EFFICACY STUDIES OF VITAMIN A FORTIFIED COOKIES IN PREGNANT AND LACTATING WOMEN

By SHAHID MAHMOOD
M.Sc.(Hons.) Food Technology (UAF)

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY
IN

FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY

UNIVERSITY OF AGRICULTURE FAISALABAD

2009

To
The Controller of Examinations, University of Agriculture, Faisalabad.

We, the members of Supervisory Committee, certify that the contents and form of thesis submitted by Mr. Shahid Mahmood , Regd. No. 96-ag-1615, have been found satisfactory and recommend that it be processed for evaluation by External Examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

Chairman (Dr. Masood Sadiq Butt)

Member (Prof. Dr. Faqir Muhammad Anjum)

Member (Dr. Haq Nawaz)

DEDICATED
TO

HOLY PROPHET MUHAMMAD

(Peace be upon Him)

TABLE OF CONTENTS
Sr. No. Acknowledgements List of Tables List of Figures List of Appendices Abstract L 2. 2.1. 2.2. 2.3. 2.3.1. 2.3.2. 2.3.3. 2.4. 2.4.1. 2.4.2. 2.5. 2.6. 2.6.1. 2.6.2. 2.6.3. 2.6.4. 2.7. 2.7.1. 2.7.2. 2.7.3. INTRODUCTION REVIEW OF LITERATURE Micronutrients and Allied Deficiencies Vitamin A; An Overview Vitamin A; Requirements and Physiological Benefits Recommended Dietary Intakes RDA for Pakistani Population Physiological Benefits Consequences of Hypo and Hypervitaminosis A Deficiency Consequences Hypervitaminosis A Vitamin A Deficiency (VAD) Intervention Strategies Food Diversification Supplementation Food Fortification Nutrition Education Vitamin A Fortification; Product Development Vitamin A Fortification Fortification of Cereal Products Quality Attributes of the Products Title Page # I II V VI VII 01 06 07 09 10 10 11 11 12 13 14 14 17 18 18 19 21 21 22 22 22

2.8. 2.8.1. 3. 3.1. 3.2. 3.2.1. 3.2.2. 3.2.2.1. 3.2.3. 3.2.3.1. 3.2.3.2. 3.2.3.3. 3.2.4. 3.2.4.1. 3.2.4.2. 3.2.4.3. 3.2.4.4. 3.2.4.5. 3.2.4.6. 3.2.4.7. 3.2.5. 3.3. 3.3.1. 3.3.2. 3.3.3. 3.4. 3.4.1. 3.4.2. 3.4.2.1

Efficacy Studies Hematology and Serology MATERIALS AND METHODS Materials Product Development Preparation of Vitamin A Fortified Cookies Stability of Vitamin A Fortificants Determination of Vitamin A content Physical Analysis of Cookies Diameter Thickness Spread Factor Chemical Composition of the Cookies Moisture Content Crude Protein Crude Fat Crude Fiber Total Ash Nitrogen Free Extract (NFE) Thiobarbituric Acid Value Sensory Evaluation Efficacy Studies of Vitamin A Fortified Cookies in Pregnant and Lactating Women Approach and Assortment of Pregnant Women Selection and Assortment of Subjects Provision and Distribution of the Fortified Cookies Blood Sampling and Determinations Whole Blood Assays Serum Assessment Liver function Test

27 31 35 35 35 35 36 36 37 37 37 38 38 38 38 38 38 39 39 39 39 40 40 41 42 42 42 43 43

3.4.2.2. 3.4.2.3. 3.4.3. 3.4.3.1. 3.4.3.2. 3.4.3.3. 3.5. 4. 4.1. 4.1.1. 4.1.2. 4.1.3. 4.2. 4.2.1. 4.2.2. 4.2.3. 4.2.4. 4.2.5. 4.2.6. 4.2.7. 4.3. 4.4. 4.4.1. 4.4.2. 4.4.3. 4.4.4. 4.4.5. 4.4.6. 4.5.

Renal Function Tests Lipid Profile Retinoids and Carotenoid Investigations Serum Retinol Assessment Serum Retinyl Esters Estimation Serum -carotene Determination Statistical Studies RESULTS AND DISCUSSIONS Physical Analysis of Fortified Cookies Diameter Thickness Spread Factor Chemical Composition of Fortified Cookies Moisture Content Ash Content Crude Protein Crude Fat Crude Fiber Nitrogen Free Extract Thiobarbituric Acid Value Baking and Storage Stability Sensory Evaluation Color Flavor Taste Texture Crispness Overall Acceptability Efficacy Studies in Pregnant Women

43 43 44 44 45 45 46 47 47 49 49 50 50 50 53 53 55 55 56 56 56 66 66 67 67 67 70 70 72

4.5.1. 4.5.2. 4.5.3. 4.5.4. 4.5.5. 4.5.6. 4.6. 4.6.1. 4.6.2. 4.6.3. 4.6.4. 4.6.5. 4.6.6. 5.

Serum Retinol, Retinyl Esters and -Carotene Profile Red Blood Cell Indices White Blood Cells Indices Liver Function Tests Renal Function Tests Lipid Profile Efficacy Studies in Lactating Women Serum Retinol, Retinyl Esters and -Carotene Profile Red Blood Cells Parameters White Blood Cells Indices Liver Function Tests Renal Function Tests Lipid Profile SUMMARY RECOMMENDATIONS LITERATURE CITED APPENDICES

72 84 97 103 111 111 120 120 128 139 143 147 152 168 173 174 196

LIST OF TABLES
Sr. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Title Different treatment used to prepare fortified cookies Mean squares for physical parameters of fortified cookies Effect of treatments on physical parameters of fortified cookies Effect of storage on physical parameters of fortified cookies Mean squares for chemical composition of fortified cookies Effect of treatments on moisture, ash and crude protein content of fortified cookies Effect of storage on moisture, ash and crude protein content of fortified cookies Effect of treatments on crude fat, crude fiber, NFE content and TBA value of fortified cookies Effect of storage period on crude fat, crude fiber, NFE content and TBA No. of fortified cookies Mean squares for effect of baking on vitamin A level in fortified cookies Effect of baking on stability of vitamin A in fortified cookies Mean squares for storage stability of vitamin A content in fortified cookies Effect of treatments and storage on stability of vitamin A (g/100g) in fortified cookies Mean squares for sensory evaluation scores of fortified cookies Effect of treatments on sensory scores of fortified cookies Effect of storage on sensory scores of fortified cookies Effect of treatments on sensory scores of fortified cookies Effect of storage on sensory scores of fortified cookies Mean squares for effect of fortified cookies on serum retinol, retinyl esters and - carotene levels of pregnant women Serum retinol level (g/dL) of pregnant women Serum retinyl esters level (g/dL) of pregnant women Serum - carotene level (g/dL) of pregnant women Mean squares for effect of fortified cookies on red blood cells indices of pregnant women Total red blood cells count (M/L) of pregnant women Hematocrit (%) of pregnant women Hemoglobin status (g/dL) of pregnant women Mean corpuscular hemoglobin (pg) of pregnant women Page # 48 51 51 52 52 54 54 57 57 59 60 61 62 68 69 69 71 71 78 79 79 80 86 87 87 88 91

Sr. No. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37 . 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Title Mean corpuscular volume (fL) of pregnant women Mean corpuscular hemoglobin concentration (g/dL) of pregnant women Platelets count (K/L) of pregnant women Erythrocytes sedimentation rate (mm/Hr) of pregnant women Mean squares for effect of fortified cookies on white blood cells indices of pregnant women Total white blood cells count (K/L) of pregnant women Neutrophiles (%) of pregnant women Lymphocytes (%) of pregnant women Monocytes (%) of pregnant women Eosinophiles (%) of pregnant women Basophiles (%) of pregnant women Mean squares for effect of fortified cookies on liver function tests of pregnant women Bilirubin total (mg/dL) of pregnant women Bilirubin direct (mg/dL) of pregnant women Bilirubin indirect (mg/dL) of pregnant women Glutamate pyruvate transaminase (U/L) of pregnant women Alkaline phosphatase (U/L) of pregnant women Mean squares for effect of fortified cookies on renal function tests of pregnant women Urea (mg/dL) of pregnant women Creatinine (mg/dL) of pregnant women Mean squares for effect of fortified cookies on lipids profile of pregnant women Cholesterol level (mg/dL) of pregnant women Low density lipoprotein (mg/dL) of pregnant women High density lipoprotein (mg/dL) of pregnant women Triglycerides level (mg/dL) of pregnant women Mean squares for effect of fortified cookies on serum retinol, retinyl esters and -carotene levels of lactating women Serum retinol level (g/dL) of lactating women Serum retinyl esters level (g/dL) of lactating women Serum -carotene level (g/dL) of lactating women Mean squares for effect of fortified cookies on red blood cells indices of lactating women Total red blood cells counts (M/L) of lactating women Hematocrit (%) of lactating women Hemoglobin status (g/dL) of lactating women

Page # 91 93 93 95 99 100 100 101 101 102 104 106 107 107 109 109 110 112 113 113 114 115 117 117 118 122 123 126 126 130 131 133 133

Sr. No. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87

Title Mean corpuscular hemoglobin (pg) of lactating women Mean corpuscular volume (fL) of lactating women Mean corpuscular hemoglobin concentration (g/dL) of lactating women Platelets count (K/L) of lactating women Erythrocytes sedimentation rate (mm/Hr) of lactating women Mean squares for effect of fortified cookies on white blood cells of lactating women Total white blood cells count (K/L) of lactating women Neutrophiles (%) of lactating women Lymphocytes (%) of lactating women Monocytes (%) of lactating women Eosinophiles (%) of lactating women Basophiles (%) of lactating women Mean squares for effect of fortified cookies on liver function tests of lactating women Bilirubin-total (mg/dL) of lactating women Bilirubin-direct (mg/dL) of lactating women Bilirubin-indirect (mg/dL) of lactating women Glutamate pyruvate transaminase (U/L) of lactating women Alkaline phosphatase (U/L) of lactating women Mean squares for effect of fortified cookies on renal function tests of lactating women Urea (mg/dL) of lactating women Creatinine (mg/dL) of lactating women Mean squares for effect of fortified cookies on lipids profile of lactating women Cholesterol level (mg/dL) of lactating women Low density lipoprotein (mg/dL) of lactating women High density lipoprotein (mg/dL) of lactating women Triglycerides (mg/dL) of lactating women Correlation Coefficients

Page # 134 134 137 137 138 140 141 141 142 142 144 144 145 146 146 148 148 149 150 151 153 153 154 154 155 157 166

LIST OF FIGURES
Sr. No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Title
Percent decrease in vitamin A of fortified cookies during baking Percent decrease in vitamin A of fortified cookies during storage Percent change in serum retinol of pregnant women Percent decrease in retinyl esters of pregnant women Percent decrease in b-carotene of pregnant women Percent change in hemoglobin of pregnant women Percent increase in serum retinol of lactating women Percent change in retinyl esters of lactating women Percent decrease in b-carotene of lactating women Percent increase in hemoglobin of lactating women Percent change in serum retinol during pregnancy and lactation Percent decrease in retinyl esters during pregnancy and lactation Percent decrease in -carotene during pregnancy and lactation Percent change in hemoglobin during pregnancy and lactation

Page #
63 64 81 82 83 90 124 127 129 135 159 161 162 164

LIST OF APPENDICES
Sr. No.
I II III IV V

Title
Sensory Evaluation Performa Population Welfare Centers Engaged in the Study Data Collection Form Volunteer Consent Performa Dietary Intake Questionnaire

Page #
196 197 198 199 200

ACKNOWLEDGEMENTS
All acclamation and appreciation are for Almighty ALLAH The Magnificent and Merciful and His prophet Muhammad (peace be upon him) who is forever a torch of guidance and knowledge for humanity. I have the honor to express my deep sense of indebtedness to ever affectionate Dr. Masood Sadiq Butt, Associate Professor, National Institute of Food Science and Technology, University of Agriculture, Faisalabad whose guidance and cooperation really helped me in the completion of this piece of work. I feel much pleasure to extend my sincere appreciation to Prof. Dr. Faqir Muhammad Anjum, Director General, National Institute of Food Science and Technology, University of Agriculture, Faisalabad for his continuous encouragement and sympathetic attitude during the course of investigation. I have no words to explain my obligation to my committee member, Dr. Haq Nawaz, Associate Professor, Institute of Animal Nutrition & Feed Technology, University of Agriculture, Faisalabad for his kind favor in this research study. I would like to thank Higher Education Commission, Pakistan, for providing funding and support for this research work. My sincere gratitude is also to the Population Welfare Department, Faisalabad for facilitating and conducting a vital part of my study. I am grateful to Mr. Tauseef Sultan, Ph. D. Scholar, National Institute of Food Science and Technology, University of Agriculture, Faisalabad for his support, extended during my research project. I will be failing in my duties if do not extend my zealous thanks to all of my friends and fellows. I am highly obliged to my wife and children for their sacrifice and consistent support during the course of my studies. Words are lacking to express my humble obligation to my loving parents, brothers and sisters for their love and wish to see me glittering high on the skies of the success. SHAHID MAHMOOD

ABSTRACT Present project was planned to alleviate the vitamin A deficiency in pregnant and lactating women by providing vitamin A fortified cookies. For the purpose, cookies were prepared by adding two vitamin A fortificants i.e. retinyl acetate and retinyl palmitate separately @ 30, 40 and 50% RDA of pregnant (750g) and lactating (1200g) women. During storage studies, moisture content and TBA no. of the cookies were increased from 2.51 to 2.84% and 0.40 to 0.69mg malenaldehyde/Kg, respectively. Vitamin A losses during baking and storage ranged from 7.95 to 15.79% and 8.02 to 9.69%, respectively, among the treatments. On the basis of physico-chemical analysis, baking & storage stability and sensoric attributes, T4 (50% of RDA; retinyl acetate) and T7 (50% of RDA; retinyl palmitate) were selected for efficacy purposes. Selected treatments (Five cookies; 50g per day) along with placebo were provided to the respective groups of vitamin A deficient pregnant women in third trimester. Retinyl acetate and retinyl palmitate fortified cookies significantly enhanced the level of serum retinol 18.51% and 21.56% in pregnant women and 9.43 and 12.84% in lactating mothers, respectively. In placebo group, the serum retinol level was significantly decreased up to 9.32% during pregnancy with a non-significant increase 1.81% during lactation. Collectively, the serum retinol level showed a significant increase of 29.69% and 37.16% in retinyl acetate and retinyl palmitate groups, respectively during six months whereas, a significant decrease (7.68%) was found in placebo group. Moreover, significant decrease was observed in retinyl esters level during pregnancy and lactation period; 35.90 and 32.00% in retinyl acetate and retinyl palmitate groups, respectively. Similarly, the placebo group also showed significant decrease in retinyl esters (64.84%). The level of carotene showed significant decrease in all treatments during pregnancy and lactation. Overall, T1 (placebo) differed entirely from other groups with 36.45% decrease as compared to groups receiving retinyl acetate (8.18%) and retinyl palmitate (6.98%) fortified cookies. Red blood cells (RBC) indices like, hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) significantly decreased in experimental groups during pregnancy, however, RBC indices depicted a significant increase in all groups during lactation. Collectively, a momentous increase in hemoglobin level was observed in women consuming retinyl acetate (12.31%) and retinyl palmitate (16.01%) in six months, while decrease of 6.15% was observed in placebo. The consumption of retinyl acetate and retinyl palmitate fortified cookies exhibited non-significant effects on renal & liver functions tests and lipid profile showing safety and suitability of these fortificants. Furthermore, retinyl palmitate was found to be more effective than retinyl acetate to uplift the serum retinol level in pregnant and lactating women. The upshots of the present investigation revealed that the cookies fortified with retinyl acetate and palmitate had potential to uplift serum vitamin A level in vulnerable segments with special reference to pregnant and lactating women.

CHAPTER 1

INTRODUCTION
In the recent era, nutritionists are more concerned regarding food security and safety, malnutrition and diet-health linkages. Over the years, they remained emphasizing upon ample availability of nutrients considering vital for humans health (Vaclavik and Christian, 2008). Micronutrients are responsible for regulating various metabolic pathways and their deficiencies lead to drastic health disparities (FAO, 2002). Food security is often conceptualized in the context of food energy or calorie intake. However, it is increasingly recognized that a large segment of the worlds population especially in developing countries consuming food that is deficient in some micronutrients like vitamin A and iron etc. Micronutrients deficiencies are also known as 'hidden hunger' and determining and aggravating factor for health status and quality of life (Long et al., 2007). A strong political commitment, nutritional education, devising programs , their implementation, regulatory control and investments for the control of micronutrients deficiencies could contribute towards the achievement of the Millennium Development Goals for reducing mother and child morbidity and mortality rates by 2/3rd between 1990 and 2015 (Aguayo and Baker, 2005). Vitamin A, iron, zinc and iodine deficiencies are global problems and affecting at least 30% of world populations. The manifestations of such micronutrients deficiencies are vitamin A deficiency syndrome, night blindness, xerophthalmia, anemia, goiter etc. In communities living in developing countries, prevalence of poor diet and infectious disease unite into a vicious circle. Moreover, they are most important risk factors for illness and death, affecting millions of pregnant women (Muller and Krawinkel, 2005).

Almost a century later after discovery, vitamin A and its precursors continued to trigger the researchers with their diverse role and profound effects on health. Knowledge of vitamin A chemistry, metabolism and deficiency consequences, developed rapidly after series of research interventions (Butt et al., 2007). Retinol, retinal and retinoic acid are biologically dynamic types of vitamin A, however, retinol is the most important type of vitamin A, originating from animal sources, efficiently used by body (Trumbo et al., 2001). The -carotene has been hooked together with vitamin A because it is converted into vitamin A within the body and often called provitamin A (Harrison, 2005). Vitamin A is essential nutrient for adults and children meant for their vision, growth, reproduction, cellular differentiation, proliferation, normal functioning of the mucosal epithelium and immune system. It also plays key role in embryogenesis and organogenesis. It can provide protection against various cancer lines and physiological threats like heart diseases (Gaines and Berliner, 2003; Perrotta et al., 2003). Vitamin A controls various important metabolic processes and its deficiency results in impairment of immune modulating mechanism like mucus secreting epithelium is replaced by keratinized epithelium thus decreasing immunity. Vitamin A level significantly affects immunoglobulin like IgG and IgM. Furthermore, total proteins, albumin and retinol binding proteins showed a significant correlation with retinol (Gazala et al., 2003; Sato et al., 2003).Vitamin A deficiency (VAD) contributes towards several health disparities like

xerophthalmia, night blindness and some others while its excess causes hypervitaminosis (Diaz et al., 2003; Underwood, 2004). Developing countries are more vulnerable to VAD as compared to developed nations and nearly half of all the vitamin A deficiencies occur in South and South East Asia and Africa (Mason et al., 2001; Bhutta et al., 2008). In Pakistan, conditions are worsened due to nutritional imbalance and consumption of inadequate diets. The prevalence of VAD risk in Pakistan is around 70% in

pregnant and lactating mothers (Kazi and Qurashi, 1998). Furthermore, stunted growth in early childhood is associated with poor nourishment of the mother during pregnancy; as a result more than 25% of newborns have low birth weight (Mahmood et al., 2006). Globally more than 7 million pregnant women suffer from VAD and 6.2 million women suffer from xerophthalmia every year (Kaleem, 2004). In South Asia, 1 to 2 million pregnant women are vitamin A deficient and nearly 60,000 women die of childbirth-related complications, could be trimmed down through better nutrition and provision of vitamin A. In Pakistan 9.4 % are facing the problem of night blindness (Anonymous, 2004; Sher, 2004). Vitamin A required to fetus and neonates comes from mothers route; therefore, an adequate availability of this vitamin to mother is imperative to cope the childs demand. In Pakistan, about 95% of lactating mothers nourished their infants by breast feeding for prolonged period. Vitamin A deficiency in females of some districts of Pakistan has been reported earlier by Pervaiz and Gilani (2001) and Mahmood et al. (2006). Occurrence of VAD in pregnant and lactating women belonging to low income group is mainly attributed to imbalance diet as highlighted in Nutrition Survey of Pakistan (Butt et al., 2007). Diet based strategies are considered to be one of the most efficient and sustainable ways to overcome VAD. There exists suitable informations and literature to efficiently address vitamin A through short and long-term interventions. The most common strategies to combat VAD include, fortification, diversification and supplementation (Ruel and Levin, 2000; Butt et al., 2007). Food fortification is a cost-effective, flexible, and generally acceptable approach to improve the nutrients intake in the vulnerable segments; contribute to provide healthier diet in many developing countries. Processed foods are often fortified with certain micronutrients accessible to large number of peoples thus playing pivotal role in prevention of deficiencies disorders (Mannar and Ameringen, 2003; Fiedler et al., 2008).

Legislative and regulatory agencies devised policies and approaches to combat vitamin A deficiency through fortifying food products of common use. The cereals, sugar, fat and oil etc. have been fortified with vitamin A in developed and developing countries; the large-scale fortifications have been focused on sugar and fats or oils in certain countries. The micronutrient malnutrition can be eliminated if fortification through suitable food vehicle containing respective micronutrients is included in national programs of the countries that are facing micronutrient deficiencies (GOP, 2001; Lazarevic et al., 2006). The important available commercial salts of vitamin A are retinyl acetate and retinyl palmitate (Lotfi et al., 1996). Fortification of cereal products with vitamin A and other micronutrient is getting higher because of high access of populations in many countries (UNICEF, 1997). Majority of the peoples in poor countries consumes cereals as dietary staple therefore, these seem to be the best vehicle for fortification (Bauernfeind and DeRitter, 1991). Among various choices, cookies are more suitable for fortification as they contain high amount of fat, comparatively cheap and liked by all age groups and classes of the society (Ranum, 2000; Jinabhai et al., 2001). Micronutrients fortification is one of the most economical means of improving the health and survival of mother and children (Long et al., 2007). By increasing intake of vitamin A of the pregnant and lactating women through food fortification programs; mother and infant morbidity and mortality related to VAD can be reduced (West and McLaren, 2003). In Pakistan, a little effort has been made to combat vitamin A deficiency through dietary approaches; therefore present project was designed to enhance the vitamin A status of pregnant and lactating women, using cookies as vehicle containing varying levels of retinyl acetate and retinyl palmitate.

OBJECTIVES Determination of physical, chemical and sensory characteristics of vitamin A fortified cookies. To assess the retention and stability of vitamin A fortificants during baking and storage To find most suitable salt of vitamin A and its dose for fortification purposes in cookies. Efficacy studies; to determine the effect of fortified cookies on the serum vitamin A level of the pregnant and lactating women.

CHAPTER 2

REVIEW OF LITERATURE
Vitamin A fortified food holds considerable potential to alleviate vitamin A deficiency by bridging the gap between dietary intake and its requirement. Adequate intake and availability of dietary essential nutrients are closely related to the normal functioning of physiological system. The vitamins, one of the frontiers of micronutrients, are organic compounds and their presence in the diet ensures well being of humans (Kennedy et al., 2006). Deficiency of one or more such nutrients imparts drastic effects on human health e.g. vitamin A deficiency (VAD) alone affects millions of peoples all over the globe. Various intervention strategies are applicable but food fortification with appropriate fortificant and vehicle can be employed to control this menace (Butt et al., 2007). Briefly, the highlights of literature available regarding VAD and its dietary control are discussed in the following subsets.

2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2.8.

Micronutrients and Allied Deficiencies Vitamin A; An Overview Vitamin A; Requirements and Physiological Benefits Consequences of Hypo and Hypervitaminosis A Vitamin A Deficiency (VAD) Intervention Strategies Vitamin A Fortification; Product Development Efficacy Studies

2.1.

Micronutrients and Allied Deficiencies There are approximately 40 vitamins and minerals, which are considered

essential for metabolic processes, physical & mental development and immune system. They are responsible for wide spectrum of functions such as antioxidants, antimutagens, anticarcinogenic and strengthen the immune system. Due to their significant importance, micronutrients are considered to be a prominent fraction of diet (Kennedy et al., 2006). Despite the fact, they are required by body in minute quantities but their absence may lead to various physiological disparities. The deficiencies lead to adverse consequences on the vigor of an individual. Women during pregnancy and lactation are at higher risk due to their additional requirements for reproduction and growth (Calloway, 1995; Darnton Hill, 1998). Micronutrient deficiencies are widespread and have far greater impacts on health, productivity and mental capacity than previously realized (FAO, 2002). Worldwide, about 852 million people are classified as undernourished by the Food and Agricultural Organization (FAO/WHO, 2004) in 2000-02. However, another 2.5 times as many people suffer from micronutrient deficiencies known as hidden hunger. The vulnerable segments appear to get enough to eat at least in terms of calories but are lacking in micro-nutrients such as vitamins and minerals (FAO/WHO, 2004; Butt et al., 2007). Industrialization and changing lifestyle led to growing reliance on one or more food components often deficient in micronutrients essential for normal growth and functioning of the body (McGuire and Beerman, 2007). Micronutrient deficiencies are quite frequent in those areas where the diet lacks variety and access to the resources denied. In particular, vitamin A, iron and iodine deficiencies are considered as major while the prevalence of zinc and folate deficiencies are thought to be minor deficiencies (Welch and Graham, 1999; van-den Briel et al., 2007).

In 2000, the World Health Organization (WHO) estimated that more than 750000 to 850000 deaths could be attributed to vitamin A, iron and zinc deficiencies. These deficiencies also lead to phenomenon of underweight that accounts for 3.7 million deaths annually (FAO/WHO, 2004). Among many challenges that developing world is facing, VAD is one of those which should be eliminated. The problem can be solved by employing certain identified, valuable and inexpensive ways. Food fortification is cost-effective intervention in developing world to control VAD (Aguayo and Baker, 2005). The health disparities arising due to micronutrient deficiencies may be devastating and life threatening. VAD appears to be a major risk factor for both maternal and child mortality (Beaton et al., 1993; West et al., 1999). Likewise, deficiency of iodine is familiar cause of mental retardation and impaired cognitive and neurological functions. Its effects are more pronounced in pregnant and lactating women and development of children (Stanbury, 1998). Similarly, iron deficiency of which anemia is one consequence, in itself is a risk for mortality especially in pregnant women (Viteri, 1994). Iron deficiency anemia has been related to fetus maldevelopment and has reversible and irreversible consequences on central nervous system (Castejon et al., 2004). Three main strategies that have been applied to address micronutrient malnutrition include supplementation, fortification and dietary modification (Kennedy et al., 2006; Butt et al., 2007). Food fortification programs are easier to design to achieve desirable objectives within timeframe while supplementation focuses on short term cure (Bulusu et al., 2007). Dietary modifications or food based strategies focus on increasing the levels of micronutrients in the diet. These all strategies are important in the context to reduce micronutrients deficiencies through nutrients availability and their absorption in human body (Ruel, 2001).

