University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION BY SYLVIA ADOMA OTENG (10336014) THIS DISSERTATION IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN DIETETICS JULY, 2018 Univ ersity of Ghana http://ugspace.ug.edu.gh DECLARATION I, Oteng Sylvia Adoma hereby declare that this dissertation is the result of my own research work carried out in the Department of Nutrition and Dietetics, School of Biomedical and Allied Health Sciences, University of Ghana, under the supervision of Mrs. Freda Intiful and Mrs Rebecca Steele- Dadzie, and neither the whole nor any part of it has been or is being or is to be submitted for another degree at this or any other university. All references cited have been fully acknowledged. ………………………………… ……...………. Miss. Sylvia Adoma Oteng Date (Student) ……………………………… …..………….. Mrs. Freda Intiful Date (Supervisor) …………………………… ……………… Mrs. Rebecca Steele-Dadzie Date (Supervisor) ii Univ ersity of Ghana http://ugspace.ug.edu.gh DEDICATION To God Almighty for His unfailing Grace and help. I am most grateful. Also to my parents Mr. George Oteng and Rita Boakye-Bugyei for always loving me and my siblings unconditionally and providing for us wholeheartedly. Thank you very much Izey and Afua Mama. God richy bless you all. Finally , I dedicate this work to my siblings Angela, Gerty, Kwaku and Ryna for their care, love and friendship. You are the best little siblings anyone could ask for. iii Univ ersity of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am very grateful to God for His Grace for a successful completion of this research and my MSc education. It has been Jesus at the centre of it all. My sincerest gratitude to my supervisors, Mrs. Freda Intiful and Mrs. Rebecca Steele- Dadzie for their dedication and help towards the completion of this study. I really appreciate it and God bless you in diverse ways. I sincerely appreciate the Head of Department of Nutrition and Dietetics, Dr. Matilda Asante and other lecturers of the department for their support and guidance. I wish to also express my gratitude to the Distict Education Officer of LEKMA and TMA, heads of the schools, parents and guardians for consenting to their wards to participate in this study. I am also grateful to all participants of this study. Special thanks to Miss. Portia Dzivenu for her immense help, Bernice Aseidu, Prof. George Asare, Obed Harrison and Mr. Samson. To my course mates, especially Faustina and Pamela thank you for the valuable memories and encouragements in this past two years. Finally to my number one fan Lukiki, thanks for being part of my life. iv Univ ersity of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: Adequate serum vitamin D levels in children has been proven to improve bone health, reduce the risk of type 1 diabetes, cardiovascular diseases and many other diseases in life. The serum 25-hydroxyvitamin D is the best biomarker in measuring vitamin D status in the body. It is able to reflect both vitamin D levels that the body receive from sunlight and dietary sources. Little is known about the vitamin D status of the population in Ghana especially among school age child. Aim: To determine the vitamin D status of school age children between 8-12 years. Methods: A cross-sectional study design was used in this study. Ninety-nine (99) participants were recruited from four (2 public and 2 private) schools in the Ledzokuku/Krowor constituency using the stratified and systematic random sampling. A structured interviewer administered questionnaire was used to collect their socio- demographic data. Participant’s dietary intakes, length of sunlight exposure and anthropometric measurement were also measured. Serum vitamin D levels were determined using an ELISA test kit. Statistical Package for Social Sciences (SPSS) Version 20.0 was used to analyze the data obtained. Chi-square test was used to determine the association between the categorical variables while independent t-test was used to find the mean differences between the vitamin D status of the school groups and gender. Statistical significance was set as p<0.05. Results: There was significant association between dietary vitamin D intakes and serum vitamin D levels of the respondents (p = 0.008). School children had very low sunlight exposure. Thirty-three (33%) spent <15mins/day in the sun everyday and 36% exposed v Univ ersity of Ghana http://ugspace.ug.edu.gh themselves to sunlight during the hours of 10am-12:59 pm. Majority (73%) of the school children exposed themselves to sunlight for more than 3 days. Almost half (49%) of the participants had deficient vitamin D levels with majority (75%) reporting from the public schools.There was no significant association between anthropometric measurement (BMI- for-age) and vitamin D status. Males were 1.35 times more likely to be vitamin D sufficient compared to females. Public school children were 0.07 times less likely to be vitamin D sufficient compared to their counterparts in private schools. Conclusion: Majority of the school children had low dietary intakes of vitamin D rich foods. Also, the school children had a low length of sunlight exposure. The results from this study shows relatively low levels of serum vitamin D in 8-12 year old Ghanaian school age children and this could be as a result of low dietary intakes of vitamin D and low sunlight exposure. More of the school children in the public schools had deficient levels of vitamin D, compared to the private school children. Furthermore, there was no observed association between serum vitamin D status and anthropometric measurement. vi Univ ersity of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ............................................................................................................. ii DEDICATION ................................................................................................................ iii ACKNOWLEDGEMENT .............................................................................................. iv ABSTRACT ......................................................................................................................v TABLE OF CONTENTS ............................................................................................... vii LIST OF TABLES .......................................................................................................... xi LIST OF FIGURES ....................................................................................................... xii LIST OF ABBREVIATIONS ....................................................................................... xiii CHAPTER ONE ...............................................................................................................1 1.0 INTRODUCTION ......................................................................................................1 1.1 Background ............................................................................................................... 1 1.2 Problem Statement .................................................................................................... 3 1.3 Significance of Study ................................................................................................ 4 1.4 Aim and Specific Objectives ..................................................................................... 4 1.4.1 Aim ..................................................................................................................... 4 1.4.2 Specific Objectives ............................................................................................. 5 CHAPTER TWO ..............................................................................................................6 2.0 LITERATURE REVIEW ...........................................................................................6 2.1 Sources of Vitamin D ................................................................................................ 6 2.2 Vitamin D Biochemistry ........................................................................................... 6 2.3 Synthesis of Vitamin D ............................................................................................. 9 2.4 Vitamin D Status In Africa...................................................................................... 11 2.5 Effects Of Vitamin D Deficiency In Africa ............................................................ 12 2.6 Vitamin D in Children ............................................................................................. 13 vii Univ ersity of Ghana http://ugspace.ug.edu.gh 2.7 Factors Affecting Serum Vitamin D Levels ............................................................ 15 2.8 Vitamin D Levels .................................................................................................... 16 2.9 Measurement of Vitamin D Levels ......................................................................... 16 2.10 Effects of Vitamin D on the Skeletal System ........................................................ 17 2.11 Effect Of Vitamin D on Non-Skeletal Systems .................................................... 18 2.11.1 Relationship between Vitamin D and Cancer................................................. 19 2.11.2 Relationship between Vitamin D and Diabetes .............................................. 20 2.11.3 Relationship between Vitamin D and Cardiovascular System ....................... 21 2.11.4 Relationship between Vitamin D and Muscle Function ................................. 21 2.11.5 Relationship between Vitamin D and the Immune System ............................ 22 2.12 Sunlight Exposure Questionnaire .......................................................................... 24 CHAPTER THREE ........................................................................................................26 3.0 METHODS ...............................................................................................................26 3.1 Study Design ........................................................................................................... 26 3.2 Study Site ................................................................................................................ 26 3.3 Study Population ..................................................................................................... 27 3.3.1 Inclusion Criteria ........................................................................................ 27 3.3.2 Exclusion Criteria ............................................................................................. 27 3.4 Sampling Technique ........................................................................................... 27 3.5 Sample Size Determination ..................................................................................... 27 3.6 Pre-testing Questionnaires....................................................................................... 29 3.7 Data Collection ........................................................................................................ 29 3.7.1 Socio-Demograhic Information ........................................................................ 29 3.7.2 Dietary Intake Assessment ............................................................................... 30 3.7.3 Sunlight Exposure Questionnaire ..................................................................... 30 3.7.4 Anthropometric Measurements ........................................................................ 30 3.7.5 Determination of Serum Concentrations of Vitamin D .................................... 31 3.8 Data Management Plan ........................................................................................... 32 viii Univ ersity of Ghana http://ugspace.ug.edu.gh 3.9 Statistical Analysis .................................................................................................. 32 3.10 Ethical Approval ................................................................................................... 33 CHAPTER FOUR ...........................................................................................................34 4.0 RESULTS .................................................................................................................34 4.1 Socio-Demographic Information............................................................................. 34 4.2 Body Mass Index for Age Classification of Participants ........................................ 37 4.2.1 Body Mass Index (BMI) for Age Categorisation based on school types ......... 37 4.3 Length of Sunlight Exposure based on school type and gender ............................. 38 4.4 Serum Vitamin D Status in Children....................................................................... 41 4.4.1 Serum vitamin D status based on school type and gender ............................... 41 4.4.2 Serum vitamin D status and Anthropometric Measurements ........................... 42 4.4.3 Serum vitamin D status and Length of sunlight Exposure ............................... 43 4.4.4 Relationship between Serum Vitamin D and School Type, Gender and Length of Sunlight Exposure ................................................................................................. 45 4.5 Dietary Vitamin D Intakes of respondents .............................................................. 48 4.5.1 Vitamin D Rich Foods Intake and Serum Vitamin D Status ............................ 48 4.5.2 Dietary vitamin D- rich foods intakes based on gender ................................... 53 4.5.3 Dietary vitamin D-rich foods intake based on school type .............................. 54 CHAPTER FIVE ............................................................................................................56 5.0 DISCUSSION ...........................................................................................................56 5.1 Dietary vitamin D intakes in school children .......................................................... 56 5.2 Length of sunlight exposure of school children ...................................................... 57 5.3 Serum vitamin D status in school children.............................................................. 59 5.4 Serum vitamin D status in private and public school children ................................ 60 5.5 Association between serum vitamin D status and anthropometric measurement ... 61 5.6 Serum vitamin D status and length of sunlight exposure ........................................ 61 5.7 Limitations .............................................................................................................. 63 CHAPTER SIX ...............................................................................................................64 ix Univ ersity of Ghana http://ugspace.ug.edu.gh CONCLUSIONS AND RECOMMENDATION ...........................................................64 6.0 CONCLUSION ....................................................................................................... 64 6.1 RECOMMENDATIONS ........................................................................................ 65 REFERENCES ...............................................................................................................66 APPENDIX I ..................................................................................................................75 APPENDIX II .................................................................................................................79 APPENDIX III ................................................................................................................81 APPENDIX IV................................................................................................................94 ETHICAL APPROVAL .................................................................................................94 x Univ ersity of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1. 1 Serum Vitamin D levels indicating Deficiency, Insufficiency and Sufficiency .........................................................................................................................16 Table 4. 1 Socio-demographic characteristics of participants (N =99) ............................ 34 Table 4. 2 Body Mass Index for age categorisation in public and private schools (N=99) ........................................................................................................................................... 38 Table 4. 3 Length of sunlight exposue based on school type and gender (N=99) ............ 40 Table 4.4. 1: Serum vitamin D status in school children (N=53). .................................... 41 Table 4.4. 2: Serum vitamin D status based on school type and gender (N=53) .............. 42 Table 4.4. 3: Serum vitamin D status based on BMI-for-Age (N=51) ............................. 43 Table 4.4. 4: Serum vitamin D status based on Length of Sunlight Exposure (N=53) .... 44 Table 4.4. 5: Relationship between Serum vitamin D status and Gender, School groupings and Length of sunlight exposure ....................................................................................... 