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A fast UPLC–HILIC method for an accurate quantification of dendrogenin A in human tissues
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
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A fast UPLC–HILIC method for an accurate quantification of dendrogenin A in human tissues
T
Régis Soulèsa,b, Fabien Audouard-Combec, Emilie Huc-Claustrea,b, Philippe de Medinaa,b, Arnaud Rivesd,e, Etienne Chatelutf,g, Florence Dalenca,b,g, Camille Francheth, ⁎ Sandrine Silvente-Poirota,b, Marc Poirota,b, Ben Allalf,g, a
Team « Cholesterol metabolism and therapeutic innovations », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France Equipe labellisée par la Ligue Nationale Contre le Cancer, France c Waters S.A.S., Saint-Quentin En Yvelines, France d Affichem, Toulouse, France e Dendrogenix, Liège, Belgium f Team “Dose individualization of anticancer drugs », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France g Institut Claudius Regaud, Institut Universitaire du Cancer-Oncopole, Toulouse, France h Service d'Anatomo-Pathologie, Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Toulouse, France b
A R T I C LE I N FO
A B S T R A C T
Keywords: Steroidal alkaloid Dendrogenin A Dendrogenin B Cholesterol Oxysterol Metabolite Method development Quantification UPLC-MS
Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.
1. Introduction
sterol that was shown to activate at low doses differentiation programs in cancer cells of different tissue origins [1–10], as well as normal neuronal progenitor cells [7,9,10]. In addition, this new re-differentiation agent was shown to activate cancer cell death through an original molecular and cellular mechanism. DDA kills cancer cells when used at μM concentrations through the induction of an autophagic cell death via the activation of the oxysterol receptor LXRβ [3,4,6,11]. In
Dendrogenin A (DDA) is a cholesterol conjugate that was recently discovered in human tissues [1]. DDA is the first mammalian steroidal alkaloid discovered to date and it results from the conjugation of the primary amine of histamine on carbon 6 of 5,6α-epoxycholesterol (5,6α-EC) by a yet unidentified enzyme (Fig. 1). DDA is an interesting
Abbreviations: Dendrogenin A (DDA), 5α-hydroxy-6β[2-(1H-imidazol-4-yl) ethylamino] cholestan-3β-ol; Hexadeuterated dendrogenin A ([2H6]-DDA), (26,26,26,27,27,27-[2H6])-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3β-ol; Dendrogenin B (DDB), 5α-hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholestan-3β-ol; 5, 6α-EC,5α,6α-epoxy-cholestan-3β-ol; OS, oxysterol; BC, breast cancer; B&D, Bligh and Dyer; QC, quality control; H, high; M, medium; L, low ⁎ Corresponding author at: Team « Cholesterol metabolism and therapeutic innovations », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France E-mail addresses: [email protected] (M. Poirot), [email protected] (B. Allal). https://doi.org/10.1016/j.jsbmb.2019.105447 Received 18 April 2019; Received in revised form 5 August 2019; Accepted 11 August 2019 Available online 12 August 2019 0960-0760/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
Fig. 1. A) Biochemical synthesis of dendrogenin A (DDA). DDA is the produce of the enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine by the DDA synthase (DDAS). B) DDA is steroidal alkaloid that contains 3 nitrogen atoms. DDA can exist through different protonated states according to the pH.
obtained with DDB suggesting that this method was not appropriated for DDB dosage if characterized as an endogenous metabolite of for pharmacokinetic purpose in case of clinical application with this compound. Recently, the hydrophilic interaction liquid chromatography (HILIC) was shown to provide an alternative approach to RP chromatography to separate small polar compounds on polar stationary phases [18]. This method was used successfully to analyze conjugated steroids [19], lipids [20] and steroidal alkaloids [21]. Because DDA and parent compounds such as DDB, C26 and C17 are steroidal alkaloids we thought that the development of a HILIC method would be interesting. The primary objective of the present study was to develop a new, fast and sensitive method of quantification of DDA in liquid and solid tissues and eventually to detect DDA analogues in human samples.
addition, it was shown that DDA inhibits breast cancer cell proliferation through the inhibition of the production of the tumor promoter 6-oxocholestan-3β,5α-diol by cancer cells at the cholesterol-5,6-epoxide hydrolase step [3,5,12]. DDA demonstrates antitumor potencies on various pre-clinical models of cancers [3–611,12], as well as neuro-regenerating potencies on a preclinical model of deafness [7,13]. DDA was detected in several mammalian organs and tissues while it was not detected in melanoma and breast cancer cell lines [1,14,15]. Preliminary studies done in patients showed that DDA levels were found strongly decreased in breast tumors from 10 patients compared to “normal “matched tissue [1], highlighting that a deregulation in DDA metabolism occurred during oncogenesis. It is thus important to develop an accurate and sensitive analytical quantification method for DDA adapted to liquid (serum and plasma) as well as solid tissues from large cohorts of patients. In our preliminary studies, the quantification of DDA was achieved after an organic solvent extraction procedure (Bligh and Dyer: B&D) [16], followed by a solid phase extraction procedure of samples on a C18 Sep-Pack cartridge and then a purification of samples by reverse phase HPLC [1]. The quantification of DDA was made by mass spectrometry and a MS/MS fragmentation of the parent peak present in the eluted fractions at the retention time that corresponding to the authentic DDA standard, and using deuterated DDA as an internal standard. The MS/MS fragmentation was used to discriminate DDA from its C17 geometric isomer that co-eluted because of their different fragmentation fingerprints. This method required almost 24 h per sample, and was not convenient for the analysis of large series of samples. We recently reported the development of a new liquid chromatography tandem mass spectrometry (LC/MS) method for the dosage of DDA and analogues based on reverse phase (RP) liquid chromatography that gave satisfactory results in terms of resolution, sensitivity and duration of the whole process. We solved an important problem of carryover using an acidic elution buffer (pH < 2), with heptafluorobutyric acid in the mobile phase that made ion pairing with cationic solutes on RP. These modifications seriously improved the resolution in our separation assay [17]. In addition, we were able to separate DDA from its regio-isomer C17, dendrogenin B (C26, DDB) and the sitosterol analog of DDA: C16 in the following order : C26 > C17 > DDA > C16 from the more hydrophilic to the more hydrophobic (Fig. 2 D, B, A, C respectively). A weak resolution was however
2. Materials and Methods 2.1. Chemicals, reagents Formic acid, chloroform, ammonium formate, (26,26,26,27,27,27[2H6]) cholesterol (98% 2H), histamine and fetal bovine serum (FBS) were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ammonia was purchased from VWR Chemicals (VWR International S.A.S. Le Périgares - Fontenay-sous-Bois cedex). 5α-hydroxy-6β-[2-(1H-imidazol4-yl)ethylamino]cholestan-3β-ol (dendrogenin A), 5α-hydroxy-6β-[2(1H-imidazol-4-yl)ethylamino]sitostan-3β-ol (C16), 5α-hydroxy-6β-[4(2-aminoethyl)imidazol-1-yl] cholestan-3β-ol (C17) and 5α-hydroxy6β-[3-(4-aminobutylamino)propylamino]cholestan-3β-ol (C26) were synthesized in our laboratory according to a previously published procedure [10] and was 99% pure. [2H6]-DDA synthesis and characterization is reported on the supplementary material section. Methanol, acetonitrile (HPLC Grade) were from Scharlau (Barcelona, Spain). Human plasma were provided by the «Etablissement Français du Sang» (EFS, Toulouse, France). A milli-Q Gradient A10 Millipore® System was used to prepare Milli-Q water. Acrodisc Nylon minispike syringe filters (13 mm, 0.2 μm) were used for sample filtration and polypropylene (12 × 32 mm Screw Neck) Vial, with cap and preslit PTFE/Silicone septa were purchased from Waters (Saint-Quentin en Yvelines, France). 2
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
Fig. 2. Separation of DDA and analogues by UPLC-MS on a HILIC column. A representative chromatogram is shown on A) for DDA, on B) for C17, on C) for C16 and on D) for C26.
