EFFECT OF HIGH DIETARY CALCIUM ON WEIGHT MANAGEMENT IN RATS

Transcription

1 EFFECT OF HIGH DIETARY CALCIUM ON WEIGHT MANAGEMENT IN RATS By MOHAMAD TAHA MOHAMAD B.Sc. Agric. Sci. (Agric. Biochemistry), Fac. Agric., Cairo Univ., 2007 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE In Agricultural Sciences (Agric. Biochemistry) Department of Agric. Biochemistry Faculty of Agriculture Cairo University EGYPT 2012

2 APPROVAL SHEET EFFECT OF HIGH DIETARY CALCIUM ON WEIGHT MANAGEMENT IN RATS M.Sc. Thesis In Agric. Sci. (Agric. Biochemistry) By MOHAMAD TAHA MOHAMAD B.Sc. Agric. Sci. (Agric. Biochemistry), Fac. Agric., Cairo Univ., 2007 Approval Committee Dr. SAMIR MUSTAFA ABD- EL-AZIZ..... Researcher Professor of Biochemistry, E. A. E. A., Cairo Dr. AHMED M. MOSTAFA ABOUL-ENEIN... Professor of Agric. Biochemistry, Fac. Agric., Cairo University Dr. GAMAL SAYED ALY EL-BAROTY Professor of Agric. Biochemistry, Fac. Agric., Cairo University Dr. RADWAN SEDKY FARAG... Professor of Agric. Biochemistry, Fac. Agric., Cairo University Date: / / 2012

3 SUPERVISION SHEET EFFECT OF HIGH DIETARY CALCIUM ON WEIGHT MANAGEMENT IN RATS M.Sc. Thesis In Agric. Sci. (Agric. Biochemistry) By MOHAMAD TAHA MOHAMAD B.Sc. Agric. Sci. (Agric. Biochemistry), Fac. Agric., Cairo Univ., 2007 SUPERVISION COMMITTEE Dr. RADWAN SEDKY FARAG Professor of Agric. Biochemistry, Fac. Agric., Cairo University Dr. GAMAL SAYED ALY EL-BAROTY Professor of Agric. Biochemistry, Fac. Agric., Cairo University Dr. GHADA IBRAHIM ISSA Researcher Professor of Radiobiochemistry, EAEA, Cairo

4 Name of Candidate: Mohamad Taha Mohamad Degree: M.Sc. Title of Thesis: Effect of High Dietary Calcium on Weight Management in Rats. Supervisors: Dr. Radwan Sedky Farag. Dr. Gamal Sayed Ali El-Baroty Dr. Ghada Ibrahim Issa. Department: Agric. Biochemistry Approval: / / 2012 ABSTRACT The present study was undertaken to find out a suitable dietary regime to maintain a lower prevalence of overweight or obesity by adjusting the diet components. Therefore, male Swiss albino rats were selected according to their ages and divided into two main groups, i.e., premature and mature groups. Each rat group was divided into 4 subgroups and each subgroup was fed on a diet of varied composition. Serum levels of lipids, calcium, phosphorous and testosterone were determined in addition to body weight measurement. The results indicate non-significant decrease of percentage of body weight gain in premature rats fed on high-calcium diets while significant decrease of percentage of body weight gain in mature rats fed on the same diet composition. The levels of serum HDL-C, LDL-C, triglycerides and testosterone were significantly decreased in premature rats fed high- calcium diets. In premature rats, only rat subgroup fed on high calcium from milk, showed a significant decrease in serum cholesterol levels. Calcium and phosphorus levels exhibited non- significant change between premature rats. In mature rats, LDL-C data demonstrate nonsignificant changes while cholesterol and triglyceride levels were significantly decreased in rats fed high -calcium diet compared to control. HDL-C level revealed a significant decrease in sera of mature rats fed on high calcium from milk. Serum testosterone levels were significantly decreased in mature rats fed low- fat diets or low fat diets supplemented with high- calcium level. In general, one would suggest to consume low fat diet (4%) supplemented with high calcium from dry skimmed milk fortified with hydroxyapatite as suitable dietary program to avoid overweight or obesity. Key words: Premature and mature rats, lipid profile, calcium, phosphorus, testosterone, obesity.

5 ACKNOWLEDGEMENT I wish to confess my thanks to Allah for everything; accomplishing this work, kind parents, the supervision board and those kind people who encouraged me during master degree journey. I wish to express my sincere thanks, deepest gratitude and appreciation to Dr. Radwan Sedky Farag, Professor of Biochemistry and Dr. Gamal Ali El-Baroty, Professor of Biochemistry, Faculty of Agriculture, Cairo University for their supervision, continued assistance and guidance throughout the course of study and revision the manuscript of this thesis. I wish to express my sincere thanks, deepest gratitude and appreciation to Dr. Ghada Ibrahim Issa, Professor of radio Biochemistry, Egyptian Atomic Energy Authority, for suggesting the subject, for providing research facilities, constructive supervision and criticism throughout the course of the work. In addition, many thanks to Dr. Osama Ahmad Abbas, lecturer of Biochemistry, Egyptian Atomic Energy Authority, for helping me all the time, guidance through the practical work and supporting me to complete this thesis. Also, Grateful appreciation to all staff members of Academy of Scientific Research and Technology for funding my master scholarship specially Dr. Tarek Hussien and Dr. Nabila El Sheikh Sheikh.

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7 اسن الطبلب: عنوان الرسالة: ح ذ طه ح ذ ػبذاىشح حأث ش ص بدة اىنبىس ى ف اىغزاء ػي ح ظ وص اىدشرا الذسجت: اى بخسخ ش الوششفوى : دكتوس : سضىا صذق فشج دكتوس : خ به س ذ ػي اىببسوط دكتوس : غبدة ابشاه ػ س قسن: اىن بء اىح ى ت تبسيخ هنح الذسجت: 2012/ / الوستخلص العشبي أخش ج اىذساست اىحبى ت إل دبد طش قت ىيحذ ا خشبس ص بدة اىىص او اىس ت ػ طش ق ضبظ بؼض نى بث اىغزاء.ح اخخ بس 08 فشد رمىس اىدشرا طبقب ألػ بسه. ح حقس اىدشرا اى د ىػخ م ب ي : 1- خشرا ب قبو اىبيىؽ: 48 رمش اىدشرا ػ ش> شهش ب خىسظ وص 5± 05.4 خ, 2- خشرا ببىغت :48 رمش اىدشرا ػ ش< 4 شهىس ب خىسظ وص ± خ. مو د ىػت ح حقس هب اى 4 ححج د ىػبث,مو ححج د ىػت بهب 18 خشرا م ب ي :اى د ىػت اىضببطت : وح حغز خهب ػي ػي قت ق بس ت خض ت, ححج د ىػت : 1 وح حغز خهب ػي ػي قت خفضت ف اىذهى ( %4(, ححج د ىػت : 2 ح حغز خهب ػي ػي قت خفضت ف اىذهى )%4( ببالضبفت اى مبىس ى ػبى )1.0 %( سخشاث اىنبىس ى, ححج د ىػت : 3 ح حغز خهب ػي ػي قت خفضت ف اىذهى )%4( ببالضبفت اى مبىس ى ػبى )1.0 %( ىب دفف ضوع اىذس ذػ به ذسومس اببح ج.ح حقذ ش سخى بث اىذهى واىنبىس ى واىفىسفىس واىخسخىسخ شو ف اىس ش ببإلضبفت إى وص اىدس. أظهشث اى خبئح قصب غ ش ؼ ى ف اى سبت اى ئى ت ىض بدة اىىص ف د ىػت اىدشرا ب قبو اىبيىؽ اىخ حغزث ػي ػالئق ػبى ت اىنبىس ى ب ب أظهشث قصب ؼ ى ب ف اى سبت اى ئى ت ىض بدة اىىص ف د ىػت اىدشرا اىببىغت اىخ حغزث ػي ػالئق ػبى ت اىنبىس ى. حذد قص ؼ ى ى سخى بث اىنىى سخشوه ػبى اىنثبفت واىنىى سخشوه خفض اىنثبفت واىذهى اىثالث ت واىخسخىسخ شو ف خشرا ب قبو اىبيىؽ اى خغز ت ػي ػالئق ػبى ت ف اىنبىس ى. ب ب مب اى قص ف سخى بث اىنىى سخشوه ؼ ى ب فقظ ف د ىػت خشرا بقبو اىبيىؽ اىخ حغزث ػي ػي قت ػبى ت اىنبىس ى ىب دفف ضوع اىذس. أظهشث اى خبئح قصب ؼ ى ب ف سخى بث اىذهى اىثالث ت واىنىى سخشوه ف اىدشرا اىببىغت اىخ حغزث ػي ػي ػالئق ػبى ت ف اىنبىس ى ب ب ى حن ه بك فشوقب ؼ ى ت ف سخى بث اىنىى سخشوه خفض اىنثبفت ب اىدشرا اىببىغت اىخ حغزث ػي ػالئق ػبى ت اىنبىس ى أو حيل اىخ حغزث ػي ػالئق ق بس ت ف اىنبىس ى. مب اى قص ؼ ى ب ف سخىي اىنىى سخشوه ػبى اىنثبفت ب اىدشرا اىببىغت اىخ حغزث ػي ػي قت ػبى ت اىنبىس ى ىب دفف ضوع اىذس أ. ظهشث اى خبئح ص بدة ؼ ى ت ف سخى اىنبىس ى فقظ ف د ىػت اىدضرا اىببىغت اىخ حغزث ػي ػي قت ػبى ت اىنبىس ى سخشاث اىنبىس ى ب ب اوضحج اىذساست قصب ؼ ى ب ف سخى بث اىفىسفىس ف اى دب غ اىخ حغزث ػي ػالئق ػبى ت ف اىنبىس ى. الكلوبث الذالت : خشرا ببىغت بقبو اىبيىؽ اىذهى ف اىذ - مبىس ى فىسفىس- اىخسخىسخ شو اىس ت.

8 تأحيش صيبدة الكبلسيوم في الغزاء على تنظين وصى الجشراى سسبلت هبجستيش في العلوم الضساعيت )الكيويبء الحيويت الضساعيت( هقذهت هي هحوذ طه هحوذ عبذالشحوي بكبلوسيوط في العلوم الضساعيت )كيويبء حيويت صساعيت(- كليت الضساعت - جبهعت القبهشة 2007 ىد ت اإلششاف دكتوس/ سضواى صذقي فشج أستبر الكيويبء الحيويت الضساعيت- كليت الضساعت - جبهعت القبهشة دكتوس/ جوبل سيذ على الببسوطي أستبر الكيويبء الحيويت الضساعيت كليت الضساعت - جبهعت القبهشة دكتوس/ غبدة ابشاهين عيسى أستبر ببحج الكيويبء الحيويت االشعبعيت هيئت الطبقت الزسيت الذقى - هصش

9 تأحيش صيبدة الكبلسيوم في الغزاء على تنظين وصى الجشراى سسبلت هبجستيش في العلوم الضساعيت )الكيويبء الحيويت الضساعيت( هقذهت هي هحوذ طه هحوذ عبذالشحوي بكبلوسيوط في العلوم الضساعيت )كيويبء حيويت صساعيت(- كليت الضساعت - جبهعت القبهشة 2007 اىحن ىد ت دكتوس/ سويش هصطفى عبذ العضيض... أستبر ببحج الكيويبء الحيويت- هشكض البحوث النوويت - هيئت الطبقت الزسيت دكتوس/أحوذ هحوود هصطفى ابوالعينيي... أستبر الكيويبء الحيويت الضساعيت - كليت الضساعت - جبهعت القبهشة دكتوس/ جوبل سيذ على الببسوطي... أستبر الكيويبء الحيويت الضساعيت - كليت الضساعت - جبهعت القبهشة دكتوس/ سضواى صذقي فشج... أستبر الكيويبء الحيويت الضساعيت - كليت الضساعت - جبهعت القبهشة 2812/ التاريخ: /

