JPH0630630B2 - Method for quantifying free fatty acid and reagent composition for quantification used therefor - Google Patents
Method for quantifying free fatty acid and reagent composition for quantification used thereforInfo
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- JPH0630630B2 JPH0630630B2 JP11815887A JP11815887A JPH0630630B2 JP H0630630 B2 JPH0630630 B2 JP H0630630B2 JP 11815887 A JP11815887 A JP 11815887A JP 11815887 A JP11815887 A JP 11815887A JP H0630630 B2 JPH0630630 B2 JP H0630630B2
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- fatty acid
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は遊離脂肪酸の定量方法、さらに詳しくは特に臨
床検査における血中遊離脂肪酸の定量に適した遊離脂肪
酸の定量方法及びそれに用いる遊離脂肪酸の定量用試薬
組成物に関するものである。TECHNICAL FIELD The present invention relates to a method for quantifying free fatty acids, and more particularly to a method for quantifying free fatty acids suitable for quantifying blood free fatty acids in clinical tests, and free fatty acids used therefor. The present invention relates to a quantitative reagent composition.
血液中の遊離脂肪酸（以下ＮＥＦＡと略す）の定量は、
生体内の脂質代謝に基づくエネルギー状態を反映するこ
とから、臨床的に有意義なものであるとされている。Quantification of free fatty acids (hereinafter abbreviated as NEFA) in blood is
It is considered clinically meaningful because it reflects the energy state based on lipid metabolism in the body.
ＮＥＦＡの定量法については、ドールの報告した滴定法
［V.P.Dole，ジャーナル オブ クリニカ インベステ
イゲーション（J.Clin.Invest.,)35,150,1956年］以来
数多くの定量法が報告されている。たとえば、ＮＥＦＡ
にコバルト［ノバク．エム．；ジャーナル オブ リピ
ッド リサーチ（Novak.M.;J.Lipid.Res..)6431 1965
年］あるいは銅［ダンコンブ ダブリュ ジー，クリニ
カ キミカ アクタ（Duncombe.W.G-,Clin.Chim.Acta)9
122,1964年］を作用させ、ＮＥＦＡのコバルト塩あるい
は銅塩として比色定量する方法、またＮＥＦＡに、アデ
ノシン３リン酸（以下ＡＴＰと略す）及びコエンザイム
Ａ（以下ＣｏＡと略す）存在下アシルコエンザイムＡ合
成酵素（以下ＡＣＳと略す）を作用させ、下記反応式Ｉ
より生成するアシルコエンザイムＡ（以下アシルＣｏＡ
と略す）やアデノシン１リン酸（以下ＡＭＰと略す）あ
るいは消費されるＣｏＡを測定する方法−いわゆる酵素
法−等が報告されている。Regarding the quantitative method of NEFA, many quantitative methods have been reported since the titration method reported by Dole [VPDole, Journal of Clinica Invest., J. Clin. Invest., 35 , 150, 1956]. For example, NEFA
Cobalt [Novak. M. ; Journal of Lipid Research (Novak.M.; J.Lipid.Res ..) 6 431 1965
Year] or copper [Duncombe W., Clin.Chim.Acta] 9
122, 1964] for colorimetric determination as a cobalt salt or copper salt of NEFA, and acyl coenzyme in the presence of adenosine triphosphate (hereinafter abbreviated as ATP) and coenzyme A (hereinafter abbreviated as CoA) in NEFA. A synthase (hereinafter abbreviated as ACS) is allowed to act on the following reaction formula I
Acyl coenzyme A produced by
And adenosine monophosphate (hereinafter abbreviated as AMP) or consumed CoA-a so-called enzyme method-and the like have been reported.
反応式Ｉ： これら定量法のうちコバルトや銅を用いる比色法は、操
作が繁雑なうえ測定精度に劣るため、ＡＣＳを利用した
上記反応式Ｉに基づく定量法が一般化した。Reaction formula I: Among these quantitative methods, the colorimetric method using cobalt or copper is complicated in operation and inferior in measurement accuracy. Therefore, the quantitative method based on the above reaction formula I using ACS has been generalized.
