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reperfusion injury in the isolated perfused langendorff heart
NUTRITION RESEARCH, Vol. 12, Suppl. 1, pp. $203-$215, 1992. 0271-5317/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
PALM OIL VITAMIN E PROTECTS AGAINST ISCHEMIA/REPERFUSION INJURY IN THE ISOLATED PERFUSED LANGENDORFF HEART E Serbinova, S Khwaja, J Catudioc, J Ericson, Z Torres, A Gapor *'1, V Kagan, L Packer Department of Molecular and Cell Biology, 251 Life Science Addition University of California at Berkeley, Berkeley, CA 94720, USA * Palm Oil Research Institute of Malaysia (PORIM), Peti Surat 10620, 50720 Kuala Lumpur, Malaysia
ABSTRACT We studied the effect of palm oil vitamin E on Langendorff perfused rat hearts subjected to 40 minutes of global ischemia. Our results demonstrated that palm oil vitamin E was more efficient in the protection of isolated Langendorff heart against ischemia/reperfusion injury than tocopherol as measured by its mechanical recovery. Palm oil vitamin E completely suppressed LDH enzyme leakage from ischemic hearts, prevented the decrease in ATP and creatine phosphate levels and inhibited the formation of endogenous lipid peroxidation products. Our data indicate that a palm oil vitamin E mixture containing both alpha-tocopherol and alpha-tocotrienol may be more efficient than alpha-tocopherol alone in the protection of the heart against oxidative stress induced by ischemia-reperfusion. KEY WORDS: Palm oil vitamin E, Langendorff heart, Ischemia-reperfusion, LDH, ATP, Creatine phosphate, Alpha-tocopherol, Alpha-tocotrienol
INTRODUCTION. Vitamin E is the generic name of a mixture of lipid-soluble phenols possessing general structural features: an aromatic chromanol head and a 16-carbon side chain. The two forms of vitamin E, tocopherols and tocotrienols, differ in their hydrocarbon tail: tocopherol has a saturated one, while tocotrienol has an unsaturated isoprenoid tail. The number and placement of methyl substituents on the chromanol nucleus produces the different alpha-,beta-, gamma- and delta- tocopherol and tocotrienol isomers. Vitamin E is believed to be the major lipid-soluble chain-breaking antioxidant found in blood plasma and membranes.
1Author to whom all correspondence should be addressed
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It has been shown that different forms of vitamin E exhibit different antioxidant protection in membranes. In vitro studies suggest that alpha-tocotrienol possesses a 40-60 times greater antioxidant activity than alpha-tocopherol against (Fe2 + + ascorbate)- and (Fe2 + + NADPH)induced lipid peroxidation in rat liver microsomal membranes. Alpha-tocotrienol also protects cytochrome P-450 against oxidative damage 6.5 times better than alpha-tocopherol (1). Tocotrienols were also shown to have a higher physiological efficiency in inhibiting growth than tocopherols. Proliferation of human and mouse tumor cells (sarcoma 180, Ehrlich carcinoma and IMC carcinoma) were suppressed after exposure to tocotrienols for 72 hours, in vitro. Tocopherol showed no significant effect (2-4). It is a well known fact that reactive oxygen species (ROS) can oxidize lipids and proteins. Recently, many studies have been conducted to investigate the role of ROS in the injury of the heart. In particular, heart ischemia/reperfusion, anthracycline, and iron studies have shown that ROS contribute to myocardial injury (5, 6). The insight into the mechanism of heart injury has suggested that administration of antioxidants may lessen oxidative damage of the heart. One highly potent antioxidant is vitamin E (7). We predict that tocotrienols may have unique health benefits for protecting heart, as well as brain, against ischemia/reperfusion injury. In the present investigation, our purpose was to study the protection afforded by palm oil vitamin E (POE), a mixture of 55% tocotrienols and 45% tocopberols, in Langendorff perfused rat hearts subjected to 40 minutes of global ischemia.
MATERIALS AND METHODS Animal and perfusion ext~eriments Male Sprague-Dawley rats (350-400 g) were used for this study. The animals were anesthetized with diethylether and intravenously injected with 400 units of heparin, before excision of the heart. The perfusion apparatus was constructed essentially as described earlier by Leipala (8) and temperature was regulated at 37~ A retrograde aortic perfusion solution was a modified Krebs-bicarbonate buffer (1.2 mM MgC12 x 6H20, 5.9 mM KC1, 1.7 mM dGlucose, 25 mM NaI-ICO3, 2.0 mM CaC12, 117 mM NaC1, pH 7.4), which was continuously bubbled with 95% 02 and 5% CO 2. After the excision of the heart, the aorta was immediately cannulated and the left ventricle perfusion was initiated for 10 minutes (perfusion period). During the perfusion stage, a cannula, which was connected to the pressure transducer, was inserted into the left ventricle through the left atrium. Hearts were then subjected to a period of global isehemia, for 40 minutes, by clamping the aortic cannula. The clamp was reopened after the ischemia period to initiate the 20 minute reoxygenation stage (reperfusion period). At the end of the perfusion experiments, all hearts were frozen and stored in liquid nitrogen. Control hearts were subjected to 60 minutes of perfusion with no ischemia.
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Left ventricular develooed vressure (LVDP) Developed pressure and heart rate were obtained with a Gould/Statham P23 pressure transducer connected to a Gilson Duograph. LVDP was calculated as the product of developed pressure and heart rate [(mm Hg/beat) x (beats/see)]. Recovery was calculated by comparing the LVDP before and after ischemia. DIET Composition of the control diet was as follows:
Substance Protein (vitamin-stripped casein) Mineral Mix A Mineral Mix B d,L-Methionine Choline Bitartarate Salt Mix Vitamin Mix Vitamin E Acetate (30 IU/kg) Potassium Acetate Tocopherol-Stripped Corn Oil Sucrose (powdered)
g/kg Diet 2~.~ 40.~ 4.~ 2.~ 10.~ 10.~ 0.06 6.27 50.~ 677.70
Supplementation of animals with vitamin E was accomplished by feeding rats the above control diets, with an addition of either 7 g of palm oil vitamin E per kg diet or 20 g vitamin E acetate per kg diet. Animals were fed either the control or vitamin E supplemented diet for 6 weeks. Lactate dehvdro~enase (LDI-I) assav LDH was assayed using discrete analyzer application kit obtained from Sigma Chemical Company. The enzyme activity was calculated in terms of IU, where 1 IU is equal to the consumption of 1 pmol of NADH per minute per litre of the coronary effluent. Extraction The frozen hearts were crushed with a chilled mortar and pestle. The formed powder was then added to 2 ml of 6% perchloric acid in 10 mM EDTA at 0~ The mixture was homogenized and centrifuged at 3600 rpm for 10 min. Pooled supernatants were neutralized to pH 7.0 with the addition of ice-cold 1 M KOH. KCIO4 precipitate was removed by centrifugation and filtration. The extracts were lyophilized and redissolved in 2.0 ml H20 and 0.5 ml D20 (5).
