JP7193114B2 - Observation method of bile acid cycle - Google Patents
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Description
本発明は、胆汁酸サイクルの経時的変化を観察する、胆汁酸サイクルの観察方法に関する。 The present invention relates to a bile acid cycle observation method for observing changes over time in the bile acid cycle.
食物、腸内細菌叢及び宿主の三者は、相互に深い影響を及ぼし、個々人の健康・栄養状況に影響を与えている。食物及び特定の栄養素は、個々の生体環境からどのように影響を受け消化吸収のプロセスを経るのか、また細胞・組織にどのような代謝の影響を及ぼすのかを理解するには、個体レベルでの代謝情報の解析が必要である。 Food, intestinal microbiota, and the host have a profound effect on each other and affect the health and nutritional status of individuals. In order to understand how food and specific nutrients are affected by the individual biological environment and go through the process of digestion and absorption, and what kind of metabolic influence on cells and tissues, it is necessary to study at the individual level. Analysis of metabolic information is required.
胆汁酸は、肝臓にてシトクロムP450の作用でコレステロールを酸化することにより産生される。胆汁酸は、タウリン、アミノ酸であるグリシンと結びついて、あるいは硫酸塩、グルクロン酸として、脱水により塩にまで濃縮されて胆嚢に蓄えられる。食事をすることにより、胆嚢に蓄えられた抱合胆汁酸は腸内に分泌され、食物脂肪の乳化を促進する。胆汁酸のその他の役割としては、体からコレステロールを排出すること、肝臓から異化生成物を胆汁分泌の際に排出すること、乳化した脂質と脂溶性ビタミンを腸内でミセル化して乳糜管系から吸収させること、界面活性剤として細菌の細胞膜を溶解する作用によって小腸内や胆管での腸内細菌叢の形成を妨げることなどが挙げられる。 Bile acids are produced in the liver by oxidizing cholesterol through the action of cytochrome P450s. Bile acids are concentrated to salts by dehydration and stored in the gallbladder as taurine, combined with the amino acid glycine, or as sulfates and glucuronic acid. Upon eating, conjugated bile acids stored in the gallbladder are secreted into the intestine and facilitate the emulsification of dietary fats. Other functions of bile acids include excretion of cholesterol from the body, excretion of catabolites from the liver during bile secretion, micellization of emulsified lipids and fat-soluble vitamins in the intestine and removal of them from the lacteal system. Absorption and inhibition of the formation of intestinal flora in the small intestine and bile ducts by the action of dissolving bacterial cell membranes as a surfactant.
近年、質量分析法を用いたメタボローム解析に注目が集まっているが、個体レベルの代謝についての研究手法は未開拓である。イメージング質量分析法(imaging mass spectrometry IMS)は、プローブを用いずに対象分子そのものを直接イオン化、検出することで、組織切片に存在する生体分子の分布を可能にした新技術である(特許文献1,2)。 In recent years, attention has been focused on metabolome analysis using mass spectrometry, but research methods on individual-level metabolism have not yet been developed. Imaging mass spectrometry IMS is a new technology that enables the distribution of biomolecules present in tissue sections by directly ionizing and detecting target molecules themselves without using probes (Patent Document 1 , 2).
この分析法は物質固有の質量を用いて可視化させるため、詳細な局在を捉えることができる。近年様々なイオン化法及び分離分析法を組み合わせた質量分析装置による可視化が試みられている。その中でもマトリックス支援レーザー脱離イオン化法(Matrix Assisted Laser Desorption / Ionization :MALDI)と飛行時間型質量分析計(Time of Flight Mass Spectrometry :TOF-MS)を組み合わせたIMSの報告が多くなってきている。MALDI-TOF-MSは汎用性の高い装置であり、検出できる物質の適応範囲や測定質量領域が広い。 Since this analysis method visualizes using the substance's peculiar mass, it is possible to capture detailed localization. In recent years, attempts have been made to visualize using a mass spectrometer that combines various ionization methods and separation analysis methods. Among them, there are increasing reports of IMS, which combines matrix-assisted laser desorption/ionization (MALDI) and time-of-flight mass spectrometry (TOF-MS). MALDI-TOF-MS is a highly versatile instrument with a wide range of detectable substances and a wide measurement mass range.
