India is situated in a tropical and subtropical region where there is abundance of sunlight. However, vitamin D deficiency has been documented commonly in both urban and rural populations of Indian neonates, infants, adolescents (especially girls) and pregnant women(Reference Sachan, Gupta and Das1–Reference Marwaha, Tandon and Reddy4). In urban Indians, indoor lifestyle and skin pigment are risk factors. In India, food is not fortified with vitamin D or Ca. Overt rickets, with softening and bending of bones, is a common problem encountered in the paediatric clinics in our country. However, only children with the most severe vitamin D and Ca deficiencies manifest overt rickets, with a larger pool of children with moderate deficiency at the bottom of the iceberg(Reference Munns, Shaw and Kiely5).
India is a large country, with a long coastline in the south and high mountains in the north. Geography and climatic conditions are diverse in this subcontinent, as are food habits. The literature documenting high prevalence of vitamin D deficiency in India is largely from inland cities or rural areas north of the Tropic of Cancer(Reference Sachan, Gupta and Das1–Reference Marwaha, Tandon and Reddy4). Particularly, literature is sparse in the toddler and pre-adolescent age group. The southern state of Kerala lies between latitudes 8°N and 12°N, entirely situated in the tropics, with abundant sunlight. There is heavy cloud cover during the South-West monsoon season from June to August, and some cloud cover also during the North-East monsoon in September and October, but the weather is warm throughout the year(6). Being a coastal area, fatty fish like sardine are available in all seasons and are relatively cheap. The majority of Keralites are fish eaters. The present study aimed to find out whether these factors protect children in Kerala from developing vitamin D deficiency.
Methods
Participants
Consecutive children, between 6 months and 12 years of age, attending the paediatric outpatient department of one unit with trivial illness like upper respiratory tract infection, first episode of urinary infection and conjunctivitis were enrolled for the study after getting informed written consent from parents. Children suffering from severe acute illness requiring hospitalization and children having any chronic illness like bronchial asthma, heart disease and renal disease were excluded from the study. Children with clinical rickets and those who had received any vitamin D supplementation, antitubercular or anticonvulsant therapy in the past year were also excluded. The study protocol was approved by the ethics committee of the Government Medical College, Kozhikode. Parents of children gave written informed consent.
Data collection: diet and sunlight exposure
A 7 d diet recall method was used for calculating dietary Ca and vitamin D intakes. The staple diet in this region is rice and fish. Ragi, a cereal rich in both Ca and Fe, is a complementary weaning food(Reference Longvah, Ananthan and Bhaskarachary7). History of clothing pattern in different seasons and activity pattern in different months was enquired about. Sun exposure (in minutes per day) from 10.00 to 16.00 hours was calculated based on typical activity pattern and clothing habits.
Blood samples and laboratory analyses
Serum Ca, inorganic phosphate and alkaline phosphatase were analysed on the same day (Erba EM 360 auto-analyser; Transasia Bio-Medicals Limited, Mumbai, Inda) and plasma was stored at −40°C until transportation on dry ice and assay for parathyroid hormone (PTH) and 25-hydroxyvitaminD (25(OH)D) by immunoradiometric assay and RIA, respectively (DiaSorin, Inc., Stillwater, MN, USA). The sensitivity of the assay for 25(OH)D was 3·75 nmol/l; the inter-assay CV in our laboratory was 14·1% and the intra-assay CV was 8·6% at 31 nmol/l. The sensitivity of the PTH assay was 0·64 pmol/l, with inter-assay CV of 10·5%.
