Substrate oxidation during exercise in childhood acute lymphoblastic leukemia survivors

Abstract Children with acute lymphoblastic leukemia (ALL) are at high risk of developing long-term cardiometabolic complications during their survivorship. Maximal fat oxidation (MFO) is a marker during exercise of cardiometabolic health, and is associated with metabolic risk factors. Our aim was to characterize the carbohydrate and fat oxidation during exercise in childhood ALL survivors. Indirect calorimetry was measured in 250 childhood ALL survivors to quantify substrate oxidation rates during a cardiopulmonary exercise test. A best-fit third-order polynomial curve was computed for fat oxidation rate (mg/min) against exercise intensity (% O2peak) and was used to determine the MFO and the peak fat oxidation (Fatmax). The crossover point was also identified. Differences between prognostic risk groups were assessed (ie, standard risk [SR], high risk with and without cardio-protective agent dexrazoxane [HR + DEX and HR]). MFO, Fatmax and crossover point were not different between the groups (p = .078; p = .765; p = .726). Fatmax and crossover point were achieved at low exercise intensities. A higher MFO was achieved by men in the SR group (287.8 ± 111.2 mg/min) compared to those in HR + DEX (239.8 ± 97.0 mg/min) and HR groups (229.3 ± 98.9 mg/min) (p = .04). Childhood ALL survivors have low fat oxidation during exercise and oxidize carbohydrates at low exercise intensities, independently of the cumulative doses of doxorubicin they received. These findings alert clinicians on the long-term impact of cancer treatments on childhood ALL survivors’ substrate oxidation.


Introduction
The energy substrate metabolism is a research field that has been particularly understudied in childhood cancer survivors compared to the general population. 1 Nonetheless, Acute lymphoblastic leukemia; cardiopulmonary exercise testing; maximal fat oxidation; pediatric cancer survivorship; substrate oxidation fatty acids and glucose are the main energy sources for humans, and their respective contributions may be influenced by several factors including sex, age, and nutritional status. 2 Metabolic risk factors such as insulin resistance, plasma triglycerides, and waist circumference are also known to influence fat and carbohydrate oxidation in the general population. 1,3,46][7][8][9] These late effects contribute to the morbidity and premature mortality observed in this population 10,11 and highlight the importance to understand their challenges to offer them an appropriate clinical follow-up.
To reach this goal, it would be beneficial to identify new outcomes related to childhood cancer survivors' metabolic health, in addition to documenting their cardiometabolic disorders and maximal fat oxidation (MFO).In fact, the MFO rate is a practical marker used during exercise to document changes in oxidation rates and energy subtract metabolism.The importance of studying energy substrate metabolism during exercise is motivated by recent findings in childhood acute lymphoblastic leukemia (ALL) survivors.Indeed, it has been observed that childhood ALL survivors had lower cardiorespiratory fitness levels compared to non-cancer population, despite a same level of daily physical activity. 12,13Thus, childhood cancer survivors' cardiorespiratory fitness levels could be associated with energy substrate oxidation impairment. 2 It has been hypothesized that childhood cancer survivors cannot achieve a normal level of energy expenditure during exercise due to physiological limitation and exercise intolerance. 14Finally, a recent pilot-controlled study showed an impairment in energy substrate oxidation during exercise in childhood ALL and acute myeloid leukemia survivors, 15 justifying the need for a larger study.
Therefore, the first aim of this study was to characterize the carbohydrate and fat oxidation during exercise in childhood ALL survivors.The second aim was to explore the associations between cancer treatments (cranial radiotherapy [CRT], doxorubicin, dexrazoxane) and substrate oxidation during exercise.The third aim was to document the associations of cardiorespiratory fitness level, level of daily physical activity, and metabolic health on substrate oxidation during exercise.

Participants
This study recruited 250 childhood ALL survivors, diagnosed between 1987 and 2010, and treated according to the DFCI-ALL 87-01 to 05-01 protocols 16 at CHU Sainte-Justine (Canada).They were initially recruited in the PETALE study and eligible survivors had to be ≤19 years old at diagnosis, ≥5 years post-diagnosis, with no history of refractory or recurrent diseases, and did not receive hematopoietic stem cell transplant, as specified in the published protocol. 17Participants who had suffered from congenital bone disease or who received osteotoxic drugs for non-ALL diseases were excluded.Written informed consent was obtained from participants or their parent/legal guardian.The study was approved by the Ethics Review Committee of CHUSJ.

