Adult Respiratory Distress Syndrome

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Adult Respiratory Distress Syndrome
Lee, George
Langkamp-Henken, Bobbi ( Mentor )
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Gainesville, Fla.
University of Florida
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Adult Respiratory Distress Syndrome

George Lee, III


Adult Respiratory Distress Syndrome (ARDS) is characterized by acute lung injury due to platelets and white blood
cells, predominantly neutrophils that accumulate in the capillaries and airspaces of the lungs. Exogenous nitric oxide (NO) is
a treatment option for ARDS because it decreases neutrophil accumulation in the lungs following septic challenge.
Increasing dietary arginine, the precursor for endogenous NO, may produce the same effects. Forty mice were fed either
a standard diet, the standard diet supplemented with 2%, 4%, or 6% arginine or the standard diet made isonitrogenous to
the 4% arginine diet for 14 days (n=4/group). Daily weights were obtained. The mice were then anesthetized, injected
with either lipopolysaccharide (to mimic early components of ARDS) or saline, and after four hours the lungs were
removed. Myeloperoxidase activity, an indicator of neutrophil accumulation, was measured. Mice on the 6% arginine diet
gained less weight than mice in the other diet groups (p<0.05). There was no significant difference in lung MPO activity
among diet and treatment groups. These preliminary data suggest that 6% dietary arginine may be excessive however,
no conclusions can be drawn regarding the effect of dietary arginine on lung neutrophil accumulation
following lipopolysaccharide treatment.


Adult Respiratory Distress Syndrome (ARDS) is a condition owing to multiple causes, one of which being sepsis. ARDS
is characterized by acute lung injury due to platelets and white blood cells, predominantly neutrophils, which accumulate in
the capillaries and airspaces of the lungs. Neutrophils are phagocytotic cells that produce free radicals and enzymes such
as myeloperoxidase (MPO) to kill bacteria [1]. Myeloperoxidase converts chloride to hypochlorous acid, which is one of
the strongest cytotoxins to be produced by phagocytes [1]. Myelperoxidase also produces halogens, chloramines,
aldehydes, and superoxide. When the neutrophils accumulate, these cytotoxins and free radicals can infiltrate the
surrounding tissue and cause damage similar to the bactericidal effects [2]. Injecting mice with lipopolysaccharide
(LPS) provides a model that mimics early components of ARDS due to sepsis [3].

Exogenous nitric oxide (NO), which is a vasodilator and attenuates platelet-endothelium and possibly neutrophil-
endothelium adhesion, is a treatment option for ARDS. Nitric oxide-induced vasodilation and disruption of cell-cell
adhesion improve pulmonary hypertension and arterial oxygenation, and attenuates neutrophil sequestration in the lung [4-
7]. It is possible that increasing endogenous NO will produce the same life saving effects [8]. Endogenous NO is
produced through arginine metabolism via nitric oxide synthase-1, 2, and 3 (NOS-1, 2, 3) in the vascular endothelium
[9]. Previous studies show that an increase in arginine has significant effects on neutrophil accumulation, pulmonary
vascular injury, and mortality in septic animals [8, 10, 11]. This could be due to the effects of NO since arginine is
readily converted to NO [12]. Under this premise, it was proposed that an increase in dietary L-arginine would
decrease neutrophil accumulation in the lungs of LPS treated mice. Neutrophil accumulation was indirectly determined
by measuring lung MPO activity. A secondary goal of this study was to determine the optimal level of dietary
arginine supplementation.


Animals and Diet

Forty male CB6F1 mice were obtained from the National Institute
of Aging at an age of one month. The mice were kept two to a cage in
a temperature-controlled room with a twelve hour light-dark cycle
at the University of Florida, Department of Food Science and
Human Nutrition, Gainesville, Florida. The mice acclimated
for fourteen days while being fed a stock diet. They were then

assigned to one of five different diets (Table 1, n=8/diet group).
Daily weights were obtained. All procedures were approved through
the University of Florida Institutional Animal Care and Use Committee.

