Junichiro Kitahara 19 , Yousuke Ohkubo 19 , Kouhei Kitajima 19 , Shin-ichi Nishio 19 Ako Oiwa¹ツ, Ai Sato¹ツ, Masanori Yamazakı¹ツ, Takahiro Sakuma²リAsami S ANO 3) , Yutaka Nishii 3) and Mitsuhisa Komatsu 1)* 1) 2) 3) Department of Internal Medicine, Division of Diabetes, Endocrinology and Metabolism, Shinshu University School of Medicine Ina Central Hospital Nagano Municipal Hospital Background : It is important to identify patients with primary aldosteronism (PA) who have a high possibility of having aldosterone-producing adenoma (APA). However, the usefulness of the adrenocorticotropic hormone (ACTH) stimulation test for such purposes is controversial. This study aimed to evaluate the diagnostic accuracy of the ACTH stimulation test, corrected for serum potassium concentration, in the identification of unilateral lesion among patients with PA. Methods : Seventy-nine patients with PA admitted to three medical centers were included in the study. ACTH stimulation tests and saline infusion tests (SITs) were performed. Plasma aldosterone concentration (PAC) was examined at 0, 30, and 60 min following ACTH stimulation and at 240 min following SIT. Receiver operator char- acteristic curve analysis was used to evaluate the diagnostic accuracy. Medical records were reviewed retro- spectively. Results : The area under the curve (AUC) at 240 min following SIT was 0.920, and the optimal cut-off value was 133.7 pg/mL, with a sensitivity of 92.3 % and a specificity of 93.1 %. For the ACTH stimulation test, the AUCs for the PAC-to-potassium concentration ratio at 0, 30, and 60 min were 0.979, 0.984, and 0.971, respectively. The most effective parameter for predicting unilateral lesion suggesting APA was the PAC-to-potassium concentra- tion ratio at 30 min, with a sensitivity of 100 % and a specificity of 95.5 %. Conclusion : These results show that the PAC-to-potassium concentration ratio at 30 min in the ACTH stimula- tion test, as well as the PAC at 240 min in the SIT, is an excellent and practical indicator for identifying unilater- al lesion in patients with PA. Shinshu Med J 68 : 159—168, 2020 (Received for publication January 8, 2020 ; accepted in revised form February 6, 2020) Key words : aldosterone, adenoma, adrenocorticotropic hormone, potassium, hyperaldosteronism
Introduction
I
Primary aldosteronism (PA), characterized by au- tonomous aldosterone hypersecretion and resultant renin suppression, is the most common cause of sec- ondary hypertension and causes frequent cardiovas- cular complications 1 ) 2 ) . There are two major types of PA : aldosterone-producing adenoma (APA), which arises from the adrenal cortex, and idiopathic aldo- steronism (IHA), which is caused by hyperplasia of the adrenocortical globular layer. It is critically im- portant to determine whether aldosterone hyperse- cretion from the adrenal glands is unilateral or bilat- eral, because the therapeutic approach differs based on laterality 3)4) PA is suspected in cases of juvenile hypertension, treatment-resistant hypertension, hypokalemia, or an incidental adrenal mass lesion. Because PA accounts for 5-10 % of all hypertension cases 516 , many endo¬ crinologists believe that PA screening should be per- formed in all patients diagnosed with hypertension. In 1981, our laboratory proposed that the aldoste¬ rone-to-renin ratio could help screen for PA, and its utility has been recognized around the world 77 . By using the aldosterone-to-renin ratio, we identified patients who should proceed with confirmatory tests such as the captopril test 318191 , saline infusion test (SIT) 2080-100 , upright furosemide test 8091 , and ACTH stimu- lation test 11) 12) As aldosterone overproduction is an underlying pathophysiology of PA, aldosterone antagonists are effective for treatment. In cases of hypersecretion from the bilateral adrenal glands, it is essential to continue treatment with aldosterone antagonists throughout the patient's lifetime. However, in cases of unilateral hypersecretion, most of which are cases of APA, surgical adrenalectomy of the affected side is recommended as a curative treatment. Therefore, in medical practice, it is extremely im¬ portant to identify both patients with PA among ubiquitous hypertensive patients, as well as those with a high possibility of unilateral lesion. In patients in whom a diagnosis of unilateral aldosterone hyper- secretion is unclear, adrenal vein sampling (AVS) is strongly recommended to determine the laterality of the lesion. However, implementation of AVS requires technical skill and is invasive and involves substan- tial costs 13)14) . Although it has recently been reported that SIT is useful not only as a confirmatory test of PA but also for the identification of unilateral PA 15), the SIT has technical limitations including increasing plasma volume and being time-consuming 13)14 . On the other hand, it has been reported that the ratio of PAC to potassium after ACTH loading in the ACTH loading test at the first visit to the hospital may be useful in diagnosing unilateral PA lesions 121 . There- fore, it was estimated that the ratio of PAC to potas- sium after loading before testing in the rapid ACTH loading test might be useful for diagnosing unilateral lesions. Thus, we aimed to discover a novice clinical index with high diagnostic accuracy, and compare the diagnostic ability for unilateral PA lesions be- tween SIT and the new index we found.
