Vitamin D deficiency is common in areas with limited sunshine and also among indoor subjects in countries where there is plenty of sunshine (1-4). The focus of current studies is to investigate the functional significance of low and insufficient serum 25(OH)D in asymptomatic apparently healthy subjects. Investigations in this area is important in view of the fact that  most of the  meta-analysis studies indicate only a limited role of vitamin D in adults i.e. modest effect on prevention of vertebral fractures, fall and dental carries (5).

There are limited studies on functional significant of vitamin D deficiency and relevance of vitamin D supplementation beyond the growing age, among asymptomatic apparently healthy adults in India. Two large placebo controlled randomized controlled trials carried out by us on functional significance of vitamin D on muscle strength among young Indians females and males have not revealed any significant effect of vitamin D/Calcium supplementation on hand grip strength, six minutes walk distance, physical quality of life after  six months and   immune function like Th1/Th2 ratio and antibacterial peptide cathelicidin  (6-8). Recently, Aloia et al., (2018) et al., no significant effect of vitamin D supplementation in prevention of bone loss among African elderly women with osteoporosis  in RCT (9). 

Notwithstanding above, currently low serum 25(OH)D  in apparently healthy normal subjects is  considered to be synonymous with vitamin D deficiency. However, to justify this statement, low serum 25(OH) D should be associated with some symptoms and/or objective alteration in body/bone composition; either in acute or chronic form. Bone mineral density (BMD) and bone markers are the standard and common bone related parameter for  assessing the effect of vitamin D supplementation (9,10). However, it has not turned out be a reliable or sensitive marker for vitamin D deficiency in asymptomatic subjects (5,9). Several reasons for these might be presence of host of confounding factors such genetic variation in genes linked with BMD and nutrition factors other than the vitamin D which can affect bone constituents relevant for osteoporosis.  Nutrients like vitamin C and K as well as protein are also important for collagen and non collagenous proteins of the bone matrix (11,12).  Malmir et al., recently reported meta-analysis of observations on dietary vitamin C intake and low risk of hip fractures in osteoporosis as well as higher BMD at femoral neck and lumbar spine (11).   

Bone fractures typical of osteoporosis i.e. vertebral and hip can occur in subjects with normal bone density (13). This is also exemplified in patients affected by chronic inflammatory disease especially if there is steroid hormone excess in the body, endogenous or exogenous (14). This has been explained by the fact that bone microarchitecture may be compromised in subjects despite normal BMD (14). Vinolas, et al., recently (2018) investigated 53 patients with endogenous Cushing’s syndrome and observed that only 24% of them had abnormal BMD, in contrast to 43% showing degraded microarchitecture (14). Similar situation might be prevalent in apparently normal subjects and could explain presence of stress and osteoporotic fractures in asymptomatic healthy subjects with normal BMD (13).

Bone microarchitecture can be assessed by bone histomorphometry. However, it is practically difficult to document the altered microstructure in large number of subjects because of the invasive nature of bone biopsy. Trabecular bone score (TBS) is a recently introduced tool for the assessment of bone microarchitecture (15-20). It is a gray-level textural metric that can be extracted from the two-dimensional lumbar spine DXA image. Bone texture in TBS correlates with bone microarchitecture such as trabecular number, trabecular separation, connectivity density, and bone volume fraction. Most of the available litretaure on TBS has been in symptomatic patients sepcific population like those with hyperparathyroidism (23). However, there is a paucity of information on TBS in asymptomatic subjects with chronic hypovitaminosis D, with no study reported from India, till date. The presnt study has been planned to assess the functional signoificance of low serum 25(OH)D on bone microarchitceture in a large cohort of apparently healthy Indian subjects with age range of 20 to 60 years including females.

