Leukocyte activation, erythrocyte damage, lipid profile and oxidative stress imposed by high competition physical exercise in adolescents

Clinica Chimica Acta 306 Ž2001. 119–126 www.elsevier.comrlocaterclinchim

Leukocyte activation, erythrocyte damage, lipid profile and oxidative stress imposed by high competition physical exercise in adolescents Alice Santos-Silva a,b,) , Maria Irene Rebelo a,b, Elisabeth Molnar Bayer Castro a,b, Luis Belo a,b, Antonio ´ Guerra c , Carla Rego c , Alexandre Quintanilha b,d a

d

Departamento de Bioquımica da Faculdade de Farmacia ´ ´ da UniÕersidade do Porto, 4050 Porto, Portugal b Instituto de Biologia Molecular e Celular da UniÕersidade do Porto, 4150 Porto, Portugal c SerÕic¸o de Pediatria do Hospital de S. Joao ˜ do Porto, 4200 Porto, Portugal Departamento de Biologia Molecular do Instituto de Ciencias Biomedicas Abel Salazar da UniÕersidade do Porto, 4050 Porto, Portugal ˆ ´ Received 7 August 2000; received in revised form 22 January 2001; accepted 26 January 2001

Abstract Background: The aim of this study was to evaluate and to compare the lipid profile and the levels of leukocyte activation, red blood cell ŽRBC. damage and of oxidative stress in two groups of adolescents, with similar body mass index: high competition swimmers and adolescents practising moderate regular physical exercise. Methods: As markers of leukocyte activation, we measured plasma lactoferrin, elastase and granulocyte–monocyte colony stimulating factor. We studied RBC membrane band 3 profile and membrane-bound hemoglobin, as markers of RBC damage and aging; total and differential leukocyte count and RBC count, hematocrit, hemoglobin concentration and hematimetric indexes were also measured. Lipid profile included the evaluation of triglycerides ŽTG., total cholesterol ŽChol., high-density lipoprotein cholesterol ŽHDLc., low-density lipoprotein cholesterol ŽLDLc., apolipoproteins AI and B ŽApo AI and B., and lipoprotein Ža. ŽLpŽa... To evaluate oxidative stress, lipoperoxidation products and total antioxidant capacity were measured. Results: We found that high competition adolescents presented increased plasma levels of leukocyte activation products, increased RBC damage suggesting aging and premature removal, and higher oxidative stress. Lipid profile showed some risk and some protective changes. Conclusions: Our data suggest that high competition exercise, by imposing a higher and sustained oxidative and proteolytic stress, may contribute in the future to a higher risk of cardiovascular disease. We believe these findings warrant a reevaluation of current views in the intensity, duration and regularity of physical exercise, and that the evaluation of leukocyte activation products, RBC damage, oxidative stress and lipid profile may represent good markers to establish putative protective thresholds. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Physical exercise; Band 3 protein; Risk factors; Cardiovascular disease; Erythrocyte; Leukocyte activation

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Corresponding author. Departamento de Bioquımica, Facul´ dade de Farmacia, Rua Anıbal Cunha, 164, 4050-047 Porto, ´ ´ Portugal. Tel.: q351-22-2078906; fax: q351-22-2003977. E-mail address: [email protected] ŽA. Santos-Silva..

