Iron bioavailability from fortified petit suisse cheese determined by the prophylactic-preventive method

In this research, we measured the iron bioavailability of ferrous gluconate stabilized with glycine (SFG) when it is used to fortify petit suisse cheese using the prophylactic-preventive method in rats. Three groups of male, weaned rats received a basal diet (control diet; 5.2 ppm Fe), a reference standard diet (SO₄Fe; 9.2 ppm Fe), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 8.8 ppm Fe) for 22d. The iron bioavailability was calculated as the ratio between the mass of iron incorporated into hemoglobin and the total iron intake per animal during the treatment. These values (BioFe) were 68% and 72% for SFG and ferrous sulfate, respectively. The value of the Relative Biological Value (RBV) was 95% for SFG in petit suisse cheese. These results show that according to this method, the iron bioavailability from industrial fortified petit suisse cheese can be considered as a high bioavailability rate.     

Iron bioavailability from fortified fluid milk and petit suisse cheese determined by the prophylactic-preventive method

In this research, we measure the iron bioavailability of micronized ferric orthophosphate when it is used to fortify low-fat fluid milk enriched with calcium and petit suisse cheese using the prophylactic-preventive method in rats. Four groups of male weaned rats received a basal diet (control diet; 6.5 ppm Fe), a reference standard diet (SO₄Fe; 18.2 ppm Fe), a basal diet using iron-fortified fluid milk as the iron source (milk diet; Fe ppm 17.9), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 18.0 ppm Fe) for 22 d. The iron bioavailability of the different sources was calculated as the ratio between the mass of iron incorporated into hemoglobin during the experiment and the total iron intake per animal. The relative biological values with regard to the reference standard (RBV%) were 61% and 69% for the milk and cheese diet, respectively. These results show that according to this method, the iron bioavailability in both fortified foods can be considered as medium bioavailability rates.     

Normal growth rate in rats is recovered after a period of zinc deficiency by restoration of zinc supply by means of a zinc-fortified Petit Suisse cheese

Fortification of a Petit Suisse cheese with zinc sulfate and zinc gluconate stabilized with glycine was used as a tool to overcome zinc-deficiency effects on total-body growth and skeletal growth. Animals were divided in 4 groups of 10 rats: basal (B), control (C), depletion-repletion 1 (DR1), and depletion-repletion 2 (DR2). These four groups were fed with four diets: basal (2 ppm Zn), control (30 ppm Zn), DR1, and DR2; they received a basal diet for 14 d and a control diet for the other 14 d of the experiment, using zinc sulfate for DR1 and zinc gluconate stabilized with glycine for DR2. After 28 d of the experiment, total-body weight and weight gain of the control and DR1 and DR2 animals were not statistically different (p<0.05), Femur weight and femur zinc content of DR1 and DR2 did not achieve the values of control animals (p<0.05), but they were higher than that of basal animals. Our results show that restoration of dietary zinc levels by means of food fortification normalized weight gain, as an indicator of total-body growth, and presented a trend to normalize bone weight, as a marker of skeletal growth, in young rats and independently of the zinc source used.     

Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats

The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5±10.5, 89.9±13.7, 118.6±13.6 and 134±19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265±49 mg; DZ, 380±40 mg; MZ, 452±54 mg; CZ, 735±66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5±5.4 ppm) was significantly different from the MZ and CZ groups (115.9±14.2 and 175.0±13.5 ppm, respectively), whereas the DZ group (62.5±11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2±9.8 mg/g; DZ, 88.0±9.2 mg/g; MZ, 90.2±13.6 mg/g; CZ, 83.1±14.7 mg/g) and magnesium (BZ, 1.82±0.13 mg/g; DZ, 1.98±0.09 mg/g; MZ, 1.93±14 mg/g; CZ, 1.83±0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected.     