Serum 25(OH)D measured in an individual for vitamin D status is usally a one time paramneter and may not adequately represent their term vitamin D status in the past.  To take this fact into account, present study also plans to assess  the  role of vitamin D in the revrsibility of poor bone microachitectre in healthy subjects. A placebo controlled RCT of vitamin D and calcium supplementaion  given for six months would be carried out in a two by two factorial mode out among subjects who have both subnormal 25(OH)D and subnormaormal TBS score as per the international norm (score <1.35).   

A) Rational of the study supported by cited literature

Vitamin D is vital for human health. Its deficiency may lead to hypocalcemic seizures, rickets in infant and impaired bone health in adults. The optimal cut-off of serum 25(OH)D for vitamin D deficiency are not known. The Endocrine Society of USA suggest 25(OH)D > 30 ng/mL as vitamin D sufficient and Institute of Medicine, defines a cut-off of 20 ng/mL (1, 24). Subnormal serum 25(OH) D are frequent in healthy infants, children, adolescents and pregnant women in urban India (2-4,25-29).  Recently, we observed that with better sunshine exposure even Indians as outdoor manual workers  (26) show normal serum 25(OH)D  with values > 40 ng/ml in several (26).  The cut-off for serum 25(OH)D for normal vitamin D is an active area of debate and research. In our previous three studies, serum 1,25(OH)D was normal in subjects with low serum 25(OH)D (2,6,8). Similar observations were observed by others (30). Therefore, a firm stand on vitamin D supplementation and/or food fortification require additional data on functional significance of low serum 25(OH)D in healthy subjects. Besides, there is no safety study after long term cholecalciferol in tropical environment.

Our center is assessing the functional aspects of low serum 25(OH)D in apparently healthy Indian subjects since 2000. Though, there was inverse correlation between serum 25(OH)D and PTH in most studies, clinical indices such as skeletal muscle strength (6,8) and other biomolecules did not show correlation with vitamin D status (7). Our centre first assessed serum 25(OH)D and thyroid autoimmunity (29) in 642 healthy subjects aged 16–60 years, with mean serum 25(OH)D of 17·5 ± 10·2 nmol/l. Though TPOAb positivity was observed in 21% of subjects, the TPOAB positivity and thyroid dysfunctions were comparable between subjects with 25(OH)D  â‰¤  25 or > 25 nmol/l.

RCT on vitamin D/Ca supplementation have not shown promising effect on muscle strength in the elderly (5). In 2012, we conducted a RCT on vitamin D and/or calcium and muscle strength in 173 young females (age, 21.7 ± 4.4 yr, 25(OH)D, 9.3 ±  3.37 ng/ml). The  groups  were 1) double placebo, 2) calcium, 3) cholecalciferol, and 4) cholecalciferol/calcium.  Cholecalciferol was given at 60,000 IU/wk for 8wk, then 60,000 IU/fortnight and elemental Ca 500 mg twice daily for 6 months. Post supplementation, serum 25(OH)D increased to 29.9 ±  8.35 ng/ml and 27.0 ± 9.54 ng/ml in two cholecalciferol groups. The hand grip strength and quality of life were comparable in groups indicating that   cholecalciferol/Ca did  not lead to improvement in the skeletal muscle strength.

We also carried out a similar RCT on Vitamin D and calcium supplementation, skeletal muscle strength and serum testosterone in 228 young healthy adult males (8). The four study  groups and cholecalciferol/Ca dose was exactly the same as described in our previous RCT in females (6). The baseline 25(OH)D was 21.5 ± 9.5 nmol/L. The parameters assessed were dominant arm handgrip and pinch-grip strength, 6 min walk test, dyspnoea-score, quality of life,  serum 25(OH)D, 1,25(OH)2D, iPTH, total testosterone and free androgen index (FAI).  After intervention, mean serum 25(OH)D was >75.0 nmol/L in cholecalciferol groups. However, the handgrip strength was comparable in the four groups. Subgroups analysis among subjects with baseline serum 25OH)D < 25.0 and <12.0 nmol/L showed similar results. The delta change in serum testosterone and FAI was also comparable in four groups.

