June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Research Paper

LC/ESI-MS/MS analysis of progesterone-derived steroids
produced in SH-SY5Y cells
Toma Shibuya, Shoichi Nishimoto-Kusunose, Ayaka Hirakawa,Kazumi Yoshizawa, Tatsuya Higashi*
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278‒8510, Japan
Abstract

Many animal studies have demonstrated that progesterone (PROG) is reduced to various metabolites in thebrain, and some of them have the regulatory eﬀect of neuronal excitation. Although the SH-SY5Y cells have been usedin various studies of neuroactive steroids as the human neuronal model cells, a complete understanding of the metabolism of PROG in these cells remains to be elucidated. The objective of this study was to identify the PROG-derived
steroids produced in the SH-SY5Y cells by high-performance liquid chromatography/electrospray ionization-tandem mass
spectrometry (LC/ESI-MS/MS).
The SH-SY5Y cells were cultured in serum-free medium supplemented with PROG for 24 h, then the steroids released
into the culture supernatant were extracted. The steroids were derivatized with Girard reagent P (GP) to enhance theirdetectability and facilitate fragmentation in the positive ESI-MS/MS. By using two diﬀerent mobile phases, allopregnanolone (AP), epiallopregnanolone (EAP), pregnenolone, 3β-dihydroprogesterone (3β-DHP), 20α-DHP and 20β-DHP
released into the supernatant were separated and detected as the GP derivatives. The peak corresponding to either 5α-DHPor 5β-DHP was also found in the chromatograms. Although the derivatized 5α-DHP and 5β-DHP were not chromatographically separated, the C5-reduced form of PROG was identiﬁed as 5α-DHP based on the presence of only AP and EAP, which
are the downstream metabolites of 5α-DHP, in the culture supernatant. Thus, this study revealed that the 5α-reduction followed by 3α-/3β-reduction, and independent 20α-/20β-reduction of PROG occurred in the SH-SY5Y cells to produce thevarious metabolites.
Key words: progesterone, SH-SY5Y cells, metabolite, Girard reagent P, LC/ESI-MS/MS

Introduction

the metabolites of PROG, acts as a positive allosteric modulator of the γ -aminobutyric acid type A (GABAA) receptor

Many animal studies have demonstrated that progesterone (PROG), which is commonly known as a luteal hor-‒3)

and strongly enhances the Cl inﬂux eﬀect of GABA ;mone, is reduced to various metabolites in the brain, some1‒3)
of which have neuroexcitatory modulatory eﬀects . Allo-

thereby, AP exhibits anxiolytic, anticonvulsant, sedative,4)and analgesic eﬀects . On the other hand, epiallopregnano-pregnanolone (AP; 3α,5α-tetrahydroprogesterone), one oflone (EAP; 3β,5α-tetrahydroprogesterone), the stereoisomerof AP, has been reported to inhibit the action of AP on the* Corresponding author

5)

GABAA receptor . Steroids that act on the central nervous
system (CNS), whether they are of endogenous or exoge-

Tatsuya Higashi
Faculty of Pharmaceutical Sciences, Tokyo University of
Science, 2641 Yamazaki, Noda-shi, Chiba 278‒8510, Japan

nous origin, are called neuroactive steroids and their associ-

Tel/Fax: ＋81‒4‒7121‒3660
E-mail: higashi@rs.tus.ac.jp

anxiety disorders, has attracted attention

ation with psychiatric disorders, such as depression and3,6).

Epub April 12, 2023.

The biosynthesis of PROG is initiated by the incorporation of cholesterol into mitochondria by the 18 kDa translo-

DOI: 10.24508/mms.2023.06.007

cator protein (TSPO) . Cholesterol is ﬁrst converted into

Received: January 6, 2023. Accepted: March 15, 2023.

