Effect of Long-Term Treatment of L-Ornithine on Visual Function and Retinal Histology in the Rats

Abstract

L-Ornithine is a non-proteinogenic amino acid, abundant in freshwater clams and commercially available as an oral nutritional supplement. L-Ornithine is metabolized by ornithine-δ-aminotransferase. Deficiency of this enzyme causes gyrate atrophy of the choroid and retina, an autosomal recessive hereditary disease characterized by the triad of progressive chorioretinal degeneration, early cataract formation, and type II muscle fiber atrophy, with hyperornithinemia. However, it is unknown whether long-term L-ornithine supplementation affects visual function and retinal histology. The aim of the present study is to determine the effect of long-term supplementation of excess amounts of L-ornithine on visual function and retinal histology in rats. Male Brown Norway rats at six weeks of age were allowed free access to chow containing 4% (w/w) L-ornithine (the high ornithine diet) or that containing 4% (w/w) casein (the control diet) for 49 weeks. The dose of L-ornithine calculated from the food intake was approximately 0.8 g/d/animal, which was 100 times higher than the recommended dose for healthy humans. The amplitude of the a-wave of the scotopic rod-cone electroretinogram and the number of cells in the ganglion cell layer in the L-ornithine-treated group were larger than those in the control group 49 weeks after initiating the test diet. No functional or histological damage to the retina was seen up to 49 weeks after the start of the high-ornithine diet. The present study demonstrated that long-term supplementation of very high doses of L-ornithine for at least 49 weeks did not induce retinal damage.

L-Ornithine (L-Orn) is one of non-proteinogenic amino acids, abundantly contained in the freshwater clam, the enoki mushroom and the cheese, and commercially available as an oral nutritional supplement. L-Orn is a central part of the urea cycle, that allows for the disposal of excess nitrogen by conversion of ammonia into urea in the liver.^1,2) A recent controlled clinical study demonstrated that L-Orn reduces blood ammonia levels by increasing urea synthesis.²⁾ Some physicians have been tried to administer L-Orn to the patients of hyperornithinaemia–hyperammonaemia–homocitrullinuria (HHH) syndrome³⁾ for detoxification of ammonia by restoration of the intramitochondrial L-Orn pool.^4,5) L-Orn has been reported to inhibit physical fatigue by modulating lipid metabolism, and to promote excretion of ammonia in humans.⁶⁾ Some nutritionists and physicians recommend taking L-Orn supplements in order to attenuate physical fatigue.⁶⁾ In addition, several findings have indicated the beneficial effects of L-Orn, such as promoting growth hormone release,^7,8) improving pressure-induced decubitus ulcer,⁹⁾ and wound-healing effects.¹⁰⁾ These observations indicate the possibility that extensive use of L-Orn as a functional food counteract fatigue and skin damages. L-Orn is partially converted to pyrroline-5-carboxylate, which is further metabolized to L-proline by pyrroline 5-carboxylate reductase, by L-ornithine : 2-oxo-acid aminotransferase (OAT).¹¹⁾ L-Proline is essential for the synthesis of many structural proteins.

In contrast, some ophthalmic researchers believe that high concentration of L-Orn is specifically toxic to the retinal pigment epithelium (RPE),¹²⁾ because OAT deficiency results in gyrate atrophy (GA) of the choroid and retina, a rare autosomal recessive genetic disorder that induces degenerative retinopathy with an increased plasma level of L-Orn.^13,14) Wang et al.¹⁵⁾ reported that RPE were the initial site that exhibited pathological changes in OAT-deficient (Oat^−/−) mice, a mouse model for GA. They also showed that Oat^−/− mice on a standard diet exhibited an increased plasma level of L-Orn (1300 µM), reduced electroretinogram (ERG) amplitude and severe retinal degeneration, whereas Oat^−/− mice on an arginine-restricted diet had a decreased L-Orn level (100–200 µM), normal ERG amplitudes and no retinal degeneration.¹³⁾ However, another mouse model for GA by dosing with 5-fluoromethylornithine, a selective inhibitor of OAT, demonstrated normal visual function and no ophthalmic toxicity, although the L-Orn level in the eye rose ten times.¹⁶⁾ In some patients, a chronic reduction of the plasma level of L-Orn to near the normal level led to a modest improvement and subsequent stabilization of visual function.^17,18) In other patients, enlargement of the atrophic area was found despite biochemical control of the plasma level of L-Orn.^13,19) Furthermore, cases of GA-like traits have been reported without any increase in the plasma level of L-Orn.^20,21) Thus, it has been unclear whether L-Orn accumulation in the plasma is a direct cause of RPE degeneration in GA. In addition, it has been unknown whether long-term L-Orn supplementation affects visual function and retinal histology.

