Significance of Discordant Results Between Confirmatory Tests in Diagnosis of Primary Aldosteronism

Abstract

Context

Current clinical guidelines recommend confirmation of a positive result in at least one confirmatory test in the diagnosis of primary aldosteronism (PA). Clinical implication of multiple confirmatory tests has not been established, especially when patients show discordant results.

Objective

The aim of the present study was to explore the role of 2 confirmatory tests in subtype diagnosis of PA.

Design and Setting

A retrospective cross-sectional study was conducted at two referral centers.

Participants and Methods

We identified 360 hypertensive patients who underwent both a captopril challenge test (CCT) and a saline infusion test (SIT) and exhibited at least one positive result. Among them, we studied 193 patients with PA whose data were available for subtype diagnosis based on adrenal vein sampling (AVS).

Main Outcome Measure

The prevalence of bilateral subtype on AVS according to the results of the confirmatory tests was measured.

Results

Of patients studied, 127 were positive for both CCT and SIT (double-positive), whereas 66 were positive for either CCT or SIT (single-positive) (n = 34 and n = 32, respectively). Altogether, 135 were diagnosed with bilateral subtype on AVS. The single-positive patients had milder clinical features of PA than the double-positive patients. The prevalence of bilateral subtype on AVS was significantly higher in the single-positive patients than in the double-positive patients. (63/66 [95.5%] vs 72/127 [56.7%], P < .01). Several clinical parameters were different between CCT single-positive and SIT single-positive patients.

Conclusion

Patients with discordant results between CCT and SIT have a high probability of bilateral subtype of PA on AVS.

aldosterone, hyperaldosteronism, hypertension, adrenal gland

Primary aldosteronism (PA) is one of the most common causes of endocrine hypertension. Its prevalence among hypertensive patients is reported to range from 5% to 20% (1-4). Chronic aldosterone excess increases arterial wall stiffness, which is known to increase the risk of cardiovascular diseases; patients with PA are at a higher risk of cardiovascular complications than those with essential hypertension even after adjusting for age and blood pressure (BP) (5-8). Clinically, subtype diagnosis of PA is critical, as the severity of cardiovascular complications and treatment plans are different between the unilateral and the bilateral subtypes. Adrenal vein sampling (AVS) is a standard procedure for subtype diagnosis. However, it is an invasive, expensive, and technically demanding procedure that cannot be performed at medical centers with limited expertise (9). Therefore, prediction of subtype diagnosis in routine clinical practice is important.

Clinical practice guidelines of the Endocrine Society and The Japan Society of Hypertension have recommended confirmation of positive result in at least one confirmatory test among the captopril challenge test (CCT), saline infusion test (SIT), oral salt-loading test, and fludrocortisone suppression test (FST) in the diagnosis of PA (10, 11). The guidelines of the Japan Endocrine Society have recommended performing at least 2 confirmatory tests (12). Therefore, in Japan, 2 confirmatory tests are often performed in routine clinical practice regardless of the result in the initial one, although not in Europe or the United States. Although CCT and SIT are commonly used to examine autonomous aldosterone secretion, the suppression mechanisms of the renin-angiotensin-aldosterone system (RAAS) are different between CCT and SIT. Hence, some patients may show discordant results between the 2 tests (13, 14). Indeed, CCT raises the renin level and suppresses aldosterone secretion through angiotensin-converting enzyme inhibition, whereas SIT results in volume overload, thereby suppressing the renin level and aldosterone secretion. However, clinical implications of discordant results between the confirmatory tests have not been addressed.

We hypothesized that a milder form of PA such as the bilateral subtype is likely to be affected by different RAAS suppression mechanisms in the diagnosis. The present study was designed to investigate the clinical implications of 2 confirmatory tests in subtype diagnosis of PA.

