Amino acid metabolism in leg muscle after an endotoxin injection in healthy volunteers

Rokhsareh F. Vesali, Maria Klaude, Olav Rooyackers, and Jan Wernerman

Department of Anesthesiology and Intensive Care, Karolinska University Hospital at Huddinge, Karolinska Institutet, Stockholm, Sweden

Submitted 11 June 2004 ; accepted in final form 30 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Decreased plasma amino acid concentrations and increased net release of amino acids from skeletal muscle, especially for glutamine, are common features in critically ill patients. A low dose of endotoxin administered to healthy volunteers was used as a human model for the initial phase of sepsis to study the early metabolic response to sepsis. Six healthy male volunteers were studied in the postabsorptive state. Blood samples from the forearm artery and femoral vein were taken during 4 h before and 4 h after an intravenous endotoxin injection (4 ng/kg body wt). In addition, muscle biopsies from the leg muscle were taken. Plasma concentration of the total sum of amino acids decreased by 19% (P = 0.001) and of glutamine by 25% (P = 0.004) the 3rd h after endotoxin administration. At the same time, muscle concentrations of the sum of amino acids and glutamine decreased by 11% (P = 0.05) and 9% (P = 0.09), respectively. In parallel, the efflux from the leg increased by 35% (P = 0.004) for the total sum of amino acids and by 43% (P = 0.05) for glutamine. In conclusion, intravenous endotoxin administration to healthy volunteers, used as a model for the initial phase of sepsis, resulted in a decrease in plasma amino acid concentrations. At the same time, amino acid concentrations in muscle tissue decreased, whereas the efflux of amino acids from leg skeletal muscle increased.

glutamine; glutamate; sepsis; skeletal muscle; protein metabolism


CRITICAL ILLNESS RESULTING FROM SEPSIS, severe trauma, or multiple organ failure is characterized by a loss of muscle mass, which is progressive over time (6, 14). This loss of muscle protein supplies extra amino acids to the splanchnic region for increased gluconeogenesis, energy demands, and production of proteins needed for the metabolic response to critical illness. Consequently, the net release of amino acids from skeletal muscle is increased in critically ill patients. However, this release does not change over the initial 2 wk of intensive care unit (ICU) treatment (19).

About two-thirds of the total amino acids released by muscle consists of glutamine and alanine. In parallel, there is a drop in muscle tissue glutamine concentrations to about one-quarter of normal. However, in contrast to the progressive loss of muscle protein, these low muscle glutamine concentrations are already established within the first 24 h of admission to the ICU and do not change any further over time (6, 18). Plasma glutamine concentrations, on the other hand, range from low to normal in ICU patients. Low levels of plasma glutamine at ICU admission have been shown to relate to ICU mortality (12).

The temporal pattern of this altered amino acid metabolism in ICU patients, and especially the early events leading to the dramatic drop in muscle glutamine, is still obscure. Administration of endotoxin can be used as a human model for the initial phase of sepsis (10). Decreased levels of plasma amino acids and an increase in the splanchnic amino acid uptake after an endotoxin injection in healthy volunteers have been reported (5). The effects on muscle tissue levels of amino acids and the exchange across the leg after endotoxin administration have not been fully elucidated before. Hence, the purpose of this study was to assess the alterations in amino acid and in particular glutamine metabolism in human leg muscle after an injection of endotoxin to provide further information about the early metabolic response to sepsis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Six healthy male volunteers participated in the study. The mean age was 27 ± 4 (SD) yr (range: 22–32), body weight was 78 ± 9 kg (range: 66–88), and body mass index was 24 ± 1 kg/m2 (range: 23–26). The local ethics committee at Karolinska Institutet approved the study protocol, and the subjects gave their written informed consent. All volunteers were healthy, as assessed by a physical examination and blood chemistry analysis. None of the volunteers was taking any medication on a regular basis.

