Assay for oxidative stress injury by detection of luminol-enhanced chemiluminescence in a freshly obtained blood sample: A study to follow the time course of oxidative injury

Fusako Takayama, Toru Egashira, Yasumitsu Yamanaka

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Abstract

To evaluate occurrence of oxidative stress in circulating blood, we developed standard methods to assess (1) granulocytes status as a source of reactive oxygen species (ROS) and (2) lipid peroxidation (LPO). A simplified and highly sensitive assay was developed by utilizing the chemiluminescence (CL) from luminol oxidized by ROS. 1. The CL, from 300 μl medium containing 1% blood, 10 μg/ml luminol and 0.025 μg/ml phorbol myristate acetate, well reflected the primed granulocyte status induced by in vitro contact with lipopolysaccharide (LPS). This CL was weakened slightly by superoxide dismutase and catalase, but markedly decreased by sodium azide. 2. We determined the optimal conditions for the t-butyl hydroperoxide (t-BuOOH)- stimulated CL method to evaluate plasma LPO in experiments on rat plasma added with phosphatidylethanolamine hydroperoxide (PEOOH). The CL from 300 μl medium containing 6.67% plasma, 10 μg/ml luminol and 5 μ mol/ml t- BuOOH was proportional to the added PEOOH amount. The integrated CL of the plasma with 0-60 nmol of PEOOH gave values of 8.280-14.213 x 106 counts/60 min/tube. 3. Only 100 μl of freshly drawn blood was enough for the two CL methods to detect the generation of ROS and the occurrence of LPO. These CL methods enabled the determination of the time course of oxidative stress occurrence in circulating blood of rats treated with 5 mg/kg LPS, i.p.

Original languageEnglish
Pages (from-to)177-186
Number of pages10
JournalFolia Pharmacologica Japonica
Volume111
Issue number3
Publication statusPublished - 1998
Externally publishedYes

Fingerprint

Luminol
Luminescence
Oxidative Stress
Wounds and Injuries
Lipid Peroxidation
Reactive Oxygen Species
Granulocytes
Lipopolysaccharides
tert-Butylhydroperoxide
Sodium Azide
Tetradecanoylphorbol Acetate
Catalase
Superoxide Dismutase

Keywords

  • Chemiluminescence
  • Free radical
  • Lipid peroxidation
  • Oxidative stress
  • Reactive oxygen species

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Assay for oxidative stress injury by detection of luminol-enhanced chemiluminescence in a freshly obtained blood sample: A study to follow the time course of oxidative injury",
abstract = "To evaluate occurrence of oxidative stress in circulating blood, we developed standard methods to assess (1) granulocytes status as a source of reactive oxygen species (ROS) and (2) lipid peroxidation (LPO). A simplified and highly sensitive assay was developed by utilizing the chemiluminescence (CL) from luminol oxidized by ROS. 1. The CL, from 300 μl medium containing 1{\%} blood, 10 μg/ml luminol and 0.025 μg/ml phorbol myristate acetate, well reflected the primed granulocyte status induced by in vitro contact with lipopolysaccharide (LPS). This CL was weakened slightly by superoxide dismutase and catalase, but markedly decreased by sodium azide. 2. We determined the optimal conditions for the t-butyl hydroperoxide (t-BuOOH)- stimulated CL method to evaluate plasma LPO in experiments on rat plasma added with phosphatidylethanolamine hydroperoxide (PEOOH). The CL from 300 μl medium containing 6.67{\%} plasma, 10 μg/ml luminol and 5 μ mol/ml t- BuOOH was proportional to the added PEOOH amount. The integrated CL of the plasma with 0-60 nmol of PEOOH gave values of 8.280-14.213 x 106 counts/60 min/tube. 3. Only 100 μl of freshly drawn blood was enough for the two CL methods to detect the generation of ROS and the occurrence of LPO. These CL methods enabled the determination of the time course of oxidative stress occurrence in circulating blood of rats treated with 5 mg/kg LPS, i.p.",
keywords = "Chemiluminescence, Free radical, Lipid peroxidation, Oxidative stress, Reactive oxygen species",
author = "Fusako Takayama and Toru Egashira and Yasumitsu Yamanaka",
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pages = "177--186",
journal = "Folia Pharmacologica Japonica",
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T1 - Assay for oxidative stress injury by detection of luminol-enhanced chemiluminescence in a freshly obtained blood sample

T2 - A study to follow the time course of oxidative injury

AU - Takayama, Fusako

AU - Egashira, Toru

AU - Yamanaka, Yasumitsu

PY - 1998

Y1 - 1998

N2 - To evaluate occurrence of oxidative stress in circulating blood, we developed standard methods to assess (1) granulocytes status as a source of reactive oxygen species (ROS) and (2) lipid peroxidation (LPO). A simplified and highly sensitive assay was developed by utilizing the chemiluminescence (CL) from luminol oxidized by ROS. 1. The CL, from 300 μl medium containing 1% blood, 10 μg/ml luminol and 0.025 μg/ml phorbol myristate acetate, well reflected the primed granulocyte status induced by in vitro contact with lipopolysaccharide (LPS). This CL was weakened slightly by superoxide dismutase and catalase, but markedly decreased by sodium azide. 2. We determined the optimal conditions for the t-butyl hydroperoxide (t-BuOOH)- stimulated CL method to evaluate plasma LPO in experiments on rat plasma added with phosphatidylethanolamine hydroperoxide (PEOOH). The CL from 300 μl medium containing 6.67% plasma, 10 μg/ml luminol and 5 μ mol/ml t- BuOOH was proportional to the added PEOOH amount. The integrated CL of the plasma with 0-60 nmol of PEOOH gave values of 8.280-14.213 x 106 counts/60 min/tube. 3. Only 100 μl of freshly drawn blood was enough for the two CL methods to detect the generation of ROS and the occurrence of LPO. These CL methods enabled the determination of the time course of oxidative stress occurrence in circulating blood of rats treated with 5 mg/kg LPS, i.p.

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