Article pubs.acs.org/JAFC

Absorption and Bioavailability of Nano-Size Reduced Calcium Citrate Fortified Milk Powder in Ovariectomized and OvariectomizedOsteoporosis Rats Arezoo Erfanian,† Hamed Mirhosseini,† Babak Rasti,§ Mohd Hair-Bejo,‡ Shuhaimi Bin Mustafa,# and Mohd Yazid Abd Manap*,# †

Department of Food Technology, Faculty of Food Science and Technology, ‡Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, and #Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Selangor, Malaysia § Faculty of Food Science and Nutrition, Universiti Malaysia Sabah (UMS), 88400 Kota Kinabalu, Sabah, Malaysia ABSTRACT: The aim of this study was to evaluate the effects of fortification and nano-size reduction on calcium absorption and bioavailability of milk powder formula in sham, ovariectomized, and ovariectomized-osteoporosis rats as a menopause and menopause-osteoporosis model. Skim milk powder and skim milk powder fortified with calcium citrate and the suitable doses of inulin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and vitamins D3, K1, and B6 were formulated based on the North American and Western European recommended dietary allowances. Optimization on cycle and pressure of highpressure homogenizer was done to produce nano-fortified milk powder. In vivo study demonstrated that fortification and calcium citrate nano-fortified milk powder increased absorption and bioavailability of calcium, as well as bone stiffness and bone strength in sham, ovariectomized, and ovariectomized-osteoporosis rats. This study successfully developed an effective fortified milk powder for food application. KEYWORDS: calcium bioavailability, calcium absorption, nanoparticle, osteoporosis, in vivo study, menopause, fortified milk



develop new healthy and more nutritious foods.9 An important requirement is that the fortified foods need to be consumed in adequate amounts by a large proportion of the target individuals in a population. Thus, this strategy can lead to relatively rapid improvements in the micronutrient status of a population.11 For example, dairy products are among the best sources of calcium, on the basis of their calcium content, other essential nutrients, absorption, and low cost relative to total nutritional value.12 Therefore, fortified dairy products are good food sources for people, especially menopausal women. Several calcium salts have been tested for the fortification of foods such as tricalcium phosphate, citrate,13 carbonate,14 chloride,15,16 lactate,17,18 and, more recently, citrate-malate.9 Calcium adequacy alone does not fully protect against bone loss, especially during aging and menopause.19 In addition, there is also a requirement to develop food components that stimulate bone formation or suppress bone resorption. Many definitions exist for nutrient bioavailability, but broadly it refers to the proportion of a nutrient that is absorbed from the diet and used for normal body functions. Chemically, the calcium compound utilized to fortify the food affects calcium bioavailability.20 Moreover, insufficient time for absorption in the gastrointestinal tract is a common cause of low bioavailability. If the mineral does not dissolve readily or cannot penetrate the epithelial membrane during its residence time in the

INTRODUCTION Calcium is an essential nutrient as all living cells require it to remain viable. Calcium is also required for a number of particular roles in the body such as bone mineralization (impregnation of the bone matrix with minerals) and growth.1 As calcium has chemical properties indispensable for skeletal function, adequate dietary calcium intake is required to achieve full accretion of bone mass.2 Calcium balance studies demonstrated that calcium requirements increase after menopause in women and the amount of calcium needed is affected by the decrease in intestinal absorption caused by aging.3 Calcium deficiency is a global public health problem, especially in developing countries.4 Several researchers5−7 showed that chronic untreated calcium deficiency can cause many severe consequences, including osteoporosis, and the subject has received worldwide attention. One of the factors that lead to osteoporosis is lack of calcium in the diet. Osteoporosis (OS) is a disease in which bones become weak and brittle; however, it is preventable by adequate recommended nutrition intake. Different health organizations worldwide recommend different intakes based on calcium balance studies, data on bone mineral content, and changes in bone mineral density.8 The Institute of Medicine of the United States recommends 1000 mg per day for females over 51,9 whereas Western European and Canadian data indicate that the amounts of recommended calcium allowance should be 1300 mg for female at the same age.10 Food fortification refers to the addition of micronutrients to processed foods. For several decades, researchers and the food industry have made many efforts and large investments to © XXXX American Chemical Society

Received: March 23, 2015 Revised: May 29, 2015 Accepted: May 29, 2015

A

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry gastrointestinal tract, its bioavailability tends to be highly variable and low.21 A possible way of slowing osteoporosis is to increase the absorption and bioavailability of calcium by using absorption enhancers or reducing the size of the compounds from microscale to nanoscale. The surface area of the nanoparticles increases, and the properties of the more reactive surface molecules lead to the new properties of the nanoparticles.22 Whereas nanodelivery systems can increase the absorption and bioavailability of nutrients, they also can change the distribution of the substances in the body.23 Previous studies by Mohanty et al.24 and Thakkar et al.25 on the effect of nanoparticle size, delivery system, and bioavailability revealed that particle size reduction is one of the most important factors to enhance absorption and bioavailability. These combined effects, namely, using suitable composition and nano-size reduction, can help to protect against the development of osteoporosis. There are different experimental techniques for measuring calcium bioavailability. One of these techniques is the use of in vivo assay. Particularly, small animals such as rats are used as their bones respond quickly to changes, in a human-like fashion, and they are easy to use and inexpensive. The ovariectomized (OVX) rat model is a widely used animal model to simulate estrogen deficiency induced bone loss and is commonly used to test calcium bioavailability for the treatment of postmenopausal osteoporosis. The current study developed a fortified milk powder containing calcium citrate and some calcium bioavailability enhancers (i.e., inulin, water-soluble and fat-soluble vitamins including B6, K1, and D3, and DHA as well as EPA). To improve the absorption and bioavailability of dietary calcium, two types of fortified milk powder have been developed and compared in vivo by OVX and OVX-OS rats as a model of menopause and menopauseOS women, respectively. It has been hypothesized that the fortified milk with specific composition and optimum preparation condition improves the absorption and bioavailability of calcium in OVX and OVX-OS rats. It is accepted that adequate amounts of calcium, vitamins D3, K1, B6, inulin, DHA, and EPA are key elements for healthy bone development, maintenance of bone density and bone strength, and prevention of osteoporosis, but fortification with micro calcium particles results in lower absorption efficiency than nano calcium particles. Among various sources of fortified foods, little information is available about nanoparticles. Therefore, for the purpose of finding another type of fortified food, we have compared nanofortified milk powder with micro- and nonfortified milk on calcium absorption and bioavailability in OVX and OVX-OS rats as a model of menopause and menopauseOS women, respectively.