なかでも反応式Ｉで生成してくるアシルＣｏＡをアシル
コエンザイムＡ酸化酵素（以下ＡＣＯＤと略す）を利用
して、下記反応式IIにしたがって酸化する方法は、測定
精度に問題のあるＡＭＰを測定する方法や、ＮＥＦＡの
定量範囲が限定されてしまうＣｏＡ消費量を測定する方
法に比べて操作性、精度、感度等の点で優れていること
から現在繁用されている方法である。Among them, the method of oxidizing acyl CoA produced in reaction formula I according to the following reaction formula II using acyl coenzyme A oxidase (hereinafter abbreviated as ACOD) measures AMP having a problem in measurement accuracy. This method is widely used because it is superior in operability, accuracy, sensitivity and the like to the method and the method for measuring the CoA consumption amount in which the quantitative range of NEFA is limited.
反応式II： ところで、ＡＣＯＤによるアシルＣｏＡの酸化反応は、
試薬として反応系に添加したＣｏＡの還元作用のため阻
害を受ける。したがってＡＣＯＤを用いる測定系を実用
化するためには、Ｎ−エチルマレイミド（以下ＮＥＭと
略す）等、ＳＨ試薬の添加によって反応式Ｉにおける未
反応のＣｏＡの影響を抑える必要があった（特公昭57-3
3955公報）。Reaction formula II: By the way, the oxidation reaction of acyl CoA by ACOD is
It is inhibited by the reducing action of CoA added to the reaction system as a reagent. Therefore, in order to put the measurement system using ACOD into practical use, it was necessary to suppress the influence of unreacted CoA in the reaction formula I by adding an SH reagent such as N-ethylmaleimide (hereinafter abbreviated as NEM) (Japanese Patent Publication No. 57-3
3955 bulletin).
しかしながら、ＡＣＯＤは活性中心にＳＨ基を有する酵
素であるため、ＳＨ試薬と長時間共存させておくと構造
的破壊を起こし、試薬自体の安定性低下につながる。However, since ACOD is an enzyme having an SH group at the active center, if it is allowed to coexist with an SH reagent for a long time, structural destruction will occur, leading to a decrease in the stability of the reagent itself.
本発明は、ＡＣＳ及びＡＣＯＤを利用するＮＥＦＡの定
量法において、従来の方法の操作性、精度、感度等を損
うことなく試薬の安定性をも高め得るＮＥＦＡの定量方
法及びそのための試薬組成物を提供することを目的とし
ている。The present invention relates to a method for quantifying NEFA that utilizes ACS and ACOD, and a method for quantifying NEFA that can enhance the stability of a reagent without impairing the operability, accuracy, sensitivity, etc. of the conventional method, and a reagent composition therefor. Is intended to provide.
本発明は、検体中の遊離脂肪酸にＡＴＰとＣｏＡの存在
下ＡＣＳを作用させ、生成するアシルＣｏＡに下記一般
式Ｉで示される脂肪酸又はその水溶性の塩の存在下でＡ
ＣＯＤを作用させ、アシルＣｏＡを酸化して生じる変化
を測定することを特徴とするＮＥＦＡの定量方法及び少
なくともＡＴＰ、ＣｏＡ及びＡＣＳを含む第１試薬と少
なくとも下記一般式Ｉで示される脂肪酸又はその水溶性
の塩及びＡＣＯＤを含む第２試薬とから構成されるＮＥ
ＦＡ定量用試薬組成物に関するものである。In the present invention, free fatty acid in a sample is reacted with ACS in the presence of ATP and CoA, and the resulting acyl CoA is reacted with A in the presence of a fatty acid represented by the following general formula I or a water-soluble salt thereof.
