protection by vitamin e, selinium, and β-carotene against oxidative damage in rat liver slices and...

Free Radical Biology & Medicine, Vol. 14, pp. 473-482, 1993 0891-5849/93 $6.00 + .00 Printed in the USA. All fights reserved. Copyright © 1993 Pergamon Press Ltd.

Original Contribution

P R O T E C T I O N BY V I T A M I N E, S E L E N I U M , A N D ~ / - C A R O T E N E

A G A I N S T O X I D A T I V E D A M A G E I N R A T L I V E R SLICES

A N D H O M O G E N A T E

H A O CHEN,* LORJ J. PELLETT,* HENRIK J. ANDERSEN, "f and A. L. TAPPEL*

*Department of Food Science, University of California, Davis, CA 95616, USA; and tRoyal Veterinary & Agriculture University, Denmark, Department of Dairy and Food Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

(Received 24 August 1992; Revised 25 November 1992; Accepted 30 November 1992)

Abstract--Male Sprague-Dawley (SD) rats were fed a vitamin E and selenium deficient diet and diets supplemented with vitamin E, selenium, B-carotene, and a combination of the three. Tissue slices and homogenate of liver were incubated at 37 °C with and without the presence of prooxidants. The effect of vitamin E, selenium, E-carotene, and the combination of the three antioxidants on the oxidative damage to rat liver tissue was studied by measuring the production of oxidized heme proteins in both tissue slices and homogenate during spontaneous and prooxidant-induced oxidation. The diet with the combination ofaU three antioxidants showed a strong protective effect against oxidative damage to heme proteins in contrast to the antioxidant-deficient diet. In general, diets with vitamin E, selenium, and ~-carotene were less effective than the combination of all three antioxidants. The protective effect of antioxidants on the heme protein oxidation was correlated with their inhibitory effect on lipid peroxidation measured as the production of thiobarbituric acid-reactive substance (TBARS). The protection of antioxidants on heine proteins was also dependent on the type of oxidation inducer. Possible mechanisms of antioxidants against oxidation in liver tissues are discussed.

Keywords--Heme proteins, Vitamin E, Selenium, B-carotene, Oxidative damage, Tissue slices, Liver homogenate, Free radicals

INTRODUCI'ION

According to their physiological functions, cellular antioxidants can be divided into three categories: preventive antioxidants, chain-breaking antioxidants, and repair and de novo compoundsJ -3 The primary role of preventive antioxidants is to reduce the rate of initiation of the free radical chain reaction. 2 When the chain reaction is initiated, the chain-breaking antioxidants will interact rapidly with the radicals and con- vert them to more stable compounds, thus delaying or inhibiting the propagation of the free radical chain reaction. 2'4 The preventive and chain-breaking antioxidants are considered as a first level of protection against oxidative damage. The second level of protection against oxidative injury in cells comes from a series of repair systems. 5 The repair systems include enzymes that attempt directly to restore biomolecules to their native confirmation, as well as catabolic en-

Address correspondence to: A. L. Tappel.

zymes that specifically degrade nonfunctional proteins, lipids, and nucleic acids.l'6

Vitamin E is a well-known chain-breaking antioxidant. 7,s a-Tocopherol donates one hydrogen to a free radical and the radical is converted to a nonradical product. Because of its stable resonance structure, the oxidized a-tocopherol is unreactive to continue the chain reaction. 2 Selenium is a trace nutrient and serves as an active site of glutathione (GSH) peroxidase. s'9 GSH peroxidase is a preventive anfioxidant since it catalyzes the conversion of hydroperoxides to stable nonradical products in which the generation of free radicals is inhibited. 2'3 Beta-carotene can be both a preventive and chain-breaking antioxidant since it effectively scavenges singlet oxygen and other reactive oxygen species to prevent the initiation of oxidation as well as trapping free radicals to break the propagation of the chain reaction. 2,1°