Study protocol. The volunteers reported to the research unit in the morning at 7:00 AM. No food was allowed after 8:00 PM the previous day. Catheters were inserted in the forearm artery and in the femoral and decubital veins. The volunteers received an intravenous injection of United States Standard Reference Escherichia coli endotoxin at 4 ng/kg body wt (Lot EC-6 US; Pharmacopeia, Rockville, MD). Heart rate, electrocardiogram, invasive blood pressure, and oxygen saturation were monitored continuously with a Datex-Engstrom Light monitor (Datex-Engstrom; Instrumentarium). Body temperature was measured in the outer ear at 15-min intervals.

Blood samples from the femoral vein and the artery were obtained during 4 h before the endotoxin bolus and 4 h after the endotoxin bolus. Blood flow was measured by venous occlusion plethysmography immediately after the venous blood samples were drawn by a minimum of 10 readings. Plasma was separated from red blood cells by centrifugation at 600 g for 15 min at 4°C. The plasma samples were frozen immediately and kept at –80°C until analysis.

Muscle biopsies of the vastus lateralis were obtained with a Bergström needle under local anesthesia (lidocaine) at –20 and 0 min before the endotoxin injection and at 160 and 180 min after the injection. The samples were weighed immediately, frozen in liquid nitrogen, and stored at –80°C until analysis.

Blood flow measurements. Venous occlusion plethysmography was performed as described previously (2, 19). Briefly, an occlusion cuff was placed around the thigh 15 cm above the knee. The pressure used was 60 mmHg. A single-strand mercury-in-rubber strain gauge was wrapped around the calf at the level of maximal circumference. The blood flow values expressed as milliliters per minute x 100 ml leg volume represent the mean of at least 10 separate readings obtained immediately after the blood sampling. The coefficient of variation for these multiple readings in the six subjects was 16.9 ± 7.0%. With this variation, a good measurement of the mean blood flow was obtained with the 10 readings, since more readings did not change the value of the mean blood flow. The coefficient of variation for the blood flow measurements obtained at different time points during the baseline period of the study for the six subjects was 11.7 ± 5.3%. The temperature of the room was constant between 21 and 22°C, and the subjects were kept in the same horizontal position throughout the blood flow measurements. Plasma flow was calculated from the blood flow and hematocrit.

Sample analysis. Amino acid concentrations were analyzed by an HPLC method described previously (19). Briefly, plasma samples were deproteinized in 3% 5-sulfosalisylic acid-2-dihydrate (SSA) containing 200 µM norvaline as internal standard. Muscle samples were homogenized and deproteinized in 4% SSA containing 200 µM norvaline as internal standard. Amino acids from plasma and skeletal muscle were analyzed using precolumn derivatization with orthophthaldialdehyde/3-mercaptopropionic acid on an HPLC system (Alliance, Waters 2690, fluorescence detector waters 474; Waters, Stockholm, Sweden).

The water content of skeletal muscle was determined by weighing the biopsies before and after freeze-drying.

Calculations. Net balances (NB) of amino acids across the leg were calculated as

where CA and CV are the amino acid concentrations (µmol/l) in the artery and the femoral vein, respectively. NB is expressed as nanomoles per minute per 100 milliliters leg tissue.

Statistics. Data are expressed as means ± SD. Student's t-test for paired samples was used to determine differences before and after endotoxin administration. ANOVA analysis was used to detect differences during the temporal patterns with Fisher's least significant difference as the post hoc test. Regression analysis was used for testing correlation. P values <0.05 were considered statistically different.


    RESULTS
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 RESULTS
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Approximately 1 h after endotoxin administration, the healthy volunteers developed shivering and headache. Shivering lasted for 30–45 min and the headache for ~2 h. The body temperature of the volunteers increased from 36.4 ± 0.15°C before to 38.0 ± 0.4°C over the 4 h after the endotoxin administration.

