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Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction
Biochimica et Biophysica Acta, 746 (1983) 209-215 Elsevier
209
BBA31662
EFFECT OF AUTO-OXIDIZED PHOSPHOLIPIDS ON OXIDATIVE ENZYME ASSAYS BASED ON TETRAZOLIUM SALT REDUCTION EDDY M. MASSA and RICARDO N. FARIAS
Departamento de Bioquimica de la Nutricirn, Instituto Superior de Investigaciones Biol6gicas (INSIBIO), CONICET-UNT, Chacabuco 461, 4.000 San Miguel de Tucumim (Argentina) (Received March 4th, 1983)
Key words: Oxidative enzyme; Phospholipia~" Hydroperoxide; Tetrazolium salt reduction
The influence of auto-oxidized phospholipids on the reduction of the tetrazolium salt MTT coupled to the NAD +-dependent lactate dehydrogenase reaction was studied. The following results were obtained: (1) peroxidized phosphatidylcholine interfered in the time-course of the lactate dehydrogenase-mediated MTT reduction; (2) there was a time-dependent decrease in the hydroperoxide content of phosphatidylcholine vesicles during the incubation; (3) the diminution of phosphatidylcholine hydroperoxides required the presence of all the components of the system except MTT; (4) hydroperoxide diminution and MTT reduction were mediated by the superoxide radical O2-, since both processes were inhibited by superoxide dismutase; (5) EDTA inhibited the hydroperoxide decrease and abolished the interference of peroxidized phosphatidylcboline with MTT reduction. It was concluded that hydroperoxides compete with M T r for the electrons coming from substrate oxidation. The superoxide radical 02- and traces of some contaminating metal ion are involved in the process. This is a potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method.
Introduction The assay of oxidative enzymes such as flavin and NAD+-dependent dehydrogenases is frequently based on the reduction of tetrazolium salts to deeply colored formazans which absorb strongly in the visible region. Thus, the time-course of the enzymatic reaction is continuously monitored by following the correspondent increase in absorbance [1-4]. Authors from different laboratories [5-8] have used this spectrophotometric assay based on the phenazine methosulfate-mediated reduction of the tetrazolium salt MTT when studying the effect of Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMPC, L-a-dimyristoylphospliatidyleholine; PC, L-a-phosphatidylcholine from egg yolk. 0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.
lipids on the activity of purified dehydrogenases. In these studies, activations have been interpreted as arising from lipid-enzyme interactions. However, we have recently shown [9] that chemical and enzyme-mediated reductions of MTT can be enhanced by the addition of a lipidic phase to the aqueous reaction mixture because the formazan derived from MTT reduction is a sparingly water-soluble compound that dissolves well in organic solvents and lipids. Thus, reduced MTT dissolves in the detergent micelles or phospholipid vesicles added to the system, favoring the reoox reaction occurring in the aqueous phase surrounding these micelles or vesicles. In addition, the absorbance of reduced MTT in lipid-like media is higher than in aqueous media [9]. Therefore, increased rate of enzyme-mediated MTT reduction when lipids are added to the incubation mixture
210 may arise from lipid-enzyme interactions a n d / o r from the direct influence of lipids on tetrazolium salt reduction itself. This points out a potential pitfall in the study of the effect of lipids on the activity of any enzyme assayed by tetrazolium salt reduction, and some previously reported papers [5-8] should be reconsidered. We have recently been studying [9] the effect of phospholipid vesicles on the reduction of M T T coupled to the N A D +-dependent lactate dehydrogenase reaction. We observed that when egg-yolk phosphatidylcholine vesicles were added to this system, the time-course of M T T reduction varied from one experiment to another. Sometimes, activations were obtained similar to those produced by DMPC vesicles, but at other times inhibitions were observed at the beginning of the reaction followed by an increased rate of MTT reduction after a variable period of time. The experiments described in the present paper were performed trying to explain this curious effect. We found that it was related to the auto-oxidation grade of the phospholipids added to the system. This is another potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method. Materials
and Methods
The following compounds were from Sigma (Saint Louis, U.S.A.): MTT, phenazine methosulfate (PMS), fl-NAD + from yeast (grade III), synthetic DMPC, PC from egg yolk (Type III-E, hexane solution), superoxide dismutase from bovine blood (Type I), and lactate dehydrogenase from bovine heart (No. 826-6, crystalline suspension in ammonium sulphate, 1000 units/ml). All other chemicals were of analytical grade. MTT reduction was coupled to the N A D +-dependent lactate dehydrogenase (LDH) reaction and followed at 570 nm as in our previous work [9]: LDH
L-lactate+ NAD + ~ pyruvate+ NADH + H +
(1)
NADH + H + + PMS ---,NAD + + reduced PMS
(2)
Reduced PMS + MTT ---,PMS+ formazan (570 nm)
(3)
The incubation media (final volume 0.5 ml) contained 50 mM sodium phosphate (pH 6), 10 mM
L-lactate, 2 mM N A D + and 1-10 /xl of suitably diluted lactate dehydrogenase. In addition, 120 /~g / m l phenazine methosulfate and 60 # g / m l MTT were present in the experiments of Fig. 1 and 2, whereas in all the other experiments phenazine methosulfate concentration was 24 # g / m l and that of MTT 30 /~g/ml. Phospholipid vesicles were included when indicated, the final phospholipid concentration being 0.25 m g / m l (about 340 #M) in all cases. Incubations were carried out at 25°C. Other experimental details are given in the figure legends. For PC auto-oxidation, appropriate aliquots of the stock solution in hexane were taken and the solvent evaporated forming a film of P C ' o n the walls of a glass vessel which was then left to stand opened at room temperature until the desired hydroperoxide content was obtained. Auto-oxidized PC was then dissolved in ethanol (25 m g / m l ) and aliquots of this solution were used to prepare PC vesicles. Phospholipid vesicles were prepared by the procedure of Batzri and Korn [10] injecting 50/~1 of an ethanolic solution of PC or DMPC (25 m g / m l ) into 2.5 ml of distilled water at about 30°C. When indicated, 0.25 ml of these phospholipid vesicles were included in the incubation media detailed above. The hydroperoxide content of PC was measured by the iodometric assay described by Buege and Aust [11] but using 3-fold smaller reagent volumes. Cumene hydroperoxide was used as the peroxide standard [ 11]. We observed that when the hydroperoxide content of PC vesicles mixed with phenazine methosulfate was determined, values about 40% lower than those in the absence of phenazine methosulfate were obtained. This effect was independent of the incubation time and the presence of the other components of the system. It was likely due to an interference in the iodometric assay caused by phenazine methosulfate or some phenazine methosulfate derivative extracted from the aqueous medium together with the PC during the partition with chloroform/methanol which precedes the hydroperoxide assay. This was supported by the fact that when an aqueous solution of phenazinc methosulfate alone was partitioned with chloroform/methanol, although most of the yellow
