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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 52 issue 4 (april 2018) : 502-507,   Doi: 10.18805/ijar.v0iOF.9146

Studying the effect of g-irradiated celery leaves on antioxidant status and cardiac enzymes in hypercholesterolemic rats

A
A.N. El shahat
A
A. M. Abdul Azeem
H
H. M. S. Mekawey
M
Mohamed H.M. Abd el-megid
1Food Irradiation Research Department, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.
Cite article:- shahat El A.N., Azeem Abdul M. A., Mekawey S. M. H., el-megid Abd H.M. Mohamed (2017). Studying the effect of g-irradiated celery leaves on antioxidant status and cardiac enzymes in hypercholesterolemic rats. Indian Journal of Animal Research. 52(4): 502-507. doi: 10.18805/ijar.v0iOF.9146.

ABSTRACT

This work aimed to study the effects of gamma (g) irradiation on the total phenols and total flavonoid contents of celery leaves and to investigate effect of g-irradiated celery leaves powder (g-Irr.CLP) against hypercholesterolemia in rats. Results obtained that g-irradiation increased the amount of total phenolic and total flavonoid contents of dried celery.  The results of biological study showed marked increases in the concentrations of serum lipid contents, activities of liver and cardiac marker enzymes and level of TBARS with a significant decrease in serum high-density lipoprotein concentration and reduction in the activities of anti-oxidant enzymes and glutathione contents of serum and heart tissues of hypercholesterolemic rats compared to control rats. Treatment of hypercholesterolemic rats with raw CLP or g-Irr.CLP showed significantly less severe damage and remarkable improvement in all of the measured parameters when compared to hypercholesterolemic rats lowered the concentrations of serum lipid contents, liver and cardiac marker enzymes and TBARS level and ameliorate anti-oxidant enzymatic status in serum and cardiac tissue. These results suggest that, treatment with g-Irr.CLP has a powerful modulating effect on hypercholesterolemia induced oxidative stress and has the potential in reducing cardiovascular complications.

