Vitamin-D in Nepal: are we really vitamin-D decient?

Background: Recently, there have been a surge in research worldwide regarding relation of vitamin D with several diseases. Many neurological diseases like multiple sclerosis, Alzheimer’s disease as well as other diseases like cardiovascular disease have shown positive correlation with vit. D deciency measured according to the international level. Most of the research on possible role of vitamin D has been carried out in America and European nations, while there is paucity of research in low- and middle-income nations (LMICS) that are nearer to the equatorial area. In countries like Nepal, geographically they lie near to the equator and sunlight exposure is well enough and milk consumption is quite moderate. Thus, special consideration for vitamin D level in Asian population seems plausible. This will not only help us to cover the vitamin D decient population but will help us to avoid excessive and unnecessary usage of it. Objectives: This study aims to detect the situation of vitamin D in Nepalese population and secondly to nd out the suitable normalized reference range for serum vitamin D in multi-ethnic Nepalese population. Methodology: This was a hospital based prospective study. A prospective study was conducted using purposive sampling technique with in-vivo and in vitro bio-physiological method to collect serum vitamin D level. After rigorous inclusive and exclusive criteria, a total of 107 subjects were collected. Result: The present study showed that 32% of participants had decit serum Vitamin-D level (<15 ng/mL), 48% of subjects had insucient serum level of Vitamin-D (15-<30ng/mL) and 20% of participants had sucient serum level of Vitamin-D (>30ng/mL). Study showed that there is lower degree of positive relationship of Body Mass Index (BMI) with Serum Vitamin-D level (r=0.162, p=0.094), History of chronic illness (χ2=0.10, p=0.03), timing of occurrence of stroke (χ2=11.41,p=0.017) and diagnosis (χ2=21.19, p=0.011) had signicant association with Serum vitamin-D level at p<0.05. Conclusion: There is direct signicant association of Serum Vitamin D with socio-demographic variables when international unit is considered. Neurological disorder showed positive association national and previous studies supported the nding as an ecological study conducted to assess association of D with socio-demographic factors in result of the study revealed that there is signicant association between 25-hydroxyvitaminD level and all the predictors i.e. age, gender and educational status (all p<0.0001). Another study on determinants of vitamin D status in young adults; inuence of lifestyle, sociodemographic and anthropometric factors among 738 subjects revealed that the relative risk (RR) for vitamin D deciency was highest for men 2.09 (1.52, 2.87); subjects who exercised 0-½ hours a week 1.88 (1.21, 2.94). A study aimed to assess the association between vitamin D deciency and depression in Nepalese population stated signicant association of gender, geographical location of residence, marital status, religion and vitamin D status with clinically signicant depression. 30 Other study has been observed a direct relationship between latitude and the prevalence of Multiple Sclerosis (MS), which suggests a role for UV radiation and vitamin D in MS development. reduced (p < 0.05). Chronic exposure to alcohol has the potential to reduce the levels of vitamin D. Several evidence suggests that vitamin D acts like a neurosteroid and is required for normal brain development and function. 35 An endocrine review on Vitamin D and neurological diseases, aimed to highlight the relationship between vitamin D and neurological diseases stated that there is association between low levels of 25(OH)D and a wide spectrum of neurodegenerative conditions such as multiple sclerosis, Alzheimer’s disease, Parkinsons’s disease and neurocognitive disorders, is supported by in vitro and in vivo data. 35 Other studies showed that the risk of MS decreases with increasing intake of vitamin D 36 , and serum 25(OH)D levels are signicantly lower in patients with MS as compared to healthy controls. 37 These studies directly support the results of our current study as history of chronic illness (χ2=0.10, p=0.03), timing of occurrence of stroke (χ2=11.41, p=0.017) and diagnosis (χ2=21.19, p=0.011) had signicant association with Serum vitamin-D level at p<0.05. There was lower degree of positive relation of chronic illness (r=0.053, p=0.579) and diagnosis (r=0.012, p=0.902) with serum Vitamin-D level. Whereas the Serum Vitamin-D level was not signicantly associated with History of stroke (χ2=11.62, p=0.06) and type of stroke (χ2=4.84, p=0.21) at p=0.05. Another cross-sectional study to evaluate the association between Vitamin-D and hypertension among 520 people supports the study as the result stated that Severe vitamin D deciency was highly prevalent in people with hypertension than in people without hypertension (p value <0.001). The study concluded that Vitamin D deciency was associated with an increased risk of having hypertension


