A. Pointillart

The Bioavailability of Dietary Calcium

This update focuses on the bioavailability of dietary calcium for humans. Fundamentals of calcium metabolism, intestinal absorption, urinary excretion and balance are recalled. Dietary factors, especially lactose and other milk components, influencing calcium bioavailability at intestinal and renal levels are reviewed. A critical examination of all the methods used for evaluating calcium bioavailability is made. This includes in vitro assays, classical and isotopic balances, urinary excretion, isotope labeling in the urine, plasma and bones, long term evaluation of bone mineralization and the use of biological bone markers. Importance and advantages of animal models are discussed. The state of the art in the comparative bioavailability of calcium in foods is detailed including a comparison of sources of calcium (dairy products and calcium salts) in human studies and in some animal studies, casein phosphopeptides, proteins, lactose and lactase and their relation with calcium bioavailability (in humans and rats). An update on the consumption of dairy products and bone mass is presented. Emphasis on peculiarities and advantages of calcium in milk and dairy products is given.

Down‐Regulation of Osteoclast Differentiation by Daidzein via Caspase 3

Phytoestrogens are plant‐derived compounds with estrogen‐like activity. Phytoestrogen‐rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17β‐estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10−8 M of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10−8 M of daidzein, 10−8 M of 17β‐estradiol for 9‐11 days. Multinucleated tartrate‐resistant acid phosphatase‐positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 ± 8% by daidzein and 52 ± 5% by estrogen (p < 0.01); these effects were reversed by 10−6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 ± 5% by daidzein and 42 ± 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3‐induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 ± 8% by daidzein and 50 ± 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase‐8 and caspase‐3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase‐3 cleavage was reversed by 10−8 M of ICI 182,780. Both compounds up‐regulated the expression of nuclear estrogen receptors ER‐α and ER‐β. Thus, daidzein, at the same concentration as 17β‐estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.

A low dose of daidzein acts as an ERβ‐selective agonist in trabecular osteoblasts of young female piglets

The role of estrogens and estrogen‐like molecules, including isoflavones, in regulating bone cell activities is essential in understanding the etiology and treatment of post‐menopausal osteoporosis. Although estrogen replacement (HRT) has been the main therapy used to prevent and treat osteoporosis, there are concerns about its safety. Isoflavones have attracted attention to their potential roles in osteoporosis prevention and treatment. We have compared the effects of the isoflavone daidzein (1 nM), which has no effect on tyrosine kinases, and 17β‐estradiol (1 nM) on the development and function of cultured osteoblasts isolated from long bones of young female piglets. Daidzein increased ALP activity, osteocalcin secretion, and mineralization, while E2 increased only ALP activity. The content of ERβ and osteoprotegerin secretion by control cells gradually increased during osteoblast differentiation, whereas the ERα and RANK‐L content decreased. Daidzein enhanced only the nuclear ERβ whereas estradiol increased both ERα and ERβ. Daidzein and estradiol increased osteoprotegerin and RANK‐L secretion. Daidzein had a more pronounced effect than did estradiol. Daidzein and estradiol increased the membrane content of RANK‐L and the nuclear content of runx2/Cbfa1. Daidzein enhanced the nuclear content of progesterone and vitamin D receptors but not as much as did estradiol. All the effects of daidzein were blocked by ICI 182,780. We conclude that a low concentration of daidzein may exert its anti‐resorptive action by increasing the activity of porcine mature osteoblasts via ERβ, by regulating runx2/Cbfa1 production, and by stimulating the secretion of key proteins involved in osteoclastogenesis, such as osteoprotegerin and RANK‐ligand.

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro

Abstract We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0.5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.