A method for quantifying NEFA, which comprises reacting COD to oxidize acyl CoA and measuring the change, and a first reagent containing at least ATP, CoA, and ACS, and at least a fatty acid represented by the following general formula I or a water-soluble thereof. NE composed of a neutral salt and a second reagent containing ACOD
The present invention relates to a FA quantification reagent composition.
一般式Ｉ： Ｒ−Ａ−（ＣＨ2）n−ＣＯＯＨ （式中、Ｒは炭素数１〜20の脂肪族基を表し、ｎは０又
は１〜２の整数を表し、 Ａは−ＣＨ＝ＣＨ−又は を表し、 Ｙ及びＺは水素原子、水酸基又は低級アルキル基を表
す、ただしＹとＺは同時に水素原子を表さない。） 本発明における上記一般式Ｉで示される脂肪酸は、下記
反応式IIIで示される反応に従って、試薬として添加し
たＣｏＡ及びＡＴＰの存在下ＡＣＳの作用を受け、ＡＣ
ＯＤの基質となり得ない物質を生成する。General formula I: R-A- (CH 2 ) in n -COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, n represents an integer of 0 or 1 to 2, A is -CH = CH- or Y and Z represent a hydrogen atom, a hydroxyl group or a lower alkyl group, provided that Y and Z do not represent a hydrogen atom at the same time. ) The fatty acid represented by the above general formula I in the present invention is subjected to the action of ACS in the presence of CoA and ATP added as reagents according to the reaction represented by the following reaction formula III to obtain AC.
It produces substances that cannot be substrates for OD.
反応式III： （式中、ＦＡは上記一般的Ｉで示される脂肪酸を示し、
ＦＡ−ＣｏＡはＡＣＯＤの基質となり得ない物質を示
す） 上記一般式Ｉで示される脂肪酸と、この脂肪酸をもとに
反応式IIIに従って生成されるＡＣＯＤの基質となり得
ない物質との関係を、具体的な物質として列挙すれば、
例えば第１表に示す如きものが挙げられる。Reaction formula III: (In the formula, FA represents a fatty acid represented by the general formula I,
FA-CoA represents a substance that cannot serve as a substrate for ACOD.) The relationship between the fatty acid represented by the above general formula I and the substance that cannot be a substrate for ACOD produced according to reaction formula III based on this fatty acid is specifically described. If it is listed as a physical substance,
For example, those as shown in Table 1 can be mentioned.
上記一般式Ｉで示される脂肪酸の使用量は、試料中の予
想されるＮＥＦＡの濃度並びに反応液中に存在するＣｏ
Ａの絶対量にもよるが、ＣｏＡに対するモル比が１以
上、好ましくは５〜50程度とするとよい。 The amount of the fatty acid represented by the above general formula I used depends on the expected concentration of NEFA in the sample and the amount of Co existing in the reaction solution.
Although it depends on the absolute amount of A, the molar ratio to CoA is 1 or more, preferably about 5 to 50.
また、ＡＣＯＤはpH6.0〜8.5の範囲内で作用させるとよ
い。Further, ACOD may be allowed to act within a pH range of 6.0 to 8.5.
本発明によるＮＥＦＡの定量方法において、ＡＣＯＤの
作用により生じる変化を検出する方法としては、次のよ
うなものが挙げられる。In the method for quantifying NEFA according to the present invention, the method for detecting the change caused by the action of ACOD includes the following.
ＡＣＯＤの作用により生成する過酸化水素を公知の方
法で測定する。Hydrogen peroxide produced by the action of ACOD is measured by a known method.
ＡＣＯＤの作用により消費される酸素を公知の方法で
測定する。The oxygen consumed by the action of ACOD is measured by a known method.
なかでも現在臨床検査の分野で繁用されている過酸化水
素の比色法による測定方法は、これまでのＮＥＦＡの定
量法にも利用されていたものなので、特別な装置や熟練
を必要としないので、好ましい方法である。In particular, the colorimetric method for measuring hydrogen peroxide, which is widely used in the field of clinical examinations, has been used for the NEFA quantification method up to now, so no special device or skill is required. Therefore, it is the preferred method.