Metabolic health assessments
Assessments of metabolic health (obesity and metabolic health outcomes) are summarized in Table 1 and detailed in the Supplementary File.Fat free mass (FFM) was calculated with weight (kg) and total body fat mass percentage.

Cardiopulmonary exercise test
After the blood sample collections and before starting the cardiopulmonary exercise test (CPET) in the morning (10:00 a.m.TO 12:00 p.m), participants were encouraged to eat a breakfast that meet the nutritional guidelines. 27The CPET was performed on an electromagnetic cycle ergometer (Ergoline, ER900, Bitz, Germany) according to the McMaster incremental cycle protocol, 28 as follows: a standard incremental procedure starting at 25 W and increasing the load by 25 W or 50 W every two minutes (pedaling cadence of 50-70 revolutions per minute), depending on the height and sex of the participant.At the end of the CPET, a three-minute active rest period at 25 W was followed by a three-minute passive rest period.The CPET protocol has been fully described elsewhere 29 and details has been provided in the Supplementary File.
All participants were monitored continuously during the CPET with a 12-lead electrocardiogram (CASE Exercise Testing, GE Marquette, Miwaukee, WI).An exercise physiologist and a cardiologist ensured that the CPET was safe and not limited by participants' symptoms, as previously described. 29The exercise physiologist determined whether the participants reached two out of three of the following criteria to validate their ɺ VO 2 peak: peak respiratory exchange ratio value ≥1.15, peak rated perceived exertion >7/10, and maximum heart rate ≥85% of the predicted heart rate value. 30

Ventilatory threshold assessment
The ventilatory threshold was visually determined by two independent operators as the point of the incremental CPET during which ventilation (VE/ ɺ VO 2 ) increased without changes in the ventilatory equivalent for CO 2 (VE/VCO 2 ). 31 A consensus was obtained when ventilatory threshold differed between the operators.For 16 participants, no consensus was achieved and were excluded from ventilatory threshold analyses.High triglycerides ≥1.7 mmol/l 23 insulin resistance at least one of three factors • Blood fasting glucose ≥6.1 mmol/l 25 • glycated hemoglobin ≥6.0% 25 • Homa-ir ≥2.86 25 at least one of two factors • Blood 25 fasting glucose ≥ 6.1 mmol/l 25 • glycated hemoglobin ≥ 6.0% 25 • Homa-ir ≥ 95th percentile 26 ldl, low density lipoprotein; Homa-ir, homoeostasis model assessment.

Indirect calorimetry assessment
Indirect calorimetry is the recognized standard method to quantify substrate oxidation rates at rest and during exercise. 32This robust method allows quantifying rates of substrate oxidation up to ∼85% of ɺ VO 2 peak. 33,34To calculate fat and carbohydrate oxidation rates over a wide range of exercise intensities (0-80% ɺ VO 2 peak), ɺ VO 2 and ɺ VCO 2 data were averaged for the last 20 s of each CPET stage in order to be representative of steady state gas exchanges. 29Fat and carbohydrate oxidation rates were determined using the Peronnet and Massicotte's equations, 32 with the assumption that protein oxidation during exercise was negligible as described by Wagenmakers 35  (L/min).
For each participant, a best-fit third order polynomial curve was constructed for fat and carbohydrate oxidation rate (mg/min) against exercise intensity ( ɺ VO 2 peak) using MATLAB versions R2015a and R2019a (MathWorks, Inc., Natick, MA).Each individual curve was used to determine the MFO.The peak fat oxidation (Fat max ) was also computed, which consists of the exercise intensity when fat oxidation rate is maximal.The individual curve was also used to determine the %power output at the MFO.Graphs of fat and carbohydrate oxidation rate (mg/min) against % relative ɺ VO 2 peak were used to illustrate fat and carbohydrate oxidation over intensities 20-80% relative ɺ VO 2 peak.Only intensities between 20 and 80% relative ɺ VO 2 peak were considered in our analyses because data <20% relative ɺ VO 2 peak represented the rest period and data >80% relative ɺ VO 2 peak represented the peak of exercise that could not be computed since it is not a documented valid method 33,34 ; this limitation has been reported by Peronnet and Massicotte. 32nergy from fat and carbohydrate (Kcal) was calculated with the fat and carbohydrate oxidation rates (g/min).The oxidation of 1 g of lipids provides approximately 9 Kcal, while the oxidation of 1 g of glucose provides 4 Kcal.Their respective contribution to the total energy was calculated over intensities 20-80% relative ɺ VO 2 peak.The crossover point was identified for each participant when 70% of the total energy (Kcal) was derived from carbohydrates and 30% from fat. 36

