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1 ORIGINAL COMMUNICATION (2002) 56, ß 2002 Nature Publishing Group All rights reserved /02 $ The effect of conventional vitamin D 2 supplementation on serum 25(OH)D concentration is weak among peripubertal Finnish girls: a 3-y prospective study M Lehtonen-Veromaa 1,TMöttönen 2 *, I Nuotio 2, K Irjala 3 and J Viikari 2 1 Paavo Nurmi Centre, Sport and Exercise Medicine Unit, Department of Physiology, University of Turku, Turku, Finland; 2 Department of Medicine, Turku University Central Hospital, Turku, Finland; and 3 Central Laboratory, Turku University Central Hospital, Turku, Finland Objectives: To study the effect of vitamin D supplementation and the impact of summer season on serum 25-hydroxyvitamin D (S-25(OH)D) in Finnish 9 15-y-old girls. Design: Three-year follow-up study with vitamin D 2 supplementation using D 2 10 mg daily from October to January for the first and from October to February for the second winter as well as 20 mg daily from October to March for the third winter. Setting: Paavo Nurmi Centre, University of Turku, Turku, Finland. Subjects: A total of 171 female volunteers aged 9 15 y. Methods: Vitamin D and calcium intakes were estimated by a semi-quantitative food frequency questionnaire (FFQ). S-25(OH)D was measured by radioimmunoassay. Results: The median daily dietary intakes of vitamin D and calcium were 3.8 mg (interquartile range (IQR) ) and 1451 mg (IQR ), respectively, over 3 y. The prevalence of severe hypovitaminosis D (S-25(OH)D < 20 nmol=l) was 14% and of moderate hypovitaminosis D (20 nmol=l S-25(OH)D 37.5 nmol=l) 75% at baseline in winter. None of the participants had severe hypovitaminosis D in summer. The effect of 10 mg ofd 2 daily was insufficient to raise S-25(OH)D from baseline. The daily supplementation of 20 mg ofd 2 increased S-25(OH)D significantly in wintertime compared with the nonsupplement users (to 45.5 vs 31.8 nmol=l; P < 0.001). None of the subjects with vitamin D 2 supplementation approximately 20 mg daily had severe hypovitaminosis D; however, 38% of those participants had moderate hypovitaminosis D at 36 months. Sun exposure in summer raised mean S-25(OH)D to 62.0 nmol=l. Both the daily supplementation of approximately 20 mg ofd 2 and summer sunlight exposure had more effect on those who had severe hypovitaminosis than those who had a normal vitamin D status (increase of 24.2 vs 0.9 nmol=l (P < 0.001), and 38.8 vs 18.2 nmol=l (P < 0.001), respectively). Conclusion: Vitamin D supplementation daily with 20 mg is needed to prevent hypovitaminosis D in peripubertal Finnish girls in winter. Sunlight exposure in summer is more effective than approximately 20 mgofd 2 supplementation daily in winter to raise S- 25(OH)D. Both the daily supplementation with 20 mg ofd 2 and summertime sunlight exposure had more effect on those who had severe hypovitaminosis D than those who had a normal vitamin D status. Sponsorship: Supported by the Yrjö Jahnsson Foundation and the Medical Research Foundation of the Turku University Central Hospital. (2002) 56, DOI: =sj=ejcn= Keywords: dietary intakes; hypovitaminosis D; serum 25-hydroxyvitamin D; supplementation of vitamin D 2 *Correspondence: T Möttönen, Department of Medicine of Turku University Central Hospital, Paimio Hospital, Paimio, Finland. timo.mottonen@tyks.fi Guarantor: TMöttönen. Contributors: All investigators contributed to the study design and writing of the paper. ML-V recruited the participants, conducted the interviews and contributed to data analysis and wrote the first draft of the manuscript. TM was the leader of the study, advised and took an active role in the study design, conducted data analysis, performed statistical analysis and revised the manuscript. KI supervised the lab work and revised the manuscript. IN contributed to data analysis and revision of the manuscript. JV contributed to the data management and revision of the manuscript and supervised the study. Received 24 April 2001; revised 22 August 2001; accepted 6 September 2001

