Arginine and ornithine kinetics in severely burned patients:
increased rate of arginine disposal
Yong-Ming
Yu,
Colleen M.
Ryan,
Leticia
Castillo,
Xiao-Ming
Lu,
Louis
Beaumier,
Ronald G.
Tompkins, and
Vernon R.
Young
Shriners Burns Hospital and Trauma Service, Massachusetts General
Hospital, Boston 02114; and Laboratory of Human Nutrition and
Clinical Research Center, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
 |
ABSTRACT |
Arginine serves multiple roles in
the pathophysiological response to burn injury. Our previous studies in
burn patients demonstrated a limited net rate of arginine de novo
synthesis despite a significantly increased arginine turnover (flux),
suggesting that this amino acid is a conditionally indispensable amino
acid after major burns. This study used
[15N2-guanidino-5,5-2H2]arginine
and [5-13C]ornithine as tracers to assess the rate of
arginine disposal via its conversion to and subsequent oxidation of
ornithine; [5,5-2H2]proline and
[5,5,5-2H3]leucine were also used to assess
proline and protein kinetics. Nine severely burned patients were
studied during a protein-free fast ("basal" or fast) and total
parenteral nutrition (TPN) feedings. Compared with values from healthy
volunteers, burn injury significantly increased 1) fluxes of
arginine, ornithine, leucine, and proline; 2)
arginine-to-ornithine conversion; 3) ornithine oxidation;
and 4) arginine oxidation. TPN increased
arginine-to-ornithine conversion and proportionally increased
irreversible arginine oxidation. The elevated arginine oxidation,
with limited net de novo synthesis from its immediate precursors,
further implies that arginine is a conditionally indispensable amino
acid in severely burned patients receiving TPN.
leucine; de novo synthesis; oxidation; basal phase; total parental
nutrition
 |
INTRODUCTION |
IN EARLIER STUDIES
in severely burned patients, we have used arginine and citrulline
tracers to explore the dynamic, metabolic interrelationships between
these amino acids (39-41). We observed that the net
rate of de novo arginine synthesis was not increased in burn patients
receiving parenteral nutrition compared with the rate found in healthy
adults. However, there was an increased plasma arginine flux that
paralleled the higher leucine fluxes and increased rates of leucine
oxidation, also when compared with estimates for healthy subjects. On
this basis, we have proposed that there are relatively higher rates of
arginine loss after burn injury and that an exogenous intake of
preformed arginine would be needed to balance this putative increased
rate of arginine oxidation and to maintain body arginine homeostasis.
To further evaluate this hypothesis, we have now determined the rate of
conversion of arginine to ornithine and the rate of ornithine oxidation
in a group of severely burned patients. Compared with data obtained in
our laboratories in healthy adults, it is concluded that net arginine
loss is increased and that a parenteral supply of arginine would be
necessary to maintain optimum amino acid balance and homeostasis in
burn patients who are fed by this route.
 |
MATERIALS AND METHODS |
Materials.
L-[15N2-guanidino-5,5-2H2]arginine
[99% atom percent excess (APE)],
L-[5,5-2H2]proline, and
L-[5-13C]ornithine (>98% 13C)
were purchased from MassTrace (Woburn, MA).
L-[5,5,5-2H3]leucine
(2H 98%) was acquired from Cambridge Isotope Laboratories
(Woburn, MA); NaH13CO3 (99% APE) was obtained
from Prochem (Summit, NJ). The isotopically labeled tracers were used
to make stock solutions at the Pharmacy of the Massachusetts General
Hospital (MGH). Before use, they were confirmed to be sterile and
pyrogen free. The total parenteral nutrition (TPN) solutions were
prepared in the Nutritional Support Unit, Department of Surgery, MGH.
Novamine (KabiVitrum, Alameda, CA) 11.4% was used as the amino acid
source; its composition is shown in Table
1.
Burn patients.
The study included nine severely burned adults (5 males, 4 females).
The general condition of the patients is shown in Table 2. The age (mean ± SE) was 51 ± 6 yr (range 32-85 yr). On the admission physical examination,
percent total body surface area burned was 61 ± 6% (SE) (range
35-90%). Smoke inhalation injury, as diagnosed by admission
bronchoscopy, was present in eight of nine patients (89%). Mortality
was 33%, consistent with the predicted mortality for this group with
severe burns and smoke inhalation (30). All patients were
treated with standard burn resuscitation and critical care measures,
including serial excision and grafting procedures beginning early in
the hospital course (mean of 2 days, range 1-4 days after
admission) (31). Enteral feedings were administered as
early as possible; however, TPN was administered when tube feedings
were poorly tolerated.
The experimental protocol was approved by the Subcommittee for Human
Studies, Committee of Research, MGH and the Partners Health Care
System. Written consent was obtained, either from the patient after
being informed of the purpose, design, and possible hazards of the
experiment or, alternatively, from the family members.
The patients were studied from 7 to 25 days after the burn, with two of
the studies at 25 days after injury, when patients had ~23% of
unhealed wound. Because patients are frequently still hypermetabolic
many weeks after the initial injury, we have included these two
patients in our database. For the present group of patients, energy
expenditure, measured via indirect calorimetry and based on
O2 consumption (see Tracer studies), was
equivalent to 38 ± 3 (SE) kcal · kg
1 · day
1 for the "basal"
state and 42 ± 4 kcal · kg
1 · day
1 for the
TPN phase. The general conditon of this group of patients is comparable
to that of the burned patients studied earlier (39, 41).
Experimental design.
Tracer studies were performed when the patients were in a relatively
stable condition, as assessed by blood pressure, heart rate and cardiac
function, respiration rate, body temperature, and liver and kidney functions.
As in previous studies (39-41), each patient was
studied twice: first during a basal or "fast" phase, when a
low glucose infusion rate was maintained to prevent
hypoglycemia, and later while they were in a "fed" (TPN) phase. The
order of the two phases was randomized (Table 2) and conducted within 1 or 2 days of each other. During the TPN conditon, patients received
nutritional support that had begun
2 days before the tracer study.