Dietary zinc intake of a group of longstay mentally handicapped women some bioavailability considerations

Dietary factors affecting zinc bioavailability were evaluated according to their relative distribution in the individual daily meals making up the basic diet of 17 institutionalized, mentally handicapped adult women. Mean intake values of zinc, phytate, nonstarch polysaccharides (NSP), calcium, protein, and energy were calculated from a dietary survey of 7 consecutive days, which also served to obtain values for the two zinc bioavailability predictor formulas, phytate/Zn millimolar ratio and [Ca] [phytate]/[Zn][energy] ratio. Mean daily zinc intake was 8.5 ±1.8 mg, with noon and evening meals accounting for the highest contribution to this value (45% and 35%, respectively), whereas breakfast meals’ contribution was 16%. The mean protein intake was 55 ±13 g, with noon and evening meals being the major contributors to total daily intake (42% and 38%, respectively). Breakfast meals accounted for 77% of daily phytate intake, giving a respective phytate/Zn millimolar ratio of 20.4 ±7.6 and a [Ca][phytate]/[Zn][energy] ratio of 336 ±127 mmol/Mcal. Values for both ratios based on noon and evening meals were negligible in comparison. The mean daily NSP intake was 9.8 ±4.2 g, with 53% of total daily intake supplied from breakfast meals, whereas noon and evening meals accounted for 30% and 14%, respectively. The results, while suggesting that zinc bioavailability is unlikely to be adversely affected, indicated that dietary fiber intake levels are probably inadequate, particularly in view of the nonambulant condition and low physical activity prevalent in such individuals, who may, as a consequence, be susceptible to health disorders associated with impaired bowel function and constipation.     

Studies on zinc bioavailability from a representative diet in Chinese urban women of childbearing age using a double label stable isotope technique

The objectives of the study were to determine dietary zinc bioavailability by using a double label stable isotope technique, and to identify the zinc content and levels of some possible factors which may affect zinc utilization in a typical representative Chinese urban diet. Twenty urban women of childbearing age were fed a diet extrinsically labeled with 67Zn and received intravenous (i.v.) injections of 70Zn. Fecal monitoring, urine monitoring and chemical balance technique were used simultaneously for comparisons. Isotopic enrichment in feces and urine after oral and i.v. administration of stable zinc isotopes was measured to determine the ways of zinc absorption. The mean (+/-SD) zinc intake from the representative Chinese diet was 10.22+/-0.80 mg/day, which is lower than the reference nutrient intake (RNI) level (11.5 mg/day), and mainly came from the subsidiary foods (63%). The mean dietary Fe/Zn weight was 1.1+/-0.1. The phytate/zinc molar ratio in the diet was about 3:1. The phytic acidxCa/zinc molar ratio in the diet was about 45:1. The apparent and true absorption rates of 67Zn were 36.2+/-9.7% and 38.2+/-10.1%, respectively, which were higher than the apparent absorption determined by balance technique (29.7+/-10.6%). The mean fractional absorption (FA) determined in urine was 35.7+/-13.3. In conclusion, the subsidiary food is the main source of dietary zinc in this typical diet. The extrinsic labeling technique by use of stable isotopes was more reliable and accurate to determine zinc absorption. Moreover, the double isotopic tracer ratio (DITR) technique applied to urine samples was a more practical and valuable way to evaluate zinc absorption than other methods. It can be speculated that iron and phytic acid might not be the main inhibitors in the representative Chinese diet.     

Changes in the content of zinc and fluoride during growth of the femur in chicken

Zinc and fluorides are capable of modifying the process of bone formation and mineralization. Statistically significant differences have been revealed in the content of zinc and fluorides between structures of the femur in chicken. The content of zinc in compact bone remained constant during the first 50 d of life. Lower and less stable contents were found in spongy bone and bone marrow. The content of fluorides in compact bone was higher than in spongy bone. The lowest concentrations of zinc and fluorides were found in articular cartilage and were further reduced at the end of observation. Correlations revealed between the content of zinc and fluorides point to structural and functional relationships between these elements in various parts of the bone.     

