doi:10.1507/endocrj.EJ24-0227

Original

Impact of the transition from radioimmunoassay (RIA) tochemiluminescent enzyme immunoassay (CLEIA) for themeasurement of plasma aldosterone concentration (PAC)on the diagnosis of primary aldosteronism (PA) via
retrospective analyses in Okinawa, Japan
Ken-ichiro Honma, Yoshiro Nakayama, Atsuko Tamaki, Moriyuki Uehara, Taiki Teruya, Takamitsu Yabiku,
Yohei Ishiki, Ken Yonaha, Rei Chinen, Tsugumi Uema, Shiki Okamoto and Hiroaki Masuzaki
Division of Endocrinology, Diabetes and Metabolism, Hematology and Rheumatology, Second Department of Internal Medicine,
Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan
Abstract. In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay(RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection
sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC
measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primaryaldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on
PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus
Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal
venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA(ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based onscreening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by
PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The
results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordantdiagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using
PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.
Key words: Hypertension, Primary aldosteronism, Plasma aldosterone concentration, Radioimmunoassay, Chemiluminescent
enzyme immunoassay

Introduction
Primary aldosteronism (PA) is a major cause of sec‐
ondary hypertension, accounting for 5–20% of hyperten‐sion cases [1-3]. Accurate diagnosis of PA is extremely
important because it is associated with a higher preva‐
lence of cerebral, cardiovascular, and renal complica‐
tions than essential hypertension, including stroke, left
Submitted Apr. 15, 2024; Accepted May 21, 2024 as EJ24-0227
Released online in J-STAGE as advance publication ??, 2024

Correspondence to: Hiroaki Masuzaki, MD, PhD, Division of
Endocrinology, Diabetes and Metabolism, Hematology andRheumatology, Second Department of Internal Medicine, Graduate
School of Medicine, University of the Ryukyus, 207 Uehara,
Nishihara, Okinawa 903-0215, Japan.
E-mail: hiroaki@med.u-ryukyu.ac.jp©The Japan Endocrine Society

ventricular hypertrophy, atrial fibrillation, coronary
artery disease, heart failure, and proteinuria [4-6].
Historically, the establishment of plasma aldosterone
concentration (PAC) assays and adoption of thealdosterone-renin ratio (ARR) for screening (SC) have
led to an increase in the number of patients diagnosed
with PA [7]. PA is diagnosed using specific confirmatory
tests including captopril challenge test (CCT) and saline
infusion test (SIT).
In Japan, PAC measurement by liquid chromatographytandem mass spectrometry (LC-MS/MS) [8] is notwidely used because it is not covered by the National
Health Insurance system. Therefore, PAC measurement
was traditionally performed using radioimmunoassay(RIA). However, RIA presents several challenges,
including the use and disposal of radioactive isotopes,

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Honma et al.

poor traceability of certified reference materials (CRM),
and reduced detection sensitivity at low concentration
ranges [9, 10]. To address these issues, a two-site sand‐wich chemiluminescent enzyme immunoassay (CLEIA)
for PAC measurements was introduced in Japan in April
2021. PAC measurement by CLEIA (PAC(CLEIA))
demonstrated good CRM traceability and aligned wellwith the LC-MS/MS values [11].
Notably, PAC(CLEIA) tends to be lower than thatmeasured by RIA (PAC(RIA)) [12], meaning that thedirect application of PAC(CLEIA) to conventional diag‐nostic criteria may lead, at least in some cases, to themisdiagnosis of PA as essential hypertension. Given thispoint, the latest clinical guidelines published by theJapan Endocrine Society (JES) in 2021 (GL2021) [13]recommend that, for SC, PAC(CLEIA) ≥60 pg/mL andARR ≥200 should be considered positive, and
PAC(CLEIA) ≥60 pg/mL and ARR 100–200 should beconsidered provisionally positive. While the previous
guidelines [14] had a PAC(RIA) cutoff value of
120 pg/mL for SC, GL2021 sets this value at half, basedon a regression analysis of PAC(RIA) and PAC(CLEIA)
against PAC measured by LC-MS/MS [9, 11, 15, 16].
Similarly, for CCT and SIT, provisional positive criteria
were established in addition to the positive criteria. How‐ever, these provisionally positive criteria were initiallyestablished pending the widespread availability of
PAC(CLEIA) and determination of optimal cutoffs, andtheir validity has not been thoroughly evaluated.
Therefore, the present study aimed to evaluate theimpact of changes in the diagnostic criteria for SC, CCT,and SIT, resulting from the transition from RIA to
CLEIA, on PAC measurement in PA diagnosis. Further‐more, it sought to identify the clinical characteristics that
distinguish positive cases of SC, CCT, and SIT fromthose that are considered provisionally positive.

