{'fraction_non_alphanumeric': 0.03760112080263931, 'fraction_numerical': 0.030324942378090115, 'mean_word_length': 4.969247369840842, 'pattern_counts': {'":': 0, '<': 8, '<?xml version=': 0, '>': 5, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effect of Vitamin D Intervention and outdoor activity on serum 25-Hydroxyvitamin D among children in North China: an observational study

Background: Living at high latitudes is one of the risk factors for vitamin D deciency in children. However, evidence on vitamin D improvement for this pediatric population to date is limited. This study aims at evaluating the effect of different vitamin D intervention methods and outdoor activity on the vitamin D status of children in North China. Methods: A total of 55,925 children aged 1 month to 18 years old were recruited from pediatric outpatient departments from July 2016 to June 2017. Data on demographics, anthropometric measurements, vitamin D intervention, and outdoor activity were recorded. The serum levels of 25-hydroxycholecalciferol (25(OH)D) were determined by high performance liquid chromatography tandem–mass spectrometry. Logistic regression analysis was performed to assess the association of vitamin D intervention or outdoor activity with blood vitamin D status, adjusted for age, gender, BMI for age, and seasons. Results: The overall rate of hypovitaminosis D was 65.60%. Of the children’s outdoor activity, 35.63%, 31.95%, and 32.42% were below 30min/d, 30-60 min/d and over 60min/d, respectively. Furthermore, the proportion of therapeutic intervention, supplementation intervention and no vitamin D intervention among the children was 16.48%, 32.87%, and 50.65%, respectively. After adjusted for confounding factors, vitamin D intervention was associated with a lower risk of hypovitaminosis D, with OR (95% CI) of 0.191 (0.180, 0.202) in children with therapeutic doses and 0.423 (0.404, 0.443) in those with supplementation doses, compared with children without vitamin D intervention. In addition, longer outdoor time was associated with a lower risk of hypovitaminosis D [0.479 (0.456, 0.504) for 60 min/d, 0.737 (0.701, 0.776) for 30–60 min/d], independent of vitamin D intervention. Conclusions: High prevalence of vitamin D deciency was found in children living at high latitudes.


Background
Vitamin D de ciency is a disorder mainly induced disorders of calcium and phosphorus metabolism, which causes defective chondrocyte differentiation, mineralization in growth plate and defects in osteoid mineralization among children [1] . Vitamin D de ciency is also related to functions of muscles and immune organs, and hematopoiesis, which are associated with multiple extra-skeletal disorders, such as myopathy, anemia, and recurrent respiratory tract infections in children [2][3][4] .
Vitamin D de ciency or insu ciency are still of global public health concerns, particularly in developing countries [5] . Approximately 30.00% of children suffer from hypovitaminosis D worldwide [6] . Academic societies of China and other regions have released guidelines to emphasize the role of outdoor time and vitamin D intervention in physical health of children and adolescents, especially those with risk factors of de ciency [7,8] . Several studies have demonstrated that vitamin D intervention signi cantly increased blood 25-hydroxyvitamin D (25(OH)D) level in children. A randomized, double-blinded study reported that vitamin D treatment in 600 IU/d for 6 weeks resulted in an increase in serum 25(OH)D in asthmatic children compared with placebo treatment (76.3 vs 48.2nmol/l) [9] . In a prospective observational study, 55 children with in ammatory bowel diseases were prescribed vitamin D 2000 IU/d for 2 to 3 months, and their serum 25(OH) D were increased from 58 nmol/L at the baseline to 85 nmol/L [10] . Uysalol et al found that serum 25(OH)D levels in asthmatic children with inadequate sun exposure and vitamin D intervention were signi cantly lower than that of the healthy controls (16.6 vs 28.2 ng/mL) in Turkey [11] . Therefore, most present studies focused on the impact of vitamin D intervention and outdoor time on pediatric patients. Yet the effect of vitamin D intervention and sun exposure on healthy children are not well documented given that few guidelines support serum 25(OH)D test for children [12] . However, previous studies suggested that circulating vitamin D level should be measured even in healthy children with a risk of vitamin D de ciency mainly due to inadequate sun exposure [13,14] , in order to early prevent vitamin D de ciency-related diseases.
Constant exposure of many parents to information on bene ts of vitamin D supplementation during pregnancy and throughout childhood may contribute to voluntary daily intake of vitamin D in children. [15,16] . Supplementation intervention of vitamin D helps prevent vitamin D de ciency in children, although few studies have been reported [17] . With the spread of child health education, children are more likely to have regular clinic visits due to early recognition of nutritional rickets-related clinical symptoms by their parents, e.g. frontal bossing, rachitic rosary, and leg deformity. 25(OH)D test is routinely performed for children in North China as part of physical examination or assessment of suspected nutritional rickets. Once the diagnosis is con rmed, individualized intervention is provided by physicians/doctors to maintain or improve vitamin D status of their pediatric patients. Nevertheless, little research has been conducted on e cacy and feasibility of therapeutic intervention to date. In our study, data of 55,925 children from our clinic were analyzed to investigate their vitamin D nutriture and possible causes, of which vitamin D intervention and outdoor time were of particular interest, to explore the necessity of regular monitoring of 25(OH)D status and intervention in children living at high latitudes.

