Bioavailability of vitamin D2 and calcium from fortified milk

Accepted Manuscript

Analytical Methods

Bioavailability of vitamin D2 and calcium from fortified milk

Ravinder Kaushik, Bhawana Sachdeva, Sumit Arora, Suman Kapila, BalbirKaur Wadhwa

PII: S0308-8146(13)01415-5DOI: http://dx.doi.org/10.1016/j.foodchem.2013.09.150Reference: FOCH 14778

To appear in: Food Chemistry

Received Date: 22 November 2012Revised Date: 4 June 2013Accepted Date: 29 September 2013

Please cite this article as: Kaushik, R., Sachdeva, B., Arora, S., Kapila, S., Wadhwa, B.K., Bioavailability of vitaminD2 and calcium from fortified milk, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.09.150

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http://dx.doi.org/10.1016/j.foodchem.2013.09.150

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1

Bioavailability of vitamin D2 and calcium from fortified milk 1

Ravinder Kaushik, Bhawana Sachdeva, Sumit Arora*, Suman Kapila and Balbir Kaur 2

Wadhwa 3

National Dairy Research Institute, Karnal, Haryana, India 4

1) Ravinder Kaushik 5

Ph.D Student 6

Dairy Chemistry Division 7


Ph.No. +91-9416962729 (M) 9

Email: [email protected] 10

2) Bhawana Sachdeva 11

Ph.D Student 12



Ph.No. +91-8930691698 (M) 15


3) Corresponding Author * 17

Dr. Sumit Arora 18

Principal Scientist 19



Ph. No. 0184-2259156 (O), +91-9896054444 (M) 22


2

FAX No. 0184-2250042 24

4) Dr. Suman Kapila 25

Senior Scientist 26

Animal Biochemistry Division 27


Ph.No. 0184-2259134 (O), +91-9416742567 (M) 29


5) Dr. Balbir Kaur Wadhwa 31

Head, Principal scientist 32



Ph. No. 0184-2259165 (O), +91-9996393876 (M) 35


37

Abstract 38

The objective of the present investigation was to determine bioavailability of calcium and 39

vitamin D2 from milk fortified with either calcium or vitamin D2 alone or when both were used 40

for preparation of multiple micronutrient fortified milk and also to study its interaction with iron 41

and zinc bioavailability. 32 weanling male rats (aged 21-28 days) were assigned into four groups 42

and were fed milk and milk fortified with calcium, vitamin D2 and calcium+vitamin D2. Vitamin 43

D2 increased calcium bioavailability. In multiple micronutrient fortified milk, the bioavailability 44

of both calcium+vitamin D2 increased in comparison to single fortification. Calcium fortification 45

decreased, whereas vitamin D2 increased the absorption of iron and zinc. However, calcium and 46

3

vitamin D2 when fortified in combination, the iron and zinc bioavailability was similar to control 47

group. There was positive association between bioavailability of calcium and vitamin D2. 48

Keywords: Fortification; calcium; vitamin D2; iron; zinc; bioavailability 49

Highlights: 50

1. Vitamin D2 increases the calcium absorption 51

2. Calcium fortification decreases the iron and zinc absorption 52

3. Vitamin D2 increases iron and zinc absorption 53

4. Milk serves as a good medium for bioavailable vitamin D2 and calcium 54

1. Introduction 55

Bone health is a major public health concern in industrialized countries (Cashman, 2002). 56

Calcium and vitamin D are both recognized as key nutrients in promoting bone health 57

(WHO/FAO, 2003). Dietary calcium deficiency has been linked epidemiologically to several 58

chronic diseases including osteoporosis, osteomalacia, hypertension, colon cancer and obesity 59

(Zemel, & Miller, 2004). Calcium alone constitutes about 2 percent of the total body weight and 60

most of this is distributed in bones. The majority of calcium (~99%) is found in bones and teeth. 61

The remaining 1% is contained in the serum, extravascular fluid, muscles and other tissues 62