Physical activity assessment
Physical activities were self-reported through questionnaires [37][38][39] and total daily minutes of moderate to vigorous leisure physical activity (MVLPA) were calculated using the Compendiums of Physical Activity for Adults and for Youth to identify reported activities with a metabolic equivalent value ≥3. 40

Statistical analyses
Statistical analyses were performed using IBM SPSS statistics, version 26.0 (IBM Corp., Armonk, NY).Analyses were conducted for all participants and separately for men and women.To compare means, a two-tailed Student t-test was performed, while comparisons of percentages were made by using a Chi-square test.Fisher exact test (performed using a univariate analysis) was used when the conditions for applying the Chi-square test were not met.Differences were considered statistically significant when the p-value was <.05.For comparisons between two means, significant differences were reported with the Cohen effect size (d) and interpreted as large (d ≥ 0.8), medium (0.5 ≤ d < 0.8) or small (d < 0.5). 41nalyses according to participants' prognostic risk group (ie, standard risk [SR], high risk with and without cardio-protective agent dexrazoxane [HR + DEX and HR]) were also performed.In the SR group, participants received a cumulative doxorubicin dosage of 60 mg/m 2 , 300 mg/m 2 for the HR + DEX group, and either 360 mg/m 2 (DFCI-ALL protocols 87-01 and 91-01) or 300 mg/m 2 (DFCI-ALL protocols 95-01 and 05-01) for the HR group. 16Group differences were explored using one-way analysis of variance and Bonferroni's post-hoc tests.

Results
Among the 250 childhood ALL survivors, 53 were excluded for the following reasons: 29 participants did not complete the CPET because there were issues with the equipment or data collection, 5 received a different treatment than the DFCI-ALL protocols, and for 19 participants, data file were corrupted preventing them from being read by the MATLAB software.Participants' characteristics (N = 197) are presented in Table 2.
The cohort (49.2% women) was mostly composed of adolescents and young adults (median age of 21 years) with an average of 15.5 ± 5.2 years after diagnosis.Metabolic rate outcomes group are presented in Table S1.

Cardiorespiratory fitness and physical activity
The CPET data showed that all 197 participants performed a maximal CPET without adverse events.They reached a ɺ VO 2 peak of 32.4 ± 8.1 mL.kg − 1.min − 1, which is 5.2 ± 6.5 mL.kg − 1.min − 1 below their predicted ɺ VO 2 peak (37.6 ± 8.3 mL.kg − 1.min − 1), as summarized in Table 2. Their mean MVLPA was 25.7 ± 25.3 min/day.We observed that participants in the SR group had a higher MVLPA (p = .008)and ɺ VO 2 peak (p < .0001)than those in the HR group.

MFO and Fat max
Absolute power output at MFO was significantly different between SR group and HR + DEX group (45.5 ± 25.5 W vs. 35.5 ± 21.1 W, p = .036,d = 0.4).Relative power output at MFO and Fat max were not different between the groups, as presented in Figure 2 and reported in Table 3. MFO was achieved by the SR group at 273.7 ± 99.5 mg/min, by the HR + DEX group at 247.3 ± 93.2 mg/min and by the HR groups at 239.2 ± 86.0 mg/ min (p = .078)(Figure 2).A higher MFO was achieved by men in the SR group (287.8 ± 111.2 mg/min) compared to those in HR + DEX (239.8 ± 97.0 mg/min) and HR groups (229.3 ± 98.9 mg/min) (p = .04).Women had a higher Fat max than men (37.3 ± 7.6 % vs. 33.0± 7.0 %, p < .001,d = 0.6).

