The ratio of ursodeoxycholyltaurine to 7‐oxolithocholyltaurine serves as a biomarker of decreased 11β‐hydroxysteroid dehydrogenase 1 activity in mouse

Background and Purpose 11β‐Hydroxysteroid dehydrogenase 1 (11β‐HSD1) regulates tissue‐specific glucocorticoid metabolism and its impaired expression and activity are associated with major diseases. Pharmacological inhibition of 11β‐HSD1 is considered a promising therapeutic strategy. This study investigated whether alternative 7‐oxo bile acid substrates of 11β‐HSD1 or the ratios to their 7‐hydroxy products can serve as biomarkers for decreased enzymatic activity. Experimental Approach Bile acid profiles were measured by ultra‐HPLC tandem‐MS in plasma and liver tissue samples of four different mouse models with decreased 11β‐HSD1 activity: global (11KO) and liver‐specific 11β‐HSD1 knockout mice (11LKO), mice lacking hexose‐6‐phosphate dehydrogenase (H6pdKO) that provides cofactor NADPH for 11β‐HSD1 and mice treated with the pharmacological inhibitor carbenoxolone. Additionally, 11β‐HSD1 expression and activity were assessed in H6pdKO‐ and carbenoxolone‐treated mice. Key Results The enzyme product to substrate ratios were more reliable markers of 11β‐HSD1 activity than absolute levels due to large inter‐individual variations in bile acid concentrations. The ratio of the 7β‐hydroxylated ursodeoxycholyltaurine (UDC‐Tau) to 7‐oxolithocholyltaurine (7oxoLC‐Tau) was diminished in plasma and liver tissue of all four mouse models and decreased in H6pdKO‐ and carbenoxolone‐treated mice with moderately reduced 11β‐HSD1 activity. The persistence of 11β‐HSD1 oxoreduction activity in the face of H6PD loss indicates the existence of an alternative NADPH source in the endoplasmic reticulum. Conclusions and Implications The plasma UDC‐Tau/7oxo‐LC‐Tau ratio detects decreased 11β‐HSD1 oxoreduction activity in different mouse models. This ratio may be a useful biomarker of decreased 11β‐HSD1 activity in pathophysiological situations or upon pharmacological inhibition. LINKED ARTICLES This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc

Besides cortisone and 11-dehydrocorticosterone, 11β-HSD1 can catalyse the carbonyl reduction of a broad range of substrates,
• Pharmacological inhibition of 11β-HSD1 is considered a promising strategy to treat glucocorticoid-dependent diseases.

What this study adds
• Ratio UDC-Tau/7oxoLC-Tau detects decreased 11β-HSD1 activity in genetically modified mouse models and upon pharmacological inhibition.
• These ratios are better markers of decreased 11β-HSD1 activity than concentrations of individual bile acids.

What is the clinical significance
• UDC-Tau/7oxoLC-Tau ratio provides a biomarker of the efficacy of pharmacological 11β-HSD1 inhibition in preclinical models.
Experiments using human liver microsomes and HEK-293 cells expressing human 11β-HSD1 and H6PD revealed that human 11β-HSD1 can convert the gut microbiota-derived 7-oxolithocholic acid (7oxoLCA) and its taurine-and glycine-conjugated forms to chenodeoxycholic acid (CDCA) and to a lesser extent to the 7β-stereoisomer ursodeoxycholic acid (UDCA) and their taurine-and glycine-conjugated forms (Odermatt et al., 2011). Unlike human 11β-HSD1, the mouse and rat enzymes are not stereo specific and were found to equally produce CDCA and UDCA (Arampatzis et al., 2005). A comparison of liver-specific 11β-HSD1 knockout (11LKO) and control (CTRL) mice showed completely abolished 7oxoLCA oxoreduction in liver microsomes from 11LKO, indicating that 11β-HSD1 is the major if not only enzyme catalysing this reaction in the liver. Plasma and intrahepatic levels of 7oxoLCA and its taurineconjugated form 7-oxolithocholyltaurine (7oxoLC-Tau) were found to be increased in 11LKO and in global 11β-HSD1 knockout (11KO) mice . Furthermore, 11KO mice exhibited increased plasma and intrahepatic levels of most bile acids, resembling a mild cholestasis phenotype.
