THE PRIMARY AIM of this study was to investigate the

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1 234 ORIGINAL ARTICLE Plasma Amino Acid Concentrations During Late Rehabilitation in Patients With Traumatic Brain Injury Elisabet Børsheim, PhD, Quynh-Uyen T. Bui, MD, Robert R. Wolfe, PhD ABSTRACT. Børsheim E, Bui Q-UT, Wolfe RR. Plasma amino acid concentrations during late rehabilitation in patients with traumatic brain injury. Arch Phys Med Rehabil 27;88: Objectives: To investigate whether the basal plasma amino acid concentrations in patients with traumatic brain injury () have returned to levels found in healthy controls at about 17 months postinjury and to determine the effect of intake of a mixture of essential amino acids (EAA) on plasma amino acid concentrations in versus healthy controls. Design: Peripheral venous amino acid concentrations in subjects with were compared with concentrations in healthy controls both at rest and for 1 hour after intake of 7g of EAA. Setting: Postacute brain injury rehabilitation center. Participants: Six men with (age standard deviation, 27 6y; months postinjury, 17 4) and 6 healthy men (age, 43 7y). Intervention: Intake of a drink consisting of 7g of EAA. Main Outcome Measures: Individual and total plasma amino acid concentrations. Results: Total amino acid concentration was about 12% lower in versus controls (P.22). Valine was reduced by 33% in the group versus controls (P.3), whereas the other EAA did not differ between groups. After intake of the EAA drink, plasma non-eaa increased to a significantly higher level in controls versus subjects (P.17). Conclusions: Plasma total amino acid concentration is still reduced 17 months postinjury in patients with versus healthy controls, mainly because of a lower valine level. This may be of importance for both brain and muscle metabolic functions, and warrant further study. Further, ingested EAA are apparently not as readily converted to non-eaa in patients as in healthy controls, suggesting that in recovery from, certain non-eaa may become provisionally essential. Key Words: Amino acids; Amino acids, essential; Brain injuries; Rehabilitation. 27 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation From the Department of Surgery, Metabolism Unit, Shriners Hospital for Children/ UTMB (Børsheim, Wolfe) and Department of Internal Medicine, UTMB (Bui), Galveston, TX. Supported by the Moody Endowment. No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the author(s) or upon any organization with which the author(s) is/are associated. Correspondence to Elisabet Børsheim, PhD, Metabolism Unit, Shriners Hospital for Children/UTMB, 815 Market St, Galveston, TX 7755, elborshe@ utmb.edu. Reprints are not available from the author /7/ $32./ doi:1.116/j.apmr THE PRIMARY AIM of this study was to investigate the plasma amino acid pattern in patients with traumatic brain injury () at a late stage of recovery ( 17mo postinjury) versus healthy controls. We hypothesized that the patients would have lower plasma amino acids than normal control subjects. Under normal conditions, the plasma amino acid profile is tightly regulated. 1 In situations in which this is not the case, disruptions in neurotransmitter synthesis and other problems may arise. For example, low plasma concentrations of the branchedchain amino acids (BCAA) leucine, isoleucine, and valine may increase the ratio of free tryptophan to BCAA. Because tryptophan and the BCAA compete for the same transporter (L-system) into the brain, and tryptophan is a precursor for serotonin, an increase in this ratio may increase serotonin production and consequently increase fatigue. 2 To our knowledge, there are no previous data on plasma amino acid concentrations in patients at a postacute stage of recovery, but disturbances in plasma amino acid concentrations have been shown in the short term after injury. For example, disruption of the plasma amino acid profile, and the accompanying increased mobilization of amino acids from muscle tissue and precipitous loss of muscle nitrogen and lean body mass, are characteristics in the initial hypermetabolic state that follows acute. 3,4 Aquilani et al 5 showed profound abnormalities of plasma amino acid concentrations in patients with severe also at later stages. Even 3 to 75 days after the acute insult, all the essential amino acids (EAA) and approximately half of the non-eaa remained reduced in patients compared with controls. Further, patients with severe admitted to rehabilitation days postinjury had lower plasma concentrations of most of the EAA compared with control subjects, and this difference was still present at discharge days after injury. 