| [Objective]1. To establish a method of ultra high performance liquid chromatography to determine the content of 25-hydroxy vitamin D3 in children, then compared with chemiluminescence method.2. To investigate and analysis the 25-(OH)D3 levels in serum of children under 14 years old in Yunnan region, and explore whether there was a correlation among 25-(OH)D3, calcium and alkaline phosphatase.3. To get a preliminary normal range of 25-hydroxy vitamin D3 content in 150 healthy children of Kunming, and further analysis the correlation among 25-(OH)D3, calcium, phosphorus, alkaline phosphatase and parathyroid hormone.[Method]1. By consulted a large number of literature, trial and error, established a method to determinate the content of 25-hydroxyl vitamin D3 in plasma by ultra high performance liquid chromatography (UPLC). Got the value of 50 cases of children’s 25-hydroxy vitamin D3 by both ultra high performance liquid chromatography (UPLC) method and chemical luminescence methods. Analysised the diffrences between ultra high performance liquid chromatography (UPLC) and chemiluminescence method by paired comparison.2.1371 children who did health examination at department of children’ health prevention of Kunming Medical University Affiliated Children’s Hospital from January 2014 to December 2014 were choosen in this research. Fasting venous blood was taken to detect the 25-(OH)D3 by UPLC, then analysised by SPSS 17.0.3. Collected samples from July to August of 2015, healthy children examinated in Kunming Medical University Affiliated Children’s Hospital. Took fasting venous blood to detect the 25-(OH)D3, the calcium, phosphorus, alkaline phosphatase and parathyroid hormone in plasma with chemiluminescence method, finally analyzed the results by SPSS 17.0 statistical software.[Results]1. Ultra high performance liquid chromatography (UPLC) method:Chromatographic column:Syncronis C18 (50 mm × 2.1 mm,1.7 um; Thermo company); flow rate:0.3 mL.min-1; mobile phase:acetonitrile-water (75:25); column temperature:30℃; sample quantity:5μL; detection wavelength:265 mn. This method had good stability, high accuracy, and its linear equation was y=66.667+284.33 x, R2=0.9995, as well as showed a good linear relationship within 10-200ug/L. Chemiluminescence method: It satisfied the criteria for methodology, with a well accuracy and practicability.No statistical difference was founded between these two methods for determininating of 25-hydroxyl vitamin D32. The results of 1371 samples belonged to the non-normal distribution,the mean level was 47.70±26.20nmol/L, the 95% 25-(OH)D3 levels of children in Yunnan region was 30.33～117.10nmol/L by percentile method. The percentage of vitamin D deficiency, vitamin D insufficiency, vitamin D normal were 55.1%,29.7% and 15.2% respectively. The results of 25-(OH)D3 of boys and girls belonged to the non-normal distribution,the mean level were 47.80±24.80,47.58±24.70nmol/L,z=-0.605,P=0.545, had no statistical difference between 25-(OH)D3 of boys and girls. The results of 25-(OH)D3 of Kunming region and other states belonged to the non-normal distribution,the mean level were 47.60±26.70,47.85±25.00nmol/L,z=-0.419,P=0.675, had no statistical difference between them. The results of 25-(OH)D3 of infant period belonged to the normal distribution(P=0.968). the mean level was 68.70±20.83nmol/L. the results of 25-(OH)D3 of toddler’s age. preschool age, school age and adolescence belonged to the non-normal distribution and the average levels of 25-(OH)D3 of these periods were 52.38±32.20.46.20±22.35.45.23±23.16.42.20±16.85nmol/L respectively. There was statistical significant difference between toddler’s age and preschool age, toddler’s age and school age, as well as toddler’s age and adolescence (P<0.05), with no significant difference between others. The result of 25-(OH)D3 in spring, summer, autumn and winter belonged to the non-normal distribution, the mean levels of 25-(OH)D3 of spring, summer, autumn and winter were 44.50±22.45,48.10±23.00, 62.00±40.45,42.90±17.20nmol/L respectively, had statistical difference between the average level of 25-(OH)D3 in autumn and others (P<0.05), with no significant difference between others. There was low positive correlation between calcium and 25-(OH)D3 (r=0.109,P<0.05), very low positive correlation between alkaline phosphatase and 25-(OH) D3 (r=0.085, P=0.002), as well as low positive correlation between calcium and alkaline phosphatase (r=0.087, P=0.001).3. The reference range of 25-(OH)D3 in healthy children of Kunming was 44.60-169.20nmol/L by expressing medical reference range with P2.5-P97.5 and the mean level of it was 88.47±45.72nmol/L as the result of 150 samples was a non-normal distribution (P<0.001).The results of 25-(OH)D3 of boys belonged to the non-normal distribution(P<0.05), mean level of 25-(OH)D3 of boys was 91.82±50.97nmol/L,the results of 25-(OH)D3 of girls belonged to the normal distribution(P=0.098), the average level of 25-(OH)D3 of girls was 89.49±29.79nmol/L, z=-1.496,P=0.135, no statistical difference for the 25-(OH)D3 levels was found between boys and girls (P>0.05).The results of 25-(OH)D3 of aged 0-3 belonged to the normal distribution(P=0.293), the average level of 25-(OH)D3 of aged 0～3 was 122.36±29.19nmol/L;the results of 25-(OH)D3 of aged 4-7 belonged to the normal distribution(P=0.179), the average level of 25-(OH)D3 of aged 4～7 was 86.11±21.58nmol/L; The results of 25-(OH)D3 of aged 8-14 belonged to the non-normal distribution(P=0.021), the mean level of 25-(OH)D3 of it was 74.33±26.95nmol/L. Significant difference was found in the level of 25-(OH)D3 between children aged 0～3 and 4～7,8～14, while no such found between children aged 4～7 and 8～14. The results of 25-(OH)D3 of picky children and balanced diet children belonged to the non-normal distribution, the average level of 25-(OH)D3 of them was 83.34±29.67,91.60±58.16nmol/L, z=-2.333,P=0.02,had statistical difference between them. The results of 25-(OH)D3 of children added vitamin D and non-added vitamin D belonged to the non-normal distribution(P>0.05),the average level of 25-(OH)D3 of them was 97.01±48.38,88.43±45.25nmol/L,z=-1.161,P=0.246, had no statistical difference between them. Significant difference was found in the level of 25-(OH)D3 between malnourished children and obese children (P=0.032). There was a low negative correlation between parathyroid hormone and 25-(OH)D3 (r=-0.181. P=0.181). No correlation among calcium, phosphorus and alkaline phosphatase with 25-(OH)D3 was found (P>0.05).[Conclusion]1.Ultra high performance liquid chromatography (UPLC) and chemiluminescence method had no statistical difference. So both of the two methods can be used for detection according to the actual clinical situation.2.The 25-(OH)D3 level of Yunnan region is low and age in years remained inversely associated with 25-(OH)D3 value, meanwhile the supplement of vitamin D should be taken seriously.3.The normal range of 25-(OH)D3 in healthy children of Kunming (44.60-169.20nmol/L) was higher than the recommend rang of 《Pediatrics》 (eighth edition) of 26.70-145.60nmol/L. |
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