Background
Research of Vitamin D in neurology has gained momentum recently. Many neurological ailments like multiple sclerosis 1 , spinal diseases, Alzheimer's disease 2 as well as other diseases like cardiovascular disease 3 have shown positive correlation with vitamin D de ciency. Probably this is one of the main reasons that family physicians and neurologists have opted for including serum vitamin D in their lab investigation. However, the reference range that is followed in all over the world comes from the western world consisting of data largely based on caucasian population and measurement and clinical interpretation is done on the basis of this reference level. 4,5 Vitamin D is a group of fat-soluble prohormones which were identi ed after the discovery of the anti-rachitic effect of cod liver oil in the early part of the 20 th century. The two major biologically inert precursors of vitamin D are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Both vitamin D precursors resulting from exposure to the sunshine and the diet are converted to 25hydroxyvitamin D [25(OH)D] (calcitriol) when they enter the liver. 25(OH)D is the major circulating form of vitamin D and is used to determine vitamin D status Vitamin D has several roles in the body, including modulation of cell growth, neuromuscular and immune function, and reduction of in ammation. Many genes encoding proteins that regulate cell proliferation, differentiation, and apoptosis are modulated in part by vitamin D. 6,7 Despite the numerous studies about the association between vitamin D and different health outcomes, there are still controversies de ning the adequate vitamin D status, daily intake needed and the potential adverse health consequences of its de ciency. Serum 25-hydroxy vitamin D concentrations are being used to de ne vitamin D de ciency, but the diagnostic test accuracy of these measurements and the reference standard used are not clearly stated. There appears to be a wide variability between different essays for its determination in different laboratories and there is not yet international consensus on the optimal concentrations in different population groups. 8 According to the epidemiologic studies, based on the internationally accepted serum vitamin D levels, about 1 billion people worldwide have vitamin D de ciency, while 50% of the population has vitamin D insu ciency. 9 The prevalence of patients with vitamin D de ciency is highest in the elderly, the obese patients, nursing home residents, and hospitalized patients. Prevalence of vitamin D de ciency was 35% higher in obese subjects irrespective of latitude and age. 10 In South-Asian region 80% of the apparently healthy population is de cient in vitamin D and up to 40% of the population is severely de cient when considered according to the international level. 11 A study on hypovitaminosis D in developing countries to assess prevalence, risk factors and outcomes revealed that though South Asia region has ultra violet B (UVB) radiation levels that are su cient for vitamin D synthesis for 11 to 12 months of the years, however, serum 25-hydroxyvitamin D levels of <25nmol/I have been reported in more than 50% of the infant, children and women. 12 Another study conducted on prevalence of Vitamin D de ciency among adult patients in a tertiary care hospital in Nepal has reported that out of total patients, vitamin D de ciency was found among total of 283 patients. Vitamin D de ciency was found to be higher in females than males. 13 Neurological disorders are an important cause of disability and death worldwide. Globally, the burden of neurological disorders has increased substantially over the past 25 years because of expanding population numbers and growing ageing, despite substantial decreases in mortality rates from stroke and communicable neurological disorders. 14 A systemic analysis for global, regional and national burden of neurological disorders during 1990-2016 stated that globally, in 2016, neurological disorders ranked as the leading cause of Disability-Adjusted Life Year (DALYs) in 2016 (i.e. 276 million, comprising 10·2% of global DALYs) and secondleading cause group of deaths (i.e. 9 million, comprising 16·8% of global deaths). The four largest contributors of neurological DALYs were Stroke (42.2%), Migraine (16.3%), Alzheimer's and other dementias (10.4%) and meningitis (7.9%). 15 As considerable Hypovitaminosis D is common in Asians, physicians often prescribe cholecalciferol at a dose of 60,000 IU/week for two months, then at 60,000 IU/month for six months for occult vitamin D de ciency. 16 Although this practice is most commonly followed, the adverse effects on excessive supplementation of vitamin D and the high serum 25(OH)D levels in Nepalese individuals have not been systematically studied.
Notably serum vitamin D is inconsistent in diverse population. Even in the homogenous population of caucasians, vitamin D genetic variability in vitamin and calcium receptors has been witnessed. 17,18 In countries like Nepal, geographically they lie near to the equator and sunlight exposure is well enough. Thus, special consideration for vitamin D level in Asian population seems plausible. This will not only help us to cover the vitamin D de cient population, 19,20 but will also help us to avoid excessive and unnecessary usage of it. We have sought to nd out whether the depletion of vitamin D in our population is for real and if these are of real, do the depletion vitamin D cause the neurological disorders?