However, dietitians and nutritionists recommend diversified diets along with some supplements and fortified products to combat various menaces (McGuire and Beerman, 2007). 2.2. Vitamin A; An Overview Vitamin A is fat soluble, found chiefly in animal foods and belongs to subgroup of compounds known as retinoids. It is found in body in three main forms differing in their oxidation states; retinol is hydroxyl form, retinal is aldehyde form and retinoic acid is the carboxylic form (Bonet et al., 2003). Vitamin A from diet or supplemental administration is stored in Ito (fat storing) cells of the liver (Stanciu et al., 2002). Retinol is the true form that is readily metabolized in human body and is called as preformed vitamin A. Its surplus amount is accumulated as retinyl esters in the liver tissues (Awan, 2005). The retinoid molecule has a cyclic and polar end group with polyene side chain. Retinoids are chromophores because of the complex conjugated system comprised of alternating C=C double bonds in polyene side chain (McGuire and Beerman, 2007). All retinoids are derived from a monocyclic parent compound containing five carbon-carbon double bonds and a functional group at the end of acyclic portion. The term vitamin A is used generically for all -ionone derivatives other than carotenoids that exhibit biological activity of all-trans retinol (McLaren and Frigg, 2001). Carotenoids are colored pigments found in several fruits and vegetables, some of them possess provitamin A activity and transform to vitamin A in human body. About 50 carotenoids are recognized as provitamin A, of which carotene is the major contribution from plant foods (Guthrie and Picciano, 1995; McLaren and Frigg, 1997; Trumbo et al., 2001; Harrison, 2005). Likewise, -carotene and -cryptoxanthin also contribute substantial amounts to some diets. The activity of -carotene and other provitamin A carotenoids were assumed 1/6th and 1/12th that of retinol, respectively

(FAO/WHO, 1988) but recent findings suggest that bioavailability of carotenoids in fruits and vegetables may be lower than expected (de Pee et al., 1995; de Pee and West, 1996; de Pee et al., 1998). Major sources of provitamin A carotenoids are carrots, cantaloupes, sweet potatoes, and spinach etc (Trumbo et al., 2001; Harrison, 2005). Additionally, carotenoids have been shown to perform as antioxidants and impart health beneficial effects (Futoryan and Gilchrest, 1994; Olson, 1996). People consuming diets rich in carotenoids are at a reduced risk of chronic illness and have low mortality rate (Vina et al., 2006; Valko et al., 2006). Vitamin A from animal sources is well absorbed and used efficiently in the body as compared to plant sources (Futoryan and Gilchrest, 1994; Olson, 1996). Fortified foods such as breakfast cereals can provide vitamin A (Guthrie and Picciano, 1995; Trumbo et al., 2001; Harrison, 2005). The sole source of vitamin A for the newborn is mothers milk; therefore, an adequate supply of vitamin A in the milk of a lactating mother is imperative for the health and appropriate growth of neonates (Pervaiz and Gilani, 2001). Requirements for vitamin A are highest for pregnant and lactating women as they act as primary source of infants nutrition (Ettyang et al., 2004). 2.3. Vitamin A; Requirements and Physiological Benefits Extensive research work has been conducted by various organizations to develop dietary reference intakes (DRIs) that can fulfill daily requirements. Dietary requirements for vitamin A vary with age and gender (Jiang et al., 2008). 2.3.1. Recommended Dietary Intakes DRIs are a general term for a set of reference values used for planning and assessing nutrient intake in healthy people. Three important types of reference values included in the DRIs are Recommended Dietary Allowances (RDA), Adequate Intakes (AIs), and Tolerable Upper Intake Levels (ULs). RDA recommends the average daily dietary intake level that is sufficient to meet nutrient requirements of nearly all healthy individuals in each age and

gender group. AIs are a set when there is insufficient scientific data to establish RDA. AIs meet or exceed the amount needed to maintain nutritional adequacy in nearly all people and ULs on the other hand, is the maximum daily intake unlikely to result in adverse health effects (Trumbo et al., 2001). RDA for vitamin A is expressed in micrograms (g) of retinol equivalents (RE) to address varying needs in different biological activities of retinol and provitamin A carotenoids. It is also expressed in International Units (IU), which is used on food and supplement labels. There is no established RDA for carotene or other provitamin A carotenoids (Trumbo et al., 2001; Awan, 2005). According to McDowell (1996), 1.0 g of retinol is equivalent to 1 RE or 3.3 IU while 1 IU of retinyl acetate, retinyl palmitate and -carotene is equal to 0.344 g, 0.55 g and 0.60 g of retinol, respectively. 2.3.2. RDA for Pakistani Population According to World Health Organization (WHO) and available data in Pakistan, RDA of vitamin A for pregnant women, lactating mother and adults is 750, 1200 and 700 g, respectively for Pakistani population (GOP, 2001; Trumbo et al., 2001). 2.3.3. Physiological Benefits Generally, vitamin A played influential roles in vision, reproduction, development, bone metabolisms, immune functions, gene transcription, reducing risk of heart diseases and cancer disorders (NIAMS, 2005; Noy, 2006; Jiang et al., 2008). It is essential constituent of rhodopsin, imperative for vision and vital for growth (Sommer et al., 1984; Flodin, 1988). The high dietary intake of vitamin A reduces the risk of cancer, cardiovascular, and other diseases (Weber, 1994). Vitamin A and its derivatives play an integral role in spermatogenesis, reproduction, cell differentiation, embryogenesis and organogenesis (Jiang et al., 2008). Moreover, cell differentiation needs adequate amount of vitamin A and retinoic acid that are considered to be essential in the segregation of white blood cells (Gaines and Berliner, 2003).

Vitamin A exhibits key role in organogenesis; almost all steps are controlled by retinoic acids, thus suggesting the necessity of retinol for proper development of embryonic tissues (Perrotta et al., 2003). The kidney, brain, liver and laryngeal development needs vitamin A because of its ability to switch on and off particular specific promoting genes (Ghoshal et al., 2003; Tateya et al., 2007). The retinoids, natural and synthetic derivatives of vitamin A, are used in treatment of vascular diseases. These have been used clinically to treat a variety of skin diseases and cancer. The recent evidence demonstrates that retinoids alleviate experimental vessel wall narrowing due to atherosclerosis, post-balloon injury stenosis and bypass graft failure (Streb and Miano, 2003). Vitamin A is also involved in protein metabolism; positive correlation of serum retinol level with that of total protein and albumin exists. Moreover, the immunoglobulin studies have shown low concentrations of IgG and IgM in subjects with low vitamin A intake (Mejia and Arroyave, 1982; Vobecky et al., 1986; Burri et al., 1990). Deficiency of vitamin A causes disorders of visions, damages respiratory, genitourinary, gastrointestinal tracts, impairs growth, bone formation and results in anemia, as well as increased susceptibility to infections (Sommer, 1989). Vitamin A is an anti malarial agent; has a direct inhibitory effect on the growth of Plasmodium falciparum in vitro (Hamzah et al., 2003). The quantity of vitamin A in Ito cells decreases resulting in liver injury (Stanciu et al., 2002). 2.4. Consequences of Hypo and Hypervitaminosis A Normal range of serum vitamin A is 20 49g/dL (Rauf et al., 2002) while for -carotene and retinyl esters is 50200g/dL and 35g/dL, respectively (Jacobs et al., 1996). Vitamin A deficiency is affirmed if serum retinol level is < 20 g/dL (WHO 1996; Rauf et al., 2002; Khan et al., 2005). Tolerable Upper Intake Levels (ULs) is the highest amount that a healthy person can utilize safely and without any risk (EFSA, 2006).

2.4.1. Deficiency Consequences The consequences underlying vitamin A deficiency include night blindness, corneal ulcers, keratomalacia, xerophthalmia, anemia through malabsorption of iron, reduced immune competence resulting in raised morbidity and mortality due to diseases like diarrhea, measles, and respiratory infections (Sommer, 2001). Night blindness is allied with a higher risk of maternal mortality and morbidity; in Nepal, the death rate was about 26/1,000 for those pregnant women who reported night blindness as compared to 3/1,000 for normal (Christian et al., 1998). During pregnancy and lactation, poor vitamin A supply results in underweight neonates with low vitamin A level at delivery that subsequently persists for few months (Azais and Pascal, 2000). The higher infant mortality rates may concerned with VAD of pregnant woman (Semba et al., 1998). During a research investigation an association between low birth weight and low maternal serum retinol status was found (Gazala et al., 2003). The absorption of vitamin A is interfered by diarrhea, parasitic infections and other intestinal disorders. The acute and chronic infections speed up its catabolism and excretion (Ross, 1992; West et al., 1992). Mother to fetus transmission of HIV is elevated during VAD .Low serum retinol level is common in HIV infection and linked with viral load, increased progression to disease (Azais and Pascal, 2000; Coutsoudis, 2000). Provision of vitamin A can reduce the rapid progression in disease effects (Stallings et al., 1997; Camp et al., 1998). Hypovitaminosis A prevails in the patients suffering from malaria as Plasmodium falciparum collects vitamin A from its host and concentrate it in the cytoplasm of late trophozoites for further use as an antioxidant (Mizuno et al., 2003).

2.4.2. Hypervitaminosis A Hypervitaminosis A is defined as the high storage of vitamin A in the body producing toxicity. Higher intake of vitamin A for longer time may produce toxicity and allied diseases. An ingestion of 660,000 and 330,000 IU of vitamin A by an adult and children, respectively induced toxic symptoms. However, intake of 10000, 25000 and 14000 IU of vitamin A daily by pregnant/lactating women, adults and children, respectively for prolonged period or 50,000 IU daily for more than 90 days stimulates toxic conditions. Toxic symptoms can also occur after taking mega doses of vitamin A for short time (Binkley and Krueger, 2000). Nausea, vomiting, headache, dizziness, blurred vision and lack of muscular coordination indicate acute toxicity of vitamin A (Udall and Greene, 1992; Trumbo et al., 2001). Major adverse effects of hypervitaminosis A are birth defects, liver abnormalities, osteoporosis and CNS disorders (Bendich 1989; Udall and Greene, 1992; Trumbo et al., 2001).

2.5. Vitamin A Deficiency (VAD)

Vitamin A deficiency is considered among major micronutrient deficiencies in the world especially in developing countries (Castejon et al., 2004; Jiang et al., 2008). VAD is consequence of depleting reservoirs of retinol from liver tissues, results in impairment of normal physiological functioning of the body (Gibson, 1990; Blomhoff et al., 1991). Strube et al., (2002) reported the occurrence of marginal VAD that frequently affects millions of people particularly children and women. Gibson, (1990) studied the reliance of VAD on inadequate dietary intakes of vitamin A and highlighted that dependence of the communities on one or two food components and high frequency of infections are mainly responsible for this syndrome. Furthermore, socioeconomic status, poverty and malnutrition also contribute their share for major outburst of VAD (Gibson, 1990; Blomhoff et al., 1991; Jiang et al., 2008).

Women, being the most susceptible group are influenced by VAD during pregnancy and lactation (Zimmermann et al., 2006). Pregnant and lactating women require higher intake of vitamin A that subsequently, plays a dominant role in maturation and development of fetus (Sato et al., 2003; Mahmood et al., 2006). In developing countries, pregnant and lactating women are consuming inadequate diets that only provide 10-30% of their dietary requirements for vitamin A (FAO/WHO, 1988). Globally, around 7.2 million pregnant women are vitamin A deficient and 13.5 million have low vitamin A status. VAD and night blindness overlap each other. About 6 million mothers suffer from night blindness during pregnancy and both conditions are related with increased threat of morbidity and death (West, 2003; Aguayo and Baker, 2005; WHO, 2006). Nearly, half of the patients suffering from VAD and xerophthalmia are reported in South and South-East Asia. In this region, 1-2 million pregnant mothers are VAD sufferer and about 60000 mothers die of childbirth associated disorders, mostly caused by complications that can be lessened through better nutrition (Kaleem, 2004; Khor, 2005; Butt et al., 2007). Worldwide, VAD affected about 140 million children and 100 million live in South Asia and Sub-Saharan Africa. Approximately, 127 million preschool aged children are vitamin A deficient and nearly 4.4 million suffered from xerophthalmia (West, 2003; WHO, 2006). An estimated 3040% of preschool children in South Asia are at heightened risk of ill health and death because of this deficiency syndrome (Mason et al., 2001). In Pakistan, over 5.7 million children aged less than five are vitamin A deficient, while 57,000 are suffering from xerophthalmia (Kaleem, 2004). Population of Pakistan, particularly the low-income segment of the community is undergoing a serious nutritional imbalance. Significant difference exists for vitamin A status between children belonging to different socioeconomic groups. Per capita consumption of milk, meat and eggs is very

low in poorer communities and they also lack in frequent intake of other dairy products like butter and butter oil (Khan et al., 2005). In another study conducted in Pakistan, it has been reported that dietary inadequacy is main cause of lower retinol level (Paracha et al., 2000; Rauf et al., 2002). Pregnant and lactating women in Pakistan are under worsening threat of VAD; can severely affect mother and child survival and nutrition (GOP, 1999). In mothers belonging to low socio-economic status, vitamin A intake is much lower than the basal required level. Women of child bearing age and lactating mothers are rigorously affected by VAD and consequently the babies. Around 95% of lactating mothers fed their babies by breast often for a prolonged period (Newman, 1994; Pervaiz and Gilani, 2001). There is a serious undetermined degree of vitamin A deficiency in Pakistan. The Nutrition Survey of Pakistan indicates an apparent deficiency in vitamin A in Pakistani diet, especially among the pregnant and lactating women and children of low-income groups (Lotfi et al., 1996). A survey conducted during 19651966, revealed that 24% rural and 13% urban population had low serum retinol levels. Ten years later, a survey indicated that about 60% of the men, 71% of the non-pregnant/lactating women and 78% of pregnant females used less than 70% of the RDA of vitamin A (Manan, 1994). Although, multivitamin supplementation levels are higher than that of twenty years ago but females are still consuming less than the required daily allowance. Thirty years after the first survey, more than 70% pregnant and lactating women of Pakistan are VAD (Kazi and Qurashi, 1998). Due to vitamin A deficiency, majority of lactating mothers (92%) are not being consumed minimum daily requirement to fulfill the retinol demands (1.05mol/L) of their babies (Liyanage et al., 2008). Furthermore, poor growth in childhood is allied directly to poor diet of pregnant mother resulting low birth weight over 25% of babies (GOP, 1999). Children belonging to different socioeconomic strata differ with regard to

vitamin A contents significantly and majority of VAD cases belong to poor families (Rauf et al., 2002). In case of Pakistan, children exhibit about 16% lower serum retinol status than those of United Kingdom due to inadequate dietary intake (Paracha et al., 2000). Diarrhea and respiratory infections are the leading causes of death in children under five years that are attributable to VAD. In Karachi region, 47% of children had low level of vitamin A in their blood serum as investigated during a study conducted in 1987 (Manan, 1994). Low serum carotene levels were observed in 64% children of 511 years, 16% of 1217 years and 20% adults of 1822 years in Multan, a district of Pakistan (Khan et al., 2005). Vitamin A deficiency syndrome is affecting millions of peoples all around the globe and provision of vitamin A through various strategies may reduce the effects of VAD.

2.6. Intervention Strategies

Micronutrient malnutrition affects more than half of the world population, particularly in developing countries (Mayer et al., 2008). Reduction in micronutrient deficiencies can contribute significantly to improve health, productivity and well-being of human. These phenomena are most important for child bearing women as their requirements for such constituents are higher and deficiencies could cause drastic effects in their neonates too. There is dire need of time to address these malnourishments before they could play havoc with human health. Micronutrients deficiencies can be overcome by adopting multifarious strategies (Adu-Afarwuah et al., 2008). Food based strategies are most effective and sustainable approach to combat VAD. These require thorough knowledge of the sources of nutrients and

their accessibility to humans. Three major strategies are food diversification, supplementation and food fortification (Bloem et al., 1998; Filteau and Tomkins, 1999; Chakarvarty, 2000). Food fortification is the key strategy to overcome VAD (Khan et al., 2007). 2.6.1. Food Diversification Food diversification involves consumption of variety of foods to cover nutrients requirements. Communities living in developing world rely on territorial specific sources of food like peoples living in South Asia and South East Asia mainly consume wheat and rice to fulfill their caloric demands but often these diets are deficient in some of the minor constituents or their bioavailability related problems. In such conditions there is need to add some other food components to bring balance in nutrients' intake. Inclusion of relatively small amounts of plant products can provide sufficient RDA of provitamin A in the form of carotenoids (Graebner et al., 2004). Likely, Haskell et al. (2004) emphasized on the consumption of some green leafy vegetables and sweet oranges to meet vitamin A requirements and International Vitamin A Consultative Group (2003) also highlighted some indigenous sources like new lines of sweet potato, sweet orange etc to enhance the dietary intake of vitamin A. 2.6.2. Supplementation Vitamin A administration to mothers with imminent preterm deliveries may augment vitamin A stores of their newborn infants at birth (Stoltzfus, 1995). Although supplementation programs are low cost, acceptable and effective in short term but do not address the primary factors resulting in deficiency i.e. inadequate dietary consumption of vitamin A to satisfy physiologic needs and high frequency of infections. It has been recommended by WHO/UNICEF/IVACG (1997) that in countries where VAD is a problem, all mothers should receive a high dose of vitamin A supplement (200000 IU) within 8 weeks of childbirth to maintain its

adequate concentration in breast milk for six months (Stoltzfus et al., 1993; IVACG, 2003). The effectiveness of supplementation programs depends on the level of coverage and compliance. The coverage of 65-85% reduced the prevalence of xerophthalmia up to 75- 85% in 1 to 4 year while coverage below 25% is unlikely to have any impact on xerophthalmia (UNACC/SCN, 1994). 2.6.3. Food Fortification Fortification of food is the addition of one or more essential nutrients to a food, whether or not it is normally contained in the food, for the purpose of preventing or correcting a demonstrated deficiency (FAO/WHO, 1994). This intervention is economical, flexible and socially acceptable to improve the nutrients intake and better nutrition in a number of developed countries e.g. fortification of milk, margarine and cereals with vitamins A has greatly reduced the occurrence of deficiency (Wahlqvist, 2008). Processed foods are reaching great number of people all over the world, and mostly are fortified. Though vitamin A has been added to a variety of foods including cereals, sugar, fat and oil, salt, tea, and monosodium glutamate, in developed and developing countries but only large-scale fortification efforts have concentrated on sugar and fats/oils in certain countries. Besides these, milk and milk powder, biscuits, porridge, rice, tea and condiments can be fortified with vitamin A with good results (Lotfi et al., 1996; Butt et al., 2007). It is documented by UNICEF (1996); here are some countries along with foods which are fortified with vitamin A namely Bolivia, Brazil, El Salvador, Guatemala, Honduras (sugar), Botswana (tsabana), Mexico (chocolate drink mix), Philippines (margarine), Venezuela (maize flour), India (cows milk) and Pakistan (cooking oil/vegetable ghee). Complementary foods are transitional foods given in addition to breast milk, following exclusive breast feeding during the first 6 months, to meet the full nutritional requirements of the infant. Strategies to improve the availability

of and accessibility to low cost complementary foods can play an important role in improving the nutritional status. Cereals are considered as the most suitable vehicle for delivering micronutrients to population at risk because of their widespread consumption, stability and versatility. To reduce the vulnerability to the health impacts of micronutrient deficiencies, several developed and developing countries have adopted various innovative, cost-effective strategies to fortify cereal based complementary foods (Bulusu et al., 2007). Monitoring systems can provide estimate average intake of vulnerable groups consuming the fortified food. This can enable corrective action in terms of expending production of the fortified food or correcting distribution bottlenecks. For example, in India, fortification of fat with vitamin A at a level of 7.5g retinol/g of fat was made compulsory in 1953. It was found that the product was mainly consumed by upper and middle class households (Manner, 1989). Furthermore, the average amount of fat consumed was 3 g/day providing 4% of RDA (22.5 g) of vitamin A (Sridhar, 1997). In Pakistan it is mandatory to fortify cooking oil/vegetable ghee with vitamin A, but no proper monitoring system exists and the law has not been implemented. Consequently, vitamin A content of the fortified oil/ghee varies considerably, and the interventions effectiveness is low (Kielmann et al., 1982). Aguayo and Baker (2005) were of the view that effective and sustained policy and program action for the control of VAD can bring about 25% reductions in mortality of pregnant and lactating women. Food fortification represents the more sustainable, potentially long lasting strategic measure. The process of elimination of micronutrients hunger can therefore be accelerated if fortification of foods with appropriate micronutrients can be included in national programs of those countries in which such deficiencies are prevalent (GOP, 2001). Concerted international fortification efforts to curb the scourge of micronutrient malnutrition are showing a positive impact but still demand

meticulousness to achieve desired goals set by international organizations (Mayer et al., 2008). 2.6.4. Nutrition Education Disseminating information among consumers regarding the importance of food containing the balanced formula is essential for alleviation of malnutrition and allied problems, mostly outcome of inadequate dietary intakes. Bringing peoples towards balanced diets comprising of cereals, meat and products with at least four to five servings of fruits and vegetables could serve the purpose. Welldesigned promotional activities using nutrition education, social marketing and mass media campaigns could possibly change consumers attitude especially pregnant and lactating mothers. Significant increase in the consumption of micronutrients and in particular vitamin A rich food can be achieved by nutrition education (Ruel, 2001; Faber, 2002). Moreover, educating medical staff is also essential because at early stages of VAD, symptoms like night-blindness are difficult to recognize by masses. Parents and medical staff need to be trained to raise awareness against various symptoms of micronutrients deficiencies. They should be informed with the dreadful consequences of vitamin A deficiency that includes immune system malfunctioning resulting in higher susceptibility to infectious disorders. Information and efforts should be focused on changing attitude of consumer regarding acceptability of fortified foods (IVACG, 2003). 2.7. Vitamin A Fortification; Product Development Ideally, a food consumed by most of the population segments should be chosen as a vehicle for vitamin A fortification. It should be designed to include high risk groups to the greatest degree possible and ensure access of these foods to them for the neediest families. That is why fortification of flour and cereal products is considered to be effective. Fortification of flour and other cereal products with vitamin A and other micronutrients is gaining attention because of high coverage of population in many countries (UNICEF, 1997). The cereals are

considered to be the best vehicle for fortification in developing countries, because about 95% of the population consumes cereals as dietary staple (Bauernfeind and DeRitter, 1991). As staples, milled cereals are relatively inexpensive; they are grown and consumed worldwide by all economic classes. They are versatile in preparation and uses. Consequently, milled cereals and their products are good vehicle for fortification. Vitamin A fortified cookies, for example, can be helpful against VAD menace (Ranum, 2000). 2.7.1. Vitamin A Fortificant The most important commercial forms of vitamin A are retinyl acetate and retinyl palmitate. These pure chemicals have primarily been added to foods as improvers and colorants, but foods can also carry them to increase vitamin A intake of the populations (Parman and Salinard, 1981; Lotfi et al., 1996). 2.7.2. Fortification of Cereal Products Being a fat soluble vitamin, it is better to develop foods containing high fat content. Among baked products, cookies, cakes and pastries contain higher amount of fat up to 20-30%. Cookies are comparatively cheap and liked by all age groups and classes of the society and it is suggested that cookies are best product for fortification `with retinyl acetate and retinyl palmitate (Ranum, 2000). Furthermore, Stringer (2000) is of the same view that cereal based food especially cookies are suitable for fortification purpose and fortification strategies with salts of vitamin A are used advantageously in developing countries. Food and Nutrition Board of the National Research Council of the USA, in 1974, proposed a new fortification formula for cereal grain products (wheat flour, semolina, maize flour and maize meal); vitamin A was added at the rate of 5000 IU/pound of flour. 2.7.3. Quality Attributes of the Products Cookies and biscuits are important cereal based products; the term biscuit is used to describe chemical leavened cereal product in European countries while

same has been designated cookies in United States of America (Hoseney, 1986). Cookies are ideal product for food fortification purpose due to their higher nutritive value, palatability, compactness and convenience. Lower moisture contents make them more suitable as they are usually free from microbial spoilage thus being more stable (Wade, 1988). Physical parameters are imperative for successful product development and marketing. Gaines and Donelson (1985) found that spread potential and size of cookies depends on flour particle size and moisture content. They reported the decrease in thickness and increase in diameter and spread factor of cookies on increasing the concentration of vitamin A (retinyl acetate). Butt et al. (2007) found non significant effect of treatments and storage periods on physical parameters in retinyl acetate fortified cookies. They reported a non-significant increase in spread factor of vitamin A fortified cookies. Spread factor was slightly increased from 48.640 to 50.483 with vitamin A fortification while value for spread factor was recorded 49.04, 49.95, and 49.04 at 0, 15 and 30 days correspondingly. The stored baked products absorb moisture from the surrounding. The increase in moisture was observed when biscuits were sealed in moisture proof packaging, small amounts of moisture present in the atmosphere within the package rapidly came in equilibrium with the product and when the biscuits were exposed to the outer environment they quickly absorbed the moisture. However, a reduction in protein content from 6.71 to 6.41% during 30 days was also observed. On sensory evaluation, an increasing tendency in texture, crispiness and overall acceptability scores with increasing level of retinyl acetate in cookies was observed while a decline in scores was observed during storage (Butt et al., 2007; Mahmood et al., 2008). Vitamin A is sensitive to heat and light; its losses occur when exposed to these physical factors. The higher storage temperature caused such losses, as it may increase oxidation of the vitamin and results in loss of ability of binding

with lipids that also results in its lower absorption capacities. Butt et al. (2007) investigated the stability of vitamins A fortificants in fortified cookies and reported that a minor and acceptable loss in vitamin A content i.e. 8.6911.11% during baking process. Likewise, storage of cookies at room temperature significantly reduced vitamin A content whereas after 15 days, the vitamin loss reached up to 5.89%. However, after 30 days, the losses were doubled as compared to those at 15 days. An increase in temperature decreases vitamin A stability and wheat flour stored at 45 C have shown 72% vitamin loss after 90 days storage. Emodi and Scialei (1980) also observed 7-10% loss of vitamin A during baking of bread. In another study, Bauernfeind (2006) also observed 90% retention of vitamin A during baking. He also observed a minimal loss of vitamin A after 6-month storage. The difference in results could be due to storage temperature. Bauernfeind and DeRitter (1991) also found slight loss in vitamin A after six months storage. Similarly a non-significant effect of fortification with vitamin A on diameter, thickness and spread factor of cookies was observed by Mahmood et al. (2008). The diameter of cookies slightly but non-significantly decreased during 3 months of storage. Vitamin A fortificants did not significantly alter the fat content (22.54, 22.43, 22.56, and 22.49%) of the fortified cookies with nonsignificant changes during storage. During a study of vitamin A fortification of cookies, 9.3% baking losses were observed. The retinyl acetate content before baking was 10.99 which reduced to 9.96g after baking. Similarly, storage of the cookies for 90 days at ambient temperature significantly reduced the vitamin A content of the cookies from 9.96 to 9.13g with a loss of 8.33%. A significant increasing trend in the diameter from 40.33 to 40.75mm of cookies by increasing rice bran oil with a non significant difference during storage was revealed by Sharif et al. (2005). They investigated a decreasing trend

in thickness of value added cookies with gradual increase in the proportionate of rice bran oil. Maximum thickness was recorded for control treatment (50.18mm) while minimum for treatment (47.97mm) containing maximum rice bran oil while non-significant variation in thickness of value added cookies was recorded 49.02 and 49.27mm, respectively for 0 and 45 days. Whereas an increasing trend in spread factor of value added cookies with gradual increase in the proportionate of rice bran oil was also reported. The rice bran oil added cookies resulted in increased moisture content varied from 1.09 to 1.26% which increased with 45 days of storage and highly significant differences in color scores due to treatments and storage of cookies were observed. The color scores decreased from 6.80 to 6.32 by different treatments while storage decreased the color scores from 7.30 to 6.54. Leelavathi and Rao (1993) and Patel and Rao (1996) reported an increase in diameter, decrease in thickness and increase in spread factor of cookies by the use of emulsifiers. Decrease in gluten development due to increased level of non wheat flours might be a contributing factor to diminished thickness of cookies (Taylor et al., 2008). It has been suggested that spread factor is affected by the competition of ingredients for the available water; flour or any other ingredient which absorbs water during dough mixing (Fuhr, 1962). OdoricaFalomir and Paredes-Lopez (1991) and Claughton and Pearce (1989) reported a reduction in spread factor of cookies by increasing the enrichment levels of flour with sunflower and safflower protein isolates. Butt et al . (2004a) also prepared cookies by supplementing sweet potato flour and reported increase in moisture content (3.44-4.04%) during 45 days storage period. Wheat flour, fat and sugar constitute the major bulk of cookies. Semwal et al. (1996) studied the nutrients composition of biscuits commercially available in Mysore, India. Total fat content of the biscuits varied from 20.1 to 24.6% and total energy values ranged from 365 to 501Kcal. Wade (1988), who observed non-

significant decrease in protein content of stored biscuits samples and concluded that the slight decrease might be due to proteolytic activity in the samples. In another study, Pasha et al. (2002) and Butt et al . (2004a) also found decreasing tendency in protein content of cookies during storage. Similar, outcomes were concluded by Anjum et al. (2003), who reported 21.64% fat contents in value added biscuits. TBA test measures deterioration in both extractable and non-extractable lipids and therefore has been more frequently applied to compound fatty foods, particularly flesh food, rather than the pure oil and fats (Kirk and Sawyer, 1999). It is documented that at moisture content greater than 12%, risk of fat oxidation and development of rancidity increases (Kent and Evers, 1994). Similarly, Fellers and Bean (1977) mentioned that cereal baked products are susceptible to lipase activity and TBA value increases, especially at higher moisture climbs. Thus, the increase in TBA value may also be the result of increasing moisture content and temperature during storage. Refined oil in good condition has TBA value of 0.020.08mg malenaldehyde/Kg where as crude oil or badly stored oils have 0.1-0.2 mg malenaldehyde/Kg. However, higher temperature of the storage room, moisture, heat and light are the key factors that further accelerate the TBA value during the storage of cookies. Sensory evaluation is important criterion in food industry and is usually performed towards the end of product development Anjum et al. (2008). Among various sensory attributes color is an important parameter. Sensory scores for all the organoleptic parameters of cookies have been reported to be decreased with storage period. This trend might be attributed to increased moisture absorption that stimulates degradation of some cookie constituents resulting in deterioration of product quality (Wade, 1998). Pasha et al. (2002) observed a decreasing trend in color during 60 days storage of cookies. Butt et al . (2004a) also found a similar trend of decrease in color of biscuits during storage of 45 days. Likewise, Pasha et al. (2002) prepared value added