TGF-β and calcitriol

Vitamin D3 and transforming growth factor-beta (TGF-beta) are molecules from unrelated families that share identical actions on cell growth and differentiation. The active metabolite of vitamin D3, calcitriol (1alpha,25-dihydroxyvitamin D3), induces an inhibitory effect on the growth of various cell types, and the expression of different markers of cell differentiation. As the receptor of vitamin D3 is ubiquitous, these effects are widespread in the organism. TGF-beta is a growth factor produced by many cell types, and is a known inhibitor of the proliferation of epithelial cells. Because of the similarity in their actions, many studies have been aimed at defining some interactions between the two substances. The purpose of this article is to illustrate the nature of the interactions, and two examples are developed. In normal or transformed epithelial cells, it has been demonstrated that the inhibitory effect of calcitriol on cell growth could be related to an induction of TGF-beta synthesis, and of a paracrine/autocrine loop. In bone, where both compounds play a very important role on the mechanisms controlling bone formation and remodeling, the interplay is more complex, and even includes the receptors of the two substances. Interest in this topic is growing and will surely lead to the establishment of new links between those two compounds.

Gonadal Steroids and Bone Metabolism in Young Castrated Male Rats

Abstract. At 45 days of age, 40 male Wistar rats were castrated, then randomly divided into four groups, S.C. injected for 60 days after surgery either with 17β-estradiol (E) 10 μg/kg BW/48 hours, progesterone (P) 140 μg/kg BW/48 hours, dihydrotestosterone (D) 2 μg/kg BW/48 hours, E + P + D same doses, or solvent alone (CX). Ten other rats were sham-operated (SH) and used as controls. Animals were put in balance to determine Ca and phosphorus (Pi) intestinal apparent absorption (IA Ca, IA Pi) and urinary pyridinium crosslinks excretion. Plasma was collected for measurement of intact-parathyroid hormone (PTH), calcitonin (CT), insulin-like growth factor I (IGF-I), 1,25 dihydroxyvitamin D (1,25(OH)2D), Ca, and Pi. Orchidectomy induced marked seminal vesicles atrophy and increased plasma CT, PTH, and Ca concentrations. IA Ca was significantly higher in P rats, however, neither castration nor any other treatment had significant effects. Orchidectomy decreased femoral length, dry weight, and Ca content, whereas E or D given alone or together with P improved endochondral growth and enhanced femoral Ca content. Again, bone mineral density was lowered by orchidectomy and reestablished by both E and EPD, even above SH values, this effect being more important at the metaphyseal levels. Urinary pyridinium cross-links excretion and plasma osteocalcin concentrations were higher in the CX animals than in the controls. Although E and D given alone did reduce both biochemical turnover markers, they showed additive effect when given together (EPD). In conclusion, in the young castrated male rat, E was more efficient than D for preventing bone loss, the most important effect being induced by a combination of E + P + D.

Effects of vitamin D on calcium regulation in vitamin-D-deficient pigs given a phytate-phosphorus diet.

1. Vitamin-D-deficient pigs were fed on a phytate-phosphorus diet and treated with vitamin D3 (+D) to examine the time-course of adaptative changes in plasma minerals, vitamin D metabolites, parathyroid hormone (PTH) and calcium balance and intestinal Ca-binding protein (CaBP). 2. The 5-week vitamin D repletion (25 micrograms cholecalciferol/kg diet) regimen restored plasma Ca, P and alkaline phosphatase (EC 3.1.3.1) to normal, decreased PTH and markedly and rapidly increased plasma 25-hydroxycholecalciferol (25-OHD, sevenfold after 4 d) and 1,25-dihydroxycholecalciferol (1, 25(OH)2D3, 1.8-fold after 4 d). 3. CaBP concentrations were markedly elevated all along the digestive tract, especially in the distal regions. 4. Ca absorption and retention were enhanced (fourfold and sixfold respectively) by the +D diet. 5. The improved Ca absorption, coupled with increased CaBP and 1,25(OH)2D3 levels, suggest that vitamin D metabolism in phytate-P-fed pigs is sensitive to the depressed Ca availability due to phytate feeding. It also indicates that CaBP may play an important role in the adaptation of Ca absorption. 6. Persistent hypercalciuria indicates that mineral metabolism was still affected by the phytate nature of the dietary P in spite of the vitamin D treatment.