Statistical analysis
Analysis was done using the statistical software package PASW Statistics version 18. Sample size of 380 was estimated for obtaining a true prevalence of vitaminD insufficiency within 95% confidence limits, with 80% power, and was calculated presuming a prevalence of 50%, lower than that reported (approximately 75%) from northern parts of the country. The Kruskal–Wallis test was for used comparison of medians of multiple groups, the Mann–Whitney U test for comparison of medians between pairs of groups, the Spearman correlation coefficient for correlation, and linear regression was done to estimate the adjusted association of each individual factor with plasma 25(OH)D. Variables that were correlated with plasma 25(OH)D at P <0·1, or were clinically relevant factors, or were significantly different on comparison of means were included in the linear regression models. Collinearity between the variables in the regression models were checked using the variance inflation factor. Since variance inflation factors for all variables were less than 4, and models excluding any of the collinear variables like fish, vitamin D and Ca intakes did not change the outcome, we used a model with all the relevant variables. P <0·05 was considered statistically significant.
Results
A total of 296 consecutive children with trivial illness presenting to the outpatient department, and who gave consent, were enrolled in the study. Baseline characteristics, including median plasma 25(OH)D in the total population and in different subgroups, are shown in Table 1. Plasma 25(OH)D was in the sufficient range (>50 nmol/l) in 160 children (54·1%), while 103 (34·8%) had insufficient levels (30–50 nmol/l) and thirty-three children (11·1%) were deficient (<30 nmol/l). Gender and egg and milk consumption patterns were not associated with plasma 25(OH)D. Although median plasma 25(OH)D was not different between toddler, mid-childhood and adolescent age groups, age was significantly negatively correlated with plasma 25(OH)D (Table 2). Significantly higher plasma 25(OH)D was observed in those consuming fish daily in comparison with those who consumed it less often than daily or never. Plasma 25(OH)D was significantly higher (median 58·7 nmol/l) among children whose blood sample was collected during the dry hot summer months (March–May) and was similar in all other seasons (median 46·6 nmol/l in December–February (dry season), 45·6 nmol/l in June–August (monsoon season) and 38·4 nmol/l in September–November (retreating monsoon season)).
Baseline characteristics of the study group of children aged 6 months–12 years (n 296), Kerala, southern India, October 2015–March 2017
25(OH)D, 25-hydroxyvitamin D; IQR, interquartile range.
Summary of quantitative measures and their correlation with plasma 25-hydroxyvitaminD (25(OH)D) among children aged 6 months–12 years (n 296), Kerala, southern India, October 2015–March 2017
PTH, parathyroid hormone.
Mean daily Ca intake was 166 (sd 147) mg and mean daily vitamin D intake was 3·8 (sd 2·5) µg (153 (sd 103) IU). Of the study children, 251 (84·7%) were exposed to sunlight for at least 60 min daily, with head, forearms, hands and legs (below knee) exposed to sunlight in the majority. Elevated alkaline phosphatase (>450 IU/l) was noted in fifteen (5·1%) children. As shown in Table 2, plasma 25(OH)D showed a significant positive correlation with Ca intake, vitamin D intake, serum P and duration of sun exposure, and a negative correlation with PTH. Ca and vitamin D intakes were strongly correlated with each other (r = 0·73, P <0·001), an expected result because the commonly eaten fish (sardine, mackerel, pomphret) are rich in vitamin D as well as Ca content. A linear regression model which included season of blood sampling, fish intake, gender, age, Ca intake, vitamin D intake and minutes of sun exposure indicated that summer season (March–May), lower age, eating fish daily and higher Ca intake were independently associated with plasma 25(OH)D (Table 3).
Linear regression results for the adjusted association of study variables with plasma 25-hydroxyvitamin D among children aged 6 months–12 years (n 296), Kerala, southern India, October 2015–March 2017
Model P <0·001, model adjusted R 2 = 0·15.
Discussion
Our data show that the mean plasma 25(OH)D of children in north Kerala is in the normal range (>50 nmol/l) and 11% have plasma 25(OH)D in the deficient range (<30 nmol/l). These values indicate better vitamin D status than found in most Indian studies conducted in various geographical regions (Table 4).
Vitamin D status in children: comparison of studies in Indian literature
SES, socio-economic status; N/A, data not available.
