FreeRadical Biology& Medicine, Vol. 12, pp. 43-52, 1992 Printed in the USA.All rightsreserved.

0891-5849/92 $5.00 + .00 Copyright© 1992PergamonPressplc

Original Contribution ENHANCEMENT

OF CHEMILUMINESCENCE ASSOCIATED PEROXIDATION BY RHODAMINE DYES

WITH

LIPID

YURIY A. VLADIMIROV,* TOKTOSLrN B. ATANAYEV,* and MICHAEL P. SHERSTNEVJf *Department of Biophysics, 2nd Moscow Medical Institute, U.S.S.R.; tInstitute for Physico-Chemical Medicine, Moscow, U.S.S.R.

(Received 25 April 1991; Revised 17 September 1991;Accepted 20 September !991)

Abstract--Rhodamine Zh in concentrations of 300-500 ~mole/l enhances FeZ+-inducedchemiluminescence (CL) in blood serum, liposome and lipoprotein suspensions by two orders of magnitude. Several different rhodamines were compared, chemiluminescence spectra were measured and relationships between dye concentration, medium composition and CL intensity were studied. Keywords--Chemiluminescence, Lipid peroxidation, Rhodamines, Free radicals

sociated with definite free radical reactions. In a large survey by Cilento '2 and subsequent reviews, the proper use of sensitizers in chemiluminescence systems has been considered. In particular, the use of sensitizers is the information they can give--provided the photochemistry of the sensitizer molecule is known--on the electronically excited states involved in the system. 13 Luminol is one of the compounds that is widely used to enhance the chemiluminescence accompanying the appearance of oxygen and lipid radicals in biochemical systems. In the presence of oxygen radicals • OH and 02- this luminophore undergoes chemical transformations accompanied by chemiluminescence, the reaction rate being virtually limited by the concentration of "OH radicals. 14'15 Unfortunately, CL of luminol is brought about not only by oxygen radicals but by many other oxidants, including C10-, I2, MnO4-, NO2 (refs. 16 & 17). Lucigenin was found to be a more selective CL enhancer insensitive to hypochlorite and hydroxyl radicals and producing intensive CL in the presence of superoxide radical 02(refs. 18-20). In previous papers, we described several compounds that enhanced CL associated with LPO. Luminol, eosin, dibromantracen, and Tb 3÷ ions enhanced CL in a suspension of liposomes subjected to

INTRODUCrlON

Measurement of CL is widely used in studies on free radical reactions occurring in cells or cellular organcUe suspensions) -5 A major source for formation of excited molecules in such systems are reactions of free radicals of unsaturated fatty acids (probably, peroxyl radicals LO2" [ref. 6]) and reactions involving active oxygen species, i.e. H202, singlet oxygen and oxygen radicals 02- and "OH (refs. 7 & 8). Unfortunately, when measuring the non-enhanced (intrinsic) CL in solutions and in cell and organelle suspensions, it is difficult to make conclusions concerning the nature of free radical reactions in the system and of the reaction rates because of low quantum yields of CL directly produced by both oxygen radical reactions in aqueous solutions and LPO reactions in biomembranes and, hence, strong effects of some compounds "occasionally" presented, for instance, such as carbonates s,9 and LPO products. 10,11 One way to make the CL method more specific for investigation of different free radical reactions in biological systems may be usage of luminescent compounds that selectively enhance the light emission asAddress correspondence to Yuriy A. Vladimirov, 2nd Moscow Medical Institute (M. Pirogovskaya la, Moscow 119828, U.S.S.R.). 43

Y.A. VLADIMIROVet al.

44

2O

x ~11,~

d~

11

It

h

50

It

4O

15

z o 1-o 10 -r"

r-

3o ~ 20 ~

CS3

).-

o -

/ \R7

5

10

RB Fig. 1. Rhodaminestructural formulae. For the substituentsR~-Rs see Table 2.

peroxidation catalyzed by iron ions. 21-24 It remains unclear whether this enhanced CL was due to lipid radicals or oxygen radicals which also may be created in the course of LPO reaction. The latter possibility was evidenced by the fact that CL was inhibited by mannitol. On the other hand, it was shown that CL produced during linoleic acid oxidation by lipoxygenase was virtually not enhanced by luminol. 2s Another fluorophore, eosin, was found to affect the kinetics of LPO process ] 1.2sand thus may not be recommended as a compound suitable to enhance luminescence intensity without interfering with the reaction course. Terbium and dibromantracen were able to enhance

6

T

* RZh

0

50

100

150

[RZh]. ,uM Fig. 3. Effect of rhodamine Zh concentration on parameters ofliposome CL flash. CL intensity (left) and the time of inductive period (right) are indicated on the ordinata, h, H, T, parameters of CL (See text). The final rhodamine Zh concentration (pM) is indicated on the abscissa. In the cuvette there were 5 ml of a buffer solution containing liposomes at a final concentration of 130 pM.

