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Enhancement of tissue lipoperoxidation in propanil-treated rats
Toxicology Letters ELSEVIER
Toxicology Letters 78 (1995) 215-218
Enhancement of tissue lipoperoxidation
in propanil-treated
rats
Mariarosaria Santillo”, Carmela Rippab, Rossella Della Morteb, Guglielmo RD. Villanib, Franc0 Santangeloa, Norma Staianob, Paolo Mondola*a ‘Istituto di Scienze Fisiologiche Umane. bDipartimento di Biochimica e Biotecnologie Mediche, Facoltci di Medicina e Chirurgia. Universitri di Napoli ‘Federico II’, Via S. Pansini, 5. 80131 Napoli, Italy
Received 8 March 1994; revision received 3 January 1995; accepted 10 January 1995
Abstract The i.p. injection of the herbicide propanil to male Sprague-Dawley rats increased the susceptibility to lipoperoxidation of liver and brain rat microsomes. A liver damage produced by propanil treatment was demonstrated by decreased serum levels of cholesterol and triglycerides as compared to serum levels of the lipids in control rats. The cellular damage of rat liver was also confirmed by the increased serum levels of aspartate aminotransferase and alkaline phosphatase activities observed in propanil-treated rats as compared to their activities in control rats. Keywork
Propanil; Lipoperoxidation;
Herbicide; Cholesterol; Triglycerides; Rat
1. Introduction Propanil (3 ‘,4’-dichloropropionanilide, CAS No. 709-98-8) is an important selective herbicide for the control of weeds in rice. It is applied after the emergence of both weeds and crops. The propanil use in Europe is limited, but a large percentage of the rice crop in the United States is treated with it [l]. The acute toxicity of propanil is expressed primarily by methemoglobin formation, which leads to cyanosis in occupationally exposed humans [2]. Propanil-induced methemoglobinemia is mediated by oxidized metabolites formed following enzymatic hydrolysis of propanil to 34dichloroaniline [3]. In addition the major * Corresponding author.
3,4-dichloroaniline metabolites, iV-hydroxy-3,C dichloroaniline and 6-hydroxy-3&dichloroaniline are able to directly oxidize hemoglobin of rat erythrocytes in vitro [4]. Since there is growing evidence demonstrating that the susceptibility of membranes to lipoperoxidation is increased in damaged tissues in many diseases and poisoning [5], we have undertaken a study in order to evaluate the induction of microsomal lipoperoxidation in different tissues of rats treated by propanil. Rat serum levels of cholesterol, triglycerides, alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities were also measured in control and propanil-treated rats, in order to ascertain the liver damage caused by this herbicide.
0378-4274/95/W9.50 @ 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03257-L
216
M. Santillo et al. / Toxicology Letters 78 (1995) 215-218
2. Materials and methods 2.1. Chemicals Propanil was purchased from Lab Service Analytica s.r.1. (Bo, Italy); malonaldehyde bis[dimethyl acetal] (MDA), 2-thiobarbituric acid (TBA), NADPH and ADP from Sigma Chemical Co. (St. Louis, MO). All the other chemicals used were highly analytical grade. 2.2. Rat treatment Thirty male Sprague-Dawley rats (Charles River, Calco Co., Italy), weighing 300 f 50 g, were group-housed 4 to a cage and were kept in boxes adequately air-conditioned and ventilated (temperature: 21 f l°C) with free access to food and water. The animals were divided in 2 groups of 15 rats each. Blood samples from the rats were collected by intracardiac puncture, after 16 h fasting, under a slight anaesthesia. Subsequently the rats of the first group were given daily 50 mg propanil dissolved in 1 ml corn oil, by i.p. injection, while the second group of animals (control group) received daily 1 ml corn oil. After 10 days of treatment, blood samples were collected as described above and rats were sacrified. 