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Decreased Physical Working Capacity in Adolescents With Nonalcoholic Fatty Liver Disease Associates With Reduced Iron Availability
Journal Pre-proof Decreased Physical Working Capacity in Adolescents With Non-Alcoholic Fatty Liver Disease Associates With Reduced Iron Availability Tim Mitchell, Elizabeth McKinnon, Oyekoya Ayonrinde, Leon A. Adams, Debbie Trinder, Anita C.G. Chua, Robert U. Newton, Leon Straker, John K. Olynyk
PII: DOI: Reference:
S1542-3565(19)31116-4 https://doi.org/10.1016/j.cgh.2019.10.017 YJCGH 56804
To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 11 October 2019 Please cite this article as: Mitchell T, McKinnon E, Ayonrinde O, Adams LA, Trinder D, Chua ACG, Newton RU, Straker L, Olynyk JK, Decreased Physical Working Capacity in Adolescents With NonAlcoholic Fatty Liver Disease Associates With Reduced Iron Availability, Clinical Gastroenterology and Hepatology (2019), doi: https://doi.org/10.1016/j.cgh.2019.10.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 by the AGA Institute
1 Title Page Title: Decreased Physical Working Capacity in Adolescents With Non-Alcoholic Fatty Liver Disease Associates With Reduced Iron Availability
Short Title: PWC in NAFLD Relates to Iron Bioavailability
Authors: Tim Mitchell1, Elizabeth McKinnon2, Oyekoya Ayonrinde1,3, Leon A. Adams3,4, Debbie Trinder3,5, Anita C. G. Chua3,5, Robert U. Newton6, Leon Straker6,7, John K. Olynyk1,8.
Affiliations: 1
Department of Gastroenterology, Fiona Stanley Fremantle Hospital Group, Perth, Australia
2
Telethon Kids Institute, Perth, Australia
3
Medical School, University of Western Australia, Perth, Australia
4
Department of Hepatology, Sir Charles Gairdner Hospital, Perth, Australia
5
Harry Perkins Institute for Medical Research, Perth, Australia
6
Exercise Medicine Research Institute, Edith Cowan University, Perth, Australia
7
School of Physiotherapy and Exercise Science, Curtin University, Perth, Australia
8
School of Medical and Health Sciences, Edith Cowan University, Perth, Australia
Grant Support: Nil
2
List of Abbreviations: NAFLD: non-alcoholic fatty liver disease MCV: mean corpuscular volume MCH: mean corpuscular haemoglobin PWC170: physical work capacity at 170 bpm PWC: physical work capacity
Correspondence: Corresponding Author: Tim Mitchell Mail: Gastroenterology Department, Fiona Stanley Hospital, 11 Robin Warren Drive, Murdoch WA 6150 Phone: (m) +61 403 781 702 (w) +61 8 6152 2827 Fax: +61 8 9335 6155 Email: [email protected]
Disclosures: Nil
Conflicts of Interest: None of the authors have any conflicts of interest to declare.
Writing Assistance: Nil
3
Author Contributions: TM: analysis and interpretation of data, drafting of the manuscript; EM: statistical analysis, drafting of the manuscript; OA, LAA, DT, ACGC, RUN, LS: critical revision of the manuscript; JKO: study concept and design, supervision and critical revision of the manuscript.
Acknowledgements The authors are grateful to the Raine Study participants and their families, the Raine Study team for cohort co-ordination and data collection and the NHMRC for their long-term contribution to funding the study. The core management of the Raine Study is funded by the University of Western Australia, Curtin University, Women and Infants Research Foundation, Telethon Kids Institute, Edith Cowan University, Murdoch University, the University of Notre Dame and the Raine Medical Research Foundation. The data collection of the Gen2-17 year follow up of the Raine Study was funded by NHMRC Grant 353514 and biological specimen collection was funded by NHMRC Grant 403981.
4 Abstract Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is common and related to obesity and insulin resistance. Iron metabolism is impaired in obese individuals and iron deficiency has been associated with physical inactivity. We investigated whether iron bioavailability is reduced in patients with NAFLD and contributes to reduced cardiorespiratory fitness.
Methods: We collected information on weight-adjusted, submaximal physical work capacity (PWC), ultrasound-determined hepatic steatosis, iron indices, and hematologic and metabolic parameters from 390 female and 458 male participants of the Raine Study—a longitudinal study of disease development in 2868 children in Western Australia. X2 and linear regression analyses were used to compare characteristics of study participants according to NAFLD status at age 17 years.
Results: Fourteen percent of the cohort had NAFLD. PWC was significantly reduced in adolescents with NAFLD compared to adolescents without NAFLD (reduction of 0.17 W/kg, P=.0003, adjusted for sex and body mass index [BMI]). Iron bioavailability (assessed by mean corpuscular volume [MCV], mean corpuscular haemoglobin [MCH], transferrin saturation, and serum levels of iron) was inversely correlated with BMI in adolescents with NAFLD (P≤.01 for all, adjusted for sex) but not in adolescents without NAFLD (P>.30). MCV and MCH correlated with PWC (MCV, P=.002 for female and P=.0003 male participants; MCH, P=.004 for female and P=.01 for male participants), irrespective of NAFLD status. Reduced PWC was associated with lower transferrin saturation in adolescents with NAFLD (reduction of 0.012 W/kg per unit decrease in transferrin saturation, P=.007) but not in
5 adolescents without NAFLD (reduction of 0.001 W/kg, P=.40), adjusted for sex. This association was independent of MCV or MCH.
Conclusions: In a well-defined cohort of adolescents, we found NAFLD to be associated with decreased cardiorespiratory fitness, independent of BMI. The relationship between transferrin saturation and PWC in adolescents with NAFLD indicates that functional iron deficiency might contribute to reductions in cardiorespiratory fitness.
Key words: steatosis, fitness, exercise, ferritin, metabolism
6 Introduction Chronic liver disease is an increasing cause of premature morbidity and mortality1. In Western countries, liver-related mortality rates have increased 400% since 1970, contrasting with substantial improvements in most other common chronic diseases1,2. A major contributor to the increasing burden is non-alcoholic fatty liver disease (NAFLD), a chronic liver disorder related to obesity and the metabolic syndrome3. The spectrum of disease ranges from simple steatosis through steatohepatitis with varying degrees of fibrosis, including cirrhosis2,4.
The prevalence of NAFLD is increasing and affects 25% of adults and 17% of adolescents5,6. The disease burden is high with NAFLD expected to become the leading indication for liver transplantation in the United States2. Current pharmacotherapy is restricted to certain highrisk groups2. Although numerous clinical trials are underway, the primary advice for patients is to modify their diet to promote weight loss and commence regular exercise7,8. Clinical trials examining exercise for NAFLD have demonstrated improvements in hepatic fat content independent of weight loss9,10. The specific exercise regimen, for example resistance training vs aerobic activity or moderate vs high intensity, does not appear to influence the efficacy of hepatic fat reduction11,12.
Unfortunately, such lifestyle advice when delivered in the clinic is rarely successful and on review, patients will often cite a lack of energy or fitness as a barrier to exercise13-15. Additionally, patients with stronger cardiorespiratory performance have been shown to respond better to lifestyle interventions and have lower cardiovascular mortality, which is the leading cause of death in NAFLD patients2,16,17. The possibility of a modifiable factor
7 inherent in NAFLD patients that reduces physical work capacity has not previously been studied, even though addressing this may improve patient engagement in exercise programs16,18,19.
Iron deficiency is a known cause of decreased physical work capacity in healthy and chronically ill individuals, even in the absence of related anaemia20. Prior studies have shown that iron deficiency is common in chronic inflammatory conditions such as congestive cardiac failure and is associated with worse outcomes, including decreased exercise tolerance20.
