JP7195521B2 - How to measure vitamin concentration - Google Patents

Description

The present invention relates to a method for measuring vitamin concentration, and more particularly to a method for measuring vitamin concentration in blood.

In the livestock industry, the amount of vitamin A in fattening cattle has been conventionally controlled in order to produce high-quality beef. Generally, when fattening cattle reach 12 months of age, the amount of vitamin A in the diet is reduced. As a result, marbling of the fattened cattle is improved.

On the other hand, if the blood vitamin A concentration of fattening cattle is too low, the health condition of fattening cattle will deteriorate (fatal accidents, edema, etc.) and productivity will decrease (poor weight gain, muscle edema, etc.). easier.

Therefore, in order to control the vitamin A concentration of fattening cattle to an appropriate concentration range that improves marbling while maintaining good health and productivity of fattening cattle, measurement of blood vitamin A concentration of fattening cattle is required. There is a need to do.

Conventionally, the HPLC method is generally used to measure the blood vitamin A concentration of fattening cattle. However, since the apparatus and the like are large and the measurement is extensive, a veterinarian or the like takes back a blood sample collected at the fattening site, and the blood sample is measured at a testing institution or the like. As a result, it takes a long time to obtain the results of measuring the vitamin A concentration after the blood sample is collected, and it may not be possible to control vitamin A in fattening cattle at the appropriate timing.

Therefore, the current vitamin A control of fattening cattle is based on the condition of the fattening cattle estimated from the producer's experience (observation of the appearance and vitality of the fattening cattle) without using the results of measuring the vitamin A concentration. It is often said that Therefore, it is difficult to avoid the occurrence of health accidents in fattening cattle due to individual differences in fattening cattle and lack of observation. Alternatively, there may be a problem that the blood vitamin A level is not lowered.

In view of the above circumstances, there is a need for a method for measuring the blood vitamin A concentration of livestock that can quantitatively measure the amount of vitamin A in the blood of fattened cattle and that provides the results of the measurement on the day of sample collection.

Therefore, for example, livestock whole blood is added to a mixture obtained by mixing a protein denaturation solution such as ethanol, a fat-soluble component extract such as hexane, and an antioxidant to obtain a stirred mixed solution, and this stirred mixed solution is obtained. A method for measuring the amount of vitamin A extracted in the fat-soluble component extract layer by measuring the absorbance of the fat-soluble component extract layer obtained by centrifugation has been proposed (Patent Document 1). However, since this method employs a centrifugal separation step, it is troublesome and difficult to carry out at the fattening site. Therefore, this method did not spread.

In addition, livestock whole blood is added to an organic solvent containing a polar organic solvent such as ethanol and a non-polar solvent such as hexane to obtain a mixed liquid, and a container holding this mixed liquid is shaken by the operator. A method has also been proposed in which phase separation is performed, the floating layer (upper layer) of the phase-separated mixed liquid is measured by fluorescence photometry, and the amount of vitamin A extracted in the floating layer is measured (Patent document 2). The method disclosed in Patent Document 2 does not require a centrifugal separation step, and can be measured near the fattening site if a small fluorometer is available.

JP 2010-230447 A Japanese Patent Publication No. 2010-503838

However, when the measurement method of Patent Document 2 was verified, as shown in FIG. 6, it was found that the correlation with the actual vitamin A concentration was poor and it was difficult to calculate the vitamin A concentration from the fluorescence intensity. In FIG. 6, the vertical axis is the fluorescence intensity obtained by the measurement method of Patent Document 2, and the fluorescence intensity of the measurement results of six cows A to F is shown in a bar graph. Cows A to C have low vitamin A concentrations, while cows D to F are samples known to have high vitamin A concentrations. rice field.

Therefore, it is an object of the present invention to provide a vitamin concentration measuring method and the like capable of quantitatively measuring the blood vitamin A concentration on-site with higher accuracy.

A first aspect of the present invention is a method for measuring vitamin concentration in blood, which contains an extraction solvent for extracting fat-soluble vitamins and blood to be measured, and does not contain a polar protic solvent. a stirring step of stirring the mixed solution; a measurement step of irradiating the extraction solvent layer of the mixed solution stirred in the stirring step with light to measure the light intensity of the light from the extraction solvent layer; and a calculating step of calculating the vitamin concentration from the light intensity measured in the step.

A second aspect of the present invention is the vitamin concentration measuring method according to the first aspect, wherein the extraction solvent is a nonpolar solvent and/or a polar aprotic solvent.

A third aspect of the present invention is the vitamin concentration measuring method according to the second aspect, wherein the extraction solvent is a non-polar solvent having a dielectric constant of 50 or less.

