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Dynamics of the protein metabolic response to burn injury
Dynamics
of the Protein
Farook Jahoor,
Metabolic
Response
Manu Desai, David N. Herndon,
to Burn Injury
and Robert R. Wolfe
The protein metabolic response to burn injury was assessed in 17 children aged 7.1 + 1 .l years (mean * SEM) and a mean burn size of 65 f 7% total body surface area (TBSA) during the acute, flow, convalescent, and recovery phases. Stable isotopes of leucine, valine, lysine, and urea were infused in postabsorptive patients in order to measure protein kinetics. The absolute rate of protein breakdown was assessed from the plasma flux of the essential amino acids (EAA), and the rate of urea production (Ra urea) was used as an index of net protein catabolism. Compared to values obtained in recovered patients, the plasma fluxes of all three EAAs were significantly increased (P < .05), indicating an increased protein breakdown, during the acute, flow, and convalescent phases of injury. Ra urea, however, was only significantly increased during the flow phase (P < .Ol), suggesting that protein breakdown was adequately counteracted in the acute and convalescent phases by elevations in protein synthesis but not in the flow phase. The protein kinetic response did not correlate with changes in the metabolic rate since resting energy expenditure (REE) was significantly increased above predicted levels during the acute and flow phases (by 40% and 50%, respectively), and returned to normal in convalescence. We conclude that (1) protein breakdown is elevated in the acute, flow, and convalescent phases of response to burn injury; (2) there is a significant increase in net loss of N only in the flow phase; and (3) the switch from net protein catabolism in the flow phase to net protein anabolism in convalescence occurs in spite of an elevated protein breakdown rate. m 1988 by Grune & Stratton, Inc.
T
HE PERSISTENT LOSS of body nitrogen (N) in response to severe injury can result in debilitating protein wasting.’ Nitrogen is lost from systemic stores, and both clinical and biochemical evidence suggest that skeletal muscle is the major source.2‘6 Since the aim of clinical care is to ensure both the survival and maximal rehabilitation of the severely burned patient in the shortest possible period of hospitalization, it is desirable to first correct the loss of N during the flow phase, and then to rapidly replenish protein stores during convalescence to facilitate an early return to normal function. In order to achieve this goal, it is first necessary to identify the underlying mechanism responsible for this disturbance in protein metabolism in the flow phase and how it changes during the transition to convalescence. Although the dynamic aspects of protein metabolism in surgical and severe trauma patients have been studied before, the results obtained are diverse and inconclusive.6-‘0 Furthermore, there is no study, of which we are aware, in which the change(s) in kinetics responsible for the shift to net protein anabolism during convalescence has been studied. For example, some workers have reported a reduction in protein synthesis but no change in breakdown in patients following elective surgery, irrespective of nutritional intakes,6u8 whereas in severe trauma, such as burn injury, when nutritional intakes were maintained or increased, there was an increased rate of protein breakdown.9“2 Since it is difficult to ascertain whether the disparity in findings is due to the different nutritional states, the peculiar response to the different pathologic states, the degree of trauma in each
From the Departments of Surgery, Anesthesiology. Biochemistry and Preventive Medicine and Community Health, University of Texas Medical Branch and Shriners Burns Institute, Galveston, TX. Supported by the Shriners of North America. Address reprint requests to Robert R. Wolfe, PhD. Shriners Burns Institute. 610 Texas Ave. Galveston, TX 77550. 0 I988 by Grune & Stratton, Inc. 0026-0495/88/3704-0006$03.00/O 330
patient, or to the different tracer methods employed by different workers,6-‘2 we decided to conduct a study in a more uniform patient population in order to minimize such variables. Young children were studied during different stages of their recovery from severe burn injury in order to define the role of changes in synthesis and breakdown in mediating the catabolic (flow phase) and then anabolic (convalescent phase) responses to injury. Patients were studied in the postabsorptive state in order to eliminate any direct dietary influence on the underlying changes in protein kinetics due to the injury. Using stable isotope tracers, the absolute rate of protein breakdown was assessed as the mean plasma flux of the essential amino acids (EEAs) lysine, leucine, and valine, and net protein catabolism was inferred from urea production rates. MATERIALS AND METHODS Patients
Twenty-two studies were conducted in 17 burned children, (I 3 males and 4 females), who were admitted to the Shriners Burns Institute in Galveston, Texas. Thirteen were treated for acute burn injury and four were recovered burn patients returning for reconstructive surgery. Characteristics of the patients in each study group are summarized in Table 1 and the individual patient characteristics are presented in Table 2. They were 7 + 1.1 years (mean + SEM) of age and had a mean burn size of 65 2 5.2% of total body surface area. Only one patient (D1 of Table 2) was febrile (core temperature 395°C) at the time of study and was treated with 10 mg/kg of acetaminophen. The patients with acute burns were admitted to the hospital within 48 hours after injury. Fluid resuscitation was provided as previously described.” They were treated with excision of the burn wound and grafting within four days of the time of injury. Excision to fascia of all full-thickness eschar was performed on the majority of patients with tangential excision reserved for deep partial-thickness burns only. Harvested autografts were meshed 4:1 and covered with cadaveric allograft meshed 2:l. Cadaveric allograft was used to cover excised areas when no autograft was available. Patients were returned to the operating room every five to seven days for reharvesting of donor sites. Allograft over the sites to be grafted was removed and 4:i autograft with 2:l allograft overlay was again utilized. This Metabolism, Vol 37, No 4 (April), 1988:
pp 330-337
PROTEIN METABOLISM IN BURN INJURY
331
Table 1. Patient Charcteristics MetabolicRate
% Phase of Injury
Acute (n = 51 Flow (n = 7)
Postburn Day
% of
Age
BSA
(Vr)
Burned
Studied
6.8 + 3.0
64 + 13
3.2 k 0.4
73 * 7
138 + 14
38.5
r 0.3
17 k 2.7
65 k 3
149 * 11
38.0
+ 0.2
51 k5
101 + 1
37.9
f 0.2
52 * 2
112 +3
37.5
* 0.1
10.3 + 1.5
63 + 7
Convalescent (n = 5)
8.1 k 2.2
61 r6
Recovered (n = 5)
6.6 * 2.2
73 f 7.5
66k
kcal/m’/h
10
277 + 77
Temperature
Predicted*
(“Cl
Values are given as mean + SEM. *Calculated from Harris-Benedict equation.
sequence was repeated until all full-thickness burn wounds were grafted. ln the acute phase (two to four days postburn) studies were performed after fluid resuscitation was completed and nutrition had started. During the flow and convalescent periods, studies were performed at least two days after the most recent surgical excision. Recovered patients were studied on the day before their reconstructive surgery. In the acute phase, nutrition was provided immediately following fluid resuscitation by tube feeding only. Thereafter, most patients received regular hospital food by mouth unless supplementation by gastric tube feeding was necessary to meet the required nutritional intake. Throughout their hospitalization patients received an average of 95 + 17 kcal kg-’ x d-’ with approximately 50% as carbohydrate. Protein intake was 3.7 + 0.6 g kg-’ x d-‘. Recovered patients received an average of 105 k 13 kcal x kg-’ d-’ and 4.0 k 0.6 g protein kg-’ x d-‘. The experimental protocol was approved by the University of Texas Medical Branch Institutional Review Board. In each case, the purpose of the study and the associated risks were explained in detail to the patient’s parents before informed consent was obtained.
infusion of “N,-urea (99% enriched, obtained from Cambridge Isotope Laboratory, Woburn, MA) was started at the rate of 0.15 pmol kg-’ x min-’ and maintained for six hours using a calibrated syringe pump. A bolus injection of 560 minutes of infusate was given over one minute as a priming dose. After three hours of “N,-urea infusion only, a primed-constant infusion of I-‘3C-leucine, 90% enriched, l-‘3C-valine, 90% enriched and alpha-15N-lysine, 99.3% enriched (Cambridge Isotope Laboratory, Woburn, MA) was started and maintained for three hours. The leucine and valine isotopes were infused at the rate of 0.08 rmol x kg-’ x min-’ and lysine at the rate of 0.1 gmol x kg-’ x mine’. For each isotope, a bolus injection of 80 minutes of infusate was given as a priming dose. The amino acid isotope infusions were delayed because of the shorter time needed for these tracers to reach an isotopic equilibrium. For each tracer an aliquot of the infusate was analyzed for the exact isotope concentration in order to calculate the actual infusion rate. A 6-mL blood sample was drawn before the 15N,-urea infusion started and at 30-minute then 15-minute intervals during the last two hours of the isotope infusions.
