Methodological approach to the intracrine study and estimation of DHEA and DHEA-S using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Please ci DHEA and http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology j ourna l h omepage: www.elsev ier .com/ locate / j sbmb Review Methodological approach to the intracrine study and estimation of DHEA and DHEA-S using liquid chromatography–tandem mass spectrometry (LC–MS/MS) YasuhiroQ1 a Department o b School of Hea a r t i c l Article history: Received 29 Ja Received in re Accepted 2 Ap Available onlin Keywords: DHEA LC–MS/MS Intracrine Isotope-labeled substrate derivatization technique compatible with each functional group for measuring 3�-hydroxy-5-enes, such as DHEA and 5�/5�-steroids, with high sensitivity and specificity is also described. Finally, we describe a newly developed method for intracrine research using stable isotope-labeled 13C-steroid substrates with tracer analysis of their metabolites by LC–MS/MS.This article is part of a Special Issue entitled’Essential role of DHEA’. © 2014 Elsevier Ltd. All rights reserved. Contents 1. Introd 2. Ioniza 2.1. 2.2. 2.3. 3. Deter 4. Analy 4.1. 4.2. 4.3. 5. Intrac 6. Bioact 7. Concl Refer ∗ Correspon E-mail add http://dx.doi.o 0960-0760/© 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 te this article in press as: Y. Shibata, et al., Methodological approach to the intracrine study and estimation of DHEA-S using liquid chromatography–tandem mass spectrometry (LC–MS/MS), J. Steroid Biochem. Mol. Biol. (2014), doi.org/10.1016/j.jsbmb.2014.04.002 uction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 tion and derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Methods of ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Chemical structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 mination of steroids using LC–MS/MS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 sis of biological samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Analysis of serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Analysis of saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Analysis of tissues and cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 rine study using stable isotope (13C or 2H)-labeled steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 ivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 usion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 ding author. Tel.: +81 27 220 8322/+81 27 220 8303; fax: +81 27 20 8318. ress: [email protected] (Y. Shibata). rg/10.1016/j.jsbmb.2014.04.002 2014 Elsevier Ltd. All rights reserved. Shibataa,∗, Seiji Araia, Seijiro Honmab f Urology, Gunma University Graduate School of Medicine, Maebashi, Japan lth Science, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan e i n f o nuary 2014 vised form 18 March 2014 ril 2014 e xxx a b s t r a c t A reliable and sensitive method for analyzing steroids using liquid chromatography tandem mass spec- trometry (LC–MS/MS) is required for research concerning dehydroepiandrosterone (DHEA), which plays a central role in steroid hormone biosynthesis and metabolism. Furthermore, after the first proposal of the concept of intracrine DHEA, stable isotope tracer analysis, which is useful for structural recognition as well as determination of steroids, has been required to evaluate physiological action and hormone biosynthe- sis/metabolism in target organs. We describe sample processing and analysis methods for simultaneous quantification of multiple hormones, including DHEA, in serum, saliva and tissue using LC–MS/MS. A Please ci app DHEA an etry http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 2 Y. Shibata et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx 1. Introduction In this section, we describe a new method of intracrine research utilizing the latest ultra-sensitive analytical technique for steroid hormones o (LC–MS/MS DHEA and i steroids an in humans. has been th metabolize cal actions precursor o the physiol and rodents adrenal gla the effect o animal mod on the phys cise method in a small a research. LC–MS/M performanc heat-labile, pounds tha By utilizing target subs LC–MS/MS derivatives has an adv precision in fields using ture sample the advant of very low and aldoste immunoass standard m utilized not but also for of endocrin In recent have been et al. prop logically ac [15]. Targe the brain a ied intracri between DH cancer tiss androstene radioactive have been identifying Therefore, w research us tracer analy 2. Ionizati 2.1. Method The ma of steroids atmospheric pressure photoionization (APPI) and electrospray ion- ization (ESI). Of these three ionization methods, ESI is easily the most sensitive for polar steroids and derivative steroids [19]. Although most steroids, including DHEA, are easy to ionize by e-mo s su ), es SI af e hig and nol ra gativ inat ckgro s spe d ne ster I is u PPI, s the vely, emic roids tane acco iffer and h tero roid h be n is cies ne, a r low I. The er li eriva mpro stero he ta drox ) or igh s e eth uctu o re tion carb roid imin tizati se hy evin /5�- was ts, in d tra tion s qu rfere 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 te this article in press as: Y. Shibata, et al., Methodological d DHEA-S using liquid chromatography–tandem mass spectrom f liquid chromatography–tandem mass spectrometry ), with a focus on dehydroepiandrosterone (DHEA). ts sulfated form (DHEA-S) are the most common serum d are mostly produced by the adrenal zona reticularis Although DHEA is known to decrease with age and ought of merely as an intermediate product that is d into active estrogens or androgens, new physiologi- of DHEA have recently received attention, since it is a f androgens [1,2]. However, it is difficult to determine ogical actions of DHEA in animal models, such as dogs , since serum DHEA (or DHEA-S) is produced not by the nds but by the gonads, making it impossible to exclude f testosterone from physiological androgenic actions in els [3]. Thus, human samples are required for studies iological actions of DHEA in humans. Moreover, a pre- for the simultaneous quantitation of several steroids mount of sample is also essential for this physiological S can easily separate quantitative targets by high- e liquid chromatography (HPLC) and can measure even non-volatile substances or isomeric steroidal com- t are difficult to measure by gas chromatography. the product ions derived from molecular ions for tances and stable isotope-labeled internal standards, can simultaneously measure targets and their chemical with high specificity and sensitivity. Thus, LC–MS–MS antage over immunoassay in terms of accuracy and the measurement of steroid levels in basic research blood, tissue (organ, hair or nail), saliva or tissue cul- s [4–8]. Many clinical researchers also have indicated age of LC–MS/MS in the clinic for the measurement levels of steroids, such as estradiol, testosterone rone, which often show higher values in traditional ays [9–11]. Hence, LC–MS/MS analysis has become the ethod for measuring steroids in the clinical field and is only for the medical treatment of endocrine diseases, the study of psychogenic depression or adverse effects e disruptors [7,12–14]. years, steroidogenesis and metabolism in target organs the center of attention in clinical research. Labrie osed the mechanism of local production of physio- tive hormones in target organs as “intracrinology” t organs of DHEA and their normal functions (e.g., nd memory, the skin and blood flow) are being stud- nologically [16,17]. The intracrinological relationship EA and disease progression is mostly studied in breast ues, where the conversion of DHEA to estrogen via dione contributes to disease progression [18]. Although steroids have been used for intracrine research, there several problems, such as difficulties in purifying and the radioactive substances because of their low levels. e describe a newly developed method for intracrine ing stable isotope-labeled 13C-steroid substrates and sis of their metabolites by LC–MS/MS. on and derivatization s of ionization in ionization methods used for LC–MS/MS analysis are atmospheric pressure chemical ionization (APCI), positiv steroid sulfate mode E to hav tures, metha The ne determ the ba of mas tive an of both APC over, A enable sensiti 2.2. Ch Ste andros (C27)— tural d group these s ene ste are hig a proto efficien nenolo of thei or APC the low 2.3. D To i target ity to t The hy (Fig. 1a gives h a stabl the str likely t as func The one ste to give deriva The in achi and 5� sitivity reagen (cis an separa plicate to inte doi.org/10.1016/j.jsbmb.2014.04.002 roach to the intracrine study and estimation of (LC–MS/MS), J. Steroid Biochem. Mol. Biol. (2014), de ESI after acetonitrile-methanol-based HPLC, several ch as steroid sulfates (including DHEA-S and estrogen trogen and aldosterone, are also ionized by negative- ter HPLC [11,20]. These negatively ionized steroids tend her log P values and acidic groups within their struc- the response is observed to be generally higher when ther than acetonitrile is used as the elution solvent [21]. e ion mode in LC–MS/MS was recently used for steroid ion in organism samples due to its ability to reduce und matrix level. Moreover, the