cookies and observed a decrease in flavor during storage. Wade (1988) affirmed that during storage of biscuits, moisture absorption results in deterioration of flavor by enhancing fat oxidation. These findings have also been supported by Bender (1996). Texture is one of the important food quality attributes, which consumers use to judge the quality of end products. Perceived texture is closely related to the structure and composition of the food (Alvarez et al., 2005). 2.8. Efficacy Studies Poor quality foods are the major cause of under nutrition, primarily the foods containing low content of absorbable micronutrients. Characteristically, poverty is allied with inadequate ingestion of fruits, vegetables and animal products which result in deficiency of vitamin A, riboflavin, vitamin B12, absorbable iron, zinc and calcium. The availability and intake of vitamin A and other micronutrients can be enhanced through food based strategies. These are associated with likelihood to sustain the intervention and potential benefits for households (Allen and Gillespie, 2001). Many food based programs have been anticipated to improve vitamin A status. The important question is; whether any of the food based programs improved vitamin A status or not. Food fortification with vitamin A improved its status as well as imparts significant impact on nutrition standing of the consumers (Gillespie and Mason, 1994; Ruel and Levin, 2000) and postfortification; positive outcomes were observed as reduction in clinical eye signs of vitamin A deficiency in the Philippines (Solon et al., 1979); reduction in night blindness in young children (Greiner and Mitra, 1995); higher serum retinol in young children in Bangladesh (de Pee et al., 1998); higher serum retinol in Thai school children (Smitasiri and Dhanamitta, 1999); and a lower prevalence of Bitot's spots and night blindness in Ethiopia (Ayalew et al., 1999). Daily consumption of vitamin A fortified wheat flour bun improved the vitamin A status significantly in subjects with marginal to low initial serum retinol concentrations (Solon et al., 1979). Vitamin A fortified biscuits were given

daily for sixty weeks during a study conducted in rural South Africa; resulted in a significant effect on serum retinol. Fortified biscuits improved micronutrient status (Jinabhai et al., 2001). Broken rice grains have been fortified with vitamin A premix by Federal University of Permanbuco, Philippines Food and Nutrition Institute, Bon Dente Nutrition and Iowa Status University and found to be effective against VAD (Lotfi et al., 1996). Muhilal et al. (1988a) conducted a study in Indonesia to determine the effects of monosodium glutamate (MSG) fortification with vitamin A on serum profile of VAD sufferers during a controlled trial. Ten villages were selected for study, fortified MSG was provided to five villages and unfortified MSG to other five villages (placebo). Serum vitamin A level was determined at base line and afterwards; increased in subjects of fortified MSG villages from 0.670.33 (base line) to 0.920.33mol/L (37.31% uplift in serum vitamin A) in 11 months. Serum levels of retinol of the subjects in control villages decreased from 0.780.35 to 0.720.33mol/L (7.69% decline). Similarly, Arroyave et al. (1981) carried out a study in Guatemala by using fortified sugar to determine the effectiveness of the fortification in uplifting the serum retinol status. Serum retinol values before and after the intervention was assessed. It was found that the addition of retinyl palmitate to sugar increased significantly serum status of retinol. It was observed that 76% of the subjects experienced an uplift of retinol in 1 year of fortification (base line serum retinol <20g/dL). Mean values increased significantly; from 16.22.9 to 30.29.7g/dL (86.42% uplift). Comparable consequences were obtained after 2 year of fortification i.e. serum retinol uplifted 57.23%; from 16.63.0 to 26.19.9g/dL. These results indicated the effectiveness of the program in raising serum retinol levels. Dary et al. (1996) reported 10% reduction in vitamin A deficiency prevalence in a study conducted in Guatemala. In this research project, fortified sugar was used by adults before and after design was used to assess the uplift

of serum retinol and similar findings were observed during a research project conducted by Solon et al. (1979) who compared the relative effectiveness of three different intervention strategies to control VAD in Cebu, Philippines. These interventions were; public health and horticulture intervention, provision of vitamin A in capsules and MSG fortification with vitamin A. Each intervention was implemented in four different areas, two urban and two rural, for about 2 years. Similar parameters were investigated before and after each intervention. Mean serum vitamin A status was 19.0, 19.6, 21.0g/dL before and 16.4, 19.6, 28.5 g/dL after public health, vitamin A capsule and fortification of MSG with A intervention, respectively. The highest serum vitamin A status (7.5g/dL; 37.71%) uplift was observed by use of fortified MSG. Hence MSG fortification was the strategy; resulted in significant reduction in clinical signs of xerophthalmia and considerable rise in serum vitamin A profile. Serum retinol status of women falls during pregnancy, even in well nourished women. This decline is due to hemo-dilution and changes in proteins profile in serum (Gebre and Vahlquist, 1984). Vitamin A is also considered to be involved in the pathogenesis of anemia through diverse biological mechanisms, such as improvement in growth and differentiation of erythrocyte progenitor cells, potentiation of immunity to infection and reduction of the anemia of infection and mobilization of iron stores from tissues. Epidemiological surveys demonstrated that occurrence of anemia is high in populations affected by VAD in developing countries. Enhancement of vitamin A status had been revealed to control anemia (Semba and Bloem, 2002). The effect of improvement in vitamin A status on biochemical indices of Fe nutrition was investigated by Mejia and Arroyave (1982). Four pairedcomparison-subgroups of subjects were studied before and after fortification of sugar with vitamin A in 6 months. The serum samples were assessed for the retinol, retinol binding protein (RBP) to indicate vitamin A profile while iron was indicated by estimating; iron, total iron binding capacity (TIBC), and percentage

saturation of transferrin (% ST) and ferritin was used as an index of iron stores. Highly significant positive correlations were found between the changes in status of retinol, profile of iron, TIBC and % ST. Several clinical studies had been conducted in which the impact of enhanced vitamin A status upon hemoglobin and anemia was investigated. The impact of improving vitamin A status through fortification was addressed in a community based trial in Indonesia by Muhilal et al. (1988b) who investigated the effect of vitamin A fortified MSG on improvements in vitamin A index accompanied by dramatic changes in health and anthropometric status. During the course of study, the prevalence of Bitots spots among subjects in program villages fell progressively from 1.2% at base line to 0.2% in 11 months whilst xerophthalmia in control remained unchanged. Linear correlation was observed between vitamin A status uplift and Hb level elevation. An increase in hemoglobin (Hb) status from 11316 to 12316g/L in 5 months was noted while mean Hb level declined - 2g/L among control subjects. A clinical study among anemic school children in Tanzania indicated that daily provision of vitamin A increased mean Hb level up to 13.5g/L at 3 months (Mwanri et al., 2000). Likewise, Mejia and Chew (1988) estimated the effect of vitamin A on Fe profile in a controlled trial. It was found that elevated vitamin A significantly increased hemoglobin, hematocrit, serum iron, and % ST. In northeast Thailand, subjects who received high dose vitamin A had higher serum Fe and % ST compared with control after 2 months (Bloem et al., 1989). Individuals with conjunctival xerosis showed a significant boost in Hb, hematocrit, Fe and % ST (Bloem et al., 1990). Semba et al. (1992) observed a significant increase in mean Hb level (21 g/L) by using vitamin A and considerable rise in plasma ferritin. Serum protein, albumin and bilirubin are often used for the evaluation of liver condition (Jung et al., 2003). Raised bilirubin had been noted in humans with hypervitaminosis A

but no case of increased bilirubin in vitamin A deficiency conditions was observed (Erickson and Mawson, 2000). Studies conducted among pregnant women recommend that vitamin A supplementation alone during pregnancy can increase Hb concentrations (Suharno et al., 1993). In West Java, Indonesia, anemic pregnant women received vitamin A and significant uplift in mean Hb profile was reported by Panth et al. (1990). Muslimatun et al. (2001) found that pregnant women who received weekly vitamin A and iron had a greater increase in hemoglobin than those received weekly iron or daily iron alone. There was an allied decrease in ferritin in women who received vitamin A and iron; suggestive of vitamin A improved the consumption of Fe for blood formation. The relatively high prevalence of marginal vitamin A status among pregnant and lactating women has raised concern about its contribution to morbidity and mortality and to the etiology of anemia among women. West et al. (1999) found a 40% reduction in maternal deaths related to pregnancy by providing vitamin A fortified food. Many biochemical indicators like serum retinol, retinol binding protein (RBP), RBP/TTR (transthyretin) molar ratio and RAG (retinoyl -glucuronide) hydrolysis test can be used to evaluate vitamin A status but serum retinol is preferred because most of the laboratories can analyze and it is well established biochemical marker of vitamin A status (McLaren and Frigg, 2001). Serum retinoids (retinol, retinyl esters) and carotenoids (-carotene) are usually analyzed through high performance liquid chromatography (HPLC) or spectrophotometry. Though spectrophotometry is simpler and inexpensive but less accurate, therefore, HPLC analysis is preferred (Arroyave et al., 1982). 2.8.1. Hematology and Serology Complete blood count (CBC) is an analysis that gives information about the cells in an individuals blood. The blood cells are; erythrocytes (RBC),

leukocytes (WBC) and thrombocytes (platelets). High or low counts may indicate some disease. Hemoglobin (Hb) is the amount of hemoglobin in blood, hematocrit (Hct)/packed cell volume (PCV) is the fraction of whole blood volume that contains RBCs, mean corpuscular volume (MCV) is the average volume of RBC; mean corpuscular hemoglobin (MCH) is the average amount of hemoglobin per RBC and mean corpuscular hemoglobin concentration (MCHC) is the average concentration of hemoglobin in RBCs (Hillman and Finch, 1985; Fischbach, 1988). The WBCs; Neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles status may rise in bacterial/acute viral infection, leukemia, tuberculosis, malaria, chronic ulcerative colitis/enteritis, parasitic infestations, asthma, allergic reaction and bone marrow related conditions. Leukocytosis, thrombocytopenia and pancytopenia may be a sign of infection, drug toxicity and low production from the bone marrow, respectively (Fischbach, 1988). The reference values for hematological and serological indices depend on age, gender, race, and state of body and may vary with lab and method of determination (Jacobs et al., 1996). The normal values for hemoglobin are 1418,1216,11.415.0,10.0 14.3,10.214.4,10.418.0, 9.515.5 and 1424g/dL for men, women, 1st , 2nd and 3rd trimester of pregnancy, postpartum (lactation), children and newborns, respectively while 4252, 3747, 3546, 3042, 3444, 30 44, 3244 and 4464% are reference ranges for hematocrit for men, women, 1st, 2nd and 3rd trimester of pregnancy, postpartum (lactation), children and newborns, respectively. The RBC count; 4.76.11012, 4.25.41012, 4.05.5 1012 and 4.87.11012 per L of blood is established standard for men, women, children and neonates, respectively and referral RBC indices are 8095fL, 2731pg and 3236g/dL and 1114.5% for MCV, MCH, MCHC and RDW, respectively. Total WBC count (standard) is 510109, 4.511109, 6.614.1109, 6.9 17.1109, 5.914.7109, 9.725.7109 per L of blood for men, women, 1st, 2nd, 3rd

trimester of pregnancy and post partum (lactation), respectively while reference WBC differential is 5570,03,2040, 28,14 and 0.51% for neutrophiles, band neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles,

respectively, however, normal values for platelets count are 150000 400000 and 150000 300000per mm3 of blood for adults/children and newborns, respectively (Hillman and Finch, 1985; Fischbach, 1988; Marieb, 1995; Pagana and Pagana, 2006). According to Widmann (1983), normal ranges for glutamate pyruvate transaminase (GPT) are 9-43 and 9-36U/L for men and women, respectively while for alkaline phosphates (ALP) are 80-306U/L for adults and 0.0-0.3, 0.0-0.8 and up to 1.1mg/dL for bilirubin direct (conjugated), bilirubin indirect

(unconjugated) and bilirubin total, respectively. Hemolysis, pernicious anemia, viral hepatitis, liver cirrhosis, biliary cirrhosis, gall bladder diseases, liver damage due to toxicity of drugs, heart disorders, alcohol, pregnancy, may alter the values of LFTs. Cella and Watson (1991) described that congestive heart failure, shock, renal diseases, starvation, diabetes, pregnancy, antibiotics, sever liver disease, liver cirrhosis, deficit /excessive protein ingestion, hypertension, Na/K salts may alter the optimal values of urea (6-20mg/dL) and creatinine i.e. 0.6-1.3 and 0.5-1.0mg/dL for men and women, respectively. Optimal status of total serum cholesterol in an adult varies from 150 to 250 mg/dL and age, sex, race, certain diseases and pregnancy affects its status. During pregnancy, its level may elevate (Chatterjea and Shinde, 2005). Familial hyperlipoproteinemia, atherosclerosis, hypertension, myocardial infarction, nephrosis, pregnancy, corticosteroids, androgens, vitamin A/D toxicity, liver diseases and pernicious anemia may alter normal serum cholesterol status (Cella and Watson, 1991). Values for triglycerides vary with age, sex, race, socioeconomic status, physical activity, dietary habits and region. Standard values of triglycerides are

50-140 and 50-150mg/dL for women and men of 20-40 years, respectively while 50-180 and 50-190mg/dL for women and men of 40-60 years, respectively. The variations in standards may be due to primary hyperlipoproteinemia, atherosclerosis, hypertension, myocardial infarction, liver cirrhosis, alcohol, steroids, niacin and ascorbic acid toxicity (Cella and Watson, 1991). Cigarette smoking, age, low HDL, hypertension, coronary heart disease, pregnancy, diabetes mellitus, hyperthyroidism, infection, inflammation and cirrhosis may affect optimal status of LDL i.e. <100mg/dL while HDL values are normally lower in men than women with optimum range 22-68mg/dL, however, alcohol, chronic hepatitis, cirrhosis and hypo/hyperthyroidism may change its standard range in serum (Cella and Watson, 1991). Future prospects of present project include the recommendations for the government to formulate legislations to make vitamin A fortification compulsory in cereals products. Prospects are being highlighted that multiple micronutrient fortification such as vitamin A and iron along with folic acid are more effective in preventing allied deficiencies that will save millions of lives.

CHAPTER 3

MATERIALS AND METHODS

Food fortification is one of the effective tools in diet based strategies to combat micronutrients deficiencies with special reference to vitamin A. Pregnant and lactating women are more vulnerable to such deficiencies and the adequate intake of micronutrients is helpful to control such health disparities. The research was conducted with the main goal to enhance the levels of vitamin A in pregnant and lactating women. Retinyl acetate and retinyl palmitate were used as fortificant while cookies as vehicle. The efficacy study was conducted initially in vitamin A deficient pregnant women in third trimester, while later fortified cookies were provided to the same; lactating mothers up to 3 months.

3.1. Materials
Commercial straight grade flour (CSGF), retinyl acetate (Fluka) and retinyl palmitate (Fluka) and other chemicals/consumables were procured from the local market.

3.2. Product Development

After conducting some initial trials, vitamin A fortified cookies with acceptable physical, chemical and sensory attributes were prepared. Cookies were evaluated for baking and storage stability of vitamin A to find out the suitable dose of each fortificant to use further in efficacy studies. 3.2.1. Preparation of Vitamin A Fortified Cookies Seven different treatments of the cookies using two fortificants with varying levels (Table 1) were prepared according to method described in AACC (2000) with some modifications using the following recipe: 500g CSGF, 250g

sugar, 250g vegetable shortening, 10g baking powder, 2 no. eggs. Creaming was done in Hobart Mixer (Model N-50, Hobart Mfg. Co. Troy, Ohio, USA) for 30 min, water was added and mixed for 8 min then eggs were added and mixed till homogeneity. Commercial straight grade flour, baking powder and vitamin A fortificants were mixed to a homogeneous mass. The cookies were baked at 1755C for 25min. The cookies were cooled, sealed in bioriented polypropylene (BOPP) wraps, packed in box and stored at room temperature for further analyses. Table 1. Different treatments used to prepare fortified cookies Treatments T1 T2 T3 T4 T5 T6 T7 Retinyl acetate % RDA 30 40 50 Retinyl palmitate % RDA 30 40 50

* Vitamin A dosages for pregnant and lactating women were calculated considering their RDAs.

3.2.2. Stability of Vitamin A Fortificants Stability of vitamin A fortificants was measured by estimating the losses occurred during baking process and storage. The batter and freshly prepared cookies were analyzed for vitamin A content. Likewise, stored cookies were also assessed fortnightly up to 3 months. 3.2.2.1. Determination of Vitamin A Content Vitamin A was contributed to batter/cookies from vegetable shortening, egg and the fortificant. Spectrophotometric estimation of vitamin A content of batter and cookies was carried out according to procedure (AOAC, 1990). Sample Preparation Batter /cookies were dried at 60C; ground and 10g was taken in a flat bottom digestion flask. Potassium hydroxide (50% solution, 10mL) and 40mL

ethanol was added. The flask was placed on a hot plate fitted with magnetic stirrer, with an air condenser on the top. The material was refluxed for 30 min. Then the flask was cooled in a water bath, ethanol: water solution (3:1) was added to make up the volume. After vigorous shaking suspended matter was allowed to settle. Extraction of Vitamin A Top layer (15mL) was taken and transferred to the separation funnel, water (6mL) and hexane (20mL) was added and mixed for 2min. After separation of layers; lower was drained off and the hexane extract containing vitamin A was collected in amber tube for further analysis. Spectrophotometer Analysis Retinyl acetate stock solution was prepared for dilutions to get standard curve. Spectrophotometer (Model: CE 7200, CECIL, UK) was adjusted to zero at 620nm using chloroform and TFA reagent as blank. Vitamin A extracts (0.2mL) and TFA reagent (2mL) was taken in a glass cuvette and absorbance was recorded. Calculations were carried out to estimate vitamin A content during baking and storage. 3.2.3. Physical Analysis of Cookies The cookies containing different levels of vitamin A fortificants were analyzed for diameter (cm), thickness (cm) and spread factor at 0 day and fortnightly up to 3 month according to the method described in AACC (2000). 3.2.3.1. Diameter Six cookies were placed edge to edge and total diameter (D) was measured in cm by using a ruler. The cookies were rotated at an angle of 90 for duplicate reading. This act repeated twice and average D was reported in cm. 3.2.3.2. Thickness Six cookies were on top of one another and total height was measured in cm with ruler. This practice was repeated thrice to get an average value and results were recorded in cm as thickness (T).

3.2.3.3. Spread Factor Spread factor (SF) was determined from D and T by employing subsequent formula; SF = (D/T X CF) X10, where CF is a correction, its value was 1.0. 3.2.4. Chemical Composition of the Cookies Proximate analysis of cookies was carried out at initiation of storage study and subsequent fortnightly up to 3 months. Proximate components i.e. crude protein, crude fat, crude fiber, total ash and nitrogen free extract (NFE) were reported on dry weight basis. 3.2.4.1. Moisture Content The moisture content in cookies was estimated by drying the sample in an Air Forced Draft Oven (Model: DO-1-30/02, PCSIR, Pakistan) at 1055C according to Method No. 44-15A as described in AACC (2000). 3.2.4.2. Crude Protein The nitrogen content of cookies was determined through Kjeltech Apparatus (Model: D-40599, Behr Labor Technik, Gmbh-Germany) according to Method No. 46-10 as given in AACC (2000). The protein percentage was calculated by multiplying nitrogen content with conversion factor i.e. 6.25. 3.2.4.3. Crude Fat Crude fat content was determined by Soxtec System (Model: H-2 1045 Extraction Unit, Hoganas, Sweden) according to Method No. 30-10 described in AACC (2000). 3.2.4.4. Crude Fiber The fat free sample of cookies was taken and treated with H2SO4 for 30 min and repeating the same procedure with NaOH solution. Crude fiber of the sample was determined through Labconco Fibertech (Labconco Corporation Kansas, USA) by following the Method No. 32-10 given in AACC (2000).

3.2.4.5. Total Ash Ash content was determined by placing the sample in Muffle Furnace (MF-1/02, PCSIR, Pakistan) at 5505C. The crucible was heated on the oxidizing flame till no fumes. Ash was calculated according to Method No. 08-01 described in AACC (2000). 3.2.4.6. Nitrogen Free Extract (NFE) NFE was calculated at the stated intervals for storage of 90 days according to the following expression NFE % = 100 - (crude protein% + crude fat% + crude fiber % + total ash %) 3.2.4.7. Thiobarbituric Acid Value To assess the development of rancidity in the product, Thiobarbituric acid number (TAB no.) of all the treatments of the cookies at stated storage intervals was determined following the method described by Kirk and Sawyer (1999). Sample (10g) and water (50mL) were macerated for 2 min in distillation flask and transferred to another containing water (47.5mL) and HCl (4 M; 2.5mL) to adjust pH (1.5). Distillate (50mL) was recovered in 10min during heating; 5mL distillate was taken in glass stopper test tube containing 5mL TBA reagent; mixed and heated in water bath for 35min along with blank sample. The tubes were cooled in water for 10min and absorbance (D) against blank was taken by adjusting spectrophotometer at 538nm. TBA no. was calculated by using the expression; TBA no = 7.8 x D (mg malenaldehyde/Kg) 3.2.5. Sensory Evaluation The sensory evaluation of different treatments of cookies for various attributes including color, flavor, taste, texture and overall acceptability was carried out fortnightly up to 3 months by trained taste panel using hedonic score system (Appendix-I) as described by Meilgaard et al. (2007). On the day of evaluation, cookies from all the treatment were placed in transparent plates, labeled with random codes.

Panelists were given distilled water and unsalted crackers to neutralize their mouth between the samples. The cookies samples were presented in random order and judges were asked to rate their acceptance by giving a score for all the parameters.

3.3.

Efficacy Studies of Vitamin A Fortified Cookies in Pregnant and Lactating Women

Considering the results of stability studies, physico-chemical assay and

sensory evaluation, two best treatments of fortified cookies; one each from fortificants along with placebo were selected and used further in the respective groups of pregnant women. The efficacy studies of vitamin A fortified cookies were conducted in vitamin A deficient pregnant women in the last trimester (commencement of 7th month) till birth and then succeeding 3 months of lactation in the same groups. 3.3.1. Approach and Assortment of Pregnant Women Efficacy studies of vitamin A fortified cookies in pregnant and lactating women was conducted with cooperation of Population Welfare Department (Ministry of Population Welfare, Pakistan), Faisalabad District. The study protocols were discussed with Family Welfare Counselor (FWC)/Family Welfare Worker (FWW); Incharge of Population Welfare Centers (PWCs). Nine PWCs (Appendix-II) were engaged in the project; their heads were given the task to assort the pregnant women in 6th month in their area to be the part of the studies. Information, education and communication (IEC) materials about vitamin A was provided. Initially, 513 pregnant women were approached (Appendix-III), 230 were agreed to take part in research project. The written consent (Appendix-IV) was taken from these volunteers. The selection of the pregnant women was carried out in accordance to convenience and purposive sampling techniques; a part of non-probability sampling methods as described by Muhammad (2000).

The dietary intake Performa was filled up by these subjects to assess their routine diet intake (Appendix-V). Their anthropometric measurements and vital signs recorded. The blood sample of each volunteer was taken in three different specified tubes i.e. Tube-I (EDTA-K3 tube: Guangzhou Improve Medical Instruments Co., Ltd), Tube-II (gel and clot activator tube: Guangzhou Improve Medical Instruments Co., Ltd) and Tube-III (Lithium heparin tube: 367884, Becton, Dickinson and Co). All the tubes containing blood sample were coded, wrapped in Aluminum foil and kept in ice box. 3.3.2. Selection and Assortment of Subjects Blood from Tube-I was used for determination of ABO blood group, Rhtyping using reagents and complete blood count (Cella and Watson, 1991) while serum was extracted from Tube-II to determine liver function tests, renal function tests, lipid profile and hepatitis B & C virus screening. Hepatitis B virus screening was carried out by HBsAg one step test device (Blumberg, 1971) while HCV one step test device was used for hepatitis C virus screening (van der Poel et al., 1991; Wilber, 1993). The serum extracted from tube-III was used for estimation of retinol, retinyl esters and -carotene. Details of protocols for these estimations are given afterward. Based on outcome of above described norms 89 women were found vitamin A deficient as their serum retinol level was <70mol/L or 20g/dL (WHO, 1996; Rauf et al., 2002; Khan et al., 2005). Prevalence of VAD was calculated 38.69% in the group under study. Out of 89 women 14 were excluded from the studies because of positive results of hepatitis B and C virus screening, 75 pregnant women were finally selected for six month studies comprising 7th, 8th and 9th month of pregnancy and then succeeding 1st, 2nd and 3rd month of lactation. These women were assorted randomly into three groups namely placebo (unfortified cookies), RA (retinyl acetate fortified cookies) and RP (retinyl palmitate fortified cookies) group; each comprised of 25 women. The

individuals each group were called on respective PWCs again and discussed in detail about the project, their role and importance, IEC material was provided to them. 3.3.3. Provision and Distribution of the fortified Cookies The selected treatments of the cookies were prepared at Ravi Foods, Hajiabad, Faisalabad. Cookies were prepared considering that five cookies (50g) provided the respective % RDA of pregnant and lactating women (750g for pregnant and 1200g for lactating). Five cookies were packed in bioriented polypropylene tikky pack and 7 such tikky packs (one week dose) were further packed in specified color and printed box containing all the information about the research project, vitamin A importance/functions, nutritional facts, date of manufacturing and best before etc. Blue color was assigned for placebo group, yellow for retinyl acetate group and red for retinyl palmitate group throughout the studies. A snap of pregnant woman and lactating women was printed on the box to avoid any confusion. Before the commencement of each month cookies were provided to PWCs for weekly distribution to the target women. Each woman came to the PWCs weekly, deposited the empty wraps and box and collected one box for next week. A diet Performa was also given to each participant at every visit to record daily diet intake. This practice was started at 1st week of 7th month till delivery and subsequent three month of lactation.

