Determination of lipid hydroperoxides by electrochemiluminescence

Adyt~a

59

Chumca Acta, 262 (1992) 59-65

Elsevler Science Pubhshers B V , Amsterdam

Determination of lipid hydroperoxides by electrochemiluminescence S Sakura * KrbunR&D

Office, 2423 Mllrkan Road, Chapel Htll, NC 27516 (USA)

J Terao Natwnal Food Research Instztute,Mumtry of Agnculture, Forestry and Fuherm, 1-2-l

Knnnondar, Tsukuba, lbarakr 305 (Japan)

(Received 21st August 1991, revlsed manuscript received 16th November 1991)

Abstract Electrochenulummesnce (ECL) was used for the detectlon of the hpld hydropcronde methyl lmoleate hydroperoxlde (MLHP) at a glassy carbon electrode m acetomtnle (AN&neutral aqueous solution The cychc voltammograms of lummol and MLHP were measured to fmd the optimum apphed potential Lummol was oxldlzed at about 0 45 V with or Hrlthout the presence of AN m the aqueous solution MLHP was oxldlzed at a fairly posItwe potential (1 10 V) The oxldatlon products were determined to be conjugated tnene, superoxlde and proton by consldermg UV spectral changes, colonmetric reactlons and reactlon with the superoxlde dlsmutase enzyme In the ECL method, lummol was oxldlzed on the electrode to the excited IntermedIate (dmzaqumone), whch reacted with MLHP and enutted hght ECL spectra and decay times were stuched In a flow-mjectlon system with pH 7 4 phosphate buffer-30% AN carrier solutlon, ermtted hght was detected at the oxldatlon potential of lunnnol The detectlon hnut was 0 3 nmol with a relative standard devlatlon of 13% (n = 5) at a agnal-to-nolsc ratio of 15 Keywords Chenulummescence, Cychc voltammetry, LIpId hydroperoxldes, Lummol, Methyl hnoleate hydroperoxlde

Llpld hydroperoxldes have attracted much attentlon because they are Involved m the regulation of prostaglandm blosynthesls, which can lead to cancer development, agmg and other patholog~cal condltlons The determmatlon of hpld hydroperoxldes m blologlcal systems IS a necessary step m the clanfication of the physlologrcal and pathologlcal effects of hpld hydroperoxldes There are several methods for the determmatlon of hpld hydroperoxldes the thlobarblturlc aad (TBA) method (mvolvmg reactlon with a decomposltlon product of hpld hydroperoxldes, malondddehyde) [ll, measurement of UV absorption of conJugated dlenes present in hpld hydroperoxtdes [21, the methylene blue (MTEQ method (oxidation of methylene blue denvatlve m the presence of hemoglobm) [31, and the oxlda-

tlon of dlchlorofluorescem [4] or of sesamol dlmer [5] by hpld hydroperoxldes More recent methods include electrochemical detection using a reduction current at a glassy carbon electrode [6,7] or chemdummescence (CL) with lummol [8,9] All these methods, except the TBA method and UV absorption of conJUgated dlenes, depend on 0x1datlon by hpld hydroperoxlde, therefore, hpld hydroperoxlde 1s reduced m these reactions The electrochemdummescence (electrolytically generated chemllummescence, ECL) method was used m thrs work In this method, the electrode potential IS applied to oxldlze substrates electrolytically to form chemdummescent excited mtermedlates as opposed to tradItiona CL, which depends on enzymatic and/or chemrcal oxldlzers ECL has been used for the detection of hydrogen

0003-2670/92/$05 00 Q 1992 - Elsevler Science Pubhshers B V All nghts reserved

S Sakura and J Terao /Anal

60

peronde at a platmum electrode [lo] and at a carbon electrode [ill, but thus IS the first reported use of ECL to detect hpld hydroperoxrdes Thus method IS expected to show as high a sensltlvlty as CL or blolummescence using photomultlpher (PM) tubes as detectors There are several advantages to usmg ECL compared with CL First, higher sensltlvlty 1s possible as the emlsslon IS concentrated around the electrodes m ECL Second, the emlsslon mechanism IS clearly known because electrolytic oxldatlon IS used Instead of an oxldlzlng reagent or enzyme In conventronal CL, mlcroperoadase [8] and cytochrome c [91are typically used as oxidants to determme hpld hydroperoxtde The mechanism resultmg m CL IS complicated, active oxygen or oxygen radical IS generated from lipid hydroperoxlde m the presence of an oxldlzmg enzyme and this mtermedlate IS then thought to excite lsolummol i-81or lummol [9] In these instances, lipid hydroperoxIde reacts as an active species In contrast, using ECL, the emlsslon mechanism can be controlled