6 Even at a delayed stage ( 1y), patients with have symptoms consistent with continued disturbances in plasma amino acid concentrations, for example, diminished exercise tolerance and profound muscle weakness. Therefore, the primary goal of this study was to compare total and individual plasma amino acid concentrations in postacute patients versus healthy controls. The second goal was to determine the response of plasma amino acid concentrations to intake of an EAA mixture in the 2 groups. We proposed that ingestion of EAA would correct blood amino acid profiles in patients with. Research has repeatedly shown that ingestion of EAA mixtures approximately formulated to mimic the EAA composition of muscle is a potent anabolic stimulus in both young and elderly healthy volunteers. 7-9 Thus, it is important to determine the plasma amino acid response to an EAA drink in patients versus control as a first step toward a possible outcome study. METHODS Participants Six healthy men and 6 men with participated in the study. Table 1 shows the characteristics of the volunteers, all of whom were white. We recruited the participants with through the Transitional Learning Center (TLC) in Galveston, TX, where they

2 AMINO ACIDS AND BRAIN INJURY, Børsheim 235 Table 1: Physical Characteristics of All Subjects Subjects Age (y) Weight (kg) Height (cm) Months Postinjury Mean 27* SD SE CV Controls Mean SD SE CV Abbreviations: CV, coefficient of variation; SD, standard deviation; SE, standard error. *P.5 vs controls. participated in a program for postacute injury rehabilitation. The volunteers were fully informed about the purpose and procedures of the study before written consent was obtained. The protocol was approved by the institutional review board of the University of Texas Medical Branch and the institutional review board at the TLC. All the patients had suffered moderate to severe diffuse brain damage (nonpenetrating head trauma that exceeds the American Congress of Rehabilitation Medicine criteria for mild ) during motor vehicle collisions. The mean initial Glasgow Coma Scale score was 5 1 (n 5; for 1 subject, data were not available). At the time of the study, all subjects had recovered sufficiently to be able to perform basic activities of daily living (1 patient with cues, 1 patient with supervision), and to ambulate independently with or without an assistive device. At the TLC, they participated in at least 1 hour of physical therapy each day. Normally they also had occupational therapy each day. Medications at the time of the study included analgesics, -blocker, stool softener, fiber pills, allergy medicine, calcium supplementation, and antiviral, antiepileptic, antidepressant, antipsychotic, antihistamine, and antianxiety medication. All the subjects ate a regular diet approved by a dietician of about 2kcal/d. The food at TLC is generally prepared according to the American Heart Association s guidelines, 1,11 with low fat and cholesterol content. The snacks eaten outside the meals were not regulated. The control subjects were normal healthy and generally active subjects, also eating a regular diet. Experimental Protocol The control subjects fasted from 1: PM the evening before the study, and reported to the Exercise Laboratory at Shriners Hospitals for Children/Galveston in the morning of the study. The volunteers with brain injuries () were clients at the TLC in Galveston, and stayed at the center overnight, fasting from 1: PM. The study was performed at the TLC the following morning. On the morning of the experiment, we inserted a 2-gauge polyethylene catheter into a vein in one of the forearms for blood sampling. Patency of the catheter was maintained by normal saline infusion. At the start of the experiment, 2 blood samples were drawn for determination of basal amino acid concentrations. Thereafter, a drink consisting of 7g of EAA was given, and blood samples were drawn after 15, 3, 45, and 6 minutes to determine plasma amino acid concentrations. The composition of the drink was:.338g of histidine,.7811g of isoleucine, g of leucine, g of lysine,.3616g of methionine,.576g of phenylalanine,.9547g of threonine, and.7377g of valine. Sample Analyses The blood samples were centrifuged for 15 minutes at 4 C and stored at 8 C until analyzed for the individual amino acid concentrations by high-performance liquid chromatography (HPLC). a For this procedure, plasma was first thoroughly mixed with 1 L internal standard and 1 L of acetonitrile (HPLC grade) and then incubated for 3 minutes on ice. Thereafter 75 L ofddh 2 O was added before the sample was centrifuged at 1 g for 1 minutes. Finally, 1 L ofthe supernatant was transferred to