47 xi Univ ersity of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 4. 1: Body Mass Index-for-age of respondents ..................................................... 37 xii Univ ersity of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS 1, 25(OH)D – 1, 25-dihydroxyvitamin D 7DHC- 7-dehydrocholecalciferol 25(OH)D – 25- hydroxyvitamin D CDK- Cyclin Dependent Kinase CKI- Cyclin Kinase Inhibitors CVDs- Cardiovascular Diseases DBS- Dried blood spot DBP- Vitamin D Binding Protein EARs- Estimated Average Requirements FG- Fibroblasts Growth Factor LC-MS/MS- Liquid chromatography –tandem mass spectrometry IOM- Institute of Medicine MAPK- Mitogen-activated protein kinase MHC- Major Histocompatibility Complex PTH- Parathyroid hormone RAS- Renin Angiotensin System RDAs- Recommended Daily Allowances xiii Univ ersity of Ghana http://ugspace.ug.edu.gh RXR- Retinoid X receptor TLRs- Toll- like receptors UV- Ultraviolet UVA- Ultra-violet A UVB- Ultra-violet B UVC- Ultra-violet C VBP – Vitamin binding protein VDR- Vitamin D receptor VDRE- Vitamin D response elements VDCC- Voltage Dependent Calcium Channels xiv Univ ersity of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Vitamin D refers to a group of fat-soluble compounds that are involved in calcium homeostasis, bone metabolism and mineralisation (Pettifor, 2014). It is usually obtained through synthesis in the skin by exposure to the sun and also through consuming food (Holick, 2009). Natural vitamin D rich foods are; egg yolk, liver, cod liver oil, sardine, mackerel, salmon, tuna, soy milk, margarine, butter, cheese, carrots, avocado, mango, pawpaw, broccoli, sweet potato, pepper, and mushrooms. Some foods are usually fortified with vitamin D and these include milk and milk products, fortified orange juice, cereals and supplements (O'Mahony, Stepien, Gibney, Nugent, & Brennan, 2011). Naturally, vitamin D occurs in two main forms: vitamin D2 (ergocalciferol) which is synthesised in plants, and vitamin D3 (cholecalciferol) also synthesised in the skin of animals and humans from 7-dehydrocholecalceferol (7DHC) in response to sunlight, particularly to ultraviolet B radiations of appropriate wavelength (270–300 nm) (Weydert, 2014). After synthesis, vitamin D is stored in the adipocytes of the body and made accessible for conversion to its active form, calcitriol [1,25(OH)2D] when needed (Weydert, 2014). Cholecalciferol is changed to 1, 25- dihydroxyvitamin D by two hydroxylation steps (Kamen & Tangpricha, 2010). In the liver, cholecalciferol (vitamin D3) is converted to calcidiol (25[OH]D), which is the best indicator of serum vitamin D 1 Univ ersity of Ghana http://ugspace.ug.edu.gh status (Kamen & Tangpricha, 2010). Serum 25(OH)D levels reflect both cutaneous synthesis and dietary intake (Weydert, 2014). In the kidneys, calcidiol [25(OH)D] is converted to calcitriol [1,25(OH)2D,], the biologically active metabolite of vitamin D. Furthermore, 1,25(OH)2D is activated by binding to the nuclear vitamin D receptor (VDR) within the cells (Shin, Shin, & Lee, 2013). The VDR is expressed all through the body and mostly found in the endocrine glands and cardiovascular tissues (Holick et al., 2011). The Vitamin D receptors are also found in hematolymphopoietic cells, which help to regulate cell differentiation and the production of immune proteins such as interleukins and cytokines (Shin et al., 2013). In addition to its significant role in calcium and bone homeostasis, vitamin D performs other cellular regulatory functions due to its numerous endocrine and paracrine functions (Weydert, 2014). The recommended daily intakes (RDIs) of vitamin D for children up to 1 year is at least 400 IU/day and above 1 year is at least 600 IU/day for optimal bone health (Holick et al., 2011). This Recommended Daily Allowances (RDA) is enough to provide all the potential non-skeletal health benefits associated with the vitamin to meet optimal bone health and muscle function (Holick et al., 2011). However, to preserve serum 25(OH)D levels consistently above 30 ng/ml (75 nmol/L) a recommended daily intake of 1000 IU/day of vitamin D is required (Holick et al., 2011). In healthy individuals, vitamin D deficiency is usually as a result of either reduced sunlight exposure, a reduced ability to synthesise vitamin D or decreased dietary intake. There is no agreement on the definition of vitamin D deficiency (Holick, 2009; Holick et al., 2011; Shin et al., 2013); however, most clinicians and researchers agree on the following 2 Univ ersity of Ghana http://ugspace.ug.edu.gh stratifications based on the serum concentration of 25(OH)D: deficiency, <50.0 nmol/L or <20.0 ng/mL; insufficiency, 50.0–74.9 nmol/L or 20.0–29.9 ng/mL; and sufficiency, ≥75.0 nmol/L or ≥30.0 ng/mL (Holick, 2009; Shin et al., 2013). The calcidiol, [25(OH) D] is the major circulating form of vitamin D and the best indicator of vitamin D status because of its longer half-life of two-three weeks. Although calcitriol [1, 25 (OH)2D] is the metabolic active form, it has a half-life of 4 hours and therefore, it is not a good indicator of vitamin D stores (Balasubramanian, Dhanalakshmi, & Amperayani, 2013). 1.2 Problem Statement It is well established that sunlight exposure improves the Vitamin D status in children. Vitamin D is essential to child growth particularly in proper bone formation and metabolism (Pettifor, 2014). It also provides extra-skeletal benefits such as in immune function and prevention of certain cancers like colorectal cancer (Pettifor, 2014). Most children obtain Vitamin D from their diets which are insufficient to meet their RDA of Vitamin D intake (Bentley, 2015). Also, children are usually vitamin D deficient especially those with dark skin require up to six times more sunlight exposure than light- skinned individuals because of their skin pigmentation (Green et al., 2015). Anecdotal evidence in recent times in Ghana suggests changing lifestyles resulting in the substitution of the former practice of children walking in the open sun to school for driving to school in air-conditioned cars with little or no exposure to sunlight. Additionally fewer and shorter breaks during school hours resulting in little play time in the open as well as replacement 3 Univ ersity of Ghana http://ugspace.ug.edu.gh of open games in the sun with in-door games at home for security and other reasons has affected the amount of sun exposure that children have in recent times (Pellegrini & Bohn- Gettler, 2013). Deficient vitamin D levels in children result in osteomalacia leading to bowed legs and rickets and skeletal deformities in children, reduced immune function, autoimmune diseases, common infections, cardiovascular diseases, cancers and even the tendency of having type 1 diabetes in adulthood (Holick et al., 2011; Weydert, 2014). In Ghana, there is scanty published information on serum vitamin D levels, especially in children. This, coupled with the observed changing lifestyles has informed the decision to investigate serum vitamin D levels among children. 1.3 Significance of Study This study will assess and provide information on the nutritional status and nutrient intake adequacy of vitamin D and the impact on their health. This will serve as the basis for dietary advice in order to improve their conditions and reduce the effect of the deficiencies. It will also contribute to knowledge by providing information that may assist dietitians to plan, develop and implement policies on nutrition and dietary practices of children. This study may also inform future research into vitamin D related conditions in Ghana. 1.4 Aim and Specific Objectives 1.4.1 Aim To determine the vitamin D status of school age children (8-12 years). 4 Univ ersity of Ghana http://ugspace.ug.edu.gh 1.4.2 Specific Objectives The specific objectives were: 1. To determine the consumption of vitamin D rich foods in the school children. 2. To determine the length of exposure to sunlight during the day. 3. To determine the serum vitamin D levels of school age children. 4. To compare vitamin D status in private and public school age children. 5. To determine the association between vitamin D status and anthropometric status in the school children. 5 Univ ersity of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Sources of Vitamin D Vitamin D is known as the ‘sunshine vitamin' because upon exposure to the sun, the body is able to provide about 10,000- 20,000 IU of vitamin D when at least thirty percent (30%) of body surface area is exposed unprotected (Weydert, 2014). Vitamin D3 (cholecalciferol) is synthesised in the skin of animals and humans from 7-dehydrocholesterol (7DHC) in response to sunlight, particularly to ultraviolet B radiations of appropriate wavelength (Wagner & Greer, 2008). In contrast, food provides a very limited amount of vitamin D to the body i.e. 100- 200IU (O'Mahony et al., 2011). Plant sources provide the body with vitamin D2 (ergocalciferol) which is photochemically synthesised in plants. Some plant sources include; mushrooms, soy milk (O'Mahony et al., 2011). The animal sources include salmon, tuna, sardine, mackerel, eel, herring, pilchards, trout, kippers, cod liver oil, egg yolk, meat, offal, milk and its products (O'Mahony et al., 2011). Some foods are also fortified with vitamin D and these include fortified orange juice, cereals, butter, cheese, margarine, yoghurt and vitamin D supplements (National Insitute of Health, 2016). 2.2 Vitamin D Biochemistry According to Weydert (2014), vitamin D is a steroid hormone. Vitamins are typically generated from foods and are normally antioxidants and co-factors of enzymatic reactions 6 Univ ersity of Ghana http://ugspace.ug.edu.gh while steroid hormones are gene expression regulators (Weydert, 2014). Vitamin D is mainly formed by the activation of plant and animal sterols, phytosterols, and cholesterol with the help of sunlight (Wagner & Greer, 2008). In plants, the sterol used in producing vitamin D is vitamin D2 but in animals and humans, it is the 7-dehydrocholesterol (7HDC). The 7HDC is a vitamin D precursor which is mainly found in the outer layer of the skin and usually stimulated by the sunlight to produce cholecalciferol which is attached to a vitamin-binding protein (VBP) (Wagner & Greer, 2008). The vitamin D3 is transported to the liver where it is converted to calcidiol [25(OH)D] by the enzyme, vitamin D-25- hydroxylase (Binkley, Ramamurthy, & Kruger, 2015). The 25-hydroxyvitamin D is also considered as an inactive innate pro-hormone (Holick, 2009). Furthermore, an enzyme 1,25 di-hydroxyvitamin D-1-α-hydroxylase converts calcidiol (25(OH)D) into the metabolically active form, calcitriol (1,25(OH)2D) (Huh & Gordon, 2008). The calcitriol regulates about two hundred genes by either acting directly or indirectly on them by binding VDR that helps in a lot of biological processes (Weydert, 2014). The conversion of the 25(OH)D to the 1,25(OH)2D mainly takes place in the kidneys and is closely controlled by the levels of parathyroid hormone (PTH), calcium and phosphorus in the body (Huh & Gordon, 2008). The 1, 25(OH)2D has extraordinary endocrine roles which also regulates both serum calcium levels and bone metabolism (Fondjo et al., 2017). Conversion of the vitamin to its biologically active form also occurs in other parts of the body such as the breast, brain, skin and other immune parts such as the monocytes and macrophages (Weydert, 2014). As a result of this production, the 1,25(OH)2D controls the differentiation, proliferation, and apoptosis of cells as well as 7 Univ ersity of Ghana http://ugspace.ug.edu.gh performing immune functions in these locations of production (Karagüzel, Sakarya, Bahadır, Yaman, & Ökten, 2016). With these functions, vitamin D affects cells either by its autocrine or paracrine functions which are usually autonomously controlled. An adequate level of 25(OH)D is important for the optimal production of calcitriol [1, 25(OH)2D]. Dietary vitamin D sources are usually limited and some sources include salmon, egg yolk, tuna, sardine, cod-liver oil, and mushrooms which usually provides about 100 to 200 IU/day (British Dietetic Association, 2013). Meanwhile, when about 30% of the body surface area is exposed to sunlight for about 15 to 30 mins/day about 10,000 to 20,000IU of vitamin D is produced (Weydert, 2014). The sunlight usually produces Ultra- violet A (UVA), Ultra-violet B (UVB) and Ultra-violet C (UVC) rays but it is only the UVB rays that stimulate the 7-dehydrocholesterol in the skin (Weydert, 2014). The UVB rays which are most effective in vitamin D production are available when the sun is at right angles to the earth's surface between the hours of 10 am and 3 pm. Sunlight is also usually affected by seasons, time of the day and the latitude (Al-Saleh et al., 2015). Also, other factors such as the use of sunscreens, dark-skinned individuals, and people in institutionalized facilities such as prisons, schools, hospitals and nursing homes are exposed to little sunlight (Weydert, 2014). Dark skinned individuals require about 10 to 15 times exposure to sunlight to produce the same amount of sunlight as in light-skinned individuals due to the presence of melanin in dark-skinned individuals (Weydert, 2014). The melanin readily absorbs UV radiations and competes for UVB rays necessary for vitamin D production. Furthermore, the use of clothing that covers the body surface area as accustomed by some cultures decreases exposure to UVB rays (Al-Saleh et al., 2015). 8 Univ ersity of Ghana http://ugspace.ug.edu.gh Obesity also affects the quantity of serum vitamin D synthesis in the skin due to the presence of subcutaneous fat which hides away synthesised vitamin D making it unavailable for conversion to its biologically active form (Flores et al., 2017). Additionally, certain drugs such as phenobarbital, valproic acid compete with vitamin D (Bikle, 2014). Medical conditions such as cystic fibrosis, Crohn's disease, and liver and kidney diseases also affect vitamin D status and use (Weydert, 2014). 2.3 Synthesis of Vitamin D Vitamin D production begins in the skin (Mostafa & Hegazy, 2015). The skin contains the pro-vitamin D which enables the skin to produce pre-vitamin D3 from absorbed UVB photons (Battault et al., 2013). Subsequently, this is followed by a thermal-dependent isomerization process which results in the production of cholecalciferol (Battault et al., 2013). In long periods of sunlight exposure, the previtamin D3 is photo-isomerized to lumisterol and tachysterol which are also biologically inactive. After cholecalciferol is formed, it binds to the vitamin D-binding protein (DBP) which enables it to be sent into the bloodstream. Also, vitamin D can be obtained from the foods we eat in the form of cholecalciferol or as ergocalciferol. Cholecalciferol is normally obtained from animal sources whilst ergocalciferol is from plant sources (O'Mahony et al., 2011). Ingested vitamin D is absorbed and transported in the chylomicrons then binds to the vitamin D binding proteins until it undergoes hydroxylation in the liver. In the liver, the hydroxylation occurs on the 25th carbon molecule by the hepatic cytochrome P-450 enzyme, CYP2R1 which is 9 Univ ersity of Ghana http://ugspace.ug.edu.gh mitochondrial and acts as the 25-hydroxylase (Battault et al., 2013). A malfunction of the CYP2R1 results in rickets. The 25(OH)D (calcidiol) is the major circulating form of vitamin D which also reflects the body's stores of vitamin D. The calcidiol reflects both the dietary and cutaneous stores of vitamin D. The serum 25(OH)D is the main biomarker of vitamin D status. Vitamin D also performs endocrinal functions in the kidneys (Holick et al., 2011). The proximal tubule is the site for CYP27B1, i.e. it supports the activities of the 1-α- hydroxylase enzyme. The 1-α-hydroxylase enzyme converts calcidiol to its active metabolite calcitriol, (1 α, 25(OH)2D) (Battault et al., 2013). The active calcitriol enters the bloodstream and acts as a hormone on distant organs and cells. Two important roles of active calcitriol are: to intensify the rate of absorption of calcium and phosphorus in the intestine and to promote the formation of pre-osteoclasts to osteoclasts (Battault et al., 2013). Calcitriol also plays an important role in the suppression of renin production in the kidneys and stimulates the secretion of insulin from the pancreas (Legarth, Grimm, Wehland, Bauer, & Krüger, 2018). In some organs and tissues such as the muscles, pancreas and colon, the 25(OH)D converts to 1-α-25(OH)2D in an extra-renal conversion process. These sites of production also provide active calcitriol in times of need (Girgis, Clifton-Bligh, Hamrick, Holick, & Gunton, 2013). The active calcitriol binds to the VDR which belongs to the nuclear receptor superfamily. The activated calcitriol also binds with the retinoid X receptor (RXR) and forms a 1α, 25(OH)2D-VDR-RXR complex. The complex attaches to vitamin D responsive elements (VDREs) which regulate the transcription of different genes in cells (Girgis et al., 2013). 