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Protective effect of l -phenylalanine on rat brain acetylcholinesterase inhibition induced by free radicals
Clinical Biochemistry, Vol. 33, No. 2, 103–106, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter
PII S0009-9120(99)00090-9
Protective Effect of L-Phenylalanine on Rat Brain Acetylcholinesterase Inhibition Induced by Free Radicals STYLIANOS TSAKIRIS,1 PANAGOULA ANGELOGIANNI,1 KLEOPATRA H. SCHULPIS,2 and JOHN C. STAVRIDIS1 1
Department of Experimental Physiology, University of Athens, Medical School, Athens, Greece, and 2Inborn Errors of Metabolism Department, Institute of Child Health, Athens, Greece Objective: To investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe) could reverse the free radical effects on brain acetylcholinesterase (AChE) activity, since it has been reported that Phe binds hydroxyl radicals (•OH). Design and methods: Two well established systems were used for production of free radicals: (a) FeSO4 (84 M) plus ascorbic acid (400 M), and (b) FeSO4, ascorbic acid and H2O2 (1 mM) at 37 °C in homogenates of adult rat whole brain. Changes in brain AChE activity were studied in the presence of each system separately. Results: AChE was inhibited (18 –28%) by both systems of free radicals. This inhibition was reversed when the brain homogenate was preincubated with Phe 1.8 mM. Conclusions: In accordance with our previous reports, Phe could protect against the direct action of •OH radicals on brain AChE and in this way it might be useful in the prevention of certain cholinergic neural dysfunctions. Copyright © 2000 The Canadian Society of Clinical Chemists
KEY WORDS: L-phenylalanine; hydroxyl radicals; rat brain; brain AChE; brain aging; pituitary AChE; hypothalamus AChE; frontal cortex AChE; hippocampus AChE.
Introduction ree radicals in brain could represent one of the main causes of cellular dysfunction that occurs during aging (1–3). Some basic evidence has suggested the above hypothesis: (a) brain contains high amounts of polyunsaturated fatty acids; (b) compared with other tissues, brain utilizes one-fifth of the total oxygen demand of the body (2), and (c) brain is not particularly enriched in any of the antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (2). In addition, it
F
has been demonstrated that a general reduction of the antioxidant protection mechanisms occurs during aging. From among the enzymatic systems, an age-dependent decrease in superoxide dismutase activity has been reported (4,5); with regard to nonenzymatic antioxidant, a decrease in brain ascorbate (6), glutathione (7,8), and ␣-tocopherol levels (9) has been shown to occur during aging. Brain AChE (EC 3.1.1.7) activity was found to be decreased in Alzheimer’s disease (10). In addition, it was shown to be lowered in aged whole brain and hypothalamus in rats (11,12). Free radical action has been shown to rise in the brain of aged rats and in that in Alzheimer’s disease (13,14). In the present study, AChE activity was estimated in homogenized whole brain, pituitary, and some specific brain areas in adult (4 mo old) and aged rats (22 mo old). The observed inhibition of AChE activity in the brain of aged rats was similar to that noticed in adult brain, in which enzyme inhibition was induced by two separate systems of free radical production: (a) FeSO4 and ascorbic acid (15,16), and (b) FeSO4, ascorbic acid and H2O2. It was also investigated whether the preincubation of brain homogenate with different Phe concentrations could reverse the free radical effects on AChE activity. It has been reported (17,18) that Phe reacts with • OH radicals to form aromatic hydroxylated products. So, we used Phe as a specific scavenger of •OH radicals, which may be produced by the two systems mentioned. Methods
