Mass spectrometry imaging for dissecting steroid intracrinology within target tissues

Article abstract of DOI:10.1021/ac402777k

Steroid concentrations within tissues are modulated by intracellular enzymes. Such steroid intracrinology influences hormone-dependent cancers and obesity and provides targets for pharmacological inhibition. However, no high resolution methods exist to quantify steroids within target tissues. We developed mass spectrometry imaging (MSI), combining matrix assisted laser desorption ionization with on-tissue derivatization with Girard T and Fourier transform ion cyclotron resonance mass spectrometry, to quantify substrate and product (11-dehydrocorticosterone and corticosterone) of the glucocorticoid-amplifying enzyme 11β-HSD1. Regional steroid distribution was imaged at 150-200 μm resolution in rat adrenal gland and mouse brain sections and confirmed with collision induced dissociation/liquid extraction surface analysis. In brains of mice with 11β-HSD1 deficiency or inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and accumulation of the alternative 11β-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues.

Full text of DOI:10.1021/ac402777k

Article
pubs.acs.org/ac
Mass Spectrometry Imaging for Dissecting Steroid Intracrinology
within Target Tissues
†
‡
§
†
Diego F. Cobice, C. Logan Mackay, Richard J. A. Goodwin, Andrew McBride,
‡
†
†
,†
Patrick R. Langridge-Smith, Scott P. Webster, Brian R. Walker, and Ruth Andrew*
†University/British Heart Foundation Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of
Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom
‡
SIRCAMS, School of Chemistry, Joseph Black Building, The King’s Buildings, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JJ, United Kingdom
§
AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TF, United Kingdom
*
S Supporting Information
ABSTRACT: Steroid concentrations within tissues are modulated
by intracellular enzymes. Such “steroid intracrinology” influences
hormone-dependent cancers and obesity and provides targets for
pharmacological inhibition. However, no high resolution methods
exist to quantify steroids within target tissues. We developed mass
spectrometry imaging (MSI), combining matrix assisted laser
desorption ionization with on-tissue derivatization with Girard T
and Fourier transform ion cyclotron resonance mass spectrometry,
to quantify substrate and product (11-dehydrocorticosterone and
corticosterone) of the glucocorticoid-amplifying enzyme 11β-
HSD1. Regional steroid distribution was imaged at 150−200 μm
resolution in rat adrenal gland and mouse brain sections and
confirmed with collision induced dissociation/liquid extraction
surface analysis. In brains of mice with 11β-HSD1 deficiency or
inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and
accumulation of the alternative 11β-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain
homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues.
easurement of steroid hormone concentrations in
plasma over more than 50 years has revealed the
concentrations therefore remain qualitative rather than
quantitative.
Mass spectrometry is widely regarded as the gold standard
for steroid quantification in plasma. Mass spectrometry imaging
M
intricate control of their secretion from endocrine glands. In
recent decades, it has been recognized that steroid concen-
trations within tissues are also modulated, independently of
circulating steroid concentrations, by local steroid-generating
and inactivating enzymes. This has been confirmed in
spontaneous human enzyme deficiency syndromes, recapitu-
lated by genetic deletion in mice and using pharmacological
inhibitors; examples include amplification of estrogen action by
(
MSI) and liquid extraction surface analysis (LESA) have
recently been developed to map spatial distribution of
molecules in tissues allowing localization, detection, identi-
fication, and quantitation of compounds in complex biological
4
matrices. The distribution of cholesterol, chemically similar to
5,6
7
steroids, has been imaged in brain and in liver while
corticosteroids and androgens have been detected in a
nonbiological matrix by cation-enhanced nanostructure-initiator
1
2
aromatase, androgen action by 5α-reductase type 2, and
3
glucocorticoid action by 11β-HSDs. Dysregulation of steroid-
metabolizing enzyme expression is thought to be important in
the pathophysiology of steroid-dependent disease, including
breast cancer, prostate disease, and obesity. Moreover,
inhibitors of these enzymes have proved useful to reduce
steroid action in these disorders. A key limitation in this
established field of “steroid intracrinology”, however, is the
inability to study steroid concentrations within tissues,
particularly in organs with region-specific expression of relevant
enzymes, such as the brain, or within heterogeneous samples,
such as from cancers. Inferences of the consequences of
enzyme (dys)regulation and inhibition for local steroid
8
mass spectrometry. However, localization of endogenous
neutral steroids in biological tissues by MSI is challenging,
not only because of their relatively low abundance but also
because of their lack of either hydrogen donor or acceptor
moieties, resulting in poor ionization yields during matrix
assisted laser desorption (MALDI). Furthermore, steroids are
Received: September 11, 2013
Accepted: October 17, 2013
Published: October 17, 2013
©
2013 American Chemical Society
11576
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2
5
susceptible to ion suppression by more abundant molecules,
such as lipids and proteins. Although steroids can be ionized by
conventional electrospray or APCI, detection in the low subpg
(male, 2−3 months) were bred in-house. The genetically
modified animals were obtained from an in house breeding
colony and were slightly older than commercial stock; however,
age-matched controls were used in each experiment. In all
studies, animals were killed by decapitation at 09:00 h; plasma
was prepared from trunk blood, collected in EDTA. Tissues
were snap frozen in liquid nitrogen and stored (−80 °C).
