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Pediatric Endocrinology |
Department of Pediatrics, Endocrinology and Metabolism Section, Texas Children’s Hospital and U.S. Department of Agriculture, Agricultural Research Service, Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Philip R. Beckett, Ph.D., Texas Children’s Hospital, 6621 Fannin, MC 3–2351, Houston, Texas 77030. E-mail: pbeckett{at}bcm.tmc.edu
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Abstract |
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We measured leucine oxidation and retention (intake minus oxidation) in
orally fed nondiabetic (n = 9) and diabetic (n = 9) human
subjects, aged 7–17 yr. Eight subjects were Tanner stage I, and 10
were Tanner stages III–V; groups were not matched for gender. After 3
days of consuming a diet containing approximately 1 g/kg · day
protein, subjects drank a commercial liquid nutrition formula,
containing L-[1-13C]leucine, every 30 min for
a total of 6 h to provide 1 g protein/kg · day. Isotopic
enrichment of CO2 was used to calculate the fractional
leucine oxidation rate and, together with 
-ketoisocaproate isotopic
enrichment, to calculate total leucine oxidation.
Leucine oxidation rates decreased with puberty in both nondiabetic subjects (36.0 ± 10.4 vs. 23.9 ± 4.2 µmol/kg fat-free mass (FFM) · h, prepubertal and pubertal, respectively; P < 0.05) and diabetic (33.6 ± 4.9% vs. 27.3 ± 3.4 µmol/kg FFM · h, prepubertal and pubertal, respectively; P < 0.1) subjects. Leucine retention increased with puberty in both nondiabetic (0.27 ± 3.2 vs. 15.7 ± 5.3 µmol/kg FFM · h, prepubertal and pubertal, respectively; P < 0.001) and diabetic (1.9 ± 4.9 vs. 13.2 ± 4.4 µmol/kg FFM · h, prepubertal and pubertal subjects, respectively; P < 0.05) subjects. The data suggest that the pubertal growth spurt is associated with a marked increase in the efficiency of dietary protein utilization for growth.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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Four groups of subjects were studied: prepubertal nondiabetic
(n = 3), pubertal nondiabetic (n = 6), prepubertal diabetic
(n = 5), and pubertal diabetic (n = 4) individuals (Table 1
). Patients ranged in age from 7–17 yr. Prepubertal
subjects were classified as Tanner stage I, and pubertal subjects as
Tanner stage III–V based on developmental stage of pubic hair. Each
subject was studied once only at the General Clinic Research Center of
Texas Children’s Hospital (Houston, TX). Each participant and a parent
or guardian gave written informed consent. The study was approved by
the Baylor College of Medicine Affiliates Review Board for Human
Subjects.
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All subjects consumed a diet containing approximately 1 g
protein/kg · day for 3 days before admission. Intake during these 3
days was assessed from written dietary records. Subjects were admitted
to the General Clinical Research Center at Texas Children’s Hospital
on the evening before the study and fasted overnight for 8 h.
Overnight euglycemia in the diabetic subjects was maintained by
following each subject’s normal routine insulin and dietary
management. Blood glucose was measured at bedtime, 0200 h, and
0600 h, but it was not necessary to administer additional insulin
or glucose to any of the subjects. Baseline samples of breath and urine
were taken the following morning. Diabetic subjects were given two
thirds their usual dose of intermediate or long-acting insulin, with no
short-acting insulin (to maintain euglycemia; blood glucose of 135
± 50 mg/dL throughout study), 30 min before isotope administration. A
commercially available complete liquid nutrition formula, Pediasure
(Ross, Columbus, OH), to which 0.3 mg/kg
L-[1-13C]leucine was added, was given orally
at a rate of 0.02 g protein/kg and 0.69 Cal/kg every 30 min for a
total of 6 h (equivalent to 1 g protein/kg · day and 33
Cal/kg · day). The leucine content of the formula was confirmed by
high performance liquid chromatography using a Beckman system 7300
amino acid analyzer (Beckman, Palo Alto, CA). The sample was defatted
and hydrolyzed with HCl before analysis, using norleucine as an
internal standard. Indirect calorimetry (Delta Trak portable
calorimeter, Sensormedics, Yorba Linda, CA) was determined over a
30-min period. Body composition was determined by total body electrical
conductivity (HA-2, EM-Scan, Springfield, IL), and Tanner staging was
determined by physical examination. Four, 5, and 6 h after the
start of isotope administration, blood was obtained from a dorsal hand
vein for -ketoisocaproic acid (-KIC) isotopic enrichment and
hormone analysis. Blood was collected as ethylenediamine tetraacetate
plasma, maintained on ice, centrifuged within 30 min, and maintained at
-20 C until analysis. Breath samples were collected in evacuated glass
Vacutainer tubes (Beckton Dickinson, Franklin Lakes, NJ) every 15 min
from 4–6 h after the start of isotope administration, and analyzed for
CO2 isotopic enrichment. Urine samples were stored at -20
C until analysis for -KIC isotopic enrichment.