2 432 Introduction Adequate vitamin D stores are essential for normal skeletal mineralization and growth. Vitamin D may be important not only for the skeleton, since, in addition to its known influence on calcium absorption, epidemiological studies have shown that hypovitaminosis D may affect other organ systems adversely (Fraser, 1995; Vieth, 1999). Vitamin D is obtained either from dietary sources or from cutaneous synthesis through a process initiated by ultraviolet radiation on the skin (Webb et al, 1988). Because ultraviolet radiation is required as a catalyst, vitamin D status shows variations dependent on season of the year and geographic latitude. Vitamin D stores can be assessed by measuring serum 25-hydroxyvitamin D (S-25(OH)D), which is the most sensitive clinical marker of vitamin D status (Hollis, 1996). Severe vitamin D deficiency resulting in rickets and osteomalacia is usually associated with S-25(OH)D concentrations below 12.5 nmol=l (Parfitt, 1994). There is considerable debate about what concentrations are desirable with regard to acquisition and maintenance of bone mass (Docio et al, 1998; Malabanan et al, 1998). Low serum concentrations of 25(OH)D are associated with reduced bone mineral density (BMD) and increased fracture risk in adults and elderly (Khaw et al, 1992; LeBoff et al, 1999). The risk of fractures in elderly can be reduced by vitamin D and calcium supplementation (Chapuy et al, 1992; Dawson-Hughes et al, 1997). The insufficient availability of vitamin D among children and adolescents has created considerable concern recently everywhere in Europe (Docio et al, 1998; Davies et al, 1999; Guillemant et al, 1999; Lehtonen-Veromaa et al, 1999). Vitamin D deficiency is not uncommon among children and adolescents, particularly during the dark seasons of the year many children and adolescents have vitamin D deficiency or even severe hypovitaminosis D owing to the lack of sunshine (Docio et al, 1998; Guillemant et al, 1999; Lehtonen-Veromaa et al, 1999). There is a lack of studies concerning the effect of vitamin D supplementation in young people (Zeghoud et al, 1995; Barger Lux et al, 1998; Lehtonen-Veromaa et al, 1999). We have earlier reported the impact of summer season on S-25(OH)D concentrations among these healthy Finnish 9 15-y-old girls (Lehtonen-Veromaa et al, 1999). The purpose of the present study was to examine the effect of vitamin D supplementation on serum 25(OH)D concentrations in winter and compare the results with the impact of summer season on serum 25(OH)D. controls were usually classmates of the athletes, or children of hospital personnel. All participants were healthy and had no chronic diseases that could affect growth or the metabolism of calcium or vitamin D. The study protocol was approved by the joint ethics committee of the Turku University and the Turku University Central Hospital. The study was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all volunteers and their parents. Study design All subjects were studied at baseline over an 8 week period in February March 1997 and followed up at 6 months intervals during a 3 y period. Height and weight were recorded. Height was measured with a fixed stadiometer (Harpenden Stadiometer, Holtain, Crymych, UK) and weight with a regularly calibrated electronic scale (EKS Exclusive, EKS International, Sweden). The body mass index (BMI) was calculated and recorded (kg=m 2 ). All measurements were made between 8 and 10 am by the same observer (ML-V). Multivitamin supplementation (Optivit 1, Leiras, Turku, Finland; containing 10 mg of vitamin D 2 ) was given to all participants with instructions to take one tablet daily from the beginning of October to January for the first winter and from October to February for the second winter to ensure the recommended dietary allowance of vitamin D. The dose was two tablets (20 mg) of vitamin D 2 daily during the winter period of the third year of the study. Calcium supplementation (Puru- Calsor 1, Orion, Espoo, Finland, containing 500 mg calcium as the carbonate salt) was given as one tablet per day to those who consumed less than 1000 mg=day calcium. The compliance of vitamin D and calcium supplementation was recorded by a questionnaire. If the participant was a young child all questionnaires, including compliance of vitamin D supplementation, were answered by one of the parents together with a child, otherwise by the participant herself. Assessment of nutrient intake Intakes of vitamin D and calcium were estimated at 6 month intervals over a 3 y period as described by Välimäki et al (1994). The interviewer administered semi-quantitative food frequency questionnaires (FFQ), which included questions on supplement use and pictures of portion sizes were used to estimate the intakes of vitamin D and calcium. Methods Subjects The study group comprised 191 healthy Caucasian girls aged 9 15 y (66 gymnasts, 65 runners and 60 nonathletic controls) who were participating in a long-term health study (Lehtonen-Veromaa et al, 1999). The participants were enrolled as volunteers who were recruited from local sports clubs and schools in the city of Turku and its vicinity. The Laboratory studies All blood samples were obtained from the participants between 08:00 and 09:00 h after an overnight fast annually and after the first summer. Local anaesthetic patches (Emla 1, Astra, Södertälje, Sweden) were used to reduce the discomfort of venipuncture. The blood samples were centrifuged (2100 g, 10 min) within 2 h of venipuncture and sera were stored frozen at 7 20 C. The serum samples for 25(OH)D