Methods
Study design, data source, and patient selection Heilongjiang is a province comprising 12 cities including Harbin, located in North China at latitude 43°3′ -53°3′ N and known as the coldest region in China, with an average annual sunshine of 1874 to 2761 h in 2016-2017, according to Heilongjiang Bureau of Statistics. The Sixth National Population Census of China indicated that the resident population in Heilongjiang was 38.31 million, in which 4.57 million (11.94%) were aged 0-14 years old.
In this cross-sectional study, children aged 0-18 years old were recruited who visited child health clinic in Harbin Children's Hospital between July 2016 and June 2017. All guardians completed questionnaires involving questions of their children's name, date of birth, date of visit, ethnic, and vitamin D intervention.
Children's outdoor time was reported by the parents as the number of hours during the last week (including the weekend) [18] .
Therapeutic intervention is de ned as vitamin D prescription in children who had physical examination or clinic visits in the past 3-6 months. Prophylactic and therapeutic dosage was recommended for pediatric patients with a normal level and in vitamin D de ciency, respectively according to the consensus of the Chinese Society of Osteoporosis and Bone Mineral Research [19] , respectively (most of them had oral vitamin D3 intake of 800-2400 IU/d). Supplementation intervention is de ned as daily vitamin D intake in children asked by their parents in last 3-6 months (most of them had oral vitamin D3 intake of 400-800 IU/d), without being monitored for their serum 25(OH)D or a prescription from doctors. No vitamin D intervention was de ned as taking no vitamin D supplements in the past 3-6 months. Children were considered being supplemented with vitamin D if they were 1) aged over 6 months and given either therapeutic or supplementation vitamin D intervention for more than 3 months and 2) aged less than 6 months and received vitamin D intervention in either aforementioned way for >1 month. Children with the following conditions were excluded from our study: 1) using vitamin D metabolites and their analogs, e.g., alfacalcidol or calcitriol, for their routine supplementation. 2) suffering from hypophosphatemic rickets, organ dysfunction, congenital disorders, inherited metabolic diseases, acute infectious diseases and chronic in ammatory disease. 3) for those over 6 months old, either aforementioned intervention suspended for >20 d in the past 3 months and 4) for children under 6 months, either aforementioned intervention suspended for >10 d in the past 1 month. Detailed information on the nature of this study was provided to the parents or guardians of participants before consent was written.

Anthropometric Measurements
Trained nurses measured twice the height and weight of the enrolled children within 0.1 cm and 0.1 kg respectively, and average the measurements for the nal value. An anthropometric calculator obtained from World Health Organization (http://www.who.int/en/) was used to determine body mass index (BMI) for age. BMI for age <90 percentile was considered normal, 90-97 percentile overweight and >97 percentile obese.

Circulating 25(OH)D Level
Blood samples (3 ml) were taken by antecubital venipuncture and then stored in a cool box for <2 h. After centrifugation at room temperature for 10 min at 1200 × g, the upper serum pipetted and stored at 4 °C for <24 h. The samples were kept at room temperature for 30 min before analysis. The serum 25(OH)D levels were determined by high performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS, API 3200; AB SCIEX, American). The lower limits of 25-(OH)D2 and 25-(OH)D3 for detection were both 1.6 ng/mL. The test sensitivity was assessed with the inter-batch coe cient of variation (CV) of 6.59% and between batches CV of 6.98%.

Diagnostic Criteria of Serum Vitamin D Status
The consensus of the Chinese Society of Osteoporosis and Bone Mineral Research, Chinese Medical Association indicated that circulating 25(OH)D <10 ng/mL was considered severely de cient, 10-20 ng/mL de cient, 20-29 ng/mL insu cient, and ≥30 ng/mL su cient [19]. High levels of circulating 25(OH)D may have bene ts for children's extra-skeletal organs [20]. Thus, the cut-off points for de ning low status of vitamin D is 30 ng/mL, which refers to hypovitaminosis D in this study [14] .