Tissue Sectioning and Mounting. The cryostat (Leica
Microsystems Inc., Bannockburn, IL, USA) was cleaned and
tissue embedded in gelatin solution 10% w/v. Coronal/
horizontal brain and adrenal cryosections (10 μm) were cut
9
range in tissues is largely out of reach, and these quantitative
approaches do not offer two-dimensional imaging. For
1
0,11
electrospray ionization (ESI), derivatization
enhances the
ionization efficiency of neutral corticosteroids, for example,
12
using Girard reagents. Similar approaches have allowed
Girard P (GirP) derivatives of oxysterols to be detected with
13
fmole sensitivity in rat brain homogenates using MALDI and
have enhanced sensitivity for cholesterol analysis using N-
alkylpyridinium isotope quaternization in analysis of human
2
6
and frozen (−80 °C), adapted from Koeniger. Briefly: (1)
fifteen tissue sections (50 μm thick) were collected for
confirmatory quantitation; (2) two adjacent sections were
thaw mounted onto glass slides (Superfrost, Thermo Scientific,
Braunschweig, Germany) and retained for histological exami-
nation; (3) four adjacent sections for MSI/LESA (10 μm) were
thaw mounted onto conductive indium tin-oxide (ITO)-coated
glass slides (Bruker Daltonics, Bremen, GmbH) precoated with
GirT-reagent (0.15 mg/cm ), and further sections for histology
×2) and confirmatory quantitation (×15) were harvested as
above. Tissue sections for confirmatory quantitation were
combined, weighed at room temperature (RT), and stored at
14
hair homogenate by MALDI-FTICR. The recent innovation
1
5,16
of on-tissue chemical derivatization (OTCD)
has paved the
way for application of derivatization techniques to MSI for
investigation of tissue steroids.
We aimed to develop MSI initially for analyzing cortico-
steroids. The major glucocorticoid in rodents, corticosterone
(
CORT), is relatively abundant and, in addition to its secretion
from the zona fasciculata of the adrenal cortex, is also generated
within target tissues from the inert precursor 11-dehydrocorti-
costerone (11DHC) by the enzyme 11β-HSD1. Inhibition of
2
(
3
1
7,18
1
1β-HSD1 in brain is associated with improved memory,
but the consequences for local steroid concentrations in the
brain subregions where 11β-HSD1 is expressed are uncertain.
Moreover, 11β-HSD1 can interconvert other keto and hydroxy
−
80 °C until LC-MS/MS analysis. Tissue sections for MSI
were stored in a vacuum desiccator (RT, 1h) and then at −80
1
9−22
°C.
substrates,
including 7-keto and 7β-hydroxycholesterol,
Instrumentation. MSI was performed using a 12T SolariX
MALDI-FTICR-MS (Bruker Daltonics, MA, US) employing a
Smartbeam 1 kHz laser, with instrument control using SolariX
control v1.5.0 (build 42.8), Hystar 3.4 (build 8), and
FlexImaging version 3.0 (build 42). On-tissue collision induced
dissociation was carried out by liquid extraction surface analysis
but it has been difficult to establish the equilibrium of these
reactions in vivo. We have applied OTCD, in combination with
MSI, for the first time to quantify and validate the detection of
CORT and 11DHC and alternative substrates within
subregions of murine brain. Neutral steroids were converted
to positively charged hydrazones derivatives using Girard
reagents T (GirT), enabling detection and quantitation by
MALDI and LESA with Fourier transform ion cyclotron MSI
(
LESA)-nanoESI-FTICR-MS using Triversa nanomate (Ad-
vion, NY, USA). Confirmatory liquid chromatography-tandem
MS (LC-MS/MS) analysis was performed using a triple-
quadrupole linear ion trap mass spectrometer (QTRAP 5500,
AB Sciex, Cheshire, UK) coupled with an ACQUITY ultra high
pressure liquid chromatography (UPLC; Waters, Manchester,
UK).
(
MALDI-FTICR-MSI). Validation was performed with LC-
MS/MS. MSI demonstrated alterations in brain glucocorticoids
and 7-ketocholesterol, an alternative substrate) in mice with
1β-HSD1 deficiency and allowed simultaneous pharmacoki-
(
1
netic/pharmacodynamic studies with an 11β-HSD1 inhibitor. A
potent, selective, and orally bioavailable piperidinyl-pyrazol
inhibitor of 11β-HSD1 (UE 2316) was used; UE 2316 crosses
the blood−brain barrier and displays potency across species
MALDI-FTICR-MSI Analysis. Optical images were taken
using a flatbed scanner (Cannon LiDE-20, Cannon, UK). MSI
analysis was performed using 250 laser shots, with a laser spot
diameter of ∼50 μm, and laser power was optimized for
consistent ion production. Ions were detected between m/z
250 and 1500, yielding a 1 Mword time-domain transient, and
with a laser spot raster spacing of 100−300 μm unless
otherwise stated. MSI data were subject to the vector
normalization method (RMS) at 0.995 as ICR noise reduction
threshold. Mass precision was typically ±0.025 Da. Average
abundances were determined by defining specific regions of
interest (ROI). A measure of the average abundance was then
assigned from the summed spectra. Neutral steroids were
analyzed (without application of derivatization reagent) in
positive and negative ion modes at +ve m/z 345.20604, −ve m/
z 343.19148 for 11DHC and +ve m/z 347.22169, −ve m/z
345.20604 for CORT. Ions formed by the derivatives were
monitored in positive mode, with +ve m/z 458.30133 (GirT-
11DHC) and +ve m/z 460.31698 (GirT-CORT) and +ve m/z
23
(
IC50: mouse, 162 nM; rat 80nM).
MATERIALS AND METHODS
Chemicals and Reagents. Internal standard (ISTD)
■
corticosterone-2,2,4,6,6,7α,21,21 (d CORT) (purity 95−97%)
8
was from Cambridge Isotopes (MA, USA); 11-dehydrocorti-
costerone (11DHC) was from Steraloids Inc. (PA, USA);
UE2316, [4-(2-chlorophenyl)-4-fluoro-1-piperidinyl][5-(1H-
pyrazol-4-yl)-3-thienyl]-methanone, and UE2346 were synthe-
23
sized by High Force Ltd., UK. Solvents were glass-distilled
HPLC grade (Fisher Scientific, Loughborough, UK). 4-Chloro-
24
α-cyanocinnamic acid (Cl-CCA) was synthesized in-house.
All other chemicals were purchased from Sigma-Aldrich
(
Dorset, UK) unless otherwise stated.
