Clinical Chemistry 67:8 Endocrinology and Metabolism

1090–1097 (2021)

Diagnostic Accuracy of Salivary Metanephrines in

Pheochromocytomas and Paragangliomas
Karin Eijkelenkamp,a,* Thamara E. Osinga,a Martijn van Faassen,b Ido P. Kema,b Michiel N. Kerstens,a
Karel Pacak,c Wim J. Sluiter,a Thera P. Links,a and Anouk N.A. van der Horst-Schriversa,d

metanephrines correlate with plasma free metanephrines

BACKGROUND: Measurements of plasma free metanephr- and are increased in patients with PPGL. At this time,
ines are recommended for diagnosing pheo-

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however, salivary metanephrines cannot replace mea-
chromocytomas and paragangliomas (PPGL). surement of plasma free metanephrines.
Metanephrines can be detected in saliva with LC-MS/
MS with sufficient analytical sensitivity and precision.
Because collecting saliva is noninvasive and less cumber-
Introduction
some than plasma or urine sampling, we assessed the di-
agnostic accuracy of salivary metanephrines in diagnos- Pheochromocytomas and sympathetic paragangliomas
ing PPGL. are rare neuroendocrine tumors arising from chromaffin
cells that can synthesize and secrete catecholamines.
METHODS: This 2-center study included 118 healthy
Approximately 40% of these tumors are associated with
participants (44 men; mean age: 33 years (range: 19–74
years)), 44 patients with PPGL, and 54 patients sus- an underlying inherited mutation (1, 2). Early detection
pected of PPGL. Metanephrines were quantified in of sympathetic pheochromocytomas and paraganglio-
plasma and saliva using LC-MS/MS. Diagnostic accu- mas (PPGL) in clinically suspected patients is warranted
racy; correlation between plasma and salivary meta- to prevent potentially lethal cardiovascular complica-
nephrines; and potential factors influencing salivary tions. Therefore, screening for PPGL is recommended
metanephrines, including age, sex, and posture during for patients with typical signs and symptoms and/or car-
sampling, were assessed. diovascular events, young lean individuals with type 2
diabetes, patients with an adrenal incidentaloma, germ-
RESULTS: Salivary metanephrines were significantly line mutation carriers, and patients with a previous his-
higher in patients with PPGL compared with healthy tory or family history of PPGL (3, 4).
participants (metanephrine (MN): 0.19 vs 0.09 nmol/L, Measurement of plasma free metanephrines is cur-
P < 0.001; normetanephrine (NMN): 2.90 vs rently considered to be the most accurate method for di-
0.49 nmol/L, P < 0.001). The diagnostic sensitivity and agnosing these tumors, with diagnostic sensitivity and
specificity of salivary metanephrines were 89% and specificity of 90%–100% and 79%–98%, respectively
87%, respectively. Diagnostic accuracy of salivary meta- (3, 5). However, the concentration of plasma free meta-
nephrines was 88%, with an area under the ROC curve nephrines can be affected by several factors, including
of 0.880. We found a significant correlation between age, sex, caffeine, medication (e.g., tricyclic antidepres-
plasma and salivary metanephrines (Pearson correlation sants), salt intake, season, adrenalectomy, smoking,
coefficient: MN, 0.86, P < 0.001; NMN, 0.83, method of sampling, and posture during blood sampling
P < 0.001). Salivary NMN concentrations were higher (3, 6–10). Blood sampling in the seated position, for ex-
when collected in a seated position compared with ample, can falsely increase plasma metanephrines, with
supine (P < 0.001) and increased with age (P < 0.001). approximately 30% due to activation of the sympathoa-
drenal system (7). Therefore, the US Endocrine Society
CONCLUSIONS: Salivary metanephrines are a promising recommends letting patients rest in the supine position
tool in the biochemical diagnosis of PPGL. Salivary for 20–30 min before blood sampling (3).Consequently,

a
Department of Endocrinology and Metabolic Diseases, University of Groningen, *Address correspondence to this author at: Department of Endocrinology, University
University Medical Center Groningen, Groningen, the Netherlands; bDepartment of Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB
Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. Fax þ31-50-3619392; e-mail k.eijkelenkamp@umcg.nl.
Groningen, the Netherlands; cEunice Kennedy Shriver National Institute of Child Health Received October 16, 2020; accepted March 24, 2021.
and Human Development, National Institutes of Health, Bethesda, MD, USA; DOI: 10.1093/clinchem/hvab064
d
Department of Internal Medicine, Division of Endocrinology, Maastricht University
Medical Center, Maastricht, the Netherlands.

