deficiency for rural than urban dwellers

Journal Pre-proof Higher risk of Vitamin D insufficiency/deficiency for rural than urban dwellers ´ P Griffin, Deirdre Wall, Liam Blake, Damian G Griffin, Tomas Stephanie Robinson, Marcia Bell, Eamon C Mulkerrin, Paula O’Shea

PII:

S0960-0760(19)30364-4

DOI:

https://doi.org/10.1016/j.jsbmb.2019.105547

Reference:

SBMB 105547

To appear in:

Journal of Steroid Biochemistry and Molecular Biology

Received Date:

18 June 2019

Revised Date:

14 November 2019

Accepted Date:

17 November 2019

Please cite this article as: Griffin TP, Wall D, Blake L, G Griffin D, Robinson S, Bell M, Mulkerrin EC, O’Shea P, Higher risk of Vitamin D insufficiency/deficiency for rural than urban dwellers, Journal of Steroid Biochemistry and Molecular Biology (2019), doi: https://doi.org/10.1016/j.jsbmb.2019.105547

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Title: Higher risk of Vitamin D insufficiency/deficiency for rural than urban dwellers. Tomás P Griffin1,2, Deirdre Wall3, Liam Blake4, Damian G Griffin1,4, Stephanie Robinson5, Marcia Bell1, Eamon C Mulkerrin5, Paula O’Shea4 1. Regenerative Medicine Institute at CÚRAM SFI Research Centre, School of Medicine, National University of Ireland Galway, Galway, Ireland. 2. Centre for Endocrinology, Diabetes and Metabolism, Galway University Hospitals, Galway, Ireland. 3. School of Mathematics, Statistics and Applied Mathematics, National University of Ireland, Galway, Ireland. 4. Department of Clinical Biochemistry, Galway University Hospitals, Galway, Ireland. 5. Department of Geriatric Medicine, Galway University Hospitals, Galway, Ireland.

Highlights:

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Corresponding author: Dr Paula M. O’Shea, Department of Clinical Biochemistry, Saolta University Health Care Group (SUHCG), Galway University Hospitals (GUH), Newcastle Road, Galway, Ireland. Email: [email protected].

Vitamin D deficiency is a major global health problem.

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Serum 25(OH)D was lower among rural v. urban dwellers irrespective of season.

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Vitamin D deficiency was greater in rural v. urban dwellers in spring, autumn and winter.

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Serum 25(OH)D was higher and the prevalence of deficiency lower in females v. males.

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Vitamin D deficiency was most prevalent among those age ≥80 years and ≤39 years.

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Abstract:

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There are many risk factors for Vitamin D deficiency. This study aimed to compare the Vitamin D status and serum 25(OH)D concentrations of adults living in an urban area to adults living in a rural

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area in the West of Ireland (latitude 53.27° North). A cross-sectional retrospective analysis of clinical records was performed. Following interrogation of the electronic laboratory information system, individuals who had Vitamin D samples analysed at Galway University Hospitals between January

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2011 and December 2015 were identified. Clinical demographics, setting and date of sampling were recorded. In total, 17,590 patients (urban n=4,824; rural n=12,766) were eligible for inclusion. Serum 25(OH)D concentrations were lower among rural compared to urban dwellers irrespective of season (spring p<0.001, summer p=0.009, autumn p=0.002, winter p<0.001). There was a significant difference in Vitamin D status between urban and rural dwellers in three of the four seasons: springdeficiency: 16%-v-23%, insufficiency: 39%-v-43%, sufficiency: 45%-v-35% (p<0.001); autumndeficiency: 11%-v-10%, insufficiency: 30%-v-35%, sufficiency: 59%-v-56% (p=0.01); winterdeficiency: 23%-v-25%, insufficiency: 35%-v-42%, sufficiency: 41%-v-33% (p<0.001). Serum 25(OH)D 1

concentrations were higher and the prevalence of deficiency lower in urban/rural females compared to urban/rural males (p<0.001). Serum 25(OH)D concentrations increased sequentially from the 1839 year age group to the 60-69 year age group in both urban (p<0.001) and rural (p<0.001) dwellers and then decreased progressively as age increased to >90 years. The odds of Vitamin D deficiency increased with age, lower daily sunshine hours, male gender, rural address and season.

Keywords: Vitamin D, Vitamin D deficiency, Vitamin D insufficiency, Urban, Rural, West of Ireland

1 Introduction:

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Vitamin D deficiency is a major global health problem [1]. Vitamin D, a fat-soluble secosteroid [2], plays an important role in calcium and phosphorous homeostasis [3], healthy bone growth and remodelling. There are 2 major forms of Vitamin D: Vitamin D2 derived from food and dietary

supplements; Vitamin D3 synthesized in the skin in response to sunlight and also from diet and dietary supplements. The primary source of Vitamin D is cutaneous biosynthesis with diet and

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dietary supplements being a lesser but significant source. Although 1,25-dihydroxyvitamin D

(1,25(OH)2D) is the active form of Vitamin D, serum 25-hydroxycholecalciferol (25(OH)D), the major

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metabolite of Vitamin D in the blood, is the best measure of Vitamin D status. 25(OH)D has a much longer half-life (approximately 3 weeks) [4] than 1,25(OH)2D (4 hours) [5] and better reflects the

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contribution of diet and cutaneous biosynthesis. The concentration of 25(OH)D is higher than 1,25(OH)2D in blood and therefore is easier to measure [5]. There is significant controversy regarding serum 25(OH)D cut-off levels to define Vitamin D deficiency, insufficiency and sufficiency [6]. For this

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study, the following thresholds are used: deficiency <25nmol/L, insufficiency 25-50nmol/L, sufficiency >50nmol/L.