本発明によるＮＥＦＡの定量用組成物における上記一般
式Ｉの脂肪酸以外の物質は、従来の酵素法に基づく定量
用組成物に使用されるものと同様のものを用いることが
できる。すなわち、第１試薬には、リン酸、ホウ酸、ク
エン酸等適当な緩衝剤や、必要に応じて防腐剤や、公知
の安定剤等を加えることが可能である。同様に第２試薬
についても、緩衝剤、防腐剤及び安定剤等を加えること
ができる。更に第１試薬あるいは第２試薬には、ＡＣＯ
Ｄの作用により生じる変化を検出し得る試薬系を共存さ
せることも可能である。As the substance other than the fatty acid of the general formula I in the composition for quantifying NEFA according to the present invention, the same substances as those used in the composition for quantifying based on the conventional enzyme method can be used. That is, it is possible to add an appropriate buffering agent such as phosphoric acid, boric acid, citric acid, an antiseptic agent, a known stabilizer, or the like to the first reagent, if necessary. Similarly, a buffer, a preservative, a stabilizer and the like can be added to the second reagent. Further, the first reagent or the second reagent contains ACO
It is also possible to coexist with a reagent system capable of detecting a change caused by the action of D.
このような試薬系として広く知られているものとして
は、ＡＣＯＤの作用で生成してくる過酸化水素の測定試
薬系が挙げられる。具体的には、ペルオキシダーゼ（以
下ＰＯＤと略す）やカタラーゼを使用するものが挙げら
れる。これらは、ＰＯＤやカタラーゼの作用により、例
えば下記反応式〜に基づいて色素を生成させ、これ
を比色定量するものである。A widely known example of such a reagent system is a reagent system for measuring hydrogen peroxide produced by the action of ACOD. Specific examples include those using peroxidase (hereinafter abbreviated as POD) and catalase. These are for producing colorants by the action of POD or catalase, for example, based on the following reaction formulas and colorimetrically quantifying them.
本発明による試薬組成物は、溶解状態のみならず、凍結
乾燥等の方法により乾燥状態で提供することも可能であ
る。 The reagent composition according to the present invention can be provided not only in a dissolved state but also in a dried state by a method such as freeze-drying.
本発明によるＮＥＦＡの定量方法及び試薬組成物におけ
る前記一般式Ｉで示される脂肪酸は、ＡＣＯＤの作用に
よって進行する前記反応式IIで示される反応をＣｏＡが
妨害するのを抑える作用を有する。すなわち、前記反応
式Ｉにおいて、ＮＥＦＡとの反応にあずからなかったＣ
ｏＡ及びＡＴＰを、前記一般式Ｉで表される脂肪酸とＡ
ＣＳの存在下で反応させてＡＣＯＤの作用しない物質と
し、反応の系外に出してしまうのである。The fatty acid represented by the general formula I in the method for quantifying NEFA and the reagent composition according to the present invention has an action of suppressing CoA from interfering with the reaction represented by the reaction formula II which proceeds by the action of ACOD. That is, in the above reaction formula I, C which was not involved in the reaction with NEFA
oA and ATP are represented by the fatty acid represented by the general formula I and A
The reaction is carried out in the presence of CS to give a substance that does not act on ACOD, and is discharged out of the reaction system.
以下、本発明を実施例により説明する。 Hereinafter, the present invention will be described with reference to examples.
実施例１ 次に示す第１試薬及び第２試薬を用いてＮＥＦＡの定量
を行った。Example 1 NEFA was quantified using the following first and second reagents.
