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Automated analysis of the serum antioxidative activities against 1047297ve differentreactive oxygen species by sequential injection system with achemiluminescence detector

Naoya Kishikawa a Kaname Ohyama a Junko Yao a Aoi Miyamoto b Takahiro Imazato ac Yukitaka Ueki cKenichiro Nakashima d Eisuke Maehata e Naotaka Kuroda a

a Department of Environmental and Pharmaceutical Sciences Graduate School of Biomedical Sciences Nagasaki University Nagasaki Japanb College of Pharmacy Nihon University Tokyo Japanc Sasebo Chuo Hospital Sasebo Japand Department of Clinical Pharmacy Graduate School of Biomedical Sciences Nagasaki University Nagasaki Japane

Showa University Fujigaoka Hospital Yokohama Japan

a b s t r a c ta r t i c l e i n f o

Article history

Received 7 January 2010

Received in revised form 23 March 2010

Accepted 7 April 2010

Available online 11 April 2010

Keywords

Antioxidative activity

Luminol chemiluminescence

Sequential injection analysis

Serum

Reactive oxygen species

Background There is a growing evidence that reactive oxygen species (ROS) may cause many pathologic

conditions including chronic diseases neurodegenerative disorders cancer and aging There are a number of methods to measure the total antioxidative activity of the serum However since the lifetime and oxidative

activity of various types of ROS are all different to measure simply the total antioxidative activity of theserum is not enough Therefore to aid the diagnosis and to improve the therapeutic strategy it is importantto develop a simple and reliable method of assaying antioxidative activity of the serumMethods A method that combines sequential injection analysis (SIA) and luminol chemiluminescence (CL)

detection was employed for the measurement of antioxidative activities of human serum We collected serafrom healthy subjects (n =42) and patients with diabetes (n =39) and rheumatoid arthritis (n =25) andtested the sensitivity reproducibility and reliability of our methodResults

Since the operation is automatically controlled by a personal computer we obtained a satisfactoryrepeatability without the need of much manpower The time required for obtaining the antioxidative activity

against one ROS for one individual is less than 3 min Although the value of antioxidative activity varies fromsubject to subject there were a certain relationship between the disease and the antioxidative values of each

type of ROS The results suggest that the measurement of antioxidative activity against different ROS mayprovide us with valuable information regarding the disease stateConclusions The evaluation of antioxidative activities against each ROS by the proposed method should bemore informative to understand the antioxidative status of biological 1047298uid

Crown Copyright copy 2010 Published by Elsevier BV All rights reserved

1 Introduction

Reactive oxygen species (ROS) generated in biological systems areinvolved in signal regulation production of energy phagocytosis anddefense mechanism against infection However the excess generationof ROS induces harmful biological oxidation Indeed ROS have been

implicated in the pathophysiology of several conditions includingdiabetes infertility rheumatoid arthritis and cardiovascular diseases

[1ndash4] Much evidence indicates that exposure to ROS cause deleteri-ous changes in cell function by a number of oxidative modi1047297cationsuch as lipid peroxidation enzyme inactivation and oxidative DNAdamage ultimately results in cell death [5ndash8] In these aspects the

evaluation of the degrees of oxidative damage caused by ROS isessential in order to clarify the contribution of ROS to several diseases

On the other hand it is known that there are different kinds of ROSincluding superoxide anion (O2

minus) nitric oxide (NO) hydrogen

peroxide (H2O2) hypochlorite anion (ClOminus) and singlet oxygen(1O2) Since each ROS has different chemical features includinglifetime and oxidative activity the oxidative damage on the livingbody and resulting disease should be different Therefore it is

important to evaluate the degrees of oxidative damage due to each

Clinica Chimica Acta 411 (2010) 1111ndash1115

Abbreviations CL chemiluminescence DMF N N -dimethylformamide HEPES N -2-

hydroxyethyl piperazine-N prime-2-ethanesulfonic acid HX Hypoxanthine LPO lactoperoxidase