Animals and Biomatrix Collection. Licensed procedures
were performed under the UK Animals (Scientific Procedures)
Act, 1986. Sprague−Dawley male rats (5−7 weeks, ∼175−200
g, source of adrenal glands) and C57BL/6 mice (6−7 weeks,
male) were from Harlan Olac Ltd. (Bicester, UK). 11β-
HSD1− mice (KO) and their wild type (WT) littermates
468.36718 (GirT-d CORT). UE2316 was monitored at +ve m/
8
z 390.08377 and GirT-7 ketocholesterol (GirT-7KC) was
monitored at +ve m/z 514.43670. α-Cyano-4-hydroxycinnamic
acid (CHCA) matrix ion at +ve m/z 417.04834 was also
/−
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monitored to assess matrix application uniformity and perform
an internal calibration.
HSD1−/− (knockout, KO) mice and their wild type littermates
(n = 6/group) and after pharmacological inhibition of 11β-
HSD1 in C57BL/6 mice (n = 6/group, killed 1, 4, and 6 h after
receiving UE2316 (20 mg/kg oral, in DMSO/PEG-400/saline
(0.9% w/v) (2:38:60)) or vehicle).
Tissues were harvested at cull, and MSI was performed as
described. The identities of ions of the steroid-GirT hydrazones
were confirmed on-tissue by CID using LESA. The average
spectral intensities of steroid derivatives in ROIs across the
cortex, hippocampus, and amygdala were recorded using MSI,
presented as ratios of CORT/11DHC and compared with
those generated by LESA and LC-MS/MS.
Statistical Analysis. Data are expressed as mean ± SEM,
and differences were analyzed using two-way ANOVA with
Fisher’s posthoc test (MSI and LESA) and Student’s t test (LC-
MS/MS). Statistical significance was accepted at p < 0.05.
Statistical calculations were performed using Statistica version
8.0, StatSoft, Inc.Tulsa, OK, USA. Data generated by different
analytical methods were compared by Bland-Altman plots.
Structure Drawing. Molecular structure drawing was
performed using MarvinSketch 5.4.0.1, Chemaxon Ltd., Buda-
pest, Hungary.
On-Tissue Chemical Derivatization (OTCD). From −80
°
C, tissues were allowed to dry in a vacuum desiccator (20
2
min). The GirT precoated glass slide (0.15 mg/cm ) was
sprayed with 2 mL of methanol containing 0.2% v/v of TFA;
then, it was placed in a sealed Petri dish (or similar container)
containing moist tissue paper to create a moisturizing reaction
environment. Two ml of distilled water was enough to produce
a suitable reaction media; more water may cause analyte
delocalization due to derivatization reagent diffusion. A moist
kim-wipe tissue was placed around the inner walls of the
container without touching the glass slide. The tissue was
incubated (60 min, 40 °C) in an oven or water bath and then
allowed to cool and dry in a vacuum desiccator (RT, 15 min) to
remove the condensed water prior to matrix deposition as
above. Other derivatization reagents were evaluated such as
isonicotinoyl and sulfonyl chlorides among other hydrazine
reagents. Details of optimization and derivatization screening
are in Supporting Information, Table S1.
Matrix Application. Matrix (CHCA; 10 mg/mL) in
acetonitrile (80% + 0.2% v/v TFA) was applied by a pneumatic
TLC sprayer (20 mL per slide with a nitrogen flow of 7.5 L/
min at a distance of 20 cm from the MALDI target). Each
manual pass took approximately 1 s, and the process was
repeated with 5−10 s between passes until a uniform matrix
coating was achieved on the tissue section. The tissue section
was then allowed to dry (RT) and stored in a desiccator until
analysis.
RESULTS
■
OTCD-MALDI-FTICR-MSI Method Development and
Optimization. To detect corticosterone (CORT) and 11-
dehydrocorticosterone (11DHC), standards were analyzed by
+
MALDI mass spectrometry. [M + H] pseudomolecular ions
were the most abundant ions detected when standards of
corticosterone (CORT) and 11-dehydrocorticosterone
Detection of Endogenous Steroids in Murine Tissue
Using Derivatization. Steroids were imaged in adrenal glands
from Sprague−Dawley rats and brains from C57Bl/6 mice (n =
(
11DHC) were analyzed, with masses at m/z 347.22101 and
m/z 345.20632, in close agreement with the theoretical masses
m/z 347.22169 and m/z 345.20604, respectively (Figure S1a,b,
Supporting Information); ions due to dehydration were not
observed. Initial imaging attempts in adrenal tissue, monitoring
6/tissue). Tissues were prepared, derivatized as described, and
matrix applied. Steroid derivatives were imaged by MALDI-
FTICR-MS.
+
Histological Staining. Cryosections were stained using
only the [M + H] ion of the glucocorticoids of interest, yielded
27
hematoxylin and eosin. After fixation in cold acetone, tissue
sections were examined using an optical microscope (40×,
Leica Microsystems Inc., Bannockburn, IL, USA) with CCD
camera (Hitachi model 3969, Japan).
ions with mass accuracy of −3.2 ppm (CORT) and −18.3 ppm
(11DHC) (Figure S1c,e,f,h,i, Supporting Information) but with
low signal-to-noise ratio. Limits of detection (LODs) of CORT
and 11DHC were ∼30 ng (off-tissue) and 1 μg (on-tissue).
To improve images of neutral steroids in tissues, a number of
approaches were pursued to enhance their low intensity signal.
Initially, matrix screening was performed using both standard
and novel matrices, to identify reagents which may enhance
energy transfer. Substantial differences were observed between
matrices; the maximum signal intensity and desorption yield
was achieved using CHCA (Figure S2a, Supporting Informa-
tion). Interestingly, the novel 4-chloro-α-cyanocinnamic acid
(Cl-CCA), which possesses a lower proton affinity than its
hydroxyl analogue and increased the signal of poorly ionizable
Liquid Extraction Surface Analysis (LESA)-ESI-FTICR-
MS. This was performed in both adrenal and brain sections.