The aim of the present study is to determine the safety of long-term supplementation of excess amount of L-Orn on visual function and retinal histology in the rats.

MATERIALS AND METHODS

Animals

Experimental procedures conformed to the Regulations for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kitasato University. Male Brown Norway rats at six weeks of age were purchased from Charles River Japan (Yokohama, Japan). The environment of the animal room was kept at 25°C with a 12 h : 12 h light–dark cycle. All animals were fed and watered ad libitum.

Study Design

Before starting to feed the test diet, ERG was taken from all of the rats as described below. The rats were allowed free accesses to the rat chow containing 4% (w/w) L-Orn (the high Orn diet) or that containing 4% (w/w) casein (the control diet) for 49 weeks. The chow containing 4% amino acid was previously used in the amino acid safety tests for 12 weeks, and the dose of amino acid is almost maximum dose for the rats in the safety test for one year.^22–24) ERG and blood samples were taken from all of the rats 16, 30, and 49 weeks after starting to feed the test diet. After measuring ERG and collection of blood samples at 49 weeks, the rats were sacrificed with an overdose of sodium pentobarbital, and both eyes were enucleated for histological analysis.

Measurement of Plasma L-Orn Concentration

In order to determine the plasma concentration of L-Orn, blood samples were collected into heparinized tubes, and the plasma was separated by centrifugation. The plasma was deproteinized with sulfosalicylic acid (50 mg/mL plasma). Amino acid concentrations were quantified using an amino acid analyzer (JLC-500/V: JEOL, Tokyo, Japan) as described previously.²⁵⁾

Electroretinography

Scotopic ERG was recorded from animals that had been dark-adapted overnight. A contact lens electroencephalogram electrode with a white light-emitting diode (Mayo, Inazawa, Japan) was placed in contact with the corneal surface and a reference electrode was placed subcutaneously on the animal’s forehead. A grounding electrode was also placed on the ipsilateral ear. The cornea was intermittently irrigated with a balanced salt solution with Hydroxyethyl cellulose (SCOPISOL®, Senju Seiyaku, Osaka, Japan) to maintain adequate electrical contact and to prevent exposure keratopathy. Long-term pupillary dilation was achieved using topical 1% atropine sulfate (Nihon Tenganyaku, Nagoya, Japan). All electrode placements and eye drop instillations were performed in dim red light so as not to reverse any dark adaptation. Responses to white light flashes (1000 cd/m², 3 ms) from a photostimulator (LS-W, Mayo) were recorded and measured by PowerLab (AD Instruments, Castle Hill, New South Wales, Australia). The a- and b-wave amplitudes were analyzed using scope software (AD Instruments).

After recording the scotopic ERGs, photopic ERGs were recorded from animals that had been light adapted for at least 10 min. A contact lens electroencephalogram electrode, a reference electrode, and a grounding electrode were placed, as described above for scotopic ERG recordings. Responses to white light flashes (1000 cd/m², 3 ms) from a photostimulator (LS-W, Mayo) were recorded and measured by PowerLab (AD Instruments). Background light intensity was 25.1 cd/m² for photopic recordings and b-wave amplitude was analyzed by scope software (AD Instruments).

