litter weight gain (rs = 0.63, n = 53, P less than 0.001), serum 1,25(OH)2D and average daily litter weight gain (rs = 0.62, n = 50, P less than 0.001), 1 alpha-hydroxylase and serum total Ca (rs = -0.52, n = 53, P less than 0.001), and average daily litter weight gain and total Ca (rs = -0.52, n = 53, P less than 0.001). Neither serum phosphorus nor immunoreactive parathyroid hormone correlated significantly with 1 alpha-hydroxylase. In addition, construction of regression models using a stepwise forward variable selection procedure revealed serum total Ca concentration to be a significant predictor for both serum 1,25(OH)2D and renal 1 alpha-hydroxylase in lactating rats. These data support the hypothesis that increasing lactational intensity leads to decreasing serum Ca concentration, resulting in stimulation of 1 alpha-hydroxylase activity and a rise in the serum 1,25(OH)2D level.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of serum 1,25-dihydroxyvitamin D3 in lactating rats.

To characterize further the mechanism(s) underlying the increased serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentration associated with lactation in the rat, we examined hormone biosynthesis [i.e., renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity] and hormone disappearance in groups of lactating Holtzman rats and age- and sex-matched nonlactating controls. 1 alpha-Hydroxylase activity was significantly greater in kidneys from lactating rats (4.0 +/- 0.42 fmol.mg-1.min-1) on a basal diet than in those from nonmated females (1.4 +/- 0.08 fmol.mg-1.min-1), an increment sufficient to account for the observed fourfold elevation of 1,25(OH)2D3 in the dams. The increase occurs despite the lower serum 1,25(OH)2D3 levels in lactating than in nonlactating rats at 12 and 24 h after a bolus injection of 1,25(OH)2D3 (2 ng/g body wt). Elevation of serum 1,25(OH)2D3 is not a requisite consequence of lactation, however, because dams receiving supplemental calcium from food (1.6%) and water (0.3%) exhibited no increase of either serum 1,25(OH)2D3 or 1 alpha-hydroxylase activity compared with controls. In contrast, lactating rats that received a diet with only 0.1% calcium had 5-fold higher serum 1,25(OH)2D3 levels and 20-fold higher 1 alpha-hydroxylase activity than nonlactating rats on the same diet. We conclude that other factors in conjunction with lactation, but not the lactating state per se, promote the changes in 1,25(OH)2D3 metabolism observed.

The ovariectomized, lactating rat as an experimental model for osteopenia: calcium metabolism and bone changes.

The ovariectomized, lactating rat (Sprague-Dawley) is proposed as an experimental model for the rapid development of osteopenia which may be used to test the effectiveness of bone-retentive drugs potentially useful in treating osteoporotic women. Rats were ovariectomized (OVX) on day 2 postpartum and were kept on a low-calcium diet (0.1%). Measurements of serum total calcium, ionic calcium, albumin and parathyroid hormone were conducted between days 4 and 21 of lactation. Serum total and ionic calcium and albumin were significantly lower and serum parathyroid hormone was significantly higher in all lactating rats at 16 days postpartum compared to nonlactating controls. Mean bone mass of the femurs of OVX lactating rats measured at day 21 was approximately 50% of that of non-lactating intact controls. The enhanced duodenal calcium absorption (in everted gut sacs) associated with lactation was not affected by OVX and neither was the average litter weight gain between 2 and 14 days of lactation. In conclusion, lactation coupled with a low-calcium diet resulted in marked osteopenia, depressed serum calcium (both total and ionic) and significantly elevated serum parathyroid hormone concentration. The rapid and extensive bone loss of this model makes it appropriate for the study of therapeutic agents designed to retain bone mass.

Parathyroid hormone is not required for normal milk composition or secretion or lactation-associated bone loss in normocalcemic rats.

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 calcium-phosphorus 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 +/- 0.2 mg/dl (mean +/- SEM) for PTX rats and 10.4 +/- 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 +/- 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypercalcemia fails to suppress elevated serum parathyroid hormone concentrations during lactation in rats.

