Vitamin D and bone mineral density status of healthy schoolchildren in northern India

¹ From the Department of Endocrinology and Thyroid Research, Institute of Nuclear Medicine and Allied Sciences, Delhi, India (RKM, RA, BS, and SS); the Departments of Endocrinology and Metabolism (NT, DHKR, and MAG) and Biostatistics (RS), All India Institute Medical Sciences, New Delhi, India; and the Department of Endocrinology, Defence Institute of Physiological Sciences, Delhi, India (RCS)

² Supported by a Task Project Grant from the Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, Ministry of Defence, Government of India.

³ Reprints not available. Address correspondence to RK Marwaha, Department of Endocrinology and Thyroid Research, Institute of Nuclear Medicine and Allied Sciences, Timarpur, New Delhi 110054, India. E-mail: marwaha_raman{at}hotmail.com.

ABSTRACT  
Background: Current data on the prevalence of vitamin D deficiency in India are scarce.

Objective: We assessed the calcium-vitamin D-parathyroid hormone axis in apparently healthy children from 2 different socioeconomic backgrounds in New Delhi, India.

Design: Clinical evaluation for evidence of vitamin D deficiency was carried out in 5137 apparently healthy schoolchildren, aged 10–18 y, attending lower (LSES) and upper (USES) socioeconomic status schools. Serum calcium, inorganic phosphorus, alkaline phosphatase, 25-hydroxyvitamin D [25(OH)D], and immunoreactive parathyroid hormone were measured in 760 children randomly selected from the larger cohort. Bone mineral density of the forearm and the calcaneum was measured in 555 children by using peripheral dual-energy X-ray absorptiometry.

Results: Clinical evidence of vitamin D deficiency was noted in 10.8% of the children. Children in the LSES group had a significantly (P < 0.01) lower 25(OH)D concentration (10.4 ± 0.4 ng/mL) than did those in the USES group (13.7 ± 0.4 ng/mL). Concentrations of 25(OH)D <9 ng/mL were seen in 35.7% of the children (42.3% in LSES; 27% in USES; P < 0.01). Boys had significantly (P = 0.004) higher 25(OH)D concentrations than did girls. There was a significant negative correlation between the mean serum immunoreactive parathyroid hormone and 25(OH) D concentrations (r = –0.202, P < 0.001). Mean forearm bone mineral density was significantly (P < 0.01) higher in the USES group than in the LSES group.

Conclusion: A high prevalence of clinical and biochemical hypovitaminosis D exists in apparently healthy schoolchildren in northern India.

Key Words: Rickets • 25-hydroxyvitamin D • 25(OH)D • immunoreactive parathyroid hormone • hypovitaminosis D • bone mineral density • children • adolescents • India

INTRODUCTION  
Vitamin D status has a profound effect on the growth and development of children and has major implications for adult bone health. Overt cases of vitamin D deficiency represent only the tip of an iceberg of vitamin D insufficiency (1). Whereas severe vitamin D deficiency, usually associated with 25-hydroxyvitamin D [25(OH)D] concentrations <5.0 ng/mL, results in rickets and osteomalacia (2), even less severe deficiency has been associated with numerous negative skeletal consequences, including secondary hyperparathyroidism, increased bone turnover, enhanced bone loss, and fracture risk (2, 3).

In assessing a person's vitamin D status, because 1,25-dihydroxyvitamin D [1,25(OH)₂D] can be normal, high, or low in vitamin D deficiency (4), the most commonly used and most sensitive index is 25(OH)D. Age, sex, pubertal status, latitude, season, race, and ethnicity influence serum concentrations of 25(OH)D (5–7).

In developing countries such as India, data on clinical and subclinical vitamin D–deficiency status are scarce. There have been scattered epidemiologic studies, but few have provided detailed clinical and biochemical information on the prevalence of hypovitaminosis D in the population. Three hospital-based studies conducted in adults showed that the mean serum 25(OH)D concentration is significantly lower than that in Western countries (8–10); the latest of these studies (10) also correlated vitamin D status and bone mineral density (BMD) measurements.