Gradient elutions were performed with an Acquity UPLC H-class system (Waters, St Quentin en Yvelines, France). The analytical column was an Acquity UPLC BEH HILIC column, 2.1 mm × 50 mm, 1.7 μm (Waters, USA). The injector and the column temperature were set at 10 °C and 25 °C respectively. The mobile phase A was 200 mM ammonium formate and 1% ammoniac (v/v) pH 9.2 and the mobile phase B was acetonitrile. The gradient elution started with 100% B for 0.25 min, decreased to 50% in 2.25 min, and stay at 50% during 1.5 min and return to 100% at a flow rate of 0.7 ml/min.
homogenizer system (Bertin technologies, Montigny-le-Bretonneux, France): briefly, frozen tissue (−80 °C) were disposed in cold tubes containing metal beads and lysis buffer (50 mM Tris−HCl, 150 mM KCl pH 7.4, 0.5% butylated hydroxytoluene (5 mg.ml−1)) 5 vol per g of tissue at 4 °C. The suspension was agitated 90 s at 10,000 r.p.m. and then submitted to two cycles of freeze/thaw (in liquid nitrogen (−196 °C) and 37 °C Block Heaters). Samples were then centrifuged (10 min at 1500 g, 4 °C), the supernatant was collected in new tubes and the protein concentration was measured by Bradford method [22] for sample normalization at 10 mg protein/ml. Sample were stored at -80°c until organic solvent extraction was performed.
2.3. Mass spectrometry
2.5. Standard solution for calibration
Compounds were detected on a Waters Xevo TQS-micro mass spectrometer equipped with an electrospray ionization ion source (ESI). Data were acquired in a multiple-reaction monitoring (MRM) mode using MassLynx software. MS collision parameters are summarized in the supplementary Table 1. Capillary and cone voltages were respectively of 3 kV and variable, source and desolvation gas were set respectively at 200 °C and 550 L/Hr. The cone gas flow was set at 30 L/h.
DDA and its [2H6]-DDA analogue were dissolved in methanol at 1 mg/ml and stored at −20 °C until used. Working solutions for calibration curves were prepared extemporaneously. 150 μl standards solutions were spiked into 600 μl of ethanol or biological matrices to obtain a concentration range from 0.01 to 100 pg/μl.
2.2. Liquid chromatography
2.6. Carry-over tests
2.4. Sample preparation
Carry-over was assessed following injection of a blank (plasma sample) immediately after three repeats of the Upper Limit Of Quantification (ULOQ). The peak area response was checked.
Clinical samples were collected from patients enrolled in the “Breasterol” trial (NCT02863900, Institut Claudius Regaud, Toulouse, France) approved by the Ethics committee of Midi-Pyrénées Region (Comité de protection des personnes Sud-Ouest et Outre-Mer III). 200 mg of human tissues were obtained by surgical resection and stored at −80 °C until used (All patients gave written informed consent). −80 °C stored tissues were homogenized at 4 °C using a Precellys 24
2.7. Retention time stability Retention time stability was tested by repeatedly injecting a mixture of four molecules (DDA, C16, C17, C26) one hundred times. The retention time was measured for each compound, and the results are expressed as a percentage of variation.
Table 1 Chromatographic parameters. tR: retention time; k: retention factor; wh: peak width at half height; N: number of plates; As5%: peak asymmetry measured at 5% peak height; α: separation factor; Rs: resolution. compound
tR
k
wh
N
As10%
α
Rs
DDA C16 C17 C26
1.86 1.86 2.10 3.16
1.91 1.91 2.28 3.93
3.0 3.2 3.5 3.1
7666 6738 7180 20723
1.0 1.2 1.3 1.0
1.05 1.11 1.19 2.06
0.68 1.33 2.29 14.76
2.8. Sample tissue and plasma extraction Two methods of sample preparation were used. The first one was the Bligh and Dyer liquid/liquid (B&D) extraction method [16] that was initially used for DDA analysis in different tissues [1]. 250 μl of samples were mixed to 500 μl chloroform and 250 μl methanol containing the internal standard [2H6]-DDA at 10 pg/μl. The suspensions were vortexed 30 s, followed by a 5 min stop and then vortexed for 30 s a second 3
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
Fig. 3. Schematic of the two different work-flows that were used for DDA analysis.
time. 400 μl of the organic layer were dried under a nitrogen flux at 37 °C. Samples were resuspended in 100 μl methanol, filtered on acrodisc Nylon minispike syringe filters into new vials for UPLC-MS analyses. In the second method, we used a methanol-protein precipitation procedure which was used cationic amphihilic drugs [23]. One volume of plasma (250 μl of standard, CQ, patient/analyte) was mixed to 5 volumes of methanol (1250 μl) spiked with the internal standard ([2H6]-DDA) at 10 pg/μl (final concentration). The suspensions were vortexed 2 x 1 min and were centrifuged 10 min at 1500 g (4 °C). 1 ml of the supernatant was collected and evaporated under a nitrogen flux at 37 °C. Dried samples were dissolved in 166 μl methanol and filtered on acrodisc Nylon minispike syringe filters into new vials for UPLC-MS analyses.
with their topological surface area (r2 = 0.9773, p < 0.05) their charge at pH 8 (r2 = 0.95, p < 0.05), their isoelectric point (r2 = 0.9908, p < 0.005) and their solubility at pH 8 (r2 = 0.9715, p < 0.05). Retention time for compounds were negatively correlated with their hydrophobicity taking into account their ionization state at pH 8 (Supplementary Table 1 and Fig. 1). No correlations were found with other parameters such as the molecular mass, the polarizability, the partition coefficient (LogP), the van der Waals volume or the molecular refractivity. 3.2. Calibration curves 3.2.1. Sample injection without matrix The method was linear over a range 0.02–100 pg/μl for DDA (Supplementary Fig. 1) as for C17, C16 and C26 (data not shown) in the absence of biological matrix.
2.9. Protocol development Quality control (QC) were set at three levels: lower (1.5 pg/μl), medium (15 pg/μl) and high (60 pg/μl) in the calibration range. QC were prepared in a plasma samples with independent standards solutions to prepare calibration curves.
3.2.2. Sample injection in a surrogate matrix According to the guidelines for Bioanalytical Method Validation Guidance for Industry – FDA (https://www.fda.gov/downloads/drugs/ guidances/ucm070107.pdf) and Guideline Bioanalytical method validation - European Medicines Agency (http://www.ema.europa.eu/ docs/en_GB/document_library/Scientific_guideline/2011/08/ WC500109686.pdf), no recommendations were made for the quantification of endogenous substances. Quantification of endogenous substances remains an unmet need for biomarker developers [24]. However, we have to satisfy two needs: the first one is to quantitate DDA in physio-pathological situations in patient samples from large cohorts of patients; the second one is to be able to perform pharmacokinetic and metabolic studies on DDA for clinical development purpose. We thus need to perform dosages in solid tissues (breast tumors and normal breast) and liquid tissues (serum or plasma). We investigated the use of a human biological matrix that contains a low amount of endogenous DDA to be used as a surrogate matrix. We first tested solid tissues extracted through B&D and MeOH precipitation. In each case we found the presence of endogenous DDA that impaired the linearity of the calibration curve and did not enabled the quantification of DDA. We next explored human plasma and tested B&D extraction and MeOH precipitation (Fig. 3). As shown in Fig. 4, a standard curve made resulting from methanol precipitation method showed a good linearity (y = 1.4x + 0.33) and (r = 0.98, representative of 3 independent experiments). The calculation of the values of QC Low (1.5 pg/μl), Medium (15 μg/μl) and High (60 μg/μl) shown for all good quantification levels of respectively: 1.31 pg/μl, 13.11 μg/μl, and 66.01 μg/μl. Our results showed that the retention time for DDA was stable at
2.10. In silico physicochemical properties prediction of compounds Prediction of the physicochemical properties was achieved using the chemicalize web platform (https://chemicalize.com) from ChemAxon. Calculator Plugins were used for structure property prediction and calculation, (2019 version release). This application enabled the calculation of the following parameters to be done: the molecular mass, the topological polar surface area, the polarizability, the isoelectric point, the charge at pH 8, the LogP, the LogD at pH 8, the solubility at pH 8, the van der Waals volume, and the molecular refractivity (see supplementary material). 3. Results 3.1. UPLC of DDA and analogues on HILIC On Fig. 2 we report that HILIC offers good capacity to separated DDA from its analogues with retention times of 1.86 min for DDA and C16, 2.10 min for C17 and 3.23 min for C26. DDA had the same retention time than C16 but compounds were discriminated because of their different parent ions (Supplementary Table T1). This separation was found robust and reproducible. The retention time for DDA gave a peak of 3 s at half height (Table 1) with a 0.1% variation after 250 injections. Retention times for compounds are positively correlated 4
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
Fig. 4. Representative chromatograms obtained for A) DDA and B) [2H6]-DDA after methanol extraction of human plasma spiked with each compounds. C) Calibration curve for the quantification of DDA. This curve was determined using increasing concentrations of DDA from 0.01 to 100 pg/μL.