Subjects
The purpose of the study was to nd out the suitable normalized reference range for serum vitamin D in multi-ethnic Nepalese population and to detect the correlation of vitamin D and Neurological disorders. This is prospective study that includes the quantitative research design selected for the dependent variables i.e. serum Vitamin D level and independent variables i.e. health history, history of chronic illness, types of stroke, smoker and alcohol consumption, physical activity and Basal Metabolic Index (BMI) of subjects, socio-demographic variables such as age, sex, education, marital status and address of subjects. Prospective purposive sampling technique was used. A total of 108 subjects were included. The study was approved by institutional review board (IRB) of our institute and informed written consent was obtained from each participant. The data collection was conducted from 1 st September till 1 st January. Inclusion and Exclusion criteria were set as the inclusion criteria for present study was the subject with: a) age 18 years and older, b) anyone willing to take part & c) presence of any neurological disease and d) any healthy person present at the time of data collection. Exclusion criteria were: a) Age below 18 years or above 90 years, b) not giving consent, c) no other chronic illness besides neurological (diabetes, other cancers, gynecologic diseases, hypertension and d) anyone taking Calcium or vitamin concentrates or supplements or has been treated for calcium recently or vitamin D supplements (within 30 days).

Clinical examination:
The Socio-demographic variables were measured using structured questionnaire that consists age, sex, educational status, permanent address, marital status of subjects. The part of same questionnaire consisted personal history such as history of stroke, type of stroke, chronic illness, physical activity, smoking habits, and alcohol consumption. All the subjects were examined with physical and neurological examination by experienced physician, and neurologist. In-vivo bio-physiological method was used to measure BMI and In-vitro bio-physiological method was used to quantify level of Vitamin-D using ELISA method with 'Mono Bind Inc.' USA. Radiological measurements were also performed to con rm the neurological disorders.
BMI, formerly called the Quetelet index, is a measure for indicating nutritional status in adults is de ned as a person's weight in kilograms divided by the square of the person's height in metres (kg/m2). The level of BMI was distributed as a) <18.5 kg/m 2 under-weight, b) 18.5-24.9 kg/m 2 normal weight, c) 25-29.9 kg/m 2 over weight and d) >30 kg/m 2 obesity. 21 In another hand the level of Vitamin-D was scored as a) <15 ng/mLde cit, b) 15-<30 ng/mL insu cient and c) >30 ng/mL su cient.

Statistical Analysis:
Statistical analysis was performed using the IBM Statistical Package for the Social Sciences (SPSS version 20). A descriptive analysis: frequency, percentage, mean, and standard deviation were used to describe the socio-demographic variables and independent variables. An inferential analysis: Chi-square test, Karl Pearson Correlation Coe cient, Multivariate Linear Regression analysis was used to nd association between Level of Vitamin-D and selected socio-demographic variables and Independent variables of Nepalese people. A nominal p-value of ≤0.05 was considered statistically signi cant, and p < 0.1 was considered a trend, using two-tailed test.

Demographic and Clinical Characteristics
The demographic characteristics of total 108 eligible subjects are presented in Table1.1, among them more than half i.e. 66% (71) were male, one third of subjects i.e. 36% (39) were 46-60years old age group, majority of participants i.e. 76% (82) were from hilly region, more than half of subjects were i.e. 62% (67) from inside the Kathmandu valley (capital of Nepal). With regards to education 44% (48) had received only primary education level (spent few years in school), 89% (96) participants were married. Majority of the participants i.e. 71% (77) were active in their life, 28% (30) were smokers and 29% (31) were alcohol consumer.
Among the participants, 40% (43) had stroke, among which, more than half 58% (63) had approached emergency department in less than 24 hours of stroke onset. Presented in table 1.2 majority of subjects i.e. 75% (81) had chronic illness with hypertension and other condition; similarly, 72% (78) of participants were suffering with chronic illness since less than 5 years. With regards to types of stroke, 64% (69) and 36% (39) had Ischemic and Hemorrhagic stroke, respectively.