10 Univ ersity of Ghana http://ugspace.ug.edu.gh 2.4 Vitamin D Status In Africa Africa is the world's second largest and populated continent after Asia, covering an area of about 30 million km2 (Prentice, Schoenmakers, Jones, Jarjou, & Goldberg, 2009). Africa is about twenty percent of the earth's total land area. It is the only continent that lies on both sides of the equator and has both northern and southern temperate zones. The distance between the most northern (*37_N in Tunisia) and most southern (*34_S in South Africa) points is 8000 km. From the most western (*17_W, Cape Verde) to the most eastern (*51_E in Somalia) points is 7400 km (Jung, Prange, & Schulz, 2016). Most large African countries lie on more than one latitude band (Jung et al., 2016). The climate ranges from tropical to subarctic and the topography includes deserts, mountains, grassland plateaus, lakes, and rivers. The weather in Africa is also very variable, and temperatures vary greatly throughout the continent. Most countries have wet and dry seasons which affects many activity levels in the continent (Prentice et al., 2009). Africa is made up of 50 countries which differ in sizes and population. Most Africans live in urban areas. Across the continent, 40% are Christians, 40% are also Muslims and the remaining 20% belong to other religious groups (Prentice et al., 2009). There is a great deal of disparity within and between countries and their inhabitants. Some spiritual views and traditional practices can impact on the customary way of dressing and dietary habits. In Africa, variations in cutaneous vitamin D synthesis would be anticipated in countries such as Morocco, Tunisia, Algeria, Libya, Egypt, and South Africa which lie at latitudes >30oN and > 30oS due to differences in seasons. In countries close to the equator, 11 Univ ersity of Ghana http://ugspace.ug.edu.gh differences in serum vitamin D levels might be expected due to differences in cloud cover (Prentice et al., 2009). In the Gambia, insignificant effects of variations in serum 25(OH)D levels were found by Prentice, Ceesay, Nigdikar, Allen, & Pettifor (2008). However in older people, low concentrations of serum 25(OH)D concentration were observed from December–February by Prentice, (2008) due to seasonality. The African diet is heterogeneous and differs from country to country (Jung et al., 2016). The African diet is also affected by the climate and geography of a population which determines what is usually consumed by the population (Prentice et al., 2009). Recently, there has been a transition from indigenous foods to westernized foods due to the influx of westerners into the continent and this differs across the continent (Prentice et al., 2009). There is scanty information on the dietary intake of vitamin D rich foods by Africans. Even though the significant effect of season on serum 25(OH)D levels were found, intakes of vitamin D rich foods provide a limited amount of vitamin D to the body, most Africans consume little or do not include them in their regular diets at all (Prentice et al., 2009). Dietary sources of both calcium and vitamin D in Africa were below recommended levels in Nigeria, Kenya and South Africa (Prentice et al., 2009). The African diet includes fewer sources of dairy products and also contains high amounts of phytates, oxalates, and tannins which reduce the absorption of calcium (Prentice et al., 2009). 2.5 Effects Of Vitamin D Deficiency In Africa The primary effects of poor vitamin D status are rickets and osteomalacia (Bakare- Odunola, Kalik-Zik, Garba, Odunola, & Bello-Mustapha, 2012), but there is also emerging 12 Univ ersity of Ghana http://ugspace.ug.edu.gh evidence for a role of vitamin D in alleviating the progression or severity of TB and HIV/AIDS (Bartley, 2010). The immunomodulatory effects of vitamin D mean there are many potential health consequences of vitamin D deficiency in Africa where the infectious disease burden is high. Vitamin D deficiency may impact on the immune system by decreasing innate immunity and immune surveillance; decreasing T lymphocyte number and function; and disrupting the Th1/Th2 balance (vitamin D normally inhibits Th1 profile) (Bartley, 2010; Kamen & Tangpricha, 2010). Vitamin D deficiency in an African setting may influence the progression of communicable diseases such as TB, HIV, and schistosomiasis. Well-established health consequences of vitamin D deficiency are rickets and osteomalacia. Although vitamin D deficiency rickets is reported in Africa, there are very little data on the burden the disease poses and on its association with malnutrition, poverty and other factors (Prentice et al., 2009). Therefore, it is challenging to ascertain the degree to which vitamin D deficiency is a cause of rickets. 2.6 Vitamin D in Children Vitamin D is essential for the absorption of calcium from the food we eat (Pettifor, 2014). It acts as a prohormone, which works on receptors in the intestine, bone, and kidneys to regulate blood calcium and phosphate levels. It also interacts with the hormones calcitonin and PTH. Vitamin D is needed for maintaining normal nerve and muscle function in children (Ritu & Gupta, 2014). It is also essential in ensuring that calcium levels are available for blood clotting (Bentley, 2015). Vitamin D and calcium helps in the ossification of bones during infancy as early as six weeks in the embryonic stages 13 Univ ersity of Ghana http://ugspace.ug.edu.gh (Chibuzor, Graham-Kalio, Meremikwu, & Adukwu, 2017). Throughout childhood to adolescence, the bone continues to develop through the remodelling process which involves a build-up of minerals such as calcium and phosphorous (Bentley, 2015; Chibuzor et al., 2017). Below optimal levels of vitamin D in childhood is well tolerated especially in a short to medium duration such as during seasonal variations in Western countries. However, a constant inadequate supply of vitamin D predisposes children to rickets (Bentley, 2015). The RDA for infants to adolescents is 400IU/day, which has been shown to be insignificant for them, hence the need for supplementation especially in dark-skinned children and pre- term babies (Wagner & Greer, 2008). The Institute of Medicine (IOM) has definite Estimated Average Requirements (EARs) and Recommended Daily Allowances (RDA) of vitamin D based on serum 25(OH)D levels of 16 and 20 ng/mL, respectively (Rathi & Rathi, 2011). The EAR and RDA for vitamin D, as suggested by the IOM, are 400 IU/day and 600 IU/day respectively, while tolerable upper level of intake is 1,000 IU/day for infants <6 months old, 1,500 IU/day for 6-12 months old, 2,500 IU/day for 1-3 years old, 3,000 IU/day for 4-8 years old and 4,000 IU/day for 9 years and above including pregnant and lactating mothers (Rathi & Rathi, 2011). These RDA estimates have been made while considering the minimal cutaneous synthesis of vitamin D. Vitamin D levels as defined by the Endocrine Society are; deficiency: <50nmol/L (20ng/ml), insufficiency: 52.5-72.5nmol/L (21-29ng/ml) and deficiency: >72.5nmol/L (30- 100ng/ml) (Holick et al., 2011). Bentley (2015) showed that many children who have deficient vitamin D levels are usually asymptomatic and this is a threat to their bone growth 14 Univ ersity of Ghana http://ugspace.ug.edu.gh and development. In the UK, the prevalence of vitamin D deficiency in children is 12% to 14% (Bentley, 2015). Globally, there is a high prevalence of vitamin D deficiency despite different types of diet consumed, skin colour and abundant sunlight (Flores et al., 2017). 2.7 Factors Affecting Serum Vitamin D Levels Nutrient deficiencies are as a result of many factors like reduced absorption, inadequate dietary intake, impaired utilization, increased demand for nutrients and elimination. Vitamin D deficiency occurs when an individual consumes less than required over an extended period of time, decreased exposure to sunlight, kidneys abnormalities resulting in failure to convert calcidiol to calcitriol or as a result of malabsorption in the gastrointestinal tract (Mostafa & Hegazy, 2015). A vitamin D deficient diet is also associated with milk allergy, lactose intolerance, ovo-vegetarianism, and veganism. Cutaneous vitamin D synthesis has many determinants. These include the environmental factors, personal variations, an influence of some practices and clothing (Lips, van Schoor, & de Jongh, 2014). Environmental factors such as geographical latitudes, seasons, time of the day, weather conditions (cloudiness) and amount of air pollution affect the amount of UVB radiations that reach the skin. Also, personal variations like the skin type of a person, being elderly, obese, clothing habits and sun avoidance practices such as the use of sunscreen creams affect one's vitamin D cutaneous production (Touvier et al., 2015). 15 Univ ersity of Ghana http://ugspace.ug.edu.gh 2.8 Vitamin D Levels Calcidiol (25(OH)D) levels can be expressed in nanograms per millilitre (ng/ml) or as nanomoles per litre (nmol/L), and nmol/L can be converted to ng/ml by dividing by 2.5 (Bentley, 2015). Vitamin D status levels are not well defined. The conventional way of defining the normal levels of vitamin D is to identify the minimum levels of 25(OH)D that reduce the secretion of PTH at a plateau of 30ng/ml. Experts argue that 20 ng/mL of 25(OH)D is considered to be vitamin D deficiency, whereas a 21-29 ng/mL of 25(OH)D is considered to be insufficient. The aim is to maintain up to 30 ng/mL in order to have efficient vitamin D stores in both adults and children (Holick, 2009). Experts have now agreed that vitamin D deficiency status should be defined as 20 ng/mL of 25(OH)D (Holick et al., 2011). Table 1. 1 Serum Vitamin D levels indicating Deficiency, Insufficiency and Sufficiency British Paediatric and Adolescent Endocrine Society Bone Group (2014) (Holick et al., 2011) Deficiency < 25nmol/L (10ng/ml) <50nmol/L (≤ 20ng/ml) Insufficiency 25-50nmol/L (10-20 ng/ml) 52.5-72.5nmol/L (21-29ng/ml) Sufficiency >50nmol/L (>20ng/ml) >72.5nmol/L (30-100ng/ml) Adopted from Bentley (2015) 2.9 Measurement of Vitamin D Levels The type of vitamin D levels to be measured is determined by what type of clinical test to be conducted. Serum 25(OH)D is the major circulating form of vitamin D and also the best indicator of overall vitamin D status because of its half-life of about two to three weeks 16 Univ ersity of Ghana http://ugspace.ug.edu.gh (Holick, 2009). 1, 25(OH)2D is the metabolically active form of the vitamin which is influenced by 25(OH)D, PTH, calcium, and phosphorus (Holick, 2009). It is used to assess metabolic disorders of calcium related to the renal production of 1,25(OH)2D with a half- life of four to six hours (Holick, 2009). Measurement techniques such as radioimmunoassay and high-performance liquid chromatography are used to evaluate vitamin D status however the liquid chromatography- tandem mass spectrometry (LC-MS/MS) has been defined as the most accurate method to quantify 25(OH)D (Enko, Kriegshauser, Stolba, Worf, & Halwachs-Baumann, 2015). It measures 25(OH)D2 and 25(OH)D3 separately (Eko et al., 2015). Recently, it has been proposed that using the LC-MS/MS or the dried blood spot (DBS) sampling method for clinical testing and screening is a less invasive and risk-free method (Li & Tse, 2010). Also, the amount of blood required for this method is less invasive and suitable for children, uses less sophisticated storage and equipment, therefore, can be used in less privileged areas (Eyles et al., 2009). The LC-MS/MS method and the DBS sampling is known to be highly sensitive and correlates with serum levels (Eyles et al., 2009). 2.10 Effects of Vitamin D on the Skeletal System Calcitriol enhances the uptake of intestinal calcium through its nuclear VDR receptors hence increases the expression of the epithelial calcium channel and a calcium- binding protein. Active calcitriol also enhances the uptake of phosphate in the intestine (Battault et al., 2013). The actions of vitamin D can also control the reabsorption of phosphate from the kidneys as calcitriol encourages the synthesis of fibroblasts growth factor (FG23) which 17 Univ ersity of Ghana http://ugspace.ug.edu.gh is a chemical that acts to intensify the excretion of renal phosphate (Girgis et al., 2013). The FG23 reduces the expression of the renal sodium-phosphate co-transporter in the proximal tubule of the kidneys (Bergwitz & Juppner, 2010). 1 α, 25(OH)2D indirectly affects the formation of osteoclasts by modulating the evolution of pre-osteoclast to multinucleated osteoclasts (Battault et al., 2013). The matured osteoclasts have an effective bone resorption activity which leads to the release of calcium and phosphate into the bloodstream and this eventually results in bone neo-mineralization (Battault et al., 2013). 2.11 Effect Of Vitamin D on Non-Skeletal Systems Further studies on the activities of the 1 α, 25(OH)2D has led to a better knowledge on how the calcitriol works in cells either through the activities of its receptors, the 1-α- hydroxylase induction or via the 24-hydroxylase induction (Battault et al., 2013). Also, vitamin D is shown to have a direct effect on innate immunity. Vitamin D receptors are present in some components of the immune system such as the macrophages, monocytes, and lymphocytes. When serum calcidiol [25(OH)D] levels are below 20ng/mL, macrophages and monocytes are unable to initiate an innate immune response (Kamen & Tangpricha, 2010). Vitamin D deficiency has been proven to contribute to the incidence and severity of some infections, for instance, lower respiratory tract infections (Bartley, 2010). In TB infections, the immune cells are able to express VDR and the enzyme, 1-α- hydroxylase which help with the synthesis of 1,25-dihydroxyvitamin D activating the transcription of other immune defense cells (Bartley, 2010). Vitamin D Receptors (VDR) 18 Univ ersity of Ghana http://ugspace.ug.edu.gh in body cells are able to change vitamin D into its metabolically active form for the body's use which is very crucial to the body's extra-skeletal benefits of vitamin D. 1,25- dihydroxyvitamin D is a steroid hormone which is able to act as a gene expresser. 1,25- dihydroxyvitamin D attaches to the VDR with other ligand-activated transcriptional factors that displace in the body's cell. In the cell, the 1,25-dihydroxyvitamin D binds to the vitamin D response elements (VDRE) (Weydert, 2014). Vitamin D is able to regulate the expression of about 200 genes of transcripted proteins enabling the proteins to adjust to cellular differentiation, proliferation, and death (Kamen & Tangpricha, 2010). Furthermore, in children, vitamin D is crucial to boost immune health and reduce the risk of acquiring respiratory infections (Weydert, 2014). With this new knowledge vitamin D status is believed to be associated with the development of some diseases: 2.11.1 Relationship between Vitamin D and Cancer Studies have shown that a good vitamin D status reduces one's risk of cancers (Vuolo, Di Somma, Faggiano, & Colao, 2012). This is achieved through the activities of the 1α, 25(OH)2D which suppresses the proliferation and differentiation of cancer cells. The cyclin/cyclin-dependent kinase (CDK) complex promotes phosphorylation of target proteins included in the cell cycle development. The cyclin/CDK is controlled by CDK inhibitors (CKIs). CDKs and CKIs prevent the formation of CDK/cyclin complexes (Battault et al., 2013; Vuolo et al., 2012). 1α,25(OH)2D encourages growth arrest by regulating the transcription of cyclins and CKIs by enhancing the expression of three CKIs; p21, p27, and p53 which are included in the G1 cell arrest by turning off the expression of the S-dependent cyclin/CDK complex phase. 1,25(OH)2D3 induces the expression of p21 19 Univ ersity of Ghana http://ugspace.ug.edu.gh and p27 and related hypo-phosphorylation of the retinoblastoma protein, leading to G0/G1 cycle cell arrest (Battault et al., 2013). Another explained procedure is that calcitriol employs its anti-proliferative benefits through its effects on the anti-Wnt-β-catenin pathway (Deeb, Trump, & Johnson, 2007). Normally, the anti-Wnt-β-catenin-TCF4 pathway regulates the transcription of the genes involved in cycle cell control (C-MYC, PPARδ.). In a study by Tuohimaa (2008), the anti- proliferative properties of vitamin D prevent the transcriptional signal by promoting the translocation of the nuclear catenin to the plasma since C-myc promotes cell growth. Vitamin D also regulates the pro- and anti-apoptotic factors in the apoptotic process. Calcitriol reduces the expression of anti-apoptotic proteins BCL2 and BCLaXl and induces the expression of pro-apoptotic proteins of BAX, BAK and BAD. It also increases telomere shortening by preventing telomerase activity (Deeb et al., 2007). Furthermore, 1 α, 25(OH)2D3 also acts as an antiangiogenic or an anti-metastatic factor. 1 α, 25(OH)2D3 preserves the normal cell phenotype by inhibiting tumour-invasive potential. 1 α, 25(OH)2D3 also up-regulate the expressions of proteins included in cell adhesion and intercellular junction (Battault et al., 2013). These chemicals help maintain cell phenotypes and tissue structure. 