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ID 11778
JaLCDOI
Sort Key
10
Title Alternative
Determination of Hematinic Acid Produced by Oxidative Cleavage of Hemoglobin Heme in Red Blood Cells
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Author
Hirota, Kazuhiro
Hirota, Takashi
Abstract
Our previous studies on the mechanism of phenylhydrazine-induced hemolytic anemia have shown that hematinic acid, one of oxidative cleavage products of heme, is formed by the reaction of hemoglobin with phenylhydrazine. Develoment of the determination of hematinic acid formed by this reaction in red blood cells (RBC) was required to study the mechanism of the hemolysis. Hemolysates prepared by lysis of fresh human RBC with water was mixed with standard hematinic acid. A solution consisting of hydrochloric acid, methanol, and acetone was added, and most of hemoglobin precipitated was removed by centrifugation. Hematinic acid was derived to the methyl ester by incubation with methanol containing sulfuric acid. The ester was passed to two type of silica gel column to remove interferences, and was analysed on a reversedphase high-performance liquid chromatographic column. Hematinic acid could be determined in the range 1.0-20.0μmol/ml RBC. Recovery from hemolysate was 65.0% ±3.5%. Standard compounds of hematinic acid and its methyl ester were prepared by the oxidation of hemin with hydrogen peroxide, and were comfirmed by elemental analyses and mass spectra.
Abstract Alternative
フェニルヒドラジン惹起性溶血貧血機構の著者らの研究において、ヘモグロビンとフェニルヒドラジンとの反応でヘムの酸化的開裂物質の1つであるヘマチン酸が生成することが示された。この反応によって赤血球(RBC)中で生じたヘマチン酸を定量する方法の確立が、溶血機構研究のため必要となった。人のRBCを水で溶血し、ヘマチン酸の標品を加えた。塩酸、メタノール、アセトンの溶液え加え、ほとんどのヘモグロビンを沈殿除去した。メタノールー硫酸溶液で加熱し、ヘマチン酸をメチルエステルに誘導した。夾雑物を2種のシリカゲルカラムを通すことによって除去し、逆相カラムを用いた高速液体クロマトグラフィーで分析した。ヘマチン酸は、1.0-20.0μmol/ml RBCの範囲で定量でき、回収率は65.0 ±3.5%であった。ヘマチン酸とこのメチルエステルの標品は、ヘミンを過酸化水素酸化することによって合成し、元素分析と質量分析によって確認した。
Keywords
hematinic acid
high-performance liquid chromatography
red blood cells
heme
Publication Title
岡山大学医療技術短期大学部紀要
Published Date
1991-03-25
Volume
volume1
Publisher
岡山大学医療技術短期大学部
Publisher Alternative
School of Health Sciences Okayama University
Start Page
77
End Page
82
ISSN
0917-4494
NCID
AN10355371
Content Type
Departmental Bulletin Paper
OAI-PMH Set
岡山大学
language
日本語
File Version
publisher
NAID
Eprints Journal Name
fhs