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Increased lipoprotein susceptibility to oxidation following long distance running in trained subjects
Clinica Chimica Acta 271 (1998) 97–103
Short communication
Increased lipoprotein susceptibility to oxidation following long distance running in trained subjects Carani Venkataraman Anuradha*, Shanmugam Dhandapani Balakrishnan Department of Biochemistry, Annamalai University, Annamalainagar — 608 002, Tamil Nadu, India Received 16 July 1997; received in revised form 9 September 1997; accepted 12 November 1997
Keywords: Lipoprotein susceptibility to oxidation; Ceruloplasmin; Albumin; Long distance running
1. Introduction Regular physical exercise improves heart functions and attenuates the risk for developing coronary artery disease by modifying the plasma lipid profile [1]. On the other hand physical exercise causes up to 10-fold increase in oxygen consumption and 10–20-fold increase in the metabolism of cells which in turn increase the formation of reactive oxygen species (ROS) [2]. ROS promote peroxidation of lipids and if the natural antioxidant mechanisms are overwhelmed, oxidative stress is said to ensue. Peroxidation products have been observed in blood after acute exercise [3]. The peroxidation of lipids in plasma alters the biological properties of lipoproteins [4]. Low density lipoprotein (LDL) which delivers cholesterol to peripheral cells is the most sensitive to peroxidation. Peroxidised LDL do not react properly with their receptors, leading to esterification of cholesterol and conversion of macrophages to foam cells thereby contributing to the development of atherosclerotic plaque. Free metal ions such as iron and copper are implicated in this lipoprotein oxidation. Addition of antioxidants (butylated hydroxy toluene, vitamin E and glutathione) or chelators of transition metal ions *Corresponding author. 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00229-5
98
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
(EDTA and desferrioxamine) prevents oxidation of LDL in vitro [5]. This strengthens the possible involvement of free radical chain reactions in the oxidative modification of LDL. Albumin and ceruloplasmin are to a large extent responsible for keeping the metal ion-induced free radical production to a minimum [6]. Structural and oxidative changes in LDL fraction were demonstrated in well-trained runners after heavy, long duration aerobic exercise [7]. This is contradictory to the protective effects of exercise against atherosclerosis. In addition, blood antioxidant status is modified in trained athletes following distance running [8]. We, therefore, determined the effect of acute long distance running on lipid peroxidation in plasma and the susceptibility of lipoproteins to peroxidation in trained subjects. We also determined the relationship between changes in lipid peroxides and ceruloplasmin and albumin which occurred during running.
2. Materials and methods The subjects were 10 well-trained male long distance runners (21–25 years old, mean 23.461.3 years). They were non-obese (body mass index ranged from 19.2–23.0 kg / m 2 , mean 20.661.3 kg / m 2 ), non-smokers and non-consumers of alcohol. The maximal oxygen consumption (VO 2 max) was 46.562.2 ml / kg / min (range 42.8–51.3 ml / kg / min). They were determined to be healthy on the basis of their medical history and from clinical examination. They were free of any medication, drugs or nutrient supplementation. They were residents of the Annamalai University mens’ hostel and their food habits were similar. The regular menu consisted of a mixed diet of Indian foods which contained the recommended dietary allowances of carbohydrates, proteins, fats and micronutrients. The daily practice schedule consisted of fertlak training, circuit training and alternate pace running.1 The subjects were involved in physical training for at least 20 h / week for a period of 2–3 years. The subjects were allowed to run a distance of 10 000 m in a circuit located in the campus, Annamalai University. They completed the run in 32.964.1 min (range 26.0–41.9 min). The subjects were fasting for at least 8 h prior to the run. Blood samples were collected immediately before and after the run in heparinised test tubes and were centrifuged at 1800 3 g for 10 min to obtain plasma.
1
These are various methods undertaken by the subjects in a training session for endurance improvement. In circuit training the subjects took part in 10 exercises one after another. In the alternate pace method the subjects performed exercises continuously with preplanned change of pace. Fertlak training is a variation of the alternate pace method in which the subjects performed exercises continuously with a change of pace which was not preplanned.
