Khin Than Myint

Khin, Than Myint

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Study on Development of Highly Sensitive Determination Method of Bile Acid 3-Sulfates in Biological Fluids by Liquid Chromatography/Tandem Mass Spectrometry

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Study on Development of Highly SensitiveDetermination Method of Bile Acid 3-Sulfatesin Biological Fluids by LiquidChromatography/Tandem Mass Spectrometry著者 Khin Than Myint号 40学位授与番号 393URL http://hdl.handle.net/10097/36672γ 氏名(国籍) 学位の種類学位記番号学位授与年月日 学位授与の要件 研究科、専攻キンタンミン 紬諭丁簸鍛My翻博  士(薬学) 薬博第393号 平成19年3月27目 学位規則第4条第1項該当東北大学大学院薬学研究科 (博士課程)医療薬科学専攻学位論文題目 S翻yoηDevelop鵬en紛f}{ig謡ySe簸sltlveDe生ermi縦loηMet藍odofBile Acid3-Sui鉛立es沁Bl()logicalFlu遍sbyLlq腫i(iC最ro瓢atogra油y/Ta船em MassSpec重rome靭 (液体クロマトグラフィー/タンデム質量分析法によるヒト体液中 胆汁酸3一サルフェートの高感度分析法の開発に関する研究)論文審査委員  (主査)教授教授助教授一一巌 贋〔盾ハ藤齋木後安鈴 一74一」 一」』lr~, ---"-'/~~ ~~~ ~C ~~ ~, ~I Bile ~~cids, the mcholic acid (CA) C-7_4 carboxyl grthem undergo decoposition by the aacid (DCA), Iithoileum and the pFoxand therefore, onglycine- and tauriis su]fation ofthethe concentrationsdiseases, they may necessary, and I human urine and serum. In a LC/ESI-MS/Mthe interfering oinvestigated the than the pK~ valuvalues of acidic gRi ~2 R2 g~t. coR3 CA 3*s~i~ate CDCA 3-suii~ate DCA 3*s&~i~ate LCA 3-sul~ate UDCA 3-s~i~ate o~-OH o~*OH Hl H ~=0H oH X oH N H H03So ~~~~ H R1 R3 ~onami~ated fo~m Gbyci~~e-con~u~ated fo~m Tauri~e-conj~gated form OH ~XCH2COOH ~!XC~{2CH2S03N Fig. ~ Str~ctwre of b~le acid 3-S~~lfates 75 JL_ respectively, and therefore, the simultaneous analysis ofthree types ofbile acid 3-sulfates was impossible under the acidic mobile phase conditions. On the other hand, although their' re]ative retention l'actors were close each other in a neutral or a weak basic condition of the mobile phase, a]1 bi]e acid 3-sulfates used in this study were clearly separated. The retention order mainly depended on the number of hydroxy] groups except for L~)CA 3-sulfate, because its hydroxyl group at 7~ position interferes the hydrophobic interaction with the ODS group. The retention order was UDCA 3-sulfate < CA 3-sulfate < CDCA 3-sulfate < DCA 3-suifate < LCA 3-sulfate. Aithough weak alkaline pH mobi]e phase was more suitab]e for separation of all bile acid 3-su]fates, it may darna*'e the silica gel support. Therefore, the pH of mobiie phase was set to 7.0. Next, the electrospray ionization and collision induced dissociation (CID) of nonamidated, **lycine- and taurine-conjugated CDCA 3-suifates were investi*~*ated when ammonium acetate was used as a vOlatile salt in the mobile phase. The only slngiy charged deprotonated moleeule, [M-H]-, was produced from nonamidated forrn during ESI process. In the case of the glycine-conjugated foun, doubly charged deprotonated mo]ecules, ~M-'_H]2-, were also prodi~ced with [M-H]-, and taurine-conjugated form more efficiently produced a doubly charged deprotonated moiecuie. These resu]ts clearly indicated that the acidity of the ionic groups at the end of the side chain greatly infiuenced the ionization efficiency and the loca]ization of the charge. Next, I measured product ion mass spectra of [M-H]- for nonamidated and glycine-conjugated forms and [M-2H]2- for taurine-conjug'ated forrn ~s precursOr