1.86 ± 0.25 min of the Total Ion Current (TIC), repeatability was good with less than 8% deviation for five injections of the same sample (results representative of 3 independent experiments). No carry over were measured. Similar results were obtained using the B&D methodology (y = 1.4x + 0.19) (not shown).
Table 3 Dosage of DDA in human breast tumors and normal matched tissue. Patient number P597
3.2.3. Accuracy, precision recovery and matrix effect The precision and the accuracy of the method were next studied for DDA. The bias (%) and the coefficient of variance (CV%) were calculated for intra- and inter-day accuracy and precision respectively (Table 2). They ranged within 15% of the target concentrations and within 20% at Lower Limit Of Quantification (LLOQ) as usually expected to fulfill the regulatory guidelines.
We next tested the detection of compounds in breast tissues. We analyzed here the DDA content in 3 tumors and in the “normal” matched tissue from patients. DDA levels are reported as ng per g of wet weight tissue and in M assuming that 1 g of tissue has a volume of 1 ml. DDA was found detectable in this normal tissues and its measured level was more than 100 fold over the limit of detection (LOD) (LOD < 0.019 pg/μl) established with plasma as a surrogate matrix (Table 3). The methanol precipitation method gave in two of the three cases a higher value in DDA levels than the B&D method of extraction. We found in the tumor from two patients (patient P597 using the methanol and the Bligh and Dyer preparation method, and patient P8860 using the B&D method of extraction) an unmeasurable amount of DDA that was under the LLOQ (LLOQ = 0.024 pg/μl) established in plasma. In a third tumor sample (p8860) we found a measurable amount of DDA and its level was hundred time lower than in the normal matched tissue (Table 3, Fig. 5). Interestingly, compound C17 was detected in some samples, although at a very low level compared to DDA (Fig. 6A). In
Nominal concentration (pg/ μl) Mean CV (%) Bias (%)
CV: coefficient of varience,
a
normal
tumor
Tumour histological status Tumor grade
HR+ HER2+
HR- HER2+
HRHER2-
III
III
III
normal
53.3 < 0.1 41.1 5.7
< 0.1 2.4 51.8 45.9
50.7 < 0.1 13.7 35.6
< 0.1 1.4 31.1 67.4
36 < 0.1 34.2 29.7
< 0.1 2.4 49.7 47.9
n.m. n.m.
9 17.5
n.m n.m.
22 43
7 3.6
11 21.5
n.m. n.m.
13 31.1
0.5 0.97
51 99
7 3.6
12 23.4
addition, dendrogenin B (C26) was detected in fetal bovine serum (Fig. 6B).
4. Discussion 4.1. UPLC of DDA and analogues on HILIC
Inter dayb
1.5
15
60
1.5
15
60
1.44 0.08 −4.00
15.44 0.18 2.93
60.62 0.14 1.03
1.44 0.10 −4.22
14.20 0.07 −5.31
63.10 0.06 5.17
b
n = 3.
n = 5,
tumor
HR+: tumor expressing hormone receptors (estrogen and progesterone receptors). HR-: tumor that does not express HR. HER2+: tumor expressing HER2. HER2-: tumor that does not express HER2. B&D: Bligh & Dyer organic extraction. DDA concentration in M was estimated assuming that 1 g of tissue has a volume of 1 ml.
Table 2 Intra-day and inter-day precision and accuracy and coefficient of variation of QC for DDA dosage. Intra daya
tumor
DDA (B&D) ng/g tissue nM DDA (MeOH precipitation) ng/g tissue nM
normal
P210
Sample type
Cellular composition of tissue samples cancer cell % epithelial cells % Fibroblast % Others (including adipocytes) %
3.3. Evaluation of DDA contents and analogues in tissues
P8860
We previously established that DDA, C16, C17 and C26 compounds can be separated by RP HPLC/MS tandem chromatography [17]. Runs were 10 min long, peaks width were between 3 s (DDA) and 4.5 s (C26) and the resolutions (Rs) were found good for DDA, C16 and C17 but insufficient for C26. The carry-over was less than 0.01% for DDA and the LLOQ was estimated to be 0.05 pg/μl in the absence of biological 5
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
Fig. 5. Chromatograms obtained after methanol precipitation of proteins from sample p8860. A) Normal matched tissue and B) tumor. DDA and [2H6]-DDA have a retention time of 1.86 min. Peak areas are given for quantification ions and [2H6]-DDA.
(r2 = 0.9773, p < 0;05), the charge at pH 8 (r2 = 0.95, p < 0.05), the isoelectric point (r2 = 0.9908, p < 0.005), the solubility of compounds in water (r2 = 0.9715, p < 0.05) and obtained an inverse correlation between the retention time of compounds and their Log D (r2 = 0.9713, p < 0.05). This is consistent with the nature of the interaction established between solutes and the stationary phase [18].
matrices. The HILIC method we report here is 4 min faster (6 min/run) than the RP method, with no carry-over, and gives a better LLOQ than our previous RP method. In addition, we obtained a good resolution for all tested compounds. Tests using different human plasma as biological matrices for DDA dosage was possible because we found plasma with very low levels of endogenous DDA. The resolution was preserved and the LLOQ measured for DDA, C16, C17 and C26 were respectively of 0.024, 0.5, 1 and 5 pg/μl. No carry over was measured. We were able to obtain positive correlations between the retention time of compounds and the calculated topological polar surfaces
4.2. Application to the dosage of DDA and other compounds in solid tissues We have applied this new UPLC-HILIC method to the quantification
Fig. 6. Chromatograms showing the detection of endogenous DDA analogues in breast tumors and fetal bovine sera (FBS). A) Compound C17 was found as a trace in some tumor samples. B) Compound C26 was detected in FBS. 6
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
R. Soulès, et al.
preparation by methanol precipitation. We report for the first time a quantification of DDA using a human matrix with a detection using an application based on liquid chromatography coupled with tandem mass spectrometry. The use of such efficient analytical tools could be used in routine practice with good possibility to be easily automated.