Oxidative damage of heme proteins involves a redox reaction of the heme group with one- or two-elec- tron(s) transfer and the denaturation of the globin structure. H-14 Under conditions of oxidative stress,

473

474 H. CHEN et al.

oxyhemoglobin is oxidized into methemoglobin due to the oxidation ofheme ferrous ion to ferric ion with either OH -z or H20 in the sixth coordination site. When the sixth coordination site is occupied by either the distal histidine or an external ligand, methemoglobin is converted to hemichrome, the main constituent of Heinz bodies. 1 i,l 2 Both methemoglobin and hemichrome can be measured by spectrophotometry. 13,~4 Since heme proteins are abundant in animal tissues and organs, their oxidized products might be a good index to measure oxidative stress in tissues.

Recently we have used a computer aided heme protein spectra analysis program (HPSAP) to determine quantitatively the composition of heme proteins in liver tissues. The HPSAP is a spreadsheet calculation program using Lotus 1, 2, 3 that is used to quantify heme proteins both in solution and in animal tissues. This program is based on the knowledge that the absorbance spectrum of a mixture of heme proteins is the sum of the spectra of the individual heme proteins including any contribution from turbidity of the biological samples. Quantitation is achieved by matching the calculated spectrum with the experimental spectrum through successive ap- proximations. The determination with HPSAP for heine proteins can be highly accurate because they closely obey Beer's Law and any interaction between individual heme proteins, such as redox reactions, will result in other measurable heme proteins.

In this investigation vitamin E, selenium,/3-carotene, as well as the combination of all three antioxidants were added to the rats' diets and the rats were fed for 6 weeks. The liver slices and homogenate were incubated at 37°C with and without the presence of various prooxidants. The absorbance spectra of liver slices and homogenate were analyzed with HPSAP and the concentrations of oxidized heme proteins were determined. The possible mechanisms of individual antioxidants and the combination of antioxidants in the protection of liver against oxidative stress are discussed.

M A T E R I A L S A N D M E T H O D S

Chemicals

The chemicals used in this study were tocopherol acid succinate (1210 IU/g); trans-/3-carotene (95%); dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO); sodium selenite (Alfa Inorganics, Beverly, MA); bromotrichloromethane (CBrC13) (Eastman Kodak Co., Rochester, NY); t-butyl hydroperoxide (TBHP) (Polysciences, Inc., Warrington, PA); and ferrous sul- fate (FeSO4) (Fisher Scientific, Fairlawn, NJ).

Table 1. An t iox idan t s Add i t i on to Die ts l

Vit. E 2 Se l en ium 3 ~-Caro tene 4 Die t G r o u p ( I U / k g diet) ( m g / k g diet) ( m g / k g diet)

1 m m _ _

2 30 - - - - 3 - - 0.3 - - 4 - - - - 45 5 30 0.3 45

1 The basal diet was bo th v i t a m i n e E a n d se len ium deficient and con t a ined 10% tocophero l - s t r ipped corn oil.

2 (+) a - tocophero l acid succinate . 3 As sod ium selenite. 4 Trans-/3-carotene.

Animals and diets

Male Sprague-Dawley rats (Bantin & Kingman, Fremont, CA) weighing 40-60 g were adapted to their surroundings for 2 d before being fed with experimental diets. The basal diet was a vitamin E and selenium deficient diet with 10% tocopherol stripped corn oil (Teklad Test Diet #TD 77068, mineral mix #170911, Teklad Test Diets, Madison, WI). Animals were housed according to NIH guidelines and had free ac- cess to deionized water and food. Dietary treatment had no effect on weight gain of the animals. The protocol for the diet study is shown in Table 1. The rats were on the experimental diets and distilled water for 6 weeks.