Tiwari and Puliyel used an ELISA (ImmunoDiagnostik), Basu et al. used a chemiluminescence assay (Roche Diagnostic) and the other studies used the DiaSorin RIA. For conversion, 1 ng/ml = 2·5 nmol/l.
* Definition of vitamin D deficiency used by Tiwari and Puliyel is 25(OH)D <35 nmol/l.
† Definition of severe vitamin D deficiency used by Sahu et al. is 25(OH)D <25 nmol/l.
‡ Definition of vitamin D deficiency used by Harinarayanan et al. is 25(OH)D <20·0 ng/ml (50·0 nmol/l)
§ Definition of moderate deficiency used by Marwaha et al. is serum 25(OH)D <25·0 nmol/l.
║ Values are medians.
Although the majority of these studies are from northern and central India, studies from regions closer to the equator, such as Tirupathi and nearby villages(Reference Harinarayan, Ramalakshmi and Venkataprasad8) and Chennai(Reference Balasubramanian, Shivbalan and Saravanakumar9), have also reported severe vitamin D deficiency to be a relevant public health and clinical problem. Despite the populations studied by Harinarayan et al. (Reference Harinarayan, Ramalakshmi and Venkataprasad8) being mainly adult, it was a large study with detailed description about the dietary pattern. The authors reported mean serum 25(OH)D of 38·9 nmol/l in urban boys and 42·4 nmol/l in rural boys. Vitamin D intake in their population was very poor. The study from Chennai reported vitamin D deficiency to be the commonest single cause of hypocalcaemic seizures in infancy, reflecting poor maternal vitamin D nutrition(Reference Balasubramanian, Shivbalan and Saravanakumar9). Chennai is a coastal city at latitude 13·1°N. However, pregnant women in India, and their infants, are among the most vulnerable to vitamin D deficiency due to modest clothing pattern and increased physiological demand(Reference Sachan, Gupta and Das1, Reference Seth, Marwaha and Singla2). The authors did not comment on the fish intake of the mothers of the infants in their study.
International studies from geographical regions situated close to the equator like Kerala also report vitamin D ‘deficiency’. A study from Sri Lanka of 340 pre-school children aged 2–5 years showed mean serum 25(OH)D to be 57·5 nmol/l, with 5% of children having serum 25(OH)D <25 nmol/l(Reference Eshani, Surekha and Chrishantha10). With regard to the cut-off for the definition of vitamin D deficiency, a recent global consensus statement has recommended that, for children, deficiency be defined as serum 25(OH)D <30 nmol/l and insufficiency as serum 25(OH)D between 30 and 50 nmol/l(Reference Munns, Shaw and Kiely5). It is in this context that recent studies in children from equatorial countries should be viewed. The four-nation SEANUTS (South East Asian Nutrition Survey) reports also mention widespread vitamin D ‘deficiency’, with about 40% of children aged between 6 months and 12 years having serum 25(OH)D <50 nmol/l(Reference Sandjaja, Basuki and Heryudarini11–Reference Poh, Ng and Haslinda14). However, mean serum 25(OH)D was above 50 nmol/l in all four countries. Like us, all studies noted highest serum 25(OH)D in the youngest age groups. The prevalence of serum 25(OH)D <30 nmol/l and the availability of serum PTH would have added value to the interpretation of these studies.
Vitamin D intake would be expected to be generous in all these countries, fish and seafood forming an important part of their diet. Intake was calculated only in the Malaysian SEANUTS study(Reference Poh, Ng and Haslinda14). Diet contributed about 5–7 µg vitamin D daily, variably in different age groups. This finding is similar to ours. Fatty fish like sardine (mathi) and mackerel (aila) are rich sources of vitamin D(Reference Longvah, Ananthan and Bhaskarachary7) and are part of the staple diet in Kerala. Furthermore, fish is also a rich source of dietary Ca in this region.