Iron deficiency is associated with inflammation and obesity but little is known about iron metabolism in NAFLD21. The assessment of iron stores in NAFLD is complex, given that elevated ferritin is common in NAFLD patients22. The high prevalence of obesity in NAFLD can make it difficult to delineate the impacts of NAFLD and iron metabolism on each other23. It is thought that the effects of obesity and NAFLD on impaired iron metabolism are mediated via dysregulated production of the key iron regulatory hormone hepcidin23. Production of hepcidin is stimulated by chronic inflammation and is associated with obesity, thereby reducing iron bioavailability24,25.
In this study, we aimed to determine if NAFLD was associated with reduced cardiorespiratory fitness in a well-defined cohort of community-dwelling individuals in late adolescence. Thereafter, we investigated whether impaired iron bioavailability as measured by mean cell volume (MCV), mean cell haemoglobin (MCH), serum iron and transferrin saturation was associated with impaired cardiorespiratory fitness, and may represent a
8 potential future therapeutic target to improve weight reduction and exercise treatment in NAFLD.
9 Methods Study population This project was a cross-sectional study using the Raine Study, a well described, prospectively enrolled cohort of 2868 live-born children from 2900 pregnancies26. The Raine Study examines the evolution of health or disease during pregnancy, early childhood and adolescence and is representative of the broader Western Australian population26. The background and available data of the Raine Study Gen2 participants have been previously described26. All active members of the Raine Study Gen2 were invited to participate in the 17-year old assessment, conducted between July 2006 and June 2009. Exclusion criteria included a diagnosis of haemochromatosis or coeliac disease, active inflammation or chronic illness, and pregnancy. Institutional ethics committee approval was obtained from the human research ethics committee of Princess Margaret Hospital for Children. Signed informed parental consent and adolescent assent were obtained prior to study participation.
Assessment The 17-year follow-up comprised detailed health questionnaires, abdominal ultrasound, anthropometric (height, weight, skin fold thickness, waist circumference), cardiovascular (resting heart rate, systolic and diastolic blood pressure) and biochemical assessments. Information on alcohol intake over the previous year was documented by self-reporting and by the completion of a validated, semiquantitative food frequency questionnaire27. Physical activity was assessed using the International Physical Activity Questionnaire, a validated, self-administered questionnaire which recalls physical activity in the previous week26. Comprehensive medication and medical histories were documented to exclude secondary
10 causes of NAFLD and concomitant liver disease. Testing for hepatitis B or C virus infections was not performed because notification rates for hepatitis B and C virus infections were on average less than 24/100000 and 23/100000, respectively, for Western Australian teenagers between the ages of 15 and 19 over the study period28. Body mass index (BMI) z-scores were derived from age- and gender-adjusted growth curves that accord with the World Health Organisation Child Growth Standards for school-aged children and adolescents and BMI cut-offs for adults29. Participants with z-scores from -2 to 1 were classified as healthy weight, 1-2 as overweight and >2 as obese29. Metabolic syndrome was defined according to age- and gender-specific criteria of the International Diabetes Federation30. Laboratory assessments were performed with venous blood samples taken after an overnight fast. All laboratory assays were performed at an accredited central laboratory (PathWest Laboratories, Perth, Western Australia).
Ultrasound Hepatic steatosis was determined using previously described ultrasound characteristics which provide 92% sensitivity and 100% specificity for the histological diagnosis of >10% hepatic steatosis31. Trained ultrasonographers performed liver ultrasound assessments28. The ultrasound images were reviewed and a total fatty liver score was determined from the captured images by a single radiologist who was blinded to the clinical and laboratory characteristics of the subjects28. The scoring system has been previously reported and assesses three parameters: liver echotexture (0-3), deep attenuation (0-2) and vessel blurring (0-1)28. The diagnosis of hepatic steatosis required a total score of ≥2, which included an echotexture score of ≥128. Severity of hepatic steatosis was classified by the total fatty liver score, as no steatosis (0-1), mild steatosis (2-3) or moderate to severe
11 steatosis (4-6)28. NAFLD diagnosis required sonographic evidence of hepatic steatosis and the exclusion of significant alcohol consumption, defined as weekly alcohol intake of less than 210g and 140g for males and females, respectively2.
Physical Work Capacity Cardiorespiratory fitness was assessed using a standardised protocol for submaximal cycle ergometry using the Monark cycle ergometer32,33. Participants cycled at 50-60 revolutions per minute throughout the test, initially at a workload of 25 watts for two minutes, which increased to 50 watts for two minutes and finally 75 watts for two minutes33. Workload output was regressed against heart rate and the maximal steady-state power attained for a heart rate of 170 beats per minute (PWC170) to determine cardiorespiratory fitness33. PWC170, which correlated strongly with body weight in healthy-weight participants (P<.0001) was divided by weight to normalise the effects of body size on workload output. The normalised PWC170 result, which we refer to as physical work capacity, was used for analysis.
Statistical analysis Chi-squared tests were used to compare demographic characteristics of study participants according to NAFLD status. Physical work capacity, anthropometric measures and biochemical characteristics were compared using Student t-tests, after log-transformation of those variables having a skewed distribution, and relationships assessed by correlation and multivariable linear regression. Analysis was undertaken using RStudio (RStudio, Inc., Boston, Massachusetts).
12 Results Data including PWC170 results and a valid NAFLD diagnosis were available from 848 Raine Study participants at 17 years of age (390 females, 458 males). Compared with other participants of the 17-year follow-up who lacked either PWC170 results or a valid NAFLD assessment (N=200), study participants did not differ significantly in either mean BMI (females: P=.10, males: P=.60) or the proportion of Caucasian European ethnicity (P>.30).
Overall, NAFLD was diagnosed in 14% of participants: 66/390 females (16.9%) and 53/458 males (11.6%). NAFLD and non-NAFLD participants were similar with respect to Caucasian ethnicity, smoking status and recent participation in vigorous exercise (P>.30, Table 1). Overall, BMI z-scores were greater in NAFLD participants, however the distribution of these scores varied between male and female NAFLD adolescents (P=.003). Nearly 50% of NAFLD females were in the healthy weight range with 29% and 24% classified as overweight and obese, respectively (Table 1). More than 50% of NAFLD males were obese. In keeping with this disproportionate prevalence of obesity, 17% of NAFLD males satisfied criteria for a diagnosis of metabolic syndrome, significantly more than non-NAFLD males (2%, P<.0001) and marginally more than NAFLD females (6%, P=.08). On average, adolescents with NAFLD had greater suprailiac and abdominal skinfold thickness measures than non-NAFLD adolescents with the same BMI. In males, the mean difference in suprailiac skin fold thickness was 5.6 cm (P<.0001) and abdominal skinfold thickness was 3.6 cm (P<.0001), adjusted for BMI. In females, the mean difference in suprailiac skinfold thickness was 3.3 cm (P=.002) and abdominal skinfold thickness was 2.1 cm (P=.01), adjusted for BMI.
13 Physical work capacity (PWC), as measured by PWC170 normalized by body weight, was significantly lower, on average, for females compared with males (females: mean (SE) 1.59 (0.4) W/kg; males 2.17 (0.5) W/kg, P<.0001), and was markedly reduced in those with NAFLD compared to those without (Table 2; females: mean (SE) difference -0.23 (0.05) W/kg, P<.0001; males: -0.53 (0.07) W/kg, P<.0001). This reduction in capacity remained significant when controlling for BMI and gender (mean (SE) difference -0.17 (0.05) W/kg, P=.0003). Reduced physical work capacity was strongly associated with greater adiposity for all measures considered (Table 3), and this was observed in both non-NAFLD and NAFLD participants. However, an increasing disparity in fitness between those with and without NAFLD was evident across BMI classifications (Figure 1).