Preparation of liver tissue slices and homogenate

The rats were decapitated and livers were immediately dissected and immersed in ice-cold Krebs- Ringer phosphate (KRP) buffer (pH 7.4). Liver was cut in 0.5 cm 3 cubes by a sharp surgical knife and sliced into 0.5 mm thick pieces (80-100 mg) by a Sta- die-Riggs tissue slicer (Thomas Scientific, Philadel- phia, PA) according to a method described by Gavino et al. ~5

Homogenate was prepared by homogenizing 1 g of liver and 9 ml of oxygenated KRP buffer containing glucose (10 mmol) (pH 7.4) using a motor-driven tissue homogenizer.

Oxidative reactions in liver slices and homogenate

About 90 mg of liver slices in a 10-ml glass serum bottle containing 5 ml of oxygenated KRP-glucose buffer (concentration of glucose was 10 mM) were incubated in a gyrotory water bath shaker (New Brunswick Scientific Co., Inc., New Brunswick, N J) at 37°C with continuous shaking (180 cycle/min). CBrC13 and TBHP were dissolved in dimethylsulfox-

Antioxidants against heme protein oxidation 475

ide, and FeSO4 was dissolved in distilled water. Proox- idants were added to the serum bottles immediately before the incubation.

Liver homogenate (1.5 ml) was transferred to a 10- ml glass serum bottle and incubated at 37°C in the same manner as the tissue slices.

Spectrophotometric measurement of tissue slices and homogenate

After incubation of both tissue slices and homogenate, the absorbance spectrum of each sample was obtained with a Beckman DU-50 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). For tissue slices, a 50-mg sample was blotted with a filter paper and then transferred to a spectrophotometer cell of 5.5 mm i.d. and a light path of 2.0 mm. In the air-tight cell the heme proteins in the tissue slices came to a redox equilibrium determined by the physiological conditions of the tissues. Four layers of parafilm representing turbidity were used as a background to blank the spectrophotometer and to subtract some of the absorbance of turbidity caused by the tissues. Spectra can be corrected for scattered light loss by using a reference material with similar scattering prop- erties to blank the spectrophotometer. ~6 We have found parafilm to be a useful reference material for tissues over the wavelength range of 500 nm to 640 nm. The cell was sealed by a microscope cover glass and mounted on the center of the window of the spectrophotometer near the photoreceptor to reduce the light scattering caused by the tissue. The sample was scanned from 500 nm to 640 nm and the absorbance versus wavelength at 5-nm intervals was automati- cally recorded by a scan program in the spectrophotometer.

For the liver homogenate, 0.6 ml of sample was transferred to a microcuvette with a light path of 10 mm and mixed well with 0.6 ml glycerol. Four layers ofparafilm were also used as a background. The spectrophotometric measurement of liver homogenate followed the same protocol as the measurement of tissue slices.

Analysis of absorbance spectra of heme proteins of liver tissue slices and homogenate with HPSAP

HPSAP is a spreadsheet program written with Lotus 1, 2, 3, (Lotus Development Corp., Cambridge, MA) that contains visible spectra of individual heme proteins from 500 to 640 nm. Micromolar extinction coefficients were obtained from the literature ~4 for hemoglobin, oxyhemoglobin, methemoglobin, ferryl hemoglobin, and hemichromes. Micromolar extinction

coefficients for reduced and oxidized mitochondrial and microsomal cytochromes were calculated by ob- taining micromolar extinction coefficients of the major mitochondrial and microsomal cytochromes from the literature and summing their contributions based on their proportions in specific tissues. Thus for rat liver tissues at each wavelength the extinction coefficient for the mitochondrial cytochromes is the sum of 29% cytochrome aa3, 30% cytochrome b, 18% cytochrome c~, and 23% cytochrome c;, the extinction coefficient for microsomal cytochromes is the sum of 40% cytochrome b and 60% cytochrome P-450. Other chromophores are not important as shown by their maximum absorption. For instance, bilirubin, ravin, and beta-carotene all have maximum absorption at 450 nm.