CL intensity but no more than by one order of magnitude. 23The complex of Eu 3+ ions with antibiotic tetracycline (a Eu:T ratio of 3:4 mol/mol) was found to be the most promising. 2t'22'24'25 In the presence of Eu-T, CL intensity in the suspension of liposomes peroxidized in the presence of Fe ions increased by three orders of magnitude, and in the linoleic acid-lipoxygenase system by two orders of magnitude. 25Unfortunately, the Eu-T complex is not stable and dissociates in the presence of competing anions, in particular, phosphates. This does not make the complex very useful in biochemical systems, such as cell organelles, homogenates, blood plasma, etc. In the present work, it was found that a dye rhodamine Zh enhances CL in blood serum, liposome and lipoprotein suspensions by two orders of magnitude. The capacities of several other rhodamines to enhance CL were compared, the spectra of initial and enhanced CL were measured, and relationships between the dye concentration, medium composition, and CL intensity were studied. MATERIALS AND METHODS

Chemica& -'Pt 0 i

i

i

i

i

i

t

0

B

12

18

24

30

36

TI ME, r a i n Fig. 2. Kinetics ofrhodamine Zh-aetivated ]iposome CL in the presence of Fe 2+ ions. +Pi = the phosphate concentration in the cuvette

was 200 #M; -Pi without phosphate; +RZh, - R Z h with rhodamine Zh in a concentration of 20 pM and without it.

The following chemicals were used: Tris-buffer (hydroxymethyl)aminomethane and human serum albumin (Sigma, USA); K O H (Chemapol, Czechoslovakia); xanthine and xanthine oxidase (Serva, Germany); rhodamine B, rhodamine G and rhodamine F3B (BASF, France); sulforhodamine B (Hoehst, Germany). The Soviet analogue of rhodamines rhodamine Zh (shown in Fig. 1) was also used. Other chemicals of reagent grade were Soviet.

Chemiluminescence and lipid peroxidation

45

Table I. Effects of Rhodamine Zh on the Amplitudes of Chem/Luminescence and Accumulation of Malondialdehyde CL intensity, 105 photons/s.4~Components Liposomes Liposomes + Fe2+ Liposomes + RZh Liposomes + Fe2+ + RZh

AMDA, ttM

h

H

43 _+ 15 112 + 11"* 20 + l 1 113 _+ 16"*

-19.0 + 4.5 -47.7 _+ 11.5"

-22.0 ___3.3 -54.0 _+5.5*

Note: *p < 0.05 as compared to the system Liposomes + Fe2+; **p < 0.05 as compared to the corresponding system without Fe2+.

Liposomes C r u d e p h o s p h o l i p i d fraction was e x t r a c t e d f r o m egg y o l k b y t h e r o u t i n e m e t h o d s 26 w i t h s u b s e q u e n t removal of neutral lipids and pigments by cooled water-free acetone. L i p o s o m e s were f o r m e d b y t h e f r e e z e - t h a w i n g m e t h o d . 27 2.0 m l o f 0.01 M Tris-HC1 buffer, p H 7.4, was a d d e d to 6.0 m l o f a c h l o r o f o r m s o l u t i o n o f p h o s p h o l i p i d s c o n t a i n i n g 6.7 m g o f p h o s p h o l i p i d s / m l . T h e s a m p l e was p l a c e d i n t o a w a t e r therm o s t a t at 3 7 ° C a n d c h l o r o f o r m p h a s e was e v a p o r a t e d u n d e r n i t r o g e n flow. T h e o b t a i n e d s u s p e n s i o n was t h e n d i l u t e d w i t h t h e s a m e T r i s - H C l buffer to a final p h o s p h o l i p i d c o n c e n t r a t i o n o f 20 m g / m l .