F I G U R E 1 Schematic overview of bile acid homeostasis and a role for 11β-HSD1. 11β-HSD1 catalyses the carbonyl reduction of the substrates cortisone and 7oxoLCA to the corresponding products cortisol, and UDCA and CDCA, respectively. 11β-HSD1 activity requires regeneration of cofactor NADPH from NADP + by H6PD-dependent conversion of glucose-6-phosphate (G6P) to 6phosphogluconate (6PG). The formation of muricholic acid metabolites by murine Cyp2c70 is indicated Because we previously observed marked inter-animal variation in circulating bile acid levels, we hypothesized that the ratios of 7βhydroxy-to 7-oxo-bile acids might serve as biomarkers for decreased 11β-HSD1 activity and that such ratios may be superior markers than individual metabolite levels. We analysed plasma and liver tissue bile acids in 11KO and 11LKO mice in order to calculate the ratios of UDCA/7oxoLCA, CDCA/7oxoLCA, ursodeoxycholyltaurine (UDC-Tau)/7oxoLC-Tau and chenodeoxycholyltaurine (CDC-Tau)/7oxoLC-Tau (Penno et al., 2014;Penno, Morgan, et al., 2013). Furthermore, we analysed bile acid composition in plasma and liver tissue samples from global H6pd knockout (H6pdKO) mice as a model of decreased
The injection volume for bile acid detection was 2 μl (plasma and cell culture supernatant) or 3 μl (liver) and for plasma steroids 5 μl. Samples were stored at −20 C until analysis by UHPLC-MS/ MS as described earlier (Penno, Arsenijevic, et al., 2013)

| In vivo 11β-HSD1 activity assessment
Mice were injected i.p. with 5 mgÁkg −1 of cortisone (in DMSO). After 10 min, mice were killed by CO 2 asphyxiation and cardiac puncture was performed immediately to collect blood. Plasma was prepared and stored as described above. Plasma was extracted, and cortisone and cortisol levels were measured by UHPLC-MS/MS as described above.

| Ex vivo activity assay
Freshly isolated liver tissue samples (50-100 mg) were placed in tubes, followed by injection of radiolabelled substrate mixture (10 μl   Absorbance of total reduced pyridine nucleotides was measured against buffer in the absence of microsomes. To determine NADPH content, the absorbance of buffer containing 1.4 IU and 0.75 mmolÁL −1 GSSG was subtracted from the absorbance obtained from the microsomal preparation. All samples were tested at least in duplicate. 3.2 | The UDC-Tau/7oxoLC-Tau ratio in plasma and liver tissue detects the lack of 11β-HSD1 activity in 11KO mice

| Statistical analysis
The concentrations of the 11β-HSD1 substrates 7oxoLCA and 7oxoLC-Tau increased about 20-fold and 40-fold, respectively, in plasma of 11KO compared to CTRL (Table 1, Figure S1), with large inter-individual variations, as reported earlier (Penno, Morgan, et al., 2013). It needs to be noted that no outliers were excluded from the analysis. The respective products of 11β-HSD1, that is, CDCA, UDCA and their taurine-conjugated forms, also were higher in 11KO plasma compared to CTRL, although clearly less pronounced than the 7-oxo metabolites. In liver tissue, 7oxoLCA was 3.7-fold and 7oxoLC-Tau 15-fold higher in 11KO compared to CTRL (Table 1, Figure S2).