REFERENCES

  1. Abd El-Mageed, N.M. (2011). Hepatoprotective effect of feeding celery leaves mixed with chicory leaves and barley grains to hypercholesterolemic rats. Pharmacogn Mag., 7(26): 151-6.
  2. Akinyemi, A. J., Oboh, G., Ademiluyi, A.O., Boligon, A. A. and Athayde M.L. (2016). Effect of two ginger varieties on arginase activity in hypercholesterolemic rats. J Acupunct Meridian Stud, 9(2):80-87.
  3. Alhat, A.G, Gunjal M.A, and Juvekar A.R. (2007). Proctective effect of Apium graveolens against experimental myocardial oxidative stress induced injury in rats. 40W”± annual conference of Indian Pharmacological Society., 123:117.
  4. Allain, C. C. , Poon, L. S., Chan, C. S., Richmond, W. and Fu, P. C. (1974). Enzymatic determination of total serum cholesterol. Clin.Chem. 20, 470.
  5. Anila, L. and Vijayalakshmi, N.R. (2003). Antioxidant action of flavonoids from Mangifera indica and Emblica officinalis in hypercholesterolemic rats. Food Chem., 83: 569–574.
  6. Aravind, R., Rajesh, B., Gopakumar, D., Verghese, S., Pillai, N.R. and Nair, H.J (2013). Effect of hydro - alcoholic extract of apium graveolens linn on isoprenaline induced myocardial infarction in albino rats. J. of Pharma Search, 8(3):13
  7. Beutler, E., Duron, O. and Kelly, B.M. (1963). Improved method for the determination of blood glutathione. J Lab Clin Med. 61: 882-888.
  8. Božin, B., Mimica-Dukiæ, N., Samojlik, I., Anaèkov, G. and Igiæ, R. 2008. Phenolic in garlic (Allium sativum L., Alliacea). Food Chem 111:925-929.
  9. Demacker, P. N. , Vos-Janssen, H. E. , Hifmans, A. G. M. , Van’t Laar, A. and Jansen, A. P. 1980. Measurement of high- density lipoprotein cholesterol in serum: Comparison of six isolation methods combined with enzymatic cholesterol analysis. Clin. Chem., 26:1780.
  10. Dikshit, M., Rastogi, L., Shukla, R. and Srimal, R.C. (1995). Prevention of ischaemia-induced biochemical changes by curcumin and quinidine in the cat heart. Indian J. Med. Res. 101:31–35
  11. Dutta, K. and Bishayi, B. (2009). Escherichia coli lipopolysaccharide administration alters antioxidant profile during hypercholesterolemia. Indian J. Clin. Biochem., 24, 179–183.
  12. Fossati, P. and Prencipe, L. (1982). Serum triglycerides determined calorimetrically with an enzyme that produce hydrogen peroxide. Clin. Chem., 28:2077.
  13. Friedwald, W.T., Levy, R. I. and Fredrickson, D. (1972). Estimation of concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem. 18(6):499. 
  14. Fukumoto, L. and Mazza, G. (2000). Asessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48: 3597-3604.
  15. Harnafi, H., Aziz, M. and Amrani, S. (2009). Sweet basil (Ocimum basilicum L.) improves lipid metabolism in hypercholesterolemic rats. Span the European J. of Clin. Nut. and Metabolism, 4: e181-e186.
  16. Huang S.J. and Mau J.L. (2006). Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of g-irradiation”. LWT-    Food Science and Technology, 39:707-716.
  17. Khoun O.S. (2012). Pharmacological Effect of Aqueous Leaves Extract of Ocimum Basilicum Against Liver Toxicity Induced by Acetaminophen in Male Rats. Thi-Qar J. Agric. Res., 1 (1) 13-27. 
  18. IFT. (1983). Radiation preservation of foods. Food Technol 37(2):55-60.
  19. Jain,G. C., Pareek, H., Khajja, B. S., Jain, K., J. halani, S., Agarwal, S. and Sharma, S. (2009). Modulation of di- (2- ethylhexyl) phthalate induced hepatic toxicity by Apium graveolens L. seeds extract in rats African J. Biochem.Res., 3 (5): 222-225.
  20. Jia, Z., Tang, M. and Wu, J. (1999). The determination of flavonoids content in mullbery and their scavenging effects on superoxide radicals. Food Chem. 64:555-559.
  21. Johansson, L. H. and Borg, L.A.H. (1988). A spectrophotometric method for determination of catalase activity in small tissue samples. Anal. Biochem. 74: 331.
  22. King J (1965) (ed). The dehydrogenases or oxidoreductases - lactate dehydrogenase. In: Practical Clinical Enzymology. Van Nostrand Company Ltd, London, 83 93.
  23. Kooti, W., Ali-Akbari, S., Asadi-Samani, M., Ghadery, H. and Ashtary-Larky, D. (2014a). A review on medicinal plant of Apium graveolens Advanced Herbal Medicine, 1 (1): 48-59.
  24. Kooti, W., Ghasemiboroon, M., Asadi-Samani, M., Ahangarpoor, A., Abadi, M. N.A., Afrisham, R. and Dashti N. (2014b). The effects of hydro-alcoholic extract of celery on lipid profile of rats fed a high fat diet. Adv. Environ. Biol.,8(9), 325-330. 