Average intakes were 0.36 ± 0.02 g
N · kg
1 · day
1, with
nonprotein calories equivalent to 32 ± 1 kcal · kg
1 · day
1 being
supplied by glucose. The basal condition was created by terminating the TPN about 10 h before the labeled tracer studies were begun. It was not possible to completely fast these very sick
patients, and because it was necessary to prevent hypoglycemia, they
received an infusion of 5% dextrose at the average rate of glucose
intake of 0.06 ± 0.01 g · kg
1 · h
1 during this
basal state. After the tracer infusion studies were completed, TPN
feedings were either resumed or replaced by enteral feeding in
accordance with orders written by the attending clinicians.
Tracer studies.
Primed constant intravenous infusions of
L-[15N2-guanidino-5,5-2H2]arginine
(M+4 arginine), L-[5-13C]ornithine
(M+1 ornithine), [5,5-2H2]proline
(M+2 proline), and
L-[5,5,5-2H3]leucine
(M+3 leucine) were used to determine the plasma kinetics of
arginine, ornithine, proline, and leucine, respectively. The tracer
studies were generally started between 0600 and 0700 and lasted
for 330 min. In our previous studies on citrulline and arginine
kinetics (39), we observed a slight increment in the enrichment of M+1 ornithine (~0.5 APE) after the infusion
of
L-[15N2-guanidino-5,5-2H2]arginine
(M+4) at the rate of 0.2 µmol · kg
1 · min
1; a
plateau was reached between 90 and 120 min after the start of the
tracer infusion. Therefore, in the present study, for a more accurate
estimate of the plasma [5-13C]ornithine enrichment
arising exclusively from the infusion of [5-13C]ornithine
(M+1) tracer, the
L-[15N2-guanidino-5,5-2H2]arginine
(M+4) and [5,5-2H2]proline
(M+2) tracers were infused for a total of 330 min, and then
the primed constant infusion of
L-[5-13C]ornithine and
L-[5,5,5-2H3]leucine was given
during the last 180 min of this 5.5-h period. Blood samples were taken
at 120 and 150 min before the administration of
L-[5-13C]ornithine. They served to estimate
the baseline enrichments of
L-[5-13C]ornithine infused between 150 and
330 min. Before the isotope infusion was started, arterial blood and
expired air samples were taken for measurements of background isotopic
levels in plasma arginine, ornithine, proline, and
-ketoisocaproate,
which serves as a surrogate of intracellular leucine enrichment
(24), and of the 13CO2 in expired
air. Blood samples (~3 ml) were taken at 120, 150, 285, 300, 315, and
330 min after the commencement of the tracer infusion. Four sets of
expired air samples were also taken at intervals of 15 min between 285 and 330 min, concomitant with the time points for blood sampling, for
determination of the isotopic steady-state level of
13CO2 enrichment in the expired air. Timed
expired air samples also were collected for determination of total
O2 consumption and CO2 production, as described
previously (39, 41). The targeted, but known, infusion
rates of labeled arginine, ornithine, proline, and leucine were,
respectively, 0.15, 0.07, 0.09, and 0.07 µmol · kg
1 · min
1.
Priming doses of these tracers were 4.5, 4.2, 5.4, and 4.2 µmol/kg, respectively. The tracer study protocol is shown in Fig.
1.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Outline of the tracer infusion protocol, including the period of
tracer infusion and time points of blood and air sampling.
|
|
Analytic methods.
Samples for isotopic abundance of arginine, ornithine, proline, and
-ketoisocaproate were measured in duplicate and determined against
calibration standards. The methods have been described, in detail,
previously (10, 39, 41). In brief, for determination of
the isotopic enrichment of arginine, ornithine, and citrulline, 200 µl of plasma were passed through an ion exchange resin
(H+ form, AG 50W, 100-200 mesh, Bio-Rad, Hercules,
CA), and a methyl ester trifluoroacetyl derivative was formed, first by
esterification with acetyl chloride and methanol and then by acylation
with trifluoroacetic acid and dichloroethane. Analysis of the
derivatives was performed with the aid of an HP 5890 series II gas
chromatograph, using on-column injection, coupled to an HP 5988 A mass
spectrometer (Hewlett-Packard). We used negative ion chemical
ionization, with methane as reagent gas. Ornithine, citrulline, and
arginine eluted at 5.2, 7.2, and 7.7 min, respectively. Selective ion
monitoring (SIM) was carried out at mass-to-charge (m/z)
338, m/z 339, and m/z 340 for natural ornithine
(M+0), [13C]- or [2H]ornithine
(M+1), and [5,5-2H2]ornithine
(M+2), respectively. For the arginine isotopologs, SIM was
carried out at m/z 456 (M
20), m/z
457, m/z 458, and m/z 460 for natural
arginine (M+0), [5-13C]- or
[5-2H]arginine (M+1),
[5,5-2H2]arginine (M+2), and
[15N2-guanidino,5,5-2H2]arginine
(M+4), respectively. The ion clusters of all tracers determined in the negative ion chemical ionization mode overlapped with
the labeled products of the other tracers. Thus a multiple linear
regression approach was used to calculate the isotopic abundance of the
amino acids from the mass spectrometry data, as we have described in
detail for isotopologs of tyrosine and leucine (37).
Plasma proline enrichment was measured using a heptafluorobutyric acid
derivative and was monitored at nominal mass m/z ratios of
335/333 and 334/333 for [5,5-2H2]proline
(M+2) and [5-2H]proline (M+1).
Measurement of plasma
-[5,5,5-2H3]ketoisocaproate (KIC) was
conducted as previously described for analysis of
[1-13C]KIC (30, 32, 33). The natural KIC and
[5,5,5-2H3]KIC were monitored, using electron
impact mass spectrometry, at m/z 259 and m/z 262, respectively.
Plasma free amino acid levels and concentrations of arginine,
citrulline, proline, and leucine in the infusates were measured by an
automated high-performance liquid chromatograph (Beckman System Gold
model 126, with model 506A Autosampler, controlled by System Gold
Chromatography Software, Beckman Instrument, San Ramon, CA), using a
post column derivatization reaction with ninhydrin (Trione) and
quantitation with the aid of a programmable detector model 168.
