George Lee, III

Journal of Undergraduate Research
Volume 5, Issue 5 - February 2004

Adult Respiratory Distress Syndrome

George Lee, III

ABSTRACT

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.

INTRODUCTION

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.

METHODS AND MATERIALS

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
Diets

Ingredients
g/kg ‡
AIN 93G
[13] 2%
Arginine 4%
Arginine 6%
Arginine Isonitrogenous**

AIN 93G
(modified) 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% hexadecyltrimethylammonium 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 μl of 0.8 M KH2PO4 (pH 5.4), 25 μl 10% HETAH, and 50 μl 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 μl of 30 mM hydrogen peroxide was added and sample was incubated for three minutes. The reaction was stopped by adding 10 μl 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 pyrogallol 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.

RESULTS

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 neutrophil accumulation (Fig. 3). However, there was an overall trend toward increased MPO activity with LPS treated mice (p=0.0577)

Figure 1. Percent weight change. *p < 0.05 vs. all other diet groups.

Figure 2. Lung weight as a percent of body weight.

Figure 3. Myeloperoxidaseactivity.

DISCUSSION

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 (n=4/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 neutrophil 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.

REFERENCES

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. J Biol Chem, 2002. 277(34): p. 30463-8.

Shepherd, V.L., The role of the respiratory burst of phagocytes in host defense. Semin Respir Infect, 1986. 1(2): p. 99-106.

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.

Conti, C.R., Nitric oxide as a therapeutic agent. Clin Cardiol, 1994. 17(5): p. 227-8.

Roberts, J.D., Jr., et al., Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. The Inhaled Nitric Oxide Study Group. N Engl J Med, 1997. 336(9): p. 605-10.

Sato, Y., et al., Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression. Am J Respir Crit Care Med, 1999. 159(5 Pt 1): p. 1469-76.

Kubes, P., M. Suzuki, and D.N. Granger, Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A, 1991. 88(11): p. 4651-5.

Sheridan, B.C., et al., L-arginine prevents lung neutrophil accumulation and preserves pulmonary endothelial function after endotoxin. Am J Physiol, 1998. 274(3 Pt 1): p. L337-42.

Nieves, C., Jr. and B. Langkamp-Henken, Arginine and immunity: a unique perspective. Biomed Pharmacother, 2002. 56(10): p. 471-82.

Calkins, C.M., et al., L-arginine attenuates lipopolysaccharide-induced lung chemokine production. Am J Physiol Lung Cell Mol Physiol, 2001. 280(3): p. L400-8.

Gianotti, L., et al., Arginine-supplemented diets improve survival in gut-derived sepsis and peritonitis by modulating bacterial clearance. The role of nitric oxide. Ann Surg, 1993. 217(6): p. 644-53; discussion 653-4.

Wu, G., Intestinal mucosal amino acid catabolism. J Nutr, 1998. 128(8): p. 1249-52.

Grisham, M.B., J.N. Benoit, and D.N. Granger, Assessment of leukocyte involvement during ischemia and reperfusion of intestine. Methods Enzymol, 1990. 186: p. 729-42.

Lowry, O.H., et al., Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 1951.

Lefer, A.M., Nitric oxide: nature's naturally occurring leukocyte inhibitor. Circulation, 1997. 95(3): p. 553-4. Chemistry, 1951.

Wu, G. and S.M. Morris, Jr., Arginine metabolism: nitric oxide and beyond. Biochem J, 1998. 336(Pt 1): p. 1-17.

Gaut, J.P., et al., Myeloperoxidase produces nitrating oxidants in vivo. J Clin Invest, 2002. 109(10): p. 1311-9.

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Ingredients g/kg ‡	AIN 93G [13]	2% Arginine	4% Arginine	6% Arginine	Isonitrogenous**
Table 1 Diets
*AIN 93G (modified)	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% hexadecyltrimethylammonium 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 μl of 0.8 M KH2PO4 (pH 5.4), 25 μl 10% HETAH, and 50 μl 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 μl of 30 mM hydrogen peroxide was added and sample was incubated for three minutes. The reaction was stopped by adding 10 μl 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 pyrogallol 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. RESULTS 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 neutrophil accumulation (Fig. 3). However, there was an overall trend toward increased MPO activity with LPS treated mice (p=0.0577) *Figure 1. Percent weight change. p < 0.05 vs. all other diet groups. Figure 2. Lung weight as a percent of body weight. Figure 3. Myeloperoxidaseactivity. DISCUSSION 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 (n=4/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 neutrophil 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. REFERENCES 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. J Biol Chem, 2002. 277(34): p. 30463-8. Shepherd, V.L., The role of the respiratory burst of phagocytes in host defense. Semin Respir Infect, 1986. 1(2): p. 99-106. 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. Conti, C.R., Nitric oxide as a therapeutic agent.* Clin Cardiol, 1994. 17(5): p. 227-8. Roberts, J.D., Jr., et al., Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. The Inhaled Nitric Oxide Study Group. N Engl J Med, 1997. 336(9): p. 605-10. Sato, Y., et al., Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression. Am J Respir Crit Care Med, 1999. 159(5 Pt 1): p. 1469-76. Kubes, P., M. Suzuki, and D.N. Granger, Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A, 1991. 88(11): p. 4651-5. Sheridan, B.C., et al., L-arginine prevents lung neutrophil accumulation and preserves pulmonary endothelial function after endotoxin. Am J Physiol, 1998. 274(3 Pt 1): p. L337-42. Nieves, C., Jr. and B. Langkamp-Henken, Arginine and immunity: a unique perspective. Biomed Pharmacother, 2002. 56(10): p. 471-82. Calkins, C.M., et al., L-arginine attenuates lipopolysaccharide-induced lung chemokine production. Am J Physiol Lung Cell Mol Physiol, 2001. 280(3): p. L400-8. Gianotti, L., et al., Arginine-supplemented diets improve survival in gut-derived sepsis and peritonitis by modulating bacterial clearance. The role of nitric oxide. Ann Surg, 1993. 217(6): p. 644-53; discussion 653-4. Wu, G., Intestinal mucosal amino acid catabolism. J Nutr, 1998. 128(8): p. 1249-52. Grisham, M.B., J.N. Benoit, and D.N. Granger, Assessment of leukocyte involvement during ischemia and reperfusion of intestine. Methods Enzymol, 1990. 186: p. 729-42. Lowry, O.H., et al., Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 1951. Lefer, A.M., Nitric oxide: nature's naturally occurring leukocyte inhibitor. Circulation, 1997. 95(3): p. 553-4. Chemistry, 1951. Wu, G. and S.M. Morris, Jr., Arginine metabolism: nitric oxide and beyond. Biochem J, 1998. 336(Pt 1): p. 1-17. Gaut, J.P., et al., Myeloperoxidase produces nitrating oxidants in vivo. J Clin Invest, 2002. 109(10): p. 1311-9. --top-- Back to the Journal of Undergraduate Research College of Liberal Arts and Sciences \| University Scholars Program \| University of Florida \| © University of Florida, Gainesville, FL 32611; (352) 846-2032.