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
99
The fractionation of plasma lipoproteins was made according to the method of Wilson and Spiger [9]. To an aliquot of plasma, heparin-manganese chloride mixture was added which caused precipitation of VLDL and LDL. The supernatant represented the HDL fraction. To another aliquot of plasma, sodium dodecylsulphate was added and allowed to stand for 2 h at 378C. The contents were centrifuged in a refrigerated centrifuge at 10 000 3 g for 10 min. This caused aggregation of VLDL as a pellicle at the top. This was removed and the remaining fraction represented the HDL 1 LDL fraction. Aliquots from these two fractions were used for lipid peroxidation studies. Lipid peroxidation in whole plasma was determined by measuring the TBA reactivity of plasma (TBARS) [10]. For this an aliquot of plasma was deproteinised with N / 12 sulphuric acid and 10% phosphotungstic acid and then heated with TBA in sodium hydroxide at 908C for 1 h. The pink colour formed gave a measure of TBARS. 1,19,3,39-Tetramethoxypropane was used as the standard and the values were expressed as m mol / l plasma. The susceptibility of lipoprotein fractions to peroxidation was studied by quantitating TBARS after incubation with copper sulphate (CuSO 4 ). For this 0.5 ml aliquots of the lipoprotein fractions (HDL, HDL 1 LDL) were incubated with 10 m M CuSO 4 in a final volume of 2 ml in 0.1 M phosphate buffer, pH 7.4. The mixtures were incubated in a shaking water bath at 378C for 1 h and then were deproteinised with N / 12 H 2 SO 4 and 10% phosphotungstic acid and heated with TBA in sodium hydroxide at 908C for 1 h. The colour formed was read at 535 nm after centrifugation. The values were expressed as nmol / dl / h. The difference between the two was computed and taken as TBARS released by LDL. LDL 2 TBARSrelease 5 [(HDL 1 LDL) 2 TBARS] 2 [(HDL) 2 TBARS] Albumin was determined by the method of Reinhold [11], ceruloplasmin by the method of Ravin [12] and total protein by the method of Lowry et al. [13]. The variability in repeated measurements was 2.3% for plasma TBARS, 4.2% for HDL-TBARS and 3.9% for LDL-TBARS. For albumin, ceruloplasmin and total protein the coefficient of variation was less than 2% (coefficient of variation for six replicates). Differences between the mean values before and after the run were tested for significance by Student’s t-test for paired comparisons. Pearson’s correlation coefficients were determined to study the association between various parameters.
3. Results Heart rate was elevated twofold after running (before — 66.662.4; after — 144.8614.3 beats / min). Significant increase in plasma TBARS (61%, P,0.01)
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C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
Table 1 Effect of long distance running on plasma variables in 10 male athletes (mean6S.D. and range)
Plasma TBARS ( m mol / l) HDL-TBARS (nmol / dl / h) LDL-TBARS (nmol / dl / h) Total protein (g / dl) Albumin (g / dl) Ceruloplasmin (mg / dl)
Before
After
3.2561.7 (1.8–6.6) 104.0614.0 (80.4–121.0) 154.0623.0 (115.0–195.0) 6.9660.30 (6.6–7.6) 4.6960.50 (3.9–5.4) 22.3961.30 (20.5–24.5)
5.2361.5* (3.3–7.4) 118.0614.0* (95.6–135.5) 270.0635.0** (225.0–335.6) 6.1760.4* (5.8–6.6) 4.0460.1* (4.15–3.90) 34.9961.1** (33.5–36.8)
*Significant at P,0.01. **Signifcant at P,0.001.
was obtained after running (Table 1). The susceptibility to copper sulphateinduced oxidation was enhanced after running in the HDL and LDL fractions. There was a significant increase in TBARS release from HDL (P,0.01) and LDL (P,0.001) in post-exercise samples compared to pre-exercise samples. Albumin and total protein after running were lower (13% and 11% respectively, P,0.001) and ceruloplasmin was higher than the basal level (20%, P, 0.001). Fig. 1 and Fig. 2 show the relationship between the changes observed during the run in various parameters. The increase in plasma TBARS was positively associated with the concomitant changes in plasma ceruloplasmin and negatively associated with that of albumin (plasma TBARS and ceruloplasmin, r50.95, P,0.001; plasma TBARS and albumin, r5 20.92, P,0.001). The differences between the LDL-TBARS release before and after the run was positively associated with the concomitant changes in plasma ceruloplasmin. A negative correlation was obtained with albumin (LDL-TBARS and ceruloplasmin, r50.88, P,0.001; LDL-TBARS and albumin, r5 20.86, P, 0.001). No significant association was obtained between the changes in HDLTBARS release with the changes in plasma antioxidants.