A fourth aspect of the present invention is the vitamin concentration measuring method of the second aspect, wherein the nonpolar solvent is a compound group consisting of alkanes, cycloalkanes, aromatic compounds, chloroform, ethyl acetate, and methylene chloride. including at least one of

A fifth aspect of the present invention is the method for measuring vitamin concentration according to any one of the second to fourth aspects, wherein the polar aprotic solvent is nitrile, ketone, tetrahydrofuran, dimethylsulfoxide, N,N-dimethyl At least one compound group consisting of formamide and ether is included.

A sixth aspect of the present invention is the vitamin concentration measuring method of the first aspect, wherein the extraction solvent contains at least one of the compound group consisting of normal hexane, toluene, tetrahydrofuran, and acetonitrile.

A seventh aspect of the present invention is the vitamin concentration measuring method according to the sixth aspect, wherein the extraction solvent is normal hexane.

An eighth aspect of the present invention is the vitamin concentration measuring method according to any one of the first to seventh aspects, wherein the blood contains blood cells.

A ninth aspect of the present invention is the vitamin concentration measuring method according to the eighth aspect, wherein the blood is whole blood.

A tenth aspect of the present invention is the vitamin concentration measuring method according to any one of the first to ninth aspects, wherein in the measuring step, the irradiated light has a wavelength of 300 nm to 350 nm, and the light to be measured is is light with a wavelength of 400 nm to 600 nm.

An eleventh aspect of the present invention is the vitamin concentration measuring method according to the tenth aspect, wherein in the measuring step, light to be measured has a wavelength of 470 nm to 560 nm.

A twelfth aspect of the present invention is the vitamin concentration measuring method according to any one of the first to eleventh aspects, wherein the vitamin is vitamin A and the blood is blood derived from fattening cattle.

According to each aspect of the present invention, it is possible to quantitatively and accurately measure vitamin concentrations in blood on-site. As a result, the number of times and the number of cows whose vitamin A concentration is measured can be increased, and fattening of cattle with uniform meat quality becomes possible.

In conventional measurement methods, when analyzing low-molecular-weight components in biological samples such as blood, it was considered necessary to remove proteins in the sample using ethanol, which is a polar protic solvent. Even in Patent Document 1, although there is a description in paragraph 0012 that fluorescence decreases due to the influence of the protein denaturation solution, the measurement is performed by adding the protein denaturation solution, and the protein denaturation step is performed to increase the measurement accuracy. thought to have been necessary.

However, as shown in FIG. 1, the vitamin A concentration measurement method of the present invention, which does not involve a protein denaturation step, is better than the conventional measurement method, which involves a protein denaturation step using ethanol, which is a polar protic solvent. Good correlation with results. This is because the contamination of unnecessary contaminants (inhibitory substances) due to the addition of ethanol is suppressed, resulting in an increase in the S/N ratio. That is, in the case of hexane+whole blood, although the measurement signal obtained is small, highly accurate measurement can be realized by adopting a fluorescence method capable of highly sensitive measurement as an optical measurement method. When ethanol was used as in the past, blood cells were destroyed and contaminants were mixed, but it is thought that the destruction of blood cells was suppressed by using a solvent other than a polar protic solvent such as ethanol. This was first clarified by the inventors of the present invention, and can be said to be an advantageous effect compared with the prior art.

In particular, according to the eighth and ninth aspects of the present invention, no special device for filtering blood is required. Therefore, it is possible to quickly measure the vitamin concentration in a place such as a fattening site where installation of an apparatus cannot be expected.

Moreover, according to the tenth and eleventh aspects of the present invention, the S/N ratio in the measurement of vitamin concentration is improved, and more accurate measurement becomes possible.

Here, in fattening cattle, grass is not mixed in the feed during the time of vitamin concentration measurement, and the blood concentration of β-carotene, which is a precursor of vitamin A and can cause measurement noise, is lowered. Therefore, according to the twelfth aspect of the present invention, the S/N ratio is particularly improved, and the concentration of vitamin A can be measured with higher accuracy.

FIG. 4 is a diagram showing the correlation between the measurement results by the fluorescence method and the measurement results by the HPLC method; FIG. 4 is a diagram showing fluorescence intensities of cows A to F according to the vitamin A concentration measuring method of the present invention. FIG. 2 is a diagram showing the strength of correlation with blood vitamin A concentration by the method for measuring vitamin A concentration of the present invention. FIG. 4 is a diagram showing the correlation between fluorescence intensity in the wavelength range of 470 to 560 nm and HPLC measurement results. It is a figure which shows the fluorescence spectrum at the time of using (a) hexane, (b) toluene, (c) THF, and (d) acetonitrile. FIG. 10 is a diagram showing fluorescence intensities of cows A to F according to a conventional vitamin A concentration measurement method.

Examples of the vitamin A concentration measuring method of the present invention are described below with reference to the drawings.