10 تأحيش صيبدة الكبلسيوم في الغزاء على تنظين وصى الجشراى سسبلت هقذهت هي هحوذ طه هحوذ عبذالشحوي بكبلوسيوط في العلوم الضساعيت )كيويبء حيويت صساعيت(- كليت الضساعت جبهعت القبهشة 2007 ىيحصىه ػي دسخت الوبجستيش في العلوم الضساعيت )كيويبء حيويت صساعيت( قسن الكيويبء الحيويت الضساعيت كليت الضساعت جبهعت القبهشة هصش 2012

11 CONTENTS Page INTRODUCTION... 1 REVIEW OF LITERATURE Definition of obesity Overweight and obesity prevalence Obesity consequences: health consequences Obesity consequences: non health consequences 5 5. Causes of obesity Role of some nutrients in overweight Anti-obesity effects of calcium and dairy products Advantages of dairy calcium Other physiological roles of calcium Calcium sources and bioavailability Calcium absorption and bioavailability Intestinal calcium absorption Methods for measuring calcium bioavailability Other calcium salts Role of calcium in apoptosis.. 24 MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES ARABIC SUMMARY. i

12 No LIST OF TABLES Title Diet composition % of different subgroup rats... Composition of minerals mixture.. Composition of vitamin mixture.. Lipid profile of premature experimental rat subgroups.. Lipid profile of mature experimental rat subgroups.. Changes in body weight of different premature rat subgroups.. Changes in body weight of different mature rat groups Testosterone, phosphorus and calcium levels in sera of premature experimental rat subgroups. Testosterone, phosphorus and calcium levels in sera of mature experimental rat subgroups.. Kidney and liver functions of different premature experimental rat groups... Kidney and liver functions of different mature experimental rat groups... Correlation between calcium, phosphorus, testosterone and lipid profile parameters in premature rats... Correlation between calcium, phosphorus, testosterone and lipid profile parameters in mature rats. Page ii

13 LIST OF FIGURES No Title A schematic representation of transepithelial Ca 2+ transport in the intestine.... Serum cholesterol and triglyceride levels in premature rats.... Serum cholesterol and triglyceride levels in mature rats. Serum total testosterone levels in premature and mature rats. Serum calcium and phosphorus levels in mature rats. Serum urea level in premature and mature rats. Serum creatinine level in premature and mature rats... Serum alanine transaminase activity levels in premature and mature rats Serum aspartate transaminase activities in premature and mature rats. Page Serum total protein levels in premature and mature rats iii

14 زيادة تأثير الملخص العربي الكالسيوم في الغذاء على تنظين وزن الجرذان رؼذ اىس خ أحذ اى شبمو اىصح خ اىؼب خ ف اىؼبى. ىزىل أخش ذ اىذساسخ اىحبى خ إل دبد طش قخ ىيحذ ص بدح اى ص أ اىس خ ػ طش ق ضجظ ثؼض ن بد اىغزاء.ر اخز بس 88 فشد رم س اىدشرا طجقب ألػ بس. ر رقس اىدشرا إى د ػز م ب ي : 1- خشرا ب قجو اىجي ؽ: 48 رمش اىدشرا ػ ش> ش ش ث ز سظ ص 5± 85.4 خ, 2- خشرا ثبىغخ :48 رمش اىدشرا ػ ش< 4 ش س ث ز سظ ص 18 ±288 خ.مو د ػخ ر رقس ب اى 4 رحذ د ػخ,مو رحذ د ػخ ث ب 18 خشرا م ب ي :اى د ػخ اىضبثطخ : ر رغز ز ب ػي ػي قخ ق بس خ زض خ, رحذ د ػخ : 1 ر رغز ز ب ػي ػي قخ خفضخ ف اىذ ( %4(, رحذ د ػخ : 2 ر رغز ز ب ػي ػي قخ خفضخ ف اىذ )%4( ثبالضبفخ اى مبىس ػبى )1.8 %( سزشاد اىنبىس, رحذ د ػخ : 3 ر رغز ز ب ػي ػي قخ خفضخ ف اىذ )%4( ثبالضبفخ اى مبىس ػبى )1.8 %( ىج دفف ض ع اىذس ذػ ث ذس مس اثبر ذ. ر سحت ػ بد اىذ شر )قجو ثؼذ ذح اىزدشثخ ) ث ػ و طشد شمض ىفصو اىس ش. ر رقذ ش سز بد اىذ ( اىن ى سزش ه اىني اىذ اىثالث خ اىن ى سزش ه ػبى اىنثبفخ اىن ى سزش ه خفض اىنثبفخ( اىنبىس اىف سف س اىزسز سز ش ظبئف اىنجذ اىني ف اىس ش ثبإلضبفخ إى ص اىدس. ويمكه تلخيص النتائج فيما يلي : 1- أظ شد اى زبئح قصب غ ش ؼ ب ف اى سجخ اى ئ خ ىض بدح اى ص ف د ػخ اىدشرا ب قجو اىجي ؽ اىز رغزد ػي ػالئق ػبى خ اىنبىس ث ب أظ شد قصب ؼ ب ف اى سجخ اى ئ خ ىض بدح اى ص ف د ػخ اىدشرا اىجبىغخ اىز رغزد ػي ػالئق ػبى خ اىنبىس. 2- حذس قص ؼ ى سز بد اىن ى سزش ه ػبى اىنثبفخ اىن ى سزش ه خفض اىنثبفخ اىذ اىثالث خ اىزسز سز ش ف خشرا ب قجو اىجي ؽ اى زغز خ ػي ػالئق ػبى خ ف اىنبىس. 1

15 3- مب اى قص ف سز بد اىن ى سزش ه ؼ ب فقظ ف د ػخ خشرا بقجو اىجي ؽ اىز رغزد ػي ػي قخ ػبى خ اىنبىس ىج دفف ض ع اىذس. 4- أظ شد اى زبئح قصب ؼ ب ف سز بد اىذ اىثالث خ اىن ى سزش ه ف اىدشرا اىجبىغخ اىز رغزد ػي ػالئق ػبى خ ف اىنبىس ث ب ى رن بك فش قب ؼ خ ف سز بد اىن ى سزش ه خفض اىنثبفخ ث اىدشرا اىجبىغخ اىز رغزد ػي ػالئق ػبى خ اىنبىس أ ريل اىز رغزد ػي ػالئق ق بس خ ف اىنبىس. 5- مب اى قص ؼ ب ف سز اىن ى سزش ه ػبى اىنثبفخ ث اىدشرا اىجبىغخ اىز رغزد ػي ػي قخ ػبى خ اىنبىس ىج دفف ض ع اىذس. 6- اظ شد اى زبئح ص بدح ؼ خ ف سز اىنبىس فقظ ف د ػخ اىدضرا اىجبىغخ اىز رغزد ػي ػي قخ ػبى خ اىنبىس سزشاد اىنبىس ث ب أ ضحذ اىذساسخ قصب ؼ ب ف سز بد اىف سف س ف اى دب غ اىز رغزد ػي ػالئق ػبى خ ف اىنبىس. 7- أظ شد سز بد اى س ب اىجش ر اىني رغ شاد غ ش ؼ خ ف مو اىدشرا اىجبىغخ خشرا ب قجو اىجي ؽ. 8- أظ شد زبئح اىنش بر قصب ؼ ب ف د ػخ خشرا بقجو اىجي ؽ اىز رغزد ػي ػالئق خفضخ اىذ مزىل ريل اىز رغزد ػي ػي قخ ػبى خ اىنبىس ىج دفف ث ب أظ شد اسرفبػب ؼ ب ف سز بر ف د ػز خشرا بقجو اىجي ؽ اى زغز خ ػي ػي قخ ػبى خ ف سزشاد اىنبىس رىل ثبى قبس خ ىي د ػخ اىضبثطخ. خب ت آخش مب بك اسرفبػب ؼ ب ف سز بد اىنش بر ى دب غ اىدشرا اىجبىغخ اى زغز خ ػي ػالئق ػبى خ اىنبىس قبس خ ثبى د ػخ اىضبثطخ اى د ػخ اى زغز خ ػي ػي قخ خفضخ اىذ. 9- أ ضحذ سز بد شبط ا ض اال اى بقو ى د ػخ األ قصب ؼ ب ف مو دب غ خشرا ب قجو اىجي ؽ رىل قبس خ ثبى د ػخ اىضبثطخ. ػي اىدب ت ا خش ف اىدشرا اىجبىغخ أظ شد مو د ػز اىدشرا اى زغز خ ػي ػي قخ ػبى خ اىنبىس اىيج ريل اى زغز خ ػي ػي قخ خفضخ اىنبىس قصب ؼ ب ف شبط ا ض اال اى بقو ى د ػخ األ ث ب أظ شد د ػخ اىدشرا اى زغز خ ػي ػي قخ ػبى خ اىنبىس سزشاد اىنبىس ص بدح ؼ خ ف شبط زا اال ض. 2

16 18- أظ شد اى زبئح قصب غ ش ؼ ب ف شبط ا ض اسجبسر ذ اى بقو ى د ػخ األ ف مو دب غ خشرا ب قجو اىجي ؽ قبس خ ثبى د ػخ اىضبثطخ ث ب ف اىدشرا اىجبىغخ أظ شد د ػخ اىدشرا اى زغز خػي ػي قخ خفضخ اىذ قصب ؼ ب ف شبط زا اال ض ف اى قذ اىز ى رن بك فش قب ؼ خ ث اى دب غ اى غز خ ػي ػالئق ػبى خ اىنبىس اى د ػخ اىضبثطخ. 11- ثشنو ػب فإ اى زبئح ز اىشسبىخ اىؼي خ رقزشذ اسز الك غزاء خفض ف اىذ )%4( غ مبىس ػبى ىج دفف ض ع اىذس اى ذػ ث ذس مس اثبر ذ م ظب غزائ بست ىزد ت ص بدح اى ص أ اىس خ ثبإلضبفخ إى أ اسز الك م بد آ خ اىنبىس ) يد مبىس \ ( ر ب ه 6-3 ام اة اىضثبد أ اىيج ن أ ن ظب ب خ ذا ىزد ت ص بدح اى ص. رطج ق زا اى ظب ػي غ ش اىجبىغ سث ب حزبج ى ض ذ اىذساسخ حز ن رأم ذ. 3

17 INTRODUCTION Obesity is recognized as one of the most significant public health problems in the world (Sakhaee and Maalouf, 2005; Bougoulia et al., 2006 and McLaughlin et al., 2006). It is a risk factor for chronic diseases as heart disease, cancer, stroke and diabetes (Kumada et al., 2003; Teegarden, 2003 and Barbato et al., 2006). The prevalence of obesity has reached alarming levels, affecting virtually both developed and developing countries of all socioeconomic groups, irrespective of age, sex or ethnicity (Kosti and Panagiotakos, 2006). It is known that dietary restriction and increased physical activity can lead to weight loss; such life style changes may be difficult to implement and maintain. Thus, the high rate of recidivism is among weight losers (Anderson et al., 2001). Maintenance of a constant body weight requires a balance between energy intake and energy expenditure, and even light imbalance in this energy equilibrium can lead to significant changes in body weight over time and may eventually result in obesity (Hill et al., 2003). Even small changes in energy balance may lead to such a weight gain, which therefore may be prevented by slight modification in food intake, such as the inclusion of functional foods that affects energy

18 metabolism and fat repartitioning may be helpful adjuncts to a dietary approach to body weight control. At a time when the obesity epidemic is growing, dietary manipulation such as an increase in calcium intake represents a simple intervention that may result in substantial rewards on a public health level. Therefore, it will be important to continue to pursue the skinny on the link between calcium intake and weight control (Gordon and Mantzoros, 2012). The current study was conducted to envisage the role of calcium in dairy products as one of the main factors to prevent the increase of rat body weight gain. In order to envisage the role of diet of varied components on rat weight, some parameters were performed on rat sera such as lipid profile, phosphorus, calcium, testosterone, liver and kidney functions. 2