The objectives of the current study were to assess the prevalence of clinical and biochemical vitamin D deficiency in healthy children and adolescents aged 10–18 y during their period of most rapid growth, to compare the biochemical variables of the calcium–vitamin D axis between 2 socioeconomic groups, and to study the effect of hypovitaminosis D on BMD.

SUBJECTS AND METHODS  
The study was conducted in 5137 apparently healthy schoolchildren (aged 10–18 y) of both sexes in urban New Delhi, India, which is geographically located at 28°N. The children attended 2 state-run schools that catered to children of lower socioeconomic status (LSES) and 2 private schools that enroll children of upper socioeconomic status (USES). Socioeconomic stratification of the subjects was based on the type of school attended. Of the above 5137 subjects, 3089 (1079 boys, 2010 girls) from state-run schools made up the LSES group, and 2048 (968 boys, 1080 girls) from private schools made up the USES group. The subjects were further divided into 3 age groups: 10–12, 13–15, and 16–18 y old.

This entire cohort of 5137 children and adolescents underwent clinical examination and anthropometric assessment, including a recording of the stigmata of vitamin D deficiency. A sunlight exposure questionnaire was administered to each child. Clinical vitamin D deficiency was diagnosed if a subject had either genu varum (bowlegs) or genu valgum (knock-knees). Genu varum and genu valgum were defined by intercondylar and intermalleolar distances >6 cm and >8 cm, respectively (11). We also looked for other clinical signs of vitamin D deficiency, eg, frontal bossing and epiphyseal enlargement of the wrists, but these signs were not considered because they are more subjective. We excluded from the study the subjects with clinical features that were suggestive of any concurrent systemic illness; intake of any drug that interferes with bone mineral metabolism, such as glucocorticoids, anticonvulsants, and antituberculous drugs; or any other skeletal disease.

Of this large cohort, 760 children (430 from LSES and 330 from USES groups) selected by randomization from each class of the school underwent further laboratory assessment. Each class was divided into 4 groups according to the number of sections in the class, and all children from one randomly selected section (cluster) were called for blood sampling the next day. Blood samples were collected from subjects in the fasting state at 0800 without venostasis under basal conditions for estimation of total calcium, phosphorus, alkaline phosphatase (AP) activity, 25(OH)D, and immunoreactive parathyroid hormone (iPTH). The serum was centrifuged at 4°C for 15 min at 1200 x g and divided into 5 aliquots, which were refrigerated. Serum calcium, phosphorus, and AP were estimated on the same day, and the remaining aliquots were stored at –20°C until 25(OH)D and iPTH were estimated. Serum calcium (Randox Laboratory Ltd, Crumlin, UK) and inorganic phosphorus (Clonital; Ampli Medical SPA, Milan, Italy) were measured by colorimetric methods. Serum AP was measured by a liquid kinetic method (Clonital). The normal laboratory range in adults for total serum calcium is 2.02–2.60 mmol/L (8.10–10.04 mg/dL), and that for serum phosphorus is 0.81–1.55 mmol/L (2.5–4.8 mg/dL), according to the kit manufacturers. It is also known that the upper limit of serum phosphorous in mid-childhood is 5.8 mg/dL (12). The normal laboratory range for serum AP at 37°C is 100–275 IU/L in adults and 180–1200 IU/L in children before epiphyseal closure.

Dietary assessment of total energy, protein, carbohydrate, fat, calcium, and phytate was done in 349 subjects randomly selected from the cohort of 760 (171 from the LSES and 178 from the USES groups) through a 24-h recall of their food intake. The serum concentrations of 25(OH)D (reference range: 9.0–37.6 ng/mL) and iPTH (reference range: 13–66 pg/mL) were measured by radioimmunoassay and immunoradiometric assay (Diasorin, Stillwater, MN), respectively. The lowest concentration of vitamin D measurable by this kit, defined as the lowest quantity differentiated from zero at 2 SDs below the mean counts per min of the zero standard, is 1.5 ng/mL.

Distal forearm and calcaneal BMDs (g/cm²) were measured by using portable densitometry with dual-energy X-ray absorptiometry (DXA) in 555 children (PIXI-1.34; Lunar Corp, Madison, WI).