Table 2. Individual Patient Characteristics
Experimental
Design
Patients were studied during the acute, flow, convalescent, and recovered phases of injury. The acute phase was considered to be any time between 48 and 96 hours postburn, when the patients’ fluid resuscitation was completed, their nutrition had started, and their respiration and hemodynamics were stabilized. The flow phase was considered to be any time between 2 and 4 weeks postburn. The convalescent phase was considered as any time after the achievement of full epithelialization of all burn wounds. In this stage, the patients were no longer in danger of dying, but had not recovered sufficiently to be discharged from the hospital. This period ranged from 40 days in a patient with a 39% TBSA burn to 94 days in a patient with a 70% TBSA burn. The recovered children were studied after complete healing of all wounds and grafts. Most of them had been discharged from the hospital at least 6 months before the isotopic study. These patients were being readmitted for reconstructive surgery. Seven patients were studied in the flow phase and five in each of the other phases. In two patients studies were completed in the acute, flow. and convalescent phases. Studies were performed in the postabsorptive state. All acute burn patients had venous and arterial catheters already in place for clinical purposes. Isotopes were infused through the venous catheter and blocId sampled from the arterial catheter. In the patients admitted for reconstructive surgery, indwelling catheters were placed in the antecubital vein of one arm for infusion, and in the vein draining the other hand for blood sampling. The hand was heated to 68°C in order to arterialize venous blood.14 After the study was completed the patients were taken to the operating room for surgery and the same catheters were utilized. After a baseline blood sample was drawn, a primed-constant
Postburn % BSA Patient
Burned
DW Studied
Metabolic
Rate % of
Temperature
kcal/m2/h
Predicted
1°C)
Acute A
17
95
108
38.6
B
84
65
125
38.0
C
90
76
142
38.0
D,
66
79
192
39.5
E, Flow
65
51
125
38.2
D,
66
13
80
196
39.0
E, F
65
12
71
173
37.5
61
23
57
117
37.8
G
71
26
61
117
37.5
H
93
25
68
158
37.8
I
35
10
54
131
38.2
J
50
10
66
156
38.4
D, K
66
68 94
58
100
37.6
69
44
100
38.0
L
60
81
45
101
38.2
M
39
38
67
104
38.2
N
70
50
42
100
37.4
Convalescent
Recovered E, 0
65
102
48
120
37.2
89
303
59
109
37.7
P
50
303
0
so
139
48
108
37.8
R
71
540
54
111
37.5
37.6
JAHOOR ET AL
332 4. 3.5 4 3.oJ
i:j
)
c
i
,3 y-y-““t
&-_-++--
wea
,
1
Le”clne
“aline
!2iiAezz:sine 240
Minutes
Of
300
330
360
IsotopsInfusion
Fig 1. Enrichments (mean * SEMl of plasma urea, leucine. valine, and lysine during the last two hours of a primed-constant infusion of [‘6N,]-urea, [l-‘3C]-leucine, [1-“C]-valine and [alpha“N]-@sine in five burned children during the acute phase of injury.
During the course of a study the patients’ metabolic rate was measured with a Horizon Metabolic Cart (Beckman Instruments, Inc, Fullerton, CA).
Analysis of Samples Blood samples were collected in ice-cold heparinized tubes and stored on ice until the end of the experiment, when plasma was separated by centrifugation at 4OC. Aliquots of plasma were placed in tubes containing EDTA and trasylol and stored at -20°C for hormone determinations. For the determination of amino acid concentrations, aliquots of plasma were immediately deproteinized with ice-cold 15% sulfosalicylic acid and stored at -2OOC for analysis the following day. The rest of plasma was stored at -2OV
1.4
270
Fig 3. Enrichments (mean + SEM) of plasma urea, leucine. valine, and lysine during the lest two hours of a primed-constant infusion of [“N,]-urea, [l -‘k]-leucine, [l -“Cl-valine, end [alpha‘6N]-lysine in five burned children during the convalescent phase of injury.
to be analyzed later for the isotopic enrichments of urea, valine, leucine, and lysine. Amino acid concentrations were determined by column chromatography on a 121M autoanalyzer (Beckman Instruments, Inc. Fullerton, CA). All hormones were determined by radioimmunoassay (RIA). Glucagon was determined as described by Faloona and Unger” using Unger’s 30K antibody. Insulin was determined with an insulin RIA kit (Amersham Corp, Arlington Heights, IL), and cortisol was determined with a cortisol RIA kit (Micromedic Systems, Inc. Horsham, PA). The enrichments of plasma urea, leucine, valine, and lysine were determined with a Hewlett-Packard (Palo Alto, CA) 5985B GCMS system. Urea enrichment was measured by GCMS analysis of its N, N’-bistrimethylsilyl derivative during electron impact ionization. Ions at m/e 189 and 191 were monitored.‘6 Enrichments of the
J_,
3.5J
Fig 2. Enrichments (mean i SEMI of plasma urea, leucine, valine, end lysine during the last two hours of a primed-constant infusion of [‘6N,]-urea, [1-“C]-leucine, [1-“C]-valine, and [alpha“N]-lysine in seven burned children during the flow phase of injury.
240
270
300
330
360 1
Enrichments (mean t SEMI of plasma urea. leucine, valine, and lysine during the last two hours of a primed-constant infusion of [“N,]-urea, [1-‘“C]-leucine. [l-‘PC]-valine, end [elpha‘6N]-lysine in five burned children during the recovered phase of injury.
PROTEIN METABOLISM IN BURN INJURY
Table 3. Urea Production
333
Rates and Leucine, Valine, and Lysine Fluxes in Children During Different
Ra (pmol x kg-’ x mine’)
ACUte
FIOW
Phases of Recovery From Burn Injury Recovered
Convalescent
Leucine
2.29
* 0.19’
2.70
+ 0.26t
2.40
* 0.10
1.66 * 0.09
Valine
2.14
* 0.18”
2.56
+ 0.17t
1.92 * 0.11
1.60 + 0.09
Lysine Mean Leucine + Wine
+ Lysine
2.22
+ 0.14”
2.63
t 0.20t
2.06
t 0.04
1.45 + 0.14
2.22
+ 0.13*
2.63
t 0.20t
2.12
* 0.11
1.57 * 0.15
Urea
5.2 + 0.3
8.6 2 0.9t
3.8 r 0.2
4.5 * 0.4
*Significantly greater than recovered value (P < .05). tsignificantly greater than recovered value (P < .O 1).
amino acids were determined on their N-acetyl propyl ester derivatives. The chemical ionization spectrums were used and ions at m/e 217.2 and 216.2 were monitored for leucine, m/e 203.3 and 202.3 for valine, and m/e 274.2 and 273.2 for lysine.16
Calculations In all experiments an isotopic steady state was achieved for all the tracers infused. This is shown in Figs 1-4, in which the average plasma enrichment of urea, leucine, valine, and lysine are presented for the patients studied in the acute, flow, convalescent, and recovered phases of injury, respectively. This permitted use of the steady state equation to calculate urea production rate and the fluxes of leucine, valine, and lysine”: Ra = MPE Infusion (
MPE Plasma
-1
xF, 1
where Ra is the rate of production (or flux) of the substrate, MPE = mole percent excess, and F = rate of infusion of the isotope (pmol kg-’ x min-‘). To compare data from two different groups, for example acute v recovered patients, the nonpaired t-test was employed. All results are presented as mean + SEM.
