What Is Known
- Cardiometabolic dysregulation is reported in up to 25% of children post-liver transplantation.
- Cardiometabolic dysregulation is typically expressed in obesity, diabetes mellitus, hypertension, and dyslipidemia in youth post-liver transplantation.
- Cardiometabolic dysregulation risk is potentiated by long-term immunosuppression in children post-liver transplantation.
- Most studies examining cardiometabolic dysregulation risk in children post-liver transplantation are cross-sectional, with limited information regarding longitudinal evolution of cardiometabolic dysregulation risk factors in youth post-liver transplantation.
What Is New
- In children with healthy body weights post-liver transplantation, declining fat mass in the presence of low lean body mass is associated with healthy body weights and low cardiometabolic dysregulation risk over 10 years.
Pediatric liver transplantation (LTx) is a lifesaving procedure for children and adults with end-stage liver disease. One of the major comorbid conditions in adults post-LTx has been the increasing prevalence of obesity and associated development of cardiometabolic dysregulation (CMD) leading to reduced graft survival and increased morbidity and mortality in adult LTx recipients (1–3). More recent data indicate that risk for CMD is prevalent in pediatric LTx recipients; affecting up to 25% of children in the post-LTx period (4–9). CMD in children post-LTx has typically been expressed in the clustering of dyslipidemia, diabetes, and hypertension with central and total obesity; all of which has potential implications for long-term cardiovascular (CVD) risk, mortality, and morbidity in children post-LTx. Most of these studies have, however, been short-term, cross-sectional by design (4–10), with no data available regarding the longitudinal evolution of CMD and the associations with growth and body composition in pediatric liver LTx recipients.
The major factors that have been identified as risk factors contributing to increased CMD risk in pediatric LTx recipients include post-LTx obesity, diabetes and immunosuppressive medications such as corticosteroids (CSTs), tacrolimus, and sirolimus. Tacrolimus, a calcineurin inhibitor, may contribute to impairment of skeletal muscle insulin sensitivity by suppressing insulin secretion from pancreatic β-cells, muscle growth, and muscle regeneration; and have longer-lasting effects than other immunosuppressive therapies (11–13). In turn, CST may contribute to increasing insulin resistance, reduced protein turnover, and visceral adiposity by promoting alterations in protein and fat metabolism both in the skeletal muscle and adipose tissue (14). Recent evidence suggests that mycophenolate mofetil (MMF) has a lower risk of late CVD complications in adults post-LTx (14). The use of CST-free regimens combined with tacrolimus and MMF has been associated with modest CVD risk reduction, without any changes in rejection risk in adults post-LTx (14). In children, limited information is available regarding CMD risk in children who have undergone LTx without CST exposure, particularly in the long term. In 2003, our centre implemented a CST-free protocol enabling the ability to evaluate the effect of this type of protocol on CMD risk in children and adolescents post-LTx (15). The study purpose was to examine the associations between longitudinal CMD expression, growth, and body composition in children post-LTx. We hypothesized that children post-LTx with healthy body weights and CST minimization would have a low expression of CMD over 10 years.
A retrospective chart review was conducted of all medical records and dual x-ray absorptiometry (DXA) reports between 1994 and 2015 of infants and children (n = 82, 1–17.9 years at time of LTx) who have undergone LTx at the Pediatric Liver Transplant Clinic at the Stollery Children's Hospital in Edmonton, Alberta, Canada. This program services geographically the largest and most remote referral for LTx in North America. Thirty-four charts were excluded from the review due to lack of available DXA scan and/or missing serum data related to CMD risk at multiple time points (n < 2 time points). There were no significant differences in age at transplant, graft type, weight for age z scores, and height for age z scores observed between children included and excluded in this review (P > 0.05).
Data were collected at time of liver transplant assessment, LTx and at time of yearly clinic appointments (n = 47) for 10 years after the date of LTx. Primary outcome variables included fasting markers of metabolic dysregulation (serum concentrations of insulin, glucose, hemoglobin A1C [A1C], homeostasis model assessment for insulin resistance [HOMA-IR] (abnormal values > 3), lipid panel [triglycerides (TGs), total cholesterol [TC], high-density lipoprotein cholesterol [HDL-cholesterol], low-density lipoprotein cholesterol [LDL cholesterol]) and resting blood pressure [BP]. These were collected for years 1 to 10 post-LTx at time of the annual follow-up LTx clinic visits. The HOMA-IR (glucose mmol/L × insulin mU/L/22.5) was used as an index of insulin resistance (16). Systolic/diastolic BP was converted to percentiles and classified as normal, hypertension according to the National High Blood Pressure Education Program Working group standards (17,18). Systolic/diastolic hypertension was defined as BP >95th percentile for age-sex and/or use of antihypertensive medications in the presence of normal/abnormal pressures (18).