The parents of each participant were informed about the study protocol and gave written informed consent to their children's participation. All subjects whose parents gave consent to blood sampling underwent the biochemical evaluation. The study protocol was approved by the institutional ethics committee of the Institute of Nuclear Medicine and Allied Sciences.

Definition of hypovitaminosis D
We classified hypovitaminosis D on the basis of the measurement of serum 25(OH)D concentrations, as recommended by Lips (3). Concentrations of 10–20, 5–10, and < 5 ng 25(OH)D/mL were classified as mild, moderate, and severe hypovitaminosis D, respectively.

Statistical analysis
Data are presented as means ± SDs. The independent-sample t test was used to compare differences between the 2 socioeconomic groups for continuous variables. Data were also analyzed with a 3-factor analysis of variance (ANOVA) with age, sex, and SES as factors. A chi-square test was performed for categorical variables. Pearson rank tests were used for correlation analysis when necessary. P values < 0.05 were considered significant. We used SPSS statistical software (version 10.0; SPSS Inc, Chicago, IL) for the analysis. Because BMD is affected by body mass, the means used in 3-factor ANOVA were adjusted for height and weight for BMD only. The other biochemical and hormonal variables are unaffected by height, weight, and body mass, and thus the means of these variables were not adjusted for those factors.

RESULTS  
A total of 5137 children were examined, and a 10.8% prevalence of clinical evidence of vitamin D deficiency was noted in 556 children. Boys had a prevalence of 10.4% and girls had a prevalence of 11.1%, and there was no significant difference between the 2 groups (P = 0.46). The prevalence of genu valgum was 3.3% (boys: 2.4%; girls: 3.9%; P = <0.01) and that of genu varum was 7.5% (boys: 8.0%; girls: 7.2%; P = 0.39). The prevalence of clinical evidence of vitamin D deficiency was 11.6% in the LSES group and 9.7% in the USES group, values that were not significantly different (P = 0.07). All of the children had a daily sunlight exposure of 30 min and had exposure of a minimum of 30% of the body surface area. There was a significant difference in the mean calcium intake between the 2 groups (314 ± 194 mg in LSES and 713 ± 241 mg in USES; P < 0.01), but there was no significant difference in the intake of phytate.

A total of 760 children were assessed biochemically—430 from the LSES group (167 males; 263 females) and 330 from the USES group (158 males; 172 females). The mean BMI of children in the 2 groups was significantly (P < 0.01) different (LSES group: 17.1 ± 2.9; USES group: 21.0 ± 4.7). The unadjusted mean concentrations of calcium, phosphorus, AP, iPTH, and 25(OH)D in children in the LSES and USES groups are shown in Table 1. A sequential analysis of each of these variables by 3-factor ANOVA with interaction is given below.

View this table:
TABLE 1. Comparison of unadjusted means of variables in the 2 socioeconomic groups¹

 
Calcium
The overall unadjusted mean for serum calcium was 9.4 ± 0.97 mg/dL. There were no significant interactions or main effects of sex, age, and SES on mean serum calcium concentration.

Phosphorus
Adjusted mean ± SE values of serum phosphorus for LSES and USES were 3.9 ± 0.04 and 4.2 ± 0.5 mg/dL, respectively (P < 0.01). In the 2 younger categories, the mean phosphorus concentration was higher in the USES than in the LSES group. However, in the oldest category, the mean phosphorus concentration was lower in the USES than in the LSES group. Mean serum phosphorus concentration was significantly higher in the males than in the females in both socioeconomic groups.

Alkaline phosphatase
Adjusted mean ± SE values of serum AP in the LSES and USES groups were 387.1 ± 8.9 and 299.2 ± 10.3 IU/L, respectively (P < 0.01). Among males, a trend for higher AP was seen in the LSES group; this trend attained significance only in the 16–18- y-old category. There was a significant positive correlation of mean serum AP with iPTH (r = 0.330, P < 0.01).

Immunoreactive parathyroid hormone
Adjusted mean ± SE values of serum iPTH for LSES and USES were 42.8 ± 1.6 and 23.5 ± 1.9 pg/mL, respectively (P < 0.01). There was a significant negative correlation of iPTH with 25(OH)D (r = –0.202, P < 0.01) (Figure 1).