4. Discussion Physical exercise could induce peroxidation of lipids in cellular membranes and of the lipid moiety of lipoproteins by promoting free radical formation. The increased level of TBARS in plasma observed in the post-exercise sample is a
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
101
Fig. 1. Correlation between the changes in plasma TBA-reactivity and the changes in ceruloplasmin and albumin during running. Left panel: plasma TBARS with albumin. Right panel: plasma TBARS with ceruloplasmin.
consequence of leakage of peroxides from tissues, especially muscle into plasma. Oxidative modification of plasma constituents is an expression of oxidative damage occurring in tissues [14]. Bonnefont et al. [15] have shown that TBARS are already present in LDL in the serum of healthy volunteers. In our study, we have measured the release of TBARS after CuSO 4 stimulation from the lipoprotein fractions. Copper is very effective in promoting the oxidative modification of lipoproteins. The release of
Fig. 2. Correlation between the changes in LDL-TBARS release after CuSO 4 stimulation and the concomitant changes in ceruloplasmin and albumin during running. Left panel: LDL-TBARS with albumin. Right panel: LDL-TBARS with ceruloplasmin.
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C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
TBARS was higher in the LDL fraction than in the HDL fraction in blood samples taken before and after running. The release was found to be maximum in the LDL fraction of the post-exercise sample. Accordingly the increase in the plasma TBARS is due more to that in the LDL fraction than to that in the HDL fraction. The possible explanation for the increased susceptibility of the LDL fraction is that increased free radical production can affect the oxidative state of the circulating LDL particles. Physical exercise acutely promotes oxidative changes in circulating LDL increasing the proportion of the electronegative LDLB form [7]. During aerobic exercise, studied herein, the LDL particles showed increased susceptibility to oxidation. Depletion of antioxidant content is implicated in this increased susceptibility. Takanami et al. [16] have reported that LDL isolated from individuals after maximal exercise are found to be oxidatively modified and this could be prevented by antioxidant supplementation. It is interesting to note that albumin and total protein are lower and ceruloplasmin is higher than the resting levels. Ceruloplasmin, a ferroxidase, is an acute phase protein and has direct anti-inflammatory properties. Elevation in ceruloplasmin can be linked to its ferroxidase action and to the endogenous response to an acute phase reaction. The reduction in serum albumin may be the consequence of extravasation of albumin due to an inflammatory tissue response to prolonged exercise and the fall in albumin may be responsible for increased peroxidation in post-exercise samples. Significant positive correlation was obtained between the increase in plasma TBARS and LDL-TBARS release and the changes in ceruloplasmin. A negative association between the increase in plasma TBARS and LDL-TBARS release after CuSO 4 stimulation with the changes in albumin was observed. This suggests that the albumin and ceruloplasmin play a significant role in regulating the oxidation of plasma constituents. Training reduces the production of lipid peroxide products and prevents oxidative damage in tissues by inducing the antioxidant system in athletes [17]. Training therefore represents a protective mechanism against oxidation. However, despite the small sample size, the findings of the present study show that in trained individuals plasma TBARS is increased immediately after long distance running. This could modify the lipoproteins increasing the susceptibility to in vitro oxidation. This appears to be an undesirable effect of exercise since oxidised LDL forms are shown to be responsible for the formation of atherosclerotic plaque. The increase in TBA reactivity of plasma and LDL fraction correlated with the alterations in albumin and ceruloplasmin. Intake of antioxidant-rich foods or supplementation may minimise the undesirable effects and optimise the beneficial effects of such exercise in trained subjects. Further experiments are needed to elucidate the mechanisms responsible for this increased susceptibility and to assay the turnover of lipid soluble antioxidants contained in the lipoprotein particles.
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
103
Acknowledgements The authors thank Dr. G. Ravindran, Reader, Department of Physical Education and Sports Sciences, Annamalai University for providing the blood samples of sports persons.