○第１試薬 ・リン酸緩衝液（pH6.8） 50mM ・ＡＴＰ ２mM ・塩化マグネシウム・ ６水和物 ５mM ・４−アミノアンチピリン ２mM ・ＣｏＡリチウム塩 0.2mM ・ＡＣＳ（比活性２Ｕ／mg タンパク） 0.3mg／ml ・トリトンＸ−100 0.1ｇ／dl ○第２試薬 ・リン酸緩衝液（pH6.8） 50mM ・Ｎ−エチル−Ｎ−（Ｏ− メチルスルホンアミドエチル） ｍ−トルイジン ２mM ・前記一般式Ｉで示される 脂肪酸（第２表参照） ２mM ・ＰＯＤ（比活性 100Ｕ／mg タンパク） 0.04mg／ml ・ＡＣＯＤ（比活性20Ｕ／mg タンパク） 0.25mg／ml ・トリトンＸ−100 0.05ｇ／dl 尚試料としては、オレイン酸ナトリウム30.45mgをトリ
トンＸ−100５ｇ／dl溶液10mlに溶解後、同溶液で全量1
00mlとしたものを用いた。測定操作は次のとおりであ
る。○ First reagent ・ Phosphate buffer (pH6.8) 50 mM ・ ATP 2 mM ・ Magnesium chloride ・ hexahydrate 5 mM ・ 4-aminoantipyrine 2 mM ・ CoA lithium salt 0.2 mM ・ ACS (specific activity 2U / mg protein) 0.3 mg / ml ・ Triton X-100 0.1 g / dl ○ Second reagent ・ Phosphate buffer (pH 6.8) 50 mM ・ N-ethyl-N- (O-methylsulfonamidoethyl) m-toluidine 2 mM ・ The above general formula Fatty acid represented by I (see Table 2) 2 mM ・ POD (specific activity 100 U / mg protein) 0.04 mg / ml ・ ACOD (specific activity 20 U / mg protein) 0.25 mg / ml ・ Triton X-100 0.05 g / dl As a sample, after dissolving 30.45 mg of sodium oleate in 10 ml of Triton X-1005 g / dl solution, the total amount of 1
What was made into 00 ml was used. The measurement operation is as follows.
試料50μと第１試薬１mlを混合し、37℃にて５分間
反応させる。50 μm of sample and 1 ml of the first reagent are mixed and reacted at 37 ° C. for 5 minutes.
更に第２試薬２mlを加え、37℃にて５分間反応させた
後555nmにおける吸光度を測定する。Further, 2 ml of the second reagent was added, the mixture was reacted at 37 ° C. for 5 minutes, and then the absorbance at 555 nm was measured.
従来法として、第２試薬中の脂肪酸にかえてＮＥＭ１mM
を加えたものを用いて同様の操作に従い定量を行って、
得られた結果を比較した。As a conventional method, NEM 1 mM was used instead of the fatty acid in the second reagent.
Quantification is performed according to the same procedure with the addition of
The results obtained were compared.
結果を第２表に示す。The results are shown in Table 2.
尚ＮＥＭを利用した場合の光学的密度は0.26程度であっ
た。 The optical density when NEM was used was about 0.26.
実施例２ 前記一般式Ｉで示される脂肪酸として３−オクテン酸を
利用した本発明によるＮＥＦＡ定量法と、ＮＥＭを利用
した従来法とで得られる結果の相関々係を調査した。Example 2 The correlation between the results obtained by the NEFA quantification method according to the present invention using 3-octenoic acid as the fatty acid represented by the general formula I and the conventional method using NEM was investigated.
本発明によるＮＥＦＡの定量法は実施例１と同様の操作
に従い、従来法はＮＥＭを含む市販のＮＥＦＡ定量用試
薬を利用し、操作は取扱い説明書の記載に従って行っ
た。The method for quantifying NEFA according to the present invention was performed in the same manner as in Example 1, the conventional method was using a commercially available reagent for quantifying NEFA containing NEM, and the operation was performed according to the instruction manual.
尚各々の定量法について、試料の定量とは別に50μの
精製水を用いて同様の操作を行って試薬ブランクの光学
的密度を求め、下記計算式を用いてＮＥＦＡ値（μＥｑ
／）を算出した。試料としては、ヒト血清15検体を、
標準試料としてはオレイン酸の1000μＥｑ／（30.45m
g／dl、実施例１で調製したものと同一）溶液を用い
た。For each quantification method, the optical density of the reagent blank was determined by performing the same operation using 50 μ of purified water separately from the quantification of the sample, and the NEFA value (μEq was calculated using the following formula.
