Dietary saturated fats do not significantly decrease intestinal calcium absorption. This explains the good bioavailability of the calcium from milk and dairy products, although these provide large amounts of long chain saturated fatty acids. Conversely, high calcium intakes markedly reduce lipid absorption due to the formation of insoluble calcium soaps, such as calcium stearate or palmitate, depending on the glyceride structure of the ingested lipids. The calcium absorption mainly occurs in the upper part (duodenum) of the digestive tract, whereas the insoluble calcium soaps are formed with the remaining unabsorbed calcium in the distal parts of the small intestine. This explains the apparent contradiction between the two opposite effects, i.e. the calcium-related decrease of lipid absorption and the unchanged Ca absorption by lipids.The polyinsaturated fatty acids and their by-products seem to enhance active calcium absorption and bone mineralization. But, some discrepancies are observed concerning the effects of arachidonic acid and conjugated, linoleic acid (CLA) upon bone remodeling.Some epidemiological observations and a few experiments suggest an inverse relationship between high dietary intake of calcium or of dairy products, and weight gain and/or obesity. According to mainly one group of investigators, the weight loss might be explained by a modulation of adipocyte functions by high calcium. However, this needs to be confirmed by further well-controlled clinical studies before practical applications. Nevertheless, it is well established that high dietary calcium, by diminishing saturated lipid absorption and favourably modifying serum lipid profile, may have protective effects upon cardiovascular diseases.

The Calcium Recommendations ("ANC") are calculated as the sum of the requirements for maintenance, growth, pregnancy and lactation, modified by a coefficient which takes into account the intestinal calcium absorption, and a margin of safety. However, this approach has led to criticisms pointing out that the values obtained could be too high for many individuals. This can be addressed by introducing the notion of "guide-values" representing 90% of FCaR (ANC) which equal to 800 mg per day in adults and approximately 1 gram in adolescents, post menopausal women (> 55 years), and elderly men and women (see Sci. Aliments, 26, 115-122, 2006).Cow milk, which contains more than 1 g Ca per litre, and dairy products are by far the most important contributory foods for calcium consumption since they account for 70% of the average total calcium intake. Is it possible to fulfil the ANC without using dairy products? A simple calculation of a daily diet combining usual food ingredients according to the recommended energy intakes shows that non-dairy part of the diet provides usually less than 450 mg Ca per day. Using the "guide-values", this indicates that 400 to 600 mg calcium a day (equivalent to 2 to 3 "portions" of dairy products containing 200 mg each) are missing according to age and physiologic status of the person. By choosing the Ca-richest ingredients (avoiding milk and milk products), it is feasible to increase calcium intakes. For example, a diet composed of rich Ca foods such as small fishes with spines (sardines), shellfish, almonds, some specific vegetables, dry fruits and a rich Ca mineral water may reach 1000 mg Ca a day. So, it is possible to answer "yes" to the early question. However, this is not a plain "yes". In fact, almonds, legumes (beans, soy, lentil...), whole-wheat by-products, spinach, watercress which are the Ca-richest vegetables also provide anti-nutritional factors like phytates or oxalates... These decrease Ca bioavailability, thus increase the amounts of calcium to provide. For those vegetables devoid of the inhibiting factors (cabbage, broccoli), their calcium is absorbed similarly to that of dairy product. However, it would be necessary to consume at least one kg of cabbage (or 2.5 kg of oranges) to provide as much calcium as does a litre of milk. Finally, some mineral waters are also interesting sources of non-dairy calcium (up to 500 mg/L). In this paper, acid/base equilibrium relative to urinary calcium loss is also discussed. In conclusion, milk and dairy products are not compulsory to ensure ANC, but the choice of other Ca-rich foods is very limited, the menu to deliver these is more difficult to establish and would be fastidious to eat on a long-term basis. Finally, these other foods may also cause some nutritional problems concerning their calcium bioavailability, which is not so "guaranteed" than that of the milk calcium.

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. © 2004 Wiley‐Liss, Inc.

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 17beta-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 D-3 [1,25(OH)(2)D-3], with or without 10-8 M of daidzein, 10-8 M of 17beta-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 h +/- 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)(2)D-3-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-alpha and ER-beta. Thus, daidzein, at the same concentration as 17beta-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.

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.

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 hone 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 OD vs. controls) and osteoclastic (+31%, p < 0.1 OD vs. controls) surfaces. This was not paralleled by increased hone 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 OD vs. controls) acid 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 OD 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, (C) 2000 by Elsevier Science Inc. All rights reserved.