Materials and Methods
Setting and participantsThis study retrospectively reviewed the data of 190
patients admitted to the Second Department of Internal
Medicine at the University of the Ryukyus Hospital
between April 2012 and March 2021. Patients were diag‐
nosed with PA and underwent adrenal venous sampling(AVS). Data were extracted from electronic medical
records, including age, sex, body mass index (BMI), sys‐
tolic and diastolic blood pressure, heart rate, random
blood glucose level, HbA1c, serum creatinine, serum
sodium, serum potassium, serum chloride, semiqualitative test for urinary protein, presence of adrenal
nodules (defined as a tumor diameter ≥10 mm) on
abdominal CT, and results of SC, CCT and SIT. PAC

was determined using RIA (SPAC-S Aldosterone kits;
Fuji Rebio Co., Ltd., Tokyo, Japan). All participants who
underwent AVS also underwent CCT and/or SIT; there‐
fore, none were excluded. Hypokalemia was defined as aserum potassium level of <3.5 mEq/L. The study proto‐
col was approved by the Ethics Review Committee forLife Sciences and Medical Research Involving Human
Subjects at the University of the Ryukyus (approval
number: 23-2138-01-00-00). Informed consent was obtained
through an opt-out option on the hospital website.

Diagnosis of PA using PAC (RIA)
PA diagnosis was based on a consensus statement on
the clinical practice of Primary Aldosteronism in Japan(CS) [14], a guideline previously developed by the JES.
For SC, a PAC(RIA) >120 pg/mL and ARR >200 wereconsidered positive; for CCT, an ARR >200 at 60 or 90
min after 50 mg captopril intake was considered positive;
and for SIT, a PAC(RIA) >60 pg/mL after 240 min of a
2 L saline infusion was considered positive. CCT and
SIT were performed not only in patients with a positive
SC result but also in those with a negative SC result if
PA was suspected based on the physician’s judgment.
Prior to testing, antihypertensive treatment with calcium
channel blockers (CCBs) and/or alpha-blockers was
attempted. Mineralocorticoid receptor antagonists(MRAs) and/or angiotensin receptor blockers (ARBs)
were also used in some patients with inadequate bloodpressure control.
PA subtype differentiation (adrenal venous sampling)
Plasma samples were collected from both the right andleft adrenal veins and the inferior vena cava 30 minutes
after a 250 μg bolus injection of cosyntropin. The PACto-plasma-cortisol concentration ratio (A/C ratio) was
calculated for each sample. Successful catheterization of
the adrenal vein was determined by the selectivity index(SI), defined as the ratio of plasma cortisol concentrationin the adrenal vein to that in the inferior vena cava, witha value ≥5 indicating successful catheterization [13,17-19]. The lateralized ratio (LR) was used for subtype
differentiation and was calculated by dividing the A/C
ratio on the dominant side by that on the non-dominantside, with an LR ≥4 indicating unilateral PA (uPA) [13,14, 20, 21]. If the LR was between 2 and 4, it was con‐sidered borderline, and subtype differentiation was deter‐
mined concerning an adrenal vein PAC(RIA) ≥14,000
pg/mL or the contralateral ratio (CR) [13, 14]. The CR
was calculated by dividing the A/C ratio on the nondominant side by that of the inferior vena cava, with aCR ≤1 indicating uPA. Patients diagnosed with uPAunderwent laparoscopic adrenalectomy, whereas those
not diagnosed with uPA were diagnosed with bilateral

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New aldosterone assay impacts diagnosis

PA (bPA) and treated with MRAs. Histopathologicaldiagnosis was performed by a well-trained pathologist at
our university hospital following laparoscopic adrenalec‐
tomy. In the present study, patients diagnosed with bPA
and those with uPA but found to have adrenal hyperpla‐
sia rather than aldosterone-producing adenoma (APA) on
histopathological examination were defined as having
idiopathic hyperaldosteronism (IHA).

Calculating estimated PAC(CLEIA) (ePAC(CLEIA))
PAC(RIA) was converted to the estimated
PAC(CLEIA) (ePAC(CLEIA)) using the following con‐version formula:ePAC(CLEIA) = 0.852 × PAC(RIA) – 36.0 [9]This formula was derived from a regression analysis
comparing PAC measured by RIA and PAC measured by
CLEIA using PAC measured by LC-MS/MS as a refer‐ence [9].
Simulating determinations in GL2021 using
ePAC(CLEIA)
Initially, the SC, CCT, and SIT determinations inGL2021 were simulated using ePAC(CLEIA), and theoutcomes were compared with those obtained from the
CS. The cutoff values of PAC(RIA) in CS, the corre‐
sponding cutoff values of ePAC(CLEIA) in CS, and thecutoff values of PAC(CLEIA) in GL2021 for SC, CCT,and SIT are shown in Supplementary Table S1. We cal‐culated the percentage of discrepant results for each of
the SC, CCT, and SIT tests between the GL2021 and CS.
Subsequently, the process was extended to simulate PA
diagnosis in GL2021 using ePAC(CLEIA). In the present
study, a PA diagnosis was defined as either SC positiveor provisionally positive, and positive in at least one of
the CCT or SIT. SC positive or provisionally positiveand provisionally positive on at least one of the CCT or
SIT was defined as a provisional diagnosis of PA. Dis‐
crepancies in diagnosis were then assessed, and patientsdiagnosed with PA or provisionally diagnosed with PA in
GL2021, who also received a PA diagnosis in CS, wereclassified as having concordant diagnoses. In contrast,cases diagnosed or provisionally diagnosed with PA in
GL2021, but not in CS, were considered discordant.
Similarly, patients with a negative PA diagnosis in
GL2021 who were not diagnosed with PA by CS werelabeled as concordant; however, cases with a negative PA
diagnosis in GL2021 but diagnosed as PA by CS wereidentified as discordant. Finally, the clinical characteris‐tics of the patients with discordant diagnoses werereviewed.
Additional analysis
We conducted an in-depth analysis of the clinical char‐acteristics of patients with confirmed, provisional, andnegative PA diagnoses using the GL2021. This included
a comparison of uPA and bPA in terms of clinical param‐
eters. We further subdivided the patients diagnosed with
PA based on the presence or absence of hypokalemia or
adrenal nodules to compare the prevalence of APA
among these subgroups.

