Vitamin D status in COPD patients at Chris Hani Baragwanath Hospital Imraan Kola Imraan Kola Student number 362488 A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Medicine Johannesburg, 2023 ii DECLARATION I, Imraan Kola, declare that this research report is my own, unaided work. It is being submitted for the Degree of Master of Medicine in the branch of Internal Medicine at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University. 27th day of July 2023 in Johannesburg Supervisors: Dr SA van Blydenstein MBBCh, DCH, FCP(SA), MMed(IntMed), Cert Pulm(SA) Specialist Physician, Division of Pulmonology, Department of Internal Medicine, Chris Hani Baragwanath Academic Hospital University of the Witwatersrand Prof S Omar MBChB, FCpath(SA)CHEM, DA(SA), Critical Care (SA) ICU Department Chris Hani Baragwanath Academic Hospital University of the Witwatersrand iii DEDICATION I dedicate this work to my mother, Zareena Kola and father, Mohamed Kola for unwavering moral and financial support, encouragement and prayers. iv PRESENTATIONS ARISING FROM THE RESEARCH PROJECT Nil presentations. PUBLICATIONS ARISING FROM THE RESEARCH PROJECT Submitted for publication to the African Journal of Thoracic and Critical Care Medicine – awaiting editorial review. v ABSTRACT Background: There has been a growing interest in nutritional/ lifestyle factors, including vitamin D, that may impact Chronic Obstructive Pulmonary Disease (COPD). Most data are in Caucasian populations and temperate climates with minimal African (ethnicity and geographical) data. Objectives: The primary objective was to determine amongst COPD patients the prevalence of vitamin D deficiency (25 [OH]D ≤ 20 ng/ml) and insufficiency (25[OH]D - 21-29 ng/ml). Secondary objectives were to investigate the association between vitamin D and demographic/lifestyle factors; lung function parameters; markers of COPD severity and corticosteroid usage. Methods: A prospective, cross-sectional study of 76 COPD patients was conducted at a tertiary Johannesburg hospital. Patients were interviewed regarding demographic/lifestyle factors, COPD severity markers and corticosteroid therapy. The most recent spirometry was recorded. Blood samples were taken for calcium, alkaline phosphatase and vitamin D. Patients were stratified according to vitamin D status (deficiency and non-deficiency) and statistical analysis was performed to assess for associations. Results: The sample included 72% males and 63% black/ African patients. The prevalence of vitamin D deficiency and insufficiency were 48% (95% confidence interval [CI] 42 -54) and 35% (CI 30-41), respectively. The Modified Medical Research Council (MMRC) dyspnoea scale of ≥2 was associated with a relative risk of 1.34 (CI 1.05-1.7) for vitamin D deficiency in univariate analysis. In multivariate regression only sunlight exposure (<1 hour/day) was an independent predictor of vitamin D deficiency (Odds ratio 2.4, CI 1.3 -4.5) Conclusion: A high prevalence of suboptimal vitamin D levels exists within this sample COPD population. A higher MMRC score was associated with an increased risk of vitamin D deficiency while low sunlight exposure was the only independent predictor of vitamin D deficiency. vi ACKNOWLEDGEMENTS The author would like to thank Professor M. Wong, Head of Pulmonology at Chris Hani Baragwanath Academic Hospital (CHBAH) for allowing the study to happen and assisting at the Respiratory Outpatient Department (OPD). Special thanks are also extended to my supervisors, Dr S.A. van Blydenstein and Prof S. Omar, for their constant guidance. A special thanks and acknowledgement to all the patients that elected to join the study, without which this research would not be possible. A special thanks to Mohau La-Donna Kapa, Amore Visagie and Annie Maphthu, respiratory technicians that assisted with spirometry and data collection. Thanks are extended to Jean Johnstone for assisting with formatting. vii TABLE OF CONTENTS DECLARATION .................................................................................................................. II DEDICATION ..................................................................................................................... III PRESENTATIONS ARISING FROM THE RESEARCH PROJECT .................................. IV PUBLICATIONS ARISING FROM THE RESEARCH PROJECT ..................................... IV ABSTRACT ......................................................................................................................... V ACKNOWLEDGEMENTS ................................................................................................. VI TABLE OF CONTENTS .................................................................................................... VII LIST OF FIGURES ............................................................................................................. IX LIST OF TABLES ................................................................................................................ X NOMENCLATURE ............................................................................................................ XI CHAPTER 1 .......................................................................................................................... 1 INTRODUCTION AND EXTENDED LITERATURE REVIEW .......................................... 1 1.1 Epidemiology ........................................................................................................... 1 1.2 Vitamin D in calcium and phosphate homeostasis .................................................... 1 1.3 Non-calcaemic vitamin D effects and role in COPD ................................................. 2 1.4 Vitamin D and other respiratory illnesses ................................................................. 2 1.5 Vitamin D deficiency definition and normal levels ................................................... 2 1.6 Vitamin D and airflow/ spirometry parameters ......................................................... 3 1.7 Vitamin D deficiency prevalence and associations in COPD patients ........................ 4 1.8 Vitamin D and exacerbations in COPD patients ........................................................ 4 1.9 COPD treatment and vitamin D associations ............................................................ 5 1.10 Effect of vitamin D supplementation on COPD clinical parameters .......................... 5 1.11 Mechanisms postulated to explain COPD vitamin D associations ............................. 6 1.12 The rationale for the current study ............................................................................ 7 CHAPTER 2 .......................................................................................................................... 8 METHODOLOGY ................................................................................................................ 8 2.1 Objectives ................................................................................................................ 8 2.2 Study design ............................................................................................................. 8 2.3 Study population ...................................................................................................... 8 2.4 Study setting............................................................................................................. 8 2.5 Sample size .............................................................................................................. 8 2.6 Data collection ......................................................................................................... 9 viii 2.7 Definitions ............................................................................................................... 9 2.8 Vitamin D testing and definition ............................................................................. 10 2.9 Statistical analysis .................................................................................................. 10 2.10 Ethical considerations ............................................................................................. 11 CHAPTER 3 ........................................................................................................................ 12 RESULTS ............................................................................................................................ 12 3.1 Sample description ................................................................................................. 12 3.2 Primary objective ................................................................................................... 13 3.3 Secondary objectives .............................................................................................. 14 3.3.1 Univariate analysis ................................................................................................. 14 3.3.2 Multivariate analysis .............................................................................................. 15 CHAPTER 4 ........................................................................................................................ 17 DISCUSSION ...................................................................................................................... 17 CHAPTER 5 ........................................................................................................................ 20 CONCLUSION AND RECOMMENDATIONS .................................................................. 20 CHAPTER 6 ........................................................................................................................ 21 REFERENCES .................................................................................................................... 21 CHAPTER 7 ........................................................................................................................ 25 APPENDICES ..................................................................................................................... 25 Appendix 1 – Research protocol........................................................................................... 25 Appendix 2 – Patient informed consent form........................................................................ 43 Appendix 3 – Patient information leaflet .............................................................................. 45 Appendix 4 – Data collection sheet ...................................................................................... 48 Appendix 5 – MMRC dyspnoea scale .................................................................................. 50 Appendix 6 – Gold grade of COPD based on FEV1 % predicted .......................................... 51 Appendix 7 – Gold groups based on exacerbation and symptom scores ................................ 