7)60

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Medical Mass Spectrometry Vol. 7 No. 1

pregnenolone (PREG) by the action of the cholesterol side-

However, a complete understanding of metabolism of
PROG in the SH-SY5Y cells remains to be elucidated. Clar-chain cleavage enzyme (P450scc) in mitochondria, then
PREG is converted into PROG by 3β-hydroxysteroid dehy-

iﬁcation of the overall picture of this metabolism could
help to characterize this cell line, and eventually, to evalu-

drogenase (3β-HSD). PROG is reduced to 5α-dihydroprogesterone (5α-DHP) by 5α-reductase (5α-Red), then further

ate its utility as an in vitro model of the human CNS in the

reduced to AP by 3α-HSD or to EAP by 3β-HSD as neces-

neuroactive steroid studies. Although other cell lines

2)

sary . In addition, PROG is known to be metabolized to its
3α-/3β- and 20α-/20β-reduced forms, and accordingly, vari-derived from rodents, such as the rat cerebellar granule(Fig. 1).
ous metabolites are present in the human body
The SH-SY5Y cells are a cell line that has been often

also used in the neuroactive steroid studies, these cells may

15)

cells

8‒10)

16,17)

and rat brain glioma cells (C6 cells), have been

fail to reﬂect the PROG metabolism in the human CNS due

11)

used as an in vitro model of the human CNS . This cellto the species diﬀerence.

line was obtained by isolating and subcloning the original
cell line called SK-N-SH from a medullary biopsy of a

The steroids examined in this study were AP, EAP, pregnanolone (P; 3α,5β-tetrahydroprogesterone), epipregnano-4-year-old female patient with neuroblastoma . There is areport suggesting the presence of 5α-Red and 3α-HSD inlone (EP; 3β,5β-tetrahydroprogesterone), 3α-/3β-/5α-/5β-/20α-/20β-DHP and PREG (precursor of PROG) (Fig. 1).

the SH-SY5Y cells , therefore, PROG would likely beconverted to 5α-DHP and AP in the cells. Another study

Due to the structural similarity of these steroids, a highly
speciﬁc method is required for the analysis; high-perfor-

demonstrated that 3β-HSD is expressed in the SH-SY5Y

mance liquid chromatography/electrospray ionization-tandem
mass spectrometry (LC/ESI-MS/MS) is considered suitable

12)

13)

14)

cells , which suggests the production of EAP in the cells.

Fig. 1.

Metabolic pathways of PROG in humans.

* represents the steroids detected in the culture supernatant of SH-SY5Y cells in this study.

61June 2023

Medical Mass Spectrometry Vol. 7 No. 1

then harvested with 0.025% trypsin-0.1 mM EDTA solution

due to the high separation power of LC and high discrimination capability of MS/MS. However, LC/ESI-MS/MS

4

2

and plated in a culture dish at a density of 2×10 cells/cm .

does not demonstrate the required sensitivity and identiﬁca-

Cell treatment

tion capability for some steroids with a low ESI eﬃciency

42

and poor fragmentation behavior in MS/MS. It well known

The cells were plated at a density of 5×10 cells/cm inthat derivatization is an eﬀective procedure to improve thethe complete DMEM/F12 in a petri dish and incubated for

detectability and fragmentation behavior of steroids during
18)
LC/ESI-MS/MS . Among the various derivatization

24 h. After the medium was removed, the cells were gently
washed with serum-free DMEM/F12, then incubated again

reagents, Girard reagent P (GP) has been widely used for19)
the LC/ESI-MS/MS of ketosteroids .

in serum-free DMEM/F12 containing 100 nM PROG for 24
h. The culture supernatant was then collected and used forBased on this background information, the objective of

the subsequent experiments.

this study was to clarify the major metabolic pathway of
PROG in the SH-SY5Y cells. To achieve this objective, we

Extraction of steroids from cell culture supernatant
Ethyl acetate (1.5 mL) was mixed with 1.5 mL of the cellﬁrst developed a method for the separation and detection of
eleven PROG-related steroids by LC/ESI-MS/MS com-

culture supernatant to extract the steroids, then 1.0 mL ofbined with the GP derivatization. The steroids released in
the culture supernatant of the SH-SY5Y cells were thenthe ethyl acetate layer was collected after centrifugation

identiﬁed using the developed method.

an N2 gas stream.