recent development ctrometers with high-speed switching between posi- gative ion modes enabled simultaneous determination oids and their sulfates [20]. sed for steroids that are difficult to ionize by ESI. More- which was recently developed as an alternative to APCI, mass to charge ratios of samples to be analyzed more compared with APCI [22]. al structures are divided into to five skeletal types—estrane (C18), (C19), pregnane (C21), cholane (C24) and cholestane rding to the number of C-atoms. Characteristic struc- ences, such as the position of double bonds, carbonyl ydroxyl groups, influence the ionization efficiencies of ids. For instance, the ionization efficiencies of 3-oxo-4- s, such as androstenedione, progesterone and corticoid, cause they possess a 3-oxo-4-ene structure to which easily added by ESI. On the other hand, the ionization of 3�-hydroxy-5-ene steroids, such as DHEA and preg- nd of 5�/5� androstane and pregnane are low because proton affinities, resulting in poor responses using ESI sensitivity of estimation for these steroids is low, with mit of the estimation range being 50–100 pg [23,24]. tization ve sensitivity, derivatization of the functional groups of ids was achieved by adding a moiety with proton affin- rget steroids using reagents, as shown in Table 1 [25]. yl groups of DHEA or DHT are reacted with picolinic acid fusaric acid (Fig. 1b), and the ester derivative obtained ensitivity [26,27]. The reagent pyridinium (Fig. 1c) gives er derivative [28]. Specifically, the hydroxyl groups in res of several steroids, including DHEA (position 3), are act with reagents with electron-donating groups, such al nitrogen and acidic groups (Fig. 1). onyl groups of DHEA, pregnenolone, DHT or 4-ene-3- s are reacted with 2-hydrazino-1-methylpiridine (HMP) o derivatives [23,29,30]. The other reagents are used for on [31]. droxyl and carbonyl group derivatives are very effective g the high sensitivity required to detect 5-ene steroids reduced steroids. A 20–200-fold improvement in sen- achieved for the target steroid DHEA. However, most cluding HMP and hydroxylamine, form two isomeric ns) products with 3-oxo-steroid. The chromatographic of DHEA derivatives from these isomers not only com- antitative analysis but also increases the susceptibility nce by endogenous compounds. 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 Please ci DHEA and http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 Y. Shibata et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx 3 Table 1 Effect of derivatization on steroid measurements using LC–ESI–MS/MS. Function Reagent Ion Mode Low limit quantitation Field Ref Intact Derivative Dehydroepiandrosterone (DHEA) OH PIC + 50–100 2 25–50 8 OH FUS + 50–100 0.5 100–200 28 Carbonyl HMP + 2000 1 2000 24 Carbonyl etylamine + 50–100 3 15–30 32 – – 50–100 4 Dihydrotestosterone (DHT) OH PIC + 50–100 1 50–100 27 Carbonyl HMP + 50–100 20 2–5 31 Carbonyl HTP + 50–100 4–10 10 32 – – 50–100 27 Testosterone OH PIC + 10 1 10 27 OH FUS + 10 0.5 20 28 Carbonyl HMP + 40 4 10 24 Carbonyl HTP + 10 1 4 31 Carbonyl etylamine + 10 3 3 32 Androstenedione DHEA-Sulfate Carbonyl etylamine + 10 3 3 45 – – 10 7 – – 10–20 39 3. Determination of steroids using LC–MS/MS analysis As suggested by FDA, validation of assay accuracy, precision, reproducibility and quantification range, and of sample stabil- ity, are required for analytical methods such as immunoassay, GC–MS and LC–MS/MS [32]. Even though validated immunoas- says have been used for steroid research, it is well known that the dose-response curves for immunoassay data are non-linear at the quantification limit. Thus, low levels of steroid hormones are usually measured in a section of the calibration curve where the variance is high. Moreover, matrix effects, such as lipids in tissues and the cross-reaction of antibodies with non-target steroids that have similar structures, are also well known to limit immunoassay methods. In contrast, LC–MS/MS calibration curves are linear, and consequently LC–MS/MS avoids the bias observed at low steroid hormone levels in the calibration curves. The most common tandem mass spectrometer consists of a triple quadrupole. After HPLC separation, the sample steroids are applied to the first quadrupole, which monitors the precursor ions of the sample steroids. Then, the second quadrupole (collision cell) performs collision between the sample steroids and a collision gas. Finally, the third quadrupole monitors the product ions of the sam- ple steroids. To develop a new determination method, a targeted standard reference sample and its stable isotope-labeled internal