Long-term effects of lactational zinc deficiency on bone mineral composition in rats fed a commercially modified Luecke diet

The purpose of this study was threefold: 1. to determine the long-term effects of interactions between lactational zinc deficiency and gender on bone mineral composition in repleted rat offspring, 2. to determine the nutritional efficacy of the second of two commercially designed, modified Luecke diets (ML2) during the gestational and lactational stress, and 3. determine the ultratrace element contents of Ralston Rodent Laboratory Chow #5001. The ML2 basal diet, based on dextrose, sprayed egg white, and corn oil contained 0.420 μg Zn/g, was supplemented with Zn (as zinc acetate) at 0 (diet 0ML2) or 30 (diet 30ML2) μg/g, and was mixed and pelleted commercially. all rat dams were fed the 30ML2 diet ad libitum during gestation. Beginning at parturition, the dams were fed either the 1. 0ML2, 2. 30ML2 (food restricted), or 3. 30ML2 (ad libitum) diets. All pups were fed the 30ML2 diet ad libitum from 23 to 40 d of age. From d 40 to 150, all pups were fed Ralston Rodent Laboratory Chow. The 30ML2 diet was found to be nutritionally efficacious; litter size and pup growth were normal and pup mortality was only 1.2%. Pups (ZD) with access to the 0ML2 diet until 23 d of age and nursed by dams fed the 0ML2 diet, when compared to pups (PF) fed restricted amounts of the 30ML2 diet, exhibited increased mortality and decreased concentrations of tibial zinc but no change in growth. Inadequate zinc nutriture during infancy, despite postlactational zinc repletion, induced imbalances in adult bone mineral metabolism. Thus, at 150 d of age, the ZD pups exhibited increased levels of bone P and Mg and decreased concentrations of K as compared to the PF pups.     

Effect of chitooligosaccharides on calcium bioavailability and bone strength in ovariectomized rats

Chitosan polymer with deacetylation degree of 93% was hydrolyzed with an endo-type chitosanase (35,000 U/g protein) with substrate to enzyme ratio of 1 to 1.5 for 18 h in a batch reactor, and then the resultant hydrolysates were fractionated into four different molecular weights using an ultrafiltration (UF) membrane reactor system. An in vitro study elucidated that four kinds of chitooligosaccharides (COSs) could efficiently inhibit the formation of insoluble calcium salts in the neutral pH. In vivo effects of COSs on Ca bioavailability were further studied in the osteoporosis modeling rats induced by ovariectomy and concurrent low calcium intake. During the experimental period corresponding to the menopause with the osteoporosis disease, calcium retention was increased and bone turnover was decreased by COS IV supplementation in the ovariectomized (OVX) rats. After the low Ca diet, COS IV diet including both normal level of calcium and vitamin D significantly decreased calcium loss in feces and increased calcium retention compared to the control diet. The levels of femoral total calcium, bone mineral density (BMD), and femoral strength were also significantly increased by the COS IV diet in a similar level to those of CPP diet group. In the present study, the results proved the beneficial effects of low molecular chitooligosaccharide (COS IV) in preventing negative mineral balance.     

Calcium exacerbates the inhibitory effects of phytic acid on zinc bioavailability in rats

