Tandem Mass Spectrometry Imaging Reveals Distinct Accumulation

Article Cite This: Anal. Chem. 2019, 91, 8918−8925

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Tandem Mass Spectrometry Imaging Reveals Distinct Accumulation Patterns of Steroid Structural Isomers in Human Adrenal Glands Emi Takeo,† Yuki Sugiura,‡ Tatsuki Uemura,§ Koshiro Nishimoto,‡,∥ Masanori Yasuda,⊥ Eiji Sugiyama,‡ Sumio Ohtsuki,§ Tatsuya Higashi,⧧ Tetsuo Nishikawa,¶ Makoto Suematsu,‡ Eiichiro Fukusaki,† and Shuichi Shimma*,† †

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biochemistry, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan § Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan ∥ Department of Uro-Oncology, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan ⊥ Department of Pathology, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan ⧧ Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ¶ Endocrinology and Diabetes Center, Yokohama Rosai Hospital, 3211 Kozukuecho, Kohoku-ku, Yokohama, Kanagawa 222-0036, Japan

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S Supporting Information *

ABSTRACT: Visualizing tissue distribution of steroid hormones is a promising application of MALDI mass spectrometry imaging (MSI). On-tissue chemical derivatization using Girard’s T reagent has enhanced the ionization efficiency of steroids. However, discriminating between structural isomers with distinct bioactivities remains a challenge. Herein, we used ion trap MS/tandem MS (MS3) to distinguish a mineralcorticoid aldosterone (Aldo) and a glucocorticoid cortisol (F), from their structural isomers. Our method is also useful to detect hybrid steroids (18-hydroxycortisol [18OHF] and 18-oxocortisol) with sufficient signal-to-noise ratio. The clinical applicability of the tandem MS method was evaluated by analyzing F, Aldo, and 18-OHF distributions in human adrenal glands. In such clinical specimens, small Aldo-producing cell clusters (APCCs) were identified and were first found to produce a high level of Aldo and not to contain F. Moreover, a part of APCCs produced 18-OHF, presumably converted from F by APCC-specific CYP11B2 activity. Catecholamine species were also visualized with another derivatization reagent (TAHS), and those profiling successfully discriminated pheochromocytoma species. These tandem MSI-methods, coupled with on-tissue chemical derivatization has proven to be useful for detecting low-abundance steroids, including Aldo and hybrid steroids and thus identifying steroid hormone-producing lesions.

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Imaging with isomeric resolution has been reported recently in lipid study.9,10 This outstanding challenge is also encountered in steroid imaging. Steroid synthesis pathways consist of multiple oxidoreductase reactions mainly involving cytochrome P450 family members and produce large numbers of structurally different isomers11−13 with distinct bioactivities. For example, aldosterone (Aldo)a mineralocorticoid that plays a vital role in the homeostatic regulation of blood pressure and plasma sodium levels14has the same molecular weight as cortisone, which is an inactivated metabolite of cortisol that released by the body in response to stress and

ALDI mass spectrometry imaging (MALDI-MSI) has been widely used in clinical and pharmaceutical applications.1−4 This method combines chemical processingincluding derivatization and ionizationof analytes on the tissue surface, followed by MS detection,1 and can, in theory, detect various steroid hormone species in tissues. Ontissue chemical derivatization (OTCD) using Girard’s T (GirT) reagent has enabled the detection and visualization of several steroid hormones by enhancing their ionization efficiency via introduction of a permanent cationic charged trimethylamine;5 the hydrazine group of GirT reagent reacts with the ketone group at the C3 position of the A ring of steroid hormones, which is conjugated with a C4−5 double bond6 in some steroids, such as glucocorticoids in murine brain and androgens in testis.5,7,8 © 2019 American Chemical Society

Received: February 1, 2019 Accepted: June 16, 2019 Published: June 16, 2019 8918

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Analytical Chemistry increases blood pressure to prepare the organism for fight or fright.15,16 It should be noted that, although its high mass resolving power allows ion peaks to be assigned to a chemical formula, Fourier transform ion cyclotron resonance (FT-ICR) MS instruments cannot discriminate between structural isomers of a steroid species (see Figure S1). In this study, we used ion trap MS in conjunction with tandem MSthat is, MS2 and MS3to distinguish Aldo and cortisol (F) from their structural isomers cortisone (E) and 18hydroxycorticosterone (18-OHB), respectively. MS3 developed in the current study has also allowed us to visualize the hybrid steroids 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18oxoF),17 collectively revealing spatial heterogeneity of these structurally different steroids in adrenal glands in vivo. As proof of concept, we used the MS3-based technique to confirm the difference in Aldo distribution in the adrenal tissue of rats fed a sodium-deficient (SDef) diet and control rats. Additionally, we used the technique to measure F, Aldo, and 18-OHF levels in human adrenal grand to evaluate its clinical applicability.

