JP2009126800A - Method for assaying liver function of equine animal, reagent and kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and simple method for assaying the liver function of an equine animal. <P>SOLUTION: The method for assaying the liver function of an equine animal by an expiratory test includes using a<SP>13</SP>C-labeled aromatic amino acid. The reagent and the kit are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method, a reagent, and a kit for examining the liver function of a horse.

  The equine digestive tract has anatomical and physiologic factors that tend to cause colic, and occurs most frequently among equine diseases following motor organ disease. Colic often includes many diseases with abdominal pain, and if left untreated, it can worsen and take the life of the horse. For this reason, appropriate prevention and treatment of colic is important in raising and managing horses. For this purpose, it is expected that the causative disease can be differentially diagnosed.

  Among them, the differential diagnosis of equine liver disease is difficult because the symptoms are similar to those of general colic caused by gastrointestinal tract such as stomach and intestine. However, there are reports that liver disease was discovered by autopsy of horses who died of unexplained colic, and because there are many horses that actually show abnormal serum enzyme levels indicating organic liver diseases, It is considered important to differentiate liver diseases.

  Conventionally, for liver disease in horses, biochemical diagnosis of blood has been performed by measuring liver deviation enzymes such as aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyltranspeptidase (GGTP). Has been used. However, these enzymes deviate from the tissue, and in the case of mild injury without tissue degeneration, an increase may not be observed. In human medicine, there are effective image diagnostic apparatuses such as ultrasound and MRI, but since there are large horses, there are no effective image diagnostic apparatuses. Therefore, it is considered necessary to establish a diagnostic method with higher sensitivity and accuracy.

In recent years, research on the ¹³ C phenylalanine (L- [1- ¹³ C] phenylalanine) breath test method has been promoted as a safe and simple liver function evaluation method in human medicine that is non-invasive and non-radioactive ( Patent Document 1, Non-Patent Documents 1 and 2). From these studies, the correlation between ¹³ C breath test method using ¹³ C phenylalanine and liver disease is shown. From this, the effectiveness of ¹³ C breath test method for liver disease is expected.
However, for equine animals, basic studies of ¹³ C breath test using ¹³ C phenylalanine have not been conducted and its effectiveness has not been confirmed.

U.S. Pat.No. 5,386,832 Digestion 2001; 63: 130-138 Journal of Surgical Research 112, 38-42 (2003)

  An object of this invention is to provide the safe and simple method for test | inspecting the liver function of a horse.

The present inventors have found, as a result of extensive studies to achieve the above object, ¹³ C-labeled aromatic dose for use ¹³ C breath test method using an amino acid liver function tests equine, Sampling time, Successful determination of the evaluation criteria has led to the completion of the present invention.
The gist of the present invention is as follows.

(1) A method for examining the liver function of equine animals by a breath test using ¹³ C-labeled aromatic amino acids.
(2) The method according to (1), wherein the ¹³ C-labeled aromatic amino acid is ¹³ C-labeled phenylalanine.
(3) The method according to (1) or (2), wherein the carbon at position 1 of the ¹³ C-labeled aromatic amino acid is ¹³ C-labeled.
(4) The method according to (2) or (3), wherein the ¹³ C-labeled phenylalanine is L- [1- ¹³ C] -phenylalanine.

(5) The method according to any one of (1) to (4), wherein the equine family belongs to the genus Horse.
(6) The method according to (5), wherein the animal belonging to the genus Horse is Equus caballus.
(7) A reagent for examining the liver function of equine animals in the breath test, containing ¹³ C-labeled aromatic amino acid as an active ingredient.
(8) A kit for examining an equine liver function in a breath test, comprising the reagent according to (7).

According to the present invention, it is possible to examine the liver function of equine animals by the ¹³ C breath test method. This test method is safe and simple.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention provides a method of examining equine liver function in a breath test using ¹³ C-labeled aromatic amino acids.
In the breath test, after the administration of a compound labeled with carbon stable isotope ¹³ C (natural abundance of about 1.1%), at regular time intervals (for example, intervals of 5 minutes, 10 minutes, 15 minutes, etc.) or continuously, By collecting exhaled air until a predetermined time (eg, 20 min, 60 min, 180 min, 240 min, etc.) and measuring the ¹³ C concentration in exhaled CO ₂ , the pathology from the digestion, absorption, metabolic pathway of the compound This method is also called a ¹³ C breath test.

