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Screening Cutoff Values for the Detection of Aldosterone Producing Adenoma by LC-MS/MS

and a Nobel Non-competitive CLEIA

Yoshikiyo Ono,

1,2

Yuta Tezuka,

1,2

Kei Omata,

1,2

Ryo Morimoto,

Yuto Yamazaki,

Sota Oguro,

Kei Takase,

Akihiro Ito,

Tatsunari Yoshimi,

Satoshi Kojima,

Sadayoshi Ito,

Hironobu

Sasano,

Takashi Suzuki,

Tetsuhiro Tanaka

, Hideki Katagiri,

and Fumitoshi Satoh

2,3

Department of Diabetes, Metabolism and Endocrinology, Tohoku University Hospital, 1-1

Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan

Division of Nephrology, Rheumatology and Endocrinology, Tohoku University Graduate

School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan

Department of Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,

Aoba-ku, Sendai, Miyagi, 980-8575, Japan

Department of Diagnostic Radiology, Tohoku University Graduate School of Medicine, 2-1

Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan

Department of Urology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,

Aoba-ku, Sendai, Miyagi, 980-8575, Japan

Fujirebio, Inc., 51, Komiya-machi, Hachioji, Tokyo, 192-0031, Japan

Key words: primary aldosteronism, aldosterone, assay, screening, diagnosis

Conflicts of Interest:

T.K., T.S. and F.S. received support through grants from the Ministry of

Health, Labour, and Welfare, Japan (No. 23FC1041). The remaining authors have nothing to

disclose.

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Source of Funding:

This study was conducted in part by the expenses provided by the research

contract between Tohoku University Hospital and Fujirebio Inc. (Tokyo, Japan).

Word count: 6529 words

Word count of abstract: 250 words

Number of figures and tables: 3 figures and 1 table with supplementary data

Author to whom correspondence and reprint requests should be addressed:

Fumitoshi Satoh, M.D., Ph.D.

Department of Pathology, Tohoku University Graduate School of Medicine

2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan

Phone: +81-22-717-7163

Fax: +81-22-717-7168

e-mail:

fsatoh@med.tohoku.ac.jp

ORCID ID: 0000-0003-0720-0470

Abstract

Context: Detecting patients with surgically curable aldosterone-producing adenoma (APA)

among hypertensive subjects is clinically pivotal. Liquid chromatography-tandem mass

spectrometry (LC-MS/MS) is the ideal method of measuring plasma aldosterone concentration

(PAC) because of the inaccuracy of conventional chemiluminescent enzyme immunoassay

(CLEIA). However, LC-MS/MS is expensive and requires expertise. We have developed a novel

non-competitive CLEIA (NC-CLEIA) for measuring PAC in 30 min.

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Objective:

To validate NC-CLEIA PAC measurements by comparing them with LC-MS/MS

measurements and determining screening cutoffs for both measurements detecting APA.

Method: We retrospectively measured PAC using LC-MS/MS and NC-CLEIA in 133 patients

with APA, 100 with bilateral hyperaldosteronism, and 111 with essential hypertension to explore

the accuracy of NC-CLEIA PAC measurements by comparing with LC-MS/MS measurements

and determined the cutoffs for detecting APA.

Results: Passing-Bablok analysis revealed that the values by NC-CLEIA (the regression slope,

intercept, and correlation coefficient were 0.962, -0.043, and 0.994, respectively) were

significantly correlated and equivalent to those by LC-MS/MS. Bland-Altman plot analysis of

NC-CLEIA and LC-MS/MS also demonstrated smaller systemic errors (a bias of -0.348 ng/dL

with limits of agreement of -4.390 and 3.694 within a 95% confidence interval) in NC-CLEIA

than LC-MS/MS. The receiver-operating characteristic analysis demonstrated that cutoff values

for aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5

(ng/dL per ng/mL/hr), with a sensitivity of 91.0% and 90.2% and specificity of 75.4% and 76.8%,

respectively, to differentiate APA from non-APA.

Conclusion: This newly developed NC-CLEIA for measuring PAC could serve as a clinically

reliable alternative to LC-MS/MS.

Introduction

Primary aldosteronism (PA) is one of the most frequent causes of secondary hypertension, and

hypertension caused by unilateral PA is generally curable or at least can be improved after

surgical treatment [1–6].

It has become pivotal for clinicians to detect surgically curable PA

among hypertensive individuals. The Endocrine Society recommended measuring plasma

aldosterone concentration (PAC) and plasma renin activity (PRA) or active renin concentration

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and calculation of the aldosterone-to-renin ratio (ARR) to screen hypertensive patients for the

clinical detection of PA [1]. The diagnostic workup of PA also included confirmatory testing by

saline infusion, captopril challenge, or fludrocortisone suppression, and determining the PA

subtypes according to adrenal CT and adrenal venous sampling (AVS) [1–6].