standard are separately subjected to LC–MS/MS analysis, and then its precursor and product ions are monitored. The masses of the precursor and product ions represent fundamental properties of the molecules: molecular weight and structure. Reliance on these Fig. 1. Deriva N-methylpyrid Hydroxylamin 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 te this article in press as: Y. Shibata, et al., Methodological app DHEA-S using liquid chromatography–tandem mass spectrometry doi.org/10.1016/j.jsbmb.2014.04.002 tive reagents widely used for hydroxyl and carbonyl groups in steroids in LC–MS inuium. (d) Pyridine. (e) 2-Hydrazino-1-methylpyridine. (f) 2-Hydrazino-4-(trifluoro e. roach to the intracrine study and estimation of (LC–MS/MS), J. Steroid Biochem. Mol. Biol. (2014), /MS estimation. (a) Picolinic acid. (b) Fusaric acid. (c) 2-Fluoro- methyl)-pyrimidine. (g) Girard’s reagent P. (h) Methoxyamine. (i) Please ci app DHEA an etry http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 4 Y. Shibata et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx fundamental properties is the basis for the high degree of analytical specificity of LC–MS/MS. Due to the highly specific and sensitive quantification of target samples, specific transition ions (precur- sor/product ion pairs) that consist of each partial structure of the sample ster over, appro to LC–MS/M After a cali ratio (targe targeted sa calibration 4. Analysis 4.1. Analys LC–MS/M Moreover, formed rela (0.2–0.4 mL to examine validation ( the approxi sample num was perform ing the lowe spiked with high). Tests reproducibi mine the lo were within within 15%. Since hu tions, the using organ column, an taneously w can be mea more cost-e steroids sho (100 �L), as surement a target stero enables sim ple steroids 11� of hydr ries. DHEA l is therefore tized using using a soli be measure In anoth human seru and the su DHEA-S an simultaneo and free co ions, respec 4.2. Analys Salivary be collected to adulthoo of several s children and chimpanzees, because saliva, in contrast to blood, can be sampled painlessly [35]. Almost all serum steroid hor- mones are bound to proteins such as corticoid-binding globulin (CBG), sex hormone-binding globulin (SHBG) and albumin. By con- –5% nd to eme tion nes ce, si r dia o us ine d idatio d as ne-fr twee rmin male cifici , sin matr pg/m y de saric s ana on in diffe EA is at th ent r egar sure tion, alys mon tion to ab equa an a sotop s en . It is spec e sho s an ts. E ding is of shou e, pr mou reco In so form me eigh eniz IKA, S dded l (3 m itate d (at ator 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 te this article in press as: Y. Shibata, et al., Methodological d DHEA-S using liquid chromatography–tandem mass spectrom oids and their internal standard are essential. More- priate HPLC conditions and sample pretreatment prior S are also required for highly sensitive quantification. bration curve is constructed by plotting the peak area ted sample/internal standard) against the amount of mple, the level of the sample is determined using this curve, and validation is carried out subsequently. of biological samples is of serum S is over 10 times as sensitive as radioimmunoassay. it has better precision, and validation can be per- tively easy. First, charcoal-treated hormone-free serum ) and serum of analytical volume (or greater) were used specificity and sensitivity by performing simple pre- n = 1–2) of the reproducibility in order to determine mate lower limit of quantification (LOQ) [25]. Then, the ber was increased, and a test for repeatability (n = 5) ed to evaluate the accuracy and precision for determin- r LOQ. This repeatability test used hormone-free serum three known levels of the target hormone (low, middle, for within-run reproducibility (n = 5) and between-run lity (n = 5, 3 different days) were performed to deter- wer LOQ. The accuracy and precision at the lower LOQ 20%, and all levels except for the low level were set to man serum has relatively high hormone concentra- steroidal fraction was extracted from 200 �L serum ic solvent. The extract was purified using a solid-phase d then 5–10 steroids were measured directly ad simul- ithout derivatization [33,34]. Since multiple steroids sured simultaneously using LC–MA/MS, the method is ffective than immunoassay. However, when multiple uld be measured using only a small amount of serum in infants and neonates, it is best to perform the mea- fter picolinic acid (or fusaric acid) derivatization of the ids [7]. This method minimizes the matrix effect and ultaneous and highly sensitive measurement of multi- all bearing hydroxyl