Plasma Amino Acid Concentrations During Late Rehabilitation in Patients With Traumatic Brain Injury

Participants 

Six healthy men and 6 men with TBI participated in the study. Table 1 shows the characteristics of the volunteers, all of whom were white.

Table 1. Physical Characteristics of All Subjects

	Subjects	Age (y)	Weight (kg)	Height (cm)	Months Postinjury
	TBI
	1	37	89.0	185.0	13
	2	29	78.9	180.3	19
	3	20	88.0	188.0	22
	4	23	97.5	182.9	15
	5	29	98.0	172.7	11
	6	23	77.6	175.3	20
	Mean	27⁎	88.2	180.7	17
	SD	6	8.7	5.8	4
	SE	3	3.6	2.4	2
	CV	0.23	0.10	0.03	0.26
	Controls
	1	48	77.3	167.6
	2	48	70.0	177.0
	3	48	105.0	183.0
	4	33	70.0	188.0
	5	43	74.1	175.0
	6	35	96.0	188.0
	Mean	43	82.1	179.8
	SD	7	14.8	8.1
	SE	3	6.0	3.3
	CV	0.16	0.18	0.04

Abbreviations: CV, coefficient of variation; SD, standard deviation; SE, standard error.

⁎ P<.05 vs controls.

We recruited the participants with TBI through the Transitional Learning Center (TLC) in Galveston, TX, where they 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 TBI) 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, β-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 2000kcal/d. The food at TLC is generally prepared according to the American Heart Association’s guidelines,10, 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 10:00 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 (TBI) were clients at the TLC in Galveston, and stayed at the center overnight, fasting from 10:00 pm. The study was performed at the TLC the following morning.

On the morning of the experiment, we inserted a 20-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, 30, 45, and 60 minutes to determine plasma amino acid concentrations. The composition of the drink was: 0.3038g of histidine, 0.7811g of isoleucine, 1.7213g of leucine, 1.6988g of lysine, 0.3616g of methionine, 0.5076g of phenylalanine, 0.9547g of threonine, and 0.7377g of valine.

Sample Analyses 

The blood samples were centrifuged for 15 minutes at 4°C and stored at −80°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 100μL internal standard and 100μL of acetonitrile (HPLC grade) and then incubated for 30 minutes on ice. Thereafter 750μL of ddH2O was added before the sample was centrifuged at 1500×g for 10 minutes. Finally, 100μL of the supernatant was transferred to a 0.2cm Ultrafree-MC centrifugal filterb and spun at 3000×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 TBI 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 TBI 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 .05 was considered statistically significant. Results are expressed as mean ± standard error unless otherwise noted.

Differences in the Basal State 

The TBI group had a lower total amino acid concentration at baseline compared with controls (2158±72nmol/mL vs 2445±78nmol/mL, P=.022) (fig 1A).

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Fig 1. (A) Total plasma amino acid, (B) EAA, and (C) non-EAA concentrations in brain injury patients (TBI; n=6) and healthy controls (CON; n=6). *Average baseline values P<.05 between groups; †average postdrink values P<.05 between groups; ‡delta average postdrink vs average basal values P<.05 between groups.

This difference was partly caused by a lower EAA concentration in TBI versus controls (802±38nmol/mL vs 932±14nmol/mL, P=.010) (fig 1B), which again was mainly caused by a 33% lower valine concentration in TBI versus controls (P=.003) (fig 2). No other significant differences in individual EAA concentrations were found in the basal state.

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Fig 2. Plasma valine concentration in brain injury patients (TBI; n=6) and healthy controls (CON; n=6). *Average baseline values P<.05 between groups; †average postdrink values P<.05 between groups.

There was only a tendency to a lower total non-EAA concentration between groups in the basal state (P=.072) (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 concentration observed in the basal state remained also after the drink (P=.016) (see fig 1A).

Total EAA concentration increased after intake of the EAA mixture, and the response was similar in both groups (see fig 1B). However, in the postdrink period, the total EAA concentration was no longer significantly different between groups (P=.086), even though the difference in valine concentration persisted in this period (P=.002) (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 TBI subjects following ingestion of EAA (P=.022) (see fig 1C). The level of non-EAA increased to a higher level in TBI versus controls after EAA intake (P=.017). The greatest discrepancy between groups was that of alanine and glutamine, which increased to a greater extent in controls versus TBI (post-pre, between groups: P=.035 and P=.025, respectively) (table 2). There were also different responses to the drink in arginine (P=.012), asparagine (P=.033), and glutamic acid (P=.038) concentrations in the 2 groups (see table 2).

Table 2. Plasma Non-EAA Concentrations in Subjects With TBI (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	30 Minutes Postdrink	45 Minutes Postdrink	60 Minutes Postdrink
	Alanine⁎†	TBI	270±15	283±16	302±24	299±19	269±23
		Controls	353±38	388±43	398±38	408±38	415±45
	Arginine†	TBI	106±14	113±14	116±15	111±10	106±9
		Controls	83±4	95±5	102±6	109±5	100±6
	Asparagine⁎†	TBI	41±2	41±2	42±3	41±2	46±3
		Controls	46±3	48±3	48±4	51±4	50±4
	Glutamic acid†	TBI	47±9	50±10	49±12	50±11	44±8
		Controls	43±10	35±11	37±10	38±11	29±6
	Glutamine†	TBI	522±39	536±37	573±33	573±35	525±62
		Controls	578±17	622±16	640±29	667±30	678±28

NOTE. Data are mean nmol/mL ± SE.

⁎ Average postdrink value P<.05 between groups; † delta average postdrink vs basal value P<.05 between groups.

Differences in the Basal State 

The greatest disturbance in plasma amino acid pattern occurs in the period immediately after TBI, but profound abnormalities in plasma amino acid concentrations are observed also during the first months after injury.5, 6 Patients with TBI on artificial nutrition (studied 12h after artificial nutrition was interrupted) had lower plasma concentrations of the EAA tyrosine, leucine, valine, methionine, and phenylalanine both 110 days after acute injury and after 70 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 TBI. 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 shown2 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 result12). 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 TBI 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 TBI is the accompanying and predominant inactivity. Immediately postinjury, patients are bedridden for extended periods. Even during rehabilitation, TBI 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 TBI 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 TBI,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 TBI (see fig 1C). The non-EAA with the greatest increases after drink in the control group, but not in the TBI 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 TBI 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 TBI, 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 TBI.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.

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 TBI 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 60 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 TBI, in which more subjects with a wider range in disabilities are studied.