3.4. Blood Sampling and Determinations

During last week of every month each woman was called on Population Welfare Centers and blood was colleted in the specified tubes as discussed earlier and analyzed for stated parameters. 3.4.1. Whole Blood Assays Whole blood from Tube-I was analyzed for complete blood count (CBC) included total red blood cells count, hemoglobin (Hb), and hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean

corpuscular hemoglobin concentration (MCHC) as reported by Widmann (1983) and Hillman and Finch (1985). Platelets count and erythrocytes sedimentation rate (ESR) also investigated as determined by Thompson and Harker (1983) and Widmann (1983). Total white blood cells (WBC), neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles were also investigated as described by Bogs and Winkelstein (1983) and Widmann (1983). All these determinations were conducted through Fully Automatic Blood Analyzer, Nihon Kohden, Japan. 3.4.2. Serum Assessment Serum was extracted by centrifugation of blood sample from Tube-II through centrifugal machine (Model: 800, Centrifugal Machine, China) at 4000 rpm for 6min (Uchida et al., 2001). That serum was used for investigations of succeeding parameters using commercial kits through Microlab-300, Merck, Germany. 3.4.2.1. Liver Function Tests Liver function tests including bilirubin total, bilirubin direct, bilirubin indirect, glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were assessed. The extracted serum was analyzed for bilirubin total, direct and indirect by Jendrassik-Grof method (Thomas, 1998; Tolman and Rej, 1999) GPT by IFFC-method and ALP by Alkaline PhosphatesDGKC method (Thomas, 1998; Moss and Henderson, 1999). 3.4.2.2. Renal Function Tests The extracted serum was analyzed for urea by GLDH-method and creatinine by Jaffe-method (Thomas, 1998). 3.4.2.3. Lipid Profile Lipid profile including total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) was estimated. The extracted serum was analyzed for TC by CHODPAP method (Stockbridge et al., 1989), TG by GPO-PAP method (Annoni et al., 1982), HDL by HDL-

Cholesterol Precipitant method (Assmann, 1979) and LDL as described by McNamara et al. (1990). 3.4.3. Retinoids and Carotenoid Investigations Serum was extracted (4000rpm, 5min) from blood sample in Tube-III, transferred in the amber tube (BT 620-A-1-PK, Biobasic Inc) and stored at -20C till further analysis (Jacobs et al., 1996). Serum was analyzed for estimation of retinol, retinyl esters and -carotene through HPLC instrumentation; from Perkin Elmer Series 200 HPLC Systems, USA, comprised of analytical pump, vacuum degasser, UV/Vis detector, auto sampler, column oven, refractive index, 600 series link chromatography interface, C-18 column (25cm 4.6mm, 5m particle size) protected by a guard column. Injection volume was 20L. Each sample was injected into HPLC apparatus by filtering through Minisart single use syringe (0.20m) filter (Sartorius Vivascience AG 30625 Hannover, Germany). Centrifugation of all the samples during extraction was carried out through Sigma Laborzentrifugen (Model: 3K30, Sigma, D-37520, Osterode am Harz, Germany). Gyro mixer (Model: TKA-0300-100, China) was used for vortex mixing at 2500rpm. All the mobile phases, hexane and ethanol were filtered twice prior to use through Vacuum Filtration Assembly (Model: Rocker-300, TODAYS, OilLess Vacuum Pump, Taiwan) by employing specified filters. 3.4.3.1. Serum Retinol Assessment Serum (100L) and deionised water (200l) was taken in a glass test tube (Tube-A), mixed vigorously on a vortex mixer (15sec) and ethanol (100l) was taken in it; mixed as above, internal standard in hexane i.e. retinyl acetate (50l) was also added and mixed (20sec). Hexane (750L) was pipeted in Tube-A ; mixed on vortex mixer (30sec), centrifuged (2000g, 10min) and top layer was transferred in another test tube (Tube-B). Hexane was added again in remaining contents of the Tube-A, centrifuged and top layer was transferred in Tube-B and

repeated this step once again. Hexane of Tube-B was dried out with Nitrogen. Methanol (mobile phase) was filtered twice through 0.45m (pore size) cellulose acetate filter (Sartorius AG 37070 Goettingen Germany). Methanol (50l) was added in Tube-B and its contents were injected to HPLC apparatus. The flow rate, sensitivity and run time was adjusted at 0.7mL/min, 0.04 AUFS, 10min. Serum retinol was estimated at 325nm (Bieri et al., 1979; Driskell and Neese 1982; Furr et al., 1992). 3.4.3.2. Serum Retinyl Esters Estimation Serum (100L), ethanol (100L) and internal standard i.e. retinyl acetate (70ng/15L hexane) was transferred to a test tube and mixed on vortex mixer (10 sec), then 150L; dioxane : hexane (mobile phase) was filtered through 0.45m nylon filter added and vortex-mixed (30sec). This mixture was centrifuged (350g, 2min, 4C) and hexane layer was drawn off in another test tube. The extraction process was repeated and the sample collected was injected to HPLC instrumentation. The detection wavelength and flow rate was 325nm and 2.5mL/min respectively (Bankson et al., 1986). 3.4.3.3. Serum -carotene Determination Serum (100L), absolute ethanol (500L), methanol (25L/91mmol BHT/L), hexane (2mL) and -carotene internal standard was taken in a glass tube and shook vigorously for 30 sec. Then water (400L) was added, vortexmixed (30sec) and centrifuged (500g, 2min). Upper layer was transferred to another test tube; evaporated with Nitrogen and the mobile phase i.e. isopropanol: acetonitrile (50L) was added and injected into HPLC instruments. An isocratic solvent system of acetonitrile: isopropanol: methanol (68:20:12 v/v), modified with 0.02% ammonium acetate (w/v), pumped at 1.0mL/min, run time was 25min and detection wavelength was 450nm (Lunetta et al., 2002).

3.5. Statistical Studies

Complete Randomized Design (CRD) was applied and data obtained were subjected to statistical analysis using statistical package Costat-2003 (Cohort v6.1) to determine the level of significance (analysis of variance technique). Means were compared through Duncans multiple range test (DMRt) according to the method described by Steel et al. (1997).

CHAPTER-4

RESULTS AND DISCUSSION

Mandate of the present study was to enhance the serum retinol level of pregnant and lactating women by providing vitamin A fortified cookies in their diet. The study was comprised of two parts; in the first phase, cookies were prepared with varying level of vitamin A fortificants (retinyl acetate and retinyl palmitate). Baking and storage stability of both fortificants were also assessed. In the second part of study, pregnant women were selected on the basis of anthropometric measurements, vital sign recording, hepatitis B and C virus screening, hematological and serological aspects and indeed their vitamin A status. The vitamin A deficient pregnant women were provided cookies in predetermined quantities in their last trimester and for further 3 months during lactation. The results obtained during the course of the study are presented below.

4.1.

Physical Analysis of the Fortified Cookies

Physical analysis of cookies is important from consumer as well as bakers

point of view and it is desirable that cookies should retain their shape during baking. Vitamin A fortified cookies along with control were analyzed for physical characteristics including diameter, thickness and spread factor during storage of 90 days. Means squares for aforementioned traits are presented in Table 2. Varying levels of fortificants and storage exhibited non-significant differences on the physical parameters.

4.1.1. Diameter The mean values for diameter of fortified cookies (Table 3) showed that diameter of vitamin A fortified cookies ranged from 4.610.020 to 4.790.034cm. Storage had also non-significant impact on this trait. The mean value at 0 days was 4.810.032cm which indicated a non-significant decline with passage of time and diameter 4.600.028cm was recorded at 90 days (Table 4). The non-significant behavior of fortification might be due to the reason that fortificants did not affect the dough/batter properties responsible for determining the diameter of the cookies. The deterioration in physical parameters usually depends upon the prevailing storage conditions. In this study, cookies were packed in bioriented polypropylene wraps and the reactions leading to deterioration in quality proceeded slowly thus retaining diameter of cookies. Moreover, Butt et al. (2007) found non-significant variations among treatments and storage for this trait in retinyl acetate fortified cookies. Similarly, non-significant effect of fortification of the cookies with retinyl acetate on diameter of the cookies was found by Mahmood et al. (2008) with maximum diameter observed by cookies containing 325 g of the salt. 4.1.2. Thickness Mean values for thickness of fortified cookies are explicated in Table 3. The thickness of fortified cookies ranged from 0.520.002 to 0.530.003cm. There were non-significant differences among the treatments during storage (Table 4). The cookies were packed in bioriented polypropylene and during storage no such reaction was proceeded that could alter the thickness of cookies. Physical parameters are dependent on gluten strength of flour and shortening used (Gaines and Donelson, 1985; Taylor et al., 2008). Likewise, Butt et al. (2007) determined a non-significant change in thickness of vitamin A fortified cookies as a function of treatments and storage. They observed 4.99mm thickness in T1 (control) while 4.92mm for T4 (25.85 g of retinyl acetate) that support present results; non-momentous decrease in

thickness was recorded as a function of varying levels of fortificants. Furthermore, similar findings were reported by Mahmood et al. (2008) that addition of vitamin A fortificant did not alter the value of thickness significantly. 4.1.3. Spread Factor Spread factor is the ratio, which depends upon the values of width and thickness. Means for spread factor revealed (Table 3) non-significant differences among vitamin A fortified cookies. The spread factor of cookies ranged from 87.140.662 to 91.390.441. Likewise, storage of cookies for 90 days did not impart any significant difference (Table 4). Spread factor of cookies depends on flour particle size, moisture content and shortening used (Gaines and Donelson, 1985; Sharif et al., 2005). It is also dependent on values of diameter and thickness and is affected by the competition of ingredients for the available water; flour or any other ingredient for water absorption during dough mixing (Fuhr, 1962). Present results are in conformity with previous researchers who reported non-significant changes in vitamin A fortified cookies during storage (Butt et al. 2007; Mahmood et al., 2008).

4.2. Chemical Composition of the Fortified Cookies

Vitamin A fortified cookies were analyzed for moisture, ash, crude protein, crude fat, crude fiber, nitrogen free extract and thiobarbituric acid (TBA) value during storage of 90 days. The mean squares for proximate composition and TBA value are given in Table 5. It is evident from the statistical analysis that only moisture content and TBA value differed significantly (P<0.01) as a function of storage. Moreover, treatments and interaction remained non-significant in their expression for all traits. 4.2.1. Moisture Content The moisture content of a food is of great significance for many scientific, technical and economic reasons. Lesser the moisture content of the cookies, better its storage stability. The mean values for moisture content (Table 6) elucidated statistically non-significant variations among the fortified cookies; ranged from

2.660.047 to 2.760.039%. However, an increasing trend was observed during three months storage (Table 7). At the initiation of study, the moisture content was 2.510.026% that increased progressively to 2.840.024% during storage. The phenomenon of moisture incorporation during storage period is also supported by Wade (1988) who explored that when cookies are sealed in moisture proof packaging, minute amount of moisture present in the air within the package rapidly come in equilibrium with the product, resulting increase in moisture content of cookies. Similar increasing trend of moisture was observed in red palm oil fortified cookies (Butt et al., 2004b), rice bran oil cookies (Sharif et al., 2005) and biscuits containing whole egg powder (Rao et al., 1995) during storage. 4.2.2. Ash Content Ash content in a food substance indicates inorganic remains after the organic matter has been burnt away. The mean values for ash content of fortified cookies are given in Table 6 and 7. Ash content varied non-significantly from 0.560.007 to 0.590.011% in fortified cookies. Similarly, there was nonsignificant decrease in ash content during storage. The results are in accordance with that of reported by Pasha et al. (2002), Anjum et al. (2003), Sharif et al. (2005) and Butt et al. (2007). 4.2.3. Crude Protein Means for protein content of vitamin A fortified cookies illustrated nonsignificant effect of fortification; the crude protein content ranged from 6.590.025 to 6.660.026% in all treatments (Table 6). In a similar study, varying level of retinyl acetate fortification did not alter the protein content of cookies (Mahmood et al., 2008). During 90 days storage, there was non-significant decline in protein content of cookies (Table 7). In freshly prepared cookies, the average protein content was 6.690.022%, which was reduced to 6.570.024% after three months storage. The non-significant decrease in protein content was certainly due to the increase in moisture of cookies that accelerates proteolytic activity

during storage. Vitamin A fortificants might not interfere with the nitrogenous substances of fortified cookies so there is no significant alteration in crude protein content due to treatments and storage. The results of the present investigation are in line with the findings of Wade (1988), Pasha et al. (2002), Butt et al. (2004b) and Sharif et al. (2005). 4.2.4. Crude Fat The means for fat content of fortified cookies (Table 8) explicated nonsignificant differences with varying levels of retinyl acetate and retinyl palmitate ranged from 22.370.081 to 22.510.053%. There were statistically nonmomentous variations (22.600.032 to 22.260.046%) in fortified cookies during storage (Table 9). However, the slight decrease in fat content was perhaps due to increase in moisture content of cookies, accelerating oxidation of fatty acids. In the current study, cookies were packed in bioriented polypropylene films, acting as barrier against atmospheric variables during storage thus preserving the composition of final product. Results of present research are in accordance with the findings of Mahmood et al. (2008), who reported non-significant variations in fat content of the fortified cookies during storage. 4.2.5. Crude Fiber The means for crude fiber of vitamin A fortified cookies (Table 8) showed that fiber content ranged from 0.250.002 to 0.260.004%. It is obvious from the results that vitamin A fortificants exhibited non-significant effect on crude fiber content because retinyl acetate and retinyl palmitate did not interact with fibrous matter of the cookies. Similarly, storage had non-significant effect on various treatments of fortified cookies (Table 9). The results of present study are in close agreement with the findings of Pasha et al. (2002), Anjum et al. (2003) and Butt et al. (2007) who observed non-significant impact of storage on fiber content of cookies.

4.2.6. Nitrogen Free Extract The means for NFE content of fortified cookies (Table 8) depicted that NFE content in different treatments ranged from 67.300.061 to 67.470.064%. Similarly, there were non-significant variations in NFE during storage (Table 9). These results are also supported by the findings of Butt et al. (2007); who determined non-significant variations in NFE content of vitamin A fortified cookies due to varying level of retinyl acetate. 4.2.7. Thiobarbituric Acid Value Thiobarbituric acid number is a test based on the reaction of thiobarbituric acid with the oxidation products of oils to form a red color. Means for TBA no. (Table 8) indicated non-significant differences among the cookies fortified with retinyl acetate and retinyl palmitate; TBA no. ranged from 0.0490.004 to 0.0530.004mg malenaldehyde/Kg. However, there was an increase in TBA no. of cookies during 90 days storage (Table 9); minimum value was recorded at the initiation of study (0.0400.001mg malenaldehyde/Kg) that was gradually increased to 0.0410.002, 0.0440.002, 0.0490.001, 0.0540.002, 0.0600.003 and 0.0690.002mg malenaldehyde/Kg, respectively, after 15, 30, 45, 60, 75 and 90 days storage. However, all treatments were within safe limit (Table 9). Refined oil in good state has TBA value 0.02-0.08 whereas badly stored oils have 0.10.2mg malenaldehyde/Kg (Kirk and Sawyer, 1999). Higher temperature, moisture, heat and light are the key factors that further accelerate the TBA value during storage (Kent and Evers, 1994). In another study, increase in TBA no. was observed in cereal based baked products, might be due to inherent enzymatic activity, accelerated due to increase in moisture during storage (Fellers and Bean, 1977).

4.3. Baking and Storage Stability

Baking and storage stability are of important considerations while defining the final dosage for any micronutrient. In the present study, baking and storage stability of various doses of retinyl acetate and retinyl palmitate were

estimated. Means squares for baking stability of fortified cookies are presented in the Table 10; revealed the significant effects of baking, fortificants and their interaction on stability of vitamin A. Vitamin A content decreased significantly as a function of baking from 9.982.18 to 9.051.98g/100g, treatments also exhibited momentous effects on the said parameter. Before baking maximum vitamin A was recorded in T4 (50% of RDA; retinyl acetate) 18.18 followed by 14.59g/100g in T3 (40% of RDA; retinyl acetate) whilst the lowest amount (0.19g/100g) was estimated in T1 (placebo) as indicated in Table 11. Vitamin A losses during baking ranged from 7.95 and 10.20% in retinyl acetate and retinyl palmitate fortified cookies, respectively, as compared to 15.79% in placebo as illustrated in Figure 1. Vitamin A fortificants were also assessed for their storage stability; mean squares presented in Table 12 elucidated that treatments and storage imparted momentous impact on vitamin A content of the cookies while interaction remained non-significant. The results in Table 13 indicated that vitamin A content vary significantly with increasing level of retinyl acetate and retinyl palmitate; maximum amount of vitamin A (15.680.181g/100g) was recorded in T4 (50% of RDA; retinyl acetate) whilst minimum level (0.140.006g/100g) was observed in T1 (placebo). Storage also affected vitamin A content significantly and declining trend was observed from 9.051.98g/100g at the initiation of study to 8.261.83g/100g after 3 months. Storage losses ranged from 8.02 to 9.69% among the treatments. However, overall storage loss was recorded to be 8.71% as depicted in Figure 2. Vitamin A is heat and light sensitive and losses take place when exposed to these physical factors. The high temperature may accelerate oxidation of vitamin A resulting in loss of ability to bind with lipids thus lowering absorption capacities (Butt et al., 2007). Losses of vitamin A are due to its vulnerability to lipid oxidation during baking and storage. Present results are in corroborated with Butt et al. (2007) who investigated the stability of retinyl acetate in fortified

cookies and affirmed that loss in vitamin A content occurred i.e. 8.6911.11% during baking process. Likewise, storage of cookies at room temperature significantly decreased vitamin A content; after 15 days, the loss was 5.89%. Nevertheless, among the treatments losses were non-significant. However, after 30 days, the loss was almost doubled than that of fortnight results. Mahmood et al. (2008) has conducted a study of vitamin A fortification in cookies and observed 9.3% loss during baking. The retinyl acetate content before baking was 10.99 that reduced to 9.96g/100g after baking. Similarly, storage of the cookies for 90 days at ambient temperature significantly reduced the vitamin A content from 9.05 to 8.26 g/100g and the loss was 8.33%. Likewise, Emodi and Scialei (1980) studied the retention of vitamin A during baking of bread and observed a loss of 710% during baking. In another study Bauernfeind (2006) found 90% retention of vitamin A during baking and minimal loss after 6 month storage. The differences in results could be due to storage temperature. Increasing storage temperature decreases retention of vitamin A. Wheat flour stored at 45C had 72% loss in vitamin A content after 3 months of storage (Anonymous, 1996; Butt et al ., 2007). Bauernfeind and DeRitter (1991) also observed slight variations in vitamin A after six months of storage that strengthened the present findings. Retinyl acetate and retinyl palmitate have greater stability than retinol. The acetate has comparable properties to palmitate except that it is less stable in the presence of moisture. Vitamin A is surprisingly stable during bread baking. Palmitate can be used with resulting stability of 90-100% after baking white bread by the standard AACC procedure. However, during 5 days of storage at room temperature, observed retention was 85-95% (Furia, 1968). Baking fortified bread causes limited losses of vitamin A (10-20%), while frying has an adverse effect on the stability. After an initial frying of vitamin A

fortified soybean oil, the vitamin A retention was about 65% (Anonymous, 1996). Stability of vitamin A is affected by oxidation, heat and light that are the potent factors destroying vitamin A.

4.4. Sensory Evaluation

Sensory evaluation is carried out to evaluate the response of judges towards the end product and they rate the liking on a scale. Cookies were evaluated by trained taste panel for various sensory attributes including color, flavor, taste, texture and overall acceptability on fortnightly basis up to 90 days of storage. The cookies were analyzed on 9-point Hedonic scale to find out the most suitable treatments for efficacy purposes by following the procedure of Meilgaard et al. (2007). Means squares (Table 14) indicated that storage affected all sensory attributes significantly. Treatments and their interaction imparted non-differential impact on these traits. 4.4.1. Color Color serves as a cue for the doneness of foods. Means for color scores explicated non-significant differences in cookies fortified with retinyl acetate and retinyl palmitate. Overall the color scores ranged from 6.770.13 to 7.090.17 (Table 15). In a similar study, there was non-significant decrease in color of cookies fortified with retinyl acetate (Butt et al., 2007). However, cookies were significantly affected during storage (Table 16); maximum score was noted in fresh cookies (7.300.09) that was gradually decreased (6.190.06) during three months that might be due to increase in moisture content of cookies. The migration or absorption of moisture results in color loss during storage (Bender, 1996). There is also a natural trend of color fading with progressive storage which ultimately affects the appearance (Manley, 2002). These results are in close agreement with the findings of Wade (1998), Pasha et al. (2002) and Sharif et al. (2005). Likewise, significant decrease in color scores (7.25 to 6.00) was observed in cookies fortified with retinyl acetate (Mahmood et al., 2008).

4.4.2. Flavor Perceptions of flavor are synthesis of taste and smell impressions, along with texture and also influenced by appearance. Means for flavor scores (Table 15) exhibited non-significant variations in cookies fortified with various levels of retinyl acetate and retinyl palmitate; ranged from 6.600.18 to 6.770.17. However, storage significantly affected flavor perception of panelists and tends to decrease (7.140.04 to 6.030.03) with the passage of time (Table 16). The decrease in flavor of cookies was might be due to increase in moisture content of cookies during storage that further accelerated flavor deterioration, probably due to fat oxidation (Wade, 1988). In earlier studies, decreasing trend in flavor scores of retinyl acetate fortified cookies was observed (Butt et al., 2007) as a function of storage. Similar decrease in flavor of cookies was recorded during storage intervals (Sharif et al., 2005). Bakery products stale rapidly and lose their flavor because staling transforms the rich aroma and flavor of the fresh product to a bland or off-flavor (Setser, 1996). 4.4.3. Taste Taste is a sensation perceived by the taste buds and influenced by the texture, flavor and composition of the foods. It is one of the most essential parameters related to the acceptability of any product. Mean scores for taste of cookies are given in Table 15; non-significant changes in taste scores of fortified cookies were observed by varying level of retinyl acetate and retinyl palmitate ranged from 6.860.20 to 7.140.14. However, storage imparted differential declining impact on the taste of the cookies (Table 16). Maximum scores (7.380.05) were obtained by fresh cookies that subsequently declined (6.380.09) after three months storage, probably due to lipolytic changes, hasten by increase in moisture content of cookies (Wade, 1998; Butt et al., 2007). 4.4.4. Texture Texture is combine sensation of all the rheological and structural traits of the product perceptible by mechanical, tactile and where appropriate, visual and auditory receptors. Means for texture scores (Table 17) explained non-significant

differences in vitamin A fortified cookies; ranged from 7.000.27 to 7.370.17. Nevertheless, a significant reduction in the texture scores was noted with the passage of time from 7.830.05 to 6.330.10 (Table 18). The decrease in quality score for texture was due to absorption of moisture that has negative impact on texture (McWatters, 2003; Sharif et al., 2005). 4.4.5. Crispness The quality score in response to crispness of the cookies (Table 17) exhibited non-significant changes in this trait. It was observed that cookies fortified with different levels of fortificants behaved alike. Overall, the score for crispness ranged from 7.000.27 to 7.290.27. Nonetheless, there was decrease in crispness of the fortified cookies during storage i.e. 7.910.04 to 6.140.07 (Table 18). The decreasing trend in quality scores for crispness of cookies was certainly due to increase in moisture that has an inverse correlation with the crispness (Wade, 1988; Butt et al., 2007). 4.4.6. Overall Acceptability Means for overall acceptability of cookies are presented in Table 17 & 18. The scores for overall acceptability ranged from 7.100.23 to 7.240.19.There were non-significant differences in cookies fortified with different vitamin A fortificants at various levels. Moreover, it is worth mentioning that vitamin A fortified cookies were liked by the judges without any discrimination of salt and its various levels. There was declining trend in the overall acceptability of cookies during storage. Maximum score was obtained by fresh cookies (7.720.05) that decreased to 6.190.12 at the end of storage. The decrease in score for over all acceptability was probably due to increase in moisture during storage, promoting proteolytic and lipolytic changes, leading to decrease in quality score for color, flavor, taste, texture and crispness of cookies. Similar, decreasing trend in scores for overall acceptability was observed by Pasha et al. (2002), Sharif et al. (2005) and Mahmood et al. (2008) in cookies during various storage intervals.

In present study, maximum levels of fortificants did not show any detrimental changes in physico-chemical or sensoric attributes of fortified cookies. After product development, two treatments with maximum fortification levels of retinyl acetate and retinyl palmitate i.e. T4 (50% RDA, retinyl acetate) and T7 (50% RDA, retinyl palmitate) were selected to meet the 50% requirement of vitamin A in pregnant and lactating women.

4.5. Efficacy Studies in Pregnant Women

Two best treatments, one from each; retinyl acetate and retinyl palmitate along with control were selected on the basis of results of physico-chemical, sensory and stability analysis. For the purpose, fortified cookies were prepared considering the RDA of pregnant women (750g) in Ravi Foods, Hajiabad, Faisalabad. During the research study, five cookies (50g) per day from selected treatments including T4 (50% RDA; retinyl acetate), T7 (50% RDA; retinyl palmitate) and T1 (placebo) were provided to the respective groups of vitamin A deficient pregnant women in third trimester. Blood samples of the subjects were collected on monthly basis and analyzed for specified hematological and serological parameters to evaluate the impact of fortification on the vitamin A status of the pregnant women. The results obtained are discussed as below. 4.5.1. Serum Retinol, Retinyl Esters and -Carotene Profile Vitamin A status of the body can be estimated from serum retinol level, however, level of retinyl esters and -carotene also provide some indications about vitamin A deficiency. Serum Retinol Mean squares presented in Table 19 explicated that treatments, stated intervals and their interaction affected serum retinol level of pregnant women significantly. Means for treatments, course of pregnancy and their interaction is presented in Table 20. The groups of pregnant women consuming retinyl palmitate fortified cookies showed the highest serum retinol level

(18.920.79g/dL) that was statistically at par with retinyl acetate group

(18.750.68g/dL) while the lowest value (16.310.34bg/dL) was observed in placebo group. Means for months of pregnancy depicted that there was significant increase in serum retinol level with passage of time and maximum concentration (18.641.59g/dL) was observed near delivery while the lowest retinol level at baseline (16.920.02g/dL). Treatments and period of pregnancy interact significantly to produce varying serum retinol levels. A gradual and significant drop in serum retinol level was only observed in placebo (T1) declining from 17.06g/dL at baseline to 15.47g/dL at the end of three months. Retinyl acetate fortified cookies (T4) showed a considerable uplift in serum retinol level of pregnant women over time from initial value of 16.91 to 20.04g/dL while retinyl palmitate improved serum retinol level from 16.97 to 20.41g/dL. Percent change in serum retinol level of pregnant women is presented in Figure 3. It is evident that there was a momentous increase in serum retinol level in group consuming fortified cookies. In placebo, serum retinol level declined by 9.32% during the whole study duration. Retinyl acetate group had shown 18.51% increase while in group consuming retinyl palmitate fortified cookies, 21.56% increase in their serum retinol level was observed. Present research project was designed to uplift serum retinol level in pregnant women as they are at greater risk of vitamin A deficiency due to the raising demand during pregnancy to meet their requirements. Communities living in developing world are more vulnerable due to diet inadequacy. Normal reference range of serum retinol is 20 49g/dL (Rauf et al., 2002). In the present project, women with vitamin A deficiency were selected on the basis of marginal vitamin A status (30g/dL) and deficient vitamin A status (<20g/dL), defined by Ahmed (1999). Consumption of fortified cookies in the present study resulted in marked increase in serum retinol level that could be attributed to phenomenon of absorption and re-absorption. Ingested retinyl

acetate or retinyl palmitate is broken down into retinol in digestive tract,

absorbed in mucosal membrane and transported to liver. During VAD state, conversion of retinol into its esters might be reduced. Retinol binding proteins tend to increase during deficiency thus result in sustainability of serum retinol levels in humans (Blomhoff, 1994). Fortification strategies are imperative to bring positive changes in vitamin A status of the body. In order to provide access to the fortified products to all populations, Steyn et al. (2008) are of the view that foods which are in common use by all communities should be used as vehicle for vitamin A fortification. Such products can ensure proper access to populations and easier acceptability in subjects. A wide range of products has been tested for vitamin A fortification that includes sugar, cereals, oil and fat and common salt. Muhilal et al. (1988a) conducted a study in Indonesia to determine the effects of monosodium glutamate (MSG) fortification with vitamin A on serum profile of VAD sufferers during a controlled trial. They observed serum vitamin A level at beginning and afterwards; reported 37.31% uplift in serum vitamin A in subjects receiving fortified MSG from 0.67 (base line) to 0.92mol/L in 11 months. However, in placebo, 7.69% decrease in serum retinol level was observed; declined from 0.78 to 0.72mol/L. Similarly, Arroyave et al. (1981) coined observations by providing fortified sugar with retinyl palmitate in Guatemala and found that one year of fortification increased serum status of retinol significantly in 76% of the subjects from 16.2 to 30.2g/dL (86.42% uplift). However, adopting same strategies for next year, they observed 57.23% of increase in serum retinol level from 16.6 to 26.1g/dL. Later on, Dary et al. (1996) reported 10% reduction in vitamin A deficiency prevalence in a study conducted in Guatemala and similar findings were also observed during a research project conducted by Solon et al. (1979) who compared the relative effectiveness of three different intervention strategies to control VAD in Cebu, Philippines. These interventions were; public health and horticulture intervention, provision vitamin A in capsules and MSG fortification

with vitamin A. Each intervention was implemented in four different areas, two urban and two rural, for about 2 years. Similar parameters were investigated before and after each intervention. Mean serum vitamin A status was 19.0, 19.6, 21.0g/dL before and 16.4, 19.6, 28.5g/dL after public health, vitamin A capsule and fortification of MSG, respectively. The highest serum vitamin A status (7.5g/dL; 37.71%) uplift was observed by use of fortified MSG. Hence fortification of food items was the strategy; resulted in significant reduction in clinical signs of xerophthalmia and considerable rise in serum vitamin A profile. Similar work has been also conducted in other parts of the globe; fortification of cereal flour with vitamin A was carried out in Venezuela and Philippines. Vitamin A fortification of wheat flour buns was effective in raising serum retinol level of targeted subjects in Philippines, (Solon et al., 2000). Similarly, in Venezuela, vitamin A fortification of precooked maize flour to reduce malnutrition was carried out with favorable outputs for future applications (Layrisse et al., 2000). Likewise, Stuijvenberg et al. (1999) determined the effect of micronutrient fortified biscuits on micronutrient status of selected subjects. They observed significant differences in serum retinol level of target group and the prevalence of low serum retinol (<0.70mol/L) decreased from 39.1 to 12.2%. Recently, Vinodkumar and Rajagopalan (2008) assessed the efficacy of a salt fortified with multiple micronutrients in Chennai, India. They reported significant

improvement in serum vitamin A (5.6g/dL) in experimental group, while recorded significant drop in the placebo. Similar type of community based research study conducted for the first time in Pakistan using fortified cookies. Provision of cookies fortified with retinyl acetate and retinyl palmitate to vitamin A deficient (VAD) pregnant women during last trimester enhanced their serum retinol level. Serum retinol concentration increased with fortification intervention. It is suggested that to

fulfill the gap in VAD patients, vitamin A should be provided through diet based strategy. Serum Retinyl Esters and -carotene Index Statistical results showed that retinyl esters are significantly affected as a function of months while treatments and interaction remained non-significant (Table 19). Means for treatments, months and their interaction regarding level of serum retinyl esters (Table 21) explicated that the treatments varied nonsignificantly however; maximum retinyl esters level (0.740.09g/dL) was observed in the group consuming unfortified cookies. Period of pregnancy decreased the retinyl esters significantly from baseline value of 0.860.04 to 0.520.01g/dL. Interaction of treatments and months showed non-momentous effects nevertheless, retinyl esters decreased in all groups irrespective of fortificants; in placebo reduced from 0.91 to 0.51g/dL, retinyl acetate group decreased from 0.87 to 0.54g/dL while in retinyl palmitate group declined from 0.79 to 0.50g/dL. In all groups of pregnant women, retinyl esters declined throughout the study period as evident from Figure 4. Considering the baseline values, percent decline in placebo was observed to be 43.95% as compared to retinyl acetate and retinyl palmitate groups i.e. 37.93 and 36.71%, respectively. It is obvious from the mean squares (Table 19) that treatments and months affected serum -carotene level of pregnant women significantly however; interactive effect of treatments and months remained non-significant. Statistically analyzed results regarding means for serum -carotene level are presented in Table 22. Intake of retinyl acetate and retinyl palmitate fortified cookies improved the -carotene level of pregnant women significantly with mean contents 31.250.43 and 30.830.39g/dL, respectively while value 27.472.32g/dL was recorded for placebo. During last trimester, -carotene content of pregnant women significantly declined from baseline value of