Histological Evaluation

Histological evaluation methods have been described previously.^26–30) Briefly, animals were euthanized with an overdose of sodium pentobarbital. Both eyes were enucleated and fixed with a Davidson solution (37.5% ethanol, 9.3% paraformaldehyde, 12.5% acetic acid) for 24 h at room temperature. Fixed eyes were dissected through the optic nerve head in the vertical meridian with a microtome blade (PATH BLADE+PRO by Kai, Matsunami Glass, Kishiwada, Japan) and embedded in paraffin after the lens had been removed. We used a microtome (HM325, Microm International, Walldorf, Germany) and a microtome blade (PATH BLADE+PRO by Kai, Matsunami Glass) to make 4-µm thickness, horizontal sections through the optic nerve head. Sections were stained with hematoxylin and eosin and examined for morphometry. Using a light microscope (Optiphot-2, Nicon, Tokyo, Japan), the total number of the cells in the retinal ganglion cell layer (GCL) was manually counted in a region beginning 1 mm from the center of the optic nerve head and ending 1.25 mm from the center of the optic nerve head (for a retinal length of 0.25 mm). Digital photographs [digital camera (Senamal, Micronet, Kawaguchi, Japan) connected to a light microscope] were taken so that ca. 0.25 mm of retina appeared in each photograph, with sections ca. 1 mm from the center of the optic nerve head chosen. The thickness of the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), and the outer nuclear layer (ONL) was then measured.

Statistical Analyses

All data are presented as mean±standard error of the mean (S.E.M.). Student’s t-test was used to compare the means of two groups. Tukey–Kramer test was used for multiple comparisons. Differences were considered to be statistically significant if p<0.05.

RESULTS

Changes of Body Weight, Food Intake and Plasma L-Orn Concentration

There was no significant difference in the body weight at the end the experimental period (Fig. 1A) and daily food intake throughout the experimental period (Fig. 1B) between the control diet group and the High Orn diet group. The mean food intake during the experimental period in the control diet group was 18.8±1.1 g/d/animal, and that in the high Orn diet group was 19.0±1.2 g/d/animal. The dose of L-Orn calculated from the food intake was approximately 0.8 g/d/animal. The plasma L-Orn concentration in the high Orn diet group was higher than that in the control diet group 16, 30 and 49 weeks after starting to feed the test diet (Fig. 1C).

Fig. 1. Effects of the Rat Chow Containing 4% (w/w) Casein (Control) and That Containing 4% (w/w) L-Ornithine (L-Orn) on Body Weight of the Rats before Starting to Feed the Test Diet (Pre) and at the End of Experiment (49 Weeks) (A), on Food Intake during the Experimental Period (B), and on Plasma L-Orn Levels 16, 30 and 49 Weeks after Starting to Feed the Test Diet (C)

The data represent the means±S.E.M. of five (Pre), nine (Control) or 11 (L-Orn) rats per group. * p<0.05 vs. Control.

Electroretinography

There was no significant difference between the control diet group and high Orn diet group in the amplitudes of scotopic rod-cone a-wave and b-wave, and photopic cone b-wave, before starting to feed the test diet (Pre in Fig. 2). The amplitude of scotopic rod-cone a-wave in the high Orn diet group was significantly higher than that in the control diet group, 16 and 49 weeks after staring to feed the test diet (Fig. 2A). The similar tendency was observed 30 weeks after staring to feed the test diet. The amplitude of scotopic rod-cone b-wave in the high Orn diet group tended to be higher than that in the control diet group, 16, 30 and 49 weeks after starting to feed the test diet (Fig. 2B). In contrast, there is no significant difference in the amplitude of photopic cone b-wave between the two groups, 16, 30 and 49 weeks after starting to feed the test diet (Fig. 2C).

Fig. 2. A: Typical Scotopic Rod-Cone and Photopic Cone Electroretinograms Recorded from the Rats Fed the Control Diet (Control) or the High L-Ornithine (L-Orn) Diet before Starting to Feed the Test Diet and 16, 30 and 49 Weeks after Starting to Feed the Test Diet B–D: The a-Wave (B) and b-Wave (C) Amplitudes of Scotopic Rod-Cone Electroretinograms and the b-Wave (D) Amplitudes of Photopic Cone Electroretinograms from the Rats before Starting to Feed the Test Diet (Pre) and 16, 30 and 49 Weeks after Starting to Feed the Test Diet

The data represent the means±S.E.M. of five (Pre), nine (Control) or 11 (L-Orn) rats per group. * p<0.05 vs. Control

E: Histological Analysis of Retinas from before Starting to Feed the Test Diet (Pre 6 Weeks) and 49 Weeks after Starting to Feed the Rat Chow Containing 4% (w/w) Casein (Control 49 Weeks) and That Containing 4% (w/w) L-Ornithine (L-Orn 49 Weeks)

All sections were stained with hematoxylin-eosin and photographed at the same magnification (200×). Scale bar represents 50 µm.