NOR1 (plusmn)-(E )-4-methyl-2-[(E )-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide ROS

reactive oxygen species SIA sequential injection analysis XOD xanthine oxidase

Corresponding author Department of Environmental and Pharmaceutical Sciences

Course of Pharmaceutical Sciences Graduate School of Biomedical Sciences Nagasaki

University 1-14 Bunkyo-machi Nagasaki 852-8521 Japan Tel +81 95 819 2894

fax +81 95 819 2444

E-mail address n-kuronagasaki-uacjp (N Kuroda)

0009-8981$ ndash see front matter Crown Copyright copy 2010 Published by Elsevier BV All rights reserved

doi101016jcca201004005

Contents lists available at ScienceDirect

Clinica Chimica Acta

j o u r n a l h o m e p a g e w w w e l s ev i e r c o m l o c a t e c l i n c h i m

8142019 1-s20-S0009898110002615-main


ROS in order to understand the relationship between ROS generationand pathogenesis

In this study we focused on the endogenous antioxidative activityof human serum as an indicator of oxidative damage due to each ROS

It is known that human serum possess antioxidants and antioxidativeenzymes to protect the body against oxidation by ROS Theconcentrations andor activities of them should decrease by the

scavenging of excessively generated ROS in the body of patients As a

result the antioxidative activity of serum of patients might be lowerthan that of healthy humanThe task of quantifying antioxidative activity of biological samples

can be approached in three different ways Firstly the concentrationsof all of the individual molecules that are currently recognized asantioxidants are measured However it does not account for thein1047298uence of undiscovered antioxidants or the substances that aretechnically dif 1047297cult to be assayed Secondly the antioxidative activity

can be measured by the decreasing ratio of 11-diphenyl-2-picrylhy-drazyl radical or peroxy radical scavenging activities [910] Howeverthese methods can be applied only for speci1047297c radicals Thirdly ferricreducing ability of plasma method [11] and total radical-trapping

potential method [12] are famous for assay of antioxidative activity of biological sample however they are based on the total reductionability of samples Since ROS differ from each other with respect tochemical features including reactivity and life-time antioxidativeactivities of biological samples against each ROS should be different

However as mentionedabove allthe assays for thetotal antioxidativeactivity of biological samples cannot meetthe requirement Thereforethe method for determination of antioxidative activities against eachROS will be required

In our laboratory antioxidative activity measurement method forplant extracts has been developed based on batch method withluminol chemiluminescence (CL) detection [13] Luminol emits lightwhen it reacts with ROS In this method the attenuation of luminol CL

due to scavenging of ROS by sample was measured as theantioxidative activity Successively luminol CL detection was hy-phenated with sequential injection analysis (SIA) for determination of antioxidative activities against ClOminus 1O2 O2

minus and NO [14ndash16]

Employing SIA makes it possible to reduce sample volume reagentconsumption and human error and obtain repeatable resultscomparing with 1047298ow injection analysis [17]

In this study we exploit the SIA-CL method to automated analysisof the serum antioxidative activity against 1047297ve different ROS Themethod has an advantage for determination of antioxidative activities

against each ROS because different ROS can be generated by alteringthe reagents in the SIA system The developed method was applied to

the serum samples collected from healthy subjects diabetic andrheumatoid patients and the measured antioxidative activities werecompared

2 Material and methods

21 Reagents and solution

Hypoxanthine (HX) L -ascorbic acid boric acid sodium hydroxideH2O2 and sodium acetate were purchased from Wako Pure Chemical

(Osaka Japan) xanthine oxidase (XOD) N N -dimethylformamide(DMF) luminol and sodium hypochlorite were from Nacalai Tesque(Kyoto Japan) (plusmn)-(E )-4-methyl-2-[(E )-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1) was from Dojindo (Kumamoto

Japan) N -2-hydroxyethyl piperazine-N prime-2-ethanesulfonic acid(HEPES) lactoperoxidase (LPO) and sodium bromide were fromSigma (St Louis MO USA) Water was deionized using an AutostillWG 220 (Yamato Kagaku Tokyo Japan) and passed through a Puric-Z(Organo Tokyo Japan) All other solvents and reagents were of

analytical grade Luminol was dissolved in DMF and then diluted withwater (DMF content was 1) The reagents usedfor generation of each