Yothyl

linolomto

hydroporoxido

by the choice of applied potential Third, It IS possible to select reaction condltlons so that only lummol IS oxldlzed to the excited state without any oxldatlon of lipid hydroperoxlde Fourth, the emitted light near the small surface area of the electrode can be easily measured, especially m the case of a short lifetime Fifth, another Important merit of ECL IS that the carrier solution 1s of neutral pH, as opposed to solutions m CL [8,9] which need to be basic (e g, sodium borate buffer solution, pH 10) Fmally, m ECL, there IS no waste of oxrdlzmg reagents or enzymes, m conventlonal CL methods the enzymes are dissolved m the carrier solution, which requires large amounts of the expensive enzymes The purpose of this work was to determine whether the proposed method IS suitable for the assay of lipid hydroperoxldes m foods and blologlcal systems Methyl lmoleate hydroperoxlde (MLHP) was adopted as a convenient model of hpld hydroperoxldes from esterlfled lipids such as triacylglycerol

isorors

OOH

-ccma -

MOOOCH,

OOH

WLRP, 9-isomer

Ooasiblo NLW

(WIP)

Chun Acta 262 (1992) 59-65

MLHP, 13-isomer

(I) oxidationproduats l

MLHP (I)

- s _---____--______>

02' +

2H' + R-C-C=C-C-C-R'

(A)

(II)

(B) b ---------------->

R- -CWC-CIC-R' 8

(III,

(C) 0 ________________>

R_ppC_~C_Rv O"

Rg 1 Methyl lmoleate hydroperoxlde oxdene, (D) rachcal

(Iv)

(D)

(MLHP) Isomers and thw

oxldatlon products

(A) MLHP, (B) conjugated tnene, (C)

II1 - H+ lulainol

‘+ PX.1- 6.00

H+

P&Z

+ hv

f hv

Xlf

XlV 0

z!k

T-r

0

N 2s

N N

NE-

-

H’ Xffl

0

N-

-e

C-T

xv

0

N

N

Rg 2 Structural formulae and reactions 1411 = lummol, IV = d~~~quinone, V = endo~r~lde, wtth an oxsdrzed ammo group Processes If --) N and IV -, WI arc electrolyhc ondatsons

WI = dmzaqumone denvattve

S Sakura and .I Terao/Anal

62 EXPERIMENTAL

Potanthl

(V

Chm Acta 262 (1992) 59-65 VS.

8C1)

Cyclic voltammetry (CV,,, electrochemdummescence (ECL) and flow-mjecnon analyss

Measurements were the same as m previous papers [lO,ll], except for the carrier solution, which contained hqmd chromatographlc grade acetonitrile (AN) An Ag/AgCl reference electrode (- 0 044 V vs SCE) was used for the flowinjection system Reagents

MLHP was prepared from autoxldlzed methyl lmoleate (purchased from Tokyo Kasel) [12] After the samples had been autoxldlzed at 37°C for 3 days, pure hydroperoxldes were obtained by two preparative thin-layer chromatographlc steps The pure sample obtained m this way was dissolved m ethanol and stored m a freezer The hpld hydroperoxlde produced by this method [12] contamed equal amounts of 9- and 13-isomers (Fig 1) The superoxlde dlsmutase was from bovine erythrocytes (Sigma)

Rg 3 CV of 3 80 mM lummol at a glassy carbon electrode m 0 1 M phosphate buffer (pH 7 4) and acetomtrde solutions Scan rate 20 mV s-l Acetomtrde content A, O%, B, lo%, C, 30%, D, 50%