The vitamin A concentration measuring method of the present invention is carried out according to the following procedure.
(1) Collect blood to be measured from livestock (fattened cattle).
(2) The collected whole blood is added to hexane (an example of the “extraction solvent” in the claims of the present application) to obtain a mixture.
(3) Stir the mixture (an example of the "stirring step" in the claims of the present application).
(4) Measure the separated upper layer after stirring by a fluorescence method (an example of the "measurement step" in the claims of the present application).
(5) Calculate the vitamin A concentration from the measurement results (an example of the "calculation step" in the claims of the present application).

In the fluorescence measurement of procedure (4), the fluorescence measurement was performed on the upper layer of the separation layer of the mixed liquid in which vitamin A was extracted at an excitation wavelength of 330 nm.

Moreover, when the stirring in step (3) was finished and the mixture was allowed to stand, phase separation occurred immediately. This is because no ethanol was added. Specifically, it separated in about 1 second. Therefore, optical measurement can be performed immediately after the stirring in step (3).

FIG. 1 is a diagram showing the correlation between the results of measurement of vitamin A concentration by fluorescence method and the result of measurement by HPLC method. The vertical axis is the fluorescence intensity, the horizontal axis is the measurement result of the HPLC method, and the square marker is a conventional measurement method in which ethanol is used to perform a protein denaturation step (hereinafter also referred to as "ethanol + hexane + whole blood"). , and circle markers are the results obtained by the method for measuring vitamin A concentration according to the present invention (hereinafter also referred to as "hexane + whole blood") without a protein denaturation step.

As shown in the figure, the hexane + whole blood of the present invention has a better correlation with the measurement results by the HPLC method than the conventional ethanol + hexane + whole blood. The HPLC method is a method generally used for measuring vitamin A concentration, and although the apparatus and the like are large and the measurement is large-scale, it is a highly accurate measurement method. Therefore, it is not suitable for measurement at a fattening site, but this time it was used to compare and confirm the accuracy of the conventional method and the method of the present invention. In addition, since the HPLC method is a chromatography using mass difference, unlike the photometric method, the vitamin A concentration can be measured well even if alcohol is added.

In the case of hexane+whole blood, the absolute amount of extracted vitamin A is reduced and the detection signal (fluorescence intensity) is smaller than in the case of ethanol+hexane+whole blood. However, since the contamination of unnecessary contaminants (inhibitory substances) due to the addition of ethanol is suppressed, the S/N ratio increases as a result. That is, in the case of hexane+whole blood, although the measurement signal obtained is small, highly accurate measurement can be realized by adopting a fluorescence method capable of highly sensitive measurement as an optical measurement method. This was first clarified by the inventors of the present invention, and can be said to be an advantageous effect compared with the prior art.

In FIG. 2, the vertical axis represents the fluorescence intensity obtained by the vitamin A concentration measurement method of the present invention, and the fluorescence intensity of the measurement results of six cows A to F is shown in a bar graph. Cows A to C are samples with low vitamin A concentrations, while cows D to F are samples known to have high vitamin A concentrations. As shown, cows DF showed higher fluorescence intensity than cows AC. In other words, in the case of hexane + whole blood, compared to the conventional case of ethanol + hexane + whole blood, the actual vitamin A concentration and the obtained fluorescence intensity show a good correlation, so the vitamin A concentration can be accurately calculated from the fluorescence intensity. It turned out to be possible.

Ethanol makes hydrogen bonds. In other words, it binds to a specific substance to make the specific substance more soluble in liquid. That is, ethanol controls the state in which substances are dissolved in liquid. Therefore, it is considered that inhibitors other than vitamin A (such as beta-carotene) are also dissolved in the solvent (ethanol+hexane+whole blood) by adding ethanol. Therefore, it is considered that the result that there is almost no correlation between the fluorescence intensity and the level of the vitamin A concentration was obtained.

In addition, in the case of the fattening cattle described above, it is possible to use whole blood after collection. Since whole blood can be used without separation, blood vitamin A concentration can be measured immediately after blood collection at the site of fattening. As whole blood, blood collected from fattening cattle and not treated in any way was used, but dried or frozen blood may also be used. Plasma or serum from which blood cell components have been removed by centrifugation or the like can also be measured instead of whole blood.

FIG. 3 is a diagram in which the vertical axis indicates the strength of the correlation with the blood vitamin A concentration measured in the procedure (4) of Example 1, and the horizontal axis indicates the wavelength. The excitation wavelength is 330 nm. As shown in FIG. 3, it was found that fluorescence peaked in the wavelength range of 470-560 nm.

FIG. 4 is a diagram showing the correlation between the fluorescence intensity in the wavelength range of 470 to 560 nm and the HPLC measurement results. It was found that the correlation coefficient was higher than when the fluorescence intensity of the entire wavelength range of the peak was used. Therefore, in procedure (4), the use of the wavelength range of 470 to 560 nm as the fluorescence wavelength enables more accurate measurement.

