KR101939454B1 - Metabolic biomarkers for non-alcoholic fatty liver disease using in vivo magnetic resonance spectroscopy of hyperpolarized 1-13C pyruvate - Google Patents

Abstract

The present invention relates to a method for providing information on the diagnosis of non-alcoholic fatty liver disease using hyperpolarised [1- ¹³ C] pyruvate ¹³ C magnetic resonance spectroscopy, and more particularly, (NAFLD) animal model induced by HFD in the first, second, fourth, and sixth digits of the dietary intake of the nonalcoholic fatty liver disease (NAFLD) The present invention relates to a method for providing a quantitative biomarker for the diagnosis of nonalcoholic fatty liver disease by measuring in real time unit intervals.

Description

Technical Field [0001] The present invention relates to a method for diagnosing non-alcoholic fatty liver disease using a novel quantitative biomarker using in vivo hyperpolarized C13 pyruvate magnetic resonance spectroscopy, and in vivo magnetic resonance spectroscopy of hyperpolarized 1-13C pyruvate

The present invention relates to a method for providing information on the diagnosis of non-alcoholic fatty liver disease using hyperpolarised [1- ¹³ C] pyruvate ¹³ C magnetic resonance spectroscopy, and more particularly, (NAFLD) animal model induced by HFD in the first, second, fourth, and sixth digits of the dietary intake of the nonalcoholic fatty liver disease (NAFLD) The present invention relates to a method for providing a quantitative biomarker for the diagnosis of nonalcoholic fatty liver disease by measuring in real time unit intervals.

Nonalcoholic fatty liver disease (NAFLD) is a disease characterized by simple steatosis with excessive accumulation of fat in hepatocytes, nonalcoholic steatohepatitis (NASH) with hepatocellular necrosis, inflammation and fibrosis, (Brunt EM Semin Liver Dis , 21: 3-16, 2001).

The prevalence of nonalcoholic fatty liver disease has been reported worldwide, and it is known that about 20-30% of non-alcoholic fatty liver disease among normal adults without special liver disease in Western countries. The prevalence of nonalcoholic fatty liver disease in Korean adults is reported to be about 16 to 50%. In general, nonalcoholic fatty liver disease is known to increase the prevalence of obesity, which is attributed to insulin resistance due to obesity as an important cause of liver fat deposition (Kim JH Korean J Fam Pract. , 4: 109-115, 2014).

The pathogenesis of fatty liver and the progression from fatty liver to lipid hepatitis and liver cirrhosis has not been well known yet. Recently, efforts have been made to clarify this mechanism. Heretofore, hereditary and environmental factors, fat accumulation in the liver, insulin mechanisms for such resistance, fatty acids and fat-toxic (lipotoxicity), stress and mitochondrial dysfunction and various oxidation cytokine / Adi-Napoca in (cytokines / adipokines) interactions have been reported (Sohn JH, et al. the Korean Journal of Medicine, 79: 461-474, 2010).

Several methods have been tried for screening for nonalcoholic fatty liver disease, but no established screening method is available. In general, liver function tests such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and abdominal sonography are widely used. However, hepatic function tests are performed in patients with nonalcoholic fatty liver or hepatitis The sensitivity of the screening test is low and abdominal ultrasonography is high in sensitivity, but it is disadvantageous in the cost of screening. In addition, serum ferritin and uric acid tests have been studied as screening tests, but further studies are needed to evaluate their efficacy (Kim CW, et al. Metabolism , 61: 1182-1188, 2012).

Since nonalcoholic fatty liver disease differs greatly in the prognosis according to histologic findings, it is clinically important to confirm whether the patient is accompanied by fatty liver or fibrosis. To date, the baseline test for liver inflammation and fibrosis is liver biopsy. However, liver biopsy has limitations such as high cost, invasiveness, risk of complications, possibility of error in reading, and difficulty in representing whole liver status with low liver tissue taken. In this regard, efforts to evaluate the histologic severity of nonalcoholic fatty liver disease (NSCLC) instead of liver biopsy have been conducted mainly in the fields of radiology and biochemistry. However, until now, The basic knowledge and basic techniques for cell metabolism are not established (Rockey DC, et al. Hepatology , 49: 1017-1044, 2009).

Particularly, in the clinical field, a magnetic resonance imaging (MRI) apparatus is mainly used to accurately evaluate liver function in patients with fatty liver disease, but it usually takes about 30 minutes to obtain images for diagnostic purposes. Because it is accompanied by a great deal of pain in patients with fatty liver disease, imaging techniques that can diagnose fatty liver disease within a short time are required.

The present inventors own a white paper non alcoholic fatty liver disease model is based on the world's first non-invasive (non-invasive) technique developed within the living body of the hyperpolarization Chemistry [1- ¹³ C] pyruvate ¹³ C Time Division magnetic resonance spectroscopy (in vivo ¹³ C dynamic magnetic resonance spectroscopy of hyperpolarized [1- ¹³ C] pyruvate) The biochemical mechanism of fatty tissue was investigated in terms of cellular metabolism.

