JPH0365500B2 - - Google Patents

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 globin. Blood also contains small amounts of protoporphyrin, a precursor for heme biosynthesis, coproporphyrin, a metabolite of coproporphyrinogen, and uroporphyrin. These protoporphyrin iron complexes (heme),
Protoporphyrin, coproporphyrin, and uroporphyrin (hereinafter collectively referred to as "porphyrins") are indispensable for maintaining life, and therefore, accurately quantifying their amounts in the living body is essential for the metabolism of the living body. This is clinically extremely important in understanding its function. That is, by measuring the amount of porphyrins in a living body, liver damage or porphyrin metabolic abnormality can be detected. 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 it is also possible to measure the degree of accumulation of these toxic substances. However, conventional measurements of porphyrins
The main method is to measure it in urine, feces, and serum, and it has been difficult to measure it in blood cells because it is affected by 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 protoporphyrin, coproporphyrin, and uroporphyrin are amphoteric substances. Traditionally, the method for extracting heme is Fischer
Glacial acetic acid method by et al. [Org.Synth.21, 53 (1941)],
Specifically, a method is known in which fresh blood is treated with a large amount of hot glacial acetic acid and salt to extract hemin. 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. In addition, as methods for extracting protoporphyrin, coproporphyrin, and uroporphyrin, the method of Grinstein et al., that is, the method using acetic acid and ethyl acetate, and the improved method of this method, the method of Piomeri et al., are known. There were large variations among the measurers, and the results were not satisfactory. As mentioned above, since all conventional methods perform extraction under acidic conditions, it is not possible to obtain heme, which is an acidic substance, in a solution state. Therefore, porphyrins are extracted at the same time.
It was not possible to quantify them simultaneously. Under these circumstances, the present inventor conducted intensive research to extract all of the above-mentioned porphyrins simultaneously and stably, and as a result, lower alcohols containing di-lower alkylamine decompose hemoglobin into heme and globin, and heme The present invention was completed based on the discovery that all porphyrins can be extracted simultaneously by dissolving porphyrins such as protoporphyrin, coproporphyrin, and uroporphyrin, and precipitating protein globin as a solid. That is, the present invention extracts protoporphyrin iron complex derived from hemoglobin and porphyrins originally present in blood by extracting blood with a lower alcohol containing a di-lower alkylamine. This is a method for quantifying porphyrins in blood, which is characterized by measuring the amount of protoporphyrin iron complex and porphyrins in an extract. To carry out the present invention, blood is first added to a lower alcohol containing a di-lower alkylamine and mixed and stirred. Diethylamine, diisopropylamine, etc. are used as the di-lower alkylamine, and methanol, ethanol, propanol, etc. are used as the lower alcohol. Of these, the combination of diethylamine and methanol is most preferred. The concentration of di-lower alkylamine varies depending on its type, but is generally preferably about 1.5 to 3%.
Mix and stir at room temperature to 50℃ for 1 minute to 1 minute.
Time is done. When this happens, the molecular bonds of hemoglobin are broken and it breaks down into heme and globin.
Heme is dissolved in a solvent in the form of hematin or di-lower alkylamine and is extracted, 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, high-performance liquid chromatography is performed using porous polymer styrene gel as a filler and 0.1N NaOH aqueous solution - methanol (1:9) as a developing solvent, and heme is detected by absorption in the visible region of 400 nm. However, other porphyrins can also be quantified by fluorescence detection with an excitation wavelength of 400 nm and a fluorescence wavelength of 630 nm. Next, an example will be given and explained. Example 1 (i) Centrifuging heparinized patient blood collection,
Blood cells and plasma were separated and stored frozen, thawed at 2°C, and 50μ of each was collected. To this was added 2 ml of methanol containing 2.5% diethylamine under stirring, followed by shaking for 1 minute and centrifugation to collect the supernatant. (ii) 20μ of the supernatant obtained in (i) above was subjected to high performance liquid chromatography [filler: spherical porous polymer styrene gel, developing solvent: 0.1N
NaOH 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 2
Figure Coproporphyrin sodium 2 minutes Figure 3 Uroporphyrin sodium 1 minute The results are shown in Table 1. The amount of porphyrins was expressed as the content in each 100 ml of blood cells and plasma.

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Claims (1)

[Scope of Claims] 1 Blood is extracted with a lower alcohol containing a di-lower alkylamine to extract protoporphyrin iron complex decomposed from hemoglobin and porphyrins originally present in the blood, 1. A method for quantifying porphyrins in blood, which comprises measuring the amount of protoporphyrin iron complex and porphyrins in the extract. 2. The quantitative method according to claim 1, wherein the amount of protoporphyrin iron complex and porphyrins in the extract is measured by subjecting the extract to high performance liquid chromatography.