Vitamin D deficiency impairs bone mineralization and can lead to rickets [3] in children and

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osteomalacia and osteoporosis in adults with a consequent increase in fracture incidence [7, 8]. In addition to increased fracture risk, Vitamin D deficiency is linked to proximal muscle weakness and

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increased risk of falls [9]. Deficiency is also associated with non-skeletal consequences including cardiovascular disease [10], metabolic diseases (type 2 diabetes mellitus, metabolic syndrome and insulin resistance) [11], cancer [10], autoimmune disease [12], infection [13] and overall mortality [14]. To date, a causative relationship has not been conclusively established. Risk factors for suboptimal Vitamin D status include: distance from the equator, pigmented skin, reduced sunlight, pollution in the atmosphere, skin concealing clothing, correct sunscreen use, exclusively breast fed infants not in receipt of recommended Vitamin D drops, multiple short interval pregnancies, old age, institutionalised, vegetarian diet, obesity, malabsorption, short bowel or use of medications that 2

interfere with Vitamin D absorption/metabolism such as anticonvulsants[15]. We have previously shown that Vitamin D insufficiency (serum 25(OH)D<50nmol/L) in the West of Ireland is a significant problem: 41.3% in females (≥18 years) attending a general practitioner [16], 47% in healthy white women (40–85 years) [17], 75.4% in female acute medical admissions [18], 74% in females >65 years living in the community [19], 87% in institutionalised females [19] and 50% in males (≥18 years) attending a general practitioner [16]. Among a representative sample of Irish community-dwelling adults ≥18 years, Cashman et al, as part of the National Adult Nutrition Survey (NANS), noted that the prevalence of serum 25(OH)D concentrations <50nmol/L varied between 28.9% in summer (April to October) and 55.0% in winter (November to March) [20]. Laird et al demonstrated in a large urban centre (Dublin) on the east coast of Ireland, that 31.8% of those sampled during summer

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(March to September) and 39.9% of those sampled during winter (October to February) had a

25(OH)D concentration ≤50nmol/L [21]. The prevalence of 25(OH)D ≤50nmol/L varied depending on postal region within the urban centre – ranging from 22.6% to 46.2% among participants sampled

during the summer months and from 29% to 48.8% among participants sampled during the winter

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months [21].

Urban and rural dwellers have different lifestyles which may affect their risk of Vitamin D deficiency.

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There are conflicting reports in the literature as to whether Vitamin D deficiency is more common in urban or rural dwellers. For example, deficiency was more prevalent in school children in an urban area of Ethiopia compared to school children in the surrounding rural area [22]; while in Pakistan

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total Vitamin D was lower in college students attending a rural compared to an urban university [23]. These findings suggest that local microclimates may also play a significant role in maintaining serum 25(OH)D concentrations. There are limited studies exploring how 25(OH)D concentrations differ

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between urban and rural dwellers in predominantly Caucasian European populations. The primary aim of this study was to compare the Vitamin D status and serum 25(OH)D

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concentrations of adults living in an urban area (Galway City) to the Vitamin D status and serum 25(OH)D concentrations of adults living in a rural area (Galway County) in the West of Ireland (latitude 53.27° North). The secondary aim was to determine the impact of age and gender on

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Vitamin D status and serum 25(OH)D concentrations between adult urban and adult rural dwellers.

2 Methods:

The research ethics committee at Galway University Hospitals (GUH) granted ethical approval for this study (Ref: C.A. 1482). 2.1 Study Design:

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Between January 2011 and December 2015, a cross-sectional retrospective analysis of clinical records was performed at GUH. Following interrogation of the electronic laboratory data system on the patient administration system (PAS), all patients ≥18 years who had serum samples collected for serum 25(OH)D measurement were identified. The inclusion criteria were: patients age ≥18 years with a postal address in Galway City (Urban) or Galway County (Rural). The exclusion criteria were: second and subsequent samples from an individual participant and those who had a postal address outside Galway City/County. Second and subsequent samples were excluded to reduce the potential interference of any intervention that may have been undertaken during the recruitment period to treat Vitamin D deficiency.

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2.2 Data collection: Baseline demographics, postal address and time of sampling were recorded. Seasons were defined

as: Spring: March, April, May; Summer: June, July, August; Autumn: September, October, November; Winter: December, January, February. Data regarding sunshine hours were sourced from Met

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Eireann’s Knock observation point (http://www.met.ie/climate-request/) situated approximately 60 km from GUH. Knock is the closest weather station to GUH that records daily sunshine hours. For this study 21-day sunshine is the average sunshine hours for the previous 21 days prior to sample

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collection (including day of sampling) and 42-day sunshine is the average sunshine hours for the previous 42 days prior to sample collection (including day of sampling). We selected arbitrary cut-

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offs for sunshine hours: 21-day (approximately one half-life [4]) and 42-day sunshine (approximately two half-lifes [4]). The use of 21- and 42- day sunshine hours are seen as surrogate indices for each individual’s potential sunshine exposure. They reflect the availability of UVB radiation during the 21-

2.3 Participants:

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or 42- day period prior to sampling but not necessarily each individual’s exposure.

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Galway is located on the west coast of Ireland, latitude 53.27° North, with a population of 258,552 (of whom 79,934 live in the city). Participants were divided into groups based on postal address:

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urban group (Galway City) - postal address inside the city boundary; rural group (Galway County) postal address in Galway County excluding addresses within the city boundary. Urban and rural dwellers were further divided into groups based on the season during which the serum 25(OH)D sample was collected, age and gender of participants. 2.4 Serum 25(OH)D measurement: Liquid chromatography tandem mass spectrometry (LC-MS/MS) on the Agilent HPLC 1290; Agilent 6460 Triple quadrupole MS/MS was used to measure serum 25(OH)D concentration. Vitamin D was 4

reported as total 25(OH)D concentration (the sum of 25(OH)D2 and 25(OH)D3 concentrations). The limit of quantification for 25(OH)D2 was 5nmol/L and for 25(OH)D3 was 8nmol/L. For the purpose of statistical analyses, values for 25(OH)D2 below 5nmol/L were assigned a value of 5nmol/L and for 25(OH)D3 values below 8nmol/L were assigned a value of 8nmol/L. Assay precision was ≤8.0% and bias was ≤5.0% at 25(OH)D2 concentrations of 42.2nmol/L and 94.4nmol/L and 25(OH)D3 concentrations of 39.9nmol/L and 94.4nmol/L. All analyses were conducted in the Clinical Biochemistry Laboratory at GUH (ISO 15189:2012 standards). The Clinical Biochemistry Laboratory participates in the international 25 Hydroxyvitamin D EQAS (External Quality Assessment Scheme) and meets the performance target set by the DEQAS (Vitamin D External Quality Assessment

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Scheme) advisory panel. 2.5 Statistics:

Microsoft® Excel 2016, GraphPad® Prism (Version 6.01) and R[24] were used for data recording and statistical analyses. Continuous data were reported using means (standard deviations) and

comparisons between groups made using ANOVA (with Tukey’s post hoc multiple comparison test)

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or Student’s t-test. Data which were not normally distributed were represented as median (min to

max) and comparisons between groups made using the Kruskal-Wallis or Kruskal-Wallis with Dunn’s

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post hoc multiple comparison test. Categorical variables were represented as frequencies (percentage) . Comparison of proportions were performed using the chi-squared test (with pairwise

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tests for independence for multiple comparisons). A binary logistic regression model was generated to explore how age (per 10 years), sunshine hours (42-day sunshine) (per hour sunshine/day), season and location (urban v rural) effected the prevalence of Vitamin D deficiency. Only 42-day

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sunshine and not 21-day sunshine was used as the values were collinear. A p-value <0.05 was

3 Results:

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deemed statistically significant.