References [1] Berlin JA, Colditz GA. A meta analysis of physical activity in prevention of coronary heart disease. Am J Epidemiol 1990;132:612. [2] Davies KJA, Quintanilha T, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochim Biophys Res Commun 1982;197:1198–205. [3] Kanter MM, Lesmen GR, Negnin ND et al. Serum lipid levels and lipid peroxidation in ultramarathon runners. Ann Sports Med 1986;3:39–41. [4] Jurgens G, Lang J, Esterbauer H, Holasek A. Modification of human low density lipoprotein by 4-hydroxynonenal. IRCS Med Sci 1984;12:252–3. [5] Morel DW, Corleto PE, Chisholm GM. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Atherosclerosis 1985;4:357–64. [6] Halliwell B, Gutteridge JMC. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246(2):501–14. [7] Sanchez-Quesada JL, Homs-Serradesanferm R, Serrat-Serrat J, Serra-Grima JR, GonzalezSastre F, Ordonez-Llanos J. Increase of LDL susceptibility to oxidation occurring after intense, long duration aerobic exercise. Atherosclerosis 1995;118:297–305. [8] Duthie GG, Robertson JD, Maughan RJ, Morrice PC. Blood antioxidant status and erythrocyte lipid peroxidation following distance running. Arch Biochem Biophys 1990;282:78–83. [9] Wilson DE, Spiger MJ. A dual precipitation method for quantitating plasma lipoprotein measurement without ultracentrifugation. J Lab Clin Med 1973;82:473–82. [10] Yagi K. Lipid peroxides and human diseases. Chem Phys Lipids 1987;45:337–51. [11] Reinhold JG. In: Reiner M, editor. Standard methods of clinical chemistry, vol. I. New York: Academic Press, 1953:88. [12] Ravin HA. An improved colorimetric enzymatic assay for ceruloplasmin. J Lab Clin Med 1961;58:161–8. [13] Lowry OH, Rosebrough MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–71. [14] Witt EH, Reznick AZ, Viguie CA, Starke-Reed P, Packer L. Exercise, oxidative damage and effects of antioxidant manipulation. J Nutr 1992;122:766–73. [15] Bonnefont D, Legrant A, Reynet J, Delattre J, Gall A. Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum. Clin Chem 1989;35:2054. [16] Takanami Y, Zwam H, Kawai Y et al. Vitamin E supplementation inhibits LDL oxidation during exercise in humans. Med Sci Sports Exerc 1994;53:261. [17] Robertson JD, Maughan RJ, Duthie GG, Morrice PC. Increased blood antioxidant systems of runners in response to training load. Clin Sci 1991;80:611–8.
Short communication
Increased lipoprotein susceptibility to oxidation following long distance running in trained subjects Carani Venkataraman Anuradha*, Shanmugam Dhandapani Balakrishnan Department of Biochemistry, Annamalai University, Annamalainagar — 608 002, Tamil Nadu, India Received 16 July 1997; received in revised form 9 September 1997; accepted 12 November 1997
Keywords: Lipoprotein susceptibility to oxidation; Ceruloplasmin; Albumin; Long distance running
1. Introduction Regular physical exercise improves heart functions and attenuates the risk for developing coronary artery disease by modifying the plasma lipid profile [1]. On the other hand physical exercise causes up to 10-fold increase in oxygen consumption and 10–20-fold increase in the metabolism of cells which in turn increase the formation of reactive oxygen species (ROS) [2]. ROS promote peroxidation of lipids and if the natural antioxidant mechanisms are overwhelmed, oxidative stress is said to ensue. Peroxidation products have been observed in blood after acute exercise [3]. The peroxidation of lipids in plasma alters the biological properties of lipoproteins [4]. Low density lipoprotein (LDL) which delivers cholesterol to peripheral cells is the most sensitive to peroxidation. Peroxidised LDL do not react properly with their receptors, leading to esterification of cholesterol and conversion of macrophages to foam cells thereby contributing to the development of atherosclerotic plaque. Free metal ions such as iron and copper are implicated in this lipoprotein oxidation. Addition of antioxidants (butylated hydroxy toluene, vitamin E and glutathione) or chelators of transition metal ions *Corresponding author. 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00229-5
98
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
(EDTA and desferrioxamine) prevents oxidation of LDL in vitro [5]. This strengthens the possible involvement of free radical chain reactions in the oxidative modification of LDL. Albumin and ceruloplasmin are to a large extent responsible for keeping the metal ion-induced free radical production to a minimum [6]. Structural and oxidative changes in LDL fraction were demonstrated in well-trained runners after heavy, long duration aerobic exercise [7]. This is contradictory to the protective effects of exercise against atherosclerosis. In addition, blood antioxidant status is modified in trained athletes following distance running [8]. We, therefore, determined the effect of acute long distance running on lipid peroxidation in plasma and the susceptibility of lipoproteins to peroxidation in trained subjects. We also determined the relationship between changes in lipid peroxides and ceruloplasmin and albumin which occurred during running.