F: The Number of the Cells in the Retinal Ganglion Cell Layer (Number of RGC) before Starting to Feed the Test Diet (Pre) and 49 Weeks after Starting to Feed the Rat Chow Containing 4% (w/w) Casein (Control) and That Containing 4% (w/w) L-Ornithine (L-Orn)

The data represent the means±S.E.M. of five (Pre), nine (Control) or 11 (L-Orn) rats per group. * p<0.05 between the indicated pairs.

G: The Thickness of the Retinal Layers before Starting to Feed the Test Diet (Pre) and 49 Weeks after Starting to Feed the Rat Chow Containing 4% (w/w) Casein (Control) and That Containing 4% (w/w) L-Ornithine (L-Orn)

IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. The data represent the means±S.E.M. of five (Pre), nine (Control) or 11 (L-Orn) rats per group. * p<0.05 vs. Pre.

Histological Analysis

The number of the cells in GCL 49 weeks after starting the test diet was significantly smaller than that before starting to feed the test diet (Pre) (Figs. 2D, E). High Orn diet significantly reduced the decrease of the number of the cells in GCL 49 weeks after starting to feed the test diet. The thickness of IPL, INL, OPL and ONL 49 weeks after starting to feed the test diet was smaller than that before starting to feed the test diet (Pre) (Figs. 2D, F). There is no significant difference in the thickness of IPL, INL, OPL and ONL between the control diet group and the high Orn diet group 49 weeks after starting to feed the test diet.

DISCUSSION

In the present study, we determined the effect of long-term supplementation of excess amount of L-Orn on visual function and retinal histology in the rats. The result in the present study indicated that long-term supplementation of L-Orn for at least 49 weeks did not cause accumulation of L-Orn in the plasma, nor induce the retinal damages at the dose used in the present study (approximately 0.8 g/d/animal) in the rat. Instead, long-term supplementation of excess amount of L-Orn augmented the amplitude of the a-wave of the scotopic rod-cone electroretinogram, and reduced the decreases of the number of the cells in the ganglion cell layer by aging. The present study suggested that supplementation of L-Orn would not affect the retinal function and histology in healthy subjects.

In the present study, the dose of L-Orn calculated from the food intake was approximately 0.8 g/d/animal. In human, the recommended dose of L-Orn is 1.5 g/d. Calculated from the body weight, the dose of L-Orn in the present study was 100 times higher than that recommended in human. The plasma L-Orn concentration in the high Orn diet group was ranging from 200 to 300 µM, and that in GA patients reported in the previous studies was ranging from 500 to 1000 µM, much higher than that in the high Orn diet group.³¹⁾ The present study demonstrated that long-term supplementation of very high dose of L-Orn did not induce accumulation of L-Orn that was found in GA patients, suggesting that supplementation of L-Orn at the recommended dose is safe in healthy subjects.

Very high concentration of L-Orn has been reported to induce lesions of RPE. Kuwabara et al.¹²⁾ reported that intravitreal injection of L-Orn (1 M, 10 µL) led to edema and degeneration of RPE in the rats. Because the volume of the vitreous body of the rats is approximately 50 µL,³²⁾ the concentration of L-Orn in the vitreous body is approximately 17 mM. Ando et al.³³⁾ reported that primary cultured bovine RPE were damaged severely by 10 mM L-Orn. As these concentrations of L-Orn are much higher than the plasma L-Orn concentration in GA patients, it is difficult to conclude that L-Orn causes RPE damages in GA patients. The present study demonstrated that 200–300 µM L-Orn did not induce retinal damages functionally and histologically. In some GA patients, enlargement of the atrophic area was found without any increase in the plasma level of L-Orn.^13,18–20) In addition, L-Orn-loaded GA relatives and patients of HHH syndrome did not develop retinal degeneration, although the plasma concentration of them were 600–1100 µM.³¹⁾ Thus, it is unlikely that only L-Orn accumulation in the plasma would be a direct cause of RPE degeneration in GA.