The respective products CDCA, UDCA and CDC-Tau tended to increase, whereas UDC-Tau decreased 30-fold. Importantly, the 11β-HSD1 product to substrate ratios (the ratio of CDCA and UDCA and their taurine-conjugated forms to the respective 7oxo metabolites) showed less variation than the individual metabolite concentrations ( Figures S1 and S2). UDC-Tau/7oxoLC-Tau was the most distinguishing marker for the lack of 11β-HSD1 activity when considering both plasma and liver tissue samples (Table 1, Figure 2).
Interestingly, in plasma and liver tissue of 11KO mice, the levels of the 7α-hydroxylated bile acid αMCA were 45-fold and 12-fold Note: The results represent mean ± SEM (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively). Analyte concentrations with a S/N ≤ 3 represent the LLOD of the UHPLC-MS/MS method. Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 , respectively) in the calculations of a specific analyte. Blue-and yellow-coloured boxes indicate statistically significant increases and decreases, respectively. Unequal group sizes reflect exclusion of one plasma sample due to insufficient collection of blood sample volume and the availability of only nine livers due to the use of nine randomly assigned livers for gene expression analyses in a previous study. Abbreviations: 11KO, global Hsd11b1 knockout; CTRL, control littermates. *P < 0.05 significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed).
higher than in CTRL, whereas its 7β-hydroxylated form βMCA was not different in CTRL plasma but 3.8-fold lower in liver tissue (Table 1).
The respective αMCA/βMCA ratios were 40-fold and 30-fold higher in 11KO compared to CTRL, suggesting a possible effect of 11β-HSD1 on isomerization. Due to a limitation of the applied analytical method, αMC-Tau and βMC-Tau could not be separated and therefore, the corresponding ratio not determined.

| 11LKO mice exhibit decreased plasma and liver tissue UDC-Tau/7oxoLC-Tau ratios
The liver shows the highest 11β-HSD1 expression; nevertheless, an earlier study reported 25-30% residual in vivo (whole body) 11β-HSD1 oxoreduction activity in 11LKO mice lacking 11β-HSD1 specifically in hepatocytes (Lavery et al., 2006). Thus, 11LKO mice represent a model of reduced 11β-HSD1 activity but with complete loss of activity in hepatocytes.
The free bile acids in plasma and liver tissue of 11LKO tended to be higher compared to CTRL (Table 2), an effect considerably more pronounced in 11KO (Table 1). 7oxoLCA was 11-fold higher in plasma and twofold in liver tissue ( Table 2). The plasma UDCA/7oxoLCA and CDCA/7oxoLCA ratios were sevenfold and twofold lower in 11LKO compared to CTRL, whilst remaining unchanged in liver tissue. Plasma 7oxoLC-Tau was slightly more abundant than its free form and it was 20-fold higher in 11LKO than in CTRL, whilst CDC-Tau was not different, and UDC-Tau was sixfold lower in 11LKO, resulting in significantly decreased product to substrate ratios (Table 2, Figure 3; see also Figure S3 for individual data points). In liver tissue, 7oxoLC-Tau was 5.5-fold increased, CDC-Tau not different and UDC-Tau threefold lower in 11LKO compared to CTRL (Table 2; see also Figure S4).
In agreement with 11KO, the CDC-Tau/7oxoLC-Tau and UDC-Tau/7oxoLC-Tau ratios were lower in 11LKO liver tissue compared to CTRL (fivefold and 16-fold, respectively). The αMCA/βMCA ratio was 3.3-fold higher in plasma and 7.3-fold in liver tissue of 11LKO (Table 2).