32.360.37 to 27.502.65g/dL. Interactive effect of treatments and months affected -carotene content non-significantly. However, a declining trend prevailed in all groups that were more pronounced in placebo group where carotene content decreased from 33.03 to 22.20g/dL. Intake of retinyl acetate and retinyl palmitate fortified cookies resulted in decline from 32.28 to 30.30 and 31.77 to 30.00g/dL, respectively. The values in Figure 5 depicted the percent decrease in the level of carotene of pregnant women throughout the study duration. Consumption of fortified cookies by pregnant women showed less decrease i.e. 6.13 and 5.57%, respectively as compared to placebo (32.79%). Results of present study indicate that level of retinyl esters and -carotene tends to decrease during the study period. The possible reason may be that during vitamin A deficiency (VAD), stores for vitamin A depleted and retinyl esters and carotene readily converted to retinol. It can be further assumed that level of retinyl esters and -carotene tends to decrease as a function of time during VAD syndrome. Normal ranges for -carotene and retinyl esters are 50200g/dL and 35 g/dL, respectively (Jacobs et al., 1996). However, in VAD, these compounds undergo conversion process and their level fall far below than that of their normal values. Dietary preformed vitamin A and carotenoids are proteolysed in stomach, aggregate with lipids and pass through upper part of small intestines. Lipases and esterases hydrolyze retinyl esters into retinol in intestinal lumen that is absorbed 70-90 % (Blomhoff, 1994). However, provitamin A carotenoids pass through the mucosa unaltered; a fraction of it, along with non-provitamin carotenoids enter into lymph and blood. The remainder resulted in breakage of molecule by a specific enzyme 15, 15-dioxygenase in mucosal cell (Goodman et al., 1966). Cleavage of the carotene molecule yields retinal which is mostly reduced to retinol; esterified to retinyl esters if serum retinol level is normal within the mucosal cells (Ong et al., 1987).

Retinyl esters and -carotene are catabolised into retinol in human body; conversion is accelerated in vitamin A deficient state. Recycling of retinol occurs between plasma, liver and other tissues and concentration of carotenoids and retinyl esters in plasma is dependent on diet (Olmedilla et al., 1994). In terms of vitamin A intake in diet, about 10% remains unabsorbed, 20% excreted through feces, 17% through urine, 3% released as CO2 and 50% is stored in the liver (Olson, 1994). In the liver, retinyl esters are taken up by hepatocytes and stellate cells (Rask et al., 1983). About 5080% of vitamin A of the body is stored in the liver; 9095% of it is in the stellate cells, 98% in the form of retinyl esters, mostly as palmitate (Batres and Olson, 1987a). In the present study, all women were vitamin A deficient so the dietary retinyl esters and -carotene might be converted to retinol; the most active form of vitamin A to make up its deficiency. 4.5.2. Red Blood Cells Indices Red blood cells transport gases in blood and the abnormal values indicate some disorders in the body. Red blood cell indices include total Red blood cells (RBC), hematocrit (Hct), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentrations (MCHC), platelet count and erythrocytes sedimentation rate (ESR). Statistical analysis (Table 23) showing means squares for red blood cells indices explicated that treatments and months significantly affected all parameters except red blood cells, platelet, ESR while interaction was found to be significant only in hemoglobin level. Total Red Blood Cells Count Non-significant effect of the study duration, treatments along with their interaction was observed in total RBC (Table 23). The number of total RBC was decreased non-significantly in all groups of pregnant women during 90 days of study. Mean values of total RBC at start of the assessment was found to be 4.980.05M/L that decreased to 4.700.06M/L in 90 days. It is obvious from

the results that treatments means varied non-significantly from each others as 4.720.07 for T1 (placebo), 4.910.08 in retinyl acetate and 4.860.04M/L in retinyl palmitate groups at the end of study (Table 24). Hematocrit Mean squares in Table 23 indicated that intake of fortified cookies influenced hematocrit percentage of pregnant women significantly. Moreover, hematocrit (%) was significantly decreased in the pregnant women during entire study period as mentioned in Table 25. Mean hematocrit percentage at initial level was noted to be 31.820.17 that declined to 27.101.25 % in 90 days (Table 25). The highest mean value for hematocrit was observed in group of females consuming retinyl acetate fortified cookies (30.450.88%), statistically similar to retinyl palmitate (30.020.78%) and the lowest value was observed in placebo group (28.141.51%). A significant gradual drop in hematocrit content was noticed in T1 (placebo) from 31.53 at baseline to 24.60% in the last month of pregnancy. Hematocrit content decline was found to be non-significant in retinyl acetate and retinyl palmitate groups as compared to placebo over time from 32.11 to 28.23 and 31.83 to 28.47%, respectively. Hemoglobin Mean squares in Table 23 elucidated that cookies fortified with different fortificants and their interaction with months influenced significantly on Hb level of pregnant women. However, level of this trait was affected non-significantly during period of three months. Hb level significantly improved in retinyl acetate and retinyl palmitate groups however, value for this trait was dropped in placebo. Hb level varied non-significantly during the entire study (Table 26). Interaction of treatments and months produced some varied results; gradual drop in hemoglobin level was observed in placebo group from 8.95 to 7.90g/dL, whilst, retinyl acetate group showed a slight increase in hemoglobin

from 9.34 to 9.78g/dL that differ non-significantly with retinyl palmitate group from 9.12 to 9.73g/dL in three months of pregnancy. The values in Figure 6 depict the percent change in hemoglobin level over the entire study. In placebo group, hemoglobin content decreased by 11.73% while, intake of fortified cookies resulted in a marked increase in hemoglobin level by 4.71 and 6.69% in retinyl acetate and retinyl palmitate groups of pregnant women, respectively. Mean Corpuscular Hemoglobin The cookies fortified with different fortificants affected MCH significantly. Study period also imparted differential impact on same parameter (Table 23). MCH value was significantly altered as function of treatments and high in groups taking retinyl acetate and retinyl palmitate fortified cookies (Table 27). Mean MCH content; 22.702.03pg was observed in T1 (placebo) as compared to 24.771.52 and 24.711.53pg in T4 (retinyl acetate) and T7 (retinyl palmitate), respectively. Course of pregnancy also produced significant declining results and MCH content was reduced from 28.040.10 to 20.190.99pg. Mean Corpuscular Volume Mean squares regarding mean corpuscular volume (MCV) are presented in Table 23; depicted the significant effect of treatments and months. However, interaction remained non-significant in its impact. Data regarding means of treatments, months and their interaction are presented in Table 28. The mean MCV content in the blood of pregnant women declined from 74.930.17 to 66.342.74fL at the end of study. MCV value for treatments showed declining trend but this trend was more pronounced in T1 (placebo) as compared to T4 (retinyl acetate) and T7 (retinyl palmitate) as evident from their mean values i.e. 67.302.98, 72.021.46 and 71.201.29fL, respectively.

Mean Corpuscular Hemoglobin Concentration Means squares presented in Table 23 revealed that treatments and months showed significant effect on mean corpuscular hemoglobin concentration (MCHC) of pregnant women. Provision of retinyl acetate and retinyl palmitate fortified cookies explicated higher MCHC content as compared to control (Table 29). However, MCHC content decreased significantly over time from baseline value of 32.550.35 to 28.201.33g/dL at the end of 9th month of pregnancy. MCHC value for treatments illuminated that retinyl acetate and retinyl palmitate had higher values of 31.230.74 and 30.550.54g/dL, respectively, as compared to placebo group i.e. 28.701.52g/dL. Platelets Count Platelets count was non-significantly affected as a function of treatments, months and their interaction (Table 23). Means for treatments, months and their interaction are shown in Table 30. Platelets value remained in the range of 270.41 to 362.50K/L. Course of pregnancy resulted in higher count; increased from (baseline) 294.637.96 to 319.12323.40K/L at the end of study. The platelets count in pregnant women during whole study period altered non-significantly by all the treatments of the fortified cookies. The mean platelets count value for T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups was 308.8220.83, 318.078.45 and 300.4415.83K/L, respectively. Erythrocytes Sedimentation Rate Mean squares (Table 23) indicated a non-significant impact of study duration, treatments and their interaction on the status of erythrocytes sedimentation rate (ESR) of pregnant women. The mean ESR values were in the range of 23.54 to 30.27mm/Hr. Nonsignificant differences exist among months as depicted by values of 27.662.09,

25.471.25, 26.990.18 and 27.320.10mm/Hr. at base line, 7th, 8th and 9th months of pregnancy, respectively. Likewise, mean ESR statistics for T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups of pregnant women alluded non-significant results (Table 31). Hematological study revealed that with the provision of 50% RDA of vitamin A through retinyl acetate and retinyl palmitate fortified cookies resulted a significant impact on Hct, Hb, MCH and MCV values. Vitamin A is involved in prevention of anemia through different biological means such as improvement in growth and differentiation of erythrocyte progenitor cells, potentiation of immunity, reduced infectious anemia and mobilization of iron stores from tissues. Enhancement of vitamin A status had been revealed to control anemia (Semba and Bloem, 2002). Vitamin A acts synergistically in folate metabolism and its deficiency accelerates the process of anemia. Total RBC count and Hct (%) might be decreased in pregnancy, iron deficiency anemia, vitamin B12 deficiency, folate deficiency, hemolytic anemia, hemorrhagic infections, bone marrow damage, metabolic disorders and chronic inflammation. Normally, hematocrit has a linear relationship with that of total RBCs count, thus factors influencing the total RBCs count affect the Hct (%). The factors that affect total RBCs count, Hct and Hb also influence the MCV, MCH and MCHC (Hillman and Finch, 1985; Cella and Watson, 1991). The platelets count may alter with reference to pregnancy, trauma, fractures, severe liver disease, uremia, iron deficiency anemia, vitamin B12 deficiency, folate deficiency, hemolytic anemia and some drugs (Thompson and Harker, 1983), whilst, ESR may be elevated in toxemia of pregnancy, heart diseases, severe anemia, vitamin A toxicity and liver cirrhosis (Widmann, 1983). In the present study, it was also observed that fortified cookies resulted a marked increase in Hb level as compared to control. Similar to the results of present study, Semba et al. (1992) observed a significant increase in mean Hb level (2.1g/dL) by adding vitamin A. In Tanzania, a clinical study among anemic

school children indicated that daily provision of vitamin A increased mean Hb level up to 1.35g/dL after 3 months (Mwanri et al., 2000). In pregnant women vitamin A supplementation alone can increase Hb levels (Suharno et al., 1993). In case of West Java, Indonesia, pregnant women with anemia after vitamin A supplementation showed significant uplift in mean Hb level (Panth et al. 1990; Chawla and Puri 1995). In another study Muslimatun et al. (2001) reported that intake of vitamin A and iron simultaneously in women during pregnancy had a positive effects on hemoglobin than those received iron alone. A decrease in ferritin level in the women who received vitamin A and iron suggested that vitamin A improved the consumption of Fe for blood formation. Several clinical studies have focused on the effect of enhanced vitamin A status upon hemoglobin and anemia. Muhilal et al. (1988b) while investigating the role of vitamin A fortified MSG observed improvements in vitamin A level accompanied by dramatic changes in health and anthropometric characteristics. Linear correlation was observed between vitamin A status uplift and Hb level elevation. An increase in Hb status from 11.3 to 12.3g/dL in 5 months was noted while mean Hb level declined -0.2g/dL among control subjects. Likewise, Stuijvenberg et al. (1999) observed decline in prevalence of anemia (Hb <12.0g/dL) from 29.6 to 15.6% after provision of vitamin A fortified biscuits. Recently, Vinodkumar and Rajagopalan (2008) detected significant improvement in hemoglobin (0.55g/dL), hematocrit (0.01g/dL) and red cell count (0.470M/L) in experimental groups consuming micronutrients fortified salt, while a significant drop for this parameters was observed in the placebo. Varma et al. (2007) assessed the efficacy of a premix fortified khichdi, a rice and dal mixture with vitamin A and observed significant difference in hemoglobin concentration between fortified and nonfortified khichdi groups.

Anand et al. (2007) also stated vitamin A fortified candies could be effective in improving the hemoglobin level and decreasing anemia prevalence. Seal et al. (2008) assessed variations in iron and vitamin A levels of the residents of Nangweshi refugee camp after the utilization of maize meal fortification. During the intervention mean Hb improved in children (0.87g/dL) and adolescents (0.24g/dL). Vitamin A treatment enhanced mean corpuscular volume (P< 0.001) indicating improved iron-deficient erythropoiesis (Zimmerman et al., 2006). In another research study four groups of anemic children were supplemented for 2 months with vitamin A, Fe, vitamin A plus Fe and placebo. Vitamin A supplementation produced significant increases in serum levels of retinol, Fe, hemoglobin, hematocrit and erythrocyte count (Mejia and Chew, 1988). Vitamin A provision mobilizes iron from body reserves to support accelerated erythropoiesis (Panth et al., 1990; Chawla and Puri, 1995; Zimmermann et al., 2006). In the present research, fortification of cookies increased hemoglobin level, explicating that it has positive correlation with vitamin A provision. 4.5.3. White Blood Cells Indices Mean squares in Table 32 showed a significant effect of study period on total WBC of pregnant women while treatments remained unaffected. Furthermore, the treatments and trial period affected neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles, non-significantly. The total number of WBC decreased non-significantly in all the groups of pregnant women consuming retinyl acetate and retinyl palmitate fortified cookies. However, three months of study affected total WBC count significantly (Table 33). Total WBC count ranged from 8.64 to 10.36K/L. Mean total WBC count at initiation of the assessment was found to be 9.090.24 that was

significantly elevated to 9.880.34K/L at 90th day. It is obvious that treatments means varied non-significantly from each other as 9.970.19 for T1 (placebo), 9.430.35 for T4 (retinyl acetate) and 9.650.24K/L for T7 (retinyl palmitate) groups of pregnant women. It is clearly depicted from Table 34 that non-significant varying trend in neutrophiles (%) of pregnant women consuming fortified cookies was observed throughout the stated intervals. Data regarding neutrophiles enumerated that it ranged from 61.78 to 67.19%. The neutrophiles content varied non-significantly i.e. 64.881.40, 64.340.92, 63.120.34 and 65.440.39% at stated intervals, respectively. Intake of retinyl acetate and retinyl palmitate fortified cookies varied non-significantly; T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups represented their means neutrophiles values i.e. 63.970.88, 65.400.71 and 63.981.02%, respectively. It is evident from Table 35 that non-significant tendency in lymphocytes (%) was observed in blood of all pregnant women consuming retinyl acetate and retinyl palmitate fortified cookies. Non-significant mean values for lymphocytes (%) at stated intervals were found i.e. 27.401.82, 27.630.72, 28.720.33 and 26.230.69%, respectively. Retinyl acetate and retinyl palmitate fortified cookies consumption resulted in non-significant variation and values were 27.990.77, 26.540.75 and 27.961.45% for T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups of pregnant women, respectively. Mean values regarding treatments, months and their interaction were presented in Table 36; illuminated varying tendency in monocytes (%) in blood of pregnant women that ranged from 2.77 to 4.30%. Consumption of cookies fortified with vitamin A salts resulted in non-significant variations in monocytes (%) all over the study period. It is obvious from Table 37 that eosinophiles (%) were varied nonsignificantly from 1.99 to 3.29%. The treatments also differed non-significantly;

placebo, retinyl acetate and retinyl palmitate groups had 2.650.11, 2.830.18 and 2.530.23% eosinophiles, respectively. Study period changed eosinophiles from 2.780.24 to 2.790.16%. Data regarding treatments, months and their interactive effect are presented in Table 38, depicted varying behavior in basophiles content of pregnant women ranged from 0.04 to 0.07%. The highest percentage was recorded in fortified groups (0.060.005%) while the lowest in placebo (0.050.004%). Basophiles content at baseline was observed 0.060.005% that nonsignificantly reduced to 0.050.000% at the end of the study. Pregnancy, infections, anemia, parasitic infestation may increase level of total WBCs while severe infections, uremia and malnutrition may decrease it (Boggs and Winkelstein, 1983). Neutrophiles, lymphocytes, monocytes,

eosinophiles and basophiles status may vary from standard ranges in bacterial/acute viral infection, leukemia, tuberculosis, malaria, chronic

ulcerative colitis/enteritis, parasitic infestations, asthma, allergic reaction, uremia, metabolic disorders, starvation, malnutrition, iron deficiency anemia, vitamin B12 and folate deficiency. Leukocytosis, thrombocytopenia and pancytopenia may be a sign of infection, drug toxicity and low production from the bone marrow, respectively (Boggs and Winkelstein, 1983; Fischbach, 1988). In the present research investigation, results of WBCs chemistry are quite comparable with that of their normal values, indicating that fortified cookies consumed by pregnant women did not contribute toward any adverse effects. The present results are in accordance with Qazaq et al. (2005) who also observed that mean hematological indices were not significant in the vitamin A deficient (< 20g/dL) pregnant women. 4.5.4. Liver Function Tests Liver function tests (LFTs) including bilirubin total, bilirubin direct, bilirubin indirect, glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) are also determined because of their importance to assess the

normal functioning of physiological system. Means squares regarding bilirubin total, bilirubin direct, bilirubin indirect, GPT and ALP are presented in Table 39. Statistical results elucidated non-significant differences among treatments, months and their interactions on LFTs. Data regarding means for treatments, months and their interaction are presented in Table 40. Bilirubin-total ranged from 0.64 to 0.90mg/dL. The duration of three months study affected bilirubin total content non-significantly i.e. 0.710.05, 0.760.04, 0.760.08 and 0.670.02mg/dL at baseline and at the end of 7th, 8th and 9th month of pregnancy, respectively. The treatments behaved nonsignificantly too for same trait; the highest mean content (0.750.06 0.75mg/dL) was recorded in retinyl palmitate group and the lowest (0.70 0.03mg/dL) in retinyl acetate group. Means for treatments, months and their interaction are presented in Table 41. Bilirubin-direct ranged from 0.16 to 0.22 mg/dL. Non-significant variations in bilirubin-direct was observed in blood of pregnant women consuming fortified cookies throughout the study period. Bilirubin-direct values were 0.200.01, 0.190.02, 0.190.01 and 0.180.01mg/dL at baseline, 7th, 8th and 9th month of pregnancy, respectively. Retinyl acetate and retinyl palmitate fortified cookies consumption resulted in non-significant variations in bilirubindirect. Values recorded were 0.180.01, 0.190.01 and 0.200.01mg/dL for T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups of pregnant women, respectively. In the pregnant women, bilirubin-indirect was affected non-significantly as a function of treatments, months and their interaction. Bilirubin-indirect was found in the range of 0.44 to 0.71mg/dL (Table 42). Non-significant variations due to treatments were noted on this trait and group consuming retinyl acetate fortified cookies showed some what lower value i.e. 0.510.03mg/dL as compared to other treatments (0.550.06mg/dL). The mean values for bilirubin-

indirect were noted; 0.510.05, 0.560.02, 0.570.07 and 0.490.03mg/dL at stated intervals, respectively. Statistical results regarding glutamate pyruvate transaminase (GPT) are presented in Table 43. Non-significant changes in serum GPT were observed in blood of pregnant women consuming test diets. The means values for months also showed non-significant variations i.e. 26.391.18, 26.351.79, 23.503.12 and 26.451.88U/L at baseline, 7th, 8th and 9th month of pregnancy, respectively. Likewise, GPT values were 27.291.47, 26.481.76 and 23.251.47U/L for T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups, respectively. Means for alkaline phosphatase (ALP) are explicated in Table 44 that depicted non-significant variations among different treatments and months. However, values for ALP content were in the range of 178.77 to 236.70U/L. Treatments were statistically at par, nevertheless, there are slight nonmomentous increase in ALP level in retinyl acetate and retinyl palmitate group but was in the safer limit. During first two months of last trimester of pregnant women, ALP content decreased but their level restored at the end of 9th month. According to Widmann (1983), hemolysis, pernicious anemia, viral hepatitis, liver cirrhosis, biliary cirrhosis, gall bladder diseases and liver damage due to toxicity of drugs, heart disorders, alcohol and pregnancy may alter the values of LFTs. Vitamin A is stored in the liver cells in the form of retinyl esters and transported back to the blood through retinol binding proteins. These binding proteins decreased as a function of vitamin A deficiency. Excessive intake of vitamin A results in toxicity causing elevation in aspartate transaminase, alanine transaminase and glutamyl transpeptidase (Perrotta et al., 2003). However, in the present study, levels of these indicators remained in the normal ranges indicating the safety of fortified cookies.

4.5.5. Renal Function Tests Renal function tests include urea and creatinine. Statistical results indicate that there were non-significant differences due to treatments, months and their interaction on serum urea and creatinine level (Table 45). Means for urea of women consumed retinyl acetate and retinyl palmitate fortified cookies revealed that this parameter fall in the range of 28.27 to 35.68mg/dL (Table 46). Mean values for urea was noted at stated intervals of pregnancy i.e. 31.851.41, 33.660.83, 30.551.18 and 33.981.41mg/dL,

respectively. Nevertheless, T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups had mean values 32.851.65, 33.280.72 and 31.401.05mg/dL, respectively. Mean values for creatinine varied non-significantly; 0.720.05, 0.800.03, 0.760.04 and 0.800.04mg/dL at stated intervals of pregnancy, respectively. Non-significant mean values of 0.760.05, 0.740.03 and 0.810.02mg/dL were observed in T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) groups of pregnant women, respectively (Table 47). Cella and Watson (1991) described that congestive heart failure, shock, renal diseases, starvation, diabetes, pregnancy, antibiotics, sever liver disease, liver cirrhosis, deficit /excessive protein ingestion, hypertension, Na/K salts may affect the normal values of RFTs. 4.5.6. Lipid Profile Lipid profile includes cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL) and triglycerides. Mean squares (Table 48) explicated that lipid profile is not affected significantly as a function of treatments, months and their interaction. Data regarding means of treatments, months and their interactions are presented in the Table 49. Cholesterol ranged from 140.87 to 155.36mg/dL during the entire period of study. The lowest cholesterol level was observed in groups supplied with retinyl palmitate fortified cookies (145.101.78mg/dL) that

differed non-significantly from other treatments. Selected intervals during pregnancy showed non-momentous effect however, it is obvious from the results that cholesterol tends to increase during first two months of study and than reduced non-significantly during last month. Means regarding low density lipoproteins (LDL) are presented in Table 50 indicated LDL content from 79.00 to 91.53mg/dL. Treatments vary nonsignificantly from each other and maximum LDL content (87.101.59mg/dL) was observed in T1 (placebo) while retinyl palmitate group had minimum LDL content (80.240.57mg/dL). LDL content increased non-significantly in the first month of study and then decreased in the subsequent two months. Means for treatments, months and their interaction on HDL level of pregnant women are presented in Table 51. HDL remained in range of 42.28 to 49.63mg/dL. Treatments showed non-significant variations in HDL content as; 45.990.65 (placebo), 46.041.53 (retinyl acetate) and 47.890.55mg/dL (retinyl palmitate) groups of pregnant women. Statistical results regarding triglycerides are presented in Table 52. Triglycerides ranged from 77.63 to 94.88mg/dL. Treatments did not differ significantly from each other nevertheless, group consuming retinyl palmitate fortified cookies had the lowest level of triglycerides; 84.863.72 as compared to 86.072.09 in placebo and 86.661.89mg/dL in group consuming retinyl acetate fortified cookies. Familial hyperlipoproteinemia, atherosclerosis, hypertension, myocardial infarction, nephrosis, pregnancy, corticosteroids, androgens, vitamin A/D toxicity, liver diseases, pernicious anemia may alter normal serum cholesterol status. The variations in standards may be due to primary hyperlipoproteinemia, atherosclerosis, hypertension, myocardial infarction, liver cirrhosis, alcohol, steroids, niacin and ascorbic acid toxicity (Cella and Watson, 1991). Age, sex, race, certain diseases and pregnancy affects lipid profile and during pregnancy their level may also elevate (Chatterjea and Shinde, 2005). Cigarette smoking,

age, low HDL, hypertension, coronary heart disease, pregnancy, diabetes mellitus, hyperthyroidism, infection, inflammation and cirrhosis may affect optimal status of LDL. Alcohol consumption, chronic hepatitis, cirrhosis and hypo/hyperthyroidism may change standard values of lipid profile (Cella and Watson, 1991). The findings of present study reflected non-significant variations in lipid profile of pregnant women consuming retinyl acetate and retinyl palmitate fortified cookies, elucidating that salts of vitamin A did not contribute towards any alteration in the normal values of lipid profile. Provision of cookies fortified with retinyl acetate and retinyl palmitate to pregnant women during third trimester enhanced their serum retinol level. It has been recommended that to fulfill the gap in vulnerable segment, vitamin A should be given through foods as diet based therapies are gaining popularity nowadays. Daily intake of 5 cookies (10g each) fortified with vitamin A by pregnant women resulted in marked increase in serum retinol level from 16.91 to 20.04 and 16.79 to 20.41g/dL in retinyl acetate and retinyl palmitate groups, respectively whilst, placebo showed declining trend in serum retinol. Normal deliveries had also been observed in pregnant women using fortified cookies and their neonates were not underweight. Improvement in serum retinol level is not only the single benefit that can achieve through vitamin A fortification but also tends to improve the hemoglobin level. Vitamin A acts synergistically with different iron metabolic pathways and improves iron absorption within the body. In the present study, hemoglobin level increased from 9.34 to 9.78g/dL and 9.12 to 9.73g/dL in women consuming retinyl acetate and retinyl palmitate fortified cookies, respectively. No adverse consequences of fortification have been reported in all subjects, as hematological and serological parameters like lipid profile, liver and renal function tests were remained in their normal ranges, throughout the study period.

