research article application of ftir-atr spectroscopy to...

Research ArticleApplication of FTIR-ATR Spectroscopy to Determine the Extentof Lipid Peroxidation in Plasma during Haemodialysis

Adam Oleszko12 Sylwia OlsztyNska-Janus1 Tomasz Walski12

Karolina Grzeszczuk-KuT12 Jolanta Bujok2 Katarzyna GaBecka12 Albert Czerski23

Wojciech Witkiewicz2 and MaBgorzata Komorowska12

1 Institute of Biomedical Engineering and Instrumentation Faculty of Fundamental Problems of TechnologyWrocław University of Technology Wybrzeze Wyspianskiego 27 50-370 Wrocław Poland2Regional Specialist Hospital in Wrocław Research and Development Centre Kamienskiego 73a 51-124 Wrocław Poland3Department of Animal Physiology and Biostructure Faculty of Veterinary MedicineWrocław University of Environmental and Life Sciences Norwida 31 50-375 Wrocław Poland

Correspondence should be addressed to Małgorzata Komorowska malgorzatakomorowskapwredupl

Received 24 November 2014 Accepted 29 January 2015

Academic Editor Silvia Maria Doglia

Copyright copy 2015 Adam Oleszko et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

During a haemodialysis (HD) because of the contact of blood with the surface of the dialyser the immune system becomesactivated and reactive oxygen species (ROS) are released into plasma Particularly exposed to the ROS are lipids and proteinscontained in plasma which undergo peroxidationThemain breakdown product of oxidized lipids is themalondialdehyde (MDA)A commonmethod for measuring the concentration ofMDA is a thiobarbituric acid reactive substances (TBARS)method Despitethe formation of MDA in plasma during HD its concentration decreases because it is removed from the blood in the dialyserTherefore this research proposes the Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) spectroscopy whichenables determination of primary peroxidation products We examined the influence of the amount of hydrogen peroxide added tolipid suspension that was earlier extracted fromplasma specimen on lipid peroxidationwith use of TBARS and FTIR-ATRmethodsLinear correlation between these methods was shown The proposed method was effective during the evaluation of changes in theextent of lipid peroxidation in plasma during a haemodialysis in sheep A measurement using the FTIR-ATR showed an increasein plasma lipid peroxidation after 15 and 240 minutes of treatment while the TBARS concentration was respectively lower

1 Introduction

Dialysis therapy allows for the removal of metabolic wasteproducts and excess water and electrolytes from the blood-stream of patients with renal failure The treatment is per-formed by putting the patientrsquos blood through the dialyserwhere it comes into contact with a semipermeablemembranewith dialysis fluid the content of which allows for regenera-tion of the blood buffer system

Contact of the patientrsquos blood with surfaces of drainsand the dialyser leads to activation of the immune system[1] resulting in intensified production of reactive oxygenspecies (ROS) via the activation of neutrophils and plateletsmdasha so-called ldquooxidative burstrdquo [2] Apart from the super

oxide radical one ROS whose concentration rises duringan oxidative burst is hydrogen peroxide The ROS presentin the blood causes oxidation of the compounds containedtherein Plasma lipids and phospholipids being part of theerythrocyte cell membrane are particularly susceptible toROS activity [1]

Research conducted byHaklar et al [3] confirms the pres-ence of a lipid peroxidation process during haemodialysisAfter the treatment an increase in the quantity of conjugateddienes from 375 to 531 nmol per 1 120583mol of lipids extractedfrom plasma was observed It has also been proven thatduring haemodialysis a peroxidation of cholesterol from thecell membranes of erythrocytes [4] and a decrease in theconcentration of antioxidants in plasma [5] take place

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 245607 8 pageshttpdxdoiorg1011552015245607

2 BioMed Research International

Lipid peroxidation is associated with the formation ofperoxides as primary products [6]They are brokendown intosecondary products with shorter hydrocarbon chains Onesuch product is malondialdehyde (MDA)There are methodsthat allow to determine the content of both primary andsecondary products [7]

One method of testing the primary products of oxidationis the method of iodometric titration It is based on theoxidation of iodide ions (119868minus) by lipid peroxides A standardsolution of potassium iodide is added to the lipid specimenAs a result of the reaction molecular iodine (119868

2

) is producedwhich is then titrated with a standard sodium thiosulphatesolution in the presence of starch [8]

Another method for testing primary products of lipidoxidation is based on the detection of conjugated dienesTheir UV-VIS spectra are characterised by the presence ofan ultraviolet absorption band (230ndash235 nm) The greaterthe oxidation of fatty acids in the specimen the greater theabsorbance [9]

MDA is one of the breakdownproducts of lipid peroxidesthus it constitutes a secondary oxidation product Determi-nation of its concentration is used as an indicator of lipidoxidation MDA is used in a reaction with thiobarbituricacid (TBA) during which a coloured MDA-TBA complexis formed absorbing at 530ndash535 nm [10] The disadvantageof this method is its low specificity Chemical compoundsother than MDA that are present in biological specimenscan react with TBA for example bilirubin sialic acid (foundin cell membranes) degradation products of carbohydratesand other aldehydes [11 12] Therefore it is more aboutdetermining the quantity of products that are reactive withTBA (thiobarbituric acid reactive substances TBARS) Nev-ertheless determining the concentration of MDA is one ofthe most popular methods for studying lipid oxidation andoxidative stress magnitude

A limitation of this method for studying the effects ofhaemodialysis on plasma lipid peroxidation is the removalof MDA from plasma that takes place on the dialyser duringtreatment Therefore despite the increased formation ofMDA in plasma during treatment its concentration afterdialysis is lower than before the patient was connected tothe dialysis machine [13 14] This makes it impossible toapply the method of determining TBARS to study the effectsof haemodialysis on plasma lipid peroxidation The solutionto this problem was supposed to be a commercial fastassay kit for measuring ROS metabolites named the d-ROMstest (Diacron International Grosseto Italy) [15] Howeverthis method is controversial in relation to the possibility ofinterference of ceruloplasmin on the value of the obtainedresult [16 17]