19 REVIEW OF LITERATURE Definition of obesity Obesity is one of the most significant public problems in the world (Sakhaee and Maalouf, 2005; Bougoulia et al., 2006 and McLaughlin et al., 2006). This state is a consequence of an energy imbalance, i.e. when energy intake exceeds energy expenditure over an extended period of time (Pan American Health Organization, 2003). It is an excess of body fat which creates increased risk of morbidity and/or premature mortality (Rielly, 2005). Definitions of obesity should therefore denote increased risk of adverse health consequences outcomes (Rielly, 2005). The most widely used measurement to define obesity is body mass index (BMI); weight in kilos divided by height in square meters (Kg/m 2 ) (Cole et al., 2000). Ascending BMI values have been identified as contributing to the development of breast cancer, although this relationship may only be evident in post menopausal women. In a case control study of 232 post menopausal women, those with BMIs classified as "obese" had three times the risk of breast cancer as compared to non-obese women after controlling for other characteristics (Montazeri et al., 2008). Overweight and obesity prevalence Prevalence of obesity is increasing world wide. Statistical information indicates that in the year 2000, the number of obese adults increased to 300 million (World Health Organization, 2000). Over 22 million children under the age of 5 years are severely overweight, in 3

20 addition to 155 million children of school age (World Heart Federation, 2006). Obesity has reached epidemic proportions in developed countries and rapidly increasing in many middle income and less developed countries (Chopra et al., 2002 and Swinburn et al., 2011). Overweight prevalence among youth is increasing and become a problem among the Egyptian youth (Salazer-Martinz et al., 2006). Gender is a factor associated with obesity prevalence where obesity was found to be more prevalent among women than men except Denmark and Switzerland where the contrary happens. Overweight and obesity in children is directly associated with being overweight in adulthood (Rielly et al., 2003; Rielly, 2005). A potential deluge is evident across the globe with obesity increasing two or three and four fold in Egypt, over the past 10 years (Ebbeling et al., 2002). Obesity consequences: health consequences Overweight and obesity lead to adverse metabolic effects on blood pressure, cholesterol, triglycerides and insulin resistance. Some confusion of the consequences of obesity arises because using different body mass index (BMI) cutoffs, and because the presence of many medical conditions involved in the development of obesity may confuse effects of obesity itself (World Health Organisation, 2000). The more life threatening problems are associated with cardiovascular disease (CVD), conditions associated with insulin resistance such as type 2 diabetes (Smith and Minson, 2012), certain types of cancers and gallbladder disease (WHO, 2000). 4

21 There is a range of non- fatal health problems associated with obesity which include respiratory difficulties, chronic musculoskeletal problems, skin problems and infertility the likelihood of developing type 2 diabetes and hypertension rises steeply with increasing body fatness (WHO, 2000; Smith and Minson, 2012). Confined to older adults for most of the 20 th century, this disease now affects obese children even before puberty approximately 85% of the people with diabetes are type 2 and of these, 90% are obese or overweight (WHO, 2000). The cardiovascular risk factors well known to be associated with obesity in adults are also associated with obesity in children and adolescent: hypertension, dyslipidemia, abnormalities in left ventricular mass and /or function, abnormalities in endothelial function and hyperinsulinemia / insulin resistance (Williams et al., 2002). In the general population, overweight and obesity increase the risk of hospitalization and death from cardiovascular disease and type 2 - diabetes after adjustment for other risk factors. Obesity consequences: non health consequences A part from the severe health consequences of childhood and adolescent obesity, a study demonstrated that obesity epidemic has deleterious economic consequences. For example; obesity is responsible for between 5% and 7% of the total annual medical expenditures in USA (Finkelstein et al., 2005). It has been estimated that medical cost for obese patients was 42% higher than normal weight patients and the 5

22 annual financial burden from obesity on the medical system in the United States alone was estimated to be as high as $147 billion annually (Finkelstein et al., 2009). Causes of obesity There are many causes of obesity because it is a complex condition with genetic, metabolic, behavioral and environmental factors, all contributing to its development (Baur, 2002 and Swinburn et al., 2011). However, the dramatic increase in the prevalence of obesity in the past few decades can only be due to significant changes in lifestyle influencing children and adults alike (Baur, 2002). For example sleeping habits contributes to obesity prevalence (Nishitani et al., 2012). Obesity promoting environmental factors are usually referred today under the general term of obesogenic or obesigenic (Lobstein et al., 2004). Overweight and obesity are strongly associated with certain types of diets such as processed food stuffs that contain a large amount of fat (Chopra et al., 2002). Increasing the intake of some types of carbohydrates can bring about deleterious effects on plasma lipids such as increased plasma triglyceride levels, which might be independent risk factor for cardiovascular disease, and increased concentrations of small dense LDL-cholesterol, along with decreased plasma HDL- cholesterol levels (Lichtenstein, 2006). 6

23 Role of some nutrients in overweight Specific micro or macro nutrients, dietary patterns, or both may modulate the same metabolic pathways affected by those genetic factors and thereby alter nutrient and energy partitioning (Zemel, 2004). Calcium plays a key role in a wide range of biologic functions, either in the form of its free ion or bound complexes. One of the most important functions as bound calcium is in skeletal mineralization. The vast majority of total body calcium (>99%) is present in the skeleton as calcium-phosphate complexes, primarily as hydroxyapatite, which is responsible for much of the material properties of bone (Wang et al., 2006). The remaining 1% of total body calcium is located in the blood, extracellular fluid and soft tissues. Of the total calcium in blood, the ionized fraction (45%) is the biologically functional portion and can be measured clinically. Most clinical laboratories report total serum concentrations. Forty-five percent of the total calcium in blood is bound to plasma, proteins notably albumin and up to 10% is bound to anions such as phosphate and citrate. Concentrations of total calcium in normal serum generally range between 8.5 and 10.5 mg/dl ( mm). (Patel et al., 2012). In bone, calcium serves two main purposes: it provides skeletal strength and, concurrently, provides a dynamic store to maintain the intra- and extracellular calcium pools. Dietary calcium is important in building bone mass in children as well as in preventing osteoprosis in elderly. An association between dietary calcium intake and obesity (Schrager, 2005), a negative 7

24 relationship between dietary calcium intake and body fat in a group of 8 year old children (Skinner et al., 2003). One gram difference in calcium intake is associated with a 8- Kg difference in body weight (Davies et al., 2000). The increase in consumption of low fat milk was associated with lower body fat and lower body weight (Drapeau et al., 2004).Weight loss is associated with improved in risk factors for cardiovascular disease (CVD) and diabetes mellitus (Teegarden, 2003; Barbato et al., 2006; Andrew and Selwyn, 2007). Low calcium diets lead to an increase in 1,25 dihydroxy vitamin D 3 [1,25 (OH) 2 D 3 ] which in turn stimulates calcium influx into adipocytes, resulting in stimulation of lipogenesis, inhibition of lipolysis and expansion of adipocyte triglycride stores. Suppressing 1,25 (OH) 2 D 3 levels by increasing dietary calcium would be predicted to inhibit adiposity and promote weight loss (Parikh and Yanovski, 2003; Shapses et al., 2004; Zemel, 2004; Thompson, et al., 2005). Calcium rich diets have been demonstrated to exert an antiobesity effect that appears to be mediated in part by suppressing circulating 1,25 dihydroxy cholcalciferol (Rao et al., 1994; Petrov and Lijnen, 1999; Zemel et al., 2000; Sun and Zemel, 2007). This approach, if viable becomes increasingly important as we recognize the frequent failure of individual persons and population to adhere to strategies designed to produce negative energy balance (Appel et al., 1997; McCarron and Reusser, 1999; Svetky et al., 1999). 8

25 Anti-obesity effects of calcium and dairy products Increasing dietary calcium from 0.4 to 1.0 g/day through the consumption of 2 cups of Yogurt for 1 year produced expected decrease in blood pressure and unexpected 4.9 Kg reduction in body weight (Zemel et al., 2000). Calcium in the form of dairy products may be more effective than elemental calcium (Barr, 2003; Zemel, 2005; Barba and Russu, 2006). Eighteen subjects received 3 cups of yogurt per day (1.1 g/day of calcium) lost more weight, body fat and abdominal circumference while attenuating loss of fat free mass in comparison with 16 subjects in a low calcium control group ( g /day of calcium) (Zemel et al., 2005). It was observed that dietary calcium levels are inversely associated with [Ca ++ ] i levels in white adipose tissue. Mice refed low Ca-diets exhibited high adipocytes [Ca ++ ] i while those refed high calcium diets exhibited low adipocytes [Ca ++ ] i. Adipocyte [Ca ++ ] i was determined to regulate human and murine adipocyte metabolism (Zemel, 2003). 1,25- dihydroxyvitamin D 3 [1,25 (OH) 2 D 3 ] was shown to stimulate [Ca ++ ] i in multiple cell types, including vascular smooth muscles cells, pancreatic B cells and adipocytes (Kim et al.,1996; Shi et al., 1999; Zemel et al., 2000; Shi et al., 2001). It can also act on human adipocytes to cause rapid sustained increase in [Ca ++ ] i and a coordinated activation of fatty acid synthase (FAS) and inhibition of lipolysis 9

26 (Zemel et al., 2000; Shi et al., 2001; Zemel, 2003). Increased 1, 25 (OH) 2 D 3 was also observed in obese humans (Zemel, 2000). 1,25- dihydroxyvitamin D 3 [1,25 (OH) 2 D 3 ] serves as a potent inhibitor for both murine and human adipocyte apoptosis. This effect is mediated, in part, via the inhibition of uncoupling protein 2 (UCP2) expression and a consequent increase in mitochondrial potential. And in part via 1,25 (OH) 2 D 3 regulation of cytosolic and mitochondrial Ca ++ and result in marked increase in adipocytes apoptosis in mice fed high ca-diets or high dairy products diets (Sun and Zemel, 2004). The magnitude of these effects depends on the source of dietary calcium; the effects were greater when the source was dairy (non-fat dry milk) than when it was calcium carbonate (Zemel et al., 2000). This approach supports that dairy products contain bioactive compounds help in these effects such as branched chain amino acids (Zemel, 2005) in particular leucine (Layman, 2003). A little support for the hypothesis that dietary calcium contributes to the etiology or maintenance of obesity was provided via reporting that no effect of dietary calcium on body weight of lean and obese mice and rats (Zhang and Tordoff, 2004). Advantages of dairy calcium A local increase in oxidative stress in accumulated fat causes dysregulated production of adipokines. Fat accumulation stimulates NADPH oxidase, a key factor in enzymatic cellular reactive oxygen species (ROS) production expression in white adipose tissue (Furukawa 10

27 et al., 2004). Consistent with this, high Ca-diet inhibited both NADPH oxidase expression and ROS production in mice (Zemel and sun, 2008). However, the high calcium and milk diets had identical calcium content and exerted comparable inhibition of 1,25 (OH) 2 D 3, whereas the milk diet resulted in further suppression of oxidative stress compared with the high Ca-diet (Zemel and Sun, 2008). This suggests that milk diet suppressed oxidative stress via an additional mechanism (Zemel and Sun, 2008). Dairy contains angiotensin converting enzyme inhibitory peptides (Zemel, 2005) and adipose tissues express all components of rennin-angiotensin system (Crandal et al., 1994; Jones et al., 1997; Giacchetti et al., 2000; Engeli et al., 2003), stimulation of this system promotes oxidative and inflammatory responses (Darimont et al., 1994 and Ruiz-Ortega et al., 2002). Whereas the antagonism of this system suppresses oxidative stress (Khan et al., 2001; Napoli et al., 2004). Also high leucine content in milk may indirectly contribute to the suppression of oxidative stress via promotion of skeletal muscle protein synthesis and inhibition of protein degradation (Koboyshi et al., 2006; Rennie et al., 2006). Other physiological roles of calcium Calcium is one of the most important and abundant minerals in the body (Goodman and Gilman, 1996) and its metabolism is one of the basic biologic processes in humans (Patel et al., 2012) and representing between 1.5 % and 2.55% of the body weight (Arnaud and Sanchez, 1990). It has important physiological functions with regard to muscular 11