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NMR soectroscoov NMR spectroscopy was performed at 121.5 MHz on a Bruker AM 300 spectrometer. D20 was used as a field lock and chemical shifts were assigned relative to the creatine phosphate (CP) peak (-2.25 ppm relative to 85% phosphoric acid). A 60s excitation pulse and 2.1s repetition time were used to acquire 3072 scans over 108 minutes. A 5 Hz line broadening was applied to reduce spectral noise. Quantification of the spectra was achieved by acquiring a second spectrum of each sample after addition of 0.5/~1 CP and ATP standards. The integrals of the spectra were compared (peak to peak) to determine the amount of phosphate present in the original sample. The reproducibility of the measurements of 31p metabolites was found to be 5% for identical samples. Generation of chromanoxyl radicals Chromanoxyl radicals from exogenous added tocopherols and tocotrienols in rat heart homogenates after ischemia and ischemia/reperfusion were generated using an enzymic oxidation system (soybean lipoxygenase + linoleic acid) as previously described by Packer (10) and Mehlhorn (11). The reaction medium (total volume 100 td), lipoxygenase (90 U//~I), and chromanols (8 raM) were subsequently added to the microsomal suspension (27 mg protein/ml). Ascorbate (3.0 mM) and chromanols were added simultaneously. ESR soectroscoov ESR measurements were made on a Varian E-109E spectrometer at room temeperature, in gas-permeable Teflon tubing (0.8 mm internal diameter, 0.013 mm thickness obtained from Zeus Industrial Products, Raritan, New Jersey, USA). The permeable tube (approximately 8 cm in length) was filled with 60 ~1 of the mixed sample, folded into quarters and placed in an open 3.0 mm internal diameter EPR quartz tube such that all of the sample was within the effective microwave irradiation area. The sample was flushed with oxygen. Spectra were recorded at 70 mW power and 2.5 gauss modulation, and 25 gauss/rain scan time. Levels of antioxidants Consumption of alpha-tocotrienol and alpha-tocopherol was monitored by HPLC using an in-line electrochemical detector and UV-detector (12). Tocotrienol and tocopherol were extracted and measured as previously described in (12). An internal standard of tocopherol and tocotrienol was included in the samples. Lipid peroxidation The content of endogenous lipid peroxidation products in the lipid extracts from the heart was measured spectrofluorimetricaUy (Perkin-Elmer MPF 552 Spectrofluorometer) using ~'ex = 365 nm and ~em = 425 nm (13).
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Reagents Heparin was obtained from the Upjohn Company, Kalamazoo, Michigan, USA. Other chemicals were acquired from the Sigma Chemical Company, St. Louis, Missouri, USA.
RESULTS Left Ventricular Develooed Pressure (LVDP) There was no significant difference between the pre-ischemic LVDP in hearts obtained from control animals and from animals fed the palm oil diet (75+16 mm Hg and 81+18 mm Hg respectively). After 40 minutes of ischemia and 20 minutes of reperfusion, the mechanical recovery rate of hearts from the POE-supplemented group was 90% as compared to the 24% recovery of the hearts obtained from rats fed the normal diet (Fig. 1) and to the 80% recovery of the hearts obtained from rats kept on alpha-tocopherol acetate supplemented diet. m
normal
diet
m POE-suppl.
diet
[] vitamin E suppl, diet
100 M E C H A N I C A L
R E C O V E R Y,
(%)
.g'.'l.'lll.';I .gn;lll,,n .gg_,.,_-_-,.,._,r .g.'-'gggggZg,.
,. ,
,
i
I I
.'.."i!iiiiii!ii =,nlnu=, ~.gggg--.--,,,_%,g: i'll,i!.."!, .g-'.'-'.-r 4 ; ; g ; g g g g g , -'1 .gg.,|gggggr .-'g-'~'-ggggggl
50
. . . . . .
:!ii!!iii !!i
lllllllllllliiiiilii
FIG. 1. Mechanical recovery of isolated Lagendorff hearts obtained from rats kept on different types of diet and subjected to I0 min perfusion + 40 min ischemia + min reperfusion. There were 6 animals in each experimental group. Conditions are given in the Methods section.
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LDH leakaee During the 10 minutes of perfusion, the LDH activity in the effluent was minute (40.4 + 12.0 IU) for both the normal and POE-supplemented hearts. Immediately after 40 minutes of global ischemia and initation of reperfusion, a substantial increase (4.5 times) in the LDH acitivity was detected in the coronary effluent of hearts from normal diet group, whereas, there was only 1.5 times increase in LDH activity in the coronary effluent of hearts from POEsupplemented group (Fig. 2). In both normal diet and POE-supplemented hearts subjected to only 60 minutes of perfusion, there was no elevation in the LDH activity with time. High energy p h o s p h ~ The results of the 31p-NMR determinations of ATP and creatine phosphate (CP) content in the heart extracts are shown in Table 1. Normal diet hearts subjected to 40 minutes of global ischemia and 20 minutes of reperfusion had 75% lower ATP and 85% lower CP levels. However, hearts from POE-supplemented group, subjected to similar conditions, did not have a significant decrease in the levels of ATP and CP. Inhibition of lipid peroxid~tion Content of endogenous lipid peroxidation fluorescent products in rat hearts are shown on Table 2. There was no significant difference in the amount of fluorescent products in normal diet and POE-supplemented hearts frozen immediately after excision. However, fluorescent products after 60 minutes of perfusion were doubled in normal diet hearts, while no change was detected in POE-supplemented hearts. When hearts were subjected to 40 minutes of ischemia and 20 minutes of reperfusion, fluorescent products increased by 6-fold in normal diet hearts and did not change in POE-supplemented hearts. Consumption of aloha-tocooherol and aloha-tocotrienol The concentration of endogenous vitamin E in Fat heart homogenates was 0.4 - 0.6 nmol/mg protein. After 6 weeks of dietary supplementation with POE, the concentration of endogenous alpha-tocopherol increased 3 times. The concentrations of alpha-tocotfienol and alpha-tocopherol in hearts were 1.2 + 0.2 nmol/mg protein and 1.8 + 0.3 nmol/mg protein, respectively. The data on the consumption of both forms of vitamin E in the course of ischemia/repcrfusion are shown in Table 3. There were no significant differences in the contents of alpha-tocopherol and alpha-tocotrienol, after 60 minutes of perfusion. However, 40 minutes of ischemia, followed by 20 minutes of reperfusion, caused a pronounced decrease in vitamin E levels. Only 41% of tocopherol and 21% of tocotrienol remained after ischemia/reperfusion in the POE-supplemented group. The amount of vitamin E in hearts from control group was less than 4 %.
