Protective effect of vitamin E on fetal distress induced by ischemia of the uteroplacental system in pregnant rats

Free Radical Biology & Medicine, Vol. 8, pp. 393-400, 1990

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÷

Original Contribution PROTECTIVE BY

EFFECT ISCHEMIA

OF

VITAMIN

OF

THE

E ON

FETAL

UTEROPLACENTAL

IN PREGNANT

DISTRESS

INDUCED

SYSTEM

RATS

HOZUMI 1WASA, TOSHIHIRO AONO, and KENJI FUKUZAWA* Department of Obstetrics and Gynecology, School of Medicine, and *Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, 770, Japan (Received 8 August 1989; Revised and Accepted 27 December 1989)

Abstract--The effect of dietary vitamin E on the fetal ischemic distress induced by clamping the uterotubal vessels of pregnant rats was studied. The fetal heart rate was measured by the pulsed doppler technique as an index of fetal distress induced by ischemia. On reperfusion after clamping the vessels for 9 min, the decreased fetal heart rate was restored to normal rapidly and completely in the E-supplemented group, but slowly and incompletely in the E-deficient and control groups. On reperfusion after ischemia, the amounts of lipid peroxides, measured as thiobarbituric acid (TBA)-reactive substances, were greatly increased in the fetal brain and liver and in the placenta of in the E-deficient and control groups, but not in the E-supplemented group. The vitamin E concentrations in fetal tissues were less than 10% of those in the maternal tissues. Significant differences were found in the vitamin E concentrations in the maternal serum and liver in the three groups of rats given diet containing different amounts of vitamin E for 2 weeks. No significant differences were found between the vitamin E-deficient and control groups in the levels of vitamin E in the fetal brain and liver and the placenta, but these levels were significantly lower than those in the E-supplemented group. Keywords--Vitamin E, a-Tocopherol, Ischemia, Fetal distress, Lipid peroxidation, TBA-reactive substances, Free radical, Active oxygen

clamping the maternal uterotubal arteries and veins and investigated the protective effects of dietary vitamin E on the resulting fetal distress, evaluated as decreases in the fetal heart rate. We also measured the changes in levels of lipid peroxides and oL-tocopherol in the fetal tissues induced by ischemia and reperfusion. The protective mechanism of vitamin E is discussed from the viewpoint of its antioxidant effect.

INTRODUCTION

Fetal distress is usually caused by ischemia due to decrease in intervillous blood flow. 1'2 Ischemia and subsequent reperfusion injury in various tissues, such as the brain, heart, and spinal cord, is thought to be due to reactions of active oxygen including lipid peroxidation in biomembranes. However, the direct correlation of lipid peroxidation with fetal distress due to ischemia of the uteroplacental system has not been examined. Vitamin E is reported to reduce ischemic injury of the brainfl liver, 4 heart, 6'7 and kidney, s possibly because of its antioxidant effect on lipids. Recently, we have also reported that vitamin E has protective effects on ischemic injury of the spinal cord, possibly by inhibiting lipid peroxidation. 9 In this study, we induced fetal ischemia in rats by

MATERIALS AND METHODS

Animals Vitamin E deficiency causes disturbance of reproduction. So, pregnant Wistar strain rats were maintained on the semisynthetic diet described below containing a normal level of e~-tocopherol acetate per 100 g of diet for 6 days after conception, and then divided into three groups, a vitamin E-deficient group (group D), control group (group C), and vitamin Esupplemented group (group E), given the diet described below containing o~-tocopherol acetate at 0.1 IU, 2 IU,

Address correspondence to: Hozumi Iwasa, Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima-shi, Tokushima, 770, Japan 393