2.3. Microsomal preparation Microsomes from liver, brain and kidney of control and propanil-treated rats were prepared according to the method of Yamazoe et al. [6]. Protein concentration of microsomal preparations was measured according to the method of Lowry et al. [7], using bovine serum albumin as standard. Microsomes were characterized for their content of cytochrome P450, determined by the method of Omura and Sato [8], and for the NADPHcytochrome P450 reductase activity, assayed by the method of Phillips and Langdon [9]. 2.4. Mcrosomal lipid peroxidation assay Lipid peroxidation was evaluated according to the method of Buege and Aust [lo], by measuring the formation of MDA from the breakdown of polyunsatured fatty acids which react with TBA to give a red species absorbing at 535 nm. Incubation mixtures (1 ml) contained: 0.2 mg microsomal proteins from control or treated rats, 100 PM ADP,
100 PM FeCls and 0.1 mM NADPH in 0.05 M Tris-HCl, pH 7.5. Reactions were started by the addition of NADPH; incubations were carried out at 37°C for 20 min under air atmosphere in a shaking water bath. To the reaction mixtures, 2 ml TCA-TBA-HCl solution (15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid; 0.25 N hydrochloric acid) were added. The solutions were heated for 15 min in a boiling water bath. After cooling, the flocculent precipitates were removed by centrifugation at 1000 x g for 10 min. The absorbance of the samples was measured against a blank that contained all the reagents minus the microsomal lipids. The MDA concentration was calculated using an extinction coefficient of 1.56 x 10’ M-’ cm-‘. 2.5. Analytical determinations Rat serum levels of cholesterol and triglycerides were determined by the enzymatic calorimetric methods of Siedel et al. [ 1I] and Wahlefeld and Bergmeyer [12], respectively. ALP, ALT and AST rat serum levels were measured according to the procedure described by Bergmeyer et al. [ 131. 2.6. Statistical analysis Statistical differences between values from control and propanil-treated rats were assessed using Student’s t-test for unpaired data. 3. Results and discussion The susceptibility to lipoperoxidation of liver, brain and kidney microsomes from control and propanil-treated rats is summarized in Table 1. The lipoperoxidation in liver microsomes of propanil-treated rats was increased by 95% as compared to that of the animals injected with only corn oil. A marked induction of lipoperoxidation in brain microsomes was also observed, it resulting 36% higher in the microsomes from propaniltreated rats with respect to control microsomes. Conversely, no statistically significant difference was detected in kidney microsomal lipoperoxidation between the two groups of animals. As demonstrated by Barber [14], destroyed tissues undergo lipid peroxidation faster than healthy ones; therefore, the effect of propanil on
M. Santiilo et al. /Toxicology Letters 78 (1995) 215-218
21-l
Table 1 NADPH-dependent microsomal lipid peroxidation in different tissues of control and propanil-treated rats 60-
TBARV nmol MDA/mg protein (mean f S.E.)
% variation compared to the control group
70-
60E
Liver Controls Propanil-treated rats Brain Controls Propanil-treated rats Kidney Controls Propanil-treated rats
393 f 23 765 zt 70*
x 507 40-
+95
30-
75 f 5 102 l lo** 96 f 4 88 l 1I “.s.
+36
20-
10-
-8 0--
Each group consists of 15 rats. a2-Thiobarbituric acid reactive substances. *P c 0.01; **P c 0.05; ns., not significantly different; all compared to the control group.
tissue susceptibility to lipoperoxidation could be due to the ability of the herbicide to cause tissue damage. In order to test this hypothesis, we measured the levels aofrat serum cholesterol, trigly.
AST
ALT
ALP
Fig. 1.Effect of propanil treatment on AST, ALT and ALP rat serum levels. Each bar represents theX f SE. of 15 treated animals, assayed in duplicate. Control values (rats treated only with corn oil) are represented by (m) and values of propaniltreated rats by (0). *Significant at P < 0.05.
Cholesterol
Triglycerides
Fig. 2. Effect of propanil treatment on cholesterol and triglycerides rat serum levels. Each bar represents theX f S.E. of 15 treated animals, assayed in duplicate. Control values (rats treated only with corn oil) are represented by (m while values of propanil-treated rats by (n). *Significant at P < 0.05.