28 contractility, blood clots, and cardiovascular, neurological, endocrine, renal, and gastrointestinal systems. In addition to serving as a cofactor for many extracellular enzymes and as signaling molecule (Levenson and Bockman, 1994 and Patel et al., 2012). The biggest increase in bone mass occurs during puberty age (Johnson et al., 1992) and reduction in bone mass beyond 40 years of age (Riggs et al., 1987; Baran et al., 1990; Hu et al., 1993) especially in women after menopause due to estrogen decline (Richelson et al., 1984; Johnston et al., 1985). The inadequate calcium intake during puberty may lead to a failure to reach the maximum bone mass (Matkovic, 1990). The adequate calcium intake was recommended between 0.8 and 1.5 g/day according to the age and sex (Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, 1997) while the maximum upper limit recommended of calcium per day is 2.5g (Schrager, 2005). To maintain overall skeletal integrity and attenuate the effects of osteoporosis, dietary sources of calcium should be used (Yuan et al., 1991). Milk is one of the best nutrients calcium sources because it has about 1.2 g Ca/Liter and contains the lactose which enhances the absorption in normal subjects (Levnson and Backman, 1994). Other foods like vegetables and cereals have calcium together with other compounds like oxalate or phytates, which forms insoluble complexes that decrease calcium absorption (Fleming and Hierbch, 1994; Rencker, 1985). Although dairy products are the major sources of calcium, individuals who do not prefer dairy products or those who are lactose intolerant can not use dairy as a source of calcium, and even calcium 12

29 content in foods from plant origin can not cover all the adequate requirments of calcium (Charle, 1992) or not in suitable form (Fleming and Hierbch, 1994). So one way to overcome these problems is using alternative calcium sources or calcium supplements and calcium fortified foods (Sarabia et al., 1999). Calcium sources and bioavailability The physicochemical and nutritional properties of the compounds used as calcium sources for this strategy are essential when choosing them. Some compounds like CaCO 3 or calcium phosphate are insipid but practically insoluble in water or aqueous environment. Other compounds, like calcium lactate and calcium gluconate are highly soluble in water but their strong taste makes people refuse to consume them (Sheikh et al., 1987). Other factors like absorbability and bioavailability are also important. Calcium supplements in the form of calcium carbonate, citrate and citrate malate have been tested for their absorbability in human and animal trails and have been generally found to be similar (Miller et al., 1988; Kochanowski, 1990; Weaver et al., 2002). Although some of the experiments report advantages for citrate and citrate malate (Miller et al., 1988). Calcium hydroxyapatite (HA), Ca 10 (PO 4 ) 6 (OH) 2 is an important inorganic material in biology and chemistry (Arends et al., 1987; LeGeros, 1991; Elliott, 1994). Biological apatites are the inorganic constituents of bone, tooth enamel and dentine (LeGeros, 1991). Calcium hydroxy apatite represents the 13

30 milk calcium and was used for milk fortification (Kruger et al., 2003). Bioavailability of calcium is equivalent from milk fortified either with calcium carbonate or milk calcium in growing rats and the kind of calcium salt used for fortification is not the determining factor for bioavailability (Kruger et al., 2003). There are different individual factors that affect calcium absorption like age, sex, nutritional status for this element (Lupton et al., 1996). The efficiency of calcium absorption decreases after middle age, especially after menopause due to interaction of hormones with calcium (Ettinger et al., 1987; Tsuchita and Kuwata, 1995). Diets containing whey protein fractions improved calcium absorption in rat compared to casein and soybean based diets (Pantako et al., 1994). There is no doubt that milk provides large amounts of calcium. While there is also no question of the nutritional effectiveness of the calcium provided by milk, there is still some debate as to whether this source of calcium is biologically better than other sources, such as calcium salts, certain vegetables or mineral waters (Allen, 1982). Calcium absorption and bioavailability The terms bioavailability and absorption of a nutrient are sometimes used interchangeably in the literature; however, there is an important difference between them. The absorption of a nutrient describes the process by which the nutrient is transported from the 14

31 gastrointestinal lumen, across the intestinal mucosa, to the serosa. The bioavailability of a nutrient, on the other hand, defines that fraction of the ingested nutrient that is utilized for normal physiological functions or storage. This definition recognizes that one of the major determinants of bioavailability is that proportion which is absorbed from the gastrointestinal tract, but that this is not the only factor influencing bioavailability since tissue utilization (or lack of utilization) of the absorbed nutrient may vary dramatically (Jackson, 1997). Specifically in the case of calcium, bioavailability may be defined as the amount of calcium in foods that can be absorbed and utilized by the body for normal metabolic functions. Intestinal calcium absorption Calcium in food occurs as salts or associated with other dietary constituents in the form of complexes of calcium ions (Ca 2+ ). Calcium must be released in a soluble and probably ionized form before it can be absorbed (i.e., its transfer from the intestinal lumen to the circulatory system) (Schacter et al., 1960). Cow s milk is considered to be a rich source of Ca. Ionic Ca levels in cow s milk have been reported by several researchers. Cow s milk at different stages of lactation contained mm [Ca 2+ ] (Lin et al., 2006). In middle lactation, it was reported to be 2.3 mm (De La Fuente et al., 1998) and reconstituted skimmed milk was found to be 2.04 mm (Sievanen et al., 2008). In other milk, ionised Ca in human milk varied between 2.3 and 4.0 mm across individuals at 90 days of lactation (Neville et al., 1994). The efficiency of calcium absorption (the fraction of dietary calcium absorbed) is affected by the intraluminal presence of dietary 15

32 components, by the calcium and vitamin D status of the body, and by physiological states such as growth, old age, pregnancy, and lactation (Allen, 1982). The presence of gastric acid appears to increase the solubility of calcium complexes (Mahoney et al., 1975). Most of the digestive enzymes that release calcium from its complexes are ph dependent. The ph of the intestine after food is consumed is approximately 6.0 in the proximal jejunum (range from 3.5 to 6.7), and 7.6 beyond the mid-small bowel (never below 6.5 in the normal circumstance) (Fordtran and Locklear, 1966). Calcium tends to precipitate from solutions with a ph greater than 6. 1 (Albright and Reifenstein, 1948), so that dietary calcium is in a more absorbable form in the duodenum and proximal jejunum. In addition, intestinal calcium-binding protein (CaBP) is found mainly in the duodenum, with relatively small amounts in the proximal jejunum, and almost non below this segment. The combination of acid ph and CaBP in the duodenum and upper jejunum probably explains why the efficiency of calcium absorption (as amount of calcium transferred per unit gut length) is much greater from the duodenojejunal segments of the intestine than from the ileal segments (Fig. 1) (Wensel et al., 1969). However, a more important factor may be the residence time of the calcium in a segment. This was demonstrated clearly by Marcus and Lengemann (1962), who studied the percentage of 45 Ca absorbed by the intestinal segments of the rat. For a liquid dose, the values were: stomach, 0%; duodenum 15%; jejunum 23%; ileum 62%. If the isotope was given with solid food, the corresponding values were 16

33 0, 8, 4, and 88%, respectively. A similar situation may exist in humans, since removal of the ileum in intestinal bypass procedures has a more deleterious effect on calcium absorption than does removal of the jejunum (Dano and Christiansen, 1974). Also Birge et al. (1969) found a greater absorption of 47 Ca from segments distal to the duodenum and jejunum in normal subjects. Bile salts increase the in vitro solubility of calcium salts and the absorption of calcium from the intestine (Webhing and Holdsworth,1966; Wills, 1973). The calcium salts of bile acids rarely precipitate from bile in the human (Hofmann, 1965). Bile salts are also required for the optimal absorption of vitamin D (Schacter et al., 1964; Kehayoglou et al., 1968). Bile is a major route for the secretion of calcium into the intestine (Blau et al., 1954). It has been suggested that bile calcium is preferentially absorbed, and does not mix significantly with dietary calcium (Briscor and Ragan, 1965). The efficiency of reabsorption of bile and digestive juice calcium is controversial; most studies have assumed that this secreted calcium is reabsorbed to the same extent as dietary calcium (Heaney and Skillman, 1964) although Rose et al. (1965) found that it was absorbed less efficiently. Once in a soluble form, calcium is absorbed by two routes, transcellular and paracellular transport (Bronner, 1987). The saturable, transcellular pathway is a multi-step process, involving the entry of luminal Ca 2+ across the microvillar membrane into the enterocyte, then movement through the cytosol (i.e., translocation to the basolateral membrane), followed by 17

34 active extrusion from the enterocyte into the lamina propria and, eventually, into the general circulation (Figure 1). The intracellular Ca 2+ diffusion is thought to be facilitated by a cytosolic calcium binding protein, calbindin D9K, whose biosynthesis is dependent on vitamin D. Calbindin D9K facilitates the diffusion of Ca 2+ across the cell by acting as an intracellular calcium ferry or a chaperone. The active extrusion of Ca 2+ at the basolateral membrane takes place against an electrochemical gradient and is mainly mediated by Ca- ATPase. The entry of Ca 2+ across the apical membrane of the enterocyte is strongly favoured electrochemically because the concentration of Ca 2+ within the cell (10-7 to 10-6 M) is considerably lower than that in the intestinal lumen (10-3 M), and the cell is electronegative relative to the intestinal lumen (Fullmer, 1992). Therefore, the movement of Ca 2+ across the apical membrane does not require the expenditure of energy. It has been controversial as to whether a transporter or a channel is responsible for this process. It was widely believed that, because of the impermeability of lipid membranes to Ca 2+, a Ca 2+ channel or integral membrane transporter must reside in the brush border membrane. Evidence would now suggest that the recently cloned calcium transport protein (CaT1) is a good candidate for this putative Ca 2+ channel (Peng et al., 1999). Each step in the transcellular movement of Ca 2+ has a vitamin D- dependent component, calbindin D9K is believed to be the rate-limiting molecule in vitamin D- induced transcellular calcium transport. The 18

35 paracellular route of calcium absorption involves a passive calcium transport through the tight junctions between mucosal cells (Figure 1). It is non-saturable, essentially independent of nutritional and physiological regulation, and is concentration dependent. Some debate still persists as to whether indeed the paracellular pathway is vitamin D-dependent (Chirayath et al., 1998; Fleet and Wood, 1999). Most calcium absorption in humans occurs in the small intestine, but there is some evidence (Barger-Lux et al., 1989) for a small colonic component (typically believed to be no more than 10% of total calcium absorption). However, the large intestine may represent a site of increased importance for calcium absorption when acidic fermentation takes place (Younes et al., 1996). This is important if one remembers that consumption of prebiotics (A prebiotic substance has been defined as a non- digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Cashman, 2003) will lead to acidic fermentation in the large intestine. In contrast, when dietary calcium is limited, the active, vitamin D-dependent transcellular pathway plays a major role in calcium absorption. Transcellular calcium absorption responds to calcium needs, as reflected by changes in plasma Ca 2+ concentration, by hormonemediated up- or down regulation of calbindin D9K in mucosal cells. For example, reduced plasma Ca 2+ evokes a parathyroid hormone mediated increase in plasma 1,25- dihydroxyvitamin D 3, which 19