Crossover point
We observed in Figure 3 that the crossover point was not significantly different between groups (p = .726).Women had a higher crossover point than men (48.0 ± 11.2 % vs. 40.3± 11.5%, p < .001,d = 0.7).carbohydrate oxidation rates. 15However, their small sample size may have limited the power of their analysis and while the design of the PETALE study has no control group, 17 its large sample size is a strength to perform larger analyses regarding participants' prognostic risk groups.Our results revealed a trend, although not significant, toward lower rates of fat oxidation at 20-70%V ɺ O 2 peak in HR and HR + DEX groups.Fat oxidation might be independent of the cumulative doses of doxorubicin that childhood ALL survivors received.

MFO and Fat max
Our analyses showed that man participants had a higher MFO in the SR group than in HR and HR + DEX groups, while no differences were found in women according to their prognosis groups.These findings suggest that men are at long-term risk of substrate oxidation disturbances, especially those in HR and HR + DEX groups.5][46] Nevertheless, woman participants in the SR group do not appear to be less at risk than those in the other groups.We hypothesize that woman participants are at long-term risk of substrate oxidation disturbances independently of the doxorubicin doses they received.This would suggest that woman childhood cancer survivors are more vulnerable to adverse treatment effects than men, as observed in other studies. 12,47,48Nevertheless, no significant differences in MFO were observed between man and woman participants during exercise, as observed in childhood acute leukemia survivors. 15hen considering Fat max results, no differences were also reported between men and women, and no differences were observed between groups.Nevertheless, our analyses showed that survivors reached their Fat max at very low intensities (35.1 ± 7.6 % ɺ VO 2 peak) when compared to another study 15 where childhood leukemia survivors reached their Fat max at 51.1 ± 7.7 % ɺ VO 2 peak.This aspect can be explained by the age difference between the studies since there is a decrease in Fat max with maturation. 49,502][53][54] Our findings suggest that childhood ALL survivors have substrate oxidation impairments during exercise.
During submaximal exercise (<65% ɺ VO 2 peak), fat oxidation is the principal source of energy supply. 51,55 ollowing MFO, we observed that fat oxidation decreased, and carbohydrate oxidation increased in both man and woman survivors.Moreover, we observed that when Fat max is at low intensity, fat oxidation is lower and there is a greater use of carbohydrates as an energy substrate during exercise.Thus, childhood ALL survivors' oxide more carbohydrate and less fat than children cured of acute leukemia 15 and healthy people. 51

Crossover point
The crossover point refers to the intensity at which energy derived from the oxidation of carbohydrates is predominant over fat. 36As reported in the literature, the principal source of energy substrate before 65% ɺ VO 2 max is fat oxidation. 51,55In our study, the crossover point (44.0 ± 12.0 % ɺ VO 2 peak) was below the normal range that is superior to 65% ɺ VO 2 peak. 51,55During exercise, childhood ALL survivors oxidized fat for a shorter period and at lower intensities before shifting toward a predominance of carbohydrate oxidation.Our results are similar to those observed in patients with type 2 diabetes and with growth hormone deficiency. 56,57These patients exhibit a decrease in fat oxidation and a shift toward lower intensities of the crossover during exercise when compared to their healthy peers. 56,57

