OArciugitnea hl ePpaapteicr response to diet modification Biol. Sport 2010;27:111-118 ACUTE HEPATIC RESPONSE TO DIET Accepted for publication MODIFICATION AND EXERCISE-INDUCED 3.02.2010 ENDOTOXEMIA DURING A LABORATORY- BASED DUATHLON AUTHORS: Moncada-Jiménez J.1, Plaisance E.P.2, Araya-Ramírez F.3, Taylor J. K.4, Ratcliff L.5, Reprint request to: Mestek M.L.6, Grandjean P.W.7, AragonVargas L.F.1,8 José Moncada-Jiménez,P.O.Box 239-1200 (Pavas) San Jose, Costa Rica 1School of Physical Education and Sports, University of Costa Rica, Costa Rica Tel. +506 8857-5738 2Department of Anatomy, Physiology & Pharmacology, Auburn University, AL Fax. +506 2225-0749 3Department of Kinesiology, Auburn University, AL E-mail: jose.moncada@ucr.ac.cr 4Division of Clinical Laboratory Science, Auburn University-Montgomery, AL 5Department of Health and Human Performance, University of Central Missouri, MO 6Integrative Vascular Biology Laboratory, University of Colorado at Boulder, CO 7Department of Kinesiology, Auburn University, AL 8The Gatorade Sports Science Institute, Barrington, IL ABSTRACT: The purpose of the study was to compare the acute hepatic response to diet modification and exercise-induced endotoxemia, and to determine if associations exist between liver damage markers, body core temperature, and IL-6 responses to a laboratory-based duathlon. Eleven moderately-trained healthy males followed a low-carbohydrate (CHO) and a high CHO diet to change their glycogen stores two-days before completing a duathlon. Blood samples were obtained at rest, immediately after and 1- and 2-h following the duathlon for determination of endotoxin-lipopolysaccharide binding protein (LPS-LBP) complex, IL-6, and liver integrity markers AST, ALT, and AST/ALT ratio. Hydration status and body core temperature were assessed at rest, during, and after the duathlon. Athletes were more dehydrated and had higher AST/ALT ratios in the low- compared to the high-CHO diet trial regardless of the measurement time (p<0.05). IL-6 increased from resting to immediately after, 1- and 2-h following duathlon regardless of the diet (p<0.05). A higher LPS-LBP complex concentration was observed from rest to immediately after the duathlon. No significant correlations were found between LPS-LBP complex levels and body core temperature. In conclusion, athletes on a low-CHO diet showed higher hepatic structural damage and finished more dehydrated compared to athletes on a high-CHO diet. Body core temperature and LPS-LBP complex levels were unrelated beyond the increase in body core temperature explained by exercise. No significant associations were found between body core temperature, IL-6 and LPS-LBP complex concentrations. KEY WORDS: human, cytokines, lipopolysaccharide, inflammation, exercise INTRODUCTION The gastrointestinal system is not only responsible for nutrient The mechanism for cytokine activation involves binding of LPS absorption, but also for hosting beneficial bacteria. A failure to keep to serum lipopolysaccharide-binding protein (LBP) to form the LPS- bacteria within the intestines will produce a state called endotoxemia LBP complex [62]. LBP is an acute-phase protein synthesized in in which bacterial lipopolysaccharides (LPS) or endotoxin translocates hepatocytes considered an opsonin (i.e., can enhance the uptake of to reach the liver. LPS translocation might occur when factors such bacteria by phagocytic cells) for activation of macrophages (e.g., as a reduction in splanchnic blood flow, ischemia, long stays at Kupffer cells) by LPS [38,64]. LPS-LBP complex catalyzes the transfer altitude, or increased body core temperature affect the integrity of of LPS to membrane-bound soluble CD14, and is considered a marker the intestinal wall [23,63]. Indeed, splanchnic blood flow decreases of bacterial translocation, transport, and clearance [17]. in an inversely proportional manner in relation to the percentage of Once the LPS binds to LBP, the intracellular signal transduction maximal oxygen consumption (VO2max) achieved during exercise occurs that leads to a proinflammatory response mediated by [20]. LPS elicits a cytokine-mediated pro-inflammatory response cytokines such as interleukin-6 (IL-6) released from macrophages that can eventually evolve in sepsis and heat stroke. [62]. Therefore, a direct association is hypothesized between Biology of Sport, Vol. 27 No2, 2010 111 Electronic PDF security powered by www.IndexCopernicus.com - - - - - Moncada-Jiménez J. et al. LPS-LBP complex and IL-6 [65]. Others have reported subclinical drugs (NSAID). Volunteers were not allowed to participate if they increases in circulating pro-inflammatory IL-6 during strenuous had anemia, any gastrointestinal disorders or other chronic disorders. exercise [27,60]. Participants were also excluded if they had a cardiac pacemaker or Endurance and resistance training exercise also promote changes other implanted electromedical device, were current cigarette in liver damage markers in humans, as previously reported during smokers, had an acute or chronic illness or infection, food allergies, cycling and running competitions [10,13,35,41,49,52,57]. or any vaccinations within the previous two-week period. The chronic effect of exercise in the detoxifying ability of the liver Liver energy manipulation and exercise intervention. Liver energy has been studied in animal models where rats who trained for stores were modified by diet and exercise as described elsewhere 10-weeks showed lower hepatic damage than their sedentary [36]. Briefly, two isoenergetic diets were designed for each counterparts [9]. This evidence in an animal model clearly suggests participant, a glycogen depletion diet or low carbohydrate (CHO) a training adaptation of the liver in its ability to detoxify noxious (low-CHO) and a diet high in CHO (high-CHO). The nutrient content substances such as LPS. How these findings might be extrapolated was significantly different for CHO and fat content for both diets. to humans is debatable and subject to research. Diets were prepared of commercially available pre-packaged foods In spite of the evidence showing that the liver potential to and given to each participant 72-h before an exercise trial taking metabolize and clear different drugs is reduced in the presence of into consideration allotments for breakfast, lunch, dinner, and LPS [61], the precise mechanisms involved are poorly understood. snacks. We suggest a hypothesis that explains that one of such mechanisms Forty-eight hours before completing a duathlon, participants on might be the initial endogenous energy supply of the liver since LPS the high-CHO diet were instructed to return to the laboratory to clearance is an energy-dependent process carried out by the liver complete a 60-min sub-maximal (70% VO2max) jog on the treadmill, resident macrophages, the Kupffer cells. A similar hypothesis was and 24-h before the duathlon, participants were required to rest. studied in a rat model where fasting reduced or deactivated temporarily During the 48-h in which participants consumed the low-CHO diet, the activity of the Kupffer cells [55]. However, the study was they were required to run on a treadmill for 60-min (70% VO2max) conducted using an animal organ donor model, and not a human two-days before the duathlon and 45-min at the same intensity on exercise-induced hepatotoxicity model. the day prior to the duathlon. Then, participants were instructed to Therefore, the purpose of this study was to compare the acute rest (i.e., no extra exercise) the day before the duathlon. hepatic response to initial endogenous hepatic energy levels and Duathlon. On the evening before the experimental session, exercise-induced endotoxemia, and to determine if associations exist participants were reminded to ingest a silicon-coated pill (HQ Inc., between liver damage markers, body core temperature, and IL-6 Palmetto, FL, USA). This pill was used as the sensor for determining responses to a laboratory-based duathlon. Our main hypothesis was core temperatures during the experimental duathlon. The sensor that endogenous liver energy stores, as manipulated by diet and was factory-calibrated and was designed to be ingested easily and endurance exercise, would elicit a differential acute response in voided with normal bowel movements within 48-h [31]. markers of liver damage and the LPS-LBP complex-mediated IL-6 On the day of the duathlon participants arrived at the laboratory, response. returned empty food packages and voided their bladders before body weight was measured. Then, they were instructed to sit quietly MATERIALS AND METHODS for 5-min. Next, a fasting blood sample was obtained. Following Subjects. Eleven moderately-trained healthy males the initial blood draw, participants were provided with a standardized (age=36.64±4.95 yr; VO2max=57.36±7.41 ml/kg/min; breakfast to eat before