1. Introduction Moderate physical exercise has been accepted as a cardiovascular protector w1,2x, though the underlying

0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 0 6 - 5

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mechanism is not well established. For intense exercise some controversy about the protective effect still exists w3x. Atherosclerosis is very gradual in its development. It begins in the first or second decade of life and the clinical manifestations appear decades later. Atherosclerotic vascular lesions are commonly reported in coronary arteries and aorta before age 20 w4x. Besides the well known cardiovascular risk factors Žlipids and lipoproteins., epidemiologic studies suggest that increased blood viscosity, which is determined by physical conditions Žtemperature, blood flow and shear forces. and by the composition of blood Žhematocrit, leukocyte count., is related to cardiovascular risk and to atherogenesis w5–7x. Physical exercise triggers both an increase in blood flow and in blood viscosity. Actually, during physical exercise, red blood cells ŽRBCs. must deliver oxygen to tissues at a higher flow rate in a more viscous fluid, due to a reduced plasma volume and to a leukocytosis w8x. Other changes imposed by physical exercise, such as the rise in cellular metabolism and in hemoglobin ŽHb. turnover, may favour reactive oxygen species ŽROS. production within the RBC, activation of leukocytes and the development of oxidative stress w9,10x. Whenever ROS are produced outside the RBCs, resulting from leukocyte activation or from cellular metabolism, they are also able to diffuse across the RBC membrane enhancing the oxidative stress within the cell. Activated white blood cells ŽWBCs. are important sources of ROS and proteases, both of which may impose oxidative and proteolytic changes to plasma constituents and to neighbouring cells, such as circulating RBCs w11–18x. Leukocyte activation products are known to modify RBC membrane proteins altering their antigenicity, and to induce Hb damage, membrane lipid peroxidation or even hemolysis. The RBC, with a very limited biosynthesis capacity and poor repair mechanisms, whenever it is exposed to unusual physical or chemical stresses, may suffer and accumulate physical andror molecular modifications, which may underlie an accelerated senescence or even premature removal w19,20x. Band 3, a RBC transmembrane protein, known as the anion channel, seems to mark the RBC for death by triggering the binding of a specific auto-anti-band 3 antibody, and complement activation w18,21–27x. The antigenicity

of band 3 may result from its cleavage, clustering or even from exposure of unusual epitopes. Considering the modifications associated with physical exercise, it seems reasonable to wonder whether intense and sustained physical exercise, such as that performed in high competition sports, imposes enhanced levels of continuous oxidative and proteolytic stress. The aim of this study was to evaluate and to compare the lipid profile and the levels of WBC activation, of RBC damage and of oxidative stress, imposed by a high competition sport and by a moderate and regular physical exercise, such as that performed by adolescents in their school physical education classes. The latter group was used as a control, since in Portugal it is obligatory at this age to attend physical education classes. The lipid profile included triglycerides ŽTG., total cholesterol ŽChol., high-density lipoprotein cholesterol ŽHDLc., lowdensity lipoprotein cholesterol ŽLDLc., apolipoproteins AI ŽApo AI. and B ŽApo B. and lipoprotein Ža. ŽLpŽa..; as markers of WBC activation, we evaluated plasma lactoferrin, elastase and granulocyte– monocyte colony stimulating factor ŽGM-CSF. concentration; as markers of RBC aging or damage, we evaluated the band 3 profile Ž% of band 3 monomer, aggregates and fragments. and the membrane-bound hemoglobin Ž%MBH.; the hematologic study included the total and differential WBC count; RBC count; hematocrit ŽHt.; Hb concentration; hematimetric indexes—Mean Cell Volume ŽMCV., Mean Cell Hemoglobin ŽMCH., and Mean Cell Hemoglobin Concentration ŽMCHC.. To evaluate the oxidative stress, we measured the total plasma lipoperoxidation products and the total plasma antioxidant capacity.

2. Materials and methods 2.1. Subjects This study was performed in two groups of adolescents aged 12–16 years, including a similar number of males and females with similar body mass index ŽBMI.. One group included 40 high competition swimmers who trained more than 20 hrweek;

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the other group included 42 adolescents, exercising for 2–4 hrweek during physical education classes. 2.2. Collection and preparation of blood samples Blood samples were collected at rest and fasted for 12 h. The collection was performed 48 h after the last swimming train, or after the last physical education class. Whole blood ŽEDTA as anticoagulant. was used for haematologic procedures and to obtain plasma for biochemical assays Želastase, lactoferrin, GM-CSF, lipoperoxidation products and TAS.; blood serum was used to study the lipid profile. 2.3. Assays 2.3.1. Basic hematologic study WBC and RBC count, Ht and Hb concentration were measured by an automatic blood cell counter ŽAbx Micros 60.. Blood cell morphology and WBC differential count were evaluated in Wright stained blood films. 2.3.2. Band 3 profile and membrane bound-hemoglobin To prepare the membranes, we isolated RBCs by centrifugation; the cells were washed in PBS pH 7.4 and, afterwards, lysed according to the Dodge method w28x; membranes were washed in Dodge buffer Žthe first two washes used phenylmethylsulfonyl fluoride, a protease inhibitor, at a final concentration of 0.1 mM in Dodge buffer.. Protein concentration of the membrane suspension was determined w29x. MBH was measured spectrophotometrically, after protein