Accordingly, it is an object of the present invention to provide a novel method for providing information on non-alcoholic fatty liver disease using in vivo hyperpolarized ¹³ C time-division magnetic resonance spectroscopy with relatively short image acquisition time.

Another object of the present invention is to provide a novel method of providing information that can evaluate the accurate diagnosis, prognosis, and therapeutic effect of nonalcoholic fatty liver disease as a quantitative biomarker with objectivity guaranteed.

The present invention relates to a novel diagnostic method using in vivo depolarization ¹³ C time-division magnetic resonance spectroscopy in nonalcoholic fatty liver disease (NAFLD) The objective of this study was to evaluate the accurate diagnosis, prognosis, and therapeutic effect through a quantitative biomarker with objectivity. The present inventors intend to provide a new diagnostic method for evaluating nonalcoholic fatty liver disease.

Hereinafter, the present invention will be described in more detail.

MRI is noninvasive and enables early diagnosis. In general, magnetic resonance imaging (MRI) used in clinical diagnosis detects and visualizes the signal of hydrogen ( ¹ H) elements constituting the living body's anatomical structure. Metabolic measurement using magnetic resonance imaging is based on nuclear magnetic resonance spectroscopic imaging of the substrate and metabolite labeled with ¹³ C element. However, since only ¹³ % of the ¹³ C element exists in the natural world, the sensitivity is lowered every week, and the hyperpolarization technique using the DNP (dynamic nuclear polarization) method is used for signal amplification of the ¹³ C element.

The hyperpolarization is based on the Nuclear Overhauser Effect (NOE) first proposed by Albert Overhauser in 1953, and is based on NOE in a glassy state at an absolute temperature of 2-4 degrees Celsius microwave is a polarization transfer phenomenon in which the polarization state of unpaired electron radicals is transferred to the nuclear spin of ¹³ C element.

The thermal equilibrium state transitions to the hyperpolarized equilibrium state, which increases the magnetic resonance signal intensity of about 10,000 times, resulting in a significant increase in sensitivity.

Metabolic MRI using a hyperpolarised ¹³ C-pyruvate as a substrate can quantitatively detect the production of ¹³ C-lactate by targeting the increased glucose metabolism in hepatocytes without radioactivity, .

An RF coil for a ¹³ C magnetic resonance spectrometer, which is a major accessory device of the present invention, can acquire a signal of a small tissue by closely adhering to a region of interest of a small animal, and has a signal- to-noise ratio (SNR) is high.

In particular, the SNR value is more important than the homogeneity of the magnetic field in order to acquire an image of a small object such as a mouse. Therefore, surface coils are highly suitable for accurately analyzing the composition and concentration of tissue images and biochemical metabolites in white rats. In the clinical 3T MRI system (GE MR750 3T Scanner, GE Healthcare, Waukesha WI), the proton magnetic resonance frequency of the coil was 32.12MHz and the impedance matching was 50ohm, and in vivo MRS data was obtained.

A main bag device of the present invention by applying a DNP (Dynamic Nuclear Polariztion) techniques in metabolites containing ¹³ C was used Hypersense DNP polarizer (Oxford Instruments, Abingdon , UK) to induce hyperpolarization (hyperpolarization) phenomenon.

Also, [1- ¹³ C] pyruvate was used as a sample for hyperpolarization. The internal temperature of a solid state NMR probe installed in a superconducting magnet having a magnetic field strength of about 3.35 Tesla was lowered to 1.4K using liquefied helium, which is a refrigerant capable of cooling to an absolute temperature of 0 K, and [1- ¹³ C] pyruvate (Gd-DOTA, Guerbet, France) were added to the wells of the culture supernatant (Isotec, Miamisburg, OH), 15 mM OX63 trityl (Oxford Instruments, Abingdon, UK) and 0.5 mM Dotarem.

In this case, ¹³ C hyperpolarized pyruvate was immersed in a buffer solution (40 mM Trisma Pre-Set Crystals pH 7.6, 80 mM NaOH, 50 mM NaCl, 2.69 mM Disodium EDTA). ^{The 13} C hyperpolarized pyruvate had a concentration of 80 mM, the pH was 7.2 to 8.0, and the polarization level was 17.2 to 19.8%. Finally, the ¹³ C hyperpolarisation solution was injected into the tail vein of the mice in 2 mL for 20 seconds.

