Agna Boass

Soy Isoflavones: No Effects on Bone Mineral Content and Bone Mineral Density in Healthy, Menstruating Young Adult Women after One Year

Background: The effects of isoflavone-enriched soy protein on human bone mineral content (mass) and density in healthy, menstruating young adult females have not been examined in a comparative prospective investigation. Peri- and post-menopausal women have been reported to show beneficial effects of isoflavones on bone measurements. Therefore, young women may also be able to improve their accrual of peak bone mineral content (BMC) and bone mineral density (BMD) during the early adult years of bone consolidation with an isoflavone-enriched diet. Objectives: In this controlled, double-blind intervention, we tested the hypothesis that an isoflavone-rich soy protein diet increases BMC and BMD in young adult females over a period of one year in comparison to a control group receiving soy protein that has isoflavones removed. Design: Young healthy women of any ethnic background, 21 to 25 years of age, were divided into two groups, placebo (n = 13) and supplement (n = 15). The soy protein supplement was enriched with isoflavones (∼90 mg of total isoflavones/day), whereas the control protein diet was isoflavone-deficient, even though it contained the same amount of soy protein and other ingredients as the isoflavone-rich diet. Dual-energy x-ray absorptiometric (DXA) measurements of BMC and BMD were made at baseline and at 6 and 12 months. DXA estimates of body composition, including fat mass and lean body mass, were generated from whole-body BMC measurements. BMI was calculated as weight (kg) over height (m) squared. Physical activity was assessed, and three-day dietary records were taken at entry (baseline) and at 6 and 12 months. Results: No changes in BMD after 12 months were found in either the isoflavone-treated (treatment) group or the isoflavone-deficient (control) group. Other variables also remained essentially constant over the 12-month period, including normal menstrual patterns in both the treatment and control groups. Conclusions: The isoflavone-rich soy preparation had no effects on BMC and BMD over a 12-month period in young healthy adult females with normal menses. An isoflavone-rich supplement appears to have little or no effect on bone in young adult women with normal ovarian function, at least over this 12-month study period.

Hormonal control of calcium metabolism in lactation.

Publisher Summary This chapter discusses the hormonal control of calcium metabolism in lactation. In lactating animals that secrete relatively large amounts of calcium in the milk, a number of adaptive changes in calcium metabolism occur. The efficiency of calcium absorption from the intestine increases twofold or more, and, in the rat, absorption may remain elevated for some time after weaning if dietary calcium intake is limited so that skeletal calcium stores are mobilized to meet the demands of milk secretion. Vitamin D and parathyroid hormone act in concert to mediate these changes. Vitamin D through its conversion to 1,25-(OH) 2 D 3 stimulates calcium absorption, and both parathyroid hormone and the vitamin D metabolite enhance skeletal calcium mobilization. Both hormones are also closely related to the serum calcium level. An elevated blood level of parathyroid hormone, in lactating cows, is stimulated and maintained by a low serum calcium level, but may be required to prevent an excessive drop in serum calcium in response to the calcium drain into the milk. High circulating levels of calcitonin are observed in lactating rats and sheep and in some lactating women. The urinary excretion of calcium is reduced during lactation in several species.

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.

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.

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 of jejunal 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.

Effects of vitamin D and parathyroid hormone on cyclic AMP production by bone cells isolated from rat calvariae

SummaryStudies presented here were designed to investigate further the basis for an impaired cAMP response to parathyroid hormone (PTH) in osteoblastlike calvarial bone cells isolated from vitamin D-deficient rat pups. The goal was to perturb Ca, PTH, and vitamin Din vivo in order to see which factors might be responsible for the impairedin vitro bone cell cAMP response. Pups either were parathyroidectomized (PTX) 3–5 days, implanted with osmotic minipumps delivering high doses of PTH, given repeated, high doses of 1,25(OH)2D3, or were D-deficient (-D, i.e., born and suckled by D-deficient mothers). Osteoblastlike bone cells, isolated by sequential enzyme digestion and centrifugation, were exposed to PTH for 5 min in the presence of a phosphodiesterase inhibitor. In bone cells isolated from -D rat pups, both basal and PTH-induced cAMP accumulation were significantly lower than in +D bone cells. Earlier, we had shown that two daily injections of -D pups with 50 ng 1,25(OH)2D3 restores this reduced bone cAMP response of -D pups toward normal. In the present study, neither basal nor PTH-induced bone cell cAMP accumulation was affected by subjecting D-replete pups to PTX, PTH infusion, or repeated high doses of 1,25(OH)2D3 despite the fact that each treatment markedly changed serum Ca or serum immunoreactive PTH. The results indicate that the impaired bone cell cAMP response seen in -D pups is not a direct result of chronic hypocalcemia and that the “heterologous desensitization” seenin vitro with added 1,25(OH)2D3 could not be duplicated byin vivo treatment of +D pups with supraphysiologic doses of 1,25(OH)2D3. Finally the lack of alteration in the bone cell cAMP response to PTHin vitro after chronic PTH infusionin vivo fails to support the notion that the impaired response in -D bone cells can be explained entirely by “homologous desensitization” induced by high circulating levels of PTH in the hypocalcemic, -D rat pup.

Circulating levels and function of 1,25-(OH)2D3 in lactation

During lactation in the rat, vitamin D is required for maintenance of a normal serum calcium level and maximal enhancement of active calcium transport in the duodenum. Vitamin D does not appear to be required for part of the adaptive increase in intestinal calcium transport or for calcium transport into the milk. The functions of vitamin D appear to be mediated by 1,25-(OH)2D3, the circulating level of which increases during lactation. Two days after sudden weaning, the serum level of 1,25-(OH)2D3 falls to levels below the pre-pregnant control level in parallel with a sharp increase in serum calcium; normal levels of calcium and 1,25-(OH)2D3 are observed one week after weaning. The initial stimulus for the increase in circulating 1,25-(OH)2D3 during lactation appears to be a small decrease in serum calcium which stimulates parathyroid hormone secretion, which in turn enhances synthesis of 1,25-(OH)2D3. Vitamin D is also required by the lactating rat to insure normal development of the suckling pup, since vitamin D deprivation during pregnancy and lactation causes significant decreases in body weight gain, in serum levels of 25-OHD3, calcium and phosphorus, in bone ash content and clear evidence of histological rickets by 20 days of age.

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.

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.