Statistical analysis
The serum level of 25(OH)D was expressed as mean±SD. Comparison of the 25(OH)D levels between different groups was determined by ANOVA. The prevalence of vitamin D status was expressed as n (%). The statistical difference of the prevalence of vitamin D status between different groups was determined by chi-square test. Logistic regression analysis was performed to evaluate the association of vitamin D intervention or outdoor time with blood vitamin D status, adjusted for age, sex, BMI for age, and season.
Data were expressed as odds ratio (OR) and 95% con dence interval (CI). All analyses were performed using SPSS 21.0. A p value < 0.05 was considered statistically signi cantly.

Participant Characteristics
A total of 55,925 participants from the 12 cities of Heilongjiang were included in this study, in which 30,683 (54.86%) were male. Table 1 presented the basic characteristics of the participants. The average serum level of 25(OH)D for all participants was 26.13±12.30 ng/mL. The median age of participants was 3.10 years old (interquartile range, 1.10-6.50 years old). Of the children's outdoor time, 35.63%, 31.95%, and 32.42% were below 30min/d, 30-60 min/d and over 60min/d, respectively. Furthermore, 50.65% of the subjects in no vitamin D intervention group, 32.87% in supplementation group, and 16.48% in therapeutic group. The vitamin D level was higher in boys, younger children, children recruited in summer, with prolonged outdoor time, and those with vitamin D intervention (P < 0.001).

Characteristics of Children with Different Vitamin D Statuses
The overall rate of hypovitaminosis D was 65.60%. 6.57%, 25.51%, and 33.52% of the children included were found with severe de ciency, de ciency and insu ciency, respectively. Moreover, 18.11% children with severe de ciency were overweight or obese, compared with 11.34% and 8.17% in those with de ciency and insu ciency, respectively. However, the rate of overweight and obesity decreased to 4.21% in children with vitamin D su ciency (P < 0.001). Among the in uential factors, outdoor activity time and intervention methods are possibly causal factors determine the vitamin D status in children, and also changeable factors for prevention of hypovitaminosis D.  We conducted logistic regression to further eliminate the in uence of outdoor activity time on the relationship between intervention methods and serum 25(OH)D level. Both therapeutic and supplementation intervention reduced the risk of hypovitaminosis D regardless of outdoor time compared to those with no vitamin D intervention. In children whose outdoor time was > 60 min/d, the OR was 0.196 (0.178, 0.216) in the former way, whereas 0.508 (0.470, 0.550) in the latter way, compared with those who were not given intervention (Table 4).