Steroids within tissue sections were derivatized as previously
described and analyzed immediately using LESA-nanoESI-
FTICR-MS as follows: solvent, methanol/water (50:50 with
0
.1% v/v of formic acid); pick-up volume, 1.5 μL; dispense
volume, 1.2 μL at 0.2 mm away from surface; droplet rest time
delay), 5 s; aspiration volume, 1.4 μL at 0.0 mm away from
(
surface. ESI-FTICR-MS was performed using the 12T SolariX
dual source (ESI-MALDI) with SolariX control v1.5.0 (build
24
42.8). Ions were detected between m/z 250 and 1500, yielding
peptides, demonstrated lower intensity signals in combination
with steroids than CHCA, although the background signal was
noticeably lower in the low mass range (200−400 Da). Despite
using CHCA, the LOD did not reach the required subpg levels
of endogenous corticosteroids in tissue.
a 2 Mword time-domain transient. Ions of GirT-hydrazones (as
for MALDI analysis above) were isolated for 30 s prior to
isolation (m/z 460.3 ± 5 Da) and CID experiments. CID was
carried out using 28 eV as collision energy.
Confirmatory Analysis by LC-MS/MS. Analyses of
Derivatization was explored by introducing readily ionizable
or permanently charged groups, an approach effective in
13
corticosteroids (adapted from Wang) and UE2316 in murine
brain were performed using LC-MS/MS in multiple reaction
monitoring mode (MRM), for method details see Supporting
Information.
Influence of 11β-HSD1 on Amounts of Active and
Inactive Glucocorticoids in Brain Tissue. The effect of
deficiency of the 11β-HSD1 enzyme was studied using 11β-
1
1−13
enhancing signal intensity with LC-MS/MS.
Poor
desorption or ionization by MALDI analysis was found using
derivatives targeting the primary (C21) alcohol moiety with
acyl and sulfonyl chlorides. Screening derivatization reactions,
targeting the α−β unsaturated ketone at C3 in the steroid A-
ring (Table S1, Supporting Information), improved signal
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Figure 1. Molecular imaging by MALDI-FTICR-MSI of corticosteroid derivatives prepared with Girard T reagent (GirT) in representative rat
adrenal gland sections. Molecular regional distribution maps showed GirT-CORT (m/z 460.31713) and GirT-11DHC (m/z 458.30139) in high
abundance in the zona fasciculata/reticularis (site of glucocorticoids synthesis) with mass accuracy of ±5 ppm from their theoretical monoisotopic
masses and (S/N) signal-to-noise ratios above 100. (a) Derivatization of corticosterone and 11-dehydrocorticosterone with GirT. (b) Histological
image of a cryosection of rat adrenal gland stained with hematoxylin and eosin (ZG = zona glomerulosa; ZFR = zona fasciculata reticularis). Heat
map of GirT derivatives of: (c) corticosterone (GirT-CORT) at m/z 460.31698 ± 0.025 Da and (d) 11-dehydrocorticosterone (GirT-11DHC) at
m/z 458.30133 ± 0.025 Da, collated by MALDI-FTICR-MS. Signal intensity is depicted by color on the scale shown. Simulated theoretical isotopic
distribution pattern of: (e) GirT-CORT and (f) GirT-11DHC. (g) Representative FTICR-MS spectrum of corticosteroid hydrazones in rat adrenal
gland, showing excellent agreement with theoretical mass. TFA = Trifluoroacetic acid. MeOH = Methanol. M = molecular weight (Da). cps =
w
counts per second. Scale bar (2 mm).
intensity in positive mode when the corticosteroids were
converted to derivatives either permanently charged or easily
protonated. In particular, formation of a quaternary ammonium
derivative, by a hydrazine type condensation reaction of the
ketone at both C3 and C20 to form the water-soluble
hydrazones group, greatly enhanced response (Table S1,
Supporting Information).
generated ions of similar intensities, with LODs of 0.01−0.03
pg (off-tissue) and 1−0.1 pg (on-tissue), and signals of the
endogenous CORT and 11DHC in tissues were considerably
enhanced (Figure 1c,d). GirT condensation is generally carried
out using protic solvents in a weak acid media at room
temperature with reaction times around 10−18 h achieving
28
60−80% of conversion to hydrazones. Recent studies have
The highest sensitivity was achieved with derivatives formed
using GirT reagent. Derivatives of CORT and 11DHC
shown that the reaction can be carried out in 30 min at 70
2
9,30
°C.
However, such a high temperature may cause
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Figure 2. Effect of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) deficiency on proportions of active and inactive glucocorticoids in regions
of murine brain. Girard T (GirT) corticosteroid hydrazones (corticosterone (CORT) and 11-dehydrocorticosterone (11DHC)) were most
abundant in the cortex, hippocampus, and amygdala with significant lower CORT/11DHC ratios observed upon transgenic disruption of the enzyme
primarily caused by a significant accumulation of 11DHC. (a) Histological image of cryosection of murine brain stained with hematoxylin and eosin
with the outline of the MSI-LESA regions of interest (ROIs) (cortex; hippocampus, HPC; and amygdala, Am). (b, d) Heat map distribution by
MALDI-FTICR-MSI of GirT-CORT derivative at m/z 460.31698 ± 0.025 Da in wild type (b) and 11β-HSD1−/− (d) mice. (c, e) Heat map
−
/−
distribution by MALDI-FTICR-MSI of GirT-11DHC derivative at m/z 458.30133 ± 0.025 Da in wild type (c) and 11β-HSD1
(e) mice. Signal
intensity is depicted by color on the scale shown. Scale bar (2 mm). (f) CORT/11DHC ratios measured by MALDI-FTICR-MSI (MSI), LC-MS/
MS, and LESA in regions of interest in murine brain were significantly lower in 11β-HSD1 mice (KO) than wild type (WT) (p < 0.001 overall
between genotypes). (g) Differences between genotypes in CORT/11DHC ratios were caused by a statistically significant increase of 11DHC and a
trend to a similar magnitude of reduction in CORT in 11β-HSD1 mice, measured in whole brain by LC-MS/MS. Statistical analysis was by two-
way ANOVA for LESA and MSI and Student’s t test for LC-MS/MS and absolute measurements (n = 12). Data are mean ± SEM. *P < 0.05, **P <
−/−
−/−
0.01, and ***P < 0.001.