本発明は、胆汁酸サイクルの経時的変化を観察する、胆汁酸サイクルの観察方法を提供することを目的とする。 An object of the present invention is to provide a method for observing the bile acid cycle, which observes changes over time in the bile acid cycle.
本発明にかかる胆汁酸サイクルの観察方法は、胆汁酸を構成する所定化合物についてイメージング質量分析を行うことにより、胆汁酸サイクルの経時的変化を観察することを特徴とする。 A method for observing a bile acid cycle according to the present invention is characterized by observing changes over time in the bile acid cycle by performing imaging mass spectrometry on a predetermined compound that constitutes bile acid.
本発明によれば、個体レベルでの胆汁酸サイクルの経時的変化を観察でき、これにより胆汁の***障害や胆汁の分泌異常等をいち早く発見することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to observe temporal changes in the bile acid cycle at the individual level, and thereby to quickly discover bile excretion disorders, bile secretion abnormalities, and the like.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. The embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is as follows. The scope of the present invention is not limited to the embodiments, and other embodiments in which the configurations of the following embodiments are appropriately replaced by those skilled in the art are also included in the scope of the present invention.
本実施形態にかかる胆汁酸サイクルの観察方法は、胆汁酸を構成する所定化合物についてイメージング質量分析を行うことにより、胆汁酸サイクルの経時的変化を観察する。 The bile acid cycle observation method according to the present embodiment observes changes over time in the bile acid cycle by performing imaging mass spectrometry on a predetermined compound that constitutes bile acid.
図1に示されるように、イメージング質量分析装置900は、試料導入部100と、例えばMALDI(Matrix Assisted Laser Desorption Ionization)であるイオン源200と、例えば飛行時間型質量分析計TOF-MS(Time-of-Flight Mass Spectrometry)である分離分析部300と、データ処理部400と、からなる。試料導入部100で試料が装置に導入され、イオン源200で試料がイオン化され、分離分析部300でイオンが質量の違いによって分離されて検出され、データ処理部400でデータ処理される。
As shown in FIG. 1, an
胆汁酸サイクルは以下である。即ち、まず胆汁酸は肝臓でコレステロールから合成され、胆汁の主成分として胆嚢・胆管を経て十二指腸に分泌される。肝細胞で生成された一次胆汁酸のコール酸とキノデオキシコール酸は、小腸内で腸内細菌により脱抱合・脱水素化などの代謝を受け、それぞれに二次胆汁酸のデオキシコール酸とリトコール酸に代謝される。その後、一次胆汁酸と同様に二次胆汁酸も小腸で再吸収され、合わせて胆汁酸の約95%が門脈を経由して肝臓に戻り再び胆汁中に分泌される。 The bile acid cycle is First, bile acid is first synthesized from cholesterol in the liver and secreted into the duodenum via the gallbladder and bile duct as the main component of bile. Cholic acid and quinodeoxycholic acid, which are primary bile acids produced in hepatocytes, undergo metabolism such as deconjugation and dehydrogenation by intestinal bacteria in the small intestine, and are converted into secondary bile acids, deoxycholic acid and lithocholic acid, respectively. metabolized to acid. After that, the secondary bile acids are reabsorbed in the small intestine as well as the primary bile acids, and about 95% of the total bile acids are returned to the liver via the portal vein and secreted again into the bile.
胆汁酸は一次胆汁酸又は二次胆汁酸の何れでも良いが、好ましくは一次胆汁酸である。一次胆汁酸は肝臓で生合成されたものいい、例えばタウロコール酸、グリココール酸又はコール酸である。二次胆汁酸は、一次胆汁酸の一部が腸管で微生物による変換を受けた代謝物いい、例えばデオキシコール酸、リトコール酸、ヒオデオキシコール酸である。 The bile acid may be either primary bile acid or secondary bile acid, preferably primary bile acid. Primary bile acids are those biosynthesized in the liver, eg taurocholic acid, glycocholic acid or cholic acid. Secondary bile acids refer to metabolites in which a portion of primary bile acids has undergone microbial conversion in the intestinal tract, such as deoxycholic acid, lithocholic acid, and hyodeoxycholic acid.
