![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Surgery, Maastricht University, NL-6200 MD Maastricht, the Netherlands
2To whom correspondence should be addressed. E-mail: nep.deutz{at}ah.unimaas.nl
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
intestine),
liver and hindquarter (50% skeletal muscle). All pigs received a
continuous enteral infusion of a liquid meal equivalent to 0.3 g
protein · kg bw-1 · h-1 3 d before
and 4 d after a 24-h endotoxemia period. Measurements were
performed 1 d before and 1 and 4 d after endotoxemia that was
induced by a 24-h endotoxin (3 µg · kg bw-1 ·
h-1 lipopolysaccharide, n = 7)
infusion. Controls received NaCl (n = 7). At 4 d after endotoxemia, hindquarter protein turnover was increased,
resulting in net synthesis. The amino acid output by the PDV was
increased 1 and 4 d after endotoxemia. In the liver, net protein
synthesis was enhanced 1 d after endotoxemia. Increased amino acid
transamination in hindquarter and PDV led to glutamine and alanine
effluxes that serve as substrates for liver and, possibly, the immune
system. By providing substrate, enteral nutrition can sustain elevated
amino acid demand in the postendotoxemic state by hindquarter, PDV and
liver for protein synthesis and transamination processes.
KEY WORDS: • intestine • liver • muscle • glucose • isotopes
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In clinical practice,
enteral nutritional support is often applied as a therapeutic
intervention to restore protein and energy losses in situations where
protein catabolism is manifest. The benefits and risks of early feeding
after sepsis have extensively been discussed. Although previous studies
of nutritional support in the early phase after sepsis failed to
improve nitrogen balance or protein synthesis, most studies demonstrate
reduced complications and improved clinical outcome (Jeevanandam et al. 1992
). Nutritional intervention may be a preventive
therapeutic measure to maintain gut integrity and in limiting the loss
of protein from lean body mass by sustaining substrate-dependent
protein synthesis. Furthermore, an adequate supply of amino acids in
the early phase after sepsis may be of great benefit for visceral
organs, specifically the liver, with respect to their increased
substrate demands. This may be of importance 1) by providing
amino acids for the process of hepatic gluconeogenesis, 2)
by supporting organ energy requirements and 3) by supporting
visceral immune response and protein synthesis, i.e., of
acute-phase proteins (Breuillé et al. 1998
). The loss of capacity to produce adequate amounts of endogenous immunonutrients, such as arginine and glutamine, is one of the main features in the inadequate immune response of septic patients. These amino acids may become "conditional" when the capacity to produce them is exceeded by their need. Nutritional supplementation of glutamine and arginine, often in combination with nucleotides and (n-3) fatty acids, may overcome their depletion in stress situations and has been suggested to improve clinical outcome in critically ill patients.
Studies on whole body protein metabolism under several metabolic
conditions such as starvation, feeding, after trauma and during sepsis
have been undertaken (Arnold et al. 1993
, Jackson et al. 1999
). However, data from these studies are very
difficult to interpret because they represent the sum of the turnover
of several proteins in different organs that respond in different ways.
Whole body protein studies provide insufficient insight into the
relative importance of the different organs and their participation in
protein metabolism. It seems preferable to assess the role that
individual organs play in protein kinetics. In this study, we used in
vivo techniques to obtain information regarding protein and amino acid
metabolism across the most important organs. Simultaneous measurements
across the hindquarter (50% skeletal muscle), the
portal-drained viscera
(PDV)3
(intestinal tract) and the liver allow us, through the use of
isotope techniques, to understand the long-term effects of a sepsis
period on the process of protein synthesis and degradation in these
organs and their amino acid interactions during feeding. In the
clinical situation, sepsis often is succeeded by early enteral
nutritional support, because the early provision of enteral nutrients
appears beneficial by reducing septic complications. Knowledge
concerning the aspects of protein metabolism after endotoxemia during
nutritional intervention is scarce and may be useful to devise
nutritional support strategies in disease states associated with sepsis
or inflammation.
The aim of this model was to investigate the immediate (d 1) and delayed (d 4) effects of 24-h endotoxin intervention during nutritional support on glucose and protein kinetics in a setting that mimics the postsepsis situation.
Bacterial endotoxins are often used in experimental models to study the
mechanisms involved in inflammation and sepsis. Large doses of
endotoxin lead to a hypodynamic profile, whereas very small doses of
endotoxin elicit a hypermetabolic response representative for
hypermetabolic human sepsis (Fink et al. 1990
).
Therefore, pigs received small doses of endotoxin over an extended
period. Pigs were supported by a fluid infusion to compensate for
hypovolemia to evoke a hemodynamic profile in which cardiac output and
organ perfusion are well preserved. For these reasons, we chose to
induce an experimental subacute hypermetabolic model of sepsis. To
reproduce the profile of a chronic human septic condition, endotoxin
challenge was followed by enteral infusion of a liquid
protein–enriched meal. The pig was chosen as a chronically
instrumented large animal model, because organ physiology and metabolic
changes in this animal are in many respects comparable to those
observed in humans (Dodds et al. 1982
, Miller et al. 1987
).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fourteen female Yorkshire x Dutch Landrace crossbred pigs aged 3
mo [20–22 kg body weight (bw)] were individually housed. Pigs were
fed water ad libitum and 1 kg of regular pig feed (Landbouwbelang;
Roermond, the Netherlands; 16% crude protein, for composition, see
Deutz et al. 1998
) daily in the morning, supporting a
growth rate of 300 g/d. The Animal Ethics Committee of the
Maastricht University approved the study.