Patients were classified as having CMD according to the Adult Treatment Panel III (ATP-III) criteria (19–21) with modified cut-offs for age-specific criteria (22). ATP-III defines metabolic syndrome by having any 3 of obesity, elevated TG, BP, reduced HDL-cholesterol, and impaired fasting glucose. For the purposes of this study, the parameter “impaired fasting glucose” was modified to include elevated fasting glucose, hemoglobin A1C, abnormal insulin, and/or HOMA-IR >3. Age-specific cut-off for each parameter was also defined: elevated serum TG (>95th percentile), reduced HDL cholesterol (<5th percentile), obesity (body mass index [BMI] >95th percentile), hypertension (BP>95th percentile, or on antihypertensive medication), abnormal glucose homeostasis (fasting glucose >6.1 mmol/L, HbA1c >6.1%, elevated or reduced insulin [<35 pmol/L] or HOMA-IR >3) (4,23,24).
Secondary outcome variables included medications including immunosuppressive therapy (type/dose/duration/trough levels), anthropometrics (weight, weight z, height, height z, BMI, BMI-z), and demographics (age at LTx, age at DXA, liver disease diagnosis) were collected at time of LTx assessment, LTx and for 10 years at yearly annual clinical appointments. Liver biochemistries (international normalized ratio, prothromin timed test, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase, total/conjugated bilirubin), serum urea, creatinine, Pediatric End Stage Liver Diseases and Model for End-Stage Liver Disease scores were calculated at LTx and at annual clinic appointments for 10 years according to United Network for Organ Sharing/Organ Procurement and Transplantation Network (25). This information is collected at routine annual clinical assessments according to standardized protocols within the LTx Program at the Stollery Children's Hospital. Routine clinical blood work (including lipid panel, insulin, A1C) was typically collected on the same day (±1 week) at annual clinic DXA scans and were performed in the Core Laboratory at Alberta Health Services according to standard methodologies (26). Immunosuppressive therapy was described as early (IV corticosteroid therapy) or induction therapy (eg, IV basiliximab at day 0 and day 4) or maintenance therapy (tacrolimus, cyclosporine, and mycophenolate mofetil or MMF). Immunosuppression was assessed as either early therapy/induction therapy (1–3 months) or maintenance therapy by dose (absolute or cumulative), duration, and/or trough levels where available (tacrolimus). Most subjects were weaned off of MMF after 1 year of twice daily administration. Median (interquartile range) length for MMF therapy was 0.57 (0.48–1.86) years. Sirolimus was prescribed in 4 children for chronic rejection precluding the ability to perform any data analysis related to sirolimus’ impact on CMD expression. All 4 patients had sirolimus trough levels within therapeutic targets (data not shown).
Body composition was measured using DXA (Hologic 4500A with Apex System 2.4.2.). DXA was performed yearly at annual clinical F/U visits at the same time as routine clinical blood work and anthropometric measures in all children older than 3 years. This included total/regional (absolute/T- and z scores) fat mass (FM), fat-free mass, lean mass (LM) as described previously (27,28). FM index = (FM (kg)/[height (m)]2 and lean mass index = (LST/)/[height (m)]2), were calculated and compared to reference values (29). Skeletal muscle mass was calculated based on age-sex-matched normal values (29).
Anthropometric data (weight and height) were measured according to standard methodologies (within 0.1 kg in the LTx clinic) by trained clinic personnel. All height-for-age z scores, weight-for-age z scores and BMI z scores were calculated using the World Health Organization Standards (30). Overweight and obesity was defined as a BMI-z score 85 to 94 percentile and ≥95th percentile, respectively. Growth (changes in weight/height) between annual clinic visits were calculated and weight standard deviation scores (SDS) and height-SDS calculated using normative data.
Ethics approval was obtained from the Human Research Ethics Board at the University of Alberta (Pro00064040).
Data are presented as mean ± standard deviation or median ± interquartile range for nonparametric variables. The Shapiro-Wilk test was conducted to assess the normality of distribution. Nonparametric data underwent logarithmic transformation. Repeated measures analysis of variance was used to assess the differences in primary outcomes (serum lipid panel, insulin, HOMA-IR, A1C, fasting glucose, and BMI-z) over time. In addition, univariate and multivariable analysis was conducted to assess potential associations between primary outcomes of interest (markers of metabolic dysregulation) and secondary outcomes (body composition/growth). Analysis of covariance was performed to adjust for any variables influencing primary outcomes (sex, Pediatric End Stage Liver Diseases/Model for End-Stage Liver Disease scores). Chi-square/Fisher tests were used to measure differences in categorical data. Data were analyzed using the SAS 9.0 statistical software (SAS, Version 9.4; SAS Institute Inc, Cary, NC). A P value ≤0.05 was indicative of statistical significance.