Experimental

Derivatization

Materials and chemicals

steroid standards or the culture supernatant extracts dissolved in methanol-acetic acid (99 : 1, v/v; 50 μL). The(1500×g, 20°
C, 5 min). The solvent was evaporated underGP (1 mg/mL in methanol; 20 μL) was added to the
AP was purchased from Abcam (Cambridge, MA, USA).
EAP, P, EP and 3α-/3β-/5α-/5β-/20α-/20β-DHP were frommixture was heated at 80°
C for 15 min, then the solvent
was evaporated under an N2 stream. The residue was dis-Steraloids (Newport, RI, USA). PREG, PROG and GP

solved in the mobile phase (100μL), a part of which was
injected into the LC/ESI-MS/MS.

were obtained from Tokyo Chemical Industry (Tokyo,
Japan). Stock solutions of 100μg/mL for each of the steroids were prepared in ethanol, then diluted with ethanol to

LC/ESI-MS/MS

prepare the working solutions. Dulbeccoʼs modiﬁed Eagleʼs
medium/Hamʼs F-12 (DMEM/F12) and penicillin-strepto-

LC/ESI-MS/MS was performed using a Waters Quattro
Premier XE connected to a Waters LC-e2695 chromato-

mycin solution (×100) were purchased from FUJIFILM
Wako Pure Chemical (Osaka, Japan). Fetal bovine serum(FBS) was from Capricorn Scientiﬁc (Ebsdorfergrund, Ger-graph (Milford, MA, USA). A YMC-Triart C18 columnmany). DMEM/F12 supplemented with 50 U/mL of penicillin, 50μg/mL of streptomycin and 10% heat-inactivated

the temperature of 40°
C and the ﬂow rate of 0.2 mL/min.
The mobile phase consisted of methanol-10 mM ammonium

FBS was described as “complete DMEM/F12” in this study.
“Serum-free DMEM/F12” was the complete DMEM/F12

formate (1 : 1, v/v, isocratic elution) or acetnitrile-10 mM(5 μm, 100×2.0 mm i.d.; YMC, Kyoto, Japan) was used atammonium formate (1 : 2, v/v, isocratic elution). The
ESI-MS/MS conditions for the GP-derivatized steroids

without the heat-inactivated FBS. All other reagents andwere as follows; capillary voltage: 3.00 kV, cone voltage:
50 V, CE: 30 eV (AP, EAP, P, EP, PREG, 3α-/3β-/5α-/5β-solvents were of the highest grade commercially available
or LC/MS grade.

DHP) or 32 eV (20α-/20β-DHP), source temperature:

Cell culture

120°
C, desolvation temperature: 400°
C, desolvation gas

The SH-SY5Y cells obtained from the American Type

(N2) ﬂow rate: 700 L/h and cone gas (N2) ﬂow rate: 50 L.

Culture Collection (Manassas, VA, USA) were cultured incomplete DMEM/F12 at 37°
C under 5% CO2 and saturated
humidity. The cells were cultured until 80‒90% conﬂuence,62June 2023

Medical Mass Spectrometry Vol. 7 No. 1

ESI-MS/MS spectra and fragmentation processes of the GP-derivatized steroids.

Fig. 2.

(a) 20α-DHP and (b) AP.

＋

Results

was assigned as [C6H9N2O] ; this ion was formed by thecleavage of the C13‒17 and C15‒16 bonds and elimination

GP has been widely used as the derivatization reagent for
the LC/ESI-MS/MS of ketosteroids due to the presence ofof the pyridine (Fig. 2b). These fragmentations were identi19)
cal to those previously reported . Although 5α-/5β-DHP

the permanently charged moiety that enhances the ESI-MS

are 3,20-diketosteroids, GP reacted only with their C20-ketosensitivity of the resulting derivative as well as the highreactivity to a keto group

groups under the stated derivatization conditions to yield
the C20-monohydrazones. Therefore, the ESI-MS/MS

acteristic product ions during MS/MS and their fragmenta-

behaviors of the derivatized 5α-/5β-DHP were similar to
those of the derivatized 20-ketosteroids. Thus, when GP