Surgical procedure.
After overnight food deprivation, all pigs were anesthetized with a
mixture of N2O/O2 (1:2) and halothane (0.8%)
and intubated. As antimicrobial prophylaxis, the pigs received 6.25 mg
lincomycin and 12.5 mg spectinomycin per kg bw intravenously.
Flunixine, a prostaglandin inhibitor, was administered intravenously
for postoperative analgesia and to avoid activation of the coagulation
system. A midline laparotomy was performed, and catheters were
implanted using a method reported previously (Deutz et al. 1992
). In brief, seven catheters (I.D. 1.0 mm, O.D. 1.8 mm,
Tygon; Westvaco, Cleveland, OH) were inserted. Two in the abdominal
aorta: one at a level just above the bifurcation (A1) and one at a
level above the right renal vein (A2), two in a similar manner in the
inferior caval vein (V1 and V2, respectively) and in the portal (P),
hepatic (H) and splenic (S) veins. The A1 and the S catheters were used
for the infusion of para-aminohippuric acid (PAH), the
V1 catheter was used for isotope and endotoxin infusion and the A2, P,
H and V1 catheters were used for blood sampling. In addition, a
gastrostomy catheter (I.D. 1.6 mm, O.D. 4.8 mm; Tygon) was inserted.
All catheters were tunneled through the abdominal wall and skin. To
avoid catheter tip infections and to maintain patency, catheters were
filled with a solution containing a mixture of gentamicin (20 mg
Gentamicin 5%/mL; A.U.V., Cuyk, the Netherlands) and chymotrypsin (25
U/mL; Merck, Darmstadt, Germany) in 150 mmol/L NaCl (saline).
Postoperative care.
Postoperative care was standardized (ten Have et al. 1996
). A canvas harness was fitted to each pig to protect the
catheters and to allow easy handling of the animal. From d 3 on, the
pigs were fed 1 kg of food daily. During the entire recovery and
experimental period (20 d), the pigs remained healthy without signs of
infection. At 4 wk after surgery, the positions of the catheter tips
were checked with the animals under anesthesia with the use of
fluoroscopy. Subsequently, the pigs were killed with an intravenous
overdose of a barbiturate (Euthanasate; Apharmo, Arnhem, the
Netherlands).
Experimental protocol.
At 10 d after surgery, to standardize daily food intake, a liquid
diet was enterally infused into the gastrostomy catheter via a swivel
system connected to a pump. The liquid diet was prepared by mixing 1 L
of tap water with 4 L of Nutrison Steriflo
High-Protein4
(osmolarity 255 mOsmol/L; Nutricia, Zoetermeer, the
Netherlands), which is a high-protein liquid meal composed for
clinical patients. This meal has an optimal protein/energy ratio and
contains all vitamins, minerals and other nutritional elements that
meet the requirements of growing pigs (Pond 1986
). For
that reason, no dietary supplements were added. Enteral nutrition was
given over 4 d (Fig. 1
). The first day, diet was infused at a rate of 2.5 mL ·
kg-1 · h-1, and on the
next 3 d, pigs received 5 mL liquid diet · kg
bw-1 · h-1, equivalent
to 0.3 g protein · kg bw-1 ·
h-1. The day before endotoxin infusion (d -1),
control samples of the fed state were taken from 14 pigs (CON -1
group). That night (2400 h), enteral nutrition was stopped. The next
morning (0800 h), seven pigs received 3 µg · kg
bw-1 · h-1endotoxin
(ET; lipopolysaccharide from Escherichia coli 055:B5; Sigma
Chemical Co., St. Louis, MO) in sterile pyrogen-free saline via the
V2 catheter for a 24-h period, whereas seven control (CON) pigs
received saline. All pigs received extra saline as fluid support: 30 mL
· kg bw-1 · h-1 during the
first 8 h and 20 mL · kg bw-1 ·
h-1 the next 16 h to ensure the development
of a hypermetabolic septic condition. At 6 h after the ET infusion
was stopped, enteral nutrition was restarted at 5 mL · kg
bw-1 · h-1. At 1 d after the
ET infusion was stopped, blood samples were taken from the A2, P, H and
V1 catheters from the control group (CON 1) and from the
endotoxin-treated pigs (ET 1). At 4 d after the ET period,
again blood samples were taken from both groups (CON 4 and ET 4).
|
On the morning of the trial, 1 h before start of the endotoxin
infusion, a primed infusion of PAH (25 mmol/L A 1422; Sigma Chemical
Co.) was conducted. After an initial bolus of 5 mL PAH solution, an
infusion was started at a rate of 40 mL/h per catheter through the
splenic vein and the aorta infusion catheters (ten Have et al. 1996
). Directly after the PAH bolus infusion, a priming dose
(37 kBq/kg bw) followed by a constant infusion (37 kBq · kg
bw-1 · h-1) of
L-[2,6-3H]phenylalanine and
L-[3,4-3H]valine (Amersham, Buckinghamshire,
U.K.) and a primed (18.5 kBq/kg bw) constant infusion (0.5 µCi · kg
bw-1 · h-1) of
D-[6-3H]glucose (NET-100A; DuPont-New
England Nuclear, Mechelen, Belgium) were administered via the inferior
caval vein catheter. Also via this catheter, a primed (0.1 µmol/kg
bw) infusion (0.1 µmol · kg bw-1 · h-1) of
L-[guanidino-15N2]arginine
(Mass Trace, Woburn, MA) was started to calculate arginine kinetics and
NO production. However, because of high enteral arginine infusion
rates, NO production data were not reliable.