211
phenazine methosulfate remained in the aqueous upper phase, a little of phenazine methosulfate or some phenazine methosulfate derivative dissolved in the chloroformic lower phase and after evaporation of the solvent the dried residue was able to inhibit the light-catalyzed liberation of iodine from the reagents used in the hydroperoxide assay. It was therefore important that when the hydroperoxide content of PC vesicles in incubating media containing phenazine methosulfate had to be determined, controls and blanks with the same amount of phenazine methosulfate present in the test sample were run in order to cancel out the above-mentioned interference from the other effects described in this paper. Data of representative experiments are shown in Figs. 1-5. Similar results were obtained in at least three separate experiments. Results
Fig. 1 shows the time-course of the lactate dehydrogenase-mediated reduction of MTT in the absence of lipids and in the presence of PC vesicles with an increasing grade of autooxidation. It can
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be seen in Fig. I A that as the peroxidation of PC vesicles increased, the M T r reduction rate decreased and the activation with respect to the curve without lipids was lower. It should be noted that the activating effect of phospholipid vesicles on MTT reduction has already been described [9]. Records shown in Fig. 1B were obtained maintaining the assay conditions as in Fig. 1A but with a 10-fold lower amount of enzyme. It can be seen that in this case the initial rate of MTT reduction in the presence of peroxidized PC vesicles was not only lower than in the presence of non-peroxidized PC, but also lower than in the absence of lipid. After this several minutes long initial period, the rate of MTT reduction began to increase gradually until it reached an apparent steady state. From Fig. 1, it is clear that the time-course of lactate dehydrogenase-mediated MTT reduction in the presence of PC vesicles varied with the peroxidation grade of the phospholipid and the enzyme activity. Most of the following experiments were carried out under assay conditions similar to those of curve 2 in Fig. lB. To see if the initial phase of inhibition in the reduction of MTT could be modified by pre-incubation under given conditions, the
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Fig. 1. Time-course of M T T reduction coupled to the N A D +dependent lactate dehydrogenase reaction. For records of panel A the amount of enzyme used was 10 #1 of a 10-fold dilution of the stock lactate dehydrogenase, whereas for those of panel B only 1 #1 of the same dilution was used. The other assay conditions were the same in A and B, and were detailed under Materials and Methods. Curves 1-3 were obtained in the presence of PC vesicles (340 /AM PC in each case) with an increasing degree of auto-oxidation in such a way that the hydroperoxide concentrations were: less than 20 # M in curves 1 (considered to be hydroperoxide free); 135/*M in curves 2; and 205 I~M in curves 3. Curves 4 were obtained in the absence of lipids.
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Fig. 2. "lime-course of MTI" reduction in the presence of auto-oxidized PC after pre-incubating the system, omitting o n e of the components. Auto-oxidized PC vesicles were preincubated for 25 min in the medium given under Materials and Methods, but omitting one of the components, which was added at the end of the pre-incubation when M T T reduction began (at zero time on the abscissa). This component was: M T T for curve 1; phenazine methosulfate for curve 2; and L-lactate for curve 3. When the component omitted in the pre-incubation was lactate dehydrogenas¢ or N A D +, the timecourse of M T T reduction was similar to curves 2 and 3 (not shown).
212
experiment shown in Fig. 2 was performed. As can be observed, the initial phase was shortened or abolished when pre-incubating the system in the absence of MTT, all the other components being present. Pre-incubation of the system omitting any one of the other components had no effect. These results suggested that something was happening to the peroxidized PC while it was being incubated with the redox system under consideration, and that all the components of the system except MTT were necessary for this to occur. This was confirmed in the experiment shown in Fig. 3. A time-dependent decrease in PC hydroperoxides was found when peroxidized PC vesicles were pre-incubated with all the components of the system except MTT (Fig. 3A). If any one of the other components of the system (lactate, lactate dehydrogenase, NAD + or phenazine methosulfate) was omitted, the hydroperoxide content remained un-
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Fig. 3. A. Concentration of PC hydroperoxides as a function of the time of pre-incubation with all the components of the system except MTT. Four aliquots of 0.5 ml were taken from a mixture containing auto-oxidized PC vesicles and all the components of the standard incubation medium except L-lactate and MTT. The aliquots were numbered l to 4. The preincubation was initiated in each of these samples by adding 10 #1 500 m M L-lactate. The addition of L-lactate was done in such a way that the pre-ineubation times were 0, 20, 40 and 60 min for samples number 1, 2, 3 and 4, respectively, and the pre-incubation of all samples concluded simultaneously. At the end of the preincubation, the samples were extracted with c h l o r o f o r m / methanol to determine PC hydroperoxides. B. Time-course of M T T reduction started after pre-incubating the system without M T T for different times. Samples l, 2, 3 and 4 were preincubated for 0, 20 and 60 min, respectively, in parallel with those in A. At the end of the pre-incubation (zero time on the abscissa), 5 #1 of M T T (3 m g / m l ) were added to each sample and the time course of the absorbance at 570 n m was recorded.
