Amino acids do not suppress proteolysis in premature neonates.

To determine whether increased amino acid availability can reduce proteolysis in premature neonates and to assess the capacity of infants born prematurely to acutely increase the irreversible catabolism of the essential amino acids leucine (via oxidation) and phenylalanine (via hydroxylation to form tyrosine), leucine and phenylalanine kinetics were measured under basal conditions and in response to a graded infusion of intravenous amino acids (1.2 and 2.4 g. kg(-1). day(-1)) in clinically stable premature (approximately 32 wk gestation) infants in the 1st wk of life. In contrast to the dose-dependent suppression of proteolysis seen in healthy full-term neonates, the endogenous rates of appearance of leucine and phenylalanine (reflecting proteolysis) were unchanged in response to amino acids (297 +/- 21, 283 +/- 19, and 284 +/- 31 micromol. kg(-1). h(-1) for leucine and 92 +/- 6, 92 +/- 4, and 84 +/- 7 micromol. kg(-1). h(-1) for phenylalanine). Similar to full-term neonates, leucine oxidation (40 +/- 5, 65 +/- 6, and 99 +/- 7 micromol. kg(-1). h(-1)) and phenylalanine hydroxylation (12 +/- 1, 16 +/- 1, and 20 +/- 2 micromol. kg(-1). h(-1)) increased in a stepwise fashion in response to graded amino acids. This capacity to increase phenylalanine hydroxylation may be crucial to meet tyrosine needs when exogenous supply is limited. Finally, to determine whether amino acids stimulate glucose production in premature neonates, glucose rate of appearance was measured during each study period. In response to amino acid infusion, rates of endogenous glucose production were unchanged (and near zero).

Energy expenditure after surgical repair in children with cyanotic congenital heart disease.

OBJECTIVE: Infants with cyanotic congenital heart disease (CCHD) have previously been shown to have similar resting energy expenditures (REEs) and elevated total energy expenditures (TEEs) compared with age-matched healthy infants. The purpose of this investigation was to re-examine the REE and TEE of the same individuals at 5 years of age, after surgical repair of the heart defect was done, to determine whether metabolic differences persist. STUDY DESIGN: Seven children were studied approximately 2.6 years after they underwent surgical repair of CCHD along with 10 age-matched healthy children. Indirect calorimetry was used to determine REE, and the doubly labeled water method was used to determine TEE and body composition. RESULTS: Results were compared with single-factor repeated measures analysis of variance. No significant differences were found between groups in weight or body composition. No significant differences were found between groups in REE, TEE, or the energy expended in physical activity. CONCLUSION: We conclude that differences in TEE observed during infancy are no longer present in 5-year-old children after they undergo surgical repair of CCHD. Furthermore, the individual components of energy expenditure of children with CCHD after repair are indistinguishable from those of healthy age-matched children.

Energy expenditure in the extremely low-birth weight infant.

Information about energy requirements of extremely low-birth weight infants is sparse, despite the rapidly improving survival rates of this population. Metabolizable energy intake can be estimated from energy balance studies and the percentage of caloric intake that is actually absorbed by these infants is approximately 87%. Data on energy expenditure in extremely premature infants is limited; however, energy expenditure has been shown to increase with postnatal age. Because both intake and expenditure are affected by multiple factors, there is significant variability in estimates of the energy requirements in extremely low-birth weight infants. At present, no valid recommendations can be made regarding optimal energy requirements for the extremely low-birth weight infant, except that their requirements probably exceed those of stable, growing very low-birth weight infants, currently estimated at 105 to 135 kcal.kg-1d-1.

Energy expenditure and energy intake during dexamethasone therapy for chronic lung disease.