For both males and females, the observed impact of NAFLD on PWC was closely paralleled by increasing suprailiac skinfold thickness and higher leptin levels, more so than elevated BMI, or hs-CRP as a marker of the low-level inflammation associated with obesity. Importantly, the effect of NAFLD on PWC remained when the analysis was restricted to nonobese individuals (mean difference -0.22 W/kg, P<.0001), or when considering PWC170 without normalisation but controlling for weight in the regression modelling (mean difference in PWC170, males -30.6W, P<.0001; females -8.5W, P<.0001).
Iron and haematological parameters were also examined for inter-relationships with adiposity, cardiorespiratory fitness and NAFLD. MCV, MCH, transferrin saturation and serum iron levels all showed significant reductions with increasing BMI in those with NAFLD (P≤.01, adjusted for gender) that were absent in those without NAFLD (P>.30) (Figure 2). In contrast, serum ferritin increased with increasing BMI, with trends similar for non-NAFLD
14 and NAFLD participants (P=.80 for interaction term). There was also a significant positive association of haemoglobin with BMI in non-NAFLD participants only (P≤.0001).
MCV and MCH were both positively associated with PWC (MCV: P=.002 females, P=.0003 males; MCH: P=.004 females, P=.01 males), irrespective of NAFLD status (P=.80 for interaction). The significant association between decreased PWC and reduced transferrin saturation levels, however, was evident only in those with NAFLD (NAFLD: mean (SE) change -0.012 (0.004) W/kg per unit decreased in transferrin saturation, P=.007; non-NAFLD: -0.001 (0.002) W/kg, P=.40; adjusted for gender). Moreover, even when controlling for MCV, reduced PWC was significantly associated with lower transferrin saturation levels in NAFLD adolescents (mean (SE) change -0.011 (0.005) W/kg per unit decrease in transferrin saturation, P=.03), but not in non-NAFLD participants (P>.90) (Figure 3). Joint modelling of associations between PWC and serum iron levels considered with either MCV or MCH yielded similar results.
15
Discussion Currently, few effective treatment options exist for NAFLD and the emphasis for patients seen in the clinic is on lifestyle modification2. Unfortunately, engagement with weight reduction strategies and exercise programs outside clinical trial settings is disappointing, with patients commonly citing lethargy as a barrier13. Our observed prevalence of NAFLD in this well-characterised, otherwise healthy cohort accords with other adolescent studies which range from 10% using ALT and elevated BMI34, to 17% defined as >5% steatosis at autopsy6. Notably, we have found adolescents with NAFLD have poorer physical work capacity, a recognised marker of cardiorespiratory fitness, which may contribute to poorer engagement with exercise programs. The impact of NAFLD, which becomes more marked as adiposity increases, remains significant when adjusted by weight and appears independent of low-level inflammation.
Our data suggests that patients with NAFLD are less fit than their non-NAFLD counterparts of similar BMI, indicating a high-risk chronic disease phenotype. This finding has important clinical relevance as prior research has highlighted that fitness can ameliorate some of the detrimental effects of fatness33. Clinical trials investigating the efficacy of exercise modification in NAFLD have shown that neither the intensity nor type of exercise affected outcomes in participating patients, suggesting that patients do not need a minimum level of fitness prior to engaging in exercise11,12. Unfortunately, all trials are affected by selection bias where motivated patients are more likely to both participate and engage with a clinical trial resulting in underwhelming “real-world” results in patients at highest risk of a poor outcome14. Our study suggests that despite participating in a similar amount of every-day
16 exercise, patients with NAFLD are less fit, which may limit the effectiveness of exercise interventions and contribute to high relapse rates following clinical trial conclusion. Nonetheless, the importance of encouraging physical exercise in this cohort is confirmed by our study.
Having established that patients with NAFLD have decreased physical work capacity we investigated whether a modifiable factor for this could be identified. Iron deficiency, even without anaemia, has been shown to affect energy levels and cause fatigue35,36. Functional iron deficiency is well established in pro-inflammatory conditions, most notably chronic kidney disease and is defined as a decreased transferrin saturation (<20%) in the context of adequate ferritin levels (>100 mcg/L)37. In this condition, iron stores are thought to be adequate but are unable to be mobilised when required through the reticuloendothelial system37. The complex dynamic between iron and NAFLD is not well understood but iron has been suggested as a cofactor for liver injury through oxidative stress and insulin resistance22. However, an effort to modify this effect through phlebotomy for patients with NAFLD and an elevated ferritin without hereditary haemochromatosis did not show benefit in a randomised control trial22.
In our study, despite normal or elevated ferritin levels, a decreased transferrin saturation correlated with decreased physical work capacity in adolescents with NAFLD. In adolescents without NAFLD there was no correlation between transferrin saturation and physical work capacity. These results suggest that functional iron deficiency is present in NAFLD patients and may independently contribute to decreased cardiorespiratory fitness. This finding is in keeping with data derived from patients with heart failure20. Furthermore, a decrease in
17 MCV correlated with reduced physical work capacity, and MCV levels were notably lower in NAFLD patients. MCV is a widely available and well recognised haematological parameter with low levels closely associated with iron deficiency in a Caucasian population38. This correlation with physical work capacity further suggests that iron deficiency may contribute to decreased cardiorespiratory fitness.
The role of functional iron deficiency has been assessed in other chronic inflammatory disease states such as chronic obstructive pulmonary disease and congestive cardiac failure20,39. Despite an association between iron deficiency and decreased exercise tolerance in these patient groups, intervention trials investigating iron replacement in chronic cardiac failure have had mixed outcomes for overall survival and symptomatic benefit40,41. Antiinflammatory and anti-cytokine medications, such as pentoxifylline, have been studied in anaemia of chronic kidney disease previously with mixed results42 but no such studies have been performed in NAFLD. Such an intervention may be a basis of future research, aiming to improve outcomes from lifestyle interventions.
Despite our promising results from this large, representative cohort of adolescents it should be noted that this study has some limitations. The cross-sectional study design does not allow causal relationships to be determined. Although we have controlled for known confounding factors and the cohort was well characterised, unidentified confounding factors, such as renal disease, thalassemia, excessive sedentary behaviour or dietary influences, may have affected our results. Testing for thalassemia was not performed in this cohort as the rate of this condition in a general Australian population is low. Finally, we do
18 not have data for hepcidin levels in this cohort, which would further strengthen the assessment of functional iron deficiency.
In conclusion, we have demonstrated that in a well-defined cohort of adolescents, the presence of NAFLD is independently associated with decreased cardiorespiratory fitness. Furthermore, functional iron deficiency in the NAFLD cohort, as demonstrated by the association between decreased transferrin saturation and physical work capacity, may be a novel explanation for this finding. Future therapies aiming to modify the inflammatory milieu of NAFLD may increase iron bioavailability and as such improve physical fitness and engagement in exercise programs.
19 Figure Legends: Figure 1. Mean physical work capacity ± 2 SE, estimated from a linear model adjusted for BMI z-score and sex and plotted at the combined group-average values.
Figure 2. Mean ± 2 SE of iron and haematological indices, estimated from linear models adjusted for sex and BMI z-score, and plotted at the combined group-average values. Greater disparities between indices of NAFLD and non-NAFLD participants are evident with increasing adiposity (* 0.01
Figure 3. Estimated physical work capacity (PWC) for those with and without NAFLD as a function of mean corpuscular volume (MCV) and transferrin saturation (TSAT), adjusted for sex.