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3.1 Clinical Characteristics:

In total, n=34,063 participants had serum 25(OH)D concentrations measured during the study period (Figure 1). In total, n=17,590 were eligible for inclusion in this study (urban: n=4,824 (27.4%); rural: n=12,766 (72.6%)). Overall, 15.9% (n=2,797) had Vitamin D deficiency, 35.6% (n=6,267) had Vitamin D insufficiency and 48.5% (n=8,526) had Vitamin D sufficiency. 3.2 Seasons:

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Table 1 outlines clinical demographics, serum 25(OH)D concentrations and 21- and 42-day sunshine hours for urban and rural dwellers in each season. Of note, rural dwellers sampled were older in spring (p<0.001), autumn (p=0.048) and winter (p<0.001) than urban dwellers. A greater portion of rural dwellers were male in each of the four seasons (spring p<0.001, summer p=0.009, autumn p<0.001, winter p<0.001). Total 25(OH)D and 25(OH)D3 concentrations were significantly higher in urban dwellers compared to rural dwellers in each of the four seasons (Table 1). Interestingly, 25(OH)D2 concentrations were significantly higher in rural dwellers compared to urban dwellers in summer and winter but not in spring and autumn (Table 1). Total 25(OH)D concentrations were highest in urban and rural dwellers in summer, followed by autumn, spring and winter (p<0.001) (Table 2). Figure 2 illustrates the proportion of both groups who were Vitamin D deficient,

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insufficient and sufficient in each of the four seasons. In spring, autumn and winter, a greater

proportion of participants in the rural group compared to the urban group had either Vitamin D

deficiency or insufficiency. There was no significant difference in the proportion of participants who had serum 25(OH)D concentrations >125nmol/L in the urban (1.43%, 69/4,824) versus the rural

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group (1.21%, 155/12,766) (p=0.254). Within both groups, there were no significant difference in the proportion of participants, who had serum 25(OH)D concentrations >125nmol/L between seasons

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(urban: spring 1.07% (13/1,215) v summer 2.22% (25/1,126) v autumn 1.28% (16/1,247) v winter 1.21% (15/1236) (p=0.106); rural: spring 1.41% (44/3,125) v summer 1.16% (36/3,097) v autumn

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1.19% (39/3,269) v winter 1.10% (36/3,275) (p=0.709)). 3.3 Age Range:

Table 3 shows clinical demographics, serum 25(OH)D concentrations and hours of sunshine in urban

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and rural dwellers based on age range. In rural but not urban dwellers, there was a significant difference in the proportion of males (rural p<0.001; urban p=0.702) and the 42-day (rural p=0.042; urban p=0.837) sunshine hours between the age groups. Within the rural age ranges 42-day

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sunshine hours were higher in the 18-39 than in the 60-69 year age group – suggesting that some of the differences that may exist in 25(OH)D concentrations between these age groups could be due to

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reduced UVB exposure prior to sampling. Despite these differences, Total 25(OH)D, 25(OH)D3 and 25(OH)D2 were higher in the 60-69 compared to the 18-39 year age group. Irrespective of location (urban or rural) Total serum 25(OH)D and 25(OH)D3 concentrations increased sequentially from the 18-39 year age range to the 60-69 year age range and then decreased progressively to the >90 years age range (urban p<0.001, rural p<0.001). Figure 3 illustrates that the proportion of participants (urban p<0.001, rural p<0.001) with Vitamin D deficiency decreased from the 18-39 year age group to the 50-59 year age group and then progressively increased to the >90 years age group. Total

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25(OH)D concentrations were significantly higher in urban compared to rural dwellers in the 50-59 (p<0.001), 60-69 (p<0.001), 70-79 (p<0.001) and 80-89 (p<0.001) year age ranges (Figure 4A). 3.4 Gender: Table 2 shows clinical demographics, serum 25(OH)D concentrations and hours of sunshine in urban and rural dwellers based on gender. There was no significant difference in age between urban males and urban females (p=0.631). Rural females were younger than rural males (p<0.001). Total 25(OH)D and 25(OH)D3 concentrations were higher in females than males irrespective of urban/rural location (urban Total 25(OH)D p<0.001; 25(OH)D3 p<0.001; rural Total 25(OH)D p<0.001; 25(OH)D3 p<0.001). The proportion of rural and urban males with Vitamin D deficiency was higher than rural and urban

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females (Figure 3; urban p<0.001, rural p<0.001). Figure 4B illustrates that Total serum 25(OH)D

concentrations were significantly higher in urban compared to rural female dwellers (p<0.001) but

there was no significant difference in Total serum 25(OH)D concentrations between urban and rural males (p=0.094).

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3.5 Multiple Variable Analysis:

Binary logistic regression analysis was carried out to explore which variables were associated with

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Vitamin D deficiency (Table 4). This analysis indicated that as age (p<0.001) increased the odds of being Vitamin D deficient increased. As the mean hours/day of sunshine decreased the odds of being

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Vitamin D deficient increased (p<0.001). Those who had a rural address (p=0.033) and male (p<0.001) had greater odds of being Vitamin D deficient than those who had an urban address and female. Participants sampled during spring and winter had higher odds of Vitamin D deficiency

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compared to those sampled in autumn. Participants sampled during winter had higher odds of Vitamin D deficiency compared to those sampled in summer. Participants sampled in summer and

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winter had lower odds of Vitamin D deficiency compared to those sampled in spring (p<0.001).

