title: A novel and sensitive assay for heme oxygenase activity.
abstract: Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.
authors.affiliation: Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan;
authors.forename: Saki
authors.initials: S
authors.lastname: Iwamori
authors.affiliation: Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan; Division of Nephrology, Endocrinology and Vascular Medicine, Department of Medicine, Tohoku University, Sendai, Japan;
authors.forename: Emiko
authors.initials: E
authors.lastname: Sato
authors.affiliation: Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan; and.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Saigusa
authors.affiliation: Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Yoshinari
authors.affiliation: Division of Nephrology, Endocrinology and Vascular Medicine, Department of Medicine, Tohoku University, Sendai, Japan;
authors.forename: Sadayoshi
authors.initials: S
authors.lastname: Ito
authors.affiliation: Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan; Division of Nephrology, Endocrinology and Vascular Medicine, Department of Medicine, Tohoku University, Sendai, Japan;
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sato
authors.affiliation: Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan; Division of Nephrology, Endocrinology and Vascular Medicine, Department of Medicine, Tohoku University, Sendai, Japan; ntakaha@m.tohoku.ac.jp.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D001663:Bilirubin / Q000096:biosynthesis; D001664:Biliverdine / Q000096:biosynthesis; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D006419:Heme Oxygenase (Decyclizing) / Q000032:analysis* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D006419:Heme Oxygenase (Decyclizing); D001664:Biliverdine; D001663:Bilirubin
keywords: LC-MS/MS; biliverdin; heme oxygenase