20
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Table 1. Cohort demographics. FEMALE Non-NAFLD N=324 Caucasian
NAFLD N=66
MALE P-value
#
Non-NAFLD N=405
NAFLD N=53
P-value
#
275 (84.9%)
57 (86.4%)
0.905
350 (86.42%)
43 (81.1%)
0.408
73 (22.5%)
10/65 (15.4%)
0.264
67/401 (16.71%)
11/50 (22.0%)
0.463
151/292 (51.7%)
32/63 (50.8%)
0.999
247/340 (72.65%)
35/47 (74.5%)
0.930
264 (81.5%)
31 (47.0%)
<0.001
337 (83.21%)
14 (26.4%)
<0.001
1 - 2 (overweight)
47 (14.5%)
19 (28.8%)
57 (14.07%)
10 (18.9%)
> 2 (obese)
13 (4.0%)
16 (24.2%)
11 (2.72%)
29 (54.72%)
6 (1.8%)
4 (6.1%)
8 (1.98%)
9 (16.98%)
Smoker* Vigorous exercise* BMI z-score -2 - 1 (healthy)
Metabolic syndrome #
0.122
The chi-squared test was used to compare the differences in proportions between NAFLD and non-NAFLD participants. * Data not collected from all participants BMI, body mass index.
<0.001
Table 2. Comparison of clinical characteristics between NAFLD and non-NAFLD subjects. FEMALES Non-NAFLD PWC170 (W) Physical work capacity (W/kg)
#
Physical activity (MET-minutes/wk) * ^ 2 #
Body mass index (kg/m )
#
Body mass index (z-score) Waist circumference (cm) Suprailiac skinfold (cm)
#
Abdominal skinfold (cm) Hemoglobin (g/L)
#
#
#
Energy intake (kJ) * ^ Hematocrit
#
Mean corpuscular volume (fL) Mean cell hemoglobin (pg)
#
Mean corpuscular hemoglobin # concentration (g/L) Red cell count (x 10x12 /L)
#
#
NAFLD
MALES P
Non-NAFLD
NAFLD
P
99.67 (24.30)
98.67 (20.16)
0.725
154.98 (38.85)
153.25 (42.74)
0.781
1.63 (0.38)
1.40 (0.31)
<0.001
2.23 (0.51)
1.70 (0.52)
<0.001
2717 (1313-5191)
2769 (1522-5972)
0.567
4131.67 (2.51)
0.337
22.35 (3.32)
25.94 (4.75)
<0.001
21.99 (2.97)
28.95 (5.79)
<0.001
0.28 (0.91)
1.15 (1.05)
<0.001
0.16 (0.95)
1.82 (1.25)
<0.001
76.21 (9.22)
85.72 (12.16) <0.001
78.73 (7.77)
97.64 (16.18)
<0.001
17.44 (6.96)
26.13 (9.07)
<0.001
11.60 (6.90)
28.43 (10.96)
<0.001
23.84 (7.42)
31.04 (8.44)
<0.001
15.79 (8.43)
31.82 (11.01)
<0.001
13.54 (0.93)
13.62 (0.84)
0.534
15.59 (0.89)
15.45 (0.85)
0.296
7737 (6353-9741)
7057 (5469-8966)
0.016
10812 (8286-13500)
9649 (7487-11337)
0.016
0.39 (0.03)
0.39 (0.03)
0.729
0.45 (0.03)
0.45 (0.03)
0.580
85.64 (3.80)
84.62 (3.44)
0.034
85.76 (3.28)
84.19 (2.95)
<0.001
29.75 (1.58)
29.49 (1.52)
0.205
29.88 (1.31)
29.26 (1.14)
<0.001
347.69 (8.92)
348.53 (9.22)
0.498
348.41 (8.58)
347.58 (7.89)
0.481
4.56 (0.31)
4.63 (0.32)
0.123
5.22 (0.34)
5.29 (0.36)
0.198
3588 (3)
13.25 (17.0-43.4)
28.80 (20/3-45.8)
0.631
55.0 (37.7-75.0)
57.2 (34.6-83.0)
0.463
21.01 (9.28)
20.24 (8.76)
0.517
26.84 (9.98)
22.38 (7.55)
<0.001
14.96 (6.10)
14.46 (5.86)
0.535
17.91 (6.41)
15.25 (4.93)
<0.001
284.82 (64.19)
301.89 (50.43)
0.019
257.84 (50.84)
275.58 (50.53)
0.019
Aspartate aminotransferase (U/L) *
21.0 (19.0-24.0)
21.0 (18.2-23.0)
0.365
25.0 (22.0-30.0)
27.0 (24.0-33.2)
0.022
Alanine aminotransferase (U/L) *
16.0 (12.0-22.0)
16.5 (12.0-22,8)
0.927
22.0 (15.0-26.0)
33.0 (21.0-47.0)
<0.001
Gamma-glutamyl transferase (U/L) *
11.0 (9.0-15.0)
13.0 (10.0-15.0)
0.437
14.0 (11.0-17.0)
18.0 (13.0-30.0)
<0.001
Highly sensitive C-reactive protein (mg/L) *
0.66 (0.3-1.8)
0.98 (0.4-2.0)
0.082
0.37 (0.2-0.8)
0.98 (0.5-2.3)
<0.001
HOMA-IR *
1.53 (1.0-2.2)
2.10 (1.3-3.1)
0.001
1.44 (0.9-2.2)
2.33 (1.4-4.0)
<0.001
Triglycerides (mmol/L) *
0.90 (0.7-1.2)
0.99 (0.7-1.4)
0.226
0.90 (0.7-1.2)
1.11 (0.8-1.6)
0.022
Low density lipoprotein # (mmol/L)
2.42 (0.62)
2.55 (0.72)
0.178
2.23 (0.64)
2.36 (0.80)
0.239
High density lipoprotein # (mmol/L)
1.43 (0.32)
1.34 (0.26)
0.021
1.21 (0.24)
1.10 (0.18)
<0.001
Ferritin (ug/L) * Transferrin saturation (%) Iron (umol/L)
#
#
9
Platelet count (x10 /L)
FEMALES
MALES
Non-NAFLD
NAFLD
P
Non-NAFLD
NAFLD
Adiponectin (mg/L) *
10.30 (7.9-14.1))
8.00 (6.0-11.8)
<0.001
7.40 (5.3-10.7)
6.50 (4.6-8.7)
0.013
Leptin (ug/L) *
22.30 (14.4-35.6)
39.75 (27.1-56.6)
<0.001
2.40 (1.4-5.2)
12.80 (6.2-27.456.6)
<0.001
#
P
Data are presented as mean (standard deviation), and compared across NAFLD and non-NAFLD groups with a t-test. * Data are presented as median (interquartile range), and log-transformed for a t-test comparison between NAFLD and nonNAFLD groups. ^ Data not collected from all participants. HOMA-IR calculated as fasting insulin (mIU/L) x fasting glucose (mmol/L)/22.5
Table 3. Pearson correlation coefficients (95% confidence interval) for linear relationships with physical work capacity FEMALES R -0.42 Body mass index -0.44 Suprailiac skinfold (cm) Abdominal skinfold (cm) -0.39 Waist circumference (cm) -0.38 -0.42 Leptin (ug/L) * -0.19 hs-CRP (mg/L) * -0.26 HOMA-IR * * log-transformed prior to analysis
MALES
(95% CI)
R
(95% CI)
(-0.50,-0.34) (-0.51,-0.35) (-0.47,-0.30) (-0.47,-0.30) (-0.49,-0.33) (-0.29,-0.10) (-0.35,-0.17)
-0.37 -0.49 -0.51 -0.40 -0.54 -0.18 -0.34
(-0.44,-0.29) (-0.56,-0.42) (-0.57,-0.44) (-0.48,-0.32) (-0.60,-0.47) (-0.27,-0.09) (-0.42,-0.25)
Need to Know Background: Iron metabolism is impaired in obese individuals and iron deficiency has been associated with physical inactivity. We investigated whether iron bioavailability is reduced in adolescents with non-alcoholic fatty liver disease (NAFLD) and contributes to reduced cardiorespiratory fitness. Findings: We associated NAFLD with decreased cardiorespiratory fitness, independent of body mass index. An association between transferrin saturation and physical work capacity indicates that functional iron deficiency might contribute to reductions in cardiorespiratory fitness in patients with NAFLD. Implications for patient care: Adolescents with NAFLD should be assessed for functional iron deficiency prior to instituting lifestyle interventions.