increase in metabolic rate (38%), urea production rate was only 15% higher than during the recovered phase, and in the convalescent phase, the urea production rate (3.8 + 0.2 pmol x kg-’ x min-‘) was actually 16% lower than the recovered phase value (Tables 3 and 4). Thus, the changes in net protein catabolism were unrelated to the changes in REE above predicted levels (Fig 5). The plasma levels of most amino acids were significantly lower (P -C .05) in all phases of burn injury (Table 5) when compared to values obtained in four normal postabsorptive children of comparable age (unpublished observation). In the recovered phase, although the plasma levels of most amino acids were higher than values in the acute, flow, and convalescence phases, they were not significantly different. Since the fluxes of the EAAs were increased during acute, flow, and convalescent phases, these low plasma amino acid levels are indicative of an increase in the rate of clearance of amino acids from plasma, even in recovered patients. Table 4. Urea Production Rates and Leucine, Valine. and Lysine Fluxes of Individual Patients Patient
RESULTS
In the acute phase, REE was elevated by 38% above the value predicted by the Harris-Benedict equation (Tables 1 and 2). In the flow phase this increased further to a value that was 49% above the predicted value, but by convalescence, REE returned to normal values. Fluxes of leucine, valine, and lysine, however, were consistently elevated in all phases of injury when compared to recovered phase values (Tables 3 and 4). Since in the fasted state EAAs can only appear in plasma from protein breakdown, the average flux rate of these three EAAs can be considered a good index of protein breakdown rate. This means that protein breakdown rates were significantly elevated during the acute (P < .05) and flow (P < .Ol) phases, peaking at a value 68% higher than the recovered phase value. By the convalescent phase, although breakdown had decreased to a value that was not significantly different from the recovered phase value, this still represented a 32% elevation in protein breakdown rate above recovered phase values. The rate of urea production during the flow phase (8.6 + was significantly elevated 0.9 ccmol x kg-’ x min-‘) (P -C .O1) when compared with the recovered phase value of 4.5 t 0.4 pmol x kg-’ x min-‘. Despite the 40% increase in the rate of protein breakdown (as assessed by amino acid flux) in the acute phase, which accompanied a marked
Lhll
Leucine
Valine
Lysine
2.26
Acute A
4.2
2.69
2.09
B
5.2
1.71
1.55
1.92
C
4.9
2.71
2.10
2.38
D,
5.9
2.00
2.65
1.90
E, Flow
5.8
2.35
2.33
2.64
D,
7.2
2.22
2.51
2.81
E, F
9.5
3.19
2.70
2.63
9.8
4.03
3.07
G
6.1
2.40
2.46
H
12.8
2.80
3.02
I
6.5
2.25
1.91
1.95
J
8.2
2.04
2.44
2.40
D, K
4.2
2.52
2.05
2.17
3.8
2.56
1.96
2.01
L
4.0
2.00
1.60
1.96
M
3.3
2.52
2.08
1.96
N
3.7
2.42
1.90
2.05
3.12
Convalescent
Recovered E, 0
3.5
2.00
1.60
4.6
1.50
1.54
1.40
P
3.5
1.88
1.76
1.63
Q
5.1
1.58
1.27
1.08
R
5.6
1.83
1.80
1.72
Ra values are given as wmol X kg-’
X
min-‘.
JAHDDR ET AL
l-
Table 6.
Plasma Hormone Levels of Individual Patients
Patients
d
l-
,-
!
I- -
q0v.r
Pr.dlc,,lJ
j-J
R.co”.r.d
Owr
Glucagon be/mL)
Cortisol bg/dL)
Acute
REE “,.a
Insulin W/mL)
R.
A
10
8
5
C
12
45 38 365
25 35
a
D, E,
-
Flow 15
280
E, F
12
485
42
G
17
270
43
H
15
430
52
I
18
115
22
12
140
13
D, K
15
200
30
L
20
220
42
M
12
270
28
N
17
230
20
D,
T
J Convalescent
CONVALESCENl
Fig 5. Percent changes in REE over the predicted values, and in urea Ra over recovered phase values in burned children during the acute, flow, and convalescent phases of injury.
21
Recovered
Both insulin and glucagon concentrations were significantly elevated (P < .OS) during the flow and convalescent phases of injury when compared to values obtained during the recovered phase (Tables 6 and 7). In the acute phase insulin concentration (9 + 2.3 pU/mL) was no different from the recovered value (8 t 0.9 pU/mL). Cortisol was significantly elevated (P < .OS) during the acute phase to 36 + 5.7 pg/dL, and decreased through the flow and convalescent phases to values that were not significantly elevated when compared to the recovered value of 24 * 5.3 pg/dL. DISCUSSION
The use of different labeled EAAs as tracers for the study of whole-body protein kinetics has become more established
E,
a
169
25
0
8
200
35
P
11
140
11
Q
7
170
20
R
7
170
30
in recent years.18 A principal assumption of this method is that metabolism of the tracer amino acid reflects that of the total amino acid pool of the body.‘* Since the metabolism of each amino acid is regulated by specific control mechanisms, which do not necessarily respond in the same fashion to different physiologic stimuli,‘9*20 it is not possible to select a particular amino acid, a priori, as being most ideal to trace whole-body protein kinetics in different clinical situations.
Table 5. Plasma Amino Acid Levels in Burn Patients During Different AminoAcid
Acute
FIOW
Phases of Recovery Convalescent
Recovered 14 + 1.9
13 + 2.3
13 + 3.2
277 + 38
310 + 78
320 r 61
130 + 22
150 ? 23
107 + 8.4
187 + 25
121 + 25
147 + 15
137 + 28
150 + 29
Serine
60 + 8
69 + 7
58 + 7.8
88 + 20
Proline
80 + 14
163 zk 35
85 + 14
95k
Threonine
42 + 9
78 + 6.5
62 + 19
Aspartic acid
13 + 3
Glutamic acid + glutamine
242 + 41
Alanine Glycine
Methionine
10 +_2.5
Lysine Histidine
73 t 9 44 t 6
Valine
159 2 30
62+
15
20 t 3.0 117 + 18 46 r 4 153+29
11 +3
17
10 t 1.7
82 k 24
116 + 19
40 + 8 101 k 18
177 k 28
54 k 9
lsoleucine
41 t 16
48 + 9
30 t 9
Leucine
91 + 16
91 t 14
62 + 12
97 k 15
Tyrosine
36 k 7
48 + 7
27 f 7
38 +- 4.7 37 + 3.2
62 k 8
57 k 8
28 + 5
Arginine Essential amino-N
402 16 844 -r 180
61 i 11 1.047 + 148
53 ? 22
Nonessential amino-N
927 I
134
1,146
+ 259
2,193
Phenylalanine
Total amino-N Values are given as Fmol/L (mean + SEMI.
1,772
49 + 7.5
58 + 6.1 1,014
+ 142
+ 126
767 + 198 1,036 + 200
1,267
-+ 159
2 271
1,804
2.171
+ 287
k 392
PROTElhl METABOLISM IN BURN INJURY
335
Table 7. Plasma Hormones Hormone Insulin (pU/mL)
9*
36 r 4.1
Phases of Recovery From Burn Injury
FIOW
1.5
Glucagon (pg/mL) lCortisol (pg/dL)
During Different
Acute
15 f 1.21
Convalescent
Recovered
15 + 1.7’
B f 0.9
288 k 24t
230 k 14t
170 * 10
32 + 8
30 + 5
24 + 4.1
‘Sigmficantly greater than recovered value (P -C ,011. tsignificantly greater than recovered value (P -C .05).
For this reason, in this study, three EAAs with unique metabolic fates and distinctly different sites of metabolism were chosen as tracers.2’-23 Whereas the initial metabolism of the branched-chain amino acids leucine and valine occurs principally in peripheral tissues,*’ lysine is mostly metabolized centrally by the liver and kidneys.*’ Furthermore, although leucine and valine are initially transaminated by a common enzyme complex (branched-chain amino acid transaminase), their carbon skeletons have distinct ketogenic and glucogenic metabolic fates, and the metabolic fate of lysine is both glucogenic and ketogenic.2’-24 Thus, when used together as tracers, the combined kinetics of these three EAAs should be more representative of whole-body protein metabolism. Since each amino acid is isolated by gas chromatography prior to mass spectrometry analysis, it was possible to simultaneously use different ‘3C-labeled amino acids without confusion. The validity of the use of the flux of EAAs to estimate protein kinetics in burned children is confirmed by our results (Tables 3 and 4) since the change in the plasma fluxes of all three tracers were always in the same direction (relative to urea production), in all phases of injury. Since all studies were performed in fasted patients, these three indispensable amino acids could have only appeared in plasma from protein breakdown. The mean plasma flux can therefore be considered a good index of the absolute rate of protein breakdown. Plasma flux as commonly determined does not, however, represent the actual whole-body flux of the amino acids. This value can only be calculated accurately if the intracellular enrichment of an amino acid is known for all the tissues of the body. Because of the relatively fast turnover time and small pool size of amino acids (compared to urea, for example), the true flux values can only be approximated by infusing the tracer into a central artery (or pulmonary artery) and sampling from the right heart.*’ For example, when leucine was infused into a vein and blood sampled downstream from an artery (similar to the present study) the enrichments in arterial samples were 20% higher than in samples obtained from a vein. Venous leucine enrichment, however. was similar to intracellular leucine enrichment, which was about 70% to 80% of arterial plasma enrichmerits.‘‘‘’ Thus, it is possible that the plasma flux of leucine in this study underestimates true whole-body flux by about 25%. The same is probably true for lysine and valine-plasma fluxes. Nevertheless, because the true fluxes of all three amino acids are probably underestimated to a similar extent in all patients in all phases of injury, their mean plasma flux can be used to compare the relative changes in protein breakdown rate from one phase of injury to another. It is generally assumed that the difference between the