of DDA in breast tissues and confirmed its detection in human breast. The analyses of breast tumors confirm the presence at low level of DDA. Higher levels of DDA were found in normal matched tissues from three patient samples. These levels are low compared to our previous analyses of 10 patients with ER(+) tumors using another method. In the de Medina’s study the amount of epithelial cells contained in normal matched tissues were greater than 50% of total cells [1], while in the present study the level was less than 2% which may explain the lower DDA level we found in the present study. Indeed, we reported that DDA was present in primary culture of epithelial cells from the breast and absent from breast cancer cell lines [1]. In addition, in an unpublished work dedicate to the identification of the dendrogenin A synthase, immunohistochemical studies performed on normal breast revealed that epithelial cells from lactating ducts and terminal units are the cells that expressed selectively the enzyme. Adipocytes, fibroblasts, myoepithelial cells, endothelial cells and immune cells do not expressed the enzyme (M Poirot et al manuscript in preparation). It is thus important to determine the cellular composition of tissue samples that are analyzed. The organic precipitation method is a method that is widely used for the recovery and the quantification of various drugs from human sera for pharmacokinetic and metabolism studies [25,26]. This approach has been proved to be automatable for sample preparation and LC/MS bioanalyses [27]. Results from the present study suggest that this method will applicable to the analyses of solid tissues from large cohort of patients, and in particular the analyses of the Breasterol cohort (NCT02863900, Institut Claudius Regaud, Toulouse, France) that includes 300 patients with an equivalent number of the three major breast cancer subtypes (ER(+), HER2 (+) and triple negative) with the aim of studying DDA metabolism deregulation in breast cancers. The detection of the DDA analogues C17 and C26 in breast tissue and FBS is of high interest. We have detected the presence of C17, the inactive isomer of DDA [4,6], in low amount. C17 was found as a minor side product obtained during the chemical synthesis of DDA [10], a reaction of condensation that requires a catalyst [28]. Early studies from our group didn’t find C17 in mammalian tissues using RPeLC conditions that did not separate the two isomers [1]. The fact that, using this new UPLC-HILIC method, we found DDA and a low amount of C17 suggests that the DDA synthase acts as a catalyst with a low substrate selectivity and may be a promiscuous enzyme. The present data will help us in our quest of the identification of the DDA synthase. Compound C26 correspond to a product of condensation of 5,6α-EC with spermidine. This compound was synthesized few years ago and showed a high potency to induce the differentiation of multipotent cells, and neuron progenitors into neurons [7,9,10]. As opposed to dendrogenin A, C26 is not a general inducer of cell differentiation and is not cytotoxic up to mM concentrations. The fact that C26 was detected in FBS suggest that C26 could play a role in foetal development. This discovery opens up a new field of research investigations and evidenced that 5,6α-EC may be at the center of a new metabolic branch that will produce bioactive lipids with different biological properties that deserves further investigations. The absolute quantification of endogenous substances is very difficult because it is not possible to establish calibration curves in the corresponding biological matrix. In the present study, we didn’t succeed in using solid tissues as biological matrices because of the presence of endogenous DDA, but we were able to use a human serum that contains very low levels of DDA. This is important because DDA displays very promising anticancer properties on preclinical models [1,3–611], and this new method will enable pharmaco-kinetic and metabolism studies to be done in the perspective of the clinical evaluation of DDA.
Declaration of Competing Interest The authors declare that they have no competing interest. Acknowledgements M. Poirot and S. Silvente-Poirot's team was funded by the Institut National de la Santé et de la Recherche Médicale (INSERM); the Université de Toulouse III; the Institut National du Cancer (INCA;PRTKK15-118, PLBIO18-130); the Fondation Toulouse Cancer Santé (2017CS065); and the associations “Céline” and “Flo”. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jsbmb.2019.105447. References [1] P. de Medina, M.R. Paillasse, G. Segala, M. Voisin, L. Mhamdi, F. Dalenc, M. Lacroix-Triki, T. Filleron, F. Pont, T.A. Saati, C. Morisseau, B.D. Hammock, S. Silvente-Poirot, M. Poirot, Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties, Nat. Commun. 4 (2013) 1840. [2] M. Bauriaud-Mallet, L. Vija-Racaru, S. Brillouet, A. Mallinger, P. de Medina, A. Rives, B. Payre, M. Poirot, F. Courbon, S. Silvente-Poirot, The cholesterol-derived metabolite dendrogenin A functionally reprograms breast adenocarcinoma and undifferentiated thyroid cancer cells, J. Steroid Biochem. Mol. Biol. 192 (2019) 105390. [3] S. Silvente-Poirot, F. Dalenc, M. Poirot, The effects of cholesterol-derived oncometabolites on nuclear receptor function in Cancer, Cancer Res. 78 (2018) 4803–4808. [4] M. Poirot, S. Silvente-Poirot, The tumor-suppressor cholesterol metabolite, dendrogenin A, is a new class of LXR modulator activating lethal autophagy in cancers, Biochem. Pharmacol. 153 (2018) 75–81. [5] M. Voisin, P. de Medina, A. Mallinger, F. Dalenc, E. Huc-Claustre, J. Leignadier, N. Serhan, R. Soules, G. Segala, A. Mougel, E. Noguer, L. Mhamdi, E. Bacquie, L. Iuliano, C. Zerbinati, M. Lacroix-Triki, L. Chaltiel, T. Filleron, V. Cavailles, T. Al Saati, P. Rochaix, R. Duprez-Paumier, C. Franchet, L. Ligat, F. Lopez, M. Record, M. Poirot, S. Silvente-Poirot, Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E9346–E9355. [6] G. Segala, M. David, P. de Medina, M.C. Poirot, N. Serhan, F. Vergez, A. Mougel, E. Saland, K. Carayon, J. Leignadier, N. Caron, M. Voisin, J. Cherier, L. Ligat, F. Lopez, E. Noguer, A. Rives, B. Payre, T.A. Saati, A. Lamaziere, G. Despres, J.M. Lobaccaro, S. Baron, C. Demur, F. de Toni, C. Larrue, H. Boutzen, F. Thomas, J.E. Sarry, M. Tosolini, D. Picard, M. Record, C. Recher, M. Poirot, S. SilventePoirot, Dendrogenin A drives LXR to trigger lethal autophagy in cancers, Nat. Commun. 8 (2017) 1903. [7] F. Dalenc, M. Poirot, S. Silvente-Poirot, Dendrogenin a: a mammalian metabolite of cholesterol with tumor suppressor and neurostimulating properties, Curr. Med. Chem. 22 (2015) 3533–3549. [8] S. Silvente-Poirot, M. Poirot, Cancer. Cholesterol and cancer, in the balance, Science 343 (2014) 1445–1446. [9] S.A. Khalifa, P. de Medina, A. Erlandsson, H.R. El-Seedi, S. Silvente-Poirot, M. Poirot, The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells, Biochem. Biophys. Res. Commun. 446 (2014) 681–686. [10] P. de Medina, M.R. Paillasse, B. Payre, S. Silvente-Poirot, M. Poirot, Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases, J. Med. Chem. 52 (2009) 7765–7777. [11] S. Silvente-Poirot, G. Segala, M.C. Poirot, M. Poirot, Ligand-dependent transcriptional induction of lethal autophagy: a new perspective for cancer treatment, Autophagy 14 (2018) 555–557. [12] M. Poirot, R. Soules, A. Mallinger, F. Dalenc, S. Silvente-Poirot, Chemistry, biochemistry, metabolic fate and mechanism of action of 6-oxo-cholestan-3beta,5alpha-diol (OCDO), a tumor promoter and cholesterol metabolite, Biochimie 153 (2018) 139–149. [13] A. Fransson, P. de Medina, M.R. Paillasse, S. Silvente-Poirot, M. Poirot, M. Ulfendahl, Dendrogenin A and B two new steroidal alkaloids increasing neural responsiveness in the deafened guinea pig, Front. Aging Neurosci. 7 (2015) 145. [14] S. Silvente-Poirot, P. de Medina, M. Record, M. Poirot, From tamoxifen to
5. Conclusion The UPLC-MS method described herein is simple, specific and highly sensitive, in addition it includes an easy and fast step of sample 7
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dendrogenin A: the discovery of a mammalian tumor suppressor and cholesterol metabolite, Biochimie 130 (2016) 109–114. M. Poirot, S. Silvente-Poirot, When cholesterol meets histamine, it gives rise to dendrogenin A: a tumour suppressor metabolite, Biochem. Soc. Trans. 44 (2016) 631–637. E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917. E. Noguer, R. Soules, C. Netter, C. Nagarathinam, J. Leignadier, E. Huc-Claustre, N. Serhan, A. Rives, P. de Medina, S. Silvente-Poirot, M. Poirot, Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry, Chem. Phys. Lipids 207 (2017) 81–86. B. Buszewski, S. Noga, Hydrophilic interaction liquid chromatography (HILIC)–a powerful separation technique, Anal. Bioanal. Chem. 402 (2012) 231–247. F. Qin, Y.Y. Zhao, M.B. Sawyer, X.F. Li, Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine, Anal. Chem. 80 (2008) 3404–3411. K.M. Hines, J. Herron, L. Xu, Assessment of altered lipid homeostasis by HILIC-ion mobility-mass spectrometry-based lipidomics, J. Lipid Res. 58 (2017) 809–819. J. Chen, L. Chen, M. Li, Y. Sun, Quantitative determination of six steroid alkaloids by sensitive hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic study in rats, Biomed. Chromatogr. 33 (2019) e4377. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
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Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
A fast UPLC–HILIC method for an accurate quantification of dendrogenin A in human tissues
T
Régis Soulèsa,b, Fabien Audouard-Combec, Emilie Huc-Claustrea,b, Philippe de Medinaa,b, Arnaud Rivesd,e, Etienne Chatelutf,g, Florence Dalenca,b,g, Camille Francheth, ⁎ Sandrine Silvente-Poirota,b, Marc Poirota,b, Ben Allalf,g, a
Team « Cholesterol metabolism and therapeutic innovations », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France Equipe labellisée par la Ligue Nationale Contre le Cancer, France c Waters S.A.S., Saint-Quentin En Yvelines, France d Affichem, Toulouse, France e Dendrogenix, Liège, Belgium f Team “Dose individualization of anticancer drugs », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France g Institut Claudius Regaud, Institut Universitaire du Cancer-Oncopole, Toulouse, France h Service d'Anatomo-Pathologie, Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Toulouse, France b
A R T I C LE I N FO
A B S T R A C T
Keywords: Steroidal alkaloid Dendrogenin A Dendrogenin B Cholesterol Oxysterol Metabolite Method development Quantification UPLC-MS
Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.