(動物)
動物実験は、同志社大学動物実験委員会の審査を受け同動物実験指針にのっとり施行した。実験にはICRマウス(哺乳1日目(P1)、清水実験材料)、ICRマウス(4週齢、雌、清水実験材料)を用いた。P1マウスはIsoflurane(Mylan)で麻酔を行い、液体窒素で急速凍結した。4週齢マウスは、頚椎脱臼を行い、皮を剥ぎ、液体窒素で急速凍結した。
(animal)
The animal experiments were reviewed by the Doshisha University Animal Experiment Committee and were conducted in accordance with the guidelines for animal experiments. For the experiment, ICR mice (first day of suckling (P1), Shimizu experimental materials) and ICR mice (4-week-old, female, Shimizu experimental materials) were used. P1 mice were anesthetized with Isoflurane (Mylan) and snap frozen in liquid nitrogen. Four-week-old mice underwent cervical dislocation, skinned, and snap frozen in liquid nitrogen.
(P1マウス腹部の組織形態解析)
凍結したP1マウスを固定し、クライオスタット(Leica CM3050)で10μm厚ごとに薄切していき、それぞれ断面をデジタルカメラで撮影した(図2)。また、クライオスタットで10μm厚切片を作成し(庫内温度-22℃:、試料台温度:-20℃)、HE染色を行った。
(Histomorphological analysis of P1 mouse abdomen)
A frozen P1 mouse was fixed, sliced into 10 μm-thick slices with a cryostat (Leica CM3050), and each section was photographed with a digital camera (FIG. 2). Also, a 10 μm-thick section was prepared with a cryostat (inside temperature: −22° C., sample table temperature: −20° C.), and HE staining was performed.
(HE染色)
標本を、PBSを溶媒とする4% PFA(Wako)に30分間浸漬して、流水で5分間洗浄した。その後、hematoxylin(CERTISTAIN)に5分間浸漬して、同様に5分間洗浄した。HCl(Wako)を0.1%含む70% ethanolで2秒間分別し,5分間水洗して、更にeosin(Wako)に20秒間浸漬し、ethanolで10分間脱水後、xylene(JUNSEI)で10分間透徹を行った。封入には、Malinol(JUNSEI)とxyleneの混合液を用いた。
(HE staining)
Specimens were immersed in 4% PFA (Wako) in PBS for 30 minutes and washed with running water for 5 minutes. Then, it was immersed in hematoxylin (CERTISTAIN) for 5 minutes and washed in the same manner for 5 minutes. Fractionate with 70% ethanol containing 0.1% HCl (Wako) for 2 seconds, wash with water for 5 minutes, immerse in eosin (Wako) for 20 seconds, dehydrate with ethanol for 10 minutes, and clear with xylene (JUNSEI) for 10 minutes. went. A mixture of Malinol (JUNSEI) and xylene was used for encapsulation.
(P1マウス組織切片作成)
凍結したP1マウスを固定し、クライオスタット(Leica CM3050)で10μm厚ごとに薄切していき(庫内温度-22℃:、試料台温度:-20℃)、腸管がよくみえる、腹側の皮膚から約3000μmの深度の部分から10μ厚の切片をつくり、透明伝導性コートを施したITOスライドグラスに融解接着させた(図3)。
(P1 mouse tissue section preparation)
A frozen P1 mouse was fixed and sliced every 10 μm with a cryostat (Leica CM3050) (inside temperature -22°C, sample table temperature: -20°C). A 10 μm thick section was cut from a depth of about 3000 μm from the surface and melt-bonded to an ITO glass slide with a transparent conductive coating (Fig. 3).
(4週齢マウス組織切片作成)
凍結した4週齢マウスを固定し、クライオスタット(Leica CM3050)で12μm厚ごとに薄切していき(庫内温度-23℃:、試料台温度:-16℃)、腸管がよくみえる部分から12μ厚の切片をつくり、透明伝導性コートを施したITOスライドグラスに融解接着させた。
(Preparation of 4-week-old mouse tissue section)
A frozen 4-week-old mouse was fixed and sliced into 12 μm-thick slices with a cryostat (Leica CM3050) (inside temperature: -23°C, sample table temperature: -16°C), and 12 μm from the part where the intestinal tract can be clearly seen. Thick sections were cut and melt glued to transparent conductive coated ITO glass slides.