absolute rates of protein breakdown and synthesis is reflected by the rate of urea excretion.26 The Ra urea determined isotopically is generally 10% to 20% greater than urea excretion, owing to recycling of urea N,27*28and can therefore not be considered a precise quantitation of net protein breakdown. However, it is the preferable means of estimating net protein breakdown in severely burned patients because it requires only blood sampling. Quantitative collection of excreted N is virtually impossible in such patients because of the difficulty in ascertaining the large amount of N lost through the burn wounds, which covered more than 65% of the body surface. We have recently validated the isotopic determination of urea Ra in humans.29 Most of the uncertainty regarding the exact nature of the alterations in protein kinetics that are responsible for the increased loss of nitrogen following severe trauma stems from the fact that the results of tracer studies investigating this problem suggest the existence of two distinct mechanisms. On the one hand, some studies indicate net protein catabolism results from a fall in protein synthesis below the breakdown rate,6-8 while others suggest that it is due to an elevation in protein breakdown rate.‘-‘* The results of the present study confirm that indeed two distinct phenomena are involved, but that they are operative during different phases of injury. In the acute phase, (two to four days postburn), although the absolute rate of protein breakdown is markedly elevated (40%) compared to recovered phase values, there is not a corresponding increase in the net loss of N (Tables 3 and 4), suggesting that protein synthetic rate is also elevated. During the flow phase, however, when the absolute rate of protein breakdown is further stimulated to a level 68% above recovered phase values, there is a significant increase in net loss (91%) of N relative to the recovered phase, suggesting that the further increase in protein breakdown rate is not matched by a similar increase in synthesis rate. It is therefore the inability of protein synthetic rate to keep up with the elevated rate of protein degradation, that is primarily responsible for the persistent negative N balance during the flow phase of severe injury. In the convalescent phase, on the other hand, the rate of protein breakdown remains elevated (32%) above recovered phase values, but net loss of N is now 16% less than recovered phase values, suggesting a simultaneous stimulation of protein synthesis to a level above recovered phase values. Thus, the protein anabolic response of convalescence was not due to the normalization of protein breakdown rate, but rather to a stimulation of protein synthesis above an elevated breakdown rate. From a clinical standpoint, this is an important finding since it demonstrates that it is possible to achieve a protein anabolic state in the face of an elevated protein breakdown
JAHOOR ET AL
336
rate. Therefore, treatment aimed at minimizing net protein losses in the flow phase should be directed not only at suppressing protein breakdown rate but also at stimulating synthesis. Since protein synthesis and breakdown are distinct cellular events with independent regulatory mechanisms, therapy aimed at suppressing breakdown rate may not necessarily stimulate synthesis. For example, whereas insulin administration suppresses protein breakdown rate in a dosedependent manner in vivo, it has not been shown to have an effect on synthesis rate in vivo. 30*3’Alternatively, administration of high levels of amino acids generally stimulates protein synthesis, while having less of an effect on breakdown.‘2 These findings suggest that a combined approach aimed at simultaneously suppressing protein breakdown rate while stimulating synthesis rate should be more successful in preventing protein losses in the flow phase. However, it is not yet clear if protein synthesis and breakdown are normally responsive to factors that affect those functions in normal volunteers. In this study, the consistent finding during all phases of injury is the elevated rate of protein degradation. Similarly, the plasma concentrations of the two counterregulatory hormones, glucagon and cortisol, with documented protein catabolic effects both in vivo and in vitro33*36are significantly elevated, even in convalescence (Table 7). It is therefore possible that the stimulation of protein degradation is mediated via the combined cortisol and glucagon response. Although the anabolic hormone insulin was also significantly elevated in the flow and convalescent periods, its anabolic effect must have been overwhelmed by the combined catabolic effects of the counterregulatory hormones during the flow phase. It is interesting that the increase in REE was unrelated to both the increased rate of protein degradation and the net loss on N (Fig 5). For example, in the acute phase, REE was elevated by 38%, yet urea production was not significantly elevated over the recovered value. In convalescence, REE had returned to normal, but the rate of protein degradation
was still elevated by 35% over the recovered value. These findings do not support the widely held concept that the increase in REE during the hypermetabolic phase of severe injury is directly related to an increased rate of protein catabolism. The lower plasma levels of most nonessential amino acids (except proline) in all phases of injury, was very similar to that of a group of adult burn patients in the flow phase3’ and to that of severely injured3* and postoperative patients.” The plasma profile of EAAs, however, differed from that of adult burn patients, severely injured patients, and postoperative patients. In these patients there is invariably an increase in the plasma concentration of branched-chain and other EAAs~‘-~~ and no marked reductions as is the case in these burned children. The significantly lower concentrations of nearly all amino acids compared to normal control values (unpublished data) during all phases of injury, together with the increased fluxes of three EAAs leucine, lysine, and valine in this study, and the nonessential amino acid alanine in a previous study3’ suggest that the rate of clearance of most amino acids is markedly elevated in burn injury. Thus, the inability of protein synthesis to match breakdown rate could not be attributed to an impaired uptake of amino acids into cells. From the results of this study we conclude that the catastrophic net loss of N during the flow phase of burn injury is not due only to an increased rate of protein breakdown, but also to an inability of protein synthesis rate to keep up with the rate of increase of protein breakdown. Therapy should therefore be aimed at both suppressing protein breakdown and stimulating synthesis rates in order to conserve protein. ACKNOWLEDGMENT The authors acknowledge Marta Wolfe for directing the mass spectrometry analyses, and thank Susan Fons, Esther Surriga, James Richardson, and Nitina Sehgal for their technical assistance and Judy Chadwick for preparation and typing of the manuscript.
REFERENCES 1. Alexander JW, Macmillan BG, Stinnett JD, et al: Beneficial effects of aggressive protein feeding in severely burned children. Ann Surg 192505517, 1980 2. Cuthbertson DP: The disturbance of metabolism produced by bony and non-bony injury, with notes on certain abnormal conditions of bone. Biochem J 24: 1244- 1263, 1930 3. Cuthbertson DP: Observations on the disturbance of metabolism produced by injury to the limbs. Q J Med 1:223-246, 1932 4. Bilmazes C, Kien CL, Rohrbaugh DK, et al: Quantitative contribution by skeletal muscle to elevated rates of whole-body protein breakdown in burned children as measured by N-methylhistidine output. Metabolism 27:671-676, 1978 5. Williamson DH, Farrell R, Kerr A, et al: Muscle protein catabolism after injury in man, as measured by urinary excretion of 3-methyl-histidine. Clin Sci 52:527-533, 1977 6. O’Keefe SJD, Sender PM, James WPT: “Catabolic” loss of body nitrogen in response to surgery. Lancet 1:1035-1037, 1974 7. Crane CW, Picou D, Smith R, et al: Protein turnover in patients before and after elective orthopaedic operations. Br J Surg 64:129-133, 1977
8. Kien CL, Young VR, Rohrbaugh DK, et al: Whole-body protein synthesis and breakdown rates in children before and after reconstructive surgery of the skin. Metabolism 27:27-34, 1978 9. Kien CL, Young VR, Rohrbaugh DK, et al: Increased rates of whole-body protein synthesis and breakdown in children recovering from burns. Ann Surg 187:383-391, 1978 10. Clague MB, Keir MJ, Wright PE, et al: The effects of nutrition and trauma on whole-body protein metabolism in man. Clin Sci 65:165-175, 1983 11. Birkhan RH, Long CL, Fitkin D, et al: Effects of major skeletal trauma on whole-body protein turnover in man measured by L-[1-“Cl-1eucine. Surgery 88:294-299, 1980 12. Hermann VM, Clarke D, Wilmore DW, et al: Protein metabolism: Effect of disease and altered intake on the stable 15N curve. Surg Forum 30:92-94, 1980 13. Carvajal HF: A physiologic approach to fluid therapy in severely burned children. Surg Gynecol Obstet 150:379-384, 1980 14. McGuire EAH, Helderman JH, Tobin JD, et al: Effects of arterial versus venous sampling on analysis of glucose kinetics in man. J Appl Physiol41:565-573, 1976
PROTEIN METABOLISM IN BURN INJURY
15. Faloona GR, Unger RH: Glucagon, in Jaffe BM, Behrman HR (eds): Methods of Hormone Radioimmunoassay. Orlando, FL, Academic, 1974, pp 317-330 16. Wolfe RR: Gas-chromatography mass-spectrometry methods, in ‘Tracers in Metabolic Research: Radioisotopes and Stable Isotope/Mass Spectrometry Methods. New York, Liss, 1984, pp 261-274 17. Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci 82:420-430, 1959 18. Waterlow JC, Garlick PJ, Millward DJ: Total protein turnover from measurements on plasma and on the whole-body, in Protein Turnover in Mammalian Tissues and in Whole-Body. Amsterdam, North Holland, 1978, pp 301-325 19. Robert JJ, Bier DJ, Zhao XH, et al: Glucose and insulin effects on de novo amino acid synthesis in young men: Studies with stable isotope labeled alanine, glycine, leucine and lysine. Metabolism 31:1210-1218, 1982 20. Moth KJ, Matthews DE, Bier DM, et al: Whole-body leucine and lysine metabolism: Response to dietary protein intake in young men. Am J Physiol240(Endocrinol Metab 3):E712-E721, 1981 21. Harper AE, Zapalowski C: Metabolism of branched-chain amino acids, in Waterlow JC, Stephen JMC (eds): Nitrogen Metabolism in Man. London, Applied Science, 198 1, pp 97- 115 22. Meister A: Intermediary metabolism of amino acids, in Biochemistry of the Amino Acids, vol 2. Orlando, FL, Academic, 1965, pp 756,950 23. Yamamoto H, Aikawa T, Matsutaka H: Interorganal relationships of amino acid metabolism in fed rats. Am J Physiol 226:1428-1433, 1974 24. Rothstein M, Miller LL: The metabolism of L-lysine-6-C’4. J Biol Chem 256:243-253, 1954 25. Layman DK, Wolfe RR: Sample site selection for tracer studies applying a unidirectional circulatory approach. Am J Physiol 253:E173-E178,1987 26. San Pietro A, Rittenberg D: A study of the rate of protein synthesis in humans. I. Measurement of the urea pool and urea space. J Biol Chem 201:445-455. 1953 27. L.ong CL, Jeevanandam M, Kinney JM: Metabolism and recycling of urea in man. Am J Clin Nutr 31:1367-1382, 1978