changed (not shown). As the pre-incubation time in the absence of MTT was longer, the hydroperoxide concentration was lower (Fig. 3A) and thereafter the initial phase of inhibition in the reduction of MTT was shortened (Fig. 3B). From all the above-described results it is clear that when peroxidized-PC vesicles are added to the enzymatic system under consideration, PC hydroperoxides participate in the redox reaction, being chemically transformed and interfering in the reduction of MTT. Since MTT reduction and the diminution of hydroperoxides required the presence of lactate, lactate dehydrogenase, NAD + and phenazine methosulfate, both processes depended on the reduction of phenazine methosulfate and therefore hydroperoxides seem to compete with MTT for the electrons of reduced phenazine methosulfate in Eqn. 3 given under Materials and Methods. MTT reduction coupled to the NAD+-dependent lactate dehydrogenase reaction can be inhibited by superoxide dismutase, both in the absence of lipids and in the presence of PC vesicles (Fig. 4). This indicates that MTT reduction in this system is mediated by the superoxide radical, 0 2 (see Discussion). It was important to determine whether superoxide dismutase was also able to inhibit the hydroperoxide decrease. This was actually the case, since when auto-oxidized PC vesicles were incubated for 45 min in the medium given under Materials and Methods excluding MTT, the hydroperoxide diminution was 72% in the absence and 18% in the presence of 260/~g/ml superoxide dismutase (not shown). Therefore, the superoxide radical was also involved in the diminution of PC hydroperoxides. As hydroperoxide degradation is frequently catalyzed by metal ions [ 12,13] and since traces of metal ions are often present in the reagents normally used in the laboratory (specially in phosphate buffers), we investigated whether the metal chelator EDTA had some influence on the system we are delaing with. It can be observed in Fig. 5 that addition of 0.1 mM EDTA enhanced the lactate dehydrogenase-mediated MTT reduction rate in the presence of peroxidized PC in such a way that the curve became almost identical to that obtained in the presence of DMPC vesicles. It is important to note that EDTA did not change the
213
Discussion
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Fig. 4. Inhibitory effect of superoxide dismutase on the reduction of MTT coupled to the NAD+-dependent lactate dehydrogenase reaction. Curves 1 and 3 were obtained in the absence of lipids, and curves 2 and 4 in the presence of auto-oxidized PC vesicles. Superoxide dismutase (260 #g/ml) was present in samples 3 and 4. Fig. 5. Effect of EDTA on the time-courseof MTT reduction coupled to the NAD+-dependent LDH reaction. Curves 1 and 2 were obtained in the presence of DMPC vesicles, and curves 3 and 4 in the presence of auto-oxidized PC vesicles. 0.1 mM EDTA was present in samples 2 and 3.
rate of M T T reduction in the presence of DMPC, which has no acyl chain with peroxidable doublebounds (Fig. 5), nor in the absence of lipids (not shown). F r o m these results, it was clear that E D T A abolished the interference of peroxidized PC in the time-course of the lactate dehydrogenase-mediated M T T reduction. E D T A also inhibited the hydroperoxide decrease observed when incubating peroxidized PC with the components of the system excluding M T T (not shown). Therefore, the diminution of PC hydroperoxides seems to be mediated by traces of some unidentified metal ion present in the incubation mixture. All the experiments described above were carried out at p H 6 because it was at this p H that the effect of peroxidized PC was first observed. Also at p H 8 the rate of M T T reduction in the presence of peroxidized PC was slower than in the presence of the non-peroxidable phospholipid DMPC, and this inhibition caused by PC hydroperoxides was abolished by EDTA. Thus, basically the same effect was obtained at p H 6 and 8, although it seemed to be less notable at p H 8.
In the present paper we describe interference in the reduction of M T T coupled to the NAD+-de o pendent L D H reaction observed when peroxidized PC is added to the system. Evidence has been presented indicating that PC hydroperoxides compete with M T T for the electrons coming from the substrate oxidation. Normally, electrons are transferred from L-lactate to M T T through the coupled reactions 1, 2 and 3 shown under Materials and Methods, but in the presence of auto-oxidized PC electrons from reduced phenazine methosulfate are also captured by PC hydroperoxides at the level of the reaction 3 and the rate of M T T reduction is decreased. In this regard, it is important to n o t e that when auto-oxidized PC vesicles are added to the system, two combined effects are produced: (a) the previously described [9] activating effect due to the presence of a lipidic phase in which reduced M T T is partitioned; and (b) the inhibitory effect described here due to the presence of hydroperoxides which compete for the electrons with MTT. Therefore, the apparent result depends on the relative importance of the individual effects (a) and (b), which in turn depends on the assay conditions such as the degree of auto-oxidation of the phospholipids and the rate of the electron flow, regulated by the amount of enzyme. This is illustrated in Fig. 1. When the electron flow was slow (low amount of lactate dehydrogenase, Fig. 1B), the time-course of M T T reduction in the presence of peroxidized PC showed an initial phase of great inhibition because a high proportion of the electrons were being transferred to the hydroperoxides and not to MTT; but after the hydroperoxide content had decreased sufficiently, M T T reduction rate began to increase. When the electron flow was faster (higher amount of lactate dehydrogenase, Fig. 1A), the proportion of the electrons transferred to the hydroperoxides was lower and the inhibitory effect on M T T reduction was less marked. In this regard, it should be noted that when the amount of lactate dehydrogenase was enhanced above a limit which was between the amounts used in Fig. 1B and 1A, the diminution of hydroperoxides did not increase further, while the rate of M T T reduction continued to increase proportionally to the added enzyme (not shown).