To determine the concentrations of individual heme proteins from a mixture, the experimental visible absorbance versus wavelength at 5-nm intervals from 500 nm to 640 nm was entered into cells in the spreadsheet. To match the experimental spectrum, a calculated spectrum was established. Micromolar values for expected individual heme proteins were entered into concentration cells. Values for contributions due to turbidity at 500 nm and 640 nm were also entered into the cells for turbidity. The turbidity contributions at other wavelengths were calculated by HPSAP by constructing a linear absorbance versus wavelength line between the turbidity values entered for 500 nm and 640 nm. Contributions of the individual heme proteins were calculated using Beer's Law and the extinction coefficients in cells for those specific heme compounds. We followed the techniques of addition of spectra as in multicomponent analysis. 17 All of the contributions were summed for each wavelength and the results were displayed on the spreadsheet as calculated absorbance, along with the difference between the experimental and calculated absorbance and the percent error at each wavelength. Both experimental and calculated spectra were viewed on the CRT screen. To achieve the best superposition of calculated and experimental spectrum, micromolar values of individual heme proteins and values for turbidity contributions at 500 nm and 640 nm were adjusted to minimize the differences between the two spectra. When the best match was achieved, the concentrations of individual heme proteins in the mixture were determined. Validation of this spectral analysis technique is reported.~S For validation we showed that four different mixtures of mi- tochondria and hemoglobin gave a calculated composition in agreement with that expected for the mixtures. As with the interpretation of any spectra of a mixture of components, knowledge of the approxi-

(a) mate composition of the mixture is important. Meth- ods of solving for the composition ofheme proteins in liver are further described. 18 The solution using HPSAP converges on the most probable composition.

Assay of thiobarbituric acid-reactive substance

Lipid peroxidation in liver homogenate was as- sessed by measurement ofthiobarbituric acid-reactive substance (TBARS). A 10% liver homogenate from 2 rats/diet was prepared by homogenizing 1 g liver/9 ml of KRP buffer (pH 7.4). Homogenization was con- ducted using a motor-driven tissue homogenizer. Liver homogenate was incubated in I 0-ml serum bottles in a 37°C water bath and shaken at 180 cycle/ rain. Prooxidant solutions were prepared by dissolv- ing CBrCI3 and TBHP indiethyl sulfoxide, and FeSO4 in distilled water. Prooxidants (50 raM) were added to the homogenate immediately before incubation in the water bath. Each treatment was run in duplicate. A l-ml aliquot of homogenate from each treatment was removed at 0.5, 1, and 2 h and stored at -20°C for subsequent analysis. Samples were analyzed using the method of Fraga et al.19

A

C •

476 H. CHEN el aL

500 520 540 560 580 600 620 640 Wavelength (nm)

Statistical analysis

The statistical package SAS (SAS Institute Inc., Cary, NC) was used to analyze all data. Results were expressed as means + standard deviation. Data were analyzed using analysis of variance (ANOVA). When significant F values were obtained, the method of least square means was used to determine the significant differences (P ~< .05) between treatment means.

RESULTS

Figure 1 and Table 2 show the application ofabsor- bance spectral measurement and HPSAP to study heme protein oxidation. Oxidative reactions of heme proteins in both liver slices and homogenate caused spectral changes. Even though the changes were less distinct in liver homogenate than tissue slices, Fig. 1 shows that the absorbance spectra of heme proteins can be used as a measure of heme protein oxidation. In general, the calculated spectrum using HPSAP showed good agreement with the experimental spectrum. The performance of HPSAP (i.e., the achieve- ment of best superposition between experimental and calculated spectra) is indicated by the average error of calculation. By matching with the experimental spectrum, the concentrations of individual heme proteins, expressed as percentage of micromolar concentration, at the different stages of reaction were determined

)

l~Absorbence =0.4

I I I J ~ J J 500 520 540 560 580 600 620 640

Wavelength (nm) Fig. 1. Spectral changes of heme proteins in liver slices (la) and homogenate (lb); OOOO, experimental spectrum; - - , calculated spectrum; A, fresh sample; B, after 1 h of incubation at 37°C; C, after 2 h of incubation at 37°C. CBrC13 (0.1 raM) was added as an oxidation inducer.


