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The Decisive Role of Free Fatty Acids for Protein Conservation during Fasting in Humans with and without Growth Hormone

Medical Department M (Endocrinology and Diabetes) (H.N., S.N., J.O.L.J., J.S.C.), Institute of Experimental Clinical Research (J.F., N.M.), Department of Nephrology (P.I.), Aarhus University Hospital, DK-8000 Aarhus C, Denmark; and Endocrinology Division, Mayo Clinic (K.S.N.), Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dr. Helene Nørrelund, Medical Department M, Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark. E-mail: helenenorrelund{at}dadlnet.dk.

	Abstract

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During fasting, a lack of GH increases protein loss by close to 50%, but the underlying mechanisms remain uncertain. The present study tests the hypothesis that the anabolic actions of GH depend on mobilization of lipids. Seven normal subjects were examined on four occasions during a 37-h fast with infusion of somatostatin, insulin, and glucagon for the final 15 h: 1) with GH replacement, 2) with GH replacement and antilipolysis with acipimox, 3) without GH and with antilipolysis, and 4) with GH replacement, antilipolysis, and infusion of intralipid. Urinary urea excretion, serum urea concentrations, and muscle protein breakdown (assessed by labeled phenylalanine) increased by almost 50% during fasting with suppression of lipolysis. Addition of GH during fasting with antilipolysis did not influence indexes of protein degradation, whereas restoration of high FFA levels regenerated proportionally low concentrations of urea and decreased whole body protein degradation (phenylalanine to tyrosine conversion) by 10–15%, but failed to affect muscle protein metabolism. Thus, the present data provide strong evidence that FFA are important protein-sparing agents during fasting. The finding that inhibition of lipolysis eliminates the ability of GH to restrict fasting protein loss indicates that stimulation of lipolysis is the principal protein-conserving mechanism of GH.

	Introduction

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DESPITE THE USE of modern technologies within agriculture and medicine, fasting-related states of distress (hunger, malnutrition, and wasting syndromes) continue to constitute a major health problem worldwide. In this context GH may play an important protein-conserving role.

The impact of GH on protein metabolism is complex. Previous studies have shown that GH postabsorptively primarily increases protein synthesis at the whole body level (1), and there is evidence that acute exposure to high levels of GH may directly increase muscle protein synthesis (2). It has also been reported that 6 wk of GH treatment to malnourished hemodialysis patients resulted in stimulation of muscle protein synthesis without any effect on muscle protein degradation (3). During fasting in normal subjects we have recently reported that suppression of GH leads to a 50% increase in urea-nitrogen excretion together with an increased net release and an increased appearance rate of phenylalanine across the forearm (4). In GH-deficient adults (GHDA), hyposomatotropinemia during fasting was associated with increased whole body protein loss, accounted for by a net reduction in protein synthesis (5). Furthermore, GH substitution for 8 wk in GHDA revealed increased net protein synthesis postabsorptively and unaltered total protein turnover (6), in agreement with dose-response studies (7).

The mechanism by which GH affects protein metabolism remains uncertain, and collectively the metabolic effects are complex, involving increased lipolysis, hyperglycemia, hyperinsulinemia, and an increase in IGF-I activity (8, 9, 10, 11). Previous studies have suggested that elevated concentrations of free fatty acids (FFA) and ketone bodies in the circulation may have an anabolic effect on protein metabolism (12, 13, 14). Postabsorptively, pharmacological suppression of FFA is accompanied by augmented whole body protein degradation (15). The degree to which release of lipids contributes to the anabolic actions of GH during fasting has not been specifically investigated previously, although the lipolytic responsiveness to GH may be increased during energy restriction (16). We have previously shown that GH increases circulating lipid intermediates and has important nitrogen-conserving effects during short-term fasting (4). The present study was undertaken to assess the acute effects of FFA and GH, independently of its lipolytic actions, on protein metabolism. For this purpose we studied seven normal subjects on four occasions after a 37-h fast with concomitant determination of urea excretion and whole body and regional muscle fluxes of phenylalanine and tyrosine. Circulating levels of GH and FFA were clamped by infusion of somatostatin and administration of acipimox (a nicotinic acid analog) together with GH and triglycerides/heparin.