Dexamethasone is commonly administered to ventilator-dependent preterm infants with chronic lung disease. Infants receiving dexamethasone therapy frequently exhibit decreased rates of weight gain. The purpose of this investigation was to determine whether decreased growth in infants receiving dexamethasone therapy is caused by increased energy expenditure. Twelve infants were studied: 6 received dexamethasone treatment at 2 wk of age and crossed over to receive placebo treatment at 4 wk; the treatment order was reversed in the other 6 infants. The doubly labeled water method was used to determine energy expenditure for a 1-wk period during each treatment phase. The rate of weight gain during dexamethasone treatment was 6.5+/-10.6 and 20.0+/-5.7 g/kg/d during placebo treatment. Energy expenditure was 93.1+/-34.6 kcal/kg/d during dexamethasone treatment and 88.3+/-37.1 kcal/kg/d during placebo treatment. Energy intake was 119.2+/-29.0 kcal/kg/d during dexamethasone treatment and 113.8+/-23.7 kcal/kg/d during placebo treatment. The difference between intake and expenditure, or the energy available for growth, was 26.2+/-36.8 kcal/kg/d during dexamethasone treatment and 25.5+/-37.4 kcal/kg/d during placebo treatment. No significant differences were found in energy expenditure or energy intake between the treatment phases. The reduced growth seen in infants receiving dexamethasone treatment cannot be explained by increased energy expenditure or decreased energy intake, but may be due to differences in the composition of newly accreted tissue.

Increased energy expenditure in infants with cyanotic congenital heart disease.

Infants with cyanotic congenital heart disease (CCHD) often have reduced weight gain compared with infants in control groups. Our purpose was to conduct a longitudinal study of energy intake, resting energy expenditure (REE), and total energy expenditure (TEE) of a group of infants with CCHD. We hypothesized that increased REE and TEE and decreased energy intake in these infants would lead to reduced growth. Ten infants with uncorrected CCHD and 12 infants in a control group were studied at 2 weeks of age and again at 3 months. Indirect calorimetry was used to determine REE; the doubly labeled water method was used to determine TEE and intake. At 2 weeks and 3 months of age, infants with CCHD weighed significantly less than infants in the control group. No significant difference was seen in energy intake or REE between groups during either period. TEE was slightly but not statistically increased in the CCHD group at 2 weeks (72.6 +/- 17.4 vs 59.8 +/- 10.9 kcal/kg/d) and significantly increased at 3 months (93.6 +/- 23.3 vs 72.2 +/- 13.2 kcal/kg/d, P

Total but not resting energy expenditure is increased in infants with ventricular septal defects.

OBJECTIVE: The purpose of this study was to determine the effect of left-to-right shunting on the resting energy expenditure (REE), total energy expenditure (TEE), and energy intake in a group of 3- to 5-month-old infants with moderate to large unrepaired ventricular septal defects (VSDs) compared with age-matched, healthy infants. METHODS: Eight infants with VSDs and 10 healthy controls between 3 to 5 months of age participated in the study. Indirect calorimetry was used to measure REE and the doubly-labeled water method was used to measure TEE and energy intake. An echocardiogram and anthropometric measurements were performed on all study participants. Daily urine samples were collected at home for 7 days. Samples were analyzed by isotope ratio mass spectrometry. Data were compared using analysis of variance. RESULTS: No significant differences were found in REE (VSD, 42.2 +/- 8.7 kcal/kg/d; control, 43.9 +/- 14.1 kcal/kg/d) or energy intake (VSD, 90.8 +/- 19.9 kcal/kg/d; control, 87.1 +/- 11.7 kcal/kg/d) between the groups. The percent total body water was significantly higher in the VSD infants and the percent fat mass was significantly lower. TEE was 40% higher in the VSD group (VSD, 87.6 +/- 10.8 kcal/kg/d; control, 61.9 +/- 10.3 kcal/kg/d). The difference between TEE and REE, reflecting the energy of activity, was 2.5 times greater in the VSD group. CONCLUSIONS: REE and energy intake are virtually identical between the two groups. Despite this, infants with VSDs have substantially higher TEE than age-matched healthy infants. The large difference between TEE and REE in VSD infants suggests a substantially elevated energy cost of physical activity in these infants. These results demonstrate that, although infants with VSDs may match the energy intake of healthy infants, they are unable to meet their increased energy demands, resulting in growth retardation.

Determination of the specific activity of sheep plasma amino acids using high-performance liquid chromatography: comparison study between liquid scintillation counter and on-line flow-through detector.