Before the start of the infusions, background blood samples were taken.
At 1 h after the start of the infusions, steady state conditions
for PAH (data not shown),
L-[2,6-3H]phenylalanine,
L-[3,4-3H]valine,
L-[guanidino-15N2]arginine
and D-[6-3H]glucose were obtained
(Deutz et al. 1998
), and blood was sampled from
catheters in the abdominal aorta, the portal, the hepatic and the caval
vein.
Sample processing.
Promptly after sampling, blood was distributed in prechilled, heparinized tubes (Sarstedt, Nümbrecht, Germany) on ice. Hematocrit was determined to enable calculation of plasma flow from blood flow. For blood gas analysis (arterial pH, bicarbonate, pO2 and pCO2), 200 µL blood was sealed airtight in heparinized 1-mL syringes and immediately analyzed on an automatic blood gas system [Acid Base Laboratory (ABL3); Radiometer, Copenhagen, Denmark]. Temperatures of the pigs were measured rectally. Centrifugation was performed at 4°C for 5 min at 8500 x g, plasma collected and kept on ice. For urea, glucose and lactate determinations, 900 µL plasma was added to 90 µL of 3 mmol/L trichloroacetic acid solution, ensuring stability of the substances. For amino acid analysis, 500 µL of plasma was deproteinized by mixing with 20 mg dry sulfosalicylic acid. For PAH determination, 300 µL of blood was added to 600 µL of 0.7 mmol/L trichloroacetic acid solution, thoroughly mixed and centrifuged, and the supernatant collected. All samples were frozen in liquid nitrogen and stored at -80°C until further analysis.
Biochemical analysis.
PAH was detected spectrophotometrically after deacetylation of the
supernatant at 100°C for 45 min (ten Have et al. 1996
), and plasma ammonia and urea were detected by standard
enzymatic methods on an automated analysis system (Cobas Mira-S;
Hoffmann-La Roche, Basel, Switzerland). Plasma amino acids were
determined by a fully automated HPLC (Pharmacia, Woerden, the
Netherlands) after precolumn derivatization with
o-phthaldialdehyde (van Eijk et al. 1993
). In addition, glucose and lactate concentrations were
determined by HPLC. Glucose and amino acid fractions were collected and
counted in 6 and 20 mL, respectively, of Ultima Gold XR (Packard,
Groningen, the Netherlands) for radioactivity on a liquid scintillation
spectrophotometer to obtain the specific activity. Although
phenylalanine can be converted into tyrosine and glucose into lactate,
we could not detect sufficient radioactivity in their respective
fractions to calculate a reliable specific activity. Enrichments of
arginine were calculated as tracer-to-tracee ratios and were determined
with fully automated liquid chromatography–mass spectrometry
(Thermoquest LCQ; Veenendaal, the Netherlands) (van Eijk et al. 1999
) that was, through column switching, in connection with
the HPLC.
Calculations.
Plasma flow (mL · kg bw-1 · min-1) was
calculated using PAH as an indicator in an indicator-dilution
technique (ten Have et al. 1996
). Organ amino acid
turnover was calculated in a two-compartment model (Wolfe 1992
). The substrate net balance was defined as NB (nmol · kg
bw-1 · min-1) and calculated in eq. 1
by
multiplying the mean plasma flow by the difference between [V] and
[A], which represent the venous and arterial plasma concentrations of
the substrate (µmol/L), respectively. Therefore, a positive NB
represents net release or efflux and a negative NB represents net
uptake or influx of substrate across the organ. The tracer net balance
across an organ (nb, nmol · kg bw-1 ·
min-1) was calculated in eq. 2
, where
SAart and SAven represent
the specific activities of the substrate in the arterial and venous
plasma, respectively.
 | (1) |
 | (2) |
 | (3) |
 | (4) |
The disposal and production rates (nmol · kg
bw-1 · min-1) across
the PDV, the splanchnic region and the hindquarter were calculated in
eqs. 3 and 4
, respectively. For calculations of arginine kinetics,
enrichments were used instead of specific activities. The venous
specific activity was used for the calculation of the disposal because
it best approaches the intracellular specific activity (precursor pool)
(Biolo et al. 1995
). Liver values were calculated by
subtracting PDV from splanchnic values. In the PDV and hindquarter, the
disposal of phenylalanine is a reflection of protein synthesis and
production of phenylalanine of breakdown, because these organs have
very low hydroxylase activity (Tourian et al. 1969
).
Valine disposed in these organs can either become transaminated or be
used in protein synthesis (Ooiwa et al. 1995
). In the
liver, valine is mainly used for protein synthesis, because its
transamination rate is very low (Ooiwa et al. 1995
). The
disposal of phenylalanine by the liver is a combination of protein
synthesis and hydroxylation of phenylalanine to tyrosine
(Tourian et al. 1969
). The rate of valine transamination
in the PDV and hindquarter and the rate of phenylalanine hydroxylation
in the liver were estimated from the assumption that the ratio of
valine to phenylalanine in these organs constitutes 1.7 (Wu et al. 1999
).