  25. Kooti, W., Ghasemi-boroon, M., Ghafourian, M., Asadi-Samani, M., Harizi, M., Sharafi-Ahvazi, N. and Afrisham, R. (2015). The effects of celery leave extract on male hormones in rats. J Herb Med Pharmacol, 4(2): 56-60.
  26. Kumari, N., Kumar, P., Mitra, D. , Prasad, B., Tiwary, B. N. and Varshney, L. (2009). Effects of ionizing radiation on microbial decontamination, phenolic contents, and anti-oxidant properties of Triphala. J. Food Sci. 74:M109-M1113.
  27. Lugasi, A., Hovari, J. Sagi, K. V. and Biro, L. (2003). The role of antioxidant phytonutrients in the prevention of diseases. Acta. Biol. Szegediensis., 47: 119–125
  28. Ma, J., Qiao, Z. and Xiang, X. (2011). Aqueous extract of Astragalus mongholicus ameliorates high cholesterol diet induced oxidative injury in experimental rats models. J. Med. Plants Res, 5:855–858.
  29. Manju, V. and Nalini, N. (2005). Chemopreventive potential of luteolin during colon carcinogenesis induced by 1,2-dimethylhydrazine. Ital. J. Biochem., 54(3-4):268-75.
  30. Mansi, K., Abushoffa, A.M., Disi, A. and Aburjai T. (2009). Hypolipidemic Effects of Seed Extract of Celery (Apium graveolens) in Rats. Phcog Mag, 5: 301-305.
  31. Minami, M. and Yoshikawa, H. (1979). A simplified assay method of superoxide dismutase activity for clinical use. Clin. Chim. Acta., 92: 337-342.
  32. Otunola, A., Oloyede, O.B., Oladiji, A.T. and Afolayan, A. A. (2010). Effects of diet-induced hypercholesterolemia on the lipid profile and some enzyme activities in female Wistar rats Gloria. Afr. J. of Bioch. Res. 4 (6):149-154.
  33. Parveen,S., Hussain, P.R., Mir, M.A., Shafi, F., Darakshan, S., Mushtaqand, A. and Suradkar, P. 2015. Gamma Irradiation Treatment of Cherry – Impact on Storage Quality, Decay Percentage and Post- Refrigeration Shelf-life Extension. Current Research in Nutrition and Food Science 3(1): 54-73.
  34. Popovic, M., Kaurinovic, B., Trivic, S., Mimica-Dukic, N. and Bursac, M. (2006). Effect of celery (Apium graveolens) extracts on some biochemical parameters of oxidative stress in mice treated with carbon tetrachloride. Phytother. Res., 20(7):531-7.
  35. Reitman, S. and Frankel, S. (1957). A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol . 28: 56.
  36. Sivashanmugam, M.U.A. and Jagannath P. (2011). Induction of Apoptosis and Cytotoxic Activities of Apium graveolens Linn. Using in vitro Models. Middle-East J Sci Res, 9(1): 90-4.
  37. Soanker, R., Naidu, M., Raju, S.B., Prasad, A.K. and Rao, T.R.K. (2012). Effect of beta-1-blocker, nebivolol, on central aortic pressure and arterial stiffness in patients with essential hypertension. Indian J. of Pharmaco., 44: 407-411.
  38. SPSS. (1998). Statistical Package for Social Science. Computer Software, Ver. 10. SPSS Company, London, UK.
  39. Suanarunsawat, T., Ayutthaya, W. D. N., Songsak, T., Thirawarapan S., and Poungshompoo S. (2011). Lipid-Lowering and Antioxidative Activities of Aqueous Extracts of Ocimum sanctum L. Leaves in Rats Fed with a High-Cholesterol Diet. Oxidative Medicine and Cellular Longevity Volume 2011, Article ID 962025, 9 pages
  40. Taher, M., Ghannadi, A. and Karmiyan, R. (2007). Effect of volatile oil extract of Anthum graveolens & Apium graveolens on activity of liver enzymes in rat. J. of Qazvin univ of Med. Sci. Vol. ll, NO.2.
  41. Thayer, D.W., Josephson, E.S., Brynjolfesson, A. and Giddings, G. (1996). Radiation pasteurization of food. Issue paper No. 7. Ames, Iowa: Council of Agricultural Science and Technology.
  42. Tsi, D. and Tan, B.K. (2000). The mechanism underlying the hypocholesterolaemic activity of aqueous celery extract, its butanol and aqueous fractions in genetically hypercholesterolemic Rico rats. Life Sciences, 66: 755-767.
  43. Villavicencio, A.L.C.H., Mancini-Filho, J., Delincée, H. and Greiner, R. (2000). Effect of irradiation on anti-nutrients (total phenolics, tannins and phytate) in Brazilian beans. Rad. Phy. and Chem., 57:289–293.
  44. Yoshioka, T., Kawada ,K., Shimada, T. and Mori ,M. (1979). Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am J Obstet Gynecol. 135: 372-376. 
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    Research Article