In the present study, although the amplitudes of scotopic and photopic ERG were not much affected by aging, the number of the cells in the ganglion cell layer and thickness of retinal layers at the end of the experiment (55 weeks old) were smaller than those before starting to feed the test diet (6 weeks old). These results are consistent with the results of the previous reports using Wistar³⁴⁾ and Wistar/ST rats.³⁵⁾ It is widely known that the amplitude of the a-wave in rod cone electroretinogram reflects the function of the rod and cone cells. The function of the retinal ganglion cells very little affect the amplitude of electroretinogram. The amplitude of a-wave is not always consistent with the thickness of the outer nuclear layer, i.e., the number of the nuclei of the rod and cone cells. For example, in a mouse model of retinitis pigmentosa, although there were two or three rows in the outer nuclear layer, the a-wave was almost flat.³⁶⁾ We speculate that L-Orn may conserve the function of the rod and cone cells in the aged retina.

El-Sayyad et al.³⁴⁾ demonstrated that depletion of the activities of anti-oxidant enzymes, such as superoxide dismutase, catalase and glutathione-S-transferase, occurred in the aged retina. Therefore, it is possible that the increases of reactive oxygen species cause the decrease of the number of the cells in the ganglion cell layer. As far as we know, there is no report showing that L-Orn itself has an anti-oxidant activity. However, L-Orn is a central part of the urea cycle, which allows for the disposal of excess nitrogen by conversion of ammonia into urea in the liver. It is well-known that the availability of cycle intermediates is one of the factors to control the urea cycle. When the level of L-Orn in the liver is high, the urea cycle proceeds more rapidly. Therefore, a high level of L-Orn will accelerate the detoxication of ammonia, one of neurological toxins. L-Orn is known to be converted to L-arginine and L-citrulline in the urea cycle. However, the plasma level of L-arginine and L-citrulline was not changed in the present study (data not shown), suggesting that L-arginine and L-citrulline is not likely involved in the effects of L-Orn found in the present study. L-Orn is known to be metabolized to polyamines, such as spermine and spermidine.³⁷⁾ Spermine is reported to scavenge free radicals,³⁸⁾ and to modulate the activity of N-methyl-D-aspartic acid receptor,³⁹⁾ that is involved in the mechanism of neuronal cell death in various pathophysiologic situations. In the present study, we cannot clarify the change in the plasma levels of the polyamines, and whether the polyamines have the similar effects to L-Orn. Further analyses are clearly needed to clarify the underlying mechanisms.

In conclusion, the present study first demonstrated that long-term supplementation of very high dose of L-Orn, which is 100 times higher than the recommended dose for human, for at least 49 weeks did not induce the retinal damages in the rats. The present study suggests that supplementation of L-Orn at the recommended dose is safe in healthy subjects.

