JPH072724A - 13c-labeled arachidonic acid and its derivative and method for producing the same - Google Patents

Abstract

PURPOSE:To enhance the multiplication rate of an arachidonic acid-producing fungus and efficiently produce the <13>C-labeled arachidonic acid at a <13>C labeling rate of >=95% by using a <13>C-labeled yeast extract in a medium. CONSTITUTION:An arachidonic acid-producing fungus is cultured in a medium containing a <13>C-labeled yeast extract having a <13>C-labeled rate of >=95% or the yeast extract and a <13>C-labeled organic compound, and the <13>C-labeled arachidonic acid is subsequently obtained from the cultured cells.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to ¹³ C-labeled arachidonic acid and its derivatives. ¹³ C-labeled arachidonic acid is extremely useful as a safe labeling compound for tracer experiments in medicine, pharmacy, physiology, biochemistry and the like.

[0002]

2. Description of the Related Art Arachidonic acid is an n-6 polyunsaturated fatty acid and is a very important substance as a precursor of an Icosanoid. In animals, arachidonic acid is generally ingested as an essential fatty acid into the body from a diet or the like. Arachidonic acid taken up in the body is mainly present in cell membrane phospholipids and adipose tissue, but it is acted on by phospholipase or the like to produce prostaglandin, leukotrienes, and eicosanoids such as thromboxane. To be converted. Eicosanoids have various physiological activities such as contraction of smooth muscle, regulation of steroid biosynthesis, regulation of gastric secretion, platelet aggregation, regulation of neurotransmission and mediation of inflammatory response. Thus, arachidonic acid is a very important substance as a precursor of eicosanoid.

[0003] Since it is necessary for human health that such a metabolic system normally operates as necessary, it is important to directly investigate the metabolism of arachidonic acid in vivo. Therefore, it is necessary to label arachidonic acid with an isotope. However, the presently obtained arachidonic acid labeled compound is a radioisotope label in which the carbon at the 1-position is replaced with carbon 14 ( ¹⁴ C), and the radioisotope is dangerous in human in vivo experiments. Therefore, it is prohibited by law in Japan. Therefore,
It is desired to provide arachidonic acid that is uniformally labeled with a stable isotope that does not endanger the human body. Further, for other higher fatty acids, stable isotope uniform labeled compounds are required for the same reason.

On the other hand, it is known that the fatty acid synthesis pathways of many organisms mainly extend the fatty acid chain by condensation of malonyl-CoA or malonyl-ACP using acetyl-CoA or acetyl-ACP as a primer (biochemistry Laboratory Lecture 9 Metabolism of Lipids Tokyo Kagaku Dojin (1975), deuterium produced by adding stable isotope-labeled sodium acetate to the medium or ¹³ C-labeled oleic acid has been used for stereochemical studies (K. Arai et al. .al. J. Am. Chem. So
c. 1989, 111, 3391-3399.). This means that deuterium-labeled and / or ¹³ C-labeled higher fatty acids can be prepared in deuterium and / or
It is shown that this is possible by growing the organism in a medium containing ¹³ C-labeled sodium acetate. In addition, ¹³
Since the isotope effect of C on ¹² C is smaller than the isotope effect of deuterium on hydrogen, it can be said that the ¹³ C-labeled compound generally gives more accurate tracer experiment results than the deuterium-labeled compound.

[0005]

As described above, as described above, ¹³ C
It is possible to produce ¹³ C-labeled higher fatty acid by growing the organism in a medium containing labeled sodium acetate,
It is possible to produce ¹³ C-labeled arachidonic acid by using an appropriate arachidonic acid-producing bacterium. However, in the conventional method for producing ¹³ C-labeled higher fatty acid, a commonly used medium is used.
¹³ because C is a labeled acetate was added, the total carbon atoms of ¹³ C-labeled higher fatty acids obtained from the cultured microorganisms or the like ¹³
The abundance ratio of C atoms (hereinafter referred to as ¹³ C labeling rate) is at most about 50%, and the ratio of ¹³ C labeled sodium acetate in the carbon source in the medium in order to obtain a higher ¹³ C labeling rate. When the amount of arachidonic acid is increased, the growth of microorganisms that produce arachidonic acid is reduced, so that production of ¹³ C-labeled arachidonic acid having a ¹³ C-labeling rate of 90% or more was practically impossible.