Statistical methods
All statistical analyses were conducted using EZR(version 1.64; Saitama Medical Center, Jichi Medical
University, Saitama, Japan), which is a graphical userinterface for R (version 4.3.1; R Foundation for Statisti‐
cal Computing, Vienna, Austria). It is a modified version
of R Commander, enhanced with additional statistical
functions frequently used in biostatistics [22]. Continu‐
ous variables are presented as mean ± standard deviation(SD) or median (interquartile range (IQR)), depending on
their distribution, and were analyzed using MannWhitney U test for comparisons between two groups.
One-way analysis of variance (ANOVA) was used toassess statistical differences among the three groups. For
post-hoc comparisons, the Bonferroni correction method
was used to adjust for multiple comparisons. Fisher’s
exact test was used to compare the proportions of
categorical variables among the three groups, and the
Bonferroni correction method was used for post-hoc
comparisons to adjust for multiple comparisons. The
threshold for statistical significance was set at p < 0.05.

Results
Clinical features
This study included 27 patients with APA and 163
patients with IHA who underwent AVS at our institution
between April 2012 and March 2021. The clinical char‐acteristics of all 190 patients are shown in Table 1.
Comparison of screening results using PAC(RIA)
according to CS and ePAC(CLEIA) according toGL2021
In a retrospective analysis, SC using PAC (RIA)
according to CS identified 148 positive and 42 negative
cases. However, by applying ePAC(CLEIA) and aligning
it with GL2021, we identified 117 positive, 43 provision‐
ally positive, and 30 negative cases. Of the PAC(RIA)
SC positive cases, ePAC(CLEIA) reclassified 38 cases asprovisionally positive and retained 110 cases as positive,
with no negatives. Conversely, among the SC negative
cases identified by PAC(RIA), ePAC(CLEIA) classified
seven cases as positive, five as provisionally positive,
and 30 as negative. This reclassification resulted in diag‐nostic discordance in 12 cases (6.4%), as illustrated in

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Honma et al.

Table 1

Clinical characteristics of all patients

Number190

Male (%)

70 (36.8)

Age (year)

51 ± 12

Body mass index (kg/m2)

26.5 ± 4.6

Systolic blood pressure (mmHg)

134 (124–144)

Diastolic blood pressure (mmHg)

80 (70–88)

Heart rate (/min)

75 (68–82)

Random blood glucose (mg/dL)

94 (88–102)

HbA1c (%)

5.6 (5.3–6.0)

Cre (mg/dL)

0.65 (0.55–0.78)

Na (mEq/L)

140 (139–141)

Hypokalemia (%)

30 (15.8)

K (mEq/L)

3.8 (3.5–4.0)

Cl (mEq/L)

104 (103–106)

Semi-qualitative test for urinary protein (%)–

157 (84.9)±

18 (9.7)

1+

9 (4.9)2+

1 (0.5)Adrenal nodules (%)

95 (50)

Number or antihypertensive drugs (%)
0

42 (22.1)

1

127 (66.8)

2

19 (10.0)32 (1.1)

Classification of antihypertensive drugs (%)
Calcium channel blockers

145 (76.3)

Alpha-blockers

12 (6.3)

Angiotensin receptor blockers

8 (4.2)Mineralocorticoid receptor antagonists

3 (1.6)

Beta-blockers

2 (1.1)

Diuretics

1 (0.5)

Potassium supplements (%)

27 (14.2)

Aldosterone-producing adenoma (%)

27 (14.2)

Data are expressed as mean ± SD, median (IQR), and n (%).
HbA1c, glycated hemoglobin; Cre, serum creatinine; Na, serum
sodium; K, serum potassium; Cl, serum chloride.

Fig. 1A, all of which were identified as IHA. Of these
discordant cases, only four cases showed provisional or
positive results on either CCT or SIT, leading to threecases with a provisional diagnosis and one case with aconfirmed diagnosis of PA, as detailed in Supplementary
Table S2.