2. Materials and methods The subjects were 10 well-trained male long distance runners (21–25 years old, mean 23.461.3 years). They were non-obese (body mass index ranged from 19.2–23.0 kg / m 2 , mean 20.661.3 kg / m 2 ), non-smokers and non-consumers of alcohol. The maximal oxygen consumption (VO 2 max) was 46.562.2 ml / kg / min (range 42.8–51.3 ml / kg / min). They were determined to be healthy on the basis of their medical history and from clinical examination. They were free of any medication, drugs or nutrient supplementation. They were residents of the Annamalai University mens’ hostel and their food habits were similar. The regular menu consisted of a mixed diet of Indian foods which contained the recommended dietary allowances of carbohydrates, proteins, fats and micronutrients. The daily practice schedule consisted of fertlak training, circuit training and alternate pace running.1 The subjects were involved in physical training for at least 20 h / week for a period of 2–3 years. The subjects were allowed to run a distance of 10 000 m in a circuit located in the campus, Annamalai University. They completed the run in 32.964.1 min (range 26.0–41.9 min). The subjects were fasting for at least 8 h prior to the run. Blood samples were collected immediately before and after the run in heparinised test tubes and were centrifuged at 1800 3 g for 10 min to obtain plasma.
1
These are various methods undertaken by the subjects in a training session for endurance improvement. In circuit training the subjects took part in 10 exercises one after another. In the alternate pace method the subjects performed exercises continuously with preplanned change of pace. Fertlak training is a variation of the alternate pace method in which the subjects performed exercises continuously with a change of pace which was not preplanned.
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
99
The fractionation of plasma lipoproteins was made according to the method of Wilson and Spiger [9]. To an aliquot of plasma, heparin-manganese chloride mixture was added which caused precipitation of VLDL and LDL. The supernatant represented the HDL fraction. To another aliquot of plasma, sodium dodecylsulphate was added and allowed to stand for 2 h at 378C. The contents were centrifuged in a refrigerated centrifuge at 10 000 3 g for 10 min. This caused aggregation of VLDL as a pellicle at the top. This was removed and the remaining fraction represented the HDL 1 LDL fraction. Aliquots from these two fractions were used for lipid peroxidation studies. Lipid peroxidation in whole plasma was determined by measuring the TBA reactivity of plasma (TBARS) [10]. For this an aliquot of plasma was deproteinised with N / 12 sulphuric acid and 10% phosphotungstic acid and then heated with TBA in sodium hydroxide at 908C for 1 h. The pink colour formed gave a measure of TBARS. 1,19,3,39-Tetramethoxypropane was used as the standard and the values were expressed as m mol / l plasma. The susceptibility of lipoprotein fractions to peroxidation was studied by quantitating TBARS after incubation with copper sulphate (CuSO 4 ). For this 0.5 ml aliquots of the lipoprotein fractions (HDL, HDL 1 LDL) were incubated with 10 m M CuSO 4 in a final volume of 2 ml in 0.1 M phosphate buffer, pH 7.4. The mixtures were incubated in a shaking water bath at 378C for 1 h and then were deproteinised with N / 12 H 2 SO 4 and 10% phosphotungstic acid and heated with TBA in sodium hydroxide at 908C for 1 h. The colour formed was read at 535 nm after centrifugation. The values were expressed as nmol / dl / h. The difference between the two was computed and taken as TBARS released by LDL. LDL 2 TBARSrelease 5 [(HDL 1 LDL) 2 TBARS] 2 [(HDL) 2 TBARS] Albumin was determined by the method of Reinhold [11], ceruloplasmin by the method of Ravin [12] and total protein by the method of Lowry et al. [13]. The variability in repeated measurements was 2.3% for plasma TBARS, 4.2% for HDL-TBARS and 3.9% for LDL-TBARS. For albumin, ceruloplasmin and total protein the coefficient of variation was less than 2% (coefficient of variation for six replicates). Differences between the mean values before and after the run were tested for significance by Student’s t-test for paired comparisons. Pearson’s correlation coefficients were determined to study the association between various parameters.
