羰基应激对血液粘度及生物组织荧光特性的影响
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摘要
羰基毒化衰老学说指出，羰-氨反应作为自由基氧化和非酶糖基化两大生化副反应的兼有反应，是氧化和糖基化造成缓慢生物老化过程的不可避免且大部分不可修复的核心过程。作为羰基毒化产物的典型代表，具有生物反应活性的不饱和醛酮类中间产物是造成衰老的最重要原因之一。疾病、紧张以及衰老等生物过程均伴随着不饱和醛酮类物质的制造过程。同时，在衰老和很多疾病状态下，都伴随着血液粘度的升高。血液粘度作为血液流变学的主要参数，一直都是病理生理学上的一个主要表征。研究表明，生物体内不可避免的生物副反应，特别是氧化紧张和非酶糖基化与生物体内血液流变学的改变像红细胞膜变形性的降低、血小板凝聚性的增加，甚至血管壁的硬化等等息息相关。通过对典型的羰基毒化产物丙二醛(MDA)的研究发现，丙二醛能够明显提高生物流体的各项流变性指标。丙二醛不但能提高小牛血清白蛋白溶液、血浆等牛顿流体的粘度，而且能够提高红细胞悬浮液等非牛顿流体的粘度。同时，塑性粘度作为重要的流变学指标，也随着加入丙二醛浓度的增加，也出现明显增加。氧化反应也能够明显增加红细胞悬浮液的粘度和塑性粘度。丙二醛(20mM)能迅速地降低蛋白溶液的典型荧光(280nm／350nm)，而就此产生在395nm的波长激发下，发射出波长为460nm的荧光，造成蛋白质的变构变性。这种荧光的相对强度随时间推移而明显增强，其波长特征与疾病形成的腊黄素以及随龄而增的老年色素类物质的荧光特性相吻合。同时，针对很多文章所推荐的在低剪切率下测定血液粘度的方法，本文还进行了一些在血液粘度测量方法学上的一些探讨。结果表明，仅仅在低剪切率下的结果，并不能够说明或者说并不能完全说明样品的流变学特性。
According to carbonyl stress aging theory, carbonyl-amine reaction as a common reaction of oxidation and glycosylation is an inevitable and irreversible core process during the slow aging procedure caused by oxidation and glycosylation. Reactive mediates of unsaturated aldehydes and ketons as representatives of carbonyl stress products are a group important causes of aging. Ailments, stress and aging et al all accompanied with the production of unsaturated aldehydes and ketons. And also, at these conditions, elevated blood viscosity occurred. Several unavoidable biological side-reactions, particularly oxidative stress, hyperglycemia related nonenzymatic glycosylation (glycation), were found to be correlated with the changed blood rheology, e.g. reduced deformability of erythrocytes, increased platelet adhesiveness, and enhanced blood plastic viscosity (see materials and methods) and blood vessel rigidity. MDA as a typical product of carbonyl stress can significantly elevate viscosity and plastic viscosity of blood materials such as bovine serum albumin (BSA), human plasma and erythrocyte suspensions. Oxidation due to Fe2+ can also have the same effect to the erythrocyte suspensions. MDA (20mM) reduced sharply the typical fluorescence of proteins (excitation 280nm/emission 350nm) and produced age pigment-like fluorescence with a strong emission peak at 460 nm when excited at 395 nm when incubated at 37 C. In contrast to other studies that consistent viscosity change of red cells was detected at very low shear rate (between 0-10 s-1), the data in this study, however, showed a substantial difference. With the present technique, the viscosity data at a low shear rate is scientifically meaningless.
According to carbonyl stress aging theory, carbonyl-amine reaction as a common reaction of oxidation and glycosylation is an inevitable and irreversible core process during the slow aging procedure caused by oxidation and glycosylation. Reactive mediates of unsaturated aldehydes and ketons as representatives of carbonyl stress products are a group important causes of aging. Ailments, stress and aging et al all accompanied with the production of unsaturated aldehydes and ketons. And also, at these conditions, elevated blood viscosity occurred. Several unavoidable biological side-reactions, particularly oxidative stress, hyperglycemia related nonenzymatic glycosylation (glycation), were found to be correlated with the changed blood rheology, e.g. reduced deformability of erythrocytes, increased platelet adhesiveness, and enhanced blood plastic viscosity (see materials and methods) and blood vessel rigidity. MDA as a typical product of carbonyl stress can significantly elevate viscosity and plastic viscosity of blood materials such as bovine serum albumin (BSA), human plasma and erythrocyte suspensions. Oxidation due to Fe2+ can also have the same effect to the erythrocyte suspensions. MDA (20mM) reduced sharply the typical fluorescence of proteins (excitation 280nm/emission 350nm) and produced age pigment-like fluorescence with a strong emission peak at 460 nm when excited at 395 nm when incubated at 37 C. In contrast to other studies that consistent viscosity change of red cells was detected at very low shear rate (between 0-10 s-1), the data in this study, however, showed a substantial difference. With the present technique, the viscosity data at a low shear rate is scientifically meaningless.