(IOM, 1997). Childhood and adolescence are critical times to optimize peak bone mass and 63

inadequate consumption of calcium in these years increases the risk of osteoporosis and bone 64

fractures in later life (Matkovic, 1996). The current consensus is that 1.66 million hip fractures 65

occur each year worldwide, and the incidence is predicted to increase fourfold by 2050 because 66

of the increasing number of older people (WHO/FAO, 2003). In dairy industry, enrichment of 67

milk with calcium can be carried out in order to improve the functional, technological and 68

sometimes nutritional properties of milk (Pirkul, Temia, & Erdem, 1997). 69

4

Vitamin D has long been known to play an important role in bone development by promoting 70

calcium absorption in the gut and bone mineralization (Bilodeau et al., 2011). It has been 71

estimated that 1 billion people worldwide have vitamin D deficiency or insufficiency (Holick, 72

2006). Vitamin D plays a vital role in skeletal development and maintenance throughout life by 73

upregulating calcium and phosphorous absorption in the small intestine (Olds, McKinley, Moore, 74

& Kimlin, 2008). Vitamin D3 has been shown to aid the functioning of the pancreas, fetal 75

development, immunity, muscle contraction and nerve conduction in all the body cells (Feldman, 76

Pike, & Glorieux, 2005). Without vitamin D, only 10 to 15% of dietary calcium and about 60% 77

of phosphorus is absorbed. The interaction of 1,25 dihydroxy vitamin D with the vitamin D 78

receptor increases the efficiency of intestinal calcium absorption to 30 to 40 % and phosphorous 79

absorption to approximately 80 % (Holick, 2006). 80

Dietary factors also regulates 1,25 D levels in humans, mainly calcium and phosphorous. When 81

serum levels of calcium are low, 1,25 D acts on the bones, kidney and intestines to increase 82

retention and absorption of calcium until serum levels return to a normal range. Similarly, if 83

serum levels of calcium are high, production of 1, 25 D is suppressed by reduced parathyroid 84

hormone production. Vitamin D-calcium interdependencies must be taken into account. Higher 85

intakes of both vitamin D and calcium can reduce bone resorption and higher concentrations of 86

one nutrient might compensate for insufficiency in the other (Weaver, & Fleet, 2004). 87

Therefore, present study was conducted to determine the bioavailability of calcium and vitamin 88

D2 when used alone to fortify milk and to determine the interactive effect when used in 89

combination to fortify milk. The effect of calcium and vitamin D2 fortification on iron and zinc 90

absorption and retention was also determined. 91

2. Materials and methods 92

5

2.1. Animals 93

Swiss albino rats of Wister Strain with a mean weight 36.42±4.56 g, were procured from Small 94

Animal House, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, 95

India. The present study was approved by the Institutional Animal Ethics Committee (IAEC), 96

National Dairy Research Institute, Karnal, Haryana, India. 97

The animals were placed in individual metabolic cages in an environmentally controlled 98

room with a constant temperature of 20–22 ºC, under 12 hrs light-dark cycle and 55-60 % 99

relative humidity in a well ventilated room in Small Animal House, National Dairy Research 100

Institute, Karnal, Haryana, India. 32 male rats of 28 days age were assigned into four groups with 101

eight rats each and maintained for a period of 7 days as acclimatization period and 28 days as 102

experimental period. 103

2.2. Preparation of fortified milk and lyophilate 104

Cow milk and buffalo milk were mixed in 1:1 ratio and toned milk was prepared by mixing 105

whole milk, skim milk and water. The fat and solid non fat (SNF) were adjusted to 3.0 % and 8.5 106

%, respectively using Pearson square method. Pearson’s Square method handily solves the 107

problem of how to mix two solutions of known percentages without having to solve sets of 108

simultaneous equations. Milk was assigned into four parts for preparation of control and fortified 109

milk samples. Milk samples prepared were control (unfortified milk), calcium fortified milk, 110

vitamin D2 fortified milk and calcium+vitamin D2 fortified milk. Calcium fortified milk was 111

prepared by adding calcium citrate at the levels of 600 ppm calcium. Vitamin D2 fortified milk 112

was prepared by adding vitamin D2 at the level of 600 IU/L. Calcium+vitamin D2 fortified milk 113

was prepared by adding 600 ppm calcium as calcium citrate and 600 IU/L vitamin D2. Milk 114

samples were pasteurised at 63 °C for 30 min in temperature controlled waterbath (PolyScience, 115