(マトリックス・キャリブレーション溶液の調製)
試料に塗布する9-AA溶液とキャリブレーション用にDHB(2,5-Dihydroxybenzoic Acid, 以下DHB)を調整した。これらは70%Methanolを溶媒として溶解させた。即ち70%Methanol+naptalamは、Methanol(Wako) 7ml+Water 3ml+naptalam 1μlとした。9-AA溶液4mg/mlは、9-Aminoacridine for synthesis(Merck) 4mg+70%Methanol 1mlとした。DHB溶液10mg/mlは、2,5-Dihydroxybenzoic Acid(Wako) 10mg+70%Methanol 1mlとした。
(Preparation of matrix calibration solution)
9-AA solution to be applied to the sample and DHB (2,5-Dihydroxybenzoic Acid, hereinafter DHB) for calibration were prepared. These were dissolved in 70% Methanol as a solvent. That is, 70% Methanol+naptalam was changed to Methanol (Wako) 7 ml+Water 3 ml+naptalam 1 μl. 9-AA solution 4 mg/ml was replaced with 9-aminoacridine for synthesis (Merck) 4 mg + 70% Methanol 1 ml. 10 mg/ml of DHB solution was 10 mg of 2,5-Dihydroxybenzoic Acid (Wako) + 1 ml of 70% Methanol.
(IMS測定)
測定には大気圧MALDI-QIT-TOF-MSを搭載した顕微質量計 iMScopeのプロトタイプ機(島津製作所)を用いた。装置を制御するPCでIMS solutionを起動させ、分析のアプリケーションを起動した。装置への装着前に、サンプルホルダに付着した埃をブロアで飛ばした。装置の試料扉を開き、試料台にサンプルホルダを装着した。試料台奥と左の当たり面に確実にサンプルホルダが押し当てられているか、確認した。装置の試料扉を閉じて、サンプルホルダを装置にロードした。ウィザードに従って、サンプルIDや保存先などのサンプル情報を入力した。マトリックス塗布前後の試料の位置を装置に登録するため、ウィザードに従って位置合わせ情報を取得した。撮影条件の設定を行い、試料プレートの全体像を撮影した。
(IMS measurement)
A prototype micromass spectrometer iMScope (Shimadzu Corporation) equipped with an atmospheric pressure MALDI-QIT-TOF-MS was used for the measurement. I started IMS solution on the PC that controls the device, and started the analysis application. Dust adhering to the sample holder was blown off with a blower before attachment to the device. The sample door of the device was opened, and the sample holder was attached to the sample stage. It was confirmed that the sample holder was firmly pressed against the back and left contact surfaces of the sample table. The instrument sample door was closed and the sample holder was loaded into the instrument. Following the wizard, I entered sample information such as the sample ID and storage location. Alignment information was obtained by following the wizard in order to register the position of the sample before and after matrix application in the instrument. The photographing conditions were set, and the entire image of the sample plate was photographed.
顕微鏡で試料を観察し、測定する領域を指定した。レーザーの照射位置がずれないように、顕微鏡倍率40 倍(40×)でピントが最良になる高さに合わせ、試料の高さ調節を行った。撮影したい観察倍率にし、分析画像の撮影を行った。同様にレーザー照射条件最適化のための画像も撮影した。 The sample was observed under a microscope and the area to be measured was designated. The height of the sample was adjusted so that the laser irradiation position did not shift, so that the best focus was obtained at a microscope magnification of 40 times (40x). An analysis image was taken at the desired observation magnification. Similarly, images for optimizing the laser irradiation conditions were also taken.
サンプルホルダの金属部分にマトリックスがかからないよう、マトリックスを塗布しない部分にパラフィルムを巻き、調製した9-AA溶液をエアブラシによるマトリックス溶液吹き付け法(スプレー法)により4ml程塗布した。マトリックスの塗布は、試料表面に小さな結晶を均一に生成することが重要であるため、均一にエアブラシを用いて塗布した。 Parafilm was wrapped around the metal part of the sample holder to prevent the matrix from covering the metal part, and about 4 ml of the prepared 9-AA solution was applied by the matrix solution spraying method (spray method) using an airbrush. Since it is important to uniformly generate small crystals on the surface of the sample, the matrix was applied uniformly using an airbrush.