disruption of tissue morphology due to an increase of water
vapor in the reaction chamber and can also lead to diffusion of
the water-soluble GirT derivatives and ion suppression effects
due to protein cleavage and tissue component degradation. The
best yield, on-tissue, was achieved in 1 h at 40 °C, in a
moisturizing environment using methanol/TFA as a solvent/
catalyst system (Figure S2b, Supporting Information). The
trifluoroacetate ion may facilitate the derivatization reagent
incorporation due its tissue-penetration properties. Greatest
signal intensity was achieved using 4 mL of 5 mg/mL solution
nitrogen flow, distance from the MALDI target, time between
passes, and solvent volume. Crystal homogeneity was inspected
using light microscopy showing a uniform cocrystallization
pattern. An image of the matrix ion was also recorded with each
tissue to ensure even application. “Solvent-free” approaches to
apply matrix, such as sublimation and sublimation/recrystalliza-
tion, were evaluated but, in combination with GirT steroids,
demonstrated poor signal-to-noise ratios. As previously
reported, proteolysis and tissue degradation during sample
preparation may cause ion-suppression. With the optimized
protocol in place, stability was evaluated temporally by
assessing the intensity of signal from GirT-CORT applied on
and off tissue sections; only 13% reduction in signal
suppression/degradation was observed after 20 min at RT,
within the necessary limit of tissue handling times (Figure S2c,
Supporting Information).
GirT derivatives yielded spectra dominated by the molecular
ion (Figure S3, Supporting Information), and high resolution
MS was used to overcome the challenge of selecting specific
analytes of interest from the many high abundance isobaric ions
in the low mass range. Structural confirmation by fragmenta-
tion, using LESA-ESI-FTICR-MS followed by CID, allowed
isolation of the precursor ions in the ICR cell, increasing both
31
2
for postcoating and 0.15 mg/cm for precoating. In the ultimate
protocol, slides were precoated with defined amounts of
reagents, offering the additional benefit of limiting analyte
diffusion. The reaction was quenched by dehydration to avoid
subsequent hydrolysis. When GirT is used in MS analysis
coupled with chromatography, the excess of derivatization
reagent is diverted to waste to avoid ion-suppression in the MS
2
9
ion source. In OTCD, this is not practical, so the amount of
derivatization reagent was limited to ensure maximum
conversion and minimum ion-suppression.
Matrix was deposited using as a spray, and analyte diffusion
as well as surface topology and crystal homogeneity were
controlled by standardization of all possible variables, such as
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Figure 3. Effect of pharmacological inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with UE2316 in C57BL/6 mice. Girard T
GirT) derivatives (corticosterone (CORT) and 11-dehydrocorticosterone (11DHC)) were mostly distributed across the cortex, hippocampus, and
(
amygdala; after enzyme inhibition, CORT/11DHC ratios showed a significant decline as a result of an increased 11DHC metabolite. (a) Histological
image of horizontal cryosection of murine brain stained with hematoxylin and eosin. (b−e) MSI heat map distribution of m/z 390.08377 ± 0.025 Da
representing UE2316 in brain over a 6 h time course in mice receiving Vehicle (V) or UE2316 (D). (f) Amounts of UE2316 in whole brain
measured by LC-MS/MS demonstrated the same temporal pattern as those determined by MSI. (g) Histological image of coronal cryosection of
murine brain stained with hematoxylin and eosin with the outline of the MSI regions of interest (ROIs) (cortex; hippocampus, HPC; and amygdala,
Am). (h, j) MSI heat map of GirT-CORT at m/z 460.31698 ± 0.005 Da brain from mice receiving Vehicle (h) or UE2316 (1h post dose) (j). (i, k)
GirT-11DHC at m/z 458.30133 ± 0.025 Da in brain from mice receiving Vehicle (i) or UE2316 (k). Signal intensity is depicted by color on the
scale shown. Scale bar (2 mm). (l) A significant decline (p < 0.01, overall between groups) in CORT/11DHC ratios was observed across the ROIs
by MSI in the brain after administration of UE2316, showing good agreement with data generated by LC-MS/MS in whole brain. (m) Absolute
quantification of corticosteroids in brain tissue by LC-MS/MS showed that the differences in CORT/11DHC ratios were associated with a
statistically significant increase of 11DHC and a trend to a decrease in CORT in UE2316-treated mice. Statistical analysis was performed using two-
way ANOVA for MSI and Student’s t test for LC-MS/MS and absolute measurements (n = 12). Data are mean ± SEM. *P < 0.05 and **P < 0.01.
sensitivity and selectivity. This process provided fragmentation
patterns typical of GirT hydrazones (Figure S4, Supporting
Information), concordant with the structures of steroid
hydrazones, extrapolating from GirP analogues, which may
was observed at m/z 383 corresponding to the neutral loss of
water at C11 of the m/z 401 GirT-CORT fragment ion.
In rat adrenal gland, molecular distribution maps showed
GirT-CORT (m/z 460.31713) and GirT-11DHC (m/z
458.30139) in high abundance in the zona fasciculata/
reticularis (Figure 1c,d), with mass accuracy of ±5 ppm from
their theoretical monoisotopic masses (Figure 1e−g) and
signal-to-noise ratios >100.