v a l e n t iron. 26 Egg y o l k was m i x e d w i t h a n e q u a l volu m e o f 0.85% NaC1 s o l u t i o n . T h i s s t o c k s u s p e n s i o n was s t o r e d at 4 ° C for n o m o r e t h a n five days. O n t h e d a y o f the e x p e r i m e n t 1.0 m l o f t h e s u s p e n s i o n was d i s s o l v e d in distilled w a t e r (1:50, v/v). P h o s p h o l i p i d c o n c e n t r a t i o n was d e t e r m i n e d after c r u d e p h o s p h o l i p i d f r a c t i o n h a d b e e n e x t r a c t e d f r o m egg y o l k s u s p e n sion b y the r o u t i n e m e t h o d . 26

Measurements of Absolute CL Intensity C L was m e a s u r e d w i t h a I F H M V - 1 c h e m i l u m i n o m e t e r (Soviet). z9 N e c e s s a r y t e m p e r a t u r e was m a i n tained automatically by an electronic thermostat. CL

Lipoproteins

5

Egg y o l k l i p o p r o t e i n s were u s e d as a s t a n d a r d l i p i d c o n t a i n i n g s y s t e m easily o x i d i z a b l e o n a d d i t i o n o f d i -

IFe2+

5 + RZh

4

~ 3 ~"

h T

Z O I---

o -r

40

a.

2

10 1

N

0

0 |

i

0

6

i

12

i

i

18 24 TIME, min

!

30

Fig. 4. Effect of rhodamine Zh on egg yolk lipoprotein CL in the presence of Fe2+ ions. Figures at the curves designate the final activator concentrations 0zM). The experiment was performed in TrisHCI buffer.

--

0

6

12 TI ME,

18

2/.

m[ n

Fig. 5. Kinetics of rhodamine Zh-activated CL of egg yolk lipoproteins in the presence of Fe2+ions. The experiment was carried out in phosphate buffer. +RZh, -RZh with and without 20 gM rbedamine Zh.

46

Y.A. VLADIMIROVet al. 24

h

~

2O

]

(

f

X

6.=

"~16 ul z o P-12 o -r

E u~ ~E

DL

tO 0

ml, and the mixture was incubated at 37°C for 2 min with continuous stirring during which the background CL was recorded. To initiate CL, 0.5 ml of FeC12 o r FeSO4 was added to create a final Fe 2+ concentration of 100 uM. To investigate the enhanced CL 0.1 ml of rhodamine Zh of a certain concentration had been added to the liposome suspension prior to Fe 2+ addition.

8 Y

_

/0 '''0

0

"

CL of Egg- Yolk Lipoproteins

Hs o

0

0 I

i

i

0

20

40

i

60

i

80

|

100

[0gift. pM Fig. 6. Effect of rhodamine Zh concentration on the parameters of egg yolk lipoprotein CL flash. All indications are the same as in the legend to Fig. 3.

20 mM phosphate buffer, pH 7.7, was added to lipoprotein suspension (0.25 mg of lipid) to a final volume of 5 ml. To initiate CL, 0.5 ml ofa Fe 2+ salt at a final concentration of 5 mM was added. To study the enhanced CL, rhodamine Zh (0.1 ml) at a final concentration of 20 #M had been added to the eggyolk suspension prior to Fe2+ addition.

CL of Blood Serum intensity was expressed in photons/s-47r (Icl). To measure the chemiluminometer's sensitivity, we used a secondary ethalon SFHM-1 (Soviet) which represents a radioluminescent light source made of uraniumm glass ZhS-19 (Soviet) possessing light emission band between 500 and 550 nm and known absolute intensity of emitted photons Ie, photons/s. 4~r (ref. 30). The SFHM-1 ethalon was calibrated by means of a standard Hastings-Weber radioluminescent source. 31 The sample's absolute CL intensity was calculated by the equation ICL = (IJI~th) Ic, where I, is the absolute intensity of the ethalon emission equal in our case to 8.58 × 105 photons/s- 47r; Icth is the ethalon's CL intensity measured by a luminometer and expressed in arbitrary units; Ic is CL intensity of the suspension measured at the same instrument sensitivity and expressed in the same units as Ieth.