3. Results Heart rate was elevated twofold after running (before — 66.662.4; after — 144.8614.3 beats / min). Significant increase in plasma TBARS (61%, P,0.01)
100
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
Table 1 Effect of long distance running on plasma variables in 10 male athletes (mean6S.D. and range)
Plasma TBARS ( m mol / l) HDL-TBARS (nmol / dl / h) LDL-TBARS (nmol / dl / h) Total protein (g / dl) Albumin (g / dl) Ceruloplasmin (mg / dl)
Before
After
3.2561.7 (1.8–6.6) 104.0614.0 (80.4–121.0) 154.0623.0 (115.0–195.0) 6.9660.30 (6.6–7.6) 4.6960.50 (3.9–5.4) 22.3961.30 (20.5–24.5)
5.2361.5* (3.3–7.4) 118.0614.0* (95.6–135.5) 270.0635.0** (225.0–335.6) 6.1760.4* (5.8–6.6) 4.0460.1* (4.15–3.90) 34.9961.1** (33.5–36.8)
*Significant at P,0.01. **Signifcant at P,0.001.
was obtained after running (Table 1). The susceptibility to copper sulphateinduced oxidation was enhanced after running in the HDL and LDL fractions. There was a significant increase in TBARS release from HDL (P,0.01) and LDL (P,0.001) in post-exercise samples compared to pre-exercise samples. Albumin and total protein after running were lower (13% and 11% respectively, P,0.001) and ceruloplasmin was higher than the basal level (20%, P, 0.001). Fig. 1 and Fig. 2 show the relationship between the changes observed during the run in various parameters. The increase in plasma TBARS was positively associated with the concomitant changes in plasma ceruloplasmin and negatively associated with that of albumin (plasma TBARS and ceruloplasmin, r50.95, P,0.001; plasma TBARS and albumin, r5 20.92, P,0.001). The differences between the LDL-TBARS release before and after the run was positively associated with the concomitant changes in plasma ceruloplasmin. A negative correlation was obtained with albumin (LDL-TBARS and ceruloplasmin, r50.88, P,0.001; LDL-TBARS and albumin, r5 20.86, P, 0.001). No significant association was obtained between the changes in HDLTBARS release with the changes in plasma antioxidants.
4. Discussion Physical exercise could induce peroxidation of lipids in cellular membranes and of the lipid moiety of lipoproteins by promoting free radical formation. The increased level of TBARS in plasma observed in the post-exercise sample is a
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
101
Fig. 1. Correlation between the changes in plasma TBA-reactivity and the changes in ceruloplasmin and albumin during running. Left panel: plasma TBARS with albumin. Right panel: plasma TBARS with ceruloplasmin.
consequence of leakage of peroxides from tissues, especially muscle into plasma. Oxidative modification of plasma constituents is an expression of oxidative damage occurring in tissues [14]. Bonnefont et al. [15] have shown that TBARS are already present in LDL in the serum of healthy volunteers. In our study, we have measured the release of TBARS after CuSO 4 stimulation from the lipoprotein fractions. Copper is very effective in promoting the oxidative modification of lipoproteins. The release of
Fig. 2. Correlation between the changes in LDL-TBARS release after CuSO 4 stimulation and the concomitant changes in ceruloplasmin and albumin during running. Left panel: LDL-TBARS with albumin. Right panel: LDL-TBARS with ceruloplasmin.
