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Medical Mass Spectrometry Vol. 6 No. 1

Research Paper

Eﬀects of 11β-hydroxysteroid dehydrogenase 2 activity
on the prediction accuracy of plasma unbound cortisol
concentration based on salivary cortisol
Akitomo Yokokawa*, Aya Kitano, Daichi Tanaka, Kai Higashi,
Shun Yanada, Kohei Shimizu, Tomomi Furihata, Hiromi Shibasaki
Laboratory of Clinical Pharmacy and Experimental Therapeutics, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, 1432‒1 Horinouchi, Hachioji, Tokyo 192‒0392, Japan
Abstract Plasma unbound cortisol concentration is an accurate measure of its physiological activity. As unbound cortisol is excreted from plasma into saliva, it may be possible to non-invasively predict plasma unbound cortisol levels using
salivary cortisol levels. However, part of the salivary cortisol is converted to cortisone, which can aﬀect such predictions.
Therefore, correlation between salivary cortisol and plasma unbound cortisol was analyzed in healthy subjects (n＝9) andthe eﬀect of cortisol-to-cortisone conversion on this correlation was investigated. The correlation equation between salivary
cortisol concentration (range, 0.26‒3.22 ng/mL) and plasma unbound cortisol concentration (range, 0.31‒2.74 ng/mL) forall subjects was determined to be y＝0.5311x＋0.5117 (r＝0.51); however, it showed low correlation. Then, as individual differences in the slope of the correlation equation ranged from 0.62 to 2.62, we classiﬁed subjects into two groups based ona mean slope value of 1.10 and re-evaluated the correlation. The resulting equations yielded better correlations (y＝2.0416x＋0.0279, r＝0.65, for two subjects and y＝0.7417x＋0.1195, r＝0.77, for seven subjects), and the observed variation in slope
was attributed to individual diﬀerences in salivary 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity. Importantly,
mean absolute percentage error in predicting plasma cortisol using salivary cortisol levels using the two correlation equations was 0.6%. Thus, these results suggest that deriving and classifying salivary cortisone/cortisol ratio, along with relevant correlation equations, can be used to non-invasively predict plasma unbound cortisol concentration.
Key words: cortisol, saliva concentration, plasma unbound concentration, GC-MS, 11β-hydroxysteroid dehydrogenase 2

Introduction
Cortisol is the principal corticosteroid in humans and isexhibit circadian rhythm. As more than 90% of the cortisol
is protein-bound in plasma, only less than 10% of thesecreted by the adrenal cortex. Its secretion is controlled byunbound cortisol crosses biological membranes and is bio-the adrenocorticotropic hormone (ACTH) and is known toactive*Correspondence author

cortisol is an accurate measure of its physiological activity,
its measurement is complicated as it involves multiple pro-

1,2)

Akitomo Yokokawa. Thus, while plasma unbound concentration ofcesses, including equilibrium dialysis, which limit its rou-

Laboratory of Clinical Pharmacy and Experimental Therapeu-

tine clinical application. Further, although physiologicaltics, School of Pharmacy, Tokyo University of Pharmacy andLife Sciences, 1432‒1 Horinouchi, Hachioji, Tokyo 192‒0392,

activity can be evaluated by predicting plasma unboundcortisol levels from a simple measurement of total plasmaJapanconcentration, the relationship between the total and plasma

Tel: ＋81‒42‒676‒5803, Fax: ＋81‒42‒676‒5805
E-mail: yokokawa@toyaku.ac.jp

unbound cortisol levels change due to ﬂuctuations inplasma protein concentration and binding aﬃnity. Hence, it
has been pointed out that measuring only total concentra-

Received: November 11, 2021. Accepted: February 10, 2022.
Epub April 8, 2022.

tion when evaluating adrenal function can lead to an errone-

DOI: 10.24508/mms.2022.06.003
45

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Medical Mass Spectrometry Vol. 6 No. 1

ro-n-butyric anhydride were purchased from Sigma-Aldrich(St. Louis, MO, USA). All other chemicals and solventswere of analytical-reagent grade purchased from Kanto
Chemicals (Tokyo, Japan).