4.6. Efficacy Studies in Lactating Women

Research trails were continued in the respective groups of women (lactating mothers) after child birth. Fortified cookies were prepared separately, considering the RDA of lactating women i.e.1200g. The cookies were prepared in a similar manner that 5 cookies (50g) per day provided the stated RDA. Fortified cookies prepared from selected treatments; T4 (50% RDA; retinyl acetate), T7 (50% RDA; retinyl palmitate) and T1 (placebo) were provided to the respective groups for consumption on daily basis. Blood samples were collected on monthly basis and analyzed for specified blood and serum parameters to conclude the impact of vitamin A fortification in lactating mothers. The results obtained are discussed below. 4.6.1. Serum Retinol, Retinyl Esters and -Carotene Profile Mean squares pertaining to levels of retinol, retinyl esters and -carotene in serum of lactating women presented in Table 53 explicated that treatments affected all parameters significantly. Lactation period imparted differential impact on serum retinol level and remained non-significant for serum retinyl esters and -carotene level. Interaction was found to be non-significant exception exist only in the case of retinyl esters. Serum Retinol The means in Table 54 explored that serum retinol level of lactating women increased significantly during the study phase in all the treatments. The highest mean value of serum retinol (21.940.57g/dL) was observed in the lactating women provided cookies fortified with retinyl palmitate (T7) followed by retinyl acetate (T4) with mean serum retinol level of 21.200.42g/dL, however, values of retinol of both salts significantly differed from placebo (15.650.06g/dL). The mean serum retinol level at base line was 18.641.59g/dL that mounted to 20.242.27g/dL during three months (Table 54). Interaction of treatments and duration of study showed some varying trends as slight uplift in

serum retinol level from 15.47g/dL at initiation to 15.75g/dL at stated intervals in placebo group (T1). Retinyl acetate fortified cookies (T4) showed a significant boost in serum retinol level of lactating mothers over time from 20.04g/dL (baseline) to 21.93g/dL (end of 3 months) and similar trend was observed in retinyl palmitate group (T7) with mean value of 20.41g/dL to 23.03g/dL. It is obvious from Figure 7 that serum retinol increased significantly in all treatments however, percent increase was more pronounced in retinyl palmitate group (12.84%) at the end of study. Retinyl acetate group also followed similar trend with an increase of 9.43% while, in placebo, there was an increase in serum retinol level only by 1.81%. Results are quite interesting but cultural heritage of Pakistan and consumption of fortified cookies could be two possible reasons for improvement in vitamin A status in placebo and lactating women consuming retinyl acetate and retinyl palmitate fortified cookies. Here in sub-continent, there is tradition to give food rich in meat, butter, eggs and other nutrients as postpartum diets. Enrichment of diet with meat and dairy products render vitamin A in some higher quantities that could substantially increase or maintain the level. Blomhoff (1994) gave evidences in the support of fortification programs that it could substantially increased serum retinol level significantly. Results of present investigation are corroborated with the findings of Arroyave et al. (1981), Muhilal et al. (1988a), Dary et al. (1996), Solon et al. (2000), Layrisse et al. (2000), Stuijvenberg et al. (1999), Hyder et al. (2007), Vinodkumar and Rajagopalan (2008) and Seal et al. (2008), proved that serum retinol level was directly proportionate with vitamin A intake through fortification strategies. Sibeko et al. (2004) studied the relationship of additional quantity of vitamin A with that of serum retinol level and indicated; higher the consumption of fortified products or vitamin A rich foods, greater rise would be in serum retinol. Lactating women are more vulnerable to VAD and require additional

vitamin A due to dependence of infants. The deficiency in mothers may possibly be transferred in infants; raise the mortality due to susceptibility of babies to infectious diseases. In present study, it was observed that the newborn feeding on breast milk of those women consuming retinyl acetate and retinyl palmitate fortified cookies were in better health i.e. protected from diarrhea and chest infections. Serum Retinyl Esters and -Carotene Means regarding retinyl esters are presented in Table 55. The women fed on cookies containing retinyl acetate had the highest mean retinyl esters (0.540.01g/dL) while the lowest mean value (0.420.04g/dL) was observed in placebo. In placebo group (T1), the concentration of serum retinyl esters dropped from 0.51 to 0.32g/dL in three months. A non-significant rise in retinyl esters of the lactating women provided with retinyl acetate fortified cookies (T4) was found i.e. 0.54, 0.52, 0.55 and 0.56g/dL at baseline, 30, 60 and 90 days of study, respectively. Likewise, retinyl palmitate (T7) fortified cookies followed similar trend and status of retinyl esters in lactating women was 0.50g/dL at baseline to 0.52 at 1st, 0.53 at 2nd and 0.54g/dL after 3rd month of lactation. The level of serum retinyl esters decreased non-significantly in lactating women at stated intervals from 0.520.01 to 0.47 0.07g/dL. Percent change in retinyl esters level (Figure 8) illuminated that decline was more pronounced in placebo (37.25%) while groups of lactating women consuming retinyl acetate and retinyl palmitate fortified cookies showed an increase in retinyl esters level by 3.70 and 8.00%, respectively. The lactating women provided with retinyl acetate fortified cookies had the highest concentration (29.960.14g/dL) of serum -carotene followed by the groups consuming cookies fortified with retinyl palmitate and placebo with mean values of 29.670.13 and 21.760.29g/dL, respectively (Table 56). Serum -carotene status of lactating women declined with the passage of lactation period but the change was non significant. At baseline -carotene level was

27.502.65 that decreased to 26.732.87g/dL after three months. The diminishing trend in -carotene content was found in all the groups under study. The placebo (T1) group had 22.20g/dL mean -carotene content at baseline that reduced to 20.99g/dL after 90 days. Similarly, in the lactating mothers provided with retinyl acetate (T4) and retinyl palmitate (T7) fortified cookies, -carotene level reduced from 30.30 to 29.64 and 30.00 to 29.55g/dL respectively, during three months of lactation. Pictorial elaboration of percent decrease in -carotene (Figure 9) illustrated that groups of lactating women varied in their response and decrease was more pronounced in placebo (5.45%) as compared to percent decline of 2.18 and 1.50% in retinyl acetate and retinyl palmitate groups, respectively. 4.6.2. Red Blood Cells Parameters The treatments (fortified cookies) and the study period influenced total RBC count, hematocrit (Hct), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), significantly. However, the non-significant effect of treatments and duration was observed on the levels of platelets count and erythrocytes sedimentation rate (ESR). The interactions remained non-significant for all traits as indicated in the mean squares given in Table 57. Total Red Blood Cells Count The lactating mothers provided cookies fortified with retinyl palmitate (T7) had the highest count of total red blood cells (4.900.04M/L) followed by the retinyl acetate (T4) group with total cells count of 4.830.05M/L. The women consuming unfortified cookies (placebo) were found to have the lowest cells count (4.710.05M/L). During lactation period of three months, the mean red blood cells count significantly increased from 4.700.06 to 4.920.05M/L. The escalating drift of total red blood cells was observed in all the groups of women during lactation (Table 58).

Hematocrit Means presented in Table 59 revealed that fortification significantly influenced the hematocrit (%) with the highest concentration (30.090.74%) in women provided cookies fortified with retinyl palmitate followed by those fortified with retinyl acetate (29.930.72%) and placebo (26.170.71%) groups. At baseline, the mean hematocrit was 27.101.25% which significantly increased to 30.571.27% during lactation. The rising trend was found in all the groups however, lactating women provided with fortified cookies showed more escalate in hematocrit content as compared to unfortified cookies. Hemoglobin Means presented in Table 60 elucidated that hemoglobin (Hb) level was significantly influenced by the provision of fortified cookies. The lactating mothers provided with retinyl palmitate fortified cookies had the highest mean Hb level (10.210.18g/dL) followed by groups utilizing the cookies fortified with retinyl acetate and placebo with mean values of 10.190.15 and 8.210.11g/dL, respectively. During the lactation period of three months, a significant rise in Hb level was observed in all the groups of women. The mean Hb level at baseline was 9.140.62 which increased to 9.820.71g/dL after three month of lactation. Percent increase in hemoglobin content is presented in Figure 10. It is obvious from the figure that said trait increased in all lactating women however, retinyl acetate and retinyl palmitate groups showed higher increase i.e. 7.26 and 8.74%, respectively, as compared to placebo (6.33%). Mean Corpuscular Hemoglobin Mean corpuscular hemoglobin (MCH) level was significantly affected by the consumption of fortified cookies. The mothers provided retinyl palmitate fortified cookies had the highest mean MCH status (26.232.23pg) followed by those utilizing the cookies fortified with retinyl acetate (25.652.15pg). The lowest level of MCH (21.061.25pg) was found in the women consuming

unfortified cookies (placebo). At initiation, mean MCH level was 20.190.99 that significantly increased to 28.702.39pg after three months of lactation (Table 61). Mean Corpuscular Volume Provision of fortified cookies during lactation significantly affected the mean corpuscular volume (MCV) of women. The mothers provided with fortified cookies had significantly higher MCV as compared to unfortified cookies. However, both the fortificants i.e. retinyl palmitate and retinyl acetate differed non-significantly with mean values of 73.151.52 and 72.211.52fL, respectively. During lactation period, a significant increase in MCV content; initially observed to be 66.342.74 and 73.062.82fL after three months of lactation (Table 62). Mean Corpuscular Hemoglobin Concentration Mean corpuscular hemoglobin concentration (MCHC) of lactating women was significantly affected by the fortified cookies. MCHC was momentously higher in mothers provided with retinyl palmitate and retinyl acetate fortified cookies with mean values of 31.450.86 and 31.300.70g/dL, respectively than that of placebo i.e. 27.290.76g/dL (Table 63). The MCHC increased significantly during study period from 28.201.33 at baseline to 31.791.37g/dL after three months. Platelets Count The platelets count of lactating women under study was non-significantly affected by treatments with mean values of 317.7518.01K/L in placebo group, 299.356.19 in retinyl acetate and 319.8218.05K/L in retinyl palmitate groups. During three months of lactation period, non-significant changes were observed in platelets count (Table 64). Erythrocytes Sedimentation Rate Means for erythrocytes sedimentation rate (ESR) as given in Table 65 showed non-significant variations in groups of lactating mothers during the whole span of study. The mean ESR values were 27.320.10, 26.320.24,

26.201.54 and 25.890.31mm/Hr at base line, 1st, 2nd and 3rd month of lactation, respectively. Muhilal et al. (1988b), Panth et al. (1990), Semba et al. (1992), Suharno et al. (1993), Chawla and Puri (1995), Mwanri et al. (2000) and Semba and Bloem (2002), investigated similar aspects related to vitamin A and red blood cells correlations and their outcomes are corroborated with the present findings. 4.6.3. White Blood Cells Indices A non-significant effect of lactation period and fortified cookies was found in total WBC, neutrophiles, monocytes, eosinophiles and basophiles. Only lymphocytes status of lactating mothers was significantly affected by the fortified cookies as indicated by the mean squares in Table 66. The number of total WBC was affected non-significantly by the fortified cookies with mean values of 10.280.15, 9.580.12 and 9.990.22K/L in different lactating mothers (Table 67). During lactation, a non-significant alteration in WBC concentration was found. Non-significant variation in neutrophiles content was found in blood of the lactating mothers provided with fortified and unfortified cookies during lactation (Table 68). The mean values for neutrophiles (%) at baseline, 1st, 2nd and 3rd month of lactation were 65.430.76, 65.980.82, 65.241.13 and 64.740.42%, respectively while T1, T4 and T7 had mean values 63.920.43, 65.580.50 and 66.550.61%, respectively that differed non-significantly from each other. Lymphocytes content of lactating mothers was significantly affected by fortified cookies with mean values of 28.530.77% in placebo group followed by 26.190.28 and 25.900.79% in retinyl acetate and retinyl palmitate groups, respectively. During lactation period, a non-significant change was observed in lymphocytes content (Table 69). Non-significant change was observed in monocytes content in lactating women provided with the fortified cookies (Table 70). It is apparent from Table

71 that spiky but non-significant trend in eosinophiles was observed in blood of lactating women after utilizing the fortified and unfortified cookies for a period of three months. Likewise, basophiles (%) were non-significantly affected during the lactation period of three months as well as provision of cookies also influenced non-significantly on this trait. Mean values for basophiles at baseline, 1st, 2nd and 3rd month of lactation was 0.050.003, 0.060.002, 0.060.005 and 0.070.003%, respectively (Table 72). The causes of variations in white blood cells were described by Boggs and Winkelstein (1983) and Fischbach (1988). 4.6.4. Liver Function Tests Mean squares elaborated in Table 73 depicted that there exists nonsignificant variations of treatments, months and their interaction on bilirubintotal, bilirubin-direct, bilirubin-indirect, glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP). Non-significant differences in bilirubin-total were observed in blood of lactating women consuming cookies i.e. T1, T4 and T7 in three months (Table 74). Mean values for bilirubin-total was noted at baseline, 1st , 2nd and 3rd month of lactation as 0.670.02, 0.730.07, 0.720.04 and 0.700.02mg/dL, respectively while T1, T4 and T7 group had mean values 0.720.04, 0.740.02 and

0.670.0mg/dL, correspondingly. Non-momentous variations in bilirubin-direct were examined in blood of all lactating women consuming cookies i.e. T1, T4 and T7 for 90 days as given in Table 75. Mean values for bilirubin-direct in lactating mothers were noted at stated intervals of lactation 0.180.01, 0.170.00, 0.200.01 and 0.190.01mg/dL while T1, T4 and T7 groups had mean values 0.180.01, 0.190.01 and 0.190.01 mg/dL, respectively. Varying tendency in bilirubin-indirect was observed in the blood of lactating women consuming the selected treatments of cookies for three months

(Table 76). The mean values for bilirubin-indirect was noted at stated intervals of lactation 0.490.03, 0.560.07, 0.520.04 and 0.510.02mg/dL, correspondingly while T1, T4 and T7 groups had mean values 0.530.04, 0.550.02 and 0.480.04 mg/dL, respectively. Statistical results regarding glutamate pyruvate transaminase (GPT) are presented in Table 77 that revealed that month of lactation differ nonsignificantly with mean values of 26.451.88, 27.181.62, 27.140.68 and 25.262.12U/L at baseline, 1st, 2nd and 3rd month of lactation, respectively.

Treatments with added fortificants showed non-significant value for GPT. The level of alkaline phosphatase (ALP) in serum is an indicator to determine toxicity limit and its values in the present studies ranged from 179.41 to 243.82U/L. Non-significant tendency in ALP was observed in blood of all the lactating women who consumed cookies prepared from selected treatments (Table 78). The mean values for ALP was noted at baseline, 1st , 2nd and 3rd month of lactation as 215.624.47, 234.322.91, 204.368.49 and 206.7319.22U/L, respectively while T1, T4 and T7 groups had mean values 210.149.10 , 227.585.91 and 208.0613.39U/L, respectively. Widmann (1983) pointed out factors affecting variation in liver profile. 4.6.5. Renal Function Tests Data pertaining to means squares regarding urea and creatinine are shown in Table 79. Treatments and their interactive effects with months showed non-significant expression on both parameters while months significantly affected serum urea level and remained non-significant for creatinine. Means regarding urea are presented in Table 80. Urea content remained in the range of 25.0 to 36.36mg/dL. Non-significant varying trend in urea level was found in blood of lactating mothers. However, significant changes in mean values of urea content were observed i.e. 33.981.41 and 34.211.17mg/dL at

baseline and after one month respectively, while in the last month of said study the lowest value for urea content (29.0902.19mg/dL) was recorded. Creatinine values changed non-significantly as a function of treatments, months and their interaction. Non-significant inclination in creatinine was observed in T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) during three months of study plan (Table 81). 4.6.6. Lipid Profile Mean squares in Table 82 illustrated that non-significant differences exist in lipid profile of lactating mothers due to treatments and period of lactation. Means values for cholesterol content fall in the range of 144.45 to 161.84 mg/dL (Table 83). Lactating women, consuming retinyl acetate fortified cookies possessed lower cholesterol content as compared to T1 (placebo) and T7 (retinyl palmitate) groups, however, they differed non-significantly from each other. Likewise, non-significant expression of months was also observed for this parameter. Data regarding low density lipoprotein of lactating women presented in Table 84 showed non-momentous variations ranged from 79.76 to 91.06mg/dL. Varying tendency in LDL level was observed in the lactating women by using T1 (placebo), T4 (retinyl acetate) and T7 (retinyl palmitate) in three months with pooled mean of 85.071.53, 83.962.02 and 86.282.62mg/dL, respectively. Mean values for LDL were noted at stated intervals; 84.911.61, 88.980.79, 83.983.56 and 82.530.99mg/dL. High density lipoprotein of lactating women also varied non-significantly with treatments and months. Intake of cookies containing retinyl palmitate increased HDL non-significantly as compared with that of control and retinyl acetate group. Moreover, months differed non-significantly as revealed from their mean values of 46.961.02, 49.612.00, 48.410.40 and 47.361.56mg/dL for baseline, 1st, 2nd and 3rd month of lactation, respectively (Table 85).

Levels of triglycerides varied non-significantly as a function of treatment and lactation months. Treatments did not differ significantly from each other however, group consuming retinyl palmitate fortified cookies had slight higher level of triglycerides (92.461.42mg/dL) as compared to 90.882.08mg/dL in placebo and 90.791.13mg/dL in group of lactating women consuming retinyl acetate fortified cookies. Non-significant trend was observed as a function of months (Table 86). Results of present study indicated that consumption of retinyl acetate and retinyl palmitate fortified cookies during entire study period did not produce any adverse effect on lipid profile of the subjects under study. In lactating women demands for the micronutrients including vitamin A are always higher than that of normal population because mothers have to nourish their neonates. Food based strategies can play imperative role in meeting demands of mothers and newly born too. Intake of five cookies (50g) per day by lactating women resulted in marked increase in serum retinol level from 20.04 to 21.93 and 20.41 to 23.03g/dL in retinyl acetate and retinyl palmitate groups, respectively. It is obvious from the present exploration that most of the women consumed fortified cookies were successful to get rid of vitamin A deficiency. Vitamin A is pivotal in folate absorption that is well dignified by increased hemoglobin level from 9.78 to 10.49g/dL and 9.73 to 10.58g/dL in lactating mothers consuming retinyl acetate and retinyl palmitate fortified cookies, respectively. Similar to pregnant women, values for lipid profile, liver & kidney functioning and hematological & serological aspects remained in the normal ranges. The findings of the present investigation revealed that the cookies fortified with retinyl acetate and palmitate had potential to uplift serum vitamin A level in vulnerable segments with special reference to pregnant and lactating women.

Comparative outcomes of vitamin A fortification in pregnant and lactating women

Comparison has been made between pregnant and lactating women for serum retinol, retinyl esters and -carotene levels and their impact on hemoglobin. Varying results were documented during pregnancy and lactation. Serum retinol varied significantly in pregnant and lactating women as a function of treatments. However, entire study plan of six months (Figure 11) depicted that during first month of interventions (end of the 7th month), serum retinol level mounted to 9.88 and 11.55% in pregnant women consuming retinyl acetate and retinyl palmitate fortified cookies, respectively. At the end of first phase (pregnancy period); provision of 50% RDA through retinyl acetate and retinyl palmitate fortified cookies enhanced serum retinol level 18.51 and 21.56%, respectively. Likewise, in lactating women, serum retinol level increased progressively in the subsequent months. During lactation, 9.43 and 12.84% increase in retinol concentrations was observed for selected treatments of fortified cookies. Significant differences were observed in placebo group during pregnancy and lactation periods, as in last trimester (pregnancy), retinol content declined continuously but afterwards there was a slight increasing trend during lactation however, 7.68% decline was observed in placebo during six months. Overall, increase of 29.69 and 37.16% in serum retinol level was recorded in groups of women consuming retinyl acetate and retinyl palmitate fortified cookies, respectively in the entire period. Retinyl esters varied momentously in pregnant and lactating women during the study periods. In pregnancy, retinyl esters decreased with the passage of time during last trimester but increased slightly during lactation except for placebo, owing to increasing serum retinol level in subjects postpartum. Percent decrease in retinyl esters 37.93 and 36.71% was observed in pregnant women consuming retinyl acetate and retinyl palmitate fortified cookies, respectively, while during lactation, an increase of 3.70 and 8.00% was recorded due to 196

consumption of selected treatments, respectively. In placebo group, decline in retinyl esters showed varied response i.e. 43.42 and 37.21% during pregnancy and lactation, respectively. Collectively, in both pregnant and lactating periods, decrease in retinyl esters 35.63 and 31.65% was observed in groups consuming retinyl acetate and retinyl palmitate fortified cookies, respectively as compared to placebo i.e. 64.84% (Figure 12). Level of -carotene declined in all treatments in both studies; pregnancy and lactation. However, decline was more pronounced in last trimester of pregnancy i.e. 6.13 and 5.57% in retinyl acetate and retinyl palmitate fortified cookies consuming groups, respectively. Later, during lactation same groups showed slight decline of 2.18 and 1.50% in -carotene. In placebo group, significant decrease in -carotene was observed during pregnancy (32.79%) as compared to lactation (5.45%). Overall, T1 (placebo) differed entirely with percent decrease of 36.45% in -carotene as compared to groups receiving retinyl acetate and retinyl palmitate fortified cookies i.e. 8.18 and 6.99%, respectively (Figure 13). Metabolic fate of vitamin A, retinyl esters and -carotene could explain the decreasing trend, as during vitamin A deficiency, retinyl esters and -carotene convert into retinol for maintaining its level. When retinol exceeds the upper limit of the normal range, it is stored in the form of retinyl esters. It was also observed that retinyl palmitate showed better performance to uplift the serum retinol level than that of retinyl acetate thus it can preferably be recommended for use as fortificant in food formulations. Supplementing childbearing women with vitamin A not only eliminates its deficiency, but also helps to reduce anemia. It releases the iron from its reservoirs which would increase its availability for hematopoiesis (Mejia and Arroyave, 1982). Wheat is the staple diet of Pakistan and people rely on cereal based products like chapatti, bread and cookies etc. Emphasis has been given to supplementation of staples (Steyn et al., 2008). In the present research, consumption of retinyl acetate and retinyl palmitate fortified cookies in pregnant and lactating women would also be

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beneficial for the fetus and infants. Efforts were also made during the study to evaluate the vitamin A status of newly borns but mothers and their families were reluctant to provide their blood samples. However, the neonates were under observation for three months of lactation and found to be less prone to diarrhea and chest infection; fed on mothers consuming fortified cookies. Several other studies supported present findings like Muhilal et al. (1988a), Vinodkumar and Rajagopalan (2008) and Seal et al. (2008), they all observed that fortification of staples enhanced the serum retinol level. Serological parameters regarding safety evaluation of cookies remained non-significant in both pregnant and lactating women clearly indicated that fortification of cookies is suitable and applicable in overcoming vitamin A deficiency in populations at risk. Interestingly, increase in hemoglobin level was also observed during the study. Hemoglobin level was linearly correlated with serum retinol level; increased from 9.34 to 9.78g/dL and 9.12 to 9.73g/dL in pregnancy while during lactation, it tends to increase from 9.78 to 10.49g/dL and 9.73 to 10.58g/dL in selected groups consuming retinyl acetate and retinyl palmitate fortified cookies, respectively. Percent increase in hemoglobin content was 4.71 and 6.69% in last trimester while 7.26 and 8.74% during three months of lactation in retinyl acetate and retinyl palmitate groups, respectively. Overall, during the whole period of six months, level of hemoglobin increased by 12.31 and 16.01% in group of women consuming retinyl acetate and retinyl palmitate as compared to 6.15% decline in placebo group (Figure 14). The increase in hemoglobin could possibly be due to increasing level of serum retinol thus improving immunity via modulation of immune system. Ameliorating infections could be another possible justification for increased level of hemoglobin in the body (Zimmermann et al., 2006). The improvement in hemoglobin levels with the provision of vitamin A in present exploration indicates prospects for controlling anemia which is, indeed, affecting majority of pregnant and lactating women of the region.

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Correlation coefficients among different variables derived from efficacy studies have been presented in Table 87. Serum retinol was positively correlated (P<0.01) with hemoglobin, red blood cells, mean corpuscular volume and mean corpuscular hemoglobin concentration. In addition, significant positive

correlation was also observed in hematocrit and mean corpuscular hemoglobin with retinol (P<0.05). Serum retinyl esters were negatively correlated with retinol but positive correlation was observed with other parameters like hematocrit, red blood cells, mean corpuscular hemoglobin concentration (P<0.05), mean corpuscular volume and mean corpuscular hemoglobin (P<0.05). However, retinyl esters were negatively correlated (P<0.05) with white blood cells. -carotene level was linearly correlated (P<0.01) with hemoglobin, red blood cells, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration while non-significantly correlated with white blood cells (P<0.01). Similarly, hemoglobin was significantly correlated with Hct, RBC, MCV, MCH and MCHC. Hemoglobin and serum retinol level had been well correlated in number of clinical studies. The data from surveys accomplished in Vietnam, Chile, Brazil, Venezuela and Ethiopia also supported present findings and these studies collectively exhibited a high correlation (r = 0.77, P <0.0001) between hemoglobin and plasma retinol concentration (Hodges et al, 1978). Molla et al. (1993b) also observed correlation co-efficient of r=0.367 (P<0.0001) between hemoglobin and retinol level in Pakistani population. Bloem et al. (1989) recorded that serum retinol was significantly associated with hematocrit (r=0.062, P<0.05).

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The outcomes of the present exploration revealed that the cookies fortified with retinyl acetate and retinyl palmitate had great potential to uplift serum vitamin A level with allied health benefits for the vulnerable segments with special reference to pregnant and lactating women. Vitamin A fortified cookies not only eliminated vitamin A deficiency but were also helpful for hematopoiesis that consequently reduced the anemia. No adverse effects of fortification were observed in the subjects as hematological and serological parameters were reported within normal ranges throughout the study period. The safer aspects and simultaneous boost up in vitamin A level through fortified cookies brighten the future prospects of vitamin A fortification to alleviate its deficiency in communities at risk.

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CHAPTER 5

SUMMARY
Vitamin A deficiency affects millions of people all over the globe and poses serious threat to communities in the developing world. Present project was an attempt to alleviate the vitamin A deficiency (VAD) in pregnant and lactating women of Pakistan. The dietary requirements of pregnant and lactating women are higher due to their metabolic demands and dependence of fetus and neonates on them. Efficacy studies of vitamin A fortified cookies in pregnant and lactating women were conducted with cooperation of Population Welfare Department, Ministry of Population Welfare, Pakistan. For the purpose, nine Population Welfare Centers of Faisalabad district were engaged in the study. Initially, 513 pregnant women were approached, 230 were agreed to take part in the research project. Anthropometric measurements were recorded along with specified hematological and serological parameters; 89 women were found to be vitamin A deficient. After screening for hepatitis virus, 14 subjects were further excluded. Finally, 75 women were selected for six month studies comprising 7th, 8th and 9th month of pregnancy and then succeeding 1st, 2nd and 3rd month of lactation. The women were assorted randomly into three groups, 25 in each, namely placebo (unfortified cookies), RA (retinyl acetate fortified cookies) and RP (retinyl palmitate fortified cookies) groups. Selected women in the respective groups were provided five cookies (50g) daily, fortified with retinyl acetate and retinyl palmitate along with placebo. The conclusive approach drawn from the present investigations is summarized below:

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In the product development phase, cookies were fortified with retinyl acetate and retinyl palmitate separately @ 30, 40 and 50% RDA of pregnant (750g) and lactating (1200g) women. Statistical analysis revealed that varying levels of fortificants and storage intervals exhibited non-significant differences on the physical and chemical parameters of fortified cookies with the exception of moisture and TBA no. that were increased from 2.51 to 2.84% and 0.40 to 0.69mg malenaldehyde/Kg, respectively, during 90 days storage. Baking and storage stability of vitamin A fortificants was also assessed. Vitamin A losses during baking ranged from 7.95 and 10.20% in retinyl acetate and retinyl palmitate fortified cookies, respectively, as compared to 15.79% in placebo. Storage periods also affected vitamin A content significantly and declining trend was observed from 9.05g/100g at the initiation of study to 8.26g/100g after 3 months. Storage losses ranged from 8.02 to 9.69% among the treatments. However, overall storage loss was recorded to be 8.71%. Mean squares regarding sensory scores depicted non-significant

differences as a function of fortificants while storage significantly affected the sensory traits. The overall acceptability scores of cookies decreased from 7.72 to 6.19 during storage. In present study, levels of fortificants did not indicate any detrimental changes in physico-chemical and sensoric attributes of fortified cookies. On the basis of physico-chemical analysis, baking & storage stability and sensoric attributes, two best treatments one from each fortificant i.e. T4 (50% of RDA; retinyl acetate) and T7 (50% of RDA; retinyl palmitate) were selected for efficacy purposes. Cookies prepared form selected treatments along with placebo were provided to respective groups of vitamin A deficient pregnant women in third trimester. Research trials were continued in the same groups (lactating mothers) after child birth considering their RDA requirements.