Vitamin D has been considered to have beneficial effect on maintenance of  immune system and prevention of autoimmunity and infections. In 2014, we assessed the effect of cholecalciferol and calcium supplementation on mRNA expression of cathelicidin Th1 (IFN-γ) and Th2 cytokines (IL-4 and its antagonist-IL-4δ2) and their transcription factors (T-bet, STAT4, GATA-3, STAT6) in the peripheral blood in healthy females with vitamin D deficiency (VDD) (7). 131 females with biochemical VDD were from our previous RCT among females described above on vitamin D supplementation and muscle strength. Cholecalciferol-supplemented groups showed significant rise of mean serum 25(OH)D (30.6 ± 7.51 and 28.6 ± 8.41 ng/ml. However, the mean mRNA transcripts of all the biochemical assessed were comparable in the supplemented and placebo groups.

There are limited number of RCT on the effect of vitamin D on morbidity and mortality in low birth weight infants in low income countries. Recently, Trilokpuri et al., (25) assessed 2079 low birth-weight infants (>37 weeks’ gestation) in New Delhi with primary outcome of  hospital admission for morbidity and mortality in the first six months of life. Vitamin D supplements (35 µg/week) for 6 months led to better vitamin D status and increased SD scores for weight, length, and arm circumference, but no decrease in the morbidity or mortality.

The relationship between vitamin D, BMD, prevention of fall and muscle strength especially in elderly has never been conclusively proven (5).  In a recent RCT  Aloia et al., assessed effect of vitamin D supplementation in 260 elderly black African women, aged 60 years orolder  in preventing  bone loss (9). Though, serum PTH declined in the vitamin D supplemented active arm, the rate of bone loss in subjects with serum 25(OH)D >75 nmol/L was  comparable to that of subjects with serum 25(OH)D at 50 nmol/L.

In view of widespread vitamin D deficiency in urban indoors, there is need to assess its functional parameters especially for bone health. TBS is a novel and non invasive tool to measure bone micro-architecture and has shown promising association with vitamin D status. This association might emerge more convincingly in urban Indian with chronic vitamin D deficiency. We plan  to assess and correlete bone microarchitceture using TBS with serum 25(OH)D in a large cohort of asymptomatic healthy subjects. This study would help fill lacunae in the interpretation of biochemically low serum 25(OH)D. The RCT in the proposed work on vitamin D supplementation would be a step forward in understanding the role of vitamin D in reversing degraded trabecular bone score. 

B) Hypothesis:

TBS is emerging a clinically relevant marker of bone microarchitecture and might correlate with serum 25(OH)D in Indians. Reversal of impaired TBS by vitamin D supplementation would provide the functional relevance of low serum 25(OH)D levels

C) Key Questions

1) Whether serum 25(OH)D levels correlate with  TBS at lumbar spine in healthy subjects in  age groups 20- 60 years. Lower age of 20 years has been selected because currently TBS  software is standardized only for age 20 years and above. Large number of subjects assessed in this study would also help provide normal TBS data.

(2) Whether, those with low TBS and low serum 25(OH)D show improvement in the TBS after six months of vitamin D and calcium supplementation.

Current status of research and development in subject (both International and National Status)

Trabecular bone score is a recent tool for the assessing bone microarchitecture. It is a gray-level textural metric that can be extracted from the 2D lumbar spine DXA images. TBS correlate with bone microarchitecture such as trabecular number, trabecular separation, connectivity density, and bone volume fraction (15-22, 31)