A method was developed for the determination of the specific activities of leucine and phenylalanine in plasma using a flow-through scintillation counter coupled with high-performance liquid chromatography components. Results were compared with those obtained from liquid scintillation counting. Differences in the specific activities of leucine and phenylalanine between the two methods were not statistically significant. We concluded that flow-through radioactivity detection can be used for quantitative amino acid assays. However, the minimum activity that can be detected may be prohibitively low in certain applications.

Acute changes in leucine and phenylalanine kinetics produced by parenteral nutrition in premature infants.

To determine the effect of parenteral nutrition on the balance and catabolism of leucine (by oxidation) and phenylalanine (by hydroxylation) and to assess any acute changes in proteolysis and/or protein synthesis, leucine and phenylalanine kinetics were measured by stable isotope tracer infusions in nine 32-wk gestation premature infants under both basal conditions and in response to an i.v. infusion of glucose, lipid, and amino acids. Leucine and phenylalanine balance both changed from negative to positive during parenteral nutrition. However, leucine and phenylalanine catabolism were differently affected by parenteral nutrition; the rate of leucine oxidation increased 2-fold, whereas the rate of phenylalanine hydroxylation was unchanged from basal values. Phenylalanine utilization for protein synthesis and leucine utilization for protein synthesis (based on both plasma leucine and alpha-ketoisocaproic acid enrichments) increased significantly during parenteral nutrition. The endogenous rates of release of leucine (based on plasma leucine enrichment) and phenylalanine (both reflecting proteolysis) were significantly reduced during parenteral nutrition. The endogenous rate of release of leucine (based on alpha-ketoisocaproic acid enrichment) was slightly but not significantly lower during parenteral nutrition. The substantial increase in leucine oxidation without changes in phenylalanine hydroxylation suggests a possible limitation in the phenylalanine/tyrosine supply during parenteral nutrition. In addition, these results suggest that premature infants respond to parenteral nutrition with acute increases in whole body protein synthesis as well as a probable reduction in proteolysis.

Amino acids suppress proteolysis independent of insulin throughout the neonatal period.

To determine how increased amino acid availability alters rates of whole body proteolysis and the irreversible catabolism of the essential amino acids leucine and phenylalanine throughout the neonatal period, leucine and phenylalanine kinetics were measured under basal conditions and in response to intravenous amino acids in two separate groups of healthy, full-term newborns (at 3 days and 3 wk of age). The endogenous rates of appearance of leucine and phenylalanine (reflecting proteolysis) were suppressed equally in both groups and in a dose-dependent fashion (by approximately 10% with 1.2 g x kg(-1) x day(-1) and by approximately 20% with 2.4 g x kg(-1) x day(-1)) in response to intravenous amino acid delivery. Insulin concentrations remained unchanged from basal values during amino acid administration. The irreversible catabolism of leucine and phenylalanine increased in a stepwise fashion in response to intravenous amino acids; again, no differences were observed between the two groups. This study clearly demonstrates that the capacity to acutely increase rates of leucine oxidation and phenylalanine hydroxylation is fully present early in the neonatal period in normal newborns. Furthermore, these data suggest that amino acid availability is a primary regulator of proteolysis in normal newborns throughout the neonatal period.

Effect of enteral versus parenteral feeding on leucine kinetics and fuel utilization in premature newborns.

To determine whether the route of nutrient delivery affects whole-body protein kinetics and fuel utilization, eight premature newborns were studied during both a 4-h period of enteral intake and a 4-h period of parenteral nutrition. The kinetics of the essential amino acid leucine were measured using a constant tracer infusion of 1-13C-leucine, and fuel utilization and energy expenditure were assessed by respiratory calorimetry. All leucine kinetic parameters were similar during enteral or parenteral nutrition (in mean +/- SD mumol/kg/h, flux = 233 +/- 51 enteral versus 258 +/- 42 parenteral, leucine from protein breakdown = 177 +/- 50 enteral versus 200 +/- 41 parenteral, leucine oxidation = 57 +/- 26 enteral versus 63 +/- 20 parenteral, and leucine used for protein synthesis = 176 +/- 63 enteral versus 196 +/- 50 parenteral). In addition, overall rates of energy expenditure (approximately 52 kcal/kg/d) and pattern of fuel utilization (approximately 70% carbohydrate, 13% fat, 17% protein) were unaltered by the route of feeding. Thus, as reflected by leucine kinetics, overall rates of protein turnover, synthesis, oxidation, and breakdown as well as energy expenditure and fuel utilization are similar when nutrition is provided to premature newborns by either the enteral or parenteral route. These results suggest that short-term provision of parenteral nutrition may be able to substitute appropriately for enteral intake, at least with regard to the utilization of one essential amino acid and the overall pattern of fuel utilization.