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Varying results were documented during pregnancy and lactation. Serum retinol level differed significantly in pregnant and lactating women as a function of treatments, months and their interaction. Consumption of retinyl acetate and retinyl palmitate fortified cookies during pregnancy resulted a significant improvement in serum retinol level i.e. 16.91 to 20.04g/dL and 16.79 to 20.41g/dL, respectively; whilst, it was decreased from 17.06 to 15.47g/dL in placebo group. During lactation, there was a significant boost in serum retinol level from 20.04 to 21.93g/dL and 20.41 to 23.03g/dL in retinyl acetate and retinyl palmitate groups, respectively. Provision of retinyl acetate and retinyl palmitate fortified cookies (50% RDA) enhanced serum retinol level in pregnant (18.51 and 21.56%) and lactating women (9.43 and 12.84%) of respective groups. Overall, an increase of 29.69% (retinyl acetate) and 37.16% (retinyl palmitate) in serum retinol level was recorded in women consuming fortified cookies during the entire study period, however momentous decrease of 7.68% in placebo was observed. Mean squares for retinyl esters revealed that months showed significant impact in pregnant women while in lactating women treatments and their interaction indicated a momentous increase for this trait. Retinyl esters decreased with the passage of time during last trimester; 37.93 and 36.71% decline was observed in pregnant women consuming retinyl acetate and palmitate fortified cookies, respectively. However, during lactation, there was significant improvement in retinyl esters level i.e. 3.70 and 8.00% in women consuming retinyl acetate and retinyl palmitate fortified cookies, respectively. In placebo group, decline in retinyl esters showed varied response i.e. 43.42 and 37.21% during pregnancy and lactation, respectively. Collectively, during pregnancy and lactation, 35.90 and 32.00% decrease in serum retinyl ester level was observed in groups consuming retinyl acetate and palmitate fortified cookies as compared to placebo i.e. 64.84%.

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For -carotene, during pregnancy, 6.13 and 5.56% decrease was observed in retinyl acetate and retinyl palmitate fortified groups, respectively. Likewise, same groups showed a slight decline in -carotene (2.18 and 1.51%) during lactation. Overall, T1 (placebo) differed entirely from other groups with 36.45% decrease as compared to groups consuming retinyl acetate (8.18%) and palmitate (6.98%) fortified cookies. Red blood cells (RBC) indices like total RBC, hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) decreased in experimental groups during pregnancy; however, the placebo group exhibited more decline as compared to groups consuming fortified cookies. There was significant increase in hemoglobin (Hb) level of retinyl acetate and retinyl palmitate groups; nevertheless, declining tendency was observed in placebo group during pregnancy. Interestingly, Hb level was increased from 9.34 to 9.78 g/dL and 9.12 to 9.73g/dL in pregnant women and 9.78 to 10.49 and 9.73 to 10.58g/dL in the lactating mothers in selected groups consuming fortified cookies i.e. T4 (50% of RDA; retinyl acetate) and T7 (50% of RDA; retinyl palmitate), respectively. The increase in hemoglobin content was 4.71 and 6.69% in last trimester while 7.26 and 8.74% during three months lactation in retinyl acetate and retinyl palmitate groups, respectively. Overall, during six months, there were 12.31 and 16.01% increase in hemoglobin level in groups consuming retinyl acetate and palmitate, respectively, while 6.15% decrease was observed in placebo. The improvements in hemoglobin status with present intervention also enlighten the role of vitamin A to control anemia related problems in pregnant and lactating women. Retinyl palmitate exhibited better performance than that of retinyl acetate in uplifting the serum retinol level and had positive effect on blood cell indices

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during pregnancy and lactation. Moreover, it was observed that women consuming fortified cookies gave normal birth and their neonates were in better health. Regarding liver function tests, the levels of bilirubin total, bilirubin direct, bilirubin indirect, glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were not affected significantly with treatments and duration. There were non-significant differences due to treatments, months and their interaction on renal function tests including urea and creatinine. Moreover, lipid profile was also affected non-significantly as a function of treatments, months and their interaction. Parameters regarding safety evaluation remained non-significant in both pregnant and lactating women, which clearly indicate that fortification of cookies is applicable in overcoming vitamin A deficiency in vulnerable segments. Correlation coefficients studies highlighted the linear relationship between hematological parameters with serum retinol level i.e. hemoglobin (r = 0.95), RBC (r = 0.68), MCV (r = 0.67) and MCH (r = 0.53). Likewise, -carotene level had positive correlation with all hematological attributes. Furthermore, a significant positive relationship (r = 0.76, P<0.01) prevailed between serum carotene and retinyl esters level. The upshots of the present exploration revealed that the cookies fortified with retinyl acetate and retinyl palmitate had potential to uplift serum vitamin A level in vulnerable segments with special reference to pregnant and lactating women. The rise in serum vitamin A levels through fortified cookies brightens the prospects of vitamin A fortification to combat its deficiency in the populations at risk.

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RECOMMENDATIONS
Vitamin A fortification of wheat based dietary staples should be launched at national level through government and non-government organizations to alleviate the vitamin A deficiency in masses. Relationship between vitamin A and folate should be investigated through multiple fortification approaches. Efficacy studies should be extended to infants of lactating mothers to investigate the uplift in serum vitamin A and immunity against infections. Vitamin A fortification should be carried out in combination with other micronutrients like iron to explore the synergistic effects. Fortified/supplemented diet during the course of pregnancy and lactation should be advised for mother and child care. Nutrition education should be encouraged in the vulnerable segments and stakeholders to create awareness and better understanding of the deficiency and allied consequences.

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Appendix-I

SENSORY EVALUATION OF COOKIES

Storage time__________

Date______________ To T1 T2

Characteristics / Treatments Color Flavor Taste Crispness Texture Overall all acceptability

Signature_____________ Instructions Chew a sample of cookies and sore for color, flavor, taste, crispness, texture and overall acceptability using the following scale. Extremely poor 1 Very poor 2 Poor 3 Below fair above poor 4 Fair 5 Below good above fair 6 Good 7 Very good 8 Excellent 9

Note 1. Chew a sample of cookies and score for color, flavor etc. 2. Before preceding the next sample, rinse mouth with water. 3. Make inter comparison of the sample and record the score. 4. Don't disturb the order of the samples. 196

Appendix-II

POPULATION WELFARE CENTRE ENGAGED IN THE STUDY

1. Population Welfare Center, House No. 1010, Lane No. 7, Mahdi Muhala, Nishatabad, Faisalabad. 2. Population Welfare Centre, House No. P-1011, Lane No. 27, Abdullah Pur, Faisalabad. 3. Population Welfare Centre, House No. P-49, Lane No. 3, Nisar Colony, Near Samanabad, Faisalabad. 4. Population Welfare Center, House No. P-1139/527-A, Sir Syed Town, Dijkot Road, Faisalabad. 5. Population Welfare Center, House No. P-25/D-1, Near MPA House, Sargodha Road, Faisalabad. 6. Population Welfare Center, Chak No. 263 R.B., Dijkot Samundri Road, Faisalabad. 7. Population Welfare Center, Chak No. 232 R.B., Risalaewala, Tehsil Sadar, Faisalabad. 8. Population Welfare Center, Chak No. 248 R.B., Dalowal, Samundri Road, Faisalabad. 9. Population Welfare Center, Chak No. 67 J.B., Sidhar Jhang Road, Faisalabad.

Appendix-III

DATA COLLECTION FORM

Name ____________________ W/O __________________ Age ______

Education ____________ Occupation/House Wife _______________________ Duration of your marriage ______________ Number of children ___________ Last Menstruation Date ______________ Month of Pregnancy ____________ Expected Delivery Date ____________ Is there any complication or disease during pregnancy? __________________ Delivery: Normal/Operation

How is your childrens health? ______________________________________ How many times you eat meat, fruits and milk in a week? _________________

Name of the Field staff

__________________________________________

Education and Designation __________________________________________ Catchments Area No. of Targets __________________________________________ __________________________________________

Signature: _______________________

Dated : _______________________

UNIVERSITY OF AGRICULTURE, FAISALABAD

National Institute of Food Science and Technology
Name of the Project

Efficacy Studies of Vitamin A Fortified Cookies in Pregnant and Lactating Women

I have been explained in detail the purpose and rationale of the above mentioned component of the Vitamin A Fortified Cookies Project. I understand that this project is of national significance and my full commitment and dedication with it will be of paramount importance, I am volunteering for it. I have had a chance to ask questions and answered them. I undertake that I will abide by all the instructions given by the investigators and will use the same Vitamin A Fortified Cookies given to me in the designated period. Further, I am bound to fill the questionnaire at the end of the week to best of my knowledge.

Name & Signature of the Subject

Dated

Name & Signature of the Person obtaining consent

Dated

Name & Signature of the Researcher

Dated

Name & Signature of the Principal Investigator

Dated

Appendix-V
UNIVERSITY OF AGRICULTURE, FAISALABAD National Institute of Food Science and Technology

Project:

Efficacy studies of vitamin a fortified cookies in pregnant and lactating women

Questionnaire
Name________________ w/o ________________ Age______________ Phone____________ Address_________________________________________

How many times and how much quantity of the following commodities is utilized per week?

Commodity
1. Milk and Milk Products i. Milk ii. Lassi iii. Dehi iv. Pannier v. Makhan vi. Desi Ghee vii. Others 2. Meat and Meat products i. Meat ii. Liver iii. Sari payee iv. Others 3. Vegetables (Raw and cooked) i. Carrot ii. Salad iii. Green leafy vegetables iv. Others
4. Fruits i. ii. iii. iv. v. Mangoes Apple Banana Apricot Others

No. of Times
1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7

Quantity

5. Any Medicine Name of medicines:

Does it contain vitamin A

Yes or No

Appendix-II

POPULATION WELFARE CENTRE ENGAGED IN THE STUDY

10. Population Welfare Center, House No. 1010, Lane No. 7, Mahdi Muhala, Nishatabad, Faisalabad. 11. Population Welfare Centre, House No. P-1011, Lane No. 27, Abdullah Pur, Faisalabad. 12. Population Welfare Centre, House No. P-49, Lane No. 3, Nisar Colony, Near Samanabad, Faisalabad. 13. Population Welfare Center, House No. P-1139/527-A, Sir Syed Town, Dijkot Road, Faisalabad. 14. Population Welfare Center, House No. P-25/D-1, Near MPA House, Sargodha Road, Faisalabad. 15. Population Welfare Center, Chak No. 263 R.B., Dijkot Samundri Road, Faisalabad. 16. Population Welfare Center, Chak No. 232 R.B., Risalaewala, Tehsil Sadar, Faisalabad. 17. Population Welfare Center, Chak No. 248 R.B., Dalowal, Samundri Road, Faisalabad. 18. Population Welfare Center, Chak No. 67 J.B., Sidhar Jhang Road, Faisalabad.

Appendix-III

DATA COLLECTION FORM

Name ____________________ W/O __________________ Age ______

Education ____________ Occupation/House Wife _______________________ Duration of your marriage ______________ Number of children ___________ Last Menstruation Date ______________ Month of Pregnancy ____________ Expected Delivery Date ____________ Is there any complication or disease during pregnancy? __________________ Delivery: Normal/Operation

How is your childrens health? ______________________________________ How many times you eat meat, fruits and milk in a week? _________________

Name of the Field staff

__________________________________________

Education and Designation __________________________________________ Catchments Area No. of Targets __________________________________________ __________________________________________

Signature: _______________________

Dated : _______________________

UNIVERSITY OF AGRICULTURE, FAISALABAD

National Institute of Food Science and Technology
Name of the Project

Efficacy Studies of Vitamin A Fortified Cookies in Pregnant and Lactating Women

I have been explained in detail the purpose and rationale of the above mentioned component of the Vitamin A Fortified Cookies Project. I understand that this project is of national significance and my full commitment and dedication with it will be of paramount importance, I am volunteering for it. I have had a chance to ask questions and answered them. I undertake that I will abide by all the instructions given by the investigators and will use the same Vitamin A Fortified Cookies given to me in the designated period. Further, I am bound to fill the questionnaire at the end of the week to best of my knowledge.

Name & Signature of the Subject

Dated

Name & Signature of the Person obtaining consent

Dated

Name & Signature of the Researcher

Dated

Name & Signature of the Principal Investigator

Dated

Appendix-V
UNIVERSITY OF AGRICULTURE, FAISALABAD National Institute of Food Science and Technology

Project:

Efficacy studies of vitamin a fortified cookies in pregnant and lactating women

Questionnaire
Name________________ w/o ________________ Age______________ Phone____________ Address_________________________________________

How many times and how much quantity of the following commodities is utilized per week?

Commodity
1. Milk and Milk Products i. Milk ii. Lassi iii. Dehi iv. Pannier v. Makhan vi. Desi Ghee vii. Others 2. Meat and Meat products i. Meat ii. Liver iii. Sari payee iv. Others 3. Vegetables (Raw and cooked) i. Carrot ii. Salad iii. Green leafy vegetables iv. Others
4. Fruits i. ii. iii. iv. v. Mangoes Apple Banana Apricot Others

No. of Times
1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7 1,2,3,4,5,6,7

Quantity

5. Any Medicine Name of medicines:

Does it contain vitamin A

Yes or No

Note: Encircle the commodities used and write down the quantity properly along with units.

200

200

200

T 1 = placebo

18.00

16.00

15.79

14.00

T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; retinyl palmitate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate

12.00
10.18 10.20 9.36 8.71 7.95

10.00

9.56

8.00

6.00

4.00

2.00

0.00 T1 T2 T3 T4 T5 T6 T7

Fig. 1. Percent decrease in vitamin A of fortified cookies during baking

200

12.0

15 days 60 days

30 days 75 days

45 days 90 days

T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; retinyl palmitate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate
9.69

10.0
9.06 8.04 8.02 7.23 7.06 9.29

8.93 8.16 7.72

8.71

8.0
7.25 6.80 6.04 6.40 5.21 4.04 2.93 2.61 2.51 2.13 1.55 1.22 1.66 3.19 3.45 5.10 6.76

7.17 6.60 5.94

6.0
5.14 4.63 3.95 3.12 4.99 4.87

4.48

4.0

2.90

2.0 1.81
1.27

1.56

0.0 T1 T2 T3 T4 T5 T6 T7

Fig. 2. Percent decrease in vitamin A of foritified cookies during storage

200

25.00

7th Month
20.00

8th Month

9th Month
18.51

21.56

17.69

15.08 15.00 11.55 9.88 10.00

5.00

0.00

-2.93 -5.00 -5.45

-10.00

-9.32

Placebo (T1)
-15.00

Retinyl acetate (T4) (T7)

Retinyl palmitate

Fig. 3. Percent change in serum retinol of pregnant women

200

50.00

7th Month
43.95

8th Month

9th Month

40.00

37.93 36.71

30.00

24.18 20.69 20.00 16.46

10.00

8.79

9.20 6.33

0.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 4. Percent decrease in serum retinyl esters of pregnant women

200

35.00 32.79

30.00

7th Month
25.00 22.46

8th Month

9th Month

20.00

%
15.00 12.02 10.00 6.13 5.00 2.29 4.34 2.05 4.25

5.57

0.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 5. Percent decrease in serum -carotene of pregnant women

200

10.00

7th Month

8th Month

9th Month
6.69 4.71 3.73 2.89 1.86 0.96

5.00

0.00

-2.12

-5.00

-7.04

-10.00

-11.73

Placebo (T1)
-15.00

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 6. Percent change in hemoglobin of pregant women

200

14.00

Ist Month
12.00

2nd Month

3rd Month

12.84

10.34 10.00 9.43

7.98 8.00 6.71 6.00 5.74

4.00

2.00 1.23

1.62

1.81

0.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 7. Percent increase in serum retinol of lactating women

200

15.00

Ist Month
10.00

2nd Month

3rd Month
8.00 6.00 4.00

5.00
1.85

3.70

0.00 -5.00 -10.00

%

-3.70

-9.80

-15.00 -20.00 -25.00

-25.49

-30.00 -35.00
-37.25

-40.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 8. Percent change in serum retinyl esters of lactating women

200

6.00 5.45

Ist Month

2nd Month

3rd Month

5.00

4.00

3.00 2.39 2.18 2.00 1.45 1.97 1.50

1.00

0.86

0.90

0.10 0.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 9. Percent decrease in serum -carotene of lactating women

200

10.00

Ist Month

2nd Month

3rd Month
8.74

8.00 7.26

6.33 6.00

6.27

5.32

5.21 4.73

4.18 4.00

4.09

2.00

0.00

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 10. Percent increase in hemoglobin of lactating women

200

40.00

Pregnancy 7th Month Lactation Ist Month

Pregnancy 8th Month Lactation 2nd Month

Pregnancy 9th Month Lactation 3rd Month

37.16 34.13

30.00

29.69 27.97 25.31

29.72

21.56

20.00

18.51 15.08

17.69

11.55 9.88

10.00

0.00
-2.93 -5.45 -8.21-7.85-7.68 -9.32

-10.00

Placebo (T1) -20.00

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 11. Percent change in serum retinol during pregancy and lactation

200

80.00 Pregnancy 7th Month Lactation Ist Month

64.84

Pregnancy 8th Month Lactation 2nd Month

Pregnancy 9th Month Lactation 3rd Month

60.00

58.24

49.45 43.96

40.23

40.00

37.93

36.78

35.63

36.71 34.18 32.91 31.65

24.18 20.69

20.00

16.46

8.79

9.20 6.33

0.00 Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7)

Fig. 12. Percent decrease in serum retinyl esters during pregnancy and lactation

200

40.00 Pregnancy 7th Month

36.45 34.39 32.79 32.79

Pregnancy 8th Month Lactation 2nd Month

Pregnancy 9th Month Lactation 3rd Month

Lactation Ist Month

30.00

22.46

20.00 %

12.02

10.00
6.13 4.34 2.29 6.94 7.50

8.18 5.57 4.25 2.05

7.43 6.42

6.99

0.00 Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7)

Fig. 13. Percent decrease in serum -carotene during pregnancy and lactation

200

17.00

Pregnancy 7th Month Lactation Ist Month

Pregnancy 8th Month Lactation 2nd Month

Pregnancy 9th Month Lactation 3rd Month

13.38 12.31

16.01

12.00 10.17 8.99 6.69 4.71 3.73

11.73

7.00

2.89 2.00 1.86 0.96

-3.00

-2.12

-6.15 -8.00 -7.04 -8.04 -7.04

-13.00

-11.73

Placebo (T1)

Retinyl acetate (T4)

Retinyl palmitate (T7)

Fig. 14. Percent change in hemolgobin during pregnancy and lactation

200

Table 2. Mean squares for physical parameters of fortified cookies SOV Treatments (T) Storage (S) TxS Error Total
NS = Non-significant

df 6 6 36 98 146

Diameter 0.025NS 0.080NS 0.019NS 0.040

Thickness 0.0000535NS 0.0000837NS 0.0000720NS 0.0002497

Spread factor 18.076NS 36.225NS 12.129NS 24.347

200

Table 3. Effect of treatments on physical parameters of fortified cookies Treatments T1 T2 T3 T4 T5 T6 T7 Diameter (cm) 4.680.024 4.790.034 4.710.031 4.750.028 4.690.045 4.640.023 4.610.020 Thickness (cm) 0.520.002 0.520.002 0.530.002 0.530.002 0.530.002 0.530.003 0.530.002 Spread factor 89.210.195 91.390.441 88.970.890 90.400.799 89.300.958 87.980.381 87.140.662

Values mentioned in the table are Means SEM T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; retinyl palmitate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate

Table 4. Effect of storage on physical parameters of fortified cookies Storage (days) 0 15 30 45 60 75 90 Diameter (cm) 4.810.032 4.760.030 4.720.028 4.690.029 4.670.022 4.640.024 4.600.028 Thickness (cm) 0.530.003 0.530.002 0.530.002 0.530.003 0.530.002 0.530.002 0.520.002 Spread factor 91.220.894 90.270.586 89.540.469 88.681.002 88.770.713 88.450.562 87.450.645

200

Table 5. Mean squares for chemical composition of fortified cookies SOV Treatments (T) Storage TxS Error Total SOV Treatments (T) Storage TxS Error Total
** = P 0.01 NS = Non-significant

df 6 6 36 98 146 df 6 6 36 98 146

Moisture 0.02397NS 0.25984** 0.00807NS 0.03293

Ash 0.00071 NS 0.01284 0.00021 NS 0.00046

Crude protein 0.01027NS 0.04757NS 0.00834NS 0.02639

Crude fat 0.04848NS 0.06626NS 0.04626NS 0.08701

(S)

Crude fiber 0.00011NS 0.00026NS 0.00013NS 0.00055

NFE 0.0812NS 0.2433NS 0.0723NS 0.1282

TBA value 0.000011NS 0.000013** 0.000007NS 0.000015

(S)

200

Table 6.

Effect of treatments on moisture, ash and crude protein content of fortified cookies

Treatments T1 T2 T3 T4 T5 T6 T7

Moisture (%) 2.760.039 2.720.048 2.690.048 2.700.046 2.660.047 2.690.037 2.670.052

Ash (%) 0.560.007 0.570.010 0.570.008 0.570.009 0.570.011 0.580.012 0.590.011

Crude protein (%) 6.600.030 6.610.023 6.590.025 6.610.029 6.610.025 6.660.026 6.610.023

Values mentioned in the table are Means SEM T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; retinyl palmitate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate

Table 7. Effect of storage on moisture, ash and crude protein content of fortified cookies Storage (days) 0 15 30 45 60 75 90 Moisture (%) 2.510.026d 2.630.021c 2.690.021bc 2.690.023bc 2.740.017abc 2.800.024ab 2.840.024a Ash (%) 0.600.004 0.590.016 0.590.002 0.570.015 0.560.004 0.550.004 0.540.003 Crude protein (%) 6.690.022 6.680.019 6.610.026 6.590.013 6.580.016 6.580.018 6.570.024

Mean values carrying same letters in a column are not significantly different

200

Table 8.

Effect of treatments on crude fat, crude fiber, NFE content and TBA value of fortified cookies

Treatments T1 T2 T3 T4 T5 T6 T7

Crude fat (%) 22.480.051 22.370.081 22.500.042 22.430.082 22.440.049 22.510.053 22.460.067

Crude fiber (%) 0.260.004 0.250.003 0.260.002 0.260.002 0.250.002 0.260.003 0.250.003

NFE (%) 67.340.053 67.470.064 67.400.033 67.430.075 67.460.051 67.300.061 67.420.048

TBA value 0.0530.004 0.0490.004 0.0510.004 0.0510.004 0.0500.004 0.0500.004 0.0510.004

Table 9.

Effect of storage period on crude fat, crude fiber, NFE content and TBA No. of fortified cookies

Storage (days) 0 15 30 45 60 75 90

Crude fat (%) 22.600.032 22.560.030 22.520.041 22.470.062 22.410.070 22.360.035 22.260.046

Crude fiber (%) 0.250.003 0.260.002 0.260.001 0.250.003 0.250.003 0.250.002 0.260.003

NFE (%) 67.350.029 67.280.047 67.330.038

TBA value 0.0400.001f 0.0410.002f 0.0440.002ef

67.430.066 0.0490.001de 67.460.070 67.460.047 67.530.048 0.0540.002cd 0.0600.003b 0.0690.002a

Mean values carrying same letters in a column are not significantly different

200

Table 10.

Mean squares for effect of baking on vitamin A level in fortified cookies SOV df 6 1 6 28 41 Vitamin A content 181.953** 9.035** 0.456** 0.071

Treatments (T) Baking (B) TxB Error Total

** = P 0.01

200

Table 11. Effect of baking on stability of vitamin A in fortified cookies Vitamin A content (g/100g) Treatments Before baking T1 T2 T3 T4 T5 T6 T7 Means 0.19k 10.98e 14.59c 18.18a 6.470i 8.65g 10.79e 9.982.18a After baking 0.16k 9.93f 13.43d 16.33b 5.81j 7.84h 9.85f 9.051.98b

Values mentioned in the table are Means SEM Mean values carrying same letters in a column are not significantly different T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; Retinyl palmitate T6 = 40% of RDA; Retinyl palmitate T7 = 50% of RDA; Retinyl palmitate

200

Table 12.

Mean squares for storage stability of vitamin A content in fortified cookies SOV df 6 6 36 98 146 Vitamin A content 533.008** 1.665** 0.065NS 0.045

Treatments (T) Storage (S) SxT Error Total

** = P 0.01 NS = Non-significant

200

Table 13.

Effect of treatments and storage on stability of vitamin A (g/100g) in fortified cookies Storage (days)

Treatments 0 T1 T2 T3 T4 T5 T6 T7 Means 0.16 9.93 13.43 16.33 5.81 7.84 9.85 9.05 1.98a 15 0.15 9.75 13.26 16.13 5.72 7.71 9.64 8.91 1.96b 30 0.15 9.62 13.08 15.92 5.64 7.59 9.51 8.79 1.93b 45 0.14 9.47 12.90 15.67 5.52 7.44 9.37 8.64 1.90c 60 0.13 9.33 12.74 15.48 5.40 7.31 9.20 8.52 1.88d 75 0.13 9.21 12.57 15.22 5.39 7.20 9.09 8.40 1.85d 90 0.11 9.03 12.35 15.02 5.27 7.08 8.97 8.26 1.83d

Means 0.14 0.006f 9.48 0.119c 12.90 0.145b 15.68 0.181a 5.54 0.074e 7.45 0.119d 9.38 0.104c

Mean values carrying same letters in a column and row are not significantly different

200

Table 14. Mean squares for sensory evaluation scores of fortified cookies SOV Treatments (T) Storage (S) TxS Error Total SOV Treatments (T) Storage (S) TxS Error Total
** = P 0.01 NS = Non-significant

df 6 6 36 245 293 df 6 6 36 245 293

Color 0.4406NS 10.8707** 0.0983NS 0.3374

Flavor 0.5941NS 13.7052** 0.1655NS 0.3483

Taste 0.3401NS 6.2449** 0.0967NS 0.3374

Crispness 0.5533NS 19.4739** 0.0771NS 0.3020

Texture 0.6751NS 15.7687** 0.1020NS 0.3456

Overall acceptability 0.1542NS 11.7098** 0.1013NS 0.3374

200

Table 15. Effect of treatments on sensory scores of fortified cookies Treatments T1 T2 T3 T4 T5 T6 T7 Color 7.090.17 6.900.23 6.810.19 6.770.13 6.950.14 6.860.12 6.800.16 Flavor 6.770.17 6.690.15 6.630.15 6.710.17 6.630.19 6.600.18 6.740.15 Taste 6.950.15 6.860.20 6.950.15 7.140.14 6.900.14 6.950.10 7.000.15

Values mentioned in the table are Means SEM T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T5 = 30% of RDA; retinyl palmitate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate

Table 16. Effect of storage on sensory scores of fortified cookies Storage (days) 0 15 30 45 60 75 90 Color 7.300.09a 7.270.13a 7.180.08ab 6.990.10b 6.740.09c 6.520.05c 6.190.06e Flavor 7.140.04a 7.060.04a 6.910.04a 6.890.07a 6.510.04b 6.230.05c 6.030.03c Taste 7.380.05a 7.330.06a 7.240.06ab 7.050.05b 6.760.06c 6.620.08cd 6.380.09d

Mean values carrying same letters in a column are not significantly different

200

Table 17. Effect of treatments on sensory scores of fortified cookies Treatments T1 T2 T3 T4 T5 T6 T7 Crispness 7.050.26 7.000.27 7.090.24 7.240.27 7.000.27 7.050.25 7.290.27 Texture 7.000.27 7.020.25 7.110.22 7.370.17 7.140.22 7.110.20 7.310.22 Overall acceptability 7.100.28 7.190.24 7.140.19 7.240.18 7.100.23 7.190.18 7.240.19

Mean values carrying same letters in a column are not significantly different

T1 = placebo T2 = 30% of RDA; retinyl acetate T3 = 40% of RDA; retinyl acetate T4 = 50% of RDA; retinyl acetate T6 = 40% of RDA; retinyl palmitate T7 = 50% of RDA; retinyl palmitate Table 18. Effect of storage on sensory scores of fortified cookies Storage (days) 0 15 30 45 60 75 90 Crispness 7.910.04a 7.850.06a 7.430.06b 7.090.06c 6.860.07d 6.430.06e 6.140.07f Texture 7.830.05a 7.760.06a 7.430.06b 7.190.07c 7.000.07c 6.520.10d 6.330.10d Overall acceptability 7.720.05a 7.670.06a 7.330.02b 7.280.05b 7.190.07b 6.810.07c 6.190.12d

Mean values carrying same letters in a column are not significantly different

200

Table 19.