Accurate and

reproducible measurement of peripheral PAC is generally considered pivotal in all diagnostic

workup of patients with PA regardless of their clinical stages. Peripheral aldosterone

concentrations usually varied from 1 to 100 ng/dL, and because of these relatively low values,

highly sensitive and specific assays are mandatory for the accurate measurement of PAC.

However, for the last several decades, PAC has been mainly measured by antibody-based

immunoassays which did not necessarily harbor high specificity and did more than frequently

result in over-estimation of PAC, in addition to marked variability in assay performance reported

among different laboratories [7,8]. Therefore, to overcome those difficulties in measuring PAC,

liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied. LC-MS/MS was

reported to be more reliable than conventional radioimmunoassay (RIA) and, therefore, has been

employed in some institutions in which many patients with PA were managed [9–12]. However,

LC-MS/MS needs more expensive and expertise, which has unfortunately prevented its wide

scale application in routine clinical practice. Therefore, in order to circumvent those pitfalls of

LC-MS/MS for measuring PAC, we and others recently developed fully automated

chemiluminescent enzyme immunoassay (CLEIA) [7,13]. CLEIA has markedly contributed to

shortening the workup time of patients with PA, because almost all CLEIAs of PAC have used

conventional competition curve analysis as in RIA. However, it is also true that a conventional

CLEIA was not necessarily associated with high specificity and sensitivity compared with LC-

MS/MS analysis [7,13]. Therefore, many clinical laboratories still utilized LC-MS/MS for the

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accurate analysis of PAC. LC-MS/MS could specifically measure lower values of PAC compared

to RIA or CLEIA, thus establishing that method -specific cutoff values for PA screening and

confirmatory testing is therefore required [9–12,14]. Several Australian and German research

groups very recently independently confirmed the approximately 20

％〜

50% lower values of

PAC measured by LC-MS/MS, and proposed the new cutoff values toward the clinical diagnosis

of PA compared with PAC measured by RIA or CLEIA [9–11,14]. These differences especially at

lower concentrations (<10 ng/dL) could be critical for diagnosing or screening for PA, because

these cutoff values of both baseline and confirmatory tests for PAC including ARR component

could be at lower concentration [1,9–11,14].

The SPAC-S aldosterone kit (SPAC) (Fujirebio Inc., Tokyo, Japan) has been used for

analyzing PAC in almost all clinical laboratories since 2009 in Japan, when the Japanese

Guidelines 2009 for diagnosis and treatment of PA were formulated [4]. However, the SPAC was

commercially terminated in March 2021. We recognized that SPAC also had limitations as in

other RIAs compared to LC-MS/MS. On the other hand, we developed the LC-MS/MS method

for PAC with Aska Pharma Medical Co. Ltd., (Fujisawa, Japan) in 2008 [15]. Since then, we have

used the standardized PAC values by LC-MS/MS for the diagnosis of PA [13,16]. We recently

developed a non-competitive (NC-) CLEIA for PAC (a sandwich method of two antibodies),

which is closely equivalent to LC-MS/MS, in conjunction with Fujirebio Inc. This novel assay

could swiftly provide results in approximately 30 min and has been approved and covered by the

universal health insurance system of the Japanese Government.

Therefore, in our present study, we further validated this NC-CLEIA PAC and compared the

results obtained with those by LC-MS/MS, a gold standard of measurement. We then studied its

clinically validated diagnostic capacity to differentiate between the patients with surgically

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curable aldosterone-producing adenoma (APA) and non-APA patients, including those with

bilateral hyperaldosteronism (BHA) and essential hypertension (EH). These validations were all

performed in the plasma samples of the patients cryopreserved after the pathological diagnosis of

APA.

Methods

Patient eligibility and diagnosis of PA

We screened hypertensive patients, including those with PA, who visited Tohoku University

Hospital between December 2016 to September 2019, as previously reported [13]. We measured

PAC and plasma renin activity using conventional RIA kits to clinically determine aldosterone-to-

renin ratio (ARR). At this point, we obtained residual blood samples from the patients

accompanied by research information disclosure. Following centrifugation, the plasma samples

were immediately frozen and stored at -20 degrees Celsius for future aldosterone measurement.

If the patients showed ARR values by RIA equal to or more than 20 ng/dL per ng/mL/h, they

proceeded to undergo Captopril Challenge Test (CCT) for diagnosing PA or essential

hypertension (EH) as the Japanese guideline recommended [4]. Our criterion for diagnosing PA

was based on ARRs surpassing 20 ng/dL per ng/mL/h after being captopril challenged . Those

who were confirmed to be clinically negative for PA were additionally screened for other

etiologies of secondary hypertension, such as Cushing syndrome, pheochromocytoma, and renal

artery stenosis, were excluded and finally diagnosed to have EH. We performed segmental AVS

which can accurately detect PA subtypes such as APA, which is curable by surgery, and BHA

(Supplementary Figure S1) [6,17,18,19]. Pathological study was performed to confirm the

presence of aldosterone-producing lesions including immunohistochemistry of CYP11B2

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(

RRID:AB_2650562

), as reported previously [20–24]. The eligible patients with PA were

registered as a cohort of PA Sendai Study with written informed consent, and the present study

was approved by the ethics committee of Tohoku University School of Medicine (#2019-1-591).