Sample collection
Fig. 1.

Plasma and saliva were collected from nine healthy volunteer subjects (Subjects A‒I, eight male and one female,

Schematic representation of transfer of cortisol
from blood to saliva. When cortisol is secreted
from the plasma into the saliva, a part of it is converted to cortisone by 11β-HSD2, which results ina reverse phenomenon in which salivary cortisol islower than plasma cortisol, but salivary cortisone
is higher than plasma cortisone.

age range 22‒42 years). Plasma and saliva were collected
for 3 days between 10:00 and 18:00 h randomly (6‒12times for each subject, total 81 times) according to the subject s schedule. The subjects did not smoke or take medication during the study period. Additionally, the subjects did
not have oral diseases, such as periodontal disease. The

ous diagnoses and unnecessary administration of corticoste-

study was approved by the Human Subjects Review Board

3,4). Furthermore, as adrenal function is assessedof the Tokyo University of Pharmacy and Life Sciences, andtemporally, i.e., over time during treatment, a simple

written informed consent was obtained from all participants.

method for routinely evaluating adrenal function, based on

Plasma and saliva samples were stored frozen at −20°
C untilplasma unbound cortisol levels, is useful.

analysis.

roids

Cortisol, like the unbound forms of many drugs andendogenous substances, is secreted into the saliva, and thus,
it might be possible to predict plasma unbound cortisol con-

Measurement of plasma unbound cortisol concentration

centration based on salivary levels when evaluating adrenal

Japan) was used for ultraﬁltration. After adding 0.5 mL of

A Nanosep 10K Omega Membrane (Nihon pall, Tokyo,

5,6). Notably, when cortisol is secreted from plasma
into saliva, part of it is converted to cortisone by 11β-

distilled water to the device, centrifugation was performedhydroxysteroid dehydrogenase type 2 (11β-HSD2),
which leads to a reverse phenomenon wherein salivary cor-

removal was repeated twice. Plasma samples were thawed

tisol is lower than plasma cortisol but salivary cortisone isplasma sample was added to the device and incubatedfunctionat 2500 g for 10 minutes and this process of distilled water

7,8)

and centrifuged at 800 g for 10 minutes, and 500μL of

5)higher than plasma cortisone (Fig. 1) . Consequently, whenat 37°C for 30 minutes. Unbound cortisol was ﬁltered by

predicting the plasma unbound cortisol concentration basedon salivary levels, it is necessary to consider the concentra-

centrifugation at 2500 g for 60 minutes in a centrifuge
heated to 37°
C.

tion of both cortisol and cortisone in saliva. However, as no

Measurement of saliva cortisol and cortisone

reports to date have investigated this, we investigated if
plasma unbound cortisol levels can be predicted based on

Saliva samples were collected using Salimetrics oral

salivary cortisol concentration by developing a correlationswabs (SOS) and swab storage tubes (SST) purchased from
Salimetrics (Carlsbad, CA, USA). The Salimetrics indi-

equation between these two variables using data fromhealthy subjects and by exploring the eﬀect of corti-

cated that salivary cortisol concentrations measured with

sol-to-cortisone conversion on such predictions.

the collection devices are almost identical to passive drool
concentrations (https://salimetrics.com/rigor-and-reproduc

Experimental

ibility-for-collecting-saliva-samples/). Saliva was collected

Chemicals and reagents

30 minutes after eating and 10 minutes after rinsing their[1,2,4,19- C4]cortisol (cortisol- C4) and [1,2,4,19- C4]
13
cortisone (cortisone- C4) were synthesized in our labora-

mouth. Tooth brushing was prohibited for at least 2 hours

sition of the labeled compounds were determined to begreater than ＞98%. Cortisol, cortisone, and heptaﬂuo-utes) and asking the participants to suck in saliva. The swab