3 were protected from light during processing and measured by radioimmunoassay (Incstar Corporation, Stillwater, Minnesota, USA). The samples were run in duplicate; the interassay coefficient of variation for S-25(OH)D was 8.3% at the level of 35.3 nmol=l (n ¼ 50). Routine blood chemistry including serum calcium and alkaline phosphatase was analysed by Hitachi 717 and 917 analysers (Hitachi Ltd, Tokyo, Japan). Hypovitaminosis D We defined severe hypovitaminosis D as S-25(OH)D below 20 nmol=l and moderate hypovitaminosis D as S-25(OH)D between 20 and 37.5 nmol=l. The definition of hypovitaminosis D was adapted from published data according to which the serum parathyroid hormone (PTH) concentration starts to increase in patients whose serum 25(OH)D concentration is below 37.5 nmol=l (Thomas et al, 1998). Assessment of pubertal stage The stage of pubertal development was evaluated by the method of Tanner (1962). The Tanner stage was examined and recorded by ML-V. When there were discrepancies between breast stage and pubic hair stage, the degree of breast development was decisive. Statistical analyses All analyses were performed using the Statistical Package for the Social Sciences for Windows (Release 9.0, SPSS; Norusis=SPSS, Inc., Chicago, IL, USA). The normality of the distributions was tested using the Kolmogorov Smirnov statistics with a Lilliefors significance or Shapiro Wilk statistics. For variables with a normal distribution descriptive values were expressed as means and standard deviations (s.d.). Statistical comparisons between measures or groups were made by the t-test or analysis of variance (ANOVA) with Tukey s significant difference test. If the variables were nonnormally distributed descriptive values were expressed as medians and interquartile ranges (IQR) and statistical comparisons between groups were made by using the Kruskal Wallis test with Bonferroni s adjusted Mann Whitney U-test. The significance level was set at P < Results One-hundred and ninety-one girls were originally evaluated. There were 15 dropouts during the 3 y and three participants were excluded because of onset of a chronic disease (coeliac disease, epilepsy and thyrotoxicosis). In addition, two participants were excluded owing to arriving from vacation in Egypt and Thailand immediately before the first blood sample collection. Thus, the results of 171 subjects were included in the final analysis. The baseline characteristics of the study group are presented in Table 1. There were no statistically significant differences between the athletic and nonathletic groups concerning the stage of puberty. At the time of recruitment 57% of the participants had not reached menarche. At baseline the prepubertal participants (Tanner 1) had significantly higher S-25(OH)D than the rest of the participants; later 25(OH)D concentrations were similar in each Tanner stage. There were no differences in daily intake of vitamin D or compliance of vitamin D supplementation between prepubertal participants and the subjects of other pubertal stages (data not shown). There were no differences in 25(OH)D concentrations or compliance of supplementation between the athletic and nonathletic groups (data not shown). The serum concentrations of calcium and alkaline phosphatase of all subjects were within the reference range (data not shown). The median daily intakes of vitamin D and calcium assessed by FFQ were 3.8 mg (IQR ) and 1451 mg (IQR ), respectively, during the 3 y follow-up period. The median daily intake of vitamin D was similar among athletes and controls (data not shown). The mean seasonal increase by sunlight exposure in S- 25(OH)D concentrations was significantly higher in the lowest baseline S-25(OH)D tertile than in the highest one in the whole study population (38.8 vs 18.2 nmol=l, P < 0.001, Figure 1). The effect of vitamin D 2 supplementation on the mean S- 25(OH)D over a 3 y span is shown in Table 2. At baseline, those participants who took weekly vitamin