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4 Discussion:

In our study, 51.5% of participants had serum 25(OH)D concentrations <50nmol/L, suboptimal for bone health. Serum 25(OH)D concentrations were higher in urban compared to rural dwellers in all four seasons. Vitamin D deficiency was more common in rural than urban dwellers in spring, autumn and winter but not in summer. Serum 25(OH)D concentrations were higher and the prevalence of deficiency lower in urban and rural females compared to urban and rural males. Interestingly, serum 25(OH)D concentrations were lower in female rural than female urban dwellers but there was no difference between urban and rural males. Serum 25(OH)D concentrations increased sequentially 7

from the 18-39 year age group to the 60-69 year age group in both urban and rural dwellers and then decreased progressively as age increased to >90 years. The odds of Vitamin D deficiency increased with age, lower daily sunshine hours, male gender, rural address and season. Among a nationally representative sample of community-dwelling Irish adults aged ≥18 years (NANS), the yearly prevalence of serum 25(OH)D <50nmol/L was 40.1% (55.0% in winter and 28.9% in summer) [20]. Unlike our study, there was no significant difference in serum 25(OH)D <50nmol/L between the genders [20]. Laird et al noted, in a large urban centre on the east coast of Ireland, an overall prevalence of serum 25(OH)D <50nmol/L of 39.9% in winter and 32.8% in summer [21]. Similar to our study, they noted that 25(OH)D concentrations were significantly higher in females

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than males irrespective of season [20]. Cashman et al in an analysis of 14 population studies across Europe noted that the yearly prevalence of serum 25(OH)D concentrations <50nmol/L was 40.4%

[25], lower than in our study. Although the definitions of summer and winter vary between studies, the prevalence of serum 25(OH)D concentrations <50nmol/L is more common among rural and

urban dwellers in winter and summer in our study than in other reported studies. Participants in the

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NANS were a nationally representative sample while those in the Laird et al study were patients who had serum 25(OH)D measurements requested by their primary care physician. Our study included all

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patients who had serum 25(OH)D measured either in hospital or primary care. It is probable that participants in the NANS had fewer medical co-morbidities than those in the Laird et al study who in

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turn had fewer co-morbidities than those in our study. This may go some way towards explaining the marked differences in the proportion of individuals who had serum 25(OH)D <50nmol/L among these studies.

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Similar to our study, in NANS, 1.3% (15/1132) of participants had a serum 25(OH)D >125nmol/L [20]. It is worth noting that 11 of these 15 participants were sampled in summer. Of the remaining 4 participants sampled in winter, 2 were taking Vitamin D supplements and one of the participants not

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on supplements had recently returned from a sun holiday [20]. The Institute of Medicine (IOM) have raised concerns that persistent serum 25(OH)D concentrations >125nmol/L may be associated with

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adverse events [26]. A reverse J-shaped association has been observed between 25(OH)D concentrations and all-cause [27] and cardiovascular [28] mortality, with the highest risk at lower concentrations. From the published data, inferences regarding causality cannot be made and further studies exploring the risks associated with elevated 25(OH)D are required. Until such time as definitive evidence-based information is available, serum 25(OH)D >125nmol/L should not be targeted. Patients should be closely observed to ensure that the serum 25(OH)D >125nmol/L state is not prolonged.

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Few European studies have analysed the impact of urban/rural living on serum 25(OH)D concentrations. In one such study, urban Belgian women were exposed to ozone levels three times higher than rural Belgian women and despite a higher sun exposure index, rural women had higher 25(OH)D concentrations. The authors concluded that air pollution could be an underappreciated risk factor for low Vitamin D [29]. Indeed, air pollution may go some way towards explaining why residents in large cities in the China National Nutrition and Health Survey (CNNHS) [30] and those living in large metro areas in the Bailey et al [31] studies have lower concentrations of serum 25(OH)D compared to residents in smaller urban areas. Due to the absence of European studies, that explore the differences in serum 25(OH)D

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concentrations and Vitamin D status between rural and urban dwellers, data from studies in other parts of the world are noteworthy. In the CNNHS, men but not women who lived in large cities were more likely to have a serum 25(OH)D<50nmol/L compared to those who lived in general rural areas. Those who lived in small to medium cities or poor rural areas were not at increased risk. Serum

25(OH)D<50nmol/L was associated with female gender, spring season and low ambient UVB levels

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[30]. Bailey et al compared the impact on serum 25(OH)D concentrations of living in large

metropolitan (≥250,000 people) compared to urban (smaller metropolitan, larger urban or smaller

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urban areas) compared to rural areas in the United States. Interestingly, 34.4% of large metropolitan, 23.1% of urban and 27.5% of rural residents had serum 25(OH)D<50nmol/L [31]. The

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findings in our study and the studies reviewed are similar, when one aligns our urban area to the small to medium cities classification in the CNNHS [30] and the urban area classification in the United States study [31]. In Tianjin, China, Fang et al showed that serum 25(OH)D concentrations

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were slightly higher among urban compared to rural participants. However, subgroup analyses indicated that rural participants >50 years of age had higher serum 25(OH)D concentrations than urban participants of a similar age; while rural participants <50 years of age had lower serum

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25(OH)D concentrations compared to urban participants of the same age [32]. Those results were in contrast with our findings which showed that there was no significant difference in serum 25(OH)D

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concentrations between urban and rural dwellers in those <50 years but there was a significant difference in the 50-59, 60-69, 70-79 and 80-89 year age groups. Also, in contrast to our findings, Harinarayan et al found that among healthy urban and rural Indian adults, both rural males and females had significantly higher 25(OH)D concentrations compared to urban males and females [33]. In a comparison of 80 male participants (40 rural; 40 urban) with equal exposure to sunlight in Lahore, Pakistan, urban males had lower concentrations of serum 25(OH)D compared to their rural counterparts [34]. Adjusting for age, ethnicity and BMI, mean serum 25(OH)D concentrations were higher in rural than urban Fijian women: 19% urban compared 9

to 12% rural had serum 25(OH)D≤50nmol/L [35]. Similarly, in Malaysia, rural women had higher serum 25(OH)D concentrations (69.5 (59.0–79.1) nmol/L) compared to urban women (31.9 (26.1– 45.5) nmol/L): 81.3% urban and only 11.9% rural had serum 25(OH)D<50nmol/L [36]. Vitamin D status was evaluated in a cohort of 750 postmenopausal women (>50 years of age), in urban and rural areas in Northern Iran. Again, it was found that, serum 25(OH)D concentrations were lower in urban (46.3±33.8 nmol/L) compared to rural (57.3±34.5 nmol/L) women. Interestingly, Vitamin D status was related to educational level in urban women; those with the lowest educational level had higher serum 25(OH)D concentrations than other educational levels [37]. In Pakastani women and their neonates, serum 25(OH)D<50nmol/L was present in 99.5% of women and 97.3% of neonates in urban Karachi compared to 89% of women and 82% of neonates in rural Jehlum [38]. Compared to

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Ireland, many of the Asian and Oceanic countries discussed have much higher UVB availability and temperature gradients which likely impact on the Vitamin D status of urban and rural dwellers.