Measurement of substrate isotopic enrichment
Breath CO2 isotopic enrichment was determined by gas
isotope ratio mass spectrometry (Europa, UK) by monitoring ions at m/z
44–45. Plasma -KIC was derivatized to its pentafluorobenzyl ester
and analyzed by negative chemical ionization gas chromatography-mass
spectrometry, monitoring ions at m/z 129 and 130 using an HP5988A
system (Hewlett-Packard, Palo Alto, CA) (8). Urinary -KIC was
derivatized to its pentafluorobenzyl ester derivative using a
modification of the method of Zaura et al. 1969 (9).
Briefly, 10 mL urine were treated with 0.25 g activated charcoal
for 15 min, vortexed every 5 min, then centrifuged for 5 min at
2000 x g. Protein was precipitated from the
supernatant using 2.5 mL ice-cold 30% HClO4. The sample
was then kept on ice for 10 min and centrifuged for 15 min at 2000
x g, and the supernatant was neutralized with 6 mol/L KOH
and centrifuged again for 15 min at 2000 x g. This
supernatant was loaded onto 2 mL Dowex 1 (formate form) columns (Sigma,
St. Louis, MO) and washed with 20 mL water. Keto-acids were eluted with
5 mL 12 N formic acid, and the eluant was concentrated
under vacuum (Savant Instruments, Farmingdale, NY), washing twice with
water. These samples then were derivatized as described for plasma.
Measurement of branched chain amino acid and hormone concentrations
The concentration of total branched chain amino acids in plasma was determined using a previously reported enzyme assay (10). Free insulin was isolated using the method of Nakagawa et al. (11), and insulin concentration was determined by RIA (Diagnostic Systems Laboratories, Webster, TX). Insulin-like growth factor-I (IGF-I), IGF-binding protein-1 (IGFBP-1), IGFBP-3, testosterone, and estradiol were determined by RIA (Diagnostic Systems Laboratories). Concentrations for each subject are the means of the samples taken at 4, 5, and 6 h.
Calculations
Leucine kinetics were calculated as follows: 1) leucine rate of
appearance (Ra) = i x [(Ei/E-KIC) - 1]; 2) leucine
oxidation = (VCO2 x ECO2)/(0.8 x
E-KIC); 3) nonoxidative leucine disposal (NOLD) = Ra -
oxidation; and 4) leucine retention = intake - oxidation,
where i is the intake of [13C]leucine in micromoles per
kg/h, Ei is the isotopic enrichment of the tracer, E-KIC is the
isotopic enrichment of plasma -KIC, VCO2 is carbon
dioxide excretion from calorimetry in micromoles per kg fat-free mass
(FFM)/h, ECO2 is the isotopic enrichment of expired carbon
dioxide, and 0.8 is assumed to be the recovery of labeled
CO2. Leucine kinetics were calculated based on the mean
isotopic enrichment between 4 and 6 h of isotope administration.
Carbohydrate and lipid oxidation rates were calculated from gaseous
exchange using the Weir equation and our leucine oxidation rates to
estimate protein oxidation, assuming leucine to be 8% of protein.