The present invention has been made in view of the above circumstances, and an object thereof is to perform a wide range of metabolic experiments, including in vivo experiments of human arachidonic acid having a beneficial pharmacological action, and measurement of metabolic ability. Extremely useful for high ¹³
Another object of the present invention is to provide a derivative such as ¹³ C-labeled arachidonic acid or its ester labeled with a C-labeling rate.

[0007]

The ¹³ C-labeled arachidonic acid according to the present invention is characterized in that the abundance ratio of ¹³ C atoms in all carbon atoms is 95% or more. The present invention also includes the ¹³ C-labeled arachidonic acid derivative.

Further, in the method for producing ¹³ C-labeled arachidonic acid according to the present invention, the abundance ratio of ¹³ C atoms in all carbon atoms is 9%.
5% or more of ¹³ C-labeled yeast extract or the yeast extract
^The method is characterized in that an arachidonic acid-producing bacterium is cultured in a medium containing a ¹³ C-labeled organic compound, and then ¹³ C-labeled arachidonic acid or a derivative thereof is separated from the cultured cells.

[0009] ¹³ C-labeled arachidonic acid according to the present invention, ¹³
The C-labeling rate is 95% or more. Oxygen and hydrogen, which are the constituent elements other than carbon in ¹³ C-labeled arachidonic acid, are not particularly limited, and they are ordinary oxygen ( ¹⁶ O) and hydrogen ( ¹ H), but these are deuterium which is a stable isotope. , ¹⁷ O, ¹⁸ O can also be used for labeling.

The present invention also includes derivatives of ¹³ C-labeled arachidonic acid. As its derivative, ¹³ C
Labeled arachidonic acid esters such as methyl and ethyl esters with lower alcohols, esters with higher alcohols, esters with glycerin, esters with glycols, esters with sterols, esters with sugar alcohols, esters, etc. , Na salt, K salt and the like.

[0011] To prepare the ¹³ C-labeled arachidonic acid, the production method of ¹³ C-labeled arachidonic acid according to the present invention,
¹³ C-labeled rate culturing the arachidonic acid-producing bacteria by using a medium containing the ¹³ C-labeled organic compound of 95% or more ¹³ C-labeled yeast extract or yeast extract, and then from the cultured cells ¹³ C
The labeled arachidonic acid or its derivative is separated.

The microorganism used in the method for producing ¹³ C-labeled arachidonic acid according to the present invention may be any microorganism as long as it produces arachidonic acid. For example, Mortierella filamentous fungi such as Mortierella alpina (IFO8568) and Mortierella exigua (IFO8571) which are arachidonic acid-producing filamentous fungi, Blastcladiella filamentous fungi, Conidiobolus filamentous fungi, Entomophthora filamentous fungi are known. However, it can be appropriately selected and used from these.

As the culture medium of these arachidonic acid-producing microorganisms, ¹³ C-labeled yeast extract or the yeast extract and ¹³
C-labeled organic compounds can be used. ^{The 13} C-labeled yeast extract is obtained by culturing yeast in a medium using a ¹³ C-labeled organic compound as a carbon source, and a ¹³ C-labeling rate of 95% or more can also be obtained. ^{The 13} C-labeled organic compound may be any compound that directly or indirectly participates in fatty acid synthesis as a metabolite, and those compounds are easily available as commercial products, such as sodium acetate, xylose and glucose. Amino acids such as sugars, alanine, and aspartic acid can be used. For the culture of these arachidonic acid-producing microorganisms, for example, a medium (pH 6.0) in which 2% by weight of ¹³ C-labeled glucose and 1% by weight of ¹³ C-labeled yeast extract are dissolved in distilled water is used.

After freeze-drying the cells cultured in such a medium and then methyl-esterifying or ethyl-esterifying them with methanol such as hydrochloric acid methanol or sodium methylate, the fatty acid composition of all fatty acid derivatives in the cells is GC. -Can be analyzed by MS. In addition, wet or dry bacterial cells are extracted with an appropriate organic solvent or the like to give silica gel T
After fractionating the lipids by LC, the constituent fatty acids of each lipid can be analyzed in the same manner. The ¹³ C-labeling rate of the fatty acid of the present invention cultivated by the above-mentioned method is almost 100%. The ¹³ C-labeled arachidonic acid esterified by the method described above can be derivatized to the free form by saponification and subsequent acidification according to a conventional method. Further, this ¹³ C-labeled arachidonic acid can be synthesized into various derivatives by esterification with various alcohols, etc., if necessary. ¹³ C-labeled arachidonic acid is reverse phase PLC according to a conventional method.
And silver nitrate treated silica gel or silver nitrate treated silica gel T
By combining LC, each can be isolated from the polyunsaturated fatty acids that are simultaneously produced.