Acknowledgment

Kunio Ishii received a research Grant from Kyowa Hakko Bio Co., Ltd. Masahiko Morita is an employee of Kyowa Hakko Bio Co., Ltd.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Li H, Meininger CJ, Hawker JR Jr, Haynes TE, Kepka-Lenhart D, Mistry SK, Morris SM Jr, Wu G. Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells. Am. J. Physiol. Endocrinol. Metab., 280, E75–E82 (2001).
• 2) Kowalski P, Bieniecki M. Pharmacokinetlcs and bioavailability study of L-ornithine–L-aspartate in healthy volunteers: acomparative study of two oral formulations. J. Pharm. Biomed. Anal., 41, 1061–1064 (2006).
• 3) Shih VE, Efron ML, Moser HW. Hyperornithinemia, hyperammonemia, and homocitrullinuria. A new disorder of amino acid metabolism associated with myoclonic seizures and mental retardation. Am. J. Dis. Child., 117, 83–92 (1969).
• 4) Palmieri F. Diseases caused by defects of mitochondrial carriers: a review. Biochim. Biophys. Acta, 1777, 564–578 (2008).
• 5) Torisu H. Hyperornithinemia–hyperammonemia–homocitrullinuria syndrome. Shonika-Shinryo, 72(Suppl.), 352 (2009).
• 6) Sugino T, Shirai T, Kajimoto Y, Kajimoto O. L-Ornithine supplementation attenuates physical fatigue in healthy volunteers by modulating lipid and amino acid metabolism. Nutr. Res., 28, 738–743 (2008).
• 7) Evain-Brion D, Donnadieu M, Roger M, Job JC. Simultaneous study of somatotrophic and corticotrophic pituitary secretions during ornithine infusion test. Clin. Endocrinol., 17, 119–122 (1982).
• 8) Bucci L, Hickson FJ Jr., Pivarnik JM, Wolinsky I, McMahon JC, Turner SD. Ornithine ingestion and growth hormone release in body builders. Nutr. Res., 10, 239–245 (1990).
• 9) Aoki M, Komatsu M, Ochiai M, Watanabe F, Igarashi M, Nonomura T, Tomita S, Umehara N, Nakai N, Morishita K. Healing effects of oral administration of L-citrulline or L-ornithine in a rat pressure-ulcer model—comparison with L-arginine—. Jpn. Pharmacol. Ther., 38, 807–816 (2010).
• 10) Shi HP, Fishel RS, Efron DT, Williams JZ, Fishel MH, Barbul A. Effect of supplemental ornithine on wound healing. J. Surg. Res., 106, 299–302 (2002).
• 11) Durante W, Johnson FK, Johnson RA. Arginase: acritical regulator of nitric oxide synthesis and vascular function. Clin. Exp. Pharmacol. Physiol., 34, 906–911 (2007).
• 12) Kuwabara T, Ishikawa Y, Kaiser-Kupfer MI. Experimental model of gyrate atrophy in animals. Ophthalmology, 88, 331–334 (1981).
• 13) Wang T, Steel G, Milam AH, Valle D. Correction of ornithine accumulation prevents retinal degeneration in a mouse model of gyrate atrophy of the choroid and retina. Proc. Natl. Acad. Sci. U.S.A., 97, 1224–1229 (2000).
• 14) Javadzadeh A, Gharabaghi D. Gyrate atrophy of the choroid and retina with hyper-ornithinemia responsive to vitamin B6: a case report. J. Med. Case Rep., 1, 27 (2007).
• 15) Wang T, Milam AH, Steel G, Valle D. A mouse model of gyrate atrophy of the choroid and retina. Early retinal pigment epithelium damage and progressive retinal degeneration. J. Clin. Invest., 97, 2753–2762 (1996).
• 16) Daune-Anglard G, Bonaventure N, Seiler N. Some biochemical and pathophysiological aspects of long-term elevation of brain ornithine concentrations. Pharmacol. Toxicol., 73, 29–34 (1993).
• 17) Valle D, Walser M, Brusilow SW, Kaiser-Kupfer M. Gyrate atrophy of the choroid and retina: amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet. J. Clin. Invest., 65, 371–378 (1980).
• 18) Kaiser-Kupfer MI, de Monasterio FM, Valle D, Walser M, Brusilow S. Gyrate atrophy of the choroid and retina: improved visual function following reduction of plasma ornithine by diet. Science, 210, 1128–1131 (1980).
• 19) Ohkubo Y, Ueta A, Ito T, Sumi S, Yamada M, Ozawa K, Togari H. Vitamin B6-responsive ornithine aminotransferase deficiency with a novel mutation G237D. Tohoku J. Exp. Med., 205, 335–342 (2005).
• 20) Mizuno K, Hayasaka S, Ishiguro S, Nakajima H, Yabata K, Watanabe S, Shiono M, Takahashi J, Tamai M, Fukuyo T, Tada K, Saito T, Kimura K. Studies on gyrate atrophy of the choroid and retina. Nippon Ganka Gakkai Zasshi, 85, 1864–1885 (1981).