13

13

13

before saliva collection. Saliva was collected by placing
SOS under the tongue for 1‒2 minutes (maximum 5 min-tory for use as internal standards and the isotopic compo9)

was kept immobile to prevent blood from mixing with the46

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Medical Mass Spectrometry Vol. 6 No. 1

saliva. The SOS was removed from the mouth, inserted into
the SST, and centrifuged at 800 g for 15 minutes. After cen-for each subject, and the equations were used to compare

trifugation, both SOS and SST were discarded, and the col-groups (high and low) based on the mean value of the slope

lected saliva was immediately frozen and stored at −20°
C

(1.10). Mean absolute percentage error (MAPE) was useduntil analysis. For the analysis of the stored saliva sample,
the collected saliva sample was thawed at room tempera-

for calculating the prediction accuracy and it was estimatedture and centrifuged at 800 g for 15 minutes.

used for model creation.

Extraction

Results

individual slope values. Correlations were divided into two

using an internal veriﬁcation method that reapplies the data

Measurement accuracy for salivary cortisol andcortisone, and plasma unbound cortisol, using GCMS/SIM

We have previously described a method for measuring
plasma and urinary cortisol and cortisone concentrations
10‒12)and this method was used in this study
using GC‒MS

The lower limit of detection (LOD) for cortisol and corti-

for measuring saliva and plasma unbound cortisol. Corti13
13
sol- C4 (13.60 ng) and cortisone- C4 (31.45 ng) used forsone was 10.2 pg and 5.45 pg, respectively, when the s/ninternal standards were added to the saliva sample

ratio was greater than 3. The limit of quantiﬁcation (LOQ)(0.75 mL) or the plasma unbound ultraﬁltration sample
from the Nanosep 10K Omega Membrane. A Sep-Pak C18

was set at 0.41 ng for cortisol and 0.44 ng for cortisone, andPlus column (360 mg/0.7 mL, Waters, Milford, MA, USA)was ﬁrst washed with 6 mL of ethyl acetate and 5 mL of dis-(RSD) were calculated −1.41% and 0.32%, respectively,tilled water. Next, the sample was injected, washed with
8 mL of distilled water, and eluted with 4 mL of ethyl ace-be measured with good accuracy. Calibration curves weretate. The solvent was evaporated to dryness under a stream

cortisone from 4.360 to 21.80 ng/mL, and linearity wasof nitrogen and the residue was derivatized.

obtained with a correlation coeﬃcient of 0.999 or higher.
For determining the accuracy of salivary cortisol and corti-

Gas chromatography-mass spectrometry/selective ion
monitoring (GC-MS/SIM)sone measurements, 12 pooled salivary samples (750μL

the relative error (RE) and relative standard deviation
indicating that samples near the quantiﬁcation limit could
constructed for cortisol from 0.406 to 10.16 ng/mL and foreach) were prepared, and 6 of them were spiked with

Capillary GC-MS/SIM analysis was done on a Shimadzu
QP2010 GC-MS. GC-MS used a SPB-1 fused-silica capil-

known concentrations of cortisol (0.81 ng) or cortisone(6.54 ng). The cortisol concentration in 750μL of saliva
sample was determined to be 1.95±0.10 ng while the corti-

lary column (15 m×0.25 mm i.d.) with the stationary phasesone concentration was 8.26±0.14 ng. Next, as the concen-coated to a ﬁlm thickness of 0.25μm (Supelco, Bellefonte,
13
PA, USA). As cortisol and cortisol- C4 were used as bis-

tration of cortisol and cortisone in the six spiked samples

methylenedioxy-heptaﬂuoro-n-butyryl (BMD-HFB) deriva-

was determined to be 2.62±0.06 ng/750 μL and 14.41±

tives, m/z 582 and 586 peaks (cortisol) were monitored and0.31 ng/750μL, respectively, RE was calculated to be

their peak area ratios were determined. Similarly, cortisone13
and cortisone- C4 were used as BMD-HFB derivatives, m/z−4.91% and −2.67% for cortisol and cortisone, respectively, whereas the RSD was 1.7‒4.9%.