ESI-MS/MS behaviors of GP-derivatized steroids

18,19). Furthermore, the resulting
ketosteroid derivatives with GP are known to provide char19)

tion behavior has been thoroughly analyzed . Due to thesewas used, two speciﬁc product ions were obtained from alladvantageous features, GP was employed in this study. All

the steroids, which was advantageous for the detection andthe steroids examined in this study satisfactorily reacted

identiﬁcation of trace steroids in the cell samples.
Several studies have reported that the GP-derivatization

with GP and the ensuing derivatives provided the intense＋
molecular cations ([M] ) in the ESI-MS operating in theprovided a several-fold greater detectability for the ketoster-

positive-ion mode.
All the GP-derivatized steroids produced the intense＋oids

18,19). In this study, the detectability before and after thederivatization was compared using AP as a model steroid;
the GP-derivatized AP showed a 10-fold higher sensitivity＋

product ions expressed as [M‒79] ([M‒C5H5N] ), which

compared to the intact AP. Thus, the GP-derivatization was
beneﬁcial not only for facilitating the speciﬁc fragmenta-

were formed by the neutral loss of pyridine from their＋[M] , during MS/MS (Fig. 2). Although the loss of CO＋
from the [M‒79] also occurred in all the steroids, this frag-tion but also for enhancing the sensitivity for the PROG-

mentation was more predominant in the steroids having aketo group only at the C3-position (3-ketosteroids), i.e.,

derived steroids.

20α-/20β-DHP; accordingly, the product ions expressed as＋＋[M‒107] ([M‒C5H5N‒CO] ) could be used for detecting

LC behaviors of GP-derivatized steroids
The derivatives of the ketosteroids with GP inevitably
18,19). The derivatizedconsisted of the E- and Z-isomers

these steroids (Fig. 2a). On the other hand, the derivatized
20-ketosteroids including AP, EAP, P, EP, 3α-/3β-DHP and

3-ketosteroids (20α-/20β-DHP) gave two peaks on the chromatogram due to their E- and Z-isomers, whereas the other

PREG provided a speciﬁc product ion at m/z 125, which
63

June 2023

Fig. 3.

Medical Mass Spectrometry Vol. 7 No. 1

SRM chromatograms of GP-derivatized dihydro-metabolites and PREG using (a) methanol- and (b) acetonitrile-based mobile phases.
Three diﬀerent SRM transitions (I‒III) suitable for the respective steroids were monitored. The upper and lower chromatograms
were obtained from the authentic standards and cell culture supernatant samples, respectively.

derivatized steroids (AP, EAP, P, EP, PREG, 3α-/3β-/5α-/5β-DHP) gave single peaks because their E- and Z-isomers

3α-DHP, 5α-DHP, 5β-DHP, AP and P co-eluted with theother steroids. The replacement of the mobile phase byacetnitrile-10 mM ammonium formate (1 : 2, v/v) gave a bet-

co-eluted. Thus, the use of GP somewhat complicated theter result in separation of the derivatized 3α-DHP, AP and P
from the other steroids (Figs. 3b and 4b, upper chromato-

chromatogram but its eﬀects of enhancing the sensitivity
and speciﬁcity outweighed this negative.
When the YMC-Triart C18 column was used with thegrams). Based on these results, two LC conditions with different mobile phases were complementarily used to sepa-

mobile phase consisting of methanol-10 mM ammonium
formate (1 : 1, v/v), the derivatized 3β-DHP, PREG,

rate and detect the PROG-derived steroids in the cell

20α-DHP (major isomer), 20β-DHP (major isomer), EAP
and EP were separated from the other steroids (Figs. 3a andculture supernatant samples. However, the separation of
5α-DHP and 5β-DHP from each other could not be achieved

4a, upper chromatograms). However, the derivatizedin spite of all our eﬀorts. For comparison, even by the64June 2023

Fig. 4.

