Download Alterations in basal nutrient metabolism increase resting energy

Alterations in basal nutrient metabolism increase
resting energy expenditure in sickle cell disease
MYFANWY J. BOREL,1 MACIEJ S. BUCHOWSKI,3 ERNEST A. TURNER,4
BENJAMIN B. PEELER,1 RICHARD E. GOLDSTEIN,1 AND PAUL J. FLAKOLL1,2
Departments of 1Surgery and 2Biochemistry, Vanderbilt University School of Medicine,
Nashville 37232; and 3Center for Nutrition and 4Comprehensive Sickle Cell Center,
Meharry Medical College, Nashville, Tennesse 37208
sickle cell anemia; protein metabolism; carbohydrate metabolism; lipolysis; amino acids
SICKLE CELL DISEASE (SCD) is a genetic disorder in which
abnormal hemoglobin (Hb), known as sickle Hb or HbS,
is produced due to the replacement of glutamic acid
with valine in position six on the b-chains of Hb (28).
On deoxygenation in peripheral tissues and organs, red
blood cells (RBCs) containing HbS become rigid and
sickled in shape and can obstruct blood flow through
small vessels, thereby causing painful vaso-occlusive
events that lead to bone, tissue, and organ damage (50).
Because of the structural change to the RBC caused by
HbS, the RBC lifespan is greatly reduced from the
normal 120 days to 10 days, resulting in a chronic
hemolytic anemia (50). Due to the accelerated synthesis of new RBCs and the enhanced catabolism of
irreversibly sickled RBCs, increased rates of RBC
protein turnover are obligatory in SCD patients. However, the impact of this increase in RBC turnover on
whole body protein homeostasis as well as on whole
body carbohydrate and lipid homeostasis is not well
established. Furthermore, previous research has dem-
onstrated that resting energy expenditure is elevated
in adults, adolescents, and children who have homozygous SCD (10, 24, 45, 51, 52). However, the contribution
of changes in whole body protein, carbohydrate, and
lipid metabolism to this increase in metabolic rate is
not completely understood. The breakdown and synthesis of protein, carbohydrate, and lipid are essential
metabolic processes that consume considerable energy.
For example, the formation of each peptide bond during
protein synthesis requires the energy equivalent to ,5
molecules of ATP (41, 42). Thus the purpose of the
present study was to identify the metabolic consequences of SCD on protein, carbohydrate, and lipid
homeostasis and to ascertain how these alterations
affect resting energy expenditure. To accomplish this
goal, we measured basal rates of whole body protein
synthesis and breakdown, whole body glucose utilization and production, whole body lipolysis, energy expenditure, and whole body oxidation of individual nutrients (amino acid, carbohydrate, lipid) in a group of SCD
patients and in a group of control subjects having a
normal Hb phenotype.
METHODS
Subject selection. Eight African-American SCD patients (6
male and 2 female), 18–50 yr of age and within 25% of ideal
body weight based on Metropolitan Life Insurance tables (33),
were identified and screened for participation in the study at
the Sickle Cell Clinic of the Comprehensive Sickle Cell Center
at Meharry Medical College (Nashville, TN) and at the
Vanderbilt University Medical Center (Nashville, TN). Additionally, six African-American subjects (3 male and 3 female)
who did not carry the sickle cell (HbS) gene were selected as
control subjects for the study. Each subject’s Hb phenotype
was determined using standard electrophoretic methods (2)
to confirm the presence of either 1) homozygous sickle cell
disease (HbSS; n 5 2), in which both genes coding for the
b-chains of Hb produce HbS; 2) sickle cell Hb C disease
(HbSC; n 5 3), in which one gene codes for HbS and the other
for HbC; 3) sickle cell-b thalassemia disease (HbSb thal; n 5
3), in which one gene codes for HbS and the other for reduced
or no production of normal Hb (HbA); or 4) normal Hb (HbAA;
n 5 6), in which both genes code for HbA (50). Subjects eligible
for participation in the study were provided with an explanation of the study, and informed consent (approved by both
Vanderbilt University Medical School and Meharry Medical
College) was obtained for procedures to be performed at the
Vanderbilt University General Clinical Research Center
(GCRC). Before participation in the study, subjects underwent a complete history and physical examination, including
metabolic, hematologic, hepatic, and renal function tests.