6

USA) in air tight glass bottles. The samples were immediately cooled to 4 °C. After 2 h of 116

storage at 4 °C, milk samples were freeze dried. These lyophilized milk powders were then fed to 117

the rats along with synthetic diet. 118

2.2. Diet Composition 119

During the initial seven days, rats were fed with basal diet obtained from Small Animal 120

House, National Dairy Research Institute, (Karnal, India) in order to provide an acclimatization 121

period. Thereafter, during the experimental period, they were provided with water and synthetic 122

diet as the composition displayed in table 1, in which almost one third of the diet was replaced 123

with the (un-)fortified milk lyophilate. Composition of mineral and vitamin mixture used in 124

normal basal diets was prepared according to AOAC (2005). 125

2.3. Experimental Design: 126

The animals were assigned into 4 groups of 8 animals each and fed on 4 different diets for 28 127

days. 128

Group I : Fed on synthetic diet (67 %) and milk lyophilate (33 %). 129

Group II : Fed on synthetic diet (67 %) and calcium fortified milk lyophilate (33 %). 130

Group III : Fed on synthetic diet (67 %) and vitamin D2 fortified milk lyophilate (33 %). 131

Group IV : Fed on synthetic diet (67 %) and vitamin D2+calcium fortified milk 132

lyophilate (33 %). 133

Procedure 134

Faeces and urine were collected on a daily basis, and absorption and retention of calcium and 135

vitamin D2 measured on weekly basis for four weeks. The experimental design is shown in figure 136

1. 137

2.4. Biological indices 138

7

Biological indices namely apparent digestibility coefficient and retention of calcium and 139

vitamin D2 were estimated as described by Satnarayana (2006) to check the digestive and 140

metabolic utilisation of calcium and vitamin D2. The apparent digestibility coefficient and 141

retention of iron and zinc were also determined for interactive effect of calcium and vitamin D2 142

on bioavailability of these minerals. Apparent digestibility coefficient and retention calculated as 143

follows: 144

Apparent digestibility coefficient = (I – F) × 100 145 I 146

Retention = I- (F –U) × 100 147 I 148 Where, 149

I = intake of calcium 150

F = faecal excretion of calcium 151

U = urinary excretion of calcium 152

2.5. Measurements 153

Calcium, iron and zinc were analysed with a Shimadzu AA-7000, Atomic Absorption 154

Spectrophotometer (AAS) using the method of AOAC (2005). Samples were weighed in silica 155

crucibles and dried for one hour at 100 ºC in forced air oven. Samples were charred on hot plate 156

and ashed in muffle furnace for a minimum of sixteen hours at 600 ºC. Ash was then dissolved in 157

1 ml concentrated HNO3, 100 µl/100 ml (5 %) of lanthanum solution was also added and 158

samples were diluted with deionised water to 1000 times. 159

Vitamin D2 was analysed using the method described by Kazmi, Vieth and Rousseau 160

(2007). Vitamin D2 in milk was isolated from milk by alkaline saponification followed by liquid-161

liquid extraction with n-hexane. The vitamins extracted into hexane were concentrated by 162

evaporation. Vitamin D2 analysis was carried out by further purifying the extract, using silica 163

8

cartridge and injecting it into the C-18 column to estimate vitamin D2 by RP-HPLC with UV 164

detection (λmax 254 nm). 165

2.6. Statistical analysis 166

Means, standard deviation, RSD, linear regression analysis and 95 % confidence intervals were 167

calculated using Microsoft Excel 2007 (Microsoft Corp., Redmond, WA). Data were subjected to 168

a single way analysis of variance (ANOVA) to calculate CD value. 169

3. Results and discussion 170

3.1. Apparent digestibility coefficient and retention of calcium 171

The apparent digestibility coefficient and retention of calcium were determined after every week 172

for four weeks and mean values of four weeks are presented in table 2. After four weeks the 173

apparent digestibility coefficient obtained was 64.99, 63.51, 68.23 and 65.21 % for control, 174

calcium fortified group, vitamin D2 fortified group and vitamin D2 + calcium fortified group, 175

respectively. The vitamin D2 fortified group had a significantly higher (p<0.05) apparent 176

digestibility coefficient of calcium compared with the control and calcium fortified groups, 177

which were not statistically different (p>0.05). From the above results it was evident that 178

apparent digestibility coefficient of calcium increased with fortification of milk with vitamin D2. 179