a.2cm Ultrafree-MC centrifugal filter b and spun at 3 g for 4 hours at 4 C. The samples were subsequently run on the HPLC. Statistical Methods To compare the basal amino acid concentrations in patients versus control, we calculated the average value of the 2 basal samples and compared between groups, using an unpaired t test. To compare the postdrink amino acid concentrations in patients versus control, the average value of the postdrink samples was calculated and compared between groups, using an unpaired t test. To determine if the response to the drink differed in the 2 groups, the average postdrink value minus the average baseline value was calculated and compared between the groups, using an unpaired t test. A P value less than.5 was considered statistically significant. Results are expressed as mean standard error unless otherwise noted. RESULTS Differences in the Basal State The group had a lower total amino acid concentration at baseline compared with controls ( nmol/mL vs nmol/mL, P.22) (fig 1A). This difference was partly caused by a lower EAA concentration in versus controls (82 38nmol/mL vs nmol/mL, P.1) (fig 1B), which again was mainly caused by a 33% lower valine concentration in versus controls (P.3) (fig 2). No other significant differences in individual EAA concentrations were found in the basal state. There was only a tendency to a lower total non-eaa concentration between groups in the basal state (P.72) (fig 1C), and no differences between groups in individual non-eaa concentrations. Effect of EAA Intake The response of total amino acid concentration to the drink did not differ between the 2 groups. Thus, the difference in

3 236 AMINO ACIDS AND BRAIN INJURY, Børsheim A Plasma amino acid concentration (nmol/ml) B Plasma EAA concentration (nmol/ml) C Plasma non-eaa concentration (nmol/ml) * * 1B). However, in the postdrink period, the total EAA concentration was no longer significantly different between groups (P.86), even though the difference in valine concentration persisted in this period (P.2) (see fig 2). No other differences in individual EAA concentrations were seen. In contrast to total amino acid and EAA concentrations, there was a significantly different response of total non-eaa concentration in controls versus subjects following ingestion of EAA (P.22) (see fig 1C). The level of non-eaa increased to a higher level in versus controls after EAA intake (P.17). The greatest discrepancy between groups was that of alanine and glutamine, which increased to a greater extent in controls versus (post-pre, between groups: P.35 and P.25, respectively) (table 2). There were also different responses to the drink in arginine (P.12), asparagine (P.33), and glutamic acid (P.38) concentrations in the 2 groups (see table 2). DISCUSSION The main finding of this study was that, even after several months of recovery from, plasma valine concentration was still depressed compared with healthy controls, leading to lower EAA and total amino acid concentrations in these subjects. Because the plasma EAA concentration response to a 7-g of EAA drink was similar in the patients and the controls, the difference in valine concentration between groups still persisted. Finally, whereas the concentration of several non- EAA increased in response to the EAA drink in the controls, this was not seen in the patients with. This may have been caused by a higher percentage of the ingested EAA being used for protein synthesis in the patients, whereas more was converted to non-eaa in the healthy controls. Further studies should be performed to explore the basis and physiologic significance of the present observations. Differences in the Basal State The greatest disturbance in plasma amino acid pattern occurs in the period immediately after, but profound abnormalities in plasma amino acid concentrations are observed also during the first months after injury. 5,6 Patients with on artificial nutrition (studied 12h after artificial nutrition was inter- Fig 1. (A) Total plasma amino acid, (B) EAA, and (C) non-eaa concentrations in brain injury patients (; n 6) and healthy controls (; n 6). *Average baseline values P<.5 between groups; average postdrink values P<.5 between groups; delta average postdrink vs average basal values P<.5 between groups. concentration observed in the basal state remained also after the drink (P.16) (see fig 1A). Total EAA concentration increased after intake of the EAA mixture, and the response was similar in both groups (see fig Fig 2. Plasma valine concentration in brain injury patients (; n 6) and healthy controls (; n 6). *Average baseline values P<.5 between groups; average postdrink values P<.5 between groups.