SIMC-Uro#11351) and pheochromocytoma (Case-3; SIMCUro#11352 at Yokohama Rosai Hospital were used in this study. Tissue Sectioning and Mounting. Frozen serial 8-μm sections were cut at −20 °C with a cryomicrotome (CM1950; Leica, Nussloch, Germany) and mounted on ITO-coated glass slides for MALDI-MSI and coated glass slides for IHC (two sections per specimen for each analysis). Before derivatization and immunostaining, the tubes containing the slides were placed at 25 °C for 20 min. OTCD with GirT. Animal and human tissue samples were prepared at Osaka University. GirT reagent was prepared at 10 mg/mL in 20% acetic acid,7 and the solution was sprayed over 15 cycles onto the tissue surface using an artist’s airbrush (PS270; GSI Creos, Tokyo, Japan). After all of the solution had been dispensed, the slide was maintained at room temperature for 60 min. For derivatization of the steroid standards, GirT solution was mixed with an equal volume of those samples and allowed to stand for 10 min before application of mixed solution onto ITO glass. Optimization of Matrix Compounds and Application Methods. We optimized matrix compounds and application methods for GirT-derivatized steroid detection. By examining the signal intensity of GirT-Aldo in a rat adrenal gland, we determined that a two-step α-CHCA application method had 40 times higher sensitivity than direct spraying.19 For the other matrix compound DHB, we examined the effect of adding 250 mM ammonium sulfate20 and found that this enhanced ionization of the derivatized steroids by 1.5 times (Figure S2). Since two-step α-CHCA application resulted in evident accumulation of steroids in the zona glomerulosa (ZG; see Figure S2), this method was selected in our experiments. Two-Step Matrix Application. Two-step matrix application was carried out as previously described.19 In the first step, the matrix (α-CHCA) was sublimated using the iMLayer vacuum deposition system (Shimadzu, Kyoto, Japan). The vacuum pressure inside the chamber was maintained at 1 × 10−3 Pa during deposition. The α-CHCA was heated at 250 °C, and gaseous α-CHCA was deposited onto the specimen surface at a thickness of 0.5 μm; the thickness was monitored by the transmittance of laser light. In the second step, αCHCA (10 mg/mL) was dissolved in a solution of 3:1:6 acetonitrile, isopropanol, and distilled water (all containing 0.1% formic acid). The matrix solution was applied over 15 cycles with the artist’s airbrush. MALDI-MSI Analysis for Steroid Hormones. MALDIMS and -MSI experiments were performed on a MALDI linear ion trap mass spectrometer (MALDI LTQ XL; Thermo Fisher Scientific, Bremen, Germany) equipped with a 60-Hz N2 laser (λ = 337 nm). The laser spot size was approximately 120 μm, and each pixel was irradiated 50 times at a repetition rate at 20 Hz. Mass spectra were acquired in the positive ion detection mode. The transition for MS3 analysis was as follows: m/z 474.3 → 415.2 (neutral loss of trimethylamine) for Aldo and E; m/z 476.3 → 417.2 for F and 18-OHB; m/z 490.3 → 431.2 for 18-oxoF; and m/z 492.3 → 433.2 for 18-OHF. Specific peak values for each steroid were recorded in the obtained mass spectra. The collision energy was optimized to maximize the ion intensity of the Aldo-specific peak using a derivatized Aldo standard. After sample analysis, ion images were reconstructed based on data extracted from m/z ranges of 397.1−397.3 (Aldo), 385.1−385.3 (E), 387.1−387.3 (F), 413.1−413.3 (18-oxoF), and 415.1−415.3 (18-OHF) using