View larger version (15K):
FIGURE 1.. Relation between serum immunoreactive parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] concentrations by category (n = 760). The number of subjects in each category of 25(OH)D depicted in the figure is as follows: 5 ng/mL, n = 86; 5.1–10 ng/mL, n = 276; 10.1–15 ng/mL, n = 224; 15.1–20 ng/mL, n = 107; 20.1–25 ng/mL, n = 33; and >25 ng/mL, n = 34. r = –0.202, P < 0.001.

 
25-Hydroxyvitamin D
The unadjusted mean serum concentration of 25(OH)D for the entire group was 11.8 ± 7.2 ng/mL. Adjusted mean ± SE values of serum 25(OH)D for LSES and USES were 10.4 ± 0.4 and 13.7 ± 0.4 ng/mL, respectively (P < 0.01). Age, sex, and SES independently influenced the variations in 25(OH)D concentrations. Males had significantly higher mean serum concentrations than did females (P = 0.004). Two hundred seventy-one children (35.7%) had serum concentrations <9 ng/mL—ie, below the normal range given by the manufacturer. A value <9 ng/mL was found in children in the LSES group significantly more often (42.3%) than in those in the USES group (27%; P < 0.01) and among females more often (41.6%) than among males (27.4%; P = 0.01). According to the Lips classification (3), hypovitaminosis D was seen in 92.6% of the LSES group (severe: 11.2%; moderate: 39.5%; and mild: 42.1%) and in 84.9% of the USES group (severe: 4.9%; moderate: 25.5%; and mild: 57.6%).

BMD evaluation was done at the forearm and the calcaneum in 555 children. The clinical, anthropometric, biochemical, and hormonal variables in children who underwent BMD assessment did not differ significantly from those in children who did not. The mean BMD measurements (g/cm²) at forearm and calcaneum are reported in Table 2. The 3-factor interaction was significant for the forearm, but, at the calcaneum, only the main effect of age was significant (P < 0.01). There was no significant correlation between BMD measurements and the mean serum concentration of either iPTH or 25(OH)D.

View this table:
TABLE 2. Comparison of unadjusted means of bone mineral density (BMD) in the 2 socioeconomic groups¹

 

DISCUSSION  
Metabolic bone disorders secondary to vitamin D deficiency continue to be prevalent in the Indian subcontinent, as documented by hospital-based studies. To the best of our knowledge, there are no recent, large, community-based studies that specifically evaluated the prevalence of hypovitaminosis D in children and adolescents in India. Hence, the current study was planned to investigate the extent of the problem in 2 socioeconomic groups by using clinical, biochemical, and hormonal variables and bone densitometry.

The earliest published description of adolescent rickets in India appeared in 1925 (13). In a study reported in 1997, the prevalence of clinical evidence of vitamin D deficiency in 5–15-y-old children was shown to be 0.19%; however, objective diagnostic criteria were not mentioned (14). In Indian immigrants in the United Kingdom during the 1960s and 1970s, the prevalence of clinical vitamin D deficiency in children and adolescents was shown to be 5–30% (15–18), whereas, in studies using biochemical and radiological variables, the prevalence was 12.5–66% (1, 19, 20). Later studies showed that the incidence of rickets had decreased in the immigrant population (21, 22), but so far no evidence of reduction in rickets in India has been reported. In children of Indian parentage who were born in and are residing in South Africa, the prevalence of knock knees and bow legs with gaps of 2.5 cm, reported in 1975, was 6.1–19.4% (23).

Adolescents are prone to vitamin D deficiency because of the greater mineral demands of their growing skeletons (16, 17, 20, 24, 25). Studies that provided biochemical documentation of vitamin D deficiency by estimating 25(OH)D in adolescents have renewed interest in this particular age group (26–29). The current study found clinical evidence of vitamin D deficiency in 10.8% of apparently healthy adolescents in New Delhi, India, and no significant difference between the upper and lower socioeconomic groups. In a similar age group, symptomatic rickets was observed in 68 per 100,000 child-years in Saudi Arabia (26) and in 9.4% of subjects in China (28).