214
Thus, the transfer of electrons to hydroperoxides seemed to reach its maximal rate at a much lower enzyme concentration than the electron transfer to MTT. Tetrazolium salts are reduced by the superoxide radical 0 2 , and the inhibition of their reduction by superoxide dismutase provided the basis for a test to detect the generation of this radical [14]. The sensitivity of an 0 2 detecting system toward superoxide dismutase depends upon the rate of generation of 0 2 , the concentration of the compound used to detect the 0 2 , and p H [14]. The data presented here show that the reduction of MTT was inhibited by superoxide dismutase. The sensitivity toward superoxide dismutase depended on the assay conditions. In preliminary experiments we had found that with a superoxide dismutase concentration 10-fold lower than that used in the experiment of Fig. 4, an inhibition of about 80% in the reduction of M T r could be obtained in the absence of lipid, but the inhibition was only of 25-30% in the presence of DMPC vesicles under the same conditions. The degree of inhibition in the reduction of MTT caused by a given concentration of superoxide dismutase also decreased with increasing concentrations of lactate dehydrogenase (probably because the rate of phenazine methosulfate reduction and 0 2 generation increased, see below). Thus, we decided to use a rather large concentration of superoxide dismutase (260 # g / m l ) in order to obtain a high degree of inhibition under different assay conditions. The inhibition of MTT reduction by superoxide dismutase indicated that this reduction was mediated by O~-. This radical was most likely produced in our system because under the aerobic conditions of the assay electrons were transferred from reduced phenazine methosulfate to oxygen, which was partially reduced to O~-. This is consistent with the fact that a system constituted by L-lactate, lactate dehydrogenase and N A D ÷ in phosphate buffer presented a phenazine methosulfate-dependent oxygen consumption (not shown). In addition, the generation of superoxide radical in the oxidation of reduced PMS by molecular oxygen has already been reported [15]. Therefore, under aerobic conditions the electrons are not directly transferred from reduced phenazine methosulfate to MTT as shown in the Eqn. 3, but this electron
transfer is mediated by oxygen which is reduced to 0 2 (by the reduced form of phenazine methosulfate) and then this radical reduces the MTT. Since tlae hydroperoxide diminution was also mediated by O~-, the interference of peroxidized PC in MTT reduction must occur after the formation of the superoxide radical (see the sequence of the electron transference given below). Another important aspect to be considered is the influence of EDTA on the system. It was clear that EDTA inhibited the transference of electrons to PC hydroperoxides and consequently abolished the interference of peroxidized PC on MTT reduction. Since the reduction of MTT in the absence of lipids or in the presence of nonperoxidized phospholipids was not affected by the addition of EDTA, it can be concluded that traces of some unidentified metal ion were catalyzing the transfer of electrons to hydroperoxides but not to MTT. All the findings discussed above may be better visualized assuming the following sequence for the electron transference from L-lactate to M T T and hydroperoxides (M = metal ion): 7 M TT LDH L-lactate --* N A D + ---*PMS ~ 0 2 "~A" hydroperoxides
The interference due to hydroperoxides is a potential complication in the study of the effect of lipids on the activity of any enzyme assayed by the tetrazolium salt method. In previous works [5-8] in which the effect of lipids on the activity of purified dehydrogenases was assayed by the phenazine methosulfate-mediated reduction of MTT, no mention was made regarding any precaution in order to avoid the auto-oxidation of the lipids or the presence of traces of ionic metals, and therefore the possibility of an interfering effect of hydroperoxides cannot be excluded. The findings presented in this paper may also be useful for those who are interested in the study of reactions involving lipid hydroperoxides. In this regard it is interesting to mention that contradictory results have been reported on the reactivity of the superoxide radical toward the hydroperoxides. For example, evidence for a reaction between 0 2 and hydroperoxides has been reported by Sutherland and Gebicki [16], whereas Ruddock et al. [17]
215
could not detect any interaction of the superoxide radical with peroxides. In our system, a transference of electrons from 0 2 to the lipid hydroperoxides seemed to occur, but only if catalyzed by traces of a metal ion. The ability of some ionic transition metals to serve as catalysts in the redox decomposition of lipid hydroperoxides (cycling electrons from the reductant to the hydroperoxides) has been studied by Gardner and Jursinic [131.
Acknowledgments The authors thank Susana Bustos for typing this paper. This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas and the Secretaria de Ciencia y T6cnica (Argentina). Both authors are Career Investigators of the Consejo Nacional.