/) Was calculated. As samples, 15 samples of human serum,
1000μEq / (30.45m of oleic acid as a standard sample
g / dl, same as that prepared in Example 1) solution was used.
計算式 本発明によるＮＥＦＡの定量法と従来法との間には、第
３表に示すとおり良好な相関々係が見られた。a formula As shown in Table 3, a good correlation was found between the NEFA quantification method according to the present invention and the conventional method.
ちなみに本発明によるＮＥＦＡの定量法における検量線
は、第１図に示すとおり、原点０を通る直線性の良いも
であった。 Incidentally, the calibration curve in the method for quantifying NEFA according to the present invention had good linearity passing through the origin 0 as shown in FIG.
実施例３ 第２試薬に３−オクテン酸（本発明）又はＮＥＭ（従来
法）を加えた場合のＡＣＯＤの安定性を調査した。Example 3 The stability of ACOD when 3-octenoic acid (invention) or NEM (conventional method) was added to the second reagent was investigated.
３−オクテン酸 ２mM又はＮＥＭ １mMを含む第２試薬
を、溶解状態で次の２つの条件のもとに放置し、試薬の
反応性を比較した。A second reagent containing 2-mM 3-octenoic acid or 1 mM NEM was left in a dissolved state under the following two conditions to compare the reactivity of the reagents.
○条件１：25℃で４日間放置し、実際に血清を用いて定
量操作を行い、第２試薬のタイムコースを観察した。結
果を第２図及び第３図に示す。○ Condition 1: The sample was left standing at 25 ° C for 4 days, and a quantitative operation was actually performed using serum to observe the time course of the second reagent. The results are shown in FIGS. 2 and 3.
○条件２：２〜10℃で24日間放置し、実際に血清を用い
て定量操作を行い、第２試薬のタイムコースを観察し
た。結果を第４図及び第５図に示す。-Condition 2: The sample was left to stand at 2 to 10 ° C for 24 days, a quantitative operation was actually performed using serum, and the time course of the second reagent was observed. The results are shown in FIGS. 4 and 5.
条件１及び２において、第２試薬中の過酸化水素測定用
試薬系の安定性を確認するために標準試料にかえて、過
酸化水素溶液を加えて発色状態を観察したが、各試薬間
での差はほとんどみられず、第２図ないし第５図におけ
る試薬間のＮＥＭ添加に起因する反応性のちがいは、Ａ
ＣＯＤの活性低下によるものと考えられた。Under conditions 1 and 2, in order to confirm the stability of the reagent system for measuring hydrogen peroxide in the second reagent, a hydrogen peroxide solution was added instead of the standard sample and the color development state was observed. The difference in reactivity due to the addition of NEM between the reagents in FIGS. 2 to 5 is
It was considered that the activity of COD was lowered.
又、条件１において、ＡＣＯＤ残存活性値をオースミら
によるバイオケミカル アンド バイオ フィジカル
リサーチ コミュニケーション（OSUMI et al,Biochem.
Biophys.Res.Comm.83.2.479-485(1978)）記載の方法に
従って測定し、保存中の活性変化を観察した。その結
果、試薬調製直後のＡＣＯＤ活性を100とすると、４日
後のＡＣＯＤ活性は、ＮＥＭを使った従来法では18.6
％、３−オクテン酸を使った本発明法では90.1％という
値が得られ、ＮＥＭの添加を避けることでＡＣＯＤ活性
の低下が著るしく抑制されることが明らかであった。Also, under condition 1, the residual activity value of ACOD was determined by the biochemical and biophysical method by Osumi et al.
Research Communication (OSUMI et al, Biochem.
Biophys.Res.Comm.83.2.479-485 (1978)) and the change in activity during storage was observed. As a result, assuming that the ACOD activity immediately after preparation of the reagent was 100, the ACOD activity after 4 days was 18.6 by the conventional method using NEM.