引文
[1] D. Yin and U.T. Brunk, Carbonyl toxification hypothesis of biological aging, in: Molecular basis of aging, A. Macieira-Coelho, ed., CRC Press Inc., New York, 1995, pp. 421-436.
[2] D. Yin, Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores, Free Radic. Biol. Med. 1996, 21: 871-888.
[3] B. Wierusz-Wysocka, H. Wysocki, H. Byks, et al. Metabolic control quality and free radical activity in diabetic patients. Diabetes Res. Clin. Pract. 1995, 27:193-197.
[4] K. Kikugawa, M. Beppu. Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins. Chem. Phys. Lipids 1987, 44:277-296
[5] T. Yoshikawa. Free radicals and their scavengers in Parkinson's disease. Eur Neurol. 1993, 33: 60-68.
[6] S. Chien. Hemorheology in clinical medicine, Clin. Hemorheol. 1982, 2: 137-142.
[7] K.J. Chen and Z.X. Shi, Practical Blood Stasisology, People's Hygiene Press, Beijing, 1999.
[8] U. Rauch, J.I. Osende, V. Fuster, J.J. Badimon, Z. Fayad and J.H. Chesebro, Thrombus formation on atherosclerotic plaques: pathogenesis and clinical consequences, Ann. Intern. Med. 2001, 134: 224-238.
[9] W.C. Aird, The hematologic system as a marker of organ dysfunction in sepsis, Mayo Clin. Proc. 2003, 78: 869-881.
[10] M. Piagnerelli, K.Z. Boudjeltia, M. Vanhaeverbeek and J.L. Vincent, Red blood cell rheology in sepsis, Intensive Care Med. 2003, 29: 1052-1061.
[11] K.R. Kensey, The mechanistic relationships between hemorheological characteristics and cardiovascular disease, Curr. Med. Res. Opin.
2003, 19: 587-596.
[12] K.R. Kensey, Rheology: an overlooked component of vascular disease, Clin. Appl. Thromb. Hemost. 2003, 9:93-99.
[13] J. Koscielny, R. Latza, S. Wolf, H. Kiesewetter and F. Jung, Early theological and microcirculatory changes in children with type I diabetes mellitus, Clin. Hemorbeol. Microcirc. 1998, 19: 134-139.
[14] L.O. Simpson and D.J. O'Neill, Red cell shape changes in the blood of people 60 years of age and older imply a role for blood theology in the aging process, Gerontology 2003, 49:310-315.
[15] S. Imre, M. Csornai and M. Balazs, High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging, Mech. Ageing Dev. 2001, 122: 69-76.
[16] R.S. Schwartz, J.W. Madsen, A.C. Rybicki and R.L. Nagel, Oxidation of spectrin and deformability defects in diabetic erythrocytes, Diabetes 1991, 40: 701-708.
[17] T.W. Chung, J.J. Yu and D.Z. Liu, Reducing lipid peroxidation stress of erythrocyte membrane by α-tocopherol nicotinate plays an important role in improving blood rheological properties in type 2 diabetic patients with retinopathy, Diabet. Med. 1998, 15: 380-385.
[18] C. Pfafferott, H.J. Meiselman and P. Hochstein, The effect of malonyldialdehyde on erythrocyte deformability, Blood 1982, 59: 12-15.
[19] F.M. Pulcinelli, P. Pignatelli, F. Violi and P.P. Gazzaniga, Platelets and oxygen radicals: mechanisms of functional modulation, Haematologica 2001, 86(11 Suppl 2): 31-34.