In order to obtain the spectrum from the image object using the magnetic resonance spectroscopy (MRS) of the present invention, a magnetic resonance imaging (MRI) for the determination of the orientation must be obtained. A 3T GE MR750 scanner (GE Healthcare, Waukesha, Wis.) Was used to obtain T1 weighted images (T1WI) and T2 weighted images (T2WI) as morphology and anatomical reference data. T2WI is the image of the sagittal plane, the axial plane, and the coronal plane using TR / TE = 2800msec / 106msec, and the T1WI has the repetition time (TR) / echo time (TE) = 800msec / 10msec. . In this case, the field of view (FOV) used is 16 cm x 16 cm, the size of the matrix is 256 x 192, the number of excitation is 2, the slice thickness is 5 mm, Respectively.

In the present invention, in order to obtain in vivo hyperpolorized ¹³ C dynamic MRS data in the in vivo hyperpolarized ¹³ C time-resolved magnetic resonance spectroscopy (MRS), a post-processing of the raw data of the MRS To obtain MR spectra.

In the post-treatment statistical analysis of the present invention, all measurements were expressed as mean ± standard error, and statistical analysis of data was performed using t-test (SPSS for window version 19.0). In particular, the survival comparison between the control and fatty liver models was performed using a two-sample t-test with a statistical significance of less than 0.05.

As a final step in the experiment of the present invention, the abdomen was dissected and the liver was extracted and fixed in a 4% paraformaldehyde solution for 24 hours. Then, 1.0 × 1.0 × 0.2 cm ³ sections were prepared and embedded in paraffin , And 6 μm thick to make slides. Tissue slides were prepared by removing paraffin, dripping, rehydrating and immunostaining. After obtaining slide images using a DM 3000 microscope (Leica Microsystems, Wetzlar, Germany), iSolution FL / auto software (IMT Inc., IL, USA) was used to measure the total lipid content (% Respectively.

As described above, the present invention is based on the observation that changes in various cellular metabolites in fatty liver tissues at the 0th, 2nd, 4th, and 6th weeks during the intake of the high-calorie diet (HFD) in the nonalcoholic fatty liver disease model " in vivo hyperpolarized ^13C dynamic MR spectroscopy "and quantitative biomarker to provide basic knowledge on physiological and biochemical pathogenesis of fatty liver disease, and furthermore, to diagnose and prognosis of nonalcoholic fatty liver disease And a new method of providing information on the effectiveness of the treatment.

An example of the present invention relates to a method for providing information on the diagnosis of nonalcoholic fatty liver disease comprising the following steps.

A composition comprising hyperpolarised ¹³ C-pyruvate was obtained from a ¹³ C-pyruvate ¹³ C-magnetic resonance spectroscopy image from an injected subject, ¹³ C-lactate (a metabolite of the ¹³ C-pyruvate lactate) and ¹³ C-alanine ( ¹³ C-NMR spectroscopy images;

¹³ calculating a ¹³ C- lactate metabolism conversion ratio (R _L1) divided by the area of the signal curve of the signal area of the ¹³ C- pyruvate in the curve of the C- lactate;

¹³ calculating a ¹³ C- alanine metabolism conversion ratio divided by the area of the signal curve of the C- alanine by the area of the signal curve of the ¹³ C- pyruvate (R _A1);

Comparing the ¹³ C-lactate metabolism conversion ratio (R ₁ ) to a standard value (R _L0 ); And

Comparing the ¹³ C-alanine metabolism conversion ratio (R ₂ ) to the standard value (R _A0 ).

The hyperpolarization of ¹³ C-pyruvate can be achieved by using a mixture of isotopically enriched ³ He, ¹²⁹ Xe or noble gas, or by thermodynamic equilibrium at very low temperatures and high magnetic fields, Or by physically rotating in a magnetic field after mixing the paramagnetic material, or by DNP (dynamic nuclear polarization). That is, ¹³ C-pyruvate is directly polarized to obtain hyperpolarised ¹³ C-pyruvate, or ¹³ C-pyruvic acid is polarized and then neutralized with a base to polarize the polarized ¹³ C-pyruvic acid RTI ID = 0.0 > ¹³ C-pyruvate. ≪ / RTI >

For example, the composition comprising the hyperpolarised ¹³ C-pyruvate can be prepared by mixing trityl radical with ¹³ C-pyruvic acid and depolymerizing it by DNP (dynamic nuclear polarization) But is not limited to, those obtained by dissolving them in a buffer solution (dissolution).

Specifically, DNP method ¹³ of ¹³ from an unpaired of a mixture of a paramagnetic compound in ¹³ C- pyruvate as the compound to be polarized and then, added to a microwave radiation in the form of energy and excite the paramagnetic compounds, paramagnetic compounds e C- pyruvate This is done by moving the polarization to the nucleus of C.

The paramagnetic compound may be a transition metal, a free radical, a nitroxide radical, and the like. For example, trityl radical may be used, but is not limited thereto.

At this time, water may be used as a co-solvent for dissolving the paramagnetic compound in pyruvic acid, but the present invention is not limited thereto.

The mole fraction ratio of ¹³ C-pyruvic acid and the paramagnetic compound mixed in the DNP method may be 5: 1, but is not limited thereto.