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dissociation with Triton X-100 Ž5% in Dodge buffer.; absorbance readings were made at 415 nm with correction at 700 nm; the obtained value and protein concentration were used to determine %MBH. Membranes of RBCs were treated with an equal volume of a solubilization buffer containing 0.125 M Tris–HCl pH 6.8, 4% sodium dodecyl sulfate ŽSDS., 20% glycerol, 10% 2-mercaptoethanol, heat denatured and subjected to polyacrylamide gel electrophoresis ŽSDS PAGE. Ž20 mg of protein per lane. using the discontinuous Laemmli system w30x. The slab gels were composed of a 9.0% separating gel and a 4.5% polyacrylamide stacking gel. Membrane proteins were electrophoretically transfered from SDS gels to nitrocellulose with a porosity of 0.2 mm w31x ŽSigma.. Additional reactive sites on the nitrocellulose were blocked by incubation in 3% gelatin and 0.1% Triton-X 100 dissolved in PBS pH 7.0 for 1 h with gentle rotation, at room temperature. Band 3 immunoblot was then performed w32x to determine whether erythrocyte band 3 was modified. The monoclonal anti-human band 3 used recognizes erythrocyte band 3 Ž90–100 kD. and a group of proteolytic fragments ŽFrag. w26x Žin the regions of 60, 40 and 20 kD.. Monoclonal antibodies anti-human band 3 ŽSigma. were added Ždilution 1:3000. and incubated for 4 h; the washing of the nitrocellulose was followed by the addition and incubation with anti-mouse IgG peroxidase-linked ŽVector. for 1 h Ždilution 1:4000.. Both incubations were carried out at room temperature and used PBS pH 7.0, containing 0.1% detergent and 0.5% gelatin, to prepare the dilutions of the antibodies. The washes used the same buffer without gelatin. The immunoblot was developed by

Table 1 Total and differential WBC count, plasma concentration of elastase, lactoferrin and GM-CSF. Mean value " S.D. found for adolescents and for adolescent swimmers are presented and compared Adolescents WBC Ž=10 9rl. Granulocytes Ž=10 9 rl. Lymphocytes Ž=10 9 rl. Monocytes Ž=10 9 rl. Elastase Žmgrl. Lactoferrin Žngrml. GM-CSF Žpgrml.

Adolescent swimmers

P-value

6.30 " 1.22 4.04 " 1.15 1.87 " 0.52 0.43 " 0.15

6.35 " 1.29 3.53 " 1.31 2.52 " 0.68 0.41 " 0.09

ns ns - 0.001 ns

44.95 " 19.92 555.5 " 147.2 0.061 " 0.088

73.09 " 68.50 621.8 " 380.5 0.241 " 0.324

- 0.05 ns ns

A. Santos-SilÕa et al.r Clinica Chimica Acta 306 (2001) 119–126

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Table 2 Mean value"S.D. for total RBC, Ht, Hb, MCV, MCH and MCHC, for adolescents and for adolescent swimmers are presented and compared

RBC Ž=10 12 rl. Hb Žgrdl. Ht Žlrl. MCV Žfl. MCH Žpg. MCHC Žgrdl.