RESULTS AND DISCUSSION

Lumuwl

In order to determine the optimum applied potential for the ECL study, CV measurements of lummol and MLHP were made at a glassy carbon electrode The chemical and electrochemlcal reactions of lummol and rts products are shown m Fig 2, together with the numbermg scheme for these compounds [ill Lummol (I-III 3-ammophthalhydrazlde or 5-ammo-2,3-dlhydrophthalazme-1,4-dlone) 1s a monoanion (II) m neutral solution with the two dlssoclatlon constants (pKal = 600, p& = 13 00) [13l It 1s known to be oxldlzed by one electron to a dutzaqumone (IV) with the loss of one proton, and then further oxldlzed, by hydrogen peroxide, to an endoperoxlde (V) or a hydroperoxlde, which rapidly decomposes to 3-ammophthahc acid (VII), emitting light [131 Figure 3 shows the oxldatlon behavior of lumlno1 at a glassy carbon electrode with concentratlons of 0, lo,30 and 50% AN m a pH 7 4 buffer A 70% AN solution caused salt precipitation

Similar lummol electrochenucal phenomena were observed m the absence of AN [ll] It was concluded that m the presence of 50% AN (scan rate 20 mV s-l), the two oxldatron peak potentials (0 45 and 059 V) were due to the oxldatlon process of lummol to dlazaqumone (Ii + IV m Fig 2) [14,15] The first wave IS due to adsorptive oxldatlon, as the followmg experunental results showed On repeated scannmg, the second wave height gradually decreased and became a shoulder The height of the first wave increased hnearly with change m voltage scan rate When the lummol concentration was as low as 0 013 mM, only the first wave was observed and a typlcal reduction peak was observed These phenomena coincide with known adsorption theory [161 With increased percentage of AN m the solutlon, the oxldatlon current increased because of decreased vlscos~ty m the solution, the vlscoslty of AN (0 375 CP at 15°C) 1s about one thud of that of water [171 Lummol has an aromatic amme function, which was oxldlzed at 1 14 V to produce VIII This

S Sakura and J Terao/Anal Chrm Acta 262 (1992) 59-65

aromatic am1ne 1s well known to be oxldlzed at ca 10 V following by protonatlon, depending on the solvent [181,as shown 1n the process IV + WI 1n Rg 2 There are a multiplicity of possible reaction paths to the diverse and complex final products (VIII XII-XV) by one electron transfer and/or two electron transfer This electrochemlcal reaction was totally n-reversible, so the numbers of electrons and protons involved 1n the oxldatlon process IV + VIII 1n F1g 2 were not exactly determined m these expenments MLHP The CV of MLHP was done at a glassy carbon electrode The oxtdatlon peak potential of MLHP m pH 7 4 buffer-50% AN solution was farly positwe (1 10 V), as shown 1n F1g 4a MLHP was electrolytically oxldlzed and the resulting oxldat1on products were studied Using a 3 78 mM MLHP solution, the MLHP was oxldrzed by a large carbon electrode (3 cm x 8 mm o d ) held at 13 V for 1 h Occasional CV scans (Fig 4a, l-4) show a decreased current as the MLHP was oxldlzed No MLHP could be detected after 60 mm of oxldatlon Figure 4b shows the UV spectral change at each oxldatlon stage 1n Fig 4a The possible oxldatlon products of MLHP are shown m F1g 1 MLHP (A) can be converted by oxldatlon to a coqugated triene (B) [and superoxlde and proton, process (a) 1n Ag 11 or an oxodlene CC>[process (b)] or MLHP radical (D) [process (cl1 The absorption at 232 nm 1s prmanly due to the MLHP functional group, MLHP (A), and MLHP radical (D) (hydroxydlene, the reduced form of MLHP, also has an absorption at 232 nm, but 1t cannot be present 1n this solution) The absorption at 275 nm can be due to oxodlene (C) or conlugated tnene (B) [19] With consideration of the spectral changes 1n Rg 4b, colorimetnc analysis and use of enzyme reactions, the MLHP oxldatlon process 1s concluded to be process (a) as follows If there were generation of MLHP radical (D) as an oxldatlon product, the absorption at 232 nm should remain the same, increase or decrease depending on the molecular absorption coefficient difference between MLHP and its radical wzthout an increase 1n absorption at 275 nm This 1s not the case As

(a)

I

1

I(b 1

r~voloagth

(nm)