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MATERIALS AND METHODS Chemicals. Acetic acid, acetone, acetonitrile, ethanol, formic acid, isopropanol, Mayer’s hematoxylin solution, methanol, and 1% eosin Y solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). α-Cyano-4hydroxycinnamic acid (α-CHCA, 99%), 2,5-dihydroxybenzoic acid (DHB, 99.5%), GirT, Aldo (>95%), B (>98%), F, and 18OHB were from Merck (Darmstadt, Germany). Hybrid steroids were purchased from Toronto Research Chemicals (North York, ON, Canada). Ammonium sulfate was from Nacalai Tesque (Kyoto, Japan). Indium tin oxide (ITO)coated glass slides (100 Ω/sq without adhesive material coating) for MALDI-MSI and adhesive-coated glass slides for immunohistochemistry (IHC) were from Matsunami Glass (Osaka, Japan). The antibody against human aldosterone synthase (cytochrome P450 family 11 subfamily B member 2 = CYP11B2) was prepared by an author, Koshiro Nishimoto.18 Animal Study. Experiments using rats were reviewed and approved by the Keio University School of Medicine Institutional Animal Care and Use Committee. Nine male Sprague−Dawley rats (CLEA Japan, Tokyo, Japan) older than 33 weeks were housed in a temperature-controlled environment and maintained on a 12:12-h light/dark cycle with free access to food and water. After 1 week of acclimation, the rats were randomly divided into two groups. Five rats were fed deficient sodium diet (SDef, 0.01%−0.02% sodium, CLEA Diet No. 010; CLEA Japan) and the other four were fed normal sodium diet (NS, CA-1; CLEA Japan). After 1 week on the diet, the rats were sacrificed under general sevoflurane anesthesia, and adrenal gland tissue and blood samples were collected. The adrenal glands were cleaned of adherent tissue and frozen in O.C.T. compound (Sakura Finetek, Nagano, Japan). Human Study. This study was approved by the Institutional Review Boards of Keio University School of Medicine (No. 2009-0018), Saitama Medical University International Medical Center (SIMC, No. 16-093 and 18-185) and Yokohama Rosai Hospital (No. 24-10). Written, informed consent was obtained from patients before radical nephrectomy for renal cell carcinoma at SIMC (Case-1 [SIMCUro#10294, a unique nonsequential patient control number in the Department of Uro-Oncology, SIMC]), as well as unilateral adrenalectomy for primary aldosteronism (Case-2; 8919

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Figure 1. Product ion spectra of GirT-Aldo and GirT-E standards obtained by MS2 and MS3. (A, B) MS2 spectra of m/z 474 and presumed structures obtained from GirT-Aldo (A) and GirT-E (B). (C, D) MS3 spectra of m/z 415 obtained from GirT-Aldo (C) and GirT-E (D). MS3 analysis showing different cleavage patterns at nonderivatized sites. (E, F) Proposed fragmentation pathway of GirT-Aldo (E) and GirT-E (F). 8920

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Figure 2. Product ion spectra of steroid standards obtained by MS3. (A, B) MS3 spectra of m/z 417 and presumed structures obtained from GirT-F (A) and GirT-18-OHB (B). (C, D) MS3 spectra of m/z 435 obtained from GirT-18-OHF (C) and of m/z 433 obtained from GirT-18-oxoF (D). Fragmentation patterns were the same as for GirT-Aldo and GirT-E. Specific peaks are shown in bold type.

been used for steroid isomer separation,22 but these are unavailable in MSI. Currently, MS n and ion-mobility separation23,24 coupled with MALDI or desorption electrospray ionization are potential techniques for discriminating between isomers of steroids and determining their localization; we, therefore, characterized the fragmentation patterns of GirT-Aldo and GirT-E by MS2 and MS3 analyses (Figure 1). Mixing with GirT reagent yielded GirT-hydrazone forms of Aldo and E. In the MALDI-MS analysis, both samples easily produced [M]+ ions at m/z 474. Although specific signals were not obtained by MS2 measurement of [M]+ ions, near-identical product ion spectra were obtained for the two steroids, with a dominant peak at m/z 415 corresponding to the [M − 59]+ ion dissociated from the GirT-derived trimethylamine group (Figure 1A and B). The MS2 fragmentation patterns of GirTAldo and GirT-E were consistent with those of typical steroid GirT derivatives reported in liquid chromatography (LC)-MS2 studies in which unique high-performance LC retention times for structural isomers were used for molecular identification.25 The peaks at m/z 415 and 387 represented the neutral loss of trimethylamine [M − 59]+ [loss of N+(CH3)3] and carbon monoxide [M − 87]+ [loss of N+(CH3)3 and CO] from GirTAldo and GirT-E, respectively. To obtain a molecule-specific fragmentation pattern, we examined MS3 spectra. The ion at m/z 415 was subjected to MS3 analysis and produced molecule-specific signals (Figure 1C and D); peaks at m/z 397 [M − 18]+ and 369 [M − 46]+ were observed for GirTAldo, while m/z 385 [M − 30]+ and 355 [M − 60]+ were only detected in GirT-E (Figure 1E, F); for GirT-Aldo, m/z 397 and 369 were estimated as the neutral loss of H2O at the C ring [M − 18]+ and of carbon monoxide at the GirT-