| H6pdKO mice exhibit moderately decreased 11β-HSD1 oxoreduction activity
Previous characterization of H6pdKO mice suggested, based on experiments using microsomal preparations, a complete loss of 11β-HSD1 oxoreduction activity (Lavery et al., 2006). To assess the conversion of 11-oxo-to 11β-hydroxyglucocorticoids in vivo, H6pdKO mice and control littermates received cortisone i.p. and were killed 10 min later, followed by measuring formed cortisol. 11β-HSD1 oxoreduction activity was reduced to approximately 60% of the level in control mice The ratio of oxoreduction to dehydrogenase activity was estimated to be about five in CTRL and 0.5 in H6pdKO liver tissue. Similar experiments in white adipose tissue showed approximately 40% residual oxoreduction activity in H6pdKO compared to CTRL, whilst dehydrogenase activity increased 30-fold to 40-fold ( Figure S5). Thus, in contrast to the expectation of a complete loss of 11β-HSD1 oxoreduction activity, these results revealed a moderately decreased oxoreduction in H6pdKO mice despite an increase in dehydrogenase activity.
An estimation of the impact of the lack of H6PD on NADPH levels in the ER using liver microsomes indicated that the content of total reduced pyridine nucleotides (NADPH + NADH) did not differ between control and H6pdKO (Figure 4f), but NADPH content of H6pdKO mouse liver microsomes was moderately lower by approximately 30% compared to control (Figure 4g). Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively) in the calculations of a specific analyte. The results represent mean ± SEM. *P < 0.05 significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed). Unequal group sizes reflect exclusion of one plasma sample due to insufficient collection of blood sample volume and the availability of only nine livers due to the use of nine randomly assigned livers for gene expression analysis in a previous study 3.5 | The UDC-Tau/7oxoLC-Tau ratio detects decreased 11β-HSD1 oxoreductase activity in H6pdKO mice Next, plasma and liver tissue bile acid profiles between H6pdKO and control mice were compared. Unlike in 11KO, primary and taurineconjugated bile acids were not generally elevated in plasma of H6pdKO mice (Table 3) Figure S7). The CDCA/7oxoLCA and UDCA/7oxoLCA ratios were twofold and fivefold lower, respectively, in H6pdKO liver tissue (Table 3, Figure 4i). The levels of the taurine-conjugated bile acids were about an order of magnitude higher than those of their free forms. 7oxoLC-Tau was fourfold higher in H6pdKO compared to control, but due to inter-individual variation, the value did not reach significance. CDC-Tau was not different between the two T A B L E 2 Bile acid profiles in plasma and liver of 11LKO mice Note: The results represent mean ± SEM (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively). Analyte concentrations defined by a S/N ≤ 3 represent the LLOD of the UHPLC-MS/MS method. Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 , respectively) in the calculations of a specific analyte. Blue-and yellow-coloured boxes indicate statistically significant increases and decreases, respectively. Unequal group sizes reflect exclusion of one 11LKO animal due to unexpected health issues prior to reaching the age for the experiment. Abbreviations: 11LKO, liver-specific Hsd11b1 knockout; CTRL, control littermates. *P < 0.05, significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed).
As seen in 11KO and 11LKO, the ratio of αMCA/βMCA was significantly higher in H6pdKO plasma (fourfold) and liver tissue (fivefold). In H6pdKO mice, this was due to significantly lower βMCA.
αMCA was not different between the genotypes (Table 3).
Analysis of bile acid profiles revealed that the sum of free primary bile acids tended to be lower (2.5-fold) in plasma upon carbenoxolone treatment, whereas taurine-conjugated primary bile acids seemed to be not affected (Table 4). 7oxoLCA concentrations were below the lower limit of detection (LLOD) in plasma and liver tissue samples in this mouse cohort, so the respective product/substrate ratios with UDCA and CDCA could not be calculated. In plasma of carbenoxolone-treated mice, 7oxoLC-Tau tended to increase, whilst UDC-Tau and CDC-Tau were not affected by carbenoxolone ( Figure S9). Nevertheless, CDC-Tau/7oxoLC-Tau and UDC-Tau/7oxoLC-Tau (Figure 5d; see also Figure S9) were 1.6-fold and twofold lower in plasma of carbenoxolone-treated mice. In liver tissue, only UDC-Tau/7oxoLC-Tau was predictive for decreased 11β-HSD1 activity (2.6-fold decreased; Table 4, Figures 5e and S10).