Correspondence: Stylianos Tsakiris, Department of Experimental Physiology, University of Athens, Medical School, P.O. Box 65257, GR-154 01 Athens, Greece. Email: [email protected] Manuscript received July 19, 1999; revised August 5, 1999; accepted October 26, 1999. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
ANIMALS Albino Wistar rats of both sexes (Saint Savvas Hospital, Athens, Greece) were used in all experiments. Body weight was 125 ⫾ 10 g (mean ⫾ SD) for adult (4 mo) and 326 ⫾ 34 g for aged (22 mo) rats. 103
TSAKIRIS
Adult or aged rats were housed four in a cage, at a constant room temperature (22 ⫾ 1 °C) under a 12 h:12 h L:D (light 08.00 –20.00 h) cycle and acclimated 1 week before use. Food and water were provided ad lib. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals. TISSUE
PREPARATION
Rats were sacrificed by decapitation. Rat whole brain or the pituitary, hypothalamus, frontal cortex, and hippocampus were rapidly removed, weighed and thoroughly perfused with isotonic saline. Pools of three pituitaries or individual tissues were homogenized in 10 vol ice-cold (0 – 4 °C) medium containing 50 mM Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), pH 7.4 and 300 mM sucrose using an ice-chilled glass homogenizing vessel at 900 rpm (4 –5 strokes). Then, the homogenate was centrifuged at 1,000 ⫻ g for 10 min to remove nuclei and debris. The resulting fresh supernatants were immediately used for AChE activity measurements at 37 °C. Supernatant proteins were determined according to Lowry et al. (19). FREE
RADICAL PRODUCTION AND
PHE
PARTICIPATION
Two systems were used for the production of free radicals: (a) FeSO4 (84 M) and ascorbic acid (400 M) (15,16), and (b) FeSO4, ascorbic acid and H2O2 (1 mM) at 37 °C in homogenates of adult rat whole brain. In the presence of iron, a Fenton reaction will occur between Fe2⫹ and H2O2 giving rise to the reactive •OH radicals. Proteins (and possibly AChE) are susceptible to free radical attack, especially by the •OH radical. Changes were studied in brain AChE activity in the presence of these mentioned systems. The incubation mixture (total volume about 1 mL) contained 50 mM Tris-HCl, pH 8.0, 240 mM sucrose, 120 mM NaCl, protein concentration 80 –100 g/ml and in the presence or absence (control) of FeSO4, ascorbic acid and H2O2. Different Phe concentrations (0.12–1.80 mM) were preincubated with whole brain homogenates, in order to investigate the possible competition of Phe with the free radical action on AChE. BIOCHEMICAL
DETERMINATIONS
AChE activity was determined according to Ellman’s method (20). The reaction mixture (1 mL) contained 50 mM Tris-HCl, pH 8.0, and 240 mM sucrose in the presence of 120 mM NaCl. Protein concentration was 80 –100 g/ml incubation mixture. A volume of 0.030 mL 5,5⬘-dithionitrobenzoic acid (DTNB) and 0.050 mL acetylthiocholine iodide, used as a substrate, was added and the reaction was started. The final concentrations of DTNB and substrate were 0.125 and 0.5 mM, respectively. The reaction was followed spectrophotometrically by the increase in absorbance (⌬ OD) at 412 nm. 104
ET AL.