Female subjects were not pregnant as determined by a
pregnancy test, were premenopausal with regular menstrual
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E357
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Borel, Myfanwy J., Maciej S. Buchowski, Ernest A.
Turner, Benjamin B. Peeler, Richard E. Goldstein, and
Paul J. Flakoll. Alterations in basal nutrient metabolism
increase resting energy expenditure in sickle cell disease.
Am. J. Physiol. 274 (Endocrinol. Metab. 37): E357–E364,
1998.—Basal rates of whole body protein, glucose, and lipid
metabolism and resting energy expenditure (REE) were
measured in eight African-American sickle cell disease (SCD)
patients and in six African-American controls. Catheters
were placed 1) in an antecubital vein for stable isotope
infusion and 2) in a heated hand vein for arterialized venous
blood. Breath and blood were collected during the last 30 min
of the 2.5-h study, and REE was measured by indirect
calorimetry. REE [128 6 5 vs. 111 6 1 kJ · kg fat-free mass
(FFM)21 · day21; P , 0.05 vs. controls] was 15% greater in the
SCD patients. Whole body protein breakdown (5.0 6 0.3 vs.
3.8 6 0.2 mg · kg FFM21 · min21; P , 0.05 vs. controls) and
protein synthesis (4.4 6 0.3 vs. 3.2 6 0.2 mg·kg FFM21 ·min21;
P , 0.05 vs. controls) were 32 and 38% greater, respectively,
in the SCD patients, but whole body amino acid oxidation was
similar (0.58 6 0.03 vs. 0.66 6 0.03 mg · kg FFM21 · min21 ).
Measures of whole body glucose and lipid metabolism were
not significantly different between the groups. The additional
energy required for the greater rates of whole body protein
breakdown and synthesis caused by SCD contributes significantly to the observed increase in REE, suggesting that
dietary energy and protein requirements are enhanced in
SCD patients.
E358
BASAL NUTRIENT METABOLISM IN SICKLE CELL DISEASE
Pharmaceutical, New York, NY) and was analyzed by radioimmunoassay (5) using Unger’s 30K antiserum (University of
Texas Southwestern Medical School, Dallas, TX). Immunoreactive insulin was determined using the Sephadex-bound
antibody (Pharmacia, Piscataway, NJ) method (55). Clinical
Assays Gammacoat radioimmunoassay kit (Travenol-Gentech, Cambridge, MA) was used to measure plasma cortisol
concentrations. Plasma amino acid concentrations were determined by reverse-phase HPLC after derivatization with
phenylisothiocyanate (25).
After deproteinization with Ba(OH)2 and ZnSO4 and elution over cation and anion resins, plasma [2H2]glucose enrichment was determined by gas chromatography-mass spectrometry (GC-MS) according to the method of Bier et al. (13).
Plasma enrichments of [13C]leucine and a-[13C]ketoisocaproate (KIC) were determined using GC-MS. Plasma was deproteinized with 4% perchloric acid, and the supernatant was
passed over a cation exchange resin to separate the keto and
amino acids. The keto acids were further extracted with
methylene chloride and 0.5 M ammonium hydroxide (34).
After drying under nitrogen gas, both the keto and amino acid
fractions were derivatized (47) with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide containing 1% t-butyldimethylchlorosilane (MtBSTFA11% t-BDMCS; Regis Technologies,
Morton Grove, IL). The derivatized samples were then analyzed via GC-MS (Hewlett-Packard 5890a GC and 5970 MS,
San Fernando, CA) for plasma leucine and KIC enrichments.
For determination of [2H5]glycerol enrichment, plasma was
deproteinized with 4% perchloric acid, and the supernatant
was passed over cation and anion exchange resins. The eluate
was dried overnight at 50°C, the glycerol fraction was derivatized with MtBSTFA11% t-BDMCS, and the derivatized
samples were injected into the GC-MS for determination of
plasma glycerol enrichment. Breath 13CO2 enrichment was
measured by isotope ratio-mass spectrometry (Metabolic
Solutions, Merrimack, NH) (48).