Table 1. Specification of Experimental Diets content (%)

a

specification

basal dieta

skim milkb

fortified milkc

protein fiber fat moisture ash lactose nitrogen-free extract inulin calcium phosphorus EPA and DHA K1 D3 B6

22 5.0 3.0 13.0 8.0

36.25

25.8

0.9 3.9 8.3 49.8

11.5 3.9 18.8 34.8

49.0 1.0 0.8

0.85 0.6

0.001

5 13 0.5 11 0.00055 0.0001 0.015

Gold Coin Animal Feed (702P-Pellet). bT1. cT2 and T2-nano.

Phase 1: Optimization of Preparation Condition of Calcium Nanofortified Milk Powder. High-Pressure Homogenization. Sample preparation was achieved by dispersing milk powder in deionized water (20% w/v). A mixer (Silverson L4R, Buckinghamshire, UK) was used for 5 min at 1000 rpm as prehomogenizer to prepare coarse sample. Then, the sample passed through a highpressure homogenizer (APV, Crawley, UK) with different homogenization condition (i.e. cycle, 3−5; pressure, 200−400 bar) to produce calcium citrate nanofortified milk sample (T2-nano). Polydispersity Index (PDI) and Average Particle Size (APS). A dynamic light-scattering particle analyzer (Malvern Series ZEN 1600, Malvern Instruments Ltd., Worcester, UK) was used to analyze APS and PDI of fortified milk powder. A laser ray passed through the sample and was scattered by the particles in a distinctive pattern. A photodiode array detector measured the APS. The measurement scale of APS was reported in micrometers. The experiment was carried out in triplicate for each diluted sample. The system reported a measure of particle size distribution as PDI. PDI values range from 0, for a completely monodisperse system, to 1.0, for a polydisperse particle dispersion. Response Surface Analysis. Response surface methodology (RSM) evaluated the effect of two independent variables, namely, x1 (cycle (3, 4, and 5)) and x2 (pressure (200, 300, and 400 bar)), on the response variables, Y1 (PDI) and Y2 (particle size (nm)). A central composite design (CCD) consisting of 14 treatments, including 6 center points, was created to optimize the preparation condition of nano calcium citrate fortified milk powder prepared by high-pressure homogenization and simultaneously study the main, interaction, and quadratic effects of independent variables on the dependent variables.26 Optimization and Validation Procedure. To determine the optimum level of preparation parameters leading to minimum particle size and minimum PDI, graphical (three-dimensional response surface) and numerical optimization27 were carried out (Minitab v.16, Minitab Inc., State College, PA, USA). To predict the exact optimum level of independent variables, response optimizer (Minitab v. 16) was used for numerical optimization leading to the desired response goals. The adequacy of RSM (model validation) was evaluated using the t test. Particle Morphology. The nanoparticle structure and morphology were examined using energy filtered transmission electron microscopy (EFTEM; LEO 912 AB Omega, LEO Electron Optics, Oberkochen, Germany). Air-dried samples on a copper grid were negatively stained with 2% phosphotungstic acid solution (2 min) and air-dried before being observed under EFTEM.27 The diameters of the particles were determined using the scale bar on the electron micrographs. Reproducibility of the EFTEM images was assured by taking at least four pictures of each sample.

MATERIALS AND METHODS

Chemicals and Materials. Skim milk powder (T1) was provided by Fonterra (Kuala Lumpur, Malaysia). Food grade calcium citrate, inulin, vitamins D3, K1, and B6, EPA, and DHA were procured from Finuco (Sankyo Seifun Co, Ltd., Chugoku/Okayama, Japan). A Sartorius stedim biotech system (GmbH, 37070 Gottingen, Germany) was used to obtain deionized water for all procedures. Milk Fortification. The fortified milk powder contained calcium citrate, EPA and DHA (1:1), vitamins D3, K1, and B6, and inulin based on the North American and Western European recommended dietary allowance (Table 1).10 These compounds were added to skim milk powder and mixed for 5 min with a mixer (Bachiller, Barcelona, Spain) (Table 1). The samples were kept in airtight glass bottles and stored in a refrigerator. B

