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Estimates of protein turnover in the fed state depend on knowledge or assumptions about the metabolic fate of dietary protein. During a typical peripheral venous tracer infusion protocol, if a significant fraction of the carbon skeleton of diet-derived leucine is removed on first pass by the splanchnic viscera (intestine or liver), rates of whole body protein breakdown and leucine oxidation will be underestimated and protein leucine balance will be overestimated. Thus, it is important to consider the metabolic fate of diet-derived leucine in studies of leucine kinetics in the fed state. We conducted this study to determine in children the splanchnic uptake of leucine in the fed state. We also estimated B.

Insulin action is abnormal in patients with CF because of defective insulin secretion, insulin resistance, or both(1–5). Recently, we found that feeding did not suppress protein breakdown in children with CF(6). Because of these previous interests, we studied children with CF as well as healthy children.

METHODS

Subjects. Patients with CF who met the following entrance criteria were recruited from the CF clinic: Tanner stage I; generally healthy as reflected by the absence of fever or antibiotic treatment for at least 10 d; and no recent weight loss with weight stable for 10 d before the study. All patients with CF had some degree of pancreatic insufficiency. They divided up their usual enzyme supplements among the hourly feedings during the study (see below). We also recruited healthy, prepubertal children in the same age range as the patients with CF. These healthy children were recruited mainly from families of hospital staff. The subjects were matched with patients with CF on the basis of age, maturation level, sex, and in one instance, race (African descent).

All procedures were explained to the subjects and their parents before obtaining written informed consent. The study was approved by the Human Subjects Research Committee at Children's Hospital and the Biomedical Sciences Human Subjects Review Committee at The Ohio State University. Physical characteristics of the subjects are presented in Table 1.

Table 1 Patient characteristics and tracer results*
Full size table

Diet. The diet during the study was designed to produce a eucaloric state. The energy intake matched that provided by the real food diet the week before on the basis of food diary information. Depending on personal preferences, all subjects except one consumed either a formula diet composed of 12% of total energy as protein, 44.1% fat, and 43.9% carbohydrate (n = 4, Pediasure, Ross Laboratories, Columbus, OH) or a diet composed of 14% protein, 31.5% fat, and 54.5% carbohydrate (n = 5, Ensure, Ross Laboratories, Columbus, OH). One subject (2035) ingested Sustacal (Mead Johnson, Evansville, IN) because he refused to drink either of the other two formulas. Thus, we chose to control the diet on the basis of energy intake, not protein or leucine intake. This decision, along with some compromise we had to make with formula selection, allowed for some variation in leucine intake, which in the CF group was sufficiently large to permit statistical analysis of the relationship of leucine intake to splanchnic uptake of leucine. However, leucine intake was taken into consideration in our calculation of the absolute rate of splanchnic leucine uptake.

Experimental design. Five prepubertal children with CF and five prepubertal healthy children were either admitted to the General Clinical Research Center at The Ohio State University Hospital or to the Clinical Study Center at Children's Hospital.

After an overnight fast, at 0700 catheters were inserted in opposite upper extremities for drawing blood and i.v. administration of 1-[13C]leucine. Then, subjects began ingesting a formula diet on an hourly basis equivalent to 1/15 of the estimated daily energy intake; this diet was then administered every hour for the next 11 h. After 5 h of the hourly feeding, a baseline blood sample was obtained for measurement of the enrichment of plasma KICA, and then, the subjects began ingesting a dose of 5,5,5-[2H3]leucine every 30 min until the conclusion of the study. After 9 h of the hourly feeding, a primed, constant, i.v. infusion of 1-[13C]leucine (99%, MSD Isotopes, Ontario, Canada; prime, 9.1 µmol/kg; infusion rate, 0.153 µmol·kg-1·min-1) was continued for 120 min. Blood was taken every 10 min during the last 40 min of the infusion. Blood for determining isotopic enrichment of plasma KICA was collected from a peripheral vein after warming with a commercial heating pad. The criterion for plateau was based on visual inspection and analysis of the data indicating no rise in enrichment (zero slope) and a coefficient of variation in enrichment of <10%.