52 Appendix 8 – Ethics clearance certificate ............................................................................. 53 Appendix 9 – Ethics approval of covid-19 modifications...................................................... 55 Appendix 10 – CHBAH Medical Advisory Committee permission letter ............................. 56 Appendix 11 – CHBAH Internal medicine Head of Department (HOD) permission letter .... 57 Appendix 12 – CHBAH Respiratory HOD permission letter ................................................ 57 Appendix 13 – Plagiarism/ Turnitin report ........................................................................... 59 ix LIST OF FIGURES Figure 3.1: Vitamin D Status versus increasing sunlight exposure ........................................ 12 x LIST OF TABLES Table 3.1: Study patient characteristics ............................................................................... 13 Table 3.2: The prevalence of Vitamin D deficiency and insufficiency among COPD study population ............................................................................................................................ 14 Table 3.3: Vitamin D levels and demographic/lifestyle factors (univariate analysis) ............ 15 Table 3.4: Vitamin D levels and lung function parameters (univariate analysis) ................... 15 Table 3.5: Vitamin D status and COPD severity markers, therapies (univariate analysis) ..... 16 xi NOMENCLATURE 1,25 [OH]2 D - 1,25–dihydroxy vitamin D 25[OH]D - 25 hydroxy-vitamin D ATS - American Thoracic Society BMI - Body Mass Index BOLD - Burden of Obstructive Lung Disease CAT - COPD Assessment Test CHBAH - Chris Hani Baragwanath Academic Hospital CI - 95 % confidence interval COPD - Chronic Obstructive Pulmonary Disease COVID-19 - Coronavirus Disease 2019 ERS - European Respiratory Society FEV1 - Forced Expiratory Volume in the first second FEV1 % - Forced Expiratory Volume % predicted FVC - Forced Vital Capacity FVC% - Forced Vital Capacity % predicted GMCSF - Granulocyte Monocyte Colony Stimulating Factor GOLD - Global Initiative for Obstructive Lung Disease HIV - Human Immunodeficiency Virus HOD - Head of Department IFN ɣ - Interferon ɣ IL - Interleukin IQR - Interquartile range IV - Intravenous LTOT - Long term oxygen therapy MAC - Medical Advisory Committee MMRC - Modified Medical Research Council n - Number of variable OPD - Outpatient department QOL - Quality of Life RCT - Randomised Control Trial SD - Standard deviation SGRQ - St. George’s Respiratory Questionnaire SPIROMICS - Subpopulations and Intermediate Outcome Measures in COPD Study xii T reg - Regulatory T cell Th1 - Type 1 T helper Th2 - Type 2 T helper UK - United Kingdom US - United States USA - United States of America UVB - Ultraviolet B UVS - Ultraviolet score VDR - Vitamin D Receptor 1 CHAPTER 1 INTRODUCTION AND EXTENDED LITERATURE REVIEW 1.1 Epidemiology Globally more than 170 million people are affected by Chronic Obstructive Pulmonary Disease (COPD) and COPD accounted for approximately 3.2 million deaths in 2015 (1). Low and middle-income countries bear a significant burden of COPD mortality. Limited data on the epidemiology of COPD in Africa is available. There is no robust South African (national) COPD prevalence data. The BOLD study (2) found a prevalence exceeding 20% in Cape Town. It should be noted that the demographics of Cape Town differ s significantly from the remainder of South Africa and it would be inaccurate to infer that these estimates represent a national prevalence. There is an increasing interest in lifestyle and dietary factors that may impact disease severity in the COPD population. There have been a large number of studies investigating the role of Vitamin D deficiency in COPD and its association with markers of disease severity. From the review of the available literature, there is a paucity of data available from South Africa or Africa in general concerning this subject. George et al. (3) in a healthy cohort in Johannesburg found a prevalence of vitamin D < 30 nmol/l (12 ng/ml) ranging between 5.1% (in black/African patients) to 28.6 % (in Asian/ Indian patients) emphasising the impact of race/ethnicity on vitamin D level. This finding may seem counterintuitive, owing to higher skin melanin content (and reduced vitamin D activation) in black/ African participants. The authors attribute the high vitamin D deficiency prevalence in Asians/ Indians to conservative cultural dressing practices limiting direct skin sunlight exposure. 1.2 Vitamin D in calcium and phosphate homeostasis Vitamin D3 is predominantly synthesised in the skin. Ultraviolet (UVB) radiation converts previtamin D3 to vitamin D3 (cholecalciferol). This step is influenced by melanin content in the skin and sunlight exposure. The other less significant source of vitamin D is dietary intake. Vitamin D3 is hydroxylated in the liver to 25 hydroxy-vitamin D (25[OH]D). 25 [OH] D is the major storage form and is used to ascertain vitamin D status in populations (4,5). Vitamin D is predominantly hydroxylated in the kidney to 1,25–dihydroxy vitamin D (1,25 2 [OH]2 D). This represents the active form of vitamin D which enhances gastrointestinal absorption of calcium and phosphate and impacts positively bone turnover and bone mineral density (6). 1.3 Non-calcaemic vitamin D effects and role in COPD Vitamin D exerts pleiotropic effects unrelated to its role in calcium and phosphate homeostasis. It is now evident that vitamin D plays a significant immunomodulatory effect. Antigen Presenting Cells can be inhibited by vitamin D. Vitamin D inhibits the innate and adaptive immune response through Vitamin D Receptors (VDRs) on inflammatory cells. Vitamin D deficiency fails to restrain macrophage and dendritic cell maturation through major histocompatibility class II restriction. Vitamin D also plays an important function in T cell differentiation. Vitamin D inhibits the Type 1 T helper (Th1) response and its associated cytokines i.e. interleukin (IL)-2, Granulocyte Monocyte Colony Stimulating Factor (GMCSF) and interferon ɣ (IFN ɣ) in favour of Type 2 T helper (Th2) and regulatory T cell (T reg) responses. Thus, vitamin D deficiency is postulated to result in dysregulation of the immune and inflammatory response resulting in chronic lung inflammation and structural lung changes promoting the onset and progression of COPD (6). Vitamin D also enhances macrophage production of antimicrobial peptides like cathelicidin which is an important component of the immune response to combat mycobacterial infections (6). Vitamin D deficiency results in increased respiratory infections (including mycobacterial tuberculosis infection) and airway microbe colonisation which could theoretically lead to COPD progression. Vitamin D deficiency also results in increased airway remodelling, fibroblast proliferation (and hence collagen synthesis) and alteration in gene expression resulting in smooth muscle proliferation and contraction promoting the airway changes characteristic of COPD. 1.4 Vitamin D and other respiratory illnesses Vitamin D deficiency has been associated with other respiratory conditions including tuberculosis (7), sarcoidosis (8), childhood asthma, cystic fibrosis (6) and recently COVID-19 (9). 1.5 Vitamin D deficiency definition and normal levels 3 There is some heterogeneity in the values quoted for vitamin D deficiency. This variability may occur because vitamin D levels required for the calcaemic effects (prevention of rickets and osteomalacia) are better established by robust data than for non-calcaemic effects of vitamin D; as well as variation in assays used to ascertain levels. The Endocrine Society defines vitamin D deficiency as 25[OH]D levels less than 20 ng /ml, and vitamin D insufficiency as levels between 21-29 ng/ml (10). The Institute of Medicine (IOM) (11) recommended that a level of 25[OH] vitamin D exceeding 20 ng/ml is adequate. 1.6 Vitamin D and airflow/ spirometry parameters A post hoc cross-sectional review of the Baltimore Longitudinal Study of Ageing (12) failed to demonstrate an association between vitamin D deficiency and airflow limitation. An important limitation of this study is that airflow limitation was not objectively assessed but based on self-reported patient diagnoses. Airflow limitation encompassed COPD, non-smoker COPD and asthma. The majority of studies reviewed demonstrated lower FEV1 (Forced Expiratory Volume in the first second) correlated with vitamin D deficiency (13–19). Most of these studies were in Europe (15,17-19) and South Korea (13,16) with one study (14) conducted in the United States of America (USA). The majority of participants were of Caucasian (14,15,17-19) or Asian (13,16) ethnicity. Three studies also showed no vitamin D FEV1 association (12,20,21). The majority of participants in all three studies were Caucasian and from Europe (20,21) or the USA (12). Jolliffe et al. (15), Mekov et al. (19) and Gawron et al. (21) found no association between FEV1/FVC (Forced Vital Capacity) and vitamin D. Jung et al. (13) in a South Korean longitudinal study found higher FEV1/FVC ratios in non-deficient vitamin D patients, as well as a trend towards lower exercise capacity in the deficient group. A small COPD Turkish study (22) with a high prevalence of vitamin D deficiency (60%) found lung function parameters (FEV1/FVC; FEV1; FVC and total lung capacity [TLC]) were lower in COPD vitamin D deficient patients. Hand grip and isokinetic knee strength were lower in the vitamin D deficient subgroup. 4 1.7 Vitamin D deficiency prevalence and associations in COPD patients A Bulgarian study (19) looked at vitamin D deficiency in COPD patients admitted to the hospital. Owing to the study design this study had patients with more severe disease – Global Initiative on COPD (GOLD) class C and D. This study demonstrated a high prevalence of vitamin D deficiency - < 20 ng/ml (83.6%) which is significantly higher than other studies where participants were not experiencing an exacerbation (31-77% prevalence). There existed a correlation between vitamin D and Quality of Life (QOL) indices (e.g. Modified Medical Research Council [MMRC] dyspnoea scale). No correlation existed between vitamin D level and comorbidities (Diabetes Mellitus, hypertension, metabolic syndrome and the number of exacerbations). Vitamin D deficiency was associated with a longer hospital stay. Kentson et al. (17) in a single-centre case-control study found no difference in the prevalence of vitamin D deficiency in long term oxygen therapy (LTOT) COPD patients versus non- LTOT users. Since this study wanted to stratify patients based on oxygen therapy a sicker cohort was selected. The sample included majority Caucasians. Peak annual vitamin D levels were measured in summer and early autumn as seasonal variation in Sweden may be significant. Lower 25 [OH] vitamin D levels were found in the COPD group compared with controls. 