2.11.2 Relationship between Vitamin D and Diabetes Calcitriol (1 α, 25(OH)2D) is able to elicit the expression of the human insulin receptor gene in U-937 human pro-monocytic cells (Battault et al.,2013). Calcitriol, [1α,25(OH)2D3] have an effect in pancreatic beta cell function, insulin sensitivity in 20 Univ ersity of Ghana http://ugspace.ug.edu.gh peripheral target cells and also indirectly affect systemic inflammation (Battault et al., 2013; Legarth et al., 2018). This could result in desirable insulin sensitivity hence indirectly reducing the likelihood of CVDs as a result of uncontrolled glycaemia. 2.11.3 Relationship between Vitamin D and Cardiovascular System A good vitamin D status helps reduce the occurrences of CVDs. 1 α, 25(OH)2D indirectly affect blood pressure as it reduces the effects of PTH. Vitamin D acts on the renin- angiotensin system (RAS) which controls blood pressure and reduces the production of renin (Legarth et al., 2018). Hence, 1 α, 25(OH)2D can reduce hypertension by retarding the renin-angiotensin system (RAS). In the heart, sarcomere contraction is regulated by 1α,25(OH)2D3 through the effects of the VDR in the membrane. Also, Zhao and Simpson, (2010) have shown that 1α,25(OH)2D3 also affects the interaction between VDR and caveolin-3 which triggers the pathway of cardiomyocyte contraction. 2.11.4 Relationship between Vitamin D and Muscle Function Poor vitamin D status often leads to rickets and osteomalacia (Battault et al., 2013). Vitamin D receptors can be found in the mammalian skeletal muscle cells particularly the nucleus and the plasma membrane (Girgis et al, 2013). These receptors on muscle cell surfaces enhance uptake of calcium. During vitamin D deficiency, high levels of atrophy and contractility disorders are indicated. 1α,25(OH)2D regulates the exchange of calcium between the muscle cell and intracellular calcium. Therefore a good balance of calcium is important for muscle contraction and relaxation (Girgis et al., 2013). The presence of 21 Univ ersity of Ghana http://ugspace.ug.edu.gh 1α,25(OH)2D in the muscle cell results in an enhanced uptake of calcium and a release from intracellular calcium. This is usually mediated by the voltage-dependent calcium channels (VDCC) and the store-operated Ca2+ channel (SOC) (Girgis et al., 2013). Many intracellular pathways activate the 1α,25(OH)2D-Ca2+ channel. This includes the G- protein stimulation which activates the phospholipase C and adenylyl cyclase channels consequently activating the PKC and PKA (Richardo, 2011). Furthermore, 1α,25(OH)2D regulates muscle cell proliferation and myogenesis. 1α,25(OH)2D3 activates three mitogen- activated protein kinase (MAPK) signalling pathway in muscle cells (Ceglia, 2008). These pathways result in eliciting extracellular signals to stimulate the intracellular targets leading to the regulation of proliferation, differentiation, apoptosis and gene expression (Ceglia, 2008). Vitamin D plays an important role in muscle physiology by modulating high levels of the parathyroid hormone (Girgis et al., 2013). Excess levels of PTH indicate abnormalities in muscle and tissues and functional ability of the muscles (Battault et al, 2013). 2.11.5 Relationship between Vitamin D and the Immune System Vitamin D plays a vital role in the action of an innate immune response. This is achieved by stimulating the antimicrobial properties and components of the innate system (Bartley, 2010). The skin, gastrointestinal and respiratory tract are also part of the innate immune system which acts as a barrier to prevent infections. The epithelial cells of the skin work with macrophages and neutrophils to prevent infections (Kamen & Tangpricha, 2010). 22 Univ ersity of Ghana http://ugspace.ug.edu.gh Circulating 25(OH)2D is absorbed by immune cells and hydrolysed to its active form 1,25(OH)2D. The activated 1,25(OH)2D is bonded to VDR, translocated into the nucleus and gets attach to the VDRE to form the 1,25(OH)2D-VDRE complex (Kamen & Tangpricha, 2010). Genes that expresses cathelicidin also adapt VDRE. Cathelicidins are antimicrobial peptides expressed by epithelial cells and neutrophils that help in innate immune function (Bartley, 2010). The 1,25(OH)2D-VDRE complex activates the gene responsible for the synthesis of cathelicidin (hCAP18). The cathelicidin is then segmented and transformed into its active form, IL37. IL37 exhibits immune activities against microbes (Mithal et al, 2009). 1 α, 25(OH)2D promotes the ability of macrophages to move, engulf and destroy microbes. 1 α, 25(OH)2D also activates antibacterial peptides which destroys the cell membrane of microorganisms (Prietl, Treiber, Pieber, & Amrein, 2013). The activation of macrophages by TLR ½ elicits the expression of VDR and 1-α-hydroxylase increasing the anti-microbial effects of vitamin D (Prietl et al., 2013). Also, macrophages express Toll-like receptors (TLRs) that help in the recognition of bacteria and signals the macrophage to produce cathelicidin. When the TLRs are signalled, the VDR and 1-α-hydroxylase are also activated to produce more cathelicidins (Kamen & Tangpricha, 2010). Literature has also proven that the conversion of vitamin D into its metabolically active form is a permanent process in the epithelia of the respiratory tract and it also increases during viral infections (Cannell, Hollis, Zasloff, & Heaney, 2008). A sufficient vitamin D status is important for an adequate production of cathelicidin which forms parts of the defence against respiratory infections (Bartley, 2010). 23 Univ ersity of Ghana http://ugspace.ug.edu.gh Studies by Prietl et al., (2013) showed that vitamin D is able to alleviate adaptive immune response. VDR agonists have the ability to alter the immune effects of dendritic cells. Stimulation of VDRs also activates tolerogenic dendritic cells (tol-DCs) which are characterized by low levels of MHC class II, costimulatory molecules and increased and decreased the production of IL-10 and IL-12. Tol-DCs also results in a decreased immune response of Th1 and Th17 and an increased response of T regulatory cells (Prietl et al., 2013). Furthermore, 1 α, 25(OH)2D also reduces the rate of B cells differentiation, proliferation and elicits apoptosis (Kamen & Tangpricha, 2010). The actions of vitamin D in adaptive immune responses also help to alleviate autoimmune responses (Mostafa & Hegazy, 2015). 2.12 Sunlight Exposure Questionnaire Sunlight exposure is difficult to measure because just by asking people how long they spend outside and where they live cannot really justify their UVB exposure hence their vitamin D status (McCarty, 2008). UV exposure is generally measured using the following measures: latitude of residence, environmental UV exposure, personal ambient UV exposure and the anatomical distribution of UV (Holick et al., 2011). Dosimetry is a method used to objectively quantify ambient exposure (McCarty, 2008). This method accounts for environmental factors like cloud cover. Environmental UV levels differ due to latitude, season, time of the day, cloud or tree cover. Personal ambient factors are individual factors that sunlight exposure. These include; sun screening behaviours, 24 Univ ersity of Ghana http://ugspace.ug.edu.gh clothing. Individual sunlight exposure can be calculated by being aware of the amount of time an individual spent outdoors, types of clothing and sunlight protection used. A sunlight exposure questionnaire is an inexpensive tool commonly used to estimate exposure to UVB in individuals of varied age group and occupations however the sunlight exposure questionnaire is susceptible to recall bias (McCarty, 2008). The sunlight exposure questionnaire can be used as a proxy measure for vitamin D status however it has been found to misclassify vitamin D status (McCarty, 2008). The advantage of using a sunlight exposure questionnaire to determine the vitamin D status is the low cost. It is also self-administered or interview administered. It can also be used to estimate sunlight exposure in the past (McCarty, 2008). However, the limitations of the sunlight exposure questionnaire to determine vitamin D status are the factors that affect an individual sunlight exposure and the synthesis of vitamin D in the skin. The potential for error is affected by varied environmental and personal factors (McCarty, 2008). Also, previous usages in other studies showed particularly low correlations between sunlight exposure and serum vitamin D levels influenced by factors that affect cutaneous synthesis of vitamin D. In conclusion, using a questionnaire to estimate vitamin D status is a poor measurement tool because of the imprecision of the UV estimates obtained from sunlight exposure questionnaires and the low correlation of sunlight measures with serum 25(OH)D (McCarty, 2008). 25 Univ ersity of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 METHODS 3.1 Study Design The cross-sectional design was employed in this study. 3.2 Study Site The study was conducted in four schools (two public schools: Martey Tsuru L/A & Kotobabi 1 & 2 and two private schools: Bask Academy School & Blue Bear International School), all in the Ledzokuku/Krowor constituency. The Ledzokuku Krowor constituency is in the Accra Metropolitan Assembly. It was chosen for this study because it has a good representation of people from low, middle and high socio-economic group (Ghana Statistical Service, 2014). The Ledzokuku/Krowor Municipal has a total population of 227,932 of both females and males representing 5.7% of Greater Accra region's total population (Ghana Statistical Service, 2014). Males constitute 47.9% and females represent 52.1%. 76,184 persons are currently attending schools in different levels with 50% males and 49.9% for females. About 31,000 persons of which 39.4% are males and 41.1% are females are in primary school (Ghana Statistical Service, 2014). 26 Univ ersity of Ghana http://ugspace.ug.edu.gh 3.3 Study Population The study comprised 8- 12 years old Ghanaian school children attending school at the four (4) selected schools. 3.3.1 Inclusion Criteria i. Eight to twelve-year-old school children in the selected schools. ii. Eight to twelve-year-old school children whose parents consented to and who assented to their participation. iii. Apparently healthy 8-12 year olds. 3.3.2 Exclusion Criteria i. Children whose parents refused to give consent or who refused to participate in the study. ii. Children who were not well. iii. Children outside the selected zone. 3.4 Sampling Technique Stratified sampling was employed to determine the number of students to be recruited from each school and each class in the school. This was followed by systematic sampling to identify the particular students within each class using the class register. In each class, the school children were stratified based on sex and every 4th child was given a consent form to seek permission from their parents. The school children who received approval from 27 Univ ersity of Ghana http://ugspace.ug.edu.gh their parents were recruited into the study and replacements were found for those who couldn’t seek permission from their parents. 3.5 Sample Size Determination The sample size of the study was calculated using the formula below. In studies designed to measure a characteristic in terms of a proportion, the equation for sample size is; N = 4 (Z 2Crit) p (1-p) (Eng, 2003) D2 N = the sample size, Zcrit value for 95% CI = 1.960, D = the total width of the expected CI for a two-way analysis is 0.20, p = the pre-study estimate of the proportion to be measured, (26% representing prevalence of vitamin D insufficiency, according to Poopedi, Norris, & Pettifor (2011). Hence N = 4 (1.960)2 0.26 (1- 0.26) (0.20)2 N = 74. Considering a drop-out rate of 10% and a non-response rate of 10%, the number of participants were rounded up to 100. Twenty percent was used in the calculation because the calculation is a two-tailed analysis considering both sides of a normal curve on each side of the equation. Thus, 100 participants were recruited into this study. 28 Univ ersity of Ghana http://ugspace.ug.edu.gh 3.6 Pre-testing Questionnaires The questionnaire was pre-tested at the Generation Grace International School, Spintex to ensure that the questionnaire was well understood and can be answered accurately. The questionnaire was reviewed to ensure accuracy. Twenty children were recruited for the pre- test. 3.7 Data Collection Demographic and socio-economic information of the parents/ guardians and their wards were obtained using a structured interview guide. Anthropometry (weight and height) were also measured. A Food Frequency Questionnaire (FFQ) was used to assess frequent consumption of vitamin D rich sources. Data were collected by face-to-face interview using a structured questionnaire which was developed after reviewing different literatures of similar studies The school children were shown pictures of common Vitamin D rich foods in Ghana. A sunlight exposure questionnaire was also used to estimate the participants' exposure to sunlight. 3.7.1 Socio-Demograhic Information Sociodemographic data including age, gender, occupation, employment status and the highest level of education of parents/ guardians as well as the age, gender and class of the children were obtained. 29 Univ ersity of Ghana http://ugspace.ug.edu.gh 3.7.2 Determination of Frequency of consumption of Vitamin D rich foods Dietary intakes were assessed using a validated food frequency questionnaire (Appendix III). The Food Frequency Questionnaire (FFQ) adopted from Asante and colleagues was used to obtain the frequency of food consumption over a period of one week (Asante, Pufulete, Thomas, Wiredu & Intiful, 2015). The questionnaire comprises 95 food items in 18 food categories including beverages, fish and seafood, starches, milk and milk products and supplements with 7 response options ranging from ‘once a day' to ‘never' for the frequency of consumption. The frequency was queried as servings per day and week. 3.7.3 Sunlight Exposure Questionnaire The sunlight exposure questionnaire adopted from Alshahrani (2014) was used to evaluate the participants' average length and time of sunlight exposure (Appendix III). The questions included were ‘about how many days a week', ‘how many minutes per day' and ‘what time they mostly spend outdoors under the sun.’ 3.7.4 Anthropometric Measurements Anthropometric measurements such as height and weight were obtained. These were measured using the standardized techniques of the National Health and Nutrition Examination Survey [NHANES] (NHANES, 2007). Height was measured to the nearest 0.1m using the Seca stadiometer (Seca model 213, Hamburg, Germany). In measuring the height, the child was asked to stand with legs straight against the backboard of the stadiometer, with the back of the head, shoulder blades, buttocks and heels making contact with the backboard as much as possible. Both 30 Univ ersity of Ghana http://ugspace.ug.edu.gh feet were bare and flat on the platform, with the heels together and toes apart. Each child was asked to look straightforward, with head in the Frankfort horizontal plane, and arms at the sides. The stadiometer headpiece was lowered to rest firmly on top of the child's head and the height read in the nearest 0.1m. The weight of the child was measured to the nearest 0.1kg using a weighing scale (Seca, Hamburg). Before stepping on the weighing scale, the child was asked to take off any excess clothing, remove their shoes and socks and also to empty their pockets to ensure accuracy. The child stood at the middle of the scale platform facing the recorder, hands at the sides, and looking straight ahead and the weight read in the nearest 0.1kg. 3.7.5 Determination of Serum Concentrations of Vitamin D Vitamin D was assayed using ELISA kits from Sunlong Biotech Co. Ltd (Hangzhou, China) (Catalog No: SL2762Hu) using the Sandwich-ELISA method. Two millilitres of blood was drawn from participants into serum separator tubes and serum separated. Serum was stored at -20ºC for not more than a month after sampling. The Micro-ELISA Strip plate involved a sandwich touch which was pre-coated with an antibody specific to vitamin D by the manufacturer. Standards and samples were added for antigens to bind to the antibody on the plate. A second antibody, Horseradish Peroxidase (HRP) was conjugated to an antibody specific to the hormone added, incubated and washed to remove unwanted components. For colour development, TMB solution was added to each well. Only wells containing the hormone and the HRP conjugated hormone antibody appeared blue and then yellow when 31 Univ ersity of Ghana http://ugspace.ug.edu.gh the stop solution is added. The Optical density of standards and samples was measured spectrophotometrically at 450 nm. A standard curve was drawn and used to calculate the concentrations of the samples. 