13
form a stable five member ring as previously reported. CID of
the GirT derivatives generated a series of fragment ions
+
characteristic of the loss of the quaternary amine tag [M-59]
+
and carbon monoxide [M-87] of the derivatized group at m/z
Studies of Manipulation of 11β-HSD1. Endogenous
CORT (Figure 2b,d) and 11DHC (Figure 2c,e) were
successfully detected as GirT hydrazones by MSI in sections
399, m/z 371 (GirT-11DHC) and m/z 401, m/z 373 (GirT-
CORT), respectively. Also, a GirT-CORT selective fragment
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Figure 4. MSI detection of 7-ketocholesterol, an alternative substrate for 11β-HSD1. A significant increase in Girard T 7-ketocholesterol (GirT-
KC) intensity was observed across the whole brain in mice after transgenic disruption of 11β-HSD1−/− (KO) and after administration of UE2316
1 h postdose). (a) Histological image of coronal cryosection of murine brain stained with hematoxylin and eosin. MSI heat map of GirT-KC at m/z
7
(
5
14.43670 ± 0.025 Da in (b) wild type (WT), (c) 11β-HSD1−/−, and brain from mice receiving (e) Vehicle (V) or (f) UE2316 (D). Quantification
−/−
of MSI signal intensity for GirT-7KC across the whole brain for (d) wild type versus 11β-HSD1
mice and for (g) mice treated with vehicle of
UE2316. (h) (I) Theoretical monoisotopic distribution of GirT-7KC and (II) GirT-KC in mouse brain. GirT-7KC was detected in murine brain, and
−/−
its levels increased in 11β-HSD1 mice (c) and following inhibition of the enzyme using UE2316 (f). Signal intensity is depicted by color on the
scale shown. Scale bar (2 mm). cps = counts per second. Statistical analysis was performed using two way ANOVA (n = 12). Data are mean ± SEM.
*P < 0.05 and ***P < 0.001.
of murine brain from both wild type (WT) and 11β-HSD1−/−
mice. Corticosteroid derivatives were detected in highest
tive analysis by LC-MS/MS confirmed these findings (Figure
3m).
abundance in the cortex, hippocampus, and amygdala. In
1
Correlation between Methods of Quantitation. Good
agreement was observed between MSI and LC-MS/MS (Figure
S5a, Supporting Information) and between MSI and LESA
(Figure S5b, Supporting Information), across the range of
values, although MSI-LESA demonstrated a negative bias using
Bland-Altman analysis.
Quantitation of Alternative Substrates of 11β-HSD1.
The GirT derivative of 7-ketocholesterol was detected by MSI
in brain sections (Figure 4), and its abundance increased in
mice with disruption (Figure 4b−d) or inhibition (Figure 4e−
g) of 11β-HSD1.
1β-HSD1− mice, the CORT/11DHC ratio was reduced in
/−
these regions of interest (Figure 2f). Absolute ratios (and
−/−
magnitude of difference between 11β-HSD1 and WT mice)
obtained by LC-MS/MS and LESA were similar to those
determined by MSI (Figure 2f). Absolute measurements
(
Figure 2g) suggested the change in pattern between genotypes
was driven by increased amounts of 11DHC and reduced
amounts of CORT, although only the former was statistically
significant.
Following a single oral dose of UE2316, an inhibitor of
murine 11β-HSD1, levels of the drug detected by MSI peaked
in brain 1 h postdose (Figure 3b, vehicle, Figure 3c−e,
UE2316); the MSI data were confirmed by LC-MS/MS of drug
in whole brain (Figure 3f). The regional distribution of
glucocorticoids was similar in brains of vehicle and UE2316
treated mice (Figure 3h,j (CORT), 3i,k (11DHC)) with
UE2316 reducing CORT/11DHC ratios measured by MSI in
the cortex, hippocampus, and amygdala (Figure 3l). Quantita-
DISCUSSION
■
This novel application of OTCD coupled with MSI permits
detection of poorly ionizable endogenous corticosteroid
hormones within tissues, by generating permanently charged
derivatives which yield intense signals upon MALDI-FTICR
analysis. The spatial distribution of the substrate and product of
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Analytical Chemistry
1β-HSD1, 11DHC, and CORT in rat adrenal glands and
Article
1
pharmacological 11β-HSD1 inhibitors. Changes in enzyme
activity were quantified by CORT/11DHC ratios, which is
appropriate since derivatives of both steroids possess similar
ionization yields and signal intensities when compared using
postprocessing vector normalization (RMS). An alternative
approach would have been to incorporate a labeled steroid
murine brain was visualized. Quantitation in 11β-HSD1
deficient mice allowed assessment of the consequences of
regional activity of 11β-HSD1 for local steroid levels, and the
approach also allowed pharmacodynamic and pharmacokinetic
assessment of an 11β-HSD1 inhibitor within the brain. This
technique has the potential to be applied to other steroids and
sterols to investigate steroid intracrinology in multiple organs
and in solid tumors.
(
e.g., d CORT) in the matrix, allowing signal intensity to be
8
normalized; this approach also offers the advantage of a further
read-out of uniformity of matrix application but was not
employed here. Disrupting or inhibiting 11β-HSD1 was
anticipated to increase the CORT/11DHC ratio, as previously
reported in homogenates of brain from 11β-HSD1 deficient
The novel use of GirT reagent permitted low LODs. Similar
derivatives have been used to enhance analysis of sterols and
10−14,29,30
steroids by LC-MS/MS (ESI),
but there has been
limited application of derivatization for tissue imaging, with
only two previous examples for nonsteroidal small mole-
33
mice. This was detected and quantified in brain subregions,
with an increase of the signal intensity of GirT-11DHC and a
smaller decline in GirT-CORT levels across the cortex,
hippocampus, and amygdala with 11β-HSD1 deficiency or
inhibition. These brain regions have high abundance of 11β-
1
5,16
cules.
The reaction reported here favored the α−β
unsaturated-keto moiety at C3 as the π electrons are
delocalized across the conjugated system leaving the carbonyl
susceptible to a nucleophilic attack. Indeed, monitoring the
reaction products for up to 3 h did not reveal formation of
multiple charged derivatives, indicative of reactions taking place
at other positions. The efficiency of OTCD reaction was clearly
demonstrated, as the signal intensity was considerable
enhanced after conversion of native corticosteroids to their
corresponding GirT hydrazones. The transformation of neutral
steroid to their corresponding water-soluble and charged
derivatives was achieved in a short period of time using a
simple customized reaction chamber. The moisturizing
environment was controlled by the amount of water and
reaction temperature allowing little analyte diffusion. The
derivatization allowed one to map the low levels of endogenous
steroids on both adrenal gland and brain tissue sections. The
concept of charge-tagging may be extrapolated to other neutral
keto-steroids for the generation of molecular profiles in target
tissues and, indeed here, was also used successfully to image 7-
ketocholesterol.