Estimation of amino acid kinetics.
The metabolic flux (Q) of arginine, ornithine, proline, and
leucine was estimated using steady-state dilution equations
(38). Briefly, the metabolic fluxes (represented by
Q, in
µmol · kg
1 · h
1) for
arginine, ornithine, proline, or leucine were calculated from the
equation
|
(1)
|
where I is the rate of tracer infusion, Ei is the
isotopic abundance of the tracer infusate, and Ep is the
plateau isotopic abundance of the tracer in plasma. In the case of
leucine, the plasma enrichment of
-[5,5,5-2H3]KIC was used as the index of
the intracellular
L-[5,5,5-2H3]leucine enrichment
(24).
The rate of ornithine oxidation (COrn) was calculated as
follows
|
(2)
|
where
13CO2 is the rate of total
13CO2 expiration at isotopic steady state;
Ei and Ep are the enrichments of
[5-13C]ornithine in the infusate and plasma at plateau,
respectively; and k is the value used to correct the
fraction of 13CO2 retention. This factor was
estimated as follows. Six healthy young adults (4 males and 2 females),
with mean (±SE) age 22 ± 1 yr and weight 73.9 ± 5.2 kg,
who received an adequate L-amino acid diet
(6), were admitted to the Clinical Research Center (CRC)
of the Massachusetts Institute of Technology (MIT) at ~1500. At 1600, a constant intravenous infusion of
L-[5-13C]glutamate (0.93 µmol · kg
1 · h
1) was
started that proceeded for 24 h. The percentages (means ± SE) of infused 13C recovered in expired air during the fast
state (lasting from 270 to 380 min) and fed state (lasting from 990 to
1,050 min) were 40.2 ± 1.8 and 52.3 ± 0.6%, respectively.
We have used these two correction factors to estimate the oxidation
rate of ornithine, following the same rationale as estimating the
recovery of 13CO2 from fatty acid oxidation via
the tricarboxylic acid cycle with [13C]acetate
(33).
13CO2 is calculated by
|
(3)
|
where
CO2 is the rate of total
CO2 expired in the breath, and E13CO2 is the
plateau level 13CO2 enrichment in the expired
air samples collected during the last 45 min of tracer infusion.
The rate of plasma arginine-to-ornithine conversion
(QArg-Orn) was calculated from the
appearance rate of [5,5-2H2]ornithine arising
from the infused
[15N2-guanidino-5,5-2H2]arginine,
essentially according to the phenylalanine/tyrosine tracer models of
Clarke and Bier (13) and Thompson et al.
(36). Thus
|
(4)
|
where QOrn and
QArg are the plasma fluxes of ornithine and
arginine
(µmol · kg
1 · h
1),
respectively, estimated from the primed, constant infusions of
[5-13C]ornithine and
[15N2-guanidino-5,5-2H2]arginine.
E2H2Orn and E2H2Arg are the respective plasma
isotope abundance of [5,5-2H2]ornithine and
the total enrichment of [5,5-2H2]arginine,
the latter of which is the sum of plateau level plasma enrichments of
[15N2-guanidino-5,5-2H2]arginine
and [5,5-2H2]arginine). The expression
[QArg/(IArg + QArg)] corrects for the contribution of the
arginine tracer infusion to arginine flux (36, 39).
The rate of arginine oxidation (CArg) was calculated by the
following equation
|
(5)
|
where COrn/QOrn is the
fraction of plasma ornithine oxidized.
It should be noted that the estimates of arginine and ornithine
kinetics were based on use of plasma isotopic enrichments as a
surrogate of the precursor enrichments in the pools other than the site
of the intrahepatic urea cycle. Thus the validity of our
interpretations and conclusions depends on this premise. We do offer
some support for this in the DISCUSSION, particularly with
respect to the estimates of arginine oxidation.
Evaluation of data.
Statistical evaluation of the data was done using PROSTAT software
(Poly Software International, Sandy, UT). All data were examined for
normalcy of distribution and then for possible significant covariates,
including age and open burn area. There were none. We then carried out
paired t-tests to compare the metabolic measurements between
the basal and the TPN states in burn patients. Values obtained from our
recent investigations with healthy adults for plasma amino acid fluxes
and ornithine oxidation were used here to help further evaluate the
status of amino acid metabolism in these burn patients, compared by
unpaired t-test.
 |
RESULTS |
Stable isotope abundance.
The isotopic abundance of the infused arginine (Fig.
2), ornithine (Fig.
3), and proline (Fig.
4) tracers and that of
-[5,5,5-2H3]KIC (Fig.
5) remained at relatively steady levels
(slope, P > 0.05) during the last 45 min of the tracer
protocol, when blood samples were taken. Infusion of
[15N2-guanidino-5,5-2H2]arginine
and [5,5-2H2]proline resulted in a slight
increment of M+1 ornithine during the 120- to 150-min
period, reaching 0.5 ± 0.2 (SE) APE. This value was subtracted
from the plateau value for the [5-13C]ornithine
enrichment measured for the 285- to 330-min period. Furthermore, after
the infusion of [5,5-2H2]proline, there was a
slight increment of M+1 proline enrichment between 120 and
150 min (Fig. 4), but this was not followed by a significant further
rise during infusion of the [5-13C]ornithine tracer
between 150 and 330 min.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Plateau level isotopic enrichments for M+4 and
M+2 arginine tracers (corrected for the infusion rate of
[15N-guanidino-5,5-2H2]arginine)
in burn patients. , M+4 basal; ,
M+4 total parenteral nutrition (TPN); ,
M+2 basal; , M+2 TPN.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Plateau level isotopic enrichments of M+1
ornithine (corrected for the infusion rate of
L-[5-13C]ornithine) and M+2
ornithine (corrected for the infusion rate of
[15N-guanidino-5,5-2H2]arginine)
in burn patients. , M+2 basal;
, M+2 TPN; , M+1
basal; , M+1 TPN.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Plateau level enrichment of M+1 and
M+2 proline tracers in burn patients (corrected for the
infusion rate of [5,5-2H2]proline).