An FTIR spectroscopy allows for a direct observation ofthe appearance or disappearance of bands that come fromdistinct vibrations of functional lipid groups whose changesindicate a formation of primary oxidation products [18ndash20]One of these bands is stretching vibration of C=O groupIncrease of ](C=O) band is result of three-step peroxidationprocess which undergoes according to following schemeFirstly during stage of initiation lipid molecule (LH) reactswith ROS Transfer of hydrogen from lipid to ROS and

appearance of lipid radical (L∙) are effects of this reaction(1) Secondly lipid radical undergoes reaction withmolecularoxygen (2a) giving lipid superoxide radicalmolecule (LOO∙)which is able to react with another lipid molecule (2b)

Initiation

LH+ROS 997888rarr ROSH + L∙ (1)

Propagation

L∙ + O2 minusrarr LOO∙

(2a)

LOO∙ + LH 997888rarr LOOH + L∙ (2b)

Termination

L∙ + L∙ 997888rarr L-L (3a)

LOO∙ + LOO∙ 997888rarr L=O + LOH +O2

(3b)

LOO∙ + L∙ 997888rarr L=O + LOH (3c)

Generation of lipid superoxide molecule (LOOH) and regen-eration of lipid radical (which can react with another oxygenand lipid molecule) are results of propagation stage Regen-eration of lipid radical shows chain reaction character ofperoxidation which can be terminated only during reactionof two radical molecules Lipid dimers (L-L) hydroxides(LOH) and oxides (L=O) belong to products of terminationstage Products of reactions (3b) and (3c) (lipid oxides) areresponsible for increase of absorbance of ](C=O) band

The aim of our experiments was to test the usefulness ofinfrared spectroscopy with a Fourier transform utilising theattenuated total reflectance (FTIR-ATR) to assess the extentof lipid peroxidation in the plasma of dialysed patients Invitro model tests were performed by oxidising total plasmalipid fractions using H

2

O2

Concurrently the FTIR-ATRspectra were recorded and the degree of lipid peroxidationwas determined using the classic TBARSmethod looking fora correlation between the twomethods Analysis of the FTIR-ATR spectra allowed us to define the bands corresponding tothe peroxidation process The developed procedure was usedfor the analysis of plasma taken from the blood of dialysedsheep and for determination of change in the degree of lipidperoxidation during treatment

2 Materials and Methods

21 Lipid Oxidation in Aqueous Suspension

211 Plasma Blood specimens of 20mL in volume werecollected from five clinically healthy male sheep (rams ofthe Polish Merino breed) with a body mass from 50 to60 kg A total of 100mL of blood was collected To preventclotting 38 of disodium citrate (POCH SA Poland) wasadded to the blood specimen The volume ratio of blood toanticoagulant was 9 1

BioMed Research International 3

Because of the need to transport blood specimens fromthe collection site to a laboratory within one hour aftercollection blood specimens were subjected to centrifugationfor 10 minutes at 3000 rpm after which plasma was collectedfor further testing

212 Lipid Extraction Lipid extraction was performed sepa-rately for each blood specimen based on a method developedby Folch et al [21] For this purpose a mixture of chloroformRG (POCH SA Poland) with methanol RG (POCH SAPoland) at a ratio of 2 1 was prepared After this a mixtureof chloroform-methanol and plasma in a volume ratio of 9 1was added to the tubesThe tubes content was shaken for fiveminutes Next most of the protein precipitate was removedfrom the mixture through filtration

In order to remove the rest of the proteins from thesupernatant a small amount of deionised water in the ratioof 1 10 was added Again it was shaken for five minutes andcentrifuged for 10 minutes at 3800 rpm (centrifuge MPW-350R MPW MED Instruments Poland) As a result ofcentrifugation a separation into two phases followed anupper (aqueous) containing protein and a lower (organic)containing lipid fraction The upper phase together with therest of the protein precipitate was removed after which theprocedure of removing proteins was repeated

Lipids from all specimens that were contained in theisolated organic phase were mixed The solvent was evapo-rated in vacuum After evaporation approximately 160mg ofextracted lipids remained in the dish

213 Preparation of Aqueous Lipid Suspension A suspensionof multilamellar liposomes was prepared from the extractedlipids for further oxidation After evaporation of the solventdeionised water was added to the dry weight and sonicateduntil a milky white suspension was obtained Lipid concen-tration amounted to 10mgmL

214 Oxidation of Lipids in a Suspension Using Hydrogen Per-oxide Hydrogen peroxide H

2

O2

with a 30 concentration(POCH SA Poland) was prediluted to 05

An aqueous lipid suspension with a concentration of1mgmL was oxidised using H

2

O2

The concentrations ofhydrogen peroxide that were used were 0 050 250 10and 20mM This procedure (repeated 3 times) allowed fora selection of oxidant concentrations in the subsequent partof the experiment 0 (control group) 025 050 075 and10mM The samples of lipid suspension were incubated ina thermomixer at 37∘C for 30min Then each sample wasdivided into two partsThe first part with a volume of 15mLwas intended to determine the concentration of TBARS Thesecond part with a volume of 20mL was intended for testingbymeans of the FTIR-ATR spectroscopyThe experiment wasrepeated seven times for both methods (119873 = 7)

215 Determining the TBARS Concentration To determinethe TBARS concentration a 037 solution of thiobarbituricacid TBA (AppliChem Germany) in a 025HCl molar solu-tion (Chempur Poland) was used in the test specimen

A suspension of oxidised lipids was mixed with a TBAsolution in the ratio of 1 1 and incubated in a water bathat 100∘C for 10 minutes Concurrently a reference specimenwas prepared (amixture of deionised water and TBA solutionin the ratio of 1 1) After incubation the cooled specimenswere transferred to cuvettes and with the use of a spec-trophotometer UV-VIS Evolution 60S (Thermo ScientificUSA) the absorbance at a wavelength of 535 and 600 nmwas measured with respect to the reference specimen Theabsorbance difference (119860diff = 119860535 minus 119860600) is a measure ofthe concentration of TBARS in a specimen [22]