resting in a comfortable chair. After height= 1.74±0.06 m; weight=74.47±7.66 kg; DEXA body the 60-min the rest period, participants had 10-min to warm up fat=17.22±6.63%; fat-free mass=61.42±5.85 kg) participated and then started the duathlon in the following order: a) treadmill in the study. They trained for middle and long distance events such run of 5-km (Run-1); b) 30-km stationary cycle (Bike); and c) as marathon and triathlon on an average of 11 h•wk-1, including 10-km treadmill run (Run-2). The subjects ran at 0% grade and running on average 13 km•wk-1 and cycling 24 to 40 km•wk-1. were allowed to modify only the treadmill speed. For the cycling Written informed consent was obtained from each subject prior to part of the race, participants had previously attached their own participation, and the Institutional Review Board from Auburn bicycles to a CompuTrainer™ (Racer Mate, Inc., Seattle, WA, USA). University approved the study. Based on the IL-6 response to exercise During the duathlon the participants were given the opportunity to from previous studies [6,14], and using the nQuery Advisor® drink chilled water ad libitum; solid foods were avoided at all times. statistical sample size software, it was estimated that a sample size Total volume of ingested liquid was measured for further analyses of 10 would have a power (β =0.80) to detect an effect size of 0.996 of hydration status. Body temperature was monitored during each with a 0.05 significance level [43]. stage of the duathlon and VO2 was also determined seeking to Protocol. The protocol initially involved screening with a health assure an exercise intensity of >70% VO2max. Once the experimental history questionnaire for current use of nonsteroidal anti-inflammatory session was completed, the subjects were provided with rehydration 112 Electronic PDF security powered by www.IndexCopernicus.com - - - - - Acute hepatic response to diet modification fluids, fruit, and an appointment for the next visit to the laboratory. IN/GL ratio in the low-CHO Diet (0.27±0.10) was lower (p≤0.05) Experimental exercise sessions were separated by at least 7 days. than the mean ratio on the high-CHO diet (0.39±0.20), indicating Blood sampling and analytical procedures. Antecubital venous athlete’s compliance to the dietary regimen and a change in blood samples were obtained at rest, immediately after and again endogenous hepatic energy status. 1- and 2-h after the duathlon. Blood samples were obtained for Hydration status, body core temperature and duathlon determination of hematocrit (Hct) and hemoglobin (Hb). Another performance. Analysis of hydration status indicated a higher fluid blood sample was obtained, allowed to clot, centrifugated at 1500g loss (3978.4±1222.4 vs. 3341.8±1235.6 ml) and dehydration for 15-min to prepare serum aliquots to be stored at -80°C for further (-1.6±1.2 vs. -1.0±1.4%) after the low-CHO diet trial compared analysis of hepatocyte integrity markers aspartate aminotransferase to the high-CHO diet trial (p=0.001). Body core temperature analyses (AST) and alanine aminotransferase (ALT) (Flex®Dimension®, Dade did not indicate an interaction effect between diet and measurement Behring Inc., Deerfield, IL, USA). Enzyme linked-immuno-sorbent times (p=0.626). In general, body core temperatures increased from assay (ELISA) kits were used for determination of insulin (IN), resting to the different segments of the race and post-exercise period glucagon (GL) (LINCO, St. Charles, MO, USA), LPS-LBP complex, (p≤0.001) regardless of the diet consumed (Fig. 1). No differences and a high-sensitivity human IL-6 (HsIL-6) (Cell Sciences, Inc., in mean performance time in the total duathlon were observed during Canton, MA, USA). Plasma concentration of IL-6 and LPS-LBP were the low-CHO diet (136.38±20.09 min) and the high-CHO corrected to take into consideration changes in plasma volume due (134.88±20.89 min) diet. Regardless of the dietary trial, the subjects to exercise [11]. performed the duathlon at an intensity of 71.1±2.0% of their Statistical analysis. Data were analyzed with the Statistical individually determined VO2max. Package for the Social Sciences (SPSS®), version 15.0 for Windows. Liver damage markers. Biochemical and immunological variables Descriptive data are presented as means (M) and standard deviation measured in this study are presented in table 1. Repeated measures (± SD), and statistical significance was