, M+2 basal; ,
M+2 TPN; , M+1 basal;
, M+1 TPN.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
Plateau level enrichment of M+3
-ketoisocaproate in burn patients (corrected for the infusion rate
of [5,5,5-2H2]leucine tracer).
, M+3 basal; ,
M+3 TPN.
|
|
Kinetics of arginine, ornithine, and proline.
The estimates of plasma arginine, ornithine, and proline kinetics for
the burn patients are shown in Table 3.
For further interpretation and discussion (see DISCUSSION),
these data are summarized here, together with those obtained in healthy
young adults with the same tracers after similar study protocols
(10) conducted at the MIT CRC and with the analyses
carried out as for the burn patients in the same laboratory.
The plasma arginine flux (QArg) in this group of
burn patients was 101.5 ± 10.3 µmol · kg
1 · h
1 in the
basal state, and it rose during the parenteral feeding. The change was
essentially equivalent to the arginine provided by the TPN. The plasma
ornithine flux (QOrn) in these burn patients during the basal state was 45.3 ± 4.2 µmol · kg
1 · h
1, with the
QOrn-to-QArg ratio being
~4.8%. The flux increased when patients were given parenteral
feeding; the increase was in proportion to that observed for the plasma
arginine flux (Table 3).
From the appearance of plasma
[5,5-2H2]ornithine, which is derived from the
infused
[15N2-guanidino-5,5-2H2]arginine,
and from the measured ornithine flux, we can estimate that the rate of
arginine to ornithine conversion in burn patients under the basal
condition is 38.6 ± 2.9 µmol · kg
1 · h
1. This
rate increased significantly when the patients received TPN.
The rate of ornithine oxidation in the burn patients during the basal
state was 24.1 ± 4.4 µmol · kg
1 · h
1 and did
not show significant increase during parenteral feeding. Thus, in burn
patients, ornithine oxidation rate accounts for 53.0 ± 5.8% of
the ornithine flux under the basal condition, and this fraction was
significantly reduced during the TPN feeding. Furthermore, from the
rate of arginine-to-ornithine conversion and the fraction of the
ornithine flux oxidized (Eq. 5), we can also estimate the
irreversible rate of arginine disposal via ornithine oxidation (Table
3). This was 20.0 ± 2.5 µmol · kg
1 · h
1 for the
basal state, and it increased significantly during the TPN phase. It
might be noted that this estimate of the irreversible loss of arginine
was essentially equivalent to the rate of ornithine oxidation.
The basal plasma proline flux, measured with
L-[5,5-2H2]proline in the
patients, was 155.6 ± 8.8 (SE)
µmol · kg
1 · h
1. TPN
feeding, supplying 58.9 ± 1.4 µmol
proline · kg
1 · h
1, was
associated with an almost equivalent increase in the plasma proline
flux (Table 3).
There was a detectable increment of M+1 proline during the
infusion of
L-[5,5-2H2]proline
tracer (Fig. 4) that appeared before the
L-[5-13C]ornithine
infusion, with the enrichment of M+1 proline not further increasing significantly. This suggests, but does not prove, that the
M+1 species arose as a result of the loss of a labeled
proton through formation of
'-pyrroline-5-carboxylate (P5C) via
proline oxidase and the return of P5C to proline via pyrroline
carboxylate reductase with addition of an unlabeled proton.
The plasma concentrations of arginine, ornithine, and proline are also
summarized in Table 3 for burn patients. These values are generally
comparable to those reported by us earlier (22). Compared
with healthy fasting controls (10), the mean
concentrations of proline and arginine are lower and the concentration
of ornithine is higher in burn patients.
Kinetics of leucine.
Leucine fluxes, which reflect the rate of whole body protein turnover
(25) and so were measured to characterize the increased turnover of protein in the injured patients, were based on plasma enrichments of
[5,5,5-2H2]KIC in the basal
state and TPN feeding state. The leucine intake rate was 55 ± 7 µmol · kg
1 · h
1, and the
fluxes were, respectively, 189 ± 14 and 244 ± 18 µmol · kg
1 · h
1 (Table
4). The plasma concentration of leucine
was compatible with our earlier findings (39). From the
leucine flux, with the average leucine content in whole body protein
taken to be 620 µmol/g protein (34), the estimated whole
body protein breakdown rate was ~7.3 and 7.5 g · kg
1 · day
1 in the basal
and TPN states, respectively. By subtracting the intakes of leucine and
arginine during the TPN phase from their plasma fluxes, the
"endogenous" rates of appearance approximate 102 and 114 for
arginine and 189 and 189 for leucine, respectively, for the basal and
TPN states (Table 4). These values give ratios for the endogenous
arginine-to-leucine fluxes of 0.55 ± 0.07 and 0.64 ± 0.06 for these two conditions. These two values closely approximate the
ratio of 0.58 for the mean arginine-to-leucine concentrations in whole
body mixed proteins (34). This finding further supports
the premise that there is not an "excess" arginine flux, and
therefore, on this basis, arginine de novo synthesis does not
contribute in a major way to the arginine flux; arginine de novo
synthesis is limited, therefore, in burn patients.
View this table:
[in this window]
[in a new window]
|
Table 4.
Leucine kinetics, plasma leucine concentration, and relationships of
leucine with protein and arginine kinetics in burn patients
|
|
 |
DISCUSSION |
Comparison with healthy controls.
To further help to interpret the metabolic and nutritional significance
of these findings in severely burned patients, we compare our data with
results from our published studies of leucine, ornithine, and arginine
kinetics in healthy adult controls. The analytic methods and tracer
paradigms were essentially the same for the present and earlier
studies. In metabolic clinical investigations of the kind we have
carried out here, it is necessary to depend on comparisons of this
type, because burned patients, for quite obvious reasons, cannot be
studied at the same time or in the same setting as a healthy control
group. We believe the present comparison with "historical" data is
appropriate, because 1) the arginine and leucine fluxes
estimated here are comparable to those measured for the basal state in
earlier studies in various groups of severely burned patients
(39, 41), indicating that our observations are broadly
reproducible among the various studies conducted at our hospital and in
this type of patient, and 2) the fluxes of leucine (e.g.,
see Refs. 15, 16, 19), arginine (2, 6-11), and ornithine (10, 11) are
similar among different studies that we have conducted in healthy controls.
