| ObjectiveVitamin D is a fat-soluble vitamin that not only affects bone growth and remodeling,but also plays a potential role in diabetes,metabolic syndrome,cancer,and autoimmune diseases.Vitamin D deficiency has become a common health concern worldwide.25-hydroxyvitamin D has become the current preferred biomarker to access the vitamin D status.However,recently studies revealed that serum Free-25OHD might be a more useful biomarker to access the vitamin D status than T-25OHD.Up to now,there are two methods to evaluate the Free-25OHD including directly method(ELISA)and indirectly method(calculation).Both detection methods have advantages and disadvantages,and the consistency between them was not ideal.Thus,it is needed to find a more reliable method to measure serum Free-250HD.The aim of this study was as follows:Section 1:to establish serum Free-25OHD method based on LC-MS/MS and verify it’s performance and evaluate the consistency among three Free-25OHD methods(LC-MS/MS,ELISA and calculation methods);Section 2:clinical application of serum Free-25OHD method based on LC-MS/MS was evaluated;Section 3:to explore the influence of sex,age,and season on T-25OHD.MethodSection 1:Serum was separated by ultrafiltration,and Free-25OHD was extracted by liquid-liquid extraction.After derivatization,LC-MS/MS analysis was performed.According to CLSI related documents,precision,sensitivity,accuracy,linear,carryover,matrix effect and method comparison were verified.Section 2:(1)The clinical residual serum from 120 apparently healthy individuals who departed in health check-up center from April to November 2019 were collected to evaluate the distribution of serum Free-25OHD;(2)The remaining serum samples of 312 apparently healthy elderly individuals who departed in the health check-up center from November 2018 to February 2019 were collected.To evaluate the distribution of Free-25OHD in the elderly population and correlations between Free-25OHD,vitamin D metabolites and common biochemical indicators;(3)The clinical residual serum from 65 apparently healthy elderly individuals who departed in the health check-up center from April to November 2019 at the Health Checkup Center were collected to evaluate the correlation between Free-25OHD and bone mineral density.All individuals included in the group were divided into two groups according to bone mineral density:T value>1 represents normal bone density(n=27),and T value<-1 represents bone loss(n=38).(4)Residual serum samples were collected from November,2018 to Feburary,2019 at the Health Checkup Center.Among these,62 individuals were case control,67 individuals were pre-diabetes.Section 3:T-25OHD measurements results and basic information were collected during from December 2015 to October 2018 to evaluate the effect of sex,age,and season on serum T-25OHD.ResultsSection 1:The repeatability and within laboratory precision were 6.0%—9.3%and 11.0%—17.7%based on LC-MS/MS to detect serum Free-250HD.The limit of detection(LOD)and limit of quantification(LOQ)were 1.9 pg/mL and 3.9 pg/mL,respectively.Matrix effect was 93.2%-105.4%.The concentrations of T-25OHD,Free-25OHD(LC-MS/MS,ELISA,and calculation method)of method comparison were 20.0±9.3 ng/mL,3.88 ± 2.14 pg/mL,3.10± 1.10 pg/mL,and 4.48 ± 2.25 pg/mL,respectively.Passing-Babloke equation showed the R2 was 0.62-0.72.Bland-Altman showed the mean deviation among three methods ranged from-0.8 pg/mL—1.4 pg/mL.Section 2:The serum Free-25OHD distribution(median,P25-P75)was 4.25(1.97-9.03)pg/mL.The median of serum Free-25OHD were 4.26 pg/mL and 4.25 pg/mL(P=0.8519).(2)The distribution of serum Free-25OHD in the elderly population(median,P2.5—P97.5)was 2.65(1.10—5.53)pg/mL.Serum T-25OHD were positively correlated with 25OHD3,1,25(OH)2D,24,25(OH)2D,and Free-25OHD,while negatively correlated with 25OHD/24,25(OH)2D.(3)The serum Free-25OHD level in the normal bone mineral density group was higher than that in the bone loss group,but the difference was not statistically significant(P=0.1690).According to bone density,the average serum Free-25OHD were 3.42 pg/mL(T<-2),4.59 pg/mL(-2 ≤T<-1),4.35pg/mL(-1 ≤T≤0),4.96 pg/mL(0 ≤ T<2)and 7.75 pg/mL(T≥2),respectively.(4)The serum Free-25OHD in the pre-diabetes group was significantly higher than that in the control group,and the difference was statistically significant(P=0.3868).There was no significant difference of serum T-25OHD between prediabetes and control group(P=0.1300).Section 3:The serum T-25OHD level in male was significantly higher than that in female(P<0.001).According to age,the median serum T-25OHD concentration was the lowest in the 19-29 age group and highest in the 0-10 age group.The median serum T-25OHD concentrations in spring,summer,autumn,and winter were 15.3 ng/mL,20.5 ng/mL,19.8 ng/mL,and 14.4 ng/mL,respectively.According to month,the serum T-25OHD was the lowest in March and the highest in August.The difference between the highest and lowest values was 61.1%.ConclusionSection 1:This study established a serum Free-25OHD method based on LC-MS/MS and the performance was verified.The consistency between LC-MS/MS method and calculation method in detecting serum Free-25OHD was better.Section 2:(1)The distribution of serum Free-25OHD was 4.25(1.97-9.03)pg/mL.(2)The serum Free-25OHD in elderly population was increased as the T-25OHD increased.(3)Serum Free-25OHD was associated with bone density.(4)Serum Free-25OHD could be a more suitable biomarker to access vitamin D status than T-25OHD in pre-diabetes.Section 3:Sex,age and season affect serum T-25OHD concentration,of which season has the greatest influence on serum T-25OHD level. |
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