%, The value of 90.1% was obtained by the method of the present invention using 3-octenoic acid, and it was clear that the decrease in ACOD activity was markedly suppressed by avoiding the addition of NEM.
本発明によれば、ＡＣＯＤの保存安定性に悪影響を及ぼ
すと考えられるＮＥＭ等のＳＨ試薬を用いないＮＥＦＡ
の定量方法及びそのための試薬組成物を提供し得る。試
薬成分の安定性の向上は、溶液状態で供給されることの
多い自動分析装置用の試薬において特に多大な効果を奏
するものと考えられる。According to the present invention, NEFA that does not use SH reagents such as NEM, which is considered to adversely affect the storage stability of ACOD.
And a reagent composition therefor can be provided. It is considered that the improvement of the stability of the reagent component is particularly effective in the reagent for the automatic analyzer, which is often supplied in a solution state.
第１図は本発明によるＮＥＦＡの定量法における検量線
を示すグラフ、 第２図及び第４図は従来法による、また第３図及び第５
図は本発明法によるＡＣＯＤの安定性を示すグラフであ
る。FIG. 1 is a graph showing a calibration curve in the method for quantifying NEFA according to the present invention, FIGS. 2 and 4 are according to the conventional method, and FIGS.
The figure is a graph showing the stability of ACOD according to the method of the present invention.
Claims (7)
とコエンザイムＡの存在下アシルコエンザイムＡ合成酵
素を作用させ、生成するアシルコエンザイムＡに、下記
一般式Ｉで示される脂肪酸又はその水溶性の塩の存在下
でアシルコエンザイムＡ酸化酵素を作用させ、アシルコ
エンザイムＡの酸化によって生じる変化を測定すること
を特徴とする遊離脂肪酸の定量方法。 一般式Ｉ： Ｒ−Ａ−（ＣＨ2）n−ＣＯＯＨ （式中、Ｒは炭素数１〜20の脂肪族基を表し、ｎは０又
は１〜２の整数を表し、 Ａは−ＣＨ＝ＣＨ−又は を表し、 Ｙ及びＺは水素原子、水酸基又は低級アルキル基を表
す、ただしＹとＺは同時に水素原子を表さない。）1. A free fatty acid in a sample is reacted with an acylcoenzyme A synthase in the presence of adenosine triphosphate and coenzyme A, and the resulting acylcoenzyme A has a fatty acid represented by the following general formula I or its water-soluble A method for quantifying free fatty acid, which comprises reacting an acylcoenzyme A oxidase in the presence of a salt and measuring a change caused by the oxidation of acylcoenzyme A. General formula I: R-A- (CH 2 ) in n -COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, n represents an integer of 0 or 1 to 2, A is -CH = CH- or Y and Z represent a hydrogen atom, a hydroxyl group or a lower alkyl group, provided that Y and Z do not represent a hydrogen atom at the same time. )
ン酸、３−ヘキセン酸、２−ヘプテン酸、３−ヘプテン
酸、２−オクテン酸、３−オクテン酸、２−ノネン酸、
３−ノネン酸、２−デセン酸、３−デセン酸、２−ウン
デセン酸、３−ウンデセン酸、２−ドデセン酸、２−ト
リデセン酸及び２−ヘキサデセン酸から成る群から選択
されたものであることを特徴とする特許請求の範囲第１
項記載の遊離脂肪酸の定量方法。2. The fatty acid represented by the general formula I is 2-hexenoic acid, 3-hexenoic acid, 2-heptenoic acid, 3-heptenoic acid, 2-octenoic acid, 3-octenoic acid, 2-nonenoic acid,
It is selected from the group consisting of 3-nonenoic acid, 2-decenoic acid, 3-decenoic acid, 2-undecenoic acid, 3-undecenoic acid, 2-dodecenoic acid, 2-tridecenoic acid and 2-hexadecenoic acid. Claim 1 characterized by
The method for quantifying free fatty acid according to the item.