In contrast to the other three mouse models, the αMCA/βMCA ratio was unchanged in plasma and even threefold lower in liver tissue; thus, this ratio is not indicative of altered 11β-HSD1 activity.

| DISCUSSION
This proof-of-concept study proposes that the UDC-Tau/7oxoLC-Tau ratio can serve as a biomarker for decreased 11β-HSD1 activity in vivo. The UDC-Tau/7oxoLC-Tau ratio in plasma and liver tissue successfully detected complete loss of 11β-HSD1 activity in 11KO mice, loss of hepatic 11β-HSD1 activity in 11LKO mice and moderately decreased oxoreduction activity in H6pdKO-and carbenoxolonetreated mice. Of note, the four models differed with respect to their genetic background (11KO, 11LKO and H6pdKO on a mixed C57BL/6J/129SvJ background, carbenoxolone group were C57BL/6JRj) that can affect lipid and bile acid homeostasis (Jolley et al., 1999), feeding regimen (11KO and 11LKO fasted overnight, the other two models ad libitum feeding; some differences in the composition of the chow) and environment (different animal facilities) that can Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively) in the calculations of a specific analyte. Results represent mean ± SEM. *P < 0.05 significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed). Unequal group sizes reflect exclusion of two H6pdKO animals from further analysis due to the occurrence of liver cysts impact the microbiome and thereby influence bile acid homeostasis (Rausch et al., 2016). A biomarker reporting decreased 11β-HSD1 oxoreduction activity in plasma opens the possibility for non-invasive applications in preclinical studies of pharmacological inhibitors for potential therapeutic applications; whether it can also be used to explore the pathophysiological role of 11β-HSD1 in situations of elevated activity remains to be investigated (Gathercole et al., 2013).
Determination of this ratio in liver tissue at the end of the study can provide additional information.
The plasma UDCA/7oxoLCA ratio was also a marker for decreased 11β-HSD1 activity. However, because the levels of free bile acids are lower than those of their taurine-conjugated forms and were below the LLOD in some mice, this is likely to be less useful practically than the ratio of the taurine-conjugated metabolites.
Whilst in mice and rats taurine-conjugated bile acids are predominant and the UDC-Tau/7oxoLC-Tau ratio is easier to assess, the ratio of the free UDCA/7oxoLCA has the advantage to be species independent, as glycine-conjugated bile acids are predominant in human and other higher mammals (Alnouti et al., 2008;Garcia-Canaveras et al., 2012;Penno et al., 2014). Improvements of the analytical sensitivity may be achieved by measuring just UDCA and 7oxoLCA, using larger sample volumes, and optimizing extraction specifically for these two bile acids, which should permit this ratio to be a good and reliable species-independent marker.
Although murine 11β-HSD1 converts 7oxoLC-Tau to UDC-Tau and CDC-Tau (Odermatt et al., 2011), the UDC-Tau/7oxoLC-Tau ratio was superior to the CDC-Tau/7oxoLC-Tau for detecting decreased 11β-HSD1 activity. A possible explanation may be the significant Note: The results represent mean ± SEM (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively). Analyte concentrations defined by a S/N ≤ 3 represent the LLOD of the UHPLC-MS/MS method. Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 , respectively) in the calculations of a specific analyte. Blue-and yellow-coloured boxes indicate statistically significant increases and decreases, respectively. Unequal group sizes reflect exclusion of two H6pdKO animals from further analysis due to the occurrence of liver cysts. Abbreviations: CTRL, control littermates; H6pdKO, global H6pd knockout. *P < 0.05, significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed).
contribution of de novo CDCA synthesis to the circulating and liver tissue levels of CDC-Tau, whereas UDC-Tau and 7oxoLC-Tau are primarily formed from gut microbiota-derived UDCA and 7oxoLCA.