Anthropometric and demographic data are presented in Table 1. The majority of children had body weights (BMI-z scores <2) within normal reference ranges over the 10 years studied (Fig. 1A). In years 1 to 3 post-LTx, 1 (2.1% year 1) to 7 children (14.8% year 3) were classified as obese (BMI-z scores >2), with levels declining to 0% for years 8 to 10 post-LTx (P = 0.002). Although measures of relative lean body mass measures (as a percentage/z scores) remained relatively stable over the 10 years (data not shown), relative body FM (total and segmental) decreased (P < 0.001) (Fig. 2A–D/Supplemental Table 1, Supplemental Digital Content, http://links.lww.com/MPG/B565). Serum TGs were associated with higher FM (% and absolute) for total and segmental (gynoid, trunk, peripheral extremities), but only represented 10% to 15% of the variability in serum TG concentrations (P < 0.05). No differences in fat-free mass with normal/abnormal serum concentrations with of TG, LDL, HDL, TC, insulin, HOMA-IR, or systolic/diastolic BP-z scores were observed (P > 0.05).
Longitudinal Changes in Markers of Cardiometabolic Dysregulation
Forty-seven children had repeated serum measures for fasting lipid panel, glucose, hemoglobin A1C, insulin, and HOMA-IR measured annually from time of LTx for 10 years at annual clinic appointments. The overall prevalence of CMD as defined by the ATP-III criteria was 3% respectively. No differences in CMD prevalence (0%–6%) by ATP-III criteria was noted over the 10-year period on a per year basis (P = 0.98). Abnormal findings for laboratory markers of CMD expression were independent of weight-z, BMI-z, systolic/diastolic BP (absolute/z scores), weight velocity/height velocity SDS, liver disease diagnosis, graft type (living related vs cadaveric), and rejection (number, severity) but was positively related to the child's age (>9.7 years) for serum insulin (P = 0.02) and TG (P = 0.04) only. Over the 10 years studied, there were no significant differences in systolic and diastolic z scores or fasting serum concentrations of TG, HDL-cholesterol, LDL cholesterol, TC, glucose, and A1C (Fig. 1B–D). Approximately 45% to 70% of patients had depressed serum concentrations of HDL over the 10 years, but no significant differences in prevalence were observed between years (P = 0.07). More than 80% of the cohort has serum insulin concentrations lower than normal reference ranges over the 10 years (Fig. 1E). Insulin concentrations decreased significantly over the first 4 years post-LTx (P < 0.05), but no differences in serum insulin were seen between 5 and 10 years post-LTx (Fig. 1E). These differences coincided with significant reductions in serum tacrolimus trough levels for the first 3 to 4 years post LTx (Fig. 1F), but were not related to tacrolimus dose (absolute or g/kg) or duration of tacrolimus therapy over the remaining years of study (P > 0.05).
CST therapy was used in 22 (pre-2003) versus 10 (post-2003) recipients in the first year post-LTx (P = 08). Doses of CST (>0.2 mg · kg−1 · day−1) and longer CS duration (>330 days) administration was associated with decreased serum HDL (P > 0.001) and increased TG concentrations (P = 0.02) over the 10 years. In particular, early CST administration (1–3 months) was associated with reduced height-z scores and serum HDL concentrations and higher serum TG concentrations (Fig. 3A–D), but had no apparent effects on weight-z, BMI-z, systolic BP-z, diastolic BP-z scores, or serum concentrations of TC, LDL, insulin concentrations or HOMA-IR over 10 years (P > 0.05). Comparing to a CST-free environment (post-2003 era), no significant associations between those children on or off CST therapy was observed for serum HDL-cholesterol, LDL-cholesterol, TC, insulin, HOMA-IR, A1C, BMI-z, systolic BP-z, and diastolic BP-z (P > 0.05). More children on MMF (79.2% [+MMF] vs 21.8% [−MMF]) had lower concentrations of HDL (P < 0.001). MMF weaning times <0.5 year were nonsignificantly associated with a greater percentage of children having normal serum HDL concentrations at years 1 to 3 (P = 0.07) then in children who had longer weaning times (>0.5 years). No other significant effects of MMF on markers of CMD were observed.