ions. The acidiL;- groups of the side chain strong!y influenced the CID fragnlentation. In the case of nonamidated form, HS04- and SO~-, derived from a sulfate group at C-3 position, were produced. On the other hand, glycine- and taurirle-ccnjugated forms produced steroid nuc]eus containing ions and the fragrnent ions related to the cleavage of the side chain moiety as we]1 as sulfate group-re]ated fragrnent ions. This may be due to the difference of charge localization. Afi:er the optimization of the CID condition for each forrn, HS04- formed from [M-H]- for nonamidated forms, [M-H-S03]- formed frorn [M-H]- for glycine-conjugates, and [M-H-H.SO~]~ formed from ~M-2H]2- for taurine-conjugates, respectively ~*/ere selected as the monitoring ions for SRM analysis. In mass spectrometry, the change of the ionization efficiency of the target compo~~nds decreases precision on the quantitative determination. Therefore, suitable internal standards (ISs), which have almost the same physicoche~nic~l properties as the target cornpoun~s, *~vere necessary for making a quantitative LC/ES~-MS/MS method, and I used stab]e isotopes-labeied cornpounds, 12-~l80,2H]-DCA 3-suifate and its glycine- and taurine-conjugates as ISs. The used of those ISs resu]ted in the wide linearity over ~00-fold for all bile acid 3-sulfates and good accuracy and precision of tbc method. The efficient c]e!~n-up procedure is also essential for good reproducible analysis because of removing much endogenous materia]s in biologicai sampies. I ernpioyed a polymer based hydrophi]ic-lipophilic balanced sorbcnt ~0asis HLB~ cartTidge for extraction of bile acid 3-s~:i]fates from human urine. The developed method has enough sensitivity and reproducibility for analysis of bile acid 3-su]fates in healthy human urine to analyze their dynamics. When the method was app!ied to analysis of bile acid 3-sulfates in urinc frorn 12 healthy voiunteers, they were - 76 -lr' ~L lr=, well separated without any interference by endogenous contaminants. Glycine- and taurine-conjugated secondary bile ~cid 3-sulfates were presented at high concentrations in every urine sample and nonamidated forrns were trace ievels. On the other hand, a re]atively strong" peak, v~'hich do not consistent with any peaks of standard materia]s, was clearly found on the SRM chromatogram corresponding to nonamidated and dihydroxy{ated bile acid monosulfate, e]uted between UDCA 3-su{fate and CDCA 3-sulfate. The estimated concentration of this unusual metabolite w~s approxim~rtely 100 ng/mL. I tried to identify the compound by the use of chromatographic and mass spectrometric properties coupled with microchemic~~1 reaction. fts ESI mass spectrum gave only one peak  at ;?1/_･-' 471 , and on]y one fragment ion lvas found at fn/.- 97 corresponding to the HS04- in the product ion mass spectrum. The result was very similar to those of nonamidated and dihydroxylated bile acid 3-sulfate. To confirrrl the existence of a su]fate group, the fraction corTesponding to the peak on the chromatograrn was chernica]1), solvolyzed, and the liberate compounds was ana]yzed by LC/ESI-MS. The retention time ofthe liberated compound completely corresponded with that of 3~,120~-dihydroxyl-5~-cholanoic acid (3-isoDCA), and the chromatographic behavior was