Conventional radioimmunoassay of plasma aldosterone and renin activity

Plasma samples were collected in a recumbent position early in the morning after bed rest for

30 min. PAC was measured by RIA using SPAC-S Aldosterone Kit (Fujirebio Inc., Tokyo, Japan)

and PRA by RIA of angiotensin I using the Renin Activity Kit YAMASA (Yamasa Co., Chiba,

Japan); the analytical sensitivity was 2.5 ng/dL for PAC and 0.2 ng/mL/hr for PRA.

Development of a novel chemiluminescence assay of plasma aldosterone concentration

We used the Lumipulese Presto Aldosterone (Fujirebio Inc.,

Catalog #298572, RRID:

AB_3076349

AB_3076350

) for PAC with fully automated, 2-step sandwich chemiluminescent

enzyme immunoassay for LUMIPULSE L system (Fujirebio Inc.) [25,26]. Detailed description of

the assay protocol is presented in Supplementary data [19]. The protocol is summarized briefly

as follows: 30 µL of plasma is mixed with 50 µL of anti-aldosterone monoclonal antibody coated

magnetic particle solution and incubated for 8 min at 37 degrees Celsius (1

reaction); 50 µL of

alkaline phosphatase-conjugated anti-metatype antibody is added and incubated for 8 min at 37

degrees Celsius following a wash (2nd reaction); then, a substrate solution is added after the 2

reaction followed by another washing procedure to measure chemiluminescence [27]. Results

were fully and automatically obtained in approximately 30 min (Supplementary Figure S2) [19].

Plasma samples used for validation of the novel assay and comparison to conventional

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methods

For the measurement of PAC and PRA, blood was collected from an antecubital vein into

EDTA-2Na tubes in a supine position after bed rest for 30 min, immediately centrifuged at room

temperature, and then stored at a temperature below -20 degrees Celsius.

Validation of the novel assay of plasma aldosterone

For the measurement of PAC, correlation and Passing-Bablok regression analyses and Bland-

Altman plots were performed to compare the results of the novel assay with those of the LC-

MS/MS method which we previously reported [15]. The same analyses were also performed to

compare the results of the conventional radioimmunoassay (SPAC-S kit) with those of LC-

MS/MS.

Clinical evaluation of diagnostic ability based on the novel assay system

PAC and ARR determined by LC-MS/MS, NC-CLEIA, and RIA were both evaluated using

receiver-operating characteristics (ROC) analysis to investigate the cutoff values, sensitivity, and

specificity for screening patients with APA from hypertensive patients including BHA and EH.

Statistical analysis

Values below a lower limit of detection were assigned to analytical sensitivity values of each

assay for subsequent statistical comparison. The normality of the collected data was analyzed

using Kolmogorov-Smirnov test. When continuous variables showed a normal distribution, one-

way analysis of variance was employed for comparison. Otherwise, Kruskal-Wallis test with

Dunn's multiple comparison test as a post-hoc test was used. Passing-Bablok regression and

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Bland Altman method were used to compare the measurement values of NC-CLEIA to those of

the LC-MS/MS method. The optimal cutoff value of both PAC and ARR were estimated by

analyzing the ROC curve and determining the Youden index for each cutoff value. Area under

curves (AUC) were also calculated for each PAC and ARR, respectively. Pairwise comparison of

AUCs was used to assess AUC differences in its ability of detecting APA. Statistical significance

was set at

<0.05, and statistical analysis was performed using Stat Flex software (version 7.1;

Artech Co., Ltd., Osaka, Japan).

Results

Validation of the novel assays for plasma aldosterone concentrations

The detailed results of the validation experiments were presented in the Supplementary data

[19]. Briefly, for the PAC assay, the limit of quantification at 10% coefficient of variation was

0.22 ng/dL, as shown in Supplementary Table S1 and Figure S3 [19]. Assay ranges (defined as

analytical sensitivity to upper limit of detection) were set to 0.2-180.0 ng/dL for the PAC assay.

Results of precision are presented in Supplementary Table S2 for the PAC assay [19]. Linearity

results of the PAC assay are shown in Supplementary Table S3 [19]. Detailed results of spike

recovery, endogenous interference, therapeutic and drug interference, and cross-reactivity for the

PAC assay are shown in Supplementary Tables S4, S5, S6, and S7, respectively [19].