ImageQuest, version 1.0.1 (Thermo Fisher Scientific). After MALDI-MSI, the matrix on the tissue surface was removed with 100% acetone (without cooling), and sample tissues were stained with hematoxylin and eosin according to a standard procedure. MALDI-MSI Analysis for Catecholamine Imaging Using TAHS Derivatization. To perform MALDI-MSI of catecholamine, 5 mg/mL of the TAHS reagent dissolved in acetonitrile was applied to the surface of the adrenal sections using an airbrush with a 0.2 mm nozzle caliber. The tissue sections were incubated 15 min at 55 °C, followed by application of DHB (50 mg/mL) dissolved in acetonitrile. MALDI-MSI experiments were performed by a MALDI LTQ XL in the positive ion detection mode. The transition for MS2 analysis was as follows: m/z 360.2 → 177.1 for adrenalin and m/z 346.2 → 177.1 for noradrenalin. IHC. Cryosections were fixed in cold acetone (−20 °C) for 10 min, and CYP11B2 expression was evaluated by IHC as previously reported.21 The anti-CYP11B2 antibody was used at 1:200 dilution. Envision reagent, coupled with horseradish peroxidase (Dako, Carpinteria, CA, USA) was used for immunodetection. Specimens were then stained with hematoxylin, and images were obtained with a digital microscope (BZ-X700; Keyence, Osaka, Japan).

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RESULTS AND DISCUSSION Development of Tandem MS Methods for Detection of GirT-Aldo, E, and F. Human steroid synthesis pathways are more complex than those of rodents, with several structural isomers (e.g., Aldo and E) sharing the same chemical formula (see Figure S1). Various chromatography techniques have 8921

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Figure 3. Changes in Aldo level induced by SDef in rat adrenal gland. Rats were fed with NS or SDef diet. Zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). (A) Optical image of hematoxylin and eosin (HE) staining and CYP11B2 immunoreactivity (brown) in adrenal tissue sections. (B) Distribution of GirT-Aldo (m/z 397.2) in rat adrenal gland as determined by MALDI-MSI. (C) Comparison of aldosterone intensity in ZG. (D) Comparison of aldosterone intensity in ZF and ZR. (**P < 0.01).

C ring [M − 18]+ (Figure 2C and D). On the basis of the structure of each steroid, we found that the specific fragmentation pattern depended on the hydroxyl group at positions 11 and 17. Steroids with a hydroxyl group at position 11 produced [M − 18]+ via dehydration between positions 11 and 12, whereas those with a hydroxyl group at position 17 produced [M − 30]+ and [M − 60]+ via cleavage around the carbonyl group in the side chain at position 17. We found that it is necessary to perform not only MS2 but also MS3 analysis to obtain specific signals for GirT-Aldo and GirT-F that were separate from the mass peaks of GirT-E and GirT-18-OHB, respectively. Visualization of Aldo Accumulation in Rat Adrenal Gland in an SDef Diet Model. We first used the MS3 methods established for the various steroids to detect Aldo in the adrenal gland of rats fed an SDef diet, which is known to activate the renin-angiotensin-Aldo system, resulting in the expansion of ZG layer cells and elevation of serum Aldo level.26 The ZG layer was expanded in the SDef as compared to the control group, as evidenced by the upregulation of CYP11B2 expression observed by IHC (Figure 3A).