Table 2. Analysis of Spectra Obtained from Liver Slices and Homogenate Incubated with CBrC13 at 37°C with HPSAP*

Incubation Time = 0 h Micromolar (%)

Tissue Heme Proteins Slices Homogenate

Oxyhemoglobin 0.00 74.00 Hemoglobin 73.00 0.00 Reduced mitochondrial cytochromes 16.00 15.00 Reduced microsomal cytochromes I 1.00 11.00 Total oxidized heme proteins 0.00 0.00 Average calculation error 0.83 0.16 Incubation Time = l h Oxyhemoglobin 0.00 45.00 Hemoglobin 47.00 0.00 Methemoglobin 6.00 26.00 Hemichrome 20 4.0 Reduced mitochondrial cytochromes 13.00 2.00 Oxidized mitochondrial cytochromes 3.00 I 1.00 Reduced microsomal cytochromes 8.00 4.00 Oxidized microsomal cytochromes 3.00 8.00 Total oxidized heme proteins 32.00 49.00 Average calculation error 1.00 0.15 Incubation Time = 2 h Oxyhemoglobin I 1.00 35.00 Hemoglobin 35.00 0.00 Methemoglobin 7.00 22.00 Hemichrome 20.00 18.00 Reduced mitochondrial cytochromes 4.00 0.00 Oxidized mitochondrial cytochromes 12.00 13.00 Reduced microsomal cytochromes 4.00 0.00 Oxidized microsomal cytochromes 7.00 12.00 Total oxidized heme proteins 46.00 65.00 Average calculation error 0.16 0.32

* Both liver slices and homogenate were from the rats fed with basal diet for 6 weeks. Concentration of CBrCI 3 was 0.1 mM.

with HPSAP, and the results are listed in Table 2. Hemoglobin is the major heme protein in fresh liver slices, while oxyhemoglobin is the predominant one in fresh liver homogenate. The total oxidized heme proteins, including methemoglobin, hemichrome, and oxidized mitochondrial and microsomal cytochromes, increased as incubation time increased. The total amount of hemoglobin and its oxidized products appeared to be consistent during the oxidative reaction in both tissue slices and homogenate, as did the total amounts of reduced mitochondrial and microsomal cytochromes and their oxidized products. These observations suggest that hemoglobin and oxyhemoglobin were oxidatively converted into methemoglobin and hemichrome, while reduced mitochondrial and microsomal cytochromes were converted into oxidized mitochondrial and microsomal cytochromes. Heine proteins were more susceptible to oxidative damage in the homogenate than tissue slices. The better protection of heme proteins in tissue slices was likely due to the integrity of cellular struc- tures and the antioxidant defense systems. The domi- nating reduced heine proteins were hemoglobin and

oxyhemoglobin in tissue slices and homogenate, re- spectively. Hemichrome was the major oxidized heme protein in tissue slices, while methemoglobin appeared to be the main oxidized compound in liver homogenate.

The effect of dietary treatment with antioxidants on the oxidative damage to liver slices is presented in Fig. 2. The combination of vitamin E, selenium, and 15-carotene was most protective against oxidative damage. Vitamin E, selenium, and B-carotene alone did not show an evident protective effect at the early stage of the oxidation (i.e., incubation for 0.5 h). As incubation time increased, vitamin E showed strong protection against oxidation. Selenium appeared to be an effective antioxidant between 1 and 2 h of incubation. The accumulation of oxidized heme proteins did not increase within this period in animals fed a selenium- adequate diet. Beta-carotene showed a moderate protective effect during l and 2 h of incubation.

A similar trend of protection against oxidation of heme proteins was observed in tissue slices incubated with CBrC13 (Fig. 3). At short periods of incubation (0.5 and 1 h), vitamin E, selenium, and B-carotene did not show an antioxidant effect. In contrast, the combination of the three antioxidants exhibited a protective effect on heme proteins even though the effect was rather moderate compared to that shown in the spontaneous oxidation. At longer periods of incubation, all individual antioxidants and the combination of vitamin E, selenium, and 15-carotene reduced production of oxidized heme proteins 20-30% compared to the production from basal diet treatment.

