Journal of Undergraduate Research
 Volume 2, Issue 8 - May 2001

Supplemental Dietary L-arginine has no Effect on the Activity of Omithine Decarboxylase, the Rate-Limiting Enzyme in Polyamine Synthesis

Carmelo Nieves, Jr.

ABSTRACT

The amino acid L-arginine is known to enhance immune responses, however, the mechanism for this phenomenon has not yet been fully determined. L-arginine stimulates growth hormone production, which indirectly stimulates ornithine decarboxylase activity (ODC). Additionally L-arginine can be metabolized to L-ornithine via ODC, the rate-limiting enzyme in polyamine production. Polyamines are important for proliferation and differentiation of immune cells. We propose that ODC is increased with L-arginine supplementation. Mice were fed a 2% L-arginine supplemented diet (n=13) or a control diet (n=12). After two weeks of supplementation the spleen was harvested and the lymphocytes were isolated and stimulated with phytohemagglutinin (10 µg/ml). Eighteen hours post-stimulation lymphocyte proliferation and ODC activity were measured. The mean ODC activity (± SEM) was 62.1 ± 17.3 pmole/hr/mg of protein in the 2% L-arginine group and 59.8 ± 10.2 pmole/hr/mg of protein in the control group. Splenocyte proliferation was 15325 ± 2792 disintegrations per minute (DPM) in the 2% L-arginine group and 12940 ± 1762 DPM in the control group. No significant difference was detected in ODC activity or splenocyte proliferation between the two diet groups. These data suggest that L-arginine supplementation does not enhance ODC activity or splenocyte proliferation.

INTRODUCTION

L-arginine is considered a conditionally essential amino acid in the young and those suffering from trauma [1]. Supplemental dietary L-arginine is known to increase thymic weight and cellularity [2]. A possible mechanism for these effects may be related to L-arginine-mediated growth hormone secretion [3]. Growth hormone stimulates the production of insulin-like growth factor 1 (IGF-1), which in turn stimulates ODC activity [4]. Additionally, L-arginine is metabolized to L-ornithine, the substrate for ODC and polyamine production. Polyamines are important for lymphocyte differentiation and proliferation.

In this study a 2% L-arginine diet or a balanced diet (control) were fed to mice. The rate-limiting enzyme in polyamine production, ODC and T-cell proliferation were measured.

MATERIALS AND METHODS

Subjects and Diets


Ten-month-old CB6F1 (BALB/c x C57BL/6, National Institute of Aging, Bethesda, MD) mice were housed in groups of two mice per cage in a room with constant temperature of 22º C and a twelve-hour light/dark cycle. After a seven-day acclimation period the mice were weighed and randomly divided into two groups. One group was fed a balanced AIN93M diet [5] and served as a control for the experiment; the other group received an AIN93M diet supplemented with 2% L-arginine (diets prepared by Harlan Teklad, Madison WI).

Sample Collection

After two weeks on the experimental and control diets, the mice were euthanized with halothane. Their spleens were removed and placed in 5 ml of RPMI wash (RPMI-1640 supplemented with 50 U/ml penicillin, 50 mg/ml streptomycin, 50 mM 2-Mercaptoethanol, 25 mM N-(2-Hydroxyethyl) piperazine-N_-(2-ethanesulfonic acid) and 2 mM L-glutamine).

Sample Isolation

Splenocytes were isolated from the spleen using the following method. Spleens were placed in 100 µm cell strainer and splenocytes were pushed through the strainer into a 50 ml conical tube using the plunger from a 3 cc syringe. The strainer was washed with RPMI wash until cleared of all cells. The cell suspension was then bought up to a final volume of 20 ml with RPMI wash, and centrifuge for 10 minutes at 350 x g.

After centrifugation the supernatant was discarded and the red blood cells were lysed by adding 2 ml of cold 0.034 M NaCl, mixing gently for exactly 1 minute, and adding 2 ml of cold 0.0274 NaCl. The cell suspension was centrifuged for 10 minutes at 200 x g. After this centrifugation supernatant was discarded and the cells were washed two more times in RPMI wash and centrifuged for 10 minutes at 350 x g.

After the final wash, the cells were quantified and bought to a final concentration of 4.0 x 106 cells/ml in RPMI complete (RPMI wash supplemented with 10% Fetal calf serum).

Preparation of Sample for ODC Assay


Stimulated and unstimulated splenocytes cultures for ODC activation were plated in a 6-well flat bottom culture plate at a concentration of 2.0 x 106 cells/ml with (stimulated) and without (unstimulated) phytohemagglutinin (10 mg/ml). Cells were incubated for 18 hours at 37ºC in 95% humidity and 5% CO2.

After 18-hour incubation, plates were scraped and rinsed with phosphate buffer saline (PBS) to remove cells and centrifuged for 10 minutes at 350 x g. After centrifugation supernatant was discarded and the cells were washed two more times with PBS and centrifuged for 10 minutes at 350 x g. Upon the final wash the cell pellet was re-suspended in 450 ml of ODC buffer (1 mM Tris(hydromethyl)aminomethane , 1 mM ethylenediaminetetraacetic acid , 5 mM dithiothreitol and 50 µM pyridoxal-5'-phosphate) and store at -80ºC for a maximum of three days.