Calculations. The rate of appearance of leucine using an i.v. tracer (Ra Leuiv) was calculated using a standard equation(7): (equation 1) where I13C-Leu is the intake of [13C]leucine, that is, the measured rate of i.v. infusion of 1-[13C]leucine in molar units, and IE KICA 13C is the 13C enrichment (M + 1) of plasma KICA, expressed as moles fraction excess (MPE/100)(8,9). Ra Leuiv simply measures the rate of appearance of leucine into the peripheral circulation.

The use of the two leucine tracers to estimate splanchnic uptake of leucine (measured as KICA) is based on literature evidence that these two tracers are equivalent(10). The rate of appearance (entry) of diet-derived leucine into the peripheral circulation (Ra Leuoral) was calculated using equation 2: where I 2H-Leu is the hourly rate of administration of the oral deuterated tracer in molar units, IE KICA 2H is the 2H enrichment (M + 3) of plasma KICA expressed in MPE/100, and Leu Intake is the estimated oral intake of diet protein-derived leucine. The derivation of this equation is found in the Appendix.

The rate of splanchnic uptake of leucine is equal to Leu Intake - Ra Leuoral. We also estimated the fractional splanchnic uptake of leucine by dividing the quantity (Leu Intake - Ra Leuoral) by the leucine intake.

The whole body B was first estimated using a standard equation: (equation 3)

B was also corrected for splanchnic leucine uptake (B Corr) using the following equation: (equation 4)

Analytical methods. Using isotopic standards, the plasma enrichment of KICA was measured by GC/MS using a Finnigan/MAT model 4021 GC/MS (at Children's Hospital, Columbus) or a Hewlett Packard Model 5970 (Indiana University)(11). As described previously by Biolo et al.(12), naturally occurring 13C on the unlabeled carbons of 1-[13C]leucine contributes to a significant M + 3 enrichment of leucine when this tracer is administered. Thus, using electron impact (EI) GC/MS, and the silylquinoxalinol derivative(11), we evaluated the M + 0, M + 1, and M + 3 enrichment of a fragment ion that did not contain the methyl carbon of KICA (232,233,235), and using chemical ionization, we also assessed the M + 0 and M + 3 enrichment of the molecular ion of KICA (m/z 275 and 278). The M + 3 enrichment of KICA obtained using EI was subtracted from the M + 3 enrichment using chemical ionization to obtain the plasma KICA enrichment due to the deuterated tracer. This "corrected" 2H enrichment of KICA was used in equation 2 (IE KICA 2H).

Statistical analysis. A two-sample t test and the Wilcoxon test were used to compare the two groups with respect to splanchnic uptake of leucine.

RESULTS

All subjects tolerated the protocol and none exhibited diarrhea or vomiting during the study. Table 1 presents ages and weights of the subjects along with the parameters used to calculated splanchnic uptake of leucine as described in the Methods. There was no significant difference between the groups in age. Table 1 shows the data on the uptake of oral leucine by the splanchnic viscera. The respective mean values (± SD) for the rate of splanchnic uptake of leucine were similar, 23.8 ± 24.0 and 21.5 ± 21.2 µmol·kg-1·h-1 for the CF and HC groups, respectively, an 11% difference (NS). If we recalculate the rate of splanchnic uptake of leucine assuming that it cannot be negative, the value for the control group was 23.5 ± 17.6 µmol·kg-1·h-1, indicating almost identical mean values for the two groups. Either way, the 95% confidence intervals for this sample overlap zero for both groups. Expressed as a percentage of the leucine intake, splanchnic uptake of leucine was 16.0 ± 11.2% and 24.4 ± 25.6% in the CF and control groups (NS).