STATISTICAL
ANALYSIS
The data were analyzed by using the two-tailed Student’s t-test. Results and discussion The effect of aging on AChE activity determined in homogenized whole brain, pituitary, and specific brain areas is presented in Table 1. Whole brain AChE inhibition in 22 mo old rats has been reported earlier (11). The enzyme activity in hypothalamus has been also found decreased (by 40%) (12). Moreover, it was found decreased in frontal cortex (by 25%) and in hippocampus (by 27%). Brain AChE activity has also been reported to be decreased in Alzheimer’s disease (13). Free radical action has been shown to rise in the brain of aged rats and in that in Alzheimer’s disease (13,14). Figure 1 presents time dependence of free radical production on AChE activity in adult brain homogenates. We used the system of incubation with FeSO4 (84 M) and ascorbic acid (400 M) at 37 °C, as previously reported (15,16). It has been shown that increased concentrations of free radicals can produce lipid peroxidation and decrease membrane fluidity (16). Therefore, the continuous brain AChE inhibition observed from 0.5 h up to 2.5 h of incubation may reflect a decrease in membrane fluidity due to lipid peroxidation, which may influence the enzyme activity, through lipid(s)-protein interactions. The observed AChE stimulation at 15 min was possibly due to an initial increase in membrane fluidity, when the enzyme is exposed to a minimum free radical concentration. Figure 2 shows that the addition of different H2O2 concentrations up to 0.1 mM in these mentioned system for 2.5 h was not able to influence the AChE activity (p ⬎ 0.05) more than the enzyme inhibition TABLE 1 Effect of Aging on Acetylcholinesterase Activity Determined in Homogenized Whole Brain, Pituitary, and Specific Brain Areas Acetylcholinesterase activity ⌬OD/min/mg protein Tissue
4 mo old rats
22 mo old rats
Whole brain Pituitary Hypothalamus Frontal cortex Hippocampus
0.598 ⫾ 0.009 0.096 ⫾ 0.008 0.499 ⫾ 0.035 0.196 ⫾ 0.006 0.288 ⫾ 0.012
0.456 ⫾ 0.006*** 0.108 ⫾ 0.012 0.299 ⫾ 0.030*** 0.147 ⫾ 0.010** 0.209 ⫾ 0.006***
Values represent means ⫾ SD of five independent experiments (five pools of three animals each) for adult or aged rats for the pituitary and eight independent experiments (8 rats) for 4-month-old rats and six experiments (6 rats) for 22-month-old rats for the whole brain as well as specific brain areas. The average value of each experiment came from three determinations. **p ⬍ 0.01; ***p ⬍ 0.001; compared to 4-month-old rats. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
PHE PROTECTION OF BRAIN ACHE FROM RADICALS
Figure 1 — Time-dependent effect of free radical production by the system of FeSO4 (84 M) and ascorbic acid (400 M) on AChE activity determined in homogenized brain of 4 mo old rats. Points and vertical bars represent mean values ⫾ SD for the control (E) and the system that was used (■). Values represent means ⫾ SD of three experiments. The average values of each experiment arise from two independent determinations. Control value of AChE activity was 0.598 ⫾ 0.009 ⌬OD / min ⫻ mg protein. **p ⬍ 0.01.
already caused by the first system. The addition of 1 mM H2O2 decreased the enzyme activity by about 28% (p ⬍ 0.01) as compared to control but by 12% more than that caused by the first system alone (from about 83% to 72% of activity). The enzyme activity reached an inhibition of about 80% in 10
Figure 2 — Effect of different H2O2 concentrations on AChE activity. H2O2 was added to the incubation mixture containing FeSO4 (84 M) and ascorbic acid (400 M) for 2.5 h. Points and vertical bars represent mean values ⫾ SD for the control (E) and the system of FeSO4, ascorbic acid and H2O2 (F). Values represent means ⫾ SD of three experiments. The average values of each experiment arise from three independent determinations. **p ⬍ 0.01; ***p ⬍ 0.001; as compared to control. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
Figure 3 — Effect of preincubation of brain homogenates with various Phe concentrations on the protection of AChE inhibition by free radicals. The two systems used for free radical production and inhibition of AChE activity were as follows: (a) FeSO4 (84 M), ascorbic acid (400 M) (■), and (b) FeSO4, ascorbic acid, H2O2 (1 mM) (E). Brain homogenates were preincubated with different Phe concentrations in the reaction medium for 1 h. After preincubation, the above mentioned systems were added to the incubation medium and incubation was continued for 2.5 h. Values represent means ⫾ SD of three experiments. The average values of each experiment arise from three independent determinations. **p ⬍ 0.01; ***p ⬍ 0.001.