Calculations. Regression of the variables of interest with
time during the 0.5-h basal sampling period indicated that
the slopes were not significantly different from zero, thereby
demonstrating that a metabolic steady state was achieved.
Thus steady-state values are reported as the means of all
time points. Basal steady-state rates of whole body glucose
appearance and disappearance (Ra 5 Rd ) were calculated by
dividing the [2H2]glucose infusion rate by the plasma
[2H2]glucose enrichment (43, 56). The steady-state rate of
whole body leucine Ra was calculated by dividing the [13C]leucine infusion rate by the plasma [13C]KIC enrichment (43,
56). Plasma KIC provides a better estimate of intracellular
leucine enrichment than does plasma leucine enrichment due
to the fact that KIC is derived only from intracellular leucine
metabolism (32). Breath 13CO2 production was determined by
multiplying the total CO2 production rate by breath 13CO2
enrichment (56). The rate of whole body leucine oxidation was
calculated by dividing breath 13CO2 production by 0.8 (correction factor for the retention of 13CO2 in the bicarbonate pool)
(8) and by the plasma KIC enrichment. The nonoxidative
leucine Rd (an estimate of whole body protein synthesis) was
determined indirectly by subtracting leucine oxidation from
leucine Ra. Rates of whole body protein breakdown, amino
acid oxidation, and protein synthesis were calculated from
the leucine Ra, the leucine oxidation rate, and the nonoxidative leucine Rd, respectively, assuming that 7.8% of whole
body protein is comprised of leucine (22). Because glycerol
released during lipolysis cannot be reincorporated into triglycerides in the adipose cell due to a lack of glycerol kinase
activity, the Ra of glycerol multiplied by three was used to
determine rates of whole body lipolysis (17). Glycerol Ra was
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cycles, and were studied between 1 and 14 days after the
onset of menses (follicular phase) to reduce experimental
variability.
Body composition measurements. Each subject’s body density was measured by hydrostatic weighing (6), and percent
body fat was calculated using an equation specific for African
Americans (46). Fat mass was calculated by multiplying body
weight and percent body fat, and fat-free mass (FFM) was
calculated by subtracting fat mass from body weight. Body
mass index (BMI) was calculated as the ratio of body weight
(kg) to height squared (m2 ).
Experimental protocol. On the day of the metabolic study,
subjects reported to the GCRC after a 12-h overnight fast so
that they were studied in the postabsorptive state. Indwelling
catheters were placed in a superficial hand vein for arterialized blood sampling and in an antecubital vein for the
infusion of stable isotopic tracers. The catheterized hand was
placed in a heated Plexiglas box with the temperature
automatically adjusted to 55°C for complete arterialization of
venous blood samples (1). In two subjects, placement of the
blood-sampling catheter in a hand vein was not possible;
therefore, blood samples were collected via a Port-A-Cath
providing mixed venous blood. In addition, catheter placement in the antecubital vein was not possible in one subject;
thus the external jugular vein was catheterized to allow for
the tracer infusions. At the beginning of the metabolic study,
each subject received a bolus infusion of NaH13CO3 (0.12
mg/kg), L-[1-13C]leucine (7.2 µmol/kg), and D-[6,6-2H2]glucose
(3.6 mg/kg) to prime the CO2, leucine, and glucose pools,
respectively. A continuous infusion of the leucine (0.12
µmol · kg21 · min21 ) and glucose (0.06 mg · kg21 · min21 ) isotopes as well as of [2H5]glycerol (0.12 µmol · kg21 · min21 ) was
then started and continued throughout the remainder of the
study (13, 20, 56). Each study consisted of a 2-h tracer
equilibration period followed by a 30-min basal sampling
period.
Blood samples were collected every 10 min during the basal
period for determination of plasma glucose, hormone, and
amino acid concentrations as well as isotopic enrichments.
Simultaneous to the blood sampling, breath samples were
collected from each subject via a Douglas bag with duplicate
20-ml samples placed in nonsiliconized glass vacutainer
tubes for measurement of breath 13CO2 enrichment. For the
45 min before the basal sampling period, total CO2 production, O2 consumption, and resting energy expenditure were
measured by indirect calorimetry using a Sensormedics 2900
metabolic cart (Yorba Linda, CA). At the conclusion of the
study, the catheters were removed and the subject was fed a
meal and discharged from the GCRC.