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Phase 2: In Vivo Study. The ultimate test of success of a formula depends on its in vivo performance, which was conducted in a suitable animal model (refer to graphical abstract). Approval to carry out in vivo study was obtained from the Animal Care and Use Committee of Faculty of Veterinary Medicine of Universiti Putra Malaysia (UPM, Selangor, Malaysia). The animals used for in vivo experiments were female Sprague−Dawley rats (n = 96, 7 weeks old) weighing ∼250 g obtained from UPM veterinary hospital (Serdang, Selangor, Malaysia). The animals were kept under standard laboratory condition at a temperature of 22 ± 2 °C and relative humidity of 50 ± 5%. The animals were housed in metabolic cages (a conventional, circular metabolic cage made of clear plastic was used), one per cage, with free access to standard laboratory feed (Gold Coin animal feed, Kuala Lumpur, Malaysia) (Table 1) and distilled water ad libitum. The experimental diets contained the nonfortified milk powder (T1, 850 mg/100 g) as the main protein source. Ovariectomy was performed by excising the ovaries. The animals were anesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg), intraperitoneally.28 Briefly, hair was cut over the surgical area and scrubbed with iodine. A small incision (∼1.0 cm) was made in the skin between the middle of the back, starting at the last rib. The skin was moved to one lateral side, and a small incision was made through the peritoneal lining. The entire ovary was removed with a single cut between the uterine horn and oviduct on each side. The skin was then recovered with a surgical staple.29 Sham surgery consisted of the skin incision and staple procedures only.30 After 1 week postsurgery treatment, the rats were randomly assigned to three main groups (sham, OVX, and OVX-OS). Each group of rats was randomized into four different subgroups including eight rats each and maintained for a period of 8 weeks as an experimental period. The first and second groups included sham and OVX rats that were fed treatment diets (1, basal diet, standard rat chow diet as a control diet; 2: T1; 3, T2; and 4, T2-nano). The third group included OVX rats that were fed a low-calcium diet for 6 weeks prior to experimental diets. The idea was to induce osteoporosis in the OVX rats and change them to OVX-OS rats. To ensure all groups were fed treatment diets similarly, during that 6 week period the other two groups (sham and OVX rats) were kept on basal diet. After 6 weeks of low-calcium diet feeding, the OVX-OS rats were randomly divided into four subgroups. Then they received experimental diets for 8 weeks the same as sham and OVX rats. During the 8 week experimental period, the treatment diets were given (5 mL) orally by gavage twice daily. Sample Collection. After an 8 week feeding period, following overnight fasting, rats were anesthetized with a 1:2 (v/v) mixture of ketamine (10 mg/kg) and xylazine (25 mg/kg) and sacrificed.31 Blood collected by cardiac puncture was centrifuged (Hettich EBA 20 centrifuge, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) at 5000 rpm (2850g) for 8 min at 4 °C to separate serum that was maintained at −20 °C for further chemical analysis. Feces were collected for each individual rat in the last 6 days of the experiment. Then, the feces were air-dried, finely ground, stored in a plastic tube, and kept at 4 °C until further use for calcium analysis. The bones (femurs) were cleaned by eliminating the soft tissue. However, they were initially soaked in saline solution prior to sealing in plastic bags and storing at −20 °C.32 They were saved for calcium determination, bone strength (maximum load) measurement, and bone morphology. Experimental Analysis. Mechanical Assays. Femurs were subjected to an Instron universal testing instrument (5kN model 3365, Norwood, MA, USA) for evaluation of biomechanical function of bones. Femurs were brought to room temperature before breaking in the exact center of the bones. To analyze the maximum load, the bone was placed on two supporting bars (2 mm in diameter) with a span of 15 mm, and an upper crosshead roller (6 mm diameter) was applied in the middle of the bone and advanced at 0.5 mm/min until the rupture was automatically determined by the apparatus. The midpoints of the femurs were determined by measuring the length of each bone and dividing the length using a Vernier caliper. The crosshead compressed the middle of the femur with linearly increasing

Table 2. Matrix of Central Composite Design (CCD), Independent Variables and Their Level, and the Responses for the Processing of T2-Nano dependent variablesa

independent variables

a b

Y1 (mean ± SD)

run

x1

x2

1 2b 3b 4 5 6b 7 8b 9 10b 11 12 13b 14

−1 0 0 −1 +1 0 +1 0 0 0 0 −1.414 0 +1.414

−1 0 0 +1 +1 0 −1 0 +1.414 0 −1.414 0 0 0

Y2 (mean ± SD)

0.514 ± 0.002 0.497 0.452 ± 0.004 0.350 0.439 ± 0.005 0.327 0.480 ± 0.003 0.259 0.560 ± 0.002 0.341 0.491 ± 0.004 0.326 0.320 ± 0.001 0.368 0.450 ± 0.003 0.344 0.480 ± 0.005 0.341 0.440 ± 0.002 0.360 0.370 ± 0.004 0.483 0.544 ± 0.003 0.369 0.470 ± 0.006 0.340 0.468 ± 0.007 0.319 level used, actual (coded)

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.003 0.004 0.001 0.001 0.007 0.003 0.002 0.005 0.006 0.008 0.005 0.004 0.003

independent variable

low (−1)

medium (0)

high (+1)

x1 = cycle x2 = pressure (bar)

3 200

4 300

5 400

Y1, PDI (polydispersity index); Y2, APS (average particle size) (nm). Center point of the design.

Table 3. Regression Coefficients, R2, p Value, Lack of Fit Test, and Significance Probability of Independent Variable Effects in the Reduced Response Surface Modelsa regression coefficient b0 b1 b2 b12 b11 b22 R2 regression (p value) lack of fit (p value)

variable PDI (Y1) APS (Y2)

p value F ratio p value F ratio

PDI

APS

1.5209 −0.4351 −0.0013 0.0006 0.0252 −0.0001 0.953 0.000b 0.162c interaction main effects effects

1.4756 −0.1608 −0.0046 0.0005 −0.0015 0.000 0.965 0.000b 0.128c quadratic effects

x1

x2

x1x2

x12

x22

0.00 148.23 0.18 2.21

0.00 83.27 0.00 711.98

0.00 124.93 0.00 52.19

0.01 15.99

0.00 679.85

a

PDI, polydispersity index; APS, average particle size; x1, cycle; x2, pressure. Only terms with statistical significance are included. b Significant (p < 0.05). cInsignificant (p > 0.05) force, until fracture occurred. The load deformation curve was recorded. The test results provided values for maximum load (N).32 Calcium Analysis. To measure serum calcium, an automatic blood chemical analyzer with Roche testing kits (Roche COBAS MIRA PLUS, Basel, Switzerland) was used. Total concentration of calcium in the diet, feces, and bone calcium contents were measured by an atomic absorption spectrophotometry (Thermo Scientific, S series, Waltham, MA, USA) using a standard procedure.32 Morphology Structure. Fractured surfaces of the femurs obtained from the mechanical strength test were processed for scanning electron microscopy (SEM) (LEO 1455VPSEM, London, UK). Briefly, bone was fixed (2.5% glutaraldehyde for 72 h and postfixation C

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Particle size distribution of (a) skim milk powder (T1), (b) calcium citrate fortified milk powder (T2), and (c) calcium citrate nanofortified milk powder (T2-nano).