mM H2O2 but by 60% more than that caused by the first system alone (from about 80% to 20% of activity). The incubation of brain homogenates with FeSO4 (84 M), ascorbic acid (400 M), or H2O2 (1 mM) separately did not induce AChE inhibition (p ⬎ 0.05). Therefore, Fenton reaction, which is a good source of •OH radicals, may have induced the observed enzymatic inhibition of up to 28%. Figure 3 presents the effect of different concentrations of Phe (0.12–1.80 mM) in the homogenates on the protection of AChE inhibition by free radicals. These mentioned intracellular concentrations of Phe correspond to 6 times the concentration in plasma (21) (0.72–10.8 mM). Phe concentrations of 0.3–5.4 mM are usually found in the plasma of phenylketonuric patients (22). The inhibited AChE activity by the free radical action was entirely reversed when the brain homogenate was preincubated with 1.80 mM Phe. Furthermore, preincubation with a lower concentration of the amino acid (0.3 mM Phe) resulted in a 50% protection of the inhibited enzyme activity. However, when Phe was added at the same time with or after the two systems used for free radical production, it was not able to reverse the inhibition of the enzyme activity (p ⬎ 0.05). In a recent report, it has been suggested by us that Phe acts directly on AChE (23). Therefore, Phe seems to protect the enzyme against the direct action of free radicals. This action could be exerted on cysteine, methionine, histidine and/or tyrosine residues of the AChE molecule (13,24). It has been reported (17,18) that Phe reacts with •OH radicals to form the 105
TSAKIRIS
aromatic hydroxylated products ortho-, meta-, and para- tyrosines. In Figure 3 it was observed that the inhibited AChE activity by the free radical action was entirely reversed when the brain homogenate was preincubated with 1.80 mM Phe. Therefore, Phe could protect against the direct action of •OH radicals on brain AChE. These results suggest that Phe treatment, before •OH radicals damage, might be a preventive measure in certain cholinergic neural dysfunctions (e.g., Alzheimer’s disease). Acknowledgements This work was funded by the University of Athens. Many thanks are extended to Char. Antoniades and K. Marinou for their assistance.
REFERENCES 1. Harman D. Free radical theory of aging: consequences of mitochondrial aging. Age 1983; 6: 86 –94. 2. Floyd RA, Zaleska MM, Harmon J. Free radicals in molecular biology, aging and disease. In: Armstrong D, Sohal RS, Cutler RG, Slater TF, Ed. Aging. Pp. 143– 61, New York: Raven Press, 1984. 3. Hall ED, Braughler JM. Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic Biol Med 1989; 6: 303–13. 4. Reiss U, Gershon D. Comparison of cytoplasmatic superoxide dismutase in liver, heart and brain of aging rats and mice. Biochem Biophys Res Commun 1976; 73: 255– 62. 5. Mizumo Y, Ohta K. Regional distribution of thiobarbituric acid-reactive products activities of enzymes regulating the metabolism of oxygen free radicals and some of the regulated enzymes in adult and aged rat brains. J Neurochem 1986; 46: 1344 –52. 6. Adlard BPF, De Souza SW, Moon S. The effect of age retardation and asphyxia on ascorbic acid concentrations in developing brain. J Neurochem 1973; 21: 877– 81. 7. Hazelton GA, Lang CA. Glutathione contents of tissues in the aging mouse. Biochem J 1980; 188: 25–30. 8. Benzi G, Pastoris O, Mazzatico F, Villa RF. Age related effect induced by oxidative stress on the cerebral glutathione system. Neurochem Res 1989; 14: 473-81. 9. Meydani M, Macanley JB, Blumberg JB. Influence of dietary vitamin E, selenium and age on regional distribution of ␣-tocopherol in the rat brain. Lipids 1986; 21: 786 –91.