Principle of TBS: A dense trabecular microstructure projected onto a plane generates an image of large pixel value with variations of smaller amplitude. A variogram of those projected images, calculated as the sum of the squared gray level differences between pixels at a specific distance can estimate a 3D structure. TBS is derived from the experimental variograms of 2D projection images. TBS is calculated as the slope of the log-log transform of the variogram, where the slope characterizes the rate of gray level amplitude variations. A steep variogram slope with a high TBS value is associated with better bone structure. TBS principles can be compared to an aerial view of a forest where individual elements i.e, trees cannot be separated out. The DXA image in TBS cannot discern the individual trabeculae. Although both aerial view of the forest and DXA have low resolution to sufficiently identify individual components like tree and trabeculae respectively, the missing areas i.e. ‘clearings in the forest’ and bone in the trabecular compartment or are clearly noticeable. A significant correlation in an imaging is usually preserved till a sampling size of 1000 µm. A DXA is a low resolution image with sampling size between 300 and 900 μm and image provides information on overall microarchitecture instead of individual trabeculae.

TBS iNsight: TBS iNsight is a software tool which can be installed on existing DXA scanners. TBS can be readily applied to common DXA desnitometers like Hologic (Delphi, QDR 4500, and Discovery; Waltham, MA, USA) densitometers. and GE Lunar (Prodigy and iDXA; Madison, WI, USA). The TBS is determined using the same region of interest as the BMD measurement, so that vertebrae excluded from the BMD calculation, e.g., vertebrae with fractures or osteoarthritis, are also excluded from the TBS analysis. The suggested cut-off values for TBS in postmenopausal women, as reported by TBS software is as follows:

TBS value (unitless)

< 1.200 (Degraded microarcitecture)

1.200 to 1.350 (partially degraded microarcitecture)

> 1.350 Normal

The precision of precision of TBS varies from 1.1- 2.1 % which comapare with spine BMD precison (0.7-1.7%) on DXA scan (31) . 

There are limited number of studies assessing the association between 25(OH) D status and TBS and most of the studies have been carried out in patients specific populations such as patients with hyperparathyroidism (23). Here we review briefly the relevant recent studies for our work.

Di Gregorio et al. (2015) studied 390 patients (M 72; F: 318; > 40 years (15). The groups were Naive of treatment (Naive, n = 67), calcium and vitamin D (n = 87), testosterone (n = 36), alendronate (n = 88), risedronate (n = 39), denosumab (n = 43) and teriparatide (n = 30). After 24 months, the TBS in the naive group decreased by 3.1% (p < 0.05). In contrats there was a significant improvement (p < 0.05) in the Ca and vitamin D group suggesting , TBS preservation with oral calcium and vitamin D supplementation.

Hansen et al. (2015), conducted a placebo controlled RCT in 230 postmenopausal women aged 75 years or less with baseline 25(OH) D levels of 14-27 ng/mL and no features of osteoporosis on BMD (16). Subjects were categorized in three arms i.e. placebo (n= 76), low dose vitamin D (800 IU/week, n= 75) and high dose vitamin D (50,000 IU/week, n= 79), supplementation. Participants in the high-dose cholecalciferol received a loading dose (50 000 IU/d for 15 days) to quickly raise their 25(OH) D levels to 30.0 ng/mL. After one year of supplementation there was no significant between-arm change in TBS.

Aloia et al., in 2015 (17) assessed a cohort of 518 healthy postmenopausal African American women with a mean age of 66 years and a BMI of 30.1 Kg/m².  The women were healthy based on history, physical, and laboratory screens. Mean TBS and BMD of the spine were 1.300 ± 0.10 and 1.011 ± 0.165 g/cm², respectively and revealed a significant correlation of r= 0.41, p < 0.0001. TBS values showed a trend near significance with serum 25(OH)D (P = 0.05). .

There is limited information on TBS and serum 25(OH)D in young children. Shawwa et al., (18) studied the predictors of TBS in 170 boys and 168 prepubertal girls (13.2 ± 2.1 years), in a RCT where population was randomized to weekly placebo or one of the two vitamin D3 groups (1400 IU/week and 14,000 IU/week). TBS and serum 25(OH) D were measured at baseline and after 12 months. They could not find any significant effect of vitamin D supplementation on TBS values in both genders and different  pubertal stages.