Interestingly, an increase in the ratio of αMCA/βMCA, formed by cytochrome P450 2C70 from CDCA and UDCA, respectively (Takahashi et al., 2016), nicely detected the decreased 11β-HSD1 activity in the three genetically modified mouse models. However, in carbenoxolone-treated mice, this ratio was not changed in plasma and showed an opposite change in liver. Plausibly, carbenoxolone inhibits cytochrome P450 2C70 or decreases its expression. Carbenoxolone might also affect gut microbiota as it was earlier shown to alter colonic mucus (Finnie et al., 1996). This merits future investigation.
Pharmacological treatment using carbenoxolone led to approximately 30% decreased 11β-HSD1 activity. It needs to be noted that the level of 11β-HSD1 inhibition is an estimation and it was determined at one given time point (i.e., at about 8 am) and the formation of cortisol upon injection of cortisone was determined after 10 min.
Nevertheless, inhibition of 11β-HSD1 could be demonstrated and the bile acid biomarker detected the decreased activity. The results suggested that besides direct inhibition, a reduced enzyme expression contributed to the decreased activity. carbenoxolone also inhibits 11β-HSD2 (Stewart et al., 1990). However, our preliminary observations suggest that this enzyme does not accept CDCA and UDCA as substrates, therefore unlikely affecting the bile acid ratios of interest.
(a) Conversion of cortisone to cortisol in vivo, measured after i.p. administration of 5 mgÁkg −1 of cortisone (in DMSO) (CTRL n = 7; CBX n = 5). (b) mRNA expression of H6pd and Hsd11b1 in CTRL-and CBX-treated animals (CTRL n = 8; CBX n = 9). (c) Western blot and semi-quantitative analysis of protein levels of H6PD and 11β-HSD1 in CTRL-and CBX-treated mice (CTRL n = 8; CBX n = 8). (d) UDC-Tau/7oxoLC-Tau ratios in CTRL mice treated with PBS (CTRL) or the pharmacologic 11β-HSD1 inhibitor carbenoxolone (CBX 100 mgÁkg −1 Áday −1 , i.p.) (CTRL n = 8; CBX n = 9) in plasma and (e) in liver tissue. Analyte concentrations defined by a S/N ≤ 3 represent the LLOD of the UHPLC-MS/MS method. Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively) in the calculations of a specific analyte. Results represent mean ± SEM. No outliers were excluded. *P < 0.05 significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed). Unequal group sizes reflect exclusion of one animal of the CTRL group due to unexpected health issues prior to the experiment and exclusion of two plasma samples of the CBX group due to insufficient collection of blood sample volume H6pdKO mice retained approximately 50% of 11β-HSD1 oxoreduction activity measured in control animals. This is consistent with observations made in isolated macrophage from H6pdKO mice, which also retained about 50-60% 11β-HSD1 oxoreduction activity (Marbet et al., 2018). Based on earlier experiments using liver microsomes (Lavery et al., 2006), it was anticipated that in the absence of H6PD, 11β-HSD1 would function exclusively as dehydrogenase and the effect on the respective bile acid ratios would be comparable with that in 11KO mice, yet the accumulation of 7oxo metabolites and the ratios derived from them clearly were less pronounced. These findings indicate the existence of a yet unknown mechanism generating NADPH in the ER capable of driving 11β-HSD1 reaction direction towards oxoreduction activity. This is supported by the continued presence of NADPH in the H6pdKO liver, albeit at reduced levels, seen previously (Rogoff et al., 2010) and also found here. A possible candidate for generating NADPH within the ER includes luminal 6-phosphogluconate dehydrogenase (Bublitz et al., 1987). However, the gene encoding this enzyme still remains to be identified.