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ESR measurements of the recycling efficiency of aloha-tocooherol and alpha-tocotden01 In the presence of heart homogenates obtained from rats subjected or not subjected to 40 minutes ischemia plus 20 minutes reperfusion, alpha-tocopherol and alpha-tocotrienol radicals
250 ~200
~ 150
E
Normal ~//biet
~"
~
POE-suppl.
~ 1oo o
~ 50' [5
O
, II--1 10 20
]'91~Time During Reperfusion Period (Minutes)'D~ I FIG. 2. Effect of supplementation with palm oil vitamin E on LDH release from isolated Langendorff hearts subjects to 10 min perfusion + 40 min ischemia + 20 min reperfusion. There were 6 animals in each experimental group. Conditions are given in the Methods section. TABLE 1 Effect Of Palm Oil Vitamin E (POE) On The Content Of High Energy Phosphates In Isolated Perfused Rat Hearts
ATP 60 min perfusion
Control +POE n=3,
100
31p Metabolites (% From control) Creatine Phosphate 100
10 min perfusion + 40 min ischemia + 20 min reperfusion 25 + 14 16 _+ 10 87 d: 17 91 5:12 p < 0.05
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TABLE 2 Effect Of Palm Oil Vitamin E (POE) On The Content Of Lipid Peroxidation Fluorescence Products in Rat Hearts (% of control) Normal Diet
POE Diet
100
100
60 rain perfusion
140 -b 20
100 • 10
10 min peffusion + 40 rain isehemia + 20 rain reperfusion
540 _+ 45
107 + 12
Control
n=6,
p < 0.05
TABLE 3 Effect Of Ischemia-Reperfusion On Vitamin E Content In Isolated Lagendorff Hearts From Rats Fed With Palm Oil Vitamin E (POE) Supplemented Diet Tocopherol Tocotrienol (nmoles/mg protein) 60 min perfusion
1.80 _+ 0.30
1.20 _ 0.20
10 min peffusion + 40 rain iscbemia + 20 min rcpcffusion
0.74 _+ 0.10
0.25 _ 0.05
n=6,
p<0.~
from exogenously added chromanols were generated by an enzymic oxidation system (lipoxygenase + linolenic acid) and their ESR spectra were recorded. Alpha-tocopherol and alpha-tocotrienol give characteristic pentameric chromanoxyl radical signals with g-values of the components 2.0122, 2.0092, 2.0061, 2.0028 and 1.9993 (14). The magnitude of alphatocotrienoxyl radical ESR signal was significantly higher than that of alpha-tocopheroxyl, both before and after iscbemia/repeffusion. Addition of ascorbate caused transient disappearance of ESR signals of chromanoxyl radicals in the presence of heart homogenate (Fig. 3) and appearance of ascorbyl radical signal. After ascorbate was consumed, its radical signal in the
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ESR spectrum was replaced by the growing signal of chromanoxyl radicals. The delay in the reappearance of chromanoxyl radical ESR signal was longer for alpha-tocotrienol than for alphatocopherol (Fig. 3).
ESR SIGNAL,
ESR SIGNAL,
Arbitrar 'Units
Arbitrar U n i t s
O•
30
---'O"- CONTROL SCORBATE
20
I0 .... J I
0
10
|
20
I
30
TOCOTRIENOL I
0
10
!
20
!
30
TIME (MIN)
FIG. 3 Time-course of chromanoxyl radicals of alpha-tocopherol and alpha-tocotrienol in rat hearts after 40 min ischemia + 20 min reperfusion. Conditions are given in the Methods section.