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10. Bowen DM, Dawison AN. Biochemical studies of nerve cells and energy metabolism in Alzheimer’s disease. Br Med Bull 1986; 42: 75– 80. 11. Tsakiris S, Angelogianni P, Stavridis JC. Correlation between activities of Na⫹,K⫹-ATPase and acetylcholinesterase in postnatally developing rat brain. Med Sci Res 1996; 24:155-6. 12. Tsakiris S, Angelogianni P, Stavridis JC. Effect of aging on the activities of acetylcholinesterase, Na⫹,K⫹-ATPase and Mg2⫹-ATPase in rat pituitary and hypothalamus. Z Naturforsch 1998; 53C: 168 – 72. 13. Go¨tz ME, Ku¨nig G, Riederer P, Youdim MBH. Oxidative stress: free radical production in neural degeneration. Pharmacol Ther 1994; 63: 37–122. 14. Williams LR. Oxidative stress, age-related neurodegeneration and the potential for neurotrophic treatment. Cerebrovasc Brain Metab Rev 1995; 7: 55–73. 15. Viani P, Cervato G, Fiorilli A, Cestaro B. Age-related differences in synaptosomal peroxidative damage and membrane properties. J Neurochem 1991; 56: 253– 8. 16. Ghosh C, Dick RM, Ali SF. Iron/Ascorbate-induced lipid peroxidation changes membrane fluidity and muscarinic cholinergic receptor binding in rat frontal cortex. Neurochem Int 1993; 23: 479 – 84. 17. Sun JZ, Kaur H, Halliwell B, Li XY, Bolli R. Use of aromatic hydroxylation of phenylalanine to measure production of hydroxyl radicals after myocardial ischemia in vivo. Direct evidence for a pathogenetic role of the hydroxyl radical in myocardial stunning. Circ Res 1993; 73: 534 – 49. 18. Van der Vliet A, O’Neill CA, Halliwell B, Cross CE, Kaur H. Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite. Evidence for hydroxyl radical production from peroxynitrite. FEBS Lett 1994; 339: 89 –92. 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75. 20. Ellman GL, Courtney D, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88 –95. 21. Scriver C, Rosenberg R. Aminoacid metabolism and its disorders. WB Saunders: Philadelphia, 1973. 22. Missiou-Tsagaraki S, Schulpis K, Loumakou M. Phenylketonuria in Greece: 12 years’ experience. J Mental Deficiency Res 1988; 32: 271– 87. 23. Tsakiris S, Kouniniotou-Krontiri P, Schulpis KH, Stavridis JC. L-phenylalanine effect on rat brain acetylcholinesterase and Na⫹,K⫹-ATPase. Z Naturforsch 1988; 53c: 163–7. 24. Rosenberry TL, Barnett P, Mays C. Acetylcholinesterase. Methods Enzymol 1982; 82: 325–39.
CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
PII S0009-9120(99)00090-9
Protective Effect of L-Phenylalanine on Rat Brain Acetylcholinesterase Inhibition Induced by Free Radicals STYLIANOS TSAKIRIS,1 PANAGOULA ANGELOGIANNI,1 KLEOPATRA H. SCHULPIS,2 and JOHN C. STAVRIDIS1 1
Department of Experimental Physiology, University of Athens, Medical School, Athens, Greece, and 2Inborn Errors of Metabolism Department, Institute of Child Health, Athens, Greece Objective: To investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe) could reverse the free radical effects on brain acetylcholinesterase (AChE) activity, since it has been reported that Phe binds hydroxyl radicals (•OH). Design and methods: Two well established systems were used for production of free radicals: (a) FeSO4 (84 M) plus ascorbic acid (400 M), and (b) FeSO4, ascorbic acid and H2O2 (1 mM) at 37 °C in homogenates of adult rat whole brain. Changes in brain AChE activity were studied in the presence of each system separately. Results: AChE was inhibited (18 –28%) by both systems of free radicals. This inhibition was reversed when the brain homogenate was preincubated with Phe 1.8 mM. Conclusions: In accordance with our previous reports, Phe could protect against the direct action of •OH radicals on brain AChE and in this way it might be useful in the prevention of certain cholinergic neural dysfunctions. Copyright © 2000 The Canadian Society of Clinical Chemists
KEY WORDS: L-phenylalanine; hydroxyl radicals; rat brain; brain AChE; brain aging; pituitary AChE; hypothalamus AChE; frontal cortex AChE; hippocampus AChE.