Absorption and Bioavailability. Apparent calcium absorption and relative bioavailability values were calculated using consumed calcium in the diets, fecal calcium, and total calcium in bones as explained previously.20 Statistical Analysis. Data analysis was carried out using Minitab software (version 16, Minitab Inc., State College, PA, USA). A completely randomized design was employed to generate all treatments. In each experiment, a comparison was made using one-way ANOVA within and between groups receiving the same diets. Significant (p < 0.05) differences were determined among treatment groups with Tukey’s multiple-range tests.

in 1% osmium tetroxide for 24 h), then dehydrated in ethanol, and dried with CO2 at critical point. Finally, it was attached to aluminum specimen holders with colloidal silver. This process provided a conducting path to remove charges that would result from the incident electron beam of SEM striking the specimen. The mounting stubs were placed in an oven at 70 °C for 15 min to allow evaporation of volatile solvents from the carbon element and silver paint. Then sputter was coated with palladium−gold using a sputter coater. Finally, the processed bone was analyzed at 25 kV accelerating voltage by SEM.32 Three micrographs at final magnifications (×450) were taken from each animal. D

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry



RESULTS

Response Surface Analysis. RSM is an experimental design method useful in optimization processes. The ability to experimentally explore the main and interaction effects and the relationship between several independent and dependent variables are the advantages of RSM over the conventional optimization process, where one factor is varied while the others remain constant. Using RSM reveals that just a few experimental runs are required to generate a vast amount of information for optimization, which reduces the time and cost required to obtain the optimized parameters. According to Table 2, the arrangement of CCD permits the progress of the suitable empirical equations.26 The estimated regression coefficients of two high-pressure variables, along with the corresponding R2, p values, lack of fit test, significant probability of each parameter, and their F ratios, are shown in Table 3. The final reduced models (FRMs) fitted for PDI and APS showed high coefficients of determination (0.953 and 0.965, respectively), which indicated the satisfactory adjustment of the reduced response surface models employed for describing the variation of dependent variables as a function of the main preparation parameters.33 The analyses of PDI and APS of T2-nano were significantly (p < 0.05) influenced by cycle and pressure (Table 3). Both response variables were significantly (p < 0.05) fitted by a second-order regression equation. In fact, the variability of PDI was significantly (p < 0.05) defined as a function of linear and interaction effects of both homogenization variables (Table 3). In addition, the variability of APS was significantly (p < 0.05) defined as a function of linear effects of pressure variable (p = 0.00) and interaction as well as quadratic effects of both homogenization variables (Table 3). In the present study, both high-pressure variables had the same negative (b1 and b2) significant (p < 0.05) effect on APS and PDI (Table 3). It was concluded that the APS and PDI of the fortified milk powder were significantly (p < 0.05) decreased with increasing pressure and cycle (Table 3). Effect of Homogenization Condition on Characteristics of Fortified Milk Powder. Figure 1 demonstrates the APS of T1, T2, and T2-nano. Lower PDI values for T2-nano compared with T2 and T1 are clearly presented in the figure. Figure 2 exhibits the significant (p = 0.00) interaction effects of high-pressure variables on APS and PDI of the fortified milk powder. The interaction effect of cycle and pressure decreased the APS of the fortified milk powder. This observation confirmed that the presence of the interaction effects improved the fitness of the FRM to the experimental data for the APS. The interaction between cycle and pressure had equal significant (p = 0.00) effects on the APS and PDI of the fortified milk powder (Table 3). Optimization and Validation of Homogenization Process. The multiple response optimizations leading to the minimum APS and PDI of the T2-nano were carried out. The overall optimal condition leading to the minimum PDI and APS was predicted to be obtained at the combined level of four cycles and ∼300 bar pressure (Figure 3). The average cycle and pressure led to the minimum PDI. Moreover, the region resulting in the smallest average size was predicted to be obtained by the average cycle and pressure. Under corresponding optimum condition, the desirable fortified milk powder with the predicted optimum PDI and APS was prepared to verify the accuracy of the reduced models. Thus, PDI and APS of the

Figure 2. Response surface plots showing the interaction effect of independent variables on polydispersity index (PDI) and average particle size (APS) of the fortified milk powder.

Figure 3. Response optimization, parameters, predicted responses (y), and desirability (d). PS, particle size; PDI, polydispersity index.

prepared fortified milk powder were determined and subsequently compared with the predicted values. The corresponding response values obtained from the experimental data and the ones predicted by the reduced models were observed to be close together (Table 4), and no significant (p = 0.63) difference was observed between the experimental and predicted values (Table 4), thus indicating the adequacy of the FRMs fitted by RSM. Briefly, the optimization of high-pressure homogenization of the calcium citrate fortified milk powder resulting in nanoparticles (100−1000 nm) was targeted. In this study, either increase or decrease of the pressure from 300 to 200 bar or from 300 to 400 bar resulted in the larger particle size and higher PDI. The same undesirable particle size and PDI were obtained with the application of more or fewer than four cycles. E

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

those in basal diets and T1. Moreover, significant difference (p = 0.00) was observed between T1 and basal diet in OVX rats. On the other hand, there was no significant (p = 0.18) difference between T1 and basal diet in OVX-OS rats. It is apparent from this table that the T2-nano group had the greatest femur calcium content compared with other groups in both OVX and OVX-OS rats. Furthermore, the femoral calcium in OVX rats was significantly (p = 0.00) higher than in OVXOS rats (Table 5), therefore indicating higher femoral bone mineral density. Moreover, the results in Table 5 show that the amount of bone calcium in the sham group was significantly (p = 0.00) higher than in OVX and OVX-OS rats. Effect of Experimental Diets on Mechanical Properties of Bones in Sham, OVX, and OVX-OS Rats. Maximum load (bone strength) was used to evaluate calcium bioavailability. Table 5 presents the results of maximum load in sham, OVX, and OVX-OS rats. The strength test revealed a significant (p = 0.02) positive effect for the fortified diet on the rat femurs. As the results show, the T2 intake in sham, OVX, and OVX-OS rats significantly (p = 0.02) enhanced the maximum load in femurs compared with basal diet and T1 groups. In the mechanical testing, the maximum load of T1 groups slightly increased in comparison with basal diet groups. However, there was no significant (p = 0.09) difference in maximum load between these groups after 8 weeks of feeding in sham and OVX rats, whereas there was a significant (p = 0.00) difference between T1 and basal diet groups in OVX-OS rats. There was no significant difference (p = 0.08) between the bone strength of OVX and OVX-OS rats receiving T2 during the experiment, but the present results (Table 5) demonstrated significant improvement in bone strength and maximum load in the sham group fed with fortified milk powder compared to the rest. The results obtained from this study indicated that fortified milk powder may have a preventive effect on bone loss in rats. In addition, rats in the T2-nano groups had significantly (p = 0.01) higher bone stiffness and bone strength compared with other groups in all sham, OVX, and OVX-OS rats (Table 5). Therefore, particle size reduction in the diets could increase maximum load and affect bone strength. As shown in Table 5, it was found that the bone strength of OVX rats receiving T2-nano significantly (p = 0.01) increased compared with OVX-OS rats. Interestingly, it was found that OVX rats fed T2-nano had similar maximum load with T2-nano sham group with no significant difference (p = 0.23). Effect of Experimental Diets on Bone Morphology of Sham, OVX, and OVX-OS Rats. Alterations of bone morphology can be compared among all experimental diets in Figure 5. The SEM images of basal diet groups (as control diet) of sham, OVX, and OVX-OS rats clearly showed severe reduction of bone mass in the femurs (Figure 5A,E,I). SEM of