The retention of calcium followed a similar trend as for apparent digestibility coefficient. 180

The retention was 60.71, 58.61, 64.14 and 60.83 % for control, calcium fortified group, vitamin 181

D2 fortified group and vitamin D2 + calcium fortified group (table 2). From the above results it 182

was evident that vitamin D2 increased the calcium retention. On the basis of percentage, the 183

calcium retention was similar between control and multiple micronutrient fortified milk, 184

however, quantity of total calcium which was retained increased from fortified milk. 185

9

Couzy, Kastenmayer, Vigo, Clough, Munoz-Box, & Barclay, (1995) studied calcium 186

absorption from milk and mineral water. Calcium absorption remained similar from both 187

sources, which indicated that calcium absorption was independent of the source of calcium. 188

Weaver, Martin, Costa, Saleeb, and Huth (2002) also did not observe any significant differences 189

in fractional absorption from five calcium salts in a rat model. No clear difference was observed 190

in the efficiency of calcium absorption between skimmed milk and calcium-enriched skimmed 191

milk using isotope technique in human subjects (Fairweather-Tait, Johnson, Eagles, Ganatra, 192

Kennedy, & Gurr, 1989). 193

However, it has also been reported that there was difference in the bioavailability of 194

calcium from milk and calcium salts. Ranjan, Arora, Sharma, Sindhu, Kansal, and Sangwan 195

(2005) determined the bioavailability of calcium fortified buffalo milk. Their results indicated 196

that absorption and retention increased with fortification of milk using calcium lactate and 197

calcium gluconate compared to control unfortified milk. Similar results were reported by Singh, 198

Arora, Sharma, Sindhu, Kansal, and Sangwan (2007) for calcium absorption from calcium 199

fortified milk. These discrepancies might be due to different experimental designs such as dietary 200

calcium level or the calcium status of the animal, due to different calcium salts used for 201

fortification and also matrix affected the calcium absorption. Van der Hee et al. (2009) 202

determined the bioavailability of calcium from calcium fortified ice cream and milk. Calcium 203

absorption from 3 % butterfat ice cream was 26 %, absorption from 9% coconut oil ice cream 204

was 28 % and absorption from reduced fat milk was 31 %. No significant difference in fractional 205

calcium absorption from all three samples was observed. No significant difference was observed 206

by Weaver et al. (2002) between calcium absorption from tofu and milk in premenopausal 207

women. 208

10

Kansal (1998) observed that several components viz. lactose, protein, phosphorous and 209

vitamin D and casein phosphopeptides (CPP) contribute for better availability of calcium from 210

milk. Calcium is well absorbed from human milk, with values for net calcium retention at 50 % 211

of intake for infants (Abrams, Wen, & Stuff, 1997). The majority of dietary calcium (~95 %) is 212

absorbed in the small intestine by active (vitamin D-dependent) and a passive (vitamin D-213

independent) mechanism. There is evidence that a combination of calcium and vitamin D is more 214

effective than either vitamin D or calcium alone (Fairweather-Taith, & Teucher, 2002). 215

Kansal, and Chaudhary (1982) reported higher calcium absorption and retention than our 216

results. They reported 81.1 to 88.0 % calcium absorption and 75.9 to 86.7 % retention for milk 217

and milk products. Similar calcium retention of 59, 69, 72 and 70 % was reported by Buchowski 218

(1989) using intrinsic and extrinsic tracer methods for cheese curd, milk, yogurt and calcium 219

chloride. The higher intake of nonphosphate calcium salts was suggested to increase the risk of 220

phosphorous insufficiency, which might have implications in the prevention and/or treatment of 221

osteoporosis (Fairweather-Taith, & Teucher, 2002). 222

3.2. Apparent digestibility coefficient and retention of vitamin D2 223

The vitamin D2 apparent digestibility coefficient was 78.25 and 81.29 % for vitamin D2 fortified 224

and vitamin D2 + calcium fortified group (table 3). Vitamin D2 absorption was slightly higher in 225

calcium + vitamin D2 than vitamin D2 group; however, there was no difference. Similar trend 226

was observed in case of vitamin D2 retention. The vitamin D2 retention was 76.96 % and 80.19 227