References 1 Mollereing, H., Wahlefeld, A.W. and Michal, G. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 1, pp. 136-144, Academic Press, New York 2 Bergrneyer, H.U. and Bernt, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 2, pp. 579-582, Acedemic Press, New York
3 Fried, R. and Fried, L.W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Voi. 2, pp. 644-649, Academic Press, New York 4 Abdallah, M.A. and Biellmann, J.F. (1980) Eur. J. Biochem. 112, 331-333 5 Tanaka, Y., Anraku, Y. and Futai, M. (1976) J. Biochem. 80, 821-830 6 Kimura, H. and Futai, M. (1978) J. Biol. Chem. 253, 1095-1100 7 Fung, L.W.M., Pratt, E.A. and Ho, C. (1979) Biochemistry 18, 317-324 8 Kovatchev, S., Vaz, W.L.C. and Eibl, H. (1981) J. Biol. Chem. 256, 10369-10374 9 Massa, E.M. and Farias, R.N. (1982) Biochem. Biophys. Res. Commun. 104, 1623-1629 10 Batzri, S. and Korn, E.D. (1973) Biochim. Biophys. Acta 298, 1015-1019 11 Buege, J.A. and Aust, S.D. (1978) Methods Enzymol. 52, 302- 310 12 O'Brien, P.J. (1969) Can. J. Biochem. 47, 485-492 13 Gardner, H.W. and Jursinic, P.A. (1981) Biochim. Biophys. Acta 665, 100-112 14 Beauchamp, C. and Fridovich, I. (1971) Anal. Biochem. 44, 276-287 15 Nishikimi, M., Rao, N.A. and Yagi, K. (1972) Biochem. Biophys. Res. Commun. 46, 849-854 16 Sutherland, M.W. and Gebicki, J.M. (1982) Arch. Biochem. Biophys. 214, 1-11 17 Ruddock, G.W., Raleigh, J.A. and Greenstock, C.L. (1981) Biochem. Biophys. Res. Commun. 102, 554-560
209
BBA31662
EFFECT OF AUTO-OXIDIZED PHOSPHOLIPIDS ON OXIDATIVE ENZYME ASSAYS BASED ON TETRAZOLIUM SALT REDUCTION EDDY M. MASSA and RICARDO N. FARIAS
Departamento de Bioquimica de la Nutricirn, Instituto Superior de Investigaciones Biol6gicas (INSIBIO), CONICET-UNT, Chacabuco 461, 4.000 San Miguel de Tucumim (Argentina) (Received March 4th, 1983)
Key words: Oxidative enzyme; Phospholipia~" Hydroperoxide; Tetrazolium salt reduction
The influence of auto-oxidized phospholipids on the reduction of the tetrazolium salt MTT coupled to the NAD +-dependent lactate dehydrogenase reaction was studied. The following results were obtained: (1) peroxidized phosphatidylcholine interfered in the time-course of the lactate dehydrogenase-mediated MTT reduction; (2) there was a time-dependent decrease in the hydroperoxide content of phosphatidylcholine vesicles during the incubation; (3) the diminution of phosphatidylcholine hydroperoxides required the presence of all the components of the system except MTT; (4) hydroperoxide diminution and MTT reduction were mediated by the superoxide radical O2-, since both processes were inhibited by superoxide dismutase; (5) EDTA inhibited the hydroperoxide decrease and abolished the interference of peroxidized phosphatidylcboline with MTT reduction. It was concluded that hydroperoxides compete with M T r for the electrons coming from substrate oxidation. The superoxide radical 02- and traces of some contaminating metal ion are involved in the process. This is a potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method.
Introduction The assay of oxidative enzymes such as flavin and NAD+-dependent dehydrogenases is frequently based on the reduction of tetrazolium salts to deeply colored formazans which absorb strongly in the visible region. Thus, the time-course of the enzymatic reaction is continuously monitored by following the correspondent increase in absorbance [1-4]. Authors from different laboratories [5-8] have used this spectrophotometric assay based on the phenazine methosulfate-mediated reduction of the tetrazolium salt MTT when studying the effect of Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMPC, L-a-dimyristoylphospliatidyleholine; PC, L-a-phosphatidylcholine from egg yolk. 0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.
lipids on the activity of purified dehydrogenases. In these studies, activations have been interpreted as arising from lipid-enzyme interactions. However, we have recently shown [9] that chemical and enzyme-mediated reductions of MTT can be enhanced by the addition of a lipidic phase to the aqueous reaction mixture because the formazan derived from MTT reduction is a sparingly water-soluble compound that dissolves well in organic solvents and lipids. Thus, reduced MTT dissolves in the detergent micelles or phospholipid vesicles added to the system, favoring the reoox reaction occurring in the aqueous phase surrounding these micelles or vesicles. In addition, the absorbance of reduced MTT in lipid-like media is higher than in aqueous media [9]. Therefore, increased rate of enzyme-mediated MTT reduction when lipids are added to the incubation mixture
210 may arise from lipid-enzyme interactions a n d / o r from the direct influence of lipids on tetrazolium salt reduction itself. This points out a potential pitfall in the study of the effect of lipids on the activity of any enzyme assayed by tetrazolium salt reduction, and some previously reported papers [5-8] should be reconsidered. We have recently been studying [9] the effect of phospholipid vesicles on the reduction of M T T coupled to the N A D +-dependent lactate dehydrogenase reaction. We observed that when egg-yolk phosphatidylcholine vesicles were added to this system, the time-course of M T T reduction varied from one experiment to another. Sometimes, activations were obtained similar to those produced by DMPC vesicles, but at other times inhibitions were observed at the beginning of the reaction followed by an increased rate of MTT reduction after a variable period of time. The experiments described in the present paper were performed trying to explain this curious effect. We found that it was related to the auto-oxidation grade of the phospholipids added to the system. This is another potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method. Materials
and Methods
The following compounds were from Sigma (Saint Louis, U.S.A.): MTT, phenazine methosulfate (PMS), fl-NAD + from yeast (grade III), synthetic DMPC, PC from egg yolk (Type III-E, hexane solution), superoxide dismutase from bovine blood (Type I), and lactate dehydrogenase from bovine heart (No. 826-6, crystalline suspension in ammonium sulphate, 1000 units/ml). All other chemicals were of analytical grade. MTT reduction was coupled to the N A D +-dependent lactate dehydrogenase (LDH) reaction and followed at 570 nm as in our previous work [9]: LDH
L-lactate+ NAD + ~ pyruvate+ NADH + H +
(1)
NADH + H + + PMS ---,NAD + + reduced PMS
(2)
Reduced PMS + MTT ---,PMS+ formazan (570 nm)
(3)
The incubation media (final volume 0.5 ml) contained 50 mM sodium phosphate (pH 6), 10 mM