Comparison of CCT results using PAC(RIA)
according to CS and ePAC(CLEIA) according toGL2021
CCT using PAC(RIA) according to CS identified 129
positive and 59 negative cases. Using ePAC(CLEIA)
according to the GL2021 criteria, 69 results were posi‐
tive, 49 were provisionally positive, and 70 were nega‐tive. Of the PAC(RIA) CCT positive cases, 69 werepositive, 45 were provisionally positive, and 15 were
negative with ePAC(CLEIA). Among the PAC(RIA)
CCT negative cases analyzed with ePAC(CLEIA), none
were reclassified as positive, four were identified as pro‐visionally positive, and 55 cases remained negative,
resulting in diagnostic discordance in 19 cases (10.1%)
between PAC(RIA) and ePAC(CLEIA) (Fig. 1B).
Among the 19 patients with discordant CCT results
between PAC(RIA) and ePAC(CLEIA), one was diag‐
nosed with APA, while the remaining 18 were diagnosed
with IHA. Of the 15 cases that were CCT positive by
PAC(RIA) and negative by ePAC(CLEIA), in SC, seven
were positive, one was provisionally positive, and theremaining seven were negative. In SIT, of the 11 cases,
one case was positive, three were provisionally positive,
and the remaining seven were negative (SIT was not per‐formed in four cases). None of these cases were ulti‐
mately diagnosed or provisionally diagnosed with PA
because of negative SC results, despite four cases having
at least one positive or provisionally positive CCT or SIT
result. Among the four cases that were CCT negative by
PAC(RIA) and provisionally positive by ePAC(CLEIA)
in SC, two of the four cases were positive, and two were
provisionally positive. In SIT, three of the four cases
were positive or provisionally positive, with all fourcases having at least one positive or provisionally posi‐tive CCT or SIT. This resulted in three cases being provi‐
sionally diagnosed with PA and one case being
diagnosed with PA (Supplementary Table S3).
Comparison of SIT results using PAC(RIA)
according to CS and ePAC(CLEIA) according toGL2021The SIT using PAC(RIA), in accordance with the CS,
identified 66 positive and 30 negative cases. Using
ePAC(CLEIA) according to the GL2021 criteria, 21
results were positive, 45 were provisionally positive, and30 were negative. Of the 66 SIT positive cases identified
by PAC(RIA), 21 were positive by ePAC(CLEIA), 45 were
provisionally positive, and none were negative. Amongthe 30 cases that were SIT negative by PAC(RIA), none
were identified as positive or provisionally positive byePAC(CLEIA), and all were confirmed as negative by
SIT. There were no discordant results between the
PAC(RIA) and ePAC(CLEIA) groups (Fig. 1C).

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New aldosterone assay impacts diagnosis

Fig. 15Comparison of the consensus statement on the clinical practice of primary aldosteronism in Japan and the Japan Endocrine Society
Clinical Practice Guideline for the Diagnosis and Management of Primary Aldosteronism 2021.
(A–D) Comparison of (A) screening, (B) captopril challenge test, (C) saline infusion test and (D) overall diagnosis of primaryaldosteronism results. The upper and lower bars of each figure show the number of positive (red), negative (blue) andprovisionally positive (orange) cases according to CS and GL 2021, respectively. Gradient bands indicate shifts in classification
between the two criteria, with red-to-blue or blue-to-red or orange gradients representing diagnostic discordance. CS: Consensus
Statement on the Clinical Practice of Primary Aldosteronism in Japan [14]; GL2021: Japan Endocrine Society clinical practiceguideline for the diagnosis and management of primary aldosteronism 2021 [13].

Comparison of PA diagnosis using PAC(RIA)
according to CS and ePAC(CLEIA) according toGL2021
Using PAC(RIA) and following the CS guidelines,
127 patients were diagnosed with PA and 63 were not.
Among the 63 cases diagnosed as PA negative according
to CS, 42 showed a negative SC result when strictly
applying the cutoff criteria of CS (Fig. 1A). However,owing to the strong suspicion of PA by the attending
physicians, these patients underwent CCT or SIT. As theresults of the confirmatory tests were positive, thepatients were diagnosed with PA and underwent AVS.
The remaining 21 cases showed a positive SC result but
were negative for CCT and SIT when strictly applying

the cutoff criteria for CS. However, these patients
showed CCT or SIT results very close to the cutoff val‐
ues and were diagnosed with PA by attending physicians
based on findings including serum potassium level,
blood pressure level, and the presence of adrenal nod‐ules. Consequently, the patients also underwent AVS.
Using ePAC(CLEIA) and adhering to the GL2021
guidelines, 75 patients were diagnosed with PA, 51 were
provisionally diagnosed, and 64 were not diagnosed.
Among the 127 patients diagnosed with PA using
PAC(RIA), 75 were diagnosed with PA using
ePAC(CLEIA), 48 were provisionally diagnosed, andfour were not diagnosed. Of the 63 patients not diag‐
nosed with PA using PAC(RIA), none were diagnosed

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Honma et al.

with PA using ePAC(CLEIA), three were provisionallydiagnosed, and 60 were not diagnosed. Thus, there wereseven cases in which the diagnosis differed between
PAC(RIA) and ePAC(CLEIA), all of which were IHA.
The diagnostic discordance rate was 3.7% (7/190) (Fig.
1D). Among the four patients diagnosed with PA by
PAC(RIA) but not by PAC(CLEIA), three were SC posi‐
tive, and one was provisionally positive. All four patientswere CCT negative, and three of the four cases were SIT
negative (one case did not undergo SIT). Among thethree patients not diagnosed with PA by PAC(RIA) but
provisionally diagnosed by PAC(CLEIA), one was SC
positive, and two were provisionally positive. In CCT,
two of the three cases were provisionally positive, andone case was negative. In SIT, two of the three cases
were provisionally positive, and one case was negative.
All three patients were provisionally diagnosed with PA
Table 2

because at least one of either CCT or SIT was provision‐
ally positive (Table 2).

