LC-MS/MS In Diagnosing KCNJ5 Mutant Aldosterone-producing Adenoma

BacKground:Primary aldosteronism(PA)is a group of disorders where adrenal aldosterone production and secretion is inappropriately high.The clinical manifestations are mainly hypertension,with or without hypokalemia.The guidelines recommend that patients with unilateral aldosterone-producing adenoma(APA)should undergo surgical treatment.Primary Aldosteronism Surgical Outcome(PASO)has been used to evaluate the surgical outcomes of patients.Most patients can benefit from surgery.The most common somatic mutation in APA is KCNJ5 germline mutation.APA patients with KCNJ5 mutation have better clinical outcomes after surgery.Therefore,it is urgent to diagnose KCNJ5 mutant APA and perform surgical treatment in time.Liquid-mass spectrometry/mass spectrometry(LC-MS/MS)has been utilized to measure the trace steroids in peripheral venous serum due to its high sensitivity.The steroid profile related to adrenal steroid synthesis in peripheral venous measured by LC-MS/MS helps diagnose different subtypes of PA,but this method hasn’t been used to identify KCNJ5 mutant APA in China.Steroids in the human body has great fluctuations,while scalp hair offers information about systemic steroids’ level over a long time.Currently,no research has been conducted to discriminate subtypes of APA by measuring the steroid profile in scalp hair.We hope that a novel detection method could be established to diagnose subtypes of APA more noninvasively and effectively.Methods:In this study,we enrolled 52 patients with APA(30 with KCNJ5 mutation and 22 without KCNJ5 mutation)who underwent surgical treatment in our hospital.The scalp hair and peripheral venous serum of patients before surgery,as well as tumor samples were collected by us.First-generation sequencing was used to determine whether patients have KCNJ5 gene mutations.The surgical outcomes of patients were evaluated according to PASO.We took advantage of LC-MS/MS to measure the steroid profile in patients’peripheral venous serum and scalp hair.Linear fit and correlation analysis were performed on the content of steroids measured in peripheral venous serum and scalp hair.Models were established by orthogonal projections to latent structures(OPLS)and Random Forest based on peripheral venous steroid profile and scalp hair steroid profile.These Models were constructed to help diagnose APA with or without KCNJ5 mutation.In the Random Forest model,the error rates of the two models based on peripheral venous serum steroid profile and scalp hair steroid profile respectively were compared through 10-fold cross-validation.Results:Compared with patients without KCNJ5 mutations,KCNJ5 mutant patients have lower serum potassium levels(2.88 vs 3.37 mmol/L),a smaller diameter on CT(1.49 vs 1.92 cm),and a higher percentage of clinical outcomes of complete success(58%vs 28%).For steroid profile measured in peripheral venous serum,the Q2 in OPLS model was 0.976,and the average error rate of the test set is 0.06 after 10-fold cross-validation in Random Forest model.The most important factors were 18-oxocortisol,18-hydroxycortisol and 11-deoxycorticosterone in these two models.For the first time,we quantitatively detected 18-oxocortisol(r=0.48),18-hydroxycortisol(r=0.64),aldosterone(r=0.88),11-deoxycorticosterone(r=0.73)and 11-deoxycortisol(r=0.75)in scalp hair.Except for 18-oxocortisol and 18-hydroxycortisol,the levels of other steroids in scalp hair and peripheral venous serum had a strong correlation.For steroid profile measured in scalp hair,the Q2 in OPLS model was 0.46,and the average error rate of the test set was 0.33 in Random Forest model.In these two models,the most important factors were 11-deoxycorticosterone,DHEA and DHEA-SO4.Discussion:Steroid profile in peripheral venous discriminates APA with or without KCNJ5 mutations well in our enrolled patients.We are the first to take advantage of the steroid profile in scalp hair to help diagnose APA with KCNJ5 mutation.However,the prediction value of the scalp hair model was not as good as the peripheral venous model.In subsequent studies,the measurements of scalp hair’s steroids should be further improved,and the correlation between 18-oxocortisol and 18-hydroxycortisol in scalp hair and peripheral venous serum should be investigated to help us better diagnose PA subtypes.Conclusion:The steroid profiles related to adrenal steroid synthesis in peripheral venous and scalp hair measured by LC-MS/MS help diagnose KCNJ5 mutant APA.