598 and 602 peaks were monitored, and their peak area

For determining the accuracy of plasma unbound cortisol

ratios were determined.

levels, plasma was ﬁltered with an ultraﬁltration device(Nanosep 10K Omega Membrane), and a known amount of

Data analysis

cortisol was added to the ﬁltrate. RE was calculated to be

Correlation equation for predicting plasma unbound cor-

1.58%, whereas RSD ranged from 2.8 to 3.0%.

tisol concentration from salivary cortisol levels was derived

Correlation between salivary cortisol and plasma unbound cortisol in nine healthy subjects

as a linear regression model with plasma unbound cortisol
concentration on the x-axis and salivary cortisol on the

In the nine healthy subjects (Subjects A‒I), salivary corti-

y-axis. The correlation coeﬃcient (r) was used as the index
for evaluating the strength of the correlation. A linear

sol concentration ranged from 0.257 to 3.216 ng/mL, salivary

regression model passing through the origin was derived

cortisone ranged between 3.624 and 16.95 ng/mL, whereas
47June 2022

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Table 1.

Slope values for the correlation equation andsalivary cortisone/cortisol ratio in nine healthy
subjects (Subjects A‒I). The average of salivary
cortisone/cortisol ratios was 5.17 (Subjects A
and B) and 8.60 (Subjects C‒I), respectively.

Subject

Fig. 2.

Fig. 3.

Correlation between salivary and plasma unboundcortisol concentration in nine healthy subjects(Subjects A‒I). The correlation equation between
saliva concentration and plasma unbound concentration was determined y＝0.5311x＋0.5117, andthe correlation coeﬃcient was low at 0.5068.

Slope values for individual subjects for the correlation between salivary and plasma unboundcortisol levels in nine healthy subjects (Subjects
A‒I). Prediction equations were designed for eachsubject with the intercept set as 0, and the slope
values ranged from 0.62 to 2.62.

Salivary cortisone/cortisol ratio

A

2.622

5.203±1.479

B

1.524

5.132±0.961

C

0.994

5.688±0.847

D

0.987

10.325±2.855

E

0.828

8.490±1.509

F

0.798

9.473±2.028

G

0.795

7.669±1.848

H

0.720

7.996±1.897

I

0.624

10.546±2.236

Fig. 4.

plasma unbound cortisol concentrations were between

Slope value

Correlation lines based on which the study subjects were classiﬁed into two groups for predicting
plasma unbound cortisol concentration from salivary cortisol concentration. The correlation equations for the two groups were derived, and while
this equation for the group, including Subjects A
and B was y＝2.0416x＋0.0279 (r＝0.65), the samefor the other group with Subjects C‒I was y＝0.7417x＋0.1195 (r＝0.77).

0.307 ng/mL and 2.737 ng/mL. The correlation equation
between saliva concentration and plasma unbound concentra-

value than the average) was 5.17 and 8.60, respectively

tion was determined to be y＝0.5311x＋0.5117; however, the(Table 1). Further, due to this diﬀerence in slope values,

correlation coeﬃcient was low at 0.51 (Fig. 2).

correlation equations for the two groups were derived
again, and while that for the group including Subjects A

Classiﬁcation of correlation based on slope of theequationand B was y＝2.0416x＋0.0279 (r＝0.65), the same for theother group with Subjects C‒I was y＝0.7417x＋0.1195 (r＝Prediction equations were created for each subject with0.77) (Fig. 4).

the intercept set as 0, and the slope values ranged from 0.62
to 2.62 (Fig. 3). The average slope value was 1.10, and subjects were divided into groups based on this value. The

Improvement in prediction accuracy of the correlation
after classiﬁcation based on slope values

average of salivary cortisone/cortisol ratios of two subjects

When plasma unbound concentration was predicted(Subjects A and B; greater slope value than the average)
and of the other seven subjects (Subjects C‒I; lesser slope

using the correlation equation before classiﬁcation based on
slope (y＝0.5311x＋0.5117), the MAPE value was −20.7%.
48

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However, when the plasma unbound concentrations werepredicted using correlation equations derived after classiﬁ-

537 samples and 31 subjects, respectively, and report highcation (i.e., groups with two subjects and seven subjects,

The data in these studies were obtained from the samples of
the ACTH supplementation test, and plasma levels of corti-

5,6)

correlation coeﬃcients of 0.96 and 0.91, respectively

respectively) the MAPE value was found to be 0.6%.