337
28. Jackson AA, Picou D, Landman J: The non-invasive measurement of urea kinetics in normal man by a constant infusion of ‘sN’5N-urea. Hum Nutr Clin Nutr 38C:339-354, 1984 29. Jahoor F, Wolfe RR: Re-assessment of the primed-constant infusion tracer technique as a tool for the measurement of urea production in humans. Am J Physiol 252(Endocrinol Metab 15):E557-E564, 1987 30. Tessari P, Trevisan R, lnchiostro S, et al: Dose-response curves of effects of insulin on leucine kinetics in humans. Am J Physiol251(Endocrinol Metab 14):E334-E342, 1986 31. Fukagawa NK, Minaker KL, Rowe JW, et al: Insulin mediated reduction of whole-body protein breakdown: Doseresponse effects on leucine metabolism in postabsorptive men. J Clin Invest 76:2306-23 11, 1985 32. Wolfe RR, Goodenough RD. Burke JF, et al: Response of protein and urea kinetics in burn patients to different levels of protein intake. Ann Surg 197:163-171, 1983 33. Darnaum B, Matthews DE, Bier DM: Effect of cortisol on glutamine, alanine, leucine and phenylalanine kinetics in man. Clin Res 33:702A, 1985 34. Millward DJ, Brown JG, Odedra B: Protein turnover in individual tissues with special emphasis on muscle, in Waterlow JC, Stephen JMC, (eds): Nitrogen Metabolism in Man. London, Applied Science, 1981, pp 475-494 35. Schworer CM, Mortimore GE: Glucagon-induced autophagy and proteolysis in rat liver: Mediation by selective deprivation of intracellular amino acids. Proc Nat1 Acad Sci USA 76:3169-3 173, 1979 36. Bessey PQ, Watters JM, Aoki TT, et al: Combined hormonal infusion stimulated the metabolic response to injury. Ann Surg 200:264-281, 1984 37. Jahoor F, Herndon DN, Wolfe RR: Role of insulin and glucagon in the response of glucose and alanine kinetics in burninjured patients. J Clin Invest 78:807-814, 1986 38. Askanazi J, Carpentier YA, Michelsen CB. et al: Muscle and plasma amino acids following injury. Ann Surg 192:78-85, 1980 39. Askanazi J, Elwyn DH, Kinney JM, et al: Muscle and plasma amino acids after injury: The role of inactivity. Ann Surg 188:797803,1978
of the Protein
Farook Jahoor,
Metabolic
Response
Manu Desai, David N. Herndon,
to Burn Injury
and Robert R. Wolfe
The protein metabolic response to burn injury was assessed in 17 children aged 7.1 + 1 .l years (mean * SEM) and a mean burn size of 65 f 7% total body surface area (TBSA) during the acute, flow, convalescent, and recovery phases. Stable isotopes of leucine, valine, lysine, and urea were infused in postabsorptive patients in order to measure protein kinetics. The absolute rate of protein breakdown was assessed from the plasma flux of the essential amino acids (EAA), and the rate of urea production (Ra urea) was used as an index of net protein catabolism. Compared to values obtained in recovered patients, the plasma fluxes of all three EAAs were significantly increased (P < .05), indicating an increased protein breakdown, during the acute, flow, and convalescent phases of injury. Ra urea, however, was only significantly increased during the flow phase (P < .Ol), suggesting that protein breakdown was adequately counteracted in the acute and convalescent phases by elevations in protein synthesis but not in the flow phase. The protein kinetic response did not correlate with changes in the metabolic rate since resting energy expenditure (REE) was significantly increased above predicted levels during the acute and flow phases (by 40% and 50%, respectively), and returned to normal in convalescence. We conclude that (1) protein breakdown is elevated in the acute, flow, and convalescent phases of response to burn injury; (2) there is a significant increase in net loss of N only in the flow phase; and (3) the switch from net protein catabolism in the flow phase to net protein anabolism in convalescence occurs in spite of an elevated protein breakdown rate. m 1988 by Grune & Stratton, Inc.
T
HE PERSISTENT LOSS of body nitrogen (N) in response to severe injury can result in debilitating protein wasting.’ Nitrogen is lost from systemic stores, and both clinical and biochemical evidence suggest that skeletal muscle is the major source.2‘6 Since the aim of clinical care is to ensure both the survival and maximal rehabilitation of the severely burned patient in the shortest possible period of hospitalization, it is desirable to first correct the loss of N during the flow phase, and then to rapidly replenish protein stores during convalescence to facilitate an early return to normal function. In order to achieve this goal, it is first necessary to identify the underlying mechanism responsible for this disturbance in protein metabolism in the flow phase and how it changes during the transition to convalescence. Although the dynamic aspects of protein metabolism in surgical and severe trauma patients have been studied before, the results obtained are diverse and inconclusive.6-‘0 Furthermore, there is no study, of which we are aware, in which the change(s) in kinetics responsible for the shift to net protein anabolism during convalescence has been studied. For example, some workers have reported a reduction in protein synthesis but no change in breakdown in patients following elective surgery, irrespective of nutritional intakes,6u8 whereas in severe trauma, such as burn injury, when nutritional intakes were maintained or increased, there was an increased rate of protein breakdown.9“2 Since it is difficult to ascertain whether the disparity in findings is due to the different nutritional states, the peculiar response to the different pathologic states, the degree of trauma in each
From the Departments of Surgery, Anesthesiology. Biochemistry and Preventive Medicine and Community Health, University of Texas Medical Branch and Shriners Burns Institute, Galveston, TX. Supported by the Shriners of North America. Address reprint requests to Robert R. Wolfe, PhD. Shriners Burns Institute. 610 Texas Ave. Galveston, TX 77550. 0 I988 by Grune & Stratton, Inc. 0026-0495/88/3704-0006$03.00/O 330
patient, or to the different tracer methods employed by different workers,6-‘2 we decided to conduct a study in a more uniform patient population in order to minimize such variables. Young children were studied during different stages of their recovery from severe burn injury in order to define the role of changes in synthesis and breakdown in mediating the catabolic (flow phase) and then anabolic (convalescent phase) responses to injury. Patients were studied in the postabsorptive state in order to eliminate any direct dietary influence on the underlying changes in protein kinetics due to the injury. Using stable isotope tracers, the absolute rate of protein breakdown was assessed as the mean plasma flux of the essential amino acids (EEAs) lysine, leucine, and valine, and net protein catabolism was inferred from urea production rates. MATERIALS AND METHODS Patients
Twenty-two studies were conducted in 17 burned children, (I 3 males and 4 females), who were admitted to the Shriners Burns Institute in Galveston, Texas. Thirteen were treated for acute burn injury and four were recovered burn patients returning for reconstructive surgery. Characteristics of the patients in each study group are summarized in Table 1 and the individual patient characteristics are presented in Table 2. They were 7 + 1.1 years (mean + SEM) of age and had a mean burn size of 65 2 5.2% of total body surface area. Only one patient (D1 of Table 2) was febrile (core temperature 395°C) at the time of study and was treated with 10 mg/kg of acetaminophen. The patients with acute burns were admitted to the hospital within 48 hours after injury. Fluid resuscitation was provided as previously described.” They were treated with excision of the burn wound and grafting within four days of the time of injury. Excision to fascia of all full-thickness eschar was performed on the majority of patients with tangential excision reserved for deep partial-thickness burns only. Harvested autografts were meshed 4:1 and covered with cadaveric allograft meshed 2:l. Cadaveric allograft was used to cover excised areas when no autograft was available. Patients were returned to the operating room every five to seven days for reharvesting of donor sites. Allograft over the sites to be grafted was removed and 4:i autograft with 2:l allograft overlay was again utilized. This Metabolism, Vol 37, No 4 (April), 1988:
pp 330-337
PROTEIN METABOLISM IN BURN INJURY
331
Table 1. Patient Charcteristics MetabolicRate
% Phase of Injury
Acute (n = 51 Flow (n = 7)
Postburn Day
% of
Age
BSA
(Vr)
Burned
Studied
6.8 + 3.0
64 + 13
3.2 k 0.4
73 * 7
138 + 14
38.5
r 0.3
17 k 2.7
65 k 3
149 * 11
38.0
+ 0.2
51 k5
101 + 1
37.9
f 0.2
52 * 2
112 +3
37.5
* 0.1
10.3 + 1.5
63 + 7
Convalescent (n = 5)
8.1 k 2.2
61 r6
Recovered (n = 5)
6.6 * 2.2
73 f 7.5
66k
kcal/m’/h
10
277 + 77
Temperature
Predicted*
(“Cl
Values are given as mean + SEM. *Calculated from Harris-Benedict equation.