In our study, Vitamin D deficiency was most prevalent in older age groups (>80years) followed by

those in the 18-39 year age group irrespective of the urban/rural divide. Ageing significantly effects

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the ability of the skin to produce Vitamin D3 [39] due to a decrease in the epidermal concentrations of 7-dehydrocholesterol as age increases [40]. Holick et al compared the impact of whole-body

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exposure to sunlight among six healthy white participants aged 20-30 years and six older white participants aged 62-80 years, and found that circulating Vitamin D concentrations increased within

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24 hours from 6.5 (±5) nmol/L to 75 (±25) nmol/L in the younger participants compared to a rise of only 3.8 (±2.5) nmol/L to 19 (±6.5) nmol/L in the older participants [39]. Laird et al noted that 25(OH)D concentrations were 27% lower in winter and 20.7% lower in summer in those aged 18-50

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years comparted to those over 50 years [21]. In the Korea National Health and Nutrition Examination Survey (KHANES), like our study, serum 25(OH)D increased from ages 20-29 years, peaked at ages 60-69 years and then decreased irrespective of gender. Unlike our study, serum

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25(OH)D<50nmol/L was more common in females (64.5%) than males (47.3%) and serum 25(OH)D concentrations were higher in rural compared to urban dwellers for both genders. The authors

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further show, and our findings support, that serum 25(OH)D concentrations were higher in summer and autumn than in spring and winter. Following adjustment for co-variates, urban males were 1.90 (1.22-2.95) and urban females were 1.49 (1.26-1.77) times more likely to be Vitamin D deficient than their rural counterparts [41]. The National Diet and Nutrition Survey (NDNS) in the UK (1992-2001) found that the prevalence of Vitamin D deficiency (<25nmol/L) was between 5% and 20% in most age ranges but peaked between 20-40% in men and women aged 19-24 years and in women aged >85 years. When 50nmol/L was used as the cut off for Vitamin D insufficiency, low Vitamin D status was present in 20-60% of the population but was >75% in young adults and the elderly [42]. In the 10

most recent NDNS (2014 to 2015, 2015 to 2016), the prevalence of 25(OH)D<25nmol/L varied in men aged 19-64 years from 4% in July to September to 30% in January to March and in women aged 19-64 years from 5% in July to September to 28% in January to March. In men over 65 years the prevalence ranged from 4% in July to September to 29% in January to March and in women over 65 years from 6% in July to September to 24% in January to March [43]. Cashman et al noted that serum 25(OH)D<50nmol/L was most common in the 36-50 year age group (58.7%), followed by the 51-64 (55.4%), 18-35 (54.4%) and 65-84 (48.1%) year age groups during winter. Furthermore, during the summer months these trends reversed with serum 25(OH)D<50nmol being more common among those aged 65-84 (35.8%), followed by 18-35 (31.3%) 36-50 (27.6%) and 51-64 (21.5%) year age groups [20]. Interestingly and unexpectedly, among a Japanese cohort, the prevalence of

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Vitamin D sufficiency (defined in that study as plasma 25(OH)D>75nmol/L) tended to increase as age increased in both genders [44]. As we have shown, Vitamin D deficiency is not only a problem for older people but as the evidence suggests younger generations are also at increasing risk.

There is significant controversy in the published literature regarding whether urban or rural dwellers

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are at greatest risk of Vitamin D deficiency. Multiple factors contribute to this uncertainty. There is

considerable heterogeneity and lack of detailed description between studies over what is considered

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urban and what is considered rural. In countries and within countries, there are regional variations in traditions, local customs, skin-covering clothing, sun avoidance behaviour, diets, industries and

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favoured sports which can contribute to reduced exposure to sunshine or dietary intake of Vitamin D. Notwithstanding, Vitamin D deficiency is a significant public health concern and is a greater threat to rural than urban dwellers in Ireland. Males, those >80 years and <39 years and those sampled in

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spring and winter are at particularly high risk of Vitamin D deficiency. Rural living in Ireland, has long been associated with the outdoors and sunshine. However, with a change in focus from farming to industry, rural dwellers are now commuting longer distances usually by car to work in the cities.

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Conversely, urban dwellers because of proximity to work have shorter commutes and often walk or cycle. With this change in lifestyle, urban dwellers have more exposure to sunshine, the primary

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source of Vitamin D, compared to rural dwellers. Older rural dwellers may be less active due to geographic isolation and therefore, fewer local amenities resulting in less exposure to sunshine. Furthermore, older rural dwellers may also have less interpersonal contact or access to the print and electronic media resulting in lack of knowledge and awareness of the benefits of good Vitamin D health. To our knowledge, this is the first large study evaluating differences in serum 25(OH)D concentrations and Vitamin D status between urban and rural dwellers in Ireland. It is an observational study, therefore conclusions regarding causality cannot be made. The lack of 11

information regarding calcium and Vitamin D supplements, calcium and Vitamin D intake, medications and medical history is a significant limitation. Differences in Vitamin D supplementation and intake between urban and rural dwellers that may exist are not captured in our study. 21- and 42- day sunshine hours are surrogate markers for sun/UVB exposure. Information regarding sun exposure on an individual level was not available. While our clinical laboratory is ISO 15189:2012 accredited and participates in DEQAS, there are limited data on Vitamin D assay standardisation in many of the other reported studies. Our assay has not been cross-calibrated with the Vitamin D Standardization Program (VDSP) protocols which have been used by Cashman et al in the NANS study [45]. The lack of standardization of 25(OH)D measurement between studies is a significant

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limitation for comparison purposes.

5 Conclusions:

Of the population studied, 51.5% had a serum 25(OH)D<50nmol/L. Urban dwellers had higher serum

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25(OH)D concentrations and less Vitamin D deficiency than rural dwellers. Serum 25(OH)D

concentrations were higher and the prevalence of Vitamin D insufficiency and deficiency lower in

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urban and rural females compared to urban and rural males. Serum 25(OH)D concentrations were lower in participants aged 18-39 years and >80 years compared to those aged 40-79 years in both

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urban and rural dwellers. The odds of Vitamin D deficiency increased with age, lower daily sunshine hours, male gender, rural dweller and season. Over half the population had a serum 25(OH)D<50nmol/L, which is a major concern for all healthcare workers and indeed the general

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population. Urgent public health interventions are needed to promote good Vitamin D health with a

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focus on at risk groups.