The whole body production rate (Q, µmol · kg
bw-1 · min-1) of the
substrate was calculated in eq. 5
(Wolfe 1992
), in which
I represents the rate of infused tracer (µmol · kg
bw-1 · min-1). The Q of
the substrate represents the sum of endogenous production and
production from enterally infused substrate absorbed by the gut.
 | (5) |
Also across the PDV, the total production rate of a substrate
measured is a combination of substrate derived from endogenous
production plus the output of substrate absorbed by the gut from the
enterally infused meal that is not retained for disposal and escapes
metabolism. This measured total production rate of a substrate by the
PDV can be corrected for the contribution of substrate
(PND) that is derived from absorption of enteral
infusion (EABS) and is not metabolized or
nondisposed. This gives a good approximation of the production of the
substrate that represents endogenous production
(PEND) in the PDV (Fig. 2
). Calculations of EABS are based on the
assumption that 90% of the enteral meal (E) is absorbed; therefore,
EABS = 0.9 · E (Deutz et al. 1998
). PND is the difference between
EABS and disposal, and
PEND can subsequently be calculated by
subtracting PND from the measured production.
|
-Amino nitrogen was calculated as the sum of the -amino acids.
Branched-chain amino acids (BCAA) were calculated as the sum of
valine, leucine and isoleucine. GNAA represented the sum of the
gluconeogenic -amino acids (all amino acids occurring in protein
except for leucine and lysine). Statistics.
Results are presented as means ± SEM. Because very similar results were obtained at d -1 in the CON and the ET group, pigs were pooled in one CON -1 group. The data were subjected to a two-way analysis of variance to compare group differences at d 1 and 4 after ET to test changes over time (d 1 and 4 post-ET versus d -1 pre-ET) within the CON group and interaction between time and group. Significant difference was tested by the Wilcoxon’s nonparametric test. Levels of significance were set at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The temperature and the arterial blood gas values did not change 1 or
4 d after the intervention of ET (Table 1
). ET challenge significantly decreased the arterial hemoglobin at d 1.
The plasma flow of the portal drained viscera (PDV) did not
significantly differ between the ET 1 and 4 and their CON groups. The
hepatic plasma flow was higher in the ET 1 than in the CON 1 group. ET
increased the hindquarter flow in a time-dependent manner in the ET
1 and 4 groups.
|
At 1 d after ET challenge, most amino acid concentrations were
lower compared with the control group except for the glutamate
concentration, which was significantly higher in a time-dependent
way (Table 2
). At 4 d after ET, the decreased plasma levels were maintained,
except for glutamate, glutamine, glycine, alanine and lysine. At 1 d after ET challenge, arterial concentrations of glucose and urea were
lower in the ET 1 than in the CON 1 group.
|
At 1 d after ET intervention, no significant changes were observed
in the Q of phenylalanine (representing protein degradation), valine,
arginine and glucose (Table 3
). The Q of arginine was higher in the ET 4 than in the CON 4 group. The
Q of valine decreased in time at d 4 compared with d -1.
|
At 1 and 4 d after ET infusion, the net uptake of glutamate and
arginine and the efflux of glutamine were higher by the hindquarter by
an interaction of ET and time. At d 4, the uptake of alanine and
glycine was lower in the ET-treated pigs than in the controls
(Table 4
). The period of ET infusion had no significant effect on the glucose or
lactate flux across the hindquarter.
|
Compared with controls, the ET infusion had a time-related effect
by increasing both the phenylalanine disposal (protein synthesis) and
production (protein degradation) across the hindquarter at d 4
(Fig. 3
A). The net uptake of phenylalanine was higher in the ET 4
group than in the CON 4 group. Both 1 and 4 d after ET challenge,
valine disposal and production were also significantly higher compared
with controls (Fig. 3B
). The estimated disposed valine used
in the hindquarter transamination was significantly higher in the ET 1
group than in the CON 1 group (995 versus 63 nmol · kg
bw-1 · min-1).
Similarly, the net disposal (d 1 and 4) and production (d 4) of
arginine by the hindquarter were significantly higher in the
ET-treated pigs than in their controls, resulting in higher net
influx of arginine (d 1 and 4) by the hindquarter (Fig. 3C
).
|
The ratio of the efflux of total amino acids by the PDV (Table 5
) relative to the total amino acids infused by enteral infusion
(Table 6
) was higher in the ET 1 group than in the CON 1 group (0.6 versus 0.4,
respectively) and was higher in the ET4 than in the CON 4 group (0.8
versus 0.5, respectively). At d 1 after ET treatment, the glutamate
efflux by the PDV was lower, the uptake of glutamine by the PDV
converted into release and efflux of most other amino acids was higher
compared with control pigs (Table 6)
. In the ET 1 group, the total
-amino nitrogen output by the PDV was 73% higher than in the CON 1
group. The amino acid output by the PDV was still 64% higher at d 4
after ET treatment.
|
|
At both d 1 and 4 d after ET challenge, no significant
changes in phenylalanine disposal (representing protein synthesis) or
production of phenylalanine were observed across the PDV. The net
release of phenylalanine was higher 1 d after ET than in controls.
To calculate endogenous production, we corrected the measured
production rate of phenylalanine for contribution of phenylalanine from
the enterally infused meal (Table 7
) that is escapes metabolism, as illustrated in Figure 2
. For example,
the rate of disposed or metabolized phenylalanine in the CON 1 group
was 791 nmol · kg bw-1 ·
min-1. The rate of phenylalanine from enteral
absorption was 90% · 1730 = 1557 nmol · kg
bw-1 · min-1. The rate
of phenylalanine from enteral nutrition that was not disposed
(PND) and contributed to the production rate was
1557 - 791 = 766 nmol · kg bw-1 ·
min-1. Corrected for nondisposed supply, the
phenylalanine derived from endogenous production
(PEND) was 1563 - 766 = 797 nmol ·
kg bw-1 · min-1 (Table 7)
. At 1 d after ET infusion, higher disposal, production and
efflux of valine by the PDV were observed in the ET-challenged
compared with the control pigs (Table 7)
. At 4 d after ET
infusion, efflux of valine was higher compared with the control pigs.