The DNP method may be performed at a magnetic field of 1 T to 25 T and at a temperature of 0.5 K to 8 K to achieve polarization, for example, at a magnetic field of 3.35 T and an ultra-low temperature of 1.4 K, no.

The solid phase of ¹³ C-pyruvic acid is converted to the liquid ¹³ C-pyruvate by dissolving in the buffer to which the base is added. In this case, a phosphate buffer solution may be used. For example, Tris may be used , But is not limited thereto.

As the base, an aqueous solution of sodium carbonate (Na ₂ CO ₃ ), an aqueous solution of sodium hydrogencarbonate (NaHCO ₃ ) or an aqueous solution of sodium hydroxide (NaOH) may be used. For example, an aqueous solution of sodium hydroxide (NaOH) But is not limited thereto.

Such buffers may include, but are not limited to, chelating agents such as EDTA and / or sodium chloride (NaCl) to maintain osmotic pressure.

By the above method, isotopically enriched ¹³ C-pyruvate is enriched to 90% or more, 95% or more, for example, 99% or more.

^13. The composition comprising a C- pyruvate is the main component ¹³ and the C- pyruvate, stabilizing agents, osmotic pressure controlling agent, but it may contain a and / or adding a solubilizing agent, and the like.

The hyperpolarization apparatus for polarizing ¹³ C-pyruvic acid may use, but not limited to, HyperSense.

^13. After placing the animal other than a human or a human in a MRI (Magnetic Resonance Imaging, MRI) device as an object to provide information about the C- MR non alcoholic fatty liver disease diagnosis in order to obtain a spectroscopic image, ¹³ C Place the magnetic resonance RF coil to cover the liver tissue area within the subject. A composition comprising the depolarized ¹³ C-pyruvate is then injected into the subject.

The ¹³ C-MR spectroscopic image may be obtained from a target object.

In the present invention, an animal model of nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) as a subject was injected.

The composition comprising the hyperpolarised ¹³ C-pyruvate may be injected through the blood vessels of the subject for 10 to 30 seconds, 10 to 25 seconds, 15 to 30 seconds, 15 to 25 seconds, for example, 20 seconds However, the present invention is not limited thereto.

The composition comprising the hyperpolarised ¹³ C-pyruvate may be administered via the blood vessel of the subject at a rate of 0.1 to 1.0 mL / s, 0.1 to 0.9 mL / s, 0.1 to 0.8 mL / s, 0.1 to 0.7 mL / s, 0.1 For example, 0.1 mL / s to 0.6 mL / s, 0.1 to 0.5 mL / s, 0.1 to 0.4 mL / s, 0.1 to 0.3 mL / s, 0.1 to 0.2 mL / , But is not limited thereto.

The ¹³ C-MR spectroscopic image may be obtained before the 120 seconds elapse after the composition containing ¹³ C-pyruvate. After 120 seconds after injection of the composition ¹³ is because C- pyruvate to obtain a ¹³ C signal because the recovery to its original state of equilibrium in the hyperpolarization state.

The ¹³ C-NMR spectra of ¹³ C-pyruvate, ¹³ C-lactate and ¹³ C-alanine are used to obtain signal curve data of each metabolite in the time domain using an image signal processing technique.

Specifically, the MR spectrum is obtained by performing a Gaussian line broadening filter of 20 Hz in order to increase a signal-to-noise ratio (SNR) in a free induction decay (FID) signal, FID data was subjected to zero-filling (16384 data points total) twice in order to improve the quality of the data. Then, the extended FID data was subjected to Fourier transform to obtain an MR spectrum, and the resonance signal of the metabolite of interest was confirmed after phasing and chemical shift of the spectrum.

The area of the signal curve of the ¹³ C-NMR spectrum is the area for the area under the signal curve.

The ¹³ C-lactate metabolism conversion ratio (R _L1 ) is a ratio of the amount converted to ¹³ C-lactate, which is a metabolite thereof, relative to the amount of ¹³ C-pyruvate. The larger the value, the greater the metabolism , And is used as an activity indicator of the anaerobic process.

The ¹³ C-alanine metabolism conversion ratio (R _A1 ) is a ratio of the amount converted to ¹³ C-alanine, which is a metabolite thereof, relative to the amount of ¹³ C-pyruvate. The larger the value, the greater the metabolism. It is used as an activity index of metabolic function.

The ¹³ C- lactate metabolic conversion rate of the target object (R _L1), and ¹³ compared to the C- alanine metabolism conversion ratio (R _A1) each standard value ^{_{(R L1 R A1), 13}} C than the respective standard values - Lactate metabolism conversion ratio (R _L1 ) of more than 60% and ¹³ C-alanine metabolism conversion ratio (R _A1 ) of 35% or more can be diagnosed as nonalcoholic fatty liver disease.