D 2 supplementation (10 70 mg) had significantly higher S-25(OH)D concentration than those who did not (43.3 vs 31.3 nmol=l, P < 0.001). After the summer (August September) S- 25(OH)D concentrations were similar between the vitamin D 2 supplementation and non-supplementation users. Although only two participants did not take vitamin D 2 supplementation (10 mg) at 12 months, the mean S- 25(OH)D concentration did not differ from baseline among the whole study population (33.2 vs 34.0 nmol=l, NS). Since S-25(OH)D concentration in the overall study population at 24 months was similar to those of the previous winter values of 25(OH)D (30.2 nmol=l), the dose of vitamin D 2 supplementation was advised to be doubled for the last Table 1 Characteristic Baseline characteristics of 171 peripubertal girls Age (y), mean (s.d.) 12.9 (1.7) Height (cm), mean (s.d.) (9.3) Weight (kg), mean (s.d.) 46.8 (9.7) Body mass index (kg=m 2 ), mean (s.d.) 18.6 (2.4) Dietary intake of calcium (mg=day), median (IQR) 1454 ( ) Dietary intake of vitamin D (mg=day), median (IQR) 4.0 ( ) Tanner stage, number (%) I 31 (18.1 %) II 39 (22.8 %) III 32 (18.7 %) IV 34 (19.9 %) V 35 (20.5 %) 433

4 434 Figure 1 The effect of season on serum 25(OH)D (nmol=l). Serum 25(OH)D at baseline and after 6 months (August September) in the subjects of each baseline tertile (n ¼ 171). Bonferroni comparisons of the seasonal increase in 25(OH)D between the lowest (I) and highest (III) tertile, P < 0.001; lowest (I) vs middle (II) tertile, P < winter period (from October 1999 to March 2000). The participants who took weekly vitamin D 2 supplementation mg ( IU, n ¼ 100) had significantly higher S-25(OH)D concentrations at 36 months than the participants who did not take vitamin D 2 supplementation at all (n ¼ 9; 43.5 vs 31.8 nmol=l, P < 0.001; Table 2). In the subgroup of the most frequent ( mg weekly, n ¼ 50) users of the vitamin D 2 supplementation S-25(OH)D was 45.5 nmol=l (s.d. 17.2) at 36 months. The effect of vitamin D 2 supplementation on S-25(OH)D is shown in Figure 2. The participants who took weekly mg ( IU) of D 2 during the last winter period and who had not used vitamin D 2 supplementation at all at the beginning of the study (n ¼ 77) were divided into tertiles according to baseline S-25(OH)D concentration. The mean increases by mg weekly of vitamin D 2 supplementation in S-25(OH)D concentrations were significant in the lowest and in the middle baseline S-25(OH)D tertiles but not in the highest one (24.2, 8.5 and 0.9 nmol=l, respectively; Figure 2). In this whole subgroup (n ¼ 77) S-25(OH)D at 36 months was significantly higher than at baseline (43.5 (s.d. 16.6) vs 32.2 nmol (s.d. 12.3); P < 0.001). Figure 2 Serum 25(OH)D (nmol=l) at baseline (February March, open bars) and after mg weekly of vitamin D 2 supplementation in winter at 36 months (solid bars) in the subjects of each baseline 25(OH)D tertile in the group of participants who did not take vitamin D supplementation at baseline but who took weekly at least 80 mg at 36 months (n ¼ 77). The prevalence of severe hypovitaminosis D (S- 25(OH)D < 20 nmol=l) was 14%, and of moderate hypovitaminosis D (20 nmol=l S-25(OH)D 37.5 nmol=l) 75% in the girls who did not take vitamin D supplementation (n ¼ 133) at baseline in February March. None of the subjects had severe hypovitaminosis D in summer. None of the participants with the most frequent vitamin D 2 supplementation ( mg weekly, n ¼ 50) had severe hypovitaminosis D, while the prevalence of moderate hypovitaminosis D was 38% in this subgroup at 36 months (Table 3). Discussion The main findings of this study are that the average dietary vitamin D intake is insufficient and the supplementation of D 2 should be at least mg ( IU) weekly in winter to provide a significant change in serum 25(OH)D concentrations among peripubertal girls at northern latitudes such as Finland. In addition, sunlight exposure in summer is more effective in raising S-25(OH)D than mg ofd 2 supplementation daily in winter. Our results confirm that vitamin D is a risk nutrient among adolescent girls in Finland. Usual dietary vitamin D Table 2 girls Effect of vitamin D 2 supplementation on the mean serum 25(OH)D (nmol=l) concentration (s.d.) during a 3 y period among 171 peripubertal At baseline At 6 months (August September) At 12 months At 24 months At 36 months Use of supplementation n 25(OH)D n 25(OH)D n 25(OH)D n 25(OH)D n 25(OH)D No supplementation (11.6) (13.9) (10.6) (7.7) (17.2) Supplementation (10 70 mg weekly) (14.7) (21.4) (11.1) (13.7) (15.4) Supplementation ( mg weekly) (15.4) Whole group (13.2) (14.3) (11.1) (13.2) (15.8)

