| Objective: 18-hydroxycortisol(18-OHF)as a representative steroid hormone is expected to be a biochemical marker to predict aldosterone adenoma(APA),a common subtype of primary aldosteronism(PA)can be possibly cured by surgery and needs to be differentiated by adrenal venous sampling(AVS).Further study found that 18-OHF was specifically associated with KCNJ5 somatic mutations,which may relate to overlap of the expression of glomerular-like and fasciculata-like cells and related enzymology in the tumor.The relationship between plasma steroids and the subtypes of PA,as well as somatic mutations in Chinese population remains unclear.In this study,we aim to evaluate the diagnostic value of plasma 18-OHF for the subtype of PA compared with gold standard---AVS.Meanwhile,somatic mutations of PA patients who performed unilateral adrenalectomy were measured by PCR direct sequencing,and the association between plasma 18-OHF and somatic mutations was further analyzed.Therefore,first,we aim to establish a liquid chromatography-mass spectrometry(LC-MS/MS)method for quantitative analysis of plasma 18-hydroxycortisol(18-OHF)concentration.Second,to discuss the relationship between plasma 18-OHF concentration and different subtypes of PA.Third,to investigate 5 target somatic mutations(KCNJ5,ATP1A1,ATP2B3,CACNA1 D,CTNNB1)in these PA patients and describe their clinical phenotype.Final,to clarify the relationship between plasma 18-OHF concentration and different somatic mutations.Methods: 1.The experimental conditions and technical route for measurement of plasma 18-OHF concentration by liquid chromatography-mass spectrometry(LCMS/MS)were determined by referring to literatures.Patients who diagnosed with PA and subtyped by adrenal venous blood sampling(AVS)in Hypertension center of People's Hospital of Xinjiang Autonomous Region were enrolled in preliminary experiments(10 cases with peripheral and adrenal blood samples).5500 QTRAP mass spectrometer(AB SCIEX)experiment was applied to set up a stable,reliable LCMS/MS method to measure plasma 18-OHF concentration.2.A total of 45 PA patients were enrolled in this study from January to December 2017,whom were subtyped by AVS and measured plasma 18-OHF concentration by the established LC-MS/MS method in the preliminary experiments.3.(1)35 adrenal tissues of PA patients who performed surgical treatments were measured genotypes of KCNJ5,ATP1A1,ATP2B3,CACNA1 D,CTNNB1-section1,CTNNB1 exon3 six segments by PCR direct sequencing,the mutations in these pieces and their corresponding clinical phenotype were analyzed.(2)The relationship between plasma 18-OHF concentration and different genotypes was analyzed in the 35 surgery patients.Results: 1.The established LC-MS/MS method in the preliminary experiment for the determination of plasma 18-OHF concentration has satisfactory sensitivity,precision and specificity,and can be used for the absolute quantitative determination of plasma 18-OHF,with good sample preparation repeatability and stable and reliable data,laying a foundation for further research.2.45 patients with PA were subtyped by AVS including 24 patients with unilateral PA and 21 patients with bilateral PA.(1)Compared with the patients with bilateral PA,serum potassium level was significantly lower in the patients with unilateral PA(3.21±0.45 vs 3.47±0.34 mmol/L,P=0.038),and in which plasma aldosterone concentration(PAC)was higher(26.6±8.0 vs 21.5±8.8 ng/dL,P=0.051),but the difference was not reach statistical significance.There were no significant differences in age,sex proportion,SBP,DBP,BMI,aldosterone/renin activity ratio(ARR),plasma renin activity(PRA),fasting glucose,serum sodium and serum creatinine levels between the two groups.(2)Spearman correlation analysis showed that plasma 18-OHF concentration was positively correlated with ARR(r=0.37,P=0.013),negatively correlated with PRA(r=-0.32,P=0.034)and serum potassium(r=-0.31,P=0.04).(3)Plasma 18-OHF concentration was significantly higher in patients with unilateral PA than in those with bilateral PA [2.16(0.73,3.66)vs 0.78(0.14,1.15)ng/mL,P=0.001].