During the period of shivering, blood flow measurements were not possible. Mean values of arterial plasma amino acid concentrations and net balances during the 60 min before endotoxin injection were compared with the mean values between 120 and 180 min after endotoxin administration. During these two periods, arterial and venous blood samples were collected most frequently.

Plasma amino acid concentration. The arterial concentration of all amino acids decreased continuously during the 4 h of endotoxemia (data not shown). The 3rd h after the endotoxin injection, glutamine concentration had decreased significantly by 25% (Fig. 1). Also glutamate, alanine, the sum of branched chain, the sum of essential, the sum of basic amino acids, and the total sum of amino acids significantly decreased in plasma concentration at this time point (Fig. 1).



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Fig. 1. Amino acid concentrations in arterial plasma of healthy volunteers (n = 6), during the 60 min immediately before an endotoxin injection (open bars), and between 120 and 180 min after the endotoxin injection (filled bars). Values are given as means ± SD. BAA, sum of basic amino acids; NEAA, sum of nonessential amino acids (not including glutamine, glutamate, and alanine); BCAA, sum of branched-chain amino acids; EAA, sum of essential amino acids; SUM, total sum of all amino acids. *Significantly different from baseline period.

 
Net balance of amino acids across the leg. During the 3rd h into endotoxemia, there was a significant increase in glutamine efflux, with a simultaneous decrease in glutamate uptake (Fig. 2). Despite the increased efflux of glutamine, there was a simultaneous drop in the arterial glutamine concentration during the 4 h after the endotoxin injection (Fig. 3, A and B). Also, alanine, the sum of essential amino acids, the sum of basic amino acids, and the total sum of amino acids showed increased effluxes in the 3rd h after endotoxin administration (Fig. 2). The mean plasma flow measured during the 60 min before and between 120 and 180 min after the endotoxin injection was 1.12 ± 0.31 and 1.13 ± 0.16 ml·min–1·100 ml–1, respectively.



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Fig. 2. Amino acid net balances in plasma across the leg of healthy volunteers (n = 6), during the 60 min immediately before an endotoxin injection (open bars), and between 120 and 180 min after the endotoxin injection (filled bars). Values are given as means ± SD. *Significantly different from baseline period.

 


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Fig. 3. Glutamine concentration in arterial plasma (A) and net balance across the leg skeletal muscle (B) of healthy volunteers (n = 6) during 3 h before and 4 h after an endotoxin challenge. Values are given as means ± SD. *Significantly different from 0/60 min; **significantly different from baseline period and 1st and 2nd h after endotoxin.

 
Muscle free amino acid concentration. The mean amino acid concentrations were determined from two muscle biopsies taken 20 min apart, at –20 and 0 min before and at 160 and 180 min after the endotoxin injection. Endotoxin administration resulted in a statistically significant decrease in the concentrations of phenylalanine, alanine, the sum of the branched-chain amino acids, the sum of the essential amino acids, and the sum of the basic amino acids (Fig. 4). The decrease in amino acid concentration was not a result of an increased tissue water content, which might have been caused by endotoxemia. Tissue water content of the muscle biopsies was 74 ± 2% before and 76 ± 2% after the endotoxin injection. Muscle free glutamine concentration and the total sum of all amino acids did not reach a statistically significant level of decrease after endotoxin administration, although all subjects except one showed decreased concentrations of both.



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Fig. 4. Amino acid concentrations in leg muscle tissue of healthy volunteers (n = 6) in biopsies taken at –20 and 0 min before an endotoxin injection (open bars) and at 160 and 180 min after an endotoxin injection (filled bars). Values are given as means ± SD. Glutamine and total sum of all amino acids are represented by y-axis on right. *Significantly different from baseline period.

 
Correlation. The plasma concentrations of glutamine and glutamate correlated, respectively, negatively and positively with the net exchange across the leg, r = 0.74 (P = 8 x 10–5) for glutamine and r = 0.92 (P = 1.4 x 10–9) for glutamate (Fig. 5, A and B). The correlations were based on mean values for the six subjects at all time points during the experiment, when both arterial and venous blood were obtained. All subjects showed similar correlations that reached or were close to statistical significance when their individual values were used.