Clinical characteristics of APA and IHA
The clinical characteristics of the patients with APA
and IHA are shown in Table 3. The APA group had
significantly more male cases (55.6% vs. 33.7%, p =0.034), higher serum sodium levels (141 mEq/L vs.
140 mEq/L, p < 0.001), and lower serum potassium
levels (3.3 mEq/L vs. 3.8 mEq/L, p < 0.001) than thoseof the IHA group. The IHA group had significantly
higher BMI (26.7 kg/m2 vs. 23.5 kg/m2, p = 0.02), dias‐
tolic blood pressure (80 mmHg vs. 74 mmHg, p =0.043), and HbA1c (5.7% vs. 5.4%, p = 0.013) than thoseof the APA group. There were no significant differencesin age, heart rate, systolic blood pressure, random blood
glucose level, serum creatinine level, serum chloride

Clinical characteristics of seven patients with discordant PA diagnoses between PAC (RIA) and ePAC (CLEIA)

Sex
Age (year)

Case 1Case 2

Case 3

Case 4

Case 5

Case 6

Case 7

FemaleFemaleFemaleFemaleFemaleFemaleFemale

71

46

61

56

45

35

60

2

BMI (kg/m )

21.7

22.9

22.4

24.8

19.6

26.0

28.2

sBP (mmHg)

136

128

140

133

136

140

126

dBP (mmHg)

76

70

80

88

72

86

70

HR (/min)

72

76

82

76

75

60

80

RBG (mg/dL)

92

86

87

83

83

—

150

HbA1c (%)

5.7

5.1

6.5

5.3

5.5

5.3

8.4

Cre (mg/dL)

0.63

0.53

0.57

0.66

0.48

0.60

0.53

Na (mEq/L)

141

139

141

143

137

137

140

K (mEq/L)

3.5

3.7

4.1

3.8

3.7

3.9

4.0

Cl (mEq/L)

107

105

108

107

103

107

107

Urinary protein

–

–

–

–

–

–

±

Adrenal nodules

–

–

–

–

–

+

+

Subtype

IHA

IHA

IHA

IHA

IHA

IHA

IHA

SC

CS/
GL2021

Positive/
Positive

Positive/
Positive

Positive/
Positive

Positive/
Provisionally

Negative/
Positive

Positive/
Provisionally

Negative/
Provisionally

CCT

CS/
GL2021

Positive/
Negative

Positive/
Negative

Positive/
Negative

Positive/
Negative

Negative/
Negative

Negative/
Provisionally

Positive/
Provisionally

SIT

CS/
GL2021–/–

Negative/
Negative

Negative/
Negative

Negative/
Negative

Positive/
Provisionally

Negative/
Negative

Positive/
ProvisionallyDiagnosis

CS/
GL2021

Positive/
Negative

Positive/
Negative

Positive/
Negative

Positive/
Negative

Negative/
Provisionally

Negative/
Provisionally

Negative/
Provisionally

BMI, body mass index; sBP, systolic blood pressure; dBP, diastolic blood pressure; HR, heart rate; RBG, random blood glucose; HbA1c,
glycated hemoglobin; Cre, serum creatinine; Na, serum sodium; K, serum potassium; Cl, serum chloride; CCBs, calcium channel blockers;
ARBs, angiotensin receptor blockers; MRBs, mineralocorticoid receptor antagonists; IHA, idiopathic hyperaldosteronism; SC, screening;
CCT, captopril challenge test; SIT, Saline infusion test; CS, the Consensus Statement on the Clinical Practice of Primary Aldosteronism inJapan [14]; GL2021, Japan Endocrine Society clinical practice guideline for the diagnosis and management of primary aldosteronism
2021 [13].

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New aldosterone assay impacts diagnosis

level, qualitative test for urinary protein, or number andtype of antihypertensive medications between the APA
and IHA groups. According to GL2021, using
ePAC(CLEIA) for SC, 85.2% of APA cases were posi‐
tive, compared to 57.7% of IHA cases. For CCT, 84.6%
of APA cases were positive, compared to 29.0% of IHA

Table 3cases, with an additional 29.0% provisionally positiveand 42% negative (p < 0.001). For SIT, 63.6% of APA
cases were positive and 18.2% were provisionally posi‐
tive, compared with 16.5% of IHA cases that were posi‐
tive, 50.6% provisionally positive, and 32.9% negative.

