1058 Am J C/in Nutr 1995;61:1058-61. Printed in USA. © 1995 American Society for Clinical Nutrition

Increased plasma bicarbonate and growth hormone afteran oral glutamine load13

Tomas C Welbourne

ABSTRACT An oral glutamine load was administered tonine healthy subjects to determine the effect on plasma glu-

tamine, bicarbonate, and circulating growth hormone concen-

trations. Two grams glutamine were dissolved in a cola drink

and ingested over a 20-mm period 45 mm after a light break-

fast. Forearm venous blood samples were obtained at zero time

and at 30-mm intervals for 90 mm and compared with timecontrols obtained I wk earlier. Eight of nine subjects responded

to the oral glutamine load with an increase in plasma glutamine

at 30 and 60 mm before returning to the control value at 90

mm. Ninety minutes after the glutamine administration load

both plasma bicarbonate concentration and circulating plasmagrowth hormone concentration were elevated. These findingsdemonstrate that a surprisingly small oral glutamine load is

capable of elevating alkaline reserves as well as plasma growth

hormone. A,n J Clin Nutr 1995;61:1058-61.

KEY WORDS Glutamine load, plasma bicarbonate, plasmagrowth hormone

Introduction

Glutamine homeostasis reflects the minute-to-minute bal-ance between glutamine removal from and addition to theblood stream (1). Normally, endogenous production combinedwith dietary intake suffice to meet daily growth demands so

that glutamine is classified as a nonessential amino acid (2).

However, physiological challenges, especially those generating

an acid load, may confer an essential role on glutamine under

these conditions (3). Beside its unique role in supporting renal

bicarbonate production (1), glutamine is the major metabolic

fuel for the small intestine (3). Glutamine availability also

appears to determine the rate of protein turnover in muscle (4),the major repository of mobile nitrogen stores. Thus, given thefundamental roles played by glutamine in supporting cellularand organ function it is not surprising to find multiorgan-

dependent processes such as the immune (5) and antiinflam-

matory response (6) to be dependent, in part, on glutamine. In

fact, over-training in athletes is associated with depressed

plasma glutamine concentration and immune system respon-siveness as judged by susceptibility to infection (7). From this

perspective an oral glutamine supplement might prove benefi-

cial in anticipation of such challenges, providing bicarbonatecould be generated. Furthermore, glutamine might also in-crease plasma arginine and glutamate concentrations, amino

acids both potentially capable of eliciting growth hormone

secretion (8,9). Accordingly, the purpose of this study was to

determine whether a small oral glutamine load might increasecirculating plasma glutamine and, if so, whether this would besufficient to increase the body fluid alkaline reserves as well as

increase plasma growth hormone concentration.

Subjects and methods

Nine healthy volunteers aged 32-64 y (Table 1) were se-lected because of the known decrease in human growth hor-mone secretion rates after the third decade (10,11). Studies

were performed on two consecutive Saturdays at exactly the

same time, 0800-1 100 and 45 mm after a light breakfast (toast,

coffee, and juice) designed to ensure a low basal growthhormone release (11). After an initial forearm venous blood

sample (t = 0), either vehicle (490 mL carbonated soft drink,pH = 3.8, containing 20 g glucose) or vehicle plus 2 g

L-glutamine (Sigma, St Louis) was ingested over a 20-mm

period with serial blood samples (2 mL) drawn at 30-mm

intervals: t = 30, 60, and 90 mm. The amount of glutamine

consumed ranged from 0.016 to 0.036 g/kg (Table 1). Note thatsubject A consumed 20 times this amount (0.56 g/kg) during anidentical protocol in an earlier study without untoward effects(12). The present protocol was approved by the Louisiana State

University Medical College Institutional Review Board for

Human Research.Blood samples were placed on ice and centrifuged (10 000 x

g for 10 mm at 4 #{176}C),and plasma was drawn for analysis of

growth hormone by radioimmunoassay (Nichols Institute, San

Juan Capistrano, CA), bicarbonate by microgasometry, and

glutamine by HPLC as described previously (13). All analyseswere performed the same day. Precision for determiningplasma glutamine was ± 3%, for total carbon dioxide ± 1.5%

(95% representing bicarbonate), and for growth hormone

± 5%. Intrassay growth hormone variability for five subjectswas determined to be 7.8 ± 2.8%. The sensitivity in detecting

plasma growth hormone is 0.06 �g/L, allowing for measure-

I From the Department of Physiology, Louisiana State University Col-

lege of Medicine, Shreveport, LA.2 Supported by funding from Research Corporation Technology and the

Louisiana Education Quality Support Fund RD-A-IS.

3 Address reprint requests to TC Welbourne, Department of Physiology,

Louisiana State University Medical College, P0 Box 33932, Shreveport,

LA 71130.

Received June 24, 1994.

Accepted for publication October 31, 1994.