Medical Mass Spectrometry Vol. 7 No. 1

SRM chromatograms of GP-derivatized tetrahydro-metabolites using (a) methanol- and (b) acetonitrile-based mobile
phases.
Two diﬀerent SRM transitions (I and II) were monitored. The upper and lower chromatograms were obtained from the authentic
standards and cell culture supernatant samples, respectively.

mobile phase consisting of three solvents, i.e., methanol,

Table 1.

acetonitrile and 10 mM ammonium formate, a poor result
was generated for the separation of 20α-DHP and PREG,

＋

[M] →
＋
[M‒79][M] →＋[C6H9N2O]
＋

[M] →
＋
[M‒107]

0.6

3.5

3.4

̶

0.0

2.0

2.7

̶

0.3

0.7

0.3

̶

0.4

5.1

1.9

̶

PREG
a)
3α-DHP

0.2

0.7

1.7

̶

0.5

0.4

2.4

̶

b)
3β-DHPa)
5α-DHP

0.4

1.0

1.2

̶

0.4

2.2

3.0

̶

5β-DHPb)
20α-DHP

0.4

0.8

0.4

3.6

0.8
̶

3.3

20β-DHP

0.3

0.8

̶

1.2

Steroids

AP

a)

EAP

To evaluate the robustness of the analytical system, tripli-Pcate measurements were performed for each steroid. The

b)

a)

EP

coeﬃcients of variation (CVs) of the retention times (tRs)b)b)

and peak areas did not exceed 0.6% and 5.6%, respectively,
for all the derivatized steroids, indicating that the systemenabled the reproducible analysis of the steroids (Table 1).

a)Furthermore, the limits of detection (LODs, S/N＝3) of thederivatized steroids were 0.6‒2.8 fmol, which demonstrated
that our method had suﬃcient sensitivity to detect trace

CV (%) for peak areaCV(%)
for tRand separation of 3α-DHP and 5α/β-DHP.

Performance of analytical system

Robustness of analytical system

b)

＋

̶

The steroids (1.0 ng each) were derivatized with GP, then one-tenthof which was injected into LC/ESI-MS/MS (triplicate measurements). a) Acetonitrile-10 mM ammonium formate (1 : 2, v/v) orb) methanol-10 mM ammonium formate (1 : 1, v/v) was used as themobile phase.

amounts of the steroids (Table 2).

Detection and identiﬁcation of PROG-derived steroids
in SH-SY5Y cell culture supernatantFigs. 3a and 4a (lower chromatograms) show the SRM
65

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Medical Mass Spectrometry Vol. 7 No. 1

chromatograms of the SH-SY5Y cell culture supernatants
using the methanol-based mobile phase. Several peaks were

natant (Figs. 4a-I and -II, lower chromatograms). We subsequently analyzed the cell samples using the acetoni-＋＋
observed when the [M] → [M‒79] transition was monitored (Figs. 3a-I and 4a-I, lower chromatograms). Sometrile-based mobile phase and observed the separated peakcorresponding to AP (tR 16.0 min) in the two SRM chro-＋＋＋＋
matograms ([M] → [M‒79] and [M] → [C6H9N2O] )(Figs. 4b-I and -II, lower chromatograms). The analysis

peaks were assigned as the derivatized 3β-DHP (tR
13.5 min), PREG (tR 15.1 min), 20α-DHP (tR 16.6 min),20β-DHP (tR 43.5 min) and EAP (tR 19.9 min) by co-using the acetonitrile-based mobile phase revealed that P (tR
14.8 min) and 3α-DHP (tR 12.1 min) were not present in thechromatography with the authentic standards. When the＋＋[M] → [M‒107] transition was used for detection ofcell culture supernatant (Figs. 3b-I, -III, 4b-I and -II).
As previously mentioned, the derivatized 5α-DHP and

20α-DHP and 20β-DHP, the trace peaks corresponding tothese steroids were also observed (Fig. 3a-II, lower chro-