Mean squares for effect of fortified cookies on serum retinol, retinyl esters and - carotene levels of pregnant women

SOV Treatments (T) Months (M) TxM Error Total

** = P 0.01 NS = Non-significant

df 2 3 6 252 263

Retinol 125.622** 58.743** 33.381** 8.931

Retinyl esters 0.068NS 2.173** 0.013NS 0.042

-carotene 251.245** 433.130** 107.598** 16.931

200

Table 20. Serum retinol level (g/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 17.06d 16.91d 16.79d 16.92 0.02b 7th 16.56de 18.58c 18.73c 17.96 0.70a 8th 16.13e 19.46b 19.76ab 18.45 1.16a 9th 16.31 15.47f 0.34b 18.75 20.04a 20.41a 18.64 1.59a 0.68a 18.92 0.79a Means

Means SEM Mean values carrying same letters in a column and row are not significantly different Baseline = observations recorded in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 21. Serum retinyl esters level (g/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.91 0.87 0.79 0.86 0.04a 7th 0.83 0.79 0.74 0.79 0.03b 8th 0.69 0.69 0.66 0.68 0.01c 9th 0.51 0.54 0.50 0.52 0.01d 0.07 0.67 0.06 0.09 0.72 0.74 Means

200

Mean values carrying same letters in a row are not significantly different

200

Table 22. Serum - carotene level (g/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 33.03a 32.28ab 31.77b 32.36 0.37a 7th 29.06d 31.54b 31.12b 30.57 0.77ab 8th 25.61e 9th 27.47 22.20f 2.32b 31.25 30.88bc 30.30bc 30.42bc 28.97 1.69bc 30.00c 27.50 2.65c 0.43a 30.83 0.39a Means

Mean values carrying same letters in a column and row are not significantly different

200

Table 23.

Mean squares for effect of fortified cookies on red blood cells indices of pregnant women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
** = P 0.01 NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Total RBC 0.542NS 1.528NS 0.063NS 1.003

Hematocrit 88.818** 436.133** 15.700NS 22.085

Hemoglobin 19.357** 0.017NS 3.583NS 2.229

MCH 81.427** 1123.297** 11.759NS 5.301

MCV

MCHC

Platelets count

ESR

452.693** 1434.822** 79.987NS 122.600

100.79** 342.21** 24.63NS 16.85

6839.708NS 8217.529NS 29162.234NS 17669.716

118.682NS 61.889NS 91.480NS 89.854

200

Table 24. Total red blood cells count (M/L) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 4.91 5.08 4.95 4.98 0.05 7th 4.74 4.96 4.91 4.87 0.07 8th 4.65 4.87 4.77 4.76 0.06 9th 4.72 4.58 0.07 4.91 4.71 4.80 4.70 0.06 0.08 4.86 0.04 Means

Means SEM Baseline = observations taken in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 25. Hematocrit (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 31.53 32.11 31.83 31.82 0.17a 7th 29.49 31.57 30.79 30.62 0.33b 8th 26.92 29.88 28.99 28.60 0.88c 9th 24.60 28.23 28.47 27.10 1.25d 0.88a 30.02 0.78a 28.14 1.51b 30.45 Means

Mean values carrying same letters in a column and row are not significantly different

200

200

Table 26. Hemoglobin status (g/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 8.95 9.34 9.12 9.14 0.11 7th 8.76 9.43 9.29 9.16 0.20 8th 8.32 9.61 9.46 9.13 0.41 9th 8.48 7.90 0.23b 9.54 9.78 9.73 9.14 0.62 0.10a 9.40 0.13a Means

Mean values carrying same letters in a column are not significantly different

200

Table 27. Mean corpuscular hemoglobin (pg) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 27.85 28.18 28.08 28.04 0.10a 7th 23.55 25.98 26.32 25.28 0.87b 8th 21.19 23.83 23.16 22.73 0.79c 9th 22.70 18.21 2.03b 24.77 21.08 21.29 20.19 0.99d 1.52a 24.71 1.53a Means

Mean values carrying same letters in a column and row are not significantly different

Table 28. Mean corpuscular volume (fL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 74.62 75.20 74.97 74.93 0.17a 7th 69.23 73.57 73.73 72.18 1.47a 8th 64.47 70.59 70.64 68.57 2.05b 9th 60.87 68.70 69.45 66.34 2.74b 67.30 2.98b 72.02 1.46a 71.20 1.29a Means

Mean values carrying same letters in a column and row are not significantly different

200

200

Table 29.

Mean corpuscular hemoglobin concentration (g/dL) of pregnant women Month of pregnancy

Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 32.49 33.18 31.97 32.55 0.35a 7th 29.63 31.27 30.75 30.55 0.48b 8th 27.14 30.87 30.02 29.34 1.13bc 9th

Means 28.70 25.53 29.60 29.46 28.20 1.33c 0.74a 30.55 0.54a 1.52b 31.23

Mean values carrying same letters in a column and row are not significantly different

Table 30. Platelets count (K/L) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 279.82 297.00 307.09 294.63 7.96 7th 272.18 335.36 342.04 316.52 22.26 8th 320.77 327.27 270.41 306.15 17.97 9th 362.50 312.64 282.23 319.12 23.40 8.45 300.44 15.83 308.82 20.83 318.07 Means

200

Table 31. Erythrocytes sedimentation rate (mm/Hr) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 30.27 23.54 29.18 27.66 2.09 7th 27.82 25.04 23.54 25.47 1.25 8th 26.86 27.36 26.77 26.99 0.18 9th 28.11 27.50 0.75 25.82 27.32 27.14 27.32 0.10 0.93 26.66 1.17 Means

200

Table 32.

Mean squares for effect of fortified cookies on white blood cells indices of pregnant women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
** = P 0.01 NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Total WBC 6.352NS 10.253** 4.453NS 3.496

Neutrophiles 40.640NS 53.852NS 56.261NS 44.655

Lymphocytes 97.390NS 193.889NS 231.537NS 132.532

Monocytes 1.299NS 1.034NS 8.845NS 6.990

Eosinophiles 2.227NS 2.535NS 3.005NS 3.889

Basophiles 0.002NS 0.001NS 0.001NS 0.005

200

Table 33. Total white blood cells count (K/L) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) 9.46 8.64 9.18 9.09 Means 0.24b 7th 10.00 10.32 9.27 9.86 0.31a 8th 10.04 9.54 10.09 9.89 0.18a 9th 9.97 10.36 0.19 9.43 9.23 10.04 9.88 0.34a 0.35 9.65 0.24 Means

Means SEM Mean values carrying same letters in a row are not significantly different Baseline = observations taken in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 34. Neutrophiles (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 65.68 67.19 61.78 64.88 7th 62.26 65.77 65.00 64.34 8th 62.63 63.89 62.85 63.12 9th 65.29 64.75 66.28 65.44 0.88 65.40 0.71 63.98 1.02 63.97 Means

200

1.40

0.92

0.34

0.39

200

Table 35. Lymphocytes (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 26.31 24.43 31.45 27.40 1.82 7th 29.29 26.74 26.85 27.63 0.72 8th 29.29 27.99 28.88 28.72 0.33 9th 27.99 27.07 0.77 26.54 26.98 24.65 26.23 0.69 0.75 27.96 1.45 Means

Table 36. Monocytes (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 3.43 4.20 3.50 3.71 0.21 7th 3.35 3.94 3.09 3.46 0.22 8th 4.30 2.77 4.14 3.74 0.42 9th 3.94 4.02 3.49 3.82 0.14 3.76 0.22 3.73 0.33 3.56 0.22 Means

200

Table 37. Eosinophiles (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 2.70 3.29 2.35 2.78 0.24 7th 2.71 2.81 2.72 2.75 0.03 8th 2.35 2.77 1.99 2.37 0.20 9th 2.65 2.85 0.11 2.83 2.44 3.07 2.79 0.16 0.18 2.53 0.23 Means

200

Table 38. Basophiles (%) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.06 0.07 0.05 0.06 0.005 7th 0.05 0.05 0.06 0.05 0.003 8th 0.04 0.05 0.07 0.05 0.008 9th 0.05 0.05 0.004 0.06 0.05 0.05 0.05 0.000 0.005 0.06 0.005 Means

200

Table 39.

Mean squares for effect of fortified cookies on liver function tests of pregnant women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Bilirubin total 0.062NS 0.116NS 0.192NS 0.111

Bilirubin direct 0.004NS 0.005NS 0.013NS 0.012

Bilirubin indirect 0.055NS 0.110NS 0.169NS 0.099

Glutamate pyruvate transaminase 402.606NS 138.793NS 257.778NS 157.142

Alkaline phosphatase 10942.322NS 7757.763NS 2008.463NS 7080.455

200

Table 40. Bilirubin total (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.81 0.68 0.64 0.71 0.05 7th 0.69 0.77 0.81 0.76 0.04 8th 0.74 0.64 0.90 0.76 0.08 9th 0.73 0.69 0.03 0.70 0.69 0.64 0.67 0.02 0.03 0.75 0.06 Means

Means SEM Baseline = observations taken in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 41. Bilirubin direct (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.20 0.22 0.18 0.20 0.01 7th 0.16 0.19 0.22 0.19 0.02 8th 0.20 0.17 0.19 0.19 0.01 3 0.17 0.17 0.20 0.18 0.01 0.01 0.20 0.01 0.18 0.01 0.19 Means

200

200

Table 42. Bilirubin indirect (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.60 0.46 0.46 0.51 0.05 7th 0.53 0.57 0.59 0.56 0.02 8th 0.53 0.47 0.71 0.57 0.07 9th 0.55 0.52 0.02 0.51 0.52 0.44 0.49 0.03 0.03 0.55 0.06 Means

Table 43. Glutamate pyruvate transaminase (U/L) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 26.36 28.46 24.36 26.39 1.18 7th 29.77 25.54 23.73 26.35 1.79 8th 29.50 21.96 19.04 23.50 3.12 9th 23.54 29.96 25.86 26.45 1.88 27.29 1.47 26.48 1.76 23.25 1.47 Means

200

Table 44. Alkaline phosphatase (U/L) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 212.91 236.77 205.96 218.55 9.33 7th 185.59 212.46 199.46 199.17 7.76 8th 178.77 207.04 207.36 197.72 9.48 9th 196.13 207.23 8.25 218.35 217.14 222.50 215.62 4.47 6.48 208.82 4.87 Means

200

Table 45.

Mean squares for effect of fortified cookies on renal function tests of pregnant women

SOV Treatments (T) Months (M) TxM Error Total

NS = Non-significant

df 2 3 6 252 263

Urea 85.955NS 171.822NS 104.894NS 125.584

Creatinine 0.124NS 0.093NS 0.112NS 0.073

200

Table 46. Urea (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 32.68 33.77 29.09 31.85 1.41 7th 34.77 32.04 34.18 33.66 0.83 8th 28.27 32.23 31.14 30.55 1.18 9th 32.85 35.68 1.65 33.28 35.09 31.18 33.98 1.41 0.72 31.40 1.05 Means

Means SEM Baseline = observations taken in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 47. Creatinine (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.64 0.70 0.82 0.72 0.05 7th 0.75 0.80 0.85 0.80 0.03 8th 0.77 0.69 0.82 0.76 0.04 9th 0.89 0.76 0.76 0.80 0.04 0.03 0.81 0.02 0.76 0.05 0.74 Means

200

Table 48.

Mean squares for effect of fortified cookies on lipids profile of pregnant women

SOV Treatments (T) Months (M) TxM Error Total

NS = Non-significant

df 2 3 6 252 263

Cholesterol 476.847NS 554.716NS 379.142NS 836.353

LDL 865.680NS 157.997NS 88.823NS 577.576

HDL 383.518NS 357.447NS 239.665NS 197.438

Triglycerides 1254.561NS 665.001NS 1312.186NS 988.106

200

Table 49. Cholesterol level (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 151.27 144.04 140.87 145.39 3.08 7th 155.36 151.74 144.49 150.53 3.20 8th 144.32 153.80 145.50 147.87 2.98 9th 150.31 150.27 150.56 149.55 150.13 0.30 2.11 145.10 1.78 2.28 150.04 Means

Means SEM Baseline = observations taken in the last week of 6th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

200

Table 50. Low density lipoprotein (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 86.62 85.00 79.00 83.54 2.32 7th 91.53 87.73 79.78 86.35 3.46 8th 83.93 87.20 80.47 83.87 1.94 9th 87.10 86.31 1.59 86.66 86.73 81.69 84.91 1.61 0.59 80.24 0.57 Means

Table 51. High density lipoprotein (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 47.31 42.28 46.34 45.31 1.54 7th 45.74 46.86 48.44 47.01 0.78 8th 44.31 49.63 47.91 47.28 1.57 9th 46.61 45.39 48.88 46.96 1.02 45.99 0.65 46.04 1.53 47.89 0.55 Means

200

Table 52. Triglycerides level (mg/dL) of pregnant women Month of pregnancy Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 86.68 83.81 77.63 82.71 2.67 7th 90.47 85.75 81.33 85.85 2.64 8th 80.40 84.86 85.59 83.62 1.62 9th 86.07 86.73 2.09 86.66 92.20 94.88 91.27 2.40 1.89 84.86 3.72 Means

200

Table 53.

Mean squares for effect of fortified cookies on serum retinol, retinyl esters and -carotene levels of lactating women

SOV Treatments (T) Months (M) TxM Error Total

** = P 0.01 * = P 0.05 NS = Non-significant

df 2 3 6 252 263

Retinol 692.696** 48.169** 5.982NS 10.681

Retinyl esters 0.247** 0.033NS 0.072** 0.019

-carotene 1269.684** 10.171NS 1.774NS 31.972

200

Table 54. Serum retinol level (g/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 15.47 20.04 20.41 18.64 1.59b 1st 15.66 21.19 21.78 19.54 1.95ab 2nd 15.72 21.64 22.52 19.96 2.14a 3rd 15.65 15.75 21.93 23.03 20.24 2.27a 0.06b 21.20 0.42a 21.94 0.57a Means

Means SEM Mean values carrying same letters in a column and row are not significantly different Baseline = observations recorded in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

200

Table 55. Serum retinyl esters level (g/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.51b 0.54a 0.50b 0.52 0.01 1st 0.46c 0.52ab 2nd 0.38d 0.55a 3rd 0.42 0.32e 0.56a 0.54a 0.47 0.07 0.01a 0.52 0.01a 0.49 0.05 0.04b 0.54 Means

0.52ab 0.53ab 0.50 0.02

Mean values carrying same letters in a column are not significantly different

Table 56. Serum -carotene level (g/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 22.20 30.30 30.00 27.50 2.65 1st 22.18 30.04 29.41 27.22 2.52 2nd 21.67 29.86 29.73 27.08 2.71 3rd 20.99 29.64 29.55 0.13a 26.73 2.87 21.76 0.29b 29.96 0.14a 29.67 Means

Mean values carrying same letters in a column are not significantly different

200

200

Table 57.

Mean squares for effect of fortified cookies on red blood cells indices of lactating women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
** = P 0.01 NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Total RBC 0.502** 0.902** 0.007** 0.082

Hematocrit 289.109** 204.847** 0.452NS 23.582

Hemoglobin 77.062** 8.416** 0.118NS 1.929

MCH 469.403** 1393.172** 26.476NS 43.440

MCV 1444.192** 863.468** 1.904NS 156.699

MCHC 327.246** 235.046** 0.923NS 47.567

Platelets count 11170.239NS 3817.771NS 28382.324NS 19322.573

ESR 67.648NS 25.146NS 33.021NS 72.587

200

Table 58. Total red blood cells counts (M/L) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 4.58c 4.71bc 4.80ab 4.70 0.06c 1st 4.69bc 4.79ab 4.88ab 4.79 0.05bc 2nd 4.75b 4.87ab 4.91ab 4.84 0.05ab 3rd 4.71 4.83ab 0.05b 4.83 4.95ab 4.99a 4.92 0.05a 0.05a 4.90 0.04a Means

Means SEM Mean values carrying same letters in a column and row are not significantly different Baseline = observations taken in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

200

Table 59. Hematocrit (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 24.60 28.23 28.47 27.10 1.25c 1st 25.84 29.51 29.72 28.36 1.26b 2nd 26.19 30.33 30.15 28.89 1.35b 3rd 26.17 28.04 0.71b 29.93 31.65 32.02 30.57 1.27a 0.72a 30.09 0.74a Means

Mean values carrying same letters in a column and row are not significantly different

Table 60. Hemoglobin status (g/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 7.90 9.78 9.73 9.14 0.62c 1st 8.23 10.18 10.19 9.53 0.65b 2nd 8.32 10.29 10.34 9.65 0.67ab 3rd 8.40 10.49 10.58 0.18a 9.82 0.71a 8.21 0.11b 10.19 0.15a 10.21 Means

Mean values carrying same letters in a column and row are not significantly different

200

Table 61. Mean corpuscular hemoglobin (pg) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 18.21 21.08 21.29 20.19 0.99d 1st 19.97 23.31 24.07 22.45 1.26c 2nd 22.13 27.47 28.11 25.90 1.90b 3rd 21.06 23.94 1.25b 25.65 30.73 31.43 28.70 2.39a 2.15a 26.23 2.23a Means

Mean values carrying same letters in a column and row are not significantly different

Table 62. Mean corpuscular volume (fL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 60.87 68.70 69.45 66.34 2.74c 1st 63.07 70.81 72.00 68.63 2.80bc 2nd 65.12 73.79 74.91 71.27 3.09ab 3rd 67.43 75.53 76.23 73.06 2.82a 64.12 1.40b 72.21 1.52a 73.15 1.52a Means

Mean values carrying same letters in a column and row are not significantly different

200

Table 63.

Mean corpuscular hemoglobin concentration (g/dL) of lactating women Month of lactation

Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 25.53 29.60 29.46 28.20 1.33c 1st 26.70 30.78 30.88 29.45 1.38bc 2nd 27.85 32.05 31.95 30.62 1.38ab 3rd

Means 27.29 29.07 32.78 33.51 31.79 1.37a 0.70a 31.45 0.86a 0.76b 31.30

Mean values carrying same letters in a column and row are not significantly different

Table 64. Platelets count (K/L) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 362.50 312.64 282.23 319.12 23.40 1st 329.77 283.18 295.64 302.86 13.93 2nd 281.18 297.86 349.36 309.47 20.52 3rd 297.54 303.73 352.04 317.77 17.23 6.19 319.82 18.05 317.75 18.01 299.35 Means

200

Table 65. Erythrocytes sedimentation rate (mm/Hr) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 27.50 27.32 27.14 27.32 0.10 1st 26.77 25.96 26.23 26.32 0.24 2nd 27.50 23.14 27.96 26.20 1.54 3rd 26.98 26.14 0.33 25.42 25.27 26.27 25.89 0.31 0.87 26.90 0.41 Means

200

Table 66.

Mean squares for effect of fortified cookies on white blood cells of lactating women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
* = P 0.05 NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Total WBC 11.015NS 0.731NS 3.379NS 4.277

Neutrophiles 11.322NS 32.398NS 27.973NS 45.587

Lymphocytes 438.761* 95.682NS 86.004NS 127.573

Monocytes 10.136NS 13.889NS 2.268NS 7.348

Eosinophiles 0.527NS 0.833NS 2.223NS 3.918

Basophiles 0.001NS 0.003NS 0.001NS 0.006

200

Table 67. Total white blood cells count (K/L) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 10.36 9.23 10.04 9.88 0.34 1st 9.86 9.73 10.18 9.92 0.13 2nd 10.32 9.64 10.36 10.11 0.23 3rd 10.28 10.59 0.15 9.58 9.73 9.36 9.89 0.36 0.12 9.99 0.22 Means

Means SEM Baseline = observations taken in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 68. Neutrophiles (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.76 0.82 1.13 0.42 64.87 64.29 67.15 65.43 1st 64.11 66.71 67.13 65.98 2nd 62.77 65.74 67.22 65.24 3rd 63.92 65.60 64.71 64.74 63.92 0.43 65.58 0.50 66.55 0.61 Means

200

Table 69. Lymphocytes (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 26.89 26.79 24.97 26.22 0.54 1st 27.72 25.74 26.11 26.52 0.53 2nd 30.36 25.71 24.48 26.85 1.55 3rd 28.53 29.16 0.77a 26.19 26.54 28.04 27.91 0.66 0.28b 25.90 0.79b Means

Mean values carrying same letters in a column are not significantly different

Table 70. Monocytes (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 3.92 3.99 3.54 3.81 0.12 1st 3.05 3.48 3.37 3.30 0.11 2nd 3.49 5.03 4.53 4.35 0.39 3rd 3.46 4.01 3.52 3.66 0.15 0.33 3.74 0.27 3.48 0.18 4.13 Means

200

Table 71. Eosinophiles (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 2.83 2.42 3.11 2.79 0.17 1st 2.81 2.79 2.35 2.65 0.13 2nd 2.21 2.54 2.88 2.55 0.17 3rd 2.60 2.56 0.14 2.55 2.47 2.66 2.56 0.05 0.08 2.75 0.16 Means

Table 72. Basophiles (%) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.05 0.05 0.06 0.05 0.003 1st 0.06 0.06 0.06 0.06 0.002 2nd 0.06 0.06 0.07 0.06 0.005 3rd 0.06 0.07 0.07 0.005 0.07 0.003 0.06 0.004 0.06 0.004 0.06 Means

200

Table 73.

Mean squares for effect of fortified cookies on liver function tests of lactating women

SOV Treatments (T) Months (M) TxM Error Total SOV Treatments (T) Months (M) TxM Error Total
NS = Non-significant

df 2 3 6 252 263 df 2 3 6 252 263

Bilirubin total 0.122NS 0.046NS 0.125NS 0.104

Bilirubin direct 0.001NS 0.010NS 0.004NS 0.011

Bilirubin indirect 0.128NS 0.054NS 0.110NS 0.093

Glutamate pyruvate transaminase 154.663NS 53.136NS 191.951NS 191.887

Alkaline phosphatase 10115.981NS 12207.424NS 6968.542NS 7692.567

200

Table 74. Bilirubin-total (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.69 0.69 0.64 0.67 0.02 1st 0.81 0.79 0.59 0.73 0.07 2nd 0.64 0.75 0.78 0.72 0.04 3rd 0.72 0.72 0.04 0.74 0.72 0.66 0.70 0.02 0.02 0.67 0.04 Means

Means SEM Baseline = observations taken in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 75. Bilirubin-direct (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.01 0.00 0.01 0.01 0.17 0.17 0.20 0.18 1st 0.17 0.18 0.17 0.17 2nd 0.19 0.21 0.20 0.20 3rd 0.20 0.19 0.18 0.19 0.18 0.01 0.19 0.01 0.19 0.01 Means

200

Table 76. Bilirubin-indirect (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.52 0.52 0.44 0.49 0.03 1st 0.64 0.61 0.42 0.56 0.07 2nd 0.45 0.54 0.58 0.52 0.04 3rd 0.53 0.52 0.04 0.55 0.53 0.47 0.51 0.02 0.02 0.48 0.04 Means

Table 77. Glutamate pyruvate transaminase (U/L) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 23.54 29.96 25.86 26.45 1.88 1st 28.59 23.96 29.00 27.18 1.62 2nd 26.73 28.46 26.23 27.14 0.68 3rd 22.46 29.41 23.91 25.26 2.12 25.33 1.42 27.95 1.36 26.25 1.05 Means

200

Table 78. Alkaline phosphatase (U/L) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 207.23 217.14 222.50 215.62 4.47 1st 236.64 228.54 237.77 234.32 2.91 2nd 199.73 220.82 192.54 204.36 8.49 3rd 210.14 196.96 9.10 227.58 243.82 179.41 206.73 19.22 5.91 208.06 13.39 Means

Table 79.

Mean squares for effect of fortified cookies on renal function tests of lactating women

SOV Treatments (T) Months (M) TxM Error Total

* = P 0.05 NS = Non-significant

df 2 3 6 252 263

Urea 115.807NS 433.682* 203.155NS 131.536

Creatinine 0.001NS 0.066NS 0.077NS 0.080

Table 80. Urea (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 35.68 35.09 31.18 33.98 1.41a 1st 32.32 33.96 36.36 34.21 1.17a 2nd 29.09 28.46 33.77 30.44 1.67ab 3rd 32.40 32.50 1.35 30.63 25.00 29.77 29.09 2.19b 2.37 32.77 1.46 Means

Means SEM Mean values carrying same letters in a row are not significantly different Baseline = observations taken in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 81. Creatinine (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 0.89 0.76 0.76 0.80 0.04 1st 0.77 0.73 0.79 0.76 0.02 2nd 0.74 0.83 0.84 0.80 0.03 3rd 0.80 0.81 0.03 0.80 0.88 0.83 0.84 0.02 0.03 0.81 0.02 Means

Table 82.

Mean squares for effect of fortified cookies on lipids profile of lactating women

SOV Treatments (T) Months (M) TxM Error Total

df 2 3 6 252 263

Cholesterol 607.714NS 1553.589NS 195.636NS 1230.890

LDL 78.786NS 755.958NS 332.375NS 582.491

HDL 231.007NS 261.564NS 191.322NS 214.006

Triglycerides 249.006NS 97.232NS 842.628NS 1123.894

NS = Non-significant

Table 83. Cholesterol level (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 150.27 150.56 149.55 150.13 0.30 1st 157.47 151.89 161.84 157.07 2.88 2nd 147.92 147.20 157.49 150.87 3.32 3rd 150.96 148.17 2.23 148.53 144.45 1.68 154.90 150.73 2.90 147.78 1.82 Means

Means SEM Baseline = observations taken in the last week of 9th month of pregnancy T1 = placebo T4 = 50% of RDA; retinyl acetate T7 = 50% of RDA; retinyl palmitate

Table 84. Low density lipoprotein (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 86.31 86.73 81.69 84.91 1.61 1st 88.34 88.04 90.56 88.98 0.79 2nd 81.12 79.76 91.06 83.98 3.56 3rd 84.49 81.31 81.80 82.53 0.99 2.02 86.28 2.62 85.07 1.53 83.96 Means

Table 85. High density lipoprotein (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 46.61 45.39 48.88 46.96 1.02 1st 50.60 45.76 52.46 49.61 2.00 2nd 47.62 48.89 48.73 48.41 0.40 3rd 47.71 46.02 1.02 46.41 45.59 0.83 50.14 50.47 0.87 47.36 1.56 Means

Table 86. Triglycerides (mg/dL) of lactating women Month of lactation Treatments Baseline Placebo (T1) Retinyl acetate (T4) Retinyl palmitate (T7) Means 86.73 92.20 94.88 91.27 2.40 1st 92.64 90.47 94.12 92.41 1.06 2nd 95.88 92.76 88.50 92.38 2.14 3rd 90.88 88.28 2.08 90.79 87.73 92.32 89.44 1.45 1.13 92.46 1.42 Means SEM

Table 87. Correlation Coefficients

Hct RBC MCV MCH MCHC Ret. WBC -caro. R.E. Hb 0.71** 0.85** 0.85** 0.70** 0.57* 0.95** -0.29NS 0.74** 0.21NS Hct 0.90** 0.94** 0.89** 0.94** 0.50* -0.44NS 0.79** 0.61** RBC MCV MCH MCHC Ret. WBC -caro. R.E.

0.96** 0.93** 0.84** 0.68** -0.52* 0.76** 0.42NS

0.89** 0.90** 0.67* -0.50* 0.84** 0.51*

0.84** 0.53* -0.37NS 0.56* 0.37NS

0.34NS -0.46NS 0.72** 0.58*

-0.17NS 0.57* -0.04NS

0.05NS -0.59*

0.76**

1.00

* = Significant ** = Highly significant NS = Non-significant Hct = Hematocrit RBC = Red Blood Cells MCV = Mean Corpuscular Volume MCH = Mean Corpuscular Hemoglobin MCHC = Mean Corpuscular Hemoglobin Concentration Ret. = Retinol WBC = White Blood Cells -caro. = -carotene R.E. = Retinyl esters

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