The ¹³ C- lactate metabolism conversion ratio (R _L1) ¹³ and ¹³ in the control group did not eat, high-calorie diet in other embodiments based on the C- alanine metabolism conversion ratio (R _A1) for determining the non-alcoholic fatty liver disease The non-alcoholic fatty liver disease can be diagnosed by comparison with the standard value of the C-lactate metabolism conversion ratio (R _L0 ) and the standard value of the ¹³ C-alanine metabolism conversion ratio (R _A0 ).

To calculate the ¹³ C-lactate metabolism conversion ratio (R _L1 ) and the ¹³ C-alanine metabolism conversion ratio (R _A1 ) of the subject, ¹³ C-MR spectroscopy images were obtained from statistically significant samples , And it is preferable to judge non-alcoholic fatty liver disease by using the average ¹³ C-lactate metabolism conversion ratio (R _L1 ) and the ¹³ C-alanine metabolism conversion ratio (R _A1 ).

The standard value (R _L0 ) of the ¹³ C-lactate metabolism conversion ratio (R _L1 ) refers to the conversion ratio of the ¹³ C-lactate metabolism of the same species as the non-alcoholic fatty liver disease subject.

The standard value (R _A0 ) of the ¹³ C-alanine metabolism conversion ratio (R _A1 ) means a conversion value of the ¹³ C-alanine metabolism of the same species as the subject having no nonalcoholic fatty liver disease.

The standard value (R _A0 ) of the standard value (R _L0 ) and / or the ¹³ C-alanine metabolism conversion ratio (R _A1 ) of the ¹³ C-lactate metabolism conversion ratio (R _L1 ) However, the present invention is not limited thereto.

In addition, the ¹³ C- lactate metabolism conversion ratio of the standard value (R _L1) (R _L0) and ¹³ after a period of time of ¹³ to C- alanine metabolism conversion ratio (R _A1) measures the standard value (R _A0), and C - lactate metabolism conversion ratio (R _L1 ) and ¹³ C-alanine metabolism conversion ratio (R _A1 ) are measured and compared to provide information on the prognosis of nonalcoholic liver disease.

The predetermined time may be 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week, but the present invention is not limited thereto, and can be easily modified by those skilled in the art in order to provide information on the prognosis.

The present invention relates to a method for providing information on the diagnosis of nonalcoholic fatty liver disease using ¹³ C-NMR spectroscopy, and more particularly, to a method for diagnosing non-alcoholic fatty liver disease induced by non-alcoholic We measured the quantitative changes of various cellular metabolites in real time at intervals of 0, 2, 4, and 6 during dietary intake of nonalcoholic fatty liver disease (NAFLD) And quantitative biomarker for the diagnosis of nonalcoholic fatty liver disease. It can be used for the accurate diagnosis, prognosis and therapeutic effect of fatty liver disease.

FIG. 1A is a ¹ H MR image acquired in the liver of a white paper for the sake selection in accordance with an embodiment of the present invention.
1B is a graph showing a time-division spectrum of a control group according to an embodiment of the present invention.
1C is a graph showing a time-division spectrum of a non-alcoholic fatty liver disease model according to an embodiment of the present invention.
FIG. 1D is a graph showing changes in concentration of each metabolite with respect to time in a control group according to an embodiment of the present invention. FIG.
FIG. 1E is a graph showing changes in concentration of each metabolite with time in a non-alcoholic fatty liver disease model according to an embodiment of the present invention. FIG.
Figure 1f is a graph depicting the time course of the inhibition of in vivo metabolites (lactate, pyruvate-hydrate, alanine) from the hyperpolarised [1- ¹³ C] Lt; RTI ID = 0.0 > ¹³ C < / RTI >
FIG. 1G is a graph illustrating the effect of the metabolism of a metabolite (lactate, pyruvate-hydrate, alanine) from hyperpolarised [1- ¹³ C] pyruvate over time in a nonalcoholic fatty liver disease model according to an embodiment of the present invention. Lt; RTI ID = 0.0 > ¹³ C < / RTI >
FIG. 2A is a graph showing the results of measuring the signal changes with respect to metabolites in each parking lot by injecting hyperpolarised [1- ¹³ C] pyruvate in a control group and a non-alcoholic fatty liver disease model group according to an embodiment of the present invention Fig.
FIG. 2B is a graph showing the results of measuring the signal changes with respect to the metabolites according to time by injecting hyperpolarised [1- ¹³ C] pyruvate into the control group and the non-alcoholic fatty liver disease model group according to an embodiment of the present invention. Fig.
FIG. 2c is a graph showing the results of measuring the signal changes of metabolites by time of each parking lot by injecting hyperpolarised [1- ¹³ C] pyruvate in a control group and a non-alcoholic fatty liver disease model group according to an embodiment of the present invention Fig.
FIG. 3A is a photograph showing histological changes in hepatic tissue at 4th week in a control group according to an embodiment of the present invention. FIG.
FIG. 3B is a photograph showing histopathological changes in the non-alcoholic fatty liver disease model group according to one embodiment of the present invention. FIG.
3C is a photograph showing histological changes in liver tissue at 6th week in a control group according to one embodiment of the present invention.
FIG. 3D is a photograph showing histopathological changes in the non-alcoholic fatty liver disease model group according to one embodiment of the present invention. FIG.
FIG. 3E is a graph showing a change in total lipid content (%) of fatty liver tissue between a control group and a non-alcoholic fatty liver disease model group according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental Example 1. Preparation of Control Model