Dairy products provide abundant, accessible calcium for humans, while some calcium sulfate-rich mineral waters could provide appreciable amounts of calcium. But there is little evidence that this calcium is as available as milk calcium for making bone. The availability of calcium was studied by monitoring bone parameters in 2-month-old pigs fed restricted amounts of calcium (70% RDA) for 2.5 months. The 3 main (> or = 50% Ca intake) Ca sources were either CaCO3 or CaSO4 or skim milk powder (29% of the diet). The bones of the pigs fed the "milk" diet had higher (P < 0.01) ash contents, breaking strength and density (DEXA) than those of the two others groups, in which the bone values were similar. Thus, the calcium provided by a diet containing milk appears to ensure better bone mineralization than do calcium salts included in a non-milk diet. The calcium restriction may have enhanced some milk properties to stimulate calcium absorption in these young, rapidly growing pigs.

Studies on calcium nutrition in appropriate large animal models can be directly relevant to humans. We have examined the effect of dietary Ca deficiency on various bone and bone-related variables, including plasma markers, histomorphometry, mineral content and breaking strength in pigs. Three groups of eight 38-d-old female pigs were fed adequate (0.9%; control), low (0.4%; LCa) or very low (0.1%; VLCa) Ca diets for 32 d. Plasma Ca significantly decreased over time only in the VLCa-deficient pigs. The concentrations of the parathyroid hormones (PTH) and calcitriol increased as Ca deficiency developed, and the plasma PTH and calcitriol levels varied inversely with dietary Ca. The total bone ash contents, bending moments, trabecular bone volume and the mineral apposition rate all decreased as the calcium intake decreased. The osteoclast surface areas were greater than those of controls in both Ca-deficient groups, whereas the osteoblast surface areas were greater only in the VLCa group. The plasma osteoblast-related markers (alkaline phosphatase, carboxy-terminal propeptide of type I procollagen and osteocalcin) were either greater or unaffected in the Ca-deficient pigs. The results indicate that deficient bone mineralization combined with an increased bone resorption led to bone loss and fragility. The differences in the changes in bone cells (number and activity) between LCa and VLCa groups might be due to differences (time and extent) of circulating PTH and calcitriol. The defective mineralization in both Ca-depleted groups resulted mainly from the lack of Ca because their osteoblast activity was either maintained or stimulated. The results also underline the progressive sensitivity of pigs to Ca supply and the usefulness of this model.

Three groups (n = 15/group) of 6-, 12- and 30-month-old (mature, old and senescent animals, respectively) female Wistar rats on a diet (6 g/100 g BW/day) containing 0.8% calcium and 0.8% inorganic phosphorus were studied. Within each group, 10 rats were ovariectomized surgically and 5 injected s.c. with 17β-estradiol (E rats, 10 µg/kg BW/48 h) and 5 with solvent alone (OVX rats) from day 2 until day 60 after ovariectomy. Five other rats were sham-operated (SH rats) and received solvent only. All rats were killed by exsanguination 60 days after ovariectomy. Neither ovariectomy nor estradiol treatment had a significant effect upon tibial mechanical properties in 6-, 12- and 30-month-old animals. Bone mineral density (BMD) and bone mineral content (BMC) of the distal femur and BMC of the whole femur were decreased by ovariectomy in 6- and 12-month-old rats, but were not different in the SH and E groups. In senescent animals, in which the lowest BMD and BMC were measured, estradiol treatment was more effective in increasing these parameters than in adult and old rats. Image analysis of the distal femoral diaphysis showed that estradiol treatment prevented trabecular bone loss induced by senescence and/or ovariectomy. In each group, urinary deoxypyridinoline excretion and plasma osteocalcin concentration were higher in the OVX animals than in the controls, consistent with increased bone turnover in the estrogen-deficient state. Both biochemical turnover markers were reduced in the estrogen-treated groups. These results indicate that 17β-estradiol is particularly effective at preventing high-turnover-induced osteopenia in 30-month-old animals.

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.

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.

We have demonstrated that alkaline phosphatase activity and collagen synthesis are dose-dependently stimulated by ascorbic acid in differentiated pig osteoblasts. In this study we further examined the relationship between ascorbic acid and bone metabolism by feeding young pigs large amounts of ascorbic acid. Three groups of seven 47-d-old pigs were given no ascorbic acid supplement (control), 500 (500 AA) or 1000 (1000 AA) mg ascorbic acid/kg diet for 4 mo. Calcium and P absorption and retention were evaluated by a 14-d balance trial immediately before killing in control and 1000 AA groups only (n = 6). Bones were collected at death and the bone ash and bending moment (three-point bending test) determined. Various plasma and urine indices of bone metabolism, especially those reflecting collagen degradation (hydroxyproline, deoxypyridinoline) and synthesis (carboxyterminal propeptide of type I collagen) were monitored. The plasma ascorbic acid concentrations increased with time and paralleled the dietary concentrations (P < 0.01). The Ca and P balances and the bone ash and bending moments in the ascorbic acid-supplemented pigs did not differ from those of the controls. Plasma osteocalcin was elevated (P < 0.05), whereas the other bone formation markers, alkaline phosphatase and carboxy terminal propeptide of type I collagen, were not affected by ascorbic acid. The plasma concentrations of Ca, P and 1,25-dihydroxycholecalciferol did not differ among the three groups. The unaffected urinary excretion of deoxypyridinoline and hydroxyproline in the ascorbic acid-supplemented pigs indicates that ascorbic acid does not alter bone resorption. Thus, high intakes of ascorbic acid have no positive influence on bone metabolism and bone characteristics in pigs. The in vivo long-term effects do not correlate with the short-term in vitro effects previously reported.

nisms of this effect remain to be elucidated.