The estimated endogenous valine production is shown in Table 7
. The
rate of valine disposal that is transaminated in the PDV, estimated
from the phenylalanine-to-valine molecular ratio in protein, was higher
in the ET 1 group than in the CON 1 group (1000 versus 0 nmol · kg
bw-1 · min-1). The
arginine production by the PDV was higher than in the control group 1
and 4 d after endotoxemia (Table 8)
, resulting in higher efflux of
arginine by the PDV. Also at these days, the production of estimated
endogenous production of arginine was higher in the ET-treated than
in the CON pigs.
|
|
A shift of efflux into influx of glutamine, valine and isoleucine in
the ET 1 and 4 groups was observed (Table 8
). At 1 d after ET treatment, the uptake of lactate by the liver
was significantly higher than that of CON pigs.
Phenylalanine, valine and arginine kinetics.
At 1 and 4 d after ET challenge, the phenylalanine disposal
(protein synthesis plus hydroxylation) and production (protein
degradation) by the liver did not significantly differ between the ET
and the CON groups (not shown). No differences were observed in the
estimated rate of phenylalanine hydroxylation between the two groups at
d 1 and 4 (not shown). Within the CON group, the disposal of valine
decreased at d 1 and 4 compared with CON d -1 (Fig. 4A
). At d 1 after the ET infusion, the disposal of valine
(protein synthesis) was higher and the production of valine (protein
degradation) was lower compared with the CON 1 group net,
resulting in net uptake of valine across the liver. Both the disposal
and production of arginine by the liver decreased at d 1 and 4 compared
with d -1 (Fig. 4B
). In comparison with CON pigs, ET had a
time-related effect by increasing the hepatic disposal of arginine
1 and 4 d after endotoxemia, resulting in higher net uptake of
arginine.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Mainous et al. 1994
). Under starved circumstances, nutritional supply
proved beneficial by promoting protein synthesis rates (Ogata et al. 1978
). The exact manner in which organ protein metabolism
is altered after a period of endotoxemia under conditions of adequate
nutritional substrates, however, remains unclear. In this study, the
changes in protein synthesis and breakdown and the amino acid
degradation across the main organs were explored to better understand
adaptations of protein and amino acid metabolism in a postendotoxemic
period during nutritional intervention. It must be taken into account
that protein turnover in pigs is higher compared with that in humans
(Garlick et al. 1976
), which, together with the use of a
moderate subacute model of sepsis, may lead to relative rapid
manifestation of the endotoxemia-induced modifications in protein
metabolism under feeding conditions. In this postsepsis model, many of the observed changes were attributable to both endotoxin intervention and changes over time. Between d -1 and d 1, the control pigs were subjected to an extensive experimental procedure that was identical to the procedure the endotoxin-treated group underwent except for the endotoxin infusion. Existing differences within the control group at these time points result from the intervening experimental procedure, emphasizing the importance of a control group undergoing a similar experimental procedure.
Across the hindquarter muscle at 1 d after endotoxemia during
feeding, no significant changes occurred in phenylalanine disposal or
production, indicating unchanged muscle protein metabolism. In a more
prolonged phase of endotoxemia at d 4, protein turnover appeared to
increase in such a way that the increase in protein synthesis exceeded
the increase in protein degradation, resulting in net protein
anabolism. The increased protein synthesis possibly represents
replenishment and renovation of wasted proteins. In the muscle under
anabolic circumstances, amino acids (except for glutamine) are not only
used for protein synthesis but also partly transaminated. In muscle,
amino acids, especially the BCAA, can either be transaminated,
producing glutamine and alanine, and involving glutamate as an
intermediate, or can be completely oxidized for energy supply after
transamination (Skeie et al. 1990
). At 1 d after
endotoxemia, the amount of valine disposal used in transamination
estimated from amino acid composition in protein was elevated. An
increased glutamine and alanine efflux in parallel with an increased
disposal of valine and net consumption of glutamate suggests increased
transamination activity in muscle during the acute and the prolonged
phase after endotoxemia. Glucose provides most of the pyruvate used for
alanine synthesis (Newsholme et al. 1983
). However, the
endotoxin infusion period had no effect on the glucose consumption or
lactate production by the hindquarter. In addition, the net uptake of
alanine decreased to a much lesser extent than the net release of
glutamine in the postendotoxemia period, suggesting a minor role for
the glycolytic pathway during feeding. After endotoxemia, muscle may
use both glutamate and BCAA from increased inward transport and from
increased protein breakdown preferentially for glutamine than for
alanine production.
Arginine is considered an indispensable amino acid under disease
conditions such as sepsis, and its availability to support immune
function (Wu et al. 1998
) and protein homeostasis
(Yu et al. 1996
) may become limiting under such
circumstances. Considering that the major route of entry of arginine
into the plasma is via its release from proteins (Beaumier et al. 1996
), the increased release of arginine from muscle
catabolism during endotoxemia has led to the depletion of intramuscular
arginine. Therefore, the observed enhanced net uptake of arginine by
the hindquarter may serve protein synthesis and reflect replenishment
of the muscle arginine pool.