Diagnosis of PA and EH

Endocrinological diagnosis was performed using the conventional SPAC-S Kit for PAC and

Renin Activity Kit YAMASA for PRA. A total of 344 patients were judged as eligible for the

present study, and of them, 233 had PA. Among these 233 patients, 133 (57%) were diagnosed to

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have unilateral disease by segmental AVS; after adrenalectomy, they were finally diagnosed with

APA (Table 1). One hundred patients (43%) were diagnosed with BHA and the remaining 111

patients were diagnosed with EH (Table 1). Based on the results obtained by conventional RIA

PAC, ARR and captopril challenged ARR were significantly higher in those with APA than in

those with BHA or EH (

<0.05). PRA was significantly higher in those with EH than in those

with APA or BHA (

<0.05), while comparison between those with APA and BHA showed no

significant difference in baseline PRA levels.

Validation of the novel assay of plasma aldosterone concentration

PAC were measured using NC-CLEIA in all eligible patients. PAC by NC-CLEIA were

significantly higher in those with APA when compared to those with BHA or EH (Table 1). When

the BHA subgroup was compared with the EH subgroup, PAC by NC-CLEIA was significantly

elevated in those with BHA (Table 1). The measurements of PAC in all specimens measured by

NC-CLEIA were significantly correlated with those obtained by LC-MS/MS (Spearman’s

correlation coefficient (

)=0.994,

<0.0001) with a Passing-Bablok regression model of

Y=0.962X-0.043 (Figure 1A). The concentration gradient of the correlation was almost one, and

there was almost none in the graft. Measurements of PAC by NC-CLEIA in the specimens with

<10 ng/dL and 10 ng/dL or higher PAC by LC-MS/MS were significantly correlated with those of

LC-MS/MS (

=0.969,

<0.0001 and

=0.991,

<0.0001, respectively) with a Passing-Bablok

model of Y=1.021X-0.301 (Supplementary Figure S4A) and Y=0.950X+0.205 (Supplementary

Figure S4B), respectively [19]. These inclines of the correlations in both ranges of PAC hardly

differed. Measurements of PAC in all specimens using the conventional RIA SPAC-S Kit were

also correlated with those of LC-MS/MS (

=0.971,

<0.0001), with a Passing-Bablok model of

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Y=1.339X+5.816 (Figure 1B). As compared with the former, the correlation of RIA and LC-

MS/MS showed a large graft, and the incline of the correlation was bigger as well. Measurements

of PAC by RIA in the specimens with <10 ng/dL and 10 ng/dL or higher PAC by LC-MS/MS

were significantly correlated with those of LC-MS/MS (

=0.781,

<0.0001 and

=0.962,

<0.0001, respectively), with a Passing-Bablok model of Y=1.836X+3.467 (Supplementary

Figure S4C) and Y=1.285X+6.589 (Supplementary Figure S4D), respectively [19]. Unlike the

former, the inclines of the correlations were changed at different ranges of PAC. Comparison of

NC-CLEIA PAC with RIA of PAC yielded results similar to that between LC-MS/MS PAC and

RIA of PAC (Supplementary Figure S6A-C) [19].

Bland-Altman plot analysis of PAC in all specimens by NC-CLEIA and LC-MS/MS revealed

a bias of -0.348 ng/dL, and the limits of agreement were -4.390 and 3.694 with 95% confidence

interval (CI) (Figure 1C). The comparison between NC-CLEIA and LC-MS/MS for specimens

with <10 ng/dL of PAC by LC-MS/MS showed a bias of -0.018 ng/dL and limits of agreement of

-1.402 and 1.366 with 95% CI (Supplementary Figure S5A), whereas for specimens with 10

ng/dL or higher of PAC, LC-MS/MS revealed a bias of -0.612 ng/dL with limits of agreement of -

5.842 and 4.619 with 95% CI (Supplementary Figure S5B) [19]. These biases were astonishingly

small, suggesting that the systemic errors of this assay were exceedingly small, especially in the

lower range of PAC. On the other hand, Bland -Altman plot analysis of PAC in all specimens by

RIA and LC-MS/MS revealed a larger bias of 11.750 ng/dL, and the limits of agreement were -

1.216 and 24.72 with 95% CI (Figure 1D). The comparison between PAC by RIA and LC-

MS/MS in specimens with <10 ng/dL of PAC by LC-MS/MS showed a bias of 8.131 ng/dL and

limits of agreement of 1.270 and 14.993 with 95% CI (Supplementary Figure S5C), whereas for

specimens with 10 ng/dL or higher of PAC by LC-MS/MS, the bias was 14.649 ng/dL and the

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limits of agreement were 0.760 and 28.537 with 95% CI (Supplementary Figure S5D) [19].