4 AMINO ACIDS AND BRAIN INJURY, Børsheim 237 Table 2: Plasma Non-EAA Concentrations in Subjects With (n 6) and Healthy Control Subjects (n 6) During Resting Basal Conditions and After Intake of an EAA Drink Amino Acid Group Average Basal Value 15 Minutes Postdrink 3 Minutes Postdrink 45 Minutes Postdrink 6 Minutes Postdrink Alanine* Controls Arginine Controls Asparagine* Controls Glutamic acid Controls Glutamine Controls NOTE. Data are mean nmol/ml SE. *Average postdrink value P.5 between groups; delta average postdrink vs basal value P.5 between groups. rupted) had lower plasma concentrations of the EAA tyrosine, leucine, valine, methionine, and phenylalanine both 11 days after acute injury and after 7 days at a rehabilitation center compared with control. 6 To our knowledge, this is the first study on plasma amino acid concentrations in patients more than 1 year after the. Our data show that even after a longer recovery period when the patients are partly back in society, eating a normal diet, and can perform activities of daily living, there are still some irregularities, but overall the patients have mostly recovered their plasma amino acid pattern. Thus, compared with studies in patients at earlier stages of recovery, 5,6 our patients showed improvement in plasma amino acid pattern. Tyrosine has an important role as a precursor for several brain neurotransmitters (dopamine, catecholamines), so it is of particular interest that this amino acid had recovered, indicating improvement as recovery is progressing. Valine was the only EAA that was still lower than in the control group (see fig 2). Valine is a BCAA. It has been shown 2 that the plasma free tryptophan/bcaa ratio is of importance for the development of central fatigue (reduction in neural drive to working muscle with decreased force output as a result 12 ). Tryptophan and the BCAA compete for the same transporters at the blood brain barrier, and a lower BCAA concentration will allow more tryptophan to be transported into the brain. Tryptophan is a precursor for the neurotransmitter serotonin, and an elevated serotonin production leads to central fatigue. Thus, it may be hypothesized that a low level of valine will lead to increased and/or early fatigue, but this speculation needs to be confirmed by further investigation. We did not measure the free tryptophan concentration because this is very difficult, and it is the free concentration that is of importance for the blood brain barrier transport. It is not clear from other studies in patients with whether the plasma free tryptophan concentration has been measured in these patients. Plasma amino acid concentrations are important for regulation of muscle protein metabolism. 13 A potential factor contributing to loss of amino acids from skeletal muscle after is the accompanying and predominant inactivity. Immediately postinjury, patients are bedridden for extended periods. Even during rehabilitation, patients experience substantial periods of inactivity. Extensive work has demonstrated that inactivity alone leads to loss of muscle and amino acids from the body. 14,15 It is our contention that patients lose muscle mass during acute hospitalization, and are unable to regain mass and function due to a chronic reduction in activity combined with a concomitant decrease of anabolic stimuli. It is also possible that this early loss affects the body s amino acid profile, and that this alteration is not totally resolved throughout rehabilitation. We have no measure of the loss of muscle mass in the present study. Effect of EAA Intake The nutritional intake during rehabilitation is important for restoration of the plasma amino acid pattern and ultimately protein metabolism in various tissues. Because protein and amino acid consuming processes (eg, infection, catabolic processes, hypermetabolic processes, increased hepatic uptake of amino acids for gluconeogenesis) may be present in people with prior, 16 the need for amino acids and protein in the diet may be increased. The results of the present study indicate that even when the plasma amino acid pattern for the most part is back to normal, there may still be an increased need for certain amino acids in these patients. This is evident from the lack of increase in non-eaa concentration after intake of the EAA in the patients with (see fig 1C). The non-eaa with the greatest increases after drink in the control group, but not in the group, were alanine and glutamine (see table 2). Thus, it is possible that less of the EAA in the drink were converted to non-eaa in the patients, and more were directed into amino acid-requiring processes, possibly brain and muscle protein synthesis. It is also possible that the non-eaa were lower in the patients due to accelerated utilization for protein synthesis. In any case, the low plasma levels in response to EAA intake suggest that, under these conditions, alanine and glutamine become provisionally essential in people with, meaning that de novo synthesis is insufficient for maintenance of the optimal concentrations. Intravenous supplementation with BCAA over a 15-day period has been shown to improve disability rating scale in patients at an earlier stage of recovery from. 17 The effect of long-term supplementation of amino acids in postacute recovery patients is not known. Our subjects responded normally to the drink, but did not regain the level of valine (see fig 2). Thus, it seems likely that a supplement for this patient group would need additional valine, and possibly that non-eaa should also be added to achieve optimal effect on protein synthesis. It seems reasonable to advise patients to eat a diet rich in valine, for example, from dietary sources like red meat, dairy products, eggs, cheese, soy protein products, mushrooms, and peanuts. The higher protein sources often contain ample amounts of the BCAA, including valine. A potential area for further research in this patient group is also to combine amino acids (specifically BCAA) with other anabolic factors, growth hormone, or testosterone.