    volume 52 issue 4 (april 2018) : 502-507,   Doi: 10.18805/ijar.v0iOF.9146

    Studying the effect of g-irradiated celery leaves on antioxidant status and cardiac enzymes in hypercholesterolemic rats

    A
    A.N. El shahat
    A
    A. M. Abdul Azeem
    H
    H. M. S. Mekawey
    M
    Mohamed H.M. Abd el-megid
    1Food Irradiation Research Department, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.
    Cite article:- shahat El A.N., Azeem Abdul M. A., Mekawey S. M. H., el-megid Abd H.M. Mohamed (2017). Studying the effect of g-irradiated celery leaves on antioxidant status and cardiac enzymes in hypercholesterolemic rats. Indian Journal of Animal Research. 52(4): 502-507. doi: 10.18805/ijar.v0iOF.9146.

    ABSTRACT

    This work aimed to study the effects of gamma (g) irradiation on the total phenols and total flavonoid contents of celery leaves and to investigate effect of g-irradiated celery leaves powder (g-Irr.CLP) against hypercholesterolemia in rats. Results obtained that g-irradiation increased the amount of total phenolic and total flavonoid contents of dried celery.  The results of biological study showed marked increases in the concentrations of serum lipid contents, activities of liver and cardiac marker enzymes and level of TBARS with a significant decrease in serum high-density lipoprotein concentration and reduction in the activities of anti-oxidant enzymes and glutathione contents of serum and heart tissues of hypercholesterolemic rats compared to control rats. Treatment of hypercholesterolemic rats with raw CLP or g-Irr.CLP showed significantly less severe damage and remarkable improvement in all of the measured parameters when compared to hypercholesterolemic rats lowered the concentrations of serum lipid contents, liver and cardiac marker enzymes and TBARS level and ameliorate anti-oxidant enzymatic status in serum and cardiac tissue. These results suggest that, treatment with g-Irr.CLP has a powerful modulating effect on hypercholesterolemia induced oxidative stress and has the potential in reducing cardiovascular complications.