.

sol were higher after the ACTH supplementation test.

Discussion

Therefore, the correlation coeﬃcient in these studies with a

Salivary and plasma unbound concentrations of cortisol
13,14)or immunohave been previously quantiﬁed by LC-MS

wide range of concentrations is considered good. However,. However, even though the GC-MS method has

women . In these reports without ACTH supplementation

excellent sensitivity, speciﬁcity, and robustness, no reports

test, the correlation between plasma unbound cortisol andhave used this method to measure salivary or plasma

salivary cortisol was y＝0.432x＋1.00 (r＝0.5949) and y＝unbound cortisol levels. We have previously established a0.324x＋0.55 (r＝0.6969) in the morning and evening,

method for measuring total plasma and urinary cortisol and
10‒12), and we used thiscortisone concentrations by GC-MS

respectively, which is close to our results. In the present

method to measure saliva and plasma unbound concentra-

subjects without ACTH loading test, which might havetions. As a result of validation, the sensitivity of LOD andreduced the correlation coeﬃcient. Therefore, to investigate
the cause of apparent non-conformity of data to the correla-assaysMeulenberg et al. studied the correlation in pregnant

3,15)

16)

study, the concentration range was narrow only in healthy

LOQ was suﬃcient to measure saliva and plasma unboundconcentrations. The calibration curve had a correlationcoeﬃcient of more than 0.999, indicating suﬃcient linear-tion equation, we created individual correlation equations

ity for quantiﬁcation. Importantly, the low RE and RSD values (RE −4.91% and −2.67%; RSD 1.7‒4.9%) demon-

subjects whose data did not ﬁt the correlation line, we ﬁrst
evaluated the slope of the correlation equation for each sub-strate that cortisol and cortisone in saliva can be quantiﬁed
with high accuracy using the GC-MS method. Plasma sam-ject by setting the intercept to 0 (as the concentration range

ples were ultraﬁltered using the Nanosep 10K Omega

diﬀerences (Table 1). We interpreted the large slope values

Membrane because this device can ﬁlter substances with ato indicate lower conversion of cortisol to cortisone whenmolecular weight (MW) of 10,000 or less; thus, cortisol
bound to the plasma albumin (MW 66,000) and CBG (MWunbound cortisol was secreted from plasma to saliva, i.e.,
salivary cortisol tended to be higher. Notably, this diﬀer-

52,000) is retained in the sample receiver and only unboundence in slope values also implies that the conversion of cor-cortisol is ﬁltered. Similar to saliva samples, accuracy oftisol to cortisone aﬀects the correlation between salivary

cortisol estimation in plasma after ultraﬁltration wasdeemed as high as both RE (＋1.58%) and RSD (2.8‒3.0%)

and plasma unbound cortisol in individual subjects. Further,for each subject using multiple sampling points. To identify

was narrow) and found that slope values showed individual

were low.

as the conversion of cortisol to cortisone is carried out by
11β-HSD2 present in the salivary glands, we hypothesized

Values for salivary cortisol concentration (0.257‒
3.216 ng/mL), cortisone concentration (3.624‒16.95 ng/

that the individual diﬀerence in 11β-HSD2 activity caused
the observed non-conformity to the correlation equation.

mL), and plasma unbound cortisol (0.307‒2.737 ng/mL)among the nine healthy subjects of this study are compara-