ROS are summarized in Table 1

22 Apparatus and manifold design

The SIA-CL system (Fig 1) consisted of a Cavro XL 3000 syringe

pump (volume 1 mL Cavro Scienti1047297c Instruments Inc CA USA) a

Table 1

Carrier solution and ROS generation reagent for the SIA-CL measurement

ROS Carrier solution ROS reagent

ClOminus 50 mmoll borate buffer (pH 95) 383 mmoll NaClOcarrier solution

NO 100 mmoll Hepes buffer (pH 82) 2 mmoll NOR1DMSO100 mmoll

HCl=111O2 100 mmoll sodium acetate buffer

(pH 45)

150 mmoll H2O2carrier solution

80 mgml LPOcarrier solution

250 mmoll NaBrcarrier solution

O2minus

100 mmoll Hepes buffer (pH 82) 16Uml XODcarrier solution10 mmoll HXcarrier solution

H2O2 50 mmoll borate buffer (pH 95) 100 mmoll H2O2carrier solution

Fig 1 Scheme ofthe SIA-CLsystemfor themeasurementof antioxidativeactivityof humanserumagainst eachROS SPsyringepump HC holding coilMV multiportvalve M mixingtee

P pump D detector R recorder

1112 N Kishikawa et al Clinica Chimica Acta 411 (2010) 1111ndash1115

8142019 1-s20-S0009898110002615-main


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(n =5) and between ndash day (n=3) repeatabilities of the blank CL signals were less than 65 and 86 respectively

32 Antioxidative activity of serum of diabetic and rheumatic patients

The measurement results of antioxidative activities of the serumsamples are summarized in Table 3 All the tested samples exhibited

antioxidative activity against 5 types of ROS We compared theantioxidative activities of serum samples between healthy subjectsand patients Serum from diabetic patients exhibited a signi1047297cantly

lower antioxidative activity against NO 1O2 and O2minus than that from

healthy subjects while the activity against ClOminus and H2O2 increasedcomparing with healthy subjects Also the antioxidative activity of serum against H2O2 from rheumatic patients was higher than that

from healthy subjects The proposed SIA-CL method can grasp thedifference of antioxidative activities between healthy subjects andROS-mediated diseases Since ROS are involved in the pathogenesis of

diabetes and rheumatoid arthritis [13] and a part of antioxidants in

the serum are consumed a decreasein the antioxidative activity of theserum from the patients was expected comparing with healthysubjects However the antioxidative activities against some ROS

increased in patients contrary to the expectationThe increased activity against H2O2 in diabetic patients may be due

to the low H2O2 production Alba-Loureiro et al reported that H2O2

production was decreased in impaired neutrophils from diabetic rats

[20] They found that the activities of three enzymes involved in themetabolism of glucose and glutamine were changed and concludedthat this change might play an important role in the impairedneutrophil function observed in diabetes Also it was described thatthe phorbol myristate acetate-stimulated neutrophils from normal

volunteers generated approximately 4-fold increases in H2O2 com-pared with these from diabetic patient by Inoue et al [21] Since thedecrease in H2O2 production naturally leads to decrease in ClOminus theantioxidative activity against ClOminusmight increase in diabetic patients

Our results were supported by these observationsIn the comparison of healthy subjects and rheumatic patients the

antioxidative activities against H2O2 signi1047297cantly increased in rheu-matic patients compared with healthy subjects It was reported that

the activity of catalase which quenches H2O2 was increased byin1047298ammation [22] The increase of catalase activity may increase theantioxidative activity in patients

Redox mechanisms have been implicated in the pathogenesis of numerous human diseases either by direct ROS-mediated tissue

damage or via gene transcription induced through redox sensitivetranscription factors Consequently there have been increasinginterest in measuring the antioxidative activity of biological 1047298uidsand tissues and a number of different assays of total antioxidative

activity have been developed [23ndash32] However the results obtainedwith this study indicated that the antioxidative activity of biological