Next a change in the value of the 119860diff parameter inrelation to the lipid control specimen (119860

0

) not subjectedto oxidation (H

2

O2

concentration equal to 0mM) wasdetermined according to

ΔTBARS =119860diff minus 11986001198600

sdot 100 (4)

Amean value of the parameterΔTBARS and standard devi-ation were determined for each value of H

2

O2

concentrationfrom each seven measurement series

216 Making Plasma Lipid Films on an ATR Accessory Aspecimen of the aqueous plasma lipid suspension with avolume of 2mL prepared as described in Section 214 wasadded with a 3mL mix of chloroform-methanol and thenshaken for five minutes in order to extract the lipids fromthe aqueous phase After centrifugation of the specimens for10 minutes at 3800 rpm the organic phase was separatedfrom the aqueous phaseThe organic phase was concentratedvia evaporation of the lipid solution in the mixture to avolume of about 50 120583L From the remaining volume afterconcentration 20120583L were taken out and placed on a crystalof the ATR sampling accessory and formed film Duringthe evaporation of the solvent the FTIR-ATR spectra wererecorded-up to a disappearance of the band at 760 cmminus1indicating the presence of chloroform (Figure 1) The lastspectrum was recorded two minutes after evaporation of thesolvent and used for further analysis

217 A Test Using the FTIR Spectroscopy Spectroscopic testswere performed using a spectrophotometer FTIR Nicolet6700 (Thermo Scientific USA) with an ATR accessory witha diamond crystal The recorded spectra were in the formof means of 32 spectra performed in the 4000ndash400 cmminus1range with a 4 cmminus1 resolution and atmospheric correctionswitched on at room temperature (25∘C)

Based on the literature [15ndash17] and own research twobands were selected for analysis ]as(CH3) at 2958 cm

minus1 and](C=O) at 1738 cmminus1 corresponding to a stretching vibrationof the CH

3

group and a stretching vibration of the C=Ogroup Together with lipid oxidation there is an increase ofintegral absorbance of the band ](C=O)

Values of integral absorbance of the bands ]as(CH3)in range 2982ndash2942 cmminus1 and ](C=O) in range 1787ndash1685 cmminus1 were calculated using the programme GRAMSAIIQ (parameters 119860

2958

and 1198601738

) In order to standardisethe results the ratio of the integral absorbance of the band


05

04

03

02

01

00

ATR

abso

rban

ce

825 800 775 750 725 700 675 650 625 600

Wavenumber (cmminus1)

1min2min3min

5min10min

Figure 1 ATR-FTIR spectra of phospholipids dissolved in chloro-form a decrease in band absorbance derived from chloroform at 756and 668 cmminus1 during the formation of film

](C=O) with respect to the band ]as(CH3) was calculatedaccording to

119868 =1198601738

1198602958

(5)

Next the change in the value of parameter 119868 was determinedin relation to the control specimen (119868contr) not subjected tooxidation (H

2

O2

concentration equal to 0mM) according tothe formula below

Δ119868 =119868 minus 119868contr119868contrsdot 100 (6)

Amean value of the parameterΔ119868 and standard deviationwere determined for each value of the H

2

O2


22 Degree of Lipid Peroxidation inthe Process of Haemodialysis

221 Haemodialysis Treatment Procedure Experiments wereperformed on a clinically healthy adultmale sheep (ramof thePolishMerino breed) weighing 56 plusmn 2 kg Vascular access wasobtained by percutaneous placement of 145 Fr times 28 cm dual-lumen Hemo-Flow Dialysis Catheter (Medcomp USA) intothe right jugular vein (vena jugularis) under premedication(xylazine 02mgkg body weight intravenously) and localanaesthesia by infiltration (2 lidocaine)

The experiments were performed using the Fresenius4008B haemodialysis machine on a sheep without pharma-cological taming standing in a pit The animal was subjectedto dialysis treatments lasting four hours three times a weekusing the Fresenius F4HPS polysulphone dialyser and stan-dard bloodlines (AV-Set Fresenius Poland) Before the treat-ment 5000 IU of heparin sodium (WZF Polfa SA Poland)

was administered in a 500mL Ringerrsquos solution (B BraunGermany) into the drip infusion During the experimentheparin (at a dose of 1250 IUh) was administered into thebloodline The dosage of heparin was established in prelim-inary experiments During four hours of haemodialysis 36 lof blood was circulated through an extracorporeal set (bloodflow rate 150mLmin) the total ultrafiltration was 200mLThe flow rate of dialysate (composition Na+ 1420 K+ 30Mg2+ 05 Ca2+ 15 Clminus 1100 HCO3minus 320mmolL glucose10 gL) was preset to 500mLmin Five haemodialysis treat-ments were performed as part of this experiment

222 Sampling During each haemodialysis three bloodspecimens of 6mL in volume were taken using disodiumcitrate as described in Section 211 Blood specimens weretaken from the venous line The first specimen was takenimmediately after commencement of treatment and thesecond was taken 15 minutes from the start of treatmentThe time of specimen collection was based on the literature[23] according to which the greatest changes in bloodare observed during the first 30 minutes of haemodialysisThe third specimen was taken just before the end of thecirculation after 240 minutes

223 Determining the TBARS Concentration in Plasma Todetermine the TBARS concentration a 037 solution ofthiobarbituric acid TBA (AppliChem Germany) was usedin a 025 HCl molar solution (Chempur Poland) and 15trichloroacetic acid solution TCA (Chempur Poland) in a025 HCl molar solution

To 05mL of plasma we added 05mL of TCA Specimenswere subjected to one hour of incubation at 4∘C thencentrifuged for 20minutes at 4500 rpm afterwhich 05mLofsupernatant was collected Next 05mL of TBA was added tothe supernatant afterwhich the specimenswere incubated inawater bath at 100∘C for 10minutes Concurrently a referencespecimen was prepared (deionised water a solution of TBAand TCA in the ratio of 1 1 1) A measurement of TBA usinga spectrophotometer was performed in a manner describedin Section 215

