JPH06340577A - 13c-labeled eicosapentaenoic acid, its derivative and production thereof - Google Patents

Abstract

PURPOSE:To obtain <13>C-labeled eicosapentaenoic acid having >=95% <13>C-labeling ratio. CONSTITUTION:A microorganism capable of producing eicosapentaenoic acid is cultured by using a culture medium containing <13>C-labeled yeast extract having >=95% <13>C-labeling ratio or the yeast extract and a <13>C-labeled organic compound to provide <13>C-labeled eicosapentaenoic acid. The proliferation ratio of the microorganism capable of producing the eicosapentaenoic acid can be enhanced by using the <13>C-labeled yeast extract in a culture medium to efficiently produce the <13>C-labeled eicosapentaenoic acid having >=95% <13>C-labeling ratio.

Description

Detailed Description of the Invention

[0001]

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

[0002]

BACKGROUND OF THE INVENTION Icosapentaenoic acid, as a highly unsaturated fatty acid of n-3, antagonizes an icosanoid derived from a highly unsaturated fatty acid of n-6 to suppress platelet aggregation, lower lipid in blood, anti-inflammatory action, antitumor action. It has been recognized that it is important for body homeostasis. Icosapentaenoic acid, which has such an effect, is mainly present in cell membrane phospholipids and adipose tissue, and enters the β-oxidative degradation system of mitochondria and peroxisomes to be degraded to acetyl-CoA, or chain length extension. And undergoes metabolism such as being desaturated and converted into the same n-3 docosahexaenoic acid. Recently, β of long-chain fatty acid of peroxisome
It has also been suggested that the oxidative degradation activity measurement may be used for diagnosis of diseases.

Since it is necessary for human health that such a metabolic system normally operates as needed, it is important to directly investigate the in vivo metabolism of icosapentaenoic acid. Therefore, it is necessary to label icosapentaenoic acid with an isotope. However, the currently obtained labeling compound of icosapentaenoic acid is a radioisotope label in which the carbon at the 1-position is replaced by carbon 14 ( ¹⁴ C), and the metabolism of β-oxidative decomposition system cannot be traced in principle. In addition, radioactive isotopes are dangerous in humans in vivo experiments and are prohibited by law in Japan. Therefore, it is desired to provide icosapentaenoic acid, which is uniformly 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 ( ² D) or ¹³ C-labeled oleic acid produced by adding stable isotope-labeled sodium acetate to the medium is used for stereochemical studies ( K.Arai et.al. JA
m. Chem. Soc. 1989, 111, 3391-3399.). This indicates that preparation of deuterium-labeled and / or ¹³ C-labeled higher fatty acids is possible by growing the organism in deuterium and / or in a medium containing ¹³ C-labeled sodium acetate. Furthermore, since the isotope effect of ¹³ C on ¹² C is smaller than that of deuterium on hydrogen, ¹³ C-labeled compounds generally give more accurate tracer experiment results than deuterium-labeled compounds. Can be said.

[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,
By using an appropriate icosapentaenoic acid-producing bacterium
It is possible to produce ¹³ C-labeled icosapentaenoic acid, but in the conventional method for producing ¹³ C-labeled higher fatty acid, since ¹³ C-labeled sodium acetate is added to a commonly used medium, it can be obtained from cultured microorganisms or the like. The abundance ratio of ¹³ C atoms to the total carbon atoms of the ¹³ C-labeled higher fatty acids (hereinafter referred to as ¹³ C labeling rate) is about 50% at most, and the medium has a higher ¹³ C labeling rate. ¹³ of total carbon source
If the proportion of C-labeled sodium acetate is increased, the growth of microorganisms that produce icosapentaenoic acid is reduced, and it is practically impossible to produce ¹³ C-labeled icosapentaenoic acid having a ¹³ C-labeling rate of 90% or more. It was

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

[0007]

The ¹³ C-labeled icosapentaenoic 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 icosapentaenoic acid derivative.

Further, the method for producing ¹³ C-labeled icosapentaenoic acid according to the present invention comprises a ¹³ C-labeled yeast extract or a yeast extract and a ¹³ C-labeled organic compound in which the abundance ratio of ¹³ C atoms to all carbon atoms is 95% or more. The icosapentaenoic acid-producing bacterium is cultured in a medium containing and then ¹³
The method is characterized in that C-labeled icosapentaenoic acid or its derivative is separated.

The ¹³ C-labeled icosapentaenoic acid according to the present invention is characterized by having a ¹³ C labeling rate of 95% or more. Oxygen and hydrogen, which are the constituent elements other than carbon in ¹³ C-labeled icosapentaenoic acid, are not particularly limited, and are ordinary oxygen ( ¹⁶ O) and hydrogen ¹ H). It is also possible to label with ¹⁷ O or ¹⁸ O.