cerides and ALP, AST, and ALT activities before and after corn oil or propanil rat treatment. Fig. 1 reports AST, ALT and ALP serum levels in the two groups of rats after the treatment. AST and ALP activities resulted higher in propaniltreated rats by 38 and 62%, respectively, as compared to serum levels of control rats, whereas ALT serum levels were not affected by propanil treatment. Corn oil administration to the control group of rats had no effect on AST, ALT and ALP basal serum levels (data not shown). The increased cytolysis expressed by the higher serum level of AST and ALP in propanil-treated rats suggest that the enhanced microsomal liver lipid peroxidation is associated to a damage of this tissue. Fig. 2 shows a statistically significant decrease of the levels of cholesterol and triglycerides in the blood serum of propanil-treated rats as compared to the levels of the lipids in control rat serum. Corn oil administration to the control group of rats did not affect basal serum levels of both cholesterol and triglycerides (data not shown). The low serum cholesterol levels showed by propanil-treated animals could be due to the hepatic damage which
218
hf. Santillo et al. /Toxicology
makes the liver unable to synthesize enough cholesterol from acetate. Alternatively, the low serum cholesterol levels could be directly ascribed to an oxidant effect of propanil on circulating cholesterol. In fact, as shown by some authors [ 121, cholesterol levels are directly correlated to the extent of its peroxidation into the blood. The same mechanism could be also postulated to explain the lowering effect exerted by propanil treatment on rat serum triglyceride levels. Even if further studies are necessary to better clarify the effects of propanil on cholesterol and triglycerides metabolism, our results demonstrate the damage induced by propanil in mammal tissues. Acknowledgements This work was supported by a grant from CNR, P.F. FATMA, contract no. 93.00739.PF41. We thank Mr. Giovanni Sequin0 for his competent assistance in animal starving. References 111Bartha, R. and Pramer, D. (1970) Metabolism of acylanilide herbicides. Adv. Appl. Microbial. 13, 317.
121Kimbrough, R.D. (1980) Human health effects of selected pesticides, chloroaniline derivatives. J. Environ. Sci. Health Bl5, 977. [31 Singleton, SD. and Murphy, S.D. (1973) Propanil (3,4dichloropropionanilide) induced methemoglobin formation in mice in relation to acylamidase activity. Toxicol. Appl. Pharmacol. 25, 20. [41 MC Millan, D.C., Freeman, J.P. and Hinson, J.A. (1990) Metabolism of the arylamide herbicide propanil. I. Microsomal metabolism and in vitro methemoglobinemia. Toxicol. Appl. Pharmacol. 103, 90.
Letters 78 (1995) 215-218
PI Halliwell, B. and Gutteridge, J.M.C. (Eds.) (1989) Lipid pcroxidation: a radical chain reaction. In: Free Radicals in Biology and Medicine II, Clarendon Press, Oxford, pp. 188-276. 161Yamazoe, Y., Kamataki, T. and Kato, R. (1981) Species difference in N-hydroxylation of a tryptofan pyrolysis product in relation to mutagenic activation. Cancer Res. 41, 4518. [71 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. 181 Omura, 7. and Sato, R. (1964) The carbon-monoxidebinding pigment of liver microsomes. II. Solubilization, purification and properties. J. Biol. Chem. 239, 2379. [91 Phillips, A.H. and Langdon, R.G. (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization and kinetic studies. J. Biol. Chem. 237, 2652. 1101 Buege, J.A. and Aust, S.D. (1977) Microsomal lipid peroxidation. Methods Enzymol. 52, 302. 1111 Siedel, J.H., Schlumberger, SK., Ziegenhom, J. and Wahlefeld, A.W. (1981) Improved reagent of serum cholesterol. J. Clin. Chem. 19, 838. WI Wahlefeld, A.W. and Bergmeyer, H.W. (1974) Triglycerides determination after enzymatic hydrohsis. In: Methods of Enzymatic Analysis, Berlachemie Zeinheim and Academic Press Inc., New York and London, pp. 1831-1835. u31 Bcrgmeyer, S.U., Buttner, H., Hillmann, G. et al. (1972) Recommendations of the German Society for Clinical Chemistry Standardisations of methods for the estimation of enzyme activities in biological fluids. Z. Klin. Chem. Khn. B&hem. 10, 281. 1141 Barber, A.A. (1962) Addendum: mechanisms of lipid peroxide formation in rat tissue homogenates. Radiat. Res. Suppl. 3, 33. WI Subbiah, M.T.R. and Yunker, R.L. (1990) Changes in plasma cholesterol values as estimated by enzymatic methods after deliberate peroxidation of plasma. Clin. Chem. 36. 1524.