25[OH]D levels did correlate with MMRC score and oxygen saturation. Joliffe et al. (15) in an extensive multicentre cross-sectional study in the United Kingdom (UK) explored if an association between various lifestyle factors and COPD severity markers correlated with vitamin D levels. Lifestyle factors associated with vitamin D deficiency included high Body Mass Index (BMI), lower socio-economic status, lack of vitamin D supplementation, winter sampling and absence of a summer holiday. No role of vitamin D pathway genetic polymorphisms or inhaled corticosteroid dose and vitamin D deficiency was demonstrated. 1.8 Vitamin D and exacerbations in COPD patients The literature is conflicting regarding exacerbations. Jung et al. (13); Gawron et al. (21); Kunisaki et al. (23) and Persson et al. (18) found no association between exacerbations and vitamin D status. A primary care setting study in Switzerland, Netherlands (24) with milder COPD phenotype (predominantly GOLD II) patients also found no such association. However, there was a trend toward vitamin D sufficient participants (25[OH]D > 30 ng/ml) 5 having fewer exacerbations. Malinovschi et al. (20) found an odds ratio of being a frequent exacerbator if having severe vitamin D deficiency was 18.1 (95% confidence interval [CI] 4.98 -65.8). The authors did concede that association does not infer causality as exacerbation history was retrospective (exacerbations in the year preceding vitamin D measurement). Burkes et al. (14) also found that vitamin D deficient participants had more exacerbations (including severe exacerbations) in the year preceding recruitment. Hyun et al. (16) also found that there was a higher rate of severe exacerbations in patients with low vitamin D and high fibrinogen levels. A 2016 meta- analysis of 21 studies (25) showed no association between vitamin D levels and exacerbation frequency. 1.9 COPD treatment and vitamin D associations Janssen et al. (26) and Gawron et al. (21) found no association between corticosteroid usage and vitamin D levels. Persson et al. (18) found an association between vitamin D and the use of inhaled corticosteroids in univariate, but not multivariate analysis. Jolliffe et al. (15) also showed no correlation between vitamin D status and inhaled corticosteroid dose. These findings call into question the idea that corticosteroids enhance vitamin D catabolism and thus contribute to deficiency in the COPD cohort. 1.10 Effect of vitamin D supplementation on COPD clinical parameters Several studies explored the effect of vitamin D supplementation on clinical outcomes amongst COPD patients. Martineau et al. (27) did a double-blinded Randomised Control Trial (RCT) with the intervention being vitamin D supplementation in COPD. Vitamin D supplementation had no effect on time to first moderate or severe exacerbation or upper respiratory tract infection. However in the vitamin D deficient subgroup (< 20 ng/ml) there was an improvement in time to the first moderate/ severe exacerbation with supplementation. An Iranian RCT (28) found that supplementation with vitamin D led to a marked reduction in exacerbation rates and significant FEV1 improvement. Limitations of this study include that only severe and very severe COPD patients were selected, which may reflect selection bias. This may make findings less generalisable. In addition, no baseline vitamin D levels were measured in intervention or control groups, but the authors note a high prevalence of vitamin 6 D deficiency in Iran. A meta-analysis (29) found that moderate to severe exacerbations were reduced in patients supplemented with vitamin D where baseline vitamin D was < 10 ng/ml. The meta-analysis was based on 4 trials. Raw data were re-analysed where available and queries were submitted to the primary author for clarification. One trial had no data set available. The small sample size of this component study would unlikely have a major impact on the results. 1.11 Mechanisms postulated to explain COPD vitamin D associations Zhu et al. (25) provided plausible mechanisms for some of the associations. Lower vitamin D levels in the COPD group may be due to increased illness and indoor hours (poorer functional status) resulting in reduced sunlight exposure. Additional postulated mechanisms that could be contributing to lower vitamin D levels in COPD include older age and smoking reducing the vitamin D activation in the dermis; enhanced vitamin D catabolism owing to corticosteroid usage and reduced storage capacity of vitamin D in COPD patients. In this meta-analysis (25), lower latitudes tended to have lower vitamin D levels but there was heterogeneity in this finding, contrary to what might be expected from sunlight exposure. This may be explained by the fact that many developed countries at higher latitudes fortify food with vitamin D; in addition, sunlight hours (based on latitude) may only be a significant contributory factor during the winter season at higher latitudes. Zhu et al. (25) also postulate mechanisms for older patients (including older COPD patients) having higher vitamin D deficiency rates. Older patients may have atrophic skin and hence reduced cutaneous vitamin D activation. In addition, they may have reduced mobility and sunlight exposure and a less nutrient-rich diet. It may be expected that individuals of African/black ethnicity may have reduced vitamin D levels owing to increased skin melanin content and reduced cutaneous vitamin D activation when compared with Asian/ Indian or Caucasian patients. George et al. (3) however, found higher rates of vitamin D deficiency in Asian/Indian participants in Johannesburg compared to Black/African participants. The authors attribute this to dietary differences and cultural dressing practices with reduced skin exposure amongst Indian/Asian participants. 7 The precise mechanism responsible for glucocorticosteroid administration resulting in vitamin D deficiency, in some studies, has not been elucidated. Skversky et al. (30) provide a convincing argument that glucocorticosteroids result in increased vitamin D catabolism accounting for the association. Akeno et al. (31) in 2000 demonstrated in an animal (mouse) model that the administration of dexamethasone increased the expression of the enzyme vitamin D-24-hydroxylase in mouse renal cells which resulted in increased degradation of vitamin D metabolites 25[OH]D and 1,25[OH]2D. 1.12 The rationale for the current study COPD secondary to smoking and biomass fuel exposure is a common medical diagnosis at Chris Hani Baragwanath Academic Hospital (CHBAH). This tertiary facility services a large catchment population including the largest township in South Africa, Soweto, and thus patients may be a fairly representative sample of the South African population demographics. There was scope for a study to explore vitamin D status in COPD patients and its clinical correlates at CHBAH in Johannesburg, where seasonal sunlight exposure is more consistent, and to determine if vitamin D status findings conform to those reported in the developed world. This study may serve as an epidemiological precursor to further intervention studies involving vitamin D supplementation in COPD patients in Johannesburg. 8 CHAPTER 2 METHODOLOGY 2.1 Objectives The primary objective was to determine the prevalence of vitamin D deficiency (25 [OH]D ≤ 20 ng/ml) and insufficiency (25[OH]D - 21-29 ng/ml). Secondary objectives included to investigate for an association between vitamin D and demographic/lifestyle factors; lung function parameters; markers of COPD severity and corticosteroid type and dosage. 2.2 Study design This was a prospective, cross-sectional study of COPD patients. 2.3 Study population Patients with spirometry-confirmed COPD were included, as per GOLD 2019 guidelines (31). For inclusion, participants had to have a post-bronchodilator FEV1/FVC of less than 70% combined with at least one of the classical symptoms of COPD – chronic cough, dyspnoea or chronic sputum production. Spirometry was the most recent lung function in the patient’s file or repeated on the date of the interview if none could be traced in patient records. Patients had to be over 18 years of age. Patients were excluded if had reversible airflow limitation, current active pulmonary tuberculosis; concomitant diagnosis of asthma; active malignancy; malabsorption; history of pancreatic insufficiency or if already on vitamin D supplementation. 2.4 Study setting The study was conducted at a tertiary academic hospital in Johannesburg. The hospital serves as a major referral centre in the area. Patients were recruited from the outpatient clinic and inpatient wards. The majority of patients were recruited from a specialist respiratory clinic. Johannesburg is at a latitude of 26.2 ˚S of the equator. 2.5 Sample size Based on previous studies, normal vitamin D levels among COPD patients range from 7% upwards. Using an estimate of 7%, a precision of 5% and a confidence level of 95%, a sample size of 100 was initially aimed for. Unfortunately, due to COVID-19 spirometry limitations 9 and COVID-19 clinic patient number curtailments a sample size of 76 patients was reached. Consecutive patients between November 2020 and July 2022 were recruited. 2.6 Data collection Data were collected by the investigators using a data sheet. Spirometry was performed if none was available in the records. Pulmonary functions were performed using a JAEGER © Vyntus SPIRO PC spirometer with a calculation of % predicted FVC and FEV1 values according to American Thoracic Society (ATS)/European Respiratory Society (ERS) recommendations. A 400 µg dose of salbutamol was administered for post-bronchodilator spirometry. Patient demographic/ lifestyle factors; clinical and COPD severity markers; spirometry findings, laboratory data and treatment information were collected. A 3-5 ml cuffed venous sample of blood was taken in an acid citrate dextrose tube from participants. Samples were transported under cold chain conditions to the laboratory. 