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and 50 IU per 100 g of diet, respectively, for 14 days. The diet consisted of 36% corn starch, 25% vitaminfree casein, 10% e~-starch of wheat, 8% powdered filter paper, 6% salt mixture, 5% sucrose, 2% vitamin mixture, and 10% stripped corn oil (Eastman Kodak Chemicals). The salt mixture consisted of 4.5% Na; 11.5% K; 9.7% P; 6.9% Ca; 1.4% Mg; 0.7% Fe; 0.01% Zn; 0.02% Mn; and 0.1% I. The vitamin mixture (1 g) consisted of 500 IU vitamin A, 100 IU D2, less than 0.05 IU E, 1.2 mg BI, 4 mg B2, 0.8 mg B~,, 0.5 /tg B~2, 30 mg C, 5.2 mg K, 0.02 mg biotin, 0.2 mg folic acid, 5 mg Ca-panthenate, 5 mg para-aminobenzoic acid, 6 mg niacin, 6 mg inositol, and 2000 mg cholineCI. Rats weighing about 230-280 g were used for experiments.

Ischemic .fetal distress On day 20 of pregnancy, the rats were anethetized by intraperitoneal injection of sodium pentobarbital (2.0 rag/100 g body weight), and subjected to lapa-

rotomy. The presence of fetuses in the uterus bicornis was confirmed and then ischemia was induced by clamping the uterotubal vessels of one horn at its cervical and ovarian ends (Fig. 1). The blood flow was restored after 5, 9, or 15 min. The fetuses in the horn on the unclamped side served as non-ischemic controls.

Evaluation of fetal distress The degree of fetal distress was evaluated from the changes in heart rate of the fetuses deduced from the waves of blood flow, because the heart rate was thought to reflect not only cardiac function but also the function of the central nervous system.~° Waves of blood flow were measured with a doppler blood flow jt and fetal cardiac pulse detector (Mini Dopplex D-500, Hantleigh Medical Co., England) connected to an 8 MHz transducer and recorded with a Unicorder desk-top recorder UR-2P (Unique Medical Co., Japan). The fetal heart rate was calculated from the wave frequency. Groups of 6 rats were used for experiments.

Fetus--

Doppler Utero-tubal artery and vein -4 Uterus

Fig. 1. Diagram of procedure for induction of ischemia by clamping uterotubal vessels and for measurementof the fetal heart rate by the ultrasonic doppler method.

Vitamin E protects fetal ischemic distress

Measurement of lipid peroxides Lipid peroxides in the fetal liver and brain and in the placenta of rats were measured by the TBA method ~2 and expressed as TBA-reactive substances in nmol of MDA. Excised tissues were promptly washed with 0.9% NaCI to remove blood and chilled in icecold 0.9% NaC1 for measurements, which were made within 1 h. For the assay, a 10% (w/v) homogenate of the tissue was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and an aqueous solution of thiobarbituric acid and heated at 95°C for 60 min. The mixture was then cooled, the pink pigment was extracted with n-butanol-pyridine mixture, and its absorbance at 532 nm was measured. Tetramethoxypropane was used as an external standard and the level of lipid peroxides was expressed as the amount of malondialdehyde. The value of TBARS was measured before ischemia and 6, 30, or 60 min after ischemia.

Assay of c~-tocopherol (vitamin E) c~-Tocopherol was extracted with ethanol-hexane (2 : 5) from sera and homogenates of maternal and fetal brains, livers, and placenta, c~-Tocopherol in the hexane layer was separated by HPLC with a Hitachi 655A11 apparatus and a YMC-PACK A-600-3 (NH2 column) and detected with a Hitachi-1100 fluorescence spectrometer.

RESULTS

As shown in Table 1, the maternal levels of c~-tocopherol in the brain and liver were significantly higher than the fetal levels in all three groups, C, D, and E. The a-tocopherol concentration in the maternal serum on day 20 of pregnancy was 5.4 --- 1 . 0 / t g / m l (n = 7) in group C, 2.2 -+ 0 . 3 / l g / m l (n = 7) in group D, and 13.0 +- 2 . 0 / z g / m l (n = 7) in group E. Thus administration of diet containing different vitamin E concentrations for 14 days resulted in significant differences in the serum et-tocopherol concentration ( p < 0.001) from that in control group C. It also