Even as close to the equator as 8°N to 12°N, there is a significant seasonal variation in serum 25(OH)D. This is possibly due to cloud cover during the monsoon season, which blocks UV rays from reaching the earth’s surface. Over Kozhikode, the India meteorological department describes the UV index to be 11–12 in January to March and 6–7 in June and July(15). This is an important observation which should be kept in mind while planning studies or supplementation based on vitamin D levels. Indeed, season was the strongest predictor of serum 25(OH)D in our regression analysis. Recent studies from countries close to the equator such as Cambodia(Reference Smith, Wimalawansa and Laillou16) or the nine-nation Mesoamerican study have commented on the low prevalence of vitamin D deficiency(Reference Robinson, Ramivez-zea and Roman17). Documenting serum 25(OH)D <30 nmol/l only in 2·9% of 781 Cambodian children, the authors attributed the good status to outdoor activity and sun exposure rather than to dietary intake. In the latter study, although by no means representative of the population due to small sample sizes, mean serum 25(OH)D in 287 children was as high as 79·5 nmol/l, with just one child having serum 25(OH)D <30 nmol/l and 3·6% with serum 25(OH)D <50 nmol/l(Reference Robinson, Ramivez-zea and Roman17). Although fortification of milk is prevalent in some of these countries, thereby providing dietary vitamin D to children, the adult population with the greatest outdoor activity in the same study had the highest serum 25(OH)D.
We have compared vitamin D status of children from various studies in India(Reference Sahu, Bhatia and Anjoo3, Reference Marwaha, Tandon and Reddy4, Reference Harinarayan, Ramalakshmi and Venkataprasad8–Reference Basu, Gupta and Mitra22) (Table 4). Other Indian studies have reported a steep increase in the prevalence of vitamin D deficiency during adolescence, noticeably in girls, with increased physiological demands compounding poor outdoor activity and modest clothing. In studies from Pune in central India and Barabanki in northern India, 70% of adolescent girls were found to have vitamin D deficiency(Reference Sahu, Bhatia and Anjoo3, Reference Agarwal, Mughal and Upadhyay18). We had only a small number of adolescents in our study and could not examine this issue.
Our study has some limitations. First, it is a hospital-based study and even though the children enrolled had only trivial illness, in the true sense they are not healthy subjects. Second, the same children were not sampled in all seasons of the year. Third, we did not meet our original sample size estimation; however, with a sample size of 296, we obtained a prevalence of vitamin D insufficiency of 46%, with a relatively narrow precision of 5·6%. Finally, the assay used for serum 25(OH)D estimation was not the gold-standard liquid chromatography–mass spectrometry technique, which could have introduced the confounding factor of assay drift. We tried to minimize this problem by performing all assays within a short period of time using a single lot number. Notwithstanding these limitations, we believe we can conclude that vitamin D deficiency in the pre-adolescent age is not as common or severe a public health problem in Kerala as it is in most other reports from the northern inland regions of India. There is significant seasonal variation in plasma 25(OH)D. Our study demonstrates that advantage can be taken of the rich natural resource of sunshine availability in some seasons of the year, in some parts of our country, to improve vitamin D nutritional status, before resorting to pharmacological supplements. Our results may also have implications for policy makers considering food fortification, drawing attention to the geographical diversity in our large country.
Acknowledgements
Acknowledgements: The authors acknowledge the technical assistance of P.K. Awasthi for laboratory analysis and of Arjun Singh, Divya Shajith, Rajimol Jayaraj and Sudheena Sudhi for data collection and handling and transportation of samples. Financial support: This work was supported by the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India (M.V., grant number BT/PR7429/SPD/11/1464/2013). DBT had no role in the design, analysis or writing of this article. Conflict of interest: None. Authorship: V.K. conceptualized the study, collected, collated and analysed the data, and wrote the manuscript. V.B. conceptualized the study, analysed the data and edited the manuscript. B.G. helped in the formation of the study plan and did the statistical analysis. V.K. will act as a guarantor for this paper. Ethics of human subject participation: This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all the procedures involving patients were approved by the institutional ethics committee of Government Medical College, Kozhikode, Kerala. Written informed consent was obtained from parents of all patients.