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Competing interests: The author(s) declared no potential conflict of interest that could be perceived as prejudicing the impartiality of the research, authorship, and/or publication of this article. Funding: TPG is supported by a Hardiman Scholarship from the College of Medicine, Nursing and Health Science, National University of Ireland, Galway and a bursary from the Irish Endocrine Society/Royal College of Physicians of Ireland. Ethical approval: The research ethics committee, Galway University Hospitals (GUH) granted ethical approval for this study (Ref: C.A. 1482). Guarantor: PMOS

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Acknowledgements: We wish to express our gratitude to the scientific, nursing and medical staff at the Centre for Endocrinology, Diabetes and Metabolism, and the Department of Clinical Biochemistry, Saolta University Health Care Group (SUHCG), Galway University Hospitals (GUH) and to all the volunteers and patients who made this study possible.

13

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MacLaughlin J, Holick MF. Aging decreases the capacity of human skin to produce vitamin D3. J Clin Invest 1985;76:1536-8. Choi HS, Oh HJ, Choi H, Choi WH, Kim JG, Kim KM, et al. Vitamin D insufficiency in Korea--a greater threat to younger generation: the Korea National Health and Nutrition Examination Survey (KNHANES) 2008. J Clin Endocrinol Metab 2011;96:643-51. Prentice A. Vitamin D deficiency: a global perspective. Nutr Rev 2008;66:S153-64. Results of the National Diet and Nutrition Survey (NDNS) rolling programme for 2014 to 2015 and 2015 to 2016. Available at: https://www.gov.uk/government/statistics/ndnsresults-from-years-7-and-8-combined. Accesed: 1/09/2019. Nakamura K, Kitamura K, Takachi R, Saito T, Kobayashi R, Oshiki R, et al. Impact of demographic, environmental, and lifestyle factors on vitamin D sufficiency in 9084 Japanese adults. Bone 2015;74:10-7. Cashman KD, Kiely M, Kinsella M, Durazo-Arvizu RA, Tian L, Zhang Y, et al. Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25-hydroxyvitamin D data: a case study of the program's potential for national nutrition and health surveys. Am J Clin Nutr 2013;97:1235-42.

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Figure 1: Recruitment Schematic.

17

Figure 2: Proportion of participants with Vitamin D deficiency (25(OH)D<25nmol/L) (Red), insufficiency (25(OH)D 25-50nmol/L) (Orange) and sufficiency (25(OH)D>50nmol/L) (Green).

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**p<0.001m, *p=0.01-chi squared test.

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Figure 3: Proportion of participants with Vitamin D deficiency (25(OH)D<25nmol/L) (Red), insufficiency (25(OH)D 25-50nmol/L) (Orange) and sufficiency (25(OH)D>50nmol/L) (Green) based on Age Range (years) and Gender. **p=0.01-chi squared test.

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Table 1: Clinical demographics, Serum 25(OH)D concentrations and hours of sunshine based on location and season.

Rural

p-value

1,215 52.2 (17.4) 318 (26.2) 46.7 (13.0 - 173.2) 44.3 (8.0 - 173.2) 5.0 (5.0 - 64.2) 4.2 (1.1 - 7.5) 3.8 (1.2 - 6.6)

3,125 55.1 (17.9) 1016 (32.5) 39.5 (13.0 - 300.0) 36.5 (8.0 - 300.0 5.0 (5.0 - 90.2) 4.2 (1.1 - 8.0) 3.8 (1.2 - 6.6)

<0.001 <0.001 <0.001 <0.001 0.079 0.632 0.737

1,126 52.9 (18.4) 300 (26.6) 61.3 (13.0 - 296.0) 60.2 (8.0 - 296.0) 5.0 (5.0 - 57.8) 4.4 (1.8 - 8.2) 4.8 (2.2 - 6.4)

3,097 53.8 (18.0) 991 (32.0) 58.6 (13.0 - 191.4) 56.6 (8.0 - 184.0) 5.0 (5.0 - 61.0) 4.5 (1.8 - 8.2) 4.8 (2.2 - 6.4)

0.141 0.001 0.009 0.001 0.001a 0.09 0.082

1,247 53.1 (17.7) 286 (22.9) 57.6 (13.0 - 264.0) 55.8 (8.0 - 264.0) 5.0 (5.0 - 58.3) 2.9 (1.0 - 4.9) 3.0 (1.5 - 48)

3,269 54.2 (17.7) 1068 (32.7) 54.3 (13.0 - 229.2) 52.1 (8.0 - 229.2) 5.0 (5.0 - 83.4) 2.8 (1.0 - 4.9) 3.0 (1.5 - 48)

0.048 <0.001 0.002 0.008 0.070 0.083 0.352

1,236 52.0 (17.9) 325 (26.3) 42.8 (13.0 - 174.0) 41.1 (8.0 - 174.0) 5.0 (5.0 - 32.0) 1.3 (0.5 - 2.3) 1.2 (0.7 -2.1)

3,275 54.3 (17.2) 1085 (33.1) 37.8 (13.0 - 288.0) 35.3 (8.0 - 288.0) 5.0 (5.0 - 179.8) 1.3 (0.5 - 2.3) 1.2 (0.7 -2.1)

<0.001 <0.001 <0.001 <0.001 0.002a 0.937 0.072

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Urban

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Variable Spring Numbers Age (years)* Male no. (%) ^ Total 25(OH)D (nmol/L)¥ 25(OH)D3 (nmol/L)¥ 25(OH)D2 (nmol/L)¥ 21-day sunshine (hours/day)¥ 42-day sunshine (hours/day)¥ Summer Numbers Age (years)* Male no. (%) ^ Total 25(OH)D (nmol/L)¥ 25(OH)D3 (nmol/L)¥ 25(OH)D2 (nmol/L)¥ 21-day sunshine (hours/day)¥ 42-day sunshine (hours/day)¥ Autumn Numbers Age (years)* Male no. (%) ^ Total 25(OH)D (nmol/L)¥ 25(OH)D3 (nmol/L)¥ 25(OH)D2 (nmol/L)¥ 21-day sunshine (hours/day)¥ 42-day sunshine (hours/day)¥ Winter Numbers Age (years)* Male no. (%) ^ Total 25(OH)D (nmol/L)¥ 25(OH)D3 (nmol/L)¥ 25(OH)D2 (nmol/L)¥ 21-day sunshine (h/day)¥ 42-day sunshine (h/day)¥

*Mean (standard deviation) ^Number (%) ¥Median (minimum to maximum). Comparisons are made using Student’s t-test for parametric data, Kruskal Wallis for non-parametric data, Chi-squared test for proportions. a

25(OH)D2 was higher in rural than urban dwellers.