5 Table 3 Prevalence (%) of hypovitaminosis D and the effect of vitamin D 2 supplementation Serum 25(OH)D 435 Severe hypovitaminosis D S-25(OH)D < 20 nmol=l Moderate hypovitaminosis D 20 nmol=l S-25(OH)D 37.5 nmol=l Intake of vitamin D n % n % Dietary intake (median 4 mg=day) at baseline a 19= = Dietary intake (median 3.7 mg=day) in summer (August September) b 0= =162 2 c Dietary intake (median 3.4 mg=day) in winter þ 10 mg D 2 8= =62 66 d Dietary intake (median 3.6 mg=day) in winter þ 20 mg D 2 0= =50 38 a In the girls who did not take vitamin D supplementation at baseline (n ¼ 133). b In the girls who did not take vitamin D supplementation in summer (n ¼ 162). c In the girls who took daily vitamin D supplementation 10 mg at 24 months (n ¼ 62). d In the girls who took daily vitamin D supplementation 20 mg at 36 months (n ¼ 50). intake is insufficient to maintain the vitamin D status at an adequate level in periods when the skin is not able to produce enough vitamin D. In equatorial regions exposure to the sun alone is adequate but at latitudes of about 40 and higher north people produce almost no vitamin D in the winter. In Edmonton, located at 52 north, this ineffective winter period extends from October to March (Webb et al, 1988). Finland is located at north, and thus, due to the lack of sunlight, the skin does not produce vitamin D for several months annually. The observation that the absolute rise in S-25(OH)D is related inversely to baseline S-25(OH)D and the amount of exposure to ultraviolet light has been made previously in adults (Snell et al, 1978; Mawer et al, 1984). Our findings in the adolescent girls were in line with those earlier observations in adults because the differences between the tertiles of serum 25(OH)D in winter disappeared during the summer, owing to sunshine. However, in this study sunlight exposure in summer, even at high latitudes, seems to be more effective in raising S-25(OH)D concentrations than 20 mg ofd 2 daily in winter. There is a paucity of data concerning the effect of vitamin D supplementation assayed by the same kind of technique in young people. Vitamin D injection of 2500 mg ( IU) kept S-25(OH)D concentrations in the normal range for only 1 2 months during wintertime (Zeghoud et al, 1995). Harris et al (1999) reported that 45 mg (1800 IU) of D 2 daily for 3 weeks increased S-25(OH)D by 30.4 nmol=l in a group of young men but only 7.5 nmol=l in a group of older men. In a recent study the effect of vitamin D supplementation was clear in small children (Davies et al, 1999). The serum 25(OH)D levels were raised considerably using daily 10 mg (400 IU) of D 2 in prepubertal Finnish children (Ala-Houhala et al, 1988). However, it is very often difficult to compare the results because the type of vitamin D used in the preparation is not always reported. Barger Lux et al (1998) reported the use of vitamin D 3 supplementation of 10 mg (400 IU) daily over an 8 week course to raise S-25(OH)D by 14 nmol=l in healthy adult males, although the baseline S-25(OH)D concentration was quite high (67 nmol=l). The effect of 20 mg (800 IU) of D 3 daily was 27 nmol=l on S-25(OH)D concentration in a group of young men (Barger Lux et al, 1998). In our study the effect of mgofd 2 weekly was only 11.7 nmol=l. Our modest results may be explained by the findings of Trang et al (1998), who found that D 2 had less efficacy than D 3 in raising S- 25(OH)D levels in adults. In their study 100 mg (4000 IU) of D 2 daily for 2 weeks raised S-25(OH)D 13.7 nmol=l and the same amount of D 3 elevated S-25(OH)D by 23.3 nmol=l. Our findings were in good agreement with Trang et al (1998), who reported that the effect of vitamin D supplementation diminished progressively above 50 nmol=l in increasing S-25(OH)D concentrations. In fact, in our study the mean S-25(OH)D concentration of the highest baseline S-25(OH)D tertile was 45.8 nmol=l and the increase of S-25(OH)D was only 0.9 nmol=l in this group of the girls. Severe vitamin D deficiency resulting in rickets and osteomalacia is usually associated with serum 25(OH)D concentrations below 12.5 nmol=l (Parfitt, 1994). We did not observe any subjects with clinical rickets in our study. Serum 25(OH)D concentrations below 20 nmol=l are generally regarded as indicating severe hypovitaminosis D but circulating concentrations up to 37.5 nmol=l may be associated with adverse skeletal effects (McKenna, 1992), and even higher concentrations (up to 50 