• 21) Deutman AF, Sengers RC, Trybels JM. Gyrate atrophy of the choroid and retina with reticular pigmentary dystrophy and ornithine-ketoacid-transaminase deficiency. Int. Ophthalmol., 1, 49–56 (1978).
• 22) Wren AF, Cleary M, Frantz C, Melton S, Norris L. Ninety-day oral toxicity study of a grape seed extract (IH636) in rats. J. Agric. Food Chem., 50, 2180–2192 (2002).
• 23) Tsubuku S, Hatayama K, Mawatari K, Smriga M, Kimura T. Thirteen-week oral toxicity study of L-arginine in rats. Int. J. Toxicol., 23, 101–105 (2004).
• 24) Tsubuku S, Hatayama K, Mawatari K, Smriga M, Kimura T. Thirteen-week oral toxicity study of L-glutamine in rats. Int. J. Toxicol., 23, 107–112 (2004).
• 25) Moinard C, Nicolis I, Neveux N, Darquy S, Benazeth S, Cynober L. Dose-ranging effects of citrulline administration on plasma amino acids and hormonal patterns in healthy subjects: the Citrudose pharmacokinetic study. Br. J. Nutr., 99, 855–862 (2008).
• 26) Sakamoto K, Kawakami T, Shimada M, Yamaguchi A, Kuwagata M, Saito M, Nakahara T, Ishii K. Histological protection by cilnidipine, a dual L/N-type Ca²⁺ channel blocker, against neurotoxicity induced by ischemia–reperfusion in rat retina. Exp. Eye Res., 88, 974–982 (2009).
• 27) Sakamoto K, Hiraiwa M, Saito M, Nakahara T, Sato Y, Nagao T, Ishii K. Protective effect of all-trans retinoic acid on NMDA-induced neuronal cell death in rat retina. Eur. J. Pharmacol., 635, 56–61 (2010).
• 28) Sakamoto K, Ohki K, Saito M, Nakahara T, Ishii K. Histological protection by donepezil against neurodegeneration induced by ischemia–reperfusion in the rat retina. J. Pharmacol. Sci., 112, 327–335 (2010).
• 29) Sakamoto K, Ohki K, Saito M, Nakahara T, Ishii K. Small molecule cyclin-dependent kinase inhibitors protect against neuronal cell death in the ischemic–reperfused rat retina. J. Ocul. Pharmacol. Ther., 27, 419–425 (2011).
• 30) Sakamoto K, Suzuki Y, Kurauchi Y, Mori A, Nakahara T, Ishii K. Hydrogen sulfide attenuates NMDA-induced neuronal injury via its anti-oxidative activity in the rat retina. Exp. Eye Res., 120, 90–96 (2014).
• 31) Hayasaka S, Kodama T, Ohira A. Retinal risks of high-dose ornithine supplements: a review. Br. J. Nutr., 106, 801–811 (2011).
• 32) Berkowitz BA, Lukaszew RA, Mullins CM, Penn JS. Impaired hyaloidal circulation function and uncoordinated ocular growth patterns in experimental retinopathy of prematurity. Invest. Ophthalmol. Vis. Sci., 39, 391–396 (1998).
• 33) Ando A, Ueda M, Uyama M, Masu Y, Okumura T, Ito S. Heterogeneity in ornithine cytotoxicity of bovine retinal pigment epithelial cells in primary culture. Exp. Eye Res., 70, 89–96 (2000).
• 34) El-Sayyad HI, Khalifa SA, El-Sayyad FI, Mousa SA, Mohammed EA. Analysis of fine structure and biochemical changes of retina during aging of Wistar albino rats. Clin. Experiment. Ophthalmol., 42, 169–181 (2014).
• 35) Kiyosawa I, Aoki M, Imamura T, Nagasawa T, Ito T, Sibata N, Naito J, Saito TR, Takahashi KW. Age-related changes in the retina of WBN/Kob rats—a pathological study. Exp. Anim., 43, 651–661 (1995).
• 36) Sakamoto K, McCluskey M, Wensel TG, Naggert JK, Nishina PM. New mouse models for recessive retinitis pigmentosa caused by mutations in the Pde6a gene. Hum. Mol. Genet., 18, 178–192 (2009).
• 37) Li H, Meininger CJ, Hawker JR Jr, Haynes TE, Kepka-Lenhart D, Mistry SK, Morris SM Jr, Wu G. Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells. Am. J. Physiol. Endocrinol. Metab., 280, E75–E82 (2001).
• 38) Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA Jr. The natural polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. U.S.A., 95, 11140–11145 (1998).
• 39) Masuko T, Kuno T, Kashiwagi K, Kusama T, Williams K, Igarashi K. Stimulatory and inhibitory properties of aminoglycoside antibiotics at N-methyl-D-aspartate receptors. J. Pharmacol. Exp. Ther., 290, 1026–1033 (1999).