21

1229 52.8 (17.4) N/A 45.2 (13.0 174.0)

3595 52.5 (18.0) 54.6 (13.0 296.0)

<0.001

25(OH)D3 (nmol/L)¥

43.8 (8.0 - 174.0)

52.8 (8.0 - 296.0)

<0.001

25(OH)D2 (nmol/L)¥

5.0 (5.0 -64.2)

5.0 (5.0 - 58.3)

21-day sunshine (hours/day)¥ 42-day sunshine (hours/day)¥ Rural Numbers Age (years)* Male no. (%)^

2.7 (0.5 - 8.2) 2.9 (0.7 - 6.6)

2.8 (0.6 - 8.2) 2.9 (0.7 - 6.6)

0.482 0.135

4160 56.2 (17.2) N/A 44.7 (13.0 279.0)

8606 53.5 (17.8)

<0.001

Autumn

Winter

1215 52.2 (17.4) 318 (26.2) 46.7 (13.0 173.2)b,c,d 44.3 (8.0 - 173.2)

1126 52.9 (17.9) 300 (26.7) 61.3 (13.0 296.0)a,c,d 60.2 (8.0 - 296.0)

b,c,d

a,c,d

1247 53.1 (17.7) 286 (22.9) 57.6 (13.0 264.0)a,b,d 55.8 (8.0 264.0)a,b,d

1236 52.0 (17.9) 325 (26.3) 42.8 (13.0 174.0)a,b,c 41.1 (8.0 174.0)a,b,c

5.0 (5.0 - 57.8)a,c

5.0 (5.0 - 58.3)a,b

5.0 (5.0 - 32.0)a

<0.001

4.4 (1.8 - 8.2)a,c,d 4.8 (2.2 - 6.4)a,c,d

2.9 (1.0 - 4.9)a,b,d 3.0 (1.5 - 4.8)a,b,d

1.3 (0.5 - 2.3)a,b,c 1.3 (0.5 - 2.3)a,b,c

<0.001 <0.001

e-

5.0 (5.0 64.2)b,c,d 4.2 (1.1 -7.5) b,c,d 3.8 (1.2 - 6.6) b,c,d

0.853

Pr

Total 25(OH)D (nmol/L)¥

0.631

p-value°

Summer

oo

Urban Numbers Age (years)* Male no. (%)^

Female

Season Spring

p-value≠

pr

Gender Male

Variable

f

Table 2: Clinical demographics, serum 25(OH)D concentrations and hours of sunshine based on urban/rural and gender/season.

0.375 0.118 <0.001 <0.001

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3125 3097 3269 3275 55.1 (17.9)b 53.8 (18.0)a 54.2 (17.7) 54.3 (17.2) 0.041 1016 (32.5) 991 (32.0) 1068 (32.7) 1085 (33.1) 0.814 49.6 (13.0 39.5 (13.0 58.6 (13.0 54.3 (13.0 37.8 (13.0 Total 25(OH)D (nmol/L)¥ <0.001 <0.001 300.0) 300.0)b,c 191.4)a,c,d 229.2)a,b,d 288.0)b,c 36.5 (8.0 56.6 (8.0 52.1 (8.0 35.3 (8.0 25(OH)D3 (nmol/L)¥ 42.5 (8.0 - 279.0) 46.8 (8.0 - 300.0) <0.001 <0.001 b,c a,c,d a,b,d 300.0) 184.0) 229.2) 288.0)b,c 5.0 (5.0 5.0 (5.0 25(OH)D2 (nmol/L)¥ 5.0 (5.0 - 179.8) 5.0 (5.0 - 149.6) 0.793 5.0 (5.0 - 61.0)a,d 5.0 (5.0 - 83.4)a <0.001 90.2)b,c,d 179.8)a,b 21-day sunshine (hours/day)¥ 2.8 (0.5 - 8.2) 2.8 (0.5 - 8.2) 0.944 4.2 (1.1 - 8.0)b,c,d 4.5 (1.8 - 8.2)a,c,d 2.8 (1.0 - 4.9)a,b,d 1.3 (0.5 - 2.3)a,b,c <0.001 ¥ 42-day sunshine (hours/day) 2.9 (0.7 - 6.6) 2.9 (0.7 - 6.6) 0.488 3.8 (1.2 - 6.6)b,c,d 4.8 (2.2 - 6.4)a,c,d 3.0 (1.5 - 4.8)a,b,d 1.2 (0.7 - 2.1)a,b,c <0.001 *Mean (standard deviation) ^Number (%) ¥Median (minimum to maximum). ≠p-values represent the significance levels for comparison between male and female. Student’s t-test for parametric data, Kruskal Wallis for non-parametric data, Chi-squared test for proportions. °p-values represent the significance levels for between seasons – ANOVA for parametric data (Tukey’s post hoc multiple comparison test), Kruskal Wallis for non-parametric data (Dunn’s post hoc multiple comparison test), Chi-squared test for proportions (with pairwise tests for independence for multiple comparisons). a – indicates a significant difference with spring; b– indicates a significant difference with summer; c – indicates a significant

22

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difference with autumn; d – indicates a significant difference with winter. Urban: 25(OH)D2 was higher in spring than in summer, autumn, winter. 25(OH)D2 was higher in autumn than in summer. Rural: 25(OH)D2 was higher in spring than in summer, autumn, winter. 25(OH)D2 was higher in winter than in summer.

23

Age Range (years) 18-39 40-49

25(OH)D3 (nmol/L)¥ 25(OH)D2 (nmol/L)¥

21-day sunshine (hours/day)¥ 42-day sunshine (hours/day)¥ Rural Numbers 3006 Age (years)* 31.6 (6.0)b,c,d,e,f,g 6 Male no. (%) 795 (26.5)b,c,d,e,f 46.3 (13.0 Total 25(OH)D (nmol/L)¥ 300.0)c,d,f,g 43.9 (8.0 25(OH)D3 (nmol/L)¥ 300.0)c,d,f,g

>90

866 55.1 (2.9)a,b,d,e,f,g 224 (25.9) 55.0 (13.0 174.0)a,b 53.3 (8.0 174.0)a,b

831 64.8 (2.9)a,b,c,e,f,g 219 (26.4) 60.2 (13.0 239.2)a,b,f,g 58.3 (8.0 – 239.2)a,b,f,g