We have previously reported that increased serum immunoreactive parathyroid hormone (iPTH) in the lactating (L) rat is generally accompanied by hypocalcemia when diets containing 0.4% calcium (Ca) or less are fed. However, instances were also observed in which elevated iPTH levels did not coincide with a hypocalcemic signal. To test the hypothesis that iPTH levels can remain elevated even in the presence of hypercalcemia in lactation, a diet containing 1.2% Ca and 0.4% phosphorus (P) was fed to lactating rats in three experiments (A, B, and C) to achieve serum ionized calcium (ICa) levels approximately 10% above levels for nonmated (NM) controls. The serum ICa of NM controls fed the 1.2% Ca diet was slightly, but significantly, elevated, and serum iPTH (determined by an N-terminal specific assay) was significantly suppressed compared with NM controls fed a 0.4% Ca diet. In experiment A, L rats fed a 1.2% Ca diet had 81% higher serum iPTH levels than NM controls fed the same diet in spite of a mean (+/- SEM) ICa level of 1.77 +/- 0.05 mM for L rats versus 1.46 +/- 0.01 mM for NM controls; NM controls fed a 0.4% Ca diet had serum ICa of 1.37 +/- 0.01 mM. This novel finding of significantly higher iPTH and ICa in L compared with NM rats fed a 1.2% Ca and 0.4% P diet was confirmed in experiment B with eight rats in each group of L or NM rats fed either the 1.2% or the 0.4% Ca diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural localization of tartrate-resistant, purple acid phosphatase in rat osteoclasts by histochemistry and immunocytochemistry.

The intracellular localization of the tartrate-resistant purple acid phosphatase in osteoclasts of developing rat bone has been determined immunocytochemically using an antiserum to the purified bone-derived purple acid phosphatase. The localization of the immunoreactivity was compared with the results of enzyme histochemistry using p-nitrophenylphosphate as substrate and 10 mM tartrate. Both methods revealed the presence of the enzyme in numerous vesicles of various sizes up to 2-3 microns in diameter and in granules. There was no immunoreactivity in the Golgi apparatus, and tartrate completely inhibited the histochemical activity of this organelle. No consistent extracellular activity could be detected, nor was any reaction product observed at the ruffled border. The localization of the tartrate-resistant purple acid phosphatase in osteoclasts is consistent with an intracellular function for this enzyme.

Adverse effects of a high-glucose diet on body weight and plasma calcium and 1,25-dihydroxyvitamin D3 levels in calcium-deficient growing rats.

We tested the hypothesis that dietary calcium would lead to greater impairment of body weight gain and calcium homeostasis if rats are fed a diet with a high glucose content compared with our standard diet in which the carbohydrate is supplied by whole wheat flour. Groups of female rats at 21 days of age were given either of two equivalent calcium-deficient diets with carbohydrate supplied either by glucose (LCaG) or by wheat flour (LCaW). Control rats were fed the wheat-flour diet containing 0.4% calcium. Since previous studies indicated divergent effects of glucose-based and flour-based diets on body weight in vitamin D-deficient rats, we designed a parallel study with vitamin D-deprived rats. Compared with rats fed the LCaW diet, the rats fed the LCaG diet had inferior body weight gain and more severe hypocalcemia (1-2 mg/ml lower) over a 40-day period, and no significant elevation of the plasma 1,25(OH)2D3 level at 61 days of age. Rats fed the LCaW diet maintained a 3-fold elevation of plasma 1,25(OH)2D3 relative to the level of control rats fed the 0.4% calcium diet. The dry weight and percent ash of tibias were similarly reduced in the two calcium-restricted groups compared to the control group. Among the vitamin D-deprived rats, those fed the glucose diet had poorer weight gain than those fed the wheat flour diet. However, both groups had similarly depressed serum calcium level, tibia ash content and 1,25(OH)2D3 level. Thus, a glucose diet combined with calcium restriction or vitamin D deprivation appears to accentuate the impairment of body weight gain and, when combined with calcium restriction, it also accentuates the impairment of calcium homeostasis and interferes with the adaptive increase in plasma 1,25(OH)2D3.

Modulation of serum parathyroid hormone and ionized calcium concentrations during reproduction in rats fed a low calcium diet.

Moderate dietary restriction of calcium (0.1% Ca) was used to accentuate the changes in serum immunoreactive parathyroid hormone (iPTH) that had been reported earlier in lactating rats fed 0.4% Ca diet. In addition, the effects of this low-Ca diet on serum total and ionized Ca and iPTH during pregnancy, extended lactation, and weaning were examined. The positive correlation between serum total and ionized Ca was highly significant (r = 0.88, p less than 0.001, n = 120). Serum iPTH was significantly higher (36%) in pregnant rats on the day of parturition compared to nonmated controls, and there was a concomitant decrease in both total and ionized serum Ca. Within 1 day after parturition, however, serum Ca had risen to the control level. Serum iPTH remained significantly elevated during the first 2 weeks of lactation, and increased further during the third week of lactation to a level more than twice that of nonlactating controls. Serum Ca fell gradually during the second week of lactation. The high serum iPTH levels were maintained for another 2 weeks when lactation was extended with foster litters. Within 6 hr of removal of the suckling pups on day 16 of lactation, maternal serum ionized and total Ca had risen and serum iPTH had fallen; all three parameters were at levels similar to those of nonmated controls by 24-48 hr after weaning. The data suggest that serum ionized Ca is a major factor contributing to the hyperparathyroid state during lactation in rats fed a low-Ca diet.