3.8 Data Management Plan Data obtained was kept under strict conditions by the researcher on a password-protected laptop. Completed questionnaires were coded, filed and kept under lock in the Department of Nutrition and Dietetics, University of Ghana. The recruitment ensured voluntary participation of each patient. The data collected remained confidential by being stored in a pass-worded database on a computer. The questionnaires used were kept in a locked-up cabinet throughout the study and were only accessible to the research team. 3.9 Statistical Analysis Statistical Package for Social Sciences (SPSS) Version 20.0 was used to analyse data. Categorical data were summarized using frequencies and percentages and continuous data were summarized as means and standard deviations. Pearson's chi-square test was used to determine the association between serum Vitamin D concentrations and anthropometric indices, sun exposure and dietary intakes. The Student's t-test was used to compare mean serum vitamin D concentrations between children in public and private schools and 32 Univ ersity of Ghana http://ugspace.ug.edu.gh between males and females. Logistic regression analysis was used to predict the odds of having low serum concentrations of vitamin D. Statistical significance was set at p<0.05. 3.10 Ethical Approval Ethical approval and permission were obtained from The Ethics and Protocol Review Committee (EPRC) of the College of Health Science, University of Ghana and the Ghana Education Services respectively. After approval, permission was sought from the heads of schools and consent sought from parents/guardians of participants. After which the participants were recruited and their written consent and assent sought. Results and report did not bear any participants name. Unique identification numbers were assigned right from the beginning when they are recruited. There was no direct benefit for participation but information on their vitamin D status was made known to them and they were advised to modify their diet to improve their nutrient intake and also increase their exposure to sunlight. 33 Univ ersity of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS The study yielded a 99% response rate, representing 99 pupils from the four selected schools sampled in the study. Even though 99 pupils participated in this study, only 53.5% consented for their blood to be sampled for serum vitamin D status. 4.1 Socio-Demographic Information The demographic characteristics of the participants who partook in this study is shown in table 4.1. Majority of the respondents of the study were females (58.6%), with 35.4% from the public schools and 23.2% from the private schools. The mean age of the participants was 10.09 ± 1.29 years. Almost all the pupils (93.9%) were Christians. Table 4. 1: Socio-demographic characteristics of participants (n =99) Characteristics Public schools (n=58) Private schools (n=41) Total (n=99) n (%) n (%) n (%) Gender Male 23 (23.2) 18 (18.2) 41 (41.4) Female 35 (35.4) 23 (23.2) 58 (58.6) Age of participants 8 12 (12.4) 1 (1.0) 13 (13.30 9 12 (12.4) 10 (10.2) 22 (22.4) 10 13 (13.3) 9 (9.2) 22 (22.4) 11 13 (13.3) 12 (12.4) 25 (25.5) 12 7 (7.1) 9 (9.2) 16 (16.3) 34 Univ ersity of Ghana http://ugspace.ug.edu.gh Table 4.1 Socio-demographic characteristics of participants cont’d Characteristics Public schools (n=58) Private schools (n=41) Total (n=99) n (%) n (%) n(%) Class of participants 1 9 (9.1) - 9 (9.1) 2 16 (16.2) - 16 (16.2) 3 13 (13.1) 12 (12.1) 25 (25.3) 4 14 (14.1) 10 (10.1) 24 (24.2) 5 - 11 (11.1) 11 (11.1) 6 6 (6.1) 8 (8.1) 14 (14.1) Religion Christian 55 (55.6) 38 (38.4) 93 (93.9) Muslim 3 (3.0) 3 (3.0) 6 (6.1) Ethnicity Akan 18 (18.2) 16 (16.2) 34 (34.3) Ga 7 (7.1) 14 (14.1) 21 (24.2) Ewe 25 (25.3) 8 (8.1) 33 (33.3) Other 8 (8.1) 3 (3.0) 11 (11.1) Person who child lived with Single parent 14 (14.1) 11 (11.1) 25 (25.2) Both parents 36 (36.4) 28 (28.3) 64 (64.6) Relative 6 (6.1) 2 (2.0) 8 (8.1) Guardian 1 (1.0) - 1 (1.0) 35 Univ ersity of Ghana http://ugspace.ug.edu.gh Table 4.1 Socio-demographic characteristics of participants cont’d Characteristics Public schools (n=58) Private schools (n=41) Total (n=99) n (%) n (%) n (%) Occupation of main provider Trader 24 (24.2) 14 (14.1) 38 (38.4) Self-employed 25 (25.2) 17 (17.2) 42 (42.4) Government employee 2 (2.0) 5 (5.1) 7 (7.1) Unemployed 2 (2.0) 4 (4.0) 6 (6.1) Other 4 (4.0) 1 (1.0) 5 (5.1) Type of house children lived in Self- contained 5 (5.1) 16 (16.2) 21 (21.2) Single room 9 (9.1) 1 (1.0) 10 (10.1) Compound house 4 (4.0) 14 (14.1) 18 (18.2) Chamber & hall 12 (12.1) 10 (10.1) 22 (22.2) Kiosk 26 (26.3) - 26 (26.3) Uncompleted 1 (1.0) - 1 (1.0) 36 Univ ersity of Ghana http://ugspace.ug.edu.gh 4.2 Body Mass Index for Age Classification of Participants Figure 4.1 shows the BMI-for-age classification of the children. According to the WHO classification for BMI, 85% of the participants were normal, 12.5% were overweight and 2.1% were thin. Body mass index for age Overweight 13% Thinness 2% Overweight Thinness Normal Normal 85% Figure 4. 1: Body Mass Index-for-age of participants 4.2.1 Body Mass Index (BMI) for Age Categorisation based on school types In table 4.2 below, it can be seen that a higher percentage (85.4%) of the participants had normal BMI-for-age with the highest number reporting from the private schools (92.3%). Out of the 12.5% who were overweight in both schools, the majority (17.5%) were from the public schools. 37 Univ ersity of Ghana http://ugspace.ug.edu.gh Table 4. 2: Body Mass Index for age categorisation in public and private schools (N=99) Characteristics Public school (n=58) Private school (n=41) Total (n=99) n (%) n (%) n (%) BMI-for-age Overweight 10 (17.5) 2 (5.1) 12 (12.5) Thinness 1 (1.8) 1 (2.6) 2 (2.1) Normal 46 (80.7) 36 (92.3) 82 (85.4) Thinness = BMI <-2SD, Normal Weight = BMI ≥ -2SD ≤ +1SD, Overweight = BMI > +1SD ≤ +2SD. 4.3 Length of Sunlight Exposure based on school type and gender In Table 4.3 below, a similar percentage of school children exposed themselves to sunlight during the hours of 7-9: 59 am (33.6%) and 10am -12: 59 pm (37.7%). Out of this, a higher percentage of the public school children (25.5%) exposed themselves to sunlight between the hours of 10am-12: 59 pm compared to those in private schools (12.2%). There was no significant difference in the proportion of males and females who exposed themselves to sunlight between 10am-12: 59 pm. With regards to amount of time mostly spent outdoors in the sun every day, more than one- third of the children (33.4%) spent less than 15 minutes a day in the sun and 20.2% were outdoors between 15 to 30 minutes per day. A significantly higher proportion of private school children (27.3%) were outdoors for less than 14 minutes a day compared to their counterparts in public schools (6.1%). Further, a significantly higher percentage of females (12.1%) were outdoors for 15-30 minutes per day compared to males (8.1 %, p= 0.048). 38 Univ ersity of Ghana http://ugspace.ug.edu.gh Majority of the participants (72.8%) in this study exposed themselves to the sun on an average of 3 days or more. Of this, a significantly higher proportion was from the public schools (50.0%). 39 University of Ghana http://ugspace.ug.edu.gh Table 4. 3: Length of sunlight exposue based on school type and gender (n=99) Characteristics Total (N=99) School Type X2 p-value Gender X2 p-value Public Private Males Females (n= 51) (n=48 ) (n= 41 ) (n=58 ) Time mostly spent in the suna 7-9:59am 33 (33.6) 17 (17.3) 16 (16.3) 2.426 0.489 11(11.2) 22(22.4) 4.019 0.259 10am-12:59pm 37 (37.7) 25 (25.5) 12 (12.2) 18(18.4) 19(19.4) 1-3:59pm 19 (19.4) 10 (10.2) 9 (9.2) 10(10.2) 9(9.2) 4-7 pm 9 (9.2) 6 (6.1) 3 (3.1) 2(2.0) 7(7.1) Time mostly spent outdoors in the sun everydayb < 15 minutes/day 29 (33.4) 6 (6.1) 27 (27.3) 38.054 <0.001* 14(14.1) 19(19.2) 7.920 0.048* 15-30 minutes/ day 20 (20.2) 12 (12.1) 8 (8.1) 8(8.1) 12(12.1) More than 30-60 25 (25.2) 23 (23.2) 2 (2.0) 15(15.1) 10(10.1) minutes/day More than 1 -2 hours 21 (21.2) 17(17.2) 4 (4.0) 4(4.0) 17(17.2) per day Average days exposed to sunc,d Not at all 7 (7.6) 2 (2.2) 5 (5.4) 10.245 0.017* 0 (0.0) 7 (12.7) 9.438 0.024* 1 day 7 (7.6) 2 (2.2) 5(5.4) 1 (2.7) 6 (10.9) 2 Days 11 (12.0) 4 (4.3) 7 (7.6) 7 (18.9) 4 (7.3) 3 days or more 67 (72.8) 46 (50.0) 21(22.8) 29 (78.4) 38 (69.1) Time mostly spent in the sun a =98. Total number of pupils who spent time outdoors under the sunb = 95. Average days exposed to sunc =92. Average days exposed to sund = Data missing for 3 females. *Significance set at p<0.05, Chi-square test 40 University of Ghana htt p://ugspace.ug.edu.gh 4.4 Serum Vitamin D Status in Children Fifty-three respondents consented and assented to have their serum vitamin D status tested. The mean vitamin D concentration was 27.73 ± 9.90 ng/ml. About half of the participants (49.1%) who were tested had a deficient level of vitamin D (Table 4.4.1). Table 4.4. 1: Serum vitamin D status in school children (n=53). Serum vitamin D status n (%) Deficiency (≤ 20ng/ml) 26 (49.1) Insufficiency (21-29ng/ml) 9 (17) Sufficient (30-100ng/ml) 18 (34) Total 53 (100) Mean ± SD ng/ml 27.73 ± 9.90 ng/ml SD- Standard Deviation 4.4.1 Serum vitamin D status based on school type and gender Table 4.4.2 shows the serum vitamin D status of the school children based on school type and gender. Almost half of the school children (49.1%) had deficient vitamin D status. Of this 75% were from the public schools and 9.5% from the private schools. A significantly higher proportion of the private school children (66.7%) reported sufficient vitamin D levels compared to those from public schools (12.5%). Mean serum vitamin D status was significant for school types. Based on gender, about one-third of the females (30.2%) were vitamin D deficient compared to the males (18.9%). Almost the same proportion of females (18.9%) and males (15.1%) reported sufficient vitamin D levels. Differences between mean serum vitamin D levels based on gender were insignificant. 41 University of Ghana htt p://ugspace.ug.edu.gh Table 4.4. 2: Serum vitamin D status based on school type and gender (N=53) Characteristics Total (N=53) School Type X2 p-value Public Private (n= 32) (n=21) Deficiency 26 (49.1) 24 (75) 2 (9.5) <0.001* Insufficiency 9 (17) 4 (12.5) 5 (23.8) 22.989 Sufficiency 18 (34) 4 (12.5) 14 (66.7) Mean ± SD (ng/ml) 18.84 ± 7.97 31.19 ± 7.70 <0.001* Gender X2 p-value Males Females (n= 21) (n=32) Deficiency 26 (49.1) 10 (18.9) 16 (30.2) 0.338 0.844 Insufficiency 9 (17) 3 (5.7) 6 (11.3) Sufficiency 18 (34) 8 (15.1) 10 (18.9) Mean ± SD ang/ml 23.73± 9.90 25.29 ± 11.60 22.71 ± 8.65 0.358 Chi-square test. p<0.05 is considered statistically significant. ** Mean serum vitamin D levels. Mean serum vitamin D levelsa of gender. SD= Standard Deviation, student t-test used to analyse mean serum vitamin D levels by gender and by school types. 4.4.2 Serum vitamin D status and Anthropometric Measurements Table 4.4.3 shows serum vitamin D status categories by anthropometric measurements. No significant associations were found for serum vitamin D status and anthropometric measurements. 42 University of Ghana htt p://ugspace.ug.edu.gh Table 4.4. 3: Serum vitamin D status based on BMI-for-Age (N=51) Serum vitamin D status and categorised BMI-for-age Serum Overweight Thinness Normal Total X2 p-value vitamin D category n (%) n (%) n (%) n (%) Deficiency 3 (5.9) 1(1.9) 21(41.2) 25 (49.0) 3.718 0.446 Insufficiency 2 (3.9) 1 (1.9) 6 (11.8) 9 (17.6) Sufficiency 1 (1.9) 0 16 (31.4) 17 (33.3) Total 6 (11.7) 2 ( 3 .8) 43 (84.4) 51 (100.0) Significance set at p<0.05. Chi-square test. 4.4.3 Serum vitamin D status and Length of sunlight Exposure In Table 4.4.4, a significant association was observed between serum vitamin D status and time mostly spent outdoors. Half of the school children (50%) reported deficient vitamin D levels. Of this, 36.5% were outdoors in the sun between the hours of 10 am- 12:59 pm. However, sufficient vitamin D status was reported in the group (13.5%) that were outdoors during the hours 1- 3:59 pm. With regards to time mostly spent outdoors in the sun every day, almost one-third of the children (28.3%) spent more than 30- 60 minutes; however, they reported deficient serum vitamin D status. A significant association was observed between serum vitamin D status and time spent in the sun every day. Furthermore, a significantly higher percentage (18.9%) were outdoors for less than 15 minutes per day (p= <0.001*). 43 University of Ghana htt p://ugspace.ug.edu.gh Majority of the participants (42.6%) in this study who exposed themselves to the sun on an average of 3 days or more had deficient serum vitamin D status and 23.4% had sufficient vitamin D status. Table 4.4. 4: Serum vitamin D status based on length of Sunlight Exposure (N=53) Characteristics Length of sunlight exposure Total X2 p - value Time mostly spent outdoor in the suna Serum vitamin D 7-9:59am 10am- 1-3:59pm 4-7pm N (%) category n (%) 12:59pm n (%) n (%) n (%) Deficiency 3 (5.8) 19 (36.5) 0 (0.0) 4 (7.7) 26 (50) 28.00 <0.001* Insufficiency 4 (7.7) 1 (1.9) 4 (7.7) 0 (0.0) 9 (17.3) 6 Sufficiency 6 (11.5) 2 (3.8) 7 (13.5) 2 (3.8) 17(32.7) Time spent in the sun everyday Serum Vitamin <15minute 15-30 More than More Total X2 p –value D category s/day minutes/day 30minutes- than 1-2 60minutes/ hours/day day Deficiency 0 (0.0) 3(5.7) 15 (28.3) 8 (15.1) 26(49.1) Insufficiency 1 (1.9) 3 (5.7) 0 (0.0) 5 (9.4) 9 (17.0) 33.06 <0.001* 0 Sufficiency 10 (18.9) 2 (3.8) 1 (1.9) 5 (9.4) 18(34.0) Serum vitamin D Average days exposed to sunb category Not at all 1 day 2 days 3 days Total X2 p-value Deficiency 0 0 3 (6.4) 20 (42.6) 23 (48.9) 8.663 0.193 Insufficiency 1 (2.1) 0 1 (2.1) 5 (10.6) 7 (14.9) Sufficiency 3 (6.4) 2 (4.3) 1 (2.1) 11 (23.4) 17 (36.2) Time mostly spent outdoor in the suna b =52 Average days exposed to sun =47 *Significance set at p<0.05. Chi-square test. 44 University of Ghana htt p://ugspace.ug.edu.gh 4.4.4 Relationship between Serum Vitamin D and School Type, Gender and Length of Sunlight Exposure A logistic regression was performed to ascertain the effect of school type, gender, and length of sunlight exposure on the likelihood that participants have sufficient serum vitamin D levels (Table 4.5.6). School type significantly predicted serum vitamin D levels, χ2 (1) = 17.076, p<0.001. The model explained 38.1% (Nagelkerke R2 ) of the variance in serum vitamin D levels and correctly classified 79.2% of cases. Public school children were 0.07 times less likely to be vitamin D sufficient compared to their counterparts in private schools. Gender was not a significant predictor of serum vitamin D levels, χ2 (1) = 0.263, p=0.608. The model explained only 0.7% (Nagelkerke R2) of the variance in serum vitamin D levels and correctly classified 66% of cases. Males were 1.35 times more likely to be vitamin D sufficient compared to females. Furthermore, time mostly spent in the sun every day was a significant predictor of serum vitamin D levels, χ2 (3) = 23.472, p<0.001. The model explained 49.5% (Nagelkerke R2) of the variance in serum vitamin D levels and correctly classified 83% of cases. School children who spent 15 to 30 minutes per day in the sun were 0.87 times less likely to be vitamin D sufficient compared to those who spent more than 1-2 hours per day. Children who spent 30 to 60 minutes per day were 0.17 times less likely to be vitamin D sufficient compared to those who spent more than 1-2 hours a day. However, spending 15-30 minutes (p=0.883) or 30-60 minutes (p=0.173) per day in the sun were not statistically significant. 45 University of Ghana htt p://ugspace.ug.edu.gh In this study, time mostly spent outdoors in the sun significantly predicted serum vitamin D levels, χ2 (3) = 12.318, p= 0.006. The model explained 29.4% (Nagelkerke R2) of the variance in serum vitamin D levels and correctly classified 73.1% of cases. School children who exposed themselves to sunlight within the hours of 10 am to 12:59 pm were 2.04 times more likely to be vitamin D sufficient compared to those exposed during 4-7pm, although not statistically significant (p=0.394). Children who exposed themselves during the hours of 1-3: 59 pm were 0.58 times less likely to be vitamin D sufficient compared to those exposed to the hours of 4-7pm, although not significant (p=0.601). Further, average days exposed to sunlight was not a significant predictor of serum vitamin D level, χ2 (3) = 7.694, p= 0.053. The model explained only 20.7% (Nagelkerke R2) of the variance in serum vitamin D levels and correctly classified 72.3% of cases. School children who did not expose themselves to sunlight in a week were 6.82 times less likely to have sufficient serum vitamin D levels compared to their counterparts who exposed themselves on an average of 3 days or more per week (p= 0.113). The participants who exposed themselves to sunlight on an average of 2 days per week were 0.63 times less likely to be vitamin D sufficient compared to those exposed on an average of 3 days or more per week (p= 0.630). 46 University of Ghana htt p://ugspace.ug.edu.gh Table 4.4. 5: Relationship between Serum vitamin D status and Gender, School groupings and Length of sunlight exposure Variables Odds Ratio df R2 95% CI p-value Lower Upper Gender 1 Female Ref 0.007 Male 1.354 0.426 4.298 0.607 School Type 1 Private Ref 0.381 schools Public 0.071 0.018 0.286 <0.001* schools Time spent in the sun 3 <0.001* everyday >1 – 2 hours Ref 0.495 < 15minutes 26.000 2.607 259.295 0.005* 15- 0.867 0.129 5.817 0.883 30minutes 30 – 0.173 0.018 1.681 0.131 60minutes Time mostly spent outdoor in 3 <0.001* the sun 4 -7 pm Ref 0.294 7-9:59am 0.117 0.019 0.718 0.020* 10am-12:59 2.042 0.395 10.553 0.394 pm 1-3:59 pm 0.583 0.078 4.386 0.601 Average days exposed to sun 3 0.053 3 days or Ref 0.207 more Not at all 6.818 0.636 73.059 0.113 1 day@ 0.000 0.999 2 Days 0.568 0.057 5.685 0.630 Binomial logistic regression test. *significance set at p<0.05. 