Materials and Methods

Study design and participants

The present cross-sectional study was conducted at Kyushu University Hospital and Sapporo City General Hospital, which are referral centers in Japan. It was an extension of the Kyushu Adrenal Database and Advanced medicine (Q-AND-A) study (15). We identified hypertensive patients with PA who underwent both CCT and SIT as confirmatory tests and exhibited a positive result in at least one test between January 2007 and April 2019. When patients with at least 1 positive result in 2 confirmatory tests (CCT, SIT, or both) desired surgical treatment, we took into account their clinical characteristics and performed AVS. We studied patients with PA whose data were available for subtype diagnosis based on AVS. We reviewed medical records to document clinical characteristics, laboratory findings, and AVS results. CCT and SIT both were performed as a part of routine clinical practice according to the guidelines of the Japan Endocrine Society (12). In Sapporo City General Hospital, we performed a furosemide upright test instead of SIT until 2016, and hence patients who visited there during this period were defined as having undergone only one test (CCT).

The present study was approved by the institutional review board of Kyushu University (approval study number: 2019-526) and was conducted according to the guidelines for clinical studies published by the Ministry of Health and Labor, Japan.

Diagnosis of primary aldosteronism

The screening test and the confirmatory tests for PA were performed according to the guidelines of the Japan Endocrine Society and the Japanese Society of Hypertension (11, 12). For the screening test and the confirmatory tests, antihypertensive medications were changed or discontinued according to the guidelines. Patients were treated only with calcium channel blockers and/or α-adrenergic blockers when required. The screening test was performed by determining the ratio of plasma aldosterone concentration (PAC) (measured in ng/dL) to plasma renin activity (PRA) (measured in ng/mL/h), which is termed the aldosterone-to-renin ratio (ARR). The test was considered positive when ARR was greater than 20 (ng/dL)/(ng/mL/h). Patients were diagnosed with PA when CCT, SIT, or both showed positive results.

Confirmatory tests

For CCT, patients received 50-mg captopril orally at 9 am after 30 minutes in the recumbent position. Blood samples were collected for the measurement of PAC and PRA before (time zero) and at 60 and 90 minutes after the administration of captopril, during which patients were in the recumbent position. The result was considered positive if ARR was greater than 20 (ng/dL)/(ng/mL/h) at 60 or 90 minutes. For SIT, patients were injected with 2 liters of 0.9% saline (NaCl) intravenously at 9 am in the recumbent position over 4 hours, after 30 minutes in the recumbent position. PAC was measured before and after the infusion of NaCl. The result was considered positive if PAC after the infusion was greater than 6.0 ng/dL.

Subtype diagnosis

Subtype diagnosis of PA was based on the AVS results. All AVS procedures were performed with cosyntropin stimulation. A continuous infusion of 50-μg cosyntropin per hour was administered 60 minutes before adrenal vein cannulation at Kyushu University Hospital. A bolus infusion of 250-μg cosyntropin was administered 30 minutes before collecting the blood samples at Sapporo City General Hospital. Adrenal vein cannulation was considered successful when the selectivity index was greater than 5 (10, 16). Selectivity index was defined as the ratio of plasma cortisol concentration in the adrenal vein to plasma cortisol concentration in the inferior vena cava. Unilateral subtype was diagnosed when the lateralization index was greater than 4 (10, 16). Lateralization index was defined as the ratio of aldosterone to cortisol ratio on the dominant side to that on the nondominant side.

Assay methods

PAC was measured using a commercially available radioimmunoassay (RIA) kit (SPAC-S Aldosterone Kits; Fujirebio Inc). The PAC reference range with patients in the recumbent position was 3.0 to 15.9 ng/dL (83.2-441.1 pmol/L). At Kyushu University Hospital, PRA was measured using a commercially available RIA kit (PRA-FR RIA kits; Fujirebio Inc), with a reference range of 0.3 to 2.9 ng/mL/h. At Sapporo City General Hospital, PRA was measured using a commercially available RIA kit (PRA radioimmunoassay kits; Yamasa), with a reference range of 0.2 to 2.7 ng/mL/h.

Analysis and statistics

We compared clinical characteristics between patients with positive results both in CCT and SIT (double-positive patients) and patients with positive result in either CCT or SIT (single-positive patients). The prevalence of bilateral subtype on AVS was compared between the double-positive and the single-positive patients. Among the single-positive patients, we also compared the clinical characteristics and the prevalence of bilateral subtype on AVS between patients with positive result only in CCT (CCT single-positive patients) and those with positive result only in SIT (SIT single-positive patients). Furthermore, we compared the clinical characteristics between patients with and without AVS data, and we also compared the clinical characteristics between the single-positive patients with and without subtype diagnosis determined by AVS.

