JPS59180459A - Quantitative analysis of porphyrin in blood - Google Patents

Abstract

PURPOSE:To effect measurement of quantity of porphyrin, etc. in extract of blood, by extracting and processing blood with organic solvents including basic matters. CONSTITUTION:Blood is added to organic solvents including basic substances and the mixture is given with agitation. As the basic substances, organic amine, ammonia, alkali metal hydroxides are named. Further, as the organic solvents, low-grade alcohol, ketone are used. Molecular tie of hemoglobin is disconnected into heme and hematin and the heme is dissolved in the solvent in a coordinated shape of hematin or bacic matter and extracted and at the same time, other porphyrin, etc. are extracted also.

Description

[Detailed description of the invention] The present invention relates to a method for quantifying porphyrins in blood.

Hemoglobin present in blood is a molecular compound of a protoporphyrin iron complex called heme and the protein darobin. In addition, trace amounts of protoporphyrin, which is a precursor for heme biosynthesis, cobrovolphyrin, UK taurorphyrin, which is a metabolite of cobrovolphyrinogen, and the like are contained in the blood.

These protoporphyrin iron complexes (heme), protoporphyrin, cobrovorphyrin, and uroporphyrin (hereinafter collectively referred to as "porphyrins") are essential for sustaining life, and therefore their amounts in living organisms must be controlled. Accurate quantification is clinically extremely important to understand the metabolic function of the living body.In other words, by measuring the amount of porphyrins in the living body, it is possible to detect liver damage or abnormalities in porphyrin metabolism. be able to. Porphyrin metabolic abnormalities include those that are congenital and those that are caused by the accumulation of heavy metals or halogen compounds from outside the body, and those caused by IC, and it is also possible to measure the degree of accumulation of these toxic substances.

However, conventional measurements of porphyrins mainly involve measuring porphyrins in urine or midstream and serum, and it has been difficult to measure porphyrins in blood cells because of the influence of hemoglobin.

In other words, in order to measure porphyrins in the blood, they must be separated from the blood, but as mentioned above, heme forms a molecular compound with globin, so this can be broken down to separate heme from globin. Must be separated. Further, even if heme is liberated, it is difficult to extract all of them at the same time because heme is an acidic substance, whereas glotoborphyrin, coproporphyrin, and uroporphyrin are amphoteric substances.

Conventionally, methods for extracting heme include the glacial acetic acid method (Org, 5ynth
, 21 r53 (1941)), a method of extracting hemin as hemin by treating fresh blood with a large amount of hot acetic acid and common salt is known. However, in this method, hemin is precipitated as crystals, and unprecipitated hemin remains in acetic acid, so it cannot be said that heme is quantitatively separated and extracted.

As a method for extracting crushed glotoporphyrin, coproporphyrin, and uroporphyrin,
The method of Eln et al., which uses acetic acid and ethyl acetate, and the improved method of Plom@ri et al. are known, but this method has large variations depending on the measurer.
It wasn't satisfying.

As mentioned above, all of the conventional methods perform extraction under acidic conditions, and therefore it is not possible to obtain heme, which is an acidic substance, in a solution state, and therefore it is not possible to simultaneously extract and quantify porphyrins.

Under such circumstances, the present inventor conducted extensive research to extract all of the above-mentioned porphyrins simultaneously and stably, and as a result, it was found that an organic solvent containing a basic substance decomposes hemoglobin into heme and globin, as well as heme and globin. The present invention was completed based on the discovery that all porphyrins can be extracted at the same time by dissolving porphyrins such as glotoporphyrin, cobrovorphyrin, and uroporphyrin and precipitating protein globin as a solid.

That is, the present invention is a method for quantifying porphyrins in blood, which comprises extracting blood with an organic solvent containing a basic substance and measuring porphyrins in the extract. .

To carry out the present invention, first, blood is added to an organic solvent containing a basic substance and mixed and stirred.

Examples of the basic substance include organic amines such as diethylamine, diimpropylamine, and pyridine, and alkali metal hydroxides such as ammonia, caustic soda, and caustic potash. As the organic solvent, lower alcohols such as methanol, ethanol, and grohanol, and ketones such as acetone and methyl ethyl ketone are used.

Of these, the combination of diethylamine and methanol is most preferred. The concentration of the basic substance varies depending on its type, but is generally preferably about 1.5 to 3%. Mixing and stirring is carried out at a temperature of room temperature to 50° C. for 1 minute to 1 hour. When doing this, the molecular bonds of hemoglobin are broken and decomposed into heme and globin, and heme is dissolved in a solvent and extracted in the form of hematin or a basic substance coordinated, and other porphyrins are also extracted at the same time. . The globin also precipitates as a solid and is therefore separated.

Next, the amount of porphyrins in the extract thus obtained is measured. Porphyrins can be measured by conventionally known methods, but high performance liquid chromatography is preferably used. For example, using a porous polymer styrene gel as a filler, Q as a developing solvent,
High-performance liquid chromatography was performed using lN NaOH aqueous solution-methanol (1:9), and heme was detected by absorption in the visible region of 400 nm.
00 nm, other porphyrins can be quantified by fluorescence detection at a fluorescence wavelength of 630 nm.

Next, an explanation will be given with reference to examples.

Example 1 (1) Blood collected from a patient treated with heparin was centrifuged, separated into blood cells and plasma, which were stored frozen and thawed at 2° C., and 50 μt of each was collected. To this, methanol 2- containing 2.5% diethylamine was added under stirring, shaken for 1 minute, and centrifuged to collect the supernatant.

(11) 20 μt of the supernatant obtained in (1) above was subjected to high performance liquid chromatography [filler: spherical porous polymer styrene gel, developing solvent: 0, I N NaO
H aqueous solution-methanol (1:9)).

Heme was detected at 400 nm, and other porphyrins were detected using fluorescence at an excitation wavelength of 400 nm and a fluorescence wavelength of 630 nm.

The retention times and calibration curves for each porphyrin were as follows.

Diethylamine form of heme 6.5 minutes Figure 1 Protoporphyrin sodium 15.3 minutes Figure 2 Kobroporphyrin sodium 2 minutes Figure 3 Urophorphyrin sodium 1 minute The results are shown in Table 1. The amount of porphyrins was expressed as the content in 100 m of collapsed spheres and 100 m of plasma.

[Brief explanation of drawings]

Figure 1 shows the $ amount curve for the diethylamine form of heme, Figure 2 shows the calibration curve for protoporphyrin sodium, and Figure 3 shows the calibration curve for cobrovolphyrin sodium. Applicant Sawara Pharmaceutical Research Institute Co., Ltd. Representative Patent Attorney Miyuki Ariga [IL,,,,--,-J,,) Patent Attorney Toshio Takano: l -2i(,'','l
J ”3!-1= /J゛ 6-, □ [jl(-1
,) j

Claims (1)

[Claims] 1. A method for quantifying porphyrins in blood, which comprises extracting blood with an organic solvent containing a basic substance and measuring the amount of porphyrins in the extract. 2. The quantitative method according to claim 1, wherein the basic substance is an organic amine, ammonia, or an alkali metal hydroxide. 3. The quantitative method according to claim 1, wherein the organic solvent is a lower alcohol or a ketone. 4. The quantitative method according to claim 1, wherein the amount of porphyrins in the extract is measured by subjecting the extract to high performance liquid chromatography.