Materials and Methods
II
We assessed 79 patients with PA who underwent AVS after an ACTH stimulation test and SIT be- tween April 2010 and March 2019 at Shinshu Univer- sity Hospital, Nagano Municipal Hospital, and Ina Central Hospital, Japan. Fig. 1 illustrates the patient flow chart. Two patients were excluded because al- dosterone antagonists were taken at the time of AVS. Five patients were excluded due to unsuccess- ful adrenal vein cannulation. Thus, 72 patients were included and assessed. We diagnosed PA following guidelines from the Japan Endocrine Society 16 ) and the Japan Society of Hypertension 9 . We screened for PA based on a value of >200 for the plasma aldoste- rone concentration (PAC: pg/ml)-to-plasma renin activity (ng/mL/hr) or a value of >40 for the PAC- to-active renin concentration (pg/ml). PA was diag- nosed based on at least one positive result from the confirmatory tests, which included the captopril chal- lenge test, the SIT, the upright furosemide test, and the oral salt loading test 319 171 . Patients who had re- ceived prescriptions for antihypertensive drugs re¬ lated to the renin angiotensin system were placed on calcium channel-blockers and α-adrenergic-blockers until 2 weeks (ACE inhibitor and ARB) or 4 weeks (aldosterone blocker) before the diagnostic tests were performed. Serum potassium levels at the time of di- agnosis were measured routinely. In addition, serum potassium levels were measured within 24 hours of the start of the ACTH stimulation test in 39 patients. Hypokalemia was defined as a serum potassium con- centration of <3.5 mEq/L before or at diagnosis of PA or if a patient was taking a potassium supple- ment. If hypokalemia was present, we supplied the patient with oral potassium tablets.
A
Assay methods
PAC was measured using commercial radioimmu- noassay  kits (SPAC-S  Aldosterone  Kit ; Fuji  Lebio, Tokyo, Japan) at Shinshu University Hospital until May 8, 2018, and at all other centers throughout the study. From May 9, 2018, PAC was measured using commercial chemiluminescence enzyme immunoas- say kits (Accuraseed Aldosterone Kit ; Wako Pure Chemical Industries, Osaka, Japan) at Shinshu Uni¬ versity Hospital. The new method showed good cor- relation compared to the previous method (r = 0.867).
B SIT
Patients who had uncontrolled hypertension with medicine or had suspected heart failure did not un- dergo an SIT. In the remaining patients, the SIT was carried out as follows : 1) Tests were performed be- fore breakfast; 2) Patients started this test from 8:00 AM after 30 min of lying in a supine posi¬ tion; 3) After drawing a blood sample, we infused 2 liters of saline for 4 hours into the patients ; 4) At the end of the infusion, we obtained a blood sample; 5) During the test, we asked the patients to stay in a supine position, and we monitored blood pressure and symptoms. Patients who were receiving potassi- um supplementation took the potassium after the test.
C
ACTH stimulation test
We performed the test as follows: 1) Tests were performed before breakfast ; 2) Patients started this test at 8:00 AM after lying in a supine position for 30 min, and we obtained a blood sample at the start ; 3) Patients were given 250 µg of cosyntropin by in- travenous injection ; 4) blood samples were obtained at 30 min and 60 min after the injection ; 5) patients were lying in the supine position during the test. Pa- tients who were receiving potassium supplementa- tion took the potassium after the test.