A control model was prepared by dietary intake of low calories (70% carbohydrate, 20% protein, 10% fat) (Research Diets, New Brunswick, NJ) on Sprague-Dawley rats (body weight 290-300 g). During the experimental period, the control group was individually ventilated at a temperature of 22 ± 2 at a temperature of 22 ± 2, a relative humidity of 40 to 60%, a ventilation frequency of 10 to 20 times / hour, fluorescent lamp illumination of 12 hours, illumination of 150 to 300 Lux cage; Tecniplate Sealsafe ^TM ). The water was allowed to freely ingest ultraviolet sterilized rectified water.

Experimental Example 2. Preparation of non-alcoholic fatty liver disease model

A non-alcoholic fatty liver disease model was prepared by dietary intake of high calorie (20% carbohydrate, 20% protein, 60% fat) (Sprague-Dawley rat, weight 290-300 g) Respectively. During the experimental period, the model group consisted of individual ventilation cages under conditions of temperature 22 ± 2, relative humidity 40-60%, number of ventilation 10 - 20 times / hour, fluorescent lighting 12 hours, illuminance 150 - 300 Lux, ventilated cage; Tecniplate Sealsafe ^TM ). The water was allowed to freely ingest ultraviolet sterilized rectified water.

Example 1. Measurement of the change in activation degree of body weight and 7 kinds of serum enzymes

Control group and non-alcoholic fatty liver disease models were divided into two groups according to body weights and seven types of serum enzymes (ALT: aspartate aminotransferase: LDH: lactate dehydrogenase: HDL: high LDL: low-density lipoprotein, TC: total cholesterol, glucose) were measured and the results are shown in Tables 1 to 4.

0 weeks (before the start of the test) group Control group (n = 3) The experimental group (n = 4) Weight (g) 295.0 + - 4.4 295.3 ± 1.9 ALT (U / L)  44.3 ± 6.3  38.3 ± 0.9 AST (U / L)   89.7 ± 10.9  68.0 ± 2.5 LDH (U / L)  1019.3 ± 290.5  504.3 + - 74.4 HDL (mg / dL)  48.0 ± 3.2  41.5 ± 1.1 LDL (mg / dL)   8.7 ± 0.5  10.3 ± 0.6 Total cholesterol (mg / dL)  64.3 ± 2.3  63.8 ± 1.3 Glucose (mg / dL) 234.0 ± 5.5 225.8 ± 8.6

2 parking group Control group (n = 3) The experimental group (n = 4) Weight (g) 374.0 + - 10.0 403.5 ± 5.5 ALT (U / L) 31.3 ± 2.0  35.8 ± 0.4 AST (U / L) 67.0 ± 2.4  64.0 + 1.7 LDH (U / L) 318.3 ± 21.7  397.3 + - 41.2 HDL (mg / dL) 46.7 ± 1.7  45.0 ± 1.2 LDL (mg / dL)  5.0 ± 0.6   8.0 ± 0.6 Total cholesterol (mg / dL) 65.3 ± 4.0  67.3 ± 4.3 Glucose (mg / dL) 250.3 ± 13.1  252.3 ± 13.6

4 parking group Control group (n = 3) The experimental group (n = 4) Weight (g) 428.0 + - 12.3   474.3 ± 10.2 ALT (U / L) 26.3 ± 1.3   38.0 ± 1.2 AST (U / L) 73.3 ± 6.2   77.8 ± 4.5 LDH (U / L) 262.0 + - 46.2    716.3 + - 188.6 HDL (mg / dL) 51.0 + - 4.9   50.8 ± 2.0 LDL (mg / dL)  7.3 ± 1.1   13.8 ± 0.6 Total cholesterol (mg / dL) 69.3 ± 3.4   66.8 ± 2.7 Glucose (mg / dL) 249.0 ± 18.6 219.0 ± 2.3

6 parking group Control group (n = 3) The experimental group (n = 4) Weight (g)   472.7 ± 11.9 532.8 ± 9.7 ALT (U / L)   22.3 ± 1.9  42.5 ± 2.6 AST (U / L)   57.7 ± 2.7  97.8 ± 7.5 LDH (U / L)  338.3 + - 4.2  1003.5 + - 215.6 HDL (mg / dL)   58.0 + 1.7  49.5 ± 1.9 LDL (mg / dL)    9.7 ± 0.2  16.0 ± 1.2 Total cholesterol (mg / dL)   78.0 ± 3.5  69.8 ± 6.5 Glucose (mg / dL)  223.3 ± 1.9  247.3 ± 15.4