Toxicology Letters 78 (1995) 215-218
Enhancement of tissue lipoperoxidation
in propanil-treated
rats
Mariarosaria Santillo”, Carmela Rippab, Rossella Della Morteb, Guglielmo RD. Villanib, Franc0 Santangeloa, Norma Staianob, Paolo Mondola*a ‘Istituto di Scienze Fisiologiche Umane. bDipartimento di Biochimica e Biotecnologie Mediche, Facoltci di Medicina e Chirurgia. Universitri di Napoli ‘Federico II’, Via S. Pansini, 5. 80131 Napoli, Italy
Received 8 March 1994; revision received 3 January 1995; accepted 10 January 1995
Abstract The i.p. injection of the herbicide propanil to male Sprague-Dawley rats increased the susceptibility to lipoperoxidation of liver and brain rat microsomes. A liver damage produced by propanil treatment was demonstrated by decreased serum levels of cholesterol and triglycerides as compared to serum levels of the lipids in control rats. The cellular damage of rat liver was also confirmed by the increased serum levels of aspartate aminotransferase and alkaline phosphatase activities observed in propanil-treated rats as compared to their activities in control rats. Keywork
Propanil; Lipoperoxidation;
Herbicide; Cholesterol; Triglycerides; Rat
1. Introduction Propanil (3 ‘,4’-dichloropropionanilide, CAS No. 709-98-8) is an important selective herbicide for the control of weeds in rice. It is applied after the emergence of both weeds and crops. The propanil use in Europe is limited, but a large percentage of the rice crop in the United States is treated with it [l]. The acute toxicity of propanil is expressed primarily by methemoglobin formation, which leads to cyanosis in occupationally exposed humans [2]. Propanil-induced methemoglobinemia is mediated by oxidized metabolites formed following enzymatic hydrolysis of propanil to 34dichloroaniline [3]. In addition the major * Corresponding author.
3,4-dichloroaniline metabolites, iV-hydroxy-3,C dichloroaniline and 6-hydroxy-3&dichloroaniline are able to directly oxidize hemoglobin of rat erythrocytes in vitro [4]. Since there is growing evidence demonstrating that the susceptibility of membranes to lipoperoxidation is increased in damaged tissues in many diseases and poisoning [5], we have undertaken a study in order to evaluate the induction of microsomal lipoperoxidation in different tissues of rats treated by propanil. Rat serum levels of cholesterol, triglycerides, alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities were also measured in control and propanil-treated rats, in order to ascertain the liver damage caused by this herbicide.
0378-4274/95/W9.50 @ 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03257-L
216
M. Santillo et al. / Toxicology Letters 78 (1995) 215-218
2. Materials and methods 2.1. Chemicals Propanil was purchased from Lab Service Analytica s.r.1. (Bo, Italy); malonaldehyde bis[dimethyl acetal] (MDA), 2-thiobarbituric acid (TBA), NADPH and ADP from Sigma Chemical Co. (St. Louis, MO). All the other chemicals used were highly analytical grade. 2.2. Rat treatment Thirty male Sprague-Dawley rats (Charles River, Calco Co., Italy), weighing 300 f 50 g, were group-housed 4 to a cage and were kept in boxes adequately air-conditioned and ventilated (temperature: 21 f l°C) with free access to food and water. The animals were divided in 2 groups of 15 rats each. Blood samples from the rats were collected by intracardiac puncture, after 16 h fasting, under a slight anaesthesia. Subsequently the rats of the first group were given daily 50 mg propanil dissolved in 1 ml corn oil, by i.p. injection, while the second group of animals (control group) received daily 1 ml corn oil. After 10 days of treatment, blood samples were collected as described above and rats were sacrified. 2.3. Microsomal preparation Microsomes from liver, brain and kidney of control and propanil-treated rats were prepared according to the method of Yamazoe et al. [6]. Protein concentration of microsomal preparations was measured according to the method of Lowry et al. [7], using bovine serum albumin as standard. Microsomes were characterized for their content of cytochrome P450, determined by the method of Omura and Sato [8], and for the NADPHcytochrome P450 reductase activity, assayed by the method of Phillips and Langdon [9]. 2.4. Mcrosomal lipid peroxidation assay Lipid peroxidation was evaluated according to the method of Buege and Aust [lo], by measuring the formation of MDA from the breakdown of polyunsatured fatty acids which react with TBA to give a red species absorbing at 535 nm. Incubation mixtures (1 ml) contained: 0.2 mg microsomal proteins from control or treated rats, 100 PM ADP,