All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.5.2). More precisely, it is a modified version of R Commander (version 2.5-1) designed to add statistical functions frequently used in biostatistics (17). Continuous variables were analyzed using the Mann-Whitney U test and were expressed as median and interquartile range. Categorical variables were analyzed using the Fisher exact test and were expressed as absolute numbers and percentages. All tests were 2-tailed, and P values less than .05 were considered statistically significant.

Results

Patients

Of 866 patients with a positive screening test being, 472 underwent either CCT or SIT, and 394 underwent both CCT and SIT. Among 394 patients who underwent both tests, we identified 360 patients with PA who had a positive result in at least one confirmatory test (CCT, SIT, or both) between January 2007 and April 2019. Among 360 patients with PA, 178 were double-positive and 182 were single-positive. Among these, 193 patients had available AVS data (127 double-positive, 34 CCT single-positive, and 32 SIT single-positive), 156 patients did not undergo AVS (46 double-positive, 65 CCT single-positive, and 45 SIT single-positive), and 11 patients had unavailable AVS data (5 double-positive, 4 CCT single-positive, and 2 SIT single-positive). The flow of diagnosis is shown in Fig. 1. Among 193 patients with available AVS data, 58 were diagnosed with unilateral subtype on AVS and 135 were diagnosed with bilateral subtype on AVS.

Figure 1.

Patient flowchart. AVS, adrenal venous sampling; CCT, captopril challenge test; SIT, saline infusion test.

Impact of 2 confirmatory tests in the subtype diagnosis of primary aldosteronism

Among 360 patients included, 178 were double-positive, whereas 182 were single-positive (103 CCT single-positive and 79 SIT single-positive). Clinical characteristics of the double-positive and the single-positive patients are summarized in Table 1. The single-positive patients exhibited milder clinical features of PA in terms of serum potassium, PAC, PRA, and ARR than the double-positive patients. Among 193 patients with available AVS data, 127 were double-positive, whereas 66 were single-positive (34 CCT single-positive and 32 SIT single-positive). Clinical characteristics of the double-positive and the single-positive patients are also summarized in Table 2. The single-positive patients exhibited milder clinical features of PA in terms of serum potassium, PAC, PRA, and ARR than the double-positive patients. The prevalence of bilateral subtype on AVS was higher in the single-positive patients than in the double-positive patients (63 out of 66 [95.5%] vs 72 out of 127 [56.7%], P < .01) (Fig. 2A).

Table 1.

Comparison of clinical characteristics of single-positive and double-positive patients

Variables	Single-positive (n = 182)	Double-positive (n = 178)	P
Age, y	54.0 (45.0-65.0)	54.0 (47.0-64.0)	.92
Female, n (%)	129 (70.9)	91 (51.1)	< .01
BMI, kg/m2	25.0 (22.0-26.9)	24.7 (22.1-27.1)	.96
Systolic BP, mm Hg	137 (127-149)	138 (130-147)	.70
Diastolic BP, mm Hg	87 (77-94)	88 (78-96)	.14
Serum potassium, mEq/L	3.9 (3.7-4.1)	3.6 (3.3-3.9)	< .01
PAC, ng/dL	13.6 (10.3-18.8)	19.8 (13.8-29.8)	< .01
PRA, ng/mL/h	0.4 (0.2-0.7)	0.2 (0.2-0.4)	< .01
ARR, ng/dL per ng/mL/h	33.8 (23.0-58.8)	74.3 (46.3-150.0)	< .01

Abbreviations: ARR, aldosterone to renin ratio; BMI, body mass index; BP, blood pressure; PAC, plasma aldosterone concentration; PRA, plasma renin activity.

Table 2.

