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Nutrient Requirements and Optimal Nutrition

Mild-to-Moderate Chronic Cholestatic Liver Disease Increases Leucine Oxidation in Children^1,2

Diana R. Mager^*,, Linda J. Wykes, Eve A. Roberts^,**,, Ronald O. Ball^*,, and Paul B. Pencharz^*,,,,3

^* Departments of Nutritional Sciences, Paediatrics, and ^** Pharmacology, University of Toronto, Toronto, Canada; The Research Institute, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada; School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, QC, H9X 3V9, Canada; and Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, T6G 2P5, Canada

³ To whom correspondence should be addressed. Email: paul.pencharz{at}sickkids.ca.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Malnutrition is prevalent in children with chronic cholestatic liver disease. Using the noninvasive indicator amino acid oxidation (IAAO) technique, we recently determined that mild-to-moderate chronic cholestatic (MCC) liver disease increases the need for branched-chain amino acids (BCAA) in children. To examine the underlying mechanisms responsible for this increased need for BCAA in liver disease, we measured L-[1-¹³C]-leucine oxidation in the postabsorptive and fed states in 10 children with MCC liver disease (8.8 ± 3.5 y) and in 11 healthy children (9.4 ± 2.2 y). The oxidation of L-[1-¹³C]-leucine to ¹³CO₂ [F¹³CO₂ in µmol/(kg·h)] was determined after a primed, continuous oral administration of the tracer. Total BCAA in diet was provided at 300 mg/(kg·d) to ensure that leucine oxidation was measured when leucine intake was in excess of requirements. In the postabsorptive state, the rate of release of ¹³CO₂ from ¹³C-leucine oxidation (F¹³CO₂) and whole-body leucine oxidation were significantly higher in children with MCC liver disease (P < 0.05). However, F¹³CO₂ and whole-body leucine oxidation did not differ in the fed state. We conclude that the increased need for dietary BCAA in MCC liver disease is mediated in part by increased leucine oxidation in the postabsorptive state.

KEY WORDS: • leucine metabolism • children • liver disease

Protein-energy malnutrition leading to significant growth failure is common in children with mild-to-moderate chronic cholestatic (MCC)⁴ liver disease (1,2). The etiology of this growth failure appears to be multifactorial. We showed previously using the indicator amino acid oxidation (IAAO) technique that the need for dietary total branched-chain amino acids (BCAA) increases in children with MCC liver disease (3). This finding is consistent with other investigators who demonstrated significant increases in weight, nitrogen balance, liver function, and quality of life in adults and children with chronic liver disease supplemented with dietary BCAA (4,5). The mechanism by which MCC liver disease may increase the need for the BCAA is unknown.

The characteristic plasma amino acid profiles observed in chronic liver disease, namely, depressed levels of the BCAA and elevated levels of the aromatic amino acids (phenylalanine and tyrosine), are thought to reflect changes in protein and amino acid metabolism that contribute to malnutrition in this population (2,6). Increased oxidation, in the presence of diminished release of endogenous leucine from the skeletal muscle is thought to be a major contributor to the change in plasma levels of the BCAA in liver disease (7–14). To investigate potential underlying mechanism(s) responsible for the increased need for dietary BCAA in children with MCC liver disease, we studied whole-body leucine oxidation in the fed and postabsorptive states in healthy children and children with MCC liver disease. In the current study, we were limited because we were not permitted to take blood from the healthy children we were studying as controls. However, because urine [1-¹³C]-leucine enrichment is a suitable surrogate for plasma (15), we chose to employ a minimally invasive approach to determine leucine enrichment in urine, as well as ¹³CO₂ enrichment in breath. Children in the fed state were provided dietary BCAA in excess of previously established requirements to ensure that leucine was provided in excess of needs (1,16). We hypothesized that leucine oxidation would be significantly higher in children with MCC liver disease compared with healthy children with normal liver function.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Subjects