100 PM FeCls and 0.1 mM NADPH in 0.05 M Tris-HCl, pH 7.5. Reactions were started by the addition of NADPH; incubations were carried out at 37°C for 20 min under air atmosphere in a shaking water bath. To the reaction mixtures, 2 ml TCA-TBA-HCl solution (15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid; 0.25 N hydrochloric acid) were added. The solutions were heated for 15 min in a boiling water bath. After cooling, the flocculent precipitates were removed by centrifugation at 1000 x g for 10 min. The absorbance of the samples was measured against a blank that contained all the reagents minus the microsomal lipids. The MDA concentration was calculated using an extinction coefficient of 1.56 x 10’ M-’ cm-‘. 2.5. Analytical determinations Rat serum levels of cholesterol and triglycerides were determined by the enzymatic calorimetric methods of Siedel et al. [ 1I] and Wahlefeld and Bergmeyer [12], respectively. ALP, ALT and AST rat serum levels were measured according to the procedure described by Bergmeyer et al. [ 131. 2.6. Statistical analysis Statistical differences between values from control and propanil-treated rats were assessed using Student’s t-test for unpaired data. 3. Results and discussion The susceptibility to lipoperoxidation of liver, brain and kidney microsomes from control and propanil-treated rats is summarized in Table 1. The lipoperoxidation in liver microsomes of propanil-treated rats was increased by 95% as compared to that of the animals injected with only corn oil. A marked induction of lipoperoxidation in brain microsomes was also observed, it resulting 36% higher in the microsomes from propaniltreated rats with respect to control microsomes. Conversely, no statistically significant difference was detected in kidney microsomal lipoperoxidation between the two groups of animals. As demonstrated by Barber [14], destroyed tissues undergo lipid peroxidation faster than healthy ones; therefore, the effect of propanil on
M. Santiilo et al. /Toxicology Letters 78 (1995) 215-218
21-l
Table 1 NADPH-dependent microsomal lipid peroxidation in different tissues of control and propanil-treated rats 60-
TBARV nmol MDA/mg protein (mean f S.E.)
% variation compared to the control group
70-
60E
Liver Controls Propanil-treated rats Brain Controls Propanil-treated rats Kidney Controls Propanil-treated rats
393 f 23 765 zt 70*
x 507 40-
+95
30-
75 f 5 102 l lo** 96 f 4 88 l 1I “.s.
+36
20-
10-
-8 0--
Each group consists of 15 rats. a2-Thiobarbituric acid reactive substances. *P c 0.01; **P c 0.05; ns., not significantly different; all compared to the control group.
tissue susceptibility to lipoperoxidation could be due to the ability of the herbicide to cause tissue damage. In order to test this hypothesis, we measured the levels aofrat serum cholesterol, trigly.
AST
ALT
ALP
Fig. 1.Effect of propanil treatment on AST, ALT and ALP rat serum levels. Each bar represents theX f SE. of 15 treated animals, assayed in duplicate. Control values (rats treated only with corn oil) are represented by (m) and values of propaniltreated rats by (0). *Significant at P < 0.05.
Cholesterol
Triglycerides
Fig. 2. Effect of propanil treatment on cholesterol and triglycerides rat serum levels. Each bar represents theX f S.E. of 15 treated animals, assayed in duplicate. Control values (rats treated only with corn oil) are represented by (m while values of propanil-treated rats by (n). *Significant at P < 0.05.