A note might be made, however, about the fact that we could not
completely fast our patients, whereas the data for healthy controls
shown in Table 3 were generated in subjects after an overnight fast.
Our patients received glucose, on average, at a rate of ~60
mg · kg
1 · h
1 (or ~1
mg · kg
1 · min
1) to prevent
hypoglycemia. This level of input would not have any significant effect
on basal insulin levels that are already elevated in burn patients
(17) or impact on total energy expenditure over the tracer
infusion period: the glucose provided 1.43 kcal/kg for the 330-min
study period compared with a basal energy expenditure of ~119 kcal/kg
for this same time frame. In addition, an infusion of glucose would, if
anything, tend to lower the plasma fluxes of leucine, arginine, and
proline and so reduce, rather than widen, differences between basal
state burn patients and fasting healthy controls. Furthermore, our
earlier study (29) revealed that, in healthy subjects,
intravenous infusion of glucose at a rate of 2 mg · kg
1 · min
1 did not
significantly alter leucine kinetics.
Thus, from the summary in Table 3, we conclude that basal arginine and
ornithine fluxes are distinctly higher in burn patients when compared
with data for healthy controls, confirming an accelerated turnover of
arginine after burn injury. The rate of ornithine oxidation is higher
in burned patients than in healthy controls. Furthermore, when the
values for leucine flux shown in Table 4 for burn patients are compared
with those reported for healthy controls, which approximate 120 µmol · kg
1 · h
1
(15, 19), a higher rate of whole body protein turnover is evident in the present group of burn patients. It might also be worth
noting that, relative to the mean differences observed between the
severely burned patients and healthy controls, age- and sex-related effects on amino acid kinetics are considered to be minimal and do not
confound these comparisons (15, 16, 26).
Increased loss of arginine.
Our previous intravenous tracer studies (39-41)
demonstrated an increased arginine flux and a limited endogenous
production rate of arginine in severe burn injury; a major purpose of
this study was to determine the disposal rate of arginine via its
conversion to and oxidation of the ornithine carbon skeleton in these
patients. Furthermore, in addition to confirming a high rate of
arginine turnover in response to the stress of burn injury, the present study has revealed that this increased arginine turnover correlates with its increased conversion to ornithine and subsequent oxidation. Hence, the irreversible disposal of arginine, via oxidative catabolism, is significantly increased after burn injury. Our conclusion is based
on the following observations: 1) the higher arginine flux is proportional to the higher ornithine flux in burn patients and is
similar to the ornithine-to-arginine flux ratio in healthy subjects
(Table 3); 2) the rate of ornithine oxidation is increased in burn patients, and this is in line with the increment of
arginine-to-ornithine conversion and parallel to the increased rate of
arginine oxidation. These results are also consistent with our
conclusions, drawn from studies in adult healthy subjects, that
arginine homeostasis is largely controlled by the conversion of
arginine to ornithine and its subsequent oxidation and the relative
rate of arginine intake (10). On the basis of these
findings of an increased rate of arginine disposal in burn patients,
taken together with our previous findings of a limited rate of arginine
de novo synthesis after severe burn injury, it is reasonable to propose
that arginine is a conditionally indispensable amino acid under these
conditions of major stress.
We should caution the reader that our interpretations are based on data
generated via an intravenous tracer paradigm in burn patients, where
providing nutrients via an oral route was not feasible. If the isotope
tracer and amino acid substrates had been given by the oral route, the
interrelationships and quantitative estimates could have been
different. This remains for new research to resolve, but we believe
that our findings do lead to a coherent picture, as we will discuss further.
Estimates of arginine oxidation.
The validity of the conclusions drawn above are also dependent, of
course, on the extent to which we have been able to estimate the actual
rate of arginine catabolism. We can arrive at assessment of this
problem as follows. First, the estimate of the arginine flux during the
basal state appears to be reasonable, because the ratio of arginine to
leucine flux was 0.55 in the basal state, or close to the expectation,
when we assume an approximate concentration ratio of arginine to
leucine in whole body mixed proteins to be 0.58 (34);
second, if the rate of leucine oxidation was 35 µmol · kg
1 · h
1 in burn
patients (41), then the rate of incorporation of arginine into proteins would be 85 [= (189) × 0.55]
µmol · kg
1 · h
1.
Therefore, since the flux of arginine is 102, and the rate of disappearance of arginine into protein is 85, then arginine must be
removed by conversion to ornithine and its subsequent oxidation at a
rate of 102
85 = 17 µmol · kg
1 · h
1. The
estimated rate of arginine oxidation was 20 ± 2 µmol · kg
1 · h
1, or
essentially the same as the rate of ornithine oxidation (24 ± 4 µmol · kg
1 · h
1). Thus
whole body arginine oxidation in the basal state appears to have been
estimated with a reasonable degree of accuracy, provided that the
foregoing assumptions are valid. Furthermore, during this basal state,
body arginine balance would be negative by an amount equal to the
difference between arginine oxidation and net de novo arginine
synthesis. We (39) have estimated the latter value to be 4 µmol · kg
1 · h
1, so
arginine balance would be
16
µmol · kg
1 · h
1 (=
4-20
µmol · kg
1 · h
1). On this
basis, the negative leucine balance would be expected to be about
29
µmol · kg
1 · h
1, which
compares favorably with the value of
30 to
35
µmol · kg
1 · h
1 reported
by us previously (39).