BackgroundComplementary feeding of breastfed infants with foods high in bioavailable zinc (Zn) can help meet physiological requirements for Zn. Some infant cereals contain high concentrations of phytic acid (PA) and calcium (Ca) that may reduce absorbable Zn.ObjectivesThis study measured PA, Zn and Ca concentrations in selected infant cereals sold in Canada and investigated the effects of dietary PA and Ca at concentrations present in infant cereals on Zn bioavailability in rats.Methods and resultsMale Sprague-Dawley rats (36-day old) were fed a control diet containing normal Zn (29.1 mg/kg) and Ca (4.95 g/kg) or six test diets (n = 12/diet group). Test diets were low in Zn (8.91–9.74 mg/kg) and contained low (2.16–2.17 g/kg), normal (5.00–5.11 g/kg) or high (14.6–14.9 g/kg) Ca without or with added PA (8 g/kg). After 2 weeks, rats were killed and Zn status of the rats was assessed. PA, Zn and Ca concentrations in infant cereals (n = 20) differed widely. PA concentrations ranged from undetectable to 16.0 g/kg. Zn and Ca concentrations ranged from 7.0–29.1 mg/kg and 0.8–13.4 g/kg, respectively. The [PA]/[Zn] and [PA × Ca]/[Zn] molar ratios in infants cereals with detectable PA (16 of 20 cereals) ranged from 22–75 and 0.9–14.9 mol/kg, respectively, predicting low Zn bioavailability. Body weight, body composition (lean and fat mass), right femur weight and length measurements and Zn concentrations in serum and femur indicated that diets higher in Ca had a more pronounced negative effect on Zn status of rats fed a PA-supplemented diet. Addition of PA to the diet had a greater negative effect on Zn status when Ca concentration in the diet was higher.ConclusionThese results show that, in rats, higher concentrations of dietary Ca and PA interact to potentiate a decrease in bioavailable Zn and may suggest lower Zn bioavailability in infant cereals with higher PA and Ca concentrations.     

Cadmium accumulation, zinc status, and mineral bioavailability of growing rats fed diets high in zinc with increasing amounts of phytic acid

Five groups of individually housed albino rats (n=7, initial average weight=48 g) were fed diets based on egg albumen and cornstarch (basal diet 8.2 g Ca, 6.0 g P, 0.7 g Mg, 225 mg Zn, 150 mg Fe, 60 mg Mn, 8 mg Cu, and 5 mg Cd) over a 4-wk period. Group I (control) was fed the basal diet free of phytic acid (PA). In groups II, III, IV, and V, cornstarch was replaced by 3.5, 7.0, 10.5, and 14.0 g sodium phytate/kg diet, respectively. Daily gain, feed efficiency, Zn status (Zn in plasma, femur, testes, liver and kidneys, activity of the plasma alkaline phosphatase) and apparent absorption of Zn, Fe, Cu, and Mn remained unchanged by the different dietary treatments. PA decreased apparent Mg absorption significantly and apparent absorption of Ca in tendency. Increasing the amount of phytate caused a corresponding enhancement of amount of the digestible P. Cd accumulation in the liver was not significantly altered, and kidney Cd accumulation slightly increased owing to PA. In conclusion, it was shown that under conditions of high dietary Zn, PA had only little effect on the carryover of Cd in growing rats.     

Determination of rat oral bioavailability of soy-derived phytoestrogens using an automated on-column extraction procedure and electrospray tandem mass spectrometry

In recent years, consumption of herbal supplements as an alternative to pharmaceutical drug therapy has increased. For example, with the health claims labeling which describes the link between soy-protein and a reduced risk of coronary heart disease (CHD), the consumption of soy and soy-derived phytoestrogens has increased dramatically. That being said, the oral bioavailability of only a few soy phytoestrogens such as Daidzein and Genestein have been previously estimated. In this paper, we present the calculated percent of rat oral bioavailability of five soy-derived phytoestrogens (Genistein, Daidzein, Biochanin A, Coumestrol, and Zearalenone) in male Sprague-Dawley rats. The plasma quantitation required for the bioavailability calculation is performed by using a rapid on-line plasma extraction procedure for the quantitative analysis. To further speed up the analysis the rats were dosed using the 'n-in-one' (cassette) protocol. The rapid on-line extraction/quantitation methodology coupled to the cassette dosing analysis of phytoestrogens is the key point of this paper. The limit of quantitation (LOQ) for each compound was 1-1000 ng/ml with each plasma sample analysis taking less than 2 min. In general the percent oral bioavailability was determined to be between 11 and 28%.     