5 238 AMINO ACIDS AND BRAIN INJURY, Børsheim Study Limitations and Methodologic Considerations Because we were measuring only plasma amino acid concentrations, we do not know the turnover rates of the amino acids in the present study. Nonetheless, it seems justified to assume that a lower plasma level of valine is caused by an increased cellular uptake in the patients, and not by reduced release from protein breakdown. Similarly, it seems defensible that the increase in several of the non-eaa after drink in the control group was caused by increased production from the added EAA and not by increased release from protein breakdown, whereas a lack of increase in plasma non-eaa after drink in patients with can be explained by the EAA being used in other processes. Our 2 groups were not matched regarding lifestyle (ie, activity level or diet). However, whereas a matched control group can be defended, a healthy control group will be the measure of a successful recovery. The average age in the 2 groups also turned out to be different, but this should not be of importance because differences in plasma amino acid pattern over this age range are unlikely. The diet was not controlled in this study, but the volunteers in both groups ate regular diets. Of interest, even abnormal diets do not result in disruptions of the normal profile of plasma amino acids. For example, even when obese subjects were totally fasted for 6 days, their plasma amino acid concentrations were maintained at completely normative levels. 18 The sample in the present study consisted of only 6 patients and 6 controls. However, although the sample size was small, it should be pointed out that significance takes into account the number of subjects, and significance with a small number of subjects is much harder to achieve. Therefore, only differences of large enough magnitude to be physiologically important will be significant. Even so, further studies should be performed to confirm the present observations, and explore the consequences on outcome measures. For example, we did not measure the relations between plasma amino acid pattern and the patients motor and cognitive functions and mood. This may be of interest in future studies of postacute patients with, in which more subjects with a wider range in disabilities are studied. CLUSIONS The results of this study show that plasma valine concentration is reduced in postacute patients versus healthy controls. This may be of importance for both brain and muscle metabolic functions. Further, EAA given in a drink are not converted to non-eaa in these patients, indicating that there may be an increased need of dietary amino acids even at later stages of recovery. Acknowledgments: We thank Jodie L. Tefer, BA, at the TLC in Galveston, for help in recruiting the volunteers, and Julie Booth, Department of Surgery, UTMB, in Galveston, for administrative help. We thank the nurse and staff at the TLC. Further, we thank Lyzanne Sargeant Mason, RN, Scott Schutzler, RN, Stephaine Blase, Christopher Danesi, MA, and Melissa Bailey, BS, Department of Surgery, UTMB, for skillful technical assistance. References 1. Wolfe RR, Miller SL. Amino acid availability controls muscle protein metabolism. Diabetes Nutr Metab 1999;12: Castell LM, Yamamoto T, Phoenix J, Newsholme EA. The role of tryptophan in fatigue in different conditions of stress. Adv Exp Med Biol 1999;467: Piek J, Lumenta CB, Bock WJ. Protein and amino acid metabolism after severe cerebral trauma. Intensive Care Med 1985;11: Dolp R, Fekl W, Ahnefeld W. Free amino acids in plasma in the post-traumatic period. Infusionsther Klin Ernahr 1975;2: Aquilani R, Viglio S, Iadarola P, et al. Peripheral plasma amino acid abnormalities in rehabilitation patients with severe brain injury. Arch Phys Med Rehabil 2;81: Aquilani R, Iadarola P, Boschi F, Pistarini C, Arcidiaco P, Contardi A. 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