	Materials and Methods

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Seven healthy male subjects (mean age, 23 ± 1 yr; body mass index, 24 ± 1 kg/m²) were examined on four occasions during a 37-h fast: 1) with GH replacement (+GH, -acipimox), 2) with GH replacement and inhibition of lipolysis (+GH, +acipimox), 3) without GH and with inhibition of lipolysis (-GH, +acipimox), and 4) with GH replacement, inhibition of lipolysis, and infusion of intralipid (10%, 1 ml/min) and heparin (90 mU/kg·min; +GH, +acipimox, +intralipid; Fig. 1). To control hormone concentrations, somatostatin, insulin, and glucagon were infused for the last 15 h (i.e. after 22 h of fasting) on all occasions, and GH (no. 1, 2, and 4) and acipimox (no. 2–4) were added during the last 15 h as outlined below. Somatostatin (200 µg/h) was infused together with insulin [100 IU/ml Actrapid, Novo Nordisk, Copenhagen, Denmark; infusion rate adjusted to maintain plasma glucose levels between 3–5 mmol/liter (0.1–0.05 mU/kg·min)] and glucagon [1 ng/ml GlucaGen, Novo Nordisk (1.0–1.5 ng/kg·min)]. All treatment limbs were identical in terms of hormone replacement doses, except for GH [12 IU/ml Norditropin, Novo Nordisk; 4.5 IU partly as bolus injections (0.6 IU every 4 h), partly as continuous infusion (0.15 IU/h)] and acipimox (Olbetam; 250 mg, orally, every 4 h). To avoid hypoglycemia (plasma glucose concentration <3 mmol/liter) during fasting with GH suppression and antilipolysis (-GH, +acipimox), all participants temporarily received glucose iv (0.2 mg/kg·min for a period of 0.5–1.5 h the night before the study); exactly the same amount of glucose was given in precisely the same manner in all experiments. As potential hypoglycemia was anticipated during GH suppression, the -GH, +acipimox experiment was always conducted before the rest to allow for control of exogenous glucose. The subjects were blinded to this procedure. Otherwise the experiments were random, with at least 6 wk elapsing between each experiment. During the last 3 h, substrate metabolism was investigated. No participants received glucose during these last 3 h, as glucose infusion was tapered at least 2 h before investigations of substrate metabolism were started. Before the study, subjects were on a weight-maintaining diet for at least 3 months. All subjects gave written informed consent, and the study was approved by the regional ethics committee and conducted according to the Declaration of Helsinki.

View larger version (29K): [in this window] [in a new window]   FIG. 1. The experimental design (see text for details).

All studies were performed with participants in the supine position. At 0800 h, priming doses of L-[¹⁵N]phenylalanine (0.7 mg/kg), L-[²H₄]tyrosine (0.5 mg/kg), and L-[¹⁵N]tyrosine (0.3 mg/kg; Cambridge Isotope Laboratories, Inc., Andover, MA) were given to reach an early plateau. A continuous infusion of L-[¹⁵N]phenylalanine (0.7 mg/kg·h) and L-[²H₄]tyrosine (0.5 mg/kg/h) was started and maintained for 3 h. The chemical, isotopic, and optical purities of the isotopes were tested before use. Solutions were prepared under sterile precautions and were shown to be free of bacteria and pyrogens. L-[¹⁵N]Phenylalanine, L-[²H₄]tyrosine, and L-[¹⁵N]tyrosine were measured as their t-butyldimethylsilyl ether derivatives under electron ionization conditions (17). In addition, concentrations of phenylalanine and tyrosine were measured by mass spectrometry using L-[²H₈]phenylalanine and L-[¹³C₆]tyrosine, respectively, as internal standards (17). Plasma concentrations of amino acids were determined by an HPLC system (Bio-Tek Kontron, series 525 and 465, fluorescence detector SFM25, Kontron Instruments, Milan, Italy) with precolumn O-phthalaldehyde derivatization using a slight modification of the method described by Jones and Gilligan (18). The plasma glucose level was measured in duplicate immediately after sampling on a glucose analyzer (Beckman Instruments, Palo Alto, CA). Whole blood glycerol, lactate and 3-hydroxybutyrate (3-OH-butyrate) were analyzed by autofluorimetric enzymatic methods (19). Levels of serum FFA were determined using a commercial kit (Wako Chemicals, Neuss, Germany). A double monoclonal immunofluorometric assay (Delfia, Wallac, Finland) was used to measure serum GH, whereas plasma glucagon and IGF-I levels were measured by in-house assays (20, 21). Insulin and C peptide were determined by commercial immunological kits (DAKO, Glostrup, Denmark; Immunoclear, Stillwater, MN). Catecholamines were measured by liquid chromatography (22). Urinary urea excretion was determined by an indophenol method, and serum urea by a commercial kit (Cobasintegra, Roche, Hvidovre, Denmark). Cortisol was measured using an automated chemiluminescence system (Chiron Diagnostics, Fernwald, Germany). Urea accumulation was determined assuming immediate dispersion of urea from the blood to total body water (TBW), which was estimated by the formula (23): TBW = 0.3625 x BW + 0.2239 x BH - 0.1387 x Y - 14.47, where Y is age in years, BW is body weight in kilograms, and BH is body height in centimeters. The urea-nitrogen synthesis rate (UNSR) was determined as urinary excretion of urea (E) corrected for accumulation (A) of urea in TBW and for hydrolysis in gut (L) (24): UNSR = (E + A)/(1 - L). L was estimated to be 0.14.