2.7 Definitions Ethnicity The ethnicity was recorded as self-reported by the participant. Season of sample collection The date of blood sample collection/ interview was categorised into seasons. Seasons were defined as follows: summer – December to February; autumn – March to May; winter – June to August and spring – September to November. Sunlight exposure The sunlight exposure was self-reported by the participant and based on recall in the week preceding the interview. Categories included: 1-4 hours/week; 5-6 hours/week; 1-2 hours/day; 3-5 hours/day and ≥ 6 hours/day Exacerbation An exacerbation was based on patient-reported figures/ file notes and included episodes requiring oral/intravenous (IV) corticosteroids; antibiotics; casualty visit or medical 10 practitioner consultation; hospital admission or worsening of cough, dyspnoea, or sputum production (exceeding 2 days). Smoking status A patient was regarded as an ex-smoker if they quit smoking for over 3 months. Never- smoker was defined as less than 100 cigarettes consumed throughout life. Systemic corticosteroids Included the use of IV/ oral corticosteroids in the year preceding interview. Low dose inhaled corticosteroids Included low and medium dose inhaled corticosteroids (fluticasone ≤ 250 mcg/day, budesonide 160 mcg/day, beclomethasone 200-400 mcg/day) High dose inhaled corticosteroids Included fluticasone ≥500 mcg/day, budesonide 320 mcg/day, Beclomethasone ≥400 mcg/day 2.8 Vitamin D testing and definition 25[OH]D was measured using a double sandwich immunoassay using a chemiluminescent label at a South African National Accreditation System (ISO15189) approved laboratory. The instrument used is the Abbott © Architect. This method is traceable to the reference method, namely liquid chromatography-mass spectrometry (LC-MS) and meets the required standards for clinical testing (32). There is some variation in levels quoted for vitamin D deficiency and insufficiency in the literature. Definitions used in this study for 25[OH]D were: deficiency: ≤ 20ng/mL; insufficiency: 21-29ng/mL; adequate: ≥30ng/ml (10). For statistical comparison 25[OH]D ≤ 20 ng/ml indicated vitamin D deficiency and non-deficiency was defined as 25[OH]D >20 ng/ml. 2.9 Statistical analysis All data obtained were entered onto a spreadsheet on Microsoft Excel and then entered into Statistica v13.3 (StatSoft, United States [US]). The prevalence data was provided as a percentage with 95% CI. The distribution of data was determined from histograms, the Shapiro Wilk and Lilliefor's tests. Categorical variables were presented as counts (n) and 11 percentages and comparisons were made in vitamin D deficient and non-deficient groups using the Chi-square test. Continuous variables were summarised as means with standard deviations for normally distributed data and medians with interquartile ranges for non- standard distributed data. Independent variables were compared in deficient vs non-deficient groups using Mann Whitney U test for independent medians and Student's T-test for independent means. For multivariate analysis, 8 variables were used to predict the presence of vitamin D deficiency or insufficiency. There were 6 continuous variables; age in years, BMI, waist circumference, smoking pack history in years, FEV1 in liters, MMRC dyspnea score, and two categorical variables; sunlight exposure (< 1 hour/day vs. ≥ 1 hour/day), and race (black vs. non-black). Six variables with a p<0.2 on the univariate model were selected for the final multivariate model 2.10 Ethical considerations Ethics approval was obtained by the Human Research Ethics Committee (Medical) at the University of the Witwatersrand (ethics reference number M200112) before the study commencement. Written informed consent was obtained from each participant before recruitment. National Health Research Database submission has been completed and is pending approval (reference GP_202210_034) https://nhrd.health.gov.za/Proposal/Details/112415 12 CHAPTER 3 RESULTS 3.1 Sample description A total of 80 patients met the inclusion criteria. 4 patients declined to participate in the study. 76 patients were included in our analysis. The study patient characteristics are summarised in Table 3.1. There were 55 males (72 %). The ethnicity profile consisted of black/ African ethnicity - 48 (63%); coloured (mixed race) – 17 (22%); Indian/Asian – 8 (11%); Caucasian – 3 (4%). The mean corrected calcium in our study population was 2.25 mmol/l (standard deviation [SD] 0.15) while the median alkaline phosphatase (ALP) was 92 U/l (interquartile range [IQR] 76-121). Figure 3.1 shows the sunlight exposure of study participants. Figure 3.1: Vitamin D Status versus increasing sunlight exposure 15 6 10 5 1 7 6 12 11 3 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1-4 hours/week 5-6 hours/week 1-2 hours/day 3-5 hours/day >6 hours /day P ro p o rt io n o f p ar ti ci p an ts in e ac h v it am in D c at eg o ry (b ar n u m b er s in d ic at e p ar ti ci p an t n u m b er s) Sunlight exposure categories in week preceding interview (participant reported) - ascending order Vitamin D status versus increasing sunlight exposusure Vitamin D deficient (25[OH]D ≤ 20 ng/ml) Vitamin D non-deficient (25[OH]D >20 ng/ml) 13 Table 3.1: Study patient characteristics * Variable N = 76 Median 25[OH]D, ng/ml (IQR) 21 (14.5 – 26.5) Mean age, years, (SD) 62 (10) Median BMI, kg/m2 (IQR) 21 (18-25) Median waist circumference, cm (IQR) 83 (73-92) Median smoking pack years, (IQR) 19 (5-37) Season of blood collection, n (%) Summer (December to February) Autumn (March to May) Winter (June to August) Spring (September to December) 24 (32) 30 (39) 12 (16) 10 (13) Lung function parameters, median (IQR) FEV1 (l) FEV1 (% predicted) FVC (% predicted) FEV1/FVC 1.1 (0.8 - 1.5) 41.9 (31.1 - 65.2) 86.1 (59.9 - 99.9) 44.6 (35.6 -55.5) Clinical characteristics Number of exacerbations last year, median (IQR) MMRC dyspnoea scale, n (%) MMRC 0 MMRC 1 MMRC 2 MMRC 3 MMRC 4 GOLD** grade, n (%) Grade 1 Grade 2 Grade 3 Grade 4 GOLD** group, n (%) Group A Group B Group C Group D 2 (2-3) 1 (1) 16 (21) 39 (51) 15(20) 5 (7) 7 (9) 23 (30) 30 (39) 16 (21) 7 (9) 33 (43) 9 (12) 27 (36) *- data summarised as numbers (n) and %, for normally distributed data - mean and standard deviation (SD), for non-normally distributed data median and interquartile range (IQR); ** GOLD 2019 (31) 3.2 Primary objective The prevalence of vitamin D deficiency and insufficiency (25[OH]D <30ng/ml) was 84% (CI 80-88). Table 3.2 summarises the proportion/percentages and median vitamin D level in each category. The median 25[OH]D in our sample was 21 ng/ml (IQR 14.5 – 26.5). 14 Table 3.2: The prevalence of Vitamin D deficiency and insufficiency among COPD study population Vitamin D status Prevalence (Confidence Interval) n/ total for the group Median 25[OH]D, ng/ml (IQR) Vitamin D deficiency and insufficiency (<30 ng/ml) 0.84(0.80-0.88) 64/76 18 (12.5 – 23.5) Vitamin D deficiency (≤20ng/mL) 0.48(0.42-0.54) 37/76 14 (11 – 17) Vitamin D insufficiency (21-29ng/mL) 0.35 (0.30-0.41) 27/76 25 (22-27) Adequate Vitamin D levels (≥30ng/mL) 0.16 (0.12-0.20) 12/76 37.5 (31.5 – 38.5) n – number of participants, IQR – Interquartile range 3.3 Secondary objectives 3.3.1 Univariate analysis The differences in lifestyle/ demographic factors between vitamin D deficient and non- deficient groups are provided in Table 3.3. There was a relative risk of vitamin D deficiency for patients with daily sunlight exposure of less than 1 hour/day compared with ≥ 1 hour/day of 1.62 (CI 1.02 -2.57), Figure 3.1. There was no difference in vitamin D deficiency between black/African and non-black/ African ethnicity, p = 0.11. The smoking status (never, ex- smoker, current) was not compared in the deficiency versus non-deficiency as 2 groups with a similar number of participants to facilitate meaningful comparison could not be constituted. The majority of participants were ex or current smoker: 68 (89%) and only 8 (11%) had never smoked. Table 3.4 illustrates the spirometry differences in deficiency and non-deficiency groups. There were no significant differences in spirometry parameters (FEV1, FEV1 % predicted, FVC and FVC % predicted, FEV1/FVC). Table 3.5 summarises the severity features and therapeutic differences between the vitamin D deficient and non-deficient groups. The MMRC dyspnoea scale of ≥2 was associated with a relative risk of 1.34 (CI 1.05-1.7) for vitamin D deficiency when compared with an MMRC dyspnoea scale of <2. No significant difference in the deficiency versus non-deficiency 15 groups was noted in the number of exacerbations in the preceding year; GOLD grade; GOLD group; use of inhaled, systemic corticosteroids in the preceding year and inhaled corticosteroid dosage. Table 3.3: Vitamin D levels and demographic/lifestyle factors (univariate analysis) * Vitamin D deficiency n= 37 Vitamin D sufficiency /Insufficiency n= 39 P Mean age (years) (SD) 64 (11) 61 (8) 0.10 Gender male, n/ total in group (%) 27/37 (73) 28/39 (72) 0.91 Median weight - kg (IQR) 58 (53-72) 58 (47 – 68) 0.44 Mean height - m (SD) 1.66 (0.08) 1.66 (0.09) 0.90 Median BMI - kg/m2 (IQR) 20 (19 -26) 21 (17 – 23) 0.56 Median waist circumference - cm (IQR) 83 (76 – 94) 83 (72 - 90) 0.44 Median smoking pack years (IQR) 24 (7.5 – 40) 17 (5 – 27) 0.13 *- data summarised as numbers (n) and %, for normally distributed data - mean and standard deviation (SD), for non-normally distributed data median and interquartile range (IQR) Table 3.4: Vitamin D levels and lung function parameters (univariate analysis) Vitamin D deficiency n=37 Vitamin D sufficiency/Insufficiency n=39 Variable Median IQR Median IQR P FEV1 (l) 0.94 0.69-1.49 1.11 0.77-1.75 0.18 FEV1 (% predicted) 41.20 28.50-53 42.90 32.2-70 0.22 FVC (l) 2.39 1.71-3.01 2.56 1.95-3.32 0.22 FVC (% predicted) 82 57.10-97.30 89 66.14-100.40 0.22 FEV1/FVC 42 36.27-55.50 47.98 34.14-55.56 0.72 IQR – interquartile range, FEV1 – Forced Expiratory Volume in 1st second, FVC – Forced Vital Capacity 3.3.2 Multivariate analysis We used 8 variables to predict the presence of vitamin D deficiency or insufficiency. The choice of these variables was based on pathophysiological plausibility and prominence in the literature review regarding their effect on vitamin D levels. There were 6 continuous variables; age in years, BMI, waist circumference, smoking pack history in years, FEV1 in liters, MMRC dyspnea score, and two categorical variables; sunlight exposure (< 1 hour/day vs. ≥ 1 hour/day), and race (black vs. non-black). Six variables with a p<0.2 on the univariate model 16 were selected for the final model. Only sunlight exposure was an independent predictor of vitamin D deficiency or insufficiency (Odds ratio 2.4, CI 1.3 -4.5). Table 3.5: Vitamin D status and COPD severity markers, therapies (univariate analysis) Vitamin D status (ng/ml) Deficiency (≤20) n=37 Non-deficiency (>20) n=39 X2 test MMRC dyspnoea scale Relative risk of deficiency (CI) MMRC <2 4/17 (24%) 13/17 (76%) MMRC ≥2 33/59 (60%) 26/59 (44%) 1.34 (1.05-1.7)* Exacerbations in last year ≤1 21/48 (44%) 27/48 (56%) P=0.26 >1 16/28 (57%) 12/28 (43%) X2 = 1.27 GOLD** grade GOLD 1 3/7 (43%) 4/7 (57%) GOLD 1,2 vs 3,4 GOLD 2 10/23 (43%) 13/23 (57%) p= 0.45 GOLD 3 14/30 (47%) 16/30 (53%) X2 = 0.57 GOLD 4 10/16 (63%) 6/16 (38%) GOLD** group GOLD A 2/7 (29%) 5/7 (71%) GOLD A,B vs C,D GOLD B 18/33 (55%) 15/33 (45%) p= 0.8 GOLD C 1/9 (11%) 8/9 (89%) X2 = 0.06 GOLD D 16/27 (59%) 11/27 (40%) Use of inhaled corticosteroids last year Yes 33/65 (51%) 32/65 (49%) p=0.38 No 4/11 (36%) 7/11 (64%) X2 = 0.06 Use of systemic corticosteroids last year Yes 21/42 (50%) 21/42 (50%) p=0.80 No 16/34 (47%) 18/34 (53%) X2 = 0.07 Dose of inhaled corticosteroids Low † 8/13 (62%) 5/13 (38%) p=0.39 High ‡ 25/52 (48%) 27/52 (52%) X2 = 0.75 MMRC – Modified Medical Research Council; GOLD – Global Initiative for Chronic Obstructive Disease; IV – intravenous; * - statistically significant; † - includes low and medium dose inhaled corticosteroids (fluticasone ≤ 250 mcg/day, budesonide 160 mcg/day, beclomethasone 200-400 mcg/day); ‡ - high dose inhaled corticosteroid includes fluticasone ≥500 mcg/day, budesonide 320 mcg/day, beclomethasone ≥400 mcg/day *** GOLD 2019 (31) 17 CHAPTER 4 DISCUSSION The main finding from this study is the high prevalence of vitamin D deficiency (48%) and insufficiency (35%) in this study population. In an analysis of the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort (14), with a large sample size of 1609, 20,55% and 33.2% of participants were vitamin D deficient and insufficient respectively. The same definitions for vitamin D levels were used in this study. The deficiency proportion is significantly lower than in the present study. Some reasons that may account for this discrepancy in findings include a higher proportion of milder COPD phenotype patients (majority GOLD 2 participants) and majority Caucasians (lower skin melanin content) in the USA study. In addition, our sample may reflect a selection bias representing a sicker cohort of COPD patients owing to recruitment predominantly from a referral centre respiratory (specialist) clinic and a small sample size. Kunisaki et al. (23) in another large USA study, found 40.4% of participants to be vitamin D deficient and 33.1% of participants to be insufficient. This is not dissimilar to our study findings, as the population matched our cohort’s COPD disease severity (majority GOLD grade 3 and had a high exacerbation risk). The marginally lower deficiency prevalence may be accounted for by ethnicity (majority Caucasian participants). Gawron et al. (21) in a small Polish case-control study found the highest reviewed rates of vitamin D deficiency (90.2%) in COPD patients. Controls also had similarly high vitamin D deficiency rates and the above may be accounted for by winter only, nadir vitamin D, sampling in a temperate location. These findings bring into question whether COPD patients do truly have higher vitamin D deficiency rates when contrasted with matched participants in the general population. Our study was not designed as a case-control study and hence cannot accurately elucidate this. It may however be useful to compare prevalence rates of vitamin D deficiency in a healthy cohort versus COPD patients. Holick (33) in a review article quoted vitamin D deficiency prevalence as 40 to 100% in elderly non-institutionalised healthy people in the USA and over 50% of postmenopausal osteoporotic women (without COPD) having suboptimal (25[OH]D < 30 ng/ml) vitamin D. A 18 large African meta-analysis (34) found a population prevalence of 59% for combined vitamin D deficiency and insufficiency, compared to 84% in our COPD study. Similar findings were noted in case-control studies (17,18,26). These general population vitamin D deficiency/ insufficiency prevalence estimates appear lower than in the COPD population. Some of the postulated mechanisms for higher vitamin D deficiency prevalence in COPD patients include a sicker phenotype hence less sunlight exposure; a poorer diet; smoking resulting in pigmentary skin changes and decreased cutaneous previtamin D3 activation; possible increased vitamin D catabolism due to corticosteroid usage and lower BMI and hence fat/muscle stores of the vitamin (6). Sunlight exposure was significantly associated with vitamin D deficiency in univariate analysis and remained the only independent predictor in the multivariate model. This is evident in many studies in Europe. Jolliffe et al. (15) in a multicentre cross-sectional study in London with 278 participants also showed that the absence of a recent sunny holiday correlated with vitamin D deficiency in a COPD cohort. Kentson et al. (17) in a Swedish case-control study with 38 COPD patients also found vitamin D deficiency associated with a lower Ultraviolet score (UVS). The UVS was a composite measure of seasonality and sunlight (UV) exposure. This study found a trend toward an association between vitamin D deficiency and greater smoking pack years, lower FEV1 and older age. Malinovschi et al. (20), in an Italian cohort, found no association between vitamin D level and age or smoking history. A Belgian study (26) also found no age, current smoking and vitamin D association. Burkes et al. (14) found the inverse trend to our study, with younger age associated with vitamin D deficiency. This trend from our study may be explained physiologically as age and disease severity may limit mobility resulting in decreased outdoor sunlight exposure. Also, with aging, skin pigmentation can become darker, decreasing cutaneous vitamin D UV activation. Burkes et al. (14) and Persson et al. (18) (in multivariate analysis) also showed an association between vitamin D levels and smoking status. The majority of reviewed studies showed an association between a lower FEV1 and vitamin D deficiency (13–19). Three studies also showed no vitamin D FEV1 association (12,20,21). The 19 relationship between FEV1 and vitamin D deficiency in our study may not have reached significance due to disparate sampling times and a limited sample size. Our data showed an MMRC dyspnoea scale of ≥2 was associated with a higher risk of vitamin D deficiency. Kentson et al. (17) also demonstrated the association between higher symptom scores (which include dyspnoea as a component) and vitamin D deficiency (COPD Assessment Test [CAT] and MMRC dyspnoea score if not on vitamin D supplementation). Kunisak et al. (23) made a similar association but with the St. George’s Respiratory Questionnaire (SGRQ). Persson et al. (18) in a large Norwegian case-control study also found the same association between MMRC dyspnoea score and vitamin D status in univariate analysis, but not the multivariate analysis, similar to our data. Hyun et al. (16) in a South Korean study also found high fibrinogen and low vitamin D associated with higher MMRC dyspnoea scores. The outlier in the literature (15) found no correlation between vitamin D and SGRQ (with dyspnoea as a component). Strengths of this study include a South African context (where sparse data exist) and a wide variety of factors investigated. Additionally, a diverse sample of COPD severity was included. Vitamin D samples were taken over multiple seasons in the study population, as a whole (on the date of interview for each patient) which may be more representative of vitamin D deficiency prevalence as opposed to vitamin D nadir (winter/ spring) only sampling. The majority of patients were recruited from an OPD setting reducing the confounding of acute illness. The same investigator conducted surveys and measured parameters, hence negating the effects of inter-investigator inconsistency/ variability. Limitations of this study include a smaller sample size due to COVID-19 clinic number curtailments and spirometry restrictions. This is a cross-sectional study and vitamin D levels were only measured at a single time/ season in each patient. There may be seasonal variations in vitamin D levels in each patient. No inferences about causality can be made as vitamin D deficient subjects were not followed up prospectively. No analysis of the comorbidities (including Human Immunodeficiency Virus [HIV]) of participants was done owing to the small sample size and this could be a confounder. A single-centre tertiary hospital study may limit the transferability of findings to different COPD populations. 20 CHAPTER 5 CONCLUSION AND RECOMMENDATIONS Conclusion A high prevalence of vitamin D deficiency and insufficiency exists within this COPD study population. A higher MMRC score was associated with an increased risk of vitamin D deficiency while sunlight exposure was the only independent predictor of vitamin D deficiency. Recommendations There is scope for case-control studies to evaluate vitamin D deficiency prevalence among healthy/hospitalised African participants compared with their COPD counterparts. Given the high prevalence of vitamin D deficiency in COPD patients, routine testing among high risk groups may be valuable. These findings need to be validated in a milder phenotype COPD population. Future studies should focus on the clinical impact of vitamin D replacement among deficient patients. 21 CHAPTER 6 REFERENCES 1. 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Thorax. 2010;65(3):215–220. 27. Martineau AR, James WY, Hooper RL, Barnes NC, Jolliffe DA, Greiller CL, et al. Vitamin D 3 supplementation in patients with chronic obstructive pulmonary disease (ViDiCO): a multicentre, double-blind, randomised controlled trial. The Lancet Respiratory Medicine. 2015;3(2):120–130. 28. Zendedel A, Gholami M, Anbari K, Ghanadi K, Bachari EC, Azargon A. Effects of Vitamin D Intake on FEV1 and COPD Exacerbation: A Randomized Clinical Trial Study. Glob J Health Sci. 2015;7(4):243–248. 29. Jolliffe DA, Greenberg L, Hooper RL, Mathyssen C, Rafiq R, de Jongh RT, et al. Vitamin D to prevent exacerbations of COPD: systematic review and meta-analysis of individual participant data from randomised controlled trials. Thorax. 2019;74(4):337–345. 24 30. Skversky AL, Kumar J, Abramowitz MK, Kaskel FJ, Melamed ML. Association of Glucocorticoid Use and Low 25-Hydroxyvitamin D Levels: Results from the National Health and Nutrition Examination Survey (NHANES): 2001–2006. J Clin Endocrinol Metab. 2011;96(12):3838-3845 31. Singh D, Agusti A, Anzueto A, Barnes PJ, Bourbeau J, Celli BR, et al. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease: the GOLD science committee report 2019. Eur Respir J. 2019;53(5):1900164. 32. Avci E, Demir S, Aslan D, Nar R, Şenol H. Assessment of Abbott Architect 25-OH vitamin D assay in different levels of vitamin D. J Med Biochem. 2020;39(1):100-107 33. Holick MF. Vitamin D Deficiency. N Engl J Med. 2007;357(3):266-281 34. Mogire RM, Mutua A, Kimita W, Kamau A, Bejon P, Pettifor JM, et al. Prevalence of vitamin D deficiency in Africa: a systematic review and meta-analysis. The Lancet Global Health. 2020;8(1):e134–142. 