Comparison of clinical characteristics of single-positive and double-positive patients with available adrenal vein sampling data

Variables	Single-positive (n = 66)	Double-positive (n = 127)	P
Age, y	50.0 (42.3-56.8)	54.0 (46.0-62.0)	.03
Female, n (%)	45 (68.2)	60 (47.2)	< .01
BMI, kg/m2	25.0 (22.8-27.3)	24.5 (22.0-26.9)	.61
Systolic BP, mm Hg	137 (129-150)	138 (130-147)	.86
Diastolic BP, mm Hg	88 (80-96)	89 (80-98)	.45
Serum potassium, mEq/L	3.9 (3.6-4.0)	3.5 (3.1-3.9)	< .01
PAC, ng/dL	14.0 (11.0-20.4)	22.8 (15.7-33.6)	< .01
PRA, ng/mL/h	0.4 (0.2-0.7)	0.2 (0.2-0.3)	< .01
ARR, ng/dL per ng/mL/h	36.5 (24.0-52.8)	85.8 (51.9-171.5)	< .01

Abbreviations: ARR, aldosterone to renin ratio; AVS, adrenal vein sampling; BMI, body mass index; BP, blood pressure; PAC, plasma aldosterone concentration; PRA, plasma renin activity.

Figure 2.

The prevalence of bilateral subtype on adrenal vein sampling (AVS). A, Comparison of the prevalence of bilateral subtype on AVS in single-positive patients vs double-positive patients. B, Comparison of the prevalence of bilateral subtype on AVS in captopril challenge test (CCT) single-positive patients vs saline infusion test (SIT) single-positive patients.

Difference in clinical characteristics between captopril challenge test–positive and saline infusion test–positive patients

Among 66 single-positive patients with available AVS data, CCT single-positive patients (n = 34) were older, had lower body mass index (BMI), and lower diastolic blood pressure than SIT single-positive patients (n = 32) (Table 3). Furthermore, CCT single-positive patients had lower PAC, lower PRA, and higher ARR than SIT single-positive patients. On subtype diagnosis, 31 out of 34 (91.2%) CCT single-positive patients were diagnosed with bilateral subtype on AVS, whereas all (100.0%) SIT single-positive patients were diagnosed with bilateral subtype on AVS (P = .24) (Fig. 2B).

Table 3.

Comparison of clinical characteristics of single-positive patients

Variables	CCT single-positive (n = 34)	SIT single-positive (n = 32)	P
Age, y	52.0 (48.0-63.5)	44.0 (40.0-52.3)	< .01
Female, n (%)	26 (76.5)	19 (59.4)	.19
BMI, kg/m2	24.3 (21.7-25.6)	25.4 (23.8-28.5)	.03
Systolic BP, mm Hg	137 (123-147)	138 (132-158)	.16
Diastolic BP, mm Hg	84 (73-92)	90 (86-97)	.01
Serum potassium, mEq/L	3.9 (3.6-4.0)	3.8 (3.7-3.9)	0.29
PAC, ng/dL	12.1 (9.1-17.1)	17.6 (13.7-24.2)	< .01
PRA, ng/mL/h	0.3 (0.2-0.4)	0.6 (0.5-0.8)	< .01
ARR, ng/dL per ng/mL/h	46.4 (29.9-64.9)	30.9 (21.5-40.2)	< .01

Abbreviations: ARR, aldosterone to renin ratio; BMI, body mass index; BP, blood pressure; CCT, captopril challenge test; PAC, plasma aldosterone concentration; PRA, plasma renin activity; SIT, saline infusion test.

Difference in clinical characteristics between patients with and without adrenal vein sampling data

Among 360 hypertensive patients, AVS data of subtype diagnosis were available in 193 patients. Clinical characteristics of patients with and without AVS data are summarized in Table 4. Patients with AVS data were younger and had higher diastolic blood pressure, lower serum potassium, higher PAC, and higher ARR than those without AVS data.

Table 4.