Forearm method

Catheters for measurements of forearm arteriovenous substrate balances were placed as previously described (4). In brief, one catheter was placed retrogradely in a deep antecubital vein, and one catheter was inserted retrogradely in a heated contralateral dorsal vein. Criteria for correct positioning were oxygen saturations less than 70% and more than 91%, respectively. A third catheter was placed antegradely in an antecubital vein of the heated hand for infusions. Before each deep venous sample, total ipsilateral forearm blood flow was determined by means of venous occlusion plethysmography. Hand blood flow was interrupted by a wrist cuff inflated to a pressure of 250 mm Hg immediately before each blood flow determination and 1 min before each deep venous sample.

Phenylalanine kinetics

For measurements of whole body phenylalanine kinetics, the equations of Thompson et al. (25) were used, and for regional phenylalanine kinetics, previously described equations (17) were used. Phenylalanine flux (Q_p) and tyrosine flux (Q_t) were calculated as follows: Q_flux = I[(E_i/E_p) - 1], where I is the rate of tracer infusion (micromoles per kilogram per hour), and Ei and Ep are enrichment of the tracer infused and plasma enrichment of the tracer at isotopic plateau, respectively.

The rate of phenylalanine conversion to tyrosine (I_pt) was calculated as follows: I_pt = Q_t x ([¹⁵N]Tyr_ei/[¹⁵N]phe_ei) x (Q_p/(I_p+Q_p)), where [¹⁵N]Tyr_ei and [¹⁵N]Phe_ei are the isotopic enrichments of the respective tracers in plasma, and Ip is the infusion rate of [¹⁵N]phenylalanine micromoles per kilogram per hour).

Phenylalanine incorporation into protein is calculated by subtracting I_pt from Q_p, because phenylalanine is irreversibly lost either by conversion into tyrosine or by incorporation into protein.

In the forearm study phenylalanine balance (PheBal) was calculated as follows: PheBal = (Phe_A - Phe_V) x F, where PheA and PheV are phenylalanine concentrations in arteries and veins, and F is blood flow.

Regional protein breakdown, represented by the phenylalanine rate of appearance (RaPhe), was calculated as follows (26): RaPhe = Phe_A [(Phe_EA/Phe_EV) - 1] x F, where Phe_EA and Phe_EV represent phenylalanine isotopic enrichment in arteries and veins.

The local rate of disappearance was calculated as: Rd Phe = Ra Phe + Phe Bal.

