Coenzyme Q-3 as an antioxidant

�9 1990 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163--4992/90/1612--0001502.40

Coenzyme Q-3 as an Antioxidant

Its Effect on the Composition and Structural Properties of Phospholipid Vesicles

LAURA LANDI, *'1 LUCIANA CABRINI, 1 DIANA FIORENTINI, 1 ANNA MARIA SECHI, 1 GIORGIO SARTOR, 2

PETRONIO PASQUALI, 2 AND LANFRANCO MASOTTI 2

l Dipartimento di Biochimica, Uniuersita' di Bologna, Italy; and 21stituto di Chimica Biologica, Uniuersita ' di Parma, Italy

Received August 5, 1988; Accepted December 7, 1988

ABSTRACT

Coenzyme Q-3 incorporated into the lipid bilayer at physiological concentration provided an 80% inhibition of the lipid peroxidation induced by ferrous ions. In coenzyme Q-containing vesicles, the fluorescence lifetime and the fluorescence anisotropy decay of the probe, 1,6-diphenyl-l,3,5-hexatriene, were measured in order to find out if the presence of the quinone can cause variations in the membrane organization. Our data show that two distinct populations of the probe were present and that both populations were available to quenching by coenzyme Q. The overall effects of coenzyme Q on the static and dynamic properties of the model membranes were: a very small effect in the ordering of the fatty acid chain, and a more noticeable decrease of the probe correlation time and, therefore, an increase in membrane fluidity at increasing quinone concentration. When vesicles were peroxidized in the absence of the coenzyme Q, the fluidity markedly decreased; in its presence, the fluidity was nearly unchanged. The results suggest that the antioxidant properties of coenzyme Q can be ascribed to its ability to react with free radicals. The effect on the fluidity of the lipid bilayer might imply that a requisite for a molecule to act as an efficient antioxidant could be its ability to readily diffuse within the membrane.

Index Entries: Coenzyme Q; phospholipid vesicles; lipid peroxidation; 1,6-dyphenyl-l,3,5-hexatriene; fluorescence anisotropy; membrane fluidity.

*Author to whom all correspondence and reprint requests should be addressed.

Cell Biophysics 1 Vol. 16, 1990

2 Landi et al.

Abbreviations: Q, coenzyme Q; Q-3, 2,3-Dirnethoxy-5 methyl-6- Jail trans farnesyl]-l,4-benzoquinone; DPH, 1,6-diphenyl-l,3,5-hexatriene; PL, phospholipid; MA, malonaldehyde; LOOH, lipid hydroperoxide; BHT, butylated hydroxytoluene; TBA, thiobarbituric acid.

INTRODUCTION

Coenzyme Q (Q)I plays its major role in funneling all electron equiva- lents from a different population of mitochondrial inner membrane dehy- drogenases to its known oxidant, the bcl complex (1). It has also been suggested that Q functions in a common pool (2), diffuses laterally in the membrane plane (3), and oscillates its benzoquinone head group toward both surfaces of the membrane (4,5). Evidence has also been presented that Golgi apparatus membranes are rich in Q in contrast to endoplasmic reticulum and plasma membranes, where none or a very small amount is found (6). According to Crane, coenzyme Q in Golgi membranes could be involved in electron transport (7). Q is also present in human neutro- philes, where it is necessary for the respiratory burst (8).

There are now many reports concerning the biomedical and clinical use of Q administered to humans or animals in a wide variety of patho- logical conditions. Many such conditions, in which beneficial effects of Q have been described, are among those where oxygen toxicity owing to free-radical formation has been implicated, e.g., adriamycin toxicity, ischemia, reperfusion syndromes, and inflammatory disorders (9). Q may exert these effects by acting as an antioxidant, similar to or

In order to clarify the mechanisms by which Q can prevent and over- come various forms of tissue damage caused by oxygen radicals, it seemed useful to perform experiments with model membranes. Vesicles prepared from components that occur in natural biological membranes offer oppor- tunities of studying a system of intermediate complexity, avoiding the complication of possible effects of cell components other than membrane lipids.

In the present study, we incorporated Q-3 in lipid bilayers and in- vestigated the ability of Q-containing vesicles to resist lipid peroxidation. Moreover, in the same vesicles, we measured the fluorescence lifetime and the fluorescence anisotropy decay of the probe 1,6-diphenyl-l,3,5- hexatriene (DPH), with the aim of evaluating if the quinone has any effect on the static and dynamic properties of the bilayer and if such an effect can be related to its protective action during the peroxidative insult.