Introduction ree radicals in brain could represent one of the main causes of cellular dysfunction that occurs during aging (1–3). Some basic evidence has suggested the above hypothesis: (a) brain contains high amounts of polyunsaturated fatty acids; (b) compared with other tissues, brain utilizes one-fifth of the total oxygen demand of the body (2), and (c) brain is not particularly enriched in any of the antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (2). In addition, it
F
has been demonstrated that a general reduction of the antioxidant protection mechanisms occurs during aging. From among the enzymatic systems, an age-dependent decrease in superoxide dismutase activity has been reported (4,5); with regard to nonenzymatic antioxidant, a decrease in brain ascorbate (6), glutathione (7,8), and ␣-tocopherol levels (9) has been shown to occur during aging. Brain AChE (EC 3.1.1.7) activity was found to be decreased in Alzheimer’s disease (10). In addition, it was shown to be lowered in aged whole brain and hypothalamus in rats (11,12). Free radical action has been shown to rise in the brain of aged rats and in that in Alzheimer’s disease (13,14). In the present study, AChE activity was estimated in homogenized whole brain, pituitary, and some specific brain areas in adult (4 mo old) and aged rats (22 mo old). The observed inhibition of AChE activity in the brain of aged rats was similar to that noticed in adult brain, in which enzyme inhibition was induced by two separate systems of free radical production: (a) FeSO4 and ascorbic acid (15,16), and (b) FeSO4, ascorbic acid and H2O2. It was also investigated whether the preincubation of brain homogenate with different Phe concentrations could reverse the free radical effects on AChE activity. It has been reported (17,18) that Phe reacts with • OH radicals to form aromatic hydroxylated products. So, we used Phe as a specific scavenger of •OH radicals, which may be produced by the two systems mentioned. Methods
Correspondence: Stylianos Tsakiris, Department of Experimental Physiology, University of Athens, Medical School, P.O. Box 65257, GR-154 01 Athens, Greece. Email: [email protected] Manuscript received July 19, 1999; revised August 5, 1999; accepted October 26, 1999. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
ANIMALS Albino Wistar rats of both sexes (Saint Savvas Hospital, Athens, Greece) were used in all experiments. Body weight was 125 ⫾ 10 g (mean ⫾ SD) for adult (4 mo) and 326 ⫾ 34 g for aged (22 mo) rats. 103
TSAKIRIS
Adult or aged rats were housed four in a cage, at a constant room temperature (22 ⫾ 1 °C) under a 12 h:12 h L:D (light 08.00 –20.00 h) cycle and acclimated 1 week before use. Food and water were provided ad lib. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals. TISSUE
PREPARATION
Rats were sacrificed by decapitation. Rat whole brain or the pituitary, hypothalamus, frontal cortex, and hippocampus were rapidly removed, weighed and thoroughly perfused with isotonic saline. Pools of three pituitaries or individual tissues were homogenized in 10 vol ice-cold (0 – 4 °C) medium containing 50 mM Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), pH 7.4 and 300 mM sucrose using an ice-chilled glass homogenizing vessel at 900 rpm (4 –5 strokes). Then, the homogenate was centrifuged at 1,000 ⫻ g for 10 min to remove nuclei and debris. The resulting fresh supernatants were immediately used for AChE activity measurements at 37 °C. Supernatant proteins were determined according to Lowry et al. (19). FREE
RADICAL PRODUCTION AND
PHE
PARTICIPATION
Two systems were used for the production of free radicals: (a) FeSO4 (84 M) and ascorbic acid (400 M) (15,16), and (b) FeSO4, ascorbic acid and H2O2 (1 mM) at 37 °C in homogenates of adult rat whole brain. In the presence of iron, a Fenton reaction will occur between Fe2⫹ and H2O2 giving rise to the reactive •OH radicals. Proteins (and possibly AChE) are susceptible to free radical attack, especially by the •OH radical. Changes were studied in brain AChE activity in the presence of these mentioned systems. The incubation mixture (total volume about 1 mL) contained 50 mM Tris-HCl, pH 8.0, 240 mM sucrose, 120 mM NaCl, protein concentration 80 –100 g/ml and in the presence or absence (control) of FeSO4, ascorbic acid and H2O2. Different Phe concentrations (0.12–1.80 mM) were preincubated with whole brain homogenates, in order to investigate the possible competition of Phe with the free radical action on AChE. BIOCHEMICAL
DETERMINATIONS
AChE activity was determined according to Ellman’s method (20). The reaction mixture (1 mL) contained 50 mM Tris-HCl, pH 8.0, and 240 mM sucrose in the presence of 120 mM NaCl. Protein concentration was 80 –100 g/ml incubation mixture. A volume of 0.030 mL 5,5⬘-dithionitrobenzoic acid (DTNB) and 0.050 mL acetylthiocholine iodide, used as a substrate, was added and the reaction was started. The final concentrations of DTNB and substrate were 0.125 and 0.5 mM, respectively. The reaction was followed spectrophotometrically by the increase in absorbance (⌬ OD) at 412 nm. 104
ET AL.