224 Extraction of Plasma Lipids and a Test Using the FTIR-ATR Spectroscopy Lipidswere extracted from3mLof plasmafor each one of the specimens as described in Sections 211and 212 After placing the specimenon a crystal its spectrumwas recorded until disappearance of the band at 760 cmminus1Recording of the spectra and its analysis was conducted ina manner described in Sections 216 and 217

225 Statistical Analysis For data analysis Matlab R2009bsoftware by MathWorks (USA) was used The results werepresented in the form of mean values and standard devi-ations The differences between particular parameters weretested using the parametric ANOVA test The threshold ofstatistical significance was set at 119875 lt 005

226 Ethics Committee Approval In order to conduct theexperiments authorisation was obtained in accordance with


Table 1 Assignment of organic compounds to bands on the FTIR-ATR spectrum of plasma lipids ]-stretching vibration 120575-bendingvibrations s-symmetric as-asymmetric and sh-shoulder band

Wave numbercmminus1 Assignment LiteratureOn recorded spectra According to literature3370 3280ndash3473 ](OndashH) and ](NndashH) water molecules choline [24 25]2958 sh 2952ndash2959 sh ]as(CH3) lipids cholesterol esters fatty acids [25ndash32]2920 2919ndash2925 ]as(CH2) lipids long-chain fatty acids [25ndash32]2868 sh 2871ndash2873 sh ]s(CH3) lipids fatty acids [24 25 27]2852 2850ndash2855 ]s(CH2) lipids long-chain fatty acids [25ndash32]1738 1732ndash1747 ](C=O) lipids cholesterol esters fatty acids oxides [25 27ndash30 32]1712 1712ndash1718 ](C=O) COOH group of fatty acids [26 32]1495 sh 1490 sh 120575as(NndashCH3) choline group [33]1465 1464ndash1468 120575(CH2) aliphatic chains of fatty acids [24ndash29]1377 1377ndash1381 120575(CH3) aliphatic chains of fatty acids [26ndash29]1184 1160ndash1179 ]as(CndashO) lipid ester bonds [27 28]1082 1075ndash1090 ]s(PO2) phospholipids [26 27]970 967ndash972 ]as(C=C) conformation trans [26 28 29]

the resolution number 102013 of the II Local Ethics Commit-tee in Wrocław regarding experiments on animals

3 Results

31 Results of Lipid Oxidation in Aqueous Suspension

311 FTIR-ATR Spectroscopy Figure 1 shows FTIR-ATRspectra of plasma lipids dissolved in chloroformThedecreasein band absorbance at 760 cmminus1 is related to the evaporationof chloroform after placing the specimen on a diamondcrystal All analyses were performed for spectra recordedafter the evaporation of chloroform

Figure 2 shows the FTIR-ATR spectrum of lipidsextracted from plasma A description of the assignedabsorption bands is listed in Table 1 In range from 3050to 2750 cmminus1 the bands correspond to asymmetric andsymmetric stretching vibrations of the methyl andmethylenegroups [24ndash32] The asymmetric and symmetric bands ofthe methyl groups were labelled as ]as(CH3) and ]s(CH3)respectively Their maxima occur at wavenumbers 2958 and2868 cmminus1 whereas asymmetric and symmetric bands ofmethylene groups labelled as ]as(CH2) and ]s(CH2) occur at2920 and 2852 cmminus1

At 1738 cmminus1 there is a very important band associatedwith stretching vibrations of the carbonyl group ](C=O)particularly ester bonds between fatty acids and glycerolwithin the lipid molecules However this type of bond mayalso be formed by peroxidation of fatty acid chains [34]Therefore we suggest that the increase in the intensity of thisband indicates an increase in lipid oxidation in the specimenwhich has been confirmed in the literature [25ndash30 32 35]

Subsequent bands are located at the following wavenumbers 1495 cmminus1 (a stretching vibration of the NndashCH

3

group derived from choline) 1465 and 1377 cmminus1 (bendingvibrations of themethyl andmethylene groups of fatty acids)1184 cmminus1 (a stretching vibration of the CndashO group of lipid

ATR

abso

rban

ce


0175

0150

0125

0100

0075

0050

0025

0000

3600

3400

3200

3000

2800

2600

2000

1800

1600

1400

1200

1000

800

600

2920

2868

2852

2958

(C=O) 120575(NndashCH3)

120575(CH2)120575(CH3)

(NndashCH3)

1712

1738

1465

1495

1377

1184

1082

970

(C=O)

(PO2)s

(CH2)s

(CH3)s

(CndashO)as

(CH3)as

(CH2)as

Figure 2 FTIR-ATR spectrum of extracted plasma lipids afterevaporation of chloroform

ester bond) and 1082 cmminus1 (a stretching vibration of thephosphate PO

2

group) [24ndash29 33]In the beginning the dependence of the value of param-

eter Δ119868 upon the H2

O2

concentration function was calcu-lated (Figure 3) This relationship is exponential in naturetaking into account the whole range of H

2

O2

concentrations(0ndash20mM) In some range exponential function can beapproximated by linear function We assume that the abovementioned ratio can be described by linear rapid growthup to the value of 10mM This reflects an increase inthe oxidation of lipids extracted from plasma in the H

2

O2

concentration function Therefore subsequent experimentswere performed for hydrogen peroxide with a concentrationranging from 0ndash10mM

Figure 4 shows spectra fromone series ofmeasurement ofplasma lipid oxidation in the spectral range 1800ndash1650 cmminus1with band ](C=O) Each of the spectra have been nor-malised with respect to band ]as(CH3) The increase in theintensity of band ](C=O) as a result of oxidation is visibleThe linear increase in the value of the parameter Δ119868 in


35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200

Hydrogen peroxide concentration (mM)

Figure 3 Dependence of change in the ratio of integral bandabsorbance ](C=O) with respect to ]as(CH3) upon the concentra-tion of H