Table 1
g/kg t AIN 93G 2% 4% 6%
[13] Arginine Arginine Arginine Isontroenous
*AIN 93G
(*AIN93G 900 900 900 900 900
Cornstarch 100 84 59 35 20
L-Arginine 0 16 41 65 0
L-Cystine 0 0 0 0 1
Casein 0 0 0 0 79
*AIN 93G (modified) = 1000 g AIN 93G - 100 g Cornstarch (Harlan Teklad, Madison, WI)
**Casein was added to obtain a nitrogen load equaal to the 4% arginine diet.
*All diets included equal amounts of Maltodextrin, Sucrose, Soybean oil, Cellulose, AIN-93 vitamin and mineral mix,
Chlorine bitartrate, and Tert-butylhydroquinone.

LPS Challenge

On the fourteenth day of the diet, each mouse was weighed
and anesthetized with halothane. Each mouse received
an intraperitoneal injection of either lipopolysaccharide (LPS-17.84
mg/kg) or phosphate buffer solution (PBS). After four hours,
each mouse was again anesthetized with halothane; the lungs
were removed, rinsed in cold PBS, wrapped in aluminum foil,
and stored at -80'C.

Tissue Preparation

The lung tissue from each mouse was thawed, dried, and weighed. The
tissue was then homogenized at 20% weight/volume of 20 mM
potassium phosphate dibasic (KH2PO4) with 1 mM ethylene diamine-

tetraacetic acid (EDTA), pH 7.4. Fifty micro liters was removed from
each sample and stored at -80'C for protein assay. The remaining
portion of the homogenate was brought to 1.5 times its new volume
with 20 mM KH2PO4 with 1 mM EDTA and centrifuged at 12,000 x g for

20 minutes at 4�C. The supernatant was removed and discarded. The
pellet was weighed and brought up to a final volume of 1.0 mL with 50

mM acetic acid with 0.5% hexadecyttrimethylammonium hydroxide
(HETAH). The suspensions were then vortexed, re-homogenized for 30
seconds, sonicated for 30 seconds, and then submitted to two cycles of
freezing and thawing. The samples were centrifuged for 20 minutes at
12,000 x g at 4�C. Then the supernatant was centrifuged for 5 minutes
at 22,500 x g at 4�C for the MPO assay.

MPO and Protein Assays

Myeloperoxidase activity was determined by measuring the hydrogen
peroxide-dependent oxidation of 3,3', 5,5'-tetramethylbenzidine
(TMB) [13]. A green-blue color change was observed with this
reaction, of which the absorbance was measured by
spectrophotometer (Beckman Laboratories, Irvine, CA) at 655 nm. A
reaction buffer was prepared containing 315 pl of 0.8 M KH2PO4 (pH
5.4), 25 pl 10% HETAH, and 50 pl of 16 mM TMB in
dimethylformamide. One hundred micro liters of sample was added to
this reaction buffer and the samples were placed in a shaking water
bath at 37� C for five minutes after which 10 pl of 30 mM hydrogen
peroxide was added and sample was incubated for three minutes. The
reaction was stopped by adding 10 pl of catalase. To each tube, 2 mL
of 0.2 M sodium acetate was added and the absorbance was read by
spectrophotometer at 655 nm. A standard curve, that uses peroxidase
enzyme (Sigma Chemical) as the standard enzyme, was used to
calculate the units of MPO activity, which is expressed as U/min/mg
protein. One unit is defined as forming 1.0 mg of purpurogallin from
pyrogallot in 20 seconds at pH 6.0 at 20� C. The protein concentration
of the lung homogenates was determined using the Bio-Rad DC
Protein Assay (Hercules, CA) adapted from methods of Lowry [14].
Statistical Analysis

Differences in percent change in average body weight, lung weight as
a percent of body weight, and MPO activity between diet and
treatment groups were analyzed using 2-way analysis of variance
(Statistical Analysis System, version 8.2, SAS Institute, Cary, NC) with

(p < 0.05). The data are presented as the mean � SEM.


Percent weight change in mice on the 6% arginine was significantly
lower than mice on the other four diets (p<0.05) (Fig. 1). No
significant difference in lung weight as a percent of body weight was
observed (Fig. 2). Also, there was no significant difference in MPO
activity among diet/treatment groups, leading to the conclusion that
there was no difference in neutrophit accumulation (Fig. 3).
However, there was an overall trend toward increased MPO activity
with LPS treated mice (p=0.0577)


AIN93G 2 % Arg 4% Arg 6% Arg ISO
Figure 1. Percent weight change.
diet groups.