A comparison of serum vitamin D data with other studies may not be entirely appropriate, given the fact that different studies were conducted in different seasons and using different assays. Nonetheless, according to the Lips classification (3), severe hypovitaminosis D (<5 ng/mL) was seen in 8.6% of our study population, in 23.5% of Finnish adolescents (25), and in 45.2% of Chinese adolescents in winter (28). In the latter study, severe hypovitaminosis D was present in only 6.7% of the subjects when they were evaluated in summer. In other studies from Finland, using cutoffs of 8–10 ng/mL, the prevalence of hypovitaminosis D was 13.5% (27, 29), which compares with 37% of children in the current study who had serum 25(OH)D concentrations <9 ng/mL (lower limit of manufacturer's normal range).

The mean serum concentration of 25(OH)D in the current study is 11.8 ± 7.2 ng/mL. Other studies have also noted low serum 25(OH)D concentrations among adults of Indian origin in both India and the United Kingdom (8, 9, 30). The mean 25(OH)D concentration in adolescents in the current study is significantly lower than that reported in studies from Europe (6, 29, 31) and Brazil (32) and marginally higher than that reported from China (28).

Our study found that the LSES group adolescents had significantly lower mean 25(OH)D concentrations than did the USES group adolescents. The only other study that compared low and high socioeconomic status groups did not find any significance difference in mean vitamin D concentration between the 2 groups (32). However, the difference that we found is further supported by the observation that LSES group children also had higher iPTH, higher AP, and lower serum phosphorus concentrations than did USES group children. Because serum calcium concentration and sunlight exposure did not differ significantly between the 2 groups and because dietary calcium intake was significantly lower in the LSES group than in the USES group, nutrition may play an important role, as was reported earlier (33, 34). In addition, the lower serum phosphorus concentration could be correlated with the higher concentrations of iPTH in the LSES group.

There was a negative correlation between 25(OH)D and iPTH (r = –0.202, P < 0.001), which is in agreement with the findings of other studies (6, 35). Two hundred seventy-one children (35.7%) had vitamin D concentrations below the normal range given by the manufacturer. Only 28 (10.3%) of these 271 subjects had iPTH concentrations above normal. In another study, the serum iPTH concentration was elevated in 37.5% adolescent girls with low vitamin D concentrations (29). Several studies in adults have investigated the threshold at which serum vitamin D induces an increase in iPTH concentrations, and values ranging from 20 to 38 ng/mL were reported (36-38). It is important to emphasize that the rise in iPTH seen in the current study was still within the normal range and that only when 25(OH)D concentrations fell below 5 ng/mL did iPTH values rise to or exceed the upper limit of normal. To the best of our knowledge, the current study is the first to investigate the relation between vitamin D and iPTH in apparently healthy children from different socioeconomic strata. The limited number of children with serum 25(OH)D concentrations >25 ng/mL prevents our arriving at a relation between vitamin D and PTH at higher values of vitamin D. The reasons for the lack of elevation of iPTH above the upper limit of normal, despite hypovitaminosis D, could be that, first, sufficient 25(OH)D is being converted to 1,25(OH)₂D for maintenance of calcium homeostasis and, second, that prolonged exposure to low vitamin D concentrations and the poor nutritional status in these children may have lowered the threshold for iPTH release.

Children in the LSES group had significantly lower BMD values at the forearm than did those in the USES group. This difference could be due to poor overall nutrition, as evidenced by low BMIs, low dietary calcium intakes (39), low serum 25(OH)D concentrations, and secondary hyperparathyroidism (40). No significant correlation was seen between BMD and the 25(OH)D concentration and serum calcium concentrations in the current study, which is in agreement with other studies (29, 39, 41, 42). However, some studies in different age groups showed a significant correlation (10, 43).

We conclude that there is a high prevalence of clinical and biochemical hypovitaminosis D in apparently healthy schoolchildren in India. The observation that children from low socioeconomic backgrounds have significantly higher prevalences of vitamin D deficiency and low BMD suggests that nutrition plays an important role in the causation of hypovitaminosis D.