For the TPN phase, we estimated arginine oxidation to be 28 µmol · kg
1 · h
1 when the
rate of exogenous arginine intake is 49 µmol · kg
1 · h
1. Hence,
TPN improved arginine balance by this difference plus that due to
additional net de novo arginine synthesis. This would be a higher
balance (>20
µmol · kg
1 · h
1) than
would be expected from our earlier study, showing a leucine balance of
+8 µmol · kg
1 · h
1 or
equivalent to about +5
µmol · kg
1 · h
1 for
arginine balance. It might be, therefore, that either we have
underestimated the rate of irreversible arginine loss in the TPN state
and/or the present subjects were in a greater anabolic state than for
our previous group of burned patients (39, 41). An
underestimation of the irreversible rate of arginine loss would arise
in the TPN condition if arginine were converted to and retained as
proline, which our tracer technique has not probed. Thus the present
arginine balance estimates are likely to be maximal values and actually
less positive than we have stated.
Relationship between arginine and ornithine and compartmentation of
the urea cycle.
The acceleration of urea production/excretion, and therefore of an
increased urea cycle activity, is a metabolic feature of the response
to burn injury. Our studies on arginine, ornithine, and proline
metabolism have further suggested that the urea cycle is
compartmentalized (10, 39, 41). Thus we and others
(41) have estimated the rate of urea production to be
~540 µmol · kg
1 · h
1 in
severely burned patients. Here, however, the plasma arginine-guanidino flux (99.7 µmol · kg
1 · h
1) appears
to be about one-fifth of the rate of urea production. Furthermore, the
rate of conversion of plasma arginine to ornithine is ~39
µmol · kg
1 · h
1,
indicating that the plasma arginine compartment contributes only ~7%
of the total urea production, with the remainder due to the turnover of
arginine in a segregated site of urea cycle activity in the liver. In
support of these in vivo findings, studies by Cheung et al.
(12) demonstrated that the extramitochondrial enzymes
catalyzing the cytosol portion of the urea cycle are spatially organized in such a way that the urea cycle intermediates are synthesized and metabolized in situ within the cells, exclusively for
the purpose of the urea cycle. The physiological significance of this
is presumably to maintain the availability of arginine in the
peripheral circulation for supporting visceral and muscle protein
synthesis, as well as in other metabolic pathways, including nitric
oxide formation (5). This compartmentation of arginine metabolism may be of particular importance under conditions of an
elevated urea cycle activity, as in stressed conditions or after an
ingestion of protein-rich meals in healthy subjects (11).
It is also evident that ornithine metabolism is similarly
compartmentalized between the intrahepatic urea cycle and plasma or
peripheral pools. The ornithine flux was about one-half of the plasma
arginine flux. Although the ornithine flux was significantly elevated
above that reported for healthy subjects, the quantitative relationships between the ornithine and arginine fluxes appeared to be
similar in both groups of individuals (Table 3). Therefore, the plasma
ornithine flux reflects the status of non-urea-cycle arginine
metabolism, and it appears that, when arginine turnover and oxidation
are increased, ornithine flux is also increased. When the arginine flux
is reduced below a usual, or normal rate, the ornithine flux and its
oxidation rate are similarly reduced (10). This leads us
to the perspective that the ornithine flux is a surrogate for arginine
turnover and catabolism. This interpretation is supported by the
studies of Alonso and Rubio (1) in mice, confirming the
involvement of the reversible reaction catalyzed by ornithine amino
transferase (OAT), whose apparent equilibrium favors
glutamate-
-semialdehyde synthesis with spontaneous conversion to
glutamate. Our view is also consistent with the markedly elevated plasma concentration of ornithine in adult patients who have an OAT
deficiency (32). However, we also recognize that proline turnover is increased in our burn patients, but the extent to which
proline metabolism contributes to this arginine-ornithine relationship
cannot be judged from our data.
Relationship among proline, ornithine, and glutamate.
Proline, ornithine, and glutamate each occupies one corner of a
metabolic triangle, which serves to connect these amino acids to the
urea cycle, on the one hand, and via the action of glutamic acid
dehydrogenase to the tricarboxylic acid cycle and energy metabolism.
Our previous study (22) demonstrated that, in burn patients, there was an increased proline oxidation, accompanied by a
compromised rate of proline de novo synthesis after burn injury,
suggesting the possible importance of an exogenous supply of proline
for the nourishment of burn patients. Studies by Cynober and colleagues
(14, 23) have demonstrated beneficial nutritional and immunological effects of a combined supply of
-ketoglutarate and
ornithine in burn patients. Thus the kinetic interrelationships among
these three amino acids deserve further investigation.
The appearance of the M+1 proline species in plasma before
the beginning of the infusion of
L-[5-13C]ornithine, and then the lack of a
perceptible change in the enrichment of M+1 proline after
that, suggests that this phenomenon arose via the formation of P5C and
its reconversion to proline via proline oxidase and
'-pyrroline-5-carboxylate reductase, respectively. This does not
mean that ornithine carbon is not incorporated into proline, because
had we given the ornithine tracer via the gut, where extensive amino
acid metabolism and interconversion occur during the first pass phase
of amino acid utilization (28), the findings with
[13C]ornithine and transfer of label to proline might
have been different. In addition, however, these findings may also help
to explain why the plasma proline flux measured with
L-[5,5-2H2]proline
(18) appears to be higher than that based on
L-[1-13C]proline as a tracer in healthy
subjects (21, 22) and also when the present data are
compared with the somewhat lower fluxes obtained earlier in burn
patients (21) with a [13C]proline tracer.
To conclude, we have confirmed an expected increased rate of arginine
disposal in severely burned patients compared with healthy adults. This
is reflected by equivalent increases in ornithine turnover and
oxidation. Therefore, with the limited endogenous production rate and
the increased irreversible disposal of arginine, it is further apparent
that arginine is a conditionally indispensable amino acid in the
support of parenterally fed, severely burned patients. This must now be
confirmed through nutritional supplementation studies, as well as by
additional investigations of the metabolic and kinetic relationships
among proline, ornithine, and glutamate. When the feeding modality is
parenteral nutrition, a preformed source of arginine appears to be
obligatory; however, if the enteral route is used, proline and
glutamate may serve as suitable precursors if given in high enough
amounts. This point deserves study because Brunton et al.
(4) have reported that proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal pigs.