nmol=l) may be required for optimal skeletal health, particularly in adolescents (Docio et al, 1998). Guillemant et al (1999) described in healthy male adolescents increasing PTH concentrations if serum 25(OH)D concentrations were below 30 nmol=l, and above 83 nmol=l of 25(OH)D concentrations PTH concentrations reached a plateau. Inadequate intakes of vitamin D have been reported in Finnish children and adolescents (Lamberg Allardt et al, 1986; Lehtonen-Veromaa et al, 1999). Foods in Finland are not supplemented with vitamin D except margarines (7 mg=100 g), and fat-free and 1% fat milk (0.08 mg=100 g). In the present prospective study the intake of calcium was generally more than adequate among healthy girls without any eating disorders and, if the intake was below

6 mg=day, supplementation was used. An adequate supply of dietary vitamin D is necessary for calcium accumulation in the skeleton. Although the stimulatory effect of vitamin D on intestinal calcium and phosphorus absorption is well understood, its effect on skeletal development is not completely clear. Parsons et al (1997) found that adolescent girls who had used a macrobiotic diet (low calcium and vitamin D) in early life had 8% lower bone mineral density (BMD) at the femoral neck and 5% lower BMD at the spine compared with adolescents on a normal diet, but they did not include serum 25(OH)D in their evaluation. In Australia, Jones and Dwyer (1998) found in their cross-sectional study that the amount of exposure to sunlight was associated with BMD in prepubertal girls. The difference in the bone mass was up to 10% between the lowest and highest sun exposure category in those girls. There is a lack of data regarding the effect of vitamin D supplementation on bone during adolescence. Zamora et al (1999) found that vitamin D supplementation in infancy was associated with increased BMD at specific skeletal sites in prepubertal girls. Vitamin D probably also affects other aspects of human health than its classical role in mineral metabolism. Epidemiological studies have shown that higher S-25(OH)D concentrations or environmental ultraviolet light exposure are associated with lower rates of breast, ovarian, prostate and colorectal cancers (Tangrea et al, 1997; Vieth 1999; Ahonen et al, 2000). Hypovitaminosis D also impairs immune function in humans. Multiple sclerosis is more prevalent in populations having low S-25(OH)D concentrations compared with populations with a normal vitamin D status (Hayes et al, 1997; Vieth 1999). We started our study in February March, when the baseline concentration of 25(OH)D would have been at its annual nadir. Our participants who did not take supplementation showed no change in serum 25(OH)D in wintertime indicating that endogenous production of vitamin D could not influence the outcome. We do not believe that poor compliance explains the modest change in S-25(OH)D, while the participants reported half-yearly the frequency of vitamin D supplementation by a self-administered questionnaire, and we included this information in the analysis. Vitamin D stability in tablets, which was not monitored, might have been a confounding factor in this study. However, we used vitamin D 2 supplementation which was delivered straight through us by the manufacturer immediately before the beginning of each supplementation period. It seems to be apparent that more attention should be focused on the importance of attaining sufficient and optimal serum 25(OH)D concentrations during childhood and adolescence. The nutritional factors are obviously important and dietary fortification or supplementation with vitamin D should be considered to assure an adequate wintertime vitamin D status. According to the present study a reduced store of vitamin D may demand daily approximately 20 mg (800 IU) of D 2 to raise S-25(OH)D concentration during the dark seasons of the year. Prospective studies of the effects of vitamin D supplementation and adequate 25(OH)D levels on bone development during childhood and adolescence are needed. Conclusion The prevalence of hypovitaminosis D is high in Finnish 9 15-y-old girls in winter. The daily supplementation of 10 mg (400 IU) of D 2 is insufficient, approximately 20 mg (800 IU) of D 2 is needed daily during the dark seasons of the year to prevent hypovitaminosis D in peripubertal girls. Sunlight exposure in summer is more effective than approximately 20 mg of D 2 supplementation daily in winter in raising serum 25(OH)D levels. 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