Studies that concern intestinal protein metabolism during sepsis are
relatively scarce, and few data on the long-term effects of sepsis
on intestinal protein metabolism are available. Studies with
long-term models of sepsis in rats yield contradictory results
because both enhanced (Breuillé et al. 1998
) and
decreased intestinal protein synthesis (Cooney et al. 1996
) were reported. In this postsepsis model, protein
synthesis and degradation in the PDV were not significantly affected
during feeding at 1 and 4 d after endotoxemia. Compromised
integrity of the gut wall and decreased intestinal transit, which are
known to be affected by endotoxin for several days (Wang et al. 1994
), could have resulted in decreased amino acid retention
and subsequent increased amino acid release by the PDV. The assumption
that intestinal absorption of amino acids from enteral supply is 90%
may not apply for the endotoxemia group. Nevertheless, a 10% higher
efficiency of absorption cannot explain the 60–70% higher output of
amino acids that occurs across the PDV of the ET-treated pigs.
Moreover, because many studies showed substrate absorption to impair
rather than to enhance under septic conditions (Gardiner et al. 1995
), this concept seems doubtful.
In the small intestine, BCAA are catabolized via transamination that
involves alanine production and the utilization of glutamine or
glutamate or through further oxidation to CO2
(Wu 1998
). We observed that at 1 d after endotoxin
treatment in pigs, alanine was released to a greater extent,
accompanying decreased release of glutamate. In addition to decreased
glutamate efflux, the PDV appears to acquire substrate for alanine
production by increasing valine disposal. Possibly, BCAA together with
glutamate are increasingly metabolized in the gut 1 d after
endotoxemia, resulting in alanine production. If glutamine synthesis
acts as a substrate involved in the increased transamination activity,
this explains the observed reduced influx of glutamine across the PDV
in our postendotoxemia model. The estimated rate of valine used for
transamination relative to protein synthesis in the PDV increased
1 d after endotoxemia. This observation suggests that BCAA that
are made available through disposal are increasingly used for the
transamination process. Glucose-derived pyruvate can be used for
alanine synthesis or energy requirements, but increased uptake of
glucose was not observed. Although under conditions of fasted
endotoxemia, the gut glucose disposal is increased (Meszaros et al. 1988
), during enteral nutrition, the glucose supply will be
sufficient to support increased demands by the PDV.
In the liver, the net protein anabolism that occurred during the acute
and prolonged phases after endotoxemia resulted from both increased
protein synthesis and decreased protein degradation. Although the
hepatic uptake of lactate was increased 1 d after endotoxin
treatment, the hepatic output of glucose was not altered. In addition,
the glucose production of the whole body was not changed after
endotoxemia. This implied that no significant elevated hepatic
gluconeogenesis rates were present, likely due to high rates of glucose
provided by enteral feeding. The augmented amino acid consumption by
liver occurring 1 and 4 d after endotoxemia therefore appears to
serve increased protein synthesis rather than gluconeogenesis. The
increase in hepatic protein synthesis possibly represents the synthesis
of acute-phase proteins in the acute-phase response that can
last for several days (Heegaard et al. 1998
). Under fed
circumstances, the major site of amino acid degradation to urea is the
liver, with glutamate in this process acting as intermediate. Opposite
of the control pigs, we observed a quantitative release of hepatic
glutamate into the circulation 1 and 4 d after endotoxemia. This
glutamate possibly derives from conversion of amino acids that are
increasingly consumed by the liver. A shift of nitrogen released as
urea toward glutamate seems apparent in liver after endotoxemia.
Glutamate can serve an important role as carbon and nitrogen carrier
between organs.
Although the sudden effect of endotoxin on skeletal muscle normally
constitutes accelerated protein degradation (Biolo et al. 1997
), no muscle catabolism was manifest during nutritional
intervention as soon as 1 d after endotoxemia. The long-term
effect of sepsis included increased protein turnover in muscle,
resulting in increased net protein synthesis and possibly reflecting
recovery mechanism of the muscle to regain protein mass. The effect of
endotoxemia also includes the enhanced transamination of BCAA with
glutamate into alanine but predominantly glutamine. The need for
glutamine for different processes may be sustained after endotoxemia.
The need for alanine in the liver for gluconeogenesis is relatively
low, because glucose is no longer a limiting fuel in the anabolic
state. The increased rates of amino acid retained by the liver at both
the acute phase and the prolonged phase after endotoxemia seem to be
for the purpose of hepatic protein synthesis. In the acute and more
prolonged phase after endotoxemia, the increased amino acid release by
the PDV area that possibly results from impaired amino acid retention
by the gut caused an increased portal output of amino acids. In
combination with the process of increased transamination, the PDV makes
more amino acids, specifically glutamine, glycine, alanine and
arginine, available for protein synthesis in liver and other consuming
organs (e.g., immune cell–rich tissues and kidneys) during the
postendotoxemia recovery period. The arginine production by PDV and
hindquarter predominantly account for the increase in whole body
production of arginine. Nevertheless, lower arginine plasma levels were
present, indicating an elevated need for arginine by organs such as the
liver and hindquarter.