Statistical analysis
The effects of diabetes and puberty on leucine kinetics and substrate oxidation rates were analyzed using one-way ANOVA (Minitab, State College, PA) for nondiabetic and diabetic subjects separately, except where indicated. Increased leucine retention relative to leucine intake was taken to indicate an increase in the efficiency of protein utilization. Plasma hormones were correlated with rates of leucine retention using regression analysis for all 18 subjects. All results are presented as the mean ± SD.
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Results |
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Body height and weight were not different between nondiabetic and
diabetic subjects, but both were greater in pubertal than in
prepubertal subjects (Table 1
). The percent body fat was not different
among groups (19.2 ± 8.5%); although the mean percent body fat
was greater in females (23.8 ± 5.4%) than in males (16.7 ±
9.0%), the difference did not reach statistical significance.
Glycosylated hemoglobin was greater in diabetic than in nondiabetic
subjects (11.4 ± 2.9 vs. 4.8 ± 0.5,
respectively; P < 0.001).
Carbon dioxide production and energy expenditure
Carbon dioxide production was significantly lower in pubertal than prepubertal diabetic (4.47 ± 0.36 vs. 3.36 ± 0.68 mL/kg · min, prepubertal and pubertal, respectively; P < 0.01), but not nondiabetic (4.44 ± 0.79 vs. 3.71 ± 0.48) subjects. Energy expenditure estimated from indirect calorimetry in the fed state was significantly lower in pubertal than in prepubertal subjects, nondiabetic and diabetic combined (37.0 ± 6.3 vs. 45.7 ± 5.6 Cal/kg FFM · day; P < 0.01).
Isotopic enrichments of -KIC and expired
CO2
Isotopic enrichments of -KIC and expired CO2
plateaued during the last 2 h of the 6-h isotope administration.
In two subjects who were given Pediasure without
[13C]leucine for 6 h, isotopic enrichment of expired
CO2 increased by 0.00018 atom percent excess, about 3% of
the plateau values in the subjects who were given the isotope.
Whole body leucine kinetics
Whole body leucine Ra and NOLD were not affected by puberty in the
nondiabetic or diabetic subjects (Figs. 1 and 2; raw data in Table 2). Leucine
oxidation was about 30% lower in pubertal than prepubertal nondiabetic
subjects (P < 0.05). Leucine oxidation was about 20%
lower in pubertal than prepubertal diabetic subjects, but did not reach
statistical significance (P = 0.066; Fig. 3). Leucine retention was increased markedly by puberty
in both nondiabetic (P < 0.001) and diabetic
(P < 0.05; Fig. 4) subjects.
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Total branched chain amino acid concentrations during the last 2 h of isotope administration were not affected by puberty (378 ± 30 vs. 359 ± 56 nmol/mL, prepubertal and pubertal, respectively) or diabetes (350 ± 40 vs. 384 ± 47 nmol/mL, nondiabetic and diabetic subjects, respectively).
Hormone concentrations and pubertal stages
Free plasma insulin concentrations were not different between
diabetic and nondiabetic subjects (6.3 ± 1.5 and 7.3 ± 3.7
µU/mL, respectively) and were not significantly different at puberty
in either nondiabetic or diabetic subjects. IGF-I concentrations were
higher in pubertal nondiabetic subjects (244 ± 125 vs.
686 ± 173 ng/mL, prepubertal and pubertal, respectively;
P < 0.01; Table 3) and pubertal
diabetic subjects (203 ± 84 vs. 517 ± 73 ng/mL,
prepubertal and pubertal, respectively; P < 0.001;
Table 3). There was a strong positive correlation between total IGF-I
and IGFBP-3 (r2 = 70%; P < 0.001).
IGFBP-I concentrations correlated inversely with both total IGF-I
(r2 = 47%; P < 0.01) and insulin
concentrations (r2 = 21%; P = 0.05). Serum
testosterone and estradiol concentrations reflected the expected
increases with puberty (Table 3).