25 CHAPTER 7 APPENDICES Appendix 1 – Research protocol PROTOCOL INTRODUCTION Epidemiology Globally more than 170 million people are affected by Chronic Obstructive Pulmonary Disease (COPD) and COPD accounted for approximately 3.2 million deaths in 2015 (1). Low and middle-income countries bear a significant burden of COPD mortality. Limited data on the epidemiology of COPD in Africa is available. The BOLD study (2) found a prevalence exceeding 20% in Cape Town. It should be noted that the demographics of Cape Town differ significantly from the remainder of South Africa and it would be inaccurate to infer that these estimates represent a national prevalence. There is an increasing interest in lifestyle and dietary factors that may impact disease severity in the COPD population. There have been a large number of studies investigating the role of Vitamin D deficiency in COPD and its association with markers of disease severity. From the review of the available literature, there is a paucity of data available from South Africa or Africa in general concerning this subject. George et al (3) in a healthy cohort in Johannesburg found a prevalence of vitamin D < 30 nmol/l ranging between 5.1% (in black patients) to 28.6 % (in Asian/ Indian patients) emphasising the impact of race/ethnicity on vitamin D level. Vitamin D - role in calcium and phosphate homeostasis Vitamin D3 is predominantly synthesised in the skin. Ultraviolet (UVB) radiation converts previtamin D3 to vitamin D3 (cholecalciferol). This step is influenced by melanin content in the skin and sunlight exposure. The other less significant source of vitamin D is dietary intake. Ergocalciferol (in plant material) and vitamin D3 (from animal sources or fortification) can be absorbed from the gut and incorporated into chylomicrons and transported into the lymphatics and then on to blood. This vitamin D3 is transported bound to vitamin D Binding Proteins (VBPs) in the blood to the liver where it is hydroxylated to 25 hydroxy-vitamin D (25 [OH] D). This is the major storage form and is used to ascertain 26 vitamin D status in populations. Vitamin D then undergoes further hydroxylation in the kidney and other tissues to become1,25–dihydroxy vitamin D (1,25 [OH]2 D). This is the active form of vitamin D and interacts with vitamin D receptors (VDRs). Vitamin D enhances gastrointestinal absorption of calcium and phosphate and also results in osteoclastic bone resorption (4). Non-calcaemic vitamin D effects and role in COPD Vitamin D exerts pleiotropic effects unrelated to its role in calcium and phosphate homeostasis. It is now evident that vitamin D plays a significant immunomodulatory effect. Antigen presenting cells can be inhibited by vitamin D. Vitamin D inhibits the innate and adaptive immune response through VDRs on inflammatory cells. Vitamin D deficiency fails to restrain macrophage and dendritic cell maturation through major histocompatibility class II restriction. Vitamin D also plays an important function in T cell differentiation. Vitamin D inhibits the Th1 type response and its associated cytokines i.e. interleukin (IL)-2, Granulocyte Monocyte Colony Stimulating Factor (GMCSF) and interferon ɣ (IFN ɣ) in favour of Th2 type and regulatory T cell (T reg) responses. Thus, vitamin D deficiency leads to dysregulation of the immune and inflammatory response resulting in chronic lung inflammation and structural lung changes promoting the onset and progression of COPD(4). Vitamin D also enhances macrophage production of antimicrobial peptides like cathelicidin which is an important component of the immune response to combat mycobacterial infections (4). Vitamin D deficiency results in increased respiratory infections (including mycobacterial tuberculosis infection) and airway microbe colonisation leading to COPD progression. Vitamin D deficiency also results in increased airway remodelling, fibroblast proliferation (and hence collagen synthesis) and alteration in gene expression resulting in smooth muscle proliferation and contraction promoting the airway changes characteristic of COPD. The immune dysregulation also explains the association between vitamin D deficiency and autoimmune disease. Vitamin D and other respiratory illnesses 27 Vitamin D levels have shown an association with certain respiratory illnesses. Kamphuis et al (5) in a retrospective review in the Netherlands found a negative correlation between vitamin D levels and sarcoid disease activity. In a multicentre case-control study (6) including low and middle-income countries investigating antiretroviral therapies (ART) among majority black participants, vitamin D deficiency at ART initiation was associated with a higher incidence of tuberculosis (TB) at 96 week follow up. A meta-analysis (7) demonstrated the beneficial effect of vitamin D supplementation in reducing the incidence of acute respiratory tract infections. A greater beneficial effect of the supplementation was seen in the subgroup of vitamin D deficient patients (25[OH]D < 25 nmol/l). Definitions of vitamin D deficiency and vitamin D normal values There is some heterogeneity in the values quoted for vitamin D deficiency. This may arise from the fact that vitamin D levels required for the calcaemic effects (prevention of rickets and osteomalacia) are better established by robust data than for levels necessary to maintain non-calcaemic effects of vitamin D, as well as variation in assays used to ascertain levels. The Endocrinology Society (8) defines vitamin D deficiency as 25[OH] D levels less than 50nmol/l, and vitamin D insufficiency as levels between 52.5 and 72.5 nmol/l. The Institute of Medicine (IOM) (9) recommended that a level of 25[OH] vitamin D exceeding 50nmol/l as adequate. Vitamin D and association with airflow parameters Hansen et al (10) found an association between vitamin D level and Forced Expiratory Volume in 1 second [FEV1] (especially in the vitamin D deficient subgroup) in third- generation and offspring of the original Framingham Heart Study participants. Importantly this study looked at healthy individuals (not those with COPD). Other limitations included a single vitamin D measurement and no data collected on vitamin D supplementation. A post hoc cross-sectional review (11) of the Baltimore Longitudinal Study of Ageing failed to demonstrate an association between vitamin D deficiency and airflow limitation. An important limitation of this study is that airflow limitation was not objectively assessed but based on self-reported patient diagnoses. Airflow limitation encompassed COPD, non-smoker 28 COPD and asthma. Vitamin D deficiency prevalence and associations in COPD patients A Bulgarian study (12) looked at vitamin D deficiency in COPD patients admitted to the hospital. Owing to the study design this study had patients with more severe disease – Global Initiative on COPD (GOLD) class C and D. This study demonstrated a high prevalence of vitamin D insufficiency - <50 nmol/l (83.6%) which is significantly higher than other studies where participants were not experiencing an exacerbation (31-77% prevalence). There existed a correlation between vitamin D and Quality of Life (QOL) indices (e.g. Modified Medical Research Council [MMRC] dyspnoea scale) as well as certain pulmonary function parameters (Forced Vital Capacity [FVC], FEV1, Forced Expiratory Volume in 6 seconds [FEV6]) but not the FEV1/FVC ratio. No correlation existed between vitamin D level and comorbidities (Diabetes Mellitus, hypertension, metabolic syndrome and the number of exacerbations). Vitamin D deficiency was associated with a longer hospital stay. Kentson et al (13) in a single centre case-control study found no difference in the prevalence of vitamin D deficiency in long term oxygen therapy (LTOT) COPD patients versus non- LTOT users. Since this study wanted to stratify patients based on oxygen therapy a sicker cohort was selected. The sample included majority Caucasians. Peak annual vitamin D levels were measured in summer and early autumn as seasonal variation in Sweden may be significant. Lower 25 [OH] vitamin D levels were found in the COPD group compared with controls. 25[OH]D levels did correlate with disease severity indices i.e. FEV1 % predicted, MMRC score and oxygen saturation. Other lifestyle factors such as a Mediterranean diet and omega-3 consumption correlated with vitamin D levels. The study designed a useful tool to quantify UV exposure (UV score) which incorporated the quarterly average UV index and sunlight exposure hours into a single value. Sunlight exposure time categories used in this study may be useful to employ in my proposed study. A Korean prospective study (14) demonstrated a correlation between 25[OH]D levels and FEV1/FVC in COPD. In keeping with other studies (15), vitamin D deficiency was also associated with lower FEV1 and FEV1% predicted. Vitamin D deficiency was also associated with reduced 6-minute walk distance. 29 Changhwan et al (16) assessed the decline in 6-minute walk tests annually in COPD patients and a decline exceeding 17 m/year was defined as rapid exercise decline. An association was established between low vitamin D levels and rapid exercise decline especially in the severe vitamin D deficient group (<25nmol/l). Important study limitations include that majority of subjects had some degree of vitamin D deficiency. The study was retrospective and there was no documentation of environmental exposure (e.g. use of vitamin D supplements and sunlight exposure) and this could skew the data. Joliffe et al (15) in an extensive multicentre cross-sectional study in the UK explored if an association between various lifestyle factors and COPD severity markers correlated with vitamin D levels. Lifestyle factors associated with vitamin D deficiency included high Body Mass Index (BMI), lower socio-economic status, lack of vitamin D supplementation, winter sampling and absence of a summer holiday. No role of vitamin D pathway genetic polymorphisms or inhaled corticosteroid dose and vitamin D deficiency was demonstrated. A small Turkish group (17) demonstrated a high prevalence of vitamin D deficiency (60%) in COPD patients. Lung function parameters (FEV1/FVC; FEV1; FVC and total lung capacity [TLC]) were lower in COPD vitamin D deficient patients. Hand grip and isokinetic knee strength were lower in the vitamin D deficient subgroup. Vitamin D and COPD acute exacerbation/ hospitalisation rates Malinovschi et al (18) in a small retrospective case study found no significant association between vitamin D status and FEV1 decline as well as other lung function parameters. Notably they measured vitamin D levels at a nadir (winter) and had a very high prevalence of vitamin D deficient subjects. A negative correlation was demonstrated between vitamin D levels and acute exacerbation and hospital admission rates. The researchers did concede the relationship did not necessarily imply causality. The investigators measured vitamin D levels at a time point and then retrospectively reviewed the medical records of participants for the preceding year concerning exacerbations and admissions. Therefore, a conclusion cannot be drawn that low vitamin D levels led to more exacerbations as the exacerbations were not recorded prospectively from the time of vitamin D level measurement. The PRECOVID randomised control trial (RCT) (19), currently underway, might provide insight into vitamin 30 