MATERIALS AND METHODS

Egg lecithin was obtained from Lipid Products (Redhill, UK) and used without further purification. Q-3 was a generous gift of Eisai Co. (Tokyo,

Cell Biophysics VoL 16, 1990

Q Antioxidant Effect on Vesicle Properties 3

Japan); it was stored at -20~ in absolute ethanol, at a concentration of about 10 mM. DPH was purchased from Aldrich Chemie GmBH (Stein- heim, FRG). All other chemicals used were of the highest available grade and were purchased from Merck (Darmstadt, FRG) or Sigma Chemicals Co. (St. Louis, MO).

Unilamellar vesicles were prepared following a previously published method (10). When present, Q-3 dissolved in chloroform was added to the phospholipid solution, and the solvent was removed by evaporation under nitrogen. Aliquots of 50 mM HEPES buffer, pH 7.2, were added to dried lipid to give a phospholipid concentration of 3.2 mg/mL. The suspensions were sonicated with a probe sonicator Labsonic 2000 (B. Braun, Melsungen, FRG) for 20 min at 4~ under a nitrogen atmosphere. The vesicles were then centrifuged for 15 min at 100,000g to remove any probe particles and large aggregates. The amount of Q-3 incorporated into the vesicles was determined spectrophotometrically, as published elsewhere (11).

In the vesicles freshly prepared, lipid peroxidation was stimulated by iron salts. The reaction mixtures (total vol I mL) contained: phospholipid 0.2-0.4 mg; Q-3, as specified in the figures; and FeCI2 0.3 mM. After different incubation times at 30~ lipid peroxidation was measured by the formation of thiobarbituric acid (TBA) reactive materials as malonaldehyde (MA) at 532 nm. To the TBA reagent 10/~L of 2% butylated hydroxytoluene (BHT) solution were added just prior to use (12). Lipid hydroperoxides (LOOH) were also measured on aliquots of vesicles with the thiocyanate method (13). The fatty acid composition of vesicles tested was determined by gas-liquid chromatography after methanolysis in acid- methanol (14). After lipid peroxidation, Q was determined by reversed- phase HPLC, according to Takada (15), with slight modifications. Lipid phosphorus was determined by the method of Marinetti (16).

Samples for fluorescence measurement were prepared adding 2.5 ~r of 2 mM DPH in tetrahydrofuran to 2.5 mL of membrane suspension appropriately diluted in order to reach a probe to phospholipid ratio of 1 to 500 and an absorbance lower than 0.2 at the excitation wavelength. Measurements were performed after 30 min of incubation of the sample at 25~

Static fluorescence anisotropy (rs) was measured using a Perkin Elmer MPF44A spectrofluorimeter equipped with Polaroid HPBN polarizers and a thermostatted cell holder. Time resolved fluorescence measurements were performed using a time correlated single photon counter equipped with an Edinburgh F199 nanosecond flashlamp, a Philips XP2020Q photo- multiplier, Ortec fast NIM electronics, and a Silena BS27N multichannel analyzer.

The intensity decays were found to be best described by the following equation

I(t) = c~lexp(--t/rl) + o~2exp(-t/r2) (1)


4 Landi et al.

where c~1 and 0/2 are the preexponential terms, and ;1 and "/'2, the fluorescence lifetimes.

The fractional fluorescence associated to the two components was calculated using the equation

Fi = c~i Ti / )'-~i o/i Ti (2)

For polarization measurements , polaroid film polarizers were used. For both static- and time-resolved fluorescence experiments, Mx was 337 n m (N2 line) and Mm was 440 nm.

Time-resolved anisotropy was determined by measur ing the vertical- vertical and the vertical-horizontal fluorescence intensity decays. Data were processed by using a nonlinear least-square procedure (17). Order parameters were calculated as

<P2> = / ro) '2 (3)

where r , is the limiting anisotropy and ro was assumed to be 0.395 (18). The correlation times (q~) and r= were calculated from the following re- lationship

r (t) = (ro - r . ) e x p ( - t /~ ) + r= (4)

The quenching rate constant was derived from the equation

To / r = 1 + Kq ro [Q] (5)

where ro is the lifetime in the absence of Q-3, r the lifetime in the presence of Q-3, Kq the quenching rate constant, and [Q] the concentration of the quinone expressed as nmol Q/mg PL.