1. Introduction
sterol that was shown to activate at low doses differentiation programs in cancer cells of different tissue origins [1–10], as well as normal neuronal progenitor cells [7,9,10]. In addition, this new re-differentiation agent was shown to activate cancer cell death through an original molecular and cellular mechanism. DDA kills cancer cells when used at μM concentrations through the induction of an autophagic cell death via the activation of the oxysterol receptor LXRβ [3,4,6,11]. In
Dendrogenin A (DDA) is a cholesterol conjugate that was recently discovered in human tissues [1]. DDA is the first mammalian steroidal alkaloid discovered to date and it results from the conjugation of the primary amine of histamine on carbon 6 of 5,6α-epoxycholesterol (5,6α-EC) by a yet unidentified enzyme (Fig. 1). DDA is an interesting
Abbreviations: Dendrogenin A (DDA), 5α-hydroxy-6β[2-(1H-imidazol-4-yl) ethylamino] cholestan-3β-ol; Hexadeuterated dendrogenin A ([2H6]-DDA), (26,26,26,27,27,27-[2H6])-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3β-ol; Dendrogenin B (DDB), 5α-hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholestan-3β-ol; 5, 6α-EC,5α,6α-epoxy-cholestan-3β-ol; OS, oxysterol; BC, breast cancer; B&D, Bligh and Dyer; QC, quality control; H, high; M, medium; L, low ⁎ Corresponding author at: Team « Cholesterol metabolism and therapeutic innovations », Cancer Research Center of Toulouse, UMR 1037 INSERM-University of Toulouse, Toulouse, France E-mail addresses: [email protected] (M. Poirot), [email protected] (B. Allal). https://doi.org/10.1016/j.jsbmb.2019.105447 Received 18 April 2019; Received in revised form 5 August 2019; Accepted 11 August 2019 Available online 12 August 2019 0960-0760/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. A) Biochemical synthesis of dendrogenin A (DDA). DDA is the produce of the enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine by the DDA synthase (DDAS). B) DDA is steroidal alkaloid that contains 3 nitrogen atoms. DDA can exist through different protonated states according to the pH.
obtained with DDB suggesting that this method was not appropriated for DDB dosage if characterized as an endogenous metabolite of for pharmacokinetic purpose in case of clinical application with this compound. Recently, the hydrophilic interaction liquid chromatography (HILIC) was shown to provide an alternative approach to RP chromatography to separate small polar compounds on polar stationary phases [18]. This method was used successfully to analyze conjugated steroids [19], lipids [20] and steroidal alkaloids [21]. Because DDA and parent compounds such as DDB, C26 and C17 are steroidal alkaloids we thought that the development of a HILIC method would be interesting. The primary objective of the present study was to develop a new, fast and sensitive method of quantification of DDA in liquid and solid tissues and eventually to detect DDA analogues in human samples.
addition, it was shown that DDA inhibits breast cancer cell proliferation through the inhibition of the production of the tumor promoter 6-oxocholestan-3β,5α-diol by cancer cells at the cholesterol-5,6-epoxide hydrolase step [3,5,12]. DDA demonstrates antitumor potencies on various pre-clinical models of cancers [3–611,12], as well as neuro-regenerating potencies on a preclinical model of deafness [7,13]. DDA was detected in several mammalian organs and tissues while it was not detected in melanoma and breast cancer cell lines [1,14,15]. Preliminary studies done in patients showed that DDA levels were found strongly decreased in breast tumors from 10 patients compared to “normal “matched tissue [1], highlighting that a deregulation in DDA metabolism occurred during oncogenesis. It is thus important to develop an accurate and sensitive analytical quantification method for DDA adapted to liquid (serum and plasma) as well as solid tissues from large cohorts of patients. In our preliminary studies, the quantification of DDA was achieved after an organic solvent extraction procedure (Bligh and Dyer: B&D) [16], followed by a solid phase extraction procedure of samples on a C18 Sep-Pack cartridge and then a purification of samples by reverse phase HPLC [1]. The quantification of DDA was made by mass spectrometry and a MS/MS fragmentation of the parent peak present in the eluted fractions at the retention time that corresponding to the authentic DDA standard, and using deuterated DDA as an internal standard. The MS/MS fragmentation was used to discriminate DDA from its C17 geometric isomer that co-eluted because of their different fragmentation fingerprints. This method required almost 24 h per sample, and was not convenient for the analysis of large series of samples. We recently reported the development of a new liquid chromatography tandem mass spectrometry (LC/MS) method for the dosage of DDA and analogues based on reverse phase (RP) liquid chromatography that gave satisfactory results in terms of resolution, sensitivity and duration of the whole process. We solved an important problem of carryover using an acidic elution buffer (pH < 2), with heptafluorobutyric acid in the mobile phase that made ion pairing with cationic solutes on RP. These modifications seriously improved the resolution in our separation assay [17]. In addition, we were able to separate DDA from its regio-isomer C17, dendrogenin B (C26, DDB) and the sitosterol analog of DDA: C16 in the following order : C26 > C17 > DDA > C16 from the more hydrophilic to the more hydrophobic (Fig. 2 D, B, A, C respectively). A weak resolution was however
2. Materials and Methods 2.1. Chemicals, reagents Formic acid, chloroform, ammonium formate, (26,26,26,27,27,27[2H6]) cholesterol (98% 2H), histamine and fetal bovine serum (FBS) were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ammonia was purchased from VWR Chemicals (VWR International S.A.S. Le Périgares - Fontenay-sous-Bois cedex). 5α-hydroxy-6β-[2-(1H-imidazol4-yl)ethylamino]cholestan-3β-ol (dendrogenin A), 5α-hydroxy-6β-[2(1H-imidazol-4-yl)ethylamino]sitostan-3β-ol (C16), 5α-hydroxy-6β-[4(2-aminoethyl)imidazol-1-yl] cholestan-3β-ol (C17) and 5α-hydroxy6β-[3-(4-aminobutylamino)propylamino]cholestan-3β-ol (C26) were synthesized in our laboratory according to a previously published procedure [10] and was 99% pure. [2H6]-DDA synthesis and characterization is reported on the supplementary material section. Methanol, acetonitrile (HPLC Grade) were from Scharlau (Barcelona, Spain). Human plasma were provided by the «Etablissement Français du Sang» (EFS, Toulouse, France). A milli-Q Gradient A10 Millipore® System was used to prepare Milli-Q water. Acrodisc Nylon minispike syringe filters (13 mm, 0.2 μm) were used for sample filtration and polypropylene (12 × 32 mm Screw Neck) Vial, with cap and preslit PTFE/Silicone septa were purchased from Waters (Saint-Quentin en Yvelines, France). 2
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Fig. 2. Separation of DDA and analogues by UPLC-MS on a HILIC column. A representative chromatogram is shown on A) for DDA, on B) for C17, on C) for C16 and on D) for C26.