5β-DHP could not be separated by our method. The peakmatogram). By the transition of [M] → [C6H9N2O] , thederivatized 3β-DHP, PREG and EAP were speciﬁcally＋＋was observed at 18.4 min in the cell culture supernatantsample when using the acetonitrile-based mobile phasedetected (Figs. 3a-III and 4a-II, lower chromatogram).
Although the methanol-based mobile phase could separate(Figs. 3b-I and -III, lower chromatograms); these results
suggested the occurrence of either 5α-DHP or 5β-DHP, or

the derivatized EP (tR 17.5 min, Figs. 4a-I and -II, upper
chromatograms) from the other steroids, the peak corre-both of the steroids in the cell culture supernatant. Given
the fact that AP and EAP (5α-reduced metabolites) weresponding to this steroid did not appear in the cell culture
supernatant, which indicated the absence of EP in the super-

detected in the cell culture supernatant, whereas P and EP(5β-reduced metabolites) were not, it was natural that only
5α-DHP was produced in the SH-SY5Y cells as the C5-

Table 2.

LODs of GP-derivatized steroids
Methanol-based
mobile phase

Steroids

reduced form of PROG.
To more reliably identify the PROG-derived steroids

Acetonitrile-based
mobile phasefound in the cell culture supernatant, the peak area ratios inAP

2.2 (0.7)

0.9 (0.3)

the two SRM chromatograms were compared between theEAP

1.9 (0.6)

0.9 (0.3)

steroids in the cell culture supernatant samples and theP

2.5 (0.8)

0.9 (0.3)EP

2.2 (0.7)

0.9 (0.3)

authentic standards. The results are shown in Table 3; thePREG
3α-DHP

1.3 (0.4)

0.6 (0.2)

2.5 (0.8)

1.6 (0.5)

2.2 (0.7)

0.9 (0.3)

3β-DHP
5α-DHP

peak area ratios of the seven steroids detected in the cell
culture supernatant were almost identical to those of thestandards. Thus, the analysis using the two speciﬁc SRM
transitions revealed that AP, EAP, PREG, 3β-DHP, 5α-DHP,

2.2 (0.7)

0.6 (0.2)

5β-DHP
20α-DHP

2.8 (0.9)

1.3 (0.4)

1.0 (0.3)

0.6 (0.2)

cells. For comparison, none of the steroids including PROG

20β-DHP

1.6 (0.5)

0.9 (0.3)

were detected in the supernatant collected from the
SH-SY5Y cells cultured without PROG.

20α-DHP and 20β-DHP were released from the SH-SY5Y

Unit; fmol/injection. The values in parentheses are amounts (pg)converted into intact steroids.

Table 3.
Steroid
AP
EAP
PREG
3β-DHP
5α-DHP
20α-DHP
20β-DHP

Peak area ratios in two SRM chromatograms
Peak area ratio (mean±SD, n＝3)

Transition([M] → [C6H9N2O] ) / ([M] → [M‒79] )＋＋＋＋([M] → [C6H9N2O] ) / ([M] → [M‒79] )
＋

＋

＋

＋

＋
([M] → [C6H9N2O] ) / ([M] → [M‒79] )＋＋＋([M] → [C6H9N2O] ) / ([M] → [M‒79] )＋＋＋＋([M] → [C6H9N2O] ) / ([M] → [M‒79] )
＋

＋

＋

＋

([M] → [M‒107] ) / ([M] → [M‒79] )＋＋＋＋([M] → [M‒107] ) / ([M] → [M‒79] )
＋

＋

＋

＋

66

Culture supernatantStandard

0.749±0.015

0.739±0.003

0.763±0.003

0.769±0.006

0.664±0.003

0.665±0.008

0.612±0.015

0.623±0.003

0.589±0.008

0.583±0.009

0.395±0.014

0.400±0.010

0.453±0.015

0.443±0.006

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Medical Mass Spectrometry Vol. 7 No. 1

Discussion

PROG and its metabolites exert various eﬀects for neuro-

expressed at suﬃcient levels in the SH-SY5Y cells and
5β-Red is signiﬁcantly less or not active. It is a well-known. Abundant evidence also suggeststhat AP, one of the PROG-derived neuroactive steroids, isfact that 5α-Red is expressed in the human CNS ,whereas to the best of our knowledge, the 5β-Red activity isrelevant to the antipsychotic drug action and pathophysiol-

not apparent in the human CNS. Therefore, in the stereoselective reduction of Δ4-bond of PROG, the SH-SY5Y cells