RESULTS

Unilamellar vesicles containing an amount of Q-3 approaching the physiological content of quinone in mitochondria and chloroplasts (19), i.e., 12-13 nmol /mg PL and control vesicles were incubated at 30~ for different times in the presence of 0.3 mM FeC12.

Table I shows the stimulatory effect of ferrous chloride on MA formation in the vesicles. Peroxidation increased in the first 10 min up to a maximum value of 17 nmol of MA/mg PL and, subsequently, remained fairly constant. In Q-containing vesicles, MA formation was very low and cor- responded to about 3 nmol /mg PL at the end of the incubation. In order to test whe the r degradation of Q-3 would occur in the experimental conditions employed, the quinone was extracted from the vesicles at 0 and 30 min of peroxidation and determined by reversed-phase HPLC. The chromatographic separation was necessary to avoid spectral interferences by BHT, present in the TBA reagent, with Q. Q-3 concentration, and re- tention time did not change during Fe2§ lipid peroxidation.

Cell Biophysics Vol. 16, 1990

Q Antioxidant Effect on Vesicle Properties

Table 1 Fe2+-Dependent Malonaldehyde Formation with Time

in Control Vesicles and Q-Containing Vesicles

5

Time, min

Malonaldehyde formation, nmol/mg PL

Control vesicles a Q-containing vesicles b

0 2.0 0.1 5 6.0 0.3 10 17.0 1.3 15 17.3 1.8 30 17.5 3.0

aVesicle preparations were obtained, as described in the Materials and Methods section. The reaction mixtures were incubated at 30~ Fe 2+ as FeCl2 0.3 mM.

bQ-3 incorporated was 14 nmol /mg PL. After 30 min of incubation, Q-3 content was unchanged.

15 -1500

0

i 0 10

0 0

~ \ 0 % 0 . . . . . . �9 . . . . . . . . . . . �9

- - - - 0

I I I 5 10 15

Q - 3 ( n m o l / m g P h )

1000

500

I I

J o

re O O

Fig. 1. Fe2+-dependent malonaldehyde and hydroperoxide formation vs Q-3 amount incorporated into vesicles. �9 �9 malonaldehyde formation; �9 0, lipid hydroperoxide formation. For determination of Q-3, malonaldehyde, lipid hydroperoxides, and total lipid concentration, see the Materials and Methods section.

Figure 1 repor ts the ant ioxidant effect of Q-3 vs the a m o u n t of the quinone incorporated into the vesicles, de termined both as MA and L O O H format ion in the presence of 0.3 m M FeC12. It can be seen that m a x i m u m protect ion occurred w h e n the a m o u n t of qu inone incorporated into the mode l m e m b r a n e s was in the range of the mitochondrial content . Further- more, at concentra t ions of about 3 n m o l / m g PL, both MA and L O O H format ion approximately u n d e r w e n t a 50% inhibition.


6 Landi et al.

Table 2 Fatty Acid Composition of Egg Lecithin Vesicles

Before and After Fe2+-Induced Peroxidation

Control Q-containing vesicles

Before After Before After Fatty acid a peroxidation peroxidation peroxidation peroxidation

16: 0 32.21 33.26 32.25 32.30 16:1 3.18 3.10 3.20 3.20 18:0 11.72 12.42 11.80 11.83 18:1 32.78 32.65 32.49 32.50 18:2 (n-6) 14.07 14.03 14.20 14.20 20:4 (n-6) 3.78 3.05 3.76 3.72 22:6 (n-3) 1.97 1.34 1.99 1.95

aGaschromatographic analysis was performed according to (15). The fatty acid compo- sitions (as methylesters) are expressed as percentages of the total weight of identified fatty acids. Data are the mean values of at least two analyses.

In Table 2, the percent composit ion of fatty acid residues of control and Q-containing vesicles, before and after iron-induced peroxidation, is reported. It can be observed that, in the absence of Q-3, there was a 24% decrease in the content of polyunsaturated fatty acids (20:4 and 22:6), whereas in its presence, only a 1% decrease occurred.