Unlike the former, the biases were large, suggesting greater systemic errors for RIA, even in the

lower range of PAC. Considering the above results, the accuracy of NC-CLEIA was quite

superior to that of RIA, especially when PAC was <10 ng/dL, which is a critical level for

screening and confirming PA. Comparison of NC-CLEIA PAC with RIA of PAC demonstrated

the results similar to that between LC-MS/MS PAC and RIA of PAC (Supplementary Figure

S6D-F) [19].

Screening ability of APA based on the novel assay of plasma aldosterone concentration

Baseline levels of PAC and ARR by LC-MS/MS, NC-CLEAR, and RIA were summarized in

Table 1, Figure 2, and Supplementary Figure S5, respectively [19]. Values of ARR by LC-

MS/MS, NC-CLEIA, and RIA were significantly higher in those with APA when compared with

those with BHA or EH (Table 1; Figures 2C, 2D, and Supplementary Figure S7B) [19]. The

lowest values of PAC in APA were 6.3, 6.0, and 12.6 ng/dL by LC-MS/MS, NC-CLEIA, and

RIA, respectively (Table S8; Figures 2A, 2B, and Supplementary Figure S7A) [19]. The lowest

values of ARR in APA were 15.9, 16.3, and 29.6 ng/dL per ng/mL/hr by LC-MS/MS, NC-

CLEIA, and RIA, respectively (Table S8; Figures 2C, 2D, and Supplementary Figure S7B) [19].

If we set PAC at ≥6.0 ng/dL and ARR at ≥15 ng/dL per ng/mL/hr as screening cutoff values of

PA by LC-MS/MS and NC-CLEIA, the sensitivity for APA is 100% by both, and the specificity is

58.8% and 59.2%, respectively. With either method, 44.0% of BHA patients and 72.1% and

73.0%, respectively, of EH patients would come off from the workup of PA, and would be

targeted for antihypertensive treatment with drugs that would include mineralocorticoid receptor

blockers, or not (Supplementary Figure S8) [19].

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Based on the measurements of PAC and ARR, ROC analysis was performed to investigate the

diagnostic ability of PAC and ARR as a screening test for APA among hypertensive patients,

including those with BHA or EH. The ROC plot revealed that PAC by LC-MS/MS had an area

under the curve (AUC) of 0.910 (95% CI: 0.880 to 0.940). It demonstrated a sensitivity of 80.5%

and specificity of 85.8% at a cutoff of 14.2 ng/dL (Figure 3A, Supplementary Table S9) [19].

PAC by NC-CLEIA demonstrated an AUC of 0.919 (95% CI: 0.891 to 0.947), with a sensitivity

of 83.5% and specificity of 85.3% at a cutoff of 13.4 ng/dL (Figure 3A, Supplementary Table

S10) [19]; PAC by RIA yielded an AUC of 0.906 (95% CI: 0.875 to 0.937) at a cutoff of 25.0

ng/dL and yielded a sensitivity of 81.2% and specificity of 84.8% (Figure 3A). AUC of PAC by

NC-CLEIA was significantly different from that by RIA.

The ROC analysis revealed that ARR by LC-MS/MS demonstrated an AUC of 0.924 (95%

CI: 0.897 to 0.950) at a cutoff of 31.2 ng/dL per ng/mL/hr and yielded a sensitivity of 91.0% and

specificity of 75.4% to discriminate APA from non-APA (Figure 3B, Supplementary Table S11)

[19]. While ARR by NC-CLEIA demonstrated an AUC of 0.929 (95% CI: 0.904 to 0.954) and, at

a cutoff of 31.5 ng/dL per ng/mL/hr, it yielded a sensitivity of 90.2% and specificity of 76.8%

(Figure 3B, Supplementary Table S12) [19]. ARR by RIA yielded an AUC of 0.901 (95% CI:

0.869 to 0.934) at a cutoff of 96.4 ng/dL per ng/mL/hr and yielded a sensitivity of 72.2% and

specificity of 90.5% (Figure 3B). AUC of ARR by NC-CLEIA was significantly different from

that by RIA. The sensitivity and specificity of PAC and ARR values by LC-MS/MS and NC-

CLEIA were almost equivalent and differed from those by RIA. Sequential results of the above

ROC plot analyses are shown in Supplementary data (Supplementary Table S9- S13) [19].

In the present study, ROC analysis of ARR demonstrated that ARR ≥30 ng/dL per ng/mL/hr

measured by both LC-MS/MS PAC and by this new NC-CLEIA PAC could be suitable as

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screening cutoff value to enable APA diagnosis with a sensitivity of 91.7% for both assays, with a

specificity of 73.0% and 73.9%, respectively (Supplementary Table S11, S12) [19]. On setting

PAC ≥10 ng/dL and PRA <1.0 ng/mL/hr as the screening cutoff value for APA diagnosis using

our data, the sensitivity and specificity would have been 88.0% and 73.9% with LC-MS/MS, and

those measured by NC-CLEIA would have been 85.0% and 75.8 %, respectively (Supplementary

Figure S9) [19].