Hyde et al., (2017) explored the relationship between maternal vitamin D (25(OH) D and offspring TBS (19). Maternal 25(OH)D was assessed  at 16 weeks’ gestation and at 28–32 weeks’ gestation. 11 years later (2013–2016), mother–child pairs (n = 181) were recalled. Offspring of mothers with sufficient 25(OH) D levels (≥50 nmol/L) at recruitment had a higher TBS (1.363 vs. 1.340, P = 0.04). In linear regression, a 10 nmol/L increase in maternal 25(OH)D was associated with a 0.005 (P = 0.04) increase in TBS.  TBS did not correlate with maternal 25(OH) at 28– 32 weeks.

EL Hage et al (2014) investigated the relation between serum 25-hydroxyvitamin D (25OHD) and TBS in 101 Lebanese postmenopausal women in the  ages range of  45-89 years (60.9 ± 13.6 years) (20). Mean serum (25OHD)l was 27.2  ± 12.2 ng/ml and mean BMI was 26.9 ± 5.2 kg/m². Age, weight, height, BMI and serum (25OHD) were not correlated to TBS, indicating  serum (25OHD)  not being the determinant of TBS in their group.

Mário Rui Mascarenhas et al., (21) evaluated influence of Vitamin D on TBS and BMD  at the lumbar spine  in a group of normal men ≥ 40 years. These men were divided in the normal, low BMD and osteoporosis groups, and as normal, vitamin D insufficiency and deficient. Vitamin D deficient group had lower TBS and showed significant correlation with 25(OH)D which was not there in  BMD.

Alwan et al., recently 2018 (22) assessed the relationship between serum 25(OH)D and TBS in a healthy adults involving. TBS was significantly higher in 25(OH)D-sufficient participants (≥30 ng/mL) in both males and females, thereby providing further evidence of positive relationship between  vitamin D and optimal TBS values.

The relevance of the proposed study

The present study is especially relevant from the public health point of view with the back ground information of recently recognized vitamin D deficiency in urban Indians. There is need to document functional significance of low serum 25(OH)D in order to justify food fortification and supplementation with vitamin D. Among all the body tissue, bone is expected to benefit maximally with vitamin D supplementation. In the present study, we plan  to assess the functional significance of low serum 25(OH)D on bone bone microarchitceture using TBS in a large cohort of asymptomatic subjects. The planned RCT on vitamin D supplementation would be a step forward in further understanding the role of vitamin D in reversing degraded trabecular bone score. This study would fill lacunae in the existing knowledge by assessing the effect of hypovitaminosis D on bone microarchitecture in asymptomatic healthy subjects. This would help in interpretation of biochemically low serum 25(OH)D and the intervention if any required for correcting the abnormality.  If we observe significant association of TBS with vitamin D or benefit of vitamin D supplementation in reversing degraded TBS, it might emerge as the useful marker for vitamin D deficiency in apparently healthy subjects.

The expected outcome of proposed study:

Besides, publications and training of manpower, the presents study is especially relevant from the public health point of view in the back ground of vitamin D deficiency in urban indoor Indians (2). There is need to document functional significance of low serum 25(OH)D in order to justify food fortification and supplementation with vitamin D. Among all the body tissue, bone is expected to benefit maximally with vitamin D supplementation. In case of vitamin D insufficiency, it is possible that bone microarchitecture might be impaired despite intact BMD. However, bone strength, compactness and microarchitecture is difficult to document in large numbers because of the invasive nature and complicated analysis required for its assessment by bone biopsy and histomorphometry. In the present study, we plan  to assess the functional significance of low serum 25(OH)D on bone bone microarchitceture using TBS in a large cohort of asymptomatic subjects. This study would fill lacunae in the existing knowledge by assessing the effect of hypovitaminosis D on bone microarchitecture in asymptomatic healthy subjects. This would help in interpret biochemically low serum 25(OH)D and the intervention if any required for correcting the abnormality. The planned RCT on vitamin D supplementation would be a step forward in further understanding the role of vitamin D in reversing degraded trabecular bone score. 