The effect of oxidized Q-3 on the static and dynamic properties of the lipid vesicles, as monitored by the fluorescence intensity and the anisotropy decay of DPH, was studied. The fluorescence intensity decayed with lifetimes of 1"1 --9 ns and ~-2--4 ns, in the absence of Q-3 (x 2= 1.4). The behavior of the two lifetimes as a function of the actual concentrat ion of Q-3 is shown in Fig. 2, where it can be seen that both lifetimes decreased w h e n the concentration of the quinone was increased. At increasing Q-3 concentrations, the fluorescence associated with the two lifetimes decreased for the long lifetime, whereas it increased for the short one, as shown in Fig. 3. Quenching experiments, reported in Fig. 4, demonst ra te that the long lifetime component was collisionally quenched, whereas a more complex molecular process was involved in the quenching of the short lifetime component .

The correlation time of DPH as a function of quinone concentration, both in peroxidized and not peroxidized vesicles, slightly decreased at increasing quinone concentrations (Fig. 5). A discontinuity at Q-3 values of 7 and 12 nmol /mg PL was observed. When the vesicles were sub- jected to peroxidation, in the absence of the quinone, the probe correlation time increased by about 50%, whereas it was virtually unchanged in the presence of Q-3.

Cell Biophysics Vol. 16, I ggo


i 0 . 0

8.0

co 6.0,

b- 4.0

2.0

0.0

~A~A~A

--A A

I i i I [

0 5 10 15 20 25

Q-3 (nmol/mgPL)

Fig. 2. Lifetimes of DPH vs Q-3 amount incorporated into vesicles. A A, short lifetime; �9 � 9 long lifetime. Lifetimes were obtained from nonlinear least-square analysis of decay, as described in the Materials and Methods section.

1.0

0.8

r

o 0.6

"~ 0.4. r~ 0

0 0.2.

~ A

~ A

'A ~ A j

0.0 f j t I I 0 5 10 15 20 25

Q-3 (nmol/mgPL)

Fig. 3. Fractional fluorescence of DPH associated to lifetimes. A .&, short lifetime; �9 � 9 long lifetime. Values were calculated from Eq. 2 (see Materials and Methods section).


8 Landi et al.

2.0.

b

b o

1.5

1.0

0.~

a j l

I I I f I I I 0 10 20 30 40 50 60 70

[Q-3] (rnM)

Fig. 4. Stern-Volmer plots of short (G G) and long (A A) lifetimes of DPH. Quencher concentration is the actual concentration of Q-3 incorporated into the membranes. Quenching constant for the long component is 7.32 "lO-SlM/s calculated from Eq. 6 (see Materials and Methods section).

5.0

%-,

4.0

3 .0 .

2.0

i .0

T 5 o

1 I I ! I 1

0 5 10 15 20 25

Q - 3 ( n m o l / m g P L )

Fig. 5. Rotational correlation time (4) of DPH vs Q-3 amount incorporated into vesicles, o, not peroxidized vesicles; �9 peroxidized vesicles. Error bars represent the standard error of the average of three experiments. Values of

were obtained from nonlinear least-square analysis of decay data, as described in the Materials and Methods section.



DISCUSSION

When model membranes were prepared by ultrasonic irradiation, radical production and consequent partial degradation of unsaturated fatty acid residues occurred, as it can be seen from Table 1. MA formation is negligible at time 0 in Q-containing vesicles, but evident in controls. Our previous data (20) suggest that the mechanism by which ubiquinones could serve as antioxidants during the preparation of vesicles by sonica- tion is not owing to their reactions with the radicals responsible for the initiation of lipid peroxidation. In these conditions, oxidized Qs probably act as chain-breaking antioxidants, scavenging lipid radicals that propagate the free-radical oxidation chain.

The antioxidant action of Q was also evident when tested on a peroxidative system in which the reaction was promoted by ferrous ions. In our experimental conditions, oxidized Q, incorporated into the bilayer at a concentration of 14 nmol/mg PL, provides an 80% inhibition of the lipid peroxidation (see Table 1). Such a protective effect is also demonstrated in Table 2 where, in the presence of the quinone, the content of arachidonic and docosahexaenoic acids in the vesicles was reduced by only 1% (3 nmol), whereas in control vesicles, it decreased by 24% (35 nmol). Thus, the data on MA formation (17.5 nmol) and polyunsaturated fatty acids loss, in accord with those of May and Mc Cay (21), do not support the view that the amount of MA produced corresponds stoichiometrically to the amount of polyunsaturated fatty acids peroxidized (22).