C American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.
V 1090
Salivary Metanephrines

these recommendations require determination of refer- the PPGL diagnosis. In the case of resection of the tu-
ence intervals for plasma free metanephrines in the su- mor, the histopathology had to be compatible with the
pine position and special facilities in the hospital for PPGL diagnosis.
blood sampling, which can be cumbersome and lead to Patients were eligible for inclusion at the UMCG if
increased costs (11). they were diagnosed with a sympathetic PPGL. This
Saliva has a potential diagnostic function and offers diagnosis was based on increased (according to the refer-
advantages over plasma because it can be collected easily ence intervals of the UMCG) plasma free MN and/or
and noninvasively. Saliva contains various enzymes, hor- NMN collected via direct venipuncture after 30 min of
mones, and antibodies that enter the saliva from the supine rest (upper reference limit of the UMCG: MN,
blood by active and passive diffusion or extracellular 0.28 nmol/L; NMN, 0.79 nmol/L) and imaging by CT

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ultrafiltration. Metanephrines also pass across the acinar or MRI and nuclear imaging and, in case of resection of
cells via ultrafiltration into saliva through gap junctions the tumor, the histopathology compatible with the
between cells of secretory units (12). Previously, we PPGL diagnosis.
showed that high-performance LC-MS/MS was a highly Group 3 consisted of patients suspected of and
sensitive technique that enabled the measurement of tested for a sympathetic PPGL (i.e., germline mutation
very low concentrations of catecholamines and meta- carriers) and patients with a previous history or family
nephrines in saliva (13). Because saliva can be collected history of PPGL and in whom a PPGL was ruled out.
at home, specimen collection could also be more Absence of PPGL was defined as negative CT or MRI
convenient. studies and negative results of follow-up biochemical
The primary aim of this study was to assess the testing (i.e., plasma free MN and plasma NMN both
diagnostic accuracy of salivary metanephrines in diag- within reference range).
nosing PPGL. Secondary objectives were to assess the Exclusion criteria for all 3 groups were as
correlation between plasma and salivary metanephrines follows: age <18 years and use of medications that
and to evaluate potential influencing factors like sex, could influence plasma free metanephrines, including
age, and posture during sampling on salivary tricyclic antidepressants, phenoxybenzamine, mono-
metanephrines. amine-oxidase inhibitors, sympathomimetics, cocaine,
and methyldopa. Patients in group 2 were excluded
Methods when their histology reports were not compatible with
a diagnosis of a PPGL.
STUDY DESIGN The study protocol was approved by the medical
This 2-center study was performed at the Eunice ethics committee of the UMCG and approved by the
Kennedy Shriver National Institute of Child Health and Institutional Review Board of the NICHD (protocol
Human Development (NICHD) at the National 00-CH-0093). All patients provided written informed
Institutes of Health (Bethesda, MD, USA) and the consent. Recommendations of the Standards for the
University Medical Center Groningen (UMCG; Reporting of Diagnostic Accuracy Studies were
Groningen, the Netherlands). followed.

PARTICIPANTS SPECIMEN COLLECTION

Group 1 consisted of healthy, normotensive participants Participants were asked to not drink caffeine-containing
(blood pressure 130/80 mmHg without the use of beverages, to abstain from smoking, and to fast for at
antihypertensive medication) with a negative history of least 1 h before saliva collection. Blood pressure was
cardiovascular events. measured in a seated position using an automated oscil-
Patients at the NICHD and UMCG with a con- lometric device. Saliva was collected using a polyethyl-
R
firmed sympathetic PPGL were included in group 2. ene swab (SalivetteV; Starstedt). After that, participants
Patients were eligible for inclusion at the NICHD if at the UMCG were placed in the supine position for
they were diagnosed with a sympathetic PPGL. This 30 min, and blood was drawn via venipuncture (using
R
diagnosis was based on increased (according to the refer- Becton Dickinson VacutainerV tubes containing K2-
ence intervals of the NICHD) plasma free metanephrine EDTA as an anticoagulant) together with saliva collec-
(MN) and/or normetanephrine (NMN) collected via an tion in a supine position. At the NICHD, saliva was
indwelling catheter after 30 min of supine rest (upper collected only in the supine position.
reference limit of the NICHD: MN, 0.31 nmol/L;
NMN, 0.61 nmol/L). In addition, imaging by com- ANALYTICAL METHOD
puted tomography (CT) or magnetic resonance imaging Salivettes were centrifuged at 1000g for 2 min, and the
(MRI) and nuclear imaging had to be compatible with filtrate was collected and stored at 80  C until analysis.