Meal-frequency effects on plasma hormone concentrations and cholesterol synthesis in humans.

To examine meal-frequency effects on circulating hormone concentrations and cholesterol synthesis, male subjects consumed liquid diets given as either six evenly spaced (ES) or three diurnal (DI) meals over 3 d. Deuterium oxide was given on day 2 and blood sampled every 4 h over days 2 and 3 to measure plasma cholesterol, glucose, insulin, and glucose-dependent insulinotropic polypeptide (GIP) concentrations and cholesterol synthesis. Cholesterol synthesis was determined from deuterium incorporation into plasma free cholesterol by using constrained and unconstrained curve-fit models. Plasma total cholesterol concentrations decreased in both ES and DI groups (P < 0.05). The ES group had lower insulin (P < 0.05) and GIP (P < 0.001) concentrations compared with the DI group. Cholesterol synthesis was reduced (P < 0.01) in the ES vs the DI group when determined by using constrained (0.050 +/- 0.002 vs 0.075 +/- 0.005 pools/d, respectively) and unconstrained (0.072 +/- 0.005 vs 0.119 +/- 0.011 pools/d, respectively) models. These data suggest meal frequency-dependent control of cholesterogenesis via hormonally mediated mechanisms.

Validation of doubly labeled water for measurement of caloric expenditure in collegiate swimmers.

To validate doubly labeled water (DLW) in aquatic athletes, energy expenditures (EE) were determined by energy intake/balance (IB) and DLW in free-living collegiate swimmers [n = 8, age 20.1 +/- 1.7 (SD) yr, height 186 +/- 11 cm, weight 74.1 +/- 9.3 kg] over 9 days. EEIB was determined from caloric analysis of duplicate foods to those consumed by subjects and corrected for changes in body composition across the study. For EEDLW, subjects were given 2H2O and H2(18)O on day 0 and 2H2O on day 10. Day 0 and 10 body compositions were measured by use of 2H dilution space, and elimination rates over days 1-10 were determined for 2H and 18O. EEDLW was calculated from CO2 respiratory quotient by use of a food quotient. Fat mass increased (P < 0.01) over the study period. Mean uncorrected energy intake was 3,895 +/- 621 kcal/day. When all eight subjects were compared, EEIB and EEDLW were 3,492 +/- 876 and 3,466 +/- 992 kcal/day, respectively. Data from one subject compared poorly despite repeat isotopic analyses. In the remaining seven subjects, EEIB and EEDLW were 3,444 +/- 925 and 3,556 +/- 1,025 kcal/day, respectively. These data indicate that DLW is a good indicator of EE in groups of swimmers and that energy requirements of swimmers are similar to other athletic groups.

Human cholesterol synthesis measurement using deuterated water. Theoretical and procedural considerations.

Human cholesterogenesis is measurable as the rate of incorporation of deuterium derived from deuterium oxide (D2O) within the body water pool into plasma or erythrocyte cholesterol pools. Oral D2O equilibrates across body water, thus enabling extracellular sampling of pools (such as urine) to serve as accurate indicators of intracellular deuterium enrichments at the point of synthesis. Required doses of D2O fall below the threshold associated with negative side effects. Deuterium/carbon incorporation ratios into cholesterol during biosynthesis have been established that are applicable in humans. Models using unconstrained and constrained curve fitting permit improved flexibility in interpretation of deuterium-uptake kinetics. However, sample-size restrictions presently limit the ability of the technique to examine the kinetics within individual lipoprotein species. Correction of enrichment data for proton exchange during combustion and reduction phases of sample preparation is an additional important procedural concern. In summary, the deuterated-water procedure is a useful tool in studies of human cholesterol synthesis that offers the advantages of short measurement interval, relative noninvasiveness, and provision of a direct index of synthesis in comparison with other available techniques.

