Study On The Relationship Between Serum Vitamin D Metabolites And Renal Function Injury In Chronic Kidney Disease Based On UPLC-MS/MS Technology

Objective:Vitamin D(VitD)is an essential nutrient in the human body.It has been confirmed that many diseases,including Chronic Kidney Disease(CKD),are closely related to its storage in the body.For the judgment of the nutritional status of patients with VitD,almost all existing international guidelines recommend using 25-hydroxyvitamin D[25(OH)D]for evaluation.However,many studies have found that 25(OH)D is easily affected by liver and kidney function,sex steroids,genetic background,and various diseases,resulting in qualitative and quantitative changes.Therefore,25(OH)D is likely to be difficult to correctly assess the storage status of subjects’ VitD.It is necessary to fully consider the existence of different forms of VitD metabolites to accurately evaluate the storage of VitD in subjects.Due to the limitation of detection methods,there are few clinical reports at present.At present,only some studies have confirmed that serum total 25-hydroxyvitamin D[t-25(OH)D,that is 25(OH)D]levels are associated with renal function impairment in CKD patients.The detection of different levels of VitD metabolites in CKD patients and their relationship with renal function damage have not been reported so far.In this study,we tested the levels of various 25(OH)D metabolites by UPLC-MS/MS,and used different methods to evaluate the VitD storage status of CKD patients in vivo according to its different activities,in order to accurately understand the storage status of VitD in patients with CKD,and further analysed the correlation between different VitD metabolites and renal function indicators in CKD patients,and explored different VitD metabolites possible relationship with CKD renal function injury.Methods:Randomly selected 389 healthy volunteers(healthy control group)and 401 CKD patients(CKD group)who came to Mianyang Central Hospital for treatment from January 2019 to December 2019.Based on KDIGO guidelines,the CKD patients were divided into 4 subgroups according to the eGFR level:①CKD stage 2(65 cases):eGFR is 60～89ml/min/1.73m2;②CKD stage 3(138 cases):eGFR is 30～59ml/min/1.73m2;③CKD stage 4(88 cases):eGFR is 15～29ml/min/1.73m2;④CKD stage 5(110 cases):eGFR<15ml/min/1.73m2.According to the K/DOQI clinical application guidelines,CKD patients were divided into 3 subgroups according to t-25(OH)D levels:①VitD deficiency group(52 cases):t-25(OH)D<15ng/mL;②VitD insufficient group(179 cases):15ng/mL≤t-25(OH)D<30ng/mL;③VitD sufficient group(170 cases):t-25(OH)D≥30ng/mL.Serum levels of 25(OH)D2,25(OH)D3,C3-epimer-25(OH)D3(C3-epi)were measured by UPLC-MS/MS,and free 25(OH)D[f-25(OH)D]was detected via ELISA.The t-25(OH)D,bioavailable vitamin D(BAVD),25(OH)D2/25(OH)D3[25(OH)D2/D3]ratio,C3-epi/t-25(OH)D ratio,f-25(OH)D/t-25(OH)D ratio,and BAVD/t-25(OH)D ratio were calculated,respectively.At the same time,renal function indicators(Urea,Cr,CysC,eGFR and UACR)were tested by a biochemical analyzer.Results:Compared with the healthy control group,the 25(OH)D2/D3 ratio,C3-epi level and C3-epi/t-25(OH)D ratio of the CKD group were distinctly elevated(all P<0.05).The levels of t-25(OH)D,25(OH)D3,C3-epi,f-25(OH)D and BAVD in CKD stage 5 were notably reduced than those in stages 2,3,and 4(all P<0.05).The ratios of C3-epi/t-25(OH)D,f-25(OH)D/t-25(OH)D and BAVD/t-25(OH)D in the VitD deficiency group were distinctly elevated than those in the VitD insufficient group or sufficient group(all P<0.05).The levels of Urea,Cr,CysC and UACR in the VitD deficiency group were apperently ascended than those in the VitD insufficient group and sufficient group(all P<0.05).The eGFR level of VitD deficiency group was makedly descended than that of VitD insufficient group and sufficient group(all P<0.05).The calculated VitD storage according to Method④[25(OH)D2/3+25(OH)D3]was only 32.17%,which was lower than the results of 52.87%by Method①[25(OH)D2+25(OH)D3+C3-epi],47.13%by Method②[25(OH)D2/3+25(OH)D3+C3-epi]and 42.39%by Method③[25(OH)D2+25(OH)D3].In addition,the VitD results calculated by four methods were positively associated with f-25(OH)D and BAVD.Both f-25(OH)D and BAVD were positively associated with serum 25(OH)D2,25(OH)D3,t-25(OH)D and C3-epi levels,and negatively correlated with 25(OH)D2/D3(all P<0.05).C3-epi was positively correlated with 25(OH)D2,25(OH)D3 and t-25(OH)D.The levels of 25(OH)D3,t-25(OH)D,C3-epi,f-25(OH)D and BAVD were negatively correlated with Urea,Cr,CysC and UACR,respectively,and positively related to eGFR(all P<0.05).CKD patients in three VitD states,the correlation of 25(OH)D3 or t-25(OH)D with some renal function indicators was very weak to moderate,while f-25(OH)D or BAVD was almost related to all observed renal function indicators(Urea,Cr,CysC,eGFR,UACR)had weak to moderate correlation.Conclusion:①More than 50%of CKD patients were in a state of deficiency and insufficient of VitD,which is related to the severity of CKD.②Serum levels of t-25(OH)D,25(OH)D3,f-25(OH)D and BAVD in CKD patients gradually decreased with the increase of CKD stages,suggesting t-25(OH)D,25(OH)D3,f-25(OH)D,BAVD may be associated with the development of CKD patients.③The ratio of C3-epi/t-25(OH)D increased significantly in CKD patients in the state of VitD deficiency,suggesting a compensatory relative increase in C3-epi as VitD level(i.e.,t-25(OH)D level)decrease.When the disease progressed to stage 5 of CKD,C3-epi began to decrease again,indicating that the change in C3-epi level may be related to the compensatory mechanism and pathological state of CKD patients.④ Serum t-25(OH)D,25(OH)D3,f-25(OH)D and BAVD in CKD patients were negatively correlated with related renal function indicators(Urea,Cr,CysC,UACR),and positively correlated with eGFR,indicating that the reduction of VitD metabolites may be related to the patient’s renal function damage.Among the metabolites of VitD,the relationship between f-25(OH)D and BAVD and renal function indicators(Urea,Cr,CysC,eGFR,UACR)was closer than that of 25(OH)D3 or t-25(OH)D,which indicated that f-25(OH)D and BAVD may be more useful biomarkers for assessing the status of VitD and the severity of renal damage in CKD patients.⑤When detecting VitD,if the f-25(OH)D or BAVD level with biological activity is detected at the same time,the storage status of VitD in the patient’s body can be accurately understood.Therefore,f-25(OH)D and BAVD may be used as potential clinical biomarkers for evaluating VitD nutritional status,available VitD and disease severity in CKD patients.