When the antioxidant effect of Q was evaluated as a function of its incorporated amount, a very interesting result could be observed. Q concentrations much lower than those physiologically found in mitochondria and chloroplasts were able to inhibit lipid peroxidation (Fig. 1).

As for a possible mechanism of action of Q as an antioxidant, first some general considerations have to be made. At pH 7.2, iron salts can stimulate lipid peroxidation by directly reacting with molecular oxygen to produce either hydroxyl radical or a species with similar reactive properties or by decomposing lipid peroxides to form alkoxyl and peroxyl radicals. Such radicals can initiate lipid peroxidation by abstracting a hydro- gen atom from unsaturated fatty acids, a reaction followed by 02 uptake and propagation. Experiments reported in (20) and others performed by using the thermal decomposition of a lipophilic azocompound, azobis (2,4-dimethyl-valeronitrile) (23), to ensure that initiation of the radical chain occurs within the lipid bilayer with no involvement of oxygen free radicals, show that Q scavenges lipid free radicals that propagate the oxidation chain. The present data do not clarify whether Q also inhibits the initiation step. To this end, more experiments are required.

Experiments were then carried out in order to verify: first, the effects of Q-3 on static and dynamic properties of control membranes; and second, what effect the antioxidant action of the quinone had on the structural organization of the membranes during peroxidation.


I0 Landi et al.

Our studies, in agreement with the data of Kawato et al. (18), show that two distinct populations of DPH are present within the lipid matrix: one located in the core of the membrane, as indicated by its long fluorescence lifetime, and one located more superficially, in a more polar environ- ment, that causes the lifetime to be shortened to a value of about 4 ns (Fig. 2). Both probe populations are available to quenching by Q-3, even if by different mechanisms. Furthermore, Q-3 is able to change the probe distribution between the two populations, as shown by the values of the fractional fluorescence reported in Fig. 3: as more Q-3 becomes incorporated, more DPH is dislocated toward the surface of the bilayer. The overall effects of the quinone on the static and dynamic properties of the model membranes are: a slight increase in the order parameter of DPH from 0.30 to 0.35, and, therefore, a very small effect in the ordering of the fatty acid chains; and a more noticeable variation of the probe correlation time that decreases when Q concentration increases and reaches its minimum value in the Q concentration range of 7-12 nmol/mg PL. Conversely, the bilayer fluidity, which is inversely related to the hindrance of the probe motion among the fatty acid chains, is increased by increasing amounts of incorporated Q and consequently reaches its maximum in the same concentration interval of the quinone.

Interestingly, this range is at which the maximum efficiency of Q-3 as an antioxidant is observed (see Fig. 1). When the vesicles are peroxidized in the absence of Q, the fluidity markedly decreases, whereas in its presence, it is nearly unchanged. These data well correlate with the unsaturated fatty acid composition (24-26), markedly decreased in the first case, and nearly unchanged in the second one, as shown in Table 2.

The results presented in this paper confirm that Q-3 possesses reason- ably good antioxidant properties and suggest that these can be ascribed to its ability of reacting with free radicals. They also show that the quinone incorporation within the membrane increases its fluidity and that the antioxidant action of Q-3, at physiological concentrations, results in maintaining such fluidity unperturbed during the peroxidative insult. Even if it is not demonstrated that fluidity changes per se are protective, a pro- vocative speculation seems to be that a requisite for the coenzyme to act as an efficient antioxidant could be its ability to readily diffuse within the membrane.

SUMMARY

Malonaldehyde formation promoted by ferrous ions was neligible in Q-containing vesicles, but evident in controls. Moreover, Q-3 concentrations lower than physiological ones were able to inhibit lipid peroxidation.

In Q-containing vesicles, the fluorescence lifetime and the fluorescence anisotropy decay of DPH was measured in order to verify both the


Q An t iox idan t Effect on Vesicle Properties 11

effect of Q on static and dynamic properties of control membranes and the structural organization of the membrane during peroxidation.

The present data suggest that the antioxidant properties of Q-3 can be ascribed to its ability of reacting with free radicals. They also show that quinone incorporation within the membrane increases its fluidity and that the antioxidant action of Q-3, at physiological concentrations, results in maintaining such fluidity unperturbed during the peroxidative insult.