Microdialysis

A microdialysis catheter (CMA 60, CMA, Stockholm, Sweden) was placed in the abdominal sc adipose tissue after anesthetization of the skin with 0.05 ml lidocaine at the site of perforation of the skin. The microdialysis catheter used has a molecular cut-off of 20 kDa. Immediately after placement, perfusion of the catheters with physiological perfusion fluid (perfusion fluid T1, CMA; Na⁺, 147 mmol/liter; K⁺, 4 mmol/liter; Ca²⁺, 2.3 mmol/liter; Cl^-, 156 mmol/liter; pH 6; osmolality, 290 mosmol/kg) at a flow rate of 0.3 ml/min with the use of a portable pump (CMA 106, CMA) was accomplished. At this flow rate, the rate of recovery with the microdialysis catheter is almost 100% (27). The microdialysis catheter was placed at 0 min (34 h of fasting; 0800 h). After 1 h of calibration with perfusion of the microdialysis catheter, allowing local edema and hemorrhage to subside, sampling started at 60 min and continued every 60 min. Urea and glycerol in the dialysate were measured by an automated spectrophotometric kinetic enzymatic analyzer (CMA 600, CMA).

Statistics

Results are expressed as the mean ± SEM. The Kolmogorov-Smirnov test was used to test for normal distribution. Statistical comparisons between the study periods (1) +GH, -acipimox, 2) +GH, +acipimox, 3) -GH, +acipimox, and 4) +GH, +acipimox, +intralipid) were assessed by a two-way ANOVA. If this test was positive, post hoc comparison was performed by means of a paired t test (to test: the effect of lipolysis (1 vs. 2), the effect of GH (2 vs. 3), and the effect of intralipid (2 vs. 4) or Wilcoxon test for nonparametric data. For time series, the area under the curve was calculated by the trapezoidal method, and comparisons were made by ANOVA. P < 0.05 was considered significant. Unless specified otherwise, data referred to below were obtained with arterialized blood based on triplicate measurements during the last 30 min of the study periods.

	Results

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Circulating hormones and metabolites

Inhibition of lipolysis led to an 80% increase in GH concentration despite somatostatin infusion (Table 1). Total IGF-I decreased, and the counterregulatory hormones cortisol and epinephrine increased. Addition of GH during inhibition of lipolysis was associated with an increase in glucagon and a decrease in norepinephrine. Addition of intralipid lowered concentrations of GH, cortisol, epinephrine, and norepinephrine.

Whole body. Urinary urea excretion (occasion 1, 521 ± 50 mmol/24 h; occasion 2, 634 ± 48 mmol/24 h; occasion 3, 691 ± 55 mmol/24 h; occasion 4, 589 ± 54 mmol/24 h; P < 0.05) and serum urea (occasion 1, 5.2 ± 0.3 mmol/liter; occasion 2, 7.3 ± 0.3 mmol/liter; occasion 3, 7.6 ± 0.3 mmol/liter; occasion 4, 5.2 ± 0.2 mmol/liter; P < 0.01) were significantly increased during fasting with suppression of lipolysis, and UNSR was increased as well (occasion 1, 22.2 ± 2.1 mmol/h; occasion 2, 32.1 ± 3.7 mmol/h; occasion 3, 30.7 ± 3.0 mmol/h; occasion 4, 24.8 ± 1.9 mmol/h; P < 0.05; Fig. 2). Interstitial urea was about 50% increased during fasting with antilipolysis, whereas addition of intralipid was associated with a proportionally low interstitial urea concentration (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Serum urea, UNSR, and interstitial urea (mean ± SE). *, P < 0.05 -acipimox vs. +acipimox (occasion 1 vs. occasion 2). **, P < 0.01 -acipimox vs. +acipimox (occasion 1 vs. occasion 2). ##, P < 0.01 -intralipid vs. +intralipid (occasion 2 vs. occasion 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we sought to define mechanisms participating in the protein-conserving actions of GH during fasting and, more specifically, to test the hypothesis that stimulation of lipolysis may be instrumental for such actions. This concept was based on previous studies in our laboratory showing that GH increases circulating lipid intermediates and that the lack of GH during short-term fasting in healthy volunteers increases urea-nitrogen loss by 50% (4, 5). The main findings of our study are that inhibition of lipolysis increases urea-nitrogen production and muscle protein breakdown (assessed by labeled phenylalanine) by nearly 50% and neutralizes the ability of GH to reduce ureagenesis and to restrict muscle protein breakdown, and restoration of high FFA levels (intralipid infusion) reestablishes proportional concentrations of urea and decreases whole body phenylalanine degradation by 10–15%, but fails to decrease muscle protein breakdown.