Examples of the ¹³ C-labeled aromatic amino acid include phenylalanine, tyrosine, and combinations thereof, and among these, phenylalanine is effective. The aromatic amino acid may be any of L-form, D-form and DL-form.
Aromatic amino acids may if at least one of carbon atoms constituting the aromatic amino acid is ¹³ C-labeled, preferably 1-position carbon is ¹³ C-labeled.

  The equine animal to be examined by the method of the present invention is preferably an animal belonging to the genus horse (genus Equine, genus Donkey, subgenus Azinanodon, subgenus Zebra, subgenus Grapy zebra), more preferably Is Equus caballus. The equine subgenus includes horses (livestock species; scientific name Equus caballus) and mocono horses. , Couran, Mokonoro, Indonova) and Can There is a Grappy Zebra.

In one embodiment of the invention, after the ¹³ C-labeled aromatic amino acid is administered to the equine animal, the breath of the animal is collected, the amount of ¹³ C in the breath is measured, and based on the measured value Liver function is assessed.
The animals to be tested should be fasted 12 hours before the administration of ¹³ C-labeled aromatic amino acid.
^When administering a ¹³ C-labeled aromatic amino acid to an animal, the ¹³ C-labeled aromatic amino acid is used alone or mixed with an excipient or carrier, and depending on the administration route, an oral preparation (for example, a tablet, a capsule, (Powder, granule, liquid, etc.), parenteral (for example, injection) and the like.

The present invention provides a reagent for examining the liver function of equine animals in the breath test, which contains a ¹³ C-labeled aromatic amino acid as an active ingredient. The reagent of the present invention can be used as a test agent and / or a diagnostic agent. The excipient or carrier may be any excipient or carrier that is conventionally used in the art and acceptable as an animal drug, and the type and composition thereof are appropriately changed depending on the administration route and administration method. For example, water (for example, distilled water) is used as the liquid carrier. As the solid carrier, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate, and the like are used. In the case of injections, generally sterile water, physiological saline and various buffer solutions are desirable. When the reagent of the present invention takes the form of a solution or an injection, it is administered after being dissolved in an appropriate liquid carrier (eg, distilled water, sterilized water, physiological saline, various buffers, etc.) at the time of administration. Also good.

The content of ¹³ C-labeled aromatic amino acid in the preparation varies depending on the kind of preparation, but is usually 0.1 to 100 weight. For example, in the case of an injection, it may be usually added so as to be 1 to 40% by weight. In the case of capsules, tablets, granules, powders, and liquids, it is about 0.1 to 100% by weight.
The dose of ¹³ C-labeled aromatic amino acid needs to be an amount that can confirm the increase of ¹³ CO _{2 in} exhaled breath by administration, and varies depending on the age, weight, test purpose, etc. of the animal to be tested. In the case of mature animals, the dose per administration is suitably about 0.1 to 1000 mg / kg body weight, preferably about 2.5 to 10 mg / kg body weight, more preferably about 5 to 10 mg / kg body weight.

¹³ C-labeled aromatic amino acid is administered to equine animals, and after administration, at regular time intervals (eg, 5 minutes, 10 minutes, 15 minutes, etc.) or continuously for a predetermined time (eg, 20 minutes, 60 minutes) , 180 minutes, 240 minutes, etc.), it is good to collect ¹³ C concentration in exhaled CO ₂ . This series of measurements may be performed once per animal or may be repeated a plurality of times.
To collect exhaled breath, insert a tube (for example, a silicone tube) with a sheet (for example, a plastic plate) that closes the nostril into the animal's nasal cavity, and adjust the exhaled breath bag (for example, UbiT dedicated exhaled sample) Bag 200 ml, 1.3L can be collected in Otsuka Pharmaceutical Co., Ltd., Japan).