    REFERENCES

    1. Abd El-Mageed, N.M. (2011). Hepatoprotective effect of feeding celery leaves mixed with chicory leaves and barley grains to hypercholesterolemic rats. Pharmacogn Mag., 7(26): 151-6.
    2. Akinyemi, A. J., Oboh, G., Ademiluyi, A.O., Boligon, A. A. and Athayde M.L. (2016). Effect of two ginger varieties on arginase activity in hypercholesterolemic rats. J Acupunct Meridian Stud, 9(2):80-87.
    3. Alhat, A.G, Gunjal M.A, and Juvekar A.R. (2007). Proctective effect of Apium graveolens against experimental myocardial oxidative stress induced injury in rats. 40W”± annual conference of Indian Pharmacological Society., 123:117.
    4. Allain, C. C. , Poon, L. S., Chan, C. S., Richmond, W. and Fu, P. C. (1974). Enzymatic determination of total serum cholesterol. Clin.Chem. 20, 470.
    5. Anila, L. and Vijayalakshmi, N.R. (2003). Antioxidant action of flavonoids from Mangifera indica and Emblica officinalis in hypercholesterolemic rats. Food Chem., 83: 569–574.
    6. Aravind, R., Rajesh, B., Gopakumar, D., Verghese, S., Pillai, N.R. and Nair, H.J (2013). Effect of hydro - alcoholic extract of apium graveolens linn on isoprenaline induced myocardial infarction in albino rats. J. of Pharma Search, 8(3):13
    7. Beutler, E., Duron, O. and Kelly, B.M. (1963). Improved method for the determination of blood glutathione. J Lab Clin Med. 61: 882-888.
    8. Božin, B., Mimica-Dukiæ, N., Samojlik, I., Anaèkov, G. and Igiæ, R. 2008. Phenolic in garlic (Allium sativum L., Alliacea). Food Chem 111:925-929.
    9. Demacker, P. N. , Vos-Janssen, H. E. , Hifmans, A. G. M. , Van’t Laar, A. and Jansen, A. P. 1980. Measurement of high- density lipoprotein cholesterol in serum: Comparison of six isolation methods combined with enzymatic cholesterol analysis. Clin. Chem., 26:1780.
    10. Dikshit, M., Rastogi, L., Shukla, R. and Srimal, R.C. (1995). Prevention of ischaemia-induced biochemical changes by curcumin and quinidine in the cat heart. Indian J. Med. Res. 101:31–35
    11. Dutta, K. and Bishayi, B. (2009). Escherichia coli lipopolysaccharide administration alters antioxidant profile during hypercholesterolemia. Indian J. Clin. Biochem., 24, 179–183.
    12. Fossati, P. and Prencipe, L. (1982). Serum triglycerides determined calorimetrically with an enzyme that produce hydrogen peroxide. Clin. Chem., 28:2077.
    13. Friedwald, W.T., Levy, R. I. and Fredrickson, D. (1972). Estimation of concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem. 18(6):499. 
    14. Fukumoto, L. and Mazza, G. (2000). Asessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48: 3597-3604.
    15. Harnafi, H., Aziz, M. and Amrani, S. (2009). Sweet basil (Ocimum basilicum L.) improves lipid metabolism in hypercholesterolemic rats. Span the European J. of Clin. Nut. and Metabolism, 4: e181-e186.
    16. Huang S.J. and Mau J.L. (2006). Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of g-irradiation”. LWT-    Food Science and Technology, 39:707-716.
    17. Khoun O.S. (2012). Pharmacological Effect of Aqueous Leaves Extract of Ocimum Basilicum Against Liver Toxicity Induced by Acetaminophen in Male Rats. Thi-Qar J. Agric. Res., 1 (1) 13-27. 
    18. IFT. (1983). Radiation preservation of foods. Food Technol 37(2):55-60.
    19. Jain,G. C., Pareek, H., Khajja, B. S., Jain, K., J. halani, S., Agarwal, S. and Sharma, S. (2009). Modulation of di- (2- ethylhexyl) phthalate induced hepatic toxicity by Apium graveolens L. seeds extract in rats African J. Biochem.Res., 3 (5): 222-225.
    20. Jia, Z., Tang, M. and Wu, J. (1999). The determination of flavonoids content in mullbery and their scavenging effects on superoxide radicals. Food Chem. 64:555-559.
    21. Johansson, L. H. and Borg, L.A.H. (1988). A spectrophotometric method for determination of catalase activity in small tissue samples. Anal. Biochem. 74: 331.
    22. King J (1965) (ed). The dehydrogenases or oxidoreductases - lactate dehydrogenase. In: Practical Clinical Enzymology. Van Nostrand Company Ltd, London, 83 93.
    23. Kooti, W., Ali-Akbari, S., Asadi-Samani, M., Ghadery, H. and Ashtary-Larky, D. (2014a). A review on medicinal plant of Apium graveolens Advanced Herbal Medicine, 1 (1): 48-59.