D supplementation’s effect on acute exacerbations. Zhu et al (20) in a meta-analysis of 21 studies showed that COPD patients (especially the severe COPD subgroup) had lower vitamin D levels than control patients. Lower vitamin D levels were also associated with an increased risk of COPD and markers of disease severity but not associated with increased exacerbations. In addition, lower vitamin D levels were found in exacerbation patients when compared with stable COPD patients. Individual study results had significant disparity in findings which was partly due to the vitamin D assays employed. Mechanisms postulated to explain COPD vitamin D associations Zhu et al(20) provided plausible mechanisms for some of the associations. Lower vitamin D levels in the COPD group may have been accounted for by increased illness and hence indoor hours (poorer functional status) and therefore reduced sunlight exposure. Additional postulated mechanisms that could be contributing to lower vitamin D levels in COPD include increased age and smoking reducing the vitamin D activation in the dermis; enhanced vitamin D catabolism owing to corticosteroid usage and reduced storage capacity of vitamin D in COPD patients. Lower latitudes tended to have lower vitamin D levels but there was heterogeneity in this finding, contrary to what might be expected from sunlight exposure. This may be explained by the fact that many developed countries at higher latitudes fortify food with vitamin D; in addition sunlight hours (based on latitude) may only be a significant contributory factor during the winter season at higher latitudes. The exact mechanism responsible for glucocorticosteroid administration resulting in vitamin D deficiency has not been precisely elucidated. Skversky et al (21) provide a convincing argument for the role of glucocorticosteroids resulting in increased vitamin D catabolism accounting for the association. Akeno et al (22) in 2000 demonstrated in an animal (mouse) study that the administration of dexamethasone increased expression of the enzyme vitamin D-24-hydroxylase in mouse renal cells which resulted in increased degradation of vitamin D metabolites 25[OH]D and 1,25[OH]2D. Vitamin D supplementation in COPD 31 Martineau et al (23) did a double-blinded RCT with the intervention being vitamin D supplementation in COPD. Vitamin D supplementation had no effect on time to first moderate or severe exacerbation or upper respiratory tract infection. However, in the vitamin D deficient subgroup (<50nmol/l) there was an improvement in time to the first moderate/ severe exacerbation with supplementation. An Iranian RCT (24) found that supplementation with vitamin D led to a marked reduction in exacerbation rates and significant FEV1 improvement. Limitations of this study include that only severe and very severe COPD patients were selected, which may reflect selection bias. This may make findings less applicable to the general COPD population. In addition, no baseline vitamin D levels were measured in intervention or control groups but authors note a high prevalence of vitamin D deficiency in Iran. A meta-analysis (25) found that moderate to severe exacerbations were reduced in patients supplemented with vitamin D where baseline vitamin D was < 25nmol/l. The meta-analysis was based on 4 trials where raw data was re-analysed where available and queries were submitted to the primary author for clarification. One trial had no data set available. The small sample size of this component study would unlikely have a major impact on results. Vitamin D and COPD prognosis Few studies involving COPD prognosis in relation to vitamin D could be identified. A longitudinal study (26) based on long term follow up of patients with either defined clinical or spirometric COPD was conducted. It demonstrated that COPD with reduced vitamin D levels had an increased risk of all-cause mortality. There was the suggestion made that this may be attributable to respiratory and other cause mortality (although this was not statistically significant). Limitations of this study include the use of prebronchodilator spirometric measures which do not conform to international COPD definition as well as delay in processing vitamin D specimens. Conclusion 32 From the current literature search, it is evident that vitamin D deficiency is more prevalent in the COPD population. There exists some association with vitamin D level and lung function parameters (FEV1 % predicted, FEV1/FVC ratio) as well as lifestyle factors e.g. (sunlight exposure and dietary intake) in COPD. Some of these associations can be explained by the physiology and immunomodulatory role of vitamin D. There is inconsistency in the data on the association of vitamin D deficiency and exacerbation rates in COPD. The majority of published reports are from developed countries with significant seasonal sunlight exposure variation. There were no available studies on COPD and vitamin D status in South Africa or Africa. COPD secondary to smoking and biomass fuel exposure is a common medical diagnosis at Chris Hani Baragwanath Academic Hospital (CHBAH). This tertiary facility services a large catchment population including the largest township in South Africa, Soweto, and thus patients may be a fairly representative sample of the South African population demographics. There is scope for a study to explore vitamin D status in COPD patients at CHBAH in Johannesburg, where seasonal sunlight exposure is more consistent, and to determine if vitamin D status findings conform to those reported in the developed world. This study may serve as an epidemiological precursor to further intervention studies involving vitamin D supplementation in COPD patients in Johannesburg. STUDY OBJECTIVES The primary objective of this study is 1. To determine the prevalence of vitamin D insufficiency (25-62.5 nmol/l) and .deficiency (<25 nmol/l) in the COPD population at CHBAH Secondary objectives include: 1. To determine if vitamin D levels have an association with demographic/lifestyle factors: age, gender, ethnicity, body mass index (BMI), waist circumference, smoking status, smoking pack year history and sunlight exposure in COPD patients 2. To determine the association between vitamin D levels and lung function parameters (FEV1, FVC, FEV1 % predicted, FEV1/FVC) in COPD patients. 3. To determine if any association exists between vitamin D levels and markers of 33 COPD severity (MMRC dyspnoea scale, number of exacerbations in the last year, GOLD grade or GOLD group) 4. To determine if an association exists between vitamin D level and inhaled corticosteroid dosage (low/medium, and high) or oral/intravenous corticosteroid usage in COPD patients in the preceding year METHODS Study design Prospective cross-sectional, descriptive study Study population Inclusion criteria: 1. Patients that have spirometric confirmed COPD will be included, as per GOLD 2019 guidelines. Patients who have a post-bronchodilator FEV1/FVC less than 70% combined with at least one of the classical symptoms of COPD – chronic cough, dyspnoea or chronic sputum production. 2. Patients will have to be over 18 years of age. Exclusion criteria: 1. Patients with current active pulmonary tuberculosis or a concomitant diagnosis of asthma or active malignancy 2. Patients with malabsorption or a history of pancreatic insufficiency or disease 3. Patients already on vitamin D supplementation Study setting The study will be conducted at Chris Hani Baragwanath Academic Hospital respiratory outpatient department (Respiratory OPD), a tertiary hospital in Johannesburg part of the University of the Witwatersrand academic circuit. Patients may also be recruited from the 34 Medical outpatient department (MOPD). If the sample size is difficult to reach from these locations additional patients will be recruited from the medical wards. Baragwanth Hospital is found at a latitude of 26.2 ˚S of the equator. Convenience sampling will be used. Sample size Based on previous studies, normal vitamin D levels among COPD patients range from 7% upwards. Using an estimate of 7%, a precision of 5% and a confidence level of 95%, a sample size of 100 is required. Data collection Patients will sign informed consent (Appendix 2) after being given a study information leaflet (Appendix 3) and verbal information regarding the study. Patients will have the choice to join the study. Data will be collected from the managing clinician or investigators at the listed locations using a datasheet (Appendix 4). An identifier list will issue a random study number which will be linked to the patient's hospital number, cellular number and name. This study number will appear on the data sheet and no personal identity information of the patient will be included on the sheet. The identifier list will only be accessible to the principal investigator and supervisors. Spirometry will be traced in the patient records and if not yet done will be done on the date of the visit. The machine used is JAEGER © Vyntus SPIRO PC spirometer (CareFusion GmbH, Hoechberg, Germany) with the calculation of % predicted FVC and FEV1 values according to American Thoracic Society (ATS)/European Respiratory Society (ERS) recommendations. A 400 µg dose of salbutamol will be administered for post- bronchodilatory spirometry. Data collected in the datasheet will include the date; managing clinician name and cellular number (to convey vitamin D deficiency results); study number; age; gender; ethnicity (self- reported by the patient); BMI; waist circumference; smoking status; smoking pack year history; average sunlight exposure in the preceding week (as per patient recall); lung function parameters (as per patient file or new spirometry) such as FEV1, FEV1% predicted, FEV1/ FVC; MMRC dyspnoea scale (as reported by the patient); GOLD grade and GOLD group and number of exacerbations in the last year. An exacerbation will be based on patient-reported 35 figures and will include episodes requiring oral/intravenous (IV) corticosteroids; antibiotics; casualty visit or medical practitioner consultation; hospital admission or worsening of cough, dyspnoea, or sputum production (exceeding 2 days). A patient will be regarded as an ex- smoker if quit smoking for over 3 months. Never smoker will be defined as less than 100 cigarettes consumed throughout life. Data about inhaled /IV or oral corticosteroid treatment usage in the preceding year will be obtained based on patient recollection or file data where available. A 3-5 ml cuffed venous sample of blood will be taken from the cubital fossa by the clinician/ investigator or phlebotomist in an acid citrate dextrose (ACD)/ yellow top tube. The sample will be accompanied by a requisition form and stored in the respiratory OPD fridge at a temperature of 4-8 ˚C. When convenient for the investigator, with a delay not exceeding 1 month, samples