Gradient elutions were performed with an Acquity UPLC H-class system (Waters, St Quentin en Yvelines, France). The analytical column was an Acquity UPLC BEH HILIC column, 2.1 mm × 50 mm, 1.7 μm (Waters, USA). The injector and the column temperature were set at 10 °C and 25 °C respectively. The mobile phase A was 200 mM ammonium formate and 1% ammoniac (v/v) pH 9.2 and the mobile phase B was acetonitrile. The gradient elution started with 100% B for 0.25 min, decreased to 50% in 2.25 min, and stay at 50% during 1.5 min and return to 100% at a flow rate of 0.7 ml/min.
homogenizer system (Bertin technologies, Montigny-le-Bretonneux, France): briefly, frozen tissue (−80 °C) were disposed in cold tubes containing metal beads and lysis buffer (50 mM Tris−HCl, 150 mM KCl pH 7.4, 0.5% butylated hydroxytoluene (5 mg.ml−1)) 5 vol per g of tissue at 4 °C. The suspension was agitated 90 s at 10,000 r.p.m. and then submitted to two cycles of freeze/thaw (in liquid nitrogen (−196 °C) and 37 °C Block Heaters). Samples were then centrifuged (10 min at 1500 g, 4 °C), the supernatant was collected in new tubes and the protein concentration was measured by Bradford method [22] for sample normalization at 10 mg protein/ml. Sample were stored at -80°c until organic solvent extraction was performed.
2.3. Mass spectrometry
2.5. Standard solution for calibration
Compounds were detected on a Waters Xevo TQS-micro mass spectrometer equipped with an electrospray ionization ion source (ESI). Data were acquired in a multiple-reaction monitoring (MRM) mode using MassLynx software. MS collision parameters are summarized in the supplementary Table 1. Capillary and cone voltages were respectively of 3 kV and variable, source and desolvation gas were set respectively at 200 °C and 550 L/Hr. The cone gas flow was set at 30 L/h.
DDA and its [2H6]-DDA analogue were dissolved in methanol at 1 mg/ml and stored at −20 °C until used. Working solutions for calibration curves were prepared extemporaneously. 150 μl standards solutions were spiked into 600 μl of ethanol or biological matrices to obtain a concentration range from 0.01 to 100 pg/μl.
2.2. Liquid chromatography
2.6. Carry-over tests
2.4. Sample preparation
Carry-over was assessed following injection of a blank (plasma sample) immediately after three repeats of the Upper Limit Of Quantification (ULOQ). The peak area response was checked.
Clinical samples were collected from patients enrolled in the “Breasterol” trial (NCT02863900, Institut Claudius Regaud, Toulouse, France) approved by the Ethics committee of Midi-Pyrénées Region (Comité de protection des personnes Sud-Ouest et Outre-Mer III). 200 mg of human tissues were obtained by surgical resection and stored at −80 °C until used (All patients gave written informed consent). −80 °C stored tissues were homogenized at 4 °C using a Precellys 24
2.7. Retention time stability Retention time stability was tested by repeatedly injecting a mixture of four molecules (DDA, C16, C17, C26) one hundred times. The retention time was measured for each compound, and the results are expressed as a percentage of variation.
Table 1 Chromatographic parameters. tR: retention time; k: retention factor; wh: peak width at half height; N: number of plates; As5%: peak asymmetry measured at 5% peak height; α: separation factor; Rs: resolution. compound
tR
k
wh
N
As10%
α
Rs
DDA C16 C17 C26
1.86 1.86 2.10 3.16
1.91 1.91 2.28 3.93
3.0 3.2 3.5 3.1
7666 6738 7180 20723
1.0 1.2 1.3 1.0
1.05 1.11 1.19 2.06
0.68 1.33 2.29 14.76
2.8. Sample tissue and plasma extraction Two methods of sample preparation were used. The first one was the Bligh and Dyer liquid/liquid (B&D) extraction method [16] that was initially used for DDA analysis in different tissues [1]. 250 μl of samples were mixed to 500 μl chloroform and 250 μl methanol containing the internal standard [2H6]-DDA at 10 pg/μl. The suspensions were vortexed 30 s, followed by a 5 min stop and then vortexed for 30 s a second 3
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Fig. 3. Schematic of the two different work-flows that were used for DDA analysis.
time. 400 μl of the organic layer were dried under a nitrogen flux at 37 °C. Samples were resuspended in 100 μl methanol, filtered on acrodisc Nylon minispike syringe filters into new vials for UPLC-MS analyses. In the second method, we used a methanol-protein precipitation procedure which was used cationic amphihilic drugs [23]. One volume of plasma (250 μl of standard, CQ, patient/analyte) was mixed to 5 volumes of methanol (1250 μl) spiked with the internal standard ([2H6]-DDA) at 10 pg/μl (final concentration). The suspensions were vortexed 2 x 1 min and were centrifuged 10 min at 1500 g (4 °C). 1 ml of the supernatant was collected and evaporated under a nitrogen flux at 37 °C. Dried samples were dissolved in 166 μl methanol and filtered on acrodisc Nylon minispike syringe filters into new vials for UPLC-MS analyses.
with their topological surface area (r2 = 0.9773, p < 0.05) their charge at pH 8 (r2 = 0.95, p < 0.05), their isoelectric point (r2 = 0.9908, p < 0.005) and their solubility at pH 8 (r2 = 0.9715, p < 0.05). Retention time for compounds were negatively correlated with their hydrophobicity taking into account their ionization state at pH 8 (Supplementary Table 1 and Fig. 1). No correlations were found with other parameters such as the molecular mass, the polarizability, the partition coefficient (LogP), the van der Waals volume or the molecular refractivity. 3.2. Calibration curves 3.2.1. Sample injection without matrix The method was linear over a range 0.02–100 pg/μl for DDA (Supplementary Fig. 1) as for C17, C16 and C26 (data not shown) in the absence of biological matrix.
2.9. Protocol development Quality control (QC) were set at three levels: lower (1.5 pg/μl), medium (15 pg/μl) and high (60 pg/μl) in the calibration range. QC were prepared in a plasma samples with independent standards solutions to prepare calibration curves.