The nitrogen cycle of BCAA/glutamate and glutamine/alanine between periphery and PDV on the one site and liver on the other site seems quantitatively more important in the fed condition than the carbon cycle of alanine and lactate/glucose. Organ protein metabolism after endotoxemia in the fed state is modified in such a way that net amino acid release by the PDV is enhanced, whereas in muscle and liver, amino acid uptake for net protein synthesis predominates. Elevated rates of amino acid release, from the transamination process in both PDV and muscle, may serve in providing substrate for the synthesis of various plasma proteins in liver and immune cells that are of importance for sustained tissue repair and host defense after endotoxemia. Therefore, it seems that in acute and prolonged postendotoxemic conditions, the PDV and hindquarter are entirely in service of the liver and possibly the immune system by releasing amino acid precursors used in recovery mechanisms. In such situations, substrate supply by nutritional intervention can assist the altered organ protein metabolism in a rapid recovery.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: BCAA, branched-chain
amino acids; bw, body weight; CON, control;
EABS, absorption from enterally infused meal;
ET, endotoxin; NB, net balance; PAH, para-aminohippuric
acid; PDV, portal-drained viscera; Q, whole body production rate.
4 Proprietary diet consists of the following (per
1 L food): 63 g casein protein, 141 g carbohydrates, 11 g
sugars, 128 g polysaccharides, 2 g other, 49 g fatty acids; 4 g
saturated fatty acids, 30 g monounsaturated fatty acids, 15 g
polyunsaturated fatty acids; minerals (Na, K, Cl, Ca, P and Mg); trace
elements (Fe, Zn, Cu, Mn, F, Mo, Se, Cr and I); vitamins (A, B-6, B-12,
C, D, E, K, thiamine, riboflavin, niacin, pantothenic acid and folic
acid) and 200 mg choline.
Manuscript received March 15, 2000. Initial review completed April 8, 2000. Revision accepted August 30, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Arnold J., Campbell I. T., Samuels T. A., Devlin J. C., Green C. J., Hipkin L. J., MacDonald I. A., Scrimgeour C. M., Smith K., Rennie M. J. Increased whole body protein breakdown predominates over increased whole body protein synthesis in multiple organ failure. Clin. Sci. (Colch.) 1993;84:655-661[Medline]
2. Beaumier L., Castillo L., Yu Y. M., Ajami A. M., Young V. R. Arginine: New and exciting developments for an "old" amino acid. Biomed. Environ. Sci. 1996;9:296-315[Medline]
3.
Biolo G., Fleming R.Y.D., Maggi S., Wolfe R. R. Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am. J. Physiol. 1995;268:E75-E84
4. Biolo G., Toigo G., Ciocchi B., Situlin R., Iscra F., Gullo A., Guarnieri G. Metabolic response to injury and sepsis: Changes in protein metabolism. Nutrition 1997;13:52S-57S[Medline]
5. Breuillé D., Arnal M., Rambourdin F., Bayle G., Levieux D., Obled C. Sustained modifications of protein metabolism in various tissues in a rat model of long-lasting sepsis. Clin. Sci. 1998;94:413-423[Medline]
6.
Cooney R. N., Owens E., Slaymaker D., Vary T. C. Prevention of skeletal muscle catabolism in sepsis does not impair visceral protein metabolism. Am. J. Physiol. 1996;270:E621-E626
7. Decker R., von Stuckrad-Barre S., Milakofsky L., Hofford J. M., Harris N., Vogel W. H. Effect of stress on amino acids and related compounds in various tissues of the rat. Life. Sci. 1995;57:1781-1790[Medline]
8.
Deutz N.E.P., Bruins M. J., Soeters P. B. Infusion of soy and casein protein meals affect interorgan amino acid metabolism and urea kinetics differently in pigs. J.Nutr. 1998;128:2435-2445
9. Deutz N.E.P., Reijven P.L.M., Athanasas G., Soeters P. B. Post-operative changes in hepatic, intestinal, splenic and muscle fluxes of amino acids and ammonia in pigs. Clin. Sci. 1992;83:607-614[Medline]
10. Dodds W. J. The pig model for biomedical research. Fed. Proc. 1982;41:247-256
11. Fink M. P., Heard S. O. Laboratory models of sepsis and septic shock. J. Surg. Res. 1990;49:186-196[Medline]
12. Gardiner K. R., Gardiner R. E., and Barbul A. Reduced intestinal absorption of arginine during sepsis. Crit. Care. Med. 1995;23:1227-1232[Medline]
13.
Garlick P. J., Burk T. L., Swick R. W. Protein synthesis and RNA in tissues of the pig. Am. J. Physiol. 1976;230:1108-1112
14. Heegaard P. M., Klausen J., Nielsen J. P., Gonzalez-Ramon N., Pineiro M., Lampreave F., Alava M. A. The porcine acute phase response to infection with Actinobacillus pleuropneumoniae: Haptoglobin, C-reactive protein, major acute phase protein and serum amyloid A protein are sensitive indicators of infection. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 1998;119:365-373[Medline]
15.
Jackson N. C., Carroll P. V., Russell-Jones D. L., Sonksen P. H., Treacher D. F., Umpleby A. M. The metabolic consequences of critical illness: Acute effects on glutamine and protein metabolism. Am. J. Physiol 1999;276:E163-E170
16.