STATISTICAL
ANALYSIS
The data were analyzed by using the two-tailed Student’s t-test. Results and discussion The effect of aging on AChE activity determined in homogenized whole brain, pituitary, and specific brain areas is presented in Table 1. Whole brain AChE inhibition in 22 mo old rats has been reported earlier (11). The enzyme activity in hypothalamus has been also found decreased (by 40%) (12). Moreover, it was found decreased in frontal cortex (by 25%) and in hippocampus (by 27%). Brain AChE activity has also been reported to be decreased in Alzheimer’s disease (13). Free radical action has been shown to rise in the brain of aged rats and in that in Alzheimer’s disease (13,14). Figure 1 presents time dependence of free radical production on AChE activity in adult brain homogenates. We used the system of incubation with FeSO4 (84 M) and ascorbic acid (400 M) at 37 °C, as previously reported (15,16). It has been shown that increased concentrations of free radicals can produce lipid peroxidation and decrease membrane fluidity (16). Therefore, the continuous brain AChE inhibition observed from 0.5 h up to 2.5 h of incubation may reflect a decrease in membrane fluidity due to lipid peroxidation, which may influence the enzyme activity, through lipid(s)-protein interactions. The observed AChE stimulation at 15 min was possibly due to an initial increase in membrane fluidity, when the enzyme is exposed to a minimum free radical concentration. Figure 2 shows that the addition of different H2O2 concentrations up to 0.1 mM in these mentioned system for 2.5 h was not able to influence the AChE activity (p ⬎ 0.05) more than the enzyme inhibition TABLE 1 Effect of Aging on Acetylcholinesterase Activity Determined in Homogenized Whole Brain, Pituitary, and Specific Brain Areas Acetylcholinesterase activity ⌬OD/min/mg protein Tissue
4 mo old rats
22 mo old rats
Whole brain Pituitary Hypothalamus Frontal cortex Hippocampus
0.598 ⫾ 0.009 0.096 ⫾ 0.008 0.499 ⫾ 0.035 0.196 ⫾ 0.006 0.288 ⫾ 0.012
0.456 ⫾ 0.006*** 0.108 ⫾ 0.012 0.299 ⫾ 0.030*** 0.147 ⫾ 0.010** 0.209 ⫾ 0.006***
Values represent means ⫾ SD of five independent experiments (five pools of three animals each) for adult or aged rats for the pituitary and eight independent experiments (8 rats) for 4-month-old rats and six experiments (6 rats) for 22-month-old rats for the whole brain as well as specific brain areas. The average value of each experiment came from three determinations. **p ⬍ 0.01; ***p ⬍ 0.001; compared to 4-month-old rats. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
PHE PROTECTION OF BRAIN ACHE FROM RADICALS
Figure 1 — Time-dependent effect of free radical production by the system of FeSO4 (84 M) and ascorbic acid (400 M) on AChE activity determined in homogenized brain of 4 mo old rats. Points and vertical bars represent mean values ⫾ SD for the control (E) and the system that was used (■). Values represent means ⫾ SD of three experiments. The average values of each experiment arise from two independent determinations. Control value of AChE activity was 0.598 ⫾ 0.009 ⌬OD / min ⫻ mg protein. **p ⬍ 0.01.