36 stimulates increased calbindin D9K synthesis in the intestinal mucosa (Bronner, 1987). Methods for measuring calcium bioavailability It was suggested that the bioavailability of a nutrient reflects the sum of the effects of various factors (dietary and others) on the absorption, transport, cellular organization, storage and excretion of the Fig. 1. A schematic representation of transepithelial Ca 2+ transport in the intestine. The central feature is that calcium absorption occurs by two independent processes, namely transcellular and paracellular transport of Ca 2+ across an epithelium. nutrient. Therefore, bioavailability is not definable by a single test or variable. Rather, the study of bioavailability is dependent on the strategic use of in vitro, subcellular and cellular systems, animal models 20

37 and human subjects in an integrated manner. Calcium status can be evaluated by measurements of the bone mass at different sites of the skeleton. Currently, dual energy X-ray absorptiometry (DEXA) is clearly the method of choice to assess bone mass, owing to its accuracy, precision and low radiation exposure. DEXA can be used to detect changes in the bone mass over time and thus to assess the effect of intervention measures aimed to prevent loss of bone mass or to increase this mass (Institute of Medicine, 1997). The measurement of bone mass is considered the best way to evaluate the long term effects on calcium status of factors which influence calcium metabolism or calcium absorption (Schaafsma, 1997). Calcium absorption is an important component of calcium bioavailability. The various methods of assessing intestinal calcium absorption have been reviewed by Schaafsma (1997). Assessment of intestinal absorption, based on measurements of the difference between calcium intake and calcium excretion (i.e., metabolic balance technique) has major inherent shortcomings, including errors in estimating intake, incompleteness of fecal collections, and the inability to measure true calcium absorption. The latter point is important, considering the magnitude of the endogenous fecal calcium excretion (i.e., calcium secreted into the gastrointestinal tract which is not re-absorbed) and therefore the large difference between true- (accounting for endogenous loss) and apparent calcium absorption (which does not account for endogenous calcium loss). 21

38 Measurement of true calcium absorption can be performed with radioactive tracers of calcium, such as 45 Ca and 47 Ca. They are the most commonly used isotopes. Of the two radioactive isotopes, 47 Ca is used more frequently because of its shorter half-life, 4.7 days compared to days for 45 Ca (Neer et al., 1978; Janghorbarn and Young, 1980). These tracers are relatively cheap, can be measured accurately and can be used in very small (tracer) amounts. A disadvantage, however, is the ionizing radiation. Therefore, for ethical reasons, nutrition studies with these tracers in human volunteers are not preferred. Application of stable isotopes ( 42 Ca, 44 Ca, 46 Ca and 48 Ca) to measure calcium absorption has become common place in spite of the high isotope costs. Various methods of mass spectrometry analysis of stable calcium isotope enrichment can be used with this approach. True calcium absorption from a particular food can be measured after labeling this food extrinsically or intrinsically with a stable calcium isotope. If at the same time of administration of this food another stable calcium isotope is administered intravenously, true calcium absorption can easily be measured from stable calcium enriched values in samples of serum and urine obtained hour after administration. The timing of the urine collection can be modified to take account of predominantly the small intestinal component of calcium absorption (i.e., use of 24 hour urine collection) or to include the later colonic component (36-48 hour urine collection) (Cashman and Flynn, 1996). A faecal collection method can also be used as a more laborious alternative for this dual labelling. It is also worth noting that dietary 22

39 intervention trials in humans which employ such approaches, and which are aimed at assessing the influence of a dietary component on intestinal calcium absorption, are best carried out in randomized, double-blind, crossover design format, if possible. This type of study design, which is widely used in clinical, medical and pharmaceutical research, is considered a good approach for evaluating the efficacy of functional food ingredients. While ideally calcium bioavailability can be measured by human studies, such studies are often very time consuming and expensive to run (Cashman and Flynn, 1996). As an alternative, experimental animal models, especially the laboratory rat, have been used quite extensively for assessing the impact of dietary and other factors on calcium absorption. The balance and isotope tracers approaches, mentioned above, have been used in rats to determine calcium absorption. The adult rat, for example, has been shown to be a useful model for studies on calcium bioavailability since absorption mechanisms for calcium are similar in rats and in humans and a number of dietary and physiological factors affect calcium absorption similarly in the two species (Cashman and Flynn, 1996). However, while studies using laboratory animals are less expensive than studies in humans, they are somewhat limited by uncertainties with regard to differences in metabolism between animals and humans. Furthermore, it has been shown that, 1, 25 dihyroxyvitamin D 3 treatment induces the saturable, but not diffusional, component of calcium transport and induces accumulation of calbindin D9K mrna 23

40 (Fleet and Wood, 1999). Therefore, this relatively simple in vitro method appears to be a good model for predicting calcium bioavailability in humans under certain conditions. Other Calcium salts Many forms of Ca are generally recognized as safe by US Food and Drug administration, including Ca carbonate, Ca chloride, Ca lactate, Ca phosphate, Ca citrate and Ca gluconate (Fairweather-Tait and Teucher, 2002). One major difference in these salts relates to their solubility. Inorganic salts such as Ca carbonate and Ca phosphate are cheaper, less soluble and mainly mixed into solid foods, where solubility is not an issue, whereas Ca citrate, Ca gluconate, Ca lactate are normally more soluble and better absorbed (Fabri, 2004). The salts used fell into two categories, the salts with poor solubility which have very little effect on ph and the soluble salts which significantly reduce ph. Addition of Ca carbonate caused a significant increase in ph, but addition of Ca citrate and trica phosphate showed a small decrease in ph (Pathomrungsiyounggul et al., 2010). Calcium citrate is claimed to be moderately soluble (Gerstner, 2003). The addition of Ca citrate (25 mm Ca), to improve nutritional profile, was found not to adversely affect the flavour or chalkiness of a sterilized soy beverage (Weingartner et al., 1983). Role of calcium in apoptosis Apoptosis (programmed cell death) refers to a cellular selfdestruction mechanism. This term is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the 24

41 removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca 2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca 2+ signaling. Massive accumulation of Ca 2+ in the mitochondria leads to apoptosis. The Ca 2+ dynamics of endoplasmic reticulum (ER) and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis (Jeong and Seol, 2008). The calcium ion (Ca 2+ ) regulates various cellular processes, including apoptosis, by activating or inhibiting cellular signaling pathways and Ca 2+ regulated proteins (Monteith et al., 2007). Cells possess a 20,000-fold gradient between their intracellular ( 100 nm free) and extracellular (mm) Ca 2+ concentrations (Clapham, 2007), and within the cell itself. There are further Ca 2+ concentration gradients between the cytosol and certain organelles such as the sarcoplasmic/endoplasmic reticulum (SR/ER) (Monteith et al., 2007). Under normal conditions, the level of intracellular Ca 2+ is regulated by the combined action of channels, buffers, pumps, and exchangers; however, upon apoptotic stimulus, this can be disrupted and cytosolic Ca 2+ concentrations increase. The B-cell lymphoma/leukaemia-2 gene (Bcl-2) has been associated to B-cell malignancies (Tsujimoto et al., 1985). 25

42 The specific localization of Bcl-2 in the membranes of ER, mitochondria (Lithgow et al., 1994) and organelles playing a key role in intracellular Ca 2+ homeostasis suggests that the latter process could be a target of the action of this oncogene (Landolfi, et al., 1998; Rizzuto, et al., 1998). 26

43 MATERIALS AND METHODS MATERIALS 1. Animals feed and management A total number of 80 male Swiss albino rats were obtained from National Organization for Drug Control And Research (NODCAR) farm in Kafr El-Gabal, Giza, Egypt. Rats were kept in wire cages with controlled temperature (26-32ºC), a 12 h light - dark cycle and relative humidity (60-70%). Food and water were supplied ad-libitum for 6 weeks. The animals were divided into two main groups according to their ages, i.e., premature group consists of 40 male rats < 2 months age with g body weight and mature group comprises of 40 male rats > 4 months age with g body weight. Each rat group was randomly divided into 4 subgroups; each subgroup consists of 10 rats. The rat subgroups were; control: fed on a balanced diet; sub group1 (SG1): fed on a low- fat diet (4% fat); sub group2 (SG2): fed on a lowfat diet (4% fat), and high- calcium (1.8%) from calcium citrate; sub group3 (SG3): fed on a low- fat diet (4% fat) and high calcium (1.8%) from dry skimmed milk fortified with hydroxyapatite (2.5%) obtained from Sigma Aldrich Company ( Germany). Standard rat basal diet was formulated according to A.O.A.C. (2000). The compositions of the vitamin and salt mixtures used were similar to that reported by A.O.A.C. (2000) and Reeve et al. (1993), respectively (Tables 2 and 3). 2. Diets Diet composition (%) of different rat subgroups is shown in Table (1). 27

44 Table 1. Diet composition (%) of experimental rats Component Control Subgroup 1 Subgroup 2 Subgroup 3 Proteins Fats Carbohydrates Cellulose Minerals Vitamins Calcium citrate Dry skimmed milk Hydroxyapatite Table 2. Composition of mineral mixture (100g) Component Amount in gram CaCO 3 20 K 2 HPO CaHPO 4.2H 2 O 6 MgSO4.7H 2 O 8.16 NaCl Fe (C 6 H5O 7 ).7H 2 O 2.2 KI MnSO 4.4 H 2 O 0.4 ZnCl Cu SO 4.4 H 2 O Table 3. Composition of vitamin mixture (Kg) Vitamin Nicotinic Acid D-Calcium Pantothenate Pyridoxine HCl Thiamine HCl Riboflavin Folic Acid D-Biotin Vitamin B 12 (0.1% triturated in mannitol) -Tocopherol Powder (250 U/gm) Vitamin A Palmitate (250,000 U/gm) Vitamin D 3 (400,000 U/gm) Phylloquinone Powdered Sucrose Amount in gram

45 METHODS 3. Blood sampling Blood samples were collected twice; at the beginning and at the end of experimental period (after 6 weeks). The first blood samples were collected from inner Canthus of eye done by non heparinized capillary tubes then centrifuged at 605 xg for 15 minutes to collect sera which then were frozen at -8 ºC. Second blood samples were collected from animals hearts after anesthesia and sacrificing at the end of the 6 th week. 4. Biochemical and hormonal assays a. Biochemical assays All parameters were determined using commercially prepared and packaged kits purchased form Vitrosient, Cairo. Egypt. 1. Determination of serum total cholesterol. Serum total cholesterol was determined according to Fossati and Prencipe (1982). a. Principle Cholesterol present in serum was determined according to the following reactions: Cholesterol ester Cholesterol esterase Cholesterol + Fatty acids Cholesterol + O 2 Cholesterol oxidase Cholesterol-4-en-3-one + H 2 O 2 2H 2 O 2 + Phenol+ 4-amino-4-antipyrine Peroxidase Quinoneimine+ H 2 O b. Reagents 1. Buffer: phosphate buffer ph 6.9 (0.1 mol/l), phenol (15mmol/l) and sodium cholate (3.74 mmol/l). 29

46 2. Enzymes : 4-amino-4-antipyrine (0.5mmol/l), peroxidase ( >100µ/l),cholesterol oxidase ( 200 µ/l) and Cholesterol esterase ( >125 µ/l). 3. Standard: cholesterol standard solution (200 mg/dl). c. Procedure 1. The enzymes were mixed with the buffer solution to prepare the working solution. 2. Aliquots from each sample (10 µl) or standard were transferred to different clean tubes. 3. An aliquot of the working solution (1 ml) was added to each tube and 1 ml was used as a blank. 4. All tubes were incubated for 10 minutes at 20-25ºC. 5. The absorbances (A) of all samples and standard were measured against blank at 500 nm. d. Calculation Cholesterol concentration (mg/dl) = A Sample / A Standard X Determination of serum HDL-cholesterol Serum HDL-cholesterol was determined according to Burstein et al. (1980). a. Principle Very low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) in the samples were precipitated with phosphotungstate and magnesium ions. The supernatant contains high density lipoprotein cholesterol (HDL- C). The HDL-C then was spectrophotometrically measured by means of the coupled reaction described below: 30