Clinical characteristics of patients with APA and IHA
APA

IHA

Number27

163

Male (%)

15 (55.6)

55 (33.7)

0.034

49 (41–60)

49 (42–60)

0.83

Body mass index (kg/m )23.5 (22.8–26.8)

26.7 (23.4–29.4)

0.020

Age (year)2

p value

Systolic blood pressure (mmHg)

130 (125–140)

136 (124–144)

0.18

Diastolic blood pressure (mmHg)

74 (68–82)

80 (70–89)

0.043Heart rate (/min)

74 (66–80)

76 (68–82)

0.99

HbA1c (%)

5.4 (5.3–5.7)

5.7 (5.3–6.0)

0.013

Cre (mg/dL)

0.68 (0.54–0.80)

0.65 (0.56–0.78)

0.64

Na (mEq/L)

141 (140–144)

140 (139–141)<0.00119 (70.4)

25 (15.3)<0.001

3.3 (2.7–3.6)

3.8 (3.6–4.0)<0.001

Adrenal nodules (%)

20 (74.1)

75 (46.0)

0.012

Classification of antihypertensive drugs (%)
Calcium channel blockers
Alpha-blockers
Angiotensin receptor blockers
Mineralocorticoid receptor antagonists
Beta-blockers
Diuretics

22 (81.5)
4 (14.8)
3 (11.1)000123 (75.5)
8 (4.9)5 (3.1)3 (1.8)2 (1.2)1 (0.6)

0.63
0.072
0.088
1.0
1.0
1.0

Potassium supplements (%)

13 (48.1)

14 (8.6)<0.001

Screening (%)
Positive
Provisionally positive
Negative

23 (85.2)
3 (11.1)
1 (3.7)94 (57.7)
40 (24.5)
29 (17.8)

Captopril challenge test (%)
Positive
Provisionally positive
Negative

22 (84.6)
2 (7.7)2 (7.7)47 (29.0)
47 (29.0)
68 (42.0)

Hypokalemia (%)
K (mEq/L)

0.025<0.001

Saline infusion test (%)
Positive
Provisionally positive
Negative

0.003
7 (63.6)
2 (18.2)
2 (18.2)

14 (16.5)
43 (50.6)
28 (32.9)

Decision (%)
Diagnosis
Provisionally positive
Negative

23 (85.2)2 (7.4)2 (7.4)52 (31.9)
49 (30.1)
62 (38.0)<0.001

Data are expressed as the median (IQR) or n (%). The p values were calculated using the Mann–
Whitney U test. HbA1c, glycated hemoglobin; Cre, serum creatinine; Na, serum sodium; K, serum
potassium; APA, aldosterone-producing adenoma; IHA, idiopathic hyperaldosteronism.

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Honma et al.

Clinical characteristics of PA diagnosis, provisional
diagnosis, and negative groups according to GL2021
using ePAC(CLEIA)
The clinical characteristics of the PA diagnosis, provi‐
sional diagnosis, and negative groups are shown in
Table 4. No significant differences in age were observed
among the groups. In the post-hoc analysis, the PA
diagnosis group contained significantly more malepatients than in the negative group (p = 0.0088)(Fig. 2A). Serum potassium levels were significantly
lower in the PA diagnosis group (3.6 mEq/L) than in theprovisional diagnosis group (3.9 mEq/L) and the nega‐tive group (3.8 mEq/L) (Fig. 2B). The prevalence of
APA was significantly higher in the PA diagnosis group(30.7%) than in the provisional diagnosis group (3.9%)
and the negative group (3.1%) (Fig. 2C). Subdivision of
the PA diagnosis group based on the presence of
hypokalemia revealed a significantly higher prevalenceof APA in patients with hypokalemia (63.3% vs. 8.9%, p< 0.001) (Table 5A). When categorized according to thepresence of adrenal nodules, the prevalence of APA wassignificantly higher in patients with adrenal nodules(39.6% vs. 14.8%, p = 0.036) (Table 5B). The clinicalcharacteristics of patients provisionally diagnosed or notdiagnosed with PA, including confirmed APA cases (n =4), are presented in Supplementary Table S4. None ofthese patients exhibited hypokalemia, and among thefour cases, one had adrenal nodules.