The preliminary work done so far

Our team has been investigating and has completed several projects related to vitamin D deficiency in Dehi, since 1998 (2,3,4,6-8,26-29). Our first study on vitamin D status in urban subjects of Delhi, published in Am J Clinical Nutrition in the year 2000, showed a high prevalence of low serum 25(OH)D in urban indoor Indians (2).  Subsequently, we have been assessing the extent of problem of vitamin D deficiency among urban and rural subjects of Delhi engaged in various activities such as farming, labourers engaged in construction work and street vendors in unorganised sector. These studies are showing a relatively better and normal status of vitamin D in these outdoor workers unlike urban indoors (26 ). Besides, we have been investigating the functional significance of vitamin D in asymptomatic healthy subjects who have vitamin D deficiency as per the biochemical criteria. The important studies among these relationship of serum 25(OH)D with BMD (10),  thyroid autoimmunity (29) and role of vitamin D supplementation on improvement in muscle strength among asymptomatic healthy young Indian females and males (6,8). We already have the bone densitometry facility at out centre which has been functioning well for the past 16 years. Recently, software for assessing the trabecular bone score has been procured and is being used to study altered bone microarchitecture in several endocrine disorders. Thus, our centre is well equipped to conduct both the component of the proposed study in this project.      

Scope of applications indicating anticipated project and processes

This study would fill lacunae in the existing knowledge by assessing the effect of hypovitaminosis D on bone microarchitecture in asymptomatic healthy subjects. This would help in interpretation of biochemically low serum 25(OH)D and the intervention if any required for correcting the abnormality. The planned RCT on vitamin D supplementation would be a step forward in the understanding the role of vitamin D in reversing degraded trabecular bone score.  If a significant association of TBS with vitamin D or benefit of vitamin D supplementation in reversing degraded TBS is observed, it might serve the purpose of a useful marker for vitamin D deficiency in apparently healthy subjects.

Objective, work Plan and Time Line

 The study objectives are

1: To assess the trabecular bone score with serum 25(OH)D D status  in apparently healthy subjects

2:  Randomized Control Trial : Effect of vitamin D and calcium supplementation on TBS in apparently healthy subjects with vitamin D deficiency and subnormal trabecular score  25(OH)D

Work Plan for objective 1:

A target of total of 800 subjects is planned in the study. The age range for inclusion would be 20 to 60 years. This age range has been planned because TBS software do not include subjects < 20 years of age. People with age > 60 years would be difficult regularly follow them at frequent interval and also because of their age related illness. The whole age range would be further stratified into eight groups at interval of five years (20-25 yr, 26-30 yr, 31-35 yr. 36-40 yr, 41-45 yr, 46-50 yr, 51-55 yr and 56-60 yr). A minimum of 100 subjects would be included in each of these groups. The target age range would comprise mix of outdoor and indoor workers. The selection of indoor and outdoor workers would be done bases on our past experience by using the people into following activities

Indoor age range 20-30: These would be drawn from a large pool of medical students engaged in graduate, post graduate and DM/PhD, Nursing and other paramedical professional courses at AIIMS and other surrounding Institute.  

Indoor age range 30-60: This would cover age range of 20-32 years. Subjects engaged as permanent staff as faculty, Nursing staff, clerical staff,  employees of the bank in the premises of AIIMS and healthy acquaintances of patients at AIIMS, willing to participate at AIIMS  would make for subjects in the age range 30-60 years

Outdoor age range 20-30: Street hawkers, petrol pump employees, gardeners and Traffic policemen would be contacted either individually or through the commanding authorities

Oudoor age range 30-60: People for this age range would again be drawn from the above professions. In case we are short of subjects in the age range of 50-60 years, a focussed effort concentrating on Traffic policemen and gardeners and hawkers would be made to enrich this group

All the study subjects would be  individually contacted and explained the purpose of the study. For subjects working in the organized sectors like vehicle fuel refill station, nursery, traffic police the manager of the fuel stations, supervisor of the nursery and traffic police commissioners would be contacted respectively with request to motivate their staff to come fasting next day at study centre. Multiple visits would made by the study authors on different days early in the morning to collect the necessary information. For cycle rickshaw workers, their union leaders would be contacted.   