塗布を行った後、パラフィルムをはがし、レーザー照準調整とキャリブレーションを行うために、試料プレートのマトリックスを塗布していない部分にDHBを1 μl滴下した。完全に乾いた事を確認したのち、試料観察時と同様に試料台にサンプルホルダを装着し、位置合わせを行った。顕微鏡倍率40×で、較正用サンプルのDHBの画像を撮影した。矩形で分析領域を指定し、レーザーのパラメーターを照射径0、強度20に設定し、較正を開始した。ウィザードに従って、レーザー照射位置を較正した。LCMS solutionの再解析質量較正機能で、先ほど測定したデータを用い質量較正を行った。このデータを装置に反映させ、レーザー照射条件の最適化を行った。レーザーの強度や照射径などの分析条件を設定後、測定を開始した。 After coating, the parafilm was removed and 1 μl of DHB was applied to the unmatrixed portion of the sample plate for laser aiming and calibration. After confirming that the sample was completely dry, the sample holder was mounted on the sample table and aligned in the same manner as in the sample observation. Images of the DHBs of the calibration samples were taken at 40x microscope magnification. The analysis area was designated by a rectangle, the laser parameters were set to 0 for irradiation diameter and 20 for intensity, and calibration was started. The laser irradiation position was calibrated according to the wizard. Using the reanalysis mass calibration function of LCMS solution, mass calibration was performed using the data measured earlier. This data was reflected in the device, and the laser irradiation conditions were optimized. After setting the analysis conditions such as laser intensity and irradiation diameter, the measurement was started.
P1マウスでの条件は以下であった。 The conditions for P1 mice were as follows.
4週齢マウスでの条件は以下であった。 Conditions for 4-week-old mice were as follows.
(データ解析)
得られたデータをIMS Solution(島津製作所)を用いて、解析を行った。HMDB(http://www.hmdb.ca/spectra/ms/search)のデータベースを用いて、質量スペクトラムから物質の推定を行った。階層的クラスタリング(Hierarchical Cluster Analysis, HCA)を用いて、TCAと近似性の高いスペクトル群データを収集した。
(data analysis)
The obtained data were analyzed using IMS Solution (Shimadzu Corporation). Using the database of HMDB (http://www.hmdb.ca/spectra/ms/search), substances were estimated from mass spectra. Hierarchical Cluster Analysis (HCA) was used to collect spectral group data closely related to TCA.
(P1マウス腹部の組織形態解析)
図4(A)はP1マウス腹部の光学顕微鏡画像であり、肝臓、胃、十二指腸、小腸、大腸及び膀胱の状態を示す図である。
(Histomorphological analysis of P1 mouse abdomen)
FIG. 4(A) is an optical microscopic image of the abdomen of a P1 mouse showing the states of the liver, stomach, duodenum, small intestine, large intestine and bladder.
図4(B)はP1マウス断面をデジタルカメラで撮影した画像である。胃の中の白色の内容物は母乳であり、小腸、大腸を経るごとに黄色が増している。胆汁の主成分である胆汁色素は、ヘモグロビンの代謝産物であり、Bilirubinという黄褐色の色素である。この色素により黄色が増していると考えられる。 FIG. 4(B) is an image of a P1 mouse cross-section taken with a digital camera. The white content in the stomach is breast milk, and the yellow color increases as it passes through the small and large intestines. The bile pigment, which is the main component of bile, is a metabolite of hemoglobin and is a yellow-brown pigment called Bilirubin. It is believed that the yellow color is enhanced by this pigment.
図4(C)はP1マウス腹部のHE染色画像である。肝臓、胃、十二指腸、小腸、大腸及び膀胱の状態を示す図である。 FIG. 4(C) is an HE-stained image of the abdomen of a P1 mouse. It is a figure which shows the state of a liver, a stomach, a duodenum, a small intestine, a large intestine, and a bladder.
(P1マウス腹部のIMS)
図5は、消化管内物質のIMS画像であり、P1マウスにおいて胃から大腸に至までの食物胆汁酸サイクルの経時的変化が示されている図である。マススペクトルm/z値=615.318では胃における消化活動が見られ、m/z=616.218では小腸における消化活動が見られ、m/z=639.307では大腸における消化活動が見られ消化活動が見られる。
(IMS of P1 mouse abdomen)
FIG. 5 is an IMS image of gastrointestinal substances, showing temporal changes in the food bile acid cycle from the stomach to the large intestine in P1 mice. Digestive activity in the stomach is observed at the mass spectrum m/z value = 615.318, digestive activity in the small intestine is observed at m/z = 616.218, and digestive activity is observed in the large intestine at m/z = 639.307.