2

O2

in the concentration range from 0 to 20mM meanvalue relative to control sample (119873 = 3)


025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM

Figure 4 FTIR-ATR spectra of lipid film corresponding to the band](C=O) increase in band absorbance due to H

2

O2

the H2

O2

concentration function (Figure 5) indicates thatlipid oxidation is taking place

312 Determination of TBARS A linear change in theTBARS concentrations has been observed relative to the con-trol specimen when H

2

O2

was added The results are shownin Figure 6 Therefore it can be ascertained that the amountof TBARS that were formed is proportional to the amountof agent that causes oxidation in the H

2

O2

concentrationranges up to 1mM and has a fixed lipid concentration levelin the specimen Smaller values of standard deviations wererecorded than in the case of using FTIR-ATR spectroscopy

25

20

15

10

5

0

ΔI

()

000 025 050 075 100


ΔI = 170 lowast cH2O2

R2= 0996

Figure 5 Dependence of changes in the ratio of integral bandabsorbance ](C=O) with respect to ]as(CH3) upon the concentra-tion of H

2

O2

mean value relative to control sample plusmn standarddeviation (119873 = 7)

20

15

10

5

0

000 025 050 075 100


ΔTBARS = 156 lowast cH2O2

R2= 0998Δ

TBA

RS(

)

Figure 6 Dependence of changes in TBARS concentration in asample upon the concentration of H

2

O2

mean value relative tocontrol sample plusmn standard deviation (119873 = 7)

32 Lipid Peroxidation under the Influence of HaemodialysisFigure 8 illustrates the changes in the amount of peroxidationproducts during haemodialysis A statistically significantincrease (119875 lt 005) was found in the amount of lipidperoxidation products of plasma after 15 and 240 minutesof treatment relative to the control specimen which wasdetermined by using FTIR-ATR spectroscopy This increaseamounted to 44 and 29 respectively

In the case of determining TBARS a decrease of 15and 41 was recorded respectively after 15 and 240 minutesof haemodialysis only the decrease after 240 minutes wasstatistically significant (119875 parameter was 0074 and 0006resp)


20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()

ΔTBARS = 0916 lowast ΔI

R2= 0991

Figure 7 Correlation between change of the ratio of integral bandabsorbance ](C=O) with respect to ]as(CH3) and change in theconcentration of TBARS mean value relative to control sample plusmnstandard deviation (119873 = 7)

4 Conclusions

During in vitro model tests a relationship was confirmedbetween the value of the ratio of absorbance bands ](C=O)with respect to ]as(CH3) and concentration of TBARS andthe amount of H

2

O2

added to lipid specimen extractedfrom plasma A correlation between the results obtainedusing FTIR spectroscopy and results of the TBARS methodwas investigated (Figure 7) The results obtained by bothmethods show high consistency Despite the fact that FTIR-ATR spectroscopy investigated primary products of lipidoxidation and the determination of TBARS method is basedon detection of its breakdown products (secondary oxidationproducts) the results are comparable

Although FTIR-ATR spectroscopy showed lower repro-ducibility between different series of measurements than theTBARS method which resulted in high standard deviationvalues the lipid peroxidation test technique using FTIR-ATR spectroscopy allowed for the determination of oxidativestress in plasma during haemodialysis while the methodfor determining TBARS concentrations showed that MDA iseffectively removed by the dialyser membranes

Determination of TBARS showed a decrease in theamount of secondary peroxidation products during haemo-dialysis by 15 and 41 after 15 and 240 minutes as a resultof MDArsquos removal in the dialyser which is consistent withthe literature [13 14] The results obtained in our studiesusing FTIR-ATR spectroscopy however revealed an increasein the amount of peroxidation products by 44 after 15minutes and 29 after 240 minutes of treatment indicatingthe presence of oxidative stress in haemodialysis This is alsorelated to the amount of neutrophils and monocytes in theblood which are responsible for the release of ROS It wasshown that the greatest decline in their numbers associatedwith activation and adhesion to the dialyser occurs in the first15 minutes of dialysis [23 36] After this time the numberof cells gradually returns to baseline Thus the most intense

18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074

lowastlowastP = 0006

P = 0036

lowast lowastlowast

0008P =

Figure 8 Dependence of the amount of peroxidation products rela-tive to control sample (0min HD) upon duration of the haemodial-ysis treatment determined by using FTIR-ATR spectroscopy andTBARS (119873 = 5)

release of ROS and lipid peroxidation occurs in the firstminutes of haemodialysis as confirmed by our results

Measurements of primary products of peroxidation onisolated lipids were performed without the presence ofproteins which may also undergo oxidative processes Thetests found a full correlation with the determination ofTBARS method The usefulness of the proposed method forstudying plasma lipid peroxidation during haemodialysis wasconfirmed

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research is part of a project called ldquoWROVASCmdashan Integrated Cardiovascular Centrerdquo which is cofinancedby the European Regional Development Fund within theOperational Program Innovative Economy for the years2007ndash2013 The study was carried out at the Research andDevelopment Centre of the Regional Specialist Hospital inWrocław

References

[1] S J Klebanoff ldquoOxygen metabolism and the toxic properties ofphagocytesrdquoAnnals of Internal Medicine vol 93 no 3 pp 480ndash489 1980

[2] B Halliwell S Chirico M A Crawford K S Bjerve and KF Gey ldquoLipid peroxidation its mechanism measurement andsignificancerdquoTheAmerican Journal of Clinical Nutrition vol 57no 5 pp 715ndash724 1993


[3] G Haklar I Yegenaga and A S Yalcin ldquoEvaluation of oxidantstress in chronic hemodialysis patients use of different param-etersrdquo Clinica Chimica Acta vol 234 no 1-2 pp 109ndash114 1995

[4] H Hashimoto M Takaya and K Sumino ldquoLipid abnormal-ities of erythrocyte membranes in hemodialysis patients withchronic renal failurerdquo Clinica Chimica Acta vol 252 no 2 pp137ndash145 1996