DISCUSSION Myocardial ischemia is a leading cause of mortality in the industrialized world (15) and a major contributor to health care costs in the US (16). The injuries of the ischemic heart occur during the reperfusion period, a phenomenon called the "reperfusion injury". It has been found that during reperfusion the amount of potentially salvageable myocardium is reduced and myocardial necrosis is increased, due to the destruction of the cardiomyocytes (17). Calcium overload (18), mitochondrial dysfunction (19), arachidonic acid metabolites (20) and defective myocardial lipid metabolism (21) contribute to the reperfusion injury. It has been postulated that
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reactive oxygen species (ROS) are one of the primary causes of heart injury (22). Therefore, it is suggested that administration of antioxidants may ameliorate myocardial ischemia/reperfusion injury. Since vitamin E is the primary lipid peroxidation chain-breaking antioxidant in membranes, it may exhibit both prophylactic and therapeutic roles against cardiac ischemic/reperfusional injury. Alpha-tocopherol and alpha-tocotrienol are two vitamin E constituients having the same aromatic chromanol "head" but different hydrocarbon ~tails'. Tocopherols have a saturated tail, while tocotrienols have an unsaturated isoprenoid chain. Serbinova et al. (1) reported that alphatocotrienol has markedly higher antioxidant activity in vitro than alpha-tocopherol, mainly due to (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. We hypothesized that alphatocotrienol may be more beneficial for the antioxidant protection of the ischemic/reperfused myocardium than alpha-tocopherol. To test this hypothesis, we tested the effects of POE on Langendorff hearts subjected to 40 minutes of global ischemia. Our results demonstrate that palm oil vitamin E was more efficient in the protection of the isolated Langendorff heart against ischemia/reperfusion injury than tocopherol, as measured by its mechanical recovery. Palm oil vitamin E completely suppressed LDH enzyme leakage from ischemic hearts, prevented the decrease in ATP and creatine phosphate levels and inhibited the formation of endogenous lipid peroxidation products. The results presented do not afford direct quantitative comparison of the protective effects of alpha-tocotrienol with that of alphatocopherol, since both were present in palm oil vitamin E concentrate in the proportion of 55:45. However, there is an indirect evidence of higher alpha-tocotrienol efficiency. Comparison of the consumption of alpha-tocopherol and alpha-tocotrienol, in the course of ischemia/reperfusion, showed that the alpha-tocotrienol loss was significantly more pronounced than that of alphatocopherol. In other words, alpha-tocotrienol was more active in radical scavenging and was preferentially consumed in the course of ischemia/reperfusion-induced oxidative stress. Our in vitro studies showed that recycling efficiency of alpha-tocotrienol, in the presence of ascorbate, is higher than that of alpha-tocopherol not only in control but also in ischemic/reperfused heart homogenates. If the recycling of vitamin E contributes to the maintenance of its concentration in vivo, the difference in the consumption of alpha-tocotrienol and alpha-tocopherol by ischemia/reperfusion-derived peroxyl radicals may even be higher than the one reported in Table 3, due to a more efficient regeneration of tocotrienol. Based on rat resorption-gestation test, d-alpha-tocopherol has the highest (100%) biopotency, while d-alpha-tocotrienol manifests only 30% of this acitivity (23). The significance of these estimations for health benefit is not clear, since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes. We have already noted in the introduction that other physiological activities of alpha-tocotrienol, such as antitumor and inhibition of cholesterol biosynthesis, were reported to be much higher than those of alphatocopherol. Thus alpha-tocotrienol may have higher physiological activity than alpha-tocopherol
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under conditions of oxidative stress. In particular, our data indicate that a palm oil vitamin E mixture containing both alpha-tocopherol and alpha-tocotrienol may be more efficient than alphatocopherol alone in the protection of the heart against oxidative stress induced by ischemia/reperfusion. ACKNOWLEDGEMENT Research supported by NIH (CA 47597) and the Palm Oil Research Institute of Malaysia 0'ORIM). REFERENCES .
Serbinova E, Kagan V, Han D, Packer L. Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. Free Radical Biol Meal 1991; 10:263-275.
.
Kato A, Yamaoka M, Tamaka A, Komiyama K, Umezawa L. Physiological effects of tocotrienol. Abura Kagaku 1985; 34:375-376.
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Komiyama K, Izuka K, Yamaoka M, Watanabe H, Tsuchiya N, Umezawa L. Studies on the biological activity of tocotrienols. Chem Pharm Bull 1989; 37:1369-1371. Sundram K, Khor HT, Ong AS, Pathmanathan R. Effects of dietary palm oil on mammary carcinogenesis in female rats induced by 7,12-diemthylbenz(a)anthracene. Cane Res 1989; 49:1447-1451.
.
.
Packer L, Valenza M, Serbinova E, Starke-Reed P, Frost K, Kagan V. Free radical scavenging is involved in the protective effect of L-propionyl carnitine protection of the heart against ischemia/reperfusion injury of the heart. Arch Biochem Biophys 1991; 288:(in press).
.
Reznick AZ, Kagan VE, Ramsey R, Tsuchiya M, Khwaja S, Serbinova E A, Packer L. Antiradical effects in L-propionyl camitine protection against ischemia/repeffusion injury: the possible role of iron chelation. Arch Biochem Biophys 1991; (submitted).
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Janero DR. Therapeutic potential of vitamin E against myocardial ischemia-reperfusion injury. Free Radical Biol Meal 1991; 10:315-324.
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Leipala J, Bhatanagar R, Pineda E, Najibi S, Massourmi K, Packer L. Protection of the reperfused heart by L-propionyl-carnitine. Optimal effect requires administration prior to ischemia. J Appl Physiol 1991; (in press).
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9.
Laagendorff O. Untersuchungen am Uberlebenden Saugetierherzen. Plugers Arch 1895; 61:291-332.
10.
Packer L, Maguire JJ, Mehlhorn RI, Serbinova E, Kagan V. Mitochondrial and microsomal membranes have free radical reductase activity. Biochem Biophys Res Communn 1989; 1:229-235.
11.
Mehlhorn R, Sumida S, Packer L. Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E enriched rat liver mitochondria and microsomes. J Biol Chem 1989; 264:13448-13452.
12.
Lang J, Gohil K, Packer L. Simultaneous determination of tocopherols, ubiquinones in blood, plasma, tissue homogenates and subcellular fractions. Analyt Biochem 1986; 157:106-116.
13.
Malshet VG, Tappel AL, Burns VM. Fluorescent products of lipid peroxidation. II. Methods for analysis and characterization. Lipids i974; 9:328.
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Mukai K, Fukuda K, Ishizu K. Stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having higher antioxidant activity than alpha-tocopherol. Chem Phys Lipids 1988; 46:31-36.
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Sempos C, Cooper R, Kovan MG, McMillen M. Divergence of the recent trends in coronary mortality for four major race-sex groups in the United States. Am J Public Health 1988; 78:422-1427.
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Wittels EH, Hay JW, Gotto AM. Medical cost of coronary artery disease in the United States. Am J Cardiol 1990; 65:432-444.
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Flaherty Jr, Weisfeldt ML. Reperfusion injury. Free Radical Biol Med 1988; 5:409-419.
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Opie LH. Proposed role of calcium in reperfusion injury. Int J Cardiol 1989; 23:159164.
19.
Jennings RB, Ganote CE. Mitochondrial structure and function in acute myocardial ischemia injury. Circ Res 1976; 81(suppl): 80-91.
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Mullane KM. Eicosanoids in myocardial ischemia/reperfusion injury. Advances in inflammation research (vol 12) 1989; NY Raven Press: 191-214.