It also is now quite clear that the route of nutrient administration can have an important impact on the ability of the organism to synthesize proline as well as arginine (3, 27, 35). We also speculate that the pathway of urea cycle anaplerosis may be a
determinant of the availability and need for proline, glutamate, and/or
glutamine in severe burn injury and other major stress conditions.
 |
ACKNOWLEDGEMENTS |
We thank Gael Basha, Laura Collier, and Caroline Breen for help in
conducting the study and Michael Kenneway, Sue Wong, Amy Lu, Jing Lin,
and Andrew B. Rhodes for their excellent work in sample analysis. We
appreciate the invaluable assistance given by the resident and nursing
staff of the Burn and Trauma Unit of Massachusetts General Hospital.
 |
FOOTNOTES |
This study was supported by National Institutes of Health Grants
DK-15856, GM-02700, and RR-88, and by grants from Shriners Hospitals
for Children (15843 and 15897).
Present address of L. Beaumier: Newborn Medicine Division, McGill
University Health Center, Montreal, Quebec, Canada H3H 1P3.
Address for reprint requests and other correspondence: V. R. Young, Shriners Burns Hospital, 51 Blossom St., Boston, MA 02114; and at MIT: 77 Mass. Ave., Bldg. E17-434, Cambridge, MA 02139.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2000; accepted in final form 17 November 2000.
 |
REFERENCES |
1.
Alonso, E,
and
Rubio V.
Orotic aciduria due to arginine deprivation: changes in the levels of carbamoyl phosphate and of other urea cycle intermediates in mouse liver.
J Nutr
119:
1188-1195,
1989[ISI][Medline].
2.
Beaumier, L,
Castillo L,
Ajami AM,
and
Young VR.
Urea cycle intermediate kinetics and nitrate excretion at normal and "therapeutic" intakes of arginine in humans.
Am J Physiol Endocrinol Metab
269:
E884-E896,
1995[Abstract/Free Full Text].
3.
Berthold, HK,
Reeds PJ,
and
Klein PD.
Isotopic evidence for the differential regulation of arginine and proline synthesis in man.
Metabolism
44:
466-473,
1995[ISI][Medline].
4.
Brunton, JA,
Bertolo RF,
Pencharz PB,
and
Ball RO.
Proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal piglets.
Am J Physiol Endocrinol Metab
277:
E223-E231,
1999[Abstract/Free Full Text].
5.
Carter, EA,
DeRojas-Walker T,
Tamir S,
Tannenbaum SR,
Yu YM,
and
Tompkins RG.
Nitric oxide production is intensely and persistently increased in tissue by thermal injury.
Biochem J
304:
201-204,
1994[ISI][Medline].
6.
Castillo, L,
Ajami A,
Branch S,
Chapman TE,
Yu YM,
Burke JF,
and
Young VR.
Plasma arginine kinetics in adult man: response to an arginine-free diet.
Metabolism
43:
114-122,
1994[ISI][Medline].
7.
Castillo, L,
Beaumier L,
Ajami AM,
and
Young VR.
Whole body nitric oxide synthesis in healthy men determined from [15N]-arginine to [15N]-citrulline labeling.
Proc Natl Acad Sci USA
93:
11460-11465,
1996[Abstract/Free Full Text].
8.
Castillo, L,
Chapman TE,
Sanchez M,
Yu YM,
Burke JF,
Ajami AM,
Vogt J,
and
Young VR.
Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets.
Proc Natl Acad Sci USA
90:
7749-7753,
1993[Abstract/Free Full Text].
9.
Castillo, L,
Chapman TE,
Yu YM,
Ajami A,
Burke JF,
and
Young VR.
Dietary arginine uptake by the splanchnic region in adult humans.
Am J Physiol Endocrinol Metab
265:
E532-E539,
1993[Abstract/Free Full Text].
10.
Castillo, L,
Sanchez M,
Chapman TE,
Ajami A,
Burke JF,
and
Young VR.
The plasma flux and oxidation rate of ornithine adaptively decline with restricted arginine intake.
Proc Natl Acad Sci USA
91:
6393-6397,
1994[Abstract].
11.
Castillo, L,
Sanchez M,
Vogt J,
Chapman TE,
DeRojas-Walker TC,
Tannenbaum SR,
Ajami AM,
and
Young VR.
Plasma arginine, citrulline, and ornithine kinetics in adults, with observations on nitric oxide synthesis.
Am J Physiol Endocrinol Metab
268:
E360-E367,
1995[Abstract/Free Full Text].
12.
Cheung, CW,
Cohen NS,
and
Raijman L.
Channeling of urea cycle intermediates in situ in permeabilized hepatocytes.
J Biol Chem
264:
4038-4044,
1989[Abstract/Free Full Text].
13.
Clarke, JT,
and
Bier DM.
The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C]tyrosine in the postabsorptive state.
Metabolism
31:
999-1005,
1982[ISI][Medline].
14.
Cynober, L.
Ornithine alpha-ketoglutarate in nutritional support.
Nutrition
7:
313-322,
1991[ISI][Medline].
15.
Fukagawa, NK,
Minaker KL,
Young VR,
Matthews DE,
Bier DM,
and
Rowe JW.
Leucine metabolism in aging humans: effect of insulin and substrate availability.
Am J Physiol Endocrinol Metab
256:
E288-E294,
1989[Abstract/Free Full Text].
16.
Fukagawa, NK,
Yu YM,
and
Young VR.
Methionine and cysteine kinetics at different intakes of methionine and cysteine in elderly men and women.
Am J Clin Nutr
68:
380-388,
1998[Abstract].
17.
Grecos, GP,
Abbott WC,
Schiller WR,
Long CL,
Birkhahn RH,
and
Blakemore WS.
The effect of major thermal injury and carbohydrate-free intake on serum triglycerides, insulin, and 3-methylhistidine excretion.
Ann Surg
200:
632-637,
1984[ISI][Medline].
18.
Hiramatsu, T,
Cortiella J,
Marchini JS,
Chapman TE,
and
Young VR.
Plasma proline and leucine kinetics: response to 4 wk with proline-free diets in young adults.
Am J Clin Nutr
60:
207-215,
1994[Abstract].
19.