Fig 4 (a) CV of 3 78 mM MLHP at a glassy carbon electrode m phosphate buffer (pH 7 4)-50% AN solution and results of oxldatlon at 13 V wth a large carbon electrode Oxldatlon tnne (1) 0, (2) 20, (3) 40, (4) 60 mm, (5) residual current Scan rate 20 mV s-l (b) W absorption change after oxldatlon of 3 78 mM MLJ-IP at 13 V with a large carbon electrode m phosphate buffer (pH 7 4)-50% AN solution Oxldatlon time (1) 0, (2) 20, (3) 40, (4) 60 mm

can be seen m Fig 4b, the absorption at 232 nm decreased while the absorption at 275 nm 1ncreased The decrease m absorption at 232 nm is caused by the omdatlon of MLHP without production of the radical The increase 1n the absorption at 275 nm 1s due to the oxldatlon product, which rmght be an oxodlene (C) [process (b)] or conJugated triene (B) [process (a)] (the generation reaction rate was 0 99 mm-‘, calculated by the absorption change at 275 nm) 2,4-D1mtrophenylhydrazme (DNPH) was added to an

Sakura and J Terao /Anal

64

ahquot to determine if oxodlene was present or not The reactlon of DNPH wth a carbonyl group results m an msoluble red preclpltate of 2,4-dlmtrophenylhydrazone [20] Hexanol was used as a reference sample with a carbonyl group There was no trace of carbonyl compound m the oxldanon product of MLHP under these experimental conditions The other possible product which gives an absorption maxtmum at 275 nm 1s the conJugated triene If a conlugated triene is present, there must also be a superoxlde (and proton) as shown m Fig 1 The existence of superoxlde was observed by studymg the effect of a superoxlde dlsmutase (SOD) enzyme reaction on the ECL intensity, as mentioned later These experiments showed that the oxldatlon products of MLHP are conjugated trrene, superoxlde and protons Electrochemdummescence (ECL)

Consldermg the oxldatlon potentials of lumlno1 and hpld hydroperoxtde, there 1s a cholcc of the applied potential for the ECL experiment as follows When the applied potential to oxldlze lumrnol 1sbetween 0 50 and 10 V, only lummol IS

t

I

I

Potential

-.

04

08

12

(V vs.

,

0

BCE)

Fig 5 Effect of apphed potential on ECL mtenslty at a carbon electrode 17 FM lummol m phosphate buffer (pH 7 40)-30% AN solution, flow-rate 0 5 ml mn-‘, 1 nmol MLHP

.

Chm Acta 262 (1992) 59-65

6

I

12 .

.

8.

-*W-L.

95

___C

__._

__C)

4.

.

I

0,

.

-0

00

I 200

Amount8 of auperoxide ensymo

300 dimutarro

(onryB0 unite)

Fig 6 Effect of superoxlde dlsmutase on ECL 17 PM lumlno1 m phosphate buffer (pH 7 4)-30% AN solutlon, flow-rate 05 m1In1n-I, 1 nmol MLHP (A) ECL at apphed potential of 0 7 V and (B) ECL at 12 V at a carbon electrode

oxtdlzed to dlazaqumone, which reacts with free hpld hydroperoxlde m the solution In this mstance, emlsslon 1s due to the simple fluorophore When the applied potential IS more positive than 10 V, both the ammo group of lummol and hpld hydroperoxlde are oxldlzed Electrochemdumlnescence occurs by the reactlon of dlazaqumone with an oxldlzed ammo group (VIII m Fig 2) and the oxldlzed product of hpld hydroperoxlde, which 1s a more complicated emlsslon Figure 5 shows the relatlonshlp between the applied potential and the ECL mtenslty in neutral phosphate buffer-30% AN solution It 1s seen that at potentials less than 0 4 V lummescence could not be observed, whereas at more positwe potentials the lummescence intensity mcreased steeply At apphed potentials more pontwe than 10 V, where oxldatlon reactlons of both the ammo group of dlazaqumone and MLHP occur, the luminescent mtermedlates are more complicated than that at 0 7 V, where only dlaza-