47 Cholesterol ester+ H 2 O Cholesterol esterase Cholesterol + Fatty acids Cholesterol + O 2 Cholesterol oxidase Cholesterol-4-en-3-one + H 2 O 2 2H 2 O 2 + Phenol+ 4-amino-antipyrine Peroxidase Quinoneimine+ H 2 O b. Reagents 1- Reagent A: Phosphotungstate (0.4 mmol /l) and magnesium chloride (20 mmol /l). 2- HDL-Cholesterol standard :( 50 mg /dl). c. Procedure 1. Precipitation Aliquots from each sample (200 µl) were transferred into centrifuge tubes, then reagent A (20 µl) (precipitating agent) was added to each tube. All tubes were mixed thoroughly and stand for 10 minutes at room temperature, and then all tubes were centrifuged for 10 minutes at 1075 xg. 2. Colorimetry Aliquots from sample supernatant (10 µl), standard or distilled water were transferred to clean test tubes, then 1 ml of working solution (previously prepared in total cholesterol determination) was added to each tube. All tubes were incubated for 10 minutes at 20-25ºC, then the absorbances (A) of the standard and samples were measured at 500 nm against the blank. d. Calculation The HDL-C concentration in the sample is calculated using the following general formula: HDL- C concentration (mg/dl) = A Sample / A Standard X 50 X sample dilution factor (1:1) 31

48 3. Determination of serum LDL-cholesterol Serum LDL-C was determined according to Gerard and Gerard (1981). a. Principle LDL-C can be determined as the difference between total cholesterol and the cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the presence of polyethylene-glycol monomethyl ether. b. Reagents 1- LDL-C precipitating reagent: polyvinyl sulphate (0.7 g /l), EDTA Na 2 (5.0 mm) and polyethylene-glycol monomethyl ether (stabilizers)(170 g/l). 2- LDL-C standard (38.7 mg/dl). c. Procedure 1. A portion from each sample (200µl) was transferred into clean centrifuge tube. 2. The precipitating reagent (100 µl) was added to all tubes. Mixed well, then allowed to stand for 30 minutes at 2-8ºC and centrifuged for 15 min at 605 xg. 3. An aliquot from supernatant (10 µl) of each sample was transferred to clean tube for the determination of cholesterol concentration in the supernatant by the enzymatic method as described in the determination of total cholesterol. d. Calculation LDL-C concentration (mg/dl) = Total cholesterol (mg/dl) X Supernatant cholesterol (mg/dl) 32

49 4. Determination of serum triglycerides Serum triglycerides were determined according to Megraw et al. (1979). a. Principle Triglyceride Lipase Glycerol + Fatty acids Glycerol + ATP Glycerol kinase Glycerol -3-phosphate + ADP G-3-p Glycerol-3-phosphate oxidase Dihydroxy acetone phosphate + H 2 O 2 H 2 O 2 +Parachlorophenol+4-Amino-antipyrine Peroxidase Quinoneimine + 4H 2 O +HCl b. Reagents 1. Buffer : Tris buffer ph 7.6 (50 mmol), parachlorophenol (5.4mmol/l) and magnesium (2 mmol/l). 2. Enzymes: 4-Amino-antipyrine (0.4mmol/l), ATP (0.8mmol/l), lipase ( µ/l), glycerol kinase ( 200 µ/l), glycerol-3-phosphate oxidase ( 2.000µ/l) and peroxidase ( 200µ/l). 3. Standard glycerol (200 mg/dl). c. Procedure 1. An aliquot from each sample (10 µl) or standard was transferred to clean tubes. 2. A portion of reagent mixture (1 ml) was added to all tubes, then mixed with 1 ml of reagent mixture and transferred to clean tube as blank. 3. All tubes were thoroughly mixed and incubated for 10 minutes at room temperature. 33

50 4. The absorbances (A) of sample and standard were measured against the blank at 500 nm using photometer d. Calculation The triglycerides concentration was calculated using the following equation: Triglycerides concentration (mg/dl) = A Sample / A Standard X Determination of serum creatinine Creatinine was kinetically determined according to Bartels et al. (1972). a. Principle The complex formed by creatinine and picric acid in alkaline medium (sodium hydroxide, and sodium phosphate) was measured for one minute at 492 nm. b. Reagents 1. Color reagent: Picric acid (8.8 mmol/l). 2. Alkaline reagent: Sodium hydroxide (0.4 mmol/l). 3. Sodium phosphate (50 mmol/l). 4. Creatinine standard (1.5 mg/100ml). c. Procedure 1. One volume from alkaline reagent was added to one volume of color reagent to form the working solution. 2. A portion of working solution (1 ml) was transferred to clean cuvette and placed at 25 C. 3. A known volume of serum (100 l) or standard was transferred to the cuvette and thoroughly mixed. 34

51 4. The absorbance of the produced complex was measured against air between t = 20 sec. and t = 80 sec. at 492 nm. d. Calculation Creatinine concentration in the sample (mg/100 ml) = Absorbance of the sample X 1.5 Absorbance of the standard 6. Determination of urea Urea content was determined according to method described by Patton and Crouch (1977). a. Principle Urea + H 2 O Urease 2NH 3 + CO 2 2NH NaClO 2NH 2 Cl + 2NaOH 2NH 2 Cl + 2Phenol + 2OH 2,4 aminophenol + 2Cl + 2H 2 O 2,4 aminophenol + 2Phenol + O 2 2 indophenol + 2H 2 O The blue dye indophenol produced in the Berthelot reaction absorbs light between 530 nm and 560 nm proportional to the initial urea concentration. b. Reagents 1. Urea standard (R1): (30 mg/dl). 2. Buffer enzyme reagent (R2): Urease (>10000 U/l) and phosphate buffer (50 mmol/l). 3. Color reagent (R3): Sodium nitroprussid (0.2 mmol/l) and phenol (100 mmol/l). 4. Alkaline reagent (R4): Sodium hydroxide (150 mmol/l) and sodium hypochlorite (15 mmol/l). 35

52 c. Procedure 1. An aliquot from each sample (10 µl) or standard was transferred to a clean tube. 2. A portion of reagent 2 (0.2 ml) was added to sample, blank and standard tubes. 3. All tubes were well mixed and incubated for 5 min at 37 C. 4. One ml from each reagents 3 and 4 was added to each tube, mixed and incubated for 10 min at 37 C. 5. The absorbances of sample and standard were measured against the blank at 550 nm. d. Calculation Urea concentration (mg/dl) = A sample / A standard Determination of serum aspartate transaminase (AST) activity a. Principle Aspartate aminotransferase (AST) activity was estimated using kinetic procedure (provided from Vitroscient) based on the following reactions: Oxoglutarate + L-Aspartate AST L-glutamate + oxaloacetate Oxaloacetate + NADH +H + MDH L-malate + NAD + The rate of oxidation of the coenzyme NADH, involved in the reaction, is proportional to the AST activity in the sample (German Society for Clinical Chemistry, 1970). b. Reagents Reagent 1: Tris buffer ph 7.8 (80 mmol/l), L- aspartate (240 mmol/l), LDH (900 U/l) and MDH (600 U/l). Reagent 2: NADH (0.18 mmol/l) and 2-oxoglutarate (12mmol/l). 36

53 c. Procedure 1. Working reagent was prepared by adding 2 ml of reagent 2 to 40 ml of reagent An aliquot from sample (100 µl) was transferred to a clean tube. 3. A portion of working reagent (1ml) was added to sample tube and well mixed. 4. The contents of each test tube were incubated for 1.0 minute and absorbances at 340 nm were taken exactly after 1, 2 and 3 minutes. d. Calculation The decrease of the optical density per min. is measured at 340 nm. The change of absorbance per min (A/min) is multiplied by a factor of 1746 to calculate AST activity as units per liter (U/L). 8. Determination of serum alanine transaminase (ALT) activity a. Principle Serum alanine aminotransferase (ALT) activity was determined using a kit supplied by Vitroscient based on the following reactions, Oxoglutarate + L- Alanine ALT L-glutamate + pyruvate Pyruvate + NADH. H + LDH lactate + NAD + The rate of oxidation of the coenzyme NADH involved in that reaction is proportional to the ALT activity in the sample (German Society for Clinical Chemistry, 1970). b. Reagents 37

54 Reagent 1: Tris buffer ph 7.8 (100 mmol/l), L- alanine (500 mmol/l) and LDH (1700 U/l). Reagent 2: NADH (0.18 mmol/l) and 2-oxoglutarate (18 mmol/l). c. Procedure 1) Working reagent was prepared by adding 2 ml of reagent 2 to 40 ml of reagent 1. 2) An aliquot from sample (100 µl) was transferred to a clean tube. 3) A portion of working reagent (1ml) was added to sample tube and well mixed. 4) The contents of each test tube were incubated for 1.0 minute and the absorbances were taken exactly after 1, 2 and 3 minutes. d. Calculation The decrease of the absorbance per min is measured at 340 nm and the change of absorbance per min (A/min) is multiplied by a factor of 1746 to calculate ALT activity as units per liter (U/L). 9. Determination of total proteins in serum a. Principle Total serum protein was estimated according to a method described by Henry (1964), using a kit supplied by Diamond diagnostics. The principle of the method is that, protein forms a colored complex with cupric ions in an alkaline medium. The color intensity is proportional to the concentration of protein in the sample. 38

55 b. Reagents 1. Standard albumin (5g/dl). 2. Biuret reagent: Cupric sulfate (6 mmol/l), sodium potassium tartarate (21 mmol/l), sodium hydroxide (750 mmol) and potasium iodide (6 mmol). c. Procedure 1. An aliquot from each sample (25 µl) or standard was transferred to a clean tube. 2. Portions of reagent 2 (1 ml) were added to sample, blank and standard tubes. 3. All tubes were well mixed and incubated for 5 min at 37 C. 4. The absorbances of sample and standard were measured against the blank at 550 nm. d. Calculation Blood total proteins (g/dl) = A sample / A standard Determination of calcium in serum Serum calcium was determined according to the method of Gindler and King (1972). a. Principle Calcium ion produces with methylthymol blue, in an alkaline medium, a blue color the intensity of which is in proportion to 39

56 the calcium concentration. The presence of hydroxyl 8 quinoline eliminate the interference due to magnesium ions. b. Reagents 1. Standard: (10 mg/dl =2.5 mmol/l). 2. Chromogen : methylthymol blue (0.2 mmol/l),hydroxyl 8-qiunoline (140 mmol/l) and hydrochloric acid (200 mmol/l). 3. Buffer : ethanolamine (6 mol/l). c. Procedure 1. Equal volumes of reagent 1 and reagent 2 were mixed well before use. 2. An aliquot from each sample (20 µl) or standard was transferred to clean tubes. 3. Portions of working reagent (1 ml) were added to sample, blank and standard tubes. 4. All tubes were well mixed and incubated for 5 min at room temperature. 5. The absorbances of sample and standard were measured against the blank at 585 nm. d. Calculation Serum calcium concentration (mg/dl) = A sample / A standard Determination of phosphorus in serum Serum phosphorus was determined according to the method described by El-Merzebani et al. (1977). 40

57 a. Principle Inorganic phosphorus present in serum as phosphate forms a phosphomolybdate complex with molybdic acid. The complex is reduced by stannous chloride to a blue color which can be measured calorimetrically. Formic acid used as protein solubilizer and glycerol as stabilizer for assay system. b. Reagents 1. Standard: (4 mg/dl = mmol/l). 2. Color reagent: formic acid (2 mol/l), glycerol (0.6 mol/l) and ammonium molybdate (0.32 mmol/l). 3. Reducing agent: stannous chloride (2 mmol/l). c. Procedure Reagent 3 was diluted 100 times before use. 1. Aliquots from each sample (25 µl) or standard were transferred to clean tubes. 2. A portion of reagent 2 (1 ml) was added to sample, blank and standard tubes. 3. All tubes were mixed and then 100 µl of reagent 3 were added to sample, blank and standard tubes. 4. All tubes were well mixed and incubated for 10 min at 37 C 5. The absorbances of sample and standard were measured against the blank at 640 nm. d. Calculation 41