[0015]

【Example】

Example 1 2% by weight of ¹³ C-labeled glucose in distilled water, ¹³
In 50 ml of a medium containing 1% by weight of C-labeled yeast extract,
Arachidonic acid producing bacterium Mortierella alpina (IFO
8568) was inoculated and cultured with shaking at a temperature of 25 ° C. After culturing for 1 week, the cells are harvested and lyophilized to about 1
00 mg of dried cells was obtained. The cells were suspended in 5 ml of hydrochloric acid methanol, heated at 80 ° C. for 1 hour, and then extracted with n-hexane to obtain about 14 mg of a fatty acid methyl ester mixture. The extracted fatty acid methyl ester mixture was purified by silver nitrate silica gel TLC to obtain about 1.6 mg of methyl arachidonic acid ester. The obtained arachidonic acid methyl ester was analyzed by GC-MS. The analysis result is shown in FIG. As a result, in the case of natural arachidonic acid methyl ester not labeled with ¹³ C, the parent ion M / Z
Is 318, whereas M / Z of the parent ion of arachidonic acid methyl ester ( ¹³ C-labeled arachidonic acid methyl ester) obtained in this example has three peaks of 338, 337, and 336. The ¹³ C-labeling rate was estimated to be about 98% from the intensity ratio of the peaks.

Example 2 An arachidonic acid-producing bacterium, Mortierella alpina (IFO8568), was inoculated into 5 ml of a medium prepared by dissolving 1% by weight of ¹³ C-labeled yeast extract in distilled water, and shaken at a temperature of 25 ° C. After culturing, the cells were collected and freeze-dried. Extraction of lipid fraction from this cell, about 3 mg
To obtain the lipid. The resulting lipid was suspended in 1 ml of hydrochloric acid methanol, heated at 80 ° C. for 1 hour and then extracted with n-hexane to obtain a fatty acid methyl ester mixture containing about 24 μg of arachidonic acid methyl ester. The obtained fatty acid methyl ester mixture was analyzed by GC-MS. As a result, M / Z of the parent ion of the obtained ¹³ C-labeled arachidonic acid methyl ester has three peaks of 338, 337, and 336, and the ¹³ C-labeling rate is about 97 from the intensity ratio of these peaks.
It was estimated to be%.

[0017]

As described above, ¹³ C according to the present invention
Labeled arachidonic acid has a ¹³ C labeling rate of 95% or more, so it can be safely used without endangering the human body.
Since the labeling rate is high, it can be detected with high accuracy by a nuclear magnetic resonance spectrum method or a GC-MS method. Further, since it is uniformly labeled with a high labeling rate, it is possible to measure a wide range of metabolic experiments and metabolic ability including in vivo experiments on the human body with high accuracy.

Further, in the production method according to the present invention, an arachidonic acid-producing bacterium is cultured using a ¹³ C-labeled yeast extract having a ¹³ C labeling rate of 95% or more or a medium containing the yeast extract and a ¹³ C-labeled organic compound. As a result, it becomes possible to produce ¹³ C-labeled arachidonic acid with a ¹³ C-labeling rate of 95% or more, which was impossible with the conventional method. Moreover, by using ¹³ C-labeled yeast extract in the medium, the growth rate of arachidonic acid-producing bacteria can be increased, and the ¹³ C-labeled rate of ¹³ % or more can be increased.
C-labeled arachidonic acid can be efficiently produced.

[Brief description of drawings]

FIG. 1 is a mass spectrum of ¹³ C-labeled arachidonic acid methyl ester prepared in an example of the present invention.

Claims (2)

[Claims] 1. A ¹³ C-labeled arachidonic acid and a derivative thereof, wherein the abundance ratio of ¹³ C atoms in all carbon atoms is 95% or more. 2. An arachidonic acid-producing bacterium is cultivated using a ¹³ C-labeled yeast extract or a medium containing the yeast extract and a ¹³ C-labeled organic compound, in which the abundance ratio of ¹³ C atoms in all carbon atoms is 95% or more. Then, ¹³ C-labeled arachidonic acid or its derivative is separated from the cultured cells.
Method for producing ¹³ C-labeled arachidonic acid.