キシミリスチン酸、３−ヒドロキシミリスチン酸、２−
ヒドロキシパルミチン酸、３−ヒドロキシパルミチン
酸、２−ヒドロキシステアリン酸、３−ヒドロキシステ
アリン酸から成る群から選択されたものであることを特
徴とする特許請求の範囲第１項記載の遊離脂肪酸の定量
方法。3. The fatty acid represented by the general formula I is 2-hydroxymyristic acid, 3-hydroxymyristic acid, 2-hydroxymyristic acid or 2-hydroxymyristic acid.
The method for quantifying free fatty acid according to claim 1, which is selected from the group consisting of hydroxypalmitic acid, 3-hydroxypalmitic acid, 2-hydroxystearic acid, and 3-hydroxystearic acid. .
酸化水素を測定することを特徴とする特許請求の範囲第
１項記載の遊離脂肪酸の定量方法。4. The method for quantifying free fatty acid according to claim 1, wherein hydrogen peroxide produced by oxidizing acylcoenzyme A is measured.
特徴とする特許請求の範囲第５項記載の遊離脂肪酸の定
量方法。5. The method for quantifying free fatty acid according to claim 5, wherein hydrogen peroxide is quantified by a colorimetric method.
イムＡ及びアシルコエンザイムＡ合成酵素を含む第１試
薬と、少なくとも下記一般式Ｉで示される脂肪酸又はそ
の水溶性の塩及びアシルコエンザイムＡ酸化酵素を含む
第２試薬とから構成されることを特徴とする遊離脂肪酸
定量用試薬組成物。 一般式Ｉ： Ｒ−Ａ−（ＣＨ2）n−ＣＯＯＨ （式中、Ｒは炭素数１〜20の脂肪族基を表し、ｎは０又
は１〜２の整数を表し、 Ａは−ＣＨ＝ＣＨ−又は を表し、 Ｙ及びＺは水素原子、水酸基又は低級アルキル基を表
す、ただしＹとＺは同時に水素原子を表さない。）6. A first reagent containing at least adenosine triphosphate, coenzyme A and acyl coenzyme A synthase, and at least a fatty acid represented by the following general formula I or a water-soluble salt thereof and an acyl coenzyme A oxidase. A reagent composition for quantifying free fatty acids, which comprises two reagents. General formula I: R-A- (CH 2 ) in n -COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, n represents an integer of 0 or 1 to 2, A is -CH = CH- or Y and Z represent a hydrogen atom, a hydroxyl group or a lower alkyl group, provided that Y and Z do not represent a hydrogen atom at the same time. )
試薬組成物を含むことを特徴とする特許請求の範囲第６
項記載の遊離脂肪酸定量用試薬組成物。7. The sixth reagent according to claim 6, wherein the second reagent further contains a reagent composition for hydrogen peroxide colorimetric measurement.
The reagent composition for quantifying free fatty acid according to the item.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11815887A JPH0630630B2 (en) | 1987-05-15 | 1987-05-15 | Method for quantifying free fatty acid and reagent composition for quantification used therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11815887A JPH0630630B2 (en) | 1987-05-15 | 1987-05-15 | Method for quantifying free fatty acid and reagent composition for quantification used therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63283596A JPS63283596A (en) | 1988-11-21 |
| JPH0630630B2 true JPH0630630B2 (en) | 1994-04-27 |
Family
ID=14729536
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11815887A Expired - Fee Related JPH0630630B2 (en) | 1987-05-15 | 1987-05-15 | Method for quantifying free fatty acid and reagent composition for quantification used therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0630630B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3004135A4 (en) | 2013-06-04 | 2016-11-23 | Agency Science Tech & Res | Protein purification process |
| SG11201607098SA (en) * | 2014-02-27 | 2016-09-29 | Agency Science Tech & Res | Antibody purification process |
-
1987
- 1987-05-15 JP JP11815887A patent/JPH0630630B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63283596A (en) | 1988-11-21 |
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