Children with MCC liver disease (n = 10; 8.8 ± 3.5 y) and 11 healthy children (9.4 ± 2.2 y) between the ages of 4 and 15 y were recruited for this study (Tables 1 and 2). Subjects were studied on an outpatient basis in the Clinical Investigation Unit at The Hospital for Sick Children (HSC), Toronto, Canada. A sample size of 10 subjects/group was determined to be required to detect a 25–30% difference in leucine oxidation between the 2 groups under study ( = 0.05 and ß = 0.80). Inclusion criteria for the children with MCC liver disease were as follows: 1) established diagnosis of chronic cholestatic liver disease; 2) mild-to-moderate disease severity; and 3) jaundice and/or laboratory evidence of cholestasis (total serum bile salts > 8.2 µmol/L, and/or conjugated bilirubin > 20 µmol/L and/or elevated serum -glutamyl transpeptidase (GT) (Table 3). All subjects with MCC liver disease had congenital disorders leading to chronic cholestatic liver disease. The severity of liver disease in this study was assessed clinically using the modified Child-Turcotte classification (17). None of the children with MCC liver disease had a recent history of variceal hemorrhage or ascites; all were ambulatory and appeared clinically stable. Healthy subjects did not have any known (as reported by caregivers) history of any underlying medical pathology. All children with MCC liver disease had routine measurement of liver biochemistries and coagulation tests as part of their clinical care. Serum liver biochemistry and coagulation factors were measured in the Core Laboratory at the Hospital for Sick Children, Toronto (18–25). Blood work was not performed in the healthy children. Tanner staging assessment was done by one of the investigators at the time of subject recruitment (26). Anthropometry (weight, height) and body composition (multiple skinfold thickness and bioelectrical impedance analysis) were measured on each study day using standard techniques as described previously (1,16,27–31). Subjects were excluded if they were taking medications that affect energy or protein metabolism (e.g., corticosteroid therapy), were clinically unstable, or were known to have any other primary diagnosis such as endocrine and/or metabolic disorders.

Experimental design

The study design was developed to enable measurement of L-[1-¹³C]leucine oxidation in the postabsorptive and fed states in children with MCC liver disease and in healthy children. Subjects were assigned in random order to either the fed or fasting study day. The fed study day protocol was similar in design to the noninvasive IAAO model (32). L-[1-¹³C]leucine was used as the stable isotope tracer. The postabsorptive study included measurement of leucine kinetics after a 12- to 14-h overnight fast. Urine and breath samples were collected for determination of isotope enrichment as described previously. (1,16,33).

The mean time to reach isotope plateau in the fasting state in healthy children and children with MCC liver disease was 3 h (Fig. 1). Hence breath and urine samples were collected every 30 min between 2.5 and 4 h after the first isotope bolus.

View larger version (15K): [in this window] [in a new window]   FIGURE 1  Effect of total BCAA intake on breath 13CO2 enrichment and urinary L-[1-13C]leucine enrichment in children with MCC liver disease in the postabsorptive state. Values are means ± SD, n = 10.

Before each study day, participants were preadapted to either 1.5 g protein/(kg·d) (healthy children) or 2.0 g protein/(kg·d) (children with MCC liver disease). These levels of dietary protein were chosen because they exceeded the recommended protein intakes for healthy children and children with MCC liver disease and were similar to the habitual protein intakes and energy intakes (resting metabolic rate x 1.7) of these populations (34,35). More importantly, these levels were used in earlier studies examining total BCAA requirements in healthy children and children with MCC liver disease in our laboratory (1,16). Menu plans provided by the investigator(s) consisted of typical foods consumed by each child, and food records were collected to ensure consistency of dietary intake before each study day as described previously (1,16). Energy needs were determined by measuring resting metabolic rate after a 12-h overnight fast, using open-circuit indirect calorimetry (2900 Computerized Energy Measurement System; Sensormedics) as described previously (16). Subjects were fed an amino acid based diet plus vitamin supplement as described previously (1,16,32). Total BCAA in the diet was provided at 300 mg/(kg·d) in the fed study to ensure that leucine oxidation was measured in excess of established dietary requirement (1).

Tracer protocol

    Fed study. Subjects were given a priming oral dose of NaH¹³CO₂ (2.07 µmol/kg) and an oral dose of L-[1-¹³C] leucine (22.9 µmol/kg) with the 5th meal. After this, subjects received a constant oral administration of L-[1-¹³C] leucine (14.9 µmol/kg) with each hourly meal, until the end of the study day. The amount of leucine in the last 5 meals was reduced by an amount corresponding to the amount of stable isotope given during the tracer infusion to ensure consistency of intake of leucine in each meal of the study.

    Postabsorptive study. For the postabsorptive study, subjects were given a priming oral dose of NaH¹³CO₂ (2.07 µmol/kg) and an oral dose of L-[1-¹³C] leucine (22.9 µmol/kg). After 60 min, subjects were given a constant oral dose of L-[1-¹³C] leucine (7.44 µmol/kg) every 30 min to simulate a constant hourly oral administration of the stable isotope tracer. This dose was equivalent to the hourly dose of L-[1-¹³C] leucine (14.9 µmol/kg) given in the fed study.