% for vitamin D2 fortified and vitamin D2 + calcium fortified groups (table 3). However, this 228

difference was also non significant (p>0.05). From the above results, it can be concluded that 229

apparent digestibility coefficient and retention of vitamin D2 were positively affected by calcium. 230

It has been reported that, depending on the vitamin D source, the absorption of vitamin D in 231

11

humans varies between 55 % and 99 % and that absorption does not decrease significantly with 232

age (Van der Berg, 1997). Wagner, Sidhom, Whiting, Rousseau, and Veith (2008) fortified 233

cheese with vitamin D and reported that it was equally bioavailable from fortified cheeses and 234

supplements, making cheese suitable for vitamin D fortification. Fat content of the cheese did not 235

affect vitamin D bioavailability. 236

3.3. Effect of calcium and vitamin D2 fortification on apparent digestibility coefficient and % 237

retention of iron 238

Effect of calcium and vitamin D2 fortification on iron apparent digestibility coefficient and 239

retention was studied. The apparent digestibility coefficient was 27.73, 21.16, 30.45 and 26.75 % 240

for control group, calcium fortified group, vitamin D2 fortified group and vitamin D2 + calcium 241

fortified group, respectively (table 4). There was a significant decrease (p<0.05) in apparent 242

digestibility coefficient of iron in calcium fortified group in comparison to control group. Whereas, 243

a significant increase (p<0.05) was observed in apparent digestibility coefficient of iron in vitamin 244

D2 fortified group. Non significant difference (p>0.05) was observed between control and calcium 245

+ vitamin D2 fortified group. Thus, it can be inferred that calcium fortification decreases the iron 246

absorption whereas vitamin D2 fortification increased the iron absorption. Also, in multiple 247

fortified group, vitamin D2 minimized the inhibitory role of fortified calcium. 248

Similar trend was observed in effect of calcium and vitamin D2 fortification on retention of 249

iron (table 4). The retention of iron was 23.62, 15.37, 27.49 and 23.22 %, for control group, 250

calcium fortified group, vitamin D2 fortified group and vitamin D2 + calcium fortified group, 251

respectively. Vitamin D2 increased and calcium fortification decreased the iron retention. In 252

vitamin D2 + calcium fortified group, the retention of iron was statistically similar to control, 253

indicating that vitamin D2 decreased the negative effect of calcium on bioavailability of iron. 254

12

The inhibitory effect of calcium on iron absorption is well documented (Amaro and 255

Camara, 2004). Perales, Barbera, Lagarda, and Farre (2006) reported inhibitory effect of calcium 256

from fortified milk upon iron absorption. This fact shows the possible competitive effect of 257

calcium and iron in the dialysis process, where a negative correlation between total calcium 258

content in milk and iron dialysis percentage. Grinder-Pedersen, Bukhave, Jensen, Hojgaard, and 259

Hansen (2004) found no differences in absorption of non-heme-iron absorption between the 260

control and a calcium-supplemented diet with milk, calcium lactate or milk mineral isolate. 261

3.4. Effect of calcium and vitamin D2 fortification on apparent digestibility coefficient and 262

% retention of zinc 263

Effect of calcium and vitamin D2 fortification on apparent digestibility coefficient and 264

retention of zinc was studied. The apparent digestibility coefficient of zinc was 64.84, 53.31, 265

71.24 and 68.66 % for control, calcium fortified group, vitamin D2 fortified group and vitamin 266

D2 + calcium fortified group (table 5). There was significant decrease (p<0.05) in apparent 267

digestibility coefficient of zinc in calcium fortified group in comparison to control. There was 268

significant increase (p<0.05) in zinc absorption in vitamin D2 fortified group and calcium + 269

vitamin D2 fortified group. We therefore concluded that calcium fortification decreased whereas 270

vitamin D2 fortification increased the apparent digestibility coefficient of zinc. In multiple 271

fortified group, vitamin D2 minimized the inhibitory role of fortified calcium. 272