ODC Assay


The following method was adapted from Langkamp-Henken et al. [6]. Samples prepared above were thawed and refrozen for two cycles, sonicated for 20 seconds and centrifuged for 10 minutes at 11,750 x g in 4ºC. A 400 µl aliquot of the supernatant was incubated for 30 minutes with 4.2 nmol DL-[1-14C]L-ornithine (47.7 mCi/mmol, New England Nuclear, Boston, MA). The reaction was terminated with the addition of 400 µl of 10% trichloroacetic acid. ODC activity was measured from the liberation of 14CO2 from the DL-[1-14C]L-ornithine, which was captured on filter paper that was pre-treated with 20 µl of 2 N NaOH and air dried. Filter paper was placed in liquid scintillation fluid. Radioactivity was counted and expressed as DPM. The remainder of supernatant was analyzed for protein concentration using Bradford method [7] (Bio-Rad, Hercules, CA), results expressed as mg/ml.

Splenocyte Proliferation Assay

Stimulated and unstimulated splenocyte cultures for proliferation were plated in a 96-well round bottom culture plate at a concentration of 4.0 x 105 cells/well with (stimulated) and without (unstimulated) phytohemagglutinin (10 µg/ml). Cells were incubated with 1 µCi/well of 3H-thymidine (20 Ci/mmol Dupont-NEN, Boston, MA) for 18 hours at 37ºC in 95% humidity and 5% CO2. After an 18-hour incubation the cells were harvested onto glass filer paper and placed in liquid scintillation fluid. Radioactivity was counted and expressed as DPM from stimulated cultures minus unstimulated cultures for each mouse.

Statistical Analysis

ODC activity and splenocyte proliferation were compared between diet groups using an unpaired t-test. Comparison was made using Instat (version 1.14). Only p Ä 0.05 was considered significant. All data reported as means ± SEM.

RESULTS

The mean ODC activity in the 2% L-arginine and control group is shown in figure 1. ODC activity was only present in stimulated cultures (data not shown), but was not different between diet groups in stimulated cultures. The mean splenocyte proliferation in the 2% L-arginine and control group is shown in figure 2. No significant difference was detected in splenocyte proliferation between the two diet groups.

Figure 1. No significant difference was detected in ODC activity between the diet groups.

Figure 1. No significant difference was detected in ODC activity between the diet groups.

Figure 2. No significant difference was detected in splenocyte proliferation between the diet groups.

Figure 2. No significant difference was detected in splenocyte proliferation between the diet groups.

DISCUSSION

L-arginine stimulates lymphocyte proliferation [2]. This effect may be due to L-arginine stimulated growth hormone production and the indirect stimulation of ODC activity. Additionally L-arginine can be metabolized to L-ornithine via ODC, the rate-limiting enzyme in polyamine production. Polyamines are important for proliferation and differentiation of immune cells. We proposed that ODC activity would be increased with L-arginine supplementation. Results from this study failed to show a difference in ODC activity or proliferation of splenocytes from mice fed an 2% L-arginine supplemented versus a control diet.

One explanation for these negative results may be that the 2% L-arginine supplemented diet may not have been optimal to stimulate growth hormone secretion. Preliminary, unpublished data from our lab shows no significant difference in plasma L-arginine levels between 2% L-arginine-supplemented and unsupplemented mice. In a diet study conducted by Saito et al. on immune compromised guinea pigs, L-arginine was supplemented at 0, 1, 2 and 4% of total energy intake. Immune parameters (i.e. delayed-type hypersensitivity) were optimal and mortality rates were lowest with 2% L-arginine supplementation [8]. Based on this study, 2% L-arginine supplementation was selected for the present study. However in studies using rats, a 1% L-arginine diet was optimal for enhancing thymic weight and lymphocyte proliferation [2, 9]. Differences in optimal L-arginine supplementation may be due to variation in uptake and utilization among species and / or differences in measured immune parameters.

In conclusion a 2% L-arginine diet did not enhance splenocyte ODC activity or proliferation. Future studies will determine the optimal level of L-arginine supplementation required to increase plasma concentrations of L-arginine and growth hormone and enhance lymphocyte proliferation

REFERENCES

  1. Visek, W.J., Arginine needs, physiological state and usual diets. A reevaluation. J Nutr, 1986. 116(1): p. 36-46.
  2. Barbul, A., et al., Thymic stimulatory actions of arginine. JPEN J Parenter Enteral Nutr, 1980. 4(5): p. 446-9.
  3. Merimee, T.J., et al., Plasma growth hormone after arginine infusion. Clinical experiences. N Engl J Med, 1967. 276(8): p. 434-9.
  4. Torring, N., et al., Systemic administration of insulin-like growth factor I (IGF-I) causes growth of the rat prostate. J Urol, 1997. 158(1): p. 222-7.
  5. Reeves, P.G., F.H. Nielsen, and G.C. Fahey, Jr., AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr, 1993. 123(11): p. 1939-51.
  6. Langkamp-Henken, B., et al., Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells. Biochim Biophys Acta, 1998. 1425(2): p. 337-47.
  7. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976. 72: p. 248-54.
  8. Saito, H., et al., Metabolic and immune effects of dietary arginine supplementation after burn. Arch Surg, 1987. 122(7): p. 784-9.
  9. Barbul, A., et al., Arginine: a thymotropic and wound-healing promoting agent. Surg Forum, 1977. 28: p. 101-3.

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