[20] D. Harman. Aging: a theory based on free radical and radiation chemistry [J]. J Gerontol, 1956, 11:298-300
[21] B. Halliwell, J.M.C. Gutteridge. Free radicals in biology and medicine [M]. 3rd edition. New York: Oxford University Press, 1999:617.
[22] K.S. Chio, U. Reiss, B. Fletcher, et al. Peroxidation of subcellular
organdies: formation of lipofuscin like fluorescent pigments. Science, 1969, 166: 1535-1536.
[23] A, Cerami. Hypothesis: glucose as a mediator of aging. J. Am. Geriatr. Soc. 1985, 33: 626-634.
[24] A. Yoritaka, N. Hattori, K. Uchida, et al. Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc. Natl. Acad. Sci. USA. 1996, 93:2696-2701.
[25] M.A. Smith, R.K. Kutty, P.L. Richey, et al. Heme oxygenase-1 is as sociated with the neurofibrillary pathology of Alzheimer's disease. Am. Pathol. 1994, 145: 42-47.
[26] 胡金麟．细胞流变学．科学出版社，北京2000．
[27] 赵春亭，赵子文．临床血液流变学．人民卫生出版社，北京1997。
[28] 秦任甲．血液流变学．人民卫生出版社，北京1999．
[29] 郑德先，吴克复，褚建新．现代实验血液学研究方法与技术．北京医科大学中国协和医科大学联合出版社。北京1999．
[30] R.B. Devereux, D.B. Case, M.H. Alderman, et al. Possible role of increased blood viscosity in the hemodynamics of systemic hypertension. Am. J Cardiol. 2000, 85:1265-1268.
[31] F.G. Fowkes, G.D. Lowe, A. Rumley, et al. The relationship between blood viscosity and blood pressure in a random sample of the population aged 55 to 74 years. Eur. Heart. J 1993, 14(5): 597-601.
[32] J.W.G. Yarnell, P.M. Sweetnarn, A. Rumley, G.D.O. Lowe, Lifestyle and hemostatic risk factors for ischemic heart disease. The Caerphilly study. Arterioscler Thromb Vasc. Biol. 2000, 20:271-279.
[33] E. Ernst, Haemorheological consequences of chronic cigarette smoking. J Cardiovasc. Risk 1995, 2:435-439.
[34] L. Dintenfass, Modifications of blood rheology during aging and age-related pathological conditions. Aging 1989, 1: 99-125.
[35] G.D. Sloop, A unifying theory of atherogenesis. Med. Hypotheses. 1996, 47: 321-325.
[36] G. Lowe, A. Rumley, J. Norrie, et al. Blood rheology, cardiovascular
risk factors, and cardiovascular disease: The West of Scotland Coronary Prevention Study. Thromb Haemost. 2000, 84: 553-558.
[37] G.D. Lowe, A.J. Lee, A. Rumley, et al. Blood viscosity and risk of cardiovascular events: The Edinburgh Artery Study. Br. J Haematol. 1997, 96:168-173.
[38] S.K. Jain, J.D. Ross, G.J. Levy and J. Duett, The effect of malonyldialdehyde on viscosity of normal and sickle red blood cells, Biochem. Med. Metab. Biol. 1990, 44: 37-41.
[39] M. Beppu, K. Murakami and K. Kikugawa, Fluorescent and cross-linked proteins of human erythrocyte ghosts formed by reaction with hydroperoxylinoleic acid, malonaldehyde and monofunctional aldehydes, Chem. Pharm. Bull. 1986, 34: 781-788.
[40] V.U. Buko, A.A. Artsukevich and K.V. Ignatenko, Aldehydic products of lipid peroxidation inactivate cytochrome p-450, Exp. Toxic Pathol. 1999, 51: 294-298.
[41] D. Yin and U.T. Brunk, Carbonyl toxification hypothesis of biological aging, in: Molecular b asis of aging, A. Macieira-Coelho, ed., CRC Press Inc., New York, 1995, pp. 421-436.
[42] D. Yin, Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores, Free Radio. Biol. Med. 1996, 21: 871-888.
[43] J. W. B aynes and S .R. Thorpe, Role o f oxidative stress in diabetic complications, Diabetes 1999, 48: 1-9.
[44] J.W. Baynes and S.R. Thorpe, Glycoxidation and lipoxidation in atherogenesis, Free Radic. Biol. Med. 2000, 28:1708-1716.