already caused by the first system. The addition of 1 mM H2O2 decreased the enzyme activity by about 28% (p ⬍ 0.01) as compared to control but by 12% more than that caused by the first system alone (from about 83% to 72% of activity). The enzyme activity reached an inhibition of about 80% in 10
Figure 2 — Effect of different H2O2 concentrations on AChE activity. H2O2 was added to the incubation mixture containing FeSO4 (84 M) and ascorbic acid (400 M) for 2.5 h. Points and vertical bars represent mean values ⫾ SD for the control (E) and the system of FeSO4, ascorbic acid and H2O2 (F). Values represent means ⫾ SD of three experiments. The average values of each experiment arise from three independent determinations. **p ⬍ 0.01; ***p ⬍ 0.001; as compared to control. CLINICAL BIOCHEMISTRY, VOLUME 33, MARCH 2000
Figure 3 — Effect of preincubation of brain homogenates with various Phe concentrations on the protection of AChE inhibition by free radicals. The two systems used for free radical production and inhibition of AChE activity were as follows: (a) FeSO4 (84 M), ascorbic acid (400 M) (■), and (b) FeSO4, ascorbic acid, H2O2 (1 mM) (E). Brain homogenates were preincubated with different Phe concentrations in the reaction medium for 1 h. After preincubation, the above mentioned systems were added to the incubation medium and incubation was continued for 2.5 h. Values represent means ⫾ SD of three experiments. The average values of each experiment arise from three independent determinations. **p ⬍ 0.01; ***p ⬍ 0.001.
mM H2O2 but by 60% more than that caused by the first system alone (from about 80% to 20% of activity). The incubation of brain homogenates with FeSO4 (84 M), ascorbic acid (400 M), or H2O2 (1 mM) separately did not induce AChE inhibition (p ⬎ 0.05). Therefore, Fenton reaction, which is a good source of •OH radicals, may have induced the observed enzymatic inhibition of up to 28%. Figure 3 presents the effect of different concentrations of Phe (0.12–1.80 mM) in the homogenates on the protection of AChE inhibition by free radicals. These mentioned intracellular concentrations of Phe correspond to 6 times the concentration in plasma (21) (0.72–10.8 mM). Phe concentrations of 0.3–5.4 mM are usually found in the plasma of phenylketonuric patients (22). The inhibited AChE activity by the free radical action was entirely reversed when the brain homogenate was preincubated with 1.80 mM Phe. Furthermore, preincubation with a lower concentration of the amino acid (0.3 mM Phe) resulted in a 50% protection of the inhibited enzyme activity. However, when Phe was added at the same time with or after the two systems used for free radical production, it was not able to reverse the inhibition of the enzyme activity (p ⬎ 0.05). In a recent report, it has been suggested by us that Phe acts directly on AChE (23). Therefore, Phe seems to protect the enzyme against the direct action of free radicals. This action could be exerted on cysteine, methionine, histidine and/or tyrosine residues of the AChE molecule (13,24). It has been reported (17,18) that Phe reacts with •OH radicals to form the 105
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aromatic hydroxylated products ortho-, meta-, and para- tyrosines. In Figure 3 it was observed that the inhibited AChE activity by the free radical action was entirely reversed when the brain homogenate was preincubated with 1.80 mM Phe. Therefore, Phe could protect against the direct action of •OH radicals on brain AChE. These results suggest that Phe treatment, before •OH radicals damage, might be a preventive measure in certain cholinergic neural dysfunctions (e.g., Alzheimer’s disease). Acknowledgements This work was funded by the University of Athens. Many thanks are extended to Char. Antoniades and K. Marinou for their assistance.
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