The protective effect of a combination of vitamin E, selenium, and S-carotene in the homogenate sys- tem is presented in Fig. 4. During the early stages of spontaneous oxidation (0.5 h), the antioxidants effectively deceased the rate of the reaction by reducing approximately 50% of oxidative conversion of heme proteins. Another significant feature is that the antioxidants completely inhibited further heme protein oxidation between l and 2 h of incubation. The antioxidants also showed a strong protective effect on heme protein oxidation during the incubation of tissue slices with CBrC13.

The effectiveness of antioxidants against oxidative damage of heme proteins was also dependent on the type of oxidation inducer (Fig. 5). Vitamin E seemed to be more effective in protection against iron, while selenium was most effective against TBHP. Beta-carotene showed protective effect against TBHP, but not against iron. In general, the combination of all three antioxidants had the most effective protection against oxidation induced by both TBHP and iron.

Figure 6 presents the TBARS measurements in

478 H . C . E N et al.

7 0 -

= 6O ° ~

~- 50 Q_

E 40 q~

30 ._~ ° ~

,~ 20 O

10

0

m

IT

0.5 1.0

Time (hr)

Z T " iiiiiii .

2.0

Fig. 2. Production of oxidized heine proteins during the spontaneous oxidative reaction in liver slices; U, basal diet; D, vitamin E + basal diet; II, selenium + basal diet; ~], B-carotene + basal diet; I~, vitamin E, selenium, and B-carotene + basal diet. Liver slices were incubated at 37°C. The values are expressed as mean _+ SD for four rats. The mean values marked with * were significantly smaller than the mean of basal diet group at a 95% confident level. At 0 time there was 0% oxidized heme proteins.

liver homogenate. As expected, the combination of vitamin E, selenium, and S-carotene showed an inhibitory effect on lipid peroxidation. There are many measurements of oxidative damage to animal tissues.

It is beneficial, therefore, to correlate the production of oxidized heme compounds with other measurements of oxidative stress. Figure 7 shows the correla- tions between the production of oxidized heme pro-

7 0 -

= 60 - T

I |

5O- T

40 iiii!ii 30

N z0

~ 10

0 ~ 0.5 1.0 2.0

Time (hr) Fig. 3. Production of oxidized heme proteins during CBrC13-induced (0.1 mM) oxidative reaction in liver slices; [2, basal diet; IN, vitamin E + basal diet; w, selenium + basal diet; r~, B-carotene + basal diet; [], vitamin E, selenium, and B-carotene + basal diet. Liver slices were incubated at 37°C. The values are expressed as mean ± SD for four rats. The mean values marked with * were significantly smaller than the mean of basal diet group at a 95% confident level. At 0 time there was 0% oxidized heme proteins.


0 .

40

3o

2o

o~ 20 60 80 100 120 140 160 180

Incub0ti0n Time (min) Fig. 4. Production of oxidized heme proteins during the spontaneous and CBrC13-induced (0.1 raM) oxidative reaction in liver homogenate; - - • - - , basal diet without CBrC13; - - • - - , vitamin E, selenium, and/3-carotene + basal diet without CBrC13; - - O - - , basal diet with CBrCI3; - - A - - , vitamin E, selenium, and/3-carotene + basal diet with CBrC13. The values are expressed as mean __. SD for four rats.

teins and the accumulation of TBARS during the spontaneous oxidation in liver homogenate.