ACKNOWLEDGMENTS

Q-3 was a kind gift from Eisai Co., Tokyo, Japan. We thank V. Barzanti for the determination of fatty acid composition. This work was supported by grants from MPI and CNR, Rome, Italy.

REFERENCES

1. Crane, F. L. (1977), Ann. Rev. Biochem. 46, 439. 2. Green, D. E. and Wharton, D. C. (1963), Biochem. Zeit. 338, 335. 3. Gupte, S., Wu, E. S., Hoechli, L., Hoechli, M., Jacobson, K., Sowers, A. E.,

and Hackenbrock, C. R. (1984), Proc. Natl. Acad. Sci. USA 81, 2606. 4. Stidham, M. A., McIntosh, T. J., and Siedow, J. N. (1984), Biochim. Biophys.

Acta 767, 423. 5. Aranda, F. J. and Gomez-Fernandez, J. C. (1985), Biochim. Biophys. Acta

820, 16. 6. Crane, F. L. and MorrO, D. J. (1977), Biomedical and Clinical Aspects of Coen-

zyme Q, vol. I (Folkers, K. and Yamamura, Y., eds.), Elsevier, Amsterdam, pp. 3-14.

7. Crane, F. L., Sun, I. L., Barr, R., and Morr6, D. J. (1984), Biomedical and Clini- cal Aspects of Coenzyme Q, vol. IV (Folkers, K. and Yamamura, Y., eds.), Elsevier, Amsterdam, pp. 77-86.

8. Crawford, D. R. and Schneider, D. L. (1983), J. Biol. Chem. 258, 5363. 9. Folkers, K. (1985), Coenzyme Q (Lenaz, G., ed.), Wiley, London, pp. 457-478.

10. Cabrini, L., Pasquali, P., Tadolini, B., Sechi, A. M., and Landi, L. (1986), Free Rad. Res. Comms. 2, 85.

11. Landi, L., Cabrini, L., Tadolini, B., Fahmy, T., and Pasquali, P. (1985), Appl. Biochem. Biotech. 11, 123.

12. Tien, M., Svingen, B. A., and Aust, S. D. (1982), Arch. Biochem. Biophys. 216, 142.

13. Cavallini, L., Valente, M. V., and Bindoli, A. (1982), Biochim. Biophys. Acta 752, 339.

14. Stoffel, W., Chu, F., and Ahrens, E. H. (1959), Anal. Chem. 31, 307. 15. Takada, M., Ikenoya, S., Yuzuriha, T., and Katayama, K. (1982), Biochim.

Biophys. Acta 679, 308. 16. Marinetti, G. V. (1962), J. Lipid Res. 3, 1.


12 Landi et al.

17. Chen, L. A., Dale, R. E., Roth, S., and Brand, L. (1977), J. Biol. Chem. 252, 2163.

18. Kawato, S., Kinosita, K., and Ikegami, A. (1977), Biochemistry 16, 2319. 19. Futami, A., Hurt, E., and Hauska, G. (1979), Biochim. Biophys. Acta 689, 363. 20. Landi, L., Pasquali, P., Bassi, P., and Cabrini, L. (1987), Biochim. Biophys.

Acta 902, 200. 21. May, H. E., and McCay, P. B. (1968), J. Biol. Chem. 243, 2296. 22. Fukuzawa, K., Chida, H., Tokumura, A., and Tsukatani, H. (1981), Arch.

Biochem. Biophys. 206, 173. 23. Landi, L., Fiorentini, D., Cabrini, L., Stefanelli, C., Sechi, A. M., and Pe-

dulli, G. (1989), The Biochemistry Bioenergetics and Clinical Applications of Ubiquinone. Taylor & Francis Ltd., London, in press.

24. Masotti, L., Cavatorta, P., Ferrari, M. B., Casali, E., Arcioni, A., Zannoni, C., Borrello, S., Minotti, G., and Galeotti, T. (1986), FEBS Lett. 198, 301.

25. Galeotti, T., Borrello, S., Minotti, G., and Masotti, L. (1986), Ann. NYAcad. Sci. 448, 468.

26. Masotti, L., Casali, E., and Galeotti, T. (1988), Free Radical Biology & Medicine 4, 377.

Cell Biophysics Vol. 16. 1990

Coenzyme Q-3 as an antioxidant

Documents

Transcript of Coenzyme Q-3 as an antioxidant