Subsequently, instead of hexane used in the procedure (2) of Example 1, an organic solvent (aprotic polar solvent) was used for measurement. FIG. 5 shows fluorescence spectra when using (a) normal hexane, (b) toluene, (c) tetrahydrofuran (THF), and (d) acetonitrile. Fluorescence spectra of measurement results of whole blood samples of four cows AD and fluorescence spectra of the organic solvent itself are shown, respectively. The polarity is in ascending order of normal hexane, toluene, THF, and acetonitrile.

As shown in FIG. 5, the fluorescence spectra of the bovine samples A to D with different vitamin A concentrations have different peak heights, especially in (a) hexane, the peak heights are significantly different. In order to obtain a good correlation between fluorescence intensity and vitamin A concentration, (a) hexane is the most preferable extraction solvent, but it is possible that other organic solvents may also exhibit a correlation between fluorescence intensity and vitamin A concentration. Do you get it. When any organic solvent was used as the extraction solvent, the measured fluorescence wavelength range was approximately 400 to 600 nm when the excitation wavelength was 300 to 350 nm.

Therefore, it is thought that a nonpolar solvent and/or a polar aprotic solvent with a dielectric constant of 50 or less may be used as an extraction solvent.

As non-polar solvents, for example alkanes, cycloalkanes, aromatics, chloroform, ethyl acetate and/or methylene chloride can be used. Among alkanes, it is preferable to use C5-C12 alkanes, especially normal hexane. Among cycloalkanes, it is preferred to use cyclohexane. Among aromatic compounds, it is particularly preferred to use toluene and/or benzene.

As polar aprotic solvents for example nitriles, ketones, tetrahydrofuran, dimethylsulfoxide, N,N-dimethylformamide and/or ethers can be used. Among nitriles, it is particularly preferred to use acetonitrile. Among ketones, it is preferred to use acetone in particular. Among ethers, it is particularly preferred to use diethyl ether.

In the above example, measurement of blood vitamin A concentration in fattening cattle was shown as an example, but blood vitamins other than vitamin A were obtained by mixing blood with hexane or a low-polarity aprotic solvent other than hexane. It is possible to extract by means of fluorometry and determine the concentration by fluorometry. That is, by mixing and stirring blood and an extraction solvent (nonpolar solvent and/or polar aprotic solvent with a dielectric constant of 50 or less), blood It becomes possible to measure the vitamin concentration of

Claims (12)

A vitamin concentration measuring method for measuring the blood vitamin concentration of fattening cattle , comprising:
A stirring step of stirring a mixture containing an extraction solvent for extracting fat-soluble vitamins and the blood of a fattening cattle to be measured, but not containing a polar protic solvent;
a measurement step of irradiating the extraction solvent layer of the mixture stirred in the stirring step with light to measure the light intensity of the light from the extraction solvent layer;
and a calculating step of calculating the vitamin concentration from the light intensity measured in the measuring step. The vitamin concentration measuring method according to claim 1, wherein the extraction solvent is a non-polar solvent and/or a polar aprotic solvent. 3. The method of measuring vitamin concentration according to claim 2, wherein said extraction solvent is a non-polar solvent having a dielectric constant of 50 or less. 3. The method of measuring vitamin concentration according to claim 2, wherein the nonpolar solvent includes at least one of a compound group consisting of alkanes, cycloalkanes, aromatic compounds, chloroform, ethyl acetate, and methylene chloride. 5. The polar aprotic solvent according to any one of claims 2 to 4, wherein at least one of the compound group consisting of nitriles, ketones, tetrahydrofuran, dimethylsulfoxide, N,N-dimethylformamide, and ethers is included. vitamin concentration measurement method. 2. The method of measuring vitamin concentration according to claim 1, wherein said extraction solvent contains at least one of a compound group consisting of normal hexane, toluene, tetrahydrofuran, and acetonitrile. 7. The method of measuring vitamin concentration according to claim 6, wherein said extraction solvent is normal hexane. The vitamin concentration measuring method according to any one of claims 1 to 7, wherein said blood contains blood cells. 9. The method of measuring vitamin concentration according to claim 8, wherein said blood is whole blood. 10. The vitamin concentration measuring method according to any one of claims 1 to 9, wherein in said measuring step, the light to be irradiated is light with a wavelength of 300 nm to 350 nm, and the light to be measured is light with a wavelength of 400 nm to 600 nm. 11. The vitamin concentration measuring method according to claim 10, wherein in said measuring step, the light to be measured has a wavelength of 470 nm to 560 nm. The vitamin concentration measuring method according to any one of claims 1 to 11, wherein said fat-soluble vitamin is vitamin A.

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