Discussion
This observational study was the rst to assess circulating 25(OH)D in over 50,000 children by HPLC-MS/MS, the current gold standard in vitamin D level assessment [21] . Our data showed that (1) status deteriorated with advancing age, which was in line with Korean and Canadian studies [22,23] .
Generally, outdoor activities and vitamin D supplementation in children younger than three years old depend on parental support, whereas those in school children older than six years are limited or encouraged by schools. Thus, we divided children's age into three groups and found that vitamin D intervention rate in group of age 0-3 years (66.89%) was higher than that of age 3-6 (42.04%) and above 6 years old (25.81%). The policy of physical examination by local communities is that regular physical examination free of charge is provided to children younger than 3 years old every 3-6 months and to those aged 3-6 years old every year, including screening for rickets and education on vitamin D supplementation. Nevertheless, routine physical examination is not performed in children older than 6 years old. Different health policy may be the main cause for high rate of vitamin D supplementation in younger children. Despite varying outdoor time and vitamin D interventions in each age group, an increase in outdoor time and vitamin D intervention present effectiveness in hypovitaminosis D prevention.
Latitude has a clear impact on the vitamin D status in children. However, we still lack of data for the vitamin D nutriture of children form different latitudes. Previous hospital-based cross-sectional studies indicated that 23.28% children in Huzhou (southeastern China, 30°2′-31°1′ N) [24] and 33.60% in Hangzhou (southeastern China, 29°1′-30°3′ N) [25] were found with hypovitaminosis D, whereas the prevalence increased to 65.91% in this study, which is consistent with early research focusing on children in high latitudes. Data from Hutterite communities (Canada, 49°2′-54°8′N) suggested that 76.00% of children suffered from hypovitaminosis D [26] , indicating that hypovitaminosis D might be a common and serious problem in children in high latitudes. Skin is a key organ in vitamin D synthesis because 80%-90% vitamin D that the human needs per day is produced in the skin from ultraviolet-B-activated 7dehydrocholesterol [27] . Evidence from Spain (36°0′-43°2′ N) suggested that production of over 1000 IU/d vitamin D in spring and summer depends on regular sunlight exposure [28] , making outdoor activity an optimal way of getting vitamin D. However, our results showed that still 17.75% and 21.63% of the enrolled children whose outdoor time was over 60 min/d had a de cient and insu cient vitamin D status, respectively. Moreover, excessive ultraviolet radiation exposure throughout childhood appears to be particularly harmful [29] . Children with sun protection by their parents are much less exposed to ultraviolet radiation [30] . Therefore, vitamin D supplementation might be taken into account to prevent hypovitaminosis D among children living in Heilongjiang.
Numerous programs or health policies for vitamin D intervention are available in almost all countries to prevent vitamin D de ciency or hypovitaminosis D in children [31][32][33][34] . In the guidelines aforementioned, some risk factors for vitamin D de ciency are recognized, including vegan diet, malabsorption syndromes, and reduced sunlight exposure; meanwhile, regular vitamin D supplementation and routine 25(OH)D testing are recommended for children. However, living at high latitude as an identi ed risk factor for vitamin D de ciency has not been explicitly mentioned for routine 25(OH)D testing in these guidelines.
Despite of high incidence of hypovitaminosis D among children living in high latitudes was found in previous studies [18] , few of them focused on related improvements. Approximately 30% of the total world population lives from 40° N to 60°N. Given the vitamin D status of children living at high latitudes, it is necessary to determine whether they need to be routinely screened for serum 25 (OH) D levels, followed by appropriate vitamin D intervention as required. In this study, we found that hypovitaminosis D was prevalent in children living at high latitude, regardless of seasons or outdoor time. The vast majority of children maintaining adequate vitamin D status took vitamin D supplements. Therefore, despite the recommendations by some guidelines that populations at high risk of vitamin D de ciency should be routinely screened, the results of our study support regular vitamin D intervention for local children regardless of taking the 25(OH)D test. However, more future research is needed to con rm whether all children living at high latitudes should be given routine vitamin D intervention without 25(OH)D test.
The pediatric health care system in China has been improved and consists of a proper examination interval and routine monitor network of serum 25(OH)D levels in children [24,35] . Prescriptions were given by health professionals according to vitamin D status and the supplementary recommendation in the consensus of the Chinese Society of Osteoporosis and Bone Mineral Research. To the best of our knowledge, this was the rst study to describe and evaluate the effect of therapeutic intervention on prevention of hypovitaminosis D among children, and therapeutic intervention had a 2.21-, and 5.24-fold decreased risk of hypovitaminosis D compared with supplementation intervention and no supplementation, respectively. Besides, health professionals urge the parents to keep regular vitamin D supplementation [36] . Minkowitz et al. reported that better adherence of guardians with low serum vitamin D levels to physicians' orders to remind their children of daily intake of vitamin supplements [37] . Although there were some unfavorable factors causing poor response to oral intervention [38] , pediatricians could improve the oral doses according to the follow-up serum 25(OH)D test results until the doses reached optimum. however, compliance of children with follow-up blood collection varied. No vitamin D intervention due to guardians' lack of awareness that might be the main cause [36] , was like to expose their children to the increased risk of hypovitaminosis D and acute and chronic diseases including infectious diseases, autoimmune disorders and childhood dental caries [39] . Thus, although therapeutic intervention has shown a better effect in preventing hypovitaminosis D than voluntary supplementation, which prevention strategy to follow depends on certain factors, in particular the local health policy and compliance of guardians with blood collection.
This study had several limitations. First, the vitamin D doses in the therapeutic group remained inconsistent. Individualized vitamin D intervention were prescribed by doctors according to the medical conditions of healthy children and those with vitamin D de ciency. Second, outdoor time might be a confounder. However, duration and sun-exposed skin areas of each child was not investigated in this study. Third, <10%-20% vitamin D that human needed per day was from diet [40] , which played an important role in the vitamin D status of children. However, such information was unavailable in our study. We assumed that children in the same region had similar dietary patterns. A prospective study is warranted to explore the role of diet in addition to other confounders. Fourth, as the subjects in this study were all recruited from the outpatient department instead of the general pediatric population, their parents might be more concerned about their children's vitamin D status or health condition, and their health status may be better. Therefore, demographic characteristics of the participants in this study could not represent that of the whole population of children living in North China.

Conclusions
In summary, a large number of blood tests in our study demonstrated low vitamin D status in local children. Supplementation and therapeutic vitamin D intervention were all promising strategies and prolonged outdoor time was negatively associated with pediatric vitamin D de ciency, the combination of which might be an optimal way to prevent hypovitaminosis D in children; nevertheless, guardian's compliance might be considered for a practical prevention strategy. In addition, regardless of taking a 25(OH)D test, all children living at high latitudes might need routine vitamin D intervention.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. including the collection, analysis, and preparation of the data or the drafting, editing, review, or approval of the manuscript.
Authors' contributions XZ and LN designed the study, writing, prepared the original draft, and acquired funding. XZ, YC, XB, DC, DZ and LL collected the data; HJ and SJ analysed and interpreted data. All authors read and approved the nal manuscript.