derivatized site [M − 46]+ (loss of H2O and CO), respectively (Figure 1D). On the contrary, m/z 385 and 355 in GirTcortisone were presumed to correspond to the loss of CH2O [M − 30]+ and CHOCH3O [M − 60]+ obtained by cleavage around the carbonyl group on the side chain of the D ring (Figure 1E).22 On the basis of these findings, product ions of m/z 474 > 415 > 397 and 474 > 415 > 385 were determined as the specific ion transitions for detecting GirT-Aldo and GirTE, respectively. Using the same approach, we developed a method for discriminating between F and 18-OHB by MS3. Since GirT-F produced an ion distinct from 18-OHB at m/z 387 in the MS3 spectrum (Figure 2A, B), an ion transition at m/z 476 > 417 > 387 was used for subsequent GirT-F identification. We also developed MS3 methods for 18-OHF and 18-oxoF based on GirT-18-OHF and GirT-18-oxoF ion transitions at m/z 492 > 433 > 415 and 490 > 431 > 413, respectively. Further, in the other four steroids we examined, the cleavage around the carbonyl group in the D ring yielded GirT-F-specific peaks at [M − 30]+ (loss of CH2O) and [M − 60]+ (loss of CHOCH3O), similar to GirT-E. GirT-18-OHF and GirT-18oxoF, as well as GirT-Aldo, showed neutral loss of H2O in the 8922

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Figure 4. Distribution of GirT-steroids in the healthy human adrenal gland. MS3 imaging results in whole section (A, Case-1; B, Case-2) and CYP11B2 immunoreactivity and detection of GirT-F (m/z 387.2), GirT-E (m/z 385.2), GirT-Aldo (m/z 397.2), and GirT-18-OHF (m/z 413.2) by MALDI-MSI with enlarged view (C, Case-1; D, Case-2). Arrowheads represent APCC regions.

Figure 5. Steroidogenesis in the ZG and zona fasciculata (ZF) of the human adrenal cortex. Cholesterol, pregnenolone, and progesterone are synthesized in both cortical layers. Aldo is exclusively produced in the ZG; F synthesis is restricted to the ZF; and 18-OHF is produced in both the ZG and ZF from circulating F and F, respectively, diffusing across the ZG/ZF interface.

MS3 imaging confirmed that Aldo levels were increased mostly in the ZG and not in the middle layer of the adrenal gland (outside of the medulla, i.e., zona fasciculata (ZF) and zona reticularis (ZR)) (Figure 3B), demonstrating that CYP11B2-positive cells are responsible for Aldo production. The GirT-Aldo peak intensity in the ZG of SDef adrenal tissue

was approximately two times higher than those in the ZG of NS adrenal tissue (Figure 3C). On the contrary, there was no difference in ZF and ZR regions between SDef and NS tissues (Figure 3D). Our established methods were sufficiently specific to detect elevated Aldo production in the adrenal ZG layer of SDef diet-fed rats. Although increased production of Aldo in 8923

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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Analytical Chemistry the ZG has been examined based on serum Aldo concentration and by FT-ICR imaging,26,27 our imaging results demonstrate localized steroid synthesis in situ and reveal the steroidproducing cells. Aldo-Producing Cell Clusters (APCCs) in Human Adrenal Gland Accumulate Aldo and 18-OHF but not F. We next examined the localization of F, Aldo, and 18-OHF in human adrenal tissues with the MS3 method. The human steroid production system is complex, involving multiple metabolic pathways, cell types, and tissues. Thus, we expect our approach for analyzing steroid distribution to be useful for identifying abnormalities in human adrenal steroid metabolism due to disease. Two adrenal glands surgically dissected from kidney cancer patients were serially sectioned; some of the sections were used for MS3 (Figure 4A and 4B), and others for IHC analysis of CYP11B2 (Figure 4C and 4D). Small regions were CYP11B2 positive (Figure 4C and 4D), and likely corresponding to APCCs,21 which we previously reported enriched in Aldo.27 To date, we have attempted to visualize several steroid hormones in adrenal tissues of patients with primary aldosteronism, which is the most common cause of secondary hypertension due to excessive Aldo production as described above. In our previous study, we successfully visualized the coaccumulation of Aldo and 18-oxoF, a hybrid steroid17 present in APCCs and larger adenomas. However, the mechanism by which 18-oxoF accumulates in APCCs is unknown; the upregulation of CYP11B2 but not CYP11B1 in these cells suggests that cortisol is incorporated from the outside of this lesion and is rapidly metabolized into 18-OHF or 18-oxoF through robust local activities of CYP11B2.28 If this hypothesis is true, then APCCs should contain high levels of Aldo and hybrid steroids, but low levels of F. In the present study, MS3 imaging revealed that while F was widely distributed in the entire tissue, Aldo was concentrated in APCCs (Figure 4C and 4D). Since APCCs #1−4 were more substantial enlarged as compared with others by immunohistochemistry. From the images, we determined that Aldo levels were elevated in APCCs #1−4, and that Aldo colocalized with CYP11B2. On the contrary, F was absent in APCCs. 18-OHF was also detected from APCCs, albeit at a lower level than that in 3 and 4. Our data supported this hypothesis, given our observation of the abnormal movement of cortisol from typical CYP11B1-active adrenal regions into APCCs (Figure 5). Perspective: Catecholamine Distribution in Adrenal Gland. Pheochromocytoma is another adrenal tumor that forms in the adrenal medulla. This tumor produces and releases huge amount of circulating catecholamines, which primarily endanger patients for developing hypertension, followed by severe vascular diseases. There are endocranially two types of pheochromocytoma, adrenalin, and noradrenalin secreting types depending on the origin of cell type, which have or lack dopamine beta-hydroxylase. We used two specimens (Figure 6A), and finally demonstrated the usefulness of OTCD coupled with tandem MS method to discriminate those tumor species by visualizing adrenaline (Figure 6B) and noradrenaline levels (Figure 6C). Here, we utilized TAHS reagent for the OTCD29 and monitored product ions in MS/MS to visualize adrenaline and noradrenaline, respectively. In the healthy tissue, both catecholamines are produced in the adrenal medulla. On the contrary, the pheochromocytoma specimen shows a moderate level of adrenaline as well as a drastically elevated level of noradrena-