21.
van der Vusse GJ, van Bilsen M, Reneman RS. Impairment of lipid metabolism in ischemia and reperfused myocardial tissue. Analysis and stimulation of the cardiac system ischemia 1989; Boca Raton CRC Press: 355-369.
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22.
McCord JM. Free radicals and myocardial ischemia: overview and outlook. Free Radic Biol Med 1988; 4:9-14.
23.
Leth T, Sondergaard H. Biological activity of vitamin E compounds and natural materials by the resorption gestation test and chemical determination of the vitamin E activity in foods and feeds. J Nutr 1977; 107:2236-2243.
PALM OIL VITAMIN E PROTECTS AGAINST ISCHEMIA/REPERFUSION INJURY IN THE ISOLATED PERFUSED LANGENDORFF HEART E Serbinova, S Khwaja, J Catudioc, J Ericson, Z Torres, A Gapor *'1, V Kagan, L Packer Department of Molecular and Cell Biology, 251 Life Science Addition University of California at Berkeley, Berkeley, CA 94720, USA * Palm Oil Research Institute of Malaysia (PORIM), Peti Surat 10620, 50720 Kuala Lumpur, Malaysia
ABSTRACT We studied the effect of palm oil vitamin E on Langendorff perfused rat hearts subjected to 40 minutes of global ischemia. Our results demonstrated that palm oil vitamin E was more efficient in the protection of isolated Langendorff heart against ischemia/reperfusion injury than tocopherol as measured by its mechanical recovery. Palm oil vitamin E completely suppressed LDH enzyme leakage from ischemic hearts, prevented the decrease in ATP and creatine phosphate levels and inhibited the formation of endogenous lipid peroxidation products. Our data indicate that a palm oil vitamin E mixture containing both alpha-tocopherol and alpha-tocotrienol may be more efficient than alpha-tocopherol alone in the protection of the heart against oxidative stress induced by ischemia-reperfusion. KEY WORDS: Palm oil vitamin E, Langendorff heart, Ischemia-reperfusion, LDH, ATP, Creatine phosphate, Alpha-tocopherol, Alpha-tocotrienol
INTRODUCTION. Vitamin E is the generic name of a mixture of lipid-soluble phenols possessing general structural features: an aromatic chromanol head and a 16-carbon side chain. The two forms of vitamin E, tocopherols and tocotrienols, differ in their hydrocarbon tail: tocopherol has a saturated one, while tocotrienol has an unsaturated isoprenoid tail. The number and placement of methyl substituents on the chromanol nucleus produces the different alpha-,beta-, gamma- and delta- tocopherol and tocotrienol isomers. Vitamin E is believed to be the major lipid-soluble chain-breaking antioxidant found in blood plasma and membranes.
1Author to whom all correspondence should be addressed
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It has been shown that different forms of vitamin E exhibit different antioxidant protection in membranes. In vitro studies suggest that alpha-tocotrienol possesses a 40-60 times greater antioxidant activity than alpha-tocopherol against (Fe2 + + ascorbate)- and (Fe2 + + NADPH)induced lipid peroxidation in rat liver microsomal membranes. Alpha-tocotrienol also protects cytochrome P-450 against oxidative damage 6.5 times better than alpha-tocopherol (1). Tocotrienols were also shown to have a higher physiological efficiency in inhibiting growth than tocopherols. Proliferation of human and mouse tumor cells (sarcoma 180, Ehrlich carcinoma and IMC carcinoma) were suppressed after exposure to tocotrienols for 72 hours, in vitro. Tocopherol showed no significant effect (2-4). It is a well known fact that reactive oxygen species (ROS) can oxidize lipids and proteins. Recently, many studies have been conducted to investigate the role of ROS in the injury of the heart. In particular, heart ischemia/reperfusion, anthracycline, and iron studies have shown that ROS contribute to myocardial injury (5, 6). The insight into the mechanism of heart injury has suggested that administration of antioxidants may lessen oxidative damage of the heart. One highly potent antioxidant is vitamin E (7). We predict that tocotrienols may have unique health benefits for protecting heart, as well as brain, against ischemia/reperfusion injury. In the present investigation, our purpose was to study the protection afforded by palm oil vitamin E (POE), a mixture of 55% tocotrienols and 45% tocopberols, in Langendorff perfused rat hearts subjected to 40 minutes of global ischemia.
MATERIALS AND METHODS Animal and perfusion ext~eriments Male Sprague-Dawley rats (350-400 g) were used for this study. The animals were anesthetized with diethylether and intravenously injected with 400 units of heparin, before excision of the heart. The perfusion apparatus was constructed essentially as described earlier by Leipala (8) and temperature was regulated at 37~ A retrograde aortic perfusion solution was a modified Krebs-bicarbonate buffer (1.2 mM MgC12 x 6H20, 5.9 mM KC1, 1.7 mM dGlucose, 25 mM NaI-ICO3, 2.0 mM CaC12, 117 mM NaC1, pH 7.4), which was continuously bubbled with 95% 02 and 5% CO 2. After the excision of the heart, the aorta was immediately cannulated and the left ventricle perfusion was initiated for 10 minutes (perfusion period). During the perfusion stage, a cannula, which was connected to the pressure transducer, was inserted into the left ventricle through the left atrium. Hearts were then subjected to a period of global isehemia, for 40 minutes, by clamping the aortic cannula. The clamp was reopened after the ischemia period to initiate the 20 minute reoxygenation stage (reperfusion period). At the end of the perfusion experiments, all hearts were frozen and stored in liquid nitrogen. Control hearts were subjected to 60 minutes of perfusion with no ischemia.