Comparison of clinical characteristics of patients with and without available adrenal vein sampling data

Variables	Patients with AVS data (n = 193)	Patients without AVS data (n = 167)	P
Age, y	52.0 (44.0-62.0)	57.0 (47.5-67.0)	< .01
Female, n (%)	105 (54.4)	115 (68.9)	< .01
BMI, kg/m2	24.7 (22.2-27.1)	25.0 (22.0-27.0)	.55
Systolic BP, mm Hg	138 (130-148)	137 (127-148)	.23
Diastolic BP, mm Hg	89 (80-97)	85 (76-94)	< .01
Serum potassium, mEq/L	3.7 (3.3-3.9)	3.9 (3.7-4.2)	< .01
PAC, ng/dL	19.1 (13.3-27.5)	13.8 (10.8-18.8)	< .01
PRA, ng/mL/h	0.3 (0.2-0.5)	0.3 (0.2-0.6)	.05
ARR, ng/dL per ng/mL/h	64.5 (36.0-128.0)	41.0 (26.7-65.5)	< .01

Abbreviations: ARR, aldosterone to renin ratio; AVS, adrenal vein sampling; BMI, body mass index; BP, blood pressure; PAC, plasma aldosterone concentration; PRA, plasma renin activity.

Difference in clinical characteristics between single-positive patients with and without subtype diagnosis determined by adrenal vein sampling

Among 182 single-positive patients, 72 patients underwent AVS. Clinical characteristics of patients with and without subtype diagnosis are summarized in Table 5. Patients with subtype diagnosis were younger and had lower serum potassium and high tendency of PAC than those without subtype diagnosis.

Table 5.

Comparison of clinical characteristics of single-positive patients with and without subtype diagnosis determined by adrenal vein sampling

Variables	Patients with subtype diagnosis (n = 72)	Patients without subtype diagnosis (n = 110)	P
Age, y	50.0 (42.8-57.0)	57.0 (47.0-67.0)	< .01
Female, n (%)	50 (69.4)	79 (71.8)	.74
BMI, kg/m2	25.2 (22.8-27.2)	24.9 (21.9-26.7)	.70
Systolic BP, mm Hg	137 (129-149)	138 (126-150)	.62
Diastolic BP, mm Hg	88 (80-95)	85 (75-93)	.17
Serum potassium, mEq/L	3.8 (3.6-4.0)	4.0 (3.7-4.2)	< .01
PAC, ng/dL	14.8 (11.1-20.3)	13.0 (9.8-17.3)	.08
PRA, ng/mL/h	0.4 (0.2-0.7)	0.3 (0.2-0.7)	.47
ARR, ng/dL per ng/mL/h	36.5 (23.7-54.6)	31.8 (22.7-60.0)	.60

Abbreviations: ARR, aldosterone to renin ratio; AVS, adrenal vein sampling; BMI, body mass index; BP, blood pressure; PAC, plasma aldosterone concentration; PRA, plasma renin activity.

Discussion

The present study demonstrated that patients with PA who had discordant results between CCT and SIT exhibited a high probability of being diagnosed with bilateral subtype of PA on AVS. To our knowledge, the present study is the first to assess the significance of discordant results between CCT and SIT. Recent studies have demonstrated that PA is a spectrum and the distinction between biochemically overt PA and renin-independent aldosterone production is an arbitrary construct limited to identifying the most severe PA (18-20). However, the current clinical practice guidelines by the Endocrine Society have suggested medical treatment including mineralocorticoid receptor antagonists (MRAs) when ARR-positive patients with at least one negative result in the confirmatory tests (CCT, SIT, or both) are unwilling and/or unable to undergo further investigation (10). Furthermore, recent evidence has shown that treatment with an MRA is effective in patients with resistant hypertension or low renin hypertension, who would not meet the cutoff of confirmatory tests (18, 19). It is therefore conceivable that patients with an initial negative confirmatory test are unlikely to have unilateral subtype of PA; they might be treated with an MRA without adding a second confirmatory test.

Among the single-positive patients with available AVS data, all SIT single-positive patients (n = 32) were diagnosed with a bilateral subtype on AVS, whereas 31 out of 34 CCT single-positive patients were diagnosed with bilateral subtype on AVS. Although SIT could diagnose all patients with unilateral subtype on AVS when the cutoff of PAC was set as 4.5 ng/dL, this cutoff showed low specificity (11.9%) for the diagnosis of a unilateral subtype. This finding is consistent with the findings of a previous report (21). However, there was no significant difference in the prevalence of the bilateral subtype on AVS between CCT single-positive and SIT single-positive patients (see Fig. 2B). In addition, it was reported that SIT in the recumbent position could not detect some cases of unilateral aldosterone-producing adenomas (22, 23). Patient characteristics such as severe hypertension could help choose which test is performed (24).