CL of Liposomes Tris-HC1 buffer at a concentration of 10 mM was added to 0.5 ml of liposome suspension placed in the cuvette of a chemiluminometer to a final volume of 5

20 #L of blood serum and 100 #L of I mM rhodamine Zh were added to 7 ml of potassium phosphate buffer (50 raM, pH 7.4) placed in the cuvette of the chemiluminimeter. To initiate the reaction, 0.5 ml of a Fe z+ salt was added to a final Fe 2+ concentration of 2.5 mM.

CL in Xanthine-Xanthine Oxidase System 0.5 ml ofxanthine (1 mM) was added to 0.05 ml of xanthine oxidase (1 unit/mg of protein). Phosphate buffer was added to a final volume of 5 ml; 0.1 ml of rhodamine Zh (20 #M), or luminol (20 uM) were added to study the enhanced CL. In some experiments 0.1 ml of superoxide dismutase (SOD) at a final concentration of 15,000 units was added to investigate the mechanism of the reactions responsible for light emission. Rhodamine absorbance spectra were measured on a Beckman model DU-7HS spectrophotometer (Germany) in the wave-length region between 400 and 600 nm. Rhodamine solutions in water and ethanol were

Table 2. Subsfituen~ in the Stru~ure ofDifferent Rhodamines Rhodamines

R1

R2

Ra

R4

R5

R~

R~

R8

Zh 6G F3B B G SR

C2H s C2H 5 C2H 5 C2H 5 C2H5 C2H 5

H H C2H 5 C2H 5 H C2H 5

H H C2H 5 C2H 5 C2H5 C2H 5

C2H s C2Hs C2H 5 C2H 5 C2H5 C2H 5

H CH3 H H H H

H CH3 H H H H

OC2Hs OC2Hs OC2H 5 OH OH OC2H 5

H H H H H SO 3

Chemiluminescenceand lipid peroxidation 6

47

6035

•

Rzh

II ~

R

F

3

RB, RG SR , 3

, 9

6

!

B

~ 0

i

, 12

530

550

570 590 WAVELENGTH, nm

610

Fig. 9. Rhodamine fluorescencespectra. Excitation wavelength is 525 nm. All rhodaminesweredissolvedin concentrationsofapproximately 1 ~M so that the absorption at 525 nm would be equal in all samples. Abbreviationsare the same as in Table 2.

J 15

TI ME. rain Fig. 7. Kinetics of egg yolk lipoprotein CL activated by various rhodamines in the presence of Fe2÷ ions. The structures of rhodamines are given in Fig. 1 and in Table 2.

used at concentrations o f 1.0 #M. Distilled water and ethanol were used as controls• Rhodamine fluorescence spectra in aqueous or

1,5 RZh _ •

i

1.2

RESULTS

Amplification of CL Associated With Peroxidation in Liposomes

RF3___B

0,9

SR RG

0.6

0,3[

0,0

440

ethanol solutions were measured on a Hitachi model MPF-4 spectrofluorimeter (Japan) in the wave-length region between 530 and 610 n m at an excitation wave length o f 525 nm. LPO products in the samples were determined as the malondialdehyde (MDA) concentration by the thiobarbituric acid test. 32 The MDA molar extinction coefficient at 535 nm, p H 0.9, was assumed to be 1.56. l0 s M - t c m -I. The expressed values are the mean _+ SEM, the linear correlation coefficients were calculated by the range method.

480

,

520

561

WAVELENGTH,

6O0

nm

Fig• 8. Rhodamine absorption spectra. The structures of rhodamines are given in Fig. 1 and in Table 2.

Figure 2 shows the kinetics o f liposome CL in the absence ( - R Z h ) and in the presence (+RZh) o f 20 # M rhodamine Zh. Liposome suspension was incubated in 20 m M Tris-HCl, phosphate-free (-Pi), or in the presence of 0.2 m M orthophosphate (+Pi). LPO in the suspension was initiated by addition o f divalent iron at a concentration o f 0.1 m M in the m o m e n t 0. Non-enhanced liposomal CL ( - P i , - R Z h ) is characterized by the stages identical to those described earlier for mitochondria L4 and liposomes~'24: rapid luminescence flash after iron addition accounted for by the Fe 2+ reaction with H202and lipid hydroperoxides; latent period; slow flash (the time interval T in Fig. 2 represents the time to the slow flash maximum); and, finally, slowly developing "stationary lumines-

48

Y.A. Vt.ADIMIROVet al.