(4)Logistic regression analysis was used to analyze the relationship between age,gender,serum potassium,PAC,plasma 18-OHF concentration and unilateral PA.The results showed that only plasma 18-OHF(OR=4.24,P=0.007)and serum potassium(OR=0.04,P=0.034)had statistical significance for unilateral PA.PAC,ARR,age and gender had no statistical significance.The results suggested that the odds ratio of plasma 18-OHF concentration for unilateral PA was 4.24 for every 1ng/mL increasing.(5)The ROC curve analysis showed that the area under curve(AUC)of plasma 18-OHF concentration to diagnose unilateral PA was 0.806,the optimal cutoff value is 1.74 ng/mL,sensitivity=65.2%,specificity=90.5%,Youden's index(YI)= 0.557,positive likelihood ratio=6.8,which is superior to PAC(AUC=0.710,sensitivity=73.9%,specificity=66.7%,positive likelihood ratio=4.1)and ARR(AUC=0.588,sensitivity=69.6%,specificity =57.1%,positive likelihood ratio=1.6).(4)To compare with gold standard test(AVS),the positive predictive value of plasma 18-OHF concentration for unilateral PA was 88.2%,the negative predictive value was 70.3%,and Kappa value was 0.55,P<0.001,suggesting that consistency of the two tests were acceptable.3.35 PA patients who underwent adrenalectomy were measured target gene fragments of KCNJ5,ATP1A1,ATP2B3,CACNA1 D,CTNNB1-segment 1 and CTNNB1 exon3 by PCR direct sequencing.(1)Gene sequencing results: i KCNJ5 mutations were found in 11 of 35 patients(4 cases of G151 R and 7 cases of L168R),with a total mutation rate of 31.4%.ii 8 patients had CACNA1 D mutation(rs117630105 C/T),with a mutation rate of 22.9%.iii No mutation was found in ATP1A1,ATP2B3,CTNNB1-segment 1 and CTNNB1 exon3.(2)The relationship between somatic mutation and clinical phenotype: i the proportion of unilateral PA in patients with KCNJ5 mutation was significantly higher than that in those with wild type(81.8% vs 45.8%,P=0.049).There was no significant difference in gender proportion,age,SBP,DBP,PRA,PAC,serum potassium,24-hour urinary potassium,ARR and tumor diameter between the mutant and wild-type patients(P>0.05).ii Among 8 patients with CACNA1 D mutation(rs117630105 C/T),3 were bilateral PA and 5 were unilateral PA.Patients with CACNA1 D mutation were significantly younger than those with wild-type(44.3±6.2 vs 51.9±8.6 years,P<0.05).There was no statistical difference in SBP,DBP,PRA,PAC,serum potassium,24-hour urinary potassium,ARR and tumor diameter between the two groups(P>0.05).(3)The relationship between plasma 18-OHF concentration and gene mutation: i The plasma 18-OHF concentrations of patients with KCNJ5 mutation were significantly higher than that of the wild-type patients[1.95(1.19,2.70)vs 0.73(0.42,1.41)ng/mL,P=0.034].ii There was no statistical difference of plasma 18-OHF concentration between CACNA1 D gene mutation and wild-type groups [0.59(0.32,2.70)vs 1.15(0.60,2.16)ng/mL,P=0.516].(3)Patients were divided into high 18-OHF group and low 18-OHF group according to the cutoff value of plasma 18-OHF>1.74ng/mL,and the proportion of KCNJ5 mutation in patients with high 18-OHF group was significantly higher than that in patients with low 18-OHF(58.3% vs 18.2%,P=0.017).However,there was no significant difference in proportion of CACNA1 D mutation(rs117630105 C/T)between the two groups(25.0% vs 22.7%,P=1.0).Conclusion: 1.We successfully established a method for the quantitative determination of plasma 18-OHF concentration by LC-MS/MS assay in the preliminary experiment.2.Plasma 18-OHF concentration was significantly higher in patients with unilateral PA than in patients with bilateral PA.Compared with gold standard---AVS,the diagnostic value of 18-OHF for unilateral PA is acceptable.However,due to the small sample size,its predictive value for selecting unilateral PA patients who need perform AVS still needs to be further verified by expanding the sample size.3.KCNJ5 mutations(151R,L168R)and CACNA1 D mutation(rs117630105 C/T)were found to be 31.4% and 22.9% in 35 PA patients.However,ATP1A1,ATP2B3 and CTNNB1 mutation were not found in this study.4.The elevated plasma 18-OHF is associated with KCNJ5 mutations,but not associated with CACNA1 D mutation. |
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