set a priori at p≤0.05. Paired ANOVA indicated no significant interaction between diet and samples t tests were used to determine significant mean differences measurement time in the AST, ALT, and/or the AST/ALT ratio (p>0.05). between experimental conditions in the dependent variables IN/GL For AST, the main effect diet showed significant mean differences in ratio, performance time in the duathlon, diet composition, and the low- compared to the high-CHO diet (39.23±3.52 vs. hydration status. Factorial 2 x 4 (diets x time points) repeated 29.40±1.75 U/L; p=0.007), whereas the main effect measurement measures analyses of variance (ANOVA) were computed to analyze AST, ALT, the AST/ALT ratio, LPS-LBP complex, and IL-6. Body core temperature was analyzed by a 2 x 7 (diets x time points) factorial, repeated-measures ANOVA. Percentage VO2max was analyzed by a 2 x 3 factorial, repeated-measures ANOVA. For all ANOVA tests, appropriate follow-up analyses were computed if significant interactions and/or main effects were found. Finally, a Pearson product-moment correlation was calculated between body composition and IL-6. RESULTS Diet and endogenous hepatic energy change. The two diets had p<0,05; c +2-h < a< c +1-h < b similar energy content (~11 MJ); however, the low-CHO diet provided FIG. 1. BODY CORE TEMPERATURE DURING A DUATHLON. VALUES significantly more fat (67%) and less CHO (21%) than the high-CHO ARE COLLAPSED IN SINGLE COLUMNS SINCE NO DIFFERENTIAL diet (25% fat, 63% CHO) (p<0.001). The protein content from total EFFECTS BETWEEN DIETARY CONDITIONS WERE OBSERVED. BROKEN LINES REPRESENT UPPER AND LOWER LIMITS FOR NORMAL RESTING energy for both diets was ~11% (p>0.05). The mean (SD) fasting BODY TEMPERATURE FOR ADULTS[33]. VALUES ARE MEANS ± SEM. TABLE 1. BIOCHEMICAL AND IMMUNE PLASMA MARKERS FOR THE EXPERIMENTAL CONDITIONS (M ± SD). Low-CHO diet High-CHO diet R I + 1 h + 2 h R I +1 h +2 h ALT (U/L)a 35.68 ± 5.54 37.05 ± 7.28 34.09 ± 6.37 34.05 ± 7.85 32.09 ± 5.36 34.50 ± 7.02 32.77 ± 7.06 33.61 ± 6.27 AST (U/L)a 31.82 ± 9.90 40.81 ± 11.56 39.97 ± 11.72 40.54 ± 12.50 24.41 ± 4.90 30.80 ± 6.37 29.97 ± 6.34 31.79 ± 6.33 LPS-LBP (pg/ml)a 4.59 ± 1.72 5.91 ± 4.03 4.92 ± 3.14 4.05 ± 1.91 3.84 ± 1.66 4.43 ± 2.10 3.67 ± 1.77 4.07 ± 0.85 IL-6 (pg/ml)a 0.82 ± 0.53 9.23 ± 7.38 5.84 ± 4.40 3.99 ± 3.52 0.73 ± 0.56 8.05 ± 5.19 5.77 ± 3.25 3.58 ± 1.78 Legend: R = resting; I = immediately after duathlon; + 1h = 1-h following duathlon; +2 h = 2-h following duathlon. a - p > 0.05, for interaction effects (diet condition X measurement time). Significant main effects are presented in the results section. Biology of Sport, Vol. 27 No2, 2010 113 Electronic PDF security powered by www.IndexCopernicus.com - - - - - Moncada-Jiménez J. et al. A * - p<0,05 A * - p<0,05 main effect measurement time from resting B * - p<0,05 from resting B FIG 2A-B. MAIN EFFECT ANALYSES SHOWS: FIG 3A-B. LPS-LBP COMPLEX ACUTE RESPONSE FOLLOWING A) AST/ALT RATIO FOLLOWING A LOW- AND A HIGH CHO DIET, TWO DIETARY CONDITIONS. PANEL A) SHOWS THE INTERACTION B) THE RELATIVE CHANGE (∆%) FROM RESTING FOLLOWING BETWEEN DIETARY CONDITIONS AND MEASUREMENT TIME. A DUATHLON. MAIN EFFECT MEASUREMENT TIME WAS SIGNIFICANTLY DIFFERENT FROM RESTING (p<0.05). BROKEN LINES REPRESENT CUT-OFF POINT FOR MILD ENDOTOXEMIA. PANEL time indicated significantly higher mean values immediately, 1- and B) SHOWS RELATIVE CHANGE (∆%) IN LPS-LBP COMPLEX FROM 2-h following exercise compared to resting values (p<0.001). RESTING FOLLOWING A DUATHLON. For ALT, a significant measurement time main effect was observed, with baseline values lower than immediately after exercise (p=0.010). No significant differences were found between immediately, 1- and 2-h following exercise in ALT values. For AST/ALT ratio, the main a 5 pg/ml [51], was found at baseline in three participants (27%) on the low-CHO diet compared to only one (9%) in the low and the high-CHO diets had endotoxemia. Finally, 2-h participant on the high-CHO diet. However, endotoxemia was following exercise, endotoxemia was found only in one athlete in the observed in six subjects (55%) immediately following exercise in the low- and the high-CHO diets, respectively. The highest LPS-LBP low-CHO diet, compared to five subjects (45%) on the high-CHO complex concentration found in a participant was 16.7 pg/ml diet. One-hour after exercise, four (36%) and three (30%) subjects immediately following exercise in the low-CHO diet condition. 