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Discussion |
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Our choice of an intragastric rather than an iv tracer in the orally
fed model might be open to criticism for the purpose of calculating
leucine kinetics, as entry of tracer into the systemic circulation is
not known precisely. However, in studies comparing both iv and
intragastric tracer administration (13, 14), leucine kinetics are
similar regardless of the tracer route when, as in the present study,
-KIC is used as the precursor. In addition, it should be noted that
absorption of dietary amino acids, and therefore tracer, from the
gastrointestinal tract approaches 100% (15). The bolus method of
feeding every 30 min, rather than a nasogastric infusion, may have
induced a greater postprandial response of insulin in the nondiabetic
subjects and is a limitation of this methodology. However, this
technique was adequate to maintain stable isotopic enrichment and
plasma amino acid concentrations during the final 2 h of the
study.
It is possible to estimate energy expenditure and substrate oxidation rates, albeit in the fed state with the errors that lipogenesis and gluconeogenesis introduce, from the indirect calorimetry data. From these data energy expenditure was lower in the pubertal subjects (45.7 ± 5.6 Cal/kg FFM · day) than in the prepubertal subjects (37.0 ± 6.3 Cal/kg FFM · day; P < 0.01), largely due to differences in lipid oxidation (23.6 ± 10.4 vs. 16.2 ± 6.8 Cal/kg FFM · day, prepubertal and pubertal, respectively; P = 0.088), but not in carbohydrate oxidation (16.0 ± 6.0 vs. 16.3 ± 5.1 Cal/kg FFM · day, prepubertal and pubertal, respectively). As energy intake was standardized for body weight in all subjects (33 Cal/kg · day; 42.0 ± 5.8 Cal/kg FFM · day), prepubertal subjects were in negative and pubertal subjects were in positive energy balance. Positive energy balance suppresses lipid oxidation and increases lipogenesis, leading to an underestimation of lipid oxidation by indirect calorimetry (16), perhaps explaining the differences we observed in lipid oxidation. Moreover, differences in energy balance affect leucine oxidation in the fed state (17). In the present study, energy balance (energy intake minus energy expenditure) correlated with leucine oxidation (r2 = 40%; P < 0.01). Decreased energy requirement relative to protein may be one of the mechanisms of increased protein gain during puberty.
Plasma free insulin, IGF-I, IGFBP-1, IGFBP-3, testosterone, and estrogen concentrations and their interrelationships observed in the current study are similar to those observed by other investigators during puberty and in diabetic subjects. Free insulin, total circulating IGF-I, and IGFBP-3 concentrations correlated positively with leucine retention (r2 = 27%, P < 0.05; r2 = 43%, P < 0.01; and r2 = 52%, P < 0.001, respectively), and IGFBP-1 concentrations correlated negatively with leucine retention (P < 0.01; r2 = 42%). IGF-I (and GH) decrease leucine oxidation (18) and are probable candidates for altering the efficiency of protein utilization during puberty. IGF-I also decreases energy expenditure in the parenterally fed rat, but not in the fasted rat (19), and may exert protein-sparing effects indirectly through its effects on energy metabolism during feeding. Indeed, in the present study, energy expenditure correlated negatively with IGF-I (r2 = 27%; P < 0.05).
In conclusion, rates of leucine oxidation in the fed state are decreased during puberty in both diabetic and nondiabetic subjects, resulting in increased efficiency of dietary protein utilization for growth. Changes in protein metabolism are probably related to the myriad of hormonal events that occur at puberty, including pubertal increases in insulin and IGF-I. However, apart from direct hormonal effects on protein metabolism, a decrease in energy expenditure in the fed state during puberty may spare dietary protein.
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Acknowledgments |
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Footnotes |
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Received December 6, 1996.
Revised April 18, 1997.
Accepted May 5, 1997.
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References |
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  A. Bitar, N. Fellmann, J. Vernet, J. Coudert, and M. Vermorel Variations and determinants of energy expenditure as measured by whole-body indirect calorimetry during puberty and adolescence Am. J. Clinical Nutrition, June 1, 1999; 69(6): 1209 - 1216. [Abstract] [Full Text] [PDF]
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