102
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
TBARS was higher in the LDL fraction than in the HDL fraction in blood samples taken before and after running. The release was found to be maximum in the LDL fraction of the post-exercise sample. Accordingly the increase in the plasma TBARS is due more to that in the LDL fraction than to that in the HDL fraction. The possible explanation for the increased susceptibility of the LDL fraction is that increased free radical production can affect the oxidative state of the circulating LDL particles. Physical exercise acutely promotes oxidative changes in circulating LDL increasing the proportion of the electronegative LDLB form [7]. During aerobic exercise, studied herein, the LDL particles showed increased susceptibility to oxidation. Depletion of antioxidant content is implicated in this increased susceptibility. Takanami et al. [16] have reported that LDL isolated from individuals after maximal exercise are found to be oxidatively modified and this could be prevented by antioxidant supplementation. It is interesting to note that albumin and total protein are lower and ceruloplasmin is higher than the resting levels. Ceruloplasmin, a ferroxidase, is an acute phase protein and has direct anti-inflammatory properties. Elevation in ceruloplasmin can be linked to its ferroxidase action and to the endogenous response to an acute phase reaction. The reduction in serum albumin may be the consequence of extravasation of albumin due to an inflammatory tissue response to prolonged exercise and the fall in albumin may be responsible for increased peroxidation in post-exercise samples. Significant positive correlation was obtained between the increase in plasma TBARS and LDL-TBARS release and the changes in ceruloplasmin. A negative association between the increase in plasma TBARS and LDL-TBARS release after CuSO 4 stimulation with the changes in albumin was observed. This suggests that the albumin and ceruloplasmin play a significant role in regulating the oxidation of plasma constituents. Training reduces the production of lipid peroxide products and prevents oxidative damage in tissues by inducing the antioxidant system in athletes [17]. Training therefore represents a protective mechanism against oxidation. However, despite the small sample size, the findings of the present study show that in trained individuals plasma TBARS is increased immediately after long distance running. This could modify the lipoproteins increasing the susceptibility to in vitro oxidation. This appears to be an undesirable effect of exercise since oxidised LDL forms are shown to be responsible for the formation of atherosclerotic plaque. The increase in TBA reactivity of plasma and LDL fraction correlated with the alterations in albumin and ceruloplasmin. Intake of antioxidant-rich foods or supplementation may minimise the undesirable effects and optimise the beneficial effects of such exercise in trained subjects. Further experiments are needed to elucidate the mechanisms responsible for this increased susceptibility and to assay the turnover of lipid soluble antioxidants contained in the lipoprotein particles.
C.V. Anuradha, S.D. Balakrishnan / Clinica Chimica Acta 271 (1998) 97 – 103
103
Acknowledgements The authors thank Dr. G. Ravindran, Reader, Department of Physical Education and Sports Sciences, Annamalai University for providing the blood samples of sports persons.
References [1] Berlin JA, Colditz GA. A meta analysis of physical activity in prevention of coronary heart disease. Am J Epidemiol 1990;132:612. [2] Davies KJA, Quintanilha T, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochim Biophys Res Commun 1982;197:1198–205. [3] Kanter MM, Lesmen GR, Negnin ND et al. Serum lipid levels and lipid peroxidation in ultramarathon runners. Ann Sports Med 1986;3:39–41. [4] Jurgens G, Lang J, Esterbauer H, Holasek A. Modification of human low density lipoprotein by 4-hydroxynonenal. IRCS Med Sci 1984;12:252–3. [5] Morel DW, Corleto PE, Chisholm GM. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Atherosclerosis 1985;4:357–64. [6] Halliwell B, Gutteridge JMC. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246(2):501–14. [7] Sanchez-Quesada JL, Homs-Serradesanferm R, Serrat-Serrat J, Serra-Grima JR, GonzalezSastre F, Ordonez-Llanos J. Increase of LDL susceptibility to oxidation occurring after intense, long duration aerobic exercise. Atherosclerosis 1995;118:297–305. [8] Duthie GG, Robertson JD, Maughan RJ, Morrice PC. Blood antioxidant status and erythrocyte lipid peroxidation following distance running. Arch Biochem Biophys 1990;282:78–83. [9] Wilson DE, Spiger MJ. A dual precipitation method for quantitating plasma lipoprotein measurement without ultracentrifugation. J Lab Clin Med 1973;82:473–82. [10] Yagi K. Lipid peroxides and human diseases. Chem Phys Lipids 1987;45:337–51. [11] Reinhold JG. In: Reiner M, editor. Standard methods of clinical chemistry, vol. I. New York: Academic Press, 1953:88. [12] Ravin HA. An improved colorimetric enzymatic assay for ceruloplasmin. J Lab Clin Med 1961;58:161–8. [13] Lowry OH, Rosebrough MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–71. [14] Witt EH, Reznick AZ, Viguie CA, Starke-Reed P, Packer L. Exercise, oxidative damage and effects of antioxidant manipulation. J Nutr 1992;122:766–73. [15] Bonnefont D, Legrant A, Reynet J, Delattre J, Gall A. Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum. Clin Chem 1989;35:2054. [16] Takanami Y, Zwam H, Kawai Y et al. Vitamin E supplementation inhibits LDL oxidation during exercise in humans. Med Sci Sports Exerc 1994;53:261. [17] Robertson JD, Maughan RJ, Duthie GG, Morrice PC. Increased blood antioxidant systems of runners in response to training load. Clin Sci 1991;80:611–8.