Clinical Chemistry 67:8 (2021) 1091

Plasma free and salivary metanephrines were analyzed accuracy of test performance was defined as the sum of
by LC-MS/MS with automatic solid-phase extraction true positive and true negative test results over the sum
sample preparation (14). All assays were performed in of the number of test results. ROC curves were con-
batches at the Department of Laboratory Medicine of structed based on a binary logistic regression model in-
the UMCG. cluding MN and NMN as covariates. The area under
The analytical method, extensively validated for the curve was calculated to determine the discrimination
plasma free metanephrines, was validated for saliva as power. The difference in test performance was calcu-
described previously, using saliva pools with low, me- lated using the method of Hanley and McNeil.
dium, and high concentrations of MN and NMN (13). After log transformation of the data, a parametric
Intra- and interassay CVs were 1.4%–7.0% and correlation analysis (Pearson correlation coefficient) was

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3.2%–8.3%, respectively, for salivary MN 1.7%–1.8% used to calculate the correlation between salivary and
and 1.7%–3.2%, respectively, for salivary NMN. Lower plasma free metanephrines.
limits of quantification in saliva were 0.03 and To assess several influencing factors on salivary
0.035 nmol/L for MN and NMN, respectively. metanephrines, intra- and intergroup differences were
Recovery was 106% (SD: 10%) for MN and 101% analyzed using the Wilcoxon signed-rank test and the
(SD: 5%) for NMN. Linearity was excellent over the Mann-Whitney U test. A 1-sided significance level of
different calibration ranges (MN: 0.03–10 nmol/L; 0.05 was used only to reject the null hypothesis that po-
NMN: 0.035–26 nmol/L) with R2 > 0.99 (13). sition during sampling does not influence the concentra-
tion of metanephrines.
ASSESSMENT OF SHORT-TERM STABILITY OF MN/NMN IN
SALIVA Results
For assessment of short-term stability of MN and NMN
in saliva, saliva samples from 6 volunteers were used. PARTICIPANTS
The saliva samples were divided into aliquots, which In group 1, we included 118 healthy participants
were stored for periods of 1, 3, 6 and 8 days at 20  C, (44 men and 74 women) with a mean age of 33 years
6  C, and room temperature until storage at 80 C (range: 19 - 74 years).
for batch sample processing and LC-MS/MS analysis. Forty-eight patients with PPGL were eligible for in-
The control group included aliquots that were stored at clusion at the NICHD, and 20 patients with PPGL
80  C right after obtaining the saliva samples. were eligible at the UMCG. Twenty-four patients were
Repeated-measures ANOVA was used to elucidate excluded due to the use of interfering medication, miss-
changes and differences in analyte concentrations ing data, or a pathology diagnosis incompatible with
(Bonferroni correction). PPGL (Fig. 1). Forty-four patients were included in
group 2 (24 men and 20 women) with a mean age of 51
STATISTICAL ANALYSIS years (6SD: 14 years). Twenty-four patients had sym-
Data are presented as mean (6SD) or median with inter- pathetic paraganglioma and 20 patients had pheochro-
quartile range (IQR), as appropriate. Analyses were per- mocytoma. Twenty-four patients had metastatic PPGL.
formed using SPSS statistics (version 23; IBM/SPSS). Sixty patients tested for PPGL were eligible for
Reference intervals for salivary metanephrines were inclusion in group 3. Six patients were excluded due
established in the healthy participants. Gaussian distri- to missing data. In total, 54 patients (23 men and 31
butions of salivary metanephrines were obtained after women) were included, with a mean age of 46 years
logarithmic transformation of the data. The antiloga- (6SD: 15.7 years). Fifty patients had a germline muta-
rithm of the mean (plus or minus 2 SD) of the trans- tion in one of the susceptibility genes for PPGL. Four
formed data was used to obtain the lower and upper patients had a previous history of PPGL. In all patients,
reference limits. a PPGL was ruled out after a median follow-up period
The diagnostic sensitivity of salivary and plasma of 4.5 years.
metanephrines was calculated from the percentage of The characteristics of the patients in groups 2 and
true-positive over total true-positive plus false-negative 3 are shown in Table 1.
test results in patients with PPGL (group 2). A true pos-
itive test result was defined as MN and/or NMN con- SALIVARY METANEPHRINES
centrations above the upper reference limit. Diagnostic Reference intervals for salivary metanephrines in a su-
specificity was calculated from the percentage of true- pine position were 0.04–0.25 nmol/L for MN and
negative over the total of true-negative plus false-positive 0.20–1.46 nmol/L for NMN (Table 2). Supine salivary
test results in patients with suspected PPGL and in MN concentrations in patients with PPGL, patients
whom a PPGL was excluded (group 3). Diagnostic with suspected PPGL, and healthy participants were