    24. Kooti, W., Ghasemiboroon, M., Asadi-Samani, M., Ahangarpoor, A., Abadi, M. N.A., Afrisham, R. and Dashti N. (2014b). The effects of hydro-alcoholic extract of celery on lipid profile of rats fed a high fat diet. Adv. Environ. Biol.,8(9), 325-330. 
    25. Kooti, W., Ghasemi-boroon, M., Ghafourian, M., Asadi-Samani, M., Harizi, M., Sharafi-Ahvazi, N. and Afrisham, R. (2015). The effects of celery leave extract on male hormones in rats. J Herb Med Pharmacol, 4(2): 56-60.
    26. Kumari, N., Kumar, P., Mitra, D. , Prasad, B., Tiwary, B. N. and Varshney, L. (2009). Effects of ionizing radiation on microbial decontamination, phenolic contents, and anti-oxidant properties of Triphala. J. Food Sci. 74:M109-M1113.
    27. Lugasi, A., Hovari, J. Sagi, K. V. and Biro, L. (2003). The role of antioxidant phytonutrients in the prevention of diseases. Acta. Biol. Szegediensis., 47: 119–125
    28. Ma, J., Qiao, Z. and Xiang, X. (2011). Aqueous extract of Astragalus mongholicus ameliorates high cholesterol diet induced oxidative injury in experimental rats models. J. Med. Plants Res, 5:855–858.
    29. Manju, V. and Nalini, N. (2005). Chemopreventive potential of luteolin during colon carcinogenesis induced by 1,2-dimethylhydrazine. Ital. J. Biochem., 54(3-4):268-75.
    30. Mansi, K., Abushoffa, A.M., Disi, A. and Aburjai T. (2009). Hypolipidemic Effects of Seed Extract of Celery (Apium graveolens) in Rats. Phcog Mag, 5: 301-305.
    31. Minami, M. and Yoshikawa, H. (1979). A simplified assay method of superoxide dismutase activity for clinical use. Clin. Chim. Acta., 92: 337-342.
    32. Otunola, A., Oloyede, O.B., Oladiji, A.T. and Afolayan, A. A. (2010). Effects of diet-induced hypercholesterolemia on the lipid profile and some enzyme activities in female Wistar rats Gloria. Afr. J. of Bioch. Res. 4 (6):149-154.
    33. Parveen,S., Hussain, P.R., Mir, M.A., Shafi, F., Darakshan, S., Mushtaqand, A. and Suradkar, P. 2015. Gamma Irradiation Treatment of Cherry – Impact on Storage Quality, Decay Percentage and Post- Refrigeration Shelf-life Extension. Current Research in Nutrition and Food Science 3(1): 54-73.
    34. Popovic, M., Kaurinovic, B., Trivic, S., Mimica-Dukic, N. and Bursac, M. (2006). Effect of celery (Apium graveolens) extracts on some biochemical parameters of oxidative stress in mice treated with carbon tetrachloride. Phytother. Res., 20(7):531-7.
    35. Reitman, S. and Frankel, S. (1957). A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol . 28: 56.
    36. Sivashanmugam, M.U.A. and Jagannath P. (2011). Induction of Apoptosis and Cytotoxic Activities of Apium graveolens Linn. Using in vitro Models. Middle-East J Sci Res, 9(1): 90-4.
    37. Soanker, R., Naidu, M., Raju, S.B., Prasad, A.K. and Rao, T.R.K. (2012). Effect of beta-1-blocker, nebivolol, on central aortic pressure and arterial stiffness in patients with essential hypertension. Indian J. of Pharmaco., 44: 407-411.
    38. SPSS. (1998). Statistical Package for Social Science. Computer Software, Ver. 10. SPSS Company, London, UK.
    39. Suanarunsawat, T., Ayutthaya, W. D. N., Songsak, T., Thirawarapan S., and Poungshompoo S. (2011). Lipid-Lowering and Antioxidative Activities of Aqueous Extracts of Ocimum sanctum L. Leaves in Rats Fed with a High-Cholesterol Diet. Oxidative Medicine and Cellular Longevity Volume 2011, Article ID 962025, 9 pages
    40. Taher, M., Ghannadi, A. and Karmiyan, R. (2007). Effect of volatile oil extract of Anthum graveolens & Apium graveolens on activity of liver enzymes in rat. J. of Qazvin univ of Med. Sci. Vol. ll, NO.2.
    41. Thayer, D.W., Josephson, E.S., Brynjolfesson, A. and Giddings, G. (1996). Radiation pasteurization of food. Issue paper No. 7. Ames, Iowa: Council of Agricultural Science and Technology.
    42. Tsi, D. and Tan, B.K. (2000). The mechanism underlying the hypocholesterolaemic activity of aqueous celery extract, its butanol and aqueous fractions in genetically hypercholesterolemic Rico rats. Life Sciences, 66: 755-767.
    43. Villavicencio, A.L.C.H., Mancini-Filho, J., Delincée, H. and Greiner, R. (2000). Effect of irradiation on anti-nutrients (total phenolics, tannins and phytate) in Brazilian beans. Rad. Phy. and Chem., 57:289–293.
    44. Yoshioka, T., Kawada ,K., Shimada, T. and Mori ,M. (1979). Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am J Obstet Gynecol. 135: 372-376. 
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