To verify our hypothesis, we evaluated individual diﬀerences in 11β-HSD2 activity in the salivary glands by using. The correla-

the salivary cortisone/cortisol ratio as the readout for 11β17,18). In support of our hypothesis, the saliHSD2 activity

ble to values reported for healthy subjects

13,14)

tion equation between salivary cortisol and plasma
unbound cortisol concentration among nine healthy sub-vary cortisone/cortisol ratio was lower in the group with thejects (Subjects A‒I; y＝0.5311x＋0.5117), derived using alllarger slope values (5.17, Subjects A and B) than that forthe other seven subjects (8.60, Subjects C‒I, small slope

measurement points, yielded a low correlation coeﬃcient.
Additionally, data from a few subjects did not ﬁt the cor-values). The results of the t-test showed a signiﬁcant diﬀerence between the two groups (t＝−2.726, df＝7, p＝0.0295).
These two groups can be classiﬁed as the low 11β-HSD2

relation equation, suggesting that predictions based on thisequation would lead to overestimation in some subjects.
Perogamvros et al. and Arafah et al. have evaluated the cor-and normal 11β-HSD2 activity groups. The group with low
11β-HSD2 activity may have decreased enzyme activity

relation between salivary and plasma unbound cortisol in49June 2022

Medical Mass Spectrometry Vol. 6 No. 1

due to genetic mutations or some subject-related back-plasma concentration based on salivary concentration ofground factors, such as disease, diet, etc. Moreover, it has
been reported that some genetic mutations in 11β-HSD2

cortisol and found that predictability had improved when
two correlation equations, corresponding to two sets of sub-activity cause essential hypertension and that the intake ofjects, were used because the MAPE value recovered from19)

glycyrrhetinic acid causes pseudohyperaldosteronism .

−20.7 to 0.6%.

We believe that investigating the factors related todecreased 11β-HSD2 activity in these subjects will be a

In conclusion, the results of this study show that it is necessary to consider individual diﬀerences in 11β-HSD2

challenge in the future. Based on these results, we hypothe-

activity present in the salivary glands while predicting
plasma unbound cortisol levels using salivary cortisol. Fur-

sized that the prediction accuracy could be improved byaccounting for the diﬀerence in 11β-HSD2 activity, i.e., thether, classifying subjects based on the salivary cortisone/

salivary cortisone/cortisol ratio (around 5.5), and creating
separate correlation equations for the two groups for pre-cortisol ratio and using a correlation equation can result in

dicting the plasma unbound cortisol concentration. Therefore, prediction correlation equations were re-created forto divide the correlation line when the salivary cortisone/

more accurate predictions. However, while it is conceivablecortisol ratio is approximately 5.5, further studies areneeded to classify 11β-HSD2 activity based on the salivary

two groups, i.e., for Subjects A and B with slope values
greater than the mean value of 1.10, and for Subjects C‒I,

cortisone/cortisol ratio. Undoubtedly, studies with larger

whose slope values were smaller. The resulting correlation

sample size that use external evaluation methods areequations had an intercept close to the origin and yieldedneeded to improve prediction accuracy based on correlation

higher r values of 0.65 and 0.77, respectively, implying abetter correlation between the two variables. However, sub-equations.

Fundingject C had a salivary cortisone/cortisol ratio of 5.688, which
was similar to that of Subjects A and B. Therefore, we calculated the correlation between Subjects A‒C. The correla-This research did not receive any speciﬁc grant fromfunding agencies in the public, commercial, or not-for-proﬁttion line was y＝0.5217x＋0.9468 (r＝0.52). The slope andsectors.

intercept were close to the correlation line from Subjects
A‒I, and the correlation did not improve. The fact that

Competing Interest

some subjects, such as subject C, had a large slope and a

The authors declare that they have no known competingsmall salivary cortisone/cortisol ratio suggests that further

ﬁnancial interests or personal relationships that could havestudies with more subjects are needed for grouping using

appeared to inﬂuence the work reported in this paper.

the salivary cortisone/cortisol ratio.
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