1047298uids 1047298uctuates for two reasons Namely (i) because of different

types of ROS and (ii) in accordance with the disease state Thereforethe measurement of only the total antioxidative activity may misleadthe diagnosis As we reported in this paper the antioxidative activityagainst each ROS should be separately measured Such results would

be very valuable because they could provide us valuable informationIn conclusion we developed a method that combined SIA and

luminol CL detection for the measurement of antioxidative activityagainst 1047297ve types of ROS in the human serum We tested the method

by using the sera obtained from healthy subjects and patients Theresult clearly indicated that this equipment would be a valuable toolfor research diagnosis and therapy

Acknowledgement

This work wassupported by a YamadaBee Farm Grant(2008-No 10)

References

[1] Oberley LW Free radicals and diabetes Free Radic Biol Med 19885113ndash24[2] AmaralS Oliveira PJ Ramalho-Santos J Diabetes and theimpairmentof reproductive

function Possible role of mitochondria and reactive oxygen species Curr DiabetesRev 2008446ndash54

[3] Gelderman KA Hultqvist M Olsson LM et al Rheumatoid arthritis the role of reactive oxygen species in diseasedevelopment and therapeuticstrategies AntioxidRedox Signal 200791541ndash67

[4] Ergul A Johansen JS Stroslashmhaug C et al Vascular dysfunction of venous bypassconduits is mediated byreactive oxygenspeciesin diabetes role ofendothelin-1

J Pharmacol Exp Ther 200531370ndash

7

Table 3

Antioxidative activity in the serum from healthy subjects ( n=42) ant patients with

diabetes (DM n =39) and rheumatoid arthritis (RA n =25)


Range () MeanplusmnSEa () 95 CIb () Statistical test

ClOminus Healthy 463ndash834 622 plusmn17 587ndash656 ndash

DM 517ndash758 655 plusmn09 637ndash673 HealthybDMc

RA 526ndash

745 630 plusmn10 609ndash

651 ndash

NO Healthy 317ndash896 707 plusmn16 674ndash740 ndash

DM 57ndash957 627 plusmn30 567ndash687 HealthyNDMc

RA 471ndash794 693 plusmn15 661ndash7241O2 Healthy 522ndash779 668 plusmn11 646ndash689 ndash


RA 543ndash832 694 plusmn13 667ndash720 ndash

O2minus Healthy 30ndash214 114 plusmn06 102ndash126 ndash

DM minus58ndash157 66 plusmn08 50ndash83 HealthyNDM c


H2O2 Healthy 107ndash232 189 plusmn04 180ndash197 ndash


RA 189ndash256 224 plusmn03 217ndash231 HealthybRAc

a Standard errorb Con1047297dence intervalc Steels test pb005

pb

001

Fig 2 Signal responses of scavenging capacity against 1O2 for human serum Five microL of

PBS (blank) and serum diluted 40 (125 nl) 20 (250 nl) and 10 times (500 nl) with PBS

were injected into the SIA system


8142019 1-s20-S0009898110002615-main


8142019 1-s20-S0009898110002615-main


ROS in order to understand the relationship between ROS generationand pathogenesis

In this study we focused on the endogenous antioxidative activityof human serum as an indicator of oxidative damage due to each ROS

It is known that human serum possess antioxidants and antioxidativeenzymes to protect the body against oxidation by ROS Theconcentrations andor activities of them should decrease by the

scavenging of excessively generated ROS in the body of patients As a

result the antioxidative activity of serum of patients might be lowerthan that of healthy humanThe task of quantifying antioxidative activity of biological samples

can be approached in three different ways Firstly the concentrationsof all of the individual molecules that are currently recognized asantioxidants are measured However it does not account for thein1047298uence of undiscovered antioxidants or the substances that aretechnically dif 1047297cult to be assayed Secondly the antioxidative activity

can be measured by the decreasing ratio of 11-diphenyl-2-picrylhy-drazyl radical or peroxy radical scavenging activities [910] Howeverthese methods can be applied only for speci1047297c radicals Thirdly ferricreducing ability of plasma method [11] and total radical-trapping

potential method [12] are famous for assay of antioxidative activity of biological sample however they are based on the total reductionability of samples Since ROS differ from each other with respect tochemical features including reactivity and life-time antioxidativeactivities of biological samples against each ROS should be different

