不饱和醛酮对于生物组织流变性及荧光特性的影响
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摘要
有关氧自由基、脂质过氧化和非酶糖基化的研究表明，这些过程中产生的醛类物质中有一部分具有很强的生物活性，能对生物体造成较大伤害，这些醛目前一般被认为是生化副反应损伤的“第二信使”。而我们则进一步提出这些不饱和的醛酮尤其是丙二醛(MDA)与4-羟基壬烯醛(HNE)很可能是导致与疾病及衰老相关的老年色素类荧光物质的形成及血液粘度增加的内在原因之一。通过对脂质过氧化的次级产物——各类双功能醛及其它单功能醛对不同生物溶液流变性及荧光特性的作用的对比研究。我们发现，不同浓度的丙二醛(MDA)与戊二醛均能显著提高生物流体的各项流变性指标。丙二醛不但能提高低浓度小牛血清白蛋白，及血浆等牛顿流体的粘度，而且能提高高浓度白蛋白及红细胞悬浮液等非牛顿流体的屈服应力与塑性粘度。戊二醛作为一种蛋白固定剂能特异地改变流体特性，而其它非脂质过氧化醛类，如甲醛、乙醛、乙二醛、丙烯醛、丙酮醛、水杨醛等并没有明显改变上述溶液的流变性。在同时进行的荧光测定中，我们发现，丙二醛(20 mM)能迅速地降低蛋白溶液的典型荧光(280 nm／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。而戊二醛除了能部分降低蛋白质特征荧光外，并不能在395nm处激发出460nm的荧光。这些数据表明，作为脂质过氧化形成的一类不饱和羰基化合物，丙二醛等造成的羰基应激作用于生物体时既能导致蛋白等生物大分子表面的电荷改变和粘度变化，它们造成的生物大分子交联又与衰老及疾病过程中种种生理病理的变化互为因果。而核酸在紫外照射下同样生成460nm的荧光峰，这就证明紫外产生的电离辐射能对生物造成衰老损伤。氧化AsA的化学性质非常活泼又是常见的还原酮，在我们的实验中氧化的维C与谷氨酰氨也生成类似的荧光，所以AsA与Gln生成交联产物的反应可能与衰老过程中脂褐素的生成相关。
Reactive aldehydes from lipid peroxidation such as malonaldehyde (MDA), 4-hydroxynonenal (HNE) are responsible for the modification of different biological macromolecular structures and functions by reacting with either SH-groups or amino groups of bio-molecules to form seminercaptals or Schiff bases and lead to various aging-related alterations. 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 excited at 395 nm when incubated at 37℃. On the other hand, malondialdehyde (MDA) in different concentrations significantly increased viscosity, plastic viscosity and yield-stress of blood materials such as human plasma and erythrocyte suspensions. Other non-peroxidated aldehydes, such as formaldehyde, acetaldehyde, glyoxal, acrolein, pyruvaldehyde, salicylaldehyde, all did not change rheological property of these biological materials. The effects of MDA on rheological parameters, crosslinking reaction and their relation with age pigment-like fluorescence were studied. Malonaldehyde, a marjor product of the peroxidation of polyunsaturated lipids, may play an essential role in the formation of the fluorescent products in lipofuscin-like fluorescent pigments and in the blood viscosity increase.
Fluorescence and non-enzymatic browning were observed in reactions between ascorbic acid (AsA) and amino acids (AA) as well as in reactions involving AsA autoxidation and/or polymerization in the presence of trace amounts of adventitious iron. These reaction products exhibited fluorescent spectra (400nm-490nm) akin to those of extracts from liposcin-rich tissues.
Considering that dehydroascorbic acid and MDA are active in biological system, the oxidation-enhanced carbonyl-protein cross-linking is suggested to be an important chemical reaction which may take place during ageing and be involved in lipofuscinogenesis.
Reactive aldehydes from lipid peroxidation such as malonaldehyde (MDA), 4-hydroxynonenal (HNE) are responsible for the modification of different biological macromolecular structures and functions by reacting with either SH-groups or amino groups of bio-molecules to form seminercaptals or Schiff bases and lead to various aging-related alterations. 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 excited at 395 nm when incubated at 37℃. On the other hand, malondialdehyde (MDA) in different concentrations significantly increased viscosity, plastic viscosity and yield-stress of blood materials such as human plasma and erythrocyte suspensions. Other non-peroxidated aldehydes, such as formaldehyde, acetaldehyde, glyoxal, acrolein, pyruvaldehyde, salicylaldehyde, all did not change rheological property of these biological materials. The effects of MDA on rheological parameters, crosslinking reaction and their relation with age pigment-like fluorescence were studied. Malonaldehyde, a marjor product of the peroxidation of polyunsaturated lipids, may play an essential role in the formation of the fluorescent products in lipofuscin-like fluorescent pigments and in the blood viscosity increase.