ALT: alanine aminotransferase;

AST: aspartate aminotransferase;

LDH: lactate dehydrogenase;

HDL: high-density lipoprotein; And

LDL: low-density lipoprotein:

As shown in Tables 1 to 4, in the nonalcoholic fatty liver disease model group ( n = 4), weight was significantly increased at 6th week compared to the control group ( n = 3) (p <0.05), and ALT and LDL serum enzyme (P <0.05). There was no significant difference between the two groups. The remaining serum enzymes did not show a significant difference between the two groups at each visit.

Example 2. Measurement of cellular metabolite change of non-alcoholic fatty liver disease model of white rats

The animals were anesthetized with isoflurane and then 24 gauge catheter was inserted into the tail vein and saline solution was injected into the vein. The injection path was secured. Real-time breathing and body temperature were measured by a physiological monitoring system (Model 1025, SA Instruments Inc., NY, USA), and the normal body temperature was maintained at 37 ° C using a heating system.

A Hypersense DNP polarizer (Oxford Instruments, Abingdon, UK) was used to induce hyperpolarization by applying the DNP (Dynamic Nuclear Polarization) technique to ¹³ C-containing metabolites. The internal temperature of a solid state NMR probe in a superconducting magnet having a magnetic field strength of about 3.35 Tesla was lowered to 1.4 K and 25 l of [l- ¹³ C] pyruvate (Isotec, Miamisburg, OH), 15 mM OX63 trityl (Oxford Instruments, Abingdon, UK) and 0.5 mM Dotarem (Gd-DOTA, Guerbet, France). At this time, microwave 94.1 GHz was irradiated for 1 hour to cause hyperpolarization. Then, ¹³ C hyperpolyzed pyruvate was dissolved in a buffer solution (40 mM Trizma Pre-Set Crystals pH 7.2-8.0, 80 mM NaOH, 50 mM NaCl, 2.69 mM Disodium EDTA). ^{The 13} C depolarized pyruvate had a concentration of 80 mM, a pH of 7.6 and a polarization level of 17.2 to 19.8%. Finally, 2 mL of a ¹³ C hyperpolarized solution was injected into the tail vein of the rats for 20 seconds.

A 3T GE MR750 scanner (GE Healthcare, Waukesha, Wis.) Was used to obtain T1 weighted images (T1WI) and T2 weighted images (T2WI) as morphology and anatomical reference data. Using a surface coil, T1WI was measured using a repetition time (TR) / echo time (TE) = 800 msec / 10 msec and T2WI using TR / TE = 2800 msec / 106 msec. Images were acquired in the plane direction. The image range (FOV) used was 16 cm x 16 cm, the size of the matrix was 192 x 192, the number of excitation was 2, and the slice thickness was 5 mm.

In vivo (in vivo) hyperpolarization screen ¹³ C magnetic resonance spectroscopy (hyperpolorized ¹³ C MRS) free induction decay chemical shift image of interest metabolites by (free induction decay chemical shift image, FIDCSI) using a pulse waveform in order to obtain the data was measured . The image range used was 12cm x 12cm, the flip angle was 10 °, the slice thickness was 15mm, the TR was 2000msec, the spectral width was 5000Hz, the number of data points was 4096, and the total spectral acquisition time was 2 minutes.

Data from the MR scanner was transferred to a PC and then SAGE 7 (GE Healthcare) software was used. First, in order to increase the signal-to-noise ratio (SNR) in the FID signal, a 20Hz Gaussian line broadening filter is performed. Then, in order to improve the digital frequency resolution, the FID data is zero- data points) were applied. Then, the extended FID data was subjected to Fourier transform to obtain an MR spectrum, and the resonance signal of the metabolite of interest was confirmed after phasing and chemical shift of the spectrum.

^In the qualitative and quantitative analysis of ¹³ C metabolites, [1- ¹³ C] pyruvate has been shown to produce pyruvate, alanine, lactate, pyruvate hydrate, bicarbonate, , And these metabolites were analyzed using the following resonance frequencies. [1- ¹³ C] lactate ^{(184.0ppm), [1- 13 C} ] pyruvate hydrate (180.0ppm), [1- 13 C] alanine ^{(177.0ppm), [1- 13 C} ] pyruvate (172.0ppm) , [1- ¹³ C] bicarbonate (162.0 ppm), and the change of metabolites over time was measured from the peak intensity values of the identified metabolites.

The hyperpolarised [1- ¹³ C] pyruvate was injected into a model of non-alcoholic fatty liver disease of rats, and the change in the signal for the metabolites with respect to each time of each parking was measured. The results are shown in Table 5 and FIGS. Respectively.