Associations of cardiorespiratory fitness level, level of daily physical activity, and metabolic health on substrate oxidation during exercise
Potential associations were explored and exposure to cancer treatments (doxorubicin treatments and CRT) was not associated with substrate oxidation (MFO, Fat max , and crossover point parameters).Associations were, however, observed between Fat max , crossover point and cardiorespiratory fitness level.][60] In fact, Venables et al. showed that low ɺ VO 2 peak is a significant predictor of a low MFO. 61Nevertheless, cardiorespiratory fitness levels were not associated with MFO in our study.Indeed, participants' cardiorespiratory fitness level did not impact the substrate oxidation since our data showed that they reached a ventilatory threshold at the same % ɺ VO 2 peak.However, the group with the lowest MFO (HR group) had lower ɺ VO 2 peak compared to the other groups (SR and HR + DEX groups) which could also explain our results.A study found that childhood ALL survivors had lower ɺ VO 2 peak, compared to the healthy population 12 which could affect our associations between ɺ VO 2 peak and MFO.It has been hypothesized that associations between MFO and ɺ VO 2 peak are only visible when comparing heterogenous groups 61,62 and that the level of physical activity can influence participants' MFO and Fat max . 61,63In our study, physical activity levels were not associated with fat oxidation, suggesting that other variables of interest may have impacted childhood ALL survivors' substrate oxidation.Further studies are needed to explore this avenue.
Identifying key parameters for better follow-up of long-term cardiometabolic complications of childhood cancer survivors is crucial.Our study supports that substrate oxidation impairments is a mechanism involved in the alterations of childhood ALL survivors' metabolic health. 7Indeed, metabolic risk factors observed in our cohort (e.g.insulin sensitivity, diabetes and obesity) are also found to be associated with MFO and Fat max in healthy men. 3,4,56,64,65In healthy active young adults, Fat max and MFO were found to be associated with waist circumference, plasma triglycerides, and clustered cardiometabolic risk factors. 66However, in healthy sedentary adults, MFO and Fat max were not related to cardiometabolic risk factors during a graded exercise test. 67ur findings are consistent with these studies.Overweight and obesity were highly prevalent in childhood ALL survivors enrolled in the PETALE study. 7Our results showed that only BMI and total body fat mass percentage were correlated with parameters of substrate oxidation.Our findings support the hypothesis that high BMI and total body fat mass percentage in childhood ALL survivors lead to higher period of fat oxidation during exercise before switching to carbohydrates.Thus, suggesting a better substrate oxidation for childhood ALL survivors with high BMI and total body fat mass percentage.[70][71][72][73][74][75]

Limitations
Our study must be interpreted in the context of its limitations.Our cohort was comprised almost exclusively (>95%) of European (French-Canadian) descent.This limitation is explained by the genetic aspect of the PETALE study since it was important to lessen the confounding genetic background of childhood ALL survivors, as described by Marcoux et al. 17 Nevertheless, it must be underlined that this can limit the generalizability of our results and its scope to other populations.Another limitation is the absence of a healthy control group to compare our data.Finally, using a standard incremental procedure 76,77 is only one of many ways to assess fat oxidation during exercise.In fact, Achten et al. 51 established a specific submaximal protocol with 3-min increment to favor participants' steady state gas exchanges and measure substrate oxidation.To date, there is, however, no consensus regarding the best increment duration to estimate MFO and Fat max . 78

Conclusion
This study provides a characterization of childhood ALL survivors' energy substrate oxidation during exercise in relation to their metabolic health.We demonstrated that our population has low-fat oxidation during exercise and that they oxidize carbohydrates at lower exercise intensities, independently of the cumulative doxorubicin doses they received.Men in HR and HR + DEX groups are particularly at long-term risk of substrate oxidation disturbances.Our study enhances our knowledge regarding the long-term impact of childhood cancer treatments on substrate oxidation.Researchers should further investigate the effects of exercise on cardiometabolic health and energy substrate metabolism.
This research was also supported in part by PhD study grants from Cole Foundation, Fonds de Recherche du Québec -Santé (FRQS), Sainte-Justine University Hospital Center Foundation and Foundation of Stars.The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Figure 1 .
Figure 1.Fat (a) and carbohydrate (B) oxidation rate according to the percentage of maximum oxygen consumption.(green) Standard risk; (yellow) High risk with dexrazoxane; (red) High risk without dexrazoxane.

Figure 2 .
Figure 2. comparison of maximal fat oxidation (a), Fat max (B) and power output (c).

Figure 3 .
Figure 3. crossover point between fat and carbohydrate for all participants.ɺ Vo 2 peak, maximum oxygen consumption.the dash line represents %carbohydrate of total energy and the solid line represents %fat of total energy of 197 participants.

Table 2 .
clinical characteristics of childhood all survivors according on their prognostic risk group.
Sr, standard risk; Hr + deX, high risk with dexrazoxane Hr, high risk without dexrazoxane; dFci, dana Farber cancer institute; ɺ V o 2 peak, maximum oxygen consumption; mVlPa, moderate and vigorous leisure physical activities.the p value represents the significant difference between standard risk group and high risk group with and without dexrazoxane.if the p value is (a) p < .05 between Sr and Hr + deX, (b) p < .05 between Sr and Hr, (c) p < .05 between Hr + deX and Hr.

Table 3 .
Substrate oxidation rates according on childhood all survivors' prognostic risk group.