Detailed history would be taken from each subject for their dietary habits, physical activity, sun exposure and history of any drug intake including calcium and vitamin D in the past six months. All subjects would be interviewed individually about the detail of their work, its duration, clothes worn, body surface area exposed to sunshine and duration of daily sunlight exposure. Body surface area exposed to sunshine would be assessed by the rule of nine and sun-index would be calculated as the product of sunshine exposure in h/week and fraction of body surface exposed (26). The daily dietary intake of calories, carbohydrate, protein, fat, calcium and phytin phosphorus (phytin-P) would be assessed by a trained dietician using a semi-quantitative food frequency questionnaire recording information on food groups and 40 food items commonly consumed by Indians. Following detailed history and brief examination, ten ml of blood would be drawn from each subject in fasting state for biochemical estimation. To ensure the fasting state, refreshment would be provided to all the study subjects

 The TBS would be measured at our DXA facility on the same visit. The BMD and TBS would be measured by dual energy X-ray absorptiometery (DXA), (Discovery A 84023, Hologic Inc., MA, USA) available at the Department of Endocrinology, AIIMS. BMD would be assessed at lumbar spine (L1–L4, antero-posterior), left hip and non-dominant forearm as per the guidelines of the International Society for Clinical Densitometry (ISCD). The precision would be measured by testing BMD three times in 15 study subjects using ISCD precision assessment tool. The standard deviation for each patient would be calculated, then the root mean square standard deviation for the group is calculated, which gives the precision error.  Fractured vertebrae will be excluded from the BMD analysis. BMD of the patients will be recorded in terms of absolute mineral content in g/cm2 at various sites.  For the assessment of trabecular bone score, data would be extracted from DXA images using TBS iNsight software (version 3.0.2.0, medimaps, Merignac, France).This software is calibrated with our DXA machine.

Serum total calcium, inorganic phosphorus, alkaline phosphatase and albumin would be measured by cobas-c111 analyzer (Roche, Mannheim, Germany). Serum 25(OH) D and iPTH  will be measured by chemiluminiscence assay (CLIA) using DiaSorin LIAISON® (DiaSorin, Inc., Stillwater, MN, USA). Serum 25(OH)D and iPTH assays would be performed in the Department of Cardiac Biochemistry, which has been participating in the “Randox International quality Assessment Scheme” (RIQAS) since 2014.  Serum 1,25(OH)2D would be measured by liquid chromatography and mass spectrometry. Vitamin D binding protein and bone specific alkaline phosphates would be measured by ELISA  

The maximum number of subjects would be recruited as follows (200 subjects in the first years, 400 subjects in the second year, 200 in the third years. In the fourth year, subject called for TBS would be those in whom repeat assessment would be required as per the requirement of study objective 2.

Work Plan for objective 2: Randomized Control Trial : Effect of vitamin D and calcium supplementation on TBS in apparently healthy subjects with vitamin D deficiency and subnormal trabecular score  25(OH)D.

Subjects: We would be recruiting those subjects who would be found to deficient in vitamin D with  serum 25(OH)D  < 20 ng/ml and TBS < 1.350.   These subjects would be drawn from the pool engaged in the objective 1 and with commitment to follow for one year.