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Left ventricular develooed vressure (LVDP) Developed pressure and heart rate were obtained with a Gould/Statham P23 pressure transducer connected to a Gilson Duograph. LVDP was calculated as the product of developed pressure and heart rate [(mm Hg/beat) x (beats/see)]. Recovery was calculated by comparing the LVDP before and after ischemia. DIET Composition of the control diet was as follows:
Substance Protein (vitamin-stripped casein) Mineral Mix A Mineral Mix B d,L-Methionine Choline Bitartarate Salt Mix Vitamin Mix Vitamin E Acetate (30 IU/kg) Potassium Acetate Tocopherol-Stripped Corn Oil Sucrose (powdered)
g/kg Diet 2~.~ 40.~ 4.~ 2.~ 10.~ 10.~ 0.06 6.27 50.~ 677.70
Supplementation of animals with vitamin E was accomplished by feeding rats the above control diets, with an addition of either 7 g of palm oil vitamin E per kg diet or 20 g vitamin E acetate per kg diet. Animals were fed either the control or vitamin E supplemented diet for 6 weeks. Lactate dehvdro~enase (LDI-I) assav LDH was assayed using discrete analyzer application kit obtained from Sigma Chemical Company. The enzyme activity was calculated in terms of IU, where 1 IU is equal to the consumption of 1 pmol of NADH per minute per litre of the coronary effluent. Extraction The frozen hearts were crushed with a chilled mortar and pestle. The formed powder was then added to 2 ml of 6% perchloric acid in 10 mM EDTA at 0~ The mixture was homogenized and centrifuged at 3600 rpm for 10 min. Pooled supernatants were neutralized to pH 7.0 with the addition of ice-cold 1 M KOH. KCIO4 precipitate was removed by centrifugation and filtration. The extracts were lyophilized and redissolved in 2.0 ml H20 and 0.5 ml D20 (5).
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NMR soectroscoov NMR spectroscopy was performed at 121.5 MHz on a Bruker AM 300 spectrometer. D20 was used as a field lock and chemical shifts were assigned relative to the creatine phosphate (CP) peak (-2.25 ppm relative to 85% phosphoric acid). A 60s excitation pulse and 2.1s repetition time were used to acquire 3072 scans over 108 minutes. A 5 Hz line broadening was applied to reduce spectral noise. Quantification of the spectra was achieved by acquiring a second spectrum of each sample after addition of 0.5/~1 CP and ATP standards. The integrals of the spectra were compared (peak to peak) to determine the amount of phosphate present in the original sample. The reproducibility of the measurements of 31p metabolites was found to be 5% for identical samples. Generation of chromanoxyl radicals Chromanoxyl radicals from exogenous added tocopherols and tocotrienols in rat heart homogenates after ischemia and ischemia/reperfusion were generated using an enzymic oxidation system (soybean lipoxygenase + linoleic acid) as previously described by Packer (10) and Mehlhorn (11). The reaction medium (total volume 100 td), lipoxygenase (90 U//~I), and chromanols (8 raM) were subsequently added to the microsomal suspension (27 mg protein/ml). Ascorbate (3.0 mM) and chromanols were added simultaneously. ESR soectroscoov ESR measurements were made on a Varian E-109E spectrometer at room temeperature, in gas-permeable Teflon tubing (0.8 mm internal diameter, 0.013 mm thickness obtained from Zeus Industrial Products, Raritan, New Jersey, USA). The permeable tube (approximately 8 cm in length) was filled with 60 ~1 of the mixed sample, folded into quarters and placed in an open 3.0 mm internal diameter EPR quartz tube such that all of the sample was within the effective microwave irradiation area. The sample was flushed with oxygen. Spectra were recorded at 70 mW power and 2.5 gauss modulation, and 25 gauss/rain scan time. Levels of antioxidants Consumption of alpha-tocotrienol and alpha-tocopherol was monitored by HPLC using an in-line electrochemical detector and UV-detector (12). Tocotrienol and tocopherol were extracted and measured as previously described in (12). An internal standard of tocopherol and tocotrienol was included in the samples. Lipid peroxidation The content of endogenous lipid peroxidation products in the lipid extracts from the heart was measured spectrofluorimetricaUy (Perkin-Elmer MPF 552 Spectrofluorometer) using ~'ex = 365 nm and ~em = 425 nm (13).
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Reagents Heparin was obtained from the Upjohn Company, Kalamazoo, Michigan, USA. Other chemicals were acquired from the Sigma Chemical Company, St. Louis, Missouri, USA.
RESULTS Left Ventricular Develooed Pressure (LVDP) There was no significant difference between the pre-ischemic LVDP in hearts obtained from control animals and from animals fed the palm oil diet (75+16 mm Hg and 81+18 mm Hg respectively). After 40 minutes of ischemia and 20 minutes of reperfusion, the mechanical recovery rate of hearts from the POE-supplemented group was 90% as compared to the 24% recovery of the hearts obtained from rats fed the normal diet (Fig. 1) and to the 80% recovery of the hearts obtained from rats kept on alpha-tocopherol acetate supplemented diet. m
normal
diet
m POE-suppl.
diet
[] vitamin E suppl, diet
100 M E C H A N I C A L
R E C O V E R Y,
(%)
.g'.'l.'lll.';I .gn;lll,,n .gg_,.,_-_-,.,._,r .g.'-'gggggZg,.
,. ,
,
i
I I
.'.."i!iiiiii!ii =,nlnu=, ~.gggg--.--,,,_%,g: i'll,i!.."!, .g-'.'-'.-r 4 ; ; g ; g g g g g , -'1 .gg.,|gggggr .-'g-'~'-ggggggl
50
. . . . . .
:!ii!!iii !!i
lllllllllllliiiiilii
FIG. 1. Mechanical recovery of isolated Lagendorff hearts obtained from rats kept on different types of diet and subjected to I0 min perfusion + 40 min ischemia + min reperfusion. There were 6 animals in each experimental group. Conditions are given in the Methods section.