运动对去卵巢大鼠骨元素代谢和相关激素的影响

目的:测定大鼠骨元素含量和血清相关激素及IL-6含量,研究运动对去卵巢大鼠骨元素代谢和相关激素的影响。方法:健康4月龄雌性SD大鼠24只,用抽签法随机分成4组:正常对照组;假去卵巢组;去卵巢组;去卵巢+运动组。运动组于去卵巢术后第7 d开始运动训练,每周5 d,每天连续匀速跑45 min,16 m/min,跑道倾角0°,持续10周。结果:去卵巢大鼠骨Ca,Mg,S,Co,Mn,Zn等含量降低,骨P含量升高,血清E2,P,TSH,T4,CT,Cortisol,GH等显著降低,IL-6,FSH,LH等的含量显著升高。运动训练可使去卵巢大鼠骨Ca,Mg,S,Co,Mn等含量回升,血清E2,P,TSH,T4,CT,Cortisol,GH等显著回升,IL-6,FSH,LH等显著回降。结论:运动可纠正去卵巢所致的大鼠骨元素代谢和相关激素的改变。     

Zinc and manganese bioavailability from human milk and infant formula used for very low birthweight infants,evaluated in a rat pup model

The bioavailability of zinc and manganese from diets used for very low birthweight infants was investigated in a rat pup model using radioisotopes. The effect of protein source and content and of pasteurization was evaluated, and two different approaches for evaluation of zinc and manganese bioavailability in the rat pup model were compared. Zinc and manganese bioavailability from the studied human milk and infant formula for very low birthweight infants was high. Liver uptake of⁶⁵Zn from labeled premature infant diets in sucklings rat pups was 26–29%, and absorption calculated as the difference between administered dose and nonabsorbed activity 6 h after oral intubation was 93–95%. Retention of manganese calculated as the sum of⁵⁴Mn retained by organs and carcass was 85–95% from human milk and premature infant formula and absorption calculated from nonabsorbed activity was 83–88% after 6 h. Fortification of early human milk significantly increased the bioavailability of zinc. No effect of pasteurization of human milk was found on zinc or manganese bioavailability. Liver zinc uptake was found to be a more sensitive parameter than absorption for evaluation of diets with a high zinc bioavailability. Measurement of retained activity of manganese in carcass and organs was judged to be the preferred parameter for evaluation of diets with high manganese availability.     

Bioavailability studies of a new iron source by means of the prophylactic-preventive method in rats

The bioavailability of iron from a new commercial source containing ferric gluconate stabilized with glycine sold under the trade name Bioferrico™ was studied in this work by means of the prophylactic-preventive test in rats. NaFeEDTA was also studied by the same methodology for comparative purposes and ferrous sulfate was used as the reference standard. The test was conducted for 4 wk with male weaned rats, which were randomized into four groups of at least eight animals each. A control group received a basal diet of low-iron content, whereas the other groups received the same diet with iron added at a dose of 20 mg/kg as FeSO₄·7H₂O, NaFeEDTA, and Bioferrico, respectively. Individual hemoglobin concentrations (HbC) and weights were determined at the beginning and at the end of the study and food intake was daily registered. The iron bioavailability (BioFe) of each source was calculated as the ratio between the amount of iron incorporated into hemoglobin during the treatment (HbFe) and the total iron intake per animal (ToFeIn). A relative biological value (RBV) was obtained for each iron source under study as the ratio between the BioFe of the tested compound and that of the reference standard. The RBVs were 98% and 86% for Bioferrico and NaFeEDTA, respectively. Bioferrico showed a high bioavailability and behaved inertly in relation to the sensorial properties of the fortified food when it was added to flour. These qualities emphasize Bioferrico as a promising source for iron fortification.     

Evaluation of SNPs in the chicken HMGA2 gene as markers for body weight gain

A QTL affecting body weight in chickens has been mapped to GGA1, between the markers GCT0006 and MCW0106. The gene HMGA2, which was previously identified as a candidate gene for determining body height in humans and mice, is also conspicuously close to the MCW0106 marker in chickens. Subsequently, 14 SNP markers of HMGA2 were genotyped in CAU chicken resource populations, and the associations between body weight and those SNP markers that displayed polymorphisms were analysed. Three SNPs (rs13849241, rs15231472 and rs13849381) were found to be significantly correlated with body weight in chickens (P < 0.05). Furthermore, haplotypes constructed based on these three SNPs were also discovered to be associated with body weight in chickens at the ages of 6, 7, 9 and 12 weeks. These results suggest that the chicken HMGA2 gene is indeed involved in body weight gain.     