ACKNOWLEDGMENTS  
RKM and NT contributed equally to the study and can both be considered as first author. RKM, NT, DKHR, RA, BS, and MG were responsible for the collection of data; RKM, NT, DHKR, and RS were responsible for the analysis of data; RKM, NT, and DHKR were responsible for writing the manuscript; RA, BS, and MG provided significant advice regarding conduct of study; and RCS and SS were responsible for the laboratory assays. None of the authors had any person or financial conflicts of interest.

REFERENCES  

1. Ford JA, McIntosh WB, Butterfield R, et al. Clinical and subclinical vitamin D deficiency in Bradford children. Arch Dis Child 1976;51:939–43.
2. Parfitt AM. Osteomalacia and related disorders In: Avioli LV, Krane SM, eds. Metabolic bone disease. 3rd ed. San Diego, CA: Academic Press, 1998:345–86.
3. Lips P. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr Rev 2001;22:477–501.
4. Hollis BW. Assessment of vitamin D nutritional and hormonal status: what to measure and how to do it. Calcif Tissue Int 1996;58:4–5.
5. Adams JS, Hollis BW. Vitamin D synthesis, metabolism and clinical measurement In: Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2002:157–74.
6. Guillemant J, Cabrol S, Allemandou A, Peres G, Guillemant S. Vitamin D-dependent seasonal variation of PTH in growing male adolescents. Bone 1995;17:513–6.
7. Stryd RP, Gilbertson TJ, Brunden MN. A seasonal variation study of 25-hydroxyvitamin D₃ serum levels in normal humans. J Clin Endocrinol Metab 1979;48:771–5.
8. Harinarayan CV, Gupta N, Kochupillai N. Vitamin D status in primary hyperparathyriodism in India. Clin Endocrinol (Oxf) 1995;43:351–8.
9. 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.
10. Arya V, Bhambri R, Godbole MM, Mithal A. Vitamin D status and its relationship with bone mineral density in healthy Asian Indians. Osteoporos Int 2004;15:56–61.
11. Solomon L, Warwick D, Nayagam S, eds. Apley's system of orthopedics and fractures. 8th ed. London, United Kingdom: Arnold, 2001:449–84.
12. Portale AA. Blood calcium, phosphorus and magnesium. In: Favus MJ, ed. Primer on the metabolic bone diseases and disorders of mineral metabolism. 2nd ed. New York: Raven Press, 1993:87–90.
13. Stapleton G. Late rickets and osteomalacia in Delhi. Lancet 1925;2:1119–23.
14. Awate RV, Ketkar YA, Somaiya PA. Prevalence of nutritional deficiency disorders among rural primary school children (5–15 years). J Indian Med Assoc 1997;95:410–1, 415.
15. Goel KM, Sweet EM, Logan RW, Warren JM, Arneil GC, Shanks RA. Florid and subclinical rickets among immigrant children in Glasgow. Lancet 1976;1:1141–5.
16. Arneil GC. Nutritional rickets in children in Glasgow. Proc Nutr Soc 1975;34:101–9.
17. Dunnigan MG, Paton JPJ, Haase S, McNicol GW, Gardner MD, Smith CM. Late rickets and osteomalacia in the Pakistani community in Glasgow. Scott Med J 1962;7:159–67.
18. Holmes AM, Enoch BA, Taylor JL, Jones ME. Occult rickets and osteomalacia amongst the Asian immigrant population. Q J Med 1973;42:125–49.
19. Dunnigan MG, McIntosh WB, Sutherland GR, Gardee R, Glekin B, Ford JA. Policy for prevention of Asian rickets in Britain: a preliminary assessment of the Glasgow rickets campaign. Br Med J 1981;282:357–60.
20. Ford JA, Colhoun EM, McIntosh WB, Dunnigan MG. Rickets and osteomalacia in the Glasgow Pakistani community, 1961–1971. Br Med J 1972;2:677–80.
21. Goel KM, Sweet EM, Campbell S, Attenburrow A, Logan RW, Arneil GC. Reduced prevalence of rickets in Asian children in Glasgow. Lancet 1981;2:405–6.