114 Electronic PDF security powered by www.IndexCopernicus.com - - - - - Acute hepatic response to diet modification No interaction effect between diet and measurement time on IL-6 complex concentration. This exercise intensity caused bacterial was observed (p>0.05). Main effect measurement time showed an translocation immediately following exercise similar to that reported IL-6 concentration increased from baseline to immediately following in marathon and ultra-endurance events [3,4,6,7,27,44]. duathlon, which remained elevated 1- and 2-h after the exercise The increased LPS-LBP complex marker indicated a higher sessions (Fig. 4). Pearson-product moment correlations in the low- transmission of cellular signaling capable of eliciting an increased CHO diet 2-h following exercise indicated an inverse correlation cytokine response. This finding is similar to previous research in between fat-free-mass and IL-6 (r=-0.645; p=0.032). Also, in the sedentary and athletic populations [20,46,47]. high-CHO diet 2-h following exercise, an inverse correlation was We expected to find a positive correlation between LPS-LBP found between fat-free-mass and IL-6 (r=-0.653; p=0.041). complex levels and body core temperature since endotoxins are pyrogenic agents capable of eliciting a febrile-like response [48]. No DISCUSSION significant correlation coefficients were found between core The primary findings of this study were a higher dehydration level temperature and endotoxin translocation levels measured immediately, and a mild increase in markers of hepatic damage in the low-CHO 1- and 2-h following exercise. We suggest that the pyrogenic effect diet compared to the high-CHO diet following a duathlon. We found of the increased endotoxemia was not significantly greater than the an acute exercise-induced mild endotoxemia in both dietary expected increase in body temperature associated with exercise (body conditions and did not find changes in physical performance or core temperature > 37.8°C). The highest mean core temperature IL-6 response when initial liver energy status was altered by diet recorded in our participants was 40.69°C in the high-CHO diet and exercise before a duathlon. condition who completed the run-2 (10 km) in more than 60 min. The main energy-requiring processes carried out by the liver are This figure was slightly below the proposed 41.0°C and 42.0°C shown ureagenesis, futile cycling of substrates, gluconeogenesis, protein to impair physiologic functions in humans [32]. LBP is produced in synthesis, and ketogenesis [37]. We hypothesized that under a low- hepatocytes under IL-6 stimulation to capture and present LPS to CHO diet, hepatic glycogen stores and available energy would be CD14 and induce the secretion of IL-6 [26]. Therefore, we expected reduced and therefore hepatocellular clearance of endotoxins impaired, to find a correlation between LPS-LBP complex and IL-6; however, as suggested by animal models where hepatocyte shrinkage and we were unable to demonstrate a significant correlation in either damage occurred following intense exercise [18,28,30,55]. dietary conditions up to 2-h following exercise. Thus, in the low-CHO diet trial, we expected to find a higher Immune cytokines such as IL-6 have very high metabolic rates hepatocellular damage following exercise compared to the high-CHO [5], and therefore fuel availability before, during and after exercise diet trial. Since LBP is produced by hepatocytes [59], we expected in is of greater concern for athletes. Others [21] have found that the low-CHO diet to find an inverse correlation between LPS-LBP and compared to high-CHO diets, low-CHO diets significantly impaired ALT, a serum transaminase more specific to hepatocellular injury [24]. immune response (e.g., higher cortisol release, neutrophilia, In the low-CHO condition, LPS-LBP complex values were consistently leucocytosis, neutrophil:lymphocyte ratio, and IL-6; and lower plasma higher than in the high-CHO trial at virtually all sampling times from glutamine) following 1-h of cycle ergometer exercise performed at resting up to the 2-h following exercise; however, no statistical 70% of their individual VO2max. A very low-CHO diet implies a very differences or significant associations with ALT were observed. high-fat dietary content, which has been shown detrimental to The AST/ALT ratio is considered a surrogate marker for hepatocyte immune function. We