1‒3)

2,24)

nal and glial functions

3,20‒22). Furthermore,
the AP preparation has been recently approved as a thera-ogy of depression and anxiety disorder

peutic agent for postpartum depression . Thus, it iscan be used as the in vitro model of the human CNS.
When discussing 3α-HSD and 3β-HSD, the latter activity

accepted that the PROG metabolites play important roles inneuronal regulation, but it is not fully understood what spe-

was found to be higher in the SH-SY5Y cells because EAP(3β,5α-reduced form) was more abundantly produced thanciﬁc metabolites are produced in the SH-SY5Y cells, which

AP (3α,5α-reduced form), and 3β-DHP was detected but
3α-DHP was not. There is a report suggesting the presence

23)

11)

have been used as an in vitro model of human CNS .

13)
of 3α-HSD in the SH-SY5Y cells . Moreover, 3β-HSD has

Based on this background, we attempted the identiﬁcationof the PROG-derived steroids produced in the SH-SY5Y
cells.

been reported to be expressed in the human glioblastoma
25)
14)
cells (U87 cells) as well as in the SH-SY5Y cells . To

First, to achieve this objective, we developed an LC/
ESI-MS/MS method for the separation and detection of thethe best of our knowledge, there is no study that clearly
demonstrates whether 3α-HSD or 3β-HSD is dominantly

metabolites of PROG. To enhance the detectability andfragmentation of the steroids in ESI-MS/MS, the GP deri-

expressed in the human CNS. Cruz et al. revealed that the
AP concentrations were similar to the EAP concentrationsin the orbital frontal cortex of subjects with post-traumatic

vatization was employed in this study; the GP-derivatizedsteroids produced several characteristic product ions includ-stress disorder and controls .

ing [M‒79] , [M‒107] and [C6H9N2O] with suﬃcient
intensities. These remarkable advantages of the GP derivat-

This study revealed that the SH-SY5Y cells also have the20α- and 20β-HSD activities because 20α- and 20β-DHP

ization signiﬁcantly helped to detect and identify the steroids in the culture supernatant of the SH-SY5Y cells. We

were detected in the cell culture supernatant. In humans,aldo-keto reductase 1C1 (AKR1C1) works as the 20α-HSD

unsuccessfully attempted to separate the eleven steroids ofinterest in a single LC run due to their very similar struc-for PROG and AP, and it is thought that this enzymetures and physicochemical properties. However, the com-

AP and eliminating its precursor, PROG, from the synthetic27)
pathways . However, based on the amounts of 20α- and

＋

＋

26)

＋

decreases the AP concentrations in the brain by inactivating

bined use of two mobile phases consisting of diﬀerent
organic modiﬁers provided satisfactory results for the steroid separation.

20β-DHP found in this study, the 20α- and 20β-HSD activities were not considered to be signiﬁcant in the SH-SY5Y

In this study, the SH-SY5Y cells were cultured in the
PROG-added medium because no steroids were detected incells. To summarize these results, the major metabolic pathway of PROG in the SH-SY5Y cells was the 5α-reduction

the culture supernatant without the addition of PROG. We
successfully identiﬁed AP, EAP, 3β-DHP, 20α-DHP andof the Δ 4-bond followed by the 3β-reduction of the C3-ketogroup; 5α-DHP and EAP were the most and second abun-

20β-DHP as the PROG metabolites formed in the cells by
the satisfactory separation from other steroids and discrim-

dant metabolites produced in the SH-SY5Y cells, respec-

inable detection using the double SRM transitions. Because
5α-DHP and 5β-DHP could not be chromatographically sep-

Although a validation for the quantiﬁcation of the
PROG-derived steroids in the cell culture supernatant wasarated, the SRM chromatograms shown in Figs. 3b-I and-III indicated only that the peak eluted at 18.4 min corresponded to either 5α-DHP or 5β-DHP. However, we con-

not performed, their approximate concentrations (mean,

cluded that 5α-DHP was present in the cell culture superna-

630 pg/mL for EAP, 490 pg/mL for PREG, 50 pg/mL for
3β-DHP, 1660 pg/mL for 5α-DHP, 20 pg/mL for 20α-DHP

tively.