Statistical analysis

A stochastic model was used to calculate leucine kinetics using standard equations as described previously (1,36–41). Multifactorial ANOVA with repeated measures was used to determine the effects of subject, order of study day, and type of study day (postabsorptive vs. fed study day). Body weight and body composition during the study period were compared by repeated-measures ANOVA. A general linear model (GLM) was performed to assess the relation of the rate of release of ¹³CO₂ from ¹³C-leucine oxidation (F¹³CO₂), leucine flux, leucine oxidation, nonoxidative leucine disposal (NOLD), and leucine released from endogenous proteolysis (B_leu) to the following variables: fed/postabsorptive state, order of intake, subject, group, and potential interactions. All analyses were conducted using SAS statistical software (SAS, Version 8; SAS Institute). Differences were considered significant at a P < 0.05. Data are expressed as means ± SD.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Of the 10 children with MCC liver disease, 8 completed both phases of the study, and 2 completed the fasting study day only. (Failure to complete the fed study day was due to patient refusal to consume the experimental diet.) All children with MCC liver disease had elevated liver biochemistries, but they had normal synthetic liver function (as assessed by serum levels of albumin and coagulation tests) and were clinically free of jaundice (except one child with Alagille syndrome). Body composition measures [weight, height, percentage of ideal body weight, fat-free mass (FFM), lean body mass (LBM)] and age did not differ significantly between the healthy children and the children with MCC liver disease and were within normal ranges for age (Tables 1 and 2) (27,29). Resting energy expenditure was not different between the healthy children and the children with MCC liver disease, although the fasting respiratory quotient for the children with MCC liver disease was significantly lower (P = 0.02) than that of the healthy children (Tables 1 and 2).

F¹³CO₂ production (P = 0.024) and leucine oxidation (P = 0.0473) were higher in children with MCC liver disease in the postabsorptive state (Table 4). F¹³CO₂ production increased by 21% in the children with MCC liver disease (Table 4). In contrast, in the fed state, F¹³CO₂ production and leucine oxidation did not differ. Mean leucine flux, NOLD, and endogenous leucine breakdown were not different in the fed or postabsorptive state between study groups (Table 4). There was no effect of order of assignment of fed vs. fasting between study groups or on any outcome (leucine kinetics, body composition) variables.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Leucine kinetics were measured in children who were meeting or exceeding their energy and protein needs given that both groups displayed age-appropriate growth rates. Indeed, nutrient analysis of food records showed that both groups of children had habitual energy and protein intakes in excess of recommended levels (34,35,42). Children in both groups were also fed in excess of their estimated total BCAA requirement during each study, as determined previously, to ensure that all subjects were meeting their BCAA needs during the fed state (1,16). Therefore, it is likely that the differences in leucine oxidation observed in the postabsorptive state reflect altered compensation to the fasting state in liver disease, rather than simply a change in whole-body proteolysis.

Few data are available linking an increase in whole-body leucine oxidation to an increase in leucine requirement in liver disease. Most studies focused on the effect of liver cirrhosis on leucine turnover in adults. Some studies found increased whole-body leucine oxidation in adults with compensated cirrhotic liver disease in the postabsorptive state (9,11), whereas many other studies did not show a consistent pattern or change in leucine oxidation (10,12,13,43). This may be due in part to differences in the models used for the assessment of leucine kinetics as well as the differences in body composition observed in cirrhotic patients (8–10,44,45). Neither was likely an issue in the current study because a variety of methods were used to assess body composition, and no differences in body composition were noted between methods or between the healthy children and the children with MCC liver disease (46). Leucine oxidation was increased in the fasting state, whether expressed per unit FFM or per kilogram total mass, indicating that the changes in leucine oxidation were not due to body compositional differences induced by liver disease. Moreover, we measured F¹³CO₂ production as our metabolic end-point. This end-product approach avoids problems with the leucine precursor pool approach and allowed the noninvasive study of leucine oxidation in the fed and postabsorptive state.

Increased protein and lipid utilization, along with hypermetabolism, are common in children with MCC liver disease (47). We demonstrated in a previous study that children with MCC liver disease are mildly insulin resistant and have fasting respiratory quotients indicative of increased protein and lipid utilization in the fasting state (1,16). Although we did not determine urinary nitrogen excretion and thus cannot calculate nonprotein respiratory quotients, the reduced respiratory quotient measured in the children with MCC liver disease in this study (0.80 ± 0.02) vs. the healthy children (0.84 ± 0.02) provides further evidence that children with MCC liver disease rely more on fat and protein to meet energy needs in the fasting state. These findings suggest that leucine oxidation is increased in the fasting state in chronic liver disease, thus contributing to an increased need for the leucine in chronic liver disease. Leucine, as a ketogenic acid, may be used preferentially as an energy substrate and the carbon skeletons of valine and isoleucine diverted toward gluconeogenesis.

