JP7193114B2 - Observation method of bile acid cycle - Google Patents

Description

The present invention relates to a bile acid cycle observation method for observing changes over time in the bile acid cycle.

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.

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.

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).

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.

JP 2007-157353 A JP 2014-215043 A

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.

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.

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.

It is a conceptual diagram of an imaging mass spectrometer. It is a P1 mouse sliced every 10 μm by a cryostat, and photographs are taken at 1850 μm, 3400 μm and 7350 μm out of the pictures taken every 50 μm. FIG. 10 shows coronal slices with a depth of 3000 μm and a thickness of 12 μm prepared with a cryostat and melt-bonded to ITO glass slides. Fig. 1 shows the histomorphological analysis of the abdomen of a P1 mouse, of which (A) is a photograph taken with an optical microscope, (B) is a photograph taken with a digital camera, and (C) is a HE staining photograph. . FIG. 11 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. FIG. 2 is an imaging diagram of nucleotide metabolites in the abdomen of P1 mice. (A) is an IMS image of taurocholic acid (TCA), a type of bile acid, and (B) is an overlay of optical microscope images.

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.

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.

As shown in FIG. 1, an imaging mass spectrometer 900 includes a sample introduction section 100, an ion source 200 such as MALDI (Matrix Assisted Laser Desorption Ionization), and a time-of-flight mass spectrometer TOF-MS (Time- of-Flight Mass Spectrometry) and a data processing unit 400 . A sample is introduced into the apparatus by the sample introduction unit 100 , the sample is ionized by the ion source 200 , the ions are separated and detected according to the difference in mass by the separation analysis unit 300 , and the data are processed by the data processing unit 400 .

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.

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.

(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.

(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 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 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).

(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.

(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 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.

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.

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.

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.

The conditions for P1 mice were as follows.

Conditions for 4-week-old mice were as follows.

(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.

(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.

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.

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.

(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.

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.

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.

It can be used to improve symptoms such as lifestyle-related diseases, obesity, and emaciation.

100: Sample introduction unit 200: Ion source 300: Separation analysis unit 400: Data processing unit 900: Imaging mass spectrometer

Claims (3)

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 . 2. The method for observing a bile acid cycle according to claim 1, wherein said bile acid is a primary bile acid. 3. The method for observing the bile acid cycle according to claim 2, wherein the predetermined compound of the primary bile acid is taurocholic acid, glycocholic acid or cholic acid.

Images