Influence of exogenous porcine growth hormone on vitamin D metabolism and calcium and phosphorus absorption in intact pigs

The influence of growth hormone (GH) on vitamin D metabolism and calcium and phosphorus absorption in vivo is not clear. We, therefore, measured calcium and phosphorus balance, plasma 1,25-dihydroxyvitamin D (1,25(OH)2D), and intestinal vitamin D-dependent calcium-binding protein (CaBP 9k) in intact growing pigs given exogenous GH. Six 10-week-old pigs were given two daily subcutaneous injections of 50 μg porcine GH/kg body weight for 2 months; six control pigs were given vehicle. They were all fed a diet containing 1.1% Ca, 0.6% P, and 1000 IU vitamin D3/kg. Apparent Ca and P absorption and retention were measured in a 10-day balance trial at the end of the 2 months. The plasma levels of Ca, P, 1,25(OH)2D, IGF-I, and GH were determined, and the duodenal and jejunal mucosal CaBP 9k content was measured at slaughter. The plasma Ca and P of GH-treated pigs were unchanged, but all aspects of mineral metabolism, including the plasma 1,25(OH)2D concentration (40%), Ca absorption and retention (70%), P absorption (33%) and retention (45%), and jejunal CaBP 9k (40%), were stimulated, in addition to an increase in the circulating IGF-I concentration.

Cell stage-dependent effects of ascorbic acid on cultured porcine bone cells.

Pig bone cells were isolated from fetuses or young animals. In culture, they proliferated for 6 days, became confluent and began differentiating. The effects of ascorbic acid (AsA) on cell proliferation, alkaline phosphatase (ALP) activity, non-collagenous protein (NCP) and collagen synthesis, were studied by adding AsA to the medium at different times during culture. AsA affected fetal and post-natal cells similarly: ALP activity, NCP and collagen synthesis were markedly reduced in cells treated before confluence, but were strongly and dose-dependently stimulated in cells treated after confluence. AsA also stimulated cell proliferation. The cell stage-dependent action of AsA suggests that it may interfere with differentiation. The effects of AsA on ALP activity and DNA content were not coupled to its effect on collagen synthesis, raising the question of whether AsA action is matrix-mediated.

In Vivo Bone Metabolism and Ex Vivo Bone Marrow Osteoprogenitors in Vitamin D-Deprived Pigs

Vitamin D insufficiency is still a concern in countries where there is no routine food supplementation, such as France. A low vitamin D status is clearly associated with an increased risk of fracture in the elderly, but the long-term consequences of latent vitamin D insufficiency in young people and adults are not known. We fed 26 growing pigs a high calcium diet (1.1%) with a 1000 IU cholecalciferol/kg diet (controls), or without vitamin D (0D) for 4 months. We then analyzed the overall impact of low vitamin D status on osteotropic hormones (calcitriol and immunoreactive parathyroid hormone), plasma markers of bone remodeling (alkaline phosphatase [ALP] activity, carboxyterminal propeptide of type I procollagen [PICP], osteocalcin, hydroxyproline), whole bone parameters (ash content, bending moment), histomorphometry, and the populations of marrow osteoblastic and osteoclastic precursors by ex vivo cultures. The fall in plasma 25-dihydroxyvitamin [25(OH)D] in the 0D pigs indicated severe depletion of their vitamin D stores. However, they remained normocalcemic, were mildly hyperparathyroid after 2 months of vitamin D deprivation, and showed only a slight decrease in plasma calcitriol. The bone mineral content and bending moment of metatarsals decreased and they had increased osteoblastic (+59%, p < 0.05 0D vs. controls) and osteoclastic (+31%, p < 0.1 0D vs. controls) surfaces. This was not paralleled by increased bone turnover, because plasma hydroxyproline and ALP were unchanged and PICP and osteocalcin were decreased. The adherent fraction of bone marrow cells showed a great increase in the number of total stromal colony-forming units (CFU-F; +93%, p < 0.05 0D vs. controls) and in the percent of ALP(+) CFU-F (+58%, p < 0.01 0D vs. controls) in cultures from 0D pigs. More tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated cells were generated in cultures of nonadherent marrow cells from 0D pigs, and the area of resorption was 345% greater than in controls. Thus, vitamin D deprivation caused only moderate hormonal changes in growing pigs fed a high-calcium diet, but affected their bone characteristics and greatly enhanced the pool of osteoblasts and osteoclasts by stimulating the commitment of their precursors in bone marrow.