DISCUSSION

Application of HPSAP in the determination of heme proteins from oxidative damage processes in liver tissues

Heme proteins, such as hemoglobin, are highly susceptible to oxidative stress 11,~2 and are readily converted to their oxidized forms (i.e., methemoglobin and hemichrome). 13,14 The characteristic visible absorbance spectra of oxidized heme proteins can be a measure of oxidative damage of tissues. Under conditions close to physiological, such as tissue slices and homogenate, the quantitative measurement of these compounds is difficult because of the turbidity caused by the biological tissues. In addition, the spectra obtained from tissue slices and homogenate are usually quite complex because of the presence of various heme proteins, both reduced and oxidized. The HPSAP analysis is a part of the solution for these problems. As shown in Fig. 1 and Table 2, the analysis using HPSAP shows particular strength in the quantitation ofheme proteins in biological samples. First, in contrast to other measurements of oxidative damage such as the TBARS method, sample preparation for HPSAP analysis is simple; thus artifacts from sample preparation can be eliminated. Second, analysis with HPSAP can be applied to biological samples with large turbidity such as tissue slices. Third, HPSAP contains a large number of spectra of known heme proteins; thus we are able to calculate the concentration of components of a complicated composite spec-

trum. Fourth, analysis with HPSAP directly targets the oxidatively damaged heme protein molecules rather than the oxygen radical intermediates in oxidative damage processes. Finally, using HPSAP we are able to detect oxidized heme proteins, such as methemoglobin and hemichrome, at a relative low concen-

70-

= 60

50

E 40 ¢u "1--

"¢D ¢, 30 N

~ 20

10

0

T . . / i

r / / / / /

/ /

/ / / J

/ /

/ / / / / /

/ /

/ / / / / .

TBHP

Pr00xidant

Fe +2 1

Fig. 5. Production of oxidized heme proteins in liver slices during the oxidative reaction induced by TBHP and Fe ÷2 (both 0.1 mM); D, basal diet; @, vitamin E + basal diet; I , selenium + basal diet; [], B-carotene + basal diet; @, vitamin E, selenium, and B-carotene + basal diet. Liver slices were incubated at 37°C for 1 h. The values are expressed as mean + SD for four rats. The mean values marked with * were significantly smaller than the mean of basal diet group at a 95% confident level.

480 H. CHEN et al.

70

6O

40 =< 30

2O I d

0

m

I I I I 0.5 1.0 1.5 2.0

Time (hr) Fig. 6. Lipid peroxidation in liver homogenate measured as TBARS during spontaneous oxidative reaction; - - © - - , basal diet; - - A - - , vitamin E, selenium, and/3-carotene + basal diet. The values are expressed as mean _+ SD for four rats.

tration; thus with its sensitivity this method is useful in determining oxidized heme protein in the early stage of oxidative damage processes.

Vitamin E, selenium, ~-carotene, and oxidative damage of heine proteins in fiver tissues

In general, vitamin E, selenium, and r-carotene have some protection of heme proteins during spontaneous oxidation (Fig. 2). The protective effects, however, usually only appeared during the late stages of the oxidative reaction (1 and 2 h). During the early stage of the oxidative reaction (0.5 h), the antioxidants showed little or no protective effect against the oxidation of heme proteins. These observations sug- gested that vitamin E, selenium, and ~-carotene prob- ably act more like chain-breaking antioxidants than preventive antioxidants during the oxidation of heme proteins.

Vitamin E (a-tocopherol) is the primary chain- breaking antioxidant in membranes and reduces per- oxyl, hydroxyl, and superoxide radicals and singlet oxygen. 2° Vitamin E showed strong protection to animal tissues against oxidative damage such as lipid peroxidation in vivo. 21,22 The weak preventive effect of vitamin E in heme protein oxidation may well be at- tributed to its poor solubility in aqueous phase, where heme proteins are located. Even though vitamin E is not effective in inhibiting the initiation of heme protein oxidation, its potent chain-breaking ability in

lipid peroxidation may contribute to the delay of the heme protein oxidation. Some experiments indicate that the lag time of the lipid peroxidation was much longer than that of oxidative damage of proteins, z3'24 Therefore, if vitamin E protected heme proteins by breaking the chain reaction from lipid peroxidation, vitamin E would only show a protective effect during the late stage of heme protein oxidation when lipid