Analytical procedures. During the metabolic study, plasma
glucose concentrations were measured enzymatically using a
Model II glucose analyzer (Beckman Instruments, Fullerton,
CA). Whole blood hemoglobin concentrations were measured
colorimetrically using the cyanmethemoglobin method (Procedure no. 525, Sigma, St. Louis, MO). Blood samples were
collected into separate Venoject tubes (Terumo Medical, Elkton, MD) containing 15 mg Na2EDTA. In addition, 3 ml of
blood were transferred to a tube containing ethylene glycolbis(b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid and reduced glutathione, with the plasma stored at 280°C for later
measurement of plasma epinephrine and norepinephrine
concentrations by high-performance liquid chromatography
(HPLC) (23). The remaining blood was spun in a Beckman
desktop centrifuge at 3,000 revolutions/min and 4°C for 10
min, and the plasma was collected and stored at 280°C for
later analysis. Plasma for glucagon analysis was collected in
tubes containing 50 µl of 500 kIU/ml aprotinin (Trasylol, FBA
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BASAL NUTRIENT METABOLISM IN SICKLE CELL DISEASE
calculated by dividing the [2H5]glycerol infusion rate by the
plasma glycerol enrichment (43, 56). Rates of whole body
amino acid, carbohydrate, and lipid oxidation were determined from indirect calorimetry in combination with the
leucine oxidation data. The energy expended due to amino
acid oxidation was subtracted from the total energy expenditure, and the net rates of carbohydrate and lipid oxidation
were calculated based on the nonprotein respiratory quotient
(30). The assumptions and limitations of calculating net
substrate oxidation based on indirect calorimetry measurements have been reviewed previously (30).
Statistical analysis. Comparisons between the SCD and
control groups were made using unpaired t-test analysis, with
differences significant at P # 0.05. Values presented in the
text, Tables 1 and 2, and Figs. 1 and 2 represent means 6 SE
for each group.
Subject characteristics. The SCD (n 5 8, 6 male and 2
female) and control (n 5 6, 3 male and 3 female) groups
of subjects were similar with regard to age, weight,
height, BMI, percent body fat, FFM, and fat mass
(Table 1). Furthermore, as would be expected, the Hb
concentration of the SCD group was significantly lower
than that of the control group (Table 1). SCD is a
collective term for the various Hb phenotypes, including HbSS, HbSC, and HbSb thal, in which sickle Hb
(HbS) is produced due to the replacement of glutamic
acid with valine in position six on the b-chains of Hb
(28). All of these Hb phenotypes result in the occurrence
of sickled RBCs, hemolytic anemia, and vaso-occlusive
events (50). Because results for the three Hb phenotypes in the SCD group vs. the controls were similar,
they were pooled and reported as one mean.
Plasma hormone concentrations. Basal plasma insulin (6.4 6 1.1 vs. 6.3 6 0.5 µU/ml), glucagon (57 6 5 vs.
54 6 4 pg/ml), cortisol (8.7 6 0.8 vs. 8.4 6 2.1 µg/dl),
epinephrine (26 6 3 vs. 28 6 4 pg/ml), and norepinephrine (146 6 19 vs. 154 6 16 pg/ml) concentrations were
comparable between the SCD and control groups, respectively.
Plasma amino acid concentrations. Although plasma
alanine and arginine concentrations were 35 and 28%
lower, respectively, in the SCD patients than in the
control subjects, concentrations of other individual
amino acids were similar between the two groups
Table 1. Subject characteristics
n/Gender
Age, yr
Weight, kg
Height, cm
BMI, kg/m2
%Body fat
Fat-free mass, kg
Fat mass, kg
Hemoglobin, g/l
Control
Sickle Cell
Disease
3 Male/3 Female
26 6 2
72.8 6 5.8
175.9 6 4.3
23.4 6 1.4
21.7 6 4.1
56.1 6 3.1
16.6 6 4.0
137 6 7
6 Male/2 Female
30 6 3
68.3 6 5.2
173.7 6 4.2
22.4 6 1.2
21.2 6 3.1
53.5 6 4.1
14.8 6 2.6
99 6 10*
Values are means 6 SE. BMI, body mass index. * P # 0.05 vs.