Discussion

LC-MS/MS measurement of PAC is currently considered a gold standard method to diagnose

PA at many medical centers in the world [8–12,14]. On the other hand, Japan’s universal health

insurance system does not necessarily reimburse the cost of expensive LC-MS/MS of PAC.

Therefore, to our regret, RIA has been used primarily because its cost is reimbursed, even though

its relatively poor sensitivity and reproducibility, especially at lower concentrations of PAC, were

widely recognized [28].

Therefore, a more accurate and cost-effective aldosterone assay, which is

closely equivalent to LC-MS/MS has been in demand among Japanese clinicians managing

patients with PA. In this study, we firstly demonstrated that PAC measured by newly developed

NC-CLEIA was significantly correlated with that measured by LC-MS/MS, and that the absolute

values of PAC were the same between NC-CLEIA and LC-MS/MS, whereas those obtained by

RIA were much higher. In addition, Eisenhofer et al. reported that PAC measured by LIAISON

and iSYS, which were

competitive

chemiluminescence assays, were significantly higher than that

determined by LC-MS/MS, suggestive of the questionable validity of immunoassay-based

diagnosis of PA [29]. On the other hand, results of our present study also demonstrated that NC-

CLEIA for PAC using LUMIPULSE L system, which is non-competitive chemiluminescence

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enzyme assay, provided almost equal absolute value to LC-MS/MS and could yield higher

sensitivity than conventional competitive chemiluminescence assays

[29,30].

In the NC-CLEIA

method, the specificity of each antibody employed could be combined using two different types

of antibodies, which reduced the influence of cross-reactivity compared to competitive assays

normally using only one single antibody. In addition, the two-step immunoassay allowed a

washing process after each immunoreaction, reducing the influence of various endogenous

interfering substances. In competitive assays, the response of inhibition rate was reported to be

low at high concentrations, resulting in the reduced levels of precision. On the other hand, in non-

competitive assays, the signal was extended with concentration, providing relatively good

precision and linearity (Table S2 and S3, Figure S2). Therefore, we believe that this assay could

measure aldosterone with higher accuracy than conventional immunoassays. As a result, “non-

competitive” chemiluminescence enzyme assay method was considered to cause those

differences among LUMIPULSE L, LIAISON, and iSYS systems in the value, sensitivity, and

accuracy for PAC. In addition, Bland -Altman plot analysis of NC-CLEIA aldosterone

measurements and LC-MS/MS measurements demonstrated much smaller biases and systemic

errors, indicating that NC-CLEIA could become not only a much more accurate replacement to

conventional RIA or CLEIA assays measured with competition curve, but also a clinically

reliable alternative to LC-MS/MS even when PAC is <10 ng/dL, which is very critical for

screening and confirming PA.

The Endocrine Society suggested the cutoff value of PAC determined by the saline infusion

test to be 5.0–10 ng/dL for PA [1]. Hence, the detection of lower values of PAC is critical for the

diagnosis of PA by the confirmatory tests. Results of our present study demonstrated that PAC

values determined by the newly developed NC-CLEIA were closely equivalent to those obtained

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by LC-MS/MS, even when PAC was in the lower concentration range. Therefore, this NC-CLEIA

PAC measurement, which is time efficient, cost-effective, and less labor intensive should be the

promising alternative to LC-MS/MS for the diagnosis of PA, especially in the lower aldosterone

concentration range determining the LC-MS/MS adjusted cutoff values for PA diagnosis. This

method is also considered important in countries like Japan where universal health care is

instituted.

The recommendation of guidelines to use ARR for detecting PA is considered clinically

important, particularly in selecting medications like mineralocorticoid receptor blockers for the

patients. However, various studies reported on the clinical effectiveness of surgery, especially for

preventing cardiovascular events and subsequently reducing the risk of incident atrial fibrillation

[1,31,32]. The major pathological type of unilateral PAs is APA, which is curable by

adrenalectomy. Therefore, the clinical differential diagnosis of APA before adrenalectomy is

considered pivotal for the management of patients with PA. AVS could clearly distinguish

patients with APA from non-APA patients in clinical practice. The diagnosis by AVS has also

been pathologically confirmed with immunohistochemical evaluation of CYP11B2 in the

specimens of adrenalectomy [20–23]. It is also true that medical treatment of patients with APA

for a long duration is generally considered clinically difficult because of possible cardiac or renal

complications. Therefore, the screening cutoff values of PAC and ARR measured by LC-MS/MS

and this new NC-CLEIA to detect APA could provide pivotal information regarding clinical

differential diagnosis between patients with APA and non-APA patients in conjunction with AVS.