sequence was repeated until all full-thickness burn wounds were grafted. ln the acute phase (two to four days postburn) studies were performed after fluid resuscitation was completed and nutrition had started. During the flow and convalescent periods, studies were performed at least two days after the most recent surgical excision. Recovered patients were studied on the day before their reconstructive surgery. In the acute phase, nutrition was provided immediately following fluid resuscitation by tube feeding only. Thereafter, most patients received regular hospital food by mouth unless supplementation by gastric tube feeding was necessary to meet the required nutritional intake. Throughout their hospitalization patients received an average of 95 + 17 kcal kg-’ x d-’ with approximately 50% as carbohydrate. Protein intake was 3.7 + 0.6 g kg-’ x d-‘. Recovered patients received an average of 105 k 13 kcal x kg-’ d-’ and 4.0 k 0.6 g protein kg-’ x d-‘. The experimental protocol was approved by the University of Texas Medical Branch Institutional Review Board. In each case, the purpose of the study and the associated risks were explained in detail to the patient’s parents before informed consent was obtained.
infusion of “N,-urea (99% enriched, obtained from Cambridge Isotope Laboratory, Woburn, MA) was started at the rate of 0.15 pmol kg-’ x min-’ and maintained for six hours using a calibrated syringe pump. A bolus injection of 560 minutes of infusate was given over one minute as a priming dose. After three hours of “N,-urea infusion only, a primed-constant infusion of I-‘3C-leucine, 90% enriched, l-‘3C-valine, 90% enriched and alpha-15N-lysine, 99.3% enriched (Cambridge Isotope Laboratory, Woburn, MA) was started and maintained for three hours. The leucine and valine isotopes were infused at the rate of 0.08 rmol x kg-’ x min-’ and lysine at the rate of 0.1 gmol x kg-’ x mine’. For each isotope, a bolus injection of 80 minutes of infusate was given as a priming dose. The amino acid isotope infusions were delayed because of the shorter time needed for these tracers to reach an isotopic equilibrium. For each tracer an aliquot of the infusate was analyzed for the exact isotope concentration in order to calculate the actual infusion rate. A 6-mL blood sample was drawn before the 15N,-urea infusion started and at 30-minute then 15-minute intervals during the last two hours of the isotope infusions.
Table 2. Individual Patient Characteristics
Experimental
Design
Patients were studied during the acute, flow, convalescent, and recovered phases of injury. The acute phase was considered to be any time between 48 and 96 hours postburn, when the patients’ fluid resuscitation was completed, their nutrition had started, and their respiration and hemodynamics were stabilized. The flow phase was considered to be any time between 2 and 4 weeks postburn. The convalescent phase was considered as any time after the achievement of full epithelialization of all burn wounds. In this stage, the patients were no longer in danger of dying, but had not recovered sufficiently to be discharged from the hospital. This period ranged from 40 days in a patient with a 39% TBSA burn to 94 days in a patient with a 70% TBSA burn. The recovered children were studied after complete healing of all wounds and grafts. Most of them had been discharged from the hospital at least 6 months before the isotopic study. These patients were being readmitted for reconstructive surgery. Seven patients were studied in the flow phase and five in each of the other phases. In two patients studies were completed in the acute, flow. and convalescent phases. Studies were performed in the postabsorptive state. All acute burn patients had venous and arterial catheters already in place for clinical purposes. Isotopes were infused through the venous catheter and blocId sampled from the arterial catheter. In the patients admitted for reconstructive surgery, indwelling catheters were placed in the antecubital vein of one arm for infusion, and in the vein draining the other hand for blood sampling. The hand was heated to 68°C in order to arterialize venous blood.14 After the study was completed the patients were taken to the operating room for surgery and the same catheters were utilized. After a baseline blood sample was drawn, a primed-constant
Postburn % BSA Patient
Burned
DW Studied
Metabolic
Rate % of
Temperature
kcal/m2/h
Predicted
1°C)
Acute A
17
95
108
38.6
B
84
65
125
38.0
C
90
76
142
38.0
D,
66
79
192
39.5
E, Flow
65
51
125
38.2
D,
66
13
80
196
39.0
E, F
65
12
71
173
37.5
61
23
57
117
37.8
G
71
26
61
117
37.5
H
93
25
68
158
37.8
I
35
10
54
131
38.2
J
50
10
66
156
38.4
D, K
66
68 94
58
100
37.6
69
44
100
38.0
L
60
81
45
101
38.2
M
39
38
67
104
38.2
N
70
50
42
100
37.4
Convalescent
Recovered E, 0
65
102
48
120
37.2
89
303
59
109
37.7
P
50
303
0
so
139
48
108
37.8
R
71
540
54
111
37.5
37.6
JAHOOR ET AL
332 4. 3.5 4 3.oJ
i:j
)
c
i
,3 y-y-““t
&-_-++--
wea
,
1
Le”clne
“aline
!2iiAezz:sine 240
Minutes
Of
300
330
360
IsotopsInfusion
Fig 1. Enrichments (mean * SEMl of plasma urea, leucine. valine, and lysine during the last two hours of a primed-constant infusion of [‘6N,]-urea, [l-‘3C]-leucine, [1-“C]-valine and [alpha“N]-@sine in five burned children during the acute phase of injury.
During the course of a study the patients’ metabolic rate was measured with a Horizon Metabolic Cart (Beckman Instruments, Inc, Fullerton, CA).
Analysis of Samples Blood samples were collected in ice-cold heparinized tubes and stored on ice until the end of the experiment, when plasma was separated by centrifugation at 4OC. Aliquots of plasma were placed in tubes containing EDTA and trasylol and stored at -20°C for hormone determinations. For the determination of amino acid concentrations, aliquots of plasma were immediately deproteinized with ice-cold 15% sulfosalicylic acid and stored at -2OOC for analysis the following day. The rest of plasma was stored at -2OV
1.4
270
Fig 3. Enrichments (mean + SEM) of plasma urea, leucine. valine, and lysine during the lest two hours of a primed-constant infusion of [“N,]-urea, [l -‘k]-leucine, [l -“Cl-valine, end [alpha‘6N]-lysine in five burned children during the convalescent phase of injury.
to be analyzed later for the isotopic enrichments of urea, valine, leucine, and lysine. Amino acid concentrations were determined by column chromatography on a 121M autoanalyzer (Beckman Instruments, Inc. Fullerton, CA). All hormones were determined by radioimmunoassay (RIA). Glucagon was determined as described by Faloona and Unger” using Unger’s 30K antibody. Insulin was determined with an insulin RIA kit (Amersham Corp, Arlington Heights, IL), and cortisol was determined with a cortisol RIA kit (Micromedic Systems, Inc. Horsham, PA). The enrichments of plasma urea, leucine, valine, and lysine were determined with a Hewlett-Packard (Palo Alto, CA) 5985B GCMS system. Urea enrichment was measured by GCMS analysis of its N, N’-bistrimethylsilyl derivative during electron impact ionization. Ions at m/e 189 and 191 were monitored.‘6 Enrichments of the
J_,
3.5J
Fig 2. Enrichments (mean i SEMI of plasma urea, leucine, valine, end lysine during the last two hours of a primed-constant infusion of [‘6N,]-urea, [1-“C]-leucine, [1-“C]-valine, and [alpha“N]-lysine in seven burned children during the flow phase of injury.
240
270
300
330
360 1
Enrichments (mean t SEMI of plasma urea. leucine, valine, and lysine during the last two hours of a primed-constant infusion of [“N,]-urea, [1-‘“C]-leucine. [l-‘PC]-valine, end [elpha‘6N]-lysine in five burned children during the recovered phase of injury.
