A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices

T. Kőszegi, Nikolett Sali, Maja Raknić, Zoltán Horváth-Szalai, Rita Csepregi, Marijana Zovko Končić, Nóra Papp, Miklós Poór

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Introduction Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well. Methods In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. Results and discussion The ECL method is fast (10 min) with an interassay precision of < 10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r = 0.84, 95% confidence interval, CI = 0.78–0.89, P < 0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p < 0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p < 0.01). Pearson's statistics showed the strongest positive correlation with serum uric acid (r = 0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.

Original languageEnglish
Pages (from-to)153-159
Number of pages7
JournalJournal of Pharmacological and Toxicological Methods
Volume88
DOIs
Publication statusPublished - Nov 1 2017

Fingerprint

Luminol
Chemiluminescence
Luminescence
Assays
Antioxidants
Plant Extracts
Reactive Oxygen Species
Peroxidase
Serum
Mammals
Uric Acid
Blood
Cells
Maintenance
Statistics
Confidence Intervals
Inflammation
Oxidation
Wounds and Injuries

Keywords

  • Antioxidant capacity
  • Enhanced chemiluminescence
  • Luminol
  • Plant extracts
  • Septic patients
  • Validation

ASJC Scopus subject areas

  • Toxicology
  • Pharmacology

Cite this

A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices. / Kőszegi, T.; Sali, Nikolett; Raknić, Maja; Horváth-Szalai, Zoltán; Csepregi, Rita; Končić, Marijana Zovko; Papp, Nóra; Poór, Miklós.

In: Journal of Pharmacological and Toxicological Methods, Vol. 88, 01.11.2017, p. 153-159.

Research output: Contribution to journalArticle

Kőszegi, T. ; Sali, Nikolett ; Raknić, Maja ; Horváth-Szalai, Zoltán ; Csepregi, Rita ; Končić, Marijana Zovko ; Papp, Nóra ; Poór, Miklós. / A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices. In: Journal of Pharmacological and Toxicological Methods. 2017 ; Vol. 88. pp. 153-159.
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T1 - A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices

AU - Kőszegi, T.

AU - Sali, Nikolett

AU - Raknić, Maja

AU - Horváth-Szalai, Zoltán

AU - Csepregi, Rita

AU - Končić, Marijana Zovko

AU - Papp, Nóra

AU - Poór, Miklós

PY - 2017/11/1

Y1 - 2017/11/1

N2 - Introduction Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well. Methods In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. Results and discussion The ECL method is fast (10 min) with an interassay precision of < 10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r = 0.84, 95% confidence interval, CI = 0.78–0.89, P < 0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p < 0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p < 0.01). Pearson's statistics showed the strongest positive correlation with serum uric acid (r = 0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.

AB - Introduction Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well. Methods In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. Results and discussion The ECL method is fast (10 min) with an interassay precision of < 10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r = 0.84, 95% confidence interval, CI = 0.78–0.89, P < 0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p < 0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p < 0.01). Pearson's statistics showed the strongest positive correlation with serum uric acid (r = 0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.

KW - Antioxidant capacity

KW - Enhanced chemiluminescence

KW - Luminol

KW - Plant extracts

KW - Septic patients

KW - Validation

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