Table 4. Comparison between Experimental and Predicted Values Based on Final Reduced Modelsa APSb

PDIb

run

Y0

Yi

Y0 − Yi

Y0

Yi

Y0 − Yi

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0.497 0.350 0.327 0.259 0.341 0.326 0.368 0.344 0.341 0.360 0.483 0.369 0.340 0.319

0.498 0.341 0.341 0.276 0.352 0.341 0.363 0.341 0.324 0.341 0.488 0.359 0.341 0.317

−0.001 0.009 −0.014 −0.017 −0.011 −0.356 −0.358 0.003 0.017 0.019 −0.005 0.010 −0.001 0.002

0.514 0.452 0.439 0.480 0.560 0.491 0.320 0.450 0.480 0.440 0.370 0.544 0.470 0.468

0.518 0.457 0.457 0.471 0.553 0.457 0.326 0.457 0.490 0.457 0.363 0.547 0.457 0.468

−0.004 −0.005 −0.018 −0.462 0.007 0.034 −0.006 −0.007 −0.010 −0.017 0.007 −0.003 0.013 0.000

a

APS, average particle size; PDI, polydispersity index; Y0, experimental value; Yi, predicted value; Y0 − Yi, residue. bNo significant difference (p > 0.05) between Y0 and Yi.

Particle Morphology. The transmission electron microscopy (TEM) images of T1, T2, and T2-nano are shown in Figure 4. Nanoparticles are generally characterized by their size and morphology using such advanced microscopic techniques as TEM. As Figure 4 shows, the size of particles was significantly reduced in nanofortified milk powder (C) compared with fortified milk (B) and skim milk (A) powders. The results of TEM were in agreement with particle size analyzer for all milk powders. Effect of Experimental Diets on Serum Calcium in Sham, OVX, and OVX-OS Rats. Blood was collected at the beginning of the experiment showing the serum calcium content of 2.54 mmol/L. Table 5 compares the experimental data on total serum calcium concentration of sham, OVX, and OVX-OS rats at the end of experiments. It is apparent from this table that, although the highest amount of total serum calcium was fournd for T2-nano, regardless of treatments, no significant (p = 0.21) difference was found among the experimental diets and between the groups (sham, OVX, and OVX-OS). Effect of Experimental Diets on Bone Calcium in Sham, OVX, and OVX-OS Rats. The results obtained from the analysis of bone calcium are shown in Table 5. To assess calcium bioavailability, bone calcium content was tested. In Table 5 there is a clear trend of decreasing bone calcium in T2-nano, T2, T1, and basal diet in both OVX and OVX-OS rats. Indeed, bone calcium content was significantly (p = 0.00) higher in both OVX and OVX-OS groups fed T2 compared to

Figure 4. Transmission electron micrographs (TEM) of the milk powder particles: T1 (A); T2 (B); T2-nano (C). F

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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0.05aA 0.09aA 0.07aA 0.19aA 2.84 2.95 3.01 3.04

diet

basal diet T1 T2 T2-nano

rat femurs receiving T1 illustrated no immense cracks resulting from increasing bone mass (Figure 5B,F,J). It was found that the bone morphology of T1 groups had an improvement compared to basal diet in sham, OVX, and OVX-OS rats. Treatment with T2 restored the morphological abnormalities (Figure 5C,G,K) in the groups. As compared with T2, treatment with T2-nano demonstrated better status of bones. It can be seen from Figure 5D,H,L that femur morphology and canal openings had a great improvement in T2-nano groups. After 8 weeks of feeding with experimental diets, the SEM images of the rat femurs showed that bone morphology was better improved in the OVX groups compared with the OVX-OS groups. In all sham, OVX, and OVX-OS rats, T2-nano showed better bone morphology than the rest of the diet groups. Effect of Experimental Diets on Absorption and Bioavailability of Calcium in Sham, OVX, and OVX-OS Rats. The results obtained from the calcium absorption and bioavailability analysis of sham, OVX, and OVX-OS rats are shown in Figure 6. It can be seen from the data in Figure 6a that the T2 groups had significantly (p = 0.00) more absorption than the T1 groups in both OVX and OVX-OS rats; however, the difference between T2 and T1 was not significant (p = 0.11) in the sham group. In addition, calcium absorption in T2-nano was significantly (p = 0.00) higher than T2 in all groups. As a result, the percentage of calcium absorption in the T2-nano group was at the highest level compared with the other groups for sham, OVX, and OVX-OS rats. Under experimental conditions, the absorption of calcium for T2-nano had a significant (p = 0.00) increase in sham and OVX rats compared to OVX-OS rats. On the other hand, the opposite trend was observed in calcium absorption for T2 (Figure 6a). From the data in Figure 6b, the same trends as calcium absorption were observed for the bioavailability of calcium with significant differences (p = 0.00) among the groups in sham, OVX, and OVX-OS rats. Similar to the calcium absorption, in all groups of rats, calcium bioavailability of T2 was higher than T1. Moreover, the percentage of calcium bioavailability in T2-nano was significantly (p = 0.00) higher than in T2. As clearly displayed in Figure 6b, the amount of calcium bioavailability had a positive significant (p = 0.00) impact in sham and OVX compared with OVX-OS rats.

a Means with different lower case letters (a−d) in a column are significantly (p < 0.05) different. Means with different upper case letters (A−C) in a row are significantly (p < 0.05) different. T1, skim milk; T2, fortified milk powder containing calcium citrate + vitamins (D3 + K1 + B6) + EPA and DHA + inulin.