also identical on the various mobile phases of the pH r2~r]ge from 4.0 to 8.5. Moreover, synthetic standard of 3-isODCA 3-sulfate (Fig. 2) gave the same retention time and mass spectra with the unusuai metabolite. HO g~P~ COOH H03so H Fig. 2 Str~lct~~re of 3-~~ODCA 3-s~~~fate The unusual metabolite in hurnan urine, 3-isoDCA 3-sulfate, was found as on]y nonamidated forTn, ~nd I did not find glycine- or taurine-conjugated 3-isODCA 3-sulfate in every samp]e. These facts motivated me to analyze and compare the concentrations of bile acid 3-sulfates in human urine and serum. Therefore, I validated the developed method as the method for ana]ysis of bile acid 3-sulfates in human serum. The result clearly showed that the method can analyze them without any matrix effects. Then, I analyzed bile acid 3-sulfates in both serum and urine from 14 healthy voiunteers. Glycine-conjugated forms were main components, and their concentrations reached approximately 800/0 of the total su]fated bile acid in serum. 3-lsoDCA 3-sulfate was detected in sera from 12 volunteers, and the concentration of 3-isODCA 3-sulfate was the highest among other nonamidated forms, which corresponded approximately 50/0 of total sulfated bile acids in serum: and urine. However, glycine- and taurine-conjugated 3-isODCA 3-su]fates were not found in any samples. In addition, the relative proportions of some bile acid 3-sulfates in urine were similar to those ;n serum, suggesting that urinary contents may reflect in - 77 -~L_ マ serumcon童e銀盃S. Tわedevebpedme伽dwasenable重oslmulta簸eouslydetermine18kindsofbiieacld3-sui飴tesw漁aneηough sensltivityinbumanserumandurine.丁賄eme幽・ds為・ulδbevaluabie£。runderstanding・ft強erela宅lons蜘・fbiie acid3-su亘餓esdynamicandvariousdiagηosesofbepa重oblllaryδiseases. 一78一ム7 審査結果の要旨 コレステ・一ルの異化代謝産物である胆汁酸は,通常肝臓を中心とする閉鎖的な系に局在しているが, 各種肝胆道疾患時には血中,尿中への排泄量が増大する.とりわけ3位水酸基に硫酸の結合したサル フェートの濃度が増大することが指摘されており,病態把握との関係から注目されている.これらサル フェートは,水溶性に富み,しかも構造が多岐に亘ることから,測定には液体クロマトグラフィー(LC) /タンデム質量分析法(MSIMS)が好適な手法と考えられる.本研究はこうした観点にたち,高感度測 定法の構築をはかるとともに,その生体試料への適用を試みたものである. 先ず,ヒト体内に存在すると考えられるサルフェート15種標品を用いて一斉分離の条件に吟味を加え た。ODS系の固定相を用いるとき,ステロイド核上の水酸基の数の多いサルフェートから順に溶出され たが,ウルソデオキシコール酸のみは他のジヒドロキシ胆汁酸より遙かに早く溶出された。これは,固 定相とステロイド核との問の疎水性相互作用が,軍位水酸基により阻害され,弱まった結果と考えられ る.一方,17位側鎖酸性官能基によっても溶出挙動は大きく変化し,移動相の液性が酸性になるに伴っ て遊離型,グリシン抱合型の溶出が順次遅くなり,タウリン抱合型はあまり変化しなかった。これは側 鎖官能基のgKaによるものと考えられた。このように得られた溶出挙動を基に一斉分離条件を設定した。 酸性化合物のイオン化にはエレクトロスプレーイオン化法(ES亙)が最も適当と考えられる.そこで, ES玉における効率的イオン化条件の設定に検討を加えた。その結果,遊離型では髄澱]一を,グリシン 並びにタウリン抱合型ではそれと伴に[M-H]2一を与え,特にタウリン抱合型では後者のイオンが効率 的に生成された。これは,イオン化室内で生成された液滴中における電荷移動に際し,緩衝液中の酢酸 イオンが影響し,側鎖酸性官能基の酸性度の強いものほど電荷移動を受けやすい結果と考えられる。さ らにCID条件を吟味し,固相抽出法並びに重酸素一重水素標識内標準物質と組み合わせる生体内サル フェートの高感度測定条件を確立した. 次いで,本法の健常人尿試料への適用を試みた。その結果,グリシン,タウリン抱合型サルフェート, 特に前者の相対的排泄量の多いことが判明した。一方,遊離型サルフェートは低濃度であるにもかかわ らず,ウルソデオキシコール酸とケノデオキシコール酸の問に未知のピークが強い強度で認められた。 そこで,MSにおける挙動を精査したところ,ジヒドロキシ胆汁酸3一サルフェートに相当することが 分かった。さらにこの異常胆汁酸のLCにおける溶出挙動を吟味するとともに,合成標品と比較し,3一 イソデオキシコール酸3一サルフェートであることを証明した.以上の結果から,さらに新規サルフェー トを含めた一斉分離条件を設定し,血液,並びに尿試料高感度測定法を確立し,これを健常人試料に適 用した。 以上,本研究は,胆汁酸3一サルフェートの高感度一斉分析法を構築し,ヒト生体試料への適用を計り, 胆汁酸研究に新たな知見を加えたものであり,博士(薬学)の論文として合格と認める. 一79一一 一L.

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液体クロマトグラフィー/タンデム質量分析法によるヒト体液中胆汁酸3-サルフェートの高感度分析法の開発に関する研究

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