1 day@ Odd ratio unavailable because of invariability in low response 47 University of Ghana htt p://ugspace.ug.edu.gh 4.5 Dietary Vitamin D Intakes of respondents Table 4.5.1 below shows the dietary vitamin D intakes of the school children. Table 4.5. 1: Dietary vitamin D intakes of respondents (N=94) Food item 1x /day More 1-2x a 3-4x a 5-6x a Rarely Never than once week week week a day n (%) n (%) n (%) n (%) n (%) n (%) n (%) Low-fat milk 14(15.1) 9(9.7) 21(22.6) 7(7.5) 10(10.8) 12(12.9) 20(21.5) Skimmed 10(10.8) 7(7.5) 4(4.3) 3(3.2) 1(1.1) 5(5.4) 63(67.7) milk Yoghurt 16(17.2) 14(15.1) 20(21.5) 13(14.0) 7(7.5) 8(8.6) 15(16.1) Ice cream 24(25.8) 10(10.8) 18(19.4) 9(9.7) 13(14.0) 5(5.4) 14(15.1) Cheese 9(9.7) 6(6.5) 9(9.7) 4(4.3) - 11(11.8) 54(58.1) Wagashi 13(14.0) 1(1.1) 15(16.1) 3(3.2) 1(1.1) 11(11.8) 49(52.7) Soymilk 13(14.0) 3(3.2) 11(11.8) 2(2.2) 2(2.2) 20(21.5) 42(45.2) Butter 14(15.1) 8(8.6) 15(16.1) 4(4.3) 2(2.2) 12(12.9) 38(40.9) Eggs 21(22.3) 15(16.0) 15(16.0) 10(10.6) 15(16.0) 5(5.3) 13(13.8) Canned fish 18(19.1) 10(10.6) 28(29.8) 2(2.1) 1(1.1) 10(10.6) 25(26.6) Salmon 29(30.9) 13(13.8) 19(20.2) 9(9.6) 9(9.6) 4(4.3) 11(11.7) Mackerel 18(19.1) 9(9.6) 19(20.2) 10(10.6) 3(3.2) 6(6.4) 29(30.9) Tuna 23(24.5) 8(8.5) 20(21.3) 12(12.8) 4(4.3) 6(6.4) 21(22.3) Sardine 17(18.1) 12(12.8) 27(28.7) 10(10.6) 3(3.2) 14(14.9) 11(11.7) Kippers 5(5.3) 2(2.1) 2(2.1) 3(3.2) 2(2.1) 9(9.6) 71(75.5) Pilchards 8(8.5) 4(4.3) 12(12.8) 1(1.1) 2(2.1) 10(10.6) 57(60.6) Eel 8(8.5) 1(1.1) 4(4.3) 1(1.1) 1(1.1) 9(9.6) 70(74.5) Herring 7(7.4) 4(4.3) 13(13.8) 10(10.6) 3(3.2) 15(16.0) 42(44.7) Margarine 15(16.1) 17(18.3) 20(21.5) 12(12.9) 9(9.7) 12(12.9) 8(8.6) Vitamin D 4(4.3) 1(1.1) 1(1.1) 1(1.1) 87(92.6) supplements 4.5.1 Vitamin D Rich Foods Intake and Serum Vitamin D Status Tables 4.5.2 show the association between dietary intakes of vitamin D rich foods and serum vitamin D status. A significant association was observed between serum vitamin D 48 University of Ghana htt p://ugspace.ug.edu.gh status and the intakes of salmon (p= 0.018), herrings (p= 0.033), yoghurt (p= 0.032), ice- cream (p=0.014), soy milk (p= 0.001) and margarine (p= 0.032). Also, a multiple linear regression analysis established that dietary vitamin D intake (salmon, herring, yoghurt, ice cream, soymilk, and margarine) significantly predicted serum vitamin D levels, F (6, 40) = 3.432, p=0.008. Thus, dietary vitamin D accounted for about 34 % of the observed variability in the serum vitamin D levels. Table 4.5. 2: Dietary vitamin D-rich foods intakes and serum vitamin D status (N=48) Serum Frequency of vitamin D rich foods consumption X2 p - vitamin value D category 1x/day More 1-2x 3-4x 5-6x Rarely Never Total than a week a week a week once a day n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Canned fish consumption Deficiency 1(2.1) 2(4.2) 9(18.8) 1(2.1) 1(2.1) 3(6.3) 4(8.3) 21(43.8) 19.604 0.075 Insufficiency 2(4.2) 0 0 0 0 3(6.3) 4(8.3) 9(18.8) Sufficiency 6(12.5) 0 5(10.4) 1(2.1) 0 0 6(12.5) 18(37.5) Salmon consumption Deficiency 0 3(6.3) 4(8.3) 4(8.3) 7(14.6) 0 3(6.3) 21(43.8) 24.389 0.018* Insufficiency 5(10.4) 0 2(4.2) 0 0 1(2.1) 1(2.1) 9(18.8) Sufficiency 6(12.5) 1(2.1) 6(12.5) 1(2.1) 2(4.2) 0 2(4.2) 18(37.5) Mackerel consumption Deficiency 0 1(2.1) 6(12.5) 4(8.3) 1(2.1) 1(2.1) 8(16.7) 21(43.8) 9.744 0.638 Insufficiency 2(4.2) 1(2.1) 1(2.1) 1(2.1) 0 0 4(8.3) 9(18.7) Sufficiency 5(10.4) 1(2.1) 3(6.3) 1(2.1) 1(2.1) 1(2.1) 6(12.5) 18(37.5) Tuna Consumption Deficiency 1(2.1) 1(2.1) 5(10.4) 8(16.7) 3(6.3) 1(2.1) 2(4.2) 21(43.8) 18.267 0.108 Insufficiency 3(6.3) 0 1(2.1) 0 0 1(2.1) 4(8.3) 9(18.7) Sufficiency 5(10.4) 1(2.1) 3(6.3) 2(4.2) 1(2.1) 0 6(12.5) 18(37.5) *Significance set at p<0.05. Chi-square test. 49 University of Ghana htt p://ugspace.ug.edu.gh Table 4.5.3: Dietary vitamin D-rich foods intakes and serum vitamin D status (N=48) cont’d Serum Frequency of vitamin D rich foods consumption X2 p - vitamin D value category 1x/day More 1-2x a 3-4x a 5-6x a Rarely Never Total than week week week once a day n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Sardine consumption Deficiency 0 1(2.1) 11(22.9) 2(4.2) 1(2.1) 2(4.2) 4(8.3) 21(43.8) 13.802 0.314 Insufficiency 1(2.1) 1(2.1) 3(6.3) 0 0 3(6.3) 1(2.1) 9(18.7) Sufficiency 4(8.3) 1(2.1) 7(14.6) 0 2(4.2) 1(2.1) 3(6.3) 18(37.5) Kipper Consumption Deficiency 1(2.1) 0 1(2.1) 1(2.1) 2(4.2) 3(6.3) 13(27.1) 21(43.8) 8.927 0.539 Insufficiency 0 0 0 0 0 0 9(18.8) 9(18.7) Sufficiency 1(2.1) 0 0 0 0 1(2.1) 16(33.3) 18(37.5) Pilchard consumption Deficiency 2(4.2) 1(2.1) 9(18.8) 1(2.1) 0 1(2.1) 7(14.6) 21(43.8) 15.471 0.116 Insufficiency 1(2.1) 0 0 0 0 1(2.1) 7(14.6) 9(18.7) Sufficiency 2(4.2) 0 1(2.1) 0 0 2(4.2) 13(27.1) 18(37.5) Eel consumption Deficiency 3(6.3) 1(2.1) 0 0 0 2(4.2) 15(31.3) 21(43.8) 3.824 0.700 Insufficiency 0 0 0 0 0 1(2.1) 8(16.7) 9(18.7) Sufficiency 1(2.1) 0 0 0 0 1(2.1) 16(33.3) 18(37.5) Herring consumption Deficiency 1 (2.1) 0 6(12.5) 2(4.2) 2(4.2) 5(10.4) 5(10.4) 21(43.8) 19.665 0.033* Insufficiency 0 0 0 1(2.1) 0 0 8(16.7) 9(18.7) Sufficiency 1(2.1) 0 1(2.1) 2 1(2.1) 0 13(27.1) 18(37.5) Trout consumption Deficiency 0 0 0 0 0 3(6.3) 18(37.5) 21(43.8) 5.823 0.213 Insufficiency 0 0 0 0 0 1(2.1) 8(16.7) 9(18.7) Sufficiency 2(4.2) 0 0 0 0 0 16(33.3) 18(37.5) *Significance set at p<0.05. Chi-square test. 50 University of Ghana htt p://ugspace.ug.edu.gh Table 4.5.4: Dietary vitamin D-rich foods intakes and serum vitamin D status (N=47) cont’d Serum Frequency of vitamin D rich foods consumption X2 p – vitamin value D category 1x/day More than 1-2x a 3-4x a 5-6x a Rarely Never Total once a day week week week n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Fortified breakfast cereal consumption Deficiency 0 0 0 0 0 2(4.2) 18(37.5) 20(42.6) 12.620 0.126 Insufficiency 1(2.1) 1(2.1) 0 0 0 0 7(14.6) 9(18.8) Sufficiency 3(6.3) 0 0 2(4.2) 0 2(4.2) 11(22.9) 18(37.5) Low-fat milk consumption Deficiency 0 0 8 2(4.2) 7(14.6) 2(4.2) 1(2.1) 20(42.6) 25.836 0.111 Insufficiency 0 0 5(10.4) 1(2.1) 0 0 3(6.3) 9(18.8) Sufficiency 4(8.3) 3(6.3) 4(8.3) 1(2.1) 1(2.1) 3(6.3) 2(4.2) 18(37.5) Skimmed milk consumption Deficiency 0 0 2(4.3) 1(2.1) 1(2.1) 0 16(34.0) 20(42.6) 9.272 0.506 Insufficiency 1(2.1) 0 0 1(2.1) 0 0 7(14.9) 9(18.8) Sufficiency 4(8.5) 0 1(2.1) 1(2.1) 0 1(2.1) 11(23.4) 18(37.5) Yoghurt consumption Defiiency 0 0 9(19.1) 6(12.8) 2(4.3) 1(2.1) 2(4.3) 20(42.6) 22.488 0.032* Insufficiency 2(4.3) 0 1(2.1) 1(2.1) 0 2(4.3) 3(6.4) 9(18.8) Sufficiency 3(6.4) 4(8.5) 4(8.5) 2(4.3) 0 1(2.1) 4(8.5) 18(37.5) Ice cream consumption Deficiency 0 0 3(6.4) 3(6.4) 9(19.1) 2(4.3) 3(6.4) 20(42.6) 25.069 0.014* Insufficiency 4(8.5) 0 0 1(2.1) 1(2.1) 0 3(6.4) 9(18.8) Sufficiency 5(10.6) 2(4.3) 5(10.6) 1(2.1) 1(2.1) 0 4(8.5) 18(37.5) *Significance set at p<0.05. Chi-square test. 51 University of Ghana htt p://ugspace.ug.edu.gh Table 4.5.5: Dietary vitamin D-rich foods intakes and serum vitamin D status (N=47) cont’d Serum Frequency of vitamin D rich foods consumption X2 p – vitamin D value category 1x/day More than 1-2x a 3-4x a 5-6x a Rarely Never Total once a day week week week n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Cheese consumption Deficiency 0 0 5(10.6) 0 0 2(4.3) 13(27.7) 20(42.6) 13.015 0.223 Insufficiency 0 0 0 1(2.1) 0 0 8(17.0) 9(18.8) Sufficiency 2(4.3) 1(2.1) 1(2.1) 2(4.3) 0 1(2.1) 11(23.4) 18(37.5) Wagashi consumption Deficiency 2(4.3) 0 5(10.6) 0 0 5(10.6) 8(17.0) 20(42.6) 10.658 0.222 Insufficiency 3(6.4) 0 1(2.1) 0 0 1(2.1) 4(8.5) 9(18.8) Sufficiency 4(8.5) 0 1(2.1) 2(4.3) 0 1(2.1) 10(21.3) 18(37.5) Soymilk consumption Deficiency 0 0 1(2.1) 0 0 12(25.5) 7(14.9) 20(42.6) 25.628 0.001* Insufficiency 3(6.4) 1(2.1) 0 0 0 0 5(10.6) 9(18.8) Sufficiency 4(8.5) 2(4.3) 4(8.5) 0 0 1(2.1) 7(14.9) 18(37.5) Butter consumption Deficiency 0 1(2.1) 2(4.3) 1(2.1) 1(2.1) 3(6.4) 12(25.5) 20(42.6) 9.563 0.654 Insufficiency 2(4.3) 0 2(4.3) 0 0 2(4.3) 3(6.4) 9(18.8) Sufficiency 0 1(2.1) 2(4.3) 1(2.1) 1(2.1) 3(6.4) 12(25.5) 20(42.6) Margarine consumption Deficiency 0 1(2.1) 5(10.6) 7(14.9) 4(8.5) 0 3(6.4) 20(42.6) 22.502 0.032* Insufficiency 0 0 0 0 0 1(2.1) 8(16.7) 9(18.7) Sufficiency 2(4.2) 0 0 0 0 0 16(33.3) 18(37.5) Cod liver consumption Deficiency 1(2.1) 0 0 0 1(2.1) 1(2.1) 17(36.2) 20(42.6) 3.361 0.762 Insufficiency 0 0 0 0 0 0 9(18.8) 9(18.8) Sufficiency 1(2.1) 0 0 0 0 0 17(36.2) 18(37.5) *Significance set at p<0.05. Chi-square test 52 University of Ghana htt p://ugspace.ug.edu.gh 4.5.2 Dietary vitamin D- rich foods intakes based on gender Table 4.5.3 shows dietary vitamin D rich foods consumption based on gender in this study. No significant association was observed between dietary vitamin D rich foods consumption and gender in this study. Table 4.5. 6: Dietary vitamin D-rich foods intakes based on gender (N=93) Characteristics Frequency of vitamin D rich foods consumption X2 p - value Gender 1x/day More 1-2x a 3-4x a 5-6x a Rarely Never Total than week week week once a day n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Yoghurt consumption Males 7 (7.5) 7 (7.5) 6 (6.4) 6 4 (4.3) 4(4.3) 5 (5.4) 39(41.9) 2.995 0.809 (6.4) Females 9 (9.7) 7 (7.5) 14 (15.1) 7 3(3.2) 4(4.3) 10(10.7) 54(58.1) (7.5) Ice cream consumption Males 11(11.8) 1(1.1) 10(10.7) 5(5.4) 5(5.4) 2(2.1) 5(5.4) 39(41.9) 6.690 0.350 Females 13(14.0) 9(9.7) 8(8.6) 4(4.3) 8(8.6) 3(3.2) 9(9.7) 54(58.1) Soymilk consumption Males 6(6.4) 2(2.1) 3(3.2) 1(1.1) 1(1.1) 9(9.7) 17(18.3) 39(41.9) 2.041 0.916 Females 7(7.5) 1(1.1) 8(8.6) 1(1.1) 1(1.1) 11(11.8 25(26.9) 54(58.1) ) Margarine Consumption Males 8(8.6) 5(5.4) 9(9.7) 4(4.3) 4(4.3) 5(5.4) 4(4.3) 39(41.9) 2.574 0.860 Females 7(7.5) 12(12.9) 11(11.8) 8(8.6) 5(5.4) 7(7.5) 4(4.3) 54(58.1) Significance set at p<0.05. Chi-square test. Salmon and herring consumption totala= 94. 53 University of Ghana htt p://ugspace.ug.edu.gh Table 4.5. 7: Dietary vitamin D-rich foods intakes based on gender (N=93) cont’d Characteristics Frequency of vitamin D rich foods consumption X2 p - value Gender 1x/day More 1-2x 3-4x 5-6x Rarely Never Total than a week a week a once week a day n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Salmon Consumptiona Males 12(12.8) 2(2.1) 7(7.4) 6(6.4) 6(6.4) 1(1.1) 6(6.4) 40(42.5) 9.628 0.141 Females 17(18.3) 11(11.7) 12(12.8) 3(3.2) 3(3.2) 3(3.2) 5(5.3) 54(58.1) Herring Consumptiona Males 2(2.1) 1(1.1) 9(9.6) 4(4.3) 2(2.1) 4(4.2) 18(19.1) 40(42.5) 7.139 0.308 Females 5(5.3) 3(3.2) 4(4.2) 6(6.4) 1(1.1) 11(11.7) 24(25.5) 54(58.1) Significance set at p<0.05. Chi-square test. Salmon and herring consumption totala= 94. 4.5.3 Dietary vitamin D-rich foods intake based on school type Table 4.5.4 shows the intakes of vitamin D rich foods between public and private schools. A significant association was found between yoghurt, ice cream, margarine, soymilk, and herrings. 54 University of Ghana htt p://ugspace.ug.edu.gh Table 4.5. 8: Dietary vitamin D-rich foods based on school type (N=93) Characteristics Frequency of vitamin D rich foods consumption X2 p - value School Type 1x/day More 1-2x a 3-4x a 5-6x a Rarely Never Total than week week week once a day n(%) n(%) n(%) n(%) n(%) n(%) n(%) N(%) Yoghurt consumption 12.844 0.046* Public schools 5(5.4) 6(6.4) 19(20.4 7(7.5) 5(5.4) 6(6.4) 13(14.0) 52(55.9) ) Private schools 11(11.8) 8(8.6) 19(20.4 6(6.4) 2(2.2) 2(2.2) 2(2.2) 41(44.1) ) Ice cream consumption Public schools 7(7.5) 6(6.4) 9(9.7) 6(6.4) 9(9.7) 3(3.2) 12(12.9) 52(55.9) 13.724 0.033* Private schools 17(18.3) 4(4.3) 9(9.7) 3(3.2) 4(4.3) 2(2.1) 2(2.1) 41(44.1) Soymilk consumption Public schools 4(4.3) - 5(5.4) 2(2.1) 1(1.1) 16(17.2 24(25.8) 52(55.9) 13.965 0.030* ) Private schools 9(9.7) 3(3.2) 6(6.4) - 1(1.1) 4(4.3) 18(19.3) 41(44.1) Margarine Consumption Public schools 2(2.1) 7(7.5) 13(14.0 10(10. 5(5.4) 9(9.7) 6(6.4) 52(55.9) 19.817 0.003* ) 8) Private schools 13(14.0) 10(10.8) 7(7.5) 2(2.1) 4(4.3) 3(3.2) 2(2.1) 41(44.1) Salmon Consumptiona Public schools 15(15.9) 7(7.4) 10(10.6 7(7.4) 5(5.3) 3(3.2) 6(6.4) 53(56.4) 2.655 0.851 ) Private schools 14(14.9) 6(6.4) 9(9.6) 2(2.1) 4(4.2) 1(1.1) 1(1.1) 41(43.6) Herring Consumptiona Public schools 52(2.1) 4(4.2) 10(10.6 4(4.2) 2(2.1) 11(11.7 17(18.0) 53(56.4) 13.263 0.039* ) ) Private schools 2(2.1) - 3(3.2) 6(6.4) 1(1.1) 4(4.2) 25(26.6) 41(43.6) *Significance set at p<0.05. Chi-square test. Salmon and herring consumption totala= 94. 55 University of Ghana htt p://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION The school-age period is considered the most energetic stage of a child's development. It is a period where the child undergoes various physical, mental, emotional and social development (Srivastava, Mahmood, Srivastava, Shrotriya, & Kumar, 2012) therefore an optimum health status of the child is necessary to facilitate the growth process. This study provides baseline data on the nutritional status and nutrient intake adequacy of vitamin D amongst school children in Ghana. Information provided will assist health professionals such as dietitians to plan, develop and implement policies on nutrition and dietary practices of children. This study may also inform future research into vitamin D related conditions in Ghana. 5.1 Dietary vitamin D intakes in school children The dietary vitamin D intakes of the school children were low with a significant association observed between the serum status and the dietary intakes of salmon, herrings, yogurt, ice cream, soy milk, and margarine. Even though dietary vitamin D contributes little to vitamin D status, this could adversely affect the vitamin D status of the respondents. These findings concur with other studies that showed low dietary vitamin D intakes among school-age children (Neyestani et al., 2011). This was as a result of few foods been rich in vitamin D in the Iranian diet and insufficient intakes of dairy products. 56 University of Ghana htt p://ugspace.ug.edu.gh Also, Voortman et al., (2015) found a significant association between serum vitamin D status and the intakes of margarine and cooking oils in the Netherlands diet, however, there was no significant association between vitamin D status and intakes of dairy products and cheese. In this study, females had higher frequent intakes of significant contributors of vitamin D rich foods than males. However, no significant association was observed between these foods and gender. Also, private school children had frequent intakes of the foods (yoghurt, soymilk, ice cream, salmon, herring, and margarine) which significantly contributed to serum vitamin D status compared to their counterparts from the public schools. Frequent intakes of significant contributors of vitamin D rich foods could be higher in private school children because these children could come from higher socio-economic status than their counterparts in the public schools. Khor et al., (2011) stated that a higher socio-economic status contributed to adequate micronutrient intakes of Malaysian children because their parents had formal education, earned a middle-class income on average, had access to nutritional information and quality health care services. 5.2 Length of sunlight exposure of school children In this study, similar percentage of the school children exposed themselves to sunlight during the hours of 10 am-12:59 pm. Public school children reported a higher percentage of sunlight exposure during the hours 10 am-12: 59 pm than their counterparts in the private schools. The public schools have a longer break period during those hours than the private schools hence the private school children could not expose themselves to the sun during 57 University of Ghana htt p://ugspace.ug.edu.gh those hours. With regards to gender, there was no significant difference in the percentages of males and females who exposed themselves to sunlight between the hours of 10 am-12: 59 pm. These findings are consistent with literature which shows that the best time for cutaneous synthesis of vitamin D is between the hours of 10 am to 12 pm and so school children who exposed themselves to the sun around these hours are more likely to have sufficient vitamin D status (Alshahrani et al., 2013). This study demonstrates that about one-third of the school children spend less than 15 minutes a day in the sun and 20.2% were outdoors between 15 to 30 minutes a day in the sun. Private school children significantly spent less time in the sun than their counterparts from the public schools. Also, a significantly higher percentage of females were outdoors for 15-30 minutes compared to males. This was similar to that of Alyahya (2017) in which more boys (38.7%) were outdoors in the sun compared to girls (22.6%) in Kuwait. Kuwait school girls exposed themselves to less sunlight mostly due to cultural practices and clothing styles. Furthermore, anecdotal evidence suggests that in recent times children spend less time outdoors, therefore, have limited sunlight exposure. These findings concur with that of Alyahya, (2017), in a cross-sectional study which recruited Kuwait school children between the ages of 5- 11 years during the spring to an early summer season in 2011. Alyahya (2017) also found that almost half of the children (49.7%) spent up to 5 minutes per day, 20.1% spent up to 15 minutes a day in the sun and 30.2% spent up to 30 minutes a day in the sun every day. Majority of the school children in this study (72.8%) exposed themselves to sunlight on an average of 3 days or more. Of this, significantly higher proportions were females and 58 University of Ghana htt p://ugspace.ug.edu.gh school children from the public schools. In Ghana, more sunny days are experienced compared to rainy or cloudy days, therefore children are more likely to come out to play. However, Al-Othman et al., (2012) in a cross-sectional study which recruited Saudi Arabian children aged between 6-17 years during the summer months of April to November, 2010 reported that about one- third of the participants (36.5%) had daily exposure to sunlight, almost 40% exposing themselves to sunlight once a week and 24% had no exposure at all. This result was attributed to children spending more time indoors than outdoors. 