[5] P Jackson CM Loughrey J H Lightbody P TMcNamee andI S Young ldquoEffect of hemodialysis on total antioxidant capacityand serum antioxidants in patients with chronic renal failurerdquoClinical Chemistry vol 41 no 8 pp 1135ndash1138 1995

[6] M El-Saadani H Esterbauer M El-Sayed M Goher A YNassar and G Jurgens ldquoA spectrophotometric assay for lipidperoxides in serum lipoproteins using a commercially availablereagentrdquo Journal of Lipid Research vol 30 no 4 pp 627ndash6301989

[7] E D Wills ldquoMechanisms of lipid peroxide formation in animaltissuesrdquo Biochemical Journal vol 99 no 3 pp 667ndash676 1966

[8] M Antolovich P D Prenzler E Patsalides S McDonald andK Robards ldquoMethods for testing antioxidant activityrdquo Analystvol 127 no 1 pp 183ndash198 2002

[9] J I Gray ldquoMeasurement of lipid oxidation a reviewrdquo Journal ofthe American Oil Chemistsrsquo Society vol 55 no 6 pp 539ndash5461978

[10] R Sinnhuber and T Yu ldquoCharacterization of the red pigmentformed in 2-thiobarbituric acid determination of oxidativerancidityrdquo Journal of Food Science vol 23 no 6 pp 626ndash6341958

[11] T Asakawa and S Matsushita ldquoThiobarbituric acid test fordetecting lipid peroxidesrdquo Lipids vol 14 no 4 pp 401ndash4061979

[12] J A Knight R K Pieper and L McClellan ldquoSpecificity ofthe thiobarbituric acid reaction its use in studies of lipidperoxidationrdquoClinical Chemistry vol 34 no 12 pp 2433ndash24381988

[13] M Daschner H Lenhartz D Botticher et al ldquoInfluence ofdialysis on plasma lipid peroxidation products and antioxidantlevelsrdquo Kidney International vol 50 no 4 pp 1268ndash1272 1996

[14] M Boaz Z Matas A Biro et al ldquoSerum malondialdehydeand prevalent cardiovascular disease in hemodialysisrdquo KidneyInternational vol 56 no 3 pp 1078ndash1083 1999

[15] L Lucchi A Iannone S Bergamini et al ldquoComparison betweenhydroperoxides and malondialdehyde as markers of acuteoxidative injury during hemodialysisrdquoArtificial Organs vol 29no 10 pp 832ndash837 2005

[16] K Kilk R Meitern O Harmson U Soomets and P HorakldquoAssessment of oxidative stress in serum by d-ROMs testrdquo FreeRadical Research vol 48 no 8 pp 883ndash889 2014

[17] M I Harma and O Erel ldquod-ROMs test detects ceruloplasminnot oxidative stressrdquo Chest vol 130 no 4 pp 1276ndash1277 2006

[18] B Vileno S Jeney A Sienkiewicz P R Marcoux L M Millerand L Forro ldquoEvidence of lipid peroxidation and proteinphosphorylation in cells upon oxidative stress photo-generatedby fullerolsrdquoBiophysical Chemistry vol 152 no 1ndash3 pp 164ndash1692010

[19] D J Moore R H Sills and R Mendelsohn ldquoPeroxidationof erythrocytes FTIR spectroscopy studies of extracted lipidsisolatedmembranes and intact cellsrdquo Biospectroscopy vol 1 no2 pp 133ndash140 1995

[20] C Petibois and G Deleris ldquoErythrocyte adaptation to oxidativestress in endurance trainingrdquo Archives of Medical Research vol36 no 5 pp 524ndash531 2005

[21] J Folch M Lees and S Sloane ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of biological chemistry vol 226 no 1 pp 497ndash5091957

[22] S Sturlan M Baumgartner E Roth and T Bachleitner-Hofmann ldquoDocosahexaenoic acid enhances arsenic trioxide-mediated apoptosis in arsenic trioxide-resistant HL-60 cellsrdquoBlood vol 101 no 12 pp 4990ndash4997 2003

[23] W A Nockher J Wiemer and J E Scherberich ldquoHaemodial-ysis monocytopenia differential sequestration kinetics ofCD14+CD16+ and CD14++ blood monocyte subsetsrdquo Clinicaland Experimental Immunology vol 123 no 1 pp 49ndash55 2001

[24] I N Hayati Y B C Man C P Tan and I N Aini ldquoMonitoringperoxide value in oxidized emulsions by Fourier transforminfrared spectroscopyrdquo European Journal of Lipid Science andTechnology vol 107 no 12 pp 886ndash895 2005

[25] H A Galeb J Salimon E EMEid N E Nacer N Saari andS Saadi ldquoThe impact of single and double hydrogen bonds oncrystallization and melting regimes of Ajwa and Barni lipidsrdquoFood Research International vol 48 no 2 pp 657ndash666 2012

[26] I Sanchez-Alonso P Carmona and M Careche ldquoVibrationalspectroscopic analysis of hake (Merluccius merluccius L) lipidsduring frozen storagerdquo Food Chemistry vol 132 no 1 pp 160ndash167 2012

[27] J L R Arrondo and F M Goni ldquoInfrared studies of protein-induced perturbation of lipids in lipoproteins and membranesrdquoChemistry and Physics of Lipids vol 96 no 1-2 pp 53ndash68 1998

[28] I Dreissig S Machill R Salzer and C Krafft ldquoQuantifica-tion of brain lipids by FTIR spectroscopy and partial leastsquares regressionrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 71 no 5 pp 2069ndash2075 2009

[29] A Rohman and Y B Che Man ldquoApplication of Fourier trans-form infrared (FT-IR) spectroscopy combined with chemomet-rics for authentication of cod-liver oilrdquoVibrational Spectroscopyvol 55 no 2 pp 141ndash145 2011