groups (except three classes and oxy groups). It is used in general examination laborato- evels in animal serum are very low. The steroid fraction collected using a solid-phase column and then deriva- picolinic acid or fusaric acid. Excess reagent is removed d-phase column, and the DHEA derivatives can finally d with high sensitivity. er report concerning the measurement of DHEA in m, 100 �L serum was deproteinized by adding ZnSO4, pernatant was purified using a solid-phase column. d non-conjugated components were then measured usly using positive ions [19]. In another report, DHEA-S mponents were measured using negative and positive tively [20]. is of saliva hormone analysis is gaining attention, since saliva can simply and non-invasively at any time from infancy d. Recently, a method for simultaneous determination teroids in saliva using LC–MS/MS was developed for trast, 1 unbou an extr centra hormo instan used fo It is als endocr Val metho hormo and be to dete and fe of spe matrix in the (30–80 ficity b and fu Thi study gender DH group surem In r be mea teiniza 4.3. An Hor conven order to inad obtain using i tration tissues biopsy Car human stituen depen analys effect adipos large a effect, bility. be per The ously w homog nizer ( were a ethano precip trifuge evapor doi.org/10.1016/j.jsbmb.2014.04.002 roach to the intracrine study and estimation of (LC–MS/MS), J. Steroid Biochem. Mol. Biol. (2014), of hormones are secreted in saliva as their free form, protein. The salivary hormone concentration shows ly good correlation with the total serum hormone con- [7,36]. For this reason, the measurement of salivary is employed in various studies and examinations. For multaneous analysis of cortisol and DHEA (DHEA-S) is gnosis of and treatment decisions in depression [13,14]. ed for epidemiological surveys of residents to evaluate isruption caused by dioxins [7,37]. n of saliva analysis was conducted using the same that for serum analysis, with 1.0 mL charcoal-treated ee saliva. Tests for within-run reproducibility (n = 5) n-run reproducibility (n = 5, 3 days) were performed e the lower LOQ. At least 10 samples (n = 5 for males s each) should be included for adequate examination ties and preservation of the stability of the salivary ce there is quite a high degree of individual variation ix. The salivary concentration of DHEA is relatively low L), but it can be analyzed adequately with high speci- rivatizing it to ester derivatives, such as picolinic acid acid. lytical method has been employed in an epidemiological fant growth and hormones, and the results showed a rence [37]. measured as an HMP derivative as it has a carbonyl e C-17 position. Sufficient validation within the mea- ange was performed as for serum [38]. d to sulfate in saliva, it was reported that sulfate could d all at once using ESI in negative ion mode after depro- similar to the serum assay [39,40]. is of tissues and cell cultures e quantification in very small amounts of tissue using al immunoassays requires sufficient purification in olish the effect of impurities. This purification leads te recovery and correction, which makes it difficult to ccurate analytical result. On the other hand, LC–MS/MS e dilution analysis to quantify tissue hormone concen- ables simple and precise analysis of various kinds of now used for the hormone analysis of prostate needle imens in amounts as low as 10 mg. uld be taken when handling the same organs from d animals, since there are differences in matrix con- ven in human tissues, the matrix affects the analysis on the kind of target steroid measured. Thus, in the very small amounts of tissue (e.g., 10 mg), the matrix ld be examined thoroughly. Generally, the analysis of ostate and mammary tissues, which can be obtained in nts, requires partial validation concerning the matrix very rate of reference standards and preservation sta- me cases, full validation with repeatability tests should ed, as for serum. thod for quantification analysis is as follows. Previ- ed adipose tissue samples were cut with scissors and ed in water (10 mL/g) using an Ultra-Turrax homoge- taufen, Germany) with cooling. Internal standards (ISs) to the homogenate, which was then extracted with L, 50 ◦C, 1 h), and then rested for over 2 h at 10 ◦C to the protein completely. The homogenate was then cen- 3000 × g), and the solvent was evaporated on a rotary . The residue was diluted with purified water (1 mL) and 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 Please ci app DHEA and etry http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 Y. Shibata et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx 5 extracted with EtOAc (4 mL). The organic solvent was distilled, and the extract was dissolved in 30% methanol (1 mL) and then loaded onto a cartridge column and purified. This purification step using a reverse-phase column cannot be omitted, since it removes over 90% of impu was reacted and then pr The quan tissues to o Prostate terone/dihy prostate can Reports by results [8,4 prostate ca concentrati that the pr in a single castration-r primary and of DHEA, w but the me important f on CRPC [42 Mamma of DHEA to described it thesized int the postme Hair and also accum to determin to stress or a week or a formed bef are thought For nai extracted f purified usi measured u [4]. For hair methanol fo measured u with age, bu Skin: Ma ship with s C19 steroid the skin has and activity StAR, which 5. Intracri steroids The fact in prostate higher than produced in conclusion labeled ste enzymes, su dehydrogen and pathol target organs will facilitate the development of new drugs that regulate the functions of those steroidogenic enzymes. Recently, biosynthesis of 13C-androstenedione and 13C-17- hydroxyprogesterone from the 13C stable isotope-labeled substrate oges exi y) in thesi ed a hu ed w [48] e ex lso r re, D mou itself [45,4 atme eir m rel ype inolo ne b e tiss reme jecti ]. met as fo bath ffer ( hom atan ugat teron e wa ction hen a . The stero 17 a the D e bi edge dipo and activ adre d is prod circa ) [52 , it is , 17-h m (1 he fi the inat entio g CR ired vene 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 te this article in press as: Y. Shibata, et al., Methodological DHEA-S using liquid chromatography–tandem mass spectrom rities in the matrix (mostly fats). The residue obtained with the derivatizing agent to obtain target derivatives ocessed in the same way as for serum analysis [26,41]. titative analytical method can be used in the following btain new knowledge. : Measurement of the tissue testos- drotestosterone ratio (T/DHT) by LC–MS/MS is used for cer diagnosis and the evaluation of its aggressiveness. Shibata et al. and Arai et al. showed detailed validation 1]. Nishiyama et al. reported that the aggressiveness of ncer can be determined by measuring the tissue DHT on at diagnosis [28]. Further, Shibata et al. suggested e-treatment evaluation of prostate androgen content needle biopsy specimen may help to predict future esistant prostate cancer (CRPC) development after rogen deprivation therapy [41]. The physiological role hich is abundant in the prostate, has not been clarified, asurement of DHEA in tissue has been suggested to be or evaluating the therapeutic effect of CYP17 inhibitors ]. ry gland: Many researchers confirmed the conversion estrogen via androstenedione in mammary tissue and as intracrine. In particular, the amount of DHEA syn- racrinologically has been highlighted as important for nopausal development of breast cancer [18]. nail: Steroids exists in hair and nails, and blood steroids ulate in these tissues as they grow. Thus, it is possible e changes in hormone concentration under exposure chemical agents over a certain period of time (e.g., month) [4,5]. However, large studies should be per- ore their clinical use, since lights and hair preparation to denature or decompose hormones. l hormone determination, steroid hormones are rom 2 to 15 mg of nails using buffer (45 ◦C, 2 h) and ng a solid-phase column. DHEA and cortisol are then sing positive and negative ions, as reported previously hormone determination, 20 mg of hair is soaked in r 7 days. DHEA-S, testosterone and estrogen are then sing negative ions. There was no difference in DHEA-S t a gender difference was found [5]. ny hormones are present in the skin, and a relation- kin blood flow has been suggested. The existence of s, such as DHEA, and C18 steroids, such as estrogen, in been confirmed. Inoue et al. reported gene expression of steroidogenic enzymes, and existence of the cofactor participates in the biosynthesis of steroids [17]. ne study using stable isotope (13C or 2H)-labeled that the levels of several physiologically active steroids , brain, mammary, lung, skin and adipose tissues are those in the serum suggests that those steroids are tracrinologically in target organs [15–17,43–47]. This is supported by in vitro studies using radioisotope- roids that verified the functions of steroidogenic ch as 5�-reductase, aromatase and 3�-hydroxysteroid ase in target organs. Elucidation of the physiological ogical effects of those steroidogenic enzymes in the 13C -pr ing the activit biosyn confirm ities in detect Fig. 1d Gen were a thermo large a tissue organ the tre date th