5.3 Serum vitamin D status in school children The findings of this study revealed that about half of the participants (49.1%) had deficient serum vitamin D status and 34% with sufficient serum vitamin D levels with mean vitamin D and standard deviation as 27.73 ± 9.90 ng/ml. This was similar to the findings of Roh et al., (2016), which showed that 59.1% of South Korean children aged 6 to 12 years were deficient in serum vitamin D levels with mean serum vitamin D of 19.83±7.39 ng/ml. On the contrary, a study conducted among 10-year old urban South African children revealed that 75% of the children had sufficient vitamin D levels (Poopedi et al., 2011). This sufficiency was ascribed to the fact that the children were likely to have had enough sunlight exposure, although not measured in their study. The present study found that the prevalence of vitamin D deficiency among females was higher than that of males. Also, females reported a higher sunlight exposure and frequent intakes of vitamin D rich foods than their male counterparts. In addition, the females 59 University of Ghana htt p://ugspace.ug.edu.gh recorded the highest sufficient status than the males. However, there was no significant association between gender and serum vitamin D status (p= 0.844) and no significant difference (p= 0.358) between the mean of serum vitamin D of females (22.71 ± 8.65 ng/mL) and males (25.29 ± 11.60 ng/mL). The findings of the present study are similar to a study in South Africa which found that black girls (34.8%) had higher deficiency levels of serum vitamin D than black boys (31.1%) (Poopedi et al., 2011). The observed differences reported by Poopedi et al., 2011 were attributed to males spending more time outdoors than females, and this is similar to the present study where males spent more time outdoors than females. Similar reasons could be attributed to the participants of the present study hence the lower prevalence of vitamin D deficiency among the males. Also, Khor et al.,(2011) in a cross-sectional study of 7-12 year old Malaysian children found a significant association (p=0.010) between vitamin D status and gender with a higher prevalence of deficiency among the girls (77.5%) than boys ( 66.1%) due to females having higher body fat mass and low sunlight exposure than boys. However, similar to the findings of the present study, Lee et al., (2013) in a cross-sectional study of peripubertal, non-obese children found no significant differences between serum vitamin D status and gender of the children studied. 5.4 Serum vitamin D status in private and public school children A significantly higher proportion of private school children reported sufficient vitamin D status compared to children in the public schools. These findings are unexpected because the children from the public schools reported a higher sunlight exposure than those from 60 University of Ghana htt p://ugspace.ug.edu.gh the private schools even though sunlight exposure is a major contributor of serum vitamin D status. Further, public school children had lower frequent intakes of significant vitamin D rich foods in this study and this could, however, explain their deficient vitamin D statuses. 5.5 Association between serum vitamin D status and anthropometric measurement This study showed no association between serum vitamin D and anthropometric status. Contrary to the findings of the present study, Voortman et al., (2015) found that children who were thin had deficient vitamin D status than those who were overweight and normal. The findings of Neyestani et al., (2011) showed that vitamin D status is affected by the BMI of the child particularly being obese which reduces the bioavailability of vitamin D and its synthesis to its metabolic form, calcitriol. Also, Khor et al., (2011) found that a little above half of the Malaysian children (58%) used in their study had a normal BMI- for-age, 17.9% were overweight, 16.4% obese and only 1.5% were too thin for their age. 5.6 Serum vitamin D status and length of sunlight exposure Serum vitamin D status and time spent in the sun every day was found to be significantly associated with this study and it significantly predicted serum vitamin D levels. It would have been expected that a greater majority of the children should be exposing themselves to sunlight because of the availability of sunlight in the country. This was however not the case. In Saudi Arabia, a prospective study recruiting adolescent girls demonstrates that 40.3% had no sun exposure every day, 13.5% had sun exposure between 11-20 minutes 61 University of Ghana htt p://ugspace.ug.edu.gh and only 6.0% with sunlight exposure for more than 30 minutes a day (Sulimani et al., 2016). Vitamin D deficiency was found to be more prevalent in those with no sun exposure and this was also found to be significantly associated with daily sunlight exposure. This was as a result of increased changes in the lifestyle of modern society resulting in increased indoor activities, decreasing exposure to sunlight among children (Roh et al., 2016). Furthermore, a study in Kuwait also revealed that only 30.2% of the school children exposed themselves to sunlight for up to 30 minutes a day and about 41% exposed themselves to sunlight for less than 5 minutes a day with low vitamin D status (Alyahya, 2017). Obviously, for children to reach an optimal vitamin D status, more sunlight exposure should be encouraged among school children as part of a whole, improved lifestyle. Furthermore, the majority of the school children who exposed themselves to the sun on an average of 3 days or more reported deficient serum vitamin D levels. In Korea, there is an assumption that sunlight exposure may be enough to meet the RDA of vitamin D in children, however Lee et al., (2013) found that the mean hours spent in the sun was less than 4 hours per week which was insufficient to meet the RDA of vitamin D during the winter season. Deficient vitamin D status was found to be significantly associated with low exposure to sunlight and low dietary vitamin D intakes. 62 University of Ghana htt p://ugspace.ug.edu.gh 5.7 Limitations The study had some limitations. 1. The sunlight exposure questionnaire used to estimate the length of sunlight exposure in this study could lead to respondents' bias because the school children could have over or under-reported their length of sunlight exposure. 2. Also, many parents refused to allow for their children to be sampled for serum vitamin D status due to fear and this lead to a small sample size (53) for serum vitamin D test which could affect the statistical power of the study. 63 University of Ghana htt p://ugspace.ug.edu.gh CHAPTER SIX CONCLUSIONS AND RECOMMENDATION 6.0 CONCLUSION Vitamin D deficiency is very prevalent (49.1%) among the school children studied but more prevalent in the public schools. Frequency of consumption of vitamin D rich foods were found to be low among the school children studied. A significant association was established between serum vitamin D status and the intakes of salmon, herrings, yoghurt, ice-cream, soy milk and margarine. Children from the public schools were more exposed to the sun. Males spent more time in the sun than females. Time spent outdoors in the sun impacted upon serum vitamin D status of the children. Further, a higher percentage of the participants had a normal BMI-for-age with the highest number reporting from the private schools. Out of the proportion who were overweight in both schools, the majority of them were from the public schools. Serum vitamin D was not associated with BMI-for-age. Children attending public schools were less likely to be vitamin D sufficient compared to their counterparts in private schools. Also, the males were not likely to be vitamin D sufficient compared to females. 64 University of Ghana htt p://ugspace.ug.edu.gh 6.1 RECOMMENDATIONS 1. There is a need for dietitians to educate school-age children on the need to increase sunlight exposure of children. Also, dietary intakes of vitamin D should be increased in children to supplement low exposure to sunlight. 2. Vitamin D fortification and dietary diversification strategies should also be considered to enrich foods. 3. Furthermore, more research is needed in this area of study. 65 University of Ghana htt p://ugspace.ug.edu.gh REFERENCES Al-Othman, A., Al-Musharaf, S., Al-Daghri, N. M., Krishnaswamy, S., Yusuf, D. S., Alkharfy, K. M., … Chrousos, G. P. (2012). Effect of physical activity and sun exposure on vitamin D status of Saudi children and adolescents. BioMed Central Pediatrics, 12(92), 1–6. https://doi.org/doi:10.1186/1471-2431-12-92 Al-Saleh, Y., Al-Daghri, N. M., Khan, N., Alfawaz, H., Al-Othman, A. M., Alokail, M. S., & Chrousos, G. P. (2015). Vitamin D status in Saudi school children based on knowledge. BMC Pediatrics, 15(53). https://doi.org/10.1186/s12887-015-0369-9 Alshahrani, A. A. (2014). Vitamin D Deficiency and Possible Risk Factors Among Middle Eastern University Students in London, Ontario, Canada. Electronic Thesis and Dissertation Repository. University of Western Ontario. Retrieved from http://ir.lib.uwo.ca/etd%0Ahttp://ir.lib.uwo.ca/etd/2010 Alshahrani, F. M., Almalki, M. H., Aljohani, N., Alzahrani, A., Alsaleh, Y., Holick, M. F., … Holick, M. F. (2013). Light side and best time of sunshine in Riyadh , Saudi Arabia. Dermato-Endocrinology, 5(1), 177–180. https://doi.org/10.4161/derm.23351 Alyahya, K. O. (2017). Vitamin D levels in schoolchildren : a cross- sectional study in Kuwait. BMC Pediatrics, 17(213), 1–10. https://doi.org/10.1186/s12887-017-0963-0 Bakare-Odunola, M. T., Kalik-Zik, T., Garba, M., Odunola, R. A., & Bello-Mustapha, K. (2012). Nutritional assessment of the diets in rickets prevalent communities in Kaduna State of Nigeria. International Journal of Medicine and Medical Sciences, 4(4), 110– 114. https://doi.org/10.5897/IJMMS12.027 Balasubramanian, S., Dhanalakshmi, K., & Amperayani, S. (2013). Vitamin D deficiency 66 University of Ghana htt p://ugspace.ug.edu.gh in childhood - A review of current guidelines on diagnosis and management. Indian Pediatrics, 50(7), 669–675. https://doi.org/10.1007/s13312... Bartley, J. (2010). Vitamin D , innate immunity and upper respiratory tract infection. Journal of Laryngology & Otology, 124, 465–469. https://doi.org/10.1017/S0022215109992684 Battault, S., Whiting, S. J., Peltier, S. L., Sadrin, S., Gerber, G., & Maixent, J. M. (2012). Vitamin D metabolism , functions and needs : From science to health claims. European Journal of Nutrition ·, 1–14. https://doi.org/10.1007/s00394-012-0430-5 Bentley, J. (2015). The role of vitamin D in infants, children and young people. Nursing Children and Young People, 27(1), 28–35. https://doi.org/10.7748/ncyp.27.1.28.e508 Bergwitz, C., & Juppner, H. (2010). Regulation of phosphate homeostasis by PTH, vitamin D and FGF23. Annu Rev Med, 61, 91–104. https://doi.org/10.1146/annurev.med051308.111339 Bikle, D. D. (2014). Review Vitamin D Metabolism, Mechanism of Action, and Clinical Applications. Chemistry & Biology Review, 21(3), 319–329. https://doi.org/10.1016/j.chembiol.2013.12.016 Binkley, N., Ramamurthy, R., & Kruger, D. (2015). Low Vitamin D Status: Definition, Prevalence, Consequences and Correction. Endocrinol Meta Clin North Am, 39(2), 287–305. https://doi.org/10.1016/j.ecl.2010.02.008.Low Cannell, J. J., Hollis, B. W., Zasloff, M., & Heaney, R. P. (2008). Diagnosis and treatment of vitamin D defi ciency. Expert Opin. Pharmacother. (2008), 9(1), 107–118. Ceglia, L. (2008). Vitamin D and skeletal muscle tissue and function. Molecular Aspects 67 University of Ghana htt p://ugspace.ug.edu.gh of Medicine, 29(6), 407–414. https://doi.org/10.1016/j.mam.2008.07.002 Chibuzor, M. T., Graham-Kalio, D., Meremikwu, M. M., & Adukwu, J. O. (2017). Vitamin D, calcium or a combination of vitamin D and calcium for the treatment of nutritional rickets in children. Cochrane Database of Systematic Reviews. https://doi.org/10.1002/14651858.CD012581 Deeb, K. K., Trump, D. L., & Johnson, C. S. (2007). Vitamin D signalling pathways in cancer; potential for anticancer therapeutics. Nat Rev Cancer, 7(9), 684–700. https://doi.org/10.1007/s00384010033 Eng, J. (2003). Sample size estimation: how many individuals should be studied? Radiology., 227(2), 309–13. https://doi.org/10.1148/radiol.2272012051 Enko, D., Kriegshauser, G., Stolba, R., Worf, E., & Halwachs-Baumann, G. (2015). Method evaluation study of a new generation of vitamin D assays. Biochemia Medica, 25(2), 203–212. https://doi.org/10.11613/BM.2015.020 Eyles, D., Anderson, C., Ko, P., Jones, A., Thomas, A., Burne, T., … McGrath, J. (2009). Clinica Chimica Acta A sensitive LC / MS / MS assay of 25OH vitamin D 3 and 25OH vitamin D 2 in dried blood spots. Clinica Chimica Acta, 403(1–2), 145–151. https://doi.org/10.1016/j.cca.2009.02.005 Flores, A., Flores, M., Macias, N., Hernández-Barrera, L., Rivera, M., Contreras, A., & Villalpando, S. (2017). Vitamin D deficiency is common and is associated with overweight in Mexican children aged 1–11 years. Public Health Nutrition, 20(10), 1807–1815. https://doi.org/10.1017/S1368980017000040 Fondjo, L. A., Owiredu, W. K. B. A., Sakyi, S. A., Laing, E. F., Adotey-Kwofie, M. A., 68 University of Ghana htt p://ugspace.ug.edu.gh Antoh, E. O., & Detoh, E. (2017). Vitamin D status and its association with insulin resistance among type 2 diabetics: A case -control study in Ghana. Plos One, 12(4), 1–14. https://doi.org/10.1371/journal.pone.0175388 Ghana Statistical Service. (2014). District Analytical Report Ledzokuku-Krowor Municipality. Retrieved from www.statsghana.gov.gh Girgis, C. M., Clifton-Bligh, R. J., Hamrick, M. W., Holick, M. F., & Gunton, J. E. (2013). The roles of vitamin D in skeletal muscle: Form, function, and metabolism. Endocrine Reviews, 34(1), 33–83. https://doi.org/10.1210/er.2012-1012 Green, R. J., Samy, G., Miqdady, M. S., El-Hodhod, M., Akinyinka, O. O., Saleh, G., … Salah, M. (2015). Vitamin D deficiency and insufficiency in Africa and the Middle East, despite year-round sunny days. South African Medical Journal, 105(7), 603– 605. https://doi.org/10.7196/SAMJnew.7785 Holick, M. F. (2009). Vitamin D Status: Measurement, Interpretation, and Clinical Application. Annals of Epidemiology, 19(2), 73–78. https://doi.org/10.1016/j.annepidem.2007.12.001 Holick, M. F., Binkley, N. C., Bischoff-Ferrari, H. A., Gordon, C. M., Hanley, D. A., Heaney, R. P., … Weaver, C. M. (2011). Evaluation, treatment, and prevention of vitamin D deficiency: An endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism, 96(7), 1911–1930. https://doi.org/10.1210/jc.2011-0385 Huh, S. Y., & Gordon, C. M. (2008). Vitamin D deficiency in children and adolescents: Epidemiology, impact and treatment. Reviews in Endocrine and Metabolic Disorders, 69 University of Ghana htt p://ugspace.ug.edu.gh 9(2), 161–170. https://doi.org/10.1007/s11154-007-9072-y Jung, G., Prange, M., & Schulz, M. (2016). Influence of topography on tropical African vegetation coverage. Climate Dynamics, 46(7), 2535–2549. https://doi.org/10.1007/s00382-015-2716-9 Kamen, D. L., & Tangpricha, V. (2010). Vitamin D and molecular actions on the immune system: modulation of innate and autoimmunity. J Mol Med (Berl), 88(5), 441–450. https://doi.org/10.1007/s00109-010-0590-9.Vitamin Karagüzel, G., Sakarya, N. P., Bahadır, S., Yaman, S., & Ökten, A. (2016). Vitamin D status and the effects of oral vitamin D treatment in children with vitiligo: A prospective study. Clinical Nutrition ESPEN, 15, 28–31. https://doi.org/10.1016/j.clnesp.2016.05.006 Khor, G. L., Chee, W. S. S., Shariff, Z. M., Poh, B. K., Arumugam, M., Rahman, J. A., & Theobald, H. E. (2011). High prevalence of vitamin D insufficiency and its association with BMI-for-age among primary school children in Kuala Lumpur , Malaysia. BioMed Central Public Health, 11(95), 1–8. https://doi.org/doi:10.1186/1471-2458-11-95 Lee, Y. A., Kim, J. Y., Kang, M. J., Chung, S. J., Shin, C. H., & Yang, S. W. (2013). Adequate vitamin D