58 Serum phosphorus concentration (mg/dl) = A sample / A standard 4 b. Hormonal assay 1- Determination of total testosterone in serum: a. Principle Total testosterone was estimated according to the coat- A- count procedure which is a solid-phase radioimmunoassay (Jaffe and Behrman, 1974), using a kit supplied from Siemens (USA). The procedure is a solid-phase radioimmunoassay, based on a testosterone-specific antibody immobilized to a wall of a polypropylene tube. 125 I-labeled testosterone competes, for a fixed time, with testosterone in samples and standards for antibody sites. The solution in the tubes were decanted to separate bound and free testosterone, and then counted in a gamma counter. The amount of testosterone present in samples was determined from a calibration curve. b. Calculation A standard curve for testosterone concentration was constructed in which percent bound (B/B %) was plotted against different testosterone concentrations for each of the non-zero calibrators. The testosterone concentrations of the unknown samples were derived from the calibration curve. The percent bound (B/B %) = Net counts / Net MB counts x 100 Where: 42

59 Net counts = Average CPM Average CPM of NSB Net MB counts: Average counts at maximum binding (i.e., counts at zero standard average CPM of NSB). Where NSB = blank or the non-specific binding, CPM = count per minute Statistical analysis Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Hence, all variables were tested for normality using the Kolmogorov-Smirnov and Shapiro-Wilke tests. Normally distributed continuous variables are expressed as means ± standard error (SE). Analysis of variance (One-way ANOVA) and least significant differences (LSD) tests were used to allow comparison between the mean values of the studied parameters. Statistical significance was set at P < Correlations were performed using the Pearson correlation for parametric data. 43

60 44

61 RESULTS AND DISCUSSION 1-Body weight as a function of low-fat diet and high calcium supplementation Two rat groups were increased in weight but the % of increase was variable. The subgroups of high calcium elicit less % of increase than other rat subgroups. In premature rat subgroups, the % increase of SG2 and SG3 were near or close to the percent of increase of control group (Table 4). On the other hand, in mature rat subgroups the % increase in SG1, SG2 and SG3 were decreased as compared with control rat group (Table 5). Table 4. Changes in body weight of different premature rat subgroups Rat subgroup Initial weight (g) Final weight (g) Difference (g) % increase Control 84.4 a ± a ± a ± a ± 10.8 SG a ± a ± a ± a ± 5.7 SG a ± a ± a ± a ± 8.1 SG a ± a ± a ± a ± 8.5 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. Table 5. Changes in body weight of different mature rat subgroups Rat Initial weight Final weight Difference % increase subgroup (g) (g) (g) Control a ± a ± a ± a ± 2.6 SG a ± b ± b ± ac ± 8.2 SG a ± b ± b ± bc ± 5.5 SG a ± c ± b ± bc ± 11.1 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. 45

62 2- Serum lipids a. Serum cholesterol Rat serum cholesterol levels of premature subgroups are shown in Table (6). The results demonstrate that there were insignificant changes in cholesterol level except premature rats SG3 group which showed a significant decrease in serum cholesterol level. On the contrary, significant decreases in cholesterol content of SG1 and SG2 and SG3 in mature subgroups were noticed compared with control rat group Table (7). Table 6. Lipid profile of premature experimental rat subgroups Rat subgroup Cholesterol (mg/dl) Triglycerides (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Control 76.5 a ± a ± a ± a ± 0.6 SG a,c ± b ± b ± a ± 0.9 SG a ± b ± c ± b ± 1.0 SG b,c ± c ± c ± b ± 1.1 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. b. Serum triglycerides Serum triglyceride levels of premature rat subgroup showed a significant decrease in SG3, SG1 and SG2 as compared with control rat group Table (6) and Fig. (2). In mature rat subgroups, a significant decrease was evident in SG3 as compared with control rat group and rat subgroups 1 and 2 (Table 7 and Fig. 3). 46

63 Table 7.Lipid profile of mature experimental rat subgroups Rat Cholesterol Triglycerides HDL-C subgroup (mg/dl) (mg/dl) (mg/dl) LDL-C (mg/dl) Control 75.7 a ± a ± a,d ± a ± 0.9 SG b ± a ± d ± a ± 1.0 SG b ± a ± a,c ± a ± 1.9 SG c ± b ± b,c ± a ± 0.8 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. c. Serum HDL-C Serum HDL-C levels in premature subgroups SG2, SG3 were significantly decreased as compared with control and SG1. The 47

64 levels of serum HDL-C in SG1 rat subgroup were significantly decreased as compared with control rat group (Table 4). On the other hand, SG3 mature rat subgroup showed a significant decrease in HDL-C compared to control (Table 5). d. Serum LDL-C Rat serum LDL-C levels in premature rat groups were significantly decreased in SG3 and SG2, as compared with control and SG1 rat groups (Table 4). On the other hand, insignificant changes of LDL-C were observed in rat mature subgroups (Table 5). 3- Serum testosterone level The decrease of testosterone levels for premature and mature rat groups was significant as compared with each control rat group 48

65 (Tables 8, 9 and Fig. 4). A significant great decrease of testosterone level was found in SG3 when compared to other rat subgroups (Tables 8, 9). Table 8. Testosterone, phosphorus and calcium levels in sera of premature experimental rat subgroups Rat subgroup Testosterone (ng/dl) Phosphorus (mg/dl) Calcium (mg/dl) Control a ± a ± a ± 0.3 SG b ± a ± a ± 0.1 SG b ± a ± a ± 0.6 SG c ± a ± a ± 0.4 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. Table 9. Testosterone, phosphorus and calcium levels in sera of mature experimental rats subgroups Rat subgroup Testosterone (ng/dl) Phosphorus (mg/dl) Calcium (mg/dl) Control a ± a ± a ± 0.25 SG b ± a ± a ± 0.36 SG c ± b ± b ± 0.4 SG d ± b ± a ± 0.19 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. 49

66 4-Serum calcium Serum calcium level showed insignificant changes in premature rat subgroups. In contrast serum calcium level revealed highly significant increase in SG2 mature rat group compared with control rat group and highly significant increase as compared with SG1 (Table 9). 5- Serum phosphorus The results indicate non-significant difference in phosphorus content of premature rat subgroups. On the other hand, mature rat subgroups elicited significant decrease in phosphorus level in SG2 and SG3 compared with control rat group and highly significance compared to SG1 ( Table 9 and Fig. 5). 6. Kidney function tests a. Serum urea level 50

67 In premature rat groups there were no significant differences between subgroups in serum urea levels (Table 10). In the mature rat group, the same results were obtained (Table 10 and Fig. 6). Table 10. Kidney function tests of different premature and mature rat experimental groups Premature rats Mature rats Rat subgroup Creatinine (mg/dl) Urea (mg/dl) Creatinine (mg/dl) Urea (mg/dl) Control 0.56 a ± a ± a ± a ± 2.02 SG b ± a ± a ± a ± 1.70 SG c ± a ± b ± a ± 2.04 SG b ± a ± b ± a ± 1.90 LSD 5% SG refers to rat subgroup. The data (value ± SE) are the mean values of three measurements for the same sample. Means with each column followed by the same letter are not significantly different. 51

68 b. Serum Creatinine level In premature rat group, SG1 and SG3 showed a significant decrease in serum creatinine level compared to control. While SG2 demonstrated a significant increase in serum creatinine level compared to SG1, SG3 and control rat subgroups (Table 10 and Fig. 7). On the other hand, in mature rat group, Table (10) shows that SG1 rat subgroup exhibit non-significant increase in serum creatinine level comparing to control. While SG3 and SG2 showed a significant serum creatinine increase compared to control. Also both SG3 and SG2 revealed a significant increase in serum creatinine level compared to SG1 (Table 10 and Fig. 7). 52

69 7 - Liver function tests a. ALT activity level in rat serum In premature rat subgroups, SG2, SG3 and SG1 showed significant decrease in serum ALT activity when compared with control. Serum ALT activity in premature SG3 rats showed a significant decrease compared to SG2 (Table 11). On the other hand, in mature rat groups, SG1 and SG3 showed a significant decrease in serum ALT activity compared to control (Table 11). SG2 revealed a significant increase in serum ALT activity compared to control and SG1 respectively. SG3 showed a significant decrease in ALT activity compared to control and SG2 (Table 11 and Fig. 8). 53

70 b. AST activity level in rat serum In premature rat group, there was non-significant decrease in serum AST activity between SG3, SG2, SG1 and control (Table 11). On 54

71 the other hand, in mature rat group, SG1 showed significant decrease in serum AST activity compared to control (Table 11). SG2 and SG3 showed a significant increase in serum AST activity compared to SG1 (Fig. 9). c. Rat serum total protein level There were non significant changes of serum total protein level in both mature and premature rat groups (Table 11 and Fig. 10). 55

72 Correlation between calcium and other parameters Generally speaking and as shown in Table (12), in premature rat groups, Ca level showed non-significant correlation with testosterone, cholesterol, triglycerides, phosphorus and HDL-C. Also, cholesterol level possessed non-significant correlation with testosterone and all other parameters except triglycerides. Triglycerides level exhibited a highly positive significant correlation with testosterone and HDL-C while it exhibited a positive significant correlation with cholesterol and LDL-C contents. In mature rat group as shown in Table (13), calcium data demonstrated non-significant correlation with testosterone, cholesterol, phosphorous, HDL-C, triglyceride and LDL-C levels. Cholesterol level illustrated high positive significant correlation with both of testosterone 56

73 and triglycerides and a positive significant correlation with phosphorous. Triglyceride level showed a highly positive significant correlation with testosterone, cholesterol and a positive significant correlation with HDL-C. HDL-C level exhibited a positive significant correlation with testosterone content. While, LDL-C level induced a non-significant correlation with cholesterol content and other parameters. Table 12. Correlation between calcium, phosphorus, testosterone and lipid profile parameters in premature rats. T P Ca Chol TG HDL LDL T CC ** 0.758** 0.588** P CC Ca CC Chol CC * TG CC 0.923** * ** 0.560* HDL CC 0.758** ** ** LDL CC 0.588** * 0.691** 1 CC refers to correlation coefficient, (T) testosterone, (P) phosphorus, (Ca) calcium, (Chol) cholesterol, (TG) triglycerides, (HDL) high density lipoprotein- C, (LDL) low density lipoprotein- C, * correlation is significant at 0.05 level, ** correlation is highly significant at 0.01 level. Table 13. Correlation between calcium, phosphorus, testosterone and lipid profile parameters in mature rats. T P Ca Chol TG HDL LDL T CC ** ** 0.762** 0.505* P CC ** *.526* Ca CC Chol CC 0.828** 0.534* ** TG CC 0.762**.526* ** * HDL CC 0.505* * LDL CC CC refers to correlation coefficient, (T) testosterone, (P) phosphorus, (Ca) calcium, (Chol) cholesterol, (TG) triglycerides, (HDL) high density lipoprotein- C, (LDL) low density lipoprotein- C, * correlation is significant at 0.05 level, ** correlation is highly significant at 0.01 level. 57