Discussion
To our knowledge, the present study is the first to
apply ePAC(CLEIA) to the GL2021 to examine howchanges in the measurement of PAC and, thus, the diag‐nostic criteria for PA affect its diagnosis. The transition
from RIA to CLEIA has had a considerable impact on
the diagnosis of PA, as evidenced by the results of thepresent study.
Although GL2021 defines provisional positive criteria
for SC, CCT, and SIT, evidence for these criteria remains
insufficient. We observed a non-negligible number ofcases initially identified as positive for SC, CCT, andSIT using PAC(RIA) based on CS, which were then clas‐sified as provisionally positive or negative upon reevaluation with ePAC(CLEIA) based on GL2021.
Assuming that there were no criteria for provisionally
positive results, patients originally considered provision‐
ally positive would have been considered negative andcases that should have been diagnosed with PA would
have been misdiagnosed as having essential hyperten‐sion. This highlights the challenge of determining appro‐
priate cutoff values for diagnosing PA. Moreover, certain
cases considered negative under the CS guidelines wereidentified as provisionally positive or positive using the
new criteria, emphasizing the need to reassess the valid‐
ity of provisionally positive cutoffs.
The significant impact of the transition from RIA to
CLEIA on the diagnosis of PA can be attributed to theinherent limitations of RIA and the advantages of
CLEIA. PAC measurements using RIA have limitations,particularly in terms of sensitivity and reproducibility [9,10]. In contrast, PAC measurement using CLEIA offers
improved traceability to the CRM for aldosterone andexcellent linearity across a broad range of concentra‐
tions, including lower levels. Moreover, PAC values
determined by CLEIA align well with those determined
by LC-MS/MS [11, 23, 24]. The advantages of CLEIA
over RIA underscore its potential as a standard PAC
assay in routine clinical practice. In addition, the strong
correlation between PAC measurements obtained using
CLEIA and LC-MS/MS facilitated an international com‐
parison of PA diagnoses.
Typical PA with a phenotype of hypertension,
hypokalemia, and unilateral adrenal nodules was firstdescribed by Conn [25]. This classic form of PA, which
is often associated with APA, responds well to laparo‐scopic adrenalectomy [26-28]. However, the broad
implementation of screening for PA using the ARR has
led to a considerable increase in milder forms of PA [29],which are predominantly IHA manageable with MRAs.
AVS has been recommended as the gold standard for dis‐tinguishing APA from IHA, thereby determining indica‐
tions for surgery. Over the past decade, despite a marked
increase in the number of diagnosed PA cases and theintroduction of AVS, only 20% of PA patients undergo‐
ing AVS have undergone adrenalectomy in Japan [30].
The diagnostic efficiency of AVS needs to be enhanced
given its invasive nature, technical challenges, cost, andradiation exposure.
A retrospective cohort study within the Japan Rare/
Intractable Adrenal Diseases Study (JRAS) of 3,689
patients with PA before changing PAC measurement
methods revealed that patients positive for CCT or SIT
showed higher APA rates than those who were provi‐
sionally positive [31]. The study reported that the per‐centages of APA in positive vs. provisionally positive
cases were 44% vs. 7% for CCT and 49% vs. 5% for SIT,
respectively [31]. However, it should be noted that thediagnostic criteria for SIT in the JRAS differed fromthose used in the present study.
According to the JRAS, patients with hypokalemia
diagnosed with PA using ePAC(CLEIA) seem to be ideal
AVS candidates because of the high prevalence of surgi‐
cally treatable APA. However, the study showed thatapproximately 5% of cases with a provisional diagnosis
of PA were APA [31], which is similar to the percentage

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New aldosterone assay impacts diagnosis

Table 4

Clinical characteristics of patients in the PA diagnosis, provisional diagnosis, and negative group
Diagnosis

Provisional diagnosis

negative

Number75

51

64

Male (%)

36 (48.0)

19 (37.4)

15 (23.4)

0.011

Age (year)

53 (43–63)

45 (38–58)

49 (45–60)

0.064

Body mass index (kg/m2)

25.1 (23.2–28.2)

27.7 (24.1–30.0)

25.9 (22.7–30.6)

0.092

Systolic blood pressure (mmHg)

132 (123–142)

132 (120–140)

136 (128–148)

0.16

Diastolic blood pressure (mmHg)

78 (70–84)

78 (70–89)

80 (76–90)

0.13Heart rate (/min)

74 (70–80)

75 (68–83)

76 (66–84)

0.89

Random blood glucose (mg/dL)

94 (89–102)

91 (87–102)

95 (88–102)

0.75

HbA1c (%)

5.6 (5.3–5.9)

5.6 (5.3–6.1)

5.7 (5.3–5.9)

0.84

Cre (mg/dL)

0.69 (0.55–0.86)

0.65 (0.56–0.78)

0.62 (0.56–0.74)

0.058

Na (mEq/L)

141 (140–142)

140 (139–141)

140 (139–141)

0.002

30 (40.0)

5 (9.8)9 (14.1)<0.001

3.6 (3.3–3.9)

3.9 (3.7–4.0)

3.8 (3.6–4.0)<0.001

Hypokalemia (%)
K (mEq/L)
Semi-qualitative test for urinary protein (%)

p value

0.76–

61 (84.7)

45 (88.2)

51 (82.3)±

7 (9.7)5 (9.8)6 (9.7)1+

4 (5.6)

1 (2.0)4 (6.5)2+

00

1 (1.6)48 (64.0)

20 (39.2)

27 (42.2)

0

11 (14.7)

19 (37.3)

12 (18.8)

1

51 (68.0)

28 (54.9)

48 (75.0)

2

13 (17.3)

3 (5.9)3 (4.7)30

1 (2.0)1 (1.6)

Calcium channel blockers

63 (84.0)

31 (60.8)

51 (79.7)

0.008

Alpha-blockers

8 (10.7)

3 (5.9)1 (1.6)0.088

Angiotensin receptor blockers

5 (6.7)

1 (2.0)2 (3.1)

0.38

Mineralocorticoid receptor antagonists

Adrenal nodules (%)
Number or antihypertensive drugs (%)

0.007
0.008

Classification of antihypertensive drugs (%)

1 (1.3)2 (3.9)00.24

Beta-blockers

00

2 (3.1)

0.14

Diuretics00

1 (1.6)

0.37

Potassium supplements (%)

20 (26.7)

2 (3.9)5 (7.8)<0.001

Aldosterone-producing adenoma (%)

23 (30.7)

2 (3.9)2 (3.1)<0.001

Data are expressed as the median (IQR) or n (%). The p values were calculated using one-way ANOVA. HbA1c, glycated hemoglobin; Cre,
serum creatinine; Na, serum sodium; K, serum potassium.