Measurement of human lipogenesis using deuterium incorporation.

In order to determine human triglyceride fatty acid (TG-FA) synthesis rates, the incorporation of deuterium from the plasma water pool in vivo into TG-FA was measured over 48 h in six healthy males under steady state conditions. Subjects consumed a nutritionally complete liquid diet as six evenly spaced small meals per day for 3 days and drank 0.7 g D2O/kg estimated body water, (99.8 APE) at 0800 h on day 2. D2O was added to the liquid diets to maintain plasma deuterium enrichment at plateau over days 2 and 3. Blood samples were obtained prior to dosing and every 4 h thereafter. Total TG-FA were obtained from plasma at each timepoint and combusted. The resultant water was vacuum-distilled into Zn-containing Pyrex tubes, reduced to hydrogen gas, and analyzed for deuterium abundance by isotope ratio mass spectrometry. TG-FA deuterium enrichment reached a plateau at approximately 12 h post-dose. To establish the amount of newly synthesized TG-FA in the total plasma TG-FA pool, two models were developed relating the maximum observed deuterium enrichment to the calculated theoretical maximum enrichment. In model A, it was assumed that TG-FA come solely from chylomicrons. This yields a value of 8.8 +/- 4.6 g/day (mean +/- SD) de novo synthesized TG-FA. On the opposite extreme in model B, it was assumed that all the TG-FA are derived from recycled very low density lipoprotein (VLDL); the amount of newly synthesized TG-FA calculated by this model was 1.7 +/- 0.8 g/day.(ABSTRACT TRUNCATED AT 250 WORDS)

Correspondence between plasma mevalonic acid levels and deuterium uptake in measuring human cholesterol synthesis.

To assess the validity of two techniques capable of identifying immediate changes in human cholesterol production, plasma mevalonic acid levels and the rate of uptake of deuterium into plasma free cholesterol were compared in 5 healthy individuals over 48 h. The free-living subjects self-selected three meals per day prior to and during study. At t = 0, deuterium oxide was administered orally. Blood samples were collected before and every 4 h after dosing. Total cholesterol and mevalonic acid levels were determined in plasma at each timepoint. Deuterium enrichment changes in plasma free cholesterol, relative to plasma water content, were used to calculate free cholesterol fractional synthetic rates (FSR) at each timepoint. Total plasma cholesterol levels remained constant, whereas significant circadian rhythmicity was observed in both plasma mevalonic acid and deuterium uptake methods, with nadir and peak formation rates indicated at 14.00 to 16.00 h and about midnight, respectively. It is suggested that plasma mevalonic acid levels and free cholesterol deuterium uptake rate techniques are both suitable techniques for short-term measurement of human cholesterol synthesis.

Cholesterol and triglyceride fatty acid synthesis in apolipoprotein E2-associated hyperlipidemia.

To investigate whether increased endogenous lipogenesis contributes to elevated plasma lipid levels in individuals with apolipoprotein (apo) E2-associated hyperlipidemia (E2-HL), plasma pool cholesterol and triglyceride fatty acid syntheses were measured in subjects with E2-HL and in those with normal lipid levels. Subjects were given a priming dose of deuterium oxide (D2O) followed by maintenance doses over 48 hours. During the first 24 hours, subjects consumed prepared meals, whereas during the 24-48 hour interval, they consumed water only. Blood samples were drawn every 12 hours, and cholesterol and triglyceride fatty acid formation rates were determined from the change in deuterium enrichment. The free cholesterol fractional synthesis rate over 0-24 hours of E2-HL subjects (0.057 +/- 0.010 day-1, mean +/- SEM) was not significantly different from that of normolipidemics (0.075 +/- 0.005 day-1). Calculated cholesterol net synthesis was not different between the two groups (0.56 +/- 0.07 and 0.75 +/- 0.05 g/day, respectively). Mean free cholesterol synthesis for all subjects was higher in the fed (0-24 hour) compared with the fasted (24-48-hour) condition. Initial 12-hour triglyceride fatty acid fractional synthesis was significantly (p less than 0.01) increased in E2-HL subjects (0.143 +/- 0.012 day-1) compared with controls (0.082 +/- 0.0013 day-1). These findings suggest that in E2-HL, elevated plasma cholesterol levels are due to factors other than increased sterol synthesis, while higher de novo fatty acid synthesis contributes to the observed hypertriglyceridemia.