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resulted in decrease in the o~-tocopherol concentration in the maternal liver in group D to 0.5 times the control level and its increase in group E to 7.0 times the level in group C. Similar differences were observed in the a-tocopherol contents of the placenta in the three groups. There was no significant difference in the o~tocopherol contents of the maternal brain in groups C and D, but the content in group E was 1.4 times that in group C. The a-tocopherol concentrations in the brain and liver of fetuses were not significantly different in groups C and D, but those of group E were 5.3 and 4.7 times higher, respectively, than those in group C (p < 0.001). Figure 2 shows typical results on waves of fetal blood flow before ischemia, during clamping of uterotubal vessels for 9 rain, and during subsequent reperfusion. In all three groups, the wave frequency decreased during ischemia. After reperfusion for 30 rain, group D still showed a decreased wave frequency, but the wave frequency in group E was restored to that before ischemia. Figure 3 showed the influences of the period of clamping of uterotubal vessels and the effect of dietary vitamin E on the fetal heart rate. In both group D and E, the fetal heart rate was decreased to about 70% of the initial rate by clamping for 5 min. It returned to 91.4% of the initial rate after reperfusion for 10 min in group E and to 90.9% of the initial rate in 60 min in group D, indicating that recovery was slightly slower in group D (Fig. 3a). lschemia induced by clamping for 9 min caused about 50% decrease in the fetal heart rate in all three groups (Fig. 3b). During reperfusion, the fetal heart rate returned to 79.4% and 86.6% of the initial rate in 10 min and 60 min, respectively, in group E. However, during reperfusion for 30 min, it returned to only 63.4% and 59.6% of the initial rate in groups C and D, respectively. Clamping for 15 min decreased the fetal heart rate to less than 50% of the initial value in group D and E, and scarcely any recovery was observed in these groups during reperfusion for 60 min (Fig. 3c). Figure 4 shows the time courses of TBARS for-

Table 1. a-Tocopherol Contents in Maternal and Fetal Rat Tissues Fetal (n = 14 per Group)

Maternal (n = 7 per Group)

Group C Group D Group E

Brain (/tg/g)

Liver (ug/g)

Serum (/tg/ml)

17.9 ± 1.5 16.6 ± 1.5 24.3 ± 1.2"

30.5 ± 3.4 14.1 -+- 1.4" 214.1 ± 53.8*

5.4 ± 1.0 2.2 ± 0.3* 13.0 ± 2.0*

Placenta

Significance of difference from respective value in group C: *p < 0.001.

(n

= 14 per Group) (,ug/g)

12.7 -+ 1.0 6.6 -+ 0.5* 40.4 ± 2.2*

Brain (ug/g)

Liver (ltg/g)

1.3 ± 0.3 1.0 ± 0.5 6.9 ± 2.2*

1.7 + 0.8 1.1 ± 0.3 8.0 -+ 1.7"

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Fig. 2. Typical recordings of blood flow waves during ischemia and reperfusion. Records are for blood flow waves before ischemia (A), during iscbemia for 9 rain (B), and after reperfusion for 30 rain in group C (C), group D (D), and group E (E).

marion in fetal tissues during ischemia and reperfusion. The T B A R S levels in the brain, liver, and placenta of fetuses were not significantly different immediately after ischemia from their levels before ischemia, but increased significantly to maxima after reperfusion for 30 min. The level in group D was the highest and was still significantly increased after reperfusion for 60 min. The order of the concentrations of T B A R S in fetal tissues decreased in the order liver > placenta > brain, and the levels in all organs were higher in group D than in group E (Table 1).

Figure 5 shows the amounts of T B A R S before and 30 min after ischemia. The TBARS contents of the brain, liver, and placenta significantly increased 30 rain after ischemia. The increases of TBARS in groups C and D were high and not significantly different, while those in group E were significantly lower. Changes in tissue c~-tocopherol concentration due to ischemia are shown in Fig. 6. The ¢x-tocopherol concentrations in the brain and liver of the fetuses did not change due to ischemia and subsequent reperfusion in any of the three groups, but in the placenta the concentrations were higher during ischemia and after

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Fig. 4. Effect of dietary vitamin E on the levels of TBARS in (A) fetal brain, (B) fetal liver, and (C) placenta before ischemia and after reperfusion. Points and bars are means +- SD for 6 rats. The shaded bar indicates the time of clamping. (Q)) group D; ( 0 ) group E. Significance of differences between values in the two groups: *p < 0.05, **p < 0.01, ***p < 0.001. Significance of differences in values before and after ischemia: a,p < 0.05; b,p < 0,01; c,p < 0.001.