In the present study, ROC analysis demonstrated that the cutoff values of ARR (31.2/31.5

ng/dL per ng/mL/hr) by LC-MS/MS and NC-CLEIA were almost equivalent because they

showed similar sensitivity (91.0%/90.2%) and specificity (75.4%/76.8%). Therefore, we can

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recommend an ARR ≥30.0 ng/dL per ng/mL/hr as a screening cutoff for both LC-MS/MS and

NC-CLEIA, which would discriminate APA with a sensitivity of 91.7% in both assays and with a

specificity of 73.0% and 73.9 %, respectively (Supplementary Tables S11 and S12) [19].

The newly revised Japanese guideline concerning PA recommends a PAC ≥6.0 ng/dL and

ARR ≥20 ng/dL per ng/mL/hr as A screening cutoff values for PA with three NC-CLEIA methods

including the assay used in the present study [5]. To our regret, there were no sufficient evident

data measured by the new NC-CLEIA PAC methods equivalent to that by LC-MS/MS PAC.

Results of our present study therefore could provide some evidence as to the screening cutoff

values in the newly revised Japanese guideline for the management of PA. If setting the screening

cutoff values in this guideline aimed at a higher sensitivity of catching APA, the present study

demonstrated that setting the values of PAC ≥6.0 ng/dL and ARR ≥15 ng/dL per ng/mL/hr as the

screening cutoff could be appropriate if measured by LC-MS/MS (sensitivity/specificity:

100%/58.8%), as well as by this new assay (sensitivity/specificity: 100%/59.2%) to detect APA.

Young WF Jr. recommended that hypertensives with a PAC ≥10 ng/dL and PRA <1.0

ng/mL/hr should undergo confirmatory tests for PA [33]. To detect APA using our data, the

sensitivity/specificity of these popularly-used cutoff values measured by LC-MS/MS and this

new assay would be 88.0%/73.9% and 85.0%/75.8%, respectively. These high sensitivity and

specificity suggest that the cutoffs were of clinical usefulness for detecting APA.

In addition, Supplementary Tables S9-S12 of ROC analyses to determine cutoff values to

detect APA could provide important information considering various clinical and social

conditions, including AVS availability, health insurances, and others among different countries

[19]. The newly developed NC-CLEIA could become a clinically reliable alternative to LC-

MS/MS for PAC measurement.

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Limitation

Our present study had some limitations. Our institute is one of the highest-volume center for

endocrine hypertension, including PA in Japan. In general, aldosterone and renin measurements

are routinely conducted for screening secondary hypertension at offices of general practioners in

Japan. When ARR of the patients was found to be elevated, the physician have referred them to

specialized institutions such as Tohoku University Hospital. Consequently, all the patients were

enrolled from the endocrine hypertension unit of Tohoku University Hospital, which could

possibly result in the bias due to difference from the actual prevalence of PA in the general

hypertensive patient population. In addition, the cohort of our present study was exclusively

composed of Japanese patients. This specific regional and ethnicity could also result in some

selection bias. Due to the retrospective and cross-sectional nature of this study, the possibility of

some human errors could not be completely ruled out, which could also advertently influence the

results of retrospective studies compared to prospective studies. The patients at baseline and after

CCT of ARR under 20 ng/dL per ng/mL/hr were tentatively diagnosed as EH, which was not

independently validated. Therefore, this could result in verification bias and skew estimation of

sensitivities, that is, the hypertensive patients with lower value of ARR in our cohort may not

necessarily represent typical APA at general clinical arena, such as normotensive, normal level of

serum potassium, and without macronodular in adrenal glands. It is therefore entirely true that

these limitations could impede the capacity to present a definitive proposal.

In addition, our present study was designed to screen for APA, and further validation through

confirmatory functional tests in the different groups of cohorts is definitively required for

clarification.

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Conclusion

RIA or CLEIA were the main available methods for practicing clinicians to screen patients for

PA, even though these assays lacked high specificity and sensitivity. LC-MS/MS is ideal for

measuring PAC, but it is time consuming, labor intensive, and expensive. Results of our present

study demonstrated that the newly developed NC-CLEIA could be as useful as LC-MS/MS to

measure PAC, with high specificity and sensitivity, even at a low concentration range of PAC. In

addition, the NC-CLEIA is less time-consuming and expensive than LC-MS/MS. Considering

circumstances of health care insurance and medical resources in many countries, this accurate,

less time consuming, and more cost-effective new assay to measure PAC could become a

clinically reliable substitute to LC-MS/MS for detecting APA in the great majority of the

countries with universal health care.

Acknowledgements

We thank Yasuko Tsukada, Akane Sugawara, Mika Ainoya, Kumi Kikuchi, and Hiroko Kato for

their secretarial assistance.

Data Availability

Original data generated and analyzed during this study are included in this published article or in

the data repositories listed in References.