Control.
1-Methylhistidine
3-Methylhistidine
Alanine
Arginine
Asparagine
Aspartate
Citrulline
Glutamate
Glycine
Histidine
Hydroxyproline
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Serine
Taurine
Threonine
Tryptophan
Tyrosine
Valine
Branched-chain AA
Gluconeogenic AA
Essential AA
Nonessential AA
Total AA
Control
Sickle Cell
Disease
9.5 6 3.6
4.2 6 0.7
289.2 6 38.5
85.1 6 3.8
74.0 6 6.2
4.8 6 0.6
29.6 6 2.6
65.5 6 8.7
213.4 6 14.7
53.4 6 4.0
8.4 6 1.4
50.2 6 6.2
139.3 6 15.1
158.6 6 19.0
24.9 6 2.7
44.8 6 3.5
56.3 6 5.3
179.1 6 28.8
106.8 6 3.1
97.5 6 8.6
120.5 6 10.8
35.4 6 2.0
56.7 6 3.9
198.5 6 19.3
388.0 6 39.7
729.9 6 64.3
922.2 6 57.1
1,183.4 6 66.8
2,105.6 6 84.5
4.1 6 1.4
6.8 6 1.1
188.6 6 17.9*
61.0 6 6.4*
61.7 6 3.9
5.3 6 1.0
28.2 6 2.2
77.8 6 12.2
207.6 6 27.8
39.6 6 4.7
11.6 6 2.2
46.7 6 4.7
124.7 6 12.0
137.8 6 13.0
20.1 6 1.2
54.5 6 3.6
47.7 6 2.9
200.5 6 22.2
105.2 6 10.7
62.6 6 15.1
126.1 6 13.8
31.2 6 2.4
50.7 6 3.5
160.5 6 14.5
331.9 6 28.2
627.5 6 33.9
795.3 6 47.4
1,065.1 6 38.9
1,860.4 6 74.5*
Values are means 6 SE and are in µM. Branched-chain amino
acids (AA) represents sum of leucine, isoleucine, and valine; gluconeogenic AA respresent sum of alanine, glycine, serine, and threonine;
essential AA represent sum of arginine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophan, and valine; total AA represent sum of all AA; and nonessential AA represent
difference between total AA and essential AA. * P , 0.05 vs. Control.
(Table 2). One exception was plasma histidine concentrations, which were 26% lower in the SCD patients
than in the control subjects, a difference that approached statistical significance (P 5 0.0541). Plasma
concentrations of total branched-chain, gluconeogenic,
essential, and nonessential amino acids tended to be
lower, although not significantly, in the SCD patients
than in the control group. However, the plasma concentration of total amino acids was 12% lower in the SCD
patients than in the controls.
Resting energy expenditure. Resting energy expenditure normalized to FFM was 15% greater in the SCD
patients compared with the controls (128 6 5 vs. 111 6
1 kJ · kg FFM21 · day21; Fig. 1A).
Whole body protein breakdown and synthesis. Rates
of basal whole body protein breakdown (5.0 6 0.3 vs.
3.8 6 0.2 mg · kg FFM21 · min21 ) and synthesis (4.4 6
0.3 vs. 3.2 6 0.2 mg · kg FFM21 · min21 ) were 32 and 38%
greater, respectively, in the SCD patients compared
with the control subjects (Fig. 2).
Whole body glucose utilization and production. Basal
plasma glucose concentrations were similar between
the SCD and control groups (91 6 2 vs. 92 6 3 mg/dl,
respectively). Likewise, basal rates of whole body glucose utilization and endogenous glucose production,
which are equivalent under the steady-state conditions
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RESULTS
Table 2. Plasma amino acid concentrations
E360
BASAL NUTRIENT METABOLISM IN SICKLE CELL DISEASE
DISCUSSION
of the study, were similar between the SCD patients
(2.5 6 0.2 mg · kg body wt21 · min21 ) and the controls
(2.4 6 0.2 mg · kg body wt21 · min21 ).