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Fig. 6. Tissue a-tocopherol concentrations (ES:]) before ischemia, (1~) during ischemia, and (11) after reperfusion for 30 min. Ischemia was produced by clamping the uterotubal vessels for 9 min. Columns and bars are mean values for 8 rats -+ SD, in (C) group C, (D) group D, and (E) group E. *Significant differences from a-tocopherol concentration before ischemia (p < 0.001).

Vitamin E protects fetal ischemic distress

reperfusion for 30 min in group E, being 50.3 - 8.2 /tg/g wet weight and 51.2 _+ 6.9 /tg/g wet weight ( p < 0.05) as compared to 38.6 - 2.7 ~g / g wet weight before ischemia.

DISCUSSION

In this work, we observed marked recovery of the fetal heart rate after postischemic reperfusion in group E (Fig. 3b), indicating that a high dietary level of vitamin E was effective for recovery from fetal ischemic distress. Supplementation of the diet with vitamin E resulted in marked decrease in the increased levels of lipid peroxides in fetal tissues after ischemia in parallel with rapid recovery of the decreased heart rate. These results suggest that a decreased heart rate and increase in the TBARS level are closely related with the pathogenesis of fetal ischemic distress, and that vitamin E prevents fetal ischemic distress by inhibiting lipid peroxidation. We found that the vitamin E contents of fetal tissues were less than one tenth of those of maternal tissues (Table 1). This finding is consistent with the report of Mino et al. that the vitamin E concentration of fetal human liver is about 1/ 10 of that of adult liver tissue. ~3 These results indicate that vitamin E does not readily pass across the placenta, as reported by Nitowski et al. H

No difference was found in the vitamin E levels of the brain and liver of fetuses in groups C and D, indicating that a normal dietary intake of vitamin E during a short period of pregnancy does not result in any marked increase in the vitamin E levels in the fetuses. But 14 days feeding of diet containing a large amount of vitamin E significantly increased the concentrations of eL-tocopherol in fetal brain and liver. It would be very interesting if such increased levels of vitamin E had startling beneficial effects. These facts in combination with the report of Leonard et al. ~5 that the vitamin E concentration in human maternal blood shows a positive correlation with that in cord blood, 16.17 suggest that the early recoveries of the fetal heart rate and the level of TBARS in group E may have been due to passage of vitamin E across the placenta to the fetus. The concentrations of vitamin E in the fetal brain and liver before ischemia, during ischemia, and after reperfusion were not significantly different, but increase in TBARS during ischemia and reperfusion were reduced in group E. The reason for this discrepancy is not known. The placental vitamin E content is reported to be significantly higher during ischemia and reperfusion than before ischemia. ~s This may represent a defensive response of the placenta to oxidative stress. During ischemia and reperfusion after ischemia, lipoxygenase and cyclooxygenase have been reported to