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Fig. 5. Correlation between the arterial plasma concentration (µmol/l) and the efflux (nmol·min–1·100 ml–1) of glutamine (A; r = 0.74, P = 8 x 10–5) and glutamate (B; r = 0.92, P = 1.4 x 10–9) from the leg in healthy volunteers (n = 6) receiving an endotoxin challenge. Symbols represent mean values of the group at 21 different time points before and after endotoxin injection.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we used an endotoxin injection in healthy volunteers as a human model for the initial phase of sepsis. The interest was focused on the amino acid exchange across muscle tissue. The endotoxin injection initiated a continuous decrease of all amino acid concentrations in plasma and a decrease in the free concentrations of amino acids in muscle. In parallel to the low plasma and tissue concentrations, the efflux of most amino acids from the leg was increased. The fact that muscle tissue concentrations drop despite a generally acknowledged increased protein catabolism indicates that the increased efflux of amino acids from the leg is larger than the increased release of extra amino acids from the net protein breakdown.

In catabolic situations, such as trauma and sepsis, there is a rather distinct alteration in the concentrations of free amino acids in plasma and in muscle tissue. In conjunction with elective surgery, the temporal development of this pattern is well characterized (3). In that situation, the time of insult is precisely defined, and also the character of the insult in terms of type of surgery, the amount of bleeding, and the underlying disease is well known. In sepsis, the insult is more variable, and the temporal development is poorly characterized. Therefore, we used this human model to study the initial phase of sepsis, especially since we have shown that the temporal pattern of the amino acid exchange across the leg in ICU patients with multiple organ failure is quite stable during the first 2 wk of ICU treatment (19). Already, 24 h after admission to the ICU, the amino acid pattern associated with multiple organ failure is usually fully established (6, 7, 18). The pattern we observed here after an endotoxin injection showed similarities with what is seen during the initial phase of a combined stress hormone infusion, which is also associated with a decrease in the plasma and muscle concentrations of all amino acids (9, 21).

The protocol chosen in the present study included a 4-h study period after the endotoxin injection, with more frequent sampling including muscle biopsies during the 3rd h. The results indicated that the alternations caused by the endotoxin injection continue to develop beyond this time point (Fig. 3). The time pattern is only given for glutamine, but all other amino acids show the same pattern. No tendency was seen for the changes to return back to the basal levels during the 4 h after the endotoxin injection. On the contrary, for most amino acids, the maximal change was usually seen at 4 h after endotoxin. Also, before the endotoxin injection, subjects were studied for 4 h, with more frequent blood sampling and muscle biopsies during the last hour to verify a physiological steady state.

After the endotoxin injection, plasma glutamine concentration dropped by 25%, to a level that is associated with an increased mortality in ICU patients (8, 12, 20). In contrast, plasma glutamine concentration is not altered after surgical trauma of medium size (3, 4, 13). This indicates that the endotoxin model gave an affected plasma glutamine concentration more similar to that seen in multiple organ failure patients in the ICU. In muscle, glutamine depletion seems to be a slower process. After elective surgery and also in response to a stress hormone infusion, it is not until 12 and 6 h, respectively, that the decrease in muscle glutamine concentration attains statistical significance (3, 9, 21). In the present study, muscle glutamine decreased in five of six volunteers 3 h after the endotoxin injection. In ICU patients, muscle glutamine concentration is often depleted down to 25% of the reference value already 24 h after admission (6, 7, 15). However, the time of admission to the ICU and the initial phase of a septic event may be quite far separated in time. For the tissue amino acid concentration, a possible effect of changes in water content resulting from the endotoxin administration was considered. However, no statistically significant change in water content was observed. In numerical terms, muscle water content increased by <3%, which cannot explain the 10–20% decreases seen for most of the individual amino acids.

