Formation of meso, N-Diphenylprotoporphyrin IX by an Aerobic Reaction of Phenylhydrazine with Oxyhemoglobins

Akira Nakanishi, Keiko Kinuta, Tadashi Abe, Kenta Araki, Yumi Yoshida, Shuang Liang, Shun Ai Li, Kohji Takei, Masahiro Kinuta

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Abstract

Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (1H-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl- and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.

Original languageEnglish
Pages (from-to)249-256
Number of pages8
JournalActa Medica Okayama
Volume57
Issue number5
Publication statusPublished - Oct 2003

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Keywords

  • Fast-atom-bombardment mass spectrometry (FAB-MS)
  • Hemoglobin
  • Phenylhydrazine
  • Proton nuclear magnetic resonance (H-NMR) spectrometry
  • Protoporphyrin

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Nakanishi, A., Kinuta, K., Abe, T., Araki, K., Yoshida, Y., Liang, S., Li, S. A., Takei, K., & Kinuta, M. (2003). Formation of meso, N-Diphenylprotoporphyrin IX by an Aerobic Reaction of Phenylhydrazine with Oxyhemoglobins. Acta Medica Okayama, 57(5), 249-256.