1092 Clinical Chemistry 67:8 (2021)

Salivary Metanephrines

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Fig. 1. Flowchart of the inclusion of healthy participants, patients with PPGL, and patients with suspected PPGL.

concentrations for patients with PPGL vs healthy par-

Table 1. Patient characteristics for groups 2 and 3. ticipants (P < 0.001) and between patients with PPGL
vs patients with suspected PPGL (P < 0.001). There
Patients with
was no statistically significant difference for salivary
Patients with suspected
PPGL (n ¼ 44) PPGL (n 5 54) MN concentrations between healthy participants vs
patients with suspected PPGL (P ¼ 0.726).
Age in years, 51 (13.9) 46 (15.7)
mean (SD) Supine salivary NMN concentrations in patients
Sex (M/F) 24/20 23/31
with PPGL, patients with suspected PPGL, and healthy
participants were 2.90 nmol/L (IQR: 1.54–5.57
PCC, n (%) 20 (45) 0
nmol/L), 0.63 nmol/L (IQR: 0.48–1.08 nmol/L), and
sPGL, n (%) 24 (55) 0 0.49 nmol/L (IQR: 0.34–0.71 nmol/L), respectively.
Metastatic PPGL, n (%) 24 (55) 0 There was a statistically significant difference for salivary
Germline mutation NMN concentrations of patients with PPGL vs healthy
confirmed: participants (P < 0.001) and between patients with
SDHx 8 28 PPGL vs patients with suspected PPGL (P < 0.001) and
RET 6 13 between patients with suspected PPGL and healthy par-
HIF2a 3 0 ticipants (P < 0.001) (Fig. 2).
VHL 1 11
NF1 1 1 CORRELATION OF PLASMA AND SALIVARY METANEPHRINES

MDH2 1 0 We found a statistically significant correlation be-

tween salivary and plasma concentrations of MN and
HIF2a, hypoxia-inducible factor-2a; MDH2, malate dehydrogenase 2; NF1,
neurofibromatosis; PCC, pheochromocytoma; RET, rearranged during transfec-
NMN in the supine position (Pearson correlation
tion; SDHx, succinate dehydrogenase; sPGL, sympathetic paraganglioma; VHL, coefficient: 0.86 and 0.83, respectively; P < 0.001 for
von Hippel Lindau. both) (Fig. 3).

INFLUENCING FACTORS ON SALIVARY METANEPHRINES IN

0.19 nmol/L (IQR: 0.08–1.41 nmol/L), 0.09 nmol/L HEALTHY PARTICIPANTS
(IQR: 0.06–0.15 nmol/L), and 0.09 nmol/L (IQR: Salivary MN collected in the seated position (median:
0.07–0.14 nmol/L; P < 0.001), respectively. There was 0.09 nmol/L (IQR:0.07–0.13 nmol/L)) was not statis-
a statistically significant difference for salivary MN tically different compared with salivary MN collected

Clinical Chemistry 67:8 (2021) 1093

 Table 2. Concentrations of salivary metanephrines in healthy participants (n ¼ 118).