32
HSD1 mRNA.
CORT in the brain is derived both from the circulation and
from local 11β-HSD1 activity. The proportionate contribution
of each source, however, is unknown. To date, this has been
inferred for some tissues, imprecisely, from studies using
arteriovenous sampling in combination with stable isotope
34
35
tracers, by sampling interstitial fluid by microdialysis or by
quantifying steroid concentrations in whole tissue homogenates
3
6
ex vivo. The MSI data here provides the novel insight that
there is a small decline of only 16% in active CORT with 11β-
HSD1 deficiency in regions of the brain where 11β-HSD1 is
normally most highly expressed, suggesting the contribution of
1
1β-HSD1 to the intracellular pool of corticosterone in these
experimental conditions is relatively modest.
Lastly using MSI, it was possible to image alternative
substrates of 11β-HSD1, in this case 7-ketocholesterol.
Previously, it has been difficult to establish whether 11β-
HSD1 predominantly reduces 7-ketocholesterol to 7β-hydrox-
Rat adrenal glands were selected as a model tissue for
method development, and subsequently as a positive control,
due to the anticipated high abundance of corticosteroids.
Glucocorticoids were visualized and most abundant in the zona
fasciculata, the major source of glucocorticoid synthesis, with
lower signal in the outer zona glomerulosa, where cortico-
sterone is further metabolized to aldosterone. This confirmed
that unwanted diffusion of analytes was constrained by the
tissue processing method. Spatial resolution of the laser is
adjustable, but increasing the spatial resolution decreases
sensitivity as the spot size sampled is smaller. In the adrenal,
collecting data at 200 μm resolution generated sufficient spatial
information with adequate signal (signal/noise >100, compared
with signal/noise ∼20 at 50 μm spatial resolution). In future
use of the technique, the spatial resolution may be increased
according to the region and tissue of interest but must be
balanced against robustness of signal. Implementation of
continuous accumulation of selected ions “CASI” may facilitate
these advances.
22
ycholesterol or if oxidation dominates. Indeed, changes in the
balance of circulating oxysterols are modest in 11β-HSD1−
mice. By demonstrating accumulation of 7-ketocholesterol with
11β-HSD1 deficiency, MSI has provided clear novel evidence
that reduction of oxysterols by 11β-HSD1 predominates.
/−
CONCLUSIONS
■
MSI with on-tissue derivatization is a powerful new tool to
study the regional variation in abundance of steroids within
tissues. This is the first technique capable of detecting and
quantifying corticosteroids to <200 μm resolution and at
physiological concentrations, allowing application within
region-specific areas of murine brain. We have demonstrated
its utility for measuring pharmacodynamic effects of small
molecule inhibitors of 11β-HSD1; in combination with
pharmacokinetic imaging, this will facilitate screening of the
disposition of such drugs being developed to treat Alzheimer’s
disease. Importantly, this technique opens the door for
expanding the range of steroids that can be studied by
MALDI-MSI by adapting existing chemical derivatization
methods. In the first instance, this may encompass other α−β
unsaturated keto steroids, such as testosterone and progester-
one, as well as keto-sterols, and may be applied in other tissues
such as prostate gland and breast tissue. This offers the
prospect of many novel insights into tissue-specific steroid and
sterol biology.
OTCD coupled with MALDI-FTICR-MSI was successfully
applied in murine brain, where discriminating abundance of
glucocorticoids in subregions was possible. The highest
abundance in cortex, hippocampus, and amygdala may reflect
the higher expression of corticosteroid-binding receptors as well
32
as of 11β-HSD1 in these brain regions. To evaluate use of the
technique to interpret biological variations in tissue steroid
levels, proof-of-principle experiments were performed using
brains of mice with 11β-HSD1 deficiency or receiving
1
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Article
(
20) Wamil, M.; Andrew, R.; Chapman, K. E.; Street, J.; Morton, N.
ASSOCIATED CONTENT
Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
M.; Seckl, J. R. Endocrinology 2008, 149, 5909−5918.
21) Hult, M.; Elleby, B.; Shafqat, N.; Svensson, S.; Rane, A.; Jornvall,
H.; Abrahamsen, L.; Oppermann, U. Cell. Mol. Life Sci. 2004, 61, 992−
99.
22) Mitic, T.; Shave, S.; Semjonous, N.; McNae, I.; Cobice, D. F.;
*
S
(
9
(
AUTHOR INFORMATION
Corresponding Author
E-mail: Ruth.Andrew@ed.ac.uk. Tel: +44 (0) 131 242 6763.
Fax: +44(0) 131 242 6779.
Lavery, G. G.; Webster, S. P.; Hadoke, P. W.; Walker, B. R.; Andrew,
R. Biochem. Pharmacol. 2013, 86, 146−153.
(
■
*
23) Webster, S. P.; Seckl, J. R.; Walker, B. R.; Ward, P.; Pallin, T. D.;
Dyke, H. J.; Perrior, T. R. PCT Intl. WO2011/033255, 2011.
24) Jaskolla, T. W.; Lehmann, W. D.; Karas, M. Proc. Natl. Acad. Sci.
U. S. A. 2008, 105, 12200−12205.
25) Kotelevtsev, Y. V.; Holmes, M. C.; Burchell, A.; Houston, P. M.;
(
Notes
The authors declare the following competing financial
interest(s): D.F.C., C.L.M., R..JA.G., A.M., P.R.L.S., and R.A.
do not have any conflict of interest. B.R.W. and S.P.W. are
inventors on patents owned by the University of Edinburgh
relating to 11β-HSD1 inhibitors.
(
Scholl, D.; Jamieson, P. M.; Best, R.; Brown, R. W.; Edwards, C. R. W.;
Seckl, J. R.; Mullins, J. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14924−
14929.
(26) Koeniger, S. L.; Talaty, N.; Luo, Y.; Ready, D.; Voorbach, M.;
Seifert, T.; Cepa, S.; Fagerland, J. A.; Bouska, J.; Buck, W.; Johnson, R.