Among the single-positive patients, several clinical parameters were different between CCT single-positive patients and SIT single-positive patients (see Table 3). PRA is known to decline with age (25-27), and obesity might increase aldosterone secretion by aldosterone-stimulating factors such as leptin and complement C1q tumor necrosis factor–related protein 1 (28-31). These observations might explain the older age with relatively low PRA in CCT-positive patients and higher BMI with relatively high PAC in SIT-positive patients. However, some overlaps in clinical parameters were observed between CCT single-positive patients and SIT single-positive patients. Thus, we could not determine which test (CCT or SIT) is more appropriate as the initial confirmatory test of PA in the present study.

The present study has some limitations, including first the lack of performed FSTs. However, a recent study reported that the accuracy of the CCT and SIT for the diagnosis of PA is comparable to that of the FST (32). Second, the AVS data on subtype diagnosis were unavailable in 167 patients because of the retrospective design of the present study. It is therefore possible that some of the single-positive patients without AVS data were of a unilateral subtype. However, clinical parameters related to PA were milder in patients without AVS data than in those with AVS data (see Table 4), suggesting that they had a high probability of having a bilateral subtype of PA (33). Third, in the single-positive patients, the percentage of having undergone AVS was low (single-positive: 39.6%, double-positive: 74.2%), and hence, there may be a selection bias. However, patients without a subtype diagnosis exhibited milder clinical features of PA than those with a subtype diagnosis (see Table 5), suggesting they had a high probability of having a bilateral subtype of PA. We considered the selection bias would not significantly affect the conclusion of the present study. Fourth, we used the criteria and cutoffs of the CCT and SIT according to the Japan Endocrine Society guidelines (12) in the present study. Therefore, it is possible that the prevalence of bilateral subtype would decrease in patients who have discordant results between confirmatory tests, especially when the strict criteria and/or cutoffs are used. Fifth, in the present study, we measured PAC and PRA using the respective RIA kits. It is unclear whether our findings can be extended to other institutions. Further validation of our results and the use of standardized measurement methods such as liquid chromatography and mass spectrometry are required in future studies.

In conclusion, the present study suggested that when 2 confirmatory tests were performed, patients with discordant results between the confirmatory tests had a high probability of being diagnosed with a bilateral subtype of PA on AVS. Thus, when patients have a negative result in the initial confirmatory test, they could be treated with medication for PA without further investigations, including a second confirmatory test.

Abbreviations

Abbreviations
 
• ARR

  aldosterone to renin ratio

 
• AVS

  adrenal vein sampling

 
• BMI

  body mass index

 
• CCT

  captopril challenge test

 
• FST

  fludrocortisone suppression test

 
• MRA

  mineralocorticoid receptor antagonist

 
• PA

  primary aldosteronism

 
• PAC

  plasma aldosterone concentration

 
• PRA

  plasma renin activity

 
• RAAS

  renin-angiotensin-aldosterone system

 
• RIA

  radioimmunoassay

 
• SIT

  saline infusion test

Acknowledgments

We thank the medical staff of Kyushu University Hospital Department of Endocrinology, Metabolism, and Diabetes for supporting the present study. We would like to thank Editage (www.editage.com) for English-language editing.

Financial Support: This work was supported in part by JSPS KAKENHI (Grant No. JP20K17493 to H.U. and Grant No. JP20K17514 to R.S), the Uehara Memorial Foundation (to H.U.), the Daiwa Securities Health Foundation (to H.U.), the Kaibara Morikazu Medical Science Promotion Foundation (to H.U.), the Japan Foundation for Applied Enzymology (to H.U.).

Additional Information

Disclosure Summary: The authors have nothing to disclose. The authors have no conflict of interest to disclose.

Data Availability

Some or all data sets generated during and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request.