PII: DOI: Reference:
S1542-3565(19)31116-4 https://doi.org/10.1016/j.cgh.2019.10.017 YJCGH 56804
To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 11 October 2019 Please cite this article as: Mitchell T, McKinnon E, Ayonrinde O, Adams LA, Trinder D, Chua ACG, Newton RU, Straker L, Olynyk JK, Decreased Physical Working Capacity in Adolescents With NonAlcoholic Fatty Liver Disease Associates With Reduced Iron Availability, Clinical Gastroenterology and Hepatology (2019), doi: https://doi.org/10.1016/j.cgh.2019.10.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 by the AGA Institute
1 Title Page Title: Decreased Physical Working Capacity in Adolescents With Non-Alcoholic Fatty Liver Disease Associates With Reduced Iron Availability
Short Title: PWC in NAFLD Relates to Iron Bioavailability
Authors: Tim Mitchell1, Elizabeth McKinnon2, Oyekoya Ayonrinde1,3, Leon A. Adams3,4, Debbie Trinder3,5, Anita C. G. Chua3,5, Robert U. Newton6, Leon Straker6,7, John K. Olynyk1,8.
Affiliations: 1
Department of Gastroenterology, Fiona Stanley Fremantle Hospital Group, Perth, Australia
2
Telethon Kids Institute, Perth, Australia
3
Medical School, University of Western Australia, Perth, Australia
4
Department of Hepatology, Sir Charles Gairdner Hospital, Perth, Australia
5
Harry Perkins Institute for Medical Research, Perth, Australia
6
Exercise Medicine Research Institute, Edith Cowan University, Perth, Australia
7
School of Physiotherapy and Exercise Science, Curtin University, Perth, Australia
8
School of Medical and Health Sciences, Edith Cowan University, Perth, Australia
Grant Support: Nil
2
List of Abbreviations: NAFLD: non-alcoholic fatty liver disease MCV: mean corpuscular volume MCH: mean corpuscular haemoglobin PWC170: physical work capacity at 170 bpm PWC: physical work capacity
Correspondence: Corresponding Author: Tim Mitchell Mail: Gastroenterology Department, Fiona Stanley Hospital, 11 Robin Warren Drive, Murdoch WA 6150 Phone: (m) +61 403 781 702 (w) +61 8 6152 2827 Fax: +61 8 9335 6155 Email: [email protected]
Disclosures: Nil
Conflicts of Interest: None of the authors have any conflicts of interest to declare.
Writing Assistance: Nil
3
Author Contributions: TM: analysis and interpretation of data, drafting of the manuscript; EM: statistical analysis, drafting of the manuscript; OA, LAA, DT, ACGC, RUN, LS: critical revision of the manuscript; JKO: study concept and design, supervision and critical revision of the manuscript.
Acknowledgements The authors are grateful to the Raine Study participants and their families, the Raine Study team for cohort co-ordination and data collection and the NHMRC for their long-term contribution to funding the study. The core management of the Raine Study is funded by the University of Western Australia, Curtin University, Women and Infants Research Foundation, Telethon Kids Institute, Edith Cowan University, Murdoch University, the University of Notre Dame and the Raine Medical Research Foundation. The data collection of the Gen2-17 year follow up of the Raine Study was funded by NHMRC Grant 353514 and biological specimen collection was funded by NHMRC Grant 403981.
4 Abstract Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is common and related to obesity and insulin resistance. Iron metabolism is impaired in obese individuals and iron deficiency has been associated with physical inactivity. We investigated whether iron bioavailability is reduced in patients with NAFLD and contributes to reduced cardiorespiratory fitness.
Methods: We collected information on weight-adjusted, submaximal physical work capacity (PWC), ultrasound-determined hepatic steatosis, iron indices, and hematologic and metabolic parameters from 390 female and 458 male participants of the Raine Study—a longitudinal study of disease development in 2868 children in Western Australia. X2 and linear regression analyses were used to compare characteristics of study participants according to NAFLD status at age 17 years.
Results: Fourteen percent of the cohort had NAFLD. PWC was significantly reduced in adolescents with NAFLD compared to adolescents without NAFLD (reduction of 0.17 W/kg, P=.0003, adjusted for sex and body mass index [BMI]). Iron bioavailability (assessed by mean corpuscular volume [MCV], mean corpuscular haemoglobin [MCH], transferrin saturation, and serum levels of iron) was inversely correlated with BMI in adolescents with NAFLD (P≤.01 for all, adjusted for sex) but not in adolescents without NAFLD (P>.30). MCV and MCH correlated with PWC (MCV, P=.002 for female and P=.0003 male participants; MCH, P=.004 for female and P=.01 for male participants), irrespective of NAFLD status. Reduced PWC was associated with lower transferrin saturation in adolescents with NAFLD (reduction of 0.012 W/kg per unit decrease in transferrin saturation, P=.007) but not in
5 adolescents without NAFLD (reduction of 0.001 W/kg, P=.40), adjusted for sex. This association was independent of MCV or MCH.
Conclusions: In a well-defined cohort of adolescents, we found NAFLD to be associated with decreased cardiorespiratory fitness, independent of BMI. The relationship between transferrin saturation and PWC in adolescents with NAFLD indicates that functional iron deficiency might contribute to reductions in cardiorespiratory fitness.
Key words: steatosis, fitness, exercise, ferritin, metabolism
6 Introduction Chronic liver disease is an increasing cause of premature morbidity and mortality1. In Western countries, liver-related mortality rates have increased 400% since 1970, contrasting with substantial improvements in most other common chronic diseases1,2. A major contributor to the increasing burden is non-alcoholic fatty liver disease (NAFLD), a chronic liver disorder related to obesity and the metabolic syndrome3. The spectrum of disease ranges from simple steatosis through steatohepatitis with varying degrees of fibrosis, including cirrhosis2,4.
The prevalence of NAFLD is increasing and affects 25% of adults and 17% of adolescents5,6. The disease burden is high with NAFLD expected to become the leading indication for liver transplantation in the United States2. Current pharmacotherapy is restricted to certain highrisk groups2. Although numerous clinical trials are underway, the primary advice for patients is to modify their diet to promote weight loss and commence regular exercise7,8. Clinical trials examining exercise for NAFLD have demonstrated improvements in hepatic fat content independent of weight loss9,10. The specific exercise regimen, for example resistance training vs aerobic activity or moderate vs high intensity, does not appear to influence the efficacy of hepatic fat reduction11,12.
Unfortunately, such lifestyle advice when delivered in the clinic is rarely successful and on review, patients will often cite a lack of energy or fitness as a barrier to exercise13-15. Additionally, patients with stronger cardiorespiratory performance have been shown to respond better to lifestyle interventions and have lower cardiovascular mortality, which is the leading cause of death in NAFLD patients2,16,17. The possibility of a modifiable factor
7 inherent in NAFLD patients that reduces physical work capacity has not previously been studied, even though addressing this may improve patient engagement in exercise programs16,18,19.
Iron deficiency is a known cause of decreased physical work capacity in healthy and chronically ill individuals, even in the absence of related anaemia20. Prior studies have shown that iron deficiency is common in chronic inflammatory conditions such as congestive cardiac failure and is associated with worse outcomes, including decreased exercise tolerance20.
Iron deficiency is associated with inflammation and obesity but little is known about iron metabolism in NAFLD21. The assessment of iron stores in NAFLD is complex, given that elevated ferritin is common in NAFLD patients22. The high prevalence of obesity in NAFLD can make it difficult to delineate the impacts of NAFLD and iron metabolism on each other23. It is thought that the effects of obesity and NAFLD on impaired iron metabolism are mediated via dysregulated production of the key iron regulatory hormone hepcidin23. Production of hepcidin is stimulated by chronic inflammation and is associated with obesity, thereby reducing iron bioavailability24,25.