Similar trend was observed on retention of zinc. The retention of zinc was 59.16, 45.73, 273

67.42 and 63.33 %, respectively. Vitamin D2 increased and calcium fortification suppressed the 274

zinc retention (table 5). 275

The inhibitory effect of calcium on zinc absorption was well documented (Amaro and 276

Camara 2004). Perales et al. (2006) reported inhibitory effect of calcium from fortified milks 277

13

upon zinc absorption. Zinc solubility and dialysis decreased in calcium fortified milks but 278

percentage zinc uptake remained unchanged. The decrease was particularly relevant in case of 279

the dialysis percentage, where 50 % reduction was found in calcium fortified milk when 280

compared to unfortified milk. 281

4. Conclusion 282

Milk is a suitable vehicle for calcium and vitamin D2 fortification. Apparent digestibility 283

coefficient of calcium and retention from control and calcium fortified milk remains similar, but 284

the quantity of bioavailable calcium increased. Vitamin D2 increased the bioavailability of 285

calcium, iron and zinc. However, calcium reduced the bioavailability of iron and zinc. When 286

used in combination, the bioavailability of calcium and vitamin D2 increased which indicated a 287

positive interaction between both the nutrients. 288

Acknowledgements 289

This study is part of the DBT-project financially supported by the Department of 290

Biotechnology (Delhi, India). 291

References 292

Abrams, S. A., Wen, J., & Stuff, J. E. (1997). Absorption of calcium, zinc, and iron from breast 293

milk by five- to seven-month-old infants. Pediatric Research, 41, 384–390. 294

Amaro, M. A., & Camara, F. (2004). Iron availability: an updated review. International Journal 295

of Food Science and Nutrition, 55, 597-606. 296

AOAC (2005). Official methods of analysis. The association of official analytical chemists. 18th 297

edition. 481. North Fredrick Avenue Gaithersburg, Maryland, USA 298

Bilodeau, L., Dufresne, G., Deeks, J., Clement, G., Bertrand, J., Turcotte, S., Robichaud, A., 299

Beraldin, F., & Fouquet, A. (2011). Determination of vitamin D3 and 25-hydroxyvitamin 300

14

D3 in foodstuffs by HPLC UV-DAD and LC–MS/MS. Journal of Food Composition and 301

Analysis, 24(3), 441-448. 302

Buchowski, M. S. (1989). A comparison of intrinsic and extrinsic tracer methods for estimating 303

calcium bioavailability to rats from dairy foods. Journal of Nutrition, 119, 228–234. 304

Cashman, K. D. (2002). Calcium intake, calcium bioavailability and bone health. British Journal 305

of Nutrition, 87 Suppl 2:S169-177. 306

Couzy, F., Kastenmayer, P., Vigo, M., Clough, J., Munoz-Box, R., & Barclay, D. V. (1995). 307

Calcium bioavailability from a calcium and sulphate rich mineral water, compared with 308

milk, in young adult women. The American Journal of Clinical Nutrition, 62(6), 1239-309

1244. 310

Fairweather-Tait, S. J., & Teucher, B. (2002). Iron and calcium bioavailability of fortified foods 311

and dietary supplements. Nutritional Reviews, 60(11), 360-367. 312

Fairweather-Tait, S. J., Johnson, A., Eagles, J., Ganatra, S., Kennedy, H., & Gurr, M. I. (1989). 313

Studies on calcium absorption from milk using a double-label stable isotope technique. 314

British Journal of Nutrition, 62, 379–388. 315

Feldman, D., Pike, J. W., & Glorieux, F. H. (2005). Vitamin D (2nd ed), Elsevier, Academic 316

Press, London, San Diego 317

Grinder-Pedersen, L., Bukhave, K., Jensen, M., Hojgaard, L., & Hansen, M. (2004). Calcium 318

from milk or calcium-fortified foods does not inhibit nonheme-iron absorption from a 319

whole diet consumed over a 4-d period. The American Journal of Clinical Nutrition, 80, 320