The mild cholestasis phenotype of 11KO mice with 10-fold to 20-fold increased plasma bile acids ( Note: The results represent mean ± SEM (nmolÁL −1 and fmolÁmg −1 for plasma and liver, respectively). Analyte concentrations defined by a S/N ≤ 3 represent the LLOD of the UHPLC-MS/MS method. Samples yielding a concentration below LLOD were included as LLOD/2 (nmolÁL −1 and fmolÁmg −1 , respectively) in the calculations of a specific analyte. Yellow-coloured boxes indicate statistically significant decreases. Unequal group sizes reflect exclusion of one animal of the CTRL group due to unexpected health issues prior to the experiment and exclusion of two plasma samples of the carbenoxolone (CBX group due to insufficient collection of blood sample volume. Abbreviations: CBX, mice treated with the pharmacologic 11β-HSD1 inhibitor carbenoxolone (100 mgÁkg −1 Áday −1 , i.p.); CTRL, control mice treated with PBS; NA, not analysed, if most values were below LLOD. *P < 0.05, significantly different as indicated; non-parametric, Mann-Whitney U test (two-tailed). and cholestasis, glucocorticoid treatment reversed the hepatic phenotype (Al-Hussaini et al., 2012;Cheung et al., 2003), indicating a direct role of glucocorticoids in maintaining bile acid homeostasis. However, no evidence for cholestasis was seen in the present study when 11β-HSD1 was inhibited by carbenoxolone and there was only a trend increase of total free but not conjugated bile acids in 11LKO mice and no change of total bile acids in H6pdKO mice. These observations do not support concerns of a general risk of cholestasis upon inhibition of 11β-HSD1. The more pronounced effect on plasma and liver tissue bile acid profiles in 11KO mice may be explained by the fact that they lack 11β-HSD1 during all stages of life and throughout the enterohepatic circuit and also by altered hypothalamus-pituitary-adrenal axis, whereas 11LKO only lack hepatic 11β-HSD1, and H6pdKO-and carbenoxolone-treated mice retain partial 11β-HSD1 activity.
A suitable biomarker reporting the in vivo 11β-HSD1 activity in health and disease situations or upon pharmacological interventions could greatly facilitate such studies. The currently used urinary (tetrahydrocorticosterone + allo-tetrahydrocorticosterone)/tetrahydro-11-dehydrocorticosterone ratio has limited value as it can lead to erroneous conclusions because of interference through altered 11β-HSD2 and 5α-reductase activities and feedback modulation. Furthermore, it needs 24-h urine sampling that due to small collection volume and contamination by food and faeces and the stress of metabolic cage housing may lead to erroneous results.
Although our data support the UDC-Tau/7oxoLC-Tau ratio as a useful in vivo marker of 11β-HSD1 activity, our study has several limitations:-(1) the present study included only male mice at 10-15 weeks of age and it will be important to study also mice at both young and very old age that may exhibit metabolic differences as well as female mice, being mindful of the effect of the oestrous cycle on bile acid homeostasis (Papacleovoulou et al., 2011); (2) the impact of feeding and diet should be studied; (3) samples were taken in the morning between 7 and 10 am and the influence of circadian rhythm and/or stress should be assessed; (4) in case of pharmacological inhibition, a possible interference of the compound with hepatic enzymes and transporters that also are involved in bile acid homeostasis needs to be kept in mind; (5) the impact of the microbiome on the production of UDCA and 7oxoLCA needs to be investigated; it has been shown that 11β-HSD1 deficiency alters the microbiome in a diet-specific manner (Johnson et al., 2017); (6) disease models with altered 11β-HSD1 activity should be studied and (7) the sensitivity of the LC-MS/MS-based quantification method can be increased by measuring specifically the bile acid metabolites needed for the ratio and by increasing sample volume and optimizing extraction. In follow-on research, the predictivity of the UDC-Tau/7oxoLC-Tau ratio for detecting altered 11β-HSD1 activity should be investigated in mouse models addressing the abovementioned factors. Finally, experiments in humans are required to establish whether such a bile acid ratio is a useful biomarker to detect altered 11β-HSD1 activity upon pharmacological treatment or in disease situations.