[45] J.W. Baynes, The role of AGEs in aging: causation or correlation, Exp. Gerontol. 2001, 36: 1527-1537.
[46] T. Shiga, N. Meada and K. Kon, Erythrocyte rheology, Crit. Rev. Oncol. Hematol. 1990, 10: 9-48.
[47] M.W. Rampling, Red cell aggregation and yield stress, in: Clinical Blood Rheology, G.D.O. Lowe, ed., CRC Press Inc., Boca Ration, Florida, 1988, pp. 5-64.
[2] D. Yin, Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores, Free Radic. Biol. Med. 1996, 21: 871-888.
[3] B. Wierusz-Wysocka, H. Wysocki, H. Byks, et al. Metabolic control quality and free radical activity in diabetic patients. Diabetes Res. Clin. Pract. 1995, 27:193-197.
[4] K. Kikugawa, M. Beppu. Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins. Chem. Phys. Lipids 1987, 44:277-296
[5] T. Yoshikawa. Free radicals and their scavengers in Parkinson's disease. Eur Neurol. 1993, 33: 60-68.
[6] S. Chien. Hemorheology in clinical medicine, Clin. Hemorheol. 1982, 2: 137-142.
[7] K.J. Chen and Z.X. Shi, Practical Blood Stasisology, People's Hygiene Press, Beijing, 1999.
[8] U. Rauch, J.I. Osende, V. Fuster, J.J. Badimon, Z. Fayad and J.H. Chesebro, Thrombus formation on atherosclerotic plaques: pathogenesis and clinical consequences, Ann. Intern. Med. 2001, 134: 224-238.
[9] W.C. Aird, The hematologic system as a marker of organ dysfunction in sepsis, Mayo Clin. Proc. 2003, 78: 869-881.
[10] M. Piagnerelli, K.Z. Boudjeltia, M. Vanhaeverbeek and J.L. Vincent, Red blood cell rheology in sepsis, Intensive Care Med. 2003, 29: 1052-1061.
[11] K.R. Kensey, The mechanistic relationships between hemorheological characteristics and cardiovascular disease, Curr. Med. Res. Opin.
2003, 19: 587-596.
[12] K.R. Kensey, Rheology: an overlooked component of vascular disease, Clin. Appl. Thromb. Hemost. 2003, 9:93-99.
[13] J. Koscielny, R. Latza, S. Wolf, H. Kiesewetter and F. Jung, Early theological and microcirculatory changes in children with type I diabetes mellitus, Clin. Hemorbeol. Microcirc. 1998, 19: 134-139.
[14] L.O. Simpson and D.J. O'Neill, Red cell shape changes in the blood of people 60 years of age and older imply a role for blood theology in the aging process, Gerontology 2003, 49:310-315.
[15] S. Imre, M. Csornai and M. Balazs, High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging, Mech. Ageing Dev. 2001, 122: 69-76.
[16] R.S. Schwartz, J.W. Madsen, A.C. Rybicki and R.L. Nagel, Oxidation of spectrin and deformability defects in diabetic erythrocytes, Diabetes 1991, 40: 701-708.
[17] T.W. Chung, J.J. Yu and D.Z. Liu, Reducing lipid peroxidation stress of erythrocyte membrane by α-tocopherol nicotinate plays an important role in improving blood rheological properties in type 2 diabetic patients with retinopathy, Diabet. Med. 1998, 15: 380-385.
[18] C. Pfafferott, H.J. Meiselman and P. Hochstein, The effect of malonyldialdehyde on erythrocyte deformability, Blood 1982, 59: 12-15.
[19] F.M. Pulcinelli, P. Pignatelli, F. Violi and P.P. Gazzaniga, Platelets and oxygen radicals: mechanisms of functional modulation, Haematologica 2001, 86(11 Suppl 2): 31-34.
[20] D. Harman. Aging: a theory based on free radical and radiation chemistry [J]. J Gerontol, 1956, 11:298-300
[21] B. Halliwell, J.M.C. Gutteridge. Free radicals in biology and medicine [M]. 3rd edition. New York: Oxford University Press, 1999:617.
[22] K.S. Chio, U. Reiss, B. Fletcher, et al. Peroxidation of subcellular
organdies: formation of lipofuscin like fluorescent pigments. Science, 1969, 166: 1535-1536.
[23] A, Cerami. Hypothesis: glucose as a mediator of aging. J. Am. Geriatr. Soc. 1985, 33: 626-634.