404-409. 321

Holick, M. F. (2006). Resurrection of vitamin D deficiency and rickets. Journal of Clinical 322

Investment, 116, 2062-72. 323

15

IOM (1997). Dietary reference intakes for calcium, phosphorus, magnesium vitamin D and 324

fluoride. Institute of Medicine, Washington DC. National Academy Press, 71-145. 325

Kansal, V. K. (1998). Milk offers dietary calcium in best available form. Indian Dairyman, 326

50(8), 23–27. 327

Kansal, V. K., & Choudhary, S. (1982). Biological availability of calcium, phosphorus and 328

magnesium from dairy products. Milchwissenschaft, 37, 261–263. 329

Kazmi, S. A., Vieth, R., & Rousseau, D. (2008). Vitamin D fortification and quantification in 330

processed dairy products. International Dairy Journal, 17(7), 753-759. 331

Matkovic, V. (1996). Nutrition, genetics and skeletal development. Journal of American College 332

of Nutrition, 15 (6) 556-569. 333

Olds, W. J., McKinley, A. R., Moore, M. R., & Kimlin, M. G. (2008). In vitro model of vitamin 334

D3 (cholecalciferol) synthesis by UV radiation: rose-response relationships. Journal of 335

photochemistry and photobiology B: Biology, 93, 88-93. 336

Perales, S., Barbera, R., Lagarda, M. J., & Farre, R. (2006). Fortification of Milk with Calcium: 337

Effect on Calcium Bioavailability and Interactions with Iron and Zinc. Journal of 338

Agriculture and Food Chemistry, 54(13), 4901–4906. 339

Pirkul, T., Temia, A., & Erdem, Y. K. (1997). Fortification of yoghurt with calcium salts and its 340

effect on starter microorganisms and yoghurt quality. International Dairy Journal, 7, 341

547–552. 342

Ranjan, P., Arora, S., Sharma, G. S., Sindhu, J. S., Kansal, V. K., and Sangwan, R. B. (2005). 343

Bioavailability of calcium and physicochemical properties of calcium-fortified buffalo 344

milk. International Journal of Dairy Technology, 58: 185-189. 345

Satnarayana, U. ( 2006). Biochemistry. 3rd edition. pp. 113-126. 346

16

Singh, G., Arora, S., Sharma, G. S., Sindhu, J. S., Kansal, V. K., and Sangwan, R. B. (2007). 347

Heat stability and calcium bioavailability of calcium fortified milk. LWT - Food Science 348

and Technology, 40(4), 625 - 631. 349

Van den Berge, H. (1997). Bioavailability of vitamin D. European Journal of Clinical Nutrition, 350

51(Suppl 1), S76–S79. 351

Van der Hee, R. M., Miret, S., Slettenaar, M., Duchateau, G. S. M. J. E, Rietveld, A. G., 352

Wilkinson, J. E., Quail, P. J., Berry, M. J., Dainty, J. R., & Teucher, B. (2009). Calcium 353

absorption from fortified ice cream formulation compared with calcium absorption from 354

milk. Journal of the American Dietetic Association, 109, 830-835. 355

Wagner, D., Sidhom, G., Whiting, S. J., Rousseau, D., & Veith, R. (2008). The Bioavailability of 356

Vitamin D from Fortified Cheeses and Supplements Is Equivalent in Adults. The Journal 357

of Nutrition, 138(7) 1365-1371. 358

Weaver, C. M., & Fleet, J. C. (2004). Vitamin D requirements: current and future. American 359

Journal of Clinical Nutrition, 80(6), 1735S- 1739S. 360

Weaver, C. M., Martin, B. R., Costa, N. M., Saleeb, F. Z., & Huth, P. J. (2002). Absorption 361

of calcium fumarate salts is equivalent to other calcium salts when measured in the rat 362

model. Journal of Agriculture Food Chemistry, 50 (17), 4974–4975. 363

WHO/FAO (2003). Report of a Joint WHO/FAO Expert Consultation. Diet, Nutrition and the 364