cence."~ Phosphate, enhancing iron autooxidation, reduces the latent period (Fig. 2). In the presence of rhodamine Zh we observed a sharp increase in the CL slow flash amplitude and a moderate increase of stationary luminescence (Cf. - R Z h and +RZh, Pi in Fig. 2). In the presence of phosphate the slow flash of non-enhanced CL was not pronounced being overlapped by stationary luminescence. On addition of rhodamine Zh, however, this flash increased sharply and became well pronounced. Stationary luminescence was also enhanced in the presence of rhodamine Zh though to a smaller extent (Cf. - R Z h and +RZh, +Pi in Fig. 2). The effect of rhodamine Zh on liposomal CL at the stages of rapid flash (h), slow flash (H), and stationary luminescence (Hs) is also shown in Fig. 3 where CL intensities at these stages are plotted as functions of the dye concentration. It is seen that the CL intensities are growing with the rhodamine concentrations up to 50-70 #M of the dye and then saturation occurs. Separate experiments have demonstrated that the rhodamine concentration at which saturation takes place depends on the liposome concentration approximately linearly (the data are not shown). The limit of the dye/phospholipid ratio above which the dye does not further enhance CL corresponds to 1 mole ofrhodamine per 40 moles of phospholipids. The maximal CL enhancement was higher for the rapid and slow flashes of CL (36- and 37-fold, respectively) and lower at the stage of stationary luminescence (12-fold). The latter can be accounted for by the fact that in the process of LPO accumulation of endogenic compounds enhancing CL may occur ~°,~ which results in augmentation of quantum yield of non-enhanced CL. It is noteworthy that CL enhancement in the presence ofrhodamine does not change the kinetics of the process and, in particular, does not change considerably the latent period (see Tin Fig. 3). A small reduction of the latent period occurs only at low dye concentrations when the extent of CL enhancement is not high. This can be accounted for by the fact that the dye molecules are charged positively and may change the surface charge of the liposomes. As it was previously shown, E u 3+ and Tb 3+ ions also reduce the latent period of liposome CL. 22'23 The fact that rhodamine Zh does not influence significantly the CL kinetics implies that the dye enhances the quantum yield of CL without participating in lipid free-radical oxidation reactions. This conclusion is supported by measurements of an LPO product MDA. It can be seen from Table 1 that MDA is accumulated during incubation of liposomes with ferrous ions; but the difference in the MDA contents in liposomes incubated with or without the dye is neg-

ligible, while the CL intensity (h and H) is enhanced by a factor of 2.5 with the dye. Unfortunately, it was difficult to use higher concentrations of the dye and, hence, more considerable enhancement of CL since the dye interfered with the spectrophotometric determination of MDA.

CL of Egg Yolk Lipoproteins The effect of rhodamine Zh on the CL intensity of egg yolk lipoproteins suspended in Tris-HC1 buffer is illustrated in Fig. 4. As in liposomes, small dye concentrations do not reduce considerably the latent period in development of luminescence. At the same time, rhodamine Zh brings about a great enhancement of luminescence at the CL slow flash stage and smaller enhancement at the stationary luminescence stage. The kinetics of lipoprotein non-enhanced CL in phosphate buffer differ considerably from those in Tris-HCl buffer: the slow flash is practically absent while powerful stationary luminescence is observed growing with time (Fig. 5, -RZh). Similar kinetics were observed previously in blood lipoproteins. 33,36 Apparently, the ferrophosphate suspension is formed after addition o f a Fe 2+ salt to phosphate, so that Fe z+ ion concentration in the solution and, hence, the LPO rate are kept temporary on a relatively constant level. The observed increase in CL intensity can be accounted for by the accumulation of luminescence endogenic sensitizers. 10.1~As in previous cases, a moderate enhancement of stationary luminescence occurs in the presence of rhodamine Zh, but along with that there arises a rather sharp CL burst (slow flash of CL) (Fig. 5, +RZh). This means that rhodamine Zh enhances CL intensity in this system, as well as in liposomes and egg yolk lipoproteins in Tris-HC1 buffer mainly at the stage of CL slow flash (i.e., when iron ions dissolved in the water phase are interacting with lipid hydroperoxides thus increasing the rate of LPO chain reactions). Figure 6 demonstrates the effect of rhodamine Zh concentration on the rapid and slow luminescence flash, on stationary luminescence and time-to-peak of slow flash (T). Again, it can be seen that the dye brings about only negligible enhancement of the stationary luminescence amplitude (no more than two or threefold), but it greatly enhances CL intensity at the stages of the fast and slow flashes. Therefore, the effects of rhodamine Zh on liposome and egg yolk lipoprotein CL in different buffer solutions are alike: The dye enhances the intensities of both the slow and fast flashes of CL by dozens of times, increases stationary luminescence intensity sev-