n＝3) were determined by comparison with known
amounts of the authentic standards; 80 pg/mL for AP,tant based on the presence of its downstream metabolites(AP and EAP) and absence of their 5β-isomers (P and EP)

and 10 pg/mL for 20β-DHP. As the steroids produced in the
SH-SY5Y cells were diluted with the cell culture medium,in the supernatant. These results suggested that 5α-Red is67June 2023

Medical Mass Spectrometry Vol. 7 No. 1

it was diﬃcult to speculate the steroid concentrations in thesymptoms have been reported as adverse reactions, which

localized areas of the target tissues based on the measured

are possibly due to the decreased AP concentration in the
28,29). Our assay system might be helpful to demonbrain

concentrations in the cell culture supernatant. Further
research is needed to determine whether the amounts of ste-strate the eﬀect of ﬁnasteride on the AP production in the
CNS. Furthermore, some drugs increase the brain AP con-roids produced in the SH-SY5Y cells are suﬃcient to havecentration (de novo AP biosynthesis in the brain) and exert
subsequent activation of GABAA receptors to have an anxi-

activity in the target tissues.
It is noted that a considerable amount PREG was detectedin the culture supernatant of the SH-SY5Y cells although it

3,20‒22). Our assay system will enable the screening of compounds with the anxiolytic eﬀect via the AP bioolytic eﬀect

is not metabolically formed from PROG. We interpreted
this phenomenon as follows. The SH-SY5Y cells constantly

synthesis.

produce PREG from cholesterol and also PROG from

Conclusion

PREG, but the amounts of PREG and PROG produced areusually very small. Therefore, none of the steroids weredetected in the culture supernatant of the SH-SY5Y cells

We developed a method for the separation and identiﬁcation of the PROG-derived steroids produced in the

without the addition of PROG by our method. When a signiﬁcant amount of PROG is added to the cells, the cells sus-

SH-SY5Y cells by LC/ESI-MS/MS using two mobile
phases. Using this method, AP, EAP, 3β-DHP, 5α-DHP,

pend the use of PREG to produce PROG, probably due to

20α-DHP, 20β-DHP and PREG were detected in the cellthe negative feedback mechanism, and the surplus PREG

culture supernatant. The ﬁndings of this study can be a help
not only for evaluating the SH-SY5Y cells as the in vitro

may be extracellularly released.
Most previous studies on the PROG metabolism in themodel of the human CNS, but also for understanding the15‒17).

metabolism of PROG, i.e., the biosynthesis of neuroactive

Moreover, these studies sometimes undiscriminatingly ana-

steroids, in the human CNS. Furthermore, the developed

lyze the stereoisomers, such as AP and EAP, which could

method is also expected to be a new tool for assessing thelead to an erroneous result. Additionally, many previous

mechanisms of drug action via promotion/inhibition of thestudies monitored only the mRNA and protein expressions

biosynthesis/metabolism of neuroactive steroids in theof the PROG metabolizing enzymes in the cultured cells

CNS.

cultured neuronal cells exclusively focused on AP

and did not show what steroids were actually produced in
2,14,25)the cells

Acknowledgment. As contrasted with these studies, the presentThis study was supported in part by JSPS KAKENHI

study comprehensively analyzed the PROG metabolites
produced in the SH-SY5Y cells with the discrimination

Grant Number JP21K15304 (to S. N.-K.).

between the regioisomers and stereoisomers, especially fordihydro-metabolites (3α-/3β-/5α-/5β-/20α-/20β-DHP) andConﬂicts of Interest

tetrahydro-metabolites (AP, EAP, P and EP), which resulted

The authors declare no conﬂicts of interest.

in the reliable identiﬁcation of the six PROG metabolites(AP, EAP and 3β-/5α-/20α-/20β-DHP) and PREG. Surpris-

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