When infusing intralipid we made some intriguing observations. Although we achieved FFA concentrations of 2.8 mmol/liter, i.e. in the supraphysiological range, 3-OH-butyrate concentrations remained suppressed. Some studies have suggested a rise in ketone body concentrations after intralipid infusion in the basal state (28, 29, 30), whereas others have failed to observe any rise (31). This obviously indicates that ketogenesis during fasting is regulated by factors others than FFA in the systemic circulation (32), and our failure to observe any effect on muscle amino acid metabolism during intralipid infusion is compatible with the idea that ketone bodies are instrumental in suppressing protein breakdown and amino acid release from muscle, as suggested by a number of studies (14, 31, 33).

Many counterregulatory hormones were significantly increased when lipolysis was inhibited during fasting, to some extent probably secondary to feedback mechanisms from the reduced levels of FFA and glucose. This increase may have contributed to the effects on protein metabolism. In humans, pharmacological doses of glucocorticosteroids, sufficient to increase plasma cortisol concentrations to the high physiological range, increase proteolysis (1, 34). In the present study a modest increase in cortisol was observed. Although we saw an increase in circulating catecholamine concentrations, there is, to our knowledge, no evidence that catecholamines stimulate protein breakdown.

Despite the fact that glucagon was administered in exactly the same manner in all four situations, plasma levels were paradoxically decreased during fasting with suppression of GH and inhibition of lipolysis. Little is known about the mechanisms responsible for glucagon clearance in the body, but the circulating serine protease dipeptidyl peptidase IV seems to be a primary enzyme involved in the degradation and inactivation of glucagon (35); the regulation of this peptide, however, remains to be clarified. Protein wasting and reduced amino acid concentrations are common findings in glucagonoma patients (36, 37), and glucagon has distinct protein catabolic effects (38, 39).

Antilipolysis was induced by administration of the nicotinic acid derivative acipimox, which is known to exert its antilipolytic effect by lowering the intracellular level of cAMP and thereby inhibiting the activity of the hormone-sensitive lipase (40). Any possible intrinsic effect of acipimox on protein metabolism, however, has not been verified. In our study acipimox exerted a marked impact on circulating FFA concentrations, and net protein degradation was significantly increased, as determined by UNSR and phenylalanine hydroxylation. Despite the increase in UNSR, whole body amino acid degradation (as measured by phenylalanine conversion to tyrosine) did not increase significantly. This could relate either to a type II error or to the very distinct metabolism of phenylalanine and tyrosine. Hydroxylation of phenylalanine to tyrosine occurs in liver and kidney catalyzed by phenylalanine hydroxylase, and tyrosine is the precursor for thyroid hormone and catecholamine synthesis. If any of these processes changes differentially from overall amino acid metabolism, the resulting estimate of protein degradation may be misleading (41). The fact that intralipid infusion, which was used to recreate high FFA concentrations, led to a normalization of serum urea, interstitial urea, and protein degradation is in line with the idea that acipimox acts on protein metabolism exclusively by lowering FFA levels. Our failure to detect a significant drop in UNSR with intralipid infusion (P = 0.13) could relate to inaccuracies related to the collection of urine.

Previous studies assessing the impact of GH on protein kinetics, which have been performed postabsorptively, have generally shown that GH primarily increases protein synthesis at the whole body level when administered systemically (1, 3, 6), and there is evidence that GH may increase muscle protein synthesis when directly infused into the forearm (2). Some studies have failed to detect any effect of GH on muscle protein synthesis (26, 42), whereas one study showed that protein synthesis is stimulated by daily GH substitution dose of 3.3 µg/kg·d, whereas a GH substitution dose of 2 µg/kg·d did not change protein turnover (7). During fasting, GH has been shown to inhibit muscle protein breakdown in healthy subjects (4), whereas continuation of GH substitution during fasting in GHDA conserves nitrogen through stimulation of protein synthesis (5). The apparent complexity of the impact of GH on protein metabolism is perhaps predictable when considering the diverse metabolic actions of GH, including stimulation of lipolysis and IGF-I activity, hyperinsulinemia, and, at times, hyperglycemia. In addition, many studies have employed exposure to very high levels of GH, rendering results difficult to interpret in a physiological context. On the whole, it appears that the effects of GH on protein metabolism include both stimulation of protein synthesis and inhibition of breakdown depending on the nature of GH administration, which tissues are being examined, and the physiological conditions of the subjects studied.