図6はP1マウスの組織にIMSを用いて解析した結果の平均スペクトラム(肝臓から膀胱までの平均)である。AMP(アデノシン一リン酸)m/z=346.05、ADP(アデノシン二リン酸)m/z=426.02、ATP(アデノシン三リン酸)m/z=505.99であった。図示されていないTCA m/z=514.29であり、一番相対強度が強いものはTCAであった。UMP(ウリジン一リン酸)、UDP(ウリジン二リン酸)等の他のピークも確認した。 FIG. 6 shows the average spectrum (average from liver to bladder) of the results of IMS analysis of P1 mouse tissues. AMP (adenosine monophosphate) m/z=346.05, ADP (adenosine diphosphate) m/z=426.02, and ATP (adenosine triphosphate) m/z=505.99. TCA (not shown) had m/z=514.29, and TCA had the highest relative intensity. Other peaks such as UMP (uridine monophosphate) and UDP (uridine diphosphate) were also confirmed.
図7(A)は胆汁酸の一種であるタウコロール酸taurocholic acid(TCA)のIMS画像であり、図7(B)は光学顕微鏡画像を重ね合わせたものである。胆汁酸由来のイオン化分子のシグナルは、肝臓、小腸に強く現れ、肝臓より小腸に強いシグナルが得られた。図7(A)の右下の楕円で囲まれた部分は、他の消化管の部位と比べて胆汁酸の分布が極端に少ない。これはこの部分が大腸であり、胆汁酸が回腸末端で門脈系を経て肝臓に循環したからであると推測できる。胆汁酸は肝細胞で産生され、総肝管を通って胆のうに一時貯蔵・濃縮される。食事時に胆のうが収縮され、十二指腸に排出される。十二指腸管内に一旦分泌された胆汁酸は約95%が回腸末端から再度吸収され、門脈を経て肝臓に戻る。この胆汁酸サイクルと呼ばれる現象を個体レベルで可視化することに成功した。 FIG. 7(A) is an IMS image of taurocholic acid (TCA), which is a type of bile acid, and FIG. 7(B) is a superimposed optical microscope image. Signals of bile acid-derived ionized molecules appeared strongly in the liver and small intestine, and stronger signals were obtained in the small intestine than in the liver. The portion surrounded by the oval in the lower right of FIG. 7(A) has an extremely low distribution of bile acids compared to other parts of the digestive tract. It can be assumed that this is because this part is the large intestine, and bile acids circulated to the liver via the portal vein system at the terminal ileum. Bile acids are produced in hepatocytes and temporarily stored and concentrated in the gallbladder through the common hepatic duct. During meals, the gallbladder contracts and is expelled into the duodenum. About 95% of the bile acids once secreted into the duodenum are reabsorbed from the terminal ileum and returned to the liver via the portal vein. We succeeded in visualizing this phenomenon called the bile acid cycle at the individual level.
生活習慣病、肥満、羸痩等の症状改善に利用できる。 It can be used to improve symptoms such as lifestyle-related diseases, obesity, and emaciation.
100:試料導入部
200:イオン源
300:分離分析部
400:データ処理部
900:イメージング質量分析装置
100: Sample introduction unit 200: Ion source 300: Separation analysis unit 400: Data processing unit 900: Imaging mass spectrometer
Claims (3)
胆汁酸サイクルの経時的変化を観察する、胆汁酸サイクルの観察方法であって、
肝臓、胃、十二指腸、小腸及び大腸におけるイメージング質量分析をそれぞれ行い、
それぞれにおける胆汁酸の状態及び分布を観察する、胆汁酸サイクルの観察方法。 By performing imaging mass spectrometry on a predetermined compound that constitutes bile acid,
A method for observing a bile acid cycle, which observes changes in the bile acid cycle over time, comprising:
Perform imaging mass spectrometry in the liver, stomach, duodenum, small intestine and large intestine, respectively,
A method for observing the bile acid cycle, observing the state and distribution of bile acids in each .
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