MN (nmol/L) P value NMN (nmol/L) P value

Posture
Supine 0.09 (0.07–0.14) 0.312 0.49 (0.34–0.71) <0.001
Seated 0.09 (0.07–0.13) 0.62 (0.46–0.86)
Age
<40 y 0.09 (0.06–0.15) 0.092 0.42 (0.32–0.59) <0.001
>40 y 0.10 (0.08–0.15) 0.63 (0.48–0.95)

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Sex
Male 0.10 (0.07–0.17) 0.060 0.55 (0.40–0.82) 0.071
Female 0.09 (0.07–0.12) 0.47 (0.33–0.67)

Data presented as median and interquartile ranges.

A p < 0.001 B p < 0.001

p = 0.726 p < 0.001

p < 0.001 p < 0.001

Fig. 2. Concentrations (median and IQR) of salivary MN (A) and NMN (B) in patients with PPGL, patients with suspected PPGL,
and healthy participants. The dotted line shows the upper reference limit of salivary MN (0.25 nmol/L) and NMN (1.46 nmol/L).

in the supine position (median: 0.09 nmol/L (IQR: position (median: 0.49 nmol/L (IQR: 0.34–0.71
0.07–0.13 nmol/L)) (P ¼ 0.312) (Table 2). Salivary nmol/L)) (P < 0.001) (Table 2).
NMN was statistically significant higher in the seated Salivary MN in healthy participants <40 years
position (median: 0.62 nmol/L (IQR: 0.46–0.86 (median: 0.09 nmol/L (IQR: 0.06-0.15 nmol/L)) was
nmol/L)) compared with collection in the supine not statistically different compared with salivary MN

1094 Clinical Chemistry 67:8 (2021)

Salivary Metanephrines

A B
100.000 1000.0
Healthy subjects
Patients suspected of PPGL

Salivary normetanephrine (nmol/L)

PPGL patients
PPGL patients with negative
Salivary metanephrine (nmol/L)

10.000 salivary (nor)metanephrine

100.0

1.000

10.0

.100

1.0

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.010

Pearson 0.86 Pearson 0.83

.001 p < 0.001 .1 p < 0.001

.001 .010 .100 1.000 10.000 100.000 .1 1.0 10.0 100.0 1000.0

Plasma metanephrine (nmol/L) Plasma normetanephrine (nmol/L)

Fig. 3. Correlation between salivary and plasma MN (A) and NMN (B) concentrations.

concentrations of healthy participants >40 years (me- 1.0

dian: 0.10 nmol/L (IQR: 0.08–0.15 nmol/L))
(P ¼ 0.092). Salivary NMN in healthy participants
>40 years (median: 0.63 nmol/L (IQR: 0.48–0.95
0.8
nmol/L)) was statistically higher than in participants
<40 years (median: 0.42 nmol/L (IQR: 0.32–0.59
nmol/L)) (P < 0.001).
There was a trend, albeit not statistically significant, 0.6
Sensitivity

that salivary MN and NMN concentrations were higher

in men (median: MN, 0.10 nmol/L (IQR: 0.07–
0.17 nmol/L); NMN, 0.55 nmol/L (IQR: 0.40– 0.4

0.82 nmol/L)) compared with women (median: MN,

0.09 nmol/L (IQR: 0.07–0.12 nmol/L)); NMN,
0.47 nmol/L (IQR: 0.33–0.67 nmol/L)), respectively 0.2

(P ¼ 0.060 and P ¼ 0.071).

AUC plasma metanephrines 0.948
AUC salivary metanephrines 0.880

SHORT-TERM STABILITY MN/NMN IN SALIVA

0.0
MN and NMN in saliva did not show a significant 0.0 0.2 0.4 0.6 0.8 1.0

time-dependent increase or decrease up to 8 days, even 1 - Specificity

at room temperature (online Supplemental Fig. 1).