PROTEIN METABOLISM IN BURN INJURY
Table 3. Urea Production
333
Rates and Leucine, Valine, and Lysine Fluxes in Children During Different
Ra (pmol x kg-’ x mine’)
ACUte
FIOW
Phases of Recovery From Burn Injury Recovered
Convalescent
Leucine
2.29
* 0.19’
2.70
+ 0.26t
2.40
* 0.10
1.66 * 0.09
Valine
2.14
* 0.18”
2.56
+ 0.17t
1.92 * 0.11
1.60 + 0.09
Lysine Mean Leucine + Wine
+ Lysine
2.22
+ 0.14”
2.63
t 0.20t
2.06
t 0.04
1.45 + 0.14
2.22
+ 0.13*
2.63
t 0.20t
2.12
* 0.11
1.57 * 0.15
Urea
5.2 + 0.3
8.6 2 0.9t
3.8 r 0.2
4.5 * 0.4
*Significantly greater than recovered value (P < .05). tsignificantly greater than recovered value (P < .O 1).
amino acids were determined on their N-acetyl propyl ester derivatives. The chemical ionization spectrums were used and ions at m/e 217.2 and 216.2 were monitored for leucine, m/e 203.3 and 202.3 for valine, and m/e 274.2 and 273.2 for lysine.16
Calculations In all experiments an isotopic steady state was achieved for all the tracers infused. This is shown in Figs 1-4, in which the average plasma enrichment of urea, leucine, valine, and lysine are presented for the patients studied in the acute, flow, convalescent, and recovered phases of injury, respectively. This permitted use of the steady state equation to calculate urea production rate and the fluxes of leucine, valine, and lysine”: Ra = MPE Infusion (
MPE Plasma
-1
xF, 1
where Ra is the rate of production (or flux) of the substrate, MPE = mole percent excess, and F = rate of infusion of the isotope (pmol kg-’ x min-‘). To compare data from two different groups, for example acute v recovered patients, the nonpaired t-test was employed. All results are presented as mean + SEM.
increase in metabolic rate (38%), urea production rate was only 15% higher than during the recovered phase, and in the convalescent phase, the urea production rate (3.8 + 0.2 pmol x kg-’ x min-‘) was actually 16% lower than the recovered phase value (Tables 3 and 4). Thus, the changes in net protein catabolism were unrelated to the changes in REE above predicted levels (Fig 5). The plasma levels of most amino acids were significantly lower (P -C .05) in all phases of burn injury (Table 5) when compared to values obtained in four normal postabsorptive children of comparable age (unpublished observation). In the recovered phase, although the plasma levels of most amino acids were higher than values in the acute, flow, and convalescence phases, they were not significantly different. Since the fluxes of the EAAs were increased during acute, flow, and convalescent phases, these low plasma amino acid levels are indicative of an increase in the rate of clearance of amino acids from plasma, even in recovered patients. Table 4. Urea Production Rates and Leucine, Valine. and Lysine Fluxes of Individual Patients Patient
RESULTS
In the acute phase, REE was elevated by 38% above the value predicted by the Harris-Benedict equation (Tables 1 and 2). In the flow phase this increased further to a value that was 49% above the predicted value, but by convalescence, REE returned to normal values. Fluxes of leucine, valine, and lysine, however, were consistently elevated in all phases of injury when compared to recovered phase values (Tables 3 and 4). Since in the fasted state EAAs can only appear in plasma from protein breakdown, the average flux rate of these three EAAs can be considered a good index of protein breakdown rate. This means that protein breakdown rates were significantly elevated during the acute (P < .05) and flow (P < .Ol) phases, peaking at a value 68% higher than the recovered phase value. By the convalescent phase, although breakdown had decreased to a value that was not significantly different from the recovered phase value, this still represented a 32% elevation in protein breakdown rate above recovered phase values. The rate of urea production during the flow phase (8.6 + was significantly elevated 0.9 ccmol x kg-’ x min-‘) (P -C .O1) when compared with the recovered phase value of 4.5 t 0.4 pmol x kg-’ x min-‘. Despite the 40% increase in the rate of protein breakdown (as assessed by amino acid flux) in the acute phase, which accompanied a marked
Lhll
Leucine
Valine
Lysine
2.26
Acute A
4.2
2.69
2.09
B
5.2
1.71
1.55
1.92
C
4.9
2.71
2.10
2.38
D,
5.9
2.00
2.65
1.90
E, Flow
5.8
2.35
2.33
2.64
D,
7.2
2.22
2.51
2.81
E, F
9.5
3.19
2.70
2.63
9.8
4.03
3.07
G
6.1
2.40
2.46
H
12.8
2.80
3.02
I
6.5
2.25
1.91
1.95
J
8.2
2.04
2.44
2.40
D, K
4.2
2.52
2.05
2.17
3.8
2.56
1.96
2.01
L
4.0
2.00
1.60
1.96
M
3.3
2.52
2.08
1.96
N
3.7
2.42
1.90
2.05
3.12
Convalescent
Recovered E, 0
3.5
2.00
1.60
4.6
1.50
1.54
1.40
P
3.5
1.88
1.76
1.63
Q
5.1
1.58
1.27
1.08
R
5.6
1.83
1.80
1.72
Ra values are given as wmol X kg-’
X
min-‘.
JAHDDR ET AL
l-
Table 6.
Plasma Hormone Levels of Individual Patients
Patients
d
l-
,-
!
I- -
q0v.r
Pr.dlc,,lJ
j-J
R.co”.r.d
Owr
Glucagon be/mL)
Cortisol bg/dL)
Acute
REE “,.a
Insulin W/mL)
R.
A
10
8
5
C
12
45 38 365
25 35
a
D, E,
-
Flow 15
280
E, F
12
485
42
G
17
270
43
H
15
430
52
I
18
115
22
12
140
13
D, K
15
200
30
L
20
220
42
M
12
270
28
N
17
230
20
D,
T
J Convalescent
CONVALESCENl
Fig 5. Percent changes in REE over the predicted values, and in urea Ra over recovered phase values in burned children during the acute, flow, and convalescent phases of injury.
21
Recovered
Both insulin and glucagon concentrations were significantly elevated (P < .OS) during the flow and convalescent phases of injury when compared to values obtained during the recovered phase (Tables 6 and 7). In the acute phase insulin concentration (9 + 2.3 pU/mL) was no different from the recovered value (8 t 0.9 pU/mL). Cortisol was significantly elevated (P < .OS) during the acute phase to 36 + 5.7 pg/dL, and decreased through the flow and convalescent phases to values that were not significantly elevated when compared to the recovered value of 24 * 5.3 pg/dL. DISCUSSION
The use of different labeled EAAs as tracers for the study of whole-body protein kinetics has become more established
E,
a
169
25
0
8
200
35
P
11
140
11
Q
7
170
20
R
7
170
30
in recent years.18 A principal assumption of this method is that metabolism of the tracer amino acid reflects that of the total amino acid pool of the body.‘* Since the metabolism of each amino acid is regulated by specific control mechanisms, which do not necessarily respond in the same fashion to different physiologic stimuli,‘9*20 it is not possible to select a particular amino acid, a priori, as being most ideal to trace whole-body protein kinetics in different clinical situations.
Table 5. Plasma Amino Acid Levels in Burn Patients During Different AminoAcid
Acute
FIOW
Phases of Recovery Convalescent
Recovered 14 + 1.9
13 + 2.3
13 + 3.2
277 + 38
310 + 78
320 r 61
130 + 22
150 ? 23
107 + 8.4
187 + 25
121 + 25
147 + 15
137 + 28
150 + 29
Serine
60 + 8
69 + 7
58 + 7.8
88 + 20
Proline
80 + 14
163 zk 35
85 + 14
95k
Threonine
42 + 9
78 + 6.5
62 + 19
Aspartic acid
13 + 3
Glutamic acid + glutamine
242 + 41
Alanine Glycine
Methionine
10 +_2.5
Lysine Histidine
73 t 9 44 t 6
Valine
159 2 30
62+
15
20 t 3.0 117 + 18 46 r 4 153+29
11 +3
17
10 t 1.7
82 k 24
116 + 19
40 + 8 101 k 18
177 k 28
54 k 9
lsoleucine
41 t 16
48 + 9
30 t 9
Leucine
91 + 16
91 t 14
62 + 12
97 k 15
Tyrosine
36 k 7
48 + 7
27 f 7
38 +- 4.7 37 + 3.2
62 k 8
57 k 8
28 + 5
Arginine Essential amino-N
402 16 844 -r 180
61 i 11 1.047 + 148
53 ? 22
Nonessential amino-N
927 I
134
1,146
+ 259
2,193
Phenylalanine
Total amino-N Values are given as Fmol/L (mean + SEMI.