D   Dexamethasone test
ACTH stimulation tests in this study did not in- clude dexamethasone loading before testing. We per- formed a 1 mg dexamethasone loading test on anoth- er day.
AVS diagnostic criteria
E
Patients with confirmed PA who wanted to under- go surgery underwent thin-slice contrast-enhanced computed tomography (CT) and AVS with ACTH stimulation. We diagnosed the subtype of PA based on the AVS results. Adrenal vein cannulation was defined as successful if the selectivity index was > 5 18 ). The selectivity index was defined as the ratio of cortisol concentration in the adrenal vein to that in the inferior vena cava. A lateralization index (LI) of >4 from AVS was used to define the presence of unilateral hyperaldosteronism 19 . The LI was calculat- ed by dividing the aldosterone-to-cortisol ratio (A/C) on the dominant side by that on the nondominant side. All patients who did not meet the criteria of
Data are presented as median (interquartile range) unless otherwise indicated. PAC : Plasma aldosterone concentration. eGFR ; estimated glomerular filtration rate
unilateral hypersecretion were considered to have bilateral hypersecretion of aldosterone. F We assessed the diagnostic accuracy of several outcomes using a receiver operating characteristic (ROC) curve analysis, and we calculated the area under the curve to determine the best level for de- termining the subtype. Statistical analyses were per- formed using EZR statistical software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) 201 . Continuous variables were expressed as median values and interquartile ranges. Categorical variables were expressed as numbers and percent- ages. Patient characteristics were compared between unilateral and bilateral lesions. We analyzed continu- ous variables using the Mann-Whitney U test and categorical variables using the Fisher exact test : i.e. the Mann-Whitney U test was used for age, body mass index, systolic blood pressure, diastolic blood pressure, initial PAC, serum potassium and eGFR, and the Fisher exact test was used for sex and pot- tasium supplement. A p -value <.05 was considered statistically significant. The study protocol was approved by the ethics committees of our hospitals in accordance with the Declaration of Helsinki. Informed consent from the patients was obtained using the opt-out method.
II
Results
Baseline characteristics
A
Patient characteristics are summarized in Table 1. Patients were classified as having either unilateral or bilateral PA based on the AVS findings. In the uni- lateral group, initial PAC was higher and initial po- tassium concentration was significantly lower than that in the bilateral group. The number of patients who needed potassium supplementation was also higher in the unilateral group. Table 2 shows the adrenal CT findings by group. In the unilateral group, 23 of 27 patients exhibited unilateral adrenal tumors >8 mm in diameter. Nineteen out of 45 pa- tients had no abnormalities on the CT scan.
B
SIT and AVS results
PAC after 240 min of saline infusion (PAC 240 ) is re- ported to be a good indicator for discrimination of unilateral and bilateral hypersecretion of aldoste- B : ROC curve of PAC values at 240 min in the SIT for differential diagnosis of bilateral and unilateral lesions. Data points represent the Youden index cutoff (specificity, sensitivity). PAC : plasma aldosterone concentration ; SIT : saline infusion test ; ROC, receiver operator characteristic : AUC : area under the curve rone 19 . As shown in Fig. 2A , PAC 240 was significant- ly higher in the unilateral group. Fig. 2B shows the ROC for PAC 2 to indicating unilateral hypersecretion of aldosterone, suggesting APA. The AUC of PAC 240 was 0.920 and the optimal cut-off value for PAC 240 was 133.7 pg/mL, with a sensitivity of 92.3 % and a specificity of 93.1 %. Thus, this was consistent with previous results 21)221 , indicating that PAC 240 was a useful parameter for discriminating between unilat- eral and bilateral aldosteronism. C    ACTH stimulation test The rapid ACTH stimulation tests were perform- ed among 58 patients with PA. Fig. 3 shows the serial change of PAC, PAC/cortisol, and PAC/potassium during the ACTH stimulation test. The values in the unilateral group were higher than those in the bilat- eral group at all time points. Upon stimulation with ACTH, PAC values increased in both groups, and the unilateral