[24] A. Yoritaka, N. Hattori, K. Uchida, et al. Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc. Natl. Acad. Sci. USA. 1996, 93:2696-2701.
[25] M.A. Smith, R.K. Kutty, P.L. Richey, et al. Heme oxygenase-1 is as sociated with the neurofibrillary pathology of Alzheimer's disease. Am. Pathol. 1994, 145: 42-47.
[26] 胡金麟．细胞流变学．科学出版社，北京2000．
[27] 赵春亭，赵子文．临床血液流变学．人民卫生出版社，北京1997。
[28] 秦任甲．血液流变学．人民卫生出版社，北京1999．
[29] 郑德先，吴克复，褚建新．现代实验血液学研究方法与技术．北京医科大学中国协和医科大学联合出版社。北京1999．
[30] R.B. Devereux, D.B. Case, M.H. Alderman, et al. Possible role of increased blood viscosity in the hemodynamics of systemic hypertension. Am. J Cardiol. 2000, 85:1265-1268.
[31] F.G. Fowkes, G.D. Lowe, A. Rumley, et al. The relationship between blood viscosity and blood pressure in a random sample of the population aged 55 to 74 years. Eur. Heart. J 1993, 14(5): 597-601.
[32] J.W.G. Yarnell, P.M. Sweetnarn, A. Rumley, G.D.O. Lowe, Lifestyle and hemostatic risk factors for ischemic heart disease. The Caerphilly study. Arterioscler Thromb Vasc. Biol. 2000, 20:271-279.
[33] E. Ernst, Haemorheological consequences of chronic cigarette smoking. J Cardiovasc. Risk 1995, 2:435-439.
[34] L. Dintenfass, Modifications of blood rheology during aging and age-related pathological conditions. Aging 1989, 1: 99-125.
[35] G.D. Sloop, A unifying theory of atherogenesis. Med. Hypotheses. 1996, 47: 321-325.
[36] G. Lowe, A. Rumley, J. Norrie, et al. Blood rheology, cardiovascular
risk factors, and cardiovascular disease: The West of Scotland Coronary Prevention Study. Thromb Haemost. 2000, 84: 553-558.
[37] G.D. Lowe, A.J. Lee, A. Rumley, et al. Blood viscosity and risk of cardiovascular events: The Edinburgh Artery Study. Br. J Haematol. 1997, 96:168-173.
[38] S.K. Jain, J.D. Ross, G.J. Levy and J. Duett, The effect of malonyldialdehyde on viscosity of normal and sickle red blood cells, Biochem. Med. Metab. Biol. 1990, 44: 37-41.
[39] M. Beppu, K. Murakami and K. Kikugawa, Fluorescent and cross-linked proteins of human erythrocyte ghosts formed by reaction with hydroperoxylinoleic acid, malonaldehyde and monofunctional aldehydes, Chem. Pharm. Bull. 1986, 34: 781-788.
[40] V.U. Buko, A.A. Artsukevich and K.V. Ignatenko, Aldehydic products of lipid peroxidation inactivate cytochrome p-450, Exp. Toxic Pathol. 1999, 51: 294-298.
[41] D. Yin and U.T. Brunk, Carbonyl toxification hypothesis of biological aging, in: Molecular b asis of aging, A. Macieira-Coelho, ed., CRC Press Inc., New York, 1995, pp. 421-436.
[42] D. Yin, Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores, Free Radio. Biol. Med. 1996, 21: 871-888.
[43] J. W. B aynes and S .R. Thorpe, Role o f oxidative stress in diabetic complications, Diabetes 1999, 48: 1-9.
[44] J.W. Baynes and S.R. Thorpe, Glycoxidation and lipoxidation in atherogenesis, Free Radic. Biol. Med. 2000, 28:1708-1716.
[45] J.W. Baynes, The role of AGEs in aging: causation or correlation, Exp. Gerontol. 2001, 36: 1527-1537.
[46] T. Shiga, N. Meada and K. Kon, Erythrocyte rheology, Crit. Rev. Oncol. Hematol. 1990, 10: 9-48.
[47] M.W. Rampling, Red cell aggregation and yield stress, in: Clinical Blood Rheology, G.D.O. Lowe, ed., CRC Press Inc., Boca Ration, Florida, 1988, pp. 5-64.