Fluorescence and non-enzymatic browning were observed in reactions between ascorbic acid (AsA) and amino acids (AA) as well as in reactions involving AsA autoxidation and/or polymerization in the presence of trace amounts of adventitious iron. These reaction products exhibited fluorescent spectra (400nm-490nm) akin to those of extracts from liposcin-rich tissues.
Considering that dehydroascorbic acid and MDA are active in biological system, the oxidation-enhanced carbonyl-protein cross-linking is suggested to be an important chemical reaction which may take place during ageing and be involved in lipofuscinogenesis.
引文
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2 Yoshikawa T. Free radicals and their scavengers in Parkinson's disease. Eur Neurol. 1993, 33: 60-68.
3 Yoritaka A, Hattori N, Uchida K, et al. Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci USA. 1996, 93: 2696-2701.
4 Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996, 19: 257-267.
5 Wierusz-Wysocka B, Wysocki H, Byks H, et al. Metabolic control quality and free radical activity in diabetic patients. Diabetes Res Clin Pract 1995, 27: 193-197.
6 Nacitarhan S, Ozben T, Tuncer N. Serum and urine malondialdehyde levels in NIDDM patients with and without hyperlipidemia. Free Radic Biol Med 1995, 19: 893-896.
7 Smith MA, Kutty RK, Richey PL, et al. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer's disease. Am Pathol. 1994, 145: 42-47.
8 Schmechel DE. New approaches to therapy for neurodegenerative diseases. South Med J 1991, 84:11-23.
9 Gerdemann A, Lemke H-D, Mtlnch G, et al. Low-molecular but not high-moleular advanced glycation end products (AGEs) are removed by high-flux dialysis. Clinical Nephrology 2000, 54: 276-283.
10 Andrews B, Burnand K, Paganga G, et al. Oxidisablity of low density lipoproteins in patients with carotid or femoral artery atherosclerosis. Atherosclerosis 1995, 112: 77-84.
11 童垣君，张宗玉．医学老年学--衰老与长寿．人民卫生出版社，1995．
13 吴小晶，吴丽娟，夏舒萌．线粒体DNA相对含量及基因缺失与衰老关系初探．中华老年医学杂志，1999，18(1)：38-41．
14 杨东丽．基因的差异性表达与衰老．生理科学进展，1997，28(2)：
142-144.
15 Kebe SI, Tanaka M, Ohno K. Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence. Biochem Biophys Res Commun, 1990, 170: 1044-1048.
16 张洪泉，余文新．中华抗衰老医药学．科学出版社，北京2000．
17 Dazhong Yin. Biochemical basis of lipofusion, ceroid and age piments-like fluorophores. Free Rad Biol Med, 1996, 21: 871-888.
2 Yoshikawa T. Free radicals and their scavengers in Parkinson's disease. Eur Neurol. 1993, 33: 60-68.
3 Yoritaka A, Hattori N, Uchida K, et al. Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci USA. 1996, 93: 2696-2701.
4 Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996, 19: 257-267.
5 Wierusz-Wysocka B, Wysocki H, Byks H, et al. Metabolic control quality and free radical activity in diabetic patients. Diabetes Res Clin Pract 1995, 27: 193-197.
6 Nacitarhan S, Ozben T, Tuncer N. Serum and urine malondialdehyde levels in NIDDM patients with and without hyperlipidemia. Free Radic Biol Med 1995, 19: 893-896.
7 Smith MA, Kutty RK, Richey PL, et al. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer's disease. Am Pathol. 1994, 145: 42-47.
8 Schmechel DE. New approaches to therapy for neurodegenerative diseases. South Med J 1991, 84:11-23.
9 Gerdemann A, Lemke H-D, Mtlnch G, et al. Low-molecular but not high-moleular advanced glycation end products (AGEs) are removed by high-flux dialysis. Clinical Nephrology 2000, 54: 276-283.
10 Andrews B, Burnand K, Paganga G, et al. Oxidisablity of low density lipoproteins in patients with carotid or femoral artery atherosclerosis. Atherosclerosis 1995, 112: 77-84.
11 童垣君，张宗玉．医学老年学--衰老与长寿．人民卫生出版社，1995．
13 吴小晶，吴丽娟，夏舒萌．线粒体DNA相对含量及基因缺失与衰老关系初探．中华老年医学杂志，1999，18(1)：38-41．
14 杨东丽．基因的差异性表达与衰老．生理科学进展，1997，28(2)：
142-144.
15 Kebe SI, Tanaka M, Ohno K. Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence. Biochem Biophys Res Commun, 1990, 170: 1044-1048.
16 张洪泉，余文新．中华抗衰老医药学．科学出版社，北京2000．
17 Dazhong Yin. Biochemical basis of lipofusion, ceroid and age piments-like fluorophores. Free Rad Biol Med, 1996, 21: 871-888.