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trigger the arachidonate cascade, which causes secondary functional injury. ~9.20As some arachidonic acid metabolites as well as MDA-like substances derived from lipid peroxides are responsive to TBA, 21 the marked increase in TBARS during postischemic reperfusion may reflect increase in precursors of prostaglandin F2,~, thromboxane A2, and leukotrienes. Further investigations are required on ischemia-induced metabolic changes of arachidonate in the placenta and the effect of vitamin E on these changes. REFERENCES I. Clavero, J. A.; Negueruela, J.; Ortiz, L.; Heros, J. A. D. L.; Modrego, S. P. Blood flow in the intervillous space and fetal blood flow: I. normal values in human pregnancies at term. Am. J. Obstet. Gvnecol. 116:340-346: 1973. 2. Clavero, J. A.; Ortiz, L.; Heros, J. A. D. L.; Neguerela, J. Blood flow in the intervillous space and fetal blood flow: It. Relation to placental histology and histometry in cases with and without high fetal risk. Am. J. Obstet. Gynecol. 116:1157- I 162; 1973. 3. Yoshida, S.; Busto, R.; Watson, B. D.; Santiso, M.; Ginsberg, M. D. Postischemic cerebral lipid peroxidation in vitro: modification by dietary vitamin E. J. Neurochem. 44:1593-1601; 1985. 4. Marubayashi, S.; Dohi, K.; Ochi, K.; Kawasaki, T. Role of free radicals in ischemic rat liver cell injury: prevention of damage by a-tocopherol administration. Surgeo' 99:184-192; 1986. 5. Werns, S. W.; Lucchesi, B. R. Leukocytes, oxygen radicals, and myocardial injury due to ischemia and reperfusion. Free Radical Biol. Med. 4:31-37: 1988. 6. Zweier, J. L.; Flaherty, J. T.: Weisfeldt, M. L. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc. Natl. Acad. Sci. 84:1404-1407; 1978. 7. Revis, N. W.; Armstread, B. The role of selenium and vitamin E in prevention of lipid peroxidation and myocardial necrosis induced by isoprenaline treatment or coronary artery ligation. Excepta Medica I.C.S. 2: 1029-1032; 1979. 8. Takenaka, M. ; Tatsukawa, Y. ; Dohi, K. ; Ezaki, H. : Matsukawa, K.; Kawasaki, T. Protective effects of a-tocopherol and coenzyme Q on warm ischemic damages of the rat kidney. Transplantation. 32:137 141; 1981. 9. lwasa, K.; lkata, T.; Fukuzawa, K. Protective effect of vitamin E on spinal cord injury by compression and concurrent lipid peroxidation. Free Radical Biol. Med. 6:599-606: 1989. 10. Hon, E. H. Additional observation on "pathologic" bradicardia. Am. J. Obstet. Gvnecol. 118:428-441: 1974. 11. Campbell, S.; Diaz-Recasens, J.; Grifin, D. R.; Cohen-Overbeek, T. E.; Pearce, J. M.; Willson, K. New doppler technique for assessing uteroplacental blood flow. Lancet. i:675-677; 1983. 12. Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351-358; 1979. 13. Mino, M. Physiological importance of vitamin E in the neonate, particularly in the newborn. J. Clin. Nutr. Japan. 49:429-435; 1976. 14. Nitowsky, H. M.; Cornblath, M.; Gordon, H. H. Studies of tocopherol deficiency in infants and children. Am. J. Dis. Child. 92:164-174; 1956. 15. Leonard, P. J.; Doyle, E.; Harrington, W. Levels of vitamin E in the plasma of newborn infants and of the mothers. Am. J. Clin. Nutr. 25:480-484; 1972. 16. Mino, M.; Nishino, H. Fetal and maternal relationship in serum vitamin E level. J. Nutr. Sci. Vitaminol. 19:475-482; 1973. 17. Imanishi, H. Studies on changes of lipid peroxidations and vitamin E during pregnancy. Nagoya Med. J. 25:117-130; 1981.

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18. Matuo, M. The role of vitamin E in fetal reproductive function: the reference of the uptake of the HC-labelled vitamin E in the several organs of female rats. Vitamins Japan. 39:1 10: 1969. 19. Gaudet, R. J.; Alam, 1.; Levin, L. Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid artcry occlusion. J. Neurochem. 35:653-658; 1980.

20. Moskowitz, M. A.: Kiwak, K. J.; Hekimian, K.; Levine, L. Synthesis of compounds with properties of leukotrienes C and D in gerbil brains after ischemia and reperfusion. Science 224:886-889; 1984. 21. Shimizu, T.: Kondo, K.; Hayaishi, O. Role of prostaglandin endoperoxides in the serum; thiobarbituric acid reaction. Arch. Biochem. Biophys. 206:271-276:1981.