Several lines of evidence support a protein-sparing effect of lipids. Infusion of ketone bodies has been shown to decrease circulating levels of alanine (13), leucine oxidation, and leucine incorporation in protein (12). Lowering of FFA concentrations in healthy subjects by the administration of acipimox increased total urea-nitrogen production (43), and urea excretion during fasting (44), whereas elevation of circulating levels by infusion of intralipid and heparin in humans was shown to have a hypoaminoacidemic action in man (45). The present study suggests that lowering circulating levels of FFA by pharmacological antilipolysis exerts important effects on net whole body protein degradation in terms of a 50% increase in urea-nitrogen synthesis and muscle protein breakdown. Addition of GH during fasting with antilipolysis did not influence indexes of protein degradation, whereas infusion of intralipid restored normal preexisting conditions at the whole body level. In the forearm study, however, neither the negative phenylalanine balance nor the amino acid balance was restored by infusion of intralipid, as discussed above.

Studies of the effect of IGF-I on protein metabolism have yielded conflicting results, and the mechanism by which IGF-I affects protein turnover is still a matter of controversy. It has been shown that iv infusion of IGF-I inhibits proteolysis (46, 47), whereas IGF-I during amino acid infusion may stimulate protein synthesis (48). Total IGF-I was significantly decreased during fasting with antilipolysis and was unaffected by the addition of GH, indicating either that acipimox may have direct effects on IGF-I or that substrate availability may be important for IGF-I generation.

A number of studies have shown that insulin profoundly affects protein metabolism, primarily by inhibiting proteolysis (49, 50). In the present study insulin levels were comparable throughout all study periods, and it is unlikely that differences in insulin influenced our observations.

It is possible that hyperglycemia per se may affect protein metabolism. In our study high levels of FFA were associated with modest hyperglycemia, whereas forearm glucose uptake did not differ among the four settings (data not given). Previous studies have implied that the administration of small amounts of glucose during fasting (51) and hyperglycemia (52) may be protein sparing. A more recent study, however, suggests that the decrease in whole body proteolysis during the infusion of insulin and glucose is mediated by the plasma insulin concentration and not the ambient glucose concentration or the rate of glucose utilization (53).

In summary, the present study gives strong evidence that FFA are important protein-retaining modulators during fasting. Lowering of circulating FFA concentrations leads to a 50% increase in urea-nitrogen production and both net and total phenylalanine release from the forearm and restoration of high FFA concentrations with exogenous lipid infusion normalizes these aberations at the whole body level and reduces phenylalanine to tyrosine conversion. The presence of high circulating concentrations of ketone bodies seems to be a prerequisite for FFA to specifically decrease muscle protein breakdown and amino acid release. The finding that inhibition of lipolysis blocks the ability of GH to restrict protein loss clearly suggests that stimulation of lipolysis is instrumental to the protein-conserving effects of GH under fasting conditions.

	Acknowledgments

 
GH preparations were generously supplied by Novo Nordisk (Copenhagen, Denmark). Dr. K. G. M. M. Alberti (University of Newcastle upon Tyne, Newcastle upon Tyne, UK) and Dr. Frederik Andreasen (University of Aarhus, Aarhus, Denmark) kindly provided the measurements of glycerol, 3-OH-butyrate, lactate, and catecholamines. We are grateful to Lone Svendsen and Iben Sørensen for excellent technical assistance.

	Footnotes

 
This work was supported by Novo Nordisk Fonden, Aage and Johanne Louis-Hansens Fond, the Danish Research Council, Grant 9600822 (Novo Nordisk Center for Research in Growth and Regeneration, Aarhus University), and NIH Grants RO1-DK-41973 and RR-00585.

Abbreviations: FFA, Free fatty acids; GHDA, GH-deficient adults; 3-OH-butyrate, 3-hydroxybutyrate; TBW, total body water; UNSR, urea-nitrogen synthesis rate.

Received February 19, 2003.

Accepted May 29, 2003.

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