74 Obesity is a serious health hazard; its prevalence is increasing annually and constitutes an important public health problem. Obesity has a significant role in a number of diseases such as hypertension, hyperinsulinmia, lazy, difficulty to breath, osteoprosis and Barrennss. Consequently, the present study was undertaken to find out a suitable dietary program to maintain lower prevalence of overweight or obesity by adjusting the diet components. In fact, several investigations stressed upon the role of calcium especially obtained from dairy products to adjust bodyweight. Consequently, the present study was basically relied on using calcium from different sources to see its role to maintain overweight or obesity in rats. Concerning the rat body weight, the results indicated nonsignificant decrease in % of body weight of premature rat subgroups. The lowest decrease in % of body weight of premature rats (SG3) fed on a high calcium diet and this case may be due to the maturation period which is characterized with maximum growth rates, maximum calcium utilization, maximum protein utilization and maximum bone growth (Johnston et al., 1992). Growing evidence supports a relationship between increasing calcium intake and reductions in body weight specific to fat mass (Teegarden, 2003). The lowering effect of high- calcium diets on body weight in mature rats shown in the present study agreed with the results of Petrov and Lijnen (1999), Sun and Zemel (2007) and Zhang et al. (2012). There are two main physiological mechanisms proposed to explain how calcium intake can affect body weight or body fat. The first 58

75 one is that the high dietary calcium intake suppresses the levels of parathyroid hormone (PTH) and 1,25-hydroxy vitamin D, thereby causing lower levels of intracellular calcium and inhibiting lipogenesis and stimulating lipolysis (Zemel, 2002 and 2003). Therefore, calcium intake may directly affect whether adipocytes store or break down fat. The second proposed mechanism by which calcium may impact body weight is that increased dietary calcium seems to bind more fatty acids in the colon and inhibit fat absorption. Welberg et al. (1994) showed that calcium supplementation increased the percentage of excretion of total fat as related to fat intake. On the contrary, Zhang and Tordoff (2004) data disagreed with the present results. In the present work, rats supplemented with high calcium in milk induced the greatest lowering effect on body weight, which is in agreement with the findings of Fried et al. (2012). Dairy sources of calcium markedly attenuate weight and fat gain and accelerate fat loss to a greater degree than do other supplemental sources of calcium. This augmented effect of dairy products relative to supplemental calcium is likely due to additional bioactive compound, including the angiotensinconverting enzyme inhibitors and the high concentration of branchedchain amino acids in whey (Zemel, 2004 and Schrager, 2005) and milk phospholipids which act as synergistic effect with calcium absorption bioavailabity and utilization (Scholz-Ahrens and Schrezenmeir, 2000; Tsuchita et al., 2001 and Erba et al., 2002). 59

76 Concerning serum total cholesterol, its level showed a significant decrease in premature SG3 rats which agreed with the findings of Malekzadeh al. (2007). While non- significant change of cholesterol in premature SG2 rats was noticed. Although feeding on high- calcium diet is highly needed due to the rapid growth rates of premature rats in which most of calcium is principally used in bone building at the expense of the hypocholestremic effect of calcium. Therefore, the available calcium is not adequate to cause the hypocholesteremic effect. The calcium results illustrate non-significant change on cholesterol level in premature rats which agreed with data of Shahkhalili et al. (2001). On the other hand, the significant decrease in mature subgroups SG1, SG2 and SG3 agreed with the findings of Malekzadeh al. (2007), Shalileh et al. (2009), Shidfar et al. (2010) and Zhang et al. (2012) and disagreed with work of Zhang and Tordoff (2004), Reid (2004) and Jacobsen et al. (2005). The biological interpretation for a cholesterol lowering effect is that calcium is known to bind to bile acids to form insoluble soaps and thus presumably prevent cholesterol entering into the enter hepatic circulation (Denke et al., 1993 and Shahkhalili et al., 2001). A study found that a calcium supplementation induced lowering cholesterol levels in rats, rabbits, and goats but not in pigs and associated with an increase in the excretion of fecal bile acids in most but not all studied animals. In addition, this effect was more pronounced when the diet contains higher proportions of saturated fats (Van-Meijl et al., 2008). Increased fecal loss of fat, especially saturated fatty acids (SFAs) is 60

77 important because SFAs tend to increase serum cholesterol levels when absorbed. Serum triglyceride level of premature rats showed a significant decrease in SG3, SG1 and SG2. The significant decrease of serum triglycerides in premature SG1 was expected due to feeding on a low-fat diet and also in premature rat subgroups SG2, SG3 due to the lipolytic effect of high-calcium diet [Shapses et al.( 2004), Thompson et al. ( 2005) and Zhang et al. (2012)]. In mature rats SG3, triglyceride level was significantly decreased. In both, premature and mature rats fed on high-calcium from milk which agreed with the work of Shalileh et al. (2009) and disagreed with the findings of Malekzadeh et al. (2007) and Shidfar et al. (2010). In the present study, premature rat subgroups; SG1, SG2 and SG3 induced a significant decrease of HDL-cholesterol level. These results agreed with Kirkland et al. (1987) who reported that levels of HDL-C cholesterol in males decreased during adolescence and disagreed with the results of Zhang et al. (2012). HDL-C showed a positive significant correlation with endogenous levels of testosterone which agreed with Freedman et al. (1991). On the other hand, insignificant decreases were observed in HDL-C in SG2 mature rats which agreed with the data of Malekzadeh et al. (2007). Serum LDL C in premature rat subgroups fed on high-calcium from milk or calcium citrate showed a significant decrease compared to control rats and low-fat subgroups, respectively. This finding agreed 61

78 with the results of Malekzadeh et al. (2007) and Shalileh et al. (2009) while in mature rat subgroups possessed non-significant change in this parameter. Testosterone, the main sex hormone in males, plays an important role in body like regulation of gene expression and male sex traits. So, it was important to study the hypolipidemic effect of calcium and the levels of testosterone especially in premature rat subgroups. In the present study, all rat subgroups whether mature or premature showed a significant decrease in serum total testosterone comparing to control. The diet containing low-fat or calcium supplemented subgroups induced the greatest decrease of total testosterone in SG3 followed by SG2 then SG1. These results are in line with the hypolipidemic effect of highcalcium supplementation or low-fat diets. This significant decrease in serum total testosterone can be interpreted as a result of lowered serum lipids due to administration of low-fat diets or high-calcium diets since lipids (cholesterol) are the precursor of steroid sex hormones. In addition, the high calcium supplementation from milk subgroups possessed the lowest serum testosterone levels and that might be alarming especially for premature males. The great decrease of testosterone in milk fed rat subgroups agreed with the data of Maruyama et al. (2010) who reported that this decrease might stem from that milk contains large amounts of estrogens and progesterone while the decrease in testosterone found in the present study can be related to the hypolipidemic effect of high calcium supplementation. 62

79 The lowering effect of high calcium intake on testosterone in mature rats agrees with findings of Chandra et al. (2012). Serum calcium level showed non-significant changes in premature rat group, and this finding agreed with Kruger et al.(2003) who mentioned that bioavailability of calcium is equivalent to that obtained from milk fortified either with calcium carbonate or milk calcium (hydroxyapatite) in growing rats. While in mature rat group SG2, serum calcium revealed a significant increase compared to control, SG1 and SG3 subgroups, and these data agreed with the results of Kochanowski (1990) and Weaver et al. (2002). Calcium and phosphorus homeostasis relies on a complex, tightly regulated system involving many ions and hormones. The regulation of calcium and phosphorus is controlled by the actions of these ions and hormones on the intestine, kidneys and bone (Taylor and Bushinsky, 2009). In the present study, premature rat groups showed nonsignificant differences in serum phosphorus levels which come in harmony with the data of serum calcium levels in the premature rat groups. This significant decrease of serum phosphorus agreed with the results of Aguilera-Barreiro et al. (2005). Also, this result can be interpreted in calcium / phosphorus homeostasis view. On the other hand, in mature rat group, the SG3 showed the greatest significant decrease of serum phosphorus levels. Also, this rat subgroup elicits a significant increase in serum calcium levels and this might be interpreted in calcium / phosphorus homeostasis view. 63

80 The normal results of kidney function of premature SG3 rat group agreed with the results of Hou et al. (2009), while results of liver function in premature SG3 rat group disagreed with his findings. On the other hand, both of Kidney and liver function results of mature SG3 rat group disagreed with the results of Hou et al. (2009). The nonsignificant changes of AST activity in both mature and premature SG2 rat group agreed with the results of Lagarto et al. (2011). From thesis results, it could be suggested that consuming safe amounts of calcium about ( g Ca/day) from 3-6 cups of yogurt or milk can be a suitable dietary regime to avoid overweight. Applying such regime on premature subjects may need further studies to be confirmed. 64

81 SUMMARY Obesity is recognized as one of the most significant public health problems in the world. Therefore, the present study was undertaken to find out a suitable dietary regime to maintain a lower prevalence of overweight or obesity by adjusting the diet components. Here, 80 male Swiss albino rats were selected according to their ages and divided into two main groups, i.e., premature and mature groups. Each rat group was divided into 4 subgroups and each subgroup was fed on a diet of varied composition. Premature rat group was consisted of 40 male rats < 2 months age with g body weight and mature group comprised of 40 male rats > 4 months age with g body weight. Each rat group was randomly divided into 4 subgroups; each subgroup consists of 10 rats. The rat subgroups were; control: fed on a balanced diet; sub group1 (SG1): fed on a low- fat diet (4% fat); sub group2 (SG2): fed on a low-fat diet (4% fat), and high- calcium (1.8%) from calcium citrate; sub group3 (SG3): fed on a lowfat diet (4% fat) and high calcium (1.8%) from dry skimmed milk fortified with hydroxyapatite. Blood samples were collected twice; at the beginning and at the end of experimental period (6 weeks) then centrifuged to separate sera. Serum levels of lipids (total cholesterol, triglycerides, HDL-C and LDL-C), calcium, phosphorous, testosterone, liver and kidney functions were determined in addition to body weight measurement. The results can be summarized as follows: 1- Non-significant decrease of percentage of body weight gain in premature rats fed on high-calcium diets while significant decrease 65

82 of percentage of body weight gain in mature rats fed on the same diet composition. 2- The levels of serum HDL-C, LDL-C, triglycerides and testosterone were significantly decreased in premature rats fed high- calcium diets. 3- In premature rats, only rat subgroup fed on high calcium from milk, showed a significant decrease in serum cholesterol levels. 4- Calcium and phosphorus levels exhibited non- significant change between premature rats. 5- In mature rats, LDL-C data demonstrated non-significant changes while cholesterol, triglyceride and HDL-C levels were significantly decreased in rats fed high -calcium diet compared to control. 6- Serum testosterone levels were significantly decreased in mature rats fed low- fat diets or low fat diets supplemented with highcalcium level. 7- Serum levels of urea and total proteins showed non-significant differences between subgroups in both premature and mature rats. 8- In premature rat group, rats fed only low fat diet or high milk calcium diet showed a significant decrease in serum creatinine level compared to control, while rats fed high calcium citrate diet demonstrated a significant increase in serum creatinine level compared to low fat- fed subgroup. On the other hand, mature rats group fed only low fat diet exhibited non-significant increase in serum creatinine level comparing to control. While mature rats group fed on high calcium diets showed a significant serum creatinine increase compared to control and rats fed on low-fat diet. 66

83 9- Serum ALT activity level showed significant decrease in all premature subgroups compared to their control group. On the other hand, in mature rat groups, both of rats fed on low-fat diet and rats fed on high Ca from milk showed a significant decrease in serum ALT activity compared to control while rats subgroup fed on high Ca diet from calcium citrate revealed a significant increase in serum ALT activity compared to control and low- fat subgroup, respectively. 10- Serum AST activity level showed non-significant decrease in all premature subgroups while in mature rats group, only rats subgroup fed on low-fat diet showed significant decrease of serum AST activity. Also there were no significant changes in serum AST activity between rats subgroups fed on high calcium diets and control. 11- In general, the results from the present thesis suggest to consume low fat diet (4%) supplemented with high calcium from dry skimmed milk fortified with hydroxyapatite as a suitable dietary program to avoid overweight. In addition, consuming safe amounts of calcium about ( mg Ca/day) from 3-6 cups of yogurt or milk can be a suitable dietary regime to avoid overweight. Applying such regime on premature subjects may need further studies to be confirmed. 67

84 68

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