The subjects would randomization based on preprinted list with variable block sizes  prepared for two intervention groups: (i) cholecalciferol sachets and calcium tablets. (ii) placebo sachets and placebo tablets. Interventions packets would be prepared in advance for 6 months according to the randomization numbers and arranged serially. Subjects would be given a serial number in a consecutive manner as per their entry into the study, and packets with matched serial number would be distributed to them. Subjects would be advised to take contents of the sachet with a glass of milk every week and one tablet per day with meals. Cholecalciferol sachets, calcium-carbonate tablets and lactose placebos matched with active supplements in colour, size, packing and taste would be prepared by the same manufacture as described in detail in our previous RCTs (6,8). Participants and assessors would be blinded to the intervention assigned to the participants, and codes of the supplementation packets were kept with an investigator who would not involved in intervention or analysis of the outcome. The supplementation schedule of cholecalciferol (60 000 IU/wk for 8 weeks followed by 60 000 IU/ every month for10 months) and calcium-carbonate (500 mg elemental calcium, one daily for 12 months) is based on the previous experience to bring serum 25(OH)D in the sufficient range (6,9). The intake of the first sachet of every month would be supervised by the investigators. A repeat assessment would be carried at the end of one year of supplementation and would include, all the baseline parameters including TBS and biochemical parameters.

Sample size estimation: The sample size estimation was based on the pilot TBS data obtained by us in 37 normal healthy subjects. The mean and SD of these controls was 1.3 ± 0.100. Assuming the subjects with subnormal group would have a TBS mean of 1.200 and a larger SD of  0.200 and with one year of intervention mean and SD changes to 1.300 ± 0.100, we would require  53 subjects in each of the two arms with alpha error of 0.05 and power of 0.90. With possibility of 20% drops rate we would therefore require 64 subjects in each study arm.  The sample size would be recalculated for more precision once we have the data of TBS score from the objective 1 in the first year for approximately 100 subjects.   

We expect the recruitment to be by over by the end 2 and half years. The final follow up would be available in the mid fourth year when the data would be finally analyzed. 

References:

1.      Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al; Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911-30.

2.      Goswami R, Gupta N, Goswami D, Marwaha RK, Tandon N, Kochupillai N. Prevalence and significance of low 25-hydroxyvitamin D concentrations in healthy subjects in Delhi. Am J Clin Nutr. 2000; 72:472-5.

3.      Goswami R, Kochupillai N, Gupta N, Goswami D, Singh N, Dudha A. Presence of 25(OH) D deficiency in a rural North Indian village despite abundant sunshine. J Assoc Physicians India. 2008; 56:755-7.

4.      Goswami R, Mishra SK, Kochupillai N. Prevalence & potential significance of vitamin D deficiency in Asian Indians. Indian J Med Res. 2008; 127:229-38.

5.      Theodoratou E, Tzoulaki I, Zgaga L, Ioannidis JP. Vitamin D and multiple health outcomes: umbrella review of systematic reviews and meta-analyses of observational studies and randomised trials. BMJ. 2014;1;348:g2035.

6.      Goswami R, Vatsa M, Sreenivas V, Singh U, Gupta N, Lakshmy R, Aggarwal S, Ganapathy A, Joshi P, Bhatia H. Skeletal muscle strength in young Asian Indian females after vitamin D and calcium supplementation: a double-blind randomized controlled clinical trial. J Clin Endocrinol Metab. 2012; 97:4709-16.

7.      Das M, Tomar N, Sreenivas V, Gupta N, Goswami R. Effect of vitamin D supplementation on cathelicidin, IFN-γ, IL-4 and Th1/Th2 transcription factors in young healthy females. Eur J Clin Nutr. 2014; 68:338-43.

8.      Saha S, Goswami R, Ramakrishnan L, Vishnubhatla S, Mahtab S, Kar P, Srinivasan S, Singh N, Singh U. Vitamin D and calcium supplementation, skeletal muscle strength and serum testosterone in young healthy adult males: Randomized control trial. Clin Endocrinol (Oxf). 2018; 88:217-226.

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