574 74.5 (2.9)a,b,c,d,f,g 148 (25.8) 59.6 (13.0 173.2)a,b,f,g 57.4 (8.0 – 173.2)a,b,f,g

310 84.0 (2.7)a,b,c,d,e,g 80 (25.8) 49.3 (13.0 161.5)d,e 47.8 (8.0 – 161.5)d,e

37 93.1 (3.0) a,b,c,d,e,f 5 (13.5) 36.0 (13.0 136.7) d,e 35.4 (8.0 – 136.7) d,e

5.0 (5.0 – 39.8)a

5.0 (5.0 – 58.3)

5.0 (5.0 - 29.1)a

5.0 (5.0 - 57.8)a

5.0 (5.0 - 29.2)

5.0 (5.0 - 12.9)

<0.001

2.8 (0.5 - 8.2) 2.8 (0.7 - 6.6)

2.7 (0.5 - 8.2) 2.9 (0.7 - 6.6)

2.7 (0.6 - 8.2) 2.9 (0.7 - 6.6)

2.8 (0.6 - 8.2) 3.1 (0.7 - 6.5)

2.7 (0.6 - 7.7) 2.8 (0.7 - 6.6)

2.3 (0.8 - 5.9) 2.6 (0.9 - 6.0)

0.796 0.837

2496 44.9 (2.9)a,c,d,e,f,g 778 (31.2)a,d,e 46.8 (13.0 288.0)c,d,f,g 44.9 (8.0 288.0)c,d,f,g

2393 55.0 (2.8)a,b,d,e,f,g 807 (33.7)a,e,g 49.9 (13.0 194.6)a,b,e,f,g 47.7 (8.0 194.6)a,b,e,f,g

oo

758 44.9 (3.0)a,c,d,e,f,g 196 (25.9) 47.1 (13.0 296.0)c,d,e 45.3 (8.0 296.0)c,d,e

70-79

p-value

80-89

pr

Total 25(OH)D (nmol/L)¥

1448 31.5 (5.6)b,c,d,e,f,g 357 (24.7) 45.7 (13.0 264.0)c,d,e 44.3 (8.0 264.0)c,d,e 5.0 (5.0 – 64.2)b,d,e 2.8 (0.5 - 7.9) 2.9 (0.7 - 6.6)

60-69

e-

Urban Numbers Age (years)* Male no. (%)^

50-59

Pr

Variable

f

Table 3: Clinical demographics, serum 25(OH)D concentrations and hours of sunshine based on urban/rural and age range.

<0.001 0.702 <0.001 <0.001

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2207 1499 949 216 a,b,c,e,f,g a,b,c,d,f,g a,b,c,d,e,g 64.8 (2.9 74.6 (2.9) 82.3 (2.7) 92.9 (2.3) a,b,c,d,e,f <0.001 a,b,g a,b,c,g a,g 807 (36.6) 588 (39.2) 331 (34.9) 54 (25.0) a,c,d,e,f <0.001 53.0 (13.0 47.8 (13.0 35.7 (13.0 32.0 (13.0 <0.001 279.0)a,b,e,f,g 221.4)c,d,f,g 182.5) a,b,c,d,e 147.8) a,b,c,d,e 50.4 (8.0 44.8 (8.0 33.6 (8.0 27.6 (8.0 - 147.8) <0.001 a,b,c,d,e,g 279.0)a,b,e,f,g 221.4)c,d,f,g 182.5) a,b,c,d,e,g 5.0 (5.0 5.0 (5.0 - 61.0) 25(OH)D2 (nmol/L)¥ 5.0 (5.0 - 51.8)d,f 5.0 (5.0 - 51.1)f 5.0 (5.0 - 179.8)f 5.0 (5.0 - 71.9)f 5.0 (5.0 - 53.4) d <0.001 a,f,g a,b,c,d,e 63.7) 21-day sunshine (hours/day)¥ 2.9 (0.5 - 8.2) 2.7 (0.5 - 8.2) 2.8 (0.5 - 8.2) 2.7 (0.5 - 8.2) 2.8 (0.5 - 8.2) 2.9 (0.5 - 8.2) 3.1 (0.6 -8.0) 0.066 ¥ d a 42-day sunshine (hours/day) 3.0 (0.7 - 6.6) 2.9 (0.7 - 6.6) 2.9 (0.7 - 6.6) 2.8 (0.7 - 6.6) 2.9 (0.7 - 6.6) 3.2 (0.7 - 6.6) 3.4 (0.8 -6.2) 0.042 *Mean (standard deviation) ^Number (%) ¥Median (minimum to maximum). p-values represent the significance levels for comparisons between the groups – ANOVA for parametric data (Tukey’s post hoc multiple comparison test), Kruskal Wallis for non-parametric data (Dunn’s post hoc multiple comparison test), Chi-squared test for proportions (with pairwise tests for independence for multiple comparisons). a – indicates a significant difference with 18-39 year age group; b– indicates a significant difference with 40-49 year age group; c – indicates a significant difference with 50-59 year age group; d – indicates a significant difference with 60-69 year age group; e - indicates a significant difference with 70-79 year age group; f - indicates a significant difference with 80-89 year age group; g- indicates a significant difference with >90 year age group. Urban: 25(OH)D2 was lower in 18-39 than 40-49, 60-

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Jo ur

na l

Pr

e-

pr

oo

f

69 and 70-79 year age groups. Rural: 25(OH)D2 was higher in 60-69 than in 18-39, 80-89 and >90 year age groups. 25(OH)D2 was lower in 80-89 than in 18-39, 40-49, 50-59, 60-69 and 70-79 year age groups.

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Table 4: Binary logistic regression to predict the odds of Vitamin D deficiency. Vitamin D deficiency (<25nmol/L) Odds Ratio (95% CI) p-value 1.0966 (1.0712, 1.1227) <0.001 0.8135 1.1990 0.7723 2.0032 2.0631 0.3855 1.0299 2.6713 1.1082

(0.7799, (1.0980, (0.6732, (1.6643, (1.7915, (0.3353, (0.8783, (2.3594, (1.0078,

0.8486) 1.3094) 0.8860) 2.4113) 2.3759) 0.4434) 1.2076) 3.0244) 1.2185)

<0.001 <0.001

<0.001

0.033

Jo

ur

na

lP

re

-p

ro of

Variable Age (per 10 years) 42-day sunshine (per hour/day) Male -v- Female Winter -v- Spring Winter -v- Summer Winter -v- Autumn Summer -v- Spring Summer -v- Autumn Spring -v- Autumn Rural -v- Urban

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