W.; Spanton, S. Rapid Commun. Mass Spectrom. 2011, 25, 503−510.
ACKNOWLEDGMENTS
■
(
(
(
27) Heap, B. J.; Kiernan, J. A. J. Anat. 1973, 115, 315−325.
We are grateful to the British Heart Foundation (D.F.C., R.A.,
B.R.W.) and the Wellcome Trust (A.M., B.R.W., S.P.W.) for
funding this work. We thank SIRCAMS and the Mass
Spectrometry Core of the Wellcome Trust Clinical Research
Facility for technical support. We also thank Dr. Gregorio
Naredo Gonzalez (Mass Spectrometry Core) for his help in the
synthetic chemistry.
28) Wheeler, O. Chem. Rev. 1962, 62, 205−221.
29) Khan, A. K.; Wang, Y.; Heidelberger, S.; Avelius, G.; Liu, S.;
Sjo
̈
vall, J.; Griffiths, W. J. Steroids 2006, 71, 42−53.
(30) Griffiths, W,J; Liu, S.; Avelius, G.; Sjo
̈
vall, J. Rapid Commun.
Mass Spectrom. 2003, 17, 924−935.
(31) Goodwin, R. J.; Iverson, S. L.; Andren, P. E. Rapid Commun.
Mass Spectrom. 2012, 26, 494−498.
(32) Moisan, M.-P.; Seckl, J. R.; Monder, C.; Agarwal, A. K.; White,
P. C.; Edwards, C. R. W. Neuroendocrinology 1990, 2, 853−858.
33) Sooy, K.; Webster, S. P.; Noble, J.; Binnie, M.; Walker, B. R.;
Seckl, J. R.; Yau, J. L. W. J. Neurosci. 2010, 30, 13867−13872.
34) Hughes, K. A.; Manolopoulos, K. N.; Iqbal, J.; Cruden, N. L.;
REFERENCES
■
(
(
1) Simpson, E. R.; Mahendroo, M. S.; Means, G. D.; Kilgore, M. W.;
Hinshelwood, M. M.; Graham-Lorence, S.; Amareh, B.; Ito, Y.; Fisher,
C. R.; Michael, M. D.; Mendelson, C. R.; Bulun, S. E. Endocr. Rev.
994, 15, 342−355.
2) Russell, D. W.; Wilson, J. D . Annu. Rev. Biochem. 1994, 63, 25−
1.
(
Stimson, R. H.; Reynolds, R. M.; Newby, D. E.; Andrew, R.; Karpe, F.;
1
(
6
(
(
(
1
(
Walker, B. R. Diabetes 2012, 61, 1357−1364.
(35) Wake, D. J.; Homer, N. Z. M.; Andrew, R.; Walker, B. R. J. Clin.
Endocrinol. Metab. 2006, 91, 4682−4688.
3) Seckl, J. R.; Walker, B. R. TEM 2004, 15, 418−424.
(36) Hughes, K. A.; Reynolds, R. M.; Critchley, H. O.; Andrew, R.;
4) Heeren, R. M.; Chughtai, K. Chem. Rev. 2010, 110, 3237−3277.
Walker, B. R. J. Clin. Endocrinol. Metab. 2013, 95, 4696−4702.
̈
5) Malmberg, H.; Nygren, P.; Sjovall, L. A. J. L. Spectroscopy 2013,
8, 503−511.
6) Dufresne, M.; Thomas, A.; Breault-Turcot, J.; Masson, J.-F.;
Chaurand, P. Anal. Chem. 2013, 85, 3318−3324.
7) Winograd, N.; Garrison, B. J. Annu. Rev. Phys. Chem. 2010, 61,
05−322.
8) Patti, G. J.; Woo, H. K.; Yanes, O.; Shriver, L.; Thomas, D.;
(
3
(
Uritboonthai, W.; Apon, J. V.; Steenwyk, R.; Manchester, M.; Siuzdak,
G. Anal. Chem. 2010, 82, 121−128.
(
9) Singh, G.; Gutierrez, A.; Xu, K.; Blair, I. A. Anal. Chem. 2000, 72,
3
(
1
(
007−3013.
10) Quirke, J. M.; Van Berkel, G. J. J. Mass Spectrom. 2001, 36,
294−1300.
11) Higashi, T.; Yamauchi, A.; Shimada, K. J. Chromatogr., B: Anal.
Technol. Biomed. Life Sci. 2005, 825, 214−222.
(12) Shackleton, C. J. Steroid Biochem. Mol. Biol. 2010, 121, 481−490.
(13) Wang, Y.; Hornshaw, M.; Alvelius, G.; Bodin, K.; Liu, S.; Sjovall,
J.; Griffiths, W. J. Anal. Chem. 2010, 78, 164−173.
14) Wang, H.; Wang, H.; Zhang, L.; Zhang, J.; Guo, Y. Anal. Chim.
Acta 2011, 690, 1−9.
15) Chacon, A.; Zagol-Ikapitte, I.; Amarnath, V.; Reyzer, M. L.;
Oates, J. A.; Caprioli, R. M.; Boutaud, O. J. Mass Spectrom. 2011, 46,
40−846.
16) Manier, M. L.; Reyzer, M. L.; Goh, A.; Dartois, V.; Via, L. E.;
Barry, C. E., III; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 2011, 22,
409−1419.
17) de Kloet, E. R.; Joels, M.; Holsboer, F. Nat. Rev. Neurosci. 2005,
, 463−475.
18) Yau, J. L.; Noble, J.; Seckl, J. R. J. Neurosci. 2011, 31, 4188−
193.
19) Kotelevtsev, Y. V.; Brown, R. W.; Fleming, S.; Edwards, C. R.
W.; Seckl, J. R.; Mullins, J. J. J. Clin. Invest. 1999, 103, 683−689.
(
(
8
(
1
(
6
(
4
(
1
1584
dx.doi.org/10.1021/ac402777k | Anal. Chem. 2013, 85, 11576−11584