will be taken to Global Labs offices in Johannesburg. Global labs measure 25[OH]D using a double sandwich immunoassay using a chemiluminescent label. The instrument used is the Abbott © Architect. The reference range used by the laboratory is as follows: 1. Severe deficiency- <10 ng/ml (< 25 nmol/l) 2. Mild to moderate deficiency- 10-24 ng/ml (25-60 nmol/l) 3. Normal population- 25-80 ng/ml (62.5-200 nmol/l) Laboratory testing is ISO 15189 compliant. Vitamin D levels will be obtained from Global Labs and added to the datasheets for relevant patients. If the patient is found to be vitamin D deficient, the managing clinician will be telephoned and informed of the result. A recommendation will be made that the patient be initiated on vitamin D 50 000 IU orally weekly, as recommended CHBAH endocrinology department. If the managing clinician is not reachable telephonically, the patient will be informed telephonically of the result and sent a text message with patient details and the vitamin D result and the vitamin D supplementation recommendation, and a follow up 36 consultation will be arranged at the patient’s clinic (MOPD or ROPD). The patient will be requested to show the text message to the clinician. Where available, on NHLS Labtrack the most recent (if done within last 3 months) calcium and alkaline phosphatase (ALP) will be obtained and added to the datasheet. If additional funding is procured a calcium and ALP level may be added to the blood request form. STATISTICAL ANALYSIS Once data is obtained it will be entered into an Excel spreadsheet, and it will be anonymised. From there it will be entered into a statistical program- Statistica. The patient demographics will be summarised using descriptive statistics. For dependent variables that are normally distributed means and standard deviations will be obtained. For comparisons between normally distributed variables comparing means between 2 groups students t-test will be employed. For comparing means for more than 2 groups ANOVA test will be used. If data is not normally distributed then the Mann-Whitney or Kruskal-Wallis test will be used. For correlations between continuous variables (e.g. FEV1 and vitamin D) regression modelling will be used and the Pearson correlation coefficient calculated. For statistical purposes, 95% confidence interval with p<0.05 will be considered significant. ETHICS The study participants will sign informed consent (Appendix 2) after being granted study information pamphlets (Appendix 3) and in addition have a full explanation of the study role, risks and benefits. They will sign an informed consent form and joining the study will be completely voluntary. Ethics application will be done through the Human Research Ethics Committee. Written consent will also be obtained from the Head of Department (HOD) of Baragwanath Hospital and the HOD of pulmonology. An identifier list will issue a random study number which will be linked to the patient's hospital number, cellular number and name. This study number will appear on the datasheet and no personal identity information of the patient will be included on the sheet. The identifier list will only be accessible to the principal investigator and supervisors, thus maintaining anonymity. All data will only be accessible to the principal investigator and supervisors and kept on a password-protected personal laptop computer. In the instance that the patient is vitamin D deficient, the managing clinician or patient will be informed and supplementation recommended (as elaborated on in 37 the data analysis section). TIMING J U N 19 J U L A U G S E P O C T N O V D E C J A N 2 0 F E B M A R A P R M A Y J U N J U L A U G S E P O C T N O V D E C J A N 2 1 Literature review Preparing Protocol Protocol Assessment Ethics Application Funding approval Collecting Data Data Analysis Writing up – Thesis Writing up – Paper FUNDING Basic project budget Item Cost per item Number of items Total Cost Vitamin D test R130 100 R13 000 VAT on test R19.50 100 R1 950 Printing and paper costs R400 Transport R600 R 15 950 Consumables such as needles, syringes, specimen bags, webcols and gloves will be provided by Global Laboratories complimentary and included in the per specimen test fee. Funding will be applied for from the Wits Pulmonology fund or postgraduate funding initiatives. If funding is delayed or difficult to secure Dr M and Mrs Z Kola have given a generous offer to sponsor the research project. The printing and transport costs will be financed by the investigator. LIMITATIONS 1. It is often time the case that patients seen in general medical wards with COPD are followed 38 up at MOPD or discharged to local clinics if uncomplicated and hence Respiratory OPD patients may represent a sicker cohort. 2. If sampling is done from the medical wards this may imply that the patient is admitted with an acute exacerbation which may impact on the vitamin D level. All attempts will be made to obtain patients from the respiratory OPD/MOPD except if the time allocated to data collection is expiring and the sample size is not reached. 3. Private funding can be problematic and delay study but a generous offer has been made by private donors if research funding through Wits cannot be secured. 4. Vitamin D levels will be sampled through several seasons and hence not necessarily represent annual peak /trough or average. However, compared to centres with significant seasonal sunshine hour variability where most current data is sourced from, Johannesburg has consistent sunlight throughout the year and hence seasonal variation will be less important. 5. No data regarding vitamin D supplementation doses and comorbidities will be obtained. 6. No calcium and PTH levels will be measured with vitamin D levels and these could impact vitamin D levels. Where available, the most recent calcium and ALP levels will be traced on NHLS Labtrack and added to the datasheet, but may not be available for all patients. If additional funding is available calcium and ALP may be added to the blood request form. 7. Another problem may be the delay in vitamin D sample processing but from a review of available data 25[OH]D is stable for a prolonged period if refrigerated. 39 LIST OF ABBREVIATIONS (USED IN PROTOCOL) 1,25[OH]2D – 1.25-dihydoxy vitamin D 25[OH]D – 25 hydroxy-vitamin D ALP – Alkaline Phosphatase ANOVA – Analysis of Variance ART – Antiretroviral Therapy CHBAH – Chris Hani Baragwanath Academic Hospital COPD – Chronic Obstructive Pulmonary Disease FEV1 – Forced Expiratory Volume in 1 Second FEV6 – Forced Expiratory Volume in 6 Seconds FVC – Forced Vital Capacity GMCSF –Granulocyte Monocyte Colony Stimulating Factor GOLD – Global Initiative on COPD IFN Ɣ – Interferon Ɣ IL – Interleukin IOM – Institute of Medicine IV – Intravenous LTOT – Long Term Oxygen Therapy MMRC – Modified Medical Research Council MOPD – Medical Outpatient Department OPD – Outpatient Department PTH – Parathyroid Hormone QOL – Quality of Life RCT – Randomised Control Trial ROPD – Respiratory Outpatient Department T reg – regulatory T cell TB – tuberculosis TLC – Total Lung Capacity UV – Ultraviolet VDBP – Vitamin D Binding Proteins VDR – Vitamin D Receptor 40 PROTOCOL REFERENCES 1. 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(2011) The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine: What Clinicians Need to Know. J Clin Endocrinol Metab. 96, 53–8. 10. Hansen, J. G., Gao, W., Dupuis, J., et al. (2015) Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study. Respir Res, 16, 81. 11. Moberg, M., Elango, P., Ferrucci, L., et al. (2015) Vitamin D deficiency and airflow 41 limitation in the Baltimore Longitudinal Study of Ageing. Eur J Clin Invest, 45, 955- 63. 12. Mekov, E., Slavova, Y., Tsakova, A., et al. (2015) Vitamin D Deficiency and Insufficiency in Hospitalized COPD Patients. PLoS One, 10, e0129080. 13. Kentson, M., Leanderson, P., Jacobson, P., et al. (2018) The influence of disease severity and lifestyle factors on the peak annual 25(OH)D value of COPD patients. Int J Chron Obstruct Pulmon Dis, 13, 1389-98. 14. Jung, J. Y., Kim, Y. S., Kim, S. K., et al. (2015) Relationship of vitamin D status with lung function and exercise capacity in COPD. Respirology, 20, 782-9. 15. Jolliffe, D. A., James, W. Y., Hooper, R. L., et al. (2018) Prevalence, determinants and clinical correlates of vitamin D deficiency in patients with Chronic Obstructive Pulmonary Disease in London, UK. J Steroid BiochemMolBiol, 175, 138-45. 16. Kim, C., Jung, J. Y., Kim, Y. S., et al. (2016) Vitamin D deficiency is associated with rapid decline in exercise capacity in male patients with Chronic Obstructive Pulmonary Disease. Respiration, 91, 351-8. 17. Yumrutepe, T., Aytemur, Z. A., Baysal, O., et al. (2015) Relationship between vitamin D and lung function, physical performance and balance on patients with stage I-III chronic obstructive pulmonary disease. Rev Assoc Med Bras, 61, 132-8. 18. Malinovschi, A., Masoero, M., Bellocchia, M., et al. (2014) Severe vitamin D deficiency is associated with frequent exacerbations and hospitalization in COPD patients. Respir Res, 15, 131. 19. Rafiq, R., Aleva, F. E., Schrumpf, J. A., et al. (2015) Prevention of exacerbations in patients with COPD and vitamin D deficiency through vitamin D supplementation (PRECOVID): a study protocol. BMC Pulm Med, 15, 106. 20. Zhu, M., Wang, T., Wang, C., et al. (2016) The association between vitamin D and COPD risk, severity, and exacerbation: an updated systematic review and meta- analysis. Int J Chron Obstruct Pulmon Dis, 11, 2597-607. 21. Skversky, A. L., Kumar, J., Abramowitz, M.K., et al. (2011) Association of glucocorticoid use and low 25-hydroxyvitamin D levels: results from the National Health and Nutrition Examination Survey (NHANES): 2001–2006. J Clin Endocrinol Metab, 96, 3838–45. 22. Akeno, N., Matsunuma, A., Maeda, T., et al. (2000) Regulation of vitamin D-1alpha- hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney. J Endocrinol, 164, 339–48. 23. Martineau, A. R., James, W. Y., Hooper, R. L., et al. (2015) Vitamin D3 supplementation 42 in patients with chronic obstructive pulmonary disease (ViDiCO): a multicentre, double- blind, randomised controlled trial. Lancet Respir Med, 3, 120-30. 24. Zendedel, A., Gholami, M., Anbari, K., et al. (2015) Effects of vitamin D intake on FEV1 and COPD exacerbation: a randomized clinical trial study.Glob J Health Sci, 7, 243-8. 25. Jolliffe, D. A., Greenberg, L., Hooper, R. L., et al. (2019) Vitamin D to prevent exacerbations of COPD: systematic review and meta-analysis of individual participant data from randomised controlled trials. Thorax, 74, 337-45. 26. Faerk, G., Colak, Y., Afzal, S., et al. (2018) Low concentrations of 25- hydroxyvitamin D and long-term prognosis of COPD: a prospective cohort study. Eur J Epidemiol, 33, 567- 77. 43 Appendix 2 – Patient informed consent