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TABLE 1

Summary of subject characteristics and glutamine dose’

Subject code Age Sex BMI Glutamine dose

y kg/rn2 ��zg/kg both’ tt’t

A 50 M 23.5 28

B 64 M 26.4 21

C 35 M 21.5 29

D 44 F 35.0 18

E 40 M 23.2 25

F 42 M 26.6 27

G 36 F 26.25 30

H’ 32 M 38.55 16

I 37 F 22.32 36

.� :!: SEM’ 43.5 ± 3.4 25.6 ± 1.5 27 ± 2

as shown in Figure 1. The increase was prompt, 19% comparedwith the time control at 30 mm, and sustained, it was 12% at 60

mm, with a gradual return to a value not significantly different

from the time control at 90 mm. In contrast, subject H had a lowerinitial glutamine concentration than the other eight subjects, 433

vs 744 ± 36 �moVL, and did not show an increase at either 30 or

60 mm after the glutamine load.The oral glutamine load increased the plasma bicarbonate

concentration as seen in Figure 2. After 90 mm, the glutamine

load produced a gain of 2.7 ± 0.7 mmolfL compared with -0.7

± 0.6 mmol/L for the time control, P < 0.02. Subject H, who

did not exhibit the plasma glutamine increase, also did not

show an increase in his plasma bicarbonate concentration.

The effect of the oral glutamine load on plasma growth

hormone is shown in Figure 3. For the time control, growthhormone concentrations were 0.029 ± 0.015 and 0.020 ±

0.002 nmol/L at t = 0 and 90 mm, respectively, with discern-

ible peaks (> 25% of preceding nadir) apparent in only three of

the subjects. In contrast, 90 mm after glutamine treatment the

plasma growth hormone concentration was 4.3-fold higher than

the time control 90-mm value (0.084 ± 0.04 compared with

0.019 ± 0.002 nmol/L; P = 0.09 by paired t test); seven of the

eight subjects showed a discernible peak at this time. In fact,

Time Control GIN

30

28

-J 26 H

�24 !

‘E� DS #{149}

, ii = 8: subject H not included in population means.

ment of concentrations as low as 0.1 p.g/L (catalog #40-2205;

HGH Kit, Nichols Institute). Glucose and nonesterified fattyacids were determined by using commercially available kits

(hexokinase enzymatic assay, Sigma, and colorimetric assay,

Boehringer Mannheim, Mannheim, Germany).

Statistical a,ialysi.s

Changes in plasma glutamine and glucose with time, at 0, 30,

60, and 90 mm, were analyzed by analysis of variance

(ANOVA; repeated measurements). Between-treatment time

point differences were analyzed by using a paired Student’s

test. Where directional changes were predicted a priori, a

one-tailed t test was used, ie, glutamine, total carbon dioxide,

and growth hormone; otherwise a two-tailed t test was used.

Results

Eight of the nine subjects receiving the oral glutamine load

responded with an elevation in systemic plasma concentration

-I

0 0.9

E

w 0.8C

E0�

(D0

� 06

Q_ 0.5

0.4

. L-G(utamine0 Vehicle

�I

0 30 60 90Minutes

GLUTAMINE, BICARBONATE, AND GROWTH HORMONE 1059

FIGURE 1. Peripheral plasma glutamine concentrations after glutamine

ingestion (2 g). � ± SEM; n = 8 subjects compared with their own time

control. Glutamine was consumed over 20 mm, t 0-20 mm with serial

sampling at 30-mm intervals. Significant difference between glutamine and

vehicle at 30 and 60 mm, P - 0.01 and P 0.02, respectively (paired a’

test). Changes in glutamine concentration with time were significant for

both vehicle and L-glutamine (P < 0.01, ANOVA).

I I I I

0 90 0 90Mm Mm

FIGURE 2. Plasma bicarbonate concentration before and 9() mm after

either vehicle (time control) or L-glutamine (GLN) (2 g for n = 9 subjects).

Time averages from eight subjects (0) (subject H not included).

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Time Control . L-Glutamine

0 Vehicle

0 30 60

D

Minutes

A

GiECBH

0 . 90Mm

0 90Mm

.30

.20

.10

-J�

0EC

‘-.9

0) 0.05C0E0� 0.04.C

00 0.03

0.02

0.01

0 ____________ ____________

FIGURE 3. Plasma growth hormone concentration before and 90 mm

after either vehicle (time control) or L-glutamine (GLN) (2 g for n = 8

subjects). Average for all the subjects (#{149})(subject H not included).

both subjects C and F exhibited early peak responses thataveraged increases of 67% and 78% above their time control at30 and 60 mm, respectively. In contrast, subject H did not

respond to the glutamine load with plasma growth hormoneconcentration at, or below, the time control value at 30 and 60

mm (0% and -28%, respectively). Note that the rise in plasma

growth hormone after glutamine ingestion occurs despite asmall increase in plasma glucose at 30 mm as shown in Figure4. The rise in glucose occurred in both time control and

glutamine groups, reflecting the glucose content of the vehicle.In contrast with glucose, the free fatty acid concentration inforearm venous blood was affected by the glutamine load as

seen in Figure 5. A greater fall in the plasma free fatty acidconcentration was observed over the 30-60-mm period corn-

pared with the time control (-0.29 ± 0.10 vs -0.10 ± 0.04

mrnol/L, P = 0.08, paired t test) consistent with enhancedforearm free fatty acid utilization (14).