0.21dC 1.30cC 0.57bB 0.21aB ± ± ± ±

OVX-OS

56.40 59.70 65.99 70.28 1.32cB 1.31cB 0.91bB 1.62aA ± ± ± ±

OVX

62.53 63.59 68.93 87.37 0.47cA 0.34cA 0.70bA 1.21aA ± ± ± ±

sham

68.77 69.17 72.07 90.06 0.03cC 0.01cC 0.11bC 0.08aC ± ± ± ±

OVX-OS

0.65 0.77 1.40 2.79 ± ± ± ±

0.03dB 0.04cB 0.08bB 0.07aB

OVX

± ± ± ±

1.71 2.28 4.54 5.56 sham

0.08aA 0.01aA 0.36aA 0.16aA ± ± ± ± 2.73 2.85 2.89 3.05 ± ± ± ±

0.06aA 0.12aA 0.04aA 0.18aA

OVX

± ± ± ±

2.86 2.89 2.96 3.05 sham

OVX-OS

5.26 5.31 5.40 6.42

0.12bA 0.18bA 0.06bA 0.04aA

maximum load (N) bone calcium (mmol/g) serum calcium (mmol/L)

Table 5. Serum Calcium Content, Bone Calcium Content, and Maximum Load in Sham, Ovariectomized (OVX), and OVX-Osteoporosis (OS) Rats (Mean ± SD; n =8)a

Journal of Agricultural and Food Chemistry



DISCUSSION This study set out with the aim of assessing the importance of suitable formula and reducing particle size in improving calcium absorption and calcium bioavailability. With respect to calcium, the average intakes among postmenopausal women are below the current recommended value. Some researchers34,35 have mainly been interested in calcium fortification, which is an important source of dietary calcium and a basic defense against osteoporosis. The results of the present study showed that fortification with calcium and suitable enhancers had a positive effect on femur morphology, bone strength, and bone calcium. Results of SEM are in agreement with previous findings,36 which showed that improved calcium absorption could decrease the occurrence of bone fracture and osteoporosis. The current study developed a calcium citrate fortified milk powder to improve the calcium absorption and bioavailability and to decrease the occurrences of bone fracture as well as osteoporosis. For instance, researchers have previously demonstrated that calcium absorption is determined by the chemical form of the calcium compound, other components of G

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 5. Bone SEM images of OVX rats (A−D), OVX-OS rats (E−H), and sham (I−L): basal diet (A, E, and I), T1 (B, F, and J), T2 (C, G, and K); calcium citrate + vitamins (D3 + K1 + B6) + EPA + DHA + inulin and T2-nano (D, H, and L).

the diet, and the absorption capacity of the intestines.37 The first important mentioned factor was the chemical form of the calcium compound. Calcium citrate with 21% of elemental calcium is widely used in calcium supplements and fortified foods.38 In addition, calcium citrate is absorbed equally well when taken with or without food.39 It is also the best choice for people with inflammatory bowel disease and other conditions that hamper calcium absorption.40 Therefore, it was chosen as the preferred source for fortification of skim milk powder. It has also been reported that there was difference in the bioavailability of calcium from milk and that from calcium salts.16 In the present study, observations showed that the fortified milk powder had more absorption and bioavailability compared to the skim milk powder. These differences might be due to the added absorption enhancers as the components of the diet. In fact, mineral bioavailability could be influenced by several factors in the diet. The magnitude of this influence depends on inhibitors and promoters contained in meal and the diet composition.

Some researchers observed positive effects of inulin and the other compounds (omega-3, vitamins D3, K1, and B6) on calcium absorption and bioavailability.41−45 The present results indicated that when calcium absorption enhancers were used in combination, absorption and bioavailability of calcium increased (Figure 6). These observations confirmed that bone loss caused via osteoporosis is preventable by effective calcium absorption enhancers. Furthermore, some researchers concluded that hormonal status and an adequate intake of calcium are major factors in determining calcium and bone status, whereas the particle size of the calcium supplement is not significant in this relation.46 On the other hand, Cho et al.47 demonstrated that nanoparticles had higher absorption and more extensive organ distribution when administered orally. Furthermore, Wegmüller et al.48 stated that decreasing the particle size of elemental iron powders increases iron absorption. Moreover, Singh et al.49 studied the effect of different-sized cerium oxide nanoparticles on the decomposition and hydrogen absorption kinetics of magnesium hydride. Their results showed nanoparticles had 1.5 times better absorption kinetics with respect to pure H