In this study, we aimed to determine if NAFLD was associated with reduced cardiorespiratory fitness in a well-defined cohort of community-dwelling individuals in late adolescence. Thereafter, we investigated whether impaired iron bioavailability as measured by mean cell volume (MCV), mean cell haemoglobin (MCH), serum iron and transferrin saturation was associated with impaired cardiorespiratory fitness, and may represent a
8 potential future therapeutic target to improve weight reduction and exercise treatment in NAFLD.
9 Methods Study population This project was a cross-sectional study using the Raine Study, a well described, prospectively enrolled cohort of 2868 live-born children from 2900 pregnancies26. The Raine Study examines the evolution of health or disease during pregnancy, early childhood and adolescence and is representative of the broader Western Australian population26. The background and available data of the Raine Study Gen2 participants have been previously described26. All active members of the Raine Study Gen2 were invited to participate in the 17-year old assessment, conducted between July 2006 and June 2009. Exclusion criteria included a diagnosis of haemochromatosis or coeliac disease, active inflammation or chronic illness, and pregnancy. Institutional ethics committee approval was obtained from the human research ethics committee of Princess Margaret Hospital for Children. Signed informed parental consent and adolescent assent were obtained prior to study participation.
Assessment The 17-year follow-up comprised detailed health questionnaires, abdominal ultrasound, anthropometric (height, weight, skin fold thickness, waist circumference), cardiovascular (resting heart rate, systolic and diastolic blood pressure) and biochemical assessments. Information on alcohol intake over the previous year was documented by self-reporting and by the completion of a validated, semiquantitative food frequency questionnaire27. Physical activity was assessed using the International Physical Activity Questionnaire, a validated, self-administered questionnaire which recalls physical activity in the previous week26. Comprehensive medication and medical histories were documented to exclude secondary
10 causes of NAFLD and concomitant liver disease. Testing for hepatitis B or C virus infections was not performed because notification rates for hepatitis B and C virus infections were on average less than 24/100000 and 23/100000, respectively, for Western Australian teenagers between the ages of 15 and 19 over the study period28. Body mass index (BMI) z-scores were derived from age- and gender-adjusted growth curves that accord with the World Health Organisation Child Growth Standards for school-aged children and adolescents and BMI cut-offs for adults29. Participants with z-scores from -2 to 1 were classified as healthy weight, 1-2 as overweight and >2 as obese29. Metabolic syndrome was defined according to age- and gender-specific criteria of the International Diabetes Federation30. Laboratory assessments were performed with venous blood samples taken after an overnight fast. All laboratory assays were performed at an accredited central laboratory (PathWest Laboratories, Perth, Western Australia).
Ultrasound Hepatic steatosis was determined using previously described ultrasound characteristics which provide 92% sensitivity and 100% specificity for the histological diagnosis of >10% hepatic steatosis31. Trained ultrasonographers performed liver ultrasound assessments28. The ultrasound images were reviewed and a total fatty liver score was determined from the captured images by a single radiologist who was blinded to the clinical and laboratory characteristics of the subjects28. The scoring system has been previously reported and assesses three parameters: liver echotexture (0-3), deep attenuation (0-2) and vessel blurring (0-1)28. The diagnosis of hepatic steatosis required a total score of ≥2, which included an echotexture score of ≥128. Severity of hepatic steatosis was classified by the total fatty liver score, as no steatosis (0-1), mild steatosis (2-3) or moderate to severe
11 steatosis (4-6)28. NAFLD diagnosis required sonographic evidence of hepatic steatosis and the exclusion of significant alcohol consumption, defined as weekly alcohol intake of less than 210g and 140g for males and females, respectively2.
Physical Work Capacity Cardiorespiratory fitness was assessed using a standardised protocol for submaximal cycle ergometry using the Monark cycle ergometer32,33. Participants cycled at 50-60 revolutions per minute throughout the test, initially at a workload of 25 watts for two minutes, which increased to 50 watts for two minutes and finally 75 watts for two minutes33. Workload output was regressed against heart rate and the maximal steady-state power attained for a heart rate of 170 beats per minute (PWC170) to determine cardiorespiratory fitness33. PWC170, which correlated strongly with body weight in healthy-weight participants (P<.0001) was divided by weight to normalise the effects of body size on workload output. The normalised PWC170 result, which we refer to as physical work capacity, was used for analysis.
Statistical analysis Chi-squared tests were used to compare demographic characteristics of study participants according to NAFLD status. Physical work capacity, anthropometric measures and biochemical characteristics were compared using Student t-tests, after log-transformation of those variables having a skewed distribution, and relationships assessed by correlation and multivariable linear regression. Analysis was undertaken using RStudio (RStudio, Inc., Boston, Massachusetts).
12 Results Data including PWC170 results and a valid NAFLD diagnosis were available from 848 Raine Study participants at 17 years of age (390 females, 458 males). Compared with other participants of the 17-year follow-up who lacked either PWC170 results or a valid NAFLD assessment (N=200), study participants did not differ significantly in either mean BMI (females: P=.10, males: P=.60) or the proportion of Caucasian European ethnicity (P>.30).
Overall, NAFLD was diagnosed in 14% of participants: 66/390 females (16.9%) and 53/458 males (11.6%). NAFLD and non-NAFLD participants were similar with respect to Caucasian ethnicity, smoking status and recent participation in vigorous exercise (P>.30, Table 1). Overall, BMI z-scores were greater in NAFLD participants, however the distribution of these scores varied between male and female NAFLD adolescents (P=.003). Nearly 50% of NAFLD females were in the healthy weight range with 29% and 24% classified as overweight and obese, respectively (Table 1). More than 50% of NAFLD males were obese. In keeping with this disproportionate prevalence of obesity, 17% of NAFLD males satisfied criteria for a diagnosis of metabolic syndrome, significantly more than non-NAFLD males (2%, P<.0001) and marginally more than NAFLD females (6%, P=.08). On average, adolescents with NAFLD had greater suprailiac and abdominal skinfold thickness measures than non-NAFLD adolescents with the same BMI. In males, the mean difference in suprailiac skin fold thickness was 5.6 cm (P<.0001) and abdominal skinfold thickness was 3.6 cm (P<.0001), adjusted for BMI. In females, the mean difference in suprailiac skinfold thickness was 3.3 cm (P=.002) and abdominal skinfold thickness was 2.1 cm (P=.01), adjusted for BMI.
13 Physical work capacity (PWC), as measured by PWC170 normalized by body weight, was significantly lower, on average, for females compared with males (females: mean (SE) 1.59 (0.4) W/kg; males 2.17 (0.5) W/kg, P<.0001), and was markedly reduced in those with NAFLD compared to those without (Table 2; females: mean (SE) difference -0.23 (0.05) W/kg, P<.0001; males: -0.53 (0.07) W/kg, P<.0001). This reduction in capacity remained significant when controlling for BMI and gender (mean (SE) difference -0.17 (0.05) W/kg, P=.0003). Reduced physical work capacity was strongly associated with greater adiposity for all measures considered (Table 3), and this was observed in both non-NAFLD and NAFLD participants. However, an increasing disparity in fitness between those with and without NAFLD was evident across BMI classifications (Figure 1).
For both males and females, the observed impact of NAFLD on PWC was closely paralleled by increasing suprailiac skinfold thickness and higher leptin levels, more so than elevated BMI, or hs-CRP as a marker of the low-level inflammation associated with obesity. Importantly, the effect of NAFLD on PWC remained when the analysis was restricted to nonobese individuals (mean difference -0.22 W/kg, P<.0001), or when considering PWC170 without normalisation but controlling for weight in the regression modelling (mean difference in PWC170, males -30.6W, P<.0001; females -8.5W, P<.0001).