Jeevanandam M., Shamos R. F., Petersen S. R. Substrate efficacy in early nutrition support of critically ill multiple trauma victims. J. Parenter. Enteral. Nutr. 1992;16:511-520
17. Kudsk K. A. Gut mucosal nutritional support: Enteral nutrition as primary therapy after multiple system trauma. Gut 1994;35:S52-S54
18. Mainous M. R., Block E. F., Deitch E. A. Nutritional support of the gut: How and why. New Horizons 1994;2:193-201[Medline]
19. Meszaros K., Lang C. H., Bagby G. J., Spitzer J. J. In vivo glucose utilization by individual tissues during nonlethal hypermetabolic sepsis. FASEB. J. 1988;2:3083-3086[Abstract]
20. Miller E. R., Ullrey D. E. The pig as a model for human nutrition. Annu. Rev. Nutr. 1987;7:361-382[Medline]
21. Newsholme E. A., Leech A. R. Biochemistry for the Medical Sciences 1983 John Wiley & Sons New York
22. Noguchi Y., James J. H., Fischer J. E., Hasselgren P. O. Increased glutamine consumption in small intestine epithelial cells during sepsis in rats. Am. J. Surg. 1997;173:199-205[Medline]
23. Ogata E. S., Foung S. K., and Holliday M. A. The effects of starvation and refeeding on muscle protein synthesis and catabolism in the young rat. J. Nutr. 1978;108:759-765
24. Ooiwa T., Goto H., Tsukamoto Y., Hayakawa T., Sugiyama S., Fujitsuka N., Shimomura Y. Regulation of valine catabolism in canine tissues: Tissue distributions of branched-chain aminotransferase and 2-oxo acid dehydrogenase complex, methacrylyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase. Biochim. Biophys.Acta 1995;1243:216-220[Medline]
25. Pond W. G. Life Cycle Feeding: Maternal Nutrition and Progeny Development. Swine in Biomedical Research 1986:915-930 Plenum Press New York
26. Skeie B., Kvetan V., Gil K. M., Rothkopf M. M., Newsholme E. A., Askanazi J. Branch-chain amino acids: Their metabolism and clinical utility. Crit. Care. Med. 1990;18:549-571[Medline]
27. Ten Have G.A.M., Bost M.C.F., Suyk-Wierts J.C.A.W., van den Bogaart A.E.J.M., Deutz N.E.P. Simultaneous measurement of metabolic flux in portally-drained viscera, liver, spleen, kidney and hindquarter in the conscious pig. Lab. Anim. 1996;30:347-358[Medline]
28. Tourian A., Goddard J., Puck T. T. Phenylalanine hydroxylase activity in mammalian cells. J. Cell. Physiol. 1969;73:159-170[Medline]
29. Van Eijk H.M.H., Rooyakkers D. R., Deutz N.E.P. Rapid routine determination of amino acids in plasma by high-performance liquid chromatography with a 2–3 µm Spherisorb ODS II column. J. Chromatogr. 1993;620:143-148[Medline]
30. Van Eijk H.M.H., Rooyakkers D. R., Deutz N.E.P. Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry. Anal. Biochem. 1999;271:8-17[Medline]
31. Wang Q., Pantzar N., Jeppsson B., Westrom B. R., Karlsson B. W. Increased intestinal marker absorption due to regional permeability changes and decreased intestinal transit during sepsis in the rat. Scand. J. Gastroenterol. 1994;29:1001-1008[Medline]
32. Wolfe R. R. Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis 1992 Wiley-Liss New York
33.
Wu G. Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am. J. Physiol. 1997;272:G1382-G1390
34.
Wu G. Intestinal mucosal amino acid catabolism. J. Nutr. 1998;128:1249-1252
35. Wu G., Morris S. M., Jr Arginine metabolism: Nitric oxide and beyond. Biochem. J. 1998;336:1-17
36.
Wu G., Ott T. L., Knabe D. A., Bazer F. W. Amino acid composition of the fetal pig. J. Nutr. 1999;129:1031-1038
37.
Yu Y.-M., Sheridan R. L., Burke J. F., Chapman T. E., Tompkins R. G., Young V. R. Kinetics of plasma arginine and leucine in pediatric burn patients. Am. J. Clin. Nutr. 1996;64:60-66
This article has been cited by other articles:
|
|
 |
|
  R. A. Orellana, A. Jeyapalan, J. Escobar, J. W. Frank, H. V. Nguyen, A. Suryawan, and T. A. Davis Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1416 - E1425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Orellana, P. M. J. O'Connor, J. A. Bush, A. Suryawan, M. C. Thivierge, H. V. Nguyen, M. L. Fiorotto, and T. A. Davis Modulation of muscle protein synthesis by insulin is maintained during neonatal endotoxemia Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E159 - E166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Engelen, E. P. Rutten, C. L. De Castro, E. F. Wouters, A. M. Schols, and N. E. Deutz Altered interorgan response to feeding in patients with chronic obstructive pulmonary disease Am. J. Clinical Nutrition, August 1, 2005; 82(2): 366 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. C. Luiking, M. M. Hallemeesch, W. H. Lamers, and N. E. P. Deutz NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice Am J Physiol Endocrinol Metab, June 1, 2005; 288(6): E1258 - E1264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. C. Luiking, M. Poeze, G. Ramsay, and N. E. P. Deutz The Role of Arginine in Infection and Sepsis JPEN J Parenter Enteral Nutr, January 1, 2005; 29(1_suppl): S70 - S74. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Soeters, M. C. G. van de Poll, W. G. van Gemert, and C. H. C. Dejong Amino Acid Adequacy in Pathophysiological States J. Nutr., June 1, 2004; 134(6): 1575S - 1582S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. E. Baracos Animal Models of Amino Acid Metabolism: A Focus on the Intestine J. Nutr., June 1, 2004; 134(6): 1656S - 1659S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. L. J. Vissers, M. M. Hallemeesch, P. B. Soeters, W. H. Lamers, and N. E. P. Deutz NOS2 deficiency increases intestinal metabolism both in nonstimulated and endotoxemic mice Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G747 - G751. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