3.2.2. Sample injection in a surrogate matrix According to the guidelines for Bioanalytical Method Validation Guidance for Industry – FDA (https://www.fda.gov/downloads/drugs/ guidances/ucm070107.pdf) and Guideline Bioanalytical method validation - European Medicines Agency (http://www.ema.europa.eu/ docs/en_GB/document_library/Scientific_guideline/2011/08/ WC500109686.pdf), no recommendations were made for the quantification of endogenous substances. Quantification of endogenous substances remains an unmet need for biomarker developers [24]. However, we have to satisfy two needs: the first one is to quantitate DDA in physio-pathological situations in patient samples from large cohorts of patients; the second one is to be able to perform pharmacokinetic and metabolic studies on DDA for clinical development purpose. We thus need to perform dosages in solid tissues (breast tumors and normal breast) and liquid tissues (serum or plasma). We investigated the use of a human biological matrix that contains a low amount of endogenous DDA to be used as a surrogate matrix. We first tested solid tissues extracted through B&D and MeOH precipitation. In each case we found the presence of endogenous DDA that impaired the linearity of the calibration curve and did not enabled the quantification of DDA. We next explored human plasma and tested B&D extraction and MeOH precipitation (Fig. 3). As shown in Fig. 4, a standard curve made resulting from methanol precipitation method showed a good linearity (y = 1.4x + 0.33) and (r = 0.98, representative of 3 independent experiments). The calculation of the values of QC Low (1.5 pg/μl), Medium (15 μg/μl) and High (60 μg/μl) shown for all good quantification levels of respectively: 1.31 pg/μl, 13.11 μg/μl, and 66.01 μg/μl. Our results showed that the retention time for DDA was stable at
2.10. In silico physicochemical properties prediction of compounds Prediction of the physicochemical properties was achieved using the chemicalize web platform (https://chemicalize.com) from ChemAxon. Calculator Plugins were used for structure property prediction and calculation, (2019 version release). This application enabled the calculation of the following parameters to be done: the molecular mass, the topological polar surface area, the polarizability, the isoelectric point, the charge at pH 8, the LogP, the LogD at pH 8, the solubility at pH 8, the van der Waals volume, and the molecular refractivity (see supplementary material). 3. Results 3.1. UPLC of DDA and analogues on HILIC On Fig. 2 we report that HILIC offers good capacity to separated DDA from its analogues with retention times of 1.86 min for DDA and C16, 2.10 min for C17 and 3.23 min for C26. DDA had the same retention time than C16 but compounds were discriminated because of their different parent ions (Supplementary Table T1). This separation was found robust and reproducible. The retention time for DDA gave a peak of 3 s at half height (Table 1) with a 0.1% variation after 250 injections. Retention times for compounds are positively correlated 4
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
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Fig. 4. Representative chromatograms obtained for A) DDA and B) [2H6]-DDA after methanol extraction of human plasma spiked with each compounds. C) Calibration curve for the quantification of DDA. This curve was determined using increasing concentrations of DDA from 0.01 to 100 pg/μL.
1.86 ± 0.25 min of the Total Ion Current (TIC), repeatability was good with less than 8% deviation for five injections of the same sample (results representative of 3 independent experiments). No carry over were measured. Similar results were obtained using the B&D methodology (y = 1.4x + 0.19) (not shown).
Table 3 Dosage of DDA in human breast tumors and normal matched tissue. Patient number P597
3.2.3. Accuracy, precision recovery and matrix effect The precision and the accuracy of the method were next studied for DDA. The bias (%) and the coefficient of variance (CV%) were calculated for intra- and inter-day accuracy and precision respectively (Table 2). They ranged within 15% of the target concentrations and within 20% at Lower Limit Of Quantification (LLOQ) as usually expected to fulfill the regulatory guidelines.
We next tested the detection of compounds in breast tissues. We analyzed here the DDA content in 3 tumors and in the “normal” matched tissue from patients. DDA levels are reported as ng per g of wet weight tissue and in M assuming that 1 g of tissue has a volume of 1 ml. DDA was found detectable in this normal tissues and its measured level was more than 100 fold over the limit of detection (LOD) (LOD < 0.019 pg/μl) established with plasma as a surrogate matrix (Table 3). The methanol precipitation method gave in two of the three cases a higher value in DDA levels than the B&D method of extraction. We found in the tumor from two patients (patient P597 using the methanol and the Bligh and Dyer preparation method, and patient P8860 using the B&D method of extraction) an unmeasurable amount of DDA that was under the LLOQ (LLOQ = 0.024 pg/μl) established in plasma. In a third tumor sample (p8860) we found a measurable amount of DDA and its level was hundred time lower than in the normal matched tissue (Table 3, Fig. 5). Interestingly, compound C17 was detected in some samples, although at a very low level compared to DDA (Fig. 6A). In
Nominal concentration (pg/ μl) Mean CV (%) Bias (%)
CV: coefficient of varience,
a
normal
tumor
Tumour histological status Tumor grade
HR+ HER2+
HR- HER2+
HRHER2-
III
III
III
normal
53.3 < 0.1 41.1 5.7
< 0.1 2.4 51.8 45.9
50.7 < 0.1 13.7 35.6
< 0.1 1.4 31.1 67.4
36 < 0.1 34.2 29.7
< 0.1 2.4 49.7 47.9
n.m. n.m.
9 17.5
n.m n.m.
22 43
7 3.6
11 21.5
n.m. n.m.
13 31.1
0.5 0.97
51 99
7 3.6
12 23.4
addition, dendrogenin B (C26) was detected in fetal bovine serum (Fig. 6B).
4. Discussion 4.1. UPLC of DDA and analogues on HILIC
Inter dayb
1.5
15
60
1.5
15
60
1.44 0.08 −4.00
15.44 0.18 2.93
60.62 0.14 1.03
1.44 0.10 −4.22
14.20 0.07 −5.31
63.10 0.06 5.17
b
n = 3.
n = 5,
tumor
HR+: tumor expressing hormone receptors (estrogen and progesterone receptors). HR-: tumor that does not express HR. HER2+: tumor expressing HER2. HER2-: tumor that does not express HER2. B&D: Bligh & Dyer organic extraction. DDA concentration in M was estimated assuming that 1 g of tissue has a volume of 1 ml.
Table 2 Intra-day and inter-day precision and accuracy and coefficient of variation of QC for DDA dosage. Intra daya
tumor
DDA (B&D) ng/g tissue nM DDA (MeOH precipitation) ng/g tissue nM
normal
P210
Sample type
Cellular composition of tissue samples cancer cell % epithelial cells % Fibroblast % Others (including adipocytes) %
3.3. Evaluation of DDA contents and analogues in tissues
P8860
We previously established that DDA, C16, C17 and C26 compounds can be separated by RP HPLC/MS tandem chromatography [17]. Runs were 10 min long, peaks width were between 3 s (DDA) and 4.5 s (C26) and the resolutions (Rs) were found good for DDA, C16 and C17 but insufficient for C26. The carry-over was less than 0.01% for DDA and the LLOQ was estimated to be 0.05 pg/μl in the absence of biological 5
Journal of Steroid Biochemistry and Molecular Biology 194 (2019) 105447
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Fig. 5. Chromatograms obtained after methanol precipitation of proteins from sample p8860. A) Normal matched tissue and B) tumor. DDA and [2H6]-DDA have a retention time of 1.86 min. Peak areas are given for quantification ions and [2H6]-DDA.
(r2 = 0.9773, p < 0;05), the charge at pH 8 (r2 = 0.95, p < 0.05), the isoelectric point (r2 = 0.9908, p < 0.005), the solubility of compounds in water (r2 = 0.9715, p < 0.05) and obtained an inverse correlation between the retention time of compounds and their Log D (r2 = 0.9713, p < 0.05). This is consistent with the nature of the interaction established between solutes and the stationary phase [18].
matrices. The HILIC method we report here is 4 min faster (6 min/run) than the RP method, with no carry-over, and gives a better LLOQ than our previous RP method. In addition, we obtained a good resolution for all tested compounds. Tests using different human plasma as biological matrices for DDA dosage was possible because we found plasma with very low levels of endogenous DDA. The resolution was preserved and the LLOQ measured for DDA, C16, C17 and C26 were respectively of 0.024, 0.5, 1 and 5 pg/μl. No carry over was measured. We were able to obtain positive correlations between the retention time of compounds and the calculated topological polar surfaces
4.2. Application to the dosage of DDA and other compounds in solid tissues We have applied this new UPLC-HILIC method to the quantification
Fig. 6. Chromatograms showing the detection of endogenous DDA analogues in breast tumors and fetal bovine sera (FBS). A) Compound C17 was found as a trace in some tumor samples. B) Compound C26 was detected in FBS. 6
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preparation by methanol precipitation. We report for the first time a quantification of DDA using a human matrix with a detection using an application based on liquid chromatography coupled with tandem mass spectrometry. The use of such efficient analytical tools could be used in routine practice with good possibility to be easily automated.