Iron and haematological parameters were also examined for inter-relationships with adiposity, cardiorespiratory fitness and NAFLD. MCV, MCH, transferrin saturation and serum iron levels all showed significant reductions with increasing BMI in those with NAFLD (P≤.01, adjusted for gender) that were absent in those without NAFLD (P>.30) (Figure 2). In contrast, serum ferritin increased with increasing BMI, with trends similar for non-NAFLD
14 and NAFLD participants (P=.80 for interaction term). There was also a significant positive association of haemoglobin with BMI in non-NAFLD participants only (P≤.0001).
MCV and MCH were both positively associated with PWC (MCV: P=.002 females, P=.0003 males; MCH: P=.004 females, P=.01 males), irrespective of NAFLD status (P=.80 for interaction). The significant association between decreased PWC and reduced transferrin saturation levels, however, was evident only in those with NAFLD (NAFLD: mean (SE) change -0.012 (0.004) W/kg per unit decreased in transferrin saturation, P=.007; non-NAFLD: -0.001 (0.002) W/kg, P=.40; adjusted for gender). Moreover, even when controlling for MCV, reduced PWC was significantly associated with lower transferrin saturation levels in NAFLD adolescents (mean (SE) change -0.011 (0.005) W/kg per unit decrease in transferrin saturation, P=.03), but not in non-NAFLD participants (P>.90) (Figure 3). Joint modelling of associations between PWC and serum iron levels considered with either MCV or MCH yielded similar results.
15
Discussion Currently, few effective treatment options exist for NAFLD and the emphasis for patients seen in the clinic is on lifestyle modification2. Unfortunately, engagement with weight reduction strategies and exercise programs outside clinical trial settings is disappointing, with patients commonly citing lethargy as a barrier13. Our observed prevalence of NAFLD in this well-characterised, otherwise healthy cohort accords with other adolescent studies which range from 10% using ALT and elevated BMI34, to 17% defined as >5% steatosis at autopsy6. Notably, we have found adolescents with NAFLD have poorer physical work capacity, a recognised marker of cardiorespiratory fitness, which may contribute to poorer engagement with exercise programs. The impact of NAFLD, which becomes more marked as adiposity increases, remains significant when adjusted by weight and appears independent of low-level inflammation.
Our data suggests that patients with NAFLD are less fit than their non-NAFLD counterparts of similar BMI, indicating a high-risk chronic disease phenotype. This finding has important clinical relevance as prior research has highlighted that fitness can ameliorate some of the detrimental effects of fatness33. Clinical trials investigating the efficacy of exercise modification in NAFLD have shown that neither the intensity nor type of exercise affected outcomes in participating patients, suggesting that patients do not need a minimum level of fitness prior to engaging in exercise11,12. Unfortunately, all trials are affected by selection bias where motivated patients are more likely to both participate and engage with a clinical trial resulting in underwhelming “real-world” results in patients at highest risk of a poor outcome14. Our study suggests that despite participating in a similar amount of every-day
16 exercise, patients with NAFLD are less fit, which may limit the effectiveness of exercise interventions and contribute to high relapse rates following clinical trial conclusion. Nonetheless, the importance of encouraging physical exercise in this cohort is confirmed by our study.
Having established that patients with NAFLD have decreased physical work capacity we investigated whether a modifiable factor for this could be identified. Iron deficiency, even without anaemia, has been shown to affect energy levels and cause fatigue35,36. Functional iron deficiency is well established in pro-inflammatory conditions, most notably chronic kidney disease and is defined as a decreased transferrin saturation (<20%) in the context of adequate ferritin levels (>100 mcg/L)37. In this condition, iron stores are thought to be adequate but are unable to be mobilised when required through the reticuloendothelial system37. The complex dynamic between iron and NAFLD is not well understood but iron has been suggested as a cofactor for liver injury through oxidative stress and insulin resistance22. However, an effort to modify this effect through phlebotomy for patients with NAFLD and an elevated ferritin without hereditary haemochromatosis did not show benefit in a randomised control trial22.
In our study, despite normal or elevated ferritin levels, a decreased transferrin saturation correlated with decreased physical work capacity in adolescents with NAFLD. In adolescents without NAFLD there was no correlation between transferrin saturation and physical work capacity. These results suggest that functional iron deficiency is present in NAFLD patients and may independently contribute to decreased cardiorespiratory fitness. This finding is in keeping with data derived from patients with heart failure20. Furthermore, a decrease in
17 MCV correlated with reduced physical work capacity, and MCV levels were notably lower in NAFLD patients. MCV is a widely available and well recognised haematological parameter with low levels closely associated with iron deficiency in a Caucasian population38. This correlation with physical work capacity further suggests that iron deficiency may contribute to decreased cardiorespiratory fitness.
The role of functional iron deficiency has been assessed in other chronic inflammatory disease states such as chronic obstructive pulmonary disease and congestive cardiac failure20,39. Despite an association between iron deficiency and decreased exercise tolerance in these patient groups, intervention trials investigating iron replacement in chronic cardiac failure have had mixed outcomes for overall survival and symptomatic benefit40,41. Antiinflammatory and anti-cytokine medications, such as pentoxifylline, have been studied in anaemia of chronic kidney disease previously with mixed results42 but no such studies have been performed in NAFLD. Such an intervention may be a basis of future research, aiming to improve outcomes from lifestyle interventions.
Despite our promising results from this large, representative cohort of adolescents it should be noted that this study has some limitations. The cross-sectional study design does not allow causal relationships to be determined. Although we have controlled for known confounding factors and the cohort was well characterised, unidentified confounding factors, such as renal disease, thalassemia, excessive sedentary behaviour or dietary influences, may have affected our results. Testing for thalassemia was not performed in this cohort as the rate of this condition in a general Australian population is low. Finally, we do
18 not have data for hepcidin levels in this cohort, which would further strengthen the assessment of functional iron deficiency.
In conclusion, we have demonstrated that in a well-defined cohort of adolescents, the presence of NAFLD is independently associated with decreased cardiorespiratory fitness. Furthermore, functional iron deficiency in the NAFLD cohort, as demonstrated by the association between decreased transferrin saturation and physical work capacity, may be a novel explanation for this finding. Future therapies aiming to modify the inflammatory milieu of NAFLD may increase iron bioavailability and as such improve physical fitness and engagement in exercise programs.
19 Figure Legends: Figure 1. Mean physical work capacity ± 2 SE, estimated from a linear model adjusted for BMI z-score and sex and plotted at the combined group-average values.
Figure 2. Mean ± 2 SE of iron and haematological indices, estimated from linear models adjusted for sex and BMI z-score, and plotted at the combined group-average values. Greater disparities between indices of NAFLD and non-NAFLD participants are evident with increasing adiposity (* 0.01
Figure 3. Estimated physical work capacity (PWC) for those with and without NAFLD as a function of mean corpuscular volume (MCV) and transferrin saturation (TSAT), adjusted for sex.
20
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Table 1. Cohort demographics. FEMALE Non-NAFLD N=324 Caucasian
NAFLD N=66
MALE P-value
#
Non-NAFLD N=405
NAFLD N=53
P-value
#
275 (84.9%)
57 (86.4%)
0.905
350 (86.42%)
43 (81.1%)
0.408
73 (22.5%)
10/65 (15.4%)
0.264
67/401 (16.71%)
11/50 (22.0%)
0.463
151/292 (51.7%)
32/63 (50.8%)
0.999
247/340 (72.65%)
35/47 (74.5%)
0.930
264 (81.5%)
31 (47.0%)
<0.001
337 (83.21%)
14 (26.4%)
<0.001
1 - 2 (overweight)
47 (14.5%)
19 (28.8%)
57 (14.07%)
10 (18.9%)
> 2 (obese)
13 (4.0%)
16 (24.2%)
11 (2.72%)
29 (54.72%)
6 (1.8%)
4 (6.1%)
8 (1.98%)
9 (16.98%)
Smoker* Vigorous exercise* BMI z-score -2 - 1 (healthy)
Metabolic syndrome #
0.122
The chi-squared test was used to compare the differences in proportions between NAFLD and non-NAFLD participants. * Data not collected from all participants BMI, body mass index.