The ¹³ C concentration in exhaled CO ₂ should be measured using gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy, mass spectrometry, photoacoustic spectroscopy, NMR (nuclear magnetic resonance) method Can do. The present invention is not limited to the above example, and other methods may be used as long as the ¹³ C concentration in exhaled CO ₂ can be measured. For example, an infrared spectroscopic analyzer (POC one, Otsuka Electronics Co., Ltd., Japan) can be used. POC one utilizes the difference in infrared absorption wavelength of ¹³ CO ₂ and ¹² CO ₂ is measured abundance ratio in the breath of the _{^{(13 CO 2/12 CO 2}} ). Further, a difference from the measured value of the reference exhalation gas having a natural abundance ratio is obtained, and the amount of change (hereinafter also referred to as Δ ¹³ CO ₂ ) is calculated. Δ ¹³ CO ₂ is often used for analysis of breath test ( ¹³ C-practice-practice and basic application, P102-111, 2002, Matsubayashi, Maruyama, ¹³ C Medical Application Study Group).

The time when the measured Δ ¹³ CO ₂ shows the maximum value is T _max, and the Δ ¹³ CO ₂ value at that time is C _max . Liver function can be evaluated from the data of C _max and / or ¹³ CO ₂ cumulative emission (Δ ¹³ CO ₂ integrated value). From the data obtained so far, when L- [1- ¹³ C] phenylalanine is administered at a dose of 10 mg / kg body weight, T _max is 33.3 ± 11.1 min and C _max is in the range of 57.8 ± 16.3 ‰ is a value, or ¹³ CO ₂ cumulative emissions (Δ ¹³ CO ₂ integrated value) is determined to be normal if the value of 545.3 ± 155.7 ‰ range, lower than C _max is 57.8 ± 16.3 ‰ range It can be suspected that the liver disease is reflected if it is shown, or if the ¹³ CO ₂ cumulative emission amount (Δ ¹³ CO ₂ integrated value) is lower than the 545.3 ± 155.7 ‰ range. By increasing the number of data of healthy horses and case horses and conducting detailed analysis, it will be possible to obtain criteria with higher accuracy and sensitivity.

In addition, evaluation by such ¹³ C breath test is useful alone, but clinical symptoms (eg depression, anorexia, colic, etc.), blood tests (AST, ALP, GGTP, LDH, etc.), liver biopsy It is more preferable to make a comprehensive judgment in combination with the results of
The test method of the present invention can be used for liver diseases such as cirrhosis, chronic hepatitis, acute hepatitis, and liver cancer, diagnosis of liver damage, evaluation of liver function before and after diet therapy and infusion administration, and the like.
Furthermore, the present invention provides a test for examining the liver function of an equine animal in an exhalation test, comprising a reagent for examining the liver function of an equine animal in an exhalation test, which contains a ¹³ C-labeled aromatic amino acid as an active ingredient. Kits are also provided.

The reagent for examining the liver function of equine animals containing ¹³ C-labeled aromatic amino acid as an active ingredient in the breath test is as described above.
The kit of the present invention further includes a nasogastric catheter, a tube (eg, a silicone tube) attached with a sheet (eg, a plastic plate) that closes the nostril, an exhalation collection bag, analysis software, a measurement device, an instruction manual, and the like. May be included. In addition to the method of using the kit, the instructions for use should include criteria for determining liver function.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

[Example 1]
Materials and methods 1) Basic study Six healthy thoroughbred horses were used as test horses (one male, three females, two pups, age 5.7 ± 2.1 years, weight 485.0 ± 40.9 kg (mean ± SD)). In addition, it was confirmed in advance that each horse had normal gastric emptying and no abnormal findings were detected by blood biochemistry. ¹³ C phenylalanine (C ₆ H ₅ CH ₂ CHNH ₂ ¹³ COOH), molecular weight 166, Tokyo Gas Chemical Co., Japan) at doses of 2.5 mg / kg, 5 mg / kg and 10 mg / kg, each 1 ml / Dissolved in kg of distilled water and administered using a nasogastric catheter. Two breaths were collected at intervals of 5 minutes up to 20 minutes, 10 minutes up to 60 minutes, 15 minutes up to 180 minutes and 30 minutes up to 240 minutes after administration of the reagent. The collected exhaled breath was measured with an infrared spectroscopic analyzer (POCone, Otsuka Electronics Co., Ltd., Japan), and the point where the measured Δ ¹³ CO ₂ ( ¹³ CO ₂ abundance in exhaled breath) showed the highest value was defined as T _max At that time, Δ ¹³ CO ₂ was _defined as C _max . Further, a value obtained by integrating Δ ¹³ CO ₂ at each sampling time was defined as a cumulative emission amount of ¹³ CO ₂ at each sampling time (Δ ¹³ CO ₂ integrated value).