Adolescents

Adolescent swimmers

P-value

4.83"0.35 14.8"1.7 0.41"0.03 84.4"2.9 30.8"3.1 36.3"3.0

4.75"0.34 14.1"0.9 0.41"0.03 86.3"3.8 29.7"1.3 34.4"0.8

ns - 0.05 ns - 0.05 ns - 0.01

the addition of hydrogen peroxide and horseradish peroxidase color developer reagent. The band 3 profile was quantified by densitometry ŽCybertech CS1.. 2.3.3. Lipid profile Serum Chol and TG were measured enzymatically using commercially available kits ŽBoehringer.. HDLc was removed from the supernatant following precipitation of the other lipoprotein fractions with phosphotungstic acid and magnesium chloride. LDLc concentration is given by the difference between total Chol and the free Chol remaining in the supernatant following LDL precipitation with polyvinylsulphate. Apo AI and Apo B were determined tur-

bidimetrically with specific antisera ŽRoche.. LpŽa. was evaluated by electroimmunodiffusion in agarose gel ŽHydragel LpŽa., Sebia.; anti apo Ža. monospecific antibody was incorporated in the agarose gel; the calibration was performed using standard sera calibrated to LpŽa.. Samples and standards migrate into the gel and give immunoprecipitation rockets with a height proportional to LpŽa. concentration. 2.3.4. Leukocyte actiÕation Plasma concentration of polymorphonuclear elastase, lactoferrin and GM-CSF were evaluated by enzyme immunoassays ŽPMN Elastase IMAC immunoassay, Merck; Bioxytech lacto f enzyme immunoassay, Oxis International; Human GM-CSF Quantikine HS, R & D Systems, respectively.. 2.3.5. OxidatiÕe stress We used a colorimetric assay to evaluate total antioxidant status ŽTAS, Randox Laboratories, UK.. Plasma lipid peroxidation was measured by the thiobarbituric acid ŽTBA. assay w33x. 2.4. Statistical analysis All measurements are expressed as mean value " standard deviation ŽS.D... For statistical analysis, we

Fig. 1. Immunoblots for band 3 of adolescents and adolescent swimmers are shown. Monoclonal anti-human band 3 used recognizes erthrocyte band 3, a 90–100 kD protein, its aggregates ŽHMWAg. and proteolytic fragments Ž60, 40, 20 kD..

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used the SPSS package. To evaluate the differences between groups, we used the Student’s t-test for the parameters presenting a gaussian distribution, and the Mann–Whitney test for those presenting a nongaussian distribution. A P-value lower than 0.05 was considered statistically significant.

3. Results 3.1. White blood cells and neutrophil actiÕation The total and the differential WBC count, and the concentration of some of their degranulation products, presented by the two groups, are shown in Table 1. We found similar values for total WBC count; however, the differential WBC count showed significantly higher values of lymphocytes in swimmers Ž P - 0.001.. Concerning elastase, lactoferrin and GM-CSF, we found that the mean values are higher in swimmers, though only elastase presented a statistically significant rise Ž P - 0.05.. 3.2. RBC damage Table 2 presents RBC count, Ht, Hb concentration and the hematimetric indexes for both groups. No significant differences were found for total RBCs, Ht and MCH. However, the swimmers’ RBCs are larger and with less Hb concentration per cell, as shown by the significant higher values of MCV Ž P - 0.05. and by the significant lower values of MCHC Ž P - 0.01.. Immunoblots for band 3 are shown in Fig. 1; the band 3 profile Ž% of each band. and the %MBH for Table 3 Band 3 profiles found for adolescents and for adolescent swimmers, quantified by densitometry on immunoblots, and the %MBH are presented and compared Mean values"S.D. of each band Ž% of the total bands.. Adolescents

Adolescent swimmers

P-value

HMWAg Band 3 Total frag

12.8"6.8 56.4"10.0 30.8"10.5

22.2"11.8 59.8"9.7 18.0"8.6

- 0.001 ns - 0.001

%MBH

287"73

356"173

- 0.05

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Table 4 Lipid profiles Žmean values"S.D. for TG, Chol, HDLc, LDLc, Apo AI, Apo B and Lp Ža.. found for adolescents and for adolescent swimmers are presented and compared

TG Žmgrdl. Chol Žmgrdl. HDLc Žmgrdl. LDLc Žmgrdl. Apo AI Žmgrdl. Apo B Žmgrdl. Lp Ža. Žmgrdl.