Chemiluminescence and lipid peroxidation

It is interesting to compare these results with the absorbance and fluorescence spectra of these compounds (Fig. 8 and Fig. 9, respectively). The ability to enhance CL showed those compounds which have short wave-length absorption (at 525 nm) and emission maxima (at 556 nm). These compounds have, at the same time, high fluorescence quantum yields (as it can be seen from the areas below the curves in Fig. 9, as far as the light absorbtion at the excitation wave length was equal in all samples). The reason why different substituents in a rhodamine molecule affect the dye's ability to enhance CL still remains unrevealed.

1.0 --,

L

/ /

Z Lt.I

tO

// / / / /

,,T

0.5

49

/ /

._1

5" LU .-¢-

1.0 - - ~ - - - - - ~

8

CL Spectrum

I(J Z UJ CJ

~0.5 Z

,_,1 LIJ -r (J

350

400

450

500

550

600

WAVELENOTH, nm

Fig. 10. Spectra of the intrinsic (stationary--top, unshaded) and rhodamine Zh-activated (stationary--top, shaded; slow flash--bottom, stepped) CL of egg yolk lipoproteins in the presence of Fe z+ ions. The fluorescence spectrum of rhodamine Zh in water is presented above by a smooth line. The cuvette contained 5 ml of phosphate buffer with lipoproteins at a final phospholipid concentration of 130 mcM, 20 ~M rhodamine Zh, and 5 mM Fe 2+.

The fact that the ability to amplify CL is a property only of those rhodamines that have high quantum yield of photoluminescenee (fluorescence) is in agreement with the hypothesis that CL enhancement may be a result of formation of the singlet excited state of dye molecules in the course of an LPO reaction. To test this hypothesis, we compared rhodamine Zh fluorescence spectra with the CL spectrum in the reaction of egg yolk lipoprotein peroxidation in the presence of the dye. The latter was estimated by means of a series of light filters with sharp short wave-length light transmittance limit ("cut-off'' light filters); the method was described by Yu. A. Vladimirov and A. Ya. Pota-

eral times as much, and does not much affect the latent period in luminescence development. 30

Comparison of Various Rhodamine Derivatives We comparedthe ability of six different rhodamines to enhance CL of egg yolk lipoproteins. The chemical structures of these compounds are presented in Fig. 1. Table 2 shows the structures of radicals R~Rs in Fig. 1 for different rhodamines. Typical curves of egg yolk CL in the presence of various rhodamines are shown in Fig. 7. It can be seen that three compounds, rhodamines B and G and sulforhodamine do not at all enhance CL (RB, RG, and SR in Fig. 7). Rhodamine F3B considerably distorted CL kinetics, probably by reacting with some intermediates. Considerable enhancement of the CL slow flash was observed in the presence of rhodamines Zh and 6G (RZh and R6G in Fig. 7).

tO

80

0 r-

CL

2•

\

150

oi TIME Fig. 11. Effect ofionol on rhodamine Zh-activated egg yolk lipoprorein CL in the presence of Fe 2+. Figures at the curves designate the final ionol concentrations 0zM). Ingredients in the cuvette were identical to those indicated in Fig. 10.

50

Y.A. VLADIMIROVet al. 6

"

4

i/I

V') Z 0 I-0 '1" &.

~

75

A

0.75

o

TI ME, rain Fig. 12. Effect of ethanol on rhodamine Zh-activated egg yolk lipoprotein CL in the presence of Fe 2÷ ions. Figures at the curves designate the final ethanol concentrations (.M). Ingredients in the cuvette were similar to those indicated in Fig. 10.