[30] J E Verity N Chhabra K Sinnathamby and C M YipldquoTracking molecular interactions in membranes by simultane-ous ATR-FTIR-AFMrdquo Biophysical Journal vol 97 no 4 pp1225ndash1231 2009

[31] J-C Tsai Y-L Lo C-Y Lin H-M Sheu and J-C Lin ldquoFeasi-bility of rapid quantitation of stratum corneum lipid content byFourier transform infrared spectrometryrdquo Spectroscopy vol 18no 3 pp 423ndash431 2004

[32] C Petibois and G Deleris ldquoChemical mapping of tumor pro-gression by FT-IR imaging towards molecular histopathologyrdquoTrends in Biotechnology vol 24 no 10 pp 455ndash462 2006

[33] M Schwarzott P Lasch D Baurecht D Naumann and U PFringeli ldquoElectric field-induced changes in lipids investigatedby modulated excitation FTIR spectroscopyrdquo Biophysical Jour-nal vol 86 no 1 pp 285ndash295 2004

[34] B Fuchs K Bresler and J Schiller ldquoOxidative changes of lipidsmonitored byMALDIMSrdquoChemistry and Physics of Lipids vol164 no 8 pp 782ndash795 2011

[35] B Muik B Lendl A Molina-Diaz M Valcarcel and MJ Ayora-Canada ldquoTwo-dimensional correlation spectroscopyandmultivariate curve resolution for the study of lipid oxidationin edible oils monitored by FTIR and FT-Raman spectroscopyrdquoAnalytica Chimica Acta vol 593 no 1 pp 54ndash67 2007

[36] K Grzeszczuk-Kuc J Bujok T Walski and M KomorowskaldquoBlood elements activation in hemodialysismdashanimal modelstudiesrdquo International Journal of Medical Health Pharmaceu-tical and Biomedical Engineering vol 7 pp 341ndash345 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Lipid peroxidation is associated with the formation ofperoxides as primary products [6]They are brokendown intosecondary products with shorter hydrocarbon chains Onesuch product is malondialdehyde (MDA)There are methodsthat allow to determine the content of both primary andsecondary products [7]

One method of testing the primary products of oxidationis the method of iodometric titration It is based on theoxidation of iodide ions (119868minus) by lipid peroxides A standardsolution of potassium iodide is added to the lipid specimenAs a result of the reaction molecular iodine (119868

2

) is producedwhich is then titrated with a standard sodium thiosulphatesolution in the presence of starch [8]

Another method for testing primary products of lipidoxidation is based on the detection of conjugated dienesTheir UV-VIS spectra are characterised by the presence ofan ultraviolet absorption band (230ndash235 nm) The greaterthe oxidation of fatty acids in the specimen the greater theabsorbance [9]

MDA is one of the breakdownproducts of lipid peroxidesthus it constitutes a secondary oxidation product Determi-nation of its concentration is used as an indicator of lipidoxidation MDA is used in a reaction with thiobarbituricacid (TBA) during which a coloured MDA-TBA complexis formed absorbing at 530ndash535 nm [10] The disadvantageof this method is its low specificity Chemical compoundsother than MDA that are present in biological specimenscan react with TBA for example bilirubin sialic acid (foundin cell membranes) degradation products of carbohydratesand other aldehydes [11 12] Therefore it is more aboutdetermining the quantity of products that are reactive withTBA (thiobarbituric acid reactive substances TBARS) Nev-ertheless determining the concentration of MDA is one ofthe most popular methods for studying lipid oxidation andoxidative stress magnitude

A limitation of this method for studying the effects ofhaemodialysis on plasma lipid peroxidation is the removalof MDA from plasma that takes place on the dialyser duringtreatment Therefore despite the increased formation ofMDA in plasma during treatment its concentration afterdialysis is lower than before the patient was connected tothe dialysis machine [13 14] This makes it impossible toapply the method of determining TBARS to study the effectsof haemodialysis on plasma lipid peroxidation The solutionto this problem was supposed to be a commercial fastassay kit for measuring ROS metabolites named the d-ROMstest (Diacron International Grosseto Italy) [15] Howeverthis method is controversial in relation to the possibility ofinterference of ceruloplasmin on the value of the obtainedresult [16 17]

An FTIR spectroscopy allows for a direct observation ofthe appearance or disappearance of bands that come fromdistinct vibrations of functional lipid groups whose changesindicate a formation of primary oxidation products [18ndash20]One of these bands is stretching vibration of C=O groupIncrease of ](C=O) band is result of three-step peroxidationprocess which undergoes according to following schemeFirstly during stage of initiation lipid molecule (LH) reactswith ROS Transfer of hydrogen from lipid to ROS and

appearance of lipid radical (L∙) are effects of this reaction(1) Secondly lipid radical undergoes reaction withmolecularoxygen (2a) giving lipid superoxide radicalmolecule (LOO∙)which is able to react with another lipid molecule (2b)

Initiation

LH+ROS 997888rarr ROSH + L∙ (1)

Propagation

L∙ + O2 minusrarr LOO∙

(2a)

LOO∙ + LH 997888rarr LOOH + L∙ (2b)

Termination

L∙ + L∙ 997888rarr L-L (3a)

LOO∙ + LOO∙ 997888rarr L=O + LOH +O2

(3b)

LOO∙ + L∙ 997888rarr L=O + LOH (3c)

Generation of lipid superoxide molecule (LOOH) and regen-eration of lipid radical (which can react with another oxygenand lipid molecule) are results of propagation stage Regen-eration of lipid radical shows chain reaction character ofperoxidation which can be terminated only during reactionof two radical molecules Lipid dimers (L-L) hydroxides(LOH) and oxides (L=O) belong to products of terminationstage Products of reactions (3b) and (3c) (lipid oxides) areresponsible for increase of absorbance of ](C=O) band

The aim of our experiments was to test the usefulness ofinfrared spectroscopy with a Fourier transform utilising theattenuated total reflectance (FTIR-ATR) to assess the extentof lipid peroxidation in the plasma of dialysed patients Invitro model tests were performed by oxidising total plasmalipid fractions using H