group exhibited a high concentration of PAC at 0, 30, and 60 min following ACTH stimula- tion (Fig. 4A-C). When we examined the PAC-to- cortisol ratio (PAC/cortisol), the values in both groups after ACTH stimulation were distinct (Fig. 4D-F), and the curve for the unilateral group was higher at all time points. When we plotted the PAC-to-basal potassium concentration ratio, the between-group difference was more evident (Fig. 4G-I). D   ROC curve analysis of the ACTH stimulation test for distinguishing between unilateral and bilateral primary aldosteronism We next analyzed ACTH stimulation data using an ROC curve analysis for PAC, PAC/cortisol, and PAC/ potassium at each time point (PAC 6 PAC 30 , and PAC 60 at 0, 30, and 60 min, respectively). The AUC for PAC was larger after ACTH stimulation, and the AUCs for PAC o , PAC 30 , and PAC 60 were 0.888, 0.926, and 0.948, respectively (Fig. 5A-C). Figures 5D-F illus- trate ROC curves for PAC/cortisol during the ACTH stimulation test. The AUCs for PAC 0 /cortisol, PAC 30 / cortisol, and PAC 60 /cortisol were 0.887, 0.888, and 0.922, respectively. These findings suggest that contrary to a previous report 221 , PAC/cortisol did not improve the AUC in comparison with PAC alone. In contrast, the PAC/potassium ratio was an excellent parameter for discriminating unilateral primary aldosteronism from bilateral primary aldosteronism. As shown in Fig. 5G-I, the AUCs of PAC 0 /potassium, PAC 30 / potassium, and PAC 60 /potassium were 0.979, 0.984, and 0.971, respectively. The most effective parame- ter appeared to be PAC 30 /potassium, with a sensitiv- ity of 100 % and a specificity of 95.5 %. This result suggests that unilateral primary aldosteronism can be completely excluded and a diagnosis of bilateral primary aldosteronism confirmed if PAC 3y /potassium is less than the cutoff value of 136.8 pg/mEq. Fig. 5 shows the ROC and indicates the sensitivity, specific- ity, and cutoff value for each time point. It should be noted that use of the potassium value before potassi- um supplementation resulted in a PAC o /initial potas- sium that was a relatively weak parameter, with an AUC of 0.912, sensitivity of 88.5 %, specificity of 84.1 %, and cutoff value of 48.58 pg/mEq. Therefore, po- tassium supplementation is required for hypokale- mia. um after the ACTH stimulation test (Fig. 5); AUCs were 0.929 and 0.964, respectively ( b = 0.627). E ROC curve analysis of PAC and potassium for distinguishing between unilateral and bilateral primary aldosteronism
Discussion
IV
We next compared the initial PAC, PAC 0 min in ACTH stimulation tests, initial potassium, and cor- rected potassium before the ACTH stimulation test in the patients with bilateral and unilateral PA. The AUC for PAC before the ACTH stimulation test was similar to the initial PAC, but the AUC for corrected potassium was larger than that for initial potassium. AUCs for the initial PAC, PAC 0 min in ACTH stim- ulation tests, initial potassium, and corrected potassi- um were 0.918, 0.888, 0.907, and 0.953, respectively. F Comparison of ROC curves for PAC 240 after SIT and PAC 30 /potassium after ACTH stimula- tion We performed both SIT and ACTH stimulation tests on 22 patients. Thus, we directly compared ROC curves of PAC 240 after SIT and PAC 30 /potassi- This study identifies a simple, useful indicator that distinguishes cases of unilateral aldosterone hyperse- cretion in patients with PA. Namely, in patients with PA for whom hypokalemia exists after potassium is used to normalize potassium concentrations, unilater- al adrenal lesions can be diagnosed by dividing the PAC 30 by the potassium concentration following ACTH stimulation ; this approach has a sensitivity of 100 % and a specificity of 95.5 %. SIT has long been used to diagnose PA 15)21) as intravenous injections do not sufficiently suppress PAC in patients with PA. Furthermore, in recent years, such an approach has also been proposed for the diagnosis of APA, as there is less PAC suppres- sion than in IHA 21)22). The present study confirms that SIT is useful for diagnosing unilateral lesions. PAC 240 in the SIT had a cutoff value of 133.7 pg/ mEq, sensitivity of 92.3 %, and specificity of 93.1 %. However, SIT has two disadvantages compared to the ACTH stimulation test in that first, there is some risk of increased plasma volume and second, it is time-consuming 13)14)23) The ROC curve analysis showed