Figure 6. Application OTCD, coupled with MS2 to detect catecholamines using different pathological adrenal gland tissues. (A) HE staining results of Case-1 (normal tissue) and Case-3 (pheochromocytoma). (B) Distribution of TAHS-adrenaline. (C) Distribution of TAHS-noradrenaline. In the normal tissue, both catecholamines have been accumulated inside APCC (mainly ZG). On the contrary, pheochromocytoma tissue provides homogeneous catecholamines distribution, including ZG, ZF, and ZR.

line production, homogeneously from the whole tumor. Thus, this pheochromocytoma specimen was noradrenaline secreting type and compartmentation of the adrenal gland, that is, cortex and medulla were lost entirely.

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CONCLUSIONS In this study, we described an MS-based imaging method for discriminating between structural isomers of steroids using OTCD and MS3 product ions. Using this method, we distinguished Aldo and F from their respective isomers and visualized for the first time F distribution in the human adrenal gland. We determined that Aldo was present while F was absent in all APCCs, and 18-OHF was present at a higher level in some APCCs. We expect the MS3 method to provide critical information on the distribution of specific isomers of steroids and other hormones for future basic or clinical endocrine research. This is the first report of particular visualization of Aldo, F, and 18-OHF in the same human tissue by MS3 imaging. In addition, our approach can utilize other pathological cases and molecules, such as catecholamines.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b00619. Human adrenocortical steroidogenesis pathway denoting structural isomers and comparison of matrix application procedure and matrix selection to detect aldosterone using MS3 in mice adrenal glands (PDF) 8924

DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925

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AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +81-6-6879-7418. E-mail: [email protected]. ORCID

Sumio Ohtsuki: 0000-0003-4634-7133 Shuichi Shimma: 0000-0002-4699-6590 Author Contributions

All authors confirm that they have contributed to the intellectual content of this paper and have met the following four requirements: (a) significant contribution to study conception and design and data acquisition, analysis, and interpretation; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article, thus ensuring that questions related to the accuracy or integrity of any part of the report are appropriately investigated and resolved. Funding

This study was financially supported by a Grant-in-aid for Japan Society for the Promotion of Science Fellows (to E.T., no. 18J20250); a KAKENHI grant (Y.S., no. JP 16748651); Hidaka Research Project grant (to K.N., no. 28-D-13); Yamaguchi Endocrine Research Foundation research grant (to K.N.); and a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y.S., no. 26111006). M.S. was supported by JST ERATO, Suematsu Gas Biology until March 2015. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Ms. Noriko Akiyama for her management of clinical information. REFERENCES

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DOI: 10.1021/acs.analchem.9b00619 Anal. Chem. 2019, 91, 8918−8925