man serum albumin at the same concentrations. These effects are probably due to interaction of the dye or some reaction intermediates with apoproteins rather than to catalytic action of the enzyme. It may be concluded, therefore, that no indications have been obtained that CL enhancement by rhodamine Zh may be attributed to oxygen free radicals in aqueous phase. A generally recognized method to detect participation of lipid free radicals in a reaction is application of free radical traps, among which butilated hydroxytoluene (BHT) is most commonly used. 3s The effect of BHT on the kinetics of egg yolk lipoprotein CL is shown in Fig. 11. Three effects of the antioxidant are seen: (a) decrease of CL slow flash, (b) slowing down the flash development rate (T grows as well as the flash width), and (c) decrease of stationary amplitude. All these effects may be accounted for by the interaction of BHT with lipid free radicals. 1,39 Hence, these results may be considered as evidence that rhodamine Zh enhances CL produced in reactions of lipid free radicals in hydrophobic membrane phase. Similar effects on the CL slow flash were exhibited by ethanol, with the exception that this compound did not influence stationary CL (Fig. 12). At present we are unable to give a reasonable explanation of the latter fact.

Blood Serum CL The data obtained are demonstrated in Fig. 10. It is seen that non-enhanced CL has two emission maxima around 480 nm and 560-570 nm. In the presence of rhodamine Zh the short wave-length component disappeared both at the stage of stationary luminescence and slow flash (Cf. Fig. 10, top and bottom), with the longer wave-length emission band being presented both without and with rhodamine. In Fig. 10, below the fluorescence spectrum ofrhodamine Zh is compared with the CL spectrum at the stage of CL slow flash. It can be seen that these spectra are similar. More precise measurements of CL spectra are, however, required to make the final conclusion that light emission at enhanced CL is due to dye molecules in singlet excited states. p e n k o . 37

Measurements of Fe 2÷ induced CL of blood plasma and serum were used earlier for diagnostics of

-4" gl

(,,t) z o bo "1-

Effect of Free Radical Traps Superoxide dismutase in concentrations up to 1 mg/ml did not affect significantly the CL slow flash amplitude in the suspension of egg yolk lipoproteins peroxidized in the presence of rhodamine Zh and ferrous ions (the data are not shown). Catalase inhibited the slow flash of CL only at high concentrations (about 1 mg/ml). A similar effect was shown by hu-

I

!

0

6

|

12 T I ME, rain

i

18

Fig. 13. Rhodamine Zh-activated CL of blood serum of patients with type II hyperlipoproteinemia before (1) and after (2) hemosorption and of a healthy individual (3).

Chemiluminescence and lipid peroxidation

51

Table 3. Alterations of Rhodamine Zh-Activated Blood Serum CL in Patients with Cardiovascular Disease in the Presence of Fez+ Ions (M _+ m) Stationary luminescence, l0 s quantum/s • 4 r

HslH

16

21.5 + 1.4"**

1.41 + 0.07",**

27 10

10.6 + 0.5* 6.2 + 1.6

0.91 + 0.06* 0.55 _+0.08

Groups under investigation Type II hypedipo-proteinemia Joint group of patients with ischemic heart disease and inferior limb arteries atherosclerosis Healthy persons

Note: *p < 0.05 as compared to healthy persons; **p < 0.05 as compared to the joint group of patients.

heart disease, appendicitis, cholecistitis, and pancreatitis. 4° Due to the low intensity of non-enhanced CL, a large amount of blood (0.5-1.0 ml of plasma or serum for each analysis) is necessary to measure nonenhanced CL. Hence, we are forced to take blood from the veins of patients. In the presence of rhodamine Zh CL intensity increases by a factor of 20-30 so that we may now use only 20 #L of plasma or serum taken from a finger for each measurement. A typical chemiluminogram of blood serum in the presence ofrhodamine Zh is presented in Fig. 13. The slow flash of CL and stationary CL are both presented while for non-enhanced CL only stationary CL is characteristic with the slow flash being screened, as The ratio between the amplitudes of stationary CL (Hs) and slow flash CL (H) was found to be different for different patients. The results of preliminary investigations of a group of patients with cardiovascular diseases are shown in Table 3. It is seen from the table that patients with type II hyperlipoproteinemia have the highest values of the above-mentioned parameters. It was also found that both Hs and the Hs/Hratio correlated with low-density lipoprotein levels in blood serum: correlation coefficients were +0.60 and 0.71, respectively. The registration technique ofrhodamine Zh-activated CL can, therefore, be employed in screening tests for hyperlipoproteinemia diagnostics.

6.

7.

8.

9. 10.

11.

12. 13.

14. 15. 16.

REFERENCES

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CL--chemiluminescence LPO--lipid peroxidation MDA--malon dialdehyde BHT--butylated hydroxytoluene SOD--superoxide dismutase

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