Whole body lipolysis. Although basal rates of whole
body lipolysis were 55% greater in the SCD patients
than in the controls (36.2 6 7.0 vs. 23.5 6 4.2 µmol · kg
fat21 · min21, respectively), the difference did not reach
statistical significance.
Nutrient oxidation. Basal rates of whole body nutrient oxidation were comparable between the SCD and
control groups as follows: amino acid oxidation (0.58 6
0.03 vs. 0.66 6 0.03 mg · kg FFM21 · min21; SCD vs.
control, respectively), carbohydrate oxidation (1.18 6
0.16 vs. 1.40 6 0.31 mg · kg body wt21 · min21 ), and lipid
oxidation (5.88 6 1.32 vs. 3.38 6 0.51 mg · kg
fat21 · min21 ). The percentage of resting energy expenditure derived from carbohydrate was 29 6 3 vs. 39 6 7%
(SCD vs. control, respectively), from protein was 13 6 1
vs. 17 6 1% (P # 0.05), and from fat was 58 6 3 vs.
44 6 7%.
Fig. 2. Basal rates of whole body protein breakdown and protein
synthesis for the control subjects and SCD patients. * Significant
difference (P # 0.05) vs. control group.
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Fig. 1. Resting energy expenditure (A) and estimated energy cost of
protein synthesis (B) for control subjects and sickle cell disease (SCD)
patients. * Significant difference (P # 0.05) vs. control group. For SCD
patients, their greater energy cost of protein synthesis [18 kJ · kg
fat-free mass (FFM)21 · day21 above control mean] accounted for
,50% of their enhanced resting energy expenditure (117 kJ · kg
FFM21 · day21 above control mean).
This research examined the impact of SCD on whole
body nutrient and energy metabolism in a comprehensive manner. We sought to identify the consequences of
SCD on basal whole body protein, carbohydrate, and
lipid metabolism and to ascertain how these changes
altered resting energy expenditure. Basal whole body
glucose utilization and production, whole body lipolysis, and whole body amino acid, carbohydrate, and fat
oxidation were not significantly affected by SCD. In
contrast, resting energy expenditure was increased by
15%, and basal rates of whole body protein breakdown
and protein synthesis were increased by 32 and 38%,
respectively, in the SCD patients vs. the controls. Thus
our data support the hypothesis that the dietary energy
and protein requirements of SCD patients are increased.
Previous data regarding the impact of SCD on resting energy expenditure are limited. Badaloo et al. (10)
observed that resting metabolic rate normalized to
body weight or metabolic body size was ,20% greater
in adult HbSS patients than in controls. A similar
magnitude of increase in resting energy expenditure
has been found in adolescents (51, 52) and children (24,
45) with HbSS. Our observation of enhanced resting
energy expenditure in adult SCD patients confirms
these previous findings and suggests that the energy
requirements of individuals with SCD may be increased. Although there are limited data examining the
influence of increasing energy intake in SCD patients
above normal dietary guidelines, two growth-retarded
children with HbSS given additional energy via daily
nasogastric nutritional supplementation were found to
have accelerated rates of growth with a decreased
incidence of painful vaso-occlusive crises and infections
(27).
The maintenance of protein homeostasis is important for human health and well-being, since the balance between protein synthesis and breakdown influences the function of proteins involved in many
physiological activities (e.g., enzymatic, hormonal, immunological, membrane transport). Early studies have
suggested that serious abnormalities in protein homeostasis exist in individuals with SCD. Using three
different levels of daily N intake (90, 135, and 180
mg · kg21 · day21 ), Odonkor et al. (36) demonstrated that
daily urinary and fecal N losses were 8–20 and 35–
216% greater, respectively, in nine male adolescent
HbSS patients compared with five age- and gendermatched controls. Daily N balance tended to be negative in the HbSS patients at all levels of N intake but
positive in the controls. However, interpretation of
these findings is limited by the fact that energy intake
per unit body weight was lower in the HbSS group than
in the control group. Although adult HbSS patients
consuming adequate protein and energy have been
found to have increased urea production, only a small
proportion of this urea is excreted in the urine, with a
larger proportion hydrolyzed in the bowel, suggesting
BASAL NUTRIENT METABOLISM IN SICKLE CELL DISEASE
Logic suggests that the rapid turnover of RBCs in
SCD necessitates an increase in whole body protein
turnover due to the accelerated synthesis of new RBCs
and to the enhanced breakdown of irreversibly sickled
RBCs. Although these events have not been directly
measured, Serjeant (49) made theoretical calculations
to determine that daily Hb synthesis must be increased
by approximately sevenfold in HbSS patients (40 g/day)
compared with normal adults (6.25 g/day). On the basis
of these calculations, Hb synthesis accounted for ,2.5%
of whole body nitrogen flux in the control subjects and
for ,12% in the HbSS patients studied by Badaloo et al.