Adolescents

Adolescent swimmers

P-value

66.5"24.6 149.8"26.1 48.8"11.4 84.2"5.7 184.2"34.6 111.4"22.6 0.216"0.133

63.1"16.0 164.4"29.1 51.8"12.0 95.7"24.6 128.4"14.8 74.7"12.2 0.317"0.280

ns - 0.05 ns - 0.05 - 0.001 - 0.001 ns

both groups are presented in Table 3. Swimmers presented higher values of high molecular weight aggregates ŽHMWAg. Ž P - 0.001. and lower values of total Frag Ž P - 0.001.. No significant difference was found for band 3 monomer. Swimmers presented significantly higher %MBH Ž P - 0.05.. 3.3. Lipid profiles Lipid profiles of both groups are shown in Table 4. Swimmers presented some risk changes, namely, a significant rise in Chol and LDLc Ž P - 0.05. and a significant decrease in Apo AI Ž P - 0.001.; a rise in LpŽa. and a reduction in TG were also observed, though not statistically significant. We found also protective changes, namely, a significant reduction in Apo B Ž P - 0.001. and a rise in HDLc. 3.4. OxidatiÕe stress Table 5 shows the values of TBA, TAS and the balance between them ŽTBArTAS.. Swimmers pre-

Table 5 Lipid peroxidation and total antioxidant status Žmean values" S.D.. in adolescents and in adolescent swimmers are presented and compared

TBA Ž=10y3 mM. TAS ŽmM. TBArTAS

Adolescents

Adolescent swimmers

P-value

1.34"0.21

1.84"0.29

- 0.001

1.57"0.08 0.84"0.14

1.57"0.04 1.17"0.19

ns - 0.001

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sented significantly higher values of TBA and of TBArTAS Ž P - 0.001.. No significant difference was observed in TAS. 4. Discussion Leukocytosis is a striking and consistent change associated with exercise w34x and is mainly due to a rise in neutrophils. The magnitude of this rise is determined by exercise intensity and duration. High WBC values are sustained for several hours, returning afterwards to preexercise levels or to even lower values. To exclude this immediate effect of exercise, we collected blood 48 h after the last physical exercise bout in both groups. We found ŽTable 1. that total WBC, granulocytes and monocytes were similar in both groups. Only lymphocytes were significantly higher in swimmers. These results suggest a different response of WBCs to different levels of exercise. Though the absolute values of WBC were similar, the study of leukocyte activation products showed different values in the two groups. Elastase was significantly higher in swimmers; lactoferrin and GM-CSF showed also a trend to rise. Considering that the adolescent swimmers practice physical exercise more than 20 hrweek Žless than 48 h between each training bout., we could assume that this kind of physical exercise imposes chronically increased levels of WBC activation products, namely, ROS andror proteases, which are able to damage vascular endothelium w35–37x or even other neighbouring cells, such as the RBCs w18x. According to the oxidative modification hypothesis, ROS contribute to the inception and progression of atherosclerotic lesions by favouring infiltration and accumulation of lipids in the subendothelial space. ROS produced by smooth muscle cells, endothelial cells or even by macrophages, may oxidatively modify LDL w38x, which when oxidised is chemotactic for circulating monocytes, inhibits the motility of macrophages and is more avidly taken by macrophages than native LDL. Therefore, it promotes the recruitment of circulating monocytes, accumulation of resident macrophages, accelerated uptake by macrophages and, finally, the formation of Afoam cellsB and fatty streak. During exercise, bodily oxygen consumption is greatly increased and it seems likely that more ROS