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry



REFERENCES

(1) Badaruddoza; Hundal, M. K. Comparison of anthropometric characteristics and blood pressure phenotypes between pre-and postmenopausal Punjabi women. Anthropologist 2009, 11, 271−275. (2) Shapiro, R.; Heaney, R. Co-dependence of calcium and phosphorus for growth and bone development under conditions of varying deficiency. Bone 2003, 32, 532−540. (3) O’Brien, K.; Abrams, S.; Liang, L.; Ellis, K.; Gagel, R. Increased efficiency of calcium absorption during short periods of inadequate calcium intake in girls. Am. J. Clin. Nutr. 1996, 63, 579−583. (4) Miller, G. D.; Jarvis, J. K.; McBean, L. D. The importance of meeting calcium needs with foods. J. Am. Coll. Nutr. 2001, 20, 168S− 185S. (5) Hasling, C.; Charles, P.; Jensen, F. T.; Mosekilde, L. Calcium metabolism in postmenopausal osteoporosis: the influence of dietary calcium and net absorbed calcium. J. Bone Miner. Res. 1990, 5, 939− 946. (6) Hunt, C. D.; Johnson, L. K. Calcium requirements: New estimations for men and women by cross-sectional statistical analyses of calcium balance data from metabolic studies. Am. J. Clin. Nutr. 2007, 86, 1054−1063. (7) Ma, J.; Johns, R. A.; Stafford, R. S. Americans are not meeting current calcium recommendations. Am. J. Clin. Nutr. 2007, 85, 1361− 1366. (8) Straub, D. Calcium supplementation in clinical practice: a review of forms, doses and indications. Nutr. Clin. Pract. 2007, 22, 286−296. (9) Soto, A. M.; Morales, P.; Haza, A. I.; Garcia, M. L.; Selgas, M. D. Bioavailability of calcium from enriched meat products using Caco-2 cells. Food Res. Int. 2014, 55, 263−270. (10) World Health Organization. Human Vitamin and Mineral Requirements; Joint FAO/WHO Expert Consultation, Bangkok, Thailand; WHO: Geneva, Switzerland, 2001; pp 48−197. (11) Ministry of Health and Family Welfare Government of India. Guidelines for Control of Iron Deficiency Anaemia, Adolescent Division; 2013; pp 16−18. (12) Heaney, R. P. Dietary protein and phosphorus do not affect calcium absorption. Am. J. Clin. Nutr. 2000, 72, 675−676. (13) Mekmene, O.; Le Graët, Y.; Gaucheron, F. A model for predicting salt equilibria in milk and mineral-enriched milks. Food Chem. 2009, 116, 233−239. (14) Van den Hee, R. M.; Regin, E. M.; Miret, S.; Slettenaar, M.; Duchateau, G. S.; Rietveld, A. G.; Wilkinson, J. E.; Teucher, B. Calcium absorption from fortified ice cream formulations compared with calcium absorption from milk. J. Am. Diet. Assoc. 2009, 109, 830− 835. (15) Singh, G.; Arora, S.; Sharma, G. S.; Sindhu, J. S.; Kansal, V. K.; Sangwan, R. B. Heat stability and calcium bioavailability of calciumfortified milk. LWT−Food Sci. Technol. 2007, 40, 625−631. (16) Costa, F. F.; Resende, J. V.; Abreu, L. R.; Goff, H. D. Effect of calcium chloride addition on ice cream structure and quality. J. Dairy Sci. 2008, 91, 2165−2174. (17) Daengprok, W.; Garnjanagoonchorna, W.; Mine, Y. Fermented pork sausage fortified with commercial or hen eggshell calcium lactate. Meat Sci. 2002, 62, 199−204. (18) Cáceres, E.; García, M. L.; Selgas, M. D. Design of a new cooked meat sausage enriched with calcium. Meat Sci. 2006, 73, 368−377. (19) Heaney, R. P.; Bilezikian, J. P.; Holick, M. F.; Nieves, J. W.; Weaver, C. M. The role of calcium in peri- and postmenopausal women: 2006 position statement of The North American Menopause Society. Menopause 2006, 13, 862−877. (20) Ayed, M. A.; Thannoun, A. M. Calcium bioavailability of calcium carbonate based diets formale growing rats. Mesopotamia J. Agric. 2006, 34, 1−14. (21) Waterbeemd, H. V.; Lennernäs, H.; Artursson, P. The importance of gut wall metabolism in determining drug bioavailability. In Drug Bioavailability-Estimation of Solubility Permeability, Absorption and Bioavailability; WaterBeemd, H., Testa, B., Eds.; Wiley-VCH: Weinheim, Germany, 2009; 321 pp.

Figure 6. (a) Absorption and (b) bioavailability of calcium for sham, OVX rats, and OVX-OS rats. Different lower case letters show significant difference (p < 0.05) between rats fed different diets. Different upper case letters show significant difference (p < 0.05) among rats fed the same diet.

magnesium hydride. Similarly, the results of the present study revealed that using a suitable composition and particle size reduction to nanoscale may induce high intestinal calcium absorption and decrease bone loss by ovariectomy and osteoporosis. The findings of the current study are also consistent with those of Kruger et al.,43 who indicated that improved calcium absorption could decrease the occurrences of bone fracture and osteoporosis. Thus, the data on the absorption and bioavailability indicated the importance of suitable formulation and preparation conditions in high intestinal absorption and high bioavailability of calcium in rats. Although, the current findings demonstrate the efficacy of nutrients and reducing particle size in the treatment of osteoporosis in rats, further experimental investigations are required to clarify the mechanism of this system in the human body.



Article

AUTHOR INFORMATION

Corresponding Author

*(M.Y.A.M.) Phone: +603 8947 1952. Fax: +603 8943 9745. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ABBREVIATIONS USED ANOVA, analysis of variance; APS, average particle size; CCD, central composite design; DHA, docosahexaenoic acid; EFTEM, energy filtered transmission electron microscopy; EPA, eicosapentaenoic acid; FRM, final reduced model; OS, osteoporosis; OVX, ovariectomized; PDI, polydispersity index; RSM, response surface methodology; SEM, scanning electron microscopy; TEM, transmission electron microscopy I

DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry (22) Shimomura, M.; Sawadaishi, T. Bottom-up strategy of materials fabrication: a new trend in nanotechnology of soft materials. Curr. Opin. Colloid Interface Sci. 2001, 6, 11−16. (23) Whitesides, G. M.; Grzybowski, B. Self-assembly at all scales. Science 2002, 295, 2418−2421. (24) Mohanty, A. K.; Dilnawaz, F.; Mohanty, C.; Sahoo, S. K. Etoposide-loaded biodegradable amphiphilicmethoxy (polyethylene glycol) and poly(ε-caprolactone) copolymeric micelles as drug delivery vehicle for cancer therapy. Drug Delivery 2010, 17, 330−342. (25) Thakkar, K. N.; Snehit, S.; Mhatre, M. S.; Rasesh, Y.; Parikh, M. S. Biological synthesis of metallic nanoparticles. Nanomedicine 2010, 6, 257−262. (26) Montgomery, D. C. Design and Analysis of Experiments; Wiley: New York, 2001. (27) Rasti, B.; Jinap, S.; Mozafari, M. R.; Abd-Manap, M. Y. Optimization on preparation condition of polyunsaturated fatty acids nanoliposome prepared by Mozafari method. J. Liposome Res. 2014, 24, 99−105. (28) Shirke, S. S.; Jadhav, S. R.; Jagtap, A. G. Methanolic extract of Cuminum cyminum inhibits ovariectomy-induced bone loss in rats. Exp. Biol. Med. 2008, 233, 1403−1410. (29) Park, H.; Jeon, B.; Ahn, J.; Kwak, H. Effects of nano calcium supplemented milk on bone calcium metabolism in ovariectomized rats. Asian−Australas. J. Anim. Sci. 2007, 20, 12−66. (30) Bimonte-Nelson, H. A.; Singleton, R. S.; Hunter, C. L.; Price, K. L.; Moore, A. B.; Granholm, A. E. Ovarian hormones and cognition in the aged female rat: I. Long-term, but not short-term, ovariectomy enhances spatial performance. Behav. Neurosci. 2003, 117, 1395−1406. (31) Ken, K.; Yasuhiro, T.; Hiroaki, M.; Jun-Ichi, Y.; Yasuhiro, M.; Hiroshi, K.; Akira, I.; Masayoshi, K.; Seiichiro, A.; Yukihiro, T. Milk basic protein enhances the bone strength in ovariectomized rats. J. Food Biochem. 2000, 24, 467−476. (32) Erfanian, A.; Mirhosseini, H.; AbdManap, M. H.; Rasti, B.; Hair Bejo, M. Influence of nano-size reduction on absorption and bioavailability of calcium from fortified milk powder in rats. Food Res. Int. 2014, 66, 1−11. (33) Joglekar, A.; May, A. Product excellence through design of experiments. Cereal Foods World 1987, 32, 857−868. (34) Chen, Y. C.; Chen, T. C. Mineral utilization in layers as influenced by dietary oligofructose and inulin. Int. J. Poult. Sci. 2004, 3, 442−445. (35) Kaushik, R.; Sachdeva, B.; Arora, S.; Kapila, S.; Wadhwa, B. K. Bioavailability of vitamin D2 and calcium from fortified milk. Food Chem. 2014, 147, 307−311. (36) Sunyecz, J. A. The use of calcium and vitamin D in the management of osteoporosis. Ther. Clin. Risk Manage. 2008, 4, 827− 836. (37) Deroisy, R.; Zartarian, M.; Meurmans, L.; Nelissenne, N.; Micheletti, M.; Albert, A.; Reginster, J. Acute changes in serum calcium and parathyroid hormone circulating levels induced by the oral intake of five currently available calcium salts in healthy male volunteers. Clin. Rheumatol. 1997, 16, 249−253. (38) National Institutes of Health. Optimal Calcium Intake; NIH Consensus Statement; Bethesda, MD, USA, 1994; Vol. 12, pp 1−31. (39) Straub, D. A. Calcium supplementation in clinical practice: a review of forms, doses, and indications. Nutr. Clin. Pract. 2007, 22, 286−296. (40) Committee to review dietary reference intakes for vitamin D and calcium, food and nutrition board, institute of medicine. Dietary Reference Intakes for Calcium and Vitamin D; National Academy Press: Washington, DC, USA, 2010. (41) Hunt, J. R.; Hunt, C. D.; Zito, C. A.; Idso, J. P.; Johnson, L. K. Calcium requirements of growing rats based on bone mass, structure, or biomechanical strength are similar. J. Nutr. 2008, 138, 1462−1468. (42) Probst-Hensch, N. M.; Imboden, M.; Dietrich, D. F.; Barthélemy, J. C.; Ackermann-Liebrich, U.; Berger, W.; Gaspoz, J. M.; Schwartz, J. Glutathione s-transferasepolymorphisms, passive smoking, obesity, and heart rate variability in nonsmokers. Environ. Health Perspect. 2008, 116, 1494−1499.

(43) Kruger, M. C.; Gallaher, B. W.; Schollum, L. M. The effects of fructooligosaccharides with various degrees of polymerization on calcium bioavailability in the growing rat. Exp. Biol. Med. 2003, 228, 683−688. (44) Sun, D.; Krishnan, A.; Zaman, K.; Lawrence, R.; Bhattacharya, A.; Fernandes, G. Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice. J. Bone Miner. Res. 2003, 18, 1206−1216. (45) Watkins, B. A.; Reinwald, S.; Li, Y.; Seifert, M. F. Protective actions of soy isoflavones and n-3 PUFAs on bone mass in ovariectomized rats. J. Nutr. Biochem. 2005, 16, 479−88. (46) Shahnazari, M.; Martin, B. R.; Legette, L. L.; Lachcik, P. J.; Welch, J.; Weaver, C. M. Diet calcium level but not calcium supplement particle size affects bone density and mechanical properties in ovariectomized rats. J. Nutr. 2009, 139, 1308−1314. (47) Cho, W. S.; Kang, B. C.; Lee, J. K.; Jeong, J.; Che, J. H.; Seok, S. H. Comparative absorption, distribution, and excretion of titanium dioxide and zinc oxide nano particles after repeated oral administration. Part. Fibre Toxicol. 2013, 10, 9−19. (48) Wegmüller, R.; Zimmermann, M. B.; Moretti, D.; Arnold, M.; Langhans, W.; Hurrell, R. F. Particle size reduction and encapsulation affect the bioavailability of ferric pyrophosphate in rats. J. Nutr. 2004, 134, 3301−3304. (49) Singh, R. K.; Sadhasivam, T.; Sheeja, G. I.; Singh, P.; Srivastava, O. N. Effect of different sized CeO2 nano particles on decomposition and hydrogen absorption kinetics of magnesium hydride. Int. J. Hydrogen Energy 2013, 38, 1−5.

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DOI: 10.1021/acs.jafc.5b01468 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Absorption and Bioavailability of Nano-Size Reduced Calcium Citrate Fortified Milk Powder in Ovariectomized and Ovariectomized-Osteoporosis Rats.

The aim of this study was to evaluate the effects of fortification and nano-size reduction on calcium absorption and bioavailability of milk powder fo...
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