Bone is sensitive to the removal of mechanical loading and the severity of unloading-induced bone loss may be influenced by an individual's genotype, gender, and the specific anatomical region. Whether these factors influence bone's mechanosensitivity directly or indirectly through differences in phenotypic baseline bone morphology and cellular activity is unknown. Here, we examined whether indices of baseline bone morphology and cellular activity are associated with the gender- and site-specific susceptibility of bone to unloading. Adult mice (4 months old, BALB/cByJ × C3H/HeJ) were assigned to one of six groups: male and female baseline controls (n = 20 each), age-matched controls (n = 10 each), or disuse (n = 11 males, n = 12 females). All baseline controls were sacrificed (0 day) to establish baseline bone morphology with micro-computed tomography (n = 10 each gender) or baseline cellular activities using histomorphometry and tartrate-resistant acid phosphatase staining (n = 10 each gender). Age-matched control and disuse mice were sacrificed (21 days) to determine disuse-induced bone loss by micro-computed tomography. Following 21 days of unloading, trabecular bone loss in the distal femur and proximal tibia was, on average, 3-fold greater in the metaphyses than in the epiphyses and 2-fold greater in females than in males. Disuse-induced changes in cortical bone were 2-fold smaller than trabecular bone losses and were more apparent in females (5 of 6 regions) than in males (1 of 6 regions). Bone loss was inversely related to baseline bone volume fraction (R2 = 0.51 for females and 0.43 for males) and directly related to baseline bone surface to volume ratio (R2 = 0.69 for females and 0.60 for males). Additionally, trabecular bone loss was correlated with baseline mineral apposition rates and osteoclast surface to bone surface ratios (R2 = 0.86 and 0.46, respectively, genders combined). These data demonstrate that baseline bone morphology and cellular activity modulate bone loss and that, independent of gender, anatomical regions with low bone quantity, high surface-to-volume ratios, and high levels of osteoblastic and osteoclastic activity are particularly susceptible to disuse.

The effects of three dietary levels of vitamin D (500, 1500 and 3000 IU/kg diet) on magnesium metabolism and on some bone parameters related to bone mineralization were studied in vitamin D-repleted pigs fed normal magnesium intakes (0.2 per cent) for two months. Apparent absorption and retention was measured in 10-day balance studies (prior to slaughter) in three groups of four 10-week old pigs. Except for vitamin D, the pigs received the same diet which met the recommended dietary allowances for growing pigs. The highest vitamin D level used was only three times the recommended level in French pig husbandry. At slaughter, the fibula and two main metatarsals (right hind leg) were collected to determine bone breaking strength, apparent density and bone mineral (ash, calcium, magnesium) contents. Blood was collected to determine plasma concentrations of calcium, magnesium and vitamin D metabolites. Magnesium absorption increased linearly from 28-39 per cent intake with increasing dietary vitamin D. Urinary magnesium was not affected, thus magnesium retention also increased linearly as a function of vitamin D intake. Plasma calcium and magnesium were not altered by vitamin D. Plasma 25-hydroxycholecalciferol concentrations reflected vitamin D intakes, while plasma 1,25 dihydroxycholecalciferol was unchanged. Density, breaking strength and mineral contents of the bones were lower in the pigs fed 1500 or 3000 than in those fed 500 IU vitamin D/kg diet. This suggests that bone resorption was stimulated by the higher dietary vitamin D. Thus, vitamin D at physiological doses may enhance magnesium absorption in non previously vitamin D-depleted pigs fed diets with abundant magnesium. This nutritional situation may help explain the predominant bone-resorbing effect of vitamin D supplementation.

We have further investigated the "meal effect" on mineral bioavailability in pigs by mineral balance studies and measurements of bone ash contents and bending moment. A group of seven pigs (CAA) was given all its dietary Ca as CaCO3 5 h after the first daily meal for 8 weeks. The control group of seven pigs received CaCO3 in the meal. Both groups were given normal P within the meals. Ca and P absorption and retention were evaluated by a 10-day balance trial. Several bones were collected at slaughter to determine bone ash, Ca, and P contents and bending moment (three-point bending test). Ingesting Ca after the meal did not affect Ca bioavailability or phosphorus absorption, but did reduce P retention, which in turn decreased the bone scores. Osteopenia, indicated by decreased total mineral contents of bones (and decreased ash:bone volume ratio), was associated with elevated plasma osteocalcin in the CAA group. Thus, CaCO3 need not be incorporated into a meal for high Ca absorption, provided that Ca is given after a meal, but simultaneous intakes of Ca and P are required for the best mineral retention.

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.