22. Stephens WP, Klimiuk PS, Warrington S, Taylor JL, Berry JL, Mawer EB. Observations on the natural history of vitamin D deficiency amongst Asian immigrants. Q J Med 1982;51:171–88.
23. Richardson BD, Walker ARP. Prevalences of leg and chest abnormalities in four South African schoolchild populations with special reference to vitamin D status. Postgrad Med J 1975;51:22–9.
24. Moncrieff MW, Lunt HRW, Arthur LJH. Nutritional rickets at puberty. Arch Dis Child 1973;48:221–4.
25. Ala- Houhala M, Parviainen MT, Pyykko K, Visakorpi JK. Serum 25-hydroxyvitamin D levels in Finnish children aged 2 to 17 years. Acta Pediatr Scand 1984;73:232–6.
26. Narchi H, El Jamil M, Kulaylat N. Symptomatic rickets in adolescence. Arch Dis Child 2001;84:501–3.
27. Lehtonen-Veromaa M, Mottonen T, Irjala K, et al. Vitamin D intake is low and hypovitaminosis D common in healthy 9- to 15-year-old Finnish girls. Eur J Clin Nutr 1999;53:746–75.
28. Du X, Greenfield H, Fraser DR, Ge K, Trube A, Wang Y. Vitamin D deficiency and associated factors in adolescent girls in Beijing. Am J Clin Nutr 2001;74:494–500.
29. Outila TA, Kärkkäinen MUM, Lamberg-Allardt CJE. Vitamin D status affects serum parathyroid hormone concentrations during winter in female adolescents: associations with forearm bone mineral density. Am J Clin Nutr 2001;74:206–10.
30. Serhan E, Newton P, Ali HA, Walford S, Singh BM. Prevalence of hypovitaminosis D in Indo-Asian patients attending a rheumatology clinic. Bone 1999;25:609–11.
31. Stamp TCB, Round JM. Seasonal changes in human plasma levels of 25-hydroxyvitamin D. Nature 1974;247:563–5.
32. Linhares ER, Jones DA, Round JM, Edwards RHT. Effect of nutrition on vitamin D status: studies on healthy and poorly nourished Brazilian children. Am J Clin Nutr 1984;39:625–30.
33. Raghuramulu N, Reddy V. Serum 25-hydroxy-vitamin D levels in malnourished children with rickets. Arch Dis Child 1980;55:285–7.
34. Raghuramulu N. Etiology of rickets in India. Indian Pediatr 1987;54:521–7.
35. Guillemant J, Taupin P, Le HT, et al. Vitamin D status during puberty in French healthy adolescents. Osteoporos Int 1999;10:222–5.
36. Chapuy MC, Preziosi P, Maamer M, et al. Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int 1997;7:439–43.
37. Krall EA, Sahyoun N, Tannnebaum S, Dallal GE, Dawson-Hughes B. Effect of vitamin D intake on seasonal variations in parathyroid hormone secretion in postmenopausal women. N Engl J Med 1989;321:1777–83.
38. Malabanan A, Veronikis IE, Holick MF. Redefining vitamin D deficiency. Lancet 1998;351:805–6.
39. Oliveri MB, Wittich A, Mautalen C, Chaperon A, Kizlansky A. Peripheral bone mass is not affected by winter vitamin D deficiency in children and young adults from Ushuaia. Calcif Tissue Int 2000;67:220–4.
40. Bonofiglio D, Maggiolini M, Catalano S, et al. Parathyroid hormone is elevated but bone markers and density are normal in young female subjects who consume inadequate dietary calcium. Br J Nutr 2000;84:111–6.
41. Meier DE, Luckey MM, Wallenstein S, Clemens TL, Orwoll ES, Waslien CI. Calcium, vitamin D and parathyroid hormone status in young white and black women: association with racial differences in bone mass. J Clin Endocrinol Metab 1991;72:703–10.
42. Kristinsson IO, Valdimarsson O, Sigurdsson G, Franzson L, Olafsson I, Steingrimsdottir L. Serum 25-hydroxy vitamin D levels and bone mineral density in 16–20-year-old girls: lack of association. J Int Med 1998;243:381–8.
43. Perry HM III, Horowitz M, Morley JE, et al. Aging and bone metabolism in African American and Caucasian women. J Clin Endocrinol Metab 1996;81:1108–17.