S Sakura and J Terao/Anal

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Chum Acta 262 (1992) 59-65

qumone and MLHP react Electrochenulummescence spectra at 0 7 V showed that the maxLmum emlsslon wavelength was 440 nm The lifetime was very short, with a decay time of 0 38 s When the apphed potential 1s 12 V, superoxlde 1s produced on the electrode As shown m Fig 6, the SOD suppressed the ECL intensity, because it decomposed superoxlde to hydrogen peroxide Therefore, the intensity of the ECL at 12 V with a high concentration of SOD decreased to the value of that at 0 7 V, where the ECL was due only to the reaction of dlazaqumone and lipid hydroperoxlde This ECL method was used for the detection of hpld hydroperoxlde The optnnum flow-rate (0 3-O 5 ml mm-‘) and the optimum lummol concentration (lo-20 PM), were the same as with hydrogen peroxide [lO,ll] The optnnum pH of the flow solution with the maximum lummescence intensity was found to be 7-9 Below pH 6 and above pH 10 there was little or no lummescence This experunent was repeated several times with fresh solutions and the same results were obtained each time The detection lmut at 0 7 V was 0 3 nmol with a relative standard deviation of 13% (n = 5) at a signal-to-noise ratio of 15 Concluswn It 1s possible to use ECL at a glassy carbon electrode for the detectlon of lipid hydroperoxides The great advantage of ECL 1s that it can be used m a neutral aqueous solution, as opposed to conventional CL, which requires a very basic lummol solution Because this detectlon method can be used m neutral aqueous solution (amllar to blologlcal fluids), it could be connected to an mnnobdrzed enzyme column The sensltrvlty of this technique 1s high because the emlsslon hfetime 1s short and the photons are generated on a small surface area of electrode The applied potential IS low enough to oxldlze lummol wlthout oxldlzmg the hpld hydroperoxlde, which prevents oxldatlon interference as much as possible The authors gratefully acknowledge the help of Mr H Tsuruta of Inca, Kyoto, for supplymg

the flow mstrumentatlon and useful dlscusslons They also gratefully acknowledge the help of Dr Karen Hassett of Meredith College, NC, for helpful dlscusslons Part of this paper was presented at the 12th Annual Japanese Lipid Hydroperoxlde and Free Radical Conference, Kyoto, m November 1988

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1 HI

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3 4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19

20

Rao and R 0 Recknagel, Exp Mol Path01 ,9 (1968) 271 N Ohlshl, H Ohkawa, A Mnke, T Tatano and K. Yagl, Bmchem Int , 10 (1985) 205 R Cathcart, E Schwters and B N Ames, Anal Blocbern, 134 (1983) 111 K IClkugawa,T Nakahara, Y Tamguchi and M Tanaka, hplds, 20 (1985) 475 K Yamada, J Terao and S Matsushita, LIpIds, 22 (1987) 125 J Terao, S S Shlbata and S Matsushita, Anal Buxhem, 169 (1988) 415 Y Yamamoto, M H Brodsky, I C Baker and B N Ames, Anal Bmchem, 160 (1987) 7 T Mlyazawa, K Yasuda and K Fujrmoto, Anal Lett 20 (1987) 915 S Sakura and H Imar, Anal SCI ,4 (1988) 9 S Sakura, Anal Chum Acta, 262 (1992) 49 J Terao and S Matsushita, J Am 011 Chem Sot, 54 (1977) 234 I Kamlya, Kagaku Hakkou, Kodan-sha, Tokyo, 1972, p 55 (m Japanese) P B Shevhn and HA Newfeld, .I Org Chem , 35 (1970) 2178 M M Rauhut, AM Semsel and B G Roberts, J Org Chem ,31 (1966) 2431 A J Bard and L R Faulkner, Electrochemical Methods, Fundamentals and Apphcatlons, Wdey, New York, 1980, p 527 J A RIddIck and W B Bunger, Organic Solvents, WileyInterscIence, New York, 1970, p 399 S D Ross, M Fmkelstem and E J Rudd, Anod~c Oxldation, Academr, New York, 1975, pp 189-192 K FuJmOtO, m T Kaneda and N Ueta (Eds ), Kasannka Shlshrtsu Jlkken Hou, Ishlyaku, Tokyo, 1983, pp 36-39 (in Japanese) E B Sanders and J Schubert, Anal Chem ,43 (1971) 59