JPS5844210B2 - Highly sensitive and simple colorimetric determination method for acetoacetic acid in blood - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a simple method for the differential determination of blood ketone bodies, particularly acetoacetic acid in serum.

It has been known for a long time that it is extremely important to measure the amount of ketone bodies in serum when clinically diagnosing diabetic patients.

However, the nitropurgide method and the enzyme method are currently commonly used for this purpose, but the former is a semi-quantitative method and is inaccurate, and the latter requires extremely complicated operating procedures. It has a defect called.

Therefore, the basic object of the present invention is to provide a method for colorimetrically determining acetoacetic acid in serum using a coupling reaction with a diazonium salt that has excellent sensitivity and accuracy.

A conventionally known diazonium salt that can be used for this purpose is 2,5-dichlorobenzenediazonium chloride, but since it is liquid and unstable, it must be freshly prepared on the day of measuring acetoacetic acid in serum. Moreover, since the coloring product is unstable, it has the disadvantage that the measured values vary.

Therefore, the main object of the present invention is a crystalline powdery diazonium salt, which is stable for several months when stored in a cool and dark place, is easily soluble in distilled water, is highly reactive with acetoacetic acid, and does not produce colored products. An object of the present invention is to provide a simple and highly sensitive colorimetric assay method for azetoenzymatic acid in blood using stable p-nitrophenyldiazonium fluoroporate.

According to the present invention, the above objects, as well as other objects that become apparent from understanding the present invention, are: a) deproteinizing the sample serum and reacting it with p-nitrophenyldiazonium fluoroporate in a citrate buffer; to convert acetoacetic acid in the serum into an azo compound, and then add a hypophosphorous acid solution to decompose the remaining diazonium salt and convert the azo compound into a hydrazo compound, increasing the absorbance (4) of the solution to 3.
b) measuring at a wavelength of 90 nm, and b) using a 6% albumin solution instead of the sample serum to remove 0% of lithium acetoacetate.
, 0.25, 0.5, 1.0, and 2.0 mM bath solutions were prepared, protein was removed, and the absorbance was measured at a wavelength of 390 nm after performing the same treatment as in step a) above. The process includes a step of plotting the value obtained by subtracting the above OmM absorbance (B blank value) from each absorbance value to create a standard calibration curve, (formula % formula %) [In the formula, F is the absorbance of the standard quantitative straight line. is the concentration of acetoacetate (mM/l) when 1.0.

The reaction steps that occur in the method of the present invention are as follows.

Next, the reagents used in the method of the present invention and the operations for carrying out the method of the present invention will be further explained.

Reagent 1, citric acid buffer A 0.23M citric acid solution and a 0.23M sodium citrate solution are mixed and adjusted to pH 4.5.

2. Diazonium salt reagent 1) Synthesis p-No206H4NH,,+HNO2+HBF4→p
-NO□C6H4N2BF4+2H20p- 3.4 g of nitroaniline was added to 11 ml of 42% borohydrofluoric acid with thorough stirring, and to this was added an ice-cold t-IJ solution of nitrite (1.7 g/3.4 ml). /?) is added dropwise.

Stir for several minutes, filter with suction through a glass filter, once with ice-cold borohydrofluoric acid 37711, twice with 95% methanol,
After several washes with ethyl ether and drying in a vacuum desiccator with phosphorous pentoxide, 5.6 g (95.9% yield) of the desired p-nitrophenyldiazonium fluoroporate as a yellow crystalline powder was obtained. can get.

11) Usage mode Dissolve in distilled water to a concentration of 1"L'm.

111) Storage mode The powder is relatively stable and will remain stable for several months if stored in a cool and dark place.As a general rule, if it is made into a solution, it should be used immediately.
By cooling with water and shielding from light, it can be stored stably for at least 4 to 5 hours.

36 Measurement procedure using 20% hypophosphorous acid solution 1. Collect 0.2 ml of serum in a deproteinization test tube, and add 2% perchloric acid solution 2.
．． The supernatant after mixing 0 ml and centrifuging at 300'0.9 for 15 minutes is used as the sample solution.

2. Color development and absorbance measurement of sample solution Add 0.5ml of citrate buffer to 0.5ml of sample solution. Then, 0.5 ml of diazonium salt reagent was added thereto, and the mixture was reacted for 20 minutes in a constant temperature bath at 30°C.

Next, 0.5 ml of hypophosphorous acid solution was added to decompose the remaining diazonium salt, and the generated ab compound was converted to a hydrazo compound, and the absorbance (4) of the solution was reduced to 390.
Measured at a wavelength of nm.

3. Preparation of standard calibration source 0% of lithium acetoacetate using 6% albumin solution
, 0.25, 0.5, 1.0 and 2.0mM solutions were prepared, and after removing protein with a 2% perchloric acid solution, the same procedure as in 2) above was carried out and the absorbance at 390 nm was measured. Then, subtract the above OmM absorbance (B blank value) from each absorbance value and plot this value to create a standard quantitative straight line.

4. Quantitative calculation (A-B)

Measurement results (1) Figure 1 shows the results of a study on the optimal temperature for the coupling reaction between acetoacetic acid and diazonium salt.
When the reaction was carried out at 0°C, the azo compound produced after the reaction was stable and the reaction completion time was short.

(2) Figure 2 shows the experimental results of reaction time (minutes) with diazonium salt depending on the concentration of astoacetic acid.

At all concentrations, the reaction completely reached its maximum level in 20 minutes.

(3) Figure 3 shows the absorption spectrum of the hydrazo compound produced by the reaction depending on the concentration of acetoacetic acid, and there was an absorption peak at 390 nm at all concentrations.

(4) Figure 4 shows the stability of the hydrazo compound produced from acetoacetic acid, which was stable with almost no change over time at any concentration.

(5) Figure 5 shows a standard calibration curve for acetoacetic acid, and there was clear linearity.

(6) Figure 6 shows the results of creating a standard calibration curve by dissolving lithium acetoacetate in each of serum, 6% albumin solution, and distilled water.

In both cases, the slope of the straight line was the same, and the coverage in serum was 98%.

[Brief explanation of the drawing]

In the accompanying drawings, Figures 1 and 2 are graphs showing the relationship between the coupling reaction of acetoacetic acid and diazonium salt, temperature and reaction time, respectively; Figure 3 is a graph showing the absorption spectrum of the produced hydrazo compound; Figure 4 is a graph showing the change over time of the hydrazo compound produced when the acetoacetic acid concentration is different, Figure 5 is a graph showing the acetoacetic acid standard quantitative line, and Figure 6 is a graph showing the results when using distilled water and when using a 6% aribumin solution. It is a graph showing a comparison of the acetoacetic acid standard quantitative line with the case where the acetoacetic acid is used.

Claims (1)

[Claims] 1 a) The sample serum is deproteinized and purified in a citrate buffer.
- React with nitrophenyldiazonium fluoroporate to convert acetoacetic acid in the serum into an azo compound, then add a hypophosphorous acid solution to decompose the remaining diazonium salt and convert the azo compound into a hydrazo compound to form a solution. absorbance (4) at a wavelength of 390 nm; b) measuring 0, 0.25, 0.5, 1.
Prepare 0 and 2.0mM solutions, remove protein, and then perform the same treatment as in step a) above, measure the absorbance at a wavelength of 390nm, and calculate the absorbance of OmM (B blank value) from each absorbance value. ) is plotted to create a standard calibration curve. 1. A highly sensitive and simple colorimetric method for quantifying acetoacetic acid in blood, which is characterized in that it is quantified according to [mM/l].