*p < 0.05 vs. all other

2 % Arginine
M 4 % Arginine
M 6 % Arginine
E= Isonitrogenous

Figure 2.

Lung weight as a percent of body weight.

-- T

0 0004 -
o, 3- 2 % Arginine
M 4 % Arginine
M 6 % Arginine
0.0002- IIsonitrogenous


Figure 3. Myeloperoxidaseactivity.


It was hypothesized that an increase in dietary L-arginine would
decrease neutrophil accumulation in the lungs of septic mice, yet this
could not be supported nor rejected by the data. Preliminary data
show that there was no significant difference in neutrophil
accumulation between PBS and LPS treated groups. There was only a
trend toward an increase in neutrophil accumulation with LPS
treatment (p=0.0577). Therefore, conclusions cannot be drawn
between arginine supplementation and neutrophil accumulation due
to sepsis. The lack of support for the hypothesis could be due to the
fact that this was a preliminary study with few animals per group

A secondary objective for this study was to determine the optimal
level of dietary arginine supplementation. Due to NO having a half-
life of only 10 to 20 seconds, arginine needs to be readily available to
be converted to NO [15]. To make arginine more readily available,
studies showing benefit used intraperitoneal or intravenously injected
arginine immediately before or after LPS injection [8-10]. Realizing
that it may be a very high dose of arginine that produced the positive
results in previous studies, that 40% of the arginine ingested by
healthy animals will be degraded by the intestine, and that
absorption is impaired even further due to decreased intestinal blood
flow after LPS injection, a 6% supplementation group was used [9,
16]. At the end of the fourteen days, all mice had gained weight, yet

the 6% arginine group gained significantly less (Fig. 1). The mice on
6% arginine consistently shredded their food and consumed less diet
than other mice (data not shown). This link between mouse growth
and amount of arginine in the diet suggests that adding greater
amounts of arginine to the diet may reduce palatability of the diet.
Therefore, it is possible that the only way to provide supplemental
arginine in concentrations great enough to increase systemic levels
may be through injection or tube feeding because at 6% oral arginine
is not tolerated.

While it is still unknown whether dietary arginine supplementation
attenuates sepsis-induced neutrophit accumulation in the lungs, some
conclusions can be drawn from this study. If high levels of dietary
arginine supplementation are required to increase systemic NO
production, then it may not be possible to obtain this amount of
arginine voluntarily. If arginine is infused or injected to increase NO
production, the literature suggests that care must be taken to avoid
excess NO production [3, 17]. Dietary arginine supplementation for
the treatment of ARDS is not recommended until further studies
demonstrate a benefit.

1. Rosen, H., J.R. Crowley, and J.W. Heinecke, Human neutrophils
use the myeloperoxidase-hydrogen peroxide-chloride system to
chlorinate but not nitrate bacterial proteins during phagocytosis.
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2. Shepherd, V.L., The role of the respiratory burst of phagocytes
in host defense. Semin Respir Infect, 1986. 1(2): p. 99-106.

3. Heremans, H., et al., Role of interferon-gamma and nitric oxide
in pulmonary edema and death induced by lipopolysaccharide.
Am J Respir Crit Care Med, 2000. 161(1): p. 110-7.

4. Conti, C.R., Nitric oxide as a therapeutic agent. Clin Cardiot,
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5. Roberts, J.D., Jr., et al., Inhaled nitric oxide and persistent

pulmonary hypertension of the newborn. The Inhaled Nitric Oxide
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7. Kubes, P., M. Suzuki, and D.N. Granger, Nitric oxide: an
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8. Sheridan, B.C., et at., L-arginine prevents lung neutrophil
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9. Nieves, C., Jr. and B. Langkamp-Henken, Arginine and immunity:
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10. Calkins, C.M., et at., L-arginine attenuates lipopolysaccharide-
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11. Gianotti, L., et at., Arginine-supplemented diets improve survival
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12. Wu, G., Intestinal mucosal amino acid catabolism. J Nutr, 1998.
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13. Grisham, M.B., J.N. Benoit, and D.N. Granger, Assessment of
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17. Gaut, J.P., et at., Myeloperoxidase produces nitrating oxidants in
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