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LDH leakaee During the 10 minutes of perfusion, the LDH activity in the effluent was minute (40.4 + 12.0 IU) for both the normal and POE-supplemented hearts. Immediately after 40 minutes of global ischemia and initation of reperfusion, a substantial increase (4.5 times) in the LDH acitivity was detected in the coronary effluent of hearts from normal diet group, whereas, there was only 1.5 times increase in LDH activity in the coronary effluent of hearts from POEsupplemented group (Fig. 2). In both normal diet and POE-supplemented hearts subjected to only 60 minutes of perfusion, there was no elevation in the LDH activity with time. High energy p h o s p h ~ The results of the 31p-NMR determinations of ATP and creatine phosphate (CP) content in the heart extracts are shown in Table 1. Normal diet hearts subjected to 40 minutes of global ischemia and 20 minutes of reperfusion had 75% lower ATP and 85% lower CP levels. However, hearts from POE-supplemented group, subjected to similar conditions, did not have a significant decrease in the levels of ATP and CP. Inhibition of lipid peroxid~tion Content of endogenous lipid peroxidation fluorescent products in rat hearts are shown on Table 2. There was no significant difference in the amount of fluorescent products in normal diet and POE-supplemented hearts frozen immediately after excision. However, fluorescent products after 60 minutes of perfusion were doubled in normal diet hearts, while no change was detected in POE-supplemented hearts. When hearts were subjected to 40 minutes of ischemia and 20 minutes of reperfusion, fluorescent products increased by 6-fold in normal diet hearts and did not change in POE-supplemented hearts. Consumption of aloha-tocooherol and aloha-tocotrienol The concentration of endogenous vitamin E in Fat heart homogenates was 0.4 - 0.6 nmol/mg protein. After 6 weeks of dietary supplementation with POE, the concentration of endogenous alpha-tocopherol increased 3 times. The concentrations of alpha-tocotfienol and alpha-tocopherol in hearts were 1.2 + 0.2 nmol/mg protein and 1.8 + 0.3 nmol/mg protein, respectively. The data on the consumption of both forms of vitamin E in the course of ischemia/repcrfusion are shown in Table 3. There were no significant differences in the contents of alpha-tocopherol and alpha-tocotrienol, after 60 minutes of perfusion. However, 40 minutes of ischemia, followed by 20 minutes of reperfusion, caused a pronounced decrease in vitamin E levels. Only 41% of tocopherol and 21% of tocotrienol remained after ischemia/reperfusion in the POE-supplemented group. The amount of vitamin E in hearts from control group was less than 4 %.
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ESR measurements of the recycling efficiency of aloha-tocooherol and alpha-tocotden01 In the presence of heart homogenates obtained from rats subjected or not subjected to 40 minutes ischemia plus 20 minutes reperfusion, alpha-tocopherol and alpha-tocotrienol radicals
250 ~200
~ 150
E
Normal ~//biet
~"
~
POE-suppl.
~ 1oo o
~ 50' [5
O
, II--1 10 20
]'91~Time During Reperfusion Period (Minutes)'D~ I FIG. 2. Effect of supplementation with palm oil vitamin E on LDH release from isolated Langendorff hearts subjects to 10 min perfusion + 40 min ischemia + 20 min reperfusion. There were 6 animals in each experimental group. Conditions are given in the Methods section. TABLE 1 Effect Of Palm Oil Vitamin E (POE) On The Content Of High Energy Phosphates In Isolated Perfused Rat Hearts
ATP 60 min perfusion
Control +POE n=3,
100
31p Metabolites (% From control) Creatine Phosphate 100
10 min perfusion + 40 min ischemia + 20 min reperfusion 25 + 14 16 _+ 10 87 d: 17 91 5:12 p < 0.05
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TABLE 2 Effect Of Palm Oil Vitamin E (POE) On The Content Of Lipid Peroxidation Fluorescence Products in Rat Hearts (% of control) Normal Diet
POE Diet
100
100
60 rain perfusion
140 -b 20
100 • 10
10 min peffusion + 40 rain isehemia + 20 rain reperfusion
540 _+ 45
107 + 12
Control
n=6,
p < 0.05
TABLE 3 Effect Of Ischemia-Reperfusion On Vitamin E Content In Isolated Lagendorff Hearts From Rats Fed With Palm Oil Vitamin E (POE) Supplemented Diet Tocopherol Tocotrienol (nmoles/mg protein) 60 min perfusion
1.80 _+ 0.30
1.20 _ 0.20
10 min peffusion + 40 rain iscbemia + 20 min rcpcffusion
0.74 _+ 0.10
0.25 _ 0.05
n=6,
p<0.~
from exogenously added chromanols were generated by an enzymic oxidation system (lipoxygenase + linolenic acid) and their ESR spectra were recorded. Alpha-tocopherol and alpha-tocotrienol give characteristic pentameric chromanoxyl radical signals with g-values of the components 2.0122, 2.0092, 2.0061, 2.0028 and 1.9993 (14). The magnitude of alphatocotrienoxyl radical ESR signal was significantly higher than that of alpha-tocopheroxyl, both before and after iscbemia/repeffusion. Addition of ascorbate caused transient disappearance of ESR signals of chromanoxyl radicals in the presence of heart homogenate (Fig. 3) and appearance of ascorbyl radical signal. After ascorbate was consumed, its radical signal in the
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ESR spectrum was replaced by the growing signal of chromanoxyl radicals. The delay in the reappearance of chromanoxyl radical ESR signal was longer for alpha-tocotrienol than for alphatocopherol (Fig. 3).
ESR SIGNAL,
ESR SIGNAL,
Arbitrar 'Units
Arbitrar U n i t s
O•
30
---'O"- CONTROL SCORBATE
20
I0 .... J I
0
10
|
20
I
30
TOCOTRIENOL I
0
10
!
20
!
30
TIME (MIN)
FIG. 3 Time-course of chromanoxyl radicals of alpha-tocopherol and alpha-tocotrienol in rat hearts after 40 min ischemia + 20 min reperfusion. Conditions are given in the Methods section.