2

O2

Concurrently the FTIR-ATRspectra were recorded and the degree of lipid peroxidationwas determined using the classic TBARSmethod looking fora correlation between the twomethods Analysis of the FTIR-ATR spectra allowed us to define the bands corresponding tothe peroxidation process The developed procedure was usedfor the analysis of plasma taken from the blood of dialysedsheep and for determination of change in the degree of lipidperoxidation during treatment

2 Materials and Methods

21 Lipid Oxidation in Aqueous Suspension

211 Plasma Blood specimens of 20mL in volume werecollected from five clinically healthy male sheep (rams ofthe Polish Merino breed) with a body mass from 50 to60 kg A total of 100mL of blood was collected To preventclotting 38 of disodium citrate (POCH SA Poland) wasadded to the blood specimen The volume ratio of blood toanticoagulant was 9 1








2

O2



2

O2





0


2

O2



sdot 100 (4)


2

O2






3



2958

and 1198601738



05

04

03

02

01

00

ATR

abso

rban

ce

825 800 775 750 725 700 675 650 625 600


1min2min3min

5min10min



119868 =1198601738

1198602958

(5)


2

O2




2

O2
















3 Results






3


ATR

abso

rban

ce


0175

0150

0125

0100

0075

0050

0025

0000

3600

3400

3200

3000

2800

2600

2000

1800

1600

1400

1200

1000

800

600

2920

2868

2852

2958


120575(CH2)120575(CH3)

(NndashCH3)

1712

1738

1465

1495

1377

1184

1082

970

(C=O)

(PO2)s

(CH2)s

(CH3)s

(CndashO)as

(CH3)as

(CH2)as



2



O2


2

O2


2

O2




35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200



2

O2



025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM


2

O2

the H2

O2



2

O2


2

O2


25

20

15

10

5

0

ΔI

()

000 025 050 075 100



R2= 0996


2

O2


20

15

10

5

0

000 025 050 075 100



R2= 0998Δ

TBA

RS(

)


2

O2





20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2

O2



2

O2





0


2

O2



sdot 100 (4)


2

O2






3



2958

and 1198601738



05

04

03

02

01

00

ATR

abso

rban

ce

825 800 775 750 725 700 675 650 625 600


1min2min3min

5min10min



119868 =1198601738

1198602958

(5)


2

O2




2

O2
















3 Results






3


ATR

abso

rban

ce


0175

0150

0125

0100

0075

0050

0025

0000

3600

3400

3200

3000

2800

2600

2000

1800

1600

1400

1200

1000

800

600

2920

2868

2852

2958


120575(CH2)120575(CH3)

(NndashCH3)

1712

1738

1465

1495

1377

1184

1082

970

(C=O)

(PO2)s

(CH2)s

(CH3)s

(CndashO)as

(CH3)as

(CH2)as



2



O2


2

O2


2

O2




35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200



2

O2



025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM


2

O2

the H2

O2



2

O2


2

O2


25

20

15

10

5

0

ΔI

()

000 025 050 075 100



R2= 0996


2

O2


20

15

10

5

0

000 025 050 075 100



R2= 0998Δ

TBA

RS(

)


2

O2





20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


05

04

03

02

01

00

ATR

abso

rban

ce

825 800 775 750 725 700 675 650 625 600


1min2min3min

5min10min



119868 =1198601738

1198602958

(5)


2

O2




2

O2
















3 Results






3


ATR

abso

rban

ce


0175

0150

0125

0100

0075

0050

0025

0000

3600

3400

3200

3000

2800

2600

2000

1800

1600

1400

1200

1000

800

600

2920

2868

2852

2958


120575(CH2)120575(CH3)

(NndashCH3)

1712

1738

1465

1495

1377

1184

1082

970

(C=O)

(PO2)s

(CH2)s

(CH3)s

(CndashO)as

(CH3)as

(CH2)as



2



O2


2

O2


2

O2




35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200



2

O2



025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM


2

O2

the H2

O2



2

O2


2

O2


25

20

15

10

5

0

ΔI

()

000 025 050 075 100



R2= 0996


2

O2


20

15

10

5

0

000 025 050 075 100



R2= 0998Δ

TBA

RS(

)


2

O2





20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results






3


ATR

abso

rban

ce


0175

0150

0125

0100

0075

0050

0025

0000

3600

3400

3200

3000

2800

2600

2000

1800

1600

1400

1200

1000

800

600

2920

2868

2852

2958


120575(CH2)120575(CH3)

(NndashCH3)

1712

1738

1465

1495

1377

1184

1082

970

(C=O)

(PO2)s

(CH2)s

(CH3)s

(CndashO)as

(CH3)as

(CH2)as



2



O2


2

O2


2

O2




35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200



2

O2



025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM


2

O2

the H2

O2



2

O2


2

O2


25

20

15

10

5

0

ΔI

()

000 025 050 075 100



R2= 0996


2

O2


20

15

10

5

0

000 025 050 075 100



R2= 0998Δ

TBA

RS(

)


2

O2





20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


35

30

25

20

15

10

5

0

ΔI

()

00 25 50 75 100 125 150 175 200



2

O2



025

020

015

010

005

000

Nor

mal

ized

abso

rban

ce

1800 1775 1750 1725 1700 1675 1650

000mM025mM050mM

075mM100mM


2

O2

the H2

O2



2

O2


2

O2


25

20

15

10

5

0

ΔI

()

000 025 050 075 100



R2= 0996


2

O2


20

15

10

5

0

000 025 050 075 100



R2= 0998Δ

TBA

RS(

)


2

O2





20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


20

15

10

5

0

2520151050

ΔTB

ARS

()

ΔI ()


R2= 0991


4 Conclusions


2

O2




18

16

14

12

10

08

06

04

Pero

xida

tion

prod

ucts

0 15 240

Dialysis time (min)

FTIRTBARS

lowastP = 0074


P = 0036

lowast lowastlowast

0008P =






Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article application of ftir-atr spectroscopy to...

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