(10). Additional evidence supporting the contribution of
increased RBC production to the increased protein
turnover observed in SCD patients is the finding of a
decrease in whole body protein turnover after splenectomy in HbSS children with hypersplenism (9, 11).
Whether the synthesis of other body proteins in SCD
patients is accelerated has not been directly measured.
However, the magnitude of the increase in whole body
protein turnover in the HbSS subjects studied by
Badaloo et al. was sixfold greater than the estimated
increase in RBC protein turnover, suggesting that the
turnover of non-RBC proteins, such as skeletal muscle,
cardiac muscle, hepatic, or gastrointestinal proteins,
also must be increased.
Although the mechanisms responsible for the increased resting energy expenditure observed in SCD
patients cannot be completely determined from the
present study, our data demonstrate that basal rates of
amino acid oxidation, which were unaffected by SCD,
did not contribute to the observed increase in resting
energy expenditure. In contrast, there appears to be a
close relationship between accelerated rates of whole
body protein and energy metabolism in SCD. The
processes of protein breakdown and protein synthesis
consume energy, thereby accounting for a considerable
portion of basal energy expenditure (42). Enhanced
protein turnover leads to an increase in the energy
required for peptide bond formation, amino acid transport, degradation of protein, urea formation, and excretion of protein breakdown waste products. For example, the synthesis of each peptide bond requires the
energy equivalent to ,5 molecules of ATP (41, 42). On
the basis of this fact, the energy cost of protein synthesis has been estimated to be 4.5 kJ or 1.076 kcal/g
protein (41). In the present study, the estimated energy
cost of protein synthesis was significantly greater in
the SCD patients (28.6 6 1.8 kJ · kg FFM21 · day21 ) than
in the control subjects (20.6 6 1.2 kJ · kg FFM21 · day21;
Fig. 1B). These estimates of the energy cost of protein
synthesis accounted for 22.5 and 18.7% of resting
energy expenditure in the SCD patients and the control
subjects, respectively. Furthermore, ,50% of the observed increase in resting energy expenditure in the
SCD patients could be accounted for by the enhanced
energy cost of protein synthesis (Fig. 1).
Although basal protein turnover and resting energy
metabolism are altered with SCD, data regarding the
effect of SCD on lipid and glucose metabolism are
limited. Whereas several studies have demonstrated a
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the presence of mechanisms for nitrogen conservation
(29).
Although negative nitrogen balance is suggestive of
poor protein homeostasis, the metabolic mechanisms
contributing to this impaired homeostasis in SCD
patients remain unestablished. In an early effort to
define these events, Waterlow (54) used L-[U-14C]lysine
and a limited number of subjects to measure whole
body protein turnover. Turnover of whole body protein
was approximately twofold greater in two male HbSS
patients than in a male control subject. Using primed
intermittent oral doses of [15N]glycine, Badaloo et al.
(10) found that estimates of whole body nitrogen flux,
protein synthesis, and protein breakdown were significantly elevated in six adult male HbSS patients compared with six adult male controls. Whereas the assessment of protein metabolism with [ 15N]glycine is
attractive due to its noninvasive nature, data from
these studies must be interpreted with care due to
limitations of the methodology (56). Our finding of
enhanced basal rates of whole body protein breakdown
and protein synthesis in the SCD patients supports the
previous findings of Waterlow and Badaloo et al. and
supports the hypothesis that dietary protein requirements are increased in SCD patients. In addition, our
data demonstrate that, although the basal turnover
rate of the body protein pool is increased, whole body
amino acid oxidation is not altered in adults with SCD,
a finding consistent with that in children with HbSS
(45).