DISCUSSION Myocardial ischemia is a leading cause of mortality in the industrialized world (15) and a major contributor to health care costs in the US (16). The injuries of the ischemic heart occur during the reperfusion period, a phenomenon called the "reperfusion injury". It has been found that during reperfusion the amount of potentially salvageable myocardium is reduced and myocardial necrosis is increased, due to the destruction of the cardiomyocytes (17). Calcium overload (18), mitochondrial dysfunction (19), arachidonic acid metabolites (20) and defective myocardial lipid metabolism (21) contribute to the reperfusion injury. It has been postulated that
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reactive oxygen species (ROS) are one of the primary causes of heart injury (22). Therefore, it is suggested that administration of antioxidants may ameliorate myocardial ischemia/reperfusion injury. Since vitamin E is the primary lipid peroxidation chain-breaking antioxidant in membranes, it may exhibit both prophylactic and therapeutic roles against cardiac ischemic/reperfusional injury. Alpha-tocopherol and alpha-tocotrienol are two vitamin E constituients having the same aromatic chromanol "head" but different hydrocarbon ~tails'. Tocopherols have a saturated tail, while tocotrienols have an unsaturated isoprenoid chain. Serbinova et al. (1) reported that alphatocotrienol has markedly higher antioxidant activity in vitro than alpha-tocopherol, mainly due to (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. We hypothesized that alphatocotrienol may be more beneficial for the antioxidant protection of the ischemic/reperfused myocardium than alpha-tocopherol. To test this hypothesis, we tested the effects of POE on Langendorff hearts subjected to 40 minutes of global ischemia. Our results demonstrate that palm oil vitamin E was more efficient in the protection of the isolated Langendorff heart against ischemia/reperfusion injury than tocopherol, as measured by its mechanical recovery. Palm oil vitamin E completely suppressed LDH enzyme leakage from ischemic hearts, prevented the decrease in ATP and creatine phosphate levels and inhibited the formation of endogenous lipid peroxidation products. The results presented do not afford direct quantitative comparison of the protective effects of alpha-tocotrienol with that of alphatocopherol, since both were present in palm oil vitamin E concentrate in the proportion of 55:45. However, there is an indirect evidence of higher alpha-tocotrienol efficiency. Comparison of the consumption of alpha-tocopherol and alpha-tocotrienol, in the course of ischemia/reperfusion, showed that the alpha-tocotrienol loss was significantly more pronounced than that of alphatocopherol. In other words, alpha-tocotrienol was more active in radical scavenging and was preferentially consumed in the course of ischemia/reperfusion-induced oxidative stress. Our in vitro studies showed that recycling efficiency of alpha-tocotrienol, in the presence of ascorbate, is higher than that of alpha-tocopherol not only in control but also in ischemic/reperfused heart homogenates. If the recycling of vitamin E contributes to the maintenance of its concentration in vivo, the difference in the consumption of alpha-tocotrienol and alpha-tocopherol by ischemia/reperfusion-derived peroxyl radicals may even be higher than the one reported in Table 3, due to a more efficient regeneration of tocotrienol. Based on rat resorption-gestation test, d-alpha-tocopherol has the highest (100%) biopotency, while d-alpha-tocotrienol manifests only 30% of this acitivity (23). The significance of these estimations for health benefit is not clear, since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes. We have already noted in the introduction that other physiological activities of alpha-tocotrienol, such as antitumor and inhibition of cholesterol biosynthesis, were reported to be much higher than those of alphatocopherol. Thus alpha-tocotrienol may have higher physiological activity than alpha-tocopherol
VIT. E AND ISCHEMINREPERFUSION
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under conditions of oxidative stress. In particular, our data indicate that a palm oil vitamin E mixture containing both alpha-tocopherol and alpha-tocotrienol may be more efficient than alphatocopherol alone in the protection of the heart against oxidative stress induced by ischemia/reperfusion. ACKNOWLEDGEMENT Research supported by NIH (CA 47597) and the Palm Oil Research Institute of Malaysia 0'ORIM). REFERENCES .
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Packer L, Valenza M, Serbinova E, Starke-Reed P, Frost K, Kagan V. Free radical scavenging is involved in the protective effect of L-propionyl carnitine protection of the heart against ischemia/reperfusion injury of the heart. Arch Biochem Biophys 1991; 288:(in press).
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Reznick AZ, Kagan VE, Ramsey R, Tsuchiya M, Khwaja S, Serbinova E A, Packer L. Antiradical effects in L-propionyl camitine protection against ischemia/repeffusion injury: the possible role of iron chelation. Arch Biochem Biophys 1991; (submitted).
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Janero DR. Therapeutic potential of vitamin E against myocardial ischemia-reperfusion injury. Free Radical Biol Meal 1991; 10:315-324.
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Leipala J, Bhatanagar R, Pineda E, Najibi S, Massourmi K, Packer L. Protection of the reperfused heart by L-propionyl-carnitine. Optimal effect requires administration prior to ischemia. J Appl Physiol 1991; (in press).
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Packer L, Maguire JJ, Mehlhorn RI, Serbinova E, Kagan V. Mitochondrial and microsomal membranes have free radical reductase activity. Biochem Biophys Res Communn 1989; 1:229-235.
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Mehlhorn R, Sumida S, Packer L. Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E enriched rat liver mitochondria and microsomes. J Biol Chem 1989; 264:13448-13452.
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Lang J, Gohil K, Packer L. Simultaneous determination of tocopherols, ubiquinones in blood, plasma, tissue homogenates and subcellular fractions. Analyt Biochem 1986; 157:106-116.
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Malshet VG, Tappel AL, Burns VM. Fluorescent products of lipid peroxidation. II. Methods for analysis and characterization. Lipids i974; 9:328.
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Mukai K, Fukuda K, Ishizu K. Stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having higher antioxidant activity than alpha-tocopherol. Chem Phys Lipids 1988; 46:31-36.
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Sempos C, Cooper R, Kovan MG, McMillen M. Divergence of the recent trends in coronary mortality for four major race-sex groups in the United States. Am J Public Health 1988; 78:422-1427.
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Wittels EH, Hay JW, Gotto AM. Medical cost of coronary artery disease in the United States. Am J Cardiol 1990; 65:432-444.
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Flaherty Jr, Weisfeldt ML. Reperfusion injury. Free Radical Biol Med 1988; 5:409-419.
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Opie LH. Proposed role of calcium in reperfusion injury. Int J Cardiol 1989; 23:159164.
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Jennings RB, Ganote CE. Mitochondrial structure and function in acute myocardial ischemia injury. Circ Res 1976; 81(suppl): 80-91.
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Mullane KM. Eicosanoids in myocardial ischemia/reperfusion injury. Advances in inflammation research (vol 12) 1989; NY Raven Press: 191-214.
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van der Vusse GJ, van Bilsen M, Reneman RS. Impairment of lipid metabolism in ischemia and reperfused myocardial tissue. Analysis and stimulation of the cardiac system ischemia 1989; Boca Raton CRC Press: 355-369.
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McCord JM. Free radicals and myocardial ischemia: overview and outlook. Free Radic Biol Med 1988; 4:9-14.
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Leth T, Sondergaard H. Biological activity of vitamin E compounds and natural materials by the resorption gestation test and chemical determination of the vitamin E activity in foods and feeds. J Nutr 1977; 107:2236-2243.