The nase t intracr cortiso adipos measu after in sue [51 The sues is an ice Tris bu further Supern centrif proges mixtur the rea were t umes) tissue CYP tion of from th knowl sis in a cancer 6. Bio The tion an DHEA, small decade infants terone of seru In t ence in contam ing att treatin is requ effecti doi.org/10.1016/j.jsbmb.2014.04.002 roach to the intracrine study and estimation of (LC–MS/MS), J. Steroid Biochem. Mol. Biol. (2014), terone was confirmed in human adipose tissue, prov- stence of CYP17 activity (both hydroxylase and lyase adipose tissue (Fig. 2a and b). As shown in Fig. 2c, the s of 13C-estrone from 13C-androstenedione was also using LC–MS/MS [45]. Separately, 5�-reductase activ- man castration-resistant prostate cancer cell line were ith 13C-androstenedione using LC–MS/MS, as shown in . pression and enzyme activities of CYP17 and CYP19 eported in adipose and prostate tissues [45,47]. Fur- HEA, androstenedione and estrone were detected in nts in adipose tissue and may be biosynthesized in the , suggesting that adipose tissue is another intracrine 7]. CYP17 inhibitors are currently being developed for nt of CRPC, and this analytical method is used to eluci- echanisms of action [49,50]. ationship between 11�-hydroxysteroid dehydroge- 1 (11�-HSD1) and obesity has been well researched gically (Fig. 2e). Because cortisol is synthesized from y 11�-HSD1, intracrinologically produced cortisol in ue is distinguished from serum-derived cortisol by the nt of deuterium-labeled cortisol in the adipose tissue on of deuterium-labeled cortisone into the adipose tis- hod used to prove the biosynthesis of steroids in tis- llows. The tissue samples were homogenized for 3 s in using an Ultra-Turrax homogenizer, after which 0.1 M pH 7.6, 0.5 g/mL) was added. The resulting mixture was ogenized uniformly using a Teflon-glass homogenizer. t was obtained from the homogenate by refrigerated ion (1000 rpm for 5 min, 4 ◦C). NADPH (10 mg) and 13C3- e (0.5 �M) were added to the supernatant, and the s incubated at 37 ◦C for a specific period of time. After , the mixture was placed on ice, and internal standards dded. The mixture was extracted with EtOAc (five vol- extract was purified using the same method as that for id analysis as described previously [45]. ctivity can be analyzed by a similar method via estima- HEA level using 13C-pregnenolone. The results obtained osynthetic analysis will provide us with important new concerning the relationship between steroid biosynthe- se tissue and various diseases, such as obesity, breast hypertension. ity nocortical hormone cortisol has wide circadian varia- greatly affected by stress, but not by age. By contrast, uced in the fascicular zone of the adrenal cortex, has dian variation but decreases with age (by 25% in a ,53]. To examine growth and the adrenals/gonads in essential to evaluate DHEA, androstenedione, testos- ydroxyprogesterone, simultaneously in a small volume 00 �L) [34]. eld of hormone-disrupting chemicals, a gender differ- DHEA concentration in children raised on breast milk ed by environmental dioxins was reported and is receiv- n [37]. Recently, CYP17 inhibitors were developed for PC, and measurement of cortisol, DHEA and DHEA-S to monitor the side effects [49,50]. Furthermore, the ss of DHEA and DHEA-S replenishment in preventing 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 Please ci DHEA an http://dx. ARTICLE IN PRESSG ModelSBMB 4183 1–7 6 Y. Shibata et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx Fig. 2. Steroid 19 or dehydrogenas http://www.te aging and l monitoring 7. Conclus We have tance of v derivatives including 3 and tissues evaluating 13C-steroid metabolites References [1] T.J. Fabia M.D. Zmu of DHEA Biol. Psyc [2] V.G. Kimo droepian neurons a Sci. U.S.AQ2 [3] B. Belang of residua pig after Steroid B [4] M.B. 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Terry, T. San- M. Bunker, A.R. Phanslkar, W.E. Owen, W.L. Roberts, Adrenal steroid ation in children seven to seventeen years of age, J. Pediatr. Endocrinol. (2007) 1281–1291. 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 Methodological approach to the intracrine study and estimation of DHEA and DHEA-S using liquid chromatography–tandem mass ... 1 Introduction 2 Ionization and derivatization 2.1 Methods of ionization 2.2 Chemical structures 2.3 Derivatization 3 Determination of steroids using LC–MS/MS analysis 4 Analysis of biological samples 4.1 Analysis of serum 4.2 Analysis of saliva 4.3 Analysis of tissues and cell cultures 5 Intracrine study using stable isotope (13C or 2H)-labeled steroids 6 Bioactivity 7 Conclusion References