60

A m

o E 50- e--

tJ') nw --

r n

~ - 4 0 -

- (a)

B

• 7 =0.80

/ e •

I I I I I I I 20 3 0 40 5 0

% Oxidized Heme Protein

50-

45-

o E 40-

v

OO rY 3 5 -

~ 3 0 -

(b)

|

• 7 =0.80

I I I I I 0 5 I0 15 20 25

% Oxidized Heme Protein Fig. 7. Correlat ions between the product ion o f oxidized heine proteins and l ip id peroxidat ion: (a) t reatment o f basal diet; (b) treatment of combination of vitamin E, selenium, and/3-carotene in diet.


peroxidation participates in the reaction. The results in Fig. 2 appear to support this assumption.

The selenium-glutathione peroxidase reduces both hydrogen peroxides and lipid hydroperoxides in aqueous phase, z° If heme protein oxidation is initiated by other types of free radicals, rather than free radicals generated from decomposition of hydrogen peroxides and lipid peroxides, the supplement of selenium in the diet would not have significant effect on the initiation of heme protein oxidation. When lipid peroxidation contributes to the oxidation of heme proteins, selenium becomes an important protection factor for heme proteins. The production of oxidized heme proteins was not increased between 1 and 2 h of incubation in animals fed a selenium-adequate diet.

Beta-carotene is an effective quencher of singlet oxygen and a radical-trapping antioxidant. 2 In an in vivo situation,/5-carotene significantly inhibited lipid peroxidation. 2s As shown in Fig. 3, B-carotene did not show a protective effect during 0.5 h of incubation but did show a moderate protective effect on heme proteins during 1 and 2 h of incubation. Similar to vitamin E, the protective effect of B-carotene is also expected to be exhibited in the lipid phase. Therefore, B-carotene may also protect heine proteins by the inhi- bition of lipid peroxidation in the membrane.

Studies indicate that the combination of individual antioxidants could significantly reduce the oxidative damage to animal tissues. 21'22 In this experiment the combination of vitamin E, selenium, and B-carotene gave the most protective effect of heme proteins against oxidative damage (Figs. 2 and 4). More impor- tantly, the combination of all three antioxidants re- sulted in a significant reduction of oxidized heme proteins during the early stage of the oxidation (0.5 h). This finding suggests that the initiation of heme protein oxidation may be prevented by manipulating the intake amount of all three antioxidants in the diets. However, it is not clear whether the improvement of protective effect is due to the interaction (synergism) among the antioxidants or the additive effect from the individual antioxidants.

The performance of individual antioxidants and the combination of all antioxidants in the protection of heme proteins during the CBrCla-induced reaction followed a similar trend compared to spontaneous oxidation (Fig. 3). The differences in the production of oxidized heme proteins between the antioxidant-deficient diet and the diets with antioxidant supplements were not as distinct as those shown in Fig. 2. This could be due to the strong oxidative effect of CBrCla added to the tissue slices, which overshadowed the protective effect of antioxidants.

The experimental results suggest that the lipid-sol-

uble antioxidants may protect heme proteins by in- terrupting the process of lipid peroxidation. Since heine proteins exist in aqueous phase, the lipid-soluble antioxidants may not be effective in reducing the rate of initiation of the heme protein oxidation process. The combination of vitamin E, selenium, and /~-carotene, however, showed a significant inhibitory effect on the early stage of the reaction, implying that the synergism of antioxidants may be able to prevent the initiation of oxidative damage of heme proteins.

Acknowledgements ~ This work was supported by National Insti- tute of Health research grant DK-39225 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. The au- thors would like to thank Mrs. Rowena Cramer Boyle for composition of the Heine Protein Spectra Analysis Program. H. J. A. was supported by a travel grant from the Danish Agricultural and Veteri- nary Research Council.

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protection by vitamin e, selinium, and β-carotene against oxidative damage in rat liver slices and...

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