The requirements for specific individual amino acids
during SCD have not been established. Hb is a structurally imbalanced protein, totally devoid of isoleucine
but rich in valine, leucine, glycine, and histidine (18).
Additionally, the pool of sulfur-containing amino acids
may be limited in SCD due to an increased loss of
taurine-conjugated bile salts. Thus individual amino
acid pools may be limiting in the formation of various
body proteins. Previously, Enwonwu et al. (18) observed
that plasma concentrations of valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, arginine, alanine, and total essential amino acids were significantly
lower in HbSS patients than in matched control subjects, suggesting that a ‘‘metabolic insufficiency’’ of
several amino acids occurs with HbSS. In contrast,
plasma amino acid concentrations of children with
HbSS were found to be similar to those of control
children except for significantly higher asparagine and
cysteine concentrations (45). In the present study,
plasma concentrations of individual amino acids were
similar for the SCD and control groups except for
significantly lower concentrations of alanine and arginine in the SCD patients. Furthermore, whereas plasma
total branched-chain, gluconeogenic, essential, and nonessential amino acid concentrations were comparable
between the two groups, the plasma total amino acid
concentration was significantly lower in the SCD patients. The significance of alterations in the amino acid
pool to changes in protein synthetic rates in SCD
requires further investigation.
E361
E362
BASAL NUTRIENT METABOLISM IN SICKLE CELL DISEASE
these hormones may play during metabolic conditions
such as stress, feeding, or exercise in SCD patients
cannot be determined from the present study. Given the
nature of SCD, one can speculate that our observed
alterations in basal protein and energy metabolism
might have been due to an enhanced ‘‘steady-state’’
(free from vaso-occlusive crisis) acute-phase reactant
and cytokine response in the SCD patients. However,
information in this regard has been contradictory, with
some studies demonstrating normal concentrations (7,
12) and others increased concentrations (21, 53) of
various acute-phase reactants and cytokines such as
C-reactive peptide, fibrinogen, tumor necrosis factor,
and interleukin-1 in steady-state SCD patients. Because the measurement of thyroid hormones, growth
hormone, and various acute phase reactants and cytokines was not possible in the present study, we cannot
make any conclusions regarding their potential influence on the increased whole body protein and energy
metabolism observed in the SCD patients.
In conclusion, findings from the present study clearly
demonstrate that basal rates of whole body protein
breakdown and protein synthesis are significantly increased in SCD patients, thereby contributing to the
greater resting energy expenditure observed in these
individuals. Furthermore, these data support the hypothesis that dietary energy and protein requirements
are greater in SCD patients. In addition, whereas
alterations in basal whole body glucose metabolism did
not contribute to the observed increase in resting
energy expenditure, there was a trend toward increased rates of whole body lipolysis and lipid oxidation
in the SCD patients, which may contribute to their
greater resting energy expenditure. The knowledge
gained from the present research provides a basis for
the design of optimal nutritional regimens for SCD
patients with the goal of improving their nutritional
well-being.
We thank Dr. Mark Koury, Division of Hematology, Vanderbilt
University Medical Center and Veterans Administration Medical
Center for referral of sickle cell disease patients and for input
regarding this research project and this manuscript. We also thank
the subjects for participation in the study, the nursing staff of the
General Clinical Research Center (GCRC) at Vanderbilt University
for help with this project, and Suzan Vaughan, Wanda Snead, and Li
Zheng for technical assistance in completing this research.
Funding for this research was provided by the following National
Institutes of Health grants: HL-56867, DK-26657 (Clinical Nutrition
Research Unit, Vanderbilt University), National Center for Research
Resources GCRC Program RR-00095 (GCRC, Vanderbilt University),
and DK-20593 (Diabetes Research and Training Center, Vanderbilt
University).
M. J. Borel was a recipient of the Clinical Nutrition Research Unit
Young Investigator Award.
Address for reprint requests: P. J. Flakoll, Dept. of Surgery, 751
MRB II, Vanderbilt Univ., Nashville, TN 37232.
Received 18 August 1997; accepted in final form 16 November 1997.
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