However, we did see in one individual a slightly increas- Fig. 4. ROC curve for salivary and plasma metanephrines
ing trend for NMN after 3 days at 6  C and room tem- in the supine position, constructed according to logistic
perature. Consequently, we suggest that the saliva regression model with a combination of NMN and MN, in-
should be sent to the laboratory with cooling packs and cluding patients with PPGL and patients with suspected
within 3 days after collection. PPGL in whom PPGL was excluded. AUC, area under the
curve.
DIAGNOSTIC ACCURACY OF SALIVARY AND PLASMA
METANEPHRINES patients with PPGL did not have increased supine sali-
The diagnostic sensitivity of salivary metanephrines in vary metanephrines concentrations (online
the supine position was 89% (39/44 patients with Supplemental Table 1).
PPGL), and diagnostic specificity was 87% (47/54). Because only patients with PPGL who had in-
The diagnostic accuracy of salivary metanephrines in the creased plasma metanephrines were included, the diag-
supine position was 88% (86/98). The area under the nostic sensitivity of plasma MN was 100%. The
ROC curve for combined salivary MN and NMN in diagnostic specificity of plasma metanephrines was 87%
the supine position was 0.880 (Fig. 4). Five of 44 (47/54 patients with PPGL). The area under the ROC

Clinical Chemistry 67:8 (2021) 1095

curve for combined plasma MN and NMN in the su- collection device, posture, eating, and awakening re-
pine position was 0.948 (Fig. 4), which was not statisti- sponse on salivary metanephrines (13). In line with 2
cally significantly different compared with salivary other studies, patients need proper instructions not to
metanephrines (P ¼ 0.086). chew on the Salivette, as this will lead to decreased con-
centrations of salivary metanephrines (16, 17).
Discussion Comparable to plasma free metanephrines, we
found higher salivary NMN, but not MN concentra-
In this study, we assessed the accuracy of salivary meta- tions, collected in the seated position compared with the
nephrines measured by LC-MS/MS as an alternative, non- supine position, and this result confirms our previous
invasive tool in the biochemical diagnosis of PPGL. We observation (13). Patients should be instructed to collect
showed that salivary metanephrines has diagnostic accuracy

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their saliva in the supine position.
of 88%, with diagnostic sensitivity of 89% and diagnostic The difference between salivary NMN in the seated
specificity of 87%. Furthermore, we found a strong corre- and supine positions but the lack of difference between
lation between plasma and salivary metanephrines, and the salivary MN in the seated and supine positions might be
concentration of salivary metanephrines was significantly explained by the fact that norepinephrine (but not epi-
higher in the group of patients with PPGL than in healthy nephrine) in saliva originates not only from the blood-
participants. In addition, salivary NMN concentrations stream but also from salivary sympathetic nerve endings
were higher when saliva was collected in the seated position (17). This locally released norepinephrine adds to the
compared with supine and increased with age in healthy measured salivary NMN concentration. This phenome-
participants. non also explains our finding that the NMN concentra-
In a previously published study (15), salivary meta- tion is higher in saliva than plasma. Unfortunately, this
nephrines was determined in 30 patients with pheochro- phenomenon might compromise the diagnostic accu-
mocytoma and 70 healthy controls, using ELISA. In racy of salivary metanephrines.
agreement with our data, this study found a significantly For clinical practice, saliva collection offers many
higher concentration of salivary metanephrines in advantages over plasma or urinary collection, as salivary
patients with pheochromocytoma compared with collection is noninvasive, inexpensive, and minimally
healthy controls and a strong correlation between stressful. This makes it potentially suitable for elderly
plasma and salivary metanephrines. The calculated diag- patients, patients with needle phobia, and children. In
nostic sensitivity and specificity of salivary metanephr- addition, saliva can be collected at home. Salivary NMN
ines for diagnosing pheochromocytoma were both and MN were stable up to 3 days at 6  C and room
100%. However, the previous study included only temperature.
patients with a pheochromocytoma with plasma concen- Our study has some limitations. As mentioned, the
trations of MN and NMN that were, on average, 9 and diagnostic sensitivity of salivary metanephrines was cal-
4 times higher, respectively, than the values in our study culated using a design with participation of consecutive
participants. The patients in our study were included in patients with PPGL who fulfilled our selection criteria,
2 tertiary referral centers, and >50% of patients had a including increased plasma metanephrines as the refer-
sympathetic PPGL or metastatic disease. ence test. A fair comparison between these 2 diagnostics
The diagnostic accuracy of plasma metanephrines would require a study with larger sample size, including
was higher than salivary metanephrines, although the consecutive patients with suspected PPGL.
difference was not statistically significant. The lack of Furthermore, comparable to plasma free NMN concen-
statistical significance is probably due to the low number trations, we found that higher age was associated with
of participants. In the present study, 5 patients had increased salivary NMN concentrations (18). This sug-
false-negative results for salivary metanephrines. It gests that age-adjusted reference intervals for salivary
should be noted that 3 patients had only mildly in- NMN seem justified. However, the relatively low num-
creased plasma metanephrines concentrations. Three of ber of patients in the present study did not allow assess-
the patients with false-negative test results demonstrated ment of such age-adjusted reference intervals. Reference
very low concentrations of salivary metanephrines. This intervals are also required for children because it has re-
might be explained by potential preanalytical factors on cently been demonstrated that plasma free NMN con-
salivary metanephrines, such as dilution of saliva due to centrations show dynamic changes during early
a high salivary flow rate. Until now, a marker to correct childhood (19). We did not include patients with hyper-
for this dilution phenomenon is lacking. Next to dilu- tension in group 1 to calculate specific reference inter-
tion and influencing factors that are already well known vals because another study has shown that, after
for plasma metanephrines, several preanalytical factors multivariate analysis, the differences between partici-
should be considered when measuring analytes in saliva. pants who are hypertensive and normotensive are not
Our previous study assessed the influence of the considered to be clinically relevant (6).