status and adiposity contribute to bone health in peripubertal nonobese children. Journal of Bone and Mineral Metabolism, 31(3), 337–345. https://doi.org/10.1007/s00774-012-0419-4 Legarth, C., Grimm, D., Wehland, M., Bauer, J., & Krüger, M. (2018). The Impact of Vitamin D in the Treatment of Essential Hypertension. International Journal of 70 University of Ghana htt p://ugspace.ug.edu.gh Molecular Sciences, 19(455), 1–14. https://doi.org/10.3390/ijms19020455 Li, W., & Tse, F. L. S. (2010). Special Issue : Review Dried blood spot sampling in combination with LC-MS / MS for quantitative analysis of small molecules. Biomed. Chromatogr, 24, 49–65. https://doi.org/10.1002/bmc.1367 Lips, P., van Schoor, N. M., & de Jongh, R. T. (2014). Diet, sun, and lifestyle as determinants of vitamin D status. Annals of the New York Academy of Sciences, 1317(1), 92–98. https://doi.org/10.1111/nyas.12443 McCarty, C. A. (2008). Sunlight exposure assessment: Can we accurately assess vitamin D exposure from sunlight questionnaires? American Journal of Clinical Nutrition, 87(4), 1097–1101. https://doi.org/87/4/1097S [pii] Mithal, A., Wahl, D. A., Burckhardt, P., Eisman, J. A., Fuleihan, G. E., Josse, R. G., … Dawson-Hughes, B. (2009). Global vitamin D status and determinants of hypovitaminosis D. Osteoporos Int, 20, 1807–1820. https://doi.org/10.1007/s00198- 009-0954-6 Mostafa, W. Z., & Hegazy, R. A. (2015). Vitamin D and the skin : Focus on a complex relationship : A review. Journal of Advanced Research, 6, 793–804. https://doi.org/10.1016/j.jare.2014.01.011 National Insitute of Health (2016). Vitamin D Fact Sheet for Consumers. National Institute of Health. Neyestani, T. R., Hajifaraji, M., Omidvar, N., Eshraghian, M. R., Shariatzadeh, N., Kalayi, A., … Nikooyeh, B. (2011). High prevalence of vitamin D deficiency in school-age 71 University of Ghana htt p://ugspace.ug.edu.gh children in Tehran, 2008: a red alert. Public Health Nutrition, 15(02), 324–330. https://doi.org/10.1017/S1368980011000188 NHANES. (2007). Anthropometry procedures manual. National Health and nutrition examinatory survey (NHANES). Retrieved from http://www.cdc.gov/nchs/data/nhanes/nhanes_07_08/manual_an.pdf O’Mahony, L., Stepien, M., Gibney, M. J., Nugent, A. P., & Brennan, L. (2011). The potential role of vitamin D enhanced foods in improving vitamin D status. Nutrients, 3(12), 1023–1041. https://doi.org/10.3390/nu3121023 Pellegrini, A. D., & Bohn-Gettler, C. M. (2013). The Benefits of Recess in Primary School. Scholarpedia, 8(2), 30448. https://doi.org/10.4249 Pettifor, J. M. (2014). Calcium and vitamin D metabolism in children in developing countries. Annals of Nutrition & Metabolism, 64(suppl 2), 15–22. https://doi.org/10.1159/000365124 Poopedi, M. A., Norris, S. A., & Pettifor, J. M. (2011). Factors influencing the vitamin D status of 10-year-old urban South African children. Public Health Nutrition, 14(02), 334–339. https://doi.org/10.1017/S136898001000234X Prentice, A., Ceesay, M., Nigdikar, S., Allen, S. J., & Pettifor, J. M. (2008). FGF23 is elevated in Gambian children with rickets. Bone, 42, 788–797. https://doi.org/10.1016/j.bone.2007.11.014 Prentice, A., Schoenmakers, I., Jones, K. S., Jarjou, L. M. A., & Goldberg, G. R. (2009). Vitamin D deficiency and its health consequences in Africa. Clinical Reviews in Bone and Mineral Metabolism, 7(1), 94–106. https://doi.org/10.1007/s12018-009-9038-6 72 University of Ghana htt p://ugspace.ug.edu.gh Prietl, B., Treiber, G., Pieber, T. R., & Amrein, K. (2013). Vitamin D and immune function. Nutrients, 5(7), 2502–2521. https://doi.org/10.3390/nu5072502 Rathi, N., & Rathi, A. (2011). Vitamin D and Child Health in the 21. Indian Pediatrics, 48, 619–625. Richardo, L. B. (2011). VDR activation of intracellular signalling pathways in skeletal muscle. Molecular Cellular Endocrinology, 347(1–2), 11–16. https://doi.org/10.1016/j.mce.2011.05.021 Ritu, G., & Gupta, A. (2014). Vitamin D deficiency in India: Prevalence, causalities and interventions. Nutrients, 6(2), 729–775. https://doi.org/10.3390/nu6020729 Roh, Y. E., Kim, B. R., Choi, W. B., Kim, H., Park, K. H., Kim, K. H., … Kim, S. Y. (2016). Vitamin D deficiency in children aged 6 to 12 years : single center ’ s experience in Busan. Annals of Pediatric Endocriology & Metabolism, 21, 149–154. https://doi.org/http://dx.doi.org/10.6065/apem.2016.21.3.149 Shin, Y. H., Shin, H. J., & Lee, Y.-J. (2013). Vitamin D status and childhood health. Korean Journal of Pediatrics, 56(10), 417–423. https://doi.org/10.3345/kjp.2013.56.10.417 Srivastava, A., Mahmood, S. E., Srivastava, P. M., Shrotriya, V. P., & Kumar, B. (2012). Nutritional status of school-age children - A scenario of urban slums in India. Archives of Public Health, 70(1), 2–9. https://doi.org/10.1186/0778-7367-70-8 Sulimani, R. A., Mohammed, A. G., Alfadda, A. A., Alshehri, S. N., Al-Othman, A. M., Al-Daghri, N. M., … Khan, A. A. (2016). Vitamin D deficiency and biochemical variations among urban Saudi adolescent girls according to season. Saudi Medical 73 University of Ghana htt p://ugspace.ug.edu.gh Journal, 37(9), 1002–1008. https://doi.org/10.15537/smj.2016.9.15248 Touvier, M., Deschasaux, M., Montourcy, M., Sutton, A., Charnaux, N., Kesse-Guyot, E., … Ezzedine, K. (2015). Determinants of vitamin D status in Caucasian adults: Influence of sun exposure, dietary intake, sociodemographic, lifestyle, anthropometric, and genetic factors. Journal of Investigative Dermatology, 135(2), 378–388. https://doi.org/10.1038/jid.2014.400 Tuohimaa, P. (2008). Vitamin D, aging, and cancer. Nutrition Reviews, 66(SUPPL.2), 147– 152. https://doi.org/10.1111/j.1753-4887.2008.00095.x Voortman, T., van den Hooven, E. H., Heijboer, A. C., Hofman, A., Jaddoe, V. W., & Franco, O. H. (2015). Vitamin D Deficiency in School-Age Children Is Associated with Sociodemographic and Lifestyle Factors. Journal of Nutrition, 145(4), 791–798. https://doi.org/10.3945/jn.114.208280 Vuolo, L., Di Somma, C., Faggiano, A., & Colao, A. (2012). Vitamin D and cancer. Frontiers in Endocrinology, 3(58), 1–13. https://doi.org/10.3389/fendo.2012.00058 Wagner, C. L., & Greer, F. R. (2008). Prevention of Rickets and Vitamin D Deficiency in Infants , Children, and Adolescents. American Academy of Pediatrics, 122(5), 1142– 1153. https://doi.org/10.1542/peds.2008-1862 Weydert, J. (2014). Vitamin D in Children’s Health. Children, 1(2), 208–226. https://doi.org/10.3390/children1020208 74 University of Ghana htt p://ugspace.ug.edu.gh APPENDIX I PARENT’S CONSENT FORM DEPARTMENT OF NUTRITION AND DIETITICS, SCHOOL OF BIOMEDICAL AND ALLIED HEALTH SCIENCES, COLLEGE OF HEALTH SCIENCES, UNIVERSITY OF GHANA VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION INFORMATION SHEET My name is Sylvia Adoma Oteng, a final year MSc Dietetics Student of the School Of Biomedical And Allied Health Sciences, University of Ghana. I am carrying out a research on “VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION”, as a partial fulfilment of my MSc. degree in Dietetics. This study seeks to assess the vitamin D status of Ghanaian school-age children. Your ward has been invited to partake in this study. Please note that participation is entirely voluntary: you may refuse to allow your ward to take part or withdraw him/her without anyone objecting. If you agree to his/her participation, you will please need to complete the attached questionnaire to provide a little information about your child and yourself. Your ward’s school will be visited to measure your child’s weight, height and draw 2mls (approximately half a teaspoon) of blood sample for vitamin D testing (drawing of the 75 University of Ghana htt p://ugspace.ug.edu.gh blood may cause a little discomfort and pain). Information on vitamin D rich food sources will also be obtained from your child. The vitamin D status of your child will be made known to you after the analysis. Please note that ethical clearance has been obtained from the Ethics and Protocol Review Committee of the School Of Biomedical and Allied Health Sciences (SBAHS), College of Health Sciences (CHS), University of Ghana. Permission has also been obtained from the Head of the School to carry out this study. All information will be kept with strict confidentiality and will be used for the research only. If you have any questions, please feel free to call any of the contacts below: Researcher’s Contact Sylvia Adoma Oteng Department of Nutrition and Dietetics University of Ghana 0246023527 Supervisors’ Contact Mrs. Freda Intiful M r s . R e b e c c a S t e e le-Dadzie Department of Nutrition and Dietetics D e p a r t m e n t o f N utrition and Dietetics University of Ghana University of Ghana 0243439389 0246242805 76 University of Ghana htt p://ugspace.ug.edu.gh INFORMED CONSENT FORM I……………………………………………………………………………………………. have read (or have had read to me in a language that I understand) the proposed study and that I have understood what is going to be done to my ward. Also, any concern that I have, have been fully addressed. My signature/ thumbprint below shows that I agree to allow my ward take part in the study. Signature/ Thumbprint……………………………………………………………………. Date………………………………………………………….. 77 University of Ghana htt p://ugspace.ug.edu.gh CODE…………… VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION INFORMATION ABOUT PARENTS/GUARDIAN (please tick appropriately where needed) 1. Parent/ guardian’s age: …………………………………………… 2. Parent/ guardian’s gender: Male Female 3. Highest level of education: None Primary Secondary Tertiary 4. Employment status: Employed Unemployed Retired 5. Occupation: ……………………………………………………… 78 University of Ghana htt p://ugspace.ug.edu.gh APPENDIX II ASSENT FORM VITAMIN D STATUS IN GHANAIAN SCHOOL AGE CHILDREN IN SOME SELECTED SCHOOLS IN THE GREATER ACCRA REGION CHILD ASSENT FORM My name is Sylvia Adoma Oteng, a final year MSc Dietetics Student of the School Of Biomedical And Allied Health Sciences, University of Ghana. I am carrying out a research on “VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION”, as a partial fulfilment of my MSc. degree in Dietetics. This will help me to identify any potential health risk you may be exposed to so that I can help to address them. If you agree to participate, I will measure your weight, height and draw 2mls (approximately half a teaspoon) of blood from your arm (drawing of the blood may cause a little discomfort and pain). I will also ask you some questions about some rich vitamin D foods you usually eat and how much sunlight you are exposed to. Always remember that your answers will be kept private and I will not show it to anyone, not even your parents or teachers. You may not have any direct benefit from participating in this study. However the information you provide will help me identify any dietary problems related to consuming vitamin D rich foods in school age children and the results of the Vitamin D test will be 79 University of Ghana htt p://ugspace.ug.edu.gh given to. Participation in this study is voluntary, if you do not want to be part, you are free to do so. Although your parents / guardians have consented to your participation, you can still refuse to take part. You are free to ask any questions about the study at any time. Call me on 0246023527. By providing your name below, you have understood the procedures of this study, had all your questions answered and have agreed to be part of the study. Thank you. Name of pupil……………………………………………….. Date…………………………. Name of researcher…………………………………………… Date………………………….. 80 University of Ghana htt p://ugspace.ug.edu.gh APPENDIX III QUESTIONNAIRE VITAMIN D STATUS OF GHANAIAN 8-12 YEAR OLD CHILDREN IN SELECTED SCHOOLS IN THE GREATER ACCRA REGION CODE…………… SECTION ONE: SOCIO-DEMOGRAPHIC INFORMATION 1. AGE: 2. GENDER: MALE FEMALE 3. CLASS: 1 2 3 4 5 6 4. RELIGION: CHRISTIAN MOSLEM TRADITIONALIST OTHER (specify)…………………….. 81 University of Ghana htt p://ugspace.ug.edu.gh 5. ETHNICITY: AKAN GA EWE OTHER (specify)………………….. 6. WHO DO YOU LIVE WITH? SINGLE PARENT BOTH PARENT RELATIVE GUARDIAN 7. OCCUPATION OF MAIN PROVIDER: TRADER SELF-EMPLOYED GOVERNMENT EMPLOYEE UNEMPLOYED OTHER (specify)…………… 8. NUMBER OF CHILDREN: ……………………………………….... 9. YOUR BIRTH POSITION: ……………………………….. 10. PLACE OF RESIDENCE: …………………………………………………... 11. TYPE OF HOUSE YOU LIVE IN: SELF-CONTAINED SINGLE ROOM COMPOUND HOUSE CHAMBER & HALL 12. NUMBER OF PEOPLE IN THE HOUSE: …………………….. 82 University of Ghana htt p://ugspace.ug.edu.gh SECTION TWO: ANTHROPOMETRY MEASUREMENTS PARAMETERS 1ST READING 2ND READING 3RD READING WEIGHT HEIGHT BMI SECTION THREE: SERUM 25(OH)D TEST RESULT CODE RESULT 83 University of Ghana htt p://ugspace.ug.edu.gh SECTION FOUR: DIETARY ASSESSMENT PLEASE CHECK THE FREQUENCY IN WHICH YOU CONSUME THESE FOODS. Food/dish Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week BEVERAGES Tea Coffee PORRIDGES Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Corn porridge Millet porridge Rice porridge Oats porridge Wheat porridge Weanimix Fortified Breakfast cereal e.g. corn flakes, Weetabix MILK AND MILK Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week PRODUCTS Low-fat milk e.g. carnation Skimmed milk 84 University of Ghana htt p://ugspace.ug.edu.gh Yoghurt Ice cream Cheese Wagashi Soymilk SPREADS Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Butter Margarine Peanut butter BREAD Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Whole meal Tea bread Sugar bread Butter bread DEEP FRIED FOODS Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Fried yams/plantain etc. Fried plantain Potato chips Beans cake(koose) 85 University of Ghana htt p://ugspace.ug.edu.gh OILS Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Palm oil White oil e.g. sunflower, frytol/ soybean Coconut oil Cod-liver oil VEGETABLES Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Pebble garden egg (abeduru) Cabbage Green leafy vegetables Carrots Peas Green beans (runner beans etc.) Cucumber Sweet peppers Kontomire stew Garden egg stew Gravy FISH AND SEAFOOD Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week 86 University of Ghana htt p://ugspace.ug.edu.gh Canned fish Salmon Mackerel Tuna Sardine Kippers Pilchards Eel Herring Trout MEAT AND MEAT Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week PRODUCTS Beef Poultry Pork Goat meat Pig feet Snails Offal Sausages Cow foot 87 University of Ghana htt p://ugspace.ug.edu.gh Canned meat and fish e.g. corned beef Game (Bush meat) Eggs STARCHES Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Rice (White) Rice (Brown) Pasta, macaroni, spaghetti Wheat SOUPS Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Palm soup Ground-nut soup Light soup Okro soup FRUITS Once a More than 1-2x a 3-4x a 5-6x a Rarely Never day once a day week week week Avocado pear Orange Banana Mango 88 University of Ghana htt p://ugspace.ug.edu.gh Pineapple Apple Pawpaw FRUIT JUICES SOFT DRINKS SNACKS (PASTRIES, BISCUITS ETC) SWEETS LEGUMES FAST FOODS Hamburgers, pizza, takeaway etc. VITAMIN D SUPPLEMENTS 89 University of Ghana htt p://ugspace.ug.edu.gh SECTION FIVE: SUN EXPOSURE ASSESSMENT 1. When is your first break at school? …………………………………………………….. 2. What do you usually do during the breaktime? ………………………………………… 3. When is your second break at school? …………………………………………………... 4. What do you usually do during the second breaktime? …………………………………. 5. Do you play outside? .......................................................................................................... 6. For how long? ……………………………………………………………………………. 7. Do you use a sunscreen cream during sunny days? ………………………………………. 8. Which parts of your body is usually exposed when dressed up? ……………………………………………………………………………………… ……… 90 University of Ghana htt p://ugspace.ug.edu.gh 9. How much time do you spend in the sun every day? o < 15 minutes per day o 15-30 minutes per day o more than 30 min- to 60 minutes per day o more than 1- 2 hours per day o hours per day 10. What time do you mostly spend outdoors under the sun? o 7-10 am o 10-1 pm o 1-4 pm o 4-7 pm 11. What do you normally wear outdoors? o Trousers o Short o T-shirt o Long sleeves shirt o Cap/ Hat o Short sleeves dress o Short sleeves blouse o Short skirt o Long skirt 91 University of Ghana htt p://ugspace.ug.edu.gh o Shorts and very brief top with shoulders exposed o Shorts and T-shirt or similar top o Shorts and long sleeves o Long pants and T-shirt or similar top o Long pants and long sleeves o Cover face and head with hand exposed o Cover head with arms exposed o Cover head with hand exposed o Fully covered 12. For Muslim Girls Only: When you are in the company of women only in outdoor private settings, describe your usual clothing: o Face, head and hands exposed o Shorts and very brief top with shoulders exposed o Shorts and T-shirt or similar top o Shorts and long sleeves o Long pants and T-shirt or similar top o Long pants and long sleeves 13. On the average, how many days a week do you expose yourself to the sun? o Not at all o 1 day 92 University of Ghana htt p://ugspace.ug.edu.gh o 2 days o 3 days 93 University of Ghana htt p://ugspace.ug.edu.gh APPENDIX IV ETHICAL APPROVAL 94 University of Ghana htt p://ugspace.ug.edu.gh 95 University of Ghana htt p://ugspace.ug.edu.gh 96