cerides and ALP, AST, and ALT activities before and after corn oil or propanil rat treatment. Fig. 1 reports AST, ALT and ALP serum levels in the two groups of rats after the treatment. AST and ALP activities resulted higher in propaniltreated rats by 38 and 62%, respectively, as compared to serum levels of control rats, whereas ALT serum levels were not affected by propanil treatment. Corn oil administration to the control group of rats had no effect on AST, ALT and ALP basal serum levels (data not shown). The increased cytolysis expressed by the higher serum level of AST and ALP in propanil-treated rats suggest that the enhanced microsomal liver lipid peroxidation is associated to a damage of this tissue. Fig. 2 shows a statistically significant decrease of the levels of cholesterol and triglycerides in the blood serum of propanil-treated rats as compared to the levels of the lipids in control rat serum. Corn oil administration to the control group of rats did not affect basal serum levels of both cholesterol and triglycerides (data not shown). The low serum cholesterol levels showed by propanil-treated animals could be due to the hepatic damage which
218
hf. Santillo et al. /Toxicology
makes the liver unable to synthesize enough cholesterol from acetate. Alternatively, the low serum cholesterol levels could be directly ascribed to an oxidant effect of propanil on circulating cholesterol. In fact, as shown by some authors [ 121, cholesterol levels are directly correlated to the extent of its peroxidation into the blood. The same mechanism could be also postulated to explain the lowering effect exerted by propanil treatment on rat serum triglyceride levels. Even if further studies are necessary to better clarify the effects of propanil on cholesterol and triglycerides metabolism, our results demonstrate the damage induced by propanil in mammal tissues. Acknowledgements This work was supported by a grant from CNR, P.F. FATMA, contract no. 93.00739.PF41. We thank Mr. Giovanni Sequin0 for his competent assistance in animal starving. References 111Bartha, R. and Pramer, D. (1970) Metabolism of acylanilide herbicides. Adv. Appl. Microbial. 13, 317.
121Kimbrough, R.D. (1980) Human health effects of selected pesticides, chloroaniline derivatives. J. Environ. Sci. Health Bl5, 977. [31 Singleton, SD. and Murphy, S.D. (1973) Propanil (3,4dichloropropionanilide) induced methemoglobin formation in mice in relation to acylamidase activity. Toxicol. Appl. Pharmacol. 25, 20. [41 MC Millan, D.C., Freeman, J.P. and Hinson, J.A. (1990) Metabolism of the arylamide herbicide propanil. I. Microsomal metabolism and in vitro methemoglobinemia. Toxicol. Appl. Pharmacol. 103, 90.
Letters 78 (1995) 215-218
PI Halliwell, B. and Gutteridge, J.M.C. (Eds.) (1989) Lipid pcroxidation: a radical chain reaction. In: Free Radicals in Biology and Medicine II, Clarendon Press, Oxford, pp. 188-276. 161Yamazoe, Y., Kamataki, T. and Kato, R. (1981) Species difference in N-hydroxylation of a tryptofan pyrolysis product in relation to mutagenic activation. Cancer Res. 41, 4518. [71 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. 181 Omura, 7. and Sato, R. (1964) The carbon-monoxidebinding pigment of liver microsomes. II. Solubilization, purification and properties. J. Biol. Chem. 239, 2379. [91 Phillips, A.H. and Langdon, R.G. (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization and kinetic studies. J. Biol. Chem. 237, 2652. 1101 Buege, J.A. and Aust, S.D. (1977) Microsomal lipid peroxidation. Methods Enzymol. 52, 302. 1111 Siedel, J.H., Schlumberger, SK., Ziegenhom, J. and Wahlefeld, A.W. (1981) Improved reagent of serum cholesterol. J. Clin. Chem. 19, 838. WI Wahlefeld, A.W. and Bergmeyer, H.W. (1974) Triglycerides determination after enzymatic hydrohsis. In: Methods of Enzymatic Analysis, Berlachemie Zeinheim and Academic Press Inc., New York and London, pp. 1831-1835. u31 Bcrgmeyer, S.U., Buttner, H., Hillmann, G. et al. (1972) Recommendations of the German Society for Clinical Chemistry Standardisations of methods for the estimation of enzyme activities in biological fluids. Z. Klin. Chem. Khn. B&hem. 10, 281. 1141 Barber, A.A. (1962) Addendum: mechanisms of lipid peroxide formation in rat tissue homogenates. Radiat. Res. Suppl. 3, 33. WI Subbiah, M.T.R. and Yunker, R.L. (1990) Changes in plasma cholesterol values as estimated by enzymatic methods after deliberate peroxidation of plasma. Clin. Chem. 36. 1524.