1,772
49 + 7.5
58 + 6.1 1,014
+ 142
+ 126
767 + 198 1,036 + 200
1,267
-+ 159
2 271
1,804
2.171
+ 287
k 392
PROTElhl METABOLISM IN BURN INJURY
335
Table 7. Plasma Hormones Hormone Insulin (pU/mL)
9*
36 r 4.1
Phases of Recovery From Burn Injury
FIOW
1.5
Glucagon (pg/mL) lCortisol (pg/dL)
During Different
Acute
15 f 1.21
Convalescent
Recovered
15 + 1.7’
B f 0.9
288 k 24t
230 k 14t
170 * 10
32 + 8
30 + 5
24 + 4.1
‘Sigmficantly greater than recovered value (P -C ,011. tsignificantly greater than recovered value (P -C .05).
For this reason, in this study, three EAAs with unique metabolic fates and distinctly different sites of metabolism were chosen as tracers.2’-23 Whereas the initial metabolism of the branched-chain amino acids leucine and valine occurs principally in peripheral tissues,*’ lysine is mostly metabolized centrally by the liver and kidneys.*’ Furthermore, although leucine and valine are initially transaminated by a common enzyme complex (branched-chain amino acid transaminase), their carbon skeletons have distinct ketogenic and glucogenic metabolic fates, and the metabolic fate of lysine is both glucogenic and ketogenic.2’-24 Thus, when used together as tracers, the combined kinetics of these three EAAs should be more representative of whole-body protein metabolism. Since each amino acid is isolated by gas chromatography prior to mass spectrometry analysis, it was possible to simultaneously use different ‘3C-labeled amino acids without confusion. The validity of the use of the flux of EAAs to estimate protein kinetics in burned children is confirmed by our results (Tables 3 and 4) since the change in the plasma fluxes of all three tracers were always in the same direction (relative to urea production), in all phases of injury. Since all studies were performed in fasted patients, these three indispensable amino acids could have only appeared in plasma from protein breakdown. The mean plasma flux can therefore be considered a good index of the absolute rate of protein breakdown. Plasma flux as commonly determined does not, however, represent the actual whole-body flux of the amino acids. This value can only be calculated accurately if the intracellular enrichment of an amino acid is known for all the tissues of the body. Because of the relatively fast turnover time and small pool size of amino acids (compared to urea, for example), the true flux values can only be approximated by infusing the tracer into a central artery (or pulmonary artery) and sampling from the right heart.*’ For example, when leucine was infused into a vein and blood sampled downstream from an artery (similar to the present study) the enrichments in arterial samples were 20% higher than in samples obtained from a vein. Venous leucine enrichment, however. was similar to intracellular leucine enrichment, which was about 70% to 80% of arterial plasma enrichmerits.‘‘‘’ Thus, it is possible that the plasma flux of leucine in this study underestimates true whole-body flux by about 25%. The same is probably true for lysine and valine-plasma fluxes. Nevertheless, because the true fluxes of all three amino acids are probably underestimated to a similar extent in all patients in all phases of injury, their mean plasma flux can be used to compare the relative changes in protein breakdown rate from one phase of injury to another. It is generally assumed that the difference between the
absolute rates of protein breakdown and synthesis is reflected by the rate of urea excretion.26 The Ra urea determined isotopically is generally 10% to 20% greater than urea excretion, owing to recycling of urea N,27*28and can therefore not be considered a precise quantitation of net protein breakdown. However, it is the preferable means of estimating net protein breakdown in severely burned patients because it requires only blood sampling. Quantitative collection of excreted N is virtually impossible in such patients because of the difficulty in ascertaining the large amount of N lost through the burn wounds, which covered more than 65% of the body surface. We have recently validated the isotopic determination of urea Ra in humans.29 Most of the uncertainty regarding the exact nature of the alterations in protein kinetics that are responsible for the increased loss of nitrogen following severe trauma stems from the fact that the results of tracer studies investigating this problem suggest the existence of two distinct mechanisms. On the one hand, some studies indicate net protein catabolism results from a fall in protein synthesis below the breakdown rate,6-8 while others suggest that it is due to an elevation in protein breakdown rate.‘-‘* The results of the present study confirm that indeed two distinct phenomena are involved, but that they are operative during different phases of injury. In the acute phase, (two to four days postburn), although the absolute rate of protein breakdown is markedly elevated (40%) compared to recovered phase values, there is not a corresponding increase in the net loss of N (Tables 3 and 4), suggesting that protein synthetic rate is also elevated. During the flow phase, however, when the absolute rate of protein breakdown is further stimulated to a level 68% above recovered phase values, there is a significant increase in net loss (91%) of N relative to the recovered phase, suggesting that the further increase in protein breakdown rate is not matched by a similar increase in synthesis rate. It is therefore the inability of protein synthetic rate to keep up with the elevated rate of protein degradation, that is primarily responsible for the persistent negative N balance during the flow phase of severe injury. In the convalescent phase, on the other hand, the rate of protein breakdown remains elevated (32%) above recovered phase values, but net loss of N is now 16% less than recovered phase values, suggesting a simultaneous stimulation of protein synthesis to a level above recovered phase values. Thus, the protein anabolic response of convalescence was not due to the normalization of protein breakdown rate, but rather to a stimulation of protein synthesis above an elevated breakdown rate. From a clinical standpoint, this is an important finding since it demonstrates that it is possible to achieve a protein anabolic state in the face of an elevated protein breakdown
JAHOOR ET AL
336
rate. Therefore, treatment aimed at minimizing net protein losses in the flow phase should be directed not only at suppressing protein breakdown rate but also at stimulating synthesis. Since protein synthesis and breakdown are distinct cellular events with independent regulatory mechanisms, therapy aimed at suppressing breakdown rate may not necessarily stimulate synthesis. For example, whereas insulin administration suppresses protein breakdown rate in a dosedependent manner in vivo, it has not been shown to have an effect on synthesis rate in vivo. 30*3’Alternatively, administration of high levels of amino acids generally stimulates protein synthesis, while having less of an effect on breakdown.‘2 These findings suggest that a combined approach aimed at simultaneously suppressing protein breakdown rate while stimulating synthesis rate should be more successful in preventing protein losses in the flow phase. However, it is not yet clear if protein synthesis and breakdown are normally responsive to factors that affect those functions in normal volunteers. In this study, the consistent finding during all phases of injury is the elevated rate of protein degradation. Similarly, the plasma concentrations of the two counterregulatory hormones, glucagon and cortisol, with documented protein catabolic effects both in vivo and in vitro33*36are significantly elevated, even in convalescence (Table 7). It is therefore possible that the stimulation of protein degradation is mediated via the combined cortisol and glucagon response. Although the anabolic hormone insulin was also significantly elevated in the flow and convalescent periods, its anabolic effect must have been overwhelmed by the combined catabolic effects of the counterregulatory hormones during the flow phase. It is interesting that the increase in REE was unrelated to both the increased rate of protein degradation and the net loss on N (Fig 5). For example, in the acute phase, REE was elevated by 38%, yet urea production was not significantly elevated over the recovered value. In convalescence, REE had returned to normal, but the rate of protein degradation
was still elevated by 35% over the recovered value. These findings do not support the widely held concept that the increase in REE during the hypermetabolic phase of severe injury is directly related to an increased rate of protein catabolism. The lower plasma levels of most nonessential amino acids (except proline) in all phases of injury, was very similar to that of a group of adult burn patients in the flow phase3’ and to that of severely injured3* and postoperative patients.” The plasma profile of EAAs, however, differed from that of adult burn patients, severely injured patients, and postoperative patients. In these patients there is invariably an increase in the plasma concentration of branched-chain and other EAAs~‘-~~ and no marked reductions as is the case in these burned children. The significantly lower concentrations of nearly all amino acids compared to normal control values (unpublished data) during all phases of injury, together with the increased fluxes of three EAAs leucine, lysine, and valine in this study, and the nonessential amino acid alanine in a previous study3’ suggest that the rate of clearance of most amino acids is markedly elevated in burn injury. Thus, the inability of protein synthesis to match breakdown rate could not be attributed to an impaired uptake of amino acids into cells. From the results of this study we conclude that the catastrophic net loss of N during the flow phase of burn injury is not due only to an increased rate of protein breakdown, but also to an inability of protein synthesis rate to keep up with the rate of increase of protein breakdown. Therapy should therefore be aimed at both suppressing protein breakdown and stimulating synthesis rates in order to conserve protein. ACKNOWLEDGMENT The authors acknowledge Marta Wolfe for directing the mass spectrometry analyses, and thank Susan Fons, Esther Surriga, James Richardson, and Nitina Sehgal for their technical assistance and Judy Chadwick for preparation and typing of the manuscript.
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PROTEIN METABOLISM IN BURN INJURY
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