1096 Clinical Chemistry 67:8 (2021)

Salivary Metanephrines

In conclusion, determining salivary metanephrines Study concept and design: Eijkelenkamp, Osinga, van Faassen, Kema,
is a promising, novel tool in the biochemical diagnosis Kerstens, Sluiter, Links, van der Horst-Schrivers. Acquisition, analysis,
of PPGL. Salivary metanephrines correlates with plasma or interpretation of data: Eijkelenkamp, Osinga, van Faassen, Kema,
Sluiter, Links, van der Horst-Schrivers. Drafting of the manuscript:
free metanephrines and is elevated in patients with Eijkelenkamp, van der Horst-Schrivers. Critical revision of the manu-
PPGL. At this moment, however, salivary metanephr- script for important intellectual content: all authors. Administrative,
ines cannot replace the measurement of plasma free technical, or material support: van Faassen, Kema, Pacak. Final ap-
metanephrines. proval of the version to be published: all authors.

Supplemental Material Authors’ Disclosures or Potential Conflicts of Interest: Upon manu-

script submission, all authors completed the author disclosure form.
Wim J. Sluiter’s author disclosure form was completed on his behalf by
Supplemental material is available at Clinical Chemistry

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Michiel N. Kerstens. Disclosures and/or potential conflicts of interest:
online.
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Nonstandard Abbreviations: PPGL, pheochromocytoma and sympa-
Honoraria: None declared.
thetic paraganglioma; MN, metanephrines; NICHD, National
Research Funding: This work was funded by the Von Hippel-Lindau
Institute of Child Health and Human Development; UMCG,
Alliance and supported by the Intramural Research Program of the
University Medical Center Groningen; NMN, normetanephrines; National Institutes of Health, Eunice Kennedy Shriver National
IQR, interquartile range. Institute of Child Health and Human Development.
Author Contributions: All authors confirmed they have contributed to Expert Testimony: None declared.
the intellectual content of this paper and have met the following 4 require- Patents: None declared.
ments: (a) significant contributions to the conception and design, acquisi- Role of Sponsor: The funding organizations played no role in the de-
tion of data, or analysis and interpretation of data; (b) drafting or revising sign of study, choice of enrolled patients, review and interpretation of
the article for intellectual content; (c) final approval of the published arti- data, preparation of manuscript, or final approval of manuscript.
cle; and (d) agreement to be accountable for all aspects of the article thus
ensuring that questions related to the accuracy or integrity of any part of Acknowledgments: We gratefully acknowledge the contribution of J.
the article are appropriately investigated and resolved. Seventer for her critical appraisal of the manuscript.

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Clinical Chemistry 67:8 (2021) 1097