expected to find an increase in IL-6 from necrosis and inflammation, with values >1.0 suggestive of hepatocyte baseline following exercise in both dietary conditions, as previously damage [1]. In this study, the AST/ALT ratio was > 1.0 immediately reported in measurements taken after light, moderate and hard following exercise and in the recovery phase (i.e., 1- and 2-h post- intensity exercise performed for short- and long-periods of time exercise), indicating hepatocyte and liver parenchyma structural [2,7,12,22,27,34,40,51]. In our study, IL-6 increased significantly damage. ALT increased from baseline to immediately following from baseline immediately following the duathlon regardless of the exercise, indicating a mild hepatocyte damage possibly explained by diet, and remained elevated 1- and 2-h following exercise. This finding a combination of factors such as exercise intensity, duration, and is consistent to figures shown by marathon runners and athletes reperfusion to the liver following exercise [28]. However, even in running in a treadmill for 2.5-h [40,45]. the presence of a reduced hepatic blood flow induced by intense In the present study, our athletes ran at approximately 70% of exercise, the liver is able to maintain its metabolic functions [39]; their individual VO2max and were allowed to drink water ad libitum therefore, using the AST/ALT as a true marker of hepatic damage until the last blood sample was drawn 2-h after the duathlon. during exercise may be equivocal [49]. We reported an 11-fold increase in IL-6 from baseline that also steadily In the present study, the mean exercise intensity (~70% VO2max) decreased 7- and 5-fold 1-and 2-h following exercise elicited by the subjects during the duathlon in both dietary conditions (Fig. 4). Others have found 22-fold increases from baseline immediately was high enough to cause intestinal permeability and bacterial and 1-h following 100-min of cycling exercise at 70% VO2max translocation from resting as demonstrated by the increased LPS-LBP followed by a time-trial test[8]. A mild dehydration (1.6% body mass) Biology of Sport, Vol. 27 No2, 2010 115 Electronic PDF security powered by www.IndexCopernicus.com - - - - - Moncada-Jiménez J. et al. was found in the low-CHO trial compared to the high-CHO trial that low-CHO diets, defined as those having ≥30% fat of total energy, did not influence performance. significantly reduced pre-exercise muscle and hepatic glycogen In this study we determined body composition by dual energy content and might impair exercise performance, yet we did not find X-ray absorptiometry (DEXA) method. A direct correlation between differences in duathlon performance times between the experimental FFM and IL-6 has been hypothesized [15,48], based on trials. the contention that skeletal muscle functions as an endocrine organ. Thus, skeletal muscle can account for most of exercise-induced CONCLUSIONS increase in plasma IL-6 [14]. In the present study, since one of In conclusion, athletes following a low-CHO diet showed higher the experimental conditions included a glycogen-depletion diet we acute hepatic structural damage markers and finished more expected a dramatic increase in IL-6 following exercise in such dehydrated compared to athletes on a high-CHO diet. Body core condition. We found inverse correlations between FFM and IL-6 only temperature and LPS-LBP complex levels were unrelated beyond 2-h following exercise in both, the low- and high-CHO dietary an expected increase in body core temperature explained by conditions. Our results suggest that fat tissue accounts for most of exercise. No significant associations were found between body core the exercise-induced increases in IL-6. We are aware that this finding temperature, IL-6 and LPS-LBP complex levels. Further studies must be interpreted with caution since IL-6 is not exclusively secreted are needed that look into the chronic effects of exercise training by muscle tissue but also by several cells and tissues (e.g., fibroblasts, on liver structure and function. endothelial cells, lymphocytes, macrophages, and adipose tissue during exercise). However, during exercise only a small contribution of IL-6 has been reported for the brain and peritendon [15,16,19,29,42], and therefore, we expected a higher contribution Acknowledgments of plasma IL-6 from skeletal muscle following exercise. 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