in the present study (2 of 51 cases, 3.9%). Thus, APA
cannot be ruled out, even with a provisional diagnosis of PA.
In the provisional diagnosis group in the present study,
only two patients (3.9%) were diagnosed with APA,
neither of whom exhibited hypokalemia, and adrenal
nodules were observed in just one case. The low propor‐
tion of patients with APA in the provisional diagnosis
group suggests a reduced need for AVS. The limited
number of APA cases in this group precluded the identi‐

fication of additional clinical indicators to distinguish
APA from IHA. Therefore, to identify cases with a highpre-diagnostic probability of APA within the group andto guide appropriate AVS indications, further studies
are necessary to establish clinical findings beyond
hypokalemia and adrenal nodules.
In contrast, a higher proportion of patients with APA
in the PA diagnosis group had hypokalemia and adrenal
nodules in the present study, consistent with a previous

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10

Fig. 2

Honma et al.

(A) Comparison of male-to-female ratio among the PA diagnosis, provisional diagnosis, and negative groups. (B) Box plot ofserum potassium levels among the PA diagnosis, provisional diagnosis, and negative groups. (C) Comparison of APA to IHA ratio
among the PA diagnosis, provisional diagnosis, and negative groups.
(A) The percentages of male and female cases are shown in the red band and the gray band, respectively. (B) Within each box,
horizontal black lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution ofvalues; vertical extending lines denote adjacent values (i.e., the most extreme values within 1.5 interquartile range of the 25th and75th percentile of each group). (C) The percentages of APA and IHA cases are shown in the red band and the gray band,
respectively. PA: Primary aldosteronism; APA: Aldosterone-producing adenoma; IHA: idiopathic hyperaldosteronism.

Table 5A Comparison of APA prevalence based on the presence of hypokalemia in the PA
diagnosis group
Hypokalemia

Normokalemia

30

45

APA (%)

19 (63.3)

4 (8.9)IHA (%)

11 (36.7)

41 (91.1)

N

p value<0.001

Table 5B Comparison of APA prevalence based on the presence of adrenal nodule in the PA
diagnosis group
Adrenal nodule +Adrenal nodule –48

27

APA (%)

19 (39.6)

4 (14.8)

IHA (%)

29 (60.4)

23 (85.2)

N

p value

0.036

Data are expressed as n (%). p values were calculated using Fisher’s exact test. Hypokalemia
was defined as a serum potassium level <3.5 mEq/L; adrenal nodules were defined as a tumor
diameter ≥10 mm on abdominal CT; PA, primary aldosteronism; APA, aldosterone-producing
adenoma; IHA, idiopathic hyperaldosteronism.

report [31]. Positivity rates for SC, CCT, and SIT using
ePAC(CLEIA) were considerably higher for APA thanfor IHA (85.2% vs. 57.7% for SC, 84.6% vs. 29.0% for
CCT, and 63.6% vs. 16.5% for SIT). This suggests that

SC and confirmatory tests may help distinguish APA
from IHA. As the number of cases was too small toestablish an appropriate cutoff, further studies are
required to resolve this issue.

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New aldosterone assay impacts diagnosis

Based on our findings, we propose that provisionally
positive cases should be considered a criterion for initiat‐
ing medical therapy with MRAs because of the high like‐lihood of IHA. In contrast, positive cases should be
regarded as a criterion for considering AVS because of
the increased possibility of APA.
A critical limitation of the present study was the esti‐
mation of PAC(CLEIA) based on PAC(RIA) measure‐ments using a conversion formula. Given the variability
in PAC(RIA) [32] among patients, this approach could
hinder the interpretation of results, rendering the study
speculative. Direct comparison of PAC measured using
both CLEIA and RIA in the same patient is essential for
accurate evaluation. Moreover, because only patientswith PA who underwent AVS were included in thepresent study, there might have been a selection bias,
potentially overestimating the prevalence of APA. Addi‐
tionally, the diagnosis was based on Japanese clinicalguidelines, which may have limited the applicability ofthis study to other countries.
In conclusion, the present study demonstrated that thetransition from PAC(RIA) to PAC(CLEIA) resulted inchanges in the diagnostic criteria for PA, leading to aconsiderable number of cases previously classified as
positive by CS being reclassified as provisionally posi‐tive or negative according to GL2021. These findings

highlight the necessity of establishing appropriate cutoff
values and validating provisionally positive criteria to
guide management decisions, particularly concerning
AVS, in patients with a high likelihood of surgically
remediable APA.

Acknowledgments
We thank E. Kakuta, M. Hirata, and C. Noguchi for
their excellent secretarial assistance.

Disclosure
None of the authors have any potential conflicts ofinterest associated with this research. Hiroaki Masuzaki
is a member of Endocrine Journal’s Editorial board.

Author’s contribution
All the authors contributed to this study. Data werecollected by K.H. The draft of the manuscript was writ‐ten by K.H., Y.N., A.T., M.U., T.T., T.Y., Y.I., K.Y., R.C.,
T.U., and S.O. and H.M. reorganized the final form of
the manuscript. All the authors have read and approved
the final version of the manuscript.

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Endocrine Journal Advance Publication