Discussion
Vitamin D de ciency has been mechanistically and clinically linked to neurological diseases and neuropsychological disorders, cognitive impairment, and neurodegenerative diseases. 22 Our current study has emphasized that 32% of participants had de cit serum Vitamin-D level, 48% of subjects had insu cient and 20% of participants had su cient serum level of vitamin-D.
Studies carried across different countries in South and South East Asia showed, with few exceptions, widespread preva lence of hypovitaminosis D, in both sexes and all age groups of the population. 23 High prevalence of hypovitaminosis D in South Asia can be explained by skin pigmentation and traditional clothing. Air pollution and limited outdoor activity further compounds this problem in the urban population. 24 The current study stated that there is lower degree of positive relationship of BMI with Serum Vitamin-D level (r=0.162, p=0.094), that is statistically not signi cant at p<0.05. In another way there is no statistically signi cant association between BMI and vitamin D (χ2=12.474, p=0.071). The nding is supported by a study on the effect of vitamin D supplementation on serum 25OHD in thin and obese women as the result showed a signi cant inverse relation between total body fat mass and serum 25-OHD (p<0.0001) and serum 1,25(OH) 2 D (p=034). There was no signi cant change in total body fat mass after treatment with vitamin D or calcitriol in randomized trials. 25 Similarly the nding is contradicted by the study aimed to nd association between BMI and vitamin D supplement the result stated that there was signi cant differences in mean 25(OH)D levels of vitamin D supplementation doses were consistently seen across BMI categories. 26 29 A study aimed to assess the association between vitamin D de ciency and depression in Nepalese population stated signi cant association of gender, geographical location of residence, marital status, religion and vitamin D status with clinically signi cant depression. 30 Other study has been observed a direct  29 Another study on interaction of vitamin D and smoking on in ammatory markers, the data was collected from Korean Elderly Environmental Panel Study that included 560 subjects. The result of the study was that association of vitamin D de ciency and hs-CRP in smokers was stronger than that in nonsmokers (smokers: β=-0.375, p=0.013; non-smokers: β=-0.060, p=0.150). Smoking status was an effect modi er that changed the association between vitamin D de ciency and hs-CRP (interaction estimate: β=-0.254, p=0.032). There was a stronger signi cant association of smokers and vitamin D de ciency than non-smokers. 32 A review on Vitamin D and alcohol aimed to evaluate the association between alcohol use and vitamin D serum levels alcohol intake was found to be positively associated with vitamin D status in 15 articles and negatively associated with vitamin D in 18 articles. 33 the nding is contradicted by another study on chronic ethanol exposure effects on vitamin D levels among subjects with alcohol use disorder stated that levels of inactive vitamin D (25(OH)D 3 ), active vitamin D (1, 25(OH) 2 D 3 ), cathelicidin/LL-37, and CYP27B1 proteins were signi cantly reduced (p < 0.05). Chronic exposure to alcohol has the potential to reduce the levels of vitamin D. 34 Several evidence suggests that vitamin D acts like a neurosteroid and is required for normal brain development and function. 35 An endocrine review on Vitamin D and neurological diseases, aimed to highlight the relationship between vitamin D and neurological diseases stated that there is association between low levels of 25(OH)D and a wide spectrum of neurodegenerative conditions such as multiple sclerosis, Alzheimer's disease, Parkinsons's disease and neurocognitive disorders, is supported by in vitro and in vivo data. 35 Other studies showed that the risk of MS decreases with increasing intake of vitamin D 36 , and serum 25(OH)D levels are signi cantly lower in patients with MS as compared to healthy controls. 37 These studies directly support the results of our current study as history of chronic illness (χ2=0. 10 41 Another study on Vitamin D in amyotrophic lateral sclerosis represented that in chronic neurological diseases levels of vitamin D in blood appeared low but there was no signi cant differences found between the level of vitamin D and Amyotrophic lateral sclerosis patients (18.8±12.2) and the healthy subjects (20.7±10.1). 42 Similarly, Cross-sectional associations of plasma vitamin D with cerebral β-amyloid in older adults at risk of dementia, the study didn't nd the association between baseline 25(OH)D levels and cerebral Aβ in any of the brain regions studied. 43 Vitamin D is not associated with incident dementia or cognitive impairment over an 18 years period of time another study presented as the result showed that the adjusted HR for the continuous GRS for all cause dementia was 1.04 (95% CI:0.91, 1.19). 44 A study to investigate association between serum concentration of vitamin D and 1-year mortality in stroke patients presents that out of the 382 stroke patients, 16.5% died in a year, and the mean 25(OH)D level was lower in those patients (32.3±22.0 vs. 44.6±28.7 nmol/l, p<0.001) and survival at 1 year was worse in patients in the lowest tertile of 25(OH)D levels (i.e. <25.7nmol/l). The study concluded that low level serum 25(OH)D level at stroke onset association was with higher mortality at 1 year in patient. 45 The nding from this study may supports for the implementation of measures to determine the real state of vitamin D and its implication on Neurological disorders. We recognize some limitations of our study as the study completed with small size of 108 due to rigorous inclusion and exclusion criteria. As it was a hospital-based study, sample might not be enough to represent the total population of our country. The study should be continued with similar control groups.

Conclusion
Despite abundant sunlight in Nepal several studies suggest the prevalence of Vitamin D de ciency in this region. Our question is whether the reference given globally is accurate for the land-locked country with different geographical area, multi-cultural, religion and practices. Through the study we concluded that more than half of population having Serum Vitamin-D insu ciency. Level of serum vitamin-D is signi cantly associated with socio-demographic variables as well with neurological conditions but there are lots of caveats with it. A larger population-based study in multinational (equatorial region) seems necessary.

Consent to participate
The participants were well informed about the study. The participants were well informed in written and well explained and obtained consent to participate.

Consent for publication
Not Applicable Author's Contribution All authors have made a substantial contribution to this paper.
AC contributed to the conceptualization and drafting of the article. BP and SD contributed to conceptualization of manuscript. RR and SG contributed to the study design and SA contributed to the revision of the article. PR, PS and RS contributed to the statistical analysis.

Competing Interests
None to declare.

Funding
Not available