of DDA in breast tissues and confirmed its detection in human breast. The analyses of breast tumors confirm the presence at low level of DDA. Higher levels of DDA were found in normal matched tissues from three patient samples. These levels are low compared to our previous analyses of 10 patients with ER(+) tumors using another method. In the de Medina’s study the amount of epithelial cells contained in normal matched tissues were greater than 50% of total cells [1], while in the present study the level was less than 2% which may explain the lower DDA level we found in the present study. Indeed, we reported that DDA was present in primary culture of epithelial cells from the breast and absent from breast cancer cell lines [1]. In addition, in an unpublished work dedicate to the identification of the dendrogenin A synthase, immunohistochemical studies performed on normal breast revealed that epithelial cells from lactating ducts and terminal units are the cells that expressed selectively the enzyme. Adipocytes, fibroblasts, myoepithelial cells, endothelial cells and immune cells do not expressed the enzyme (M Poirot et al manuscript in preparation). It is thus important to determine the cellular composition of tissue samples that are analyzed. The organic precipitation method is a method that is widely used for the recovery and the quantification of various drugs from human sera for pharmacokinetic and metabolism studies [25,26]. This approach has been proved to be automatable for sample preparation and LC/MS bioanalyses [27]. Results from the present study suggest that this method will applicable to the analyses of solid tissues from large cohort of patients, and in particular the analyses of the Breasterol cohort (NCT02863900, Institut Claudius Regaud, Toulouse, France) that includes 300 patients with an equivalent number of the three major breast cancer subtypes (ER(+), HER2 (+) and triple negative) with the aim of studying DDA metabolism deregulation in breast cancers. The detection of the DDA analogues C17 and C26 in breast tissue and FBS is of high interest. We have detected the presence of C17, the inactive isomer of DDA [4,6], in low amount. C17 was found as a minor side product obtained during the chemical synthesis of DDA [10], a reaction of condensation that requires a catalyst [28]. Early studies from our group didn’t find C17 in mammalian tissues using RPeLC conditions that did not separate the two isomers [1]. The fact that, using this new UPLC-HILIC method, we found DDA and a low amount of C17 suggests that the DDA synthase acts as a catalyst with a low substrate selectivity and may be a promiscuous enzyme. The present data will help us in our quest of the identification of the DDA synthase. Compound C26 correspond to a product of condensation of 5,6α-EC with spermidine. This compound was synthesized few years ago and showed a high potency to induce the differentiation of multipotent cells, and neuron progenitors into neurons [7,9,10]. As opposed to dendrogenin A, C26 is not a general inducer of cell differentiation and is not cytotoxic up to mM concentrations. The fact that C26 was detected in FBS suggest that C26 could play a role in foetal development. This discovery opens up a new field of research investigations and evidenced that 5,6α-EC may be at the center of a new metabolic branch that will produce bioactive lipids with different biological properties that deserves further investigations. The absolute quantification of endogenous substances is very difficult because it is not possible to establish calibration curves in the corresponding biological matrix. In the present study, we didn’t succeed in using solid tissues as biological matrices because of the presence of endogenous DDA, but we were able to use a human serum that contains very low levels of DDA. This is important because DDA displays very promising anticancer properties on preclinical models [1,3–611], and this new method will enable pharmaco-kinetic and metabolism studies to be done in the perspective of the clinical evaluation of DDA.
Declaration of Competing Interest The authors declare that they have no competing interest. Acknowledgements M. Poirot and S. Silvente-Poirot's team was funded by the Institut National de la Santé et de la Recherche Médicale (INSERM); the Université de Toulouse III; the Institut National du Cancer (INCA;PRTKK15-118, PLBIO18-130); the Fondation Toulouse Cancer Santé (2017CS065); and the associations “Céline” and “Flo”. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jsbmb.2019.105447. References [1] P. de Medina, M.R. Paillasse, G. Segala, M. Voisin, L. Mhamdi, F. Dalenc, M. Lacroix-Triki, T. Filleron, F. Pont, T.A. Saati, C. Morisseau, B.D. Hammock, S. Silvente-Poirot, M. Poirot, Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties, Nat. Commun. 4 (2013) 1840. [2] M. Bauriaud-Mallet, L. Vija-Racaru, S. Brillouet, A. Mallinger, P. de Medina, A. Rives, B. Payre, M. Poirot, F. Courbon, S. Silvente-Poirot, The cholesterol-derived metabolite dendrogenin A functionally reprograms breast adenocarcinoma and undifferentiated thyroid cancer cells, J. Steroid Biochem. Mol. Biol. 192 (2019) 105390. [3] S. Silvente-Poirot, F. Dalenc, M. Poirot, The effects of cholesterol-derived oncometabolites on nuclear receptor function in Cancer, Cancer Res. 78 (2018) 4803–4808. [4] M. Poirot, S. Silvente-Poirot, The tumor-suppressor cholesterol metabolite, dendrogenin A, is a new class of LXR modulator activating lethal autophagy in cancers, Biochem. Pharmacol. 153 (2018) 75–81. [5] M. Voisin, P. de Medina, A. Mallinger, F. Dalenc, E. Huc-Claustre, J. Leignadier, N. Serhan, R. Soules, G. Segala, A. Mougel, E. Noguer, L. Mhamdi, E. Bacquie, L. Iuliano, C. Zerbinati, M. Lacroix-Triki, L. Chaltiel, T. Filleron, V. Cavailles, T. Al Saati, P. Rochaix, R. Duprez-Paumier, C. Franchet, L. Ligat, F. Lopez, M. Record, M. Poirot, S. Silvente-Poirot, Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E9346–E9355. [6] G. Segala, M. David, P. de Medina, M.C. Poirot, N. Serhan, F. Vergez, A. Mougel, E. Saland, K. Carayon, J. Leignadier, N. Caron, M. Voisin, J. Cherier, L. Ligat, F. Lopez, E. Noguer, A. Rives, B. Payre, T.A. Saati, A. Lamaziere, G. Despres, J.M. Lobaccaro, S. Baron, C. Demur, F. de Toni, C. Larrue, H. Boutzen, F. Thomas, J.E. Sarry, M. Tosolini, D. Picard, M. Record, C. Recher, M. Poirot, S. SilventePoirot, Dendrogenin A drives LXR to trigger lethal autophagy in cancers, Nat. Commun. 8 (2017) 1903. [7] F. Dalenc, M. Poirot, S. Silvente-Poirot, Dendrogenin a: a mammalian metabolite of cholesterol with tumor suppressor and neurostimulating properties, Curr. Med. Chem. 22 (2015) 3533–3549. [8] S. Silvente-Poirot, M. Poirot, Cancer. Cholesterol and cancer, in the balance, Science 343 (2014) 1445–1446. [9] S.A. Khalifa, P. de Medina, A. Erlandsson, H.R. El-Seedi, S. Silvente-Poirot, M. Poirot, The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells, Biochem. Biophys. Res. Commun. 446 (2014) 681–686. [10] P. de Medina, M.R. Paillasse, B. Payre, S. Silvente-Poirot, M. Poirot, Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases, J. Med. Chem. 52 (2009) 7765–7777. [11] S. Silvente-Poirot, G. Segala, M.C. Poirot, M. Poirot, Ligand-dependent transcriptional induction of lethal autophagy: a new perspective for cancer treatment, Autophagy 14 (2018) 555–557. [12] M. Poirot, R. Soules, A. Mallinger, F. Dalenc, S. Silvente-Poirot, Chemistry, biochemistry, metabolic fate and mechanism of action of 6-oxo-cholestan-3beta,5alpha-diol (OCDO), a tumor promoter and cholesterol metabolite, Biochimie 153 (2018) 139–149. [13] A. Fransson, P. de Medina, M.R. Paillasse, S. Silvente-Poirot, M. Poirot, M. Ulfendahl, Dendrogenin A and B two new steroidal alkaloids increasing neural responsiveness in the deafened guinea pig, Front. Aging Neurosci. 7 (2015) 145. [14] S. Silvente-Poirot, P. de Medina, M. Record, M. Poirot, From tamoxifen to
5. Conclusion The UPLC-MS method described herein is simple, specific and highly sensitive, in addition it includes an easy and fast step of sample 7
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