2) Examination in cases A breath test was conducted on two animals suspected of having liver disease based on blood biochemical examination findings. In case 1 (Thoroughbred, 4 years old, 532 kg), an increase in AST (1070 U / L) was observed in blood biochemistry. Necropsy was performed and nonspecific mild changes such as infiltration of glycon sheath inflammatory cells, formation of pseudobile ducts, and vacuolar degeneration of hepatocytes throughout the tissue were observed. In case 2 (quarter hose, female 5 years old, 407 kg), an increase in AST (2000 U / L) was observed on blood biochemistry. The case horse was measured using a dose of 10 mg / kg, and the other methods were the same as those for healthy horses.

Results The results of the breath test in each healthy horse were 10 mg / kg, 5 mg / kg, and T _max was 33.3 ± 11.1 min and 32.5 min ± 14.6 min, respectively. At mg / kg, multiple time points showing T _max were observed at 30 min and 40 min, and no clear peak was observed. In addition, C _{max at} 10 mg / kg was 57.8 ± 16.3 ‰, C _{max at} 5 mg / kg was 32.1 ± 11.0 ‰, and standard deviation was lower at C _max of 5 mg / kg compared to 10 mg / kg. Values are shown (Table 1, FIG. 1).
In both cases 1 and 2, T _max was 30-40 min, which was not significantly different from healthy horses. However, in case 1, C _max was remarkably low (29.4 ‰), and in case 2 also low (47.9 ‰) (Table 2, FIG. 2). In both cases, Δ ¹³ CO ₂ showed a low value compared to healthy horses at all time points, and the cumulative emission of ¹³ CO ₂ at the last measurement time of 240 minutes for healthy horses (Δ ¹³ CO ₂ integrated value) However, Case 1 showed a markedly low value of 273.9 ‰, and Case 2 also showed a low value of 410.7 ‰ (Table 3, FIG. 3).

Consideration
A clear peak was observed at 10 mg / kg and 5 mg / kg, and the standard deviation of C _max at 5 mg / kg was low, so 5 mg / kg was ¹³ C for horses. It is considered suitable as a dose for the phenylalanine breath test.
In the results of Case 1, C _max shows a markedly lower value compared to healthy horses, and in both cases the ¹³ CO ₂ cumulative emissions (Δ ¹³ CO ₂ accumulated value) are compared to healthy horses. Since it showed a low value at each measurement time, the decrease in C _max and cumulative emissions (Δ ¹³ CO ₂ integrated value) may reflect liver disease.

  The present invention can be used for the examination of the liver function of horses.

The amount of change in ¹³ CO ₂ abundance (Δ ¹³ CO ₂ ) in exhaled breath of healthy horses (average of 6 horses) administered with ¹³ C phenylalanine at doses of 2.5 mg / kg, 5 mg / kg and 10 mg / kg is shown. The amount of change in ¹³ CO ₂ abundance (Δ ¹³ CO ₂ ) in exhaled breath of healthy horses (average of 6) and cases 1 and 2 administered with ¹³ C phenylalanine at a dose of 10 mg / kg is shown. Accumulated change of ¹³ CO ₂ abundance (Δ ¹³ CO ₂ ) in the exhaled breath of healthy horses (average of 6) treated with ¹³ C phenylalanine at a dose of 10 mg / kg and cases 1 and 2 ( ¹³ CO ₂ cumulative emissions) Amount).

Claims (8)

A method of examining the liver function of equine animals by breath test using ¹³ C-labeled aromatic amino acid. The method according to claim 1, wherein the ¹³ C-labeled aromatic amino acid is ¹³ C-labeled phenylalanine. The method according to claim 1 or 2, wherein the carbon at position 1 of the ¹³ C-labeled aromatic amino acid is ¹³ C-labeled. The method according to claim 2 or 3, wherein the ¹³ C-labeled phenylalanine is L- [1- ¹³ C] -phenylalanine. The method according to any one of claims 1 to 4, wherein the equine family belongs to the genus Horse. The method according to claim 5, wherein the animal belonging to the genus Horse is Equus caballus. A reagent containing ¹³ C-labeled aromatic amino acid as an active ingredient for examining the liver function of equine animals in the breath test. A kit for examining a liver function of an equine animal by a breath test, comprising the reagent according to claim 7.