Measurement of triglyceride synthesis in humans using deuterium oxide and isotope ratio mass spectrometry.

Short-term triglyceride (TG) synthesis was measured over 48 h in four healthy males from the incorporation rate of deuterium in body water into plasma TG. Subjects drank 0.7 g D2O kg-1 estimated body water (99.8 atom% excess), followed by water containing 1.4 g D2O kg-1 water to maintain plasma deuterium enrichment at plateau. Blood samples (20 ml) were obtained before dosing and every 4 h thereafter. Subjects self-selected three meals each day. TG from each time point were separated from plasma lipids by thin-layer chromatography and combusted to water and CO2. Combustion water was vacuum distilled into Zn-containing Pyrex tubes, reduced to hydrogen gas, and analyzed for deuterium enrichment by isotope ratio mass spectrometry. Deuterium enrichment of TG increased over the 48 h study period for all four subjects studied. Superimposed on this increase were short-term non-periodic fluctuations in enrichment reflecting dietary influx and intra-individual differences in TG metabolism. The TG fractional synthetic rate (FSR) was calculated using linear and mono-exponential models. Triglyceride FSR of the subjects over the first 24 h of the study was 0.0702 +/- 0.0048 day-1 (mean +/- SEM) by the linear model and 0.0728 +/- 0.0051 day-1 by the exponential model. Deuterium enrichment reached a plateau on day 2, indicative of continuing TG synthesis in a saturated body water pool. These results are consistent with the notion of meal-dependent variability in TG synthesis into a small rapid turnover plasma TG pool.

Validation of bioelectrical-impedance analysis as a measurement of change in body composition in obesity.

The bioelectrical-impedance-analysis (BIA) method accurately measures body composition in weight-stable subjects. This study validates the use of BIA to measure change in body composition. Twelve obese females underwent weight loss at a mean rate of 1.16 kg/wk. Body composition was measured by deuterium oxide dilution (D2O), BIA, and skinfold anthropometry (SFA) at baseline and at 5% decrements in weight. Highly significant correlations were obtained between D2O and BIA (r = 0.971) and between D2O and SFA (r = 0.932). Overall, BIA predicted change in fat-free mass with greater accuracy (to 0.4 kg) and precision (+/- 1.28 kg) than did anthropometry (to 0.8 kg and +/- 2.58 kg, respectively). We conclude that BIA is a useful clinical method for measuring change in body composition.

Sleep deprivation in the rat: V. Energy use and mediation.

We investigated the use and possible mechanisms mediating the increased energy expenditure (EE) previously described for rats subjected to total or paradoxical sleep deprivation. Bomb calorimetry of wastes showed that during deprivation the efficiency of energy utilization was not reduced. Estimates of CO2 production by the doubly labelled water method of indirect calorimetry correlated with EE estimated from the caloric value of food, weight change, and wastes and confirmed an increase in EE during deprivation. Core temperatures decreased during the later stages of deprivation, suggesting the hypothesis that excessive heat loss may have required increased EE to protect body temperature. The increased EE could not be explained by the metabolic cost of increase wakefulness, water exposure, or motor activity; an increase in resting EE was indicated. The contribution of the hypothalamic-pituitary-adrenal axis, thyroid gland, and sympathoadrenal system to the mediation of the EE increases was evaluated by measuring the plasma levels of their hormones. Results appear to rule out the first as a mediator. Evidence for the other two was equivocal.