Discussion

A surprisingly small oral glutarnine load, 2 g, was able toproduce a prompt and sustained (0.5 h) elevation in circulatingplasma glutamine, indicating that significant amounts of an

orally administered glutamine load did reach the periphery(15). Larger glutamine loads would increase plasma glutamine

even further but carry the risk of activating hepatic uptake. For

8.0

7.5

-J�‘-

0E 6.5Ejo.0

8 5.5

(D 5.0

4.5

4.0

90

FIGURE 4. Plasma glucose concentration after vehicle and glutamine

ingestion for eight subjects. Elevation in plasma glucose was significant for

both vehicle and vehicle plus glutamine, P < 0.01 (ANOVA).

example, a glutamine load 20 times higher increased plasmaglutamine 214% (12) compared with the 19% observed at 30

mm in the present study; however, this load provoked anaccelerated glutamine clearance consistent with activation of

hepatic glutamine removal (16). On the other hand, smaller oralloads, � 1 g, run the risk of being unable to significantlyelevate circulating plasma glutamine. The failure to elicit aresponse in subject H might well reflect the limiting dose inthis obese individual. Nevertheless, the present study clearly

shows that an oral glutamine load approximating the dose

range shown here appears to attain a “window” of effectivenessin achieving the desired objectives without calling into play theformidable, and even counterproductive, hepatic mechanism

for responding to a large increase in plasma glutamine (1).The effectiveness of an oral glutamine load would depend on

its ability to increase plasma bicarbonate concentration and

thus the buffering capacity of body fluid. Indeed, with the loadused, plasma bicarbonate increased in a manner consistent withthe elevation in circulating glutamine and glutamine’s role as a

-I

U-

LUz

0

E

E

C

ci)0)C0

-C0

0-30 Mm 30-60 Mm 60-90 Mm

FIGURE 5. Glutamine ingestion accelerates the decrease in forearm

free fatty acid concentration compared with vehicle. NEFA, nonesteri-

fied fatty acids.

1060 WELBOURNE

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GLUTAMINE, BICARBONATE, AND GROWTH HORMONE 1061

precursor for renal base generation (1). Note that both the rise

in plasma bicarbonate and oral glutamine would be expected to

drive up muscle cellular glutamine content (17), thereby slow-ing protein breakdown (4), a process accelerated under acido-

genic conditions (17,18).

The rise in plasma glutamine after the oral glutamine load

was also associated with an increase in plasma growth hormone

above the low basal concentrations observed in the time con-

trols. Because growth hormone concentration fluctuates with

time and food intake (10) the conditions were selected to

provide a minimal secretion in a population of age-dependent

low secreters (1 1). Under such controlled conditions, signifi-cant peaks are defined by the threshold criteria as being � 20%

more than a preceding nadir (19). In the present study the rise

in growth hormone occurred in seven of eight subjects and

exceeded the time control value by fourfold. Although this

represents a small increment in circulating growth hormone,

note that it is effective in eliciting growth hormone’s metabolic

effects (20). Indeed, the smaller ligand increments may be

more effective in receptor activation than would be largerincrements, which could exert autoinhibiting effects (21).

How an oral glutamine load might stimulate basal growth

hormone secretion was not elucidated in the present study.

However, glutamine initiates secondary amino acid fluxes that

could affect growth hormone secretion. For example, conver-

sion of glutamine to citrulline in the small intestine (22) sup-

ports renal arginine synthesis (23), a known stimulus for

growth hormone secretion (9). In addition, conversion of glu-

tamine to glutamate provides a stimulus for directly activating

somatotrophic growth hormone release (8). Thus, either one or

both effectors might play a role in stimulating growth hormone

secretion in response to an oral glutamine load.

In addition to the better-recognized role of growth hormonein shifting fuel utilization from glucose to fatty acids in muscle

(14), growth hormone was recently proposed to play a major

role in acid-base homeostasis (13). Growth hormone acceler-

ates renal acid secretion (13) and thereby facilitates eliminationof acid from the body fluids, a potentially important role duringstrenuous exercise when acidogenesis may limit performance

(24). Note that circulating growth hormone concentration in-

creases in strenuous exercise and this response can be sup-

pressed by bicarbonate administration (25). Interestingly, low-

ering plasma bicarbonate by acid loading (ammonium chloride)

increases circulating growth hormone concentration (26),

which is consistent with a role for growth hormone in acid-base

homeostasis. In summary, a surprisingly small oral glutamine

load was shown to be effective in elevating both the plasmaalkaline reserves and growth hormone responses that may

contribute to acid-base homeostasis. In addition, substitution of

the growth hormone-dependent fuel mix favoring fat oxidation

suggests a potential benefit in terms of body composition. UI thank Sudhir Joshi for technical assistance.

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