0 weeks (before the start of the test) group Control group (n = 3) The experimental group (n = 4) Alanine / pyruvate (%) 18.9 ± 0.9 21.6 ± 2.2 Lactate / Pyruvate (%) 18.8 ± 2.2 15.4 ± 0.6 Pyruvate hydrate / pyruvate (%) 11.9 ± 0.2 12.1 ± 0.4 2 parking Alanine / pyruvate (%) 18.4 ± 1.8 23.1 ± 1.0 Lactate / Pyruvate (%) 25.7 ± 2.8 26.0 ± 1.5 Pyruvate hydrate / pyruvate (%) 11.5 ± 0.4 11.3 ± 0.2 4 parking Alanine / pyruvate (%) 18.1 ± 2.1 24.2 ± 1.6 Lactate / Pyruvate (%) 22.5 ± 2.5 35.0 ± 4.1 Pyruvate hydrate / pyruvate (%) 11.1 ± 0.1 11.3 ± 0.5 6 parking Alanine / pyruvate (%) 18.7 ± 1.3 25.5 ± 0.7 Lactate / Pyruvate (%) 22.7 ± 1.8 36.8 ± 2.2 Pyruvate hydrate / pyruvate (%) 11.0 ± 0.3 11.8 ± 0.2

As shown in Table 5 and FIGS. 2A to 2C, when the changes of metabolites were compared based on the pyruvate signal in the combined spectral data, the metabolism of [1- ¹³ C] alanine in the nonalcoholic fatty liver disease model group And [1- ¹³ C] lactate were increased.

These results indicate that the hepatotoxicity model induced by 1,3-dichloro-2-propanol (Kim GW, et al. Magn The signal of [1- ¹³ C] alanine in this nonalcoholic fatty liver disease model is 1.5%, the signal of [1- ¹³ C] lactate is 7.8 (compared with Reson Imaging , 34 (2): 159-65, 2016) %, Respectively, to provide a differentiated diagnostic biomarker according to quantitative analysis of metabolic changes in liver disease models.

Example 3. Histopathology and measurement of total lipid content change in liver tissue

In order to immunohistochemically stain the fatty tissue, the abdomen was excised and the liver was further immobilized in 4% paraformaldehyde solution for 24 hours. Then, 1.0 × 1.0 × 0.2 cm ³ sections were prepared and embedded in paraffin. To make a slide. Tissue slides were prepared by deparaffinization and rehydration after removal of wax, followed by immunostaining. After obtaining slide images using a DM 3000 microscope (Leica Microsystems, Wetzlar, Germany), iSolution FL / auto software (IMT Inc., IL, USA) was used to measure the total lipid content (% Respectively. The results are shown in Figs. 3A to 3E and Table 6.

Hepatic lipid content (%) CD HFD 4 parking 5 ± 0.4 13 ± 0.3 6 parking 5 ± 0.9 21 ± 3.2

As shown in Figs. 3A to 3E and Table 6, in immunohistochemical staining, lipid droplet was most dominant in the non-alcoholic fatty liver disease model at 6th week, Lipids accounted for 21%. However, histopathologic data at 0 and 2 did not show good dyeability, especially eosin was poorly stained and not used as an experimental result.

Claims (3)

A composition comprising hyperpolarised ¹³ C-pyruvate was obtained from ¹³ C-pyruvate ¹³ C-magnetic resonance spectroscopy images obtained from the injected subjects, ¹³ C-pyruvate metabolites ¹³ C-lact Obtaining a signal curve of each metabolite from a ¹³ C-MR spectroscopy image of lactate and ¹³ C-alanine;
¹³ calculating a ¹³ C- lactate metabolism conversion ratio (R _L1) divided by the area of the signal curve of the signal area of the ¹³ C- pyruvate in the curve of the C- lactate;
¹³ calculating a ¹³ C- alanine metabolism conversion ratio divided by the area of the signal curve of the C- alanine by the area of the signal curve of the ¹³ C- pyruvate (R _A1);
Calculating the difference between the ¹³ C-lactate metabolism conversion ratio (R _L1 ) and the standard value (R _L0 ); And
Calculating the difference between the ¹³ C-alanine metabolism conversion ratio (R _A1 ) and the standard value (R _A0 );
The method comprising the steps of: The composition of claim 1, wherein the composition comprising the depolarized &lt; RTI ID = 0.0 &gt; ^13C -pyruvate &
Which is obtained by mixing trityl radical with ¹³ C-pyruvic acid, depolarizing by dynamic nuclear polarization (DNP) method, and dissolving in a buffer solution. How to provide information. The method according to claim 1, wherein the ¹³ C-MR spectroscopy image is acquired before 120 seconds elapse after injection of a composition comprising ¹³ C-pyruvate, wherein the method comprises providing information about the diagnosis of nonalcoholic fatty liver disease .

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