The efflux of glutamine from muscle tissue is elevated in trauma and sepsis, as well as in ICU patients with multiple organ failure (1, 11). In the present study, the glutamine efflux from the leg increased over time to a level similar to what is seen in ICU patients (19). This strongly suggests that the increased efflux of glutamine from the leg in ICU patients starts very early after a septic event. An interesting observation was that the leg efflux and plasma concentration of glutamine correlated negatively, indicating a larger glutamine efflux at lower plasma concentrations. This is also demonstrated in the temporal pattern, where the increase in efflux and the decrease in concentration developed in parallel. In addition, the uptake of glutamate across the leg decreased in parallel to the decrease in plasma glutamate concentration. Such a concentration-dependent balance across the leg for glutamate has been described previously (17, 19). However, a concentration-dependent balance of glutamine across the leg has not been reported before. This correlation might indicate that low plasma glutamine concentrations initiate the increased efflux of glutamine from leg muscle, supporting a pull rather than a push hypothesis. The decreases in plasma glutamine concentrations are most likely the result of a larger uptake and subsequent utilization of glutamine in the splanchnic tissues. A 50% increase in glutamine uptake by the splanchnic area has been shown after surgery (16). Also after an endotoxin challenge, as used in the present study, a 50–100% increase in glutamine uptake by the splanchnic tissues has been shown (5).

A potential difficulty sometimes encountered is that a variable leg blood flow has a large impact on the quantitative measurements of amino acid exchange, since these are always discrete values for a given time point. In the present study, the variation of the method to measure blood flow was controlled for by obtaining multiple readings at each time point. Despite this, the intraindividual variation was still high, which could possibly be explained by a physiological variation in blood flow. Because of this variation, it is difficult to conclude that the blood flow does not change due to the endotoxin challenge, although the mean values are similar. However, even with this large variation in blood flow, the even higher changes in amino acid effluxes after the endotoxin challenge cannot be attributed to changes in blood flow only.

During a combined stress hormone infusion and after elective surgery, an initial decrease in both plasma and muscle concentrations of the branched-chain and aromatic amino acids is seen, which is later followed by an increase (3, 9, 21). Also, in multiple organ failure patients, plasma and muscle concentrations of the branched-chain amino acids and of phenylalanine are increased (6, 7). In the present study, after the endotoxin challenge, a decrease of these amino acids and of the sum of essential amino acids was seen. One could hypothesize that a similar biphasic response would have been seen if subjects had been studied for a longer period after the endotoxin. The magnitude of differences might not be comparable because of variation in severity of the insults, although the pattern of changes might be similar.

In conclusion, an endotoxin injection administered to healthy volunteers resulted in a dramatic drop in plasma concentrations of all amino acids. Amino acid concentrations in muscle tissue also decreased, and in addition the efflux of amino acids from the leg increased for most amino acids. The results showed that an endotoxin injection used as a model for the initial phase of sepsis caused immediate alterations in the exchange of amino acids across muscle, indicating that the alterations in amino acid metabolism seen in multiple organ failure patients may be initiated quite early in the course of disease.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The study was supported by grants from the Swedish Medical Research Council (project nos. 04210 and 14244) and a European Society of Parenteral and Enteral Nutrition fellowship in 2001 (to R. F. Vesali).


    ACKNOWLEDGMENTS
 
We thank Lisselott Thunblad and Christina Hebert for excellent technical assistance and Viveka Gustavsson for expert nursing assistance. We thank the research volunteers for participation in the study. Analyses were performed in our laboratory at the Clinical Research Center.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. F. Vesali, Dep. of Anesthesiology & Intensive Care, Karolinska Institutet, KFC Novum, Karolinska Univ. Hospital, S-141 86 Huddinge, Sweden (E-mail: Farrah.vesali{at}cfss.ki.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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