B was not significantly different between the groups (CF versus HC) with (159 ± 18 versus 135 ± 28 µmol·kg-1·h-1) or without (135 ± 14 versus 114 ± 20 µmol·kg-1·h-1) correction for splanchnic leucine uptake. However, as expected, for the 10 cases combined, B corrected for splanchnic leucine uptake (147 ± 26 µmol·kg-1·h-1) was 18% greater than uncorrected B (124 ± 20 µmol·kg-1·h-1) (p = 0.009).

In the healthy control group, there was relatively little variation in leucine intake (Table 1), and there was no significant correlation between fractional splanchnic leucine uptake and leucine intake. However within the CF group, the range in leucine intake was almost three times that in the healthy control group, and we observed a perfect rank correlation between fractional splanchnic leucine uptake and leucine intake.

DISCUSSION

The primary purpose of this study was to assess splanchnic uptake in healthy children and in children with CF. Our results show that the rates of splanchnic uptake of leucine were similar in the two groups. Moreover, correction of individual values for B did not affect the comparison of the parameter in the two groups. Although B corrected for splanchnic leucine uptake was significantly higher than B for the 10 subjects combined, there was no significant difference between the two subject groups, with or without a correction for splanchnic uptake of leucine. Protein breakdown was 17% higher in the CF group with the correction and 18% higher without the correction, suggesting that in this study the relative (albeit small) difference in B between the controls and the children with CF was not affected by knowledge of splanchnic leucine uptake. This result lends credence to our previous studies of protein kinetics in patients with CF and healthy children(6).

This is the first study of splanchnic leucine uptake in children, and intakes could not be precisely regulated although we attempted to provide the usual energy intake for each child. Studying these children required some flexibility on the part of the investigators with respect to the choice of formula used in the study. This factor, along with our design based on studying children ingesting their usual energy intake, resulted in variation in leucine intake, although this variable was taken into account in the calculations. Fractional splanchnic uptake of leucine also varied considerably. To grasp the statistical relevance of this variation, consider that to achieve a power of about 0.8, we would need to study 90 individuals in each group. Thus, although comparative estimates of protein breakdown were not affected by including estimates of splanchnic leucine uptake, we also cannot really conclude that this process was really "normal" in children with CF. The interindividual variation in the splanchnic uptake of leucine did not seem to be because of measurement artifact as the enrichment of leucine with both isotopes reached a plateau value. The observed variation in leucine uptake and its possible relationship to leucine intake suggests that the precision of future estimates of leucine flux and oxidation in the fed state might be enhanced in some studies if estimates of splanchnic uptake of leucine were obtained in each subject, particularly if the subjects range in age or if food and leucine intake cannot be rigidly controlled for ethical or practical reasons.

We did not measure fecal nitrogen excretion. Although there is perhaps not a practical mean difference between the groups in splanchnic leucine uptake, it is relevant to consider whether relative maldigestion of protein in the CF group (despite enzyme supplementation) might affect the comparative results. The oral leucine tracer does not require digestion for absorption; therefore, there is no reason to suspect that this tracer was absorbed differently in the two groups. Lack of complete digestion of food protein would tend to raise the measured value of KICA enrichment measured using both tracers and would not affect the calculation of the percentage of oral leucine tracer reaching the peripheral blood. However, in the patients with CF, overestimation of absorbed leucine may have artifactually increased the estimate of splanchnic leucine uptake expressed as a rate and may have increased the difference between the two groups. Therefore, perhaps the arithmetic difference in this parameter was even less between the two groups.

The positive correlation of fractional splanchnic leucine uptake and leucine intake in the CF group is consistent with recently reported data(13) that indicate that splanchnic uptake of leucine is greater when protein is ingested compared with a protein-free diet. Although cause and effect cannot be determined by our experimental design, these data do raise the question about whether the liver particularly can regulate plasma concentrations of leucine by first-pass uptake and oxidation. Perhaps of practical note, these results would mean that splanchnic uptake of leucine might have a greater effect on calculations of protein balance at greater leucine intakes, resulting in an artifactual overestimation of the effect of leucine intake on leucine balance. For this additional reason, we suggest that there may be merit to conducting companion studies of splanchnic leucine uptake in studies of leucine requirements as others have done(14).