Mass spectrometry imaging for dissecting steroid intracrinology within target tissues

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.

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 haematoxylin and eosin (ZG = zona glomerulosa; ZFR = zona fasciculata reticularis). Heat map of GirT derivatives of: (c) corticosterone (GirT-CORT) at m/z 460.31698±0.025Da; and (d) 11-dehydrocorticosterone (GirT-11DHC) at m/z 458.30133±0.025Da, collated by MALDIFTICR-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. Mw = molecular weight (Da). cps = counts per second. Scale bar (2mm).

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 haematoxylin 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±0.025Da 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±0.025Da in wild type (c) and 11β-HSD1^−/− (e) mice. Signal intensity is depicted by color on the scale shown. Scale bar (2mm). cps = count per second. (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.

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 haematoxylin and eosin. (b-e) MSI heat map distribution of m/z 390.08377±0.025Da representing UE2316 in brain over a 6h 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 haematoxylin 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±0.005Da brain from mice receiving Vehicle (h) or UE2316 (1h post dose) (j). (i, k) GirT-11DHC at m/z 458.30133±0.025Da in brain from mice receiving Vehicle (i) or UE2316 (k). Signal intensity is depicted by color on the scale shown. Scale bar (2mm). cps = count per second. (h) 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. (i) 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.

Figure 4. MSI detection of 7-ketocholesterol, an alternative substrate for 11β-HSD1

A significant increase in Girard T 7-ketocholesterol (GirT-7KC) intensity was observed across the whole brain in mice after transgenic disruption of 11β-HSD1^−/− (KO) and after administration of UE2316 (1 h post-dose). (a) Histological image of coronal cryosection of murine brain stained with haematoxylin and eosin. MSI heat map of GirT-KC at m/z 514.43670±0.025Da in (b) wild type (WT), (c) 11β-HSD1^−/− and brain from mice receiving (e) Vehicle (V) or (f) UE2316. 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 (2mm). 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.