that PAC o /potas- sium at the start of the ACTH stimulation test is also a good indicator, with a sensitivity of 94.1 %, a speci- ficity of 95.5 %, and a cutoff value of 73.3 pg/mEq. The higher test accuracy gained by considering po¬ tassium in relation to PAC 0 indicates that aldoste- rone secretion is affected by the concentration of po- tassium at that time : potassium directly stimulates aldosterone secretion via cell membrane depolariza- tion 24 . Generally, in APA, autonomic aldosterone se- cretion is higher than that in IHA 251 , but the potassi¬ um concentration decreases due to the action of aldosterone, and the apparent concentration of PAC decreases. As a result, there may be cases where it is difficult to distinguish APA from IHA ; therefore, use of only PAC without consideration of potassium levels may result in misidentification. In APA, ACTH stimulates aldosterone secretion 24 ), and diurnal fluctuation of ACTH often leads to diur- nal fluctuation of PAC 26 . Building on these data, re- ports show that ACTH stimulation test results can be used to distinguish APA from IHA 12)23)26)-28) Furthermore, we considered that it would be better to correct the PAC value relative to cortisol levels. Moreover, Sonoyama et al. proposed an ACTH stim- ulation test wherein dexamethasone was used to suppress endogenous ACTH secretion beforehand 11 ; however, Inoue et al. reported that dexamethasone pre-administration did not improve diagnostic abili- ty 29 . Umakoshi et al. argued that the accuracy of the ACTH stimulation test was lower than that of SIT, and the usefulness of the ACTH stimulation test is still under discussion 30 ). However, as we clarified in the current study, the significance of the ACTH stimulation test is largely affected by correction for potassium concentration. Twenty-two of the present patients underwent both SIT and ACTH stimulation tests. We performed an ROC curve analysis for each of these patient groups. While there was no statistical difference between the two in terms of index, PAC 30 /potassium had higher sensitivity and specificity. Accordingly, we believe that both approaches have the same abili- ty to distinguish unilateral lesion. However, SIT has disadvantages in that the saline load given to pa- tients with PA leads to heart failure in some cases and the test takes over 4 hours to complete 31 ) . Therefore, we conclude that it is better to perform an ACTH stimulation test rather than SIT. Simple abdominal CT is an important examination in the process of PA diagnosis and treatment policy decisions. In the present study, all 27 patients with unilateral lesions diagnosed by AVS had abnormal adrenal glands. However, there were three cases in which CT showed bilateral lesions, and there were no cases in which aldosterone hypersecretion was observed on the side contralateral to where the mass was confirmed. Nonetheless, it is important to re- member that a clear mass on CT does not clearly in- dicate it as the cause of aldosterone hypersecre- tion 31 . The limitation of this study is that not all cases were able to undergo both the ACTH stimulation test and SIT, so the number of cases was insufficient to directly compare the two approaches. Further¬ more, to clarify and extend these findings, it is neces- sary to define protocols in advance and collect data at multiple facilities. In this study, SIT was per- formed in the supine position, but the results may be different if performed in the sitting position 32 ) . In ad- dition, unilateral or bilateral determination was made based on AVS alone, so a small APA may have been missed due to insufficient pathological examination in non-operative cases. Moreover, because the AVS di- agnostic criteria were based solely on criteria after ACTH loading, some APAs may have been missed 33 ). Finally, this study may include indication bias by treatment because AVS was performed only on pa- tients who wanted surgery according to guidelines 81 9)16) In conclusion, we showed that PAC 35 /potassium following an ACTH stimulation test in patients with PA is an excellent indicator to screen for unilateral lesions. Furthermore, we confirmed that the PAC 240 in the SIT is also a good indicator. Both indicators exhibited good sensitivity and specificity. For diagno- sis of unilateral PA, the ACTH stimulation test seems to be appropriate in consideration of safety and required time.