The present invention also includes derivatives of ¹³ C-labeled icosapentaenoic acid. Examples of the derivative include ester with lower alcohol such as methyl ester or ethyl ester of ¹³ C-labeled icosapentaenoic acid, ester with higher alcohol, ester with glycerin, ester with glycol, ester with sterol, ester with sugar alcohol. And the like, acid chlorides, Na salts, K salts and the like.

The microorganism used in the method for producing ¹³ C-labeled icosapentaenoic acid according to the present invention may be any microorganism as long as it produces icosapentaenoic acid. For example, Shiwanella putrefaciens SCR, which is a marine bacterium producing icosapentaenoic acid.
C-2874 (FERMBP-1625), Alteromonas putrifaciens SCRC-2871
(FERMBP-1624), Pseudomonas putrifaciens SCRC-2878 (FERMBP
-1623) and the like, Mortierella alpina (IFO 85, which is an arachidonic acid-producing filamentous fungus when cultured at a medium added with a precursor of icosapentaenoic acid or at a low temperature).
68) and the like.

As a medium for these microorganisms producing icosapentaenoic acid, ¹³ C-labeled yeast extract or the yeast extract and ¹³ C-labeled organic compound 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, and examples thereof include sodium acetate, xylose, Sugars such as glucose and amino acids such as alanine and aspartic acid can be used. For example, 1% by weight of a ¹³ C-labeled amino acid mixture for marine bacteria and 0.5% by weight of ¹³ C-labeled yeast extract were used for culturing.
Medium (pH 7.0) dissolved in 2 concentrations of artificial seawater, ¹³
A medium (pH 7.0) in which 1% by weight of C-labeled yeast extract is dissolved in 1/2 concentration of artificial seawater 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 obtain silica gel TL.
After fractionating the lipids at C, the constituent fatty acids of each lipid can be similarly analyzed. The ¹³ C-labeling rate of the fatty acid of the present invention cultivated by the above method is almost 100%. The ¹³ C-labeled icosapentaenoic acid esterified by the method described above can be derivatized to the free form by saponification and subsequent acidification according to a conventional method. Also, various derivatives of this ¹³ C-labeled icosapentaenoic acid can be synthesized by subjecting it to esterification with various alcohols as necessary. ¹³ C-labeled icosapentaenoic acid can be prepared by combining reverse-phase PLC and silver nitrate-treated silica gel or silver nitrate-treated silica gel TLC according to a conventional method.
Each can be isolated from the polyunsaturated fatty acids that are produced simultaneously.

[0014]

[Examples] 50 ml of a medium prepared by dissolving 1% by weight of ¹³ C-labeled yeast extract in 1/2 concentration artificial seawater was inoculated with Shiwanella putrifaciens (FERMBP-1625), which is a marine bacterium producing icosapentaenoic acid, and 10 The cells were shake-cultured at a temperature of ° C. The cells were collected in the late logarithmic growth phase, and the separated cultured cells were freeze-dried to obtain 84 mg of dried cells. The cells were suspended in 6 ml of hydrochloric acid methanol,
After heating at ℃ for 1 hour, extract with n-hexane,
mg of fatty acid methyl ester mixture was obtained. Extracted fatty acid methyl ester mixture with silver nitrate treated silica gel TL
Purification by C gave approximately 200 μg of icosapentaenoic acid methyl ester. The obtained icosapentaenoic acid methyl ester was analyzed by GC-MS. The analysis result is shown in FIG. As a result, in the case of natural icosapentaenoic acid methyl ester not labeled with ¹³ C, the parent ion M / Z
Is 316, whereas M / Z of the parent ion of icosapentaenoic acid methyl ester ( ¹³ C-labeled icosapentaenoic acid methyl ester) obtained in this example has a unique peak at 336. As a result, the ¹³ C labeling rate was estimated to be 99% or more.

[0015]

As described above, ¹³ C according to the present invention
Labeled icosapentaenoic acid has a ¹³ C labeling rate of 95% or more, so it can be used safely without endangering the human body. Due to its high labeling rate, it can be highly labeled by nuclear magnetic resonance spectroscopy or GC-MS. It can be detected with accuracy. 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.

In the production method according to the present invention, the icosapentaenoic 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 icosapentaenoic acid having a ¹³ C-labeling rate of 95% or more, which was impossible by the conventional method. Moreover, by using ¹³ C-labeled yeast extract to the medium, it is possible to increase the growth rate of eicosapentaenoic acid producing bacteria, ¹³ C-labeled rate can efficiently produce 95% of the ¹³ C-labeled eicosapentaenoic acid .

[Brief description of drawings]

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

Claims (2)

[Claims] 1. A ¹³ C-labeled icosapentaenoic acid and a derivative thereof, wherein the abundance ratio of ¹³ C atoms to all carbon atoms is 95% or more. 2. An icosapentaenoic 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 to all carbon atoms is 95% or more. Then, ¹³ C-labeled icosapentaenoic acid or a derivative thereof is separated from the cultured cells, and a method for producing ¹³ C-labeled icosapentaenoic acid.