Whole-body leucine oxidation did not increase in the fed state in the healthy children or the children with MCC liver disease. It appears that in the fed state, children with MCC liver disease can correct their leucine metabolism to normal, but not in the fasting state (42). Earlier studies in adults showed that leucine oxidation increases in the fed state once protein requirements are met (48,49). The protein intake level was chosen to be slightly above requirement. The failure of leucine oxidation to increase in the fed state in this study may be due in part to the reduced activity levels of the children on the study days, leading to positive energy balance. Motil et al. (50) showed that leucine oxidation decreases in the fed state in response to energy excess. We speculate that leucine oxidation may also have been suppressed due to the "nibbling" effect of the study diet protocol (51). Raguso and colleagues demonstrated higher rates of whole-body leucine oxidation in healthy adults fed in a bolus vs. a "nibbling" pattern, particularly at breakfast when fasting insulin levels were lower (51). This suggests that the pattern of meal feeding employed in this study may have also influenced whole-body leucine estimates in the fed state because children were fed in a meal pattern resembling a "nibbling" or "grazing" pattern. This may have permitted them to overcome mild insulin resistance in the fed studies.

Improved glucose sensitivity in the fed state in patients with compensated liver cirrhosis has been associated with suppression of peripheral leucine oxidation and improved nitrogen balance (13,43,52–54). Dietary leucine and isoleucine (but not valine) were shown to improve glucose uptake in skeletal muscle in animal models of chronic liver disease. This may also explain in part why increases in leucine oxidation were not observed in the fed state in this study (54).

In summary, this study demonstrates that whole-body leucine oxidation is higher in the postabsorptive state in children with MCC liver disease than in healthy children. It is unclear how this increase in whole-body leucine oxidation contributes to an increased need for the BCAA in chronic liver disease. However, some evidence suggests that increased protein and lipid utilization, along with hypermetabolism, may result in increased utilization of BCAA in the fasting state. We did not detect any differences in whole-body leucine oxidation between the two 2 groups in the fed state. We speculate that this may be due in part to increased insulin sensitivity with this form of meal feeding in MCC liver disease. These findings are consistent with evidence that meal "grazing or nibbling" patterns of food intake improved nitrogen balance in adults and children with chronic liver disease (42,53,55). This suggests that nutrition support strategies in children with MCC liver disease include the potential for provision of late night and/or nocturnal feeding, particularly in children with more advanced liver disease because malnutrition and growth are prevalent in this population. Further studies examining the effect of dietary BCAA supplementation on substrate utilization in the fed and fasting states are warranted in children with chronic liver disease.

	ACKNOWLEDGMENTS

 
We acknowledge the technical assistance of Mahroukh Rafii. We thank Karen Chapman for coordinating the activity in the Clinical Investigation Unit of The Hospital for Sick Children (HSC) and Linda Chow (Department of Nutrition and Food Services, The Hospital for Sick Children) for preparation of the protein-free cookies used in these studies. Mean Johnson Nutritionals (Canada) provided the protein-powder used in the experimental diet.

	FOOTNOTES

 
¹ Presented in part in abstract form at Experimental Biology 03, San Diego CA [Mager DR, Wykes JL, Ball RO, Pencharz PB. Chronic liver disease increases total branched chain amino acid requirements in children (abstract). FASEB J. 2003;17:431.4].

² Supported by the Canadian Institute of Health Research (grant MOP-10321). D.M. received doctoral support from the Canadian Liver Foundation & Clinician Scientist Fellowship, The Research Institute, The Hospital for Sick Children.

⁴ Abbreviations used: BCAA, branched-chain amino acid; F¹³CO₂, rate of release of ¹³CO₂ from ¹³C-leucine oxidation; FFM, fat-free mass; GT, -glutamyl transpeptidase; HSC, Hospital for Sick Children; IAAO, indicator amino acid oxidation; KIC, -ketoisocaproate; LBM, lean body mass; MCC, mild-to-moderate chronic cholestatic; NOLD, nonoxidative leucine disposal.

Manuscript received 16 November 2005. Initial review completed 8 December 2005. Revision accepted 28 December 2005.

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