Prevention of Chronic Diseases. WHO, Geneva, pp. 129-33. 365

Zemel, M. B., & Miller, S. L. (2004). Dietary calcium and dairy modulation of adiposity and obesity 366

risk. Nutrition Reviews, 62, 125–131. 367

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369

17

Figure caption 370

Figure 1: Experimental Design of Biological Experiment 371

Table Caption 372

Table 1. Composition of synthetic diet 373

374

Table 2. Apparent digestibility coefficient and retention of calcium 375

Table 3. Apparent digestibility coefficient and retention of vitamin D2 376

Table 4. Apparent digestibility coefficient and % retention of iron 377

Table 5. Apparent digestibility coefficient and retention of zinc 378

Table 1. Composition of synthetic diet

Ingredient %

Starch 59.00% for balance studies of added nutrients

Fat 10.00

Protein 16.00 % for balance studies of added nutrients

Cellulose 2.50

Sucrose 7.50

*Mineral mixture 3.50

*Vitamin mixture 1.00

Choline chloride 0.25

DL methionine 0.30

Final weight was made up to 1 kg with starch

*Mineral and Vitamin Mixture were prepared and mixed according to AOAC (2005)

Table 2. Apparent digestibility coefficient and retention of calcium

Groups Apparent digestibility

coefficient of calcium (%)

Retention of calcium

(%)

Control 64.99±1.56a 60.71±1.74

a

Calcium (600 ppm)

fortified group

63.51±1.45a 58.61±1.60

a

Vitamin D2 (600

IU/L) fortified

group

68.23±2.21b 64.14±2.55

b

Vitamin D2 +

calcium (600 ppm)

fortified group

65.21±1.06ab

60.83±1.47ab

Data are presented as means±SEM (n=8).

abMeans within columns with different lowercase superscript are significantly different

(p<0.05) from each other.

Table 3. Apparent digestibility coefficient and retention of vitamin D2


coefficient of vitamin D2

(%)

Retention of vitamin D2

(%)

Control group -- --

Calcium (600 ppm)

fortified group

-- --

Vitamin D2

(600IU/L) fortified

group

78.25±1.25a 76.96±1.37

a

Vitamin D2

(600IU/L) + calcium

(600 ppm) fortified

group

81.29±2.32a 80.19±2.43

a




Table 4. Apparent digestibility coefficient and % retention of iron


coefficient of Iron (%)

Retention of Iron

(%)

Control group 27.73±1.35b 23.62±1.25

b

Calcium (600 ppm)

fortified group

21.16±1.33a 15.37±1.09

a

Vitamin D2 (600IU/L)

fortified group

30.45±1.24c 27.49±1.19

c

Vitamin D2 (600IU/L) +

calcium (600 ppm)

fortified group

26.75±1.23b 23.22±1.34

b




Table 5. Apparent digestibility coefficient and retention of zinc


coefficient of zinc (%)

Retention of zinc

(%)

Control group 64.84±2.17b 59.16±2.34

b

Calcium (600 ppm)

fortified group

53.31±2.21a 45.73±2.55

a

Vitamin D2 (600IU/L)

fortified group

71.24±1.72c 67.42±1.79

d

Vitamin D2 (600IU/L) +

calcium (600 ppm)

fortified group

68.66±1.65c 63.33±1.60

c




Preliminary period Experimental Period

(7 days) (28 days)

1.

0 day

2.

Figure 1: Experimental Design of Biological Experiment

Control group (8 rats) Stock diet + unfortified

milk lyophilate

Calcium fortified group (8

rats)

Stock diet + calcium

fortified milk lyophilate Absorption and

retention studies

after every week

for four weeks

Calcium + vitamin D2

fortified group (8 rats)

Vitamin D2 fortified group

(8 rats)

Stock diet + vitamin D2

fortified milk lyophilate

Stock diet + calcium and

vitamin D2 fortified milk

lyophilate

18

Highlights: 379

1) Preparation of calcium and vitamin D2 fortified milk 380

2) Bioavailability of calcium and vitamin D2 from fortified milk 381

3) Interactive role of calcium and vitamin D2 382

4) Effect of fortification on absorption of iron and zinc. 383

384

385

Documents

Bioavailability of vitamin D2 and calcium from fortified milk