L-lactate, 2 mM N A D + and 1-10 /xl of suitably diluted lactate dehydrogenase. In addition, 120 /~g / m l phenazine methosulfate and 60 # g / m l MTT were present in the experiments of Fig. 1 and 2, whereas in all the other experiments phenazine methosulfate concentration was 24 # g / m l and that of MTT 30 /~g/ml. Phospholipid vesicles were included when indicated, the final phospholipid concentration being 0.25 m g / m l (about 340 #M) in all cases. Incubations were carried out at 25°C. Other experimental details are given in the figure legends. For PC auto-oxidation, appropriate aliquots of the stock solution in hexane were taken and the solvent evaporated forming a film of P C ' o n the walls of a glass vessel which was then left to stand opened at room temperature until the desired hydroperoxide content was obtained. Auto-oxidized PC was then dissolved in ethanol (25 m g / m l ) and aliquots of this solution were used to prepare PC vesicles. Phospholipid vesicles were prepared by the procedure of Batzri and Korn [10] injecting 50/~1 of an ethanolic solution of PC or DMPC (25 m g / m l ) into 2.5 ml of distilled water at about 30°C. When indicated, 0.25 ml of these phospholipid vesicles were included in the incubation media detailed above. The hydroperoxide content of PC was measured by the iodometric assay described by Buege and Aust [11] but using 3-fold smaller reagent volumes. Cumene hydroperoxide was used as the peroxide standard [ 11]. We observed that when the hydroperoxide content of PC vesicles mixed with phenazine methosulfate was determined, values about 40% lower than those in the absence of phenazine methosulfate were obtained. This effect was independent of the incubation time and the presence of the other components of the system. It was likely due to an interference in the iodometric assay caused by phenazine methosulfate or some phenazine methosulfate derivative extracted from the aqueous medium together with the PC during the partition with chloroform/methanol which precedes the hydroperoxide assay. This was supported by the fact that when an aqueous solution of phenazinc methosulfate alone was partitioned with chloroform/methanol, although most of the yellow
211
phenazine methosulfate remained in the aqueous upper phase, a little of phenazine methosulfate or some phenazine methosulfate derivative dissolved in the chloroformic lower phase and after evaporation of the solvent the dried residue was able to inhibit the light-catalyzed liberation of iodine from the reagents used in the hydroperoxide assay. It was therefore important that when the hydroperoxide content of PC vesicles in incubating media containing phenazine methosulfate had to be determined, controls and blanks with the same amount of phenazine methosulfate present in the test sample were run in order to cancel out the above-mentioned interference from the other effects described in this paper. Data of representative experiments are shown in Figs. 1-5. Similar results were obtained in at least three separate experiments. Results
Fig. 1 shows the time-course of the lactate dehydrogenase-mediated reduction of MTT in the absence of lipids and in the presence of PC vesicles with an increasing grade of autooxidation. It can
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be seen in Fig. I A that as the peroxidation of PC vesicles increased, the M T r reduction rate decreased and the activation with respect to the curve without lipids was lower. It should be noted that the activating effect of phospholipid vesicles on MTT reduction has already been described [9]. Records shown in Fig. 1B were obtained maintaining the assay conditions as in Fig. 1A but with a 10-fold lower amount of enzyme. It can be seen that in this case the initial rate of MTT reduction in the presence of peroxidized PC vesicles was not only lower than in the presence of non-peroxidized PC, but also lower than in the absence of lipid. After this several minutes long initial period, the rate of MTT reduction began to increase gradually until it reached an apparent steady state. From Fig. 1, it is clear that the time-course of lactate dehydrogenase-mediated MTT reduction in the presence of PC vesicles varied with the peroxidation grade of the phospholipid and the enzyme activity. Most of the following experiments were carried out under assay conditions similar to those of curve 2 in Fig. lB. To see if the initial phase of inhibition in the reduction of MTT could be modified by pre-incubation under given conditions, the
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212
experiment shown in Fig. 2 was performed. As can be observed, the initial phase was shortened or abolished when pre-incubating the system in the absence of MTT, all the other components being present. Pre-incubation of the system omitting any one of the other components had no effect. These results suggested that something was happening to the peroxidized PC while it was being incubated with the redox system under consideration, and that all the components of the system except MTT were necessary for this to occur. This was confirmed in the experiment shown in Fig. 3. A time-dependent decrease in PC hydroperoxides was found when peroxidized PC vesicles were pre-incubated with all the components of the system except MTT (Fig. 3A). If any one of the other components of the system (lactate, lactate dehydrogenase, NAD + or phenazine methosulfate) was omitted, the hydroperoxide content remained un-
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Fig. 3. A. Concentration of PC hydroperoxides as a function of the time of pre-incubation with all the components of the system except MTT. Four aliquots of 0.5 ml were taken from a mixture containing auto-oxidized PC vesicles and all the components of the standard incubation medium except L-lactate and MTT. The aliquots were numbered l to 4. The preincubation was initiated in each of these samples by adding 10 #1 500 m M L-lactate. The addition of L-lactate was done in such a way that the pre-ineubation times were 0, 20, 40 and 60 min for samples number 1, 2, 3 and 4, respectively, and the pre-incubation of all samples concluded simultaneously. At the end of the preincubation, the samples were extracted with c h l o r o f o r m / methanol to determine PC hydroperoxides. B. Time-course of M T T reduction started after pre-incubating the system without M T T for different times. Samples l, 2, 3 and 4 were preincubated for 0, 20 and 60 min, respectively, in parallel with those in A. At the end of the pre-incubation (zero time on the abscissa), 5 #1 of M T T (3 m g / m l ) were added to each sample and the time course of the absorbance at 570 n m was recorded.