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Figure legends

Figure 1. Correlation of plasma aldosterone concentration (PAC) between non-competitive

chemiluminescent enzyme immunoassay (NC-CLEIA) and liquid chromatography-tandem mass

spectrometry (LC-MS/MS) (A) and correlation of PAC between radioimmunoassay (RIA) and

LC-MS/MS (B). Bland-Altman plot analysis of PAC between LC-MS/MS and NC-CLEIA (C)

and between LC-MS/MS and RIA (D). In the Bland-Altman plot analysis, solid line indicates

mean difference (bias), and dotted lines indicate 95% limits of agreement, respectively. SD and

CI represent standard deviation and confidence interval, respectively.

Figure 2

Distribution of liquid chromatography-tandem mass spectrometry (LC-MS/MS; A) and non-

competitive chemiluminescent enzyme immunoassay (NC-CLEIA; B) of plasma aldosterone

concentration (PAC) measurements in aldosterone-producing adenoma (APA), bilateral

hyperaldosteronism (BHA), and essential hypertension (EH). Distribution of LC-MS/MS (C) and

NC-CLEIA (D) of aldosterone/renin activity ratio (ARR) in APA, BHA, and EH. * indicates

<0.05.

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Figure 3

Receiver-operating characteristics (ROC) analysis with liquid chromatography-tandem mass

spectrometry (LC-MS/MS), non-competitive chemiluminescent enzyme immunoassay (NC-

CLEIA), and radioimmunoassay (RIA) of plasma aldosterone concentration (PAC) measurements

to discriminate aldosterone-producing adenoma (APA) from non-APA (A). ROC analysis with

LC-MS/MS, NC-CLEIA, and RIA of aldosterone/renin activity ratio (ARR) to discriminate APA

from non-APA (B).

Table 1. Clinical Characteristics of the aldosterone-producing adenoma, bilateral

hyperaldosteronism, and essential hypertension Groups.

APA

BHA

va lues

Number

133

100

111

Age (yea r old)

51.7 ± 12.7

56.7 ± 10.1

49.0± 14.6

< 0.05 *†

Sex (ma le, fema le)

75, 58

33, 67

45, 66

Body ma ss index (kg/m

)

25.1 ± 4.8

24.8 ± 4.1

26.0 ± 5.6

N.S.

Systolic blood pressure (mmHg)

148.4 ± 17.0

145.8 ± 17.5

144.1 ± 25.6

N.S.

Dia stolic blood pressure (mmHg)

95.4 ± 10.3

93.6 ± 11.5

93.1 ± 19.0

N.S.

Hea rt ra te (bea t per minute)

75.7 ± 13.0

76.9 ± 10.8

80.1 ± 13.3

< 0.05 ‡

Number of a nti-hypertensive a gents per da y

1.9 ± 1.3

1.6 ± 1.0

1.1 ± 0.9

< 0.05 ‡

eGFR (mL/min/1.73m

)

79.4 ± 28.5

77.0 ± 18.1

86.3 ± 28.6

N.S.

Serum pota ssium (mM)

3.4 ± 0.5

3.9 ± 0.5

4.0 ± 0.4

< 0.05 *‡

Pota ssium repla cement (mmol/da y)

71.4 ± 42.2

18.3 ± 26.0

9.2 ± 17.9

< 0.05 *†‡

LC-MS/MS PAC (ng/dL)

32.5 ± 22.1

8.5 ± 6.1

8.6 ± 6.9

< 0.05 *‡

NC-CLEIA PAC (ng/dL)

32.1 ± 21.0

8.3 ± 5.7

8.2 ± 6.5

< 0.05 *‡

RIA PAC (ng/dL)

48.3 ± 25.9

17.6 ± 8.7

17.9 ± 10.1

< 0.05 *‡

Pla sma renin a ctivity (ng/mL/hr)

0.3 ± 0.2

0.3 ± 0.1

1.1 ± 1.0

< 0.05 †‡

ARR with LC-MS/MS PAC (ng/dL per ng/mL/hr)

133.7 ± 114.4

34.5 ± 23.4

13.4 ± 12.2

< 0.05 *†‡

ARR with NC-CLEIA PAC (ng/dL per ng/mL/hr)

131.8 ± 109.4

33.8 ± 22.1

12.6 ± 11.2

< 0.05 *†‡

ARR with RIA PAC (ng/dL per ng/mL/hr)

197.7 ± 140.5

73.0 ± 37.8

28.3 ± 20.0

< 0.05 *†‡

Ca ptopril-cha llenged ARR with RIA PAC (ng/dL per
ng/mL/hr)

225.3 ± 250.7

53.1 ± 30.7

11.0 ± 5.1

< 0.05 *†‡

Cortisol with 1mg dexa metha sone suppression test (µg/dL)

1.2 ± 0.9

0.9 ± 0.5

0.9 ± 0.6

< 0.05 *‡

Data were shown as mean ± standard deviation. APA indicates aldosterone-producing adenoma;

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