are produced in vivo, due to WBC activation, to increased cellular metabolism and to increased Hb turnover. Actually, we found in swimmers a significant rise of TBA and a significant imbalance of TBArTAS, denoting a higher oxidative stress in this group ŽTable 5.. Considering that most ROS are able to diffuse through the RBC membrane w10x and that RBC has limited repair mechanisms, it is reasonable that the oldest RBCs may not overcome the oxidative stress developed within the cell; the youngest RBCs may develop andror accumulate oxidative lesions. Proteases produced by WBC may also impose proteolytic modifications to RBC membrane. Thus, we could assume that the increased oxidative and proteolytic stress occurring during physical exercise may promote an accelerated RBC senescence or even a premature removal of the oldest RBCs. We found that ŽTable 2. swimmers presented lower values of total RBC and Hb, suggesting that, actually, intense exercise may trigger a premature removal of the oldest RBCs. In swimmers, the RBCs were larger, as shown by a higher MCV, and this may result from a rise in reticulocytes, which are larger than mature RBC, andror from the removal of the oldest RBCs, which are known to be smaller in volume. To evaluate the oxidative and proteolytic RBC damage, band 3 profile and MBH were evaluated. As Hb denatures, it binds to the intracellular surface of the membrane, leading to band 3 aggregation w23x. We found ŽTable 3. that swimmers presented significantly higher values of HMWAg and significantly lower values of Frag of band 3, suggesting that oxidative modifications are enhanced in swimmers, as shown by the rise in HMWAg and, also, in MBH. The reduction in Frag does not exclude the possibility of proteolytic stress. The production of Frag with a molecular weight under the cutoff of the nitrocellulose membrane would be lost from the immunoblot. Actually, the immunoblot ŽFig. 1. clearly suggests that proteolysis seems particularly relevant. The HMWAg band seems to increase only slightly its absolute value, while the bands of Frag clearly show a reduction in value. Therefore, the increase in %HMWAg and %band 3 monomer may, in part, arise from the absolute reduction of Frag. These fragments may cross-link, contributing to the HMWAg rise, andror, by further proteolysis, produce fragments that are lost during electrophoretic

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protein transfer. We must emphasize that similar changes in band 3 profile were reported in pathologies known as oxidative stress conditions, such as cardiovascular disease w32x, and in pathologies associated with accelerated RBC aging, such as hereditary spherocytosis w39,40x. A similar band 3 profile was found in vitro after incubation of RBCs with activated neutrophils, with elastase, and in older RBCs w18x. In lipid profile ŽTable 4., though all the values were within the normal ranges, we found in swimmers some risk changes, namely, significantly higher values for Chol and for LDLc and significantly lower values for Apo AI. Curiously enough protective changes were also observed, such as a significantly lower value of Apo B and a trend to rise in HDLc, and to reduce in TG. Our data seem to be, in part, in agreement with literature, since a decrease in TG and an increase in HDLc are reported as the major effects in lipoprotein metabolism of regular exercise, whatever its intensity w3x. We must emphasize that the higher values of LDLc and of oxidative stress may become a dangerous association, considering that the regularity and intensity of this kind of physical exercise seems to contribute to higher sustained values. Our data show that adaptation to intense physical exercise, such as that performed in high competition, seems to be associated with chronically increased plasma levels of WBC activation products, which seems to lead to an accelerated RBC aging or even to its premature removal. Considering that ROS and proteases are able to injure vascular endothelium it seems reasonable that high competition, by sustaining high levels of ROS, proteases, Chol and LDLc, could lead to the development of premature atherosclerotic lesions. Moreover, during physical exercise all the parameters are expected to rise as a result of hemoconcentration; therefore, endothelial injury and lipid infiltration are probably enhanced during that performance, specially in areas of abnormal shear stress w41x. Our data suggests that high competition physical exercise, by imposing a higher and sustained oxidative and proteolytic stress, may contribute in the future to a higher risk of cardiovascular diseases. However, it seems to lead to some protective changes, namely, to a removal of older RBCs, which are less

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efficient in the transfer of needed oxygen, to a reduction in Apo B and TG, and to a rise in HDLc. We suggest that in considering high competition exercise, a threshold may be found in the intensity, duration and regularity of physical exercise that, whenever it is exceeded, could lead to deleterious effects in terms of atherogenesis. We believe these findings warrant additional studies and that plasma levels of leukocyte activation products and the evaluation of erythrocyte damage and lipid profile may represent good markers to establish those protective thresholds.

Acknowledgements This study was in part supported by grants from the AFundac¸ao ˜ Gomes TeixeiraB, the Oporto University and Praxis XXI no. 2r2.1rSAUr1240r95 ŽJNICT.. The authors are greatful to Dr. Laura Pereira for the technical support.

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