This study demonstrates that children post-LTx with healthy body weights on CST-free regimens have a very low prevalence of CMD (3%–6%) over 10 years. Longitudinal expression of CMD risk factors such as elevated BP, alterations in glycemic control, and TG/cholesterol metabolism appeared to be largely independent of body composition and more related to tacrolimus and/or MMF therapies in this study. These results are in contrast to other pediatric studies which suggest that the risk for CMD in pediatric LTx recipients ranges between 10% and 25% (4–7,31).One of the major reasons for differences in findings is the much lower prevalence of pediatric obesity (2%–14%) in this cohort compared to other sites in which the prevalence of obesity has been reported to range between 10% and 30%.(4–7,31,32). In addition, the majority of children in this cohort were CST free which may in part explain the lower rates of child overweight/obesity. CST stimulates appetite and adversely influences protein and bone turnover, insulin sensitivity, and fat metabolism. Interestingly, although relative body fatness was within normal reference ranges over the entire period studied, the contribution to overall body weight decreased during the 10-year period. This occurred when children were growing (height and weight) within normal healthy reference ranges. These changes in body composition also may have ameliorated the potential effects of immunosuppression (tacrolimus) on insulin sensitivity and influenced the overall prevalence of dyslipidemia and insulin resistance, particularly in the longer term when relative body fatness appeared to decrease with time.
The present study has some limitations including the smaller sample size, particularly in relation to the longitudinal review of the CMD markers which may have resulted in an underestimation of the potential changes experienced. This was, however, unlikely to be a major factor since the intrasubject variability in biochemical markers of most patients was <5% over the entire 10-year study period. Although we used a modified definition of CMD risk (ATP-III) which could have potentially contributed to underestimation of CMD burden, we did not find any differences in CMD prevalence when comparing to WHO definitions or other known definitions. No information related to pubertal stage and its potential impact on CMD risk was available. This potentially would be important as children typically experience insulin resistance during puberty and this may in part potentially explain why changes in serum insulin concentrations were no longer apparent 4 years after LTx when the average age of the cohort exceeded 13 years of age. Importantly, as serum insulin concentrations were low and HOMA-IR values within reference ranges, the influences of puberty on insulin sensitivity may have been masked by the effects of immunosuppressive therapies such as tacrolimus which are known to suppress insulin secretion. In addition, the majority of children were CST free (>95%) after the first year of LTx (data not shown), which likely would have resulted in a reduced influence of CST on insulin sensitivity; particularly as the majority of children had body weights within normal reference ranges.
No information was available regarding the potential lifestyle factors (diet, physical activity) that may have contributed to CMD risk. Although recent work within our group has shown that children post-LTx have diets characterized by low diet quality and micronutrient intake and higher saturated fat intake, intakes were similar to their healthy peers and few children in this cohort were overweight or obese (33). These diets may, however, be problematic in the presence of energy excess, as diets high in saturated fat are known to contribute to CMD expression in pediatric obesity (34). Dietary modification (lower saturated fat) tailored to address the perturbations in lipid and cholesterol metabolism may potentially be beneficial in ameliorating the effects of immunosuppression over the long term, particularly in the presence of overweight and obesity. Physical inactivity due to early fatigue, reduced exercise capacity, and unwillingness to participate in routine physical activity may also be a potential CMD risk factor for youth post-LTx (35). This may be compounded by the presence of post-LTx sarcopenia, which may impair the ability to perform physical activity. We have recently shown that sarcopenia occurs in up to 40% of pediatric LTx recipients, but it is unknown if this translates to reduced muscle functionality, participation in physical activity, and risk for CMD (28).
No data were related to patient care outcomes such as onset of comorbidities (eg, hepatic steatosis, CVD), healthcare utilization, and/or health-related quality of life. Several studies in adult LTx recipients have demonstrated that onset of obesity and CMD results in reduced graft survival, reduced CV, and liver health and reduced overall health-related quality of life (1–3). Hepatic steatosis in particular may occur in up to 30% of adults in the post-LTx period and has been related to increased morbidity and mortality in adults post-LTx (36). Although hepatic steatosis often develops in children early after LTx, it rarely persists beyond 6 months (32). There are, however, no studies that have longitudinally examined the prevalence of hepatic steatosis, and what factors (lifestyle, immunosuppression) may contribute to the development of liver steatosis and CMD. Moreover data relating this to patient care outcomes such as graft survival are also sorely lacking in pediatric LTx recipients, including progression to adulthood.
In summary, children post-LTx with body weights within healthy reference ranges and age-appropriate growth on CST-free regimens have a low prevalence of CMD over 10 years. Further studies examining the longitudinal expression of CMD, lifestyle factors influencing CMD risk, and the potential impact on patient care outcomes in children post-LTx are warranted.
The authors wish to gratefully acknowledge the assistance of Jessica Stroud, RD and Don Breakwell, RTR, CDT, Medical Imaging Consultants for their assistance with data collection/data auditing. The authors gratefully acknowledge summer studentship funding to A.H. by the Canadian Liver Foundation, Alberta Transplant Institute, University of Alberta, and the Undergraduate Research Initiative, University of Alberta.
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