However as mentionedabove allthe assays for thetotal antioxidativeactivity of biological samples cannot meetthe requirement Thereforethe method for determination of antioxidative activities against eachROS will be required

In our laboratory antioxidative activity measurement method forplant extracts has been developed based on batch method withluminol chemiluminescence (CL) detection [13] Luminol emits lightwhen it reacts with ROS In this method the attenuation of luminol CL

due to scavenging of ROS by sample was measured as theantioxidative activity Successively luminol CL detection was hy-phenated with sequential injection analysis (SIA) for determination of antioxidative activities against ClOminus 1O2 O2

minus and NO [14ndash16]

Employing SIA makes it possible to reduce sample volume reagentconsumption and human error and obtain repeatable resultscomparing with 1047298ow injection analysis [17]

In this study we exploit the SIA-CL method to automated analysisof the serum antioxidative activity against 1047297ve different ROS Themethod has an advantage for determination of antioxidative activities

against each ROS because different ROS can be generated by alteringthe reagents in the SIA system The developed method was applied to

the serum samples collected from healthy subjects diabetic andrheumatoid patients and the measured antioxidative activities werecompared

2 Material and methods

21 Reagents and solution

Hypoxanthine (HX) L -ascorbic acid boric acid sodium hydroxideH2O2 and sodium acetate were purchased from Wako Pure Chemical

(Osaka Japan) xanthine oxidase (XOD) N N -dimethylformamide(DMF) luminol and sodium hypochlorite were from Nacalai Tesque(Kyoto Japan) (plusmn)-(E )-4-methyl-2-[(E )-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1) was from Dojindo (Kumamoto

Japan) N -2-hydroxyethyl piperazine-N prime-2-ethanesulfonic acid(HEPES) lactoperoxidase (LPO) and sodium bromide were fromSigma (St Louis MO USA) Water was deionized using an AutostillWG 220 (Yamato Kagaku Tokyo Japan) and passed through a Puric-Z(Organo Tokyo Japan) All other solvents and reagents were of

analytical grade Luminol was dissolved in DMF and then diluted withwater (DMF content was 1) The reagents usedfor generation of each

ROS are summarized in Table 1

22 Apparatus and manifold design

The SIA-CL system (Fig 1) consisted of a Cavro XL 3000 syringe

pump (volume 1 mL Cavro Scienti1047297c Instruments Inc CA USA) a

Table 1

Carrier solution and ROS generation reagent for the SIA-CL measurement

ROS Carrier solution ROS reagent

ClOminus 50 mmoll borate buffer (pH 95) 383 mmoll NaClOcarrier solution

NO 100 mmoll Hepes buffer (pH 82) 2 mmoll NOR1DMSO100 mmoll

HCl=111O2 100 mmoll sodium acetate buffer

(pH 45)

150 mmoll H2O2carrier solution

80 mgml LPOcarrier solution

250 mmoll NaBrcarrier solution

O2minus

100 mmoll Hepes buffer (pH 82) 16Uml XODcarrier solution10 mmoll HXcarrier solution

H2O2 50 mmoll borate buffer (pH 95) 100 mmoll H2O2carrier solution

Fig 1 Scheme ofthe SIA-CLsystemfor themeasurementof antioxidativeactivityof humanserumagainst eachROS SPsyringepump HC holding coilMV multiportvalve M mixingtee

P pump D detector R recorder


8142019 1-s20-S0009898110002615-main


8142019 1-s20-S0009898110002615-main



























Acknowledgement


References






7

Table 3







RA 526ndash


651 ndash













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001





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Acknowledgement


References






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Table 3







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Acknowledgement


References






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Table 3







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651 ndash













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8142019 1-s20-S0009898110002615-main


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