Hoerr, RA,
Matthews DE,
Bier DM,
and
Young VR.
Leucine kinetics from [2H3]- and [13C]leucine infused simultaneously by gut and vein.
Am J Physiol Endocrinol Metab
260:
E111-E117,
1991[Abstract/Free Full Text].
20.
Jaksic, T,
Wagner DA,
Burke JF,
and
Young VR.
Plasma proline kinetics and the regulation of proline synthesis in man.
Metabolism
36:
1040-1046,
1987[ISI][Medline].
21.
Jaksic, T,
Wagner DA,
Burke JF,
and
Young VR.
Proline metabolism in adult male burned patients and healthy control subjects.
Am J Clin Nutr
54:
408-413,
1991[Abstract].
22.
Jaksic, T,
Wagner DA,
and
Young VR.
Plasma proline kinetics and concentrations in young men in response to dietary proline deprivation.
Am J Clin Nutr
52:
307-312,
1990[Abstract].
23.
Le Bricon, T,
Cynober L,
and
Baracos VE.
Ornithine alpha-ketoglutarate limits muscle protein breakdown without stimulating tumor growth in rats bearing Yoshida ascites hepatoma.
Metabolism
43:
899-905,
1994[ISI][Medline].
24.
Matthews, DE,
Schwarz HP,
Yang RD,
Motil KJ,
Young VR,
and
Bier DM.
Relationship of plasma leucine and alpha-ketoisocaproate during a L-[1-13C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment.
Metabolism
31:
1105-1112,
1982[ISI][Medline].
25.
Millward, DJ,
Price GM,
Pacy PJ,
and
Halliday D.
Whole-body protein and amino acid turnover in man: what can we measure with confidence?
Proc Nutr Soc
50:
197-216,
1991[ISI][Medline].
26.
Millward, DJ,
Fereday A,
Gibson N,
and
Pacy PJ.
Aging, protein requirements, and protein turnover.
Am J Clin Nutr
66:
774-786,
1977[Abstract].
27.
Murphy, JM,
Murch SJ,
and
Ball RO.
Proline is synthesized from glutamate during intragastric infusion but not during intravenous infusion in neonatal piglets.
J Nutr
126:
878-886,
1996[ISI][Medline].
28.
Reeds, PJ,
Burrin DG,
Stoll B,
and
Jahoor F.
Intestinal glutamate metabolism.
J Nutr
130, Suppl:
978S-982S,
2000[ISI][Medline].
29.
Robert, JJ,
Bier D,
Schoeller D,
Wolfe R,
Matthews DE,
Munro HN,
and
Young VR.
Effects of intravenous glucose on whole body leucine dynamics, studied with 1-13C-leucine, in healthy young and elderly adults.
J Gerontol
39:
673-681,
1984[ISI][Medline].
30.
Ryan, CM,
Schoenfeld DA,
Thorpe WP,
Sheridan RL,
Cassem EH,
and
Tompkins RG.
Objective estimates of the probability of death from burns.
N Engl J Med
338:
362-366,
1998[Abstract/Free Full Text].
31.
Ryan, CM,
and
Tompkins RG.
Topical therapy. II. Burns.
In: Pharmacological Approach to Critically Ill Patients (3rd ed.), edited by Chernow B. Baltimore: Williams & Wilkins, 1994, p. 830-843.
32.
Shih, VE.
Regulation of ornithine metabolism.
Enzyme
26:
254-258,
1981[ISI][Medline].
33.
Sidossis, LS,
Coggan AR,
Gastaldelli A,
and
Wolfe RR.
A new correction factor for use in tracer estimations of plasma fatty acid oxidation.
Am J Physiol Endocrinol Metab
269:
E649-E656,
1995[Abstract/Free Full Text].
34.
Stegink, LD,
Bell EF,
Daabees TT,
Andersen DW,
Zike WL,
and
Filer LJ, Jr.
Factors influencing utilization of glycine, glutamate and aspartate in clinical products.
In: Amino Acids: Metabolism and Medical Applications, edited by Blackburn GL,
Grant JB,
and Young VR. Boston: Wright J PSG, 1983, p. 123-146.
35.
Stoll, B,
Henry J,
Reeds PJ,
Yu H,
Jahoor F,
and
Burrin DG.
Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets.
J Nutr
128:
606-614,
1998[Abstract/Free Full Text].
36.
Thompson, GN,
Pacy PJ,
Merritt H,
Ford GC,
Read MA,
Cheng KN,
and
Halliday D.
Rapid measurement of whole body and forearm protein turnover using a [2H5]phenylalanine model.
Am J Physiol Endocrinol Metab
256:
E631-E639,
1989[Abstract/Free Full Text].
37.
Vogt, JA,
Chapman TE,
Wagner DA,
Young VR,
and
Burke JF.
Determination of the isotope enrichment of one or a mixture of two stable labeled tracers of the same compound using the complete isotopomer distribution of an ion fragment; theory and application to in vivo human tracer studies.
Biol Mass Spectrom
22:
600-612,
1993[ISI][Medline].
38.
Wolfe, RR.
Stable and Radioactive Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. New York: Wiley-Liss, 1992, p. 19-144.
39.
Yu, YM,
Ryan CM,
Burke JF,
Tompkins RG,
and
Young VR.
Relations among arginine, citrulline, ornithine, and leucine kinetics in adult burn patients.
Am J Clin Nutr
62:
960-968,
1995[Abstract].
40.
Yu, YM,
Sheridan RL,
Burke JF,
Chapman TE,
Tompkins RG,
and
Young VR.
Kinetics of plasma arginine and leucine in pediatric burn patients.
Am J Clin Nutr
64:
60-66,
1996[Abstract].
41.
Yu, YM,
Young VR,
Castillo L,
Chapman TE,
Tompkins RG,
Ryan CM,
and
Burke JF.
Plasma arginine and leucine kinetics and urea production rates in burn patients.
Metabolism
44:
659-666,
1995[ISI][Medline].
Am J Physiol Endocrinol Metab 280(3):E509-E517
0193-1849/01 $5.00
Copyright © 2001 the American Physiological Society