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg⁻¹ day⁻¹ dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.     

Absorption and metabolism of oral zinc gluconate in humans in fasting state,during, and after a meal

The absorption and metabolism of zinc in a commercial form for oral use (Rubozinc^®, 15 mg zinc as gluconate) were investigated in 10 subjects by a kinetic study of the serum zinc profile after administration of 45 mg zinc under three conditions: after an overnight fast, during a standardized breakfast, and 2 h after this meal. The pharmacokinetic parameters were calculated by a method suitable to the characterization of rebound effects (recycling of the element in the gastrointestinal tract). In fasting state, the parameters were comparable to those previously collected in the same subjects with oral 45 mg zinc as sulfate, except with very significantly higherC _max and area under curve (AUC), showing a better bioavailability for zinc in the commercial form. The light meal perturbed the absorption process as evidenced by the significant increases in the lag time (+180%), thet _max (+57%), and the lag times for the first two cycles during the meal. However, the parameters returned to normal values 2 h after the meal. TheC _max only moderately decreased during the meal (31%) as did the AUC (?28%). An important delay in the absorption of zinc in the commercial form when taken during a meal was therefore demonstrated, but the effect on zinc bioavailability was only moderate.     

Determination of zinc in tissues of normal and dystrophic mice using electrothermal atomic absorption spectrometry and slurry sampling

An electrothermal atomic absorption spectrometric procedure for zinc determination in animal tissues is first optimized and then used to measure the metal content of different tissues from normal and dystrophic mice. The procedure involves minimal manipulation of the sample to overcome the severe contamination problems normally associated with the measurement of low zinc levels. The samples (recommended amount 10 mg) are slurried in 2 ml of a 10-mM tetramethylammonium hydroxide solution containing 0.1 g/100 ml silicone antifoam. After mild heating at 60 degrees C for 10 min and homogenization for 1 min, the suspensions are submitted to a 10-fold dilution and then injected into the electrothermal atomizer. Calibration is carried out using aqueous standard solutions of zinc prepared in the same suspension media. The reliability of the whole procedure is verified using three certified reference materials.     

Role of zinc in cellular zinc trafficking and mineralization in a murine osteoblast-like cell line

Zinc (Zn) supplementation stimulates bone growth in Zn-deficient humans and animals. A biphasic pattern of mineralization has been observed in cultured osteoblasts; an initiation phase and a progression phase. We used MC3T3-E1, a murine osteoblastic cell line, to elucidate the physiological role of Zn in osteoblast mineralization and cellular Zn trafficking during the mineralization event. Cells were cultured in media containing Chelex-treated fetal bovine serum and 1, 4, 10 and 20 μM Zn as ZnSO₄ for 14 days (early phase of mineralization) or 21 days (mid-to-late phase of mineralization). During the early phase of mineralization, Alizarin Red staining indicated that mineralization was increased by Zn in a dose-dependent manner. Although Zn exposure did not affect monolayer Zn concentration, metallothionein (MT) mRNA expression increased dose-dependently as assessed by real-time PCR. During the late phase of mineralization, mineralization was maximal at 1 μM Zn and monolayer Zn concentration reflected Zn exposure. The increase in MT mRNA expression during the late phase was similar to that during the early phase, but the difference in expression between culture Zn concentrations tended to be smaller. ZnT-2 mRNA expression decreased significantly with increasing zinc concentrations in the culture medium during the early phase, but increased significantly during the late phase. Osteocalcin mRNA levels were positively correlated to Zn exposure at both time points. Taken together, we propose that Zn may play an important role in osteoblast mineralization through Zn trafficking involving Zn storage proteins and Zn transporters.