<0.001
Table 2. Comparison of clinical characteristics between NAFLD and non-NAFLD subjects. FEMALES Non-NAFLD PWC170 (W) Physical work capacity (W/kg)
#
Physical activity (MET-minutes/wk) * ^ 2 #
Body mass index (kg/m )
#
Body mass index (z-score) Waist circumference (cm) Suprailiac skinfold (cm)
#
Abdominal skinfold (cm) Hemoglobin (g/L)
#
#
#
Energy intake (kJ) * ^ Hematocrit
#
Mean corpuscular volume (fL) Mean cell hemoglobin (pg)
#
Mean corpuscular hemoglobin # concentration (g/L) Red cell count (x 10x12 /L)
#
#
NAFLD
MALES P
Non-NAFLD
NAFLD
P
99.67 (24.30)
98.67 (20.16)
0.725
154.98 (38.85)
153.25 (42.74)
0.781
1.63 (0.38)
1.40 (0.31)
<0.001
2.23 (0.51)
1.70 (0.52)
<0.001
2717 (1313-5191)
2769 (1522-5972)
0.567
4131.67 (2.51)
0.337
22.35 (3.32)
25.94 (4.75)
<0.001
21.99 (2.97)
28.95 (5.79)
<0.001
0.28 (0.91)
1.15 (1.05)
<0.001
0.16 (0.95)
1.82 (1.25)
<0.001
76.21 (9.22)
85.72 (12.16) <0.001
78.73 (7.77)
97.64 (16.18)
<0.001
17.44 (6.96)
26.13 (9.07)
<0.001
11.60 (6.90)
28.43 (10.96)
<0.001
23.84 (7.42)
31.04 (8.44)
<0.001
15.79 (8.43)
31.82 (11.01)
<0.001
13.54 (0.93)
13.62 (0.84)
0.534
15.59 (0.89)
15.45 (0.85)
0.296
7737 (6353-9741)
7057 (5469-8966)
0.016
10812 (8286-13500)
9649 (7487-11337)
0.016
0.39 (0.03)
0.39 (0.03)
0.729
0.45 (0.03)
0.45 (0.03)
0.580
85.64 (3.80)
84.62 (3.44)
0.034
85.76 (3.28)
84.19 (2.95)
<0.001
29.75 (1.58)
29.49 (1.52)
0.205
29.88 (1.31)
29.26 (1.14)
<0.001
347.69 (8.92)
348.53 (9.22)
0.498
348.41 (8.58)
347.58 (7.89)
0.481
4.56 (0.31)
4.63 (0.32)
0.123
5.22 (0.34)
5.29 (0.36)
0.198
3588 (3)
13.25 (17.0-43.4)
28.80 (20/3-45.8)
0.631
55.0 (37.7-75.0)
57.2 (34.6-83.0)
0.463
21.01 (9.28)
20.24 (8.76)
0.517
26.84 (9.98)
22.38 (7.55)
<0.001
14.96 (6.10)
14.46 (5.86)
0.535
17.91 (6.41)
15.25 (4.93)
<0.001
284.82 (64.19)
301.89 (50.43)
0.019
257.84 (50.84)
275.58 (50.53)
0.019
Aspartate aminotransferase (U/L) *
21.0 (19.0-24.0)
21.0 (18.2-23.0)
0.365
25.0 (22.0-30.0)
27.0 (24.0-33.2)
0.022
Alanine aminotransferase (U/L) *
16.0 (12.0-22.0)
16.5 (12.0-22,8)
0.927
22.0 (15.0-26.0)
33.0 (21.0-47.0)
<0.001
Gamma-glutamyl transferase (U/L) *
11.0 (9.0-15.0)
13.0 (10.0-15.0)
0.437
14.0 (11.0-17.0)
18.0 (13.0-30.0)
<0.001
Highly sensitive C-reactive protein (mg/L) *
0.66 (0.3-1.8)
0.98 (0.4-2.0)
0.082
0.37 (0.2-0.8)
0.98 (0.5-2.3)
<0.001
HOMA-IR *
1.53 (1.0-2.2)
2.10 (1.3-3.1)
0.001
1.44 (0.9-2.2)
2.33 (1.4-4.0)
<0.001
Triglycerides (mmol/L) *
0.90 (0.7-1.2)
0.99 (0.7-1.4)
0.226
0.90 (0.7-1.2)
1.11 (0.8-1.6)
0.022
Low density lipoprotein # (mmol/L)
2.42 (0.62)
2.55 (0.72)
0.178
2.23 (0.64)
2.36 (0.80)
0.239
High density lipoprotein # (mmol/L)
1.43 (0.32)
1.34 (0.26)
0.021
1.21 (0.24)
1.10 (0.18)
<0.001
Ferritin (ug/L) * Transferrin saturation (%) Iron (umol/L)
#
#
9
Platelet count (x10 /L)
FEMALES
MALES
Non-NAFLD
NAFLD
P
Non-NAFLD
NAFLD
Adiponectin (mg/L) *
10.30 (7.9-14.1))
8.00 (6.0-11.8)
<0.001
7.40 (5.3-10.7)
6.50 (4.6-8.7)
0.013
Leptin (ug/L) *
22.30 (14.4-35.6)
39.75 (27.1-56.6)
<0.001
2.40 (1.4-5.2)
12.80 (6.2-27.456.6)
<0.001
#
P
Data are presented as mean (standard deviation), and compared across NAFLD and non-NAFLD groups with a t-test. * Data are presented as median (interquartile range), and log-transformed for a t-test comparison between NAFLD and nonNAFLD groups. ^ Data not collected from all participants. HOMA-IR calculated as fasting insulin (mIU/L) x fasting glucose (mmol/L)/22.5
Table 3. Pearson correlation coefficients (95% confidence interval) for linear relationships with physical work capacity FEMALES R -0.42 Body mass index -0.44 Suprailiac skinfold (cm) Abdominal skinfold (cm) -0.39 Waist circumference (cm) -0.38 -0.42 Leptin (ug/L) * -0.19 hs-CRP (mg/L) * -0.26 HOMA-IR * * log-transformed prior to analysis
MALES
(95% CI)
R
(95% CI)
(-0.50,-0.34) (-0.51,-0.35) (-0.47,-0.30) (-0.47,-0.30) (-0.49,-0.33) (-0.29,-0.10) (-0.35,-0.17)
-0.37 -0.49 -0.51 -0.40 -0.54 -0.18 -0.34
(-0.44,-0.29) (-0.56,-0.42) (-0.57,-0.44) (-0.48,-0.32) (-0.60,-0.47) (-0.27,-0.09) (-0.42,-0.25)
Need to Know Background: Iron metabolism is impaired in obese individuals and iron deficiency has been associated with physical inactivity. We investigated whether iron bioavailability is reduced in adolescents with non-alcoholic fatty liver disease (NAFLD) and contributes to reduced cardiorespiratory fitness. Findings: We associated NAFLD with decreased cardiorespiratory fitness, independent of body mass index. An association between transferrin saturation and physical work capacity indicates that functional iron deficiency might contribute to reductions in cardiorespiratory fitness in patients with NAFLD. Implications for patient care: Adolescents with NAFLD should be assessed for functional iron deficiency prior to instituting lifestyle interventions.