changed (not shown). As the pre-incubation time in the absence of MTT was longer, the hydroperoxide concentration was lower (Fig. 3A) and thereafter the initial phase of inhibition in the reduction of MTT was shortened (Fig. 3B). From all the above-described results it is clear that when peroxidized-PC vesicles are added to the enzymatic system under consideration, PC hydroperoxides participate in the redox reaction, being chemically transformed and interfering in the reduction of MTT. Since MTT reduction and the diminution of hydroperoxides required the presence of lactate, lactate dehydrogenase, NAD + and phenazine methosulfate, both processes depended on the reduction of phenazine methosulfate and therefore hydroperoxides seem to compete with MTT for the electrons of reduced phenazine methosulfate in Eqn. 3 given under Materials and Methods. MTT reduction coupled to the NAD+-dependent lactate dehydrogenase reaction can be inhibited by superoxide dismutase, both in the absence of lipids and in the presence of PC vesicles (Fig. 4). This indicates that MTT reduction in this system is mediated by the superoxide radical, 0 2 (see Discussion). It was important to determine whether superoxide dismutase was also able to inhibit the hydroperoxide decrease. This was actually the case, since when auto-oxidized PC vesicles were incubated for 45 min in the medium given under Materials and Methods excluding MTT, the hydroperoxide diminution was 72% in the absence and 18% in the presence of 260/~g/ml superoxide dismutase (not shown). Therefore, the superoxide radical was also involved in the diminution of PC hydroperoxides. As hydroperoxide degradation is frequently catalyzed by metal ions [ 12,13] and since traces of metal ions are often present in the reagents normally used in the laboratory (specially in phosphate buffers), we investigated whether the metal chelator EDTA had some influence on the system we are delaing with. It can be observed in Fig. 5 that addition of 0.1 mM EDTA enhanced the lactate dehydrogenase-mediated MTT reduction rate in the presence of peroxidized PC in such a way that the curve became almost identical to that obtained in the presence of DMPC vesicles. It is important to note that EDTA did not change the
213
Discussion
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Fig. 4. Inhibitory effect of superoxide dismutase on the reduction of MTT coupled to the NAD+-dependent lactate dehydrogenase reaction. Curves 1 and 3 were obtained in the absence of lipids, and curves 2 and 4 in the presence of auto-oxidized PC vesicles. Superoxide dismutase (260 #g/ml) was present in samples 3 and 4. Fig. 5. Effect of EDTA on the time-courseof MTT reduction coupled to the NAD+-dependent LDH reaction. Curves 1 and 2 were obtained in the presence of DMPC vesicles, and curves 3 and 4 in the presence of auto-oxidized PC vesicles. 0.1 mM EDTA was present in samples 2 and 3.
rate of M T T reduction in the presence of DMPC, which has no acyl chain with peroxidable doublebounds (Fig. 5), nor in the absence of lipids (not shown). F r o m these results, it was clear that E D T A abolished the interference of peroxidized PC in the time-course of the lactate dehydrogenase-mediated M T T reduction. E D T A also inhibited the hydroperoxide decrease observed when incubating peroxidized PC with the components of the system excluding M T T (not shown). Therefore, the diminution of PC hydroperoxides seems to be mediated by traces of some unidentified metal ion present in the incubation mixture. All the experiments described above were carried out at p H 6 because it was at this p H that the effect of peroxidized PC was first observed. Also at p H 8 the rate of M T T reduction in the presence of peroxidized PC was slower than in the presence of the non-peroxidable phospholipid DMPC, and this inhibition caused by PC hydroperoxides was abolished by EDTA. Thus, basically the same effect was obtained at p H 6 and 8, although it seemed to be less notable at p H 8.
In the present paper we describe interference in the reduction of M T T coupled to the NAD+-de o pendent L D H reaction observed when peroxidized PC is added to the system. Evidence has been presented indicating that PC hydroperoxides compete with M T T for the electrons coming from the substrate oxidation. Normally, electrons are transferred from L-lactate to M T T through the coupled reactions 1, 2 and 3 shown under Materials and Methods, but in the presence of auto-oxidized PC electrons from reduced phenazine methosulfate are also captured by PC hydroperoxides at the level of the reaction 3 and the rate of M T T reduction is decreased. In this regard, it is important to n o t e that when auto-oxidized PC vesicles are added to the system, two combined effects are produced: (a) the previously described [9] activating effect due to the presence of a lipidic phase in which reduced M T T is partitioned; and (b) the inhibitory effect described here due to the presence of hydroperoxides which compete for the electrons with MTT. Therefore, the apparent result depends on the relative importance of the individual effects (a) and (b), which in turn depends on the assay conditions such as the degree of auto-oxidation of the phospholipids and the rate of the electron flow, regulated by the amount of enzyme. This is illustrated in Fig. 1. When the electron flow was slow (low amount of lactate dehydrogenase, Fig. 1B), the time-course of M T T reduction in the presence of peroxidized PC showed an initial phase of great inhibition because a high proportion of the electrons were being transferred to the hydroperoxides and not to MTT; but after the hydroperoxide content had decreased sufficiently, M T T reduction rate began to increase. When the electron flow was faster (higher amount of lactate dehydrogenase, Fig. 1A), the proportion of the electrons transferred to the hydroperoxides was lower and the inhibitory effect on M T T reduction was less marked. In this regard, it should be noted that when the amount of lactate dehydrogenase was enhanced above a limit which was between the amounts used in Fig. 1B and 1A, the diminution of hydroperoxides did not increase further, while the rate of M T T reduction continued to increase proportionally to the added enzyme (not shown).
214
Thus, the transfer of electrons to hydroperoxides seemed to reach its maximal rate at a much lower enzyme concentration than the electron transfer to MTT. Tetrazolium salts are reduced by the superoxide radical 0 2 , and the inhibition of their reduction by superoxide dismutase provided the basis for a test to detect the generation of this radical [14]. The sensitivity of an 0 2 detecting system toward superoxide dismutase depends upon the rate of generation of 0 2 , the concentration of the compound used to detect the 0 2 , and p H [14]. The data presented here show that the reduction of MTT was inhibited by superoxide dismutase. The sensitivity toward superoxide dismutase depended on the assay conditions. In preliminary experiments we had found that with a superoxide dismutase concentration 10-fold lower than that used in the experiment of Fig. 4, an inhibition of about 80% in the reduction of M T r could be obtained in the absence of lipid, but the inhibition was only of 25-30% in the presence of DMPC vesicles under the same conditions. The degree of inhibition in the reduction of MTT caused by a given concentration of superoxide dismutase also decreased with increasing concentrations of lactate dehydrogenase (probably because the rate of phenazine methosulfate reduction and 0 2 generation increased, see below). Thus, we decided to use a rather large concentration of superoxide dismutase (260 # g / m l ) in order to obtain a high degree of inhibition under different assay conditions. The inhibition of MTT reduction by superoxide dismutase indicated that this reduction was mediated by O~-. This radical was most likely produced in our system because under the aerobic conditions of the assay electrons were transferred from reduced phenazine methosulfate to oxygen, which was partially reduced to O~-. This is consistent with the fact that a system constituted by L-lactate, lactate dehydrogenase and N A D ÷ in phosphate buffer presented a phenazine methosulfate-dependent oxygen consumption (not shown). In addition, the generation of superoxide radical in the oxidation of reduced PMS by molecular oxygen has already been reported [15]. Therefore, under aerobic conditions the electrons are not directly transferred from reduced phenazine methosulfate to MTT as shown in the Eqn. 3, but this electron
transfer is mediated by oxygen which is reduced to 0 2 (by the reduced form of phenazine methosulfate) and then this radical reduces the MTT. Since tlae hydroperoxide diminution was also mediated by O~-, the interference of peroxidized PC in MTT reduction must occur after the formation of the superoxide radical (see the sequence of the electron transference given below). Another important aspect to be considered is the influence of EDTA on the system. It was clear that EDTA inhibited the transference of electrons to PC hydroperoxides and consequently abolished the interference of peroxidized PC on MTT reduction. Since the reduction of MTT in the absence of lipids or in the presence of nonperoxidized phospholipids was not affected by the addition of EDTA, it can be concluded that traces of some unidentified metal ion were catalyzing the transfer of electrons to hydroperoxides but not to MTT. All the findings discussed above may be better visualized assuming the following sequence for the electron transference from L-lactate to M T T and hydroperoxides (M = metal ion): 7 M TT LDH L-lactate --* N A D + ---*PMS ~ 0 2 "~A" hydroperoxides
The interference due to hydroperoxides is a potential complication in the study of the effect of lipids on the activity of any enzyme assayed by the tetrazolium salt method. In previous works [5-8] in which the effect of lipids on the activity of purified dehydrogenases was assayed by the phenazine methosulfate-mediated reduction of MTT, no mention was made regarding any precaution in order to avoid the auto-oxidation of the lipids or the presence of traces of ionic metals, and therefore the possibility of an interfering effect of hydroperoxides cannot be excluded. The findings presented in this paper may also be useful for those who are interested in the study of reactions involving lipid hydroperoxides. In this regard it is interesting to mention that contradictory results have been reported on the reactivity of the superoxide radical toward the hydroperoxides. For example, evidence for a reaction between 0 2 and hydroperoxides has been reported by Sutherland and Gebicki [16], whereas Ruddock et al. [17]
215
could not detect any interaction of the superoxide radical with peroxides. In our system, a transference of electrons from 0 2 to the lipid hydroperoxides seemed to occur, but only if catalyzed by traces of a metal ion. The ability of some ionic transition metals to serve as catalysts in the redox decomposition of lipid hydroperoxides (cycling electrons from the reductant to the hydroperoxides) has been studied by Gardner and Jursinic [131.
Acknowledgments The authors thank Susana Bustos for typing this paper. This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas and the Secretaria de Ciencia y T6cnica (Argentina). Both authors are Career Investigators of the Consejo Nacional.
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3 Fried, R. and Fried, L.W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Voi. 2, pp. 644-649, Academic Press, New York 4 Abdallah, M.A. and Biellmann, J.F. (1980) Eur. J. Biochem. 112, 331-333 5 Tanaka, Y., Anraku, Y. and Futai, M. (1976) J. Biochem. 80, 821-830 6 Kimura, H. and Futai, M. (1978) J. Biol. Chem. 253, 1095-1100 7 Fung, L.W.M., Pratt, E.A. and Ho, C. (1979) Biochemistry 18, 317-324 8 Kovatchev, S., Vaz, W.L.C. and Eibl, H. (1981) J. Biol. Chem. 256, 10369-10374 9 Massa, E.M. and Farias, R.N. (1982) Biochem. Biophys. Res. Commun. 104, 1623-1629 10 Batzri, S. and Korn, E.D. (1973) Biochim. Biophys. Acta 298, 1015-1019 11 Buege, J.A. and Aust, S.D. (1978) Methods Enzymol. 52, 302- 310 12 O'Brien, P.J. (1969) Can. J. Biochem. 47, 485-492 13 Gardner, H.W. and Jursinic, P.A. (1981) Biochim. Biophys. Acta 665, 100-112 14 Beauchamp, C. and Fridovich, I. (1971) Anal. Biochem. 44, 276-287 15 Nishikimi, M., Rao, N.A. and Yagi, K. (1972) Biochem. Biophys. Res. Commun. 46, 849-854 16 